Progressive Proximal-to-Distal Reduction in Expression of the Tight Junction Complex in Colonic Epithelium of Virally-Suppressed HIV+ Individuals

There is no author summary for this article yet. Authors can add summaries to their articles on ScienceOpen to make them more accessible to a non-specialist audience.

Abstract

Effective antiretroviral therapy (ART) dramatically reduces AIDS-related complications, yet the life expectancy of long-term ART-treated HIV-infected patients remains shortened compared to that of uninfected controls, due to increased risk of non-AIDS related morbidities. Many propose that these complications result from translocated microbial products from the gut that stimulate systemic inflammation – a consequence of increased intestinal paracellular permeability that persists in this population. Concurrent intestinal immunodeficiency and structural barrier deterioration are postulated to drive microbial translocation, and direct evidence of intestinal epithelial breakdown has been reported in untreated pathogenic SIV infection of rhesus macaques. To assess and characterize the extent of epithelial cell damage in virally-suppressed HIV-infected patients, we analyzed intestinal biopsy tissues for changes in the epithelium at the cellular and molecular level. The intestinal epithelium in the HIV gut is grossly intact, exhibiting no decreases in the relative abundance and packing of intestinal epithelial cells. We found no evidence for structural and subcellular localization changes in intestinal epithelial tight junctions (TJ), but observed significant decreases in the colonic, but not terminal ileal, transcript levels of TJ components in the HIV+ cohort. This result is confirmed by a reduction in TJ proteins in the descending colon of HIV+ patients. In the HIV+ cohort, colonic TJ transcript levels progressively decreased along the proximal-to-distal axis. In contrast, expression levels of the same TJ transcripts stayed unchanged, or progressively increased, from the proximal-to-distal gut in the healthy controls. Non-TJ intestinal epithelial cell-specific mRNAs reveal differing patterns of HIV-associated transcriptional alteration, arguing for an overall change in intestinal epithelial transcriptional regulation in the HIV colon. These findings suggest that persistent intestinal epithelial dysregulation involving a reduction in TJ expression is a mechanism driving increases in colonic permeability and microbial translocation in the ART-treated HIV-infected patient, and a possible immunopathogenic factor for non-AIDS related complications.

Author Summary

While antiretroviral therapy for HIV-infected patients is remarkably effective in suppressing viral replication and preventing progression to AIDS, treated patients still have a shorter life expectancy due to increased risks for non-AIDS associated morbidities. Recent data showed that these complications are associated with chronic systemic inflammation, and it is hypothesized that bacterial products breaching the intestinal barrier may cause the inflammation. It is known that HIV induces persistent intestinal mucosal immunodeficiency, but evidence for structural damage to the intestinal epithelium is lacking in the antiretroviral-treated patient population. Here, we characterized the intestinal epithelial damage that leads to increased intestinal permeability in this population. We found that while the colonic epithelial layer is intact microscopically, intercellular tight junctions (TJ) are down-regulated at the transcriptional and translational levels. We observed further that TJ transcripts progressively decrease along the proximal-to-distal HIV gut. Concurrent alterations in the levels of non-TJ epithelial transcripts suggest that epithelial cells in the HIV gut are transcriptionally dysregulated. Our data provide evidence that TJ disruption is a novel mechanism for increasing colonic permeability in the antiretroviral-treated HIV patient, which may then result in systemic inflammation and associated complications.

Related collections

Most cited references 53

Record: found
Abstract: found
Article: not found

Alterations in the gut microbiota associated with HIV-1 infection.

Catherine A. Lozupone, Marcella Li, Thomas B. Campbell … (2013)

Understanding gut microbiota alterations associated with HIV infection and factors that drive these alterations may help explain gut-linked diseases prevalent with HIV. 16S rRNA sequencing of feces from HIV-infected individuals revealed that HIV infection is associated with highly characteristic gut community changes, and antiretroviral therapy does not consistently restore the microbiota to an HIV-negative state. Despite the chronic gut inflammation characteristic of HIV infection, the associated microbiota showed limited similarity with other inflammatory states and instead showed increased, rather than decreased, diversity. Meta-analysis revealed that the microbiota of HIV-infected individuals in the U.S. was most similar to a Prevotella-rich community composition typically observed in healthy individuals in agrarian cultures of Malawi and Venezuela and related to that of U.S. individuals with carbohydrate-rich, protein- and fat-poor diets. By evaluating innate and adaptive immune responses to lysates from bacteria that differ with HIV, we explore the functional drivers of these compositional differences. Copyright © 2013 Elsevier Inc. All rights reserved.

0 comments Cited 210 times – based on 0 reviews      Review now

Bookmark

Record: found
Abstract: found
Article: found

Is Open Access

Exposure to HIV-1 Directly Impairs Mucosal Epithelial Barrier Integrity Allowing Microbial Translocation

Aisha Nazli, Olivia Chan, Wendy Dobson-Belaire … (2010)

Introduction The mucosa presents a primary barrier against a multitude of micro-organisms present on the mucosal surfaces of the human body [1]. The intestinal and upper reproductive tract are lined by a continuous monolayer of columnar epithelial cells that is responsible for maintaining the physical and functional barrier to harmful microorganisms, such as bacteria and their products, including bacterial toxins as well as commensal organisms [2]–[4]. The preservation of the barrier function is dependent on the intactness of apical plasma membrane on the epithelial cells as well as the intercellular tight junctions. The disruption of the tight junctions can cause increased permeability, leading to “leakiness” such that normally excluded molecules can cross the mucosal epithelium by paracellular permeation, and could lead to inflammatory conditions in the mucosa. Various pathogenic organisms have developed strategies to either infect or traverse through the epithelial cells at mucosal surfaces, as part of the strategy to establish infection in the host. In fact, mucosal transmission account for majority of infections in humans [5]. Viruses such as rotavirus and astrovirus as well as bacteria such as enteropathogenic E. Coli and C. difficile are known to increase intestinal permeability by disrupting tight junctions, as part of their pathogenesis [6]–[9]. Increased permeability is also related to a number of other disease conditions that may or may not be related to infection by a pathogen. Crohn's disease, a chronic inflammatory condition of the intestines is characterized by defective tight junction barrier functions, manifested by increased intestinal permeability, although the etiology of the disease is not clearly understood [10]. HIV-1 infection is initiated primarily on mucosal surfaces, through heterosexual or homosexual transmission [1],[11]. In fact, mucosal transmission accounts for greater than 90% of HIV infection [12],[13]. A number of clinical studies have reported intestinal barrier dysfunction, especially during chronic stage of HIV infection [14]–[19]. However, the pathophysiologic mechanism associated with compromised barrier function and whether HIV-1 plays a direct role in this is still unclear. Currently, epithelial barrier defect during HIV-1 infection is thought to be a consequence of mucosal T cell activation following infection, which could lead to increased production of inflammatory cytokines [19],[20]. The intestinal barrier dysfunction has also been implicated as the cause of systemic immune activation during chronic phase of HIV infection, although a recent study has raised the possibility that this may not be universal phenomenon [20],[21]. Studies that have demonstrated immune activation propose this to be the main driving force for progressive immune failure leading to the immunodeficiency stage [22]–[24]. In these studies, HIV disease progression was shown to correlate with increased circulating level of LPS, considered an indicator of microbial translocation, in chronic HIV-infected individuals [25]. Interestingly, immune activation was observed in both the chronic as well as acute phase of HIV infection [25]. The source of or mechanism whereby microbial products could cross the epithelial barrier leading to immune activation have not been elucidated thus far. In the present study, we investigated the direct effects of HIV-1 exposure on intestinal and genital mucosal epithelia, where primary HIV-1 infection is frequently initiated. We show that in fact the impairment of epithelial barrier function can be a direct result of exposure to HIV-1. Using ex-vivo cultures of pure primary genital epithelium as well as an intestinal epithelial cell line, we show significantly decreased barrier functions and enhanced permeability that is not unique to the intestinal epithelium; similar increase in permeability was seen in the genital epithelium as well. Small amounts of both bacterial and viral translocation were seen following HIV-1 exposure. The mechanism appears to be mediated by increased production of inflammatory cytokines directly from the epithelial cells following exposure to HIV-1, including TNF-alpha, known to disrupt barrier functions. Further, we show that HIV-1 envelope protein gp120 was able to impair barrier functions in epithelial cells on its own. Neutralization of gp120 or exposure to HIV-1 lacking gp160 surface envelope glycoprotein did not have any effect on epithelial cells. These results provide strong evidence that exposure to HIV-1 may lead to impairment in barrier function of mucosal epithelium which could result both in translocation of HIV-1 and/or luminal bacteria that could serve as the source of immune activation during HIV-1 infection. Results Genital and intestinal epithelial monolayer transepithelial resistances (TER) are decreased following exposure to different strains of HIV-1 In order to study HIV-1 induced barrier defect in epithelial monolayers, HIV-1 (106 infectious viral units/ml) was added apically to confluent monolayers of differentiated primary female genital epithelial cells (ECs) or T84 intestinal epithelial cells grown in transwells. Transepithelial resistance (TER), a measure of epithelial monolayer integrity, was measured before and 24h post-infection and calculated as a percentage of pretreatment TER. Transepithelial resistances of primary endometrial epithelial monolayers exposed to various strains of HIV-1 were significantly reduced (p 0.05), while the wild type HIV showed significant decrease in TERs (p<0.01) (Figure 7B). This was further confirmed by intact ZO-1 staining seen in epithelial monolayers exposed to Env− HIV, similar to mock-treated cells (Figure 7C). Combined with the results from gp120 neutralizing antibody described above, these results indicate that the HIV-1 surface glycoprotein is responsible for disruption of epithelial barrier leading to increased permeability. HIV-1 exposure induces inflammatory cytokines in genital and intestinal epithelial monolayers Epithelial cells are known to secrete a variety of cytokines at constitutive levels. Many of these are upregulated or induced de novo in response to pathogens such as Neisseria gonorrhea [31]. Additionally, inflammatory cytokines have been shown to mediate enhanced permeability of intestinal epithelial cells [32]. We therefore examined the cytokine secretion profile of epithelial cells following HIV-1 exposure. Apical and basolateral supernatants of genital and intestinal monolayers were collected 24h post HIV-1 exposure and examined for presence of six cytokines known to be secreted by epithelial cells (Figure 8A–F). The T84 intestinal cell line constitutively secreted low levels of IL-10 and IL-1β. In comparison, the primary endometrial epithelial monolayers showed constitutive production of a larger array of cytokines, some of them in high amounts (IL-6, IL-8, MCP-1). Following HIV-1 exposure, there was a significant increase in production of TNF-α, IL-6, IL-8 and MCP-1 in T84 intestinal epithelial monolayers (Figures 8A–D). In primary endometrial ECs there was a significant increase in production of TNF-α, IL-6, MCP-1, IL-10, and IL-1β secretion after 24 hours of HIV-1 exposure (Figures 8A, B, E, F). 10.1371/journal.ppat.1000852.g008 Figure 8 Primary endometrial EC and T84 intestinal monolayers were exposed to HIV-1 (ADA, 106 infectious viral units/ml, p24 280ng/ml) and apical and basolateral supernatants were collected 24 hours post-exposure and assayed by Luminex multi-analyte kit for the following cytokines: (A) TNF-α (B) IL-6, (C) IL-8, (D) MCP-1, (E) IL-10, (F) IL-1β. *p<0.01, **p<0.001. Data shown is representative of three separate experiments from different tissues, each experiment had 3–5 replicate cultures for each experimental condition. HIV-1 mediated TER decrease is reversed by treatment with anti-TNF antibody Of the cytokines that showed increased production in genital and intestinal epithelial cells following HIV-1 exposure, TNF-α is well known to disrupt epithelial cell tight junction assembly and increase intestinal cell permeability [33]. Since TNF-α was significantly up-regulated following HIV-1 exposure in both genital and intestinal ECs, we neutralized TNF-α to see whether this would affect barrier function alterations. Confluent T84 intestinal epithelial monolayers were treated with TNF-α (20 ng/ml), TNF-α+anti-TNF antibody (mouse anti-human TNF-α , 25 µg/ml), TNF-α+mouse serum (control), HIV-1 alone, HIV-1+anti-TNF antibody (mouse anti-human TNF-α antibody, 25 µg/ml) and HIV-1+mouse serum (control). The TER measurements were taken prior to and 24 hours following the treatments. As expected both TNF-α and HIV-1 caused a significant drop in TER values compared to untreated control monolayers (Figure 9). When epithelial monolayers were pre-treated with anti-TNF-α antibody prior to treatment with TNF-α and HIV-1, the TER values did not decrease significantly over 24 hours of exposure. Incubation of monolayers with normal mouse serum did not show the same effect as the anti-TNF-α antibody. These results provide direct evidence that TNF-α secreted by epithelial cells in response to HIV-1 exposure contributed significantly to the disruption of barrier function in epithelial cells. 10.1371/journal.ppat.1000852.g009 Figure 9 Primary endometrial epithelial monolayers were exposed to TNF-α or HIV-1 alone; TNF-α or HIV-1 (ADA,106 infectious viral units/ml, p24 280ng/ml) in combination with anti-TNF-α neutralizing antibody; TNF-α or HIV-1 in combination with normal mouse serum for 24 hours. TER measurements were taken as a measure of change in permeability and presented as percentage of pre-treatment TER. Data shown is representative of two separate experiments, each experiment had 3–5 replicate cultures for each experimental condition. Increased permeability correlates with translocation of virus and bacteria across the epithelial monolayers To correlate barrier dysfunction with increased permeability to luminal antigens, we examined bacterial and viral translocation across the epithelial monolayers post-exposure to HIV-1. Intestinal epithelial monolayers grown to confluence were exposed to HIV-1. TNF-α was used as a positive control since it is known to disrupt tight junctions and increase permeability. Because direct exposure to TNF-α for prolonged period of time causes irreversible damage to epithelial cells, TNF-α treatment was limited to 6 hours prior to addition of non-pathogenic E. Coli to allow observation of bacterial translocation. Exposure time for HIV-1 was chosen at 6 hours (for comparison with TNF-α) and 24 hours (based on our results of maximum permeability with continued viability). Six hours after addition of E. Coli to the apical compartment, basolateral supernatants were collected and plated on LB agar and bacterial colonies were quantified. Transepithelial resistance measured prior to and 24 hours following treatments to determine if addition of E. Coli had any effect on TERs (Figure 10A). In HIV-1 exposed monolayers TER decreased significantly within 6h of exposure as expected; further reduction was seen at 24 hours. In comparison, HIV-1 unexposed monolayers only and those that were untreated except with E. Coli, TER values were maintained at 106% and 89% percent respectively, of pre-treatment TER values. Bacterial translocation was seen only in monolayers following 24hours of HIV-1 exposure and 6 hours of TNF-α treatment (Figure 10B). Bacterial translocation seen following 24 hours of HIV-1 treatment was about 50% of that seen following 6 hours of TNF treatment. No significant bacterial translocation was seen after 6h of HIV-1 exposure. 10.1371/journal.ppat.1000852.g010 Figure 10 Bacterial and viral translocation across mucosal epithelial monolayers following HIV-1 exposure. (A) Bacterial translocation was measured in T84 intestinal monolayers. Confluent monolayers were left untreated or treated for 6 hours with TNF-α (20ng/ml), E. coli (108 CFU/ml) , TNF-α (20ng/ml) +E. coli (108 CFU/ml), HIV-1 or HIV-1 (6 or 24 hours)+E. coli (108 CFU/ml). (A) TER measurements following various treatments in the presence or absence of E. Coli. * p<0.001. (B) Basolateral supernatants were collected and bacterial counts were done. (C) Viral translocation was determined in endometrial EC monolayers exposed to HIV-1 (ADA, 106 infectious viral units/ml, p24 280ng/ml) on the apical side. Basolateral supernatants were collected after different time intervals infectious and viral counts were done on TZM/b-l indicator cell line. Viral counts are depicted as percentage of inoculum added to the apical compartment of monolayers. Data shown is representative of two separate experiments, each experiment had 3–5 replicate cultures for each experimental condition. In a separate experiment, lipopolysaccaride (LPS) leakage in HIV-1 exposed endometrial monolayers was determined. LPS was added on apical side of HIV-1 exposed and control monolayers and one hour later basolateral supernatants were collected and LPS leakage was measured. The LPS levels in basolateral supernatants were increased by 47.3±0.922% in HIV-1 exposed monolayers in comparison with LPS leakage in mock-treated control monolayers. We also measured translocation of HIV-1 through the primary endometrial monolayers (Figure 10C). At various time points following HIV-1 exposure on the apical side, basolateral supernatant was collected and HIV-1 infectious viral counts were determined TZMb-1 indicator cell assay. The results are presented as percent of inoculum virus added on apical side. Infectious viral counts were seen starting at 6 hours following exposure to HIV-1 (0.03% of inoculum) and infectious virus continued to accumulate in the basolateral compartment (0.08% of inoculum) up to 48 hours time, which was the last time point examined. Discussion To summarize, we were able to demonstrate that exposure to HIV-1 directly decreased the transepithelial resistance across intestinal and genital epithelial monolayers. The reduction in TER correlated with significant decrease in tight junction protein expression and increased permeability, indicating functional impairment of the barrier. The effect was specific for HIV-1 and reached significant levels within 2–4 hours following HIV-1 exposure. Similar reduction in tight junction functioning was observed following treatment of ECs with HIV-1 envelope protein gp120 but not tat, a regulatory protein. Neutralization of gp120 and exposure to an Env− HIV significantly abrogated the impairment of epithelial barrier, indicating that the effect was mediated by HIV-1 envelope glycoprotein. We further determined that exposure of the epithelial monolayers to HIV-1 led to enhanced production of a number of inflammatory cytokines, including TNF-α, by both intestinal and genital epithelial cells. When epithelial cells were exposed to HIV-1 in presence of anti-TNF antibody, there was no significant decrease in TER, indicating that TNF played a major role in impairing the barrier functions. In experiments designed to determine whether the disruption of epithelial barrier function could be directly associated with microbial leakage across the mucosa, we found evidence for small but significant bacterial and viral translocation across epithelial monolayers following HIV-1 exposure. To the best of our knowledge, this is the first study to demonstrate that HIV-1 can directly disrupt mucosal epithelial barrier functions that can lead to enhanced microbial translocation. Previous clinical studies have documented that in HIV-1 infected patients intestinal permeability is altered, characterized by diarrhea-induction [15]–[17] . A recent study showed impairment of barrier function in intestinal biopsies of HAART-naïve patients compared to those on HAART treatment [14]. Increased production of cytokines IL-2, IL-4, IL-5 and TNF-α was found in supernatants of cultured intestinal biopsies in this study. Their conclusion was that following infection, HIV replication in target cells leads to local increase of inflammatory cytokines in the intestinal mucosa, which induce barrier impairment. This supports previous studies where PBMCs co-cultured with HIV-infected macrophages resulted in increased production of a number of cytokines, including TNF-α, IL-1β, IFN-α and IFN-γ which were shown to compromise epithelial barrier function [34]. The prevailing opinion from these studies is that the effect on epithelial barrier is likely mediated via immune cell activation due to viral replication [14],[25]. Of note are other studies that were unable to show that mononuclear cells isolated from colon of infected patients produce increased amount of cytokines [35],[36]. Thus far the cellular source of inflammatory cytokines that could lead to barrier disruption in HIV infected patients remains controversial [20]. Based on our results, we would like to propose that the primary sources of the inflammatory cytokines that disrupt the mucosal barrier are the epithelial cells themselves. Our studies demonstrate that epithelial cells respond directly and rapidly to HIV envelope glycoprotein by production of increased levels of cytokines which lead to loss of barrier functions, rather than an indirect effect mediated by immune cells following HIV replication. This provides an alternate and more direct explanation as to why decrease in viral load following HAART treatment restores intestinal barrier functions [14]. Our results that demonstrate that barrier dysfunction can allow bacterial translocation could also provide explanation for increased levels of immune activation during acute infection, an observation noted in a previous study which examined immune activation following HIV infection in North American cohorts [25]. The mechanism demonstrated in the present study does not exclude the possibility that cytokines released from immune cells in the HIV-infected intestines could also contribute to further disruption of the barrier, more likely in the chronic phase of the infection. That viral exposure could directly lead to compromised barrier function has been shown before [6],[8],[9]. Many other viruses and even bacteria have been shown to directly compromise both epithelial and endothelial barrier integrity. Astrovirus, a single stranded RNA virus and a causative organism of common diarrhea was recently shown to increase epithelial barrier permeability in Caco-2 intestinal cells, modulated by its capsid protein, independent of viral replication [6]. Coxsackievirus has also been shown to directly compromise endothelial tight junctions [8]. Previous studies have shown that HIV-1 infection can compromise the blood-brain barrier thereby leading to progression of HIV-1 encephalitis (reviewed in [37]). The functioning of the tight junctions between endothelial cells, that form the blood-brain barrier, is quite similar to those present between mucosal epithelial cells. However, the mechanism elucidated by these studies was not a direct effect of HIV, but facilitated by production of TNF-α during chronic infection that mediated opening of paracellular route in endothelial lining, for viral entry into the brain. Interestingly, a recent study elucidated that HIV-1 tat protein can directly compromise the retinal epithelial barrier function [38]. Despite this evidence, no studies have so far examined the direct effect of HIV-1 exposure on mucosal epithelium. Our results show that increased permeability is mediated directly by HIV viral envelope glycoprotein. Further, given that significant disruption of tight junction proteins and decreased TERs occurred following treatment with UV inactivated virus, this phenomenon is independent of viral replication. Whether HIV-1 entry is required for the epithelial cell response is currently being examined. In our study, both the intestinal cell line and primary genital epithelial cells showed similar response to different strains of HIV-1: disruption of tight junctions, and increased permeability. However, we found the profile of cytokines produced constitutively by intestinal and genital epithelial cells was quite distinct. While the intestinal cell line T84 did not constitutively produce TNF-α, IL-6, IL-8 and MCP-1, there was significant induction of these cytokines following HIV-1 exposure. Primary genital epithelial cultures, on the other hand, constitutively produced TNF-α, IL-6, IL-8 and MCP-1 and production of TNF-α and IL-6 was significantly upregulated following HIV-1 exposure. Both types of ECs secreted minimal levels of IL-10 and IL-1β which was upregulated following HIV-1 exposure only in primary genital epithelial cells. The differences in the constitutive cytokine profile between genital and intestinal epithelial cells could be due to distinct characteristics of primary cells compared to cell lines. Alternatively, intestinal epithelial cells are likely to be more quiescent in terms of baseline cytokine production given their microenvironment where a variety of commensal organisms are always present in the lumen [39]. In comparison, upper genital tract epithelium exist in a relatively sterile environment and are known to actively secrete an array of cytokines [2]. Nevertheless, following exposure to HIV-1 both types of ECs responded with enhanced induction of inflammatory cytokines that mediated disruption of tight junctions. This indicates that as long as the viral load and exposure times are sufficient, HIV can likely disrupt any mucosal barrier in the body, independent of infection and replication. Among the cytokines that were upregulated, the direct effect of TNF-α on disruption of intestinal epithelial tight junction and increased permeability has been extensively characterized [33]. TNF-induced increase in permeability of Caco-2 cells is known to be mediated by NF-kB activation that downregulates ZO-1 protein expression [40]. ZO-1 proteins are integral part of the tight junction assembly and function as a scaffolding protein critical in maintaining the integrity of the tight junctions. The results from ZO-1 quantification (Figure 4) indicate that the disruption of tight junctions following HIV-1 exposure likely happens in two stages: initially there may be a displacement of ZO-1 that leads to disruption of tight junction integrity followed by marked reduction in the amount of ZO-1 and other tight junction proteins due to decreased transcription. Thus, TNF-α produced by the ECs in response to HIV-1 envelope glycoprotein could induce NF-kB activation and subsequent downregulation of tight junction proteins, including ZO-1. Our ongoing studies show that NF-kB translocation occurs within 1 hour of HIV-1 exposure (Nazli and Kaushic, unpublished). Whether there are distinct steps in this process that have discrete mechanisms is currently under investigation. Regardless of the detailed mechanism, the outcome of tight junction disruption is decrease in TER and leakage across the epithelial barrier. The finding that disruption of barrier function can result in small but significant amount of both viral and bacterial translocation across ECs following exposure to HIV-1 has profound implications. Although previous studies have demonstrated presence of LPS in serum of HIV infected patients and correlated it with immune activation in North American cohorts, the inference that microbial flora in the intestines was the source of LPS was indirect [25]. Our studies provide direct evidence that both bacteria and virus present on the apical side of mucosal epithelial cells during HIV-1 exposure could leak through because of the impairment of epithelial tight junctions and increased permeability. This could allow HIV-1 access to target cells located in the lamina propria of the mucosa as well allow bacterial translocation that could cause local immune activation. While the viral-epithelial interactions described here are novel, further investigation is needed to determine what role increased barrier permeability plays in initiating HIV-1 infection. HIV-1 transmission across intestinal and genital mucosa occurs predominantly via infected semen; currently the role of seminal plasma in HIV-1 transmission is far from clear. Recent studies indicate that seminal plasma can lead to inflammatory responses and facilitate HIV-1 transmission [41]–[43]. However, seminal plasma components such as TGF-β and HGF also enhance epithelial barrier functions [44]. Further, given the low efficiency of viral translocation seen in the ex-vivo model described here, the ability of HIV-1 to cross over in significant numbers, in vivo, would be depend presence of high viral load in the semen, most likely in acute phase of infection. While plasma and semen loads show overall correlation, compartmentalization between genital and blood viral loads is well recognized and more recent studies show that seminal plasma viral load can persist following treatment with HAART [45]–[47]. While the results from the present study elucidate a new mechanism that could lead to viral translocation across the epithelial barrier, more information is needed to understand how other factors like seminal plasma, stage of the infection and viral load may influence any viral leakage across the mucosal barrier. If under physiological conditions, viral leakage does occur because of barrier disruption, it could play a critical role in initiation of infection, especially in the presence of existing inflammation from other viral or bacterial co-infections [48]. In conclusion, the current study provides evidence for the first time that HIV-1 exposure at the mucosal surface leads to direct response by the mucosal epithelium, seen by production of inflammatory cytokines. This response is rapid, independent of viral infection and likely plays a key role in initiation of mucosal damage. This information will be critical for strategies to target control of mucosal damage. Methods Primary genital epithelial and intestinal cell line cultures Reproductive tract tissues were obtained from women aged 30–59 years (mean age 42.9+7.2) undergoing hysterectomy for benign gynecological reasons at Hamilton Health Sciences Hospital. Written informed consent was obtained from all patients, with the approval of Hamilton Health Sciences Research Ethics Board. The most common reasons for surgery were uterine fibroids and heavy bleeding. Tissues were first examined by pathologists and if they were deemed free from any malignant or other clinically observed disease, coin-sized pieces were collected for further processing. Detailed protocol for isolation and culture of genital epithelial cells (GEC) has been described previously [49]. Briefly, endometrial and cervical tissues were obtained from women undergoing hysterectomy and minced into small pieces and digested in an enzyme mixture for 1 hour at 37°C. Epithelial cells (EC) were isolated by a series of separations through nylon mesh filters of different pore sizes. EC were grown onto Matrigel™ (Becton, Dickinson and Company) coated, 0.4-µm pore-size polycarbonate membrane tissue culture inserts (BD Falcon, Mississauga, Canada) with primary tissue culture medium (DMEM/F12; Invitrogen, Canada) supplemented with 10 µM HEPES (Invitrogen, Canada), 2 µM l-glutamine (Invitrogen, Canada), 100 units/ml penicillin/streptomycin (Sigma–Aldrich, Oakville, Canada), 2.5% Nu Serum culture supplement (Becton, Dickinson and company, Franklin Lakes, USA), and 2.5% Hyclone defined fetal bovine serum (Hyclone, Logan, USA). Polarized monolayers were formed within 5–7 days. The purity of GEC monolayers was between 95% and 98%. There was no trace of any hematopoietic cells in the confluent monolayers. The methodology used for monitoring the purity of the epithelial monolayers and absence of CD45 staining in confluent cultures has been described in detail before [49]. The human colon-derived crypt-like T84 epithelial cell line was maintained and cultured as described previously [50]. Briefly T84 intestinal cells were grown and maintained in a 1∶1 (vol/vol) mixture of Dulbecco's modified Eagle's medium and Ham's F-12 medium, supplemented with 10% fetal bovine serum, 1.5% HEPES, and 2% penicillin-streptomycin (Life Technologies, Grand Island, NY) at 37°C in 5% CO2. T84 cells were seeded onto filter supports (0.5×106 cells/well, 0.4-µm pore-size polycarbonate membrane tissue culture inserts, BD Falcon, Mississauga, Canada) and grown for approximately 5–6 days till they reached confluency. The confluency of EC cultures and T84 monolayers was monitored microscopically and by trans-epithelial resistance (TER) across monolayers grown on cell culture inserts, using a volt ohm meter (EVOM; World Precision Instruments, Sarasota, FL, USA). Epithelial monolayers showing TER values higher than 1000 Ω/cm2 were considered completely confluent and used for further experiments. Virus strains, propagation and infection HIV-1 R5 and X4 -tropic laboratory strains were prepared by one of two methods. R5-tropic ADA and X4-tropic laboratory strain IIIB viral stocks were prepared by infection of adherent monocytes from human PBMCs (ADA) or from chronically infected H9 cell line (IIIB), followed by virus concentration by Amicon Ultra-15 filtration system (Millipore, Billerica, US). Virus stock preparations were checked for possible contamination by cellular factors by multiplex bead-based sandwich immunoassay (Luminex Corporation, Austin, TX, USA). TNF-α, IL-6, IL-8, MCP-1, MIP-1α, MIP-1β, RANTES, IL-1α, IL-1β were not detected in any viral stock (standard range of detection limit for different factors: 0.1–4.5 pg/ml). Laboratory strains of HIV-1 virus were also prepared by ultracentrifugation method. HIV-1 R5 laboratory strains ADA, Bal, and X4 strains IIIB, MN and NL4-3 and four clinical strains 11242 (dual), 11249 (R5), 4648 (R5), 7681 (X4) (Dr. Donald R. Branch, University of Toronto) were prepared in human PBMC preparations and concentrated by ultracentrifugation over 20% sucrose for 1 hour at 19,000 rpm (33,000g). All HIV-1 stocks were titered for infectious viral count/ml by TZMb-1 indicator cell assay as described previously [51]. TZMb-1 assay is based on infection of Hela cell line that has been stably transfected with CD4, CXCR4 and CCR5 receptors for HIV-1 attachment. The cell line also carries two indicator systems (β-galactosidase and luciferase systems) under the influence of HIV-1 promoter. HIV-1 infection is detected by staining the cells for β-galactosidase activity resulting in cells turning blue, indicative of HIV-1 replication or alternately by detection of luciferase activity. Infectious viral units/ml = infectious viral counts indicated by number of blue cells per well X dilution factor/ml. For HIV-1 exposure, primary epithelial cells, isolated from human female genital tract tissues or intestinal T84 cells were grown to confluence. Epithelial cell cultures were exposed apically or basolaterally to HIV-1 virus (105 infectious viral units/well/100µl, final concentration 106 infectious viral units/ml), corresponding to MOI of 1.0 or other viral doses as mentioned in individual experiments. The p24 values corresponding to this standard concentration of virus (106 infectious viral units/ml) varied, depending on the viral strain, as determined by p24 ELISA (Zeptometrix Corp., Buffalo, NY, USA); it corresponded to p24 concentration between 0.7–1110 ng/ml for X4 tropic lab strains and between 0.3–790 ng/ml for R5 tropic viruses. For clinical strains p24 concentrations used for HIV exposures were between 49–773 ng/ml. Mock infection controls included exposure to same volume of media without HIV-1 (media control, mock) or exposure to same volume of virus (and gp120) free supernatant from PBMC (for R5 HIV-1) or H-9 (X4 HIV-1) cell line cultures (R5 and X4 controls). Env-defective mutant, Env− (kind gift of D. Johnson, NCI) was on an NL4-3 backbone (X4-tropic HIV-1 laboratory strain) and was compared to wildtype NL4-3 for its effect on epithelial cell permeability [30]. T84 intestinal epithelial monolayers were exposed to wildtype (106 infectious units/ml, p24 79ng/ml) or Env− NL4-3 (p24, 79ng/ml) and TER were measured prior to and 24 hours post-exposure. Monolayers were fixed for ZO-1 staining. UV inactivation of HIV HIV-1 R5-tropic strain ADA and X4-tropic strain IIIB were inactivated by UV exposure. 106 infectious units/ml of virus was subjected to 25–100mJ/cm2 UV with a UV cross-linker (Fisher Scientific, USA). UV inactivation of virus was confirmed by titration on TZMb-1 cells. Gp120 and Tat treatments HIV-1 proteins gp120 envelope protein and soluble Tat protein were obtained from NIH AIDS Research & Reference Reagent Program. Epithelial cell cultures were treated with HIV-1 viral proteins gp120 (0.8nM, 0.1 µg/ml) or Tat (100 nM, 1.4ug/ml). A range of Gp120 concentration (50ng–1µg/ml) was tried based on those used in previous studies [28]. Concentration of tat was consistent with that used in other studies for cultured brain endothelial cells and corneal epithelial cells [29],[38]. HIV-1 proteins were allowed to interact with the epithelial cells for 24 hours at 37°C. Gp120 neutralization assay To test the role of gp120, HIV-1 IIIB was incubated at 37C with a recombinant human monoclonal neutralizing antibody against HIV-1 gp120 (IgG1, clone 2G12, Polymun Scientific, Austria) at a concentration of 35µg/ml or an isotype control antibody (Southern Biotechnology, Birmingham, USA) at same concentration for 1 hour. TERs were measured prior to and post-exposure. Quantitative real-time reverse transcriptase polymerase chain reaction of tight junction proteins Quantitation of tight junction gene expression in epithelial cells post-HIV-1 exposure and comparison with unexposed control epithelial cells was done by real time quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) with Syber Green. The tight junction genes examined were Claudin 1, 2, 3, 4, 5, ZO-1, and Occludin. ECs were lysed by Trizol reagent, total RNA was extracted by RNeasy mini kit (Qiagen Inc., ON, Canada) and treated with DNase column (RNase-free DNase set, Qiagen Inc., ON, Canada) to remove DNA contamination. The cDNA was synthesized by qScript™ cDNA supermix (Quanta Bioscience Inc., Gaithersburg, MD, US) according to manufacturer's protocol. Real-time PCR was performed for each tight junction gene mRNA and GAPDH (internal control) in AB7700 SDS V1.7 (Applied Biosystems, Foster City, CA) with the program: 50°C 2 min, 94°C for 10 minutes and 40 cycles at 94°C for 15 s and 60°C for 1 minute. To validate the quantitative real-time RT-PCR protocol, melting curve analysis was performed to check for the absence of primer dimers. The sequence of primers targeting tight junction genes was taken from published studies (Table 1). The quantitative PCR data was analyzed using the comparative CT method [52]. Briefly the difference in cycle times, ΔCT, was determined as the difference between the tested gene and the reference house keeping gene GAPDH. ΔΔCT was obtained by finding the difference between exposed and mock-treatment groups for each gene. The fold change was calculated as FC = 2−ΔΔCT and results were expressed as fold decrease following HIV exposure compared to mock-treated control cultures. Immunofluorescent staining for tight junction proteins Following treatment, EC monolayers were fixed in 4% Paraformaldehyde, permeabilized with 0.1% Triton X-100 (Mallinckrodt Inc., Paris, KY), and blocked for 30 minutes in blocking solution (5% bovine serum albumin and 5% goat serum (Sigma-Aldrich, ON, Canada) in 0.1% Triton X-100]. Primary antibodies (rabbit anti-human claudin-2, rabbit anti-human Occludin, or rabbit anti-human ZO-1 from Zymed Laboratories, CA, USA) were diluted (2 µg/ml) in blocking solution and incubated with monolayers for 1 hour at room temperature. Normal rabbit serum was used as a negative control to check the specificity of primary antibodies. Following incubation with primary antibodies the monolayers were washed with PBS and secondary antibody, Alexa Fluor 488 goat anti-rabbit IgG (1.5 µg/ml, Molecular Probes, Eugene, OR) was added for 1 hour at room temperature. Nuclear counterstaining was done with Propidium Iodide (500nM, Molecular Probes, Eugene, OR). After extensive washing, filters were excised from the polystyrene inserts and mounted on glass slides in mounting medium (Vectashield mounting medium, Vector Lab, CA, USA). All samples were imaged on an inverted confocal laser-scanning microscope (LSM 510, Zeiss, Germany) using standard operating conditions (63× objective, optical laser thickness of 1µm, image dimension of 512×512, lasers: argon (450nm) and HeNe (543nm) for ZO-1 and nuclear staining, respectively. For each experiment, confocal microscope settings for image acquisition and processing were identical between control and treated monolayers and 3 separate, random images were acquired and analyzed for each experimental condition. Each experiment was repeated at least 3 times. Monolayers were scanned in an apical to basolateral sequence and sequential image sets were analyzed by image analysis software (Image J, NIH) to measure the areas of both fluorescently stained ZO-1 and cellular nuclei. Images are presented as either en face to illustrate the distribution of tight junction protein immunoreactivity or as a composite Z-stack reconstruction, which shows the monolayer in transverse profile with the basally located nuclei identified by propidium iodide staining (red) and tight junction proteins by fluorescein isothiocyanate labeled secondary antibodies (green). For Figure 4A–C, optical sections (XY planes) through the apical regions of monolayers were stacked to represent complete tight junction ZO-1 staining distribution in order to make direction comparison between control and experimental counterparts. MTT viability assay MTT assay was used to determine viability of HIV-1 exposed monolayers and compared to unexposed control monolayers. The assay was performed according to manufactures instructions (Biotium Inc., CA, USA). Briefly, human primary endometrial epithelial cells and T84 intestinal epithelial cells were seeded on 96-well plates at a density of 103 cells/well and allowed to attach to the plate and grown for 5 days. Triplicate wells were treated with media or exposed with laboratory strains of HIV-1 (104 infectious viral units/ml, MOI 1∶1) in 100 µl quantity. After 24 hours incubation, 10 µl of MTT solution was added and incubated for 4h at 37°C. After incubation, the medium was discarded and the purple blue sediment was dissolved in 200 µl DMSO. The relative optical density (OD)/well were determined at a test wavelength of 570 nm in a ELISA reader using a 630 nm reference wavelength. The MTT assay is based on the cleavage of the yellow tetrazolium salt (MTT) to purple formazan by metabolically active cells, based on their mitochondrial activity. Cell viability was expressed as a percentage of untreated cells, which served as a negative control group and was designated 100%; the results are expressed as % of negative control. All assays were performed in triplicate. Blue Dextran leakage assay Blue Dextran dye was dissolved in primary medium (2.3 mg/ml, [26]) and added to the apical surface of confluent epithelial cell monolayers grown on 0.4µm pore size culture inserts. At various time intervals, post-HIV-1 exposure, 50ml of basolateral medium was sampled and replaced by equal volume of primary growth media. Blue Dextran dye in basolateral samples was measured using a microplate reader (Safire, tecan, NC, USA) at 610nm and the optical density was expressed as a % of density of dye added to the apical medium at the beginning of experiment (Time “0”). Cytokine analysis Apical and basolateral supernatants were analyzed for multiple cytokines using the Luminex multianalyte technology (Luminex Corporation, Austin, TX, USA) as described before [53]. Multiplex bead-based sandwich immunoassay kits (Upstate Biotech, Millipore, MA, USA) were used to measure levels of IL-1β, IL-6, IL-8, IL-10, MCP-1 and TNF-α, as per the manufacturer's instructions. Primary endometrial EC and T84 monolayers were exposed to HIV-1 (ADA strain, 106 infectious viral units/ml) and apical and basolateral supernatants were collected after 24 hours. Minimum detection limit for the cytokines were 0.1 pg/ml for TNF-α, 0.2 pg/ml for IL-8, 0.3 pg/ml for IL-6 and IL-10, 0.4 pg/ml for IL-1β, 0.9 pg/ml for MCP-1. Levels detected at or below this limit were considered and reported as undetectable. TNF-α neutralization assay Epithelial cells were grown to confluence and treated with TNF-α (20ng/ml) or HIV-1 (ADA, 106 infectious viral units /ml ) for 24 hours. To test the role of TNF-α, mouse anti-human TNF-α neutralizing antibody (25 µg/ml) (R&D Systems, USA) or normal mouse serum (25 µg/ml) was added to confluent monolayers for 1 hour at 37C prior to treatment with TNF-α (20ng/ml) or HIV-1. Barrier function was determined by TER measurements before and after treatment. Bacterial and HIV-1 translocation For bacterial translocation experiments, non-pathogenic E. coli strain HB101 was grown and cultured in Luria-Bertani (LB) broth (Invitrogen, Canada). T84 cells were grown to confluence on 3.0-µm-pore-size filters (BD Falcon, Canada), transferred to antibiotic-free Hanks solution, and treated with TNF-α (20ng/ml), E.coli (108 CFU/ml), HIV-1 (106 infectious virus units/ml) for 6h, HIV-1 (106 infectious viral units/ml) for 24h, TNF-α+E.coli, HIV-1 + E.coli at the same time for 6h and HIV-1 for 24h + E.coli for 6h. Some wells were left untreated as negative controls. TER was measured before and after treatment and basolateral supernatants were collected 6 hours after the addition of E. Coli to detect bacterial translocation to the basolateral side. The supernatants were diluted and plated on LB agar and incubated for 24h followed by enumeration of bacterial colony counts. For viral translocation, HIV-1 was added to the apical surface of confluent EC monolayers at a concentration of 105 infectious viral units/well and basolateral supernatants were collected at different time intervals. Viral counts were determined using TZMb-1 indicator cell assay. For assessment of LPS leakage, LPS (100ng/ml; from E.coli O26:B6; Sigma-Aldrich, MO, USA) was added to the apical surface of confluent EC monolayers, 24h post-exposure to HIV and compared with unexposed controls. Basolateral supernatants were collected 1 hour after addition of LPS and LPS leakage was measured by measuring LPS levels in the basolateral supernatants by Pyrochrome LPS detection kit (Cape Cod incorporated, MA, USA) according to the manufacturer's instructions. Statistical analysis GraphPad Prism Version 4 (GraphPad Software, San Diego, CA) was used to compare three or more means by 2 way analysis of variance (ANOVA). When an overall statistically significant difference was seen, post-tests were performed to compare pairs of treatments, using the Bonferroni method to adjust the p-value for multiple comparisons. An alpha value of 0.05 was set for statistical significance. p-Values for each analysis are indicated in figure legends. Accession numbers of genes and proteins TNF-a (NCBI Accession number AAD18091), IL-8 (NCBI Accession number CAA77745), IL-6 (NCBI Accession number AAD13886), IL-10 (NCBI Accession number AAA63207), IL-1b (NCBI Accession number AAC03536), MCP-1 (NCBI Accession number AABB29926). ZO-1 (GeneBank Accession number NM_003257), Occludin (GeneBank Accession number NM_002538), Claudin-1 (Genebank Accession number NM_021101, Claudin-2 (Genebank Accession number NM_020384), Claudin-3 (Genebank Accession number NM_001306), Claudin-4 (Genebank Accession number NM_001305), Claudin-5 (Genebank Accession number NM_003277), GAPDH (Genebank Accession number NM_002046).

0 comments Cited 192 times – based on 0 reviews      Review now

Bookmark

Record: found
Abstract: found
Article: found

Is Open Access

Damaged Intestinal Epithelial Integrity Linked to Microbial Translocation in Pathogenic Simian Immunodeficiency Virus Infections

Jacob D Estes, Levelle D. Harris, Nichole Klatt … (2010)

Introduction Persistently elevated immune activation characterized by polyclonal B cell activation [1], increased T-cell turnover [2], increased frequencies of T cells with an activated phenotype [3], and increased levels of pro-inflammatory molecules [4] is a hallmark of disease progression in pathogenic HIV/SIV primate lentiviral infections and is a stronger predictor of disease progression than either CD4+ T-cell count or plasma viral load [5]. The importance of immune activation to disease progression in HIV/SIV infections is highlighted by the low levels of immune activation measured during the chronic phase of infection in natural hosts of SIV such as African green monkeys (AGMs) and Sooty mangabeys (SMs), which do not progress to AIDS [6]. While the consequences of immune activation in HIV/SIV infection are numerous and include increased numbers of activated CD4+ T-cell targets for the virus, attrition of the memory CD4+ T-cell pool and accumulation of high frequencies of terminally differentiated and exhausted memory T and B cells, the underlying mechanisms and sources of immune activation during infection are not well understood [7], [8]. Given accumulating evidence that persistent immune activation is at the heart of disease progression, understanding the mechanisms driving immune activation in chronic HIV disease will be important for the development of new adjunctive treatment strategies targeting this process. Although many factors may contribute to immune activation during chronic HIV/SIV infection, recent evidence has indicated that translocation of microbial products from the lumen of the intestine into the periphery may contribute importantly to this process [9], [10], [11], [12], [13], [14]. These microbial products can stimulate immune cells directly via pattern recognition receptors such as toll-like receptors. Indeed, immune activation related to microbial translocation occurs in other settings and has been implicated in many other pathological conditions. For example, the preconditioning chemotherapy and radiation prior to progenitor stem cell transplantation in individuals with hematological malignancies leads to damage of the tight epithelial barrier of the gastrointestinal (GI) tract resulting in microbial translocation [15]. These translocated microbial products can then stimulate the immune system, exacerbating graft versus host disease [16], [17], [18]. Microbial translocation leading to immune activation also occurs in inflammatory bowl disease [19], after invasive surgery [20], [21], and in pancreatitis [22]. While microbial translocation has been indirectly implicated in driving immune activation in chronically HIV-infected humans and SIV-infected rhesus macaques (RMs), the mechanisms underlying this phenomenon remain unclear, with enterocyte apoptosis [23], massive loss of GI tract CD4+ T cells [24] and/or preferential loss of GI tract Th17 cells [25], [26] all proposed as important contributing factors. Moreover, the timing of the onset of microbial translocation relative to infection has remained obscure, and direct evidence of translocation at the tissue level has been lacking. Here, using a quantitative image analysis approach to study large segments of tissue, we provide direct immunohistochemical evidence of translocation, define the timing of microbial translocation in pathogenic SIV infection of RMs and identify loss of the integrity of the intestinal epithelial barrier as a plausible mechanistic correlate of microbial translocation. The absence of translocation or associated immune activation in chronic SIV infection of SMs, which does not result in progressive disease, underscores the critical role this process plays in the pathogenesis of primate lentiviral infections and the potential value of limiting it as an approach to adjunctive therapy. Results In vivo detection of microbial translocation and host responses in tissues of SIV infected RMs Initially, we sought evidence of microbial translocation by staining with a monoclonal antibody against LPS-core antigen in paraffin-embedded colon tissue sections obtained at necropsy from SIV-uninfected RMs (n = 6), RMs euthanized during early acute (n = 10) or late acute (n = 3) SIV infection, and chronically SIV-infected RMs euthanized at protocol specified endpoints (“Non-AIDS”; n = 8) or clinical endpoints (AIDS defining conditions, “AIDS”; n = 5, Table 1). Prior to and during early acute SIV infection, the LPS-core antigen-specific mAb stained only rare cells within the lamina propria (LP), but in dramatic contrast, in chronically infected animals both numerous LPS+ cells and abundant extracellular core LPS antigen were observed (Figures 1 and S1). Figures show a broad spectrum of microbial tanslocation and discontinuities to the structural barrier of the GI tract (discussed in detail below) that was seen in our animal cohort, from negligible (SIV-) to most severe (AIDS). Although the amount of apparently extracellular bacterial constituents within the LP varied among our chronically SIV-infected RMs; all chronically SIV-infected RMs showed readily demonstrable evidence of microbial translocation in multifocial lesions along the GI tract, findings that were absent in the SIV-uninfected animals and in early acute SIV infection (i.e. 1–10 dpi). Importantly, microbial translocation was evident in the large bowel of RMs chronically-infected with different pathogenic strains of virus (i.e. SIVmac239, SIVmac251 and SIVsmE660; Table 1), suggesting that intestinal damage leading to microbial translocation is a common feature of pathogenic SIV infections. 10.1371/journal.ppat.1001052.g001 Figure 1 Identification of microbial translocation in large bowel of chronically SIV+ RMs. Representative images (200×) of unselected colon sections from SIV-uninfected and chronically SIV-infected RMs (Non-AIDS and AIDS) stained for LPS-core antigen (brown). The images show the array of microbial translocation and damage seen in our cohort that ranged from negligible (SIV- RMs) to regions of epithelial loss in our SIV+ Non-AIDS RMs resulting in luminal content in direct contact with the LP and increased frequency of LPS+ cells within the LP to more extensive epithelial damage in our SIV+ AIDS RMs that extended to overt ulcerations that showed significant microbial product infiltration throughout these regions; features which were absent from our SIV-uninfected RMs. Arrows point to regions of epithelial discontinuities and microbial translocation into the LP. 10.1371/journal.ppat.1001052.t001 Table 1 Animals used for study. Animal ID Species Virus Days post infection Infection Status Major Pathologic/Clinical Findings Opportunistic Infections pVLa 595 RM None Uninfected - ND None NA DBM6 RM None Uninfected - ND None NA 34572A RM None Uninfected - Pneumoconiosis None NA 04D169 RM None Uninfected - ND None NA P049 RM None Uninfected - ND None NA 27776b RM AT-2 SIVmac251 Uninfected - ND None NA 23053 RM SIVmac251 1 Early Acute Endometriosis, moderate None <125 30127 RM SIVmac251 1 Early Acute Splenomegaly, mild None <125 31373 RM SIVmac251 4 Early Acute Parathyroid Nodule None <125 31385 RM SIVmac251 4 Early Acute Gastritis None 1.5×103 30991 RM SIVmac251 6 Early Acute Cervical Ectopy None 2.3×104 31523 RM SIVmac251 6 Early Acute Generalized Lymphadenopathy, moderate None 7.3×104 26222 RM SIVmac251 7 Early Acute Lymphadenopathy, mild; Splenomegaly, mild; Intussusception, Ileocecal None 2.6×106 34498 RM SIVmac251 8 Early Acute Lymphadenopathy, generalized, marked None 2.5×106 28013 RM SIVmac251 9 Early Acute Lymphadenopathy, mild; Cervical ectopy None 2.5×106 24818 RM SIVmac251 10 Early Acute Lymphadenopathy, generalized, moderate-marked None 9.9×106 24037 RM SIVmac239 14 Late Acute Lymphadenopathy, generalized, marked; Splenomegaly; Hepatic Lipidosis; Endometritis, mild, Chronic None 2.3×108 27099 RM SIVmac239 21 Late Acute Lymphadenopathy, generalized None 6.8×106 24225 RM SIVmac239 28 Late Acute No significant findings None 1.5×106 551 RM SIVmac239 168 Chronic/Non-AIDS Lymphadenopathy, generalized, moderate; Lymphocytic portal inflammation, moderate multifocal; Pulmonary lymphoid hyperplasia, marked None 1.0×106 83I RM SIVmac239 168 Chronic/Non-AIDS Renal lymphofollicular inflammation, mild focal None 1.4×106 AD09 RM SIVmac239 161 Chronic/Non-AIDS Lymphadenopathy, generalized, moderate; Portal hapatitis, chronic suppurative with fibrosis None 2.7×106 AY25 RM SIVmac239 273 Chronic/Non-AIDS Lymphadenopathy, mild; Gastritis, minimal; Colitis, mild with mucosal hyperplasia None 3.5×105 (30)c AY44 RM SIVmac239 273 Chronic/Non-AIDS Lymphadenopathy, follicular, mild; Colitis, mild with mucosal hyperplasia; Hepatitis, mild; Nephritis, mild None 2.0106 (55)c R443d RM SIVmac239 391 Chronic/Non-AIDS No apparent clinical manifestations of disease ND 1.4×105 R457d RM SIVmac239 397 Chronic/Non-AIDS Chronic diarrhea and recurrent epistaxis ND 8.4×105 R462d RM SIVmac239 396 Chronic/Non-AIDS Weight loss ND 2.8×105 AY99 RM SIVmac239 213 Chronic/AIDS Lymphadenopathy, generalized, moderate; Gastritis, mild-moderate; Colitis; mild with multifocal lymphoid follicles; Enteritis, multifocal and focally extensive with no overt OIs. CMV meningitis, buccal Candida 1.0×107 R451e RM SIVmac239 148 Chronic/AIDS Chronic diarrhea, weight loss and cutaneous lesions; Enteritis, multifocal and focally extensive with no overt OIs. NF 4.1×107f R475e RM SIVmac239 195 Chronic/AIDS Chronic diarrhea, intermittent epistaxis and weight loss; Enteritis, multifocal and focally extensive with no overt OIs. NF 7.6×108 R268e RM SIVsmE660 56 Chronic/AIDS Chronic diarrhea, Polyuria/Polydipsia and weight loss; Enteritis, multifocal and focally extensive with no overt OIs. NF 1.4×109 R437e RM SIVsmE660 184 Chronic/AIDS Chronic diarrhea and weight loss; Enteritis, extensive with no overt OIs. NF 7.8×106 FOu SM None Uninfected - NR None NA FZr SM None Uninfected - NR None NA FAv SM SIVsmm Naturally infected Non-AIDS NR None 1.0×104g FBn SM SIVsmm Naturally infected Non-AIDS NR None 3.1×104g FBq SM SIVsmm Naturally infected Non-AIDS NR None 4.1×104g FFq SM SIVsmm Naturally infected Non-AIDS NR None 3.5×104g FKn SM SIVsmm Naturally infected Non-AIDS NR None 1.2×104g a Viral loads are SIV vRNA copies/mL of plasma and the detection limit of this assay was 125 copies of vRNA/ml of plasma as reported previously [53]. bAnimal 27776 was given i.vag. aldithriol-2 (AT-2)-inactivated SIVmac239, using the same inoculation schedule as that for the other animals inoculated i.vag. with SIVmac. This animal, had no signs of infection and was considered an uninfected control. cViral loads measured 1 week prior being placed on an ART regimen, which lasted 5 weeks. Parentheses are viral load measurements at time of euthanasia (i.e. 5 weeks post ART). dPathology reports were not available for these animals, thus only major clinical findings are reported but ehaematoxylin and eosin stains were examined on tissues when possible. However, we did have sufficient tissue to perform large bowel histopathological evaluation, which are also reported. fViral load measured 4 weeks before euthanasia. gViral load at time of tissue biopsy. ND, not determined. NR, not relevant, NF, none found. The specificity of our immunohistochemical staining directly documenting microbial translocation in gut tissue sections is supported by: (i) the remarkably low frequency of LPS+ cells within the LP in SIV-uninfected or RMs with early acute infection; (ii) the absence of staining with isotype-matched control antibodies in SIV-uninfected, acutely and chronically SIV-infected animals (data not shown); (iii) the lack of any evidence of non-cell associated LPS in the LP of our SIV-uninfected and early acute SIV-infected RMs, despite the abundant LPS staining in the residual luminal content of these same samples; (iv) the detection of microbial products within the LP of the colon of chronically SIV-infected RMs that were rarely seen in SIV-uninfected and early acute infected RMs, using a rabbit polyclonal antibody against Escherichia coli that recognizes many E. coli proteins (Figures 2 and S2), or using peptide nucleic acid fluorescent in situ hybridization to detect bacterial 16S RNA (data not shown). 10.1371/journal.ppat.1001052.g002 Figure 2 Identification of microbial translocation in large bowel of chronically SIV+ RMs. Representative images (200×) of colon stained with a polyclonal antibody against E. coli (brown) in SIV early acute infected (4 dpi) and chronically SIV-infected RMs (Non-AIDS and AIDS). Note the small regions of epithelial discontinuities in the SIV+ Non-AIDS RMs resulting in luminal E. coli + content in direct contact with the LP and increased frequency of E. coli + cells within the LP. Sections from two SIV+ AIDS RMs show the range of epithelial damage with associated microbial product infiltration, findings frequently seen in RMs with AIDS. Arrows point to regions of epithelial discontinuities and microbial translocation into the LP. To relate microbial translocation in the large bowel of chronically SIV-infected RMs to systemic microbial translocation, we stained sections from lymph nodes, identifying large numbers of LPS+ cells within both local draining lymph nodes (mesenteric; MesLN) (Figures 3 and S3), and remote peripheral lymph nodes (axillary; AxLN) (Figures 4 and S4) of chronically SIV-infected RMs, suggesting systemic dissemination of translocated microbial constituents originating from the GI tract. Consistent with what might be expected for the anatomic site of inductive T-cell immune responses, we found immunoreactive LPS within the medullary cords and paracortex of draining MesLN as well as peripheral AxLN during the chronic stage of infection. In contrast, in SIV-uninfected or early/acute SIV-infected RMs we found only low levels of LPS+ cells in the gut-draining MesLN and virtually no LPS+ cells in the peripheral AxLN of (Figures 3,4 S3 and S4). The primary localization of LPS within the medullary cords and sinuses, and to a lesser extent the paracortex and germinal centers, in SIV+ Non-AIDS RMs is consistent with microbial products traversing from the damaged intestine via the lymphatics. The presence of LPS within the paracortex and germinal centers suggests a) antigen presenting cells which have bound microbial antigens migrate into the T cell inductive site of the LN and b) possibly microbial product-immune complex deposition on follicular dendritic cells may be occurring. However, the biological relevance of finding microbial constituents in these anatomical sites, at this point, remains unclear. Moreover, consistent with the liver's important function as a “gatekeeper” between the intestine and peripheral circulation, we also found multifocal evidence of microbial products within the liver, in regions surrounding the hepatic portal veins and tracts in chronically SIV-infected Non-AIDS RMs with more extensive staining into the lobules of the liver in chronically SIV-infected AIDS RMs, consistent with increased intestinal damage correlating with more microbial dissemination (Figure 5 and Figure S5). 10.1371/journal.ppat.1001052.g003 Figure 3 Identification of microbial translocation in gut draining MesLN of chronically SIV+ RMs. Representative images (200×) of the paracortical T-cell zone of MesLN stained for LPS-core antigen (brown). Note the more extensive accumulation of microbial products within the T cell zone, the T-cell inductive parenchyma of the lymphatic tissue with progressively more severe SIV infection. 10.1371/journal.ppat.1001052.g004 Figure 4 Identification of systemic microbial translocation in distant AxLN of chronically SIV+ RMs. Representative images (200×) of the paracortex of AxLN stained for LPS-core antigen (brown). Note the increased accumulation of microbial products within the paracortex, germinal centers, and medullary chords of the lymphatic tissue, with progressive SIV infection in chronically SIV+ RMs, but the paucity of LPS staining seen in SIV-uninfected RMs. 10.1371/journal.ppat.1001052.g005 Figure 5 Identification of microbial translocation in the liver of chronically SIV+ RMs. Representative images (200×) of liver stained forE. coli constituents (brown). Note the increasing accumulation of microbial products near and surrounding the portal tract in chronically SIV+ RMs with more severe disease but lack of microbial constituent staining seen in SIV-uninfected RMs. Using these approaches, we identified unequivocal direct evidence of microbial constituents within the LP of the large bowel, and in the liver and lymph nodes of all chronically SIV-infected RMs studied, but not uninfected RMs. This finding was consistent across all 32 RMs studied, including SIV-uninfected RMs (n = 6); early acute SIV-infected RMs (1–10 dpi, n = 10); late acute RMs (14–28 dpi infection, n = 3); and chronically SIV-infected RMs (56–397 dpi, n = 13 (Non-AIDS and AIDS); Table 1) and indicate that microbial translocation involves infiltration of microbial products into the LP of the GI tract during the chronic phase of SIV infection of RMs. Quantitative image analysis of microbial translocation After demonstrating the qualitative presence of increased microbial products in the LP of large bowel, liver and lymph nodes of SIV infected RMs, we used quantitative image analysis techniques [27] to quantify the extent of microbial translocation demonstrable in high power (400×) digital scans, of tissue section whole mounts (ScanScope CS System, Aperio). Sections of colonic mucosa analyzed for each animal represented, on average, a total of 350 distinct 400× image fields per scanned tissue, providing an in-depth, systematic, assessment of segments of the GI tract that ranged from 43 to 101 linear mm of colonic epithelial lining and 16 to 39 mm2 of intestinal mucosal area. This comprehensive analysis approach, applied to randomly selected tissue sections, provided us with an unbiased and detailed evaluation of microbial translocation in representative sections of the GI tract that otherwise may not have been possible using conventional established tissue analysis methods. Using this approach, we found that the percent area of LP of the colonic mucosa containing LPS was significantly higher in chronically-infected RMs compared to uninfected and acutely (early) SIV-infected RMs (Figure 6A). 10.1371/journal.ppat.1001052.g006 Figure 6 Quantitative image analysis of microbial translocation. (A) Levels of LPS within the LP of the colon of SIV-uninfected (white squares), early acute (white circles), late acute (gray circles), chronic Non-AIDS (black circles) and chronic AIDS (red circles) RMs. Mann-Whitney U test performed between the chronically infected (both Non-AIDS and AIDS) and early acute RMs and between late acute and early acute RMs. (B) Correlation between LPS levels within the LP of the colon and the mesenteric LN for animals in which we had obtained both colon and MesLN samples. Significance determined using Spearman's rank correlation coefficient and associated p-value shown. (C) Correlation between LPS levels within MesLN and AxLN for animals in which we had obtained both MesLN and AxLN samples. Significance determined using Spearman's rank correlation coefficient and associated P-value shown. We used the same quantitative image analysis approach to evaluate the relationship between the microbial product burden within the LP of the colon and in draining (MesLN) and distant LN (AxLN) from the same animals, calculating the percent area of each tissue that contained LPS. We found a significant positive correlation between the amount of LPS within the LP of the colon and the amount of LPS within the corresponding draining MesLN (r = 0.69, P = 0.0065, Figure 6B). Moreover, we also found a significant positive correlation between LPS staining within the MesLN and within the matched AxLN (r = 0.59, P = 0.027, Figure 6C). Taken together, these data indicate that the presence of microbial products in the peripheral lymphatic tissues is intimately linked to microbial translocation from the gut. Induction of immune activation: translocated microbial products co-localize with markers of immune activation Microbial products can directly stimulate the innate immune system via interactions with Toll-like receptors (TLR) that lead to an inflammatory cascade. To evaluate the possible relationship between translocation of microbial products and inflammation we performed double-label immunohistochemical staining for microbial products and the innate proinflammatory cytokine IFNα. Reflecting the immune activation of chronic SIV infection in RMs, IFNα expression was widespread and we consistently demonstrated co-localization of IFNα and microbial products within the LP of the colon (Figure S6), and in AxLN, and MesLN (data not shown). The majority of such co-localization occurred in the absence of detectable local viral replication, as during the chronic phase of infection productively infected cells within the LP are only rarely demonstrable by in situ hybridization in most SIV-infected RMs that have not progressed to AIDS (data not shown). Indeed, in extensive double label studies using immunohistochemical staining for IFNα and in situ hybridization for SIV RNA in tissues from chronically SIV-infected Non-AIDS RMs, there was only very limited co-localization between IFNα expression and in situ hybridization for SIV RNA in the colon or MesLN (data not shown). The overwhelming majority of the abundant IFNα immunostaining was found relatively distant from local viral replication, but in close proximity to microbial products (data not shown). Moreover, the levels of IFNα immunostaining were significantly greater than the tissue level of SIV RNA. To evaluate further the role of translocated microbial products in driving immune activation in chronic SIV infection, we assessed the distribution and co-localization of interleukin-18 (IL-18) and LPS in the gut-draining MesLN in SIV-uninfected and chronically SIV-infected Non-AIDS RMs (Figure S7). IL-18 is produced by activated macrophages and dendritic cells in response to microbial product stimulation [28]. We found that before SIV infection there was a basal, low-level, expression of IL-18 in all structural compartments of MesLN (i.e. B cell follicles, T-cell zone and medullary cords) and that this expression was dramatically increased in SIV infection (data not shown). Although IL-18 was up-regulated in some regions where there were no visualized microbial products, high levels of IL-18+ cells were always found in close proximity to LPS in MesLN (Figure S7). Consistent with a role for translocated microbial products inducing immune activation and IL-18 expression, Ahmad and colleagues recently described significantly elevated levels of IL-18 in the serum of HIV-infected/AIDS patients compared to those of HIV-seronegative healthy individuals [29]. Collectively, these data strongly suggest that microbial products, which infiltrate the LP of the GI tract, and then spread systemically, can directly stimulate the immune system and contribute to chronic immune activation. Timing and mechanisms of increased microbial translocation To determine how early during infection microbial translocation occurs, we compared microbial translocation into the LP of the colon of RMs throughout the acute stage of SIV infection (1 to 28 dpi) in vaginally-challenged animals. With the exception of one animal at 8 dpi, we found only very low levels of LPS+ cells within the LP of RMs between 1–10 dpi, observations that were indistinguishable from SIV-uninfected animals (Figures 6 and data not shown). There was a statistically significant increase in LPS seen within the LP of animals infected for between 14 and 28 days compared to uninfected or early/acute animals and evidence of microbial translocation was detected at small foci associated with breaks in the epithelial lining (Figures 6 and data not shown). Importantly, during the acute phase of infection, areas of discontinuity in the epithelial barrier were relatively infrequent, while LPS staining in the LP appeared to increase into the late acute stage of SIV infection (14–28 dpi). Interestingly the extent of lesions and discontinuities were lower than might have been expected relative to the massive enterocyte apoptosis previously described as peaking at 14 dpi in these same animals [23]. We comment below on the possible mechanisms responsible for this dissociation. Although enterocyte apoptosis subsequently decreased, even as microbial translocation increased in the late acute stage of infection, the abundant evidence of LPS+ within the lamina propria at 28 dpi suggested that early damage to the integrity of the epithelial lining could facilitate translocation of microbial products (data not shown). Thus, we sought to determine directly if the compromise of the integrity of the epithelial barrier is a distinguishing feature of microbial translocation during chronic SIV infection, evaluating the integrity of the epithelial barrier by staining for the tight junction protein claudin-3. A similar technique has been used in studying human samples to assess the integrity of the epithelial barrier in diseases associated with discontinuities in the GI tract [30]. Examination of the integrity of the epithelial barrier by staining for claudin-3, revealed multifocal disruptions and epithelial loss of the normally continuous, epithelial barrier in tissues from the chronic stages of infection, but not in tissues from uninfected or early acute animals (Figure 7A and Figure S8). We recognize the possible contribution that undetected opportunistic enteric pathogens may play in the continuum of epithelial damage seen in our chronically SIV-infected (AIDS) RMs, and thus show in Figures 7 and S8 two examples of end-stage RMs demonstrating this dynamic spectrum of epithelial damage from multifocal epithelial loss to severe epithelial damage and ulceration. Importantly, confocal analysis showed that disruptions in the integrity of the epithelial barrier were directly associated with translocated microbial products (Figure 7B). Furthermore, quantitative image analysis from high power entire colonic tissue section scans, confirmed that chronically-infected animals had significantly more damage to the integrity of the epithelial barrier compared to uninfected and acutely-infected RMs (Figure 8A). Moreover, the degree to which the integrity of the epithelial barrier was compromised was significantly correlated with the amount of LPS within the LP (Figure 8B; r = 0.57, P = 0.032). 10.1371/journal.ppat.1001052.g007 Figure 7 Damage to the integrity of the epithelial barrier is associated with infiltration of microbial products into the lamina propria. (A) Representative images (200×) of colon stained for the tight junction protein claudin-3 (brown) as a marker for intact epithelial barrier. Note the multifocal regions of the epithelial barrier that are missing in SIV+ RMs, allowing direct contact of luminal contents with the LP. (B) Confocal immunofluorescent 3-D images of colon (400×) stained for cytokeratin (epithelial cells; green), E. coli (red) and DAPI (nuclei; blue). Discontinuities in the epithelial barrier allowing translocation of microbial products into the LP are present in late acute SIV+ RMs (14 dpi) and progressively more severe in chronic Non-AIDS and chronic AIDS RMs (white arrows point to epithelial discontinuities and translocated microbial products). Scale bars = 40 µm. 10.1371/journal.ppat.1001052.g008 Figure 8 Damage to the integrity of the epithelial barrier correlates with microbial translocation. (A) Quantitative image analysis showing the proportion of the epithelial lining in the large bowel that is damaged (discontinuous) in SIV-uninfected (white squares), early acute (white circles), late acute (gray circles), chronic Non-AIDS (black circles) and chronic AIDS (red circles) RMs. Mann-Whitney U test performed between the chronically infected (both Non-AIDS and AIDS) and early acute RMs and between chronically infected (both Non-AIDS and AIDS) and SIV- RMs. (B) Statistically significant positive correlation between the proportion of epithelial damage and the level of LPS within the LP of the colon. Significance determined using Spearman's rank correlation coefficient and associated P-value shown. Loss of integrity of the epithelial barrier of the GI tract would be expected to result in several host responses including polymorphonuclear neutrophil (PMN) infiltration and increased local proliferation of enterocytes within colonic crypts in an attempt to restore the integrity of the GI tract. Importantly, we observed increased levels of myeloperoxidase+ PMNs within the lamina propria of chronically SIV-infected RMs associated with damage to the epithelial barrier (Figure S9), providing strong evidence for a tissue specific response to GI epithelial damage. To assess proliferation of GI tract enterocytes we immunohistochemically stained colon tissues with a monoclonal antibody against Ki67, a cellular marker for proliferation, and performed quantitative image analysis measuring the fraction of enterocytes along the colonic crypt that were proliferating (Ki67+). We found increased levels of proliferating enterocytes (Ki67+) in chronically SIV-infected animals compared to SIV uninfected and acutely infected RMs (Figure 9A–B and Figure S10, P = 0.016). Moreover, there was a trend towards an increase of Ki67+ enterocytes in animals between 14 and 28 days post SIV infection compared to animals infected for ∼1 week (Figure 9B, P = 0.057). These data are consistent with early (14–28 dpi) and progressive damage to the integrity of the epithelial barrier, and indicate that one mechanism underlying microbial translocation likely involves breakdown of the structural barrier of the GI tract at a rate that exceeds enterocyte proliferation and other repair mechanisms, consistent with previous reports of abnormalities within the GI tracts of chronically HIV or SIV-infected individuals [31], [32], [33], [34], [35], [36], [37], [38]. Our findings of host responses to microbial-product infiltration into the lamina propria are consistent with previous findings of fibrosis within the lamina propria of the GI tract [39]. While our data and those of previous studies are consistent with increased discontinuity of the structural barrier of the GI tract during chronic SIV infection of RMs, we cannot exclude that the structural damage we observed by immunohistochemical analysis may be attributed to increased enterocyte turnover overall, leading to an apparent increase in structural damage to the GI tract. However, the increased levels of myeloperoxidase+ PMNs within the lamina propria of chronically SIV-infected RMs associated with observed damage to the epithelial barrier (Figure S9), strongly support our conclusion of GI epithelial damage. Regardless, these data suggest that the integrity of the structural barrier of the GI tract is significantly weakened in SIV-infected individuals leading to microbial translocation. 10.1371/journal.ppat.1001052.g009 Figure 9 Damage to the integrity of the epithelial barrier is associated with increased enterocyte proliferation. (A) Representative images (200×) of colon stained for the proliferative nuclear protein Ki67 (brown). Note progressive change in frequency and location of Ki67+ enterocytes, from few cells at the base of crypts during early acute infection (4 dpi), with full involvement of crypts with proliferating cells in chronically infected Non-AIDS and AIDS animals. (B) Quantitative image analysis of Ki67 expression by colonic enterocytes in early acute (white circles), late acute (gray circles), chronic Non-AIDS (black circles) and chronic AIDS (red circles) RMs demonstrates the statistically significant increase in enterocyte proliferation in chronic infection. Mann-Whitney U test performed between the chronically infected (both Non-AIDS and AIDS) and late acute RMs and between early acute and late acute RMs. Microbial translocation and phagocytosis by intestinal macrophages When microbial products cross the epithelial barrier under normal, physiological conditions, they are generally phagocytosed by specialized intestinal macrophages [40]. The relatively abundant and apparently non-cell associated microbial constituents we saw in the LP of our chronically SIV-infected RMs, particularly in animals with AIDS, suggested that this might result from microbial translocation in excess of the phagocytic capacity of these macrophages. This could reflect a saturation of the maximum capacity of these macrophages to phagocytose translocated microbial constituents or alternatively, might reflect a compromise of macrophage phagocytic function resulting in a net relative defect in microbial clearance and the extracellular accumulation of microbial products. To assess this, we performed confocal microscopy of GI tract tissue from SIV-uninfected and acute and chronically SIV-infected RMs to assess the localization of microbial constituents relative to GI tract macrophages (intracellular vs. extracellular). In the rare instances where microbial products were detected in the LP of SIV-uninfected RMs, they were virtually always within HAM56+ macrophages (Figure 10 and Figure S11). In addition, during the acute phase of infection (until 28 dpi), most microbial products were mostly found within HAM56+ macrophages (Figure S11), perhaps helping to explain why LPS levels in plasma are not elevated during acute infection while sCD14 levels were moderately raised [9]. Moreover, microbial products crossing the epithelial barrier at 14 dpi were virtually always found within macrophages, whereas abundant macrophages were juxtaposed to damaged regions of the colon in late acute (28 dpi) (Figure S11). Furthermore, as infection progressed into the chronic stage of disease, the frequency of macrophages negative for microbial products increased, even though abundant numbers of macrophages were present and adjacent to microbial products (Figure 10 and Figure S11). There was no apparent change in the overall frequency of HAM56+ macrophages at the tips of the colonic crypts between SIV-uninfected, and acutely or chronically SIV-infected RMs (data not shown). The presence of high frequencies of macrophages without internalized microbial constituents, along with abundant extracellular microbial products suggested that GI tract macrophages in the later phase of acute infection and chronic stages of disease may become increasingly incapable of phagocytosing microbial products that translocate into the LP. 10.1371/journal.ppat.1001052.g010 Figure 10 Microbial products are differentially phagocytosed in acutely and chronically SIV-infected RMs. Confocal immunofluorescent 3-D images of colon (400×) stained for HAM56 (macrophages; green), E. coli (red) and DAPI (nuclei; blue) showing the progressive decrease in the fraction of microbial products (E. coli, red) internalized by macrophages (green, highlighted by white arrows), and an increase in the proportion that were not cell associated with progressively more severe SIV infection. For specimens obtained from animals with later stages of infection, the presence of abundant HAM56+ macrophages (green) without internalized E. coli constituents (red) (highlighted by white arrow heads), despite extensive E. coli constituents present in the LP near macrophages, suggests that macrophages in chronic SIV+ RMs become less able to efficiently clear microbial products translocated into the LP. Rare microbial constituents are present in SIV-uninfected RMs but are virtually always found within macrophages. Scale bars = 40 µm. Maintenance of the epithelial barrier and lack of microbial translocation in Sooty mangabeys A distinguishing feature of non-progressive infection in natural hosts of SIV is the absence of immune activation during the chronic phase of infection [6], [41], [42], [43], [44], [45]. Because elevated plasma LPS levels are absent in both chronically SIVsmm-infected SMs and SIVagm-infected AGMs [9], [45], [46], we evaluated the structural integrity of the intestinal epithelial barrier in chronically SIVsmm-infected SMs. In contrast to findings in chronically SIV-infected RMs, and consistent with the lack of LPS within the circulation of these SIV natural host animals [9], we found no evidence of damage to the integrity of the epithelial barrier (Figure 11A) and no infiltration of microbial products into the LP of large bowel (Figure 11B) or peripheral lymph nodes (Figure 11C). These data were consistent among 7 animals studied (n = 2, SIVsmm-uninfected SMs; n = 5, chronically SIVsmm-infected SMs; Table 1). Hence preservation of the tight epithelial barrier is associated with lack of microbial translocation and immune activation in non-progressive, natural SIV infection. 10.1371/journal.ppat.1001052.g011 Figure 11 Absence of structural epithelial damage and microbial translocation in non-pathogenic infection of SMs. (A) Representative images (200×) of rectum stained for the tight junction protein claudin-3 (brown) show the complete maintenance of the epithelial barrier in SIVsmm-uninfected and SIVsmm-infected SMs. Representative images (200×) of (B) rectum and (C) peripheral lymph nodes (100×) stained for LPS-core antigen (brown) shows the absence of microbial translocation in SIVsmm-uninfected and SIVsmm-infected SMs. Discussion Indirect evidence has implicated microbial translocation from the gut as a factor contributing to pathological immune activation in chronic HIV/SIV infection, but direct evidence of translocation and demonstration of a plausible underlying mechanism have been lacking. Using an unbiased, comprehensive approach for quantitative and qualitative immunohistologic analysis of randomly selected tissue specimens obtained from non-human primates at various times relative to SIV infection, we have shown that: 1) microbial products can be found in the LP of the large bowel, in draining and distant lymph nodes, and in the liver of chronically SIV-infected RMs; 2) microbial translocation is associated with breakdown of the integrity of the epithelial barrier of SIV-infected RMs; 3) the extent of epithelial breakdown correlates with the extent of microbial translocation; 4) epithelial barrier breakdown and microbial translocation begin to be apparent during the late acute phase of infection (14 dpi); 5) the presence of microbial products in multiple anatomical sites is associated with expression of IFN-α and IL-18 in the absence of detectable local viral replication in the LP, consistent with direct induction of immune activation; 6) macrophages in chronically SIV-infected RMs appear dysfunctional with respect to their ability to phagocytose translocated microbial products; and 7) neither epithelial barrier breakdown nor infiltration of microbial products into the LP occur during the chronic phase of SIV infection of SMs. We provide two lines of evidence linking microbial translocation to immune activation. First, we show that in pathogenic SIV infection of RMs, damage to the integrity of the epithelial barrier of the GI tract is associated with microbial translocation, and that microbial translocation is linked to local immune activation, based on co-localization of microbial products and production of the immunoinflammatory cytokines IFNα and IL-18, including in lymph nodes anatomically distant from the GI tract. Second, in marked contrast, in SIV-infected SMs where immune activation is quickly resolved in the acute stage of infection [47], and chronic infection is not accompanied by persistent immune activation, we found neither damage to the intestinal barrier nor microbial translocation. Recent in vitro studies with peripheral blood lymphocytes from SMs have been interpreted to suggest that a lack of type I IFN cytokine response to SIV RNA accounts for the typically nonprogressive nature of SIV infection in SMs [48]. Furthermore, these data have been used to suggest that the raised plasma LPS levels observed in chronically SIV-infected RMs and HIV-infected humans, are simply markers of damage to the GI tract and that the microbial translocation that is reflected in elevated plasma LPS levels does not contribute significantly to causing systemic immune activation [48]. However not only were LPS levels increased in chronically-infected individuals, but sCD14 and LPS binding protein levels were also increased [9], [11], [13]. These data strongly suggested that LPS was directly stimulating the immune system in vivo. In the present study, we show directly that damage to the integrity of the epithelial barrier of the GI tract allows microbial products to infiltrate into the LP and this infiltration is associated with local immune activation demonstrated by co-localization of microbial products within the LP with IFNα and IL-18 in MesLN. As only limited viral replication is demonstrable in the LP of the GI tract of chronically SIV-infected RMs, the damage to the GI epithelial lining, microbial translocation and local immune activation are unlikely to be caused by the direct effects of local viral replication. Rather, where rare infected cells are seen in the LP underlying damaged mucosa, it is more likely that the chronic immune activation, due to translocated microbial products, has provided activated CD4+ T cell targets for the virus. Taken together, these data suggest that microbial translocation, resulting from damage to the GI tract epithelial barrier and impaired macrophage-mediated phagocytosis, results in immune activation during the chronic phase of HIV/SIV infection of humans and RMs. Importantly, we found a significant correlation between the extent of damage to the epithelial barrier of the colon and the amount of LPS within the underlying mucosa and the extent of translocated microbial products in draining and remote lymph node tissues. The extent of microbial constituents present in lymph node tissues correlated with the extent of local evidence of immune activation, further substantiating the link. Moreover, while we find that microbial translocation begins during the acute phase of infection, our previous work had indicated that elevated levels of microbial products were not seen in plasma until the chronic phase [9]. Our data suggest that microbial products are localized within tissue macrophages during the acute phase thus limiting their circulation. The causes of damage to the integrity of the epithelial barrier of the GI tract are likely to be multifaceted, but in the chronic stages of SIV infection seem unlikely to be due to direct virotoxic effects, given the lack of association with very low levels of demonstrable local viral replication in the LP relative to the extensive epithelial damage. One possible mechanism may be related to the preferential loss of Th17 cells in the GI tract in progressive immunodeficiency lentiviral infections [25], [26], because Th17 cells produce cytokines important for enterocyte proliferation and antibacterial defensins [49], [50], [51] and IL-17 has recently been shown to suppress Th1-mediated damage to gut epithelium. Importantly, preservation of this T cell subset in the gut of chronically SIVsmm-infected SMs and SIVagm-infected AGMs [26], [52] and the sparing of the epithelial barrier of SIVsmm infected SMs we show here supports this mechanism. We speculate that the association we found between immune activation, microbial translocation and chronic stages of SIV infection, and similarly, later stages of HIV-1 infection, reflects damage to the structural integrity of the GI tract and a potential “deficiency” of the GI tract macrophage-phagocytic system. Our observation that intestinal macrophages from SIV-infected RMs, which are generally not proinflammatory [40], are unable to clear translocated microbial products, within the LP, and could lead to increased proinflammatory responses locally are supported by several groups findings that showed: i) impaired monocyte phagocytosis in HIV-infected individuals [53]; ii) reduced LPS-mediated enhancement of phagocytosis in monocytes HIV-infected individuals compared to healthy donors [54]; and iii) significantly higher colonic mucosa proinflammatory mRNA expression levels (e.g. TNF-α, IFN-γ, and IL-6) in HIV-infected patients than in control patients [55]. These data certainly warrant further investigation into the functional properties of tissue macrophages from HIV/SIV-infected individuals and the mechanisms underlying their apparent dysfunction. While increased microbial translocation begins in the late acute stage of SIV infection, it was not until the later stages of infection that the capacity of macrophages for clearance was apparently affected, suggesting that microbial translocation has an increasing major contribution to immune activation as the host progresses towards disease. The evidence for this model comes from images of MesLN and AxLN stained for bacterial products in the chronic stage that showed dramatically increased extracellular bacterial constituents in the late AIDS stage of SIV infection, versus the mainly cellular staining of LPS at earlier stages. Taken together, these data strongly suggest that in SIV infection of RMs, and by extension, HIV infection of humans, damage to the epithelial barrier of the GI tract leads to levels of microbial translocation that exceed the capacity of host defense mechanisms to sequester away microbial constituents from secondary lymphatic tissues, resulting in persistent immune activation that contributes importantly to pathogenesis during the chronic phase of infection. Understanding the factors underlying damage to the integrity of the epithelial barrier and macrophage deficiencies that we report may lead to novel therapeutic interventions that aim to reduce microbial translocation and the deleterious effects of the consequent immune activation. Materials and Methods Animals and tissues To characterize the extent of microbial translocation in the gastrointestinal tract in SIV infection, we studied tissues from an assembled cohort of SIV-uninfected and infected RMs and SIVsmm-infected and uninfected SMs originally involved in separate studies (summarized in Table 1). Tissues (colon and LNs) were obtained at necropsy from 13 rhesus macaques (Macaca mulatta) of Indian origin euthanized 1–28 days after atraumatic intravaginal infection with SIVmac251 or SIVmac239 as described elsewhere [56]. Six additional SIV-negative RMs were used as controls. In a separate study, tissues were obtained at necropsy from 13 adult RMs chronically infected with SIVmac239 or SIVsmE660 that were sacrificed either because of end-stage disease (AIDS; defined by opportunistic infections, lymphomas, or a diagnosis of wasting, based on greater than 15% body mass weight loss, n = 5) or protocol specified experimental end point (Non-AIDS; n = 8). For immunohistochemistry studies, samples were very quickly processed into fixative to avoid potential artifacts associated with post-mortem tissue changes. The post mortem interval, the time from euthanasia until collection of lymph nodes and GI tract segments were placed into fixative, ranged from ∼10–30 minutes and was consistent over four independent primate facilities contributing tissues to the present study. The GI tract segments sampled at necropsy were representative and were not selected with regard to any visually apparent lesions or other pathology. In a third study, LNs and rectal biopsies were obtained from 2 SIVsmm-negative SMs (Cercocebus atys) and 5 SMs that were naturally infected with SIVsmm as previously described [47]. Animals were housed and cared in accordance with American Association for Accreditation of Laboratory Animal Care standards in AAALAC accredited facilties, and all animal procedures were performed according to protocols approved by the Institutional Animal Care and Use Committees of the National Cancer Institute, California National Primate Research Center or Yerkes National Primate Research Center (Table 1). Unfortunately, paraffin-embedded tissue from colon, mesLN, and axLN samples were not available from all animals. Plasma viral loads Plasma samples were analyzed for SIV vRNA by using a quantitative branched DNA (bDNA) assay [53] or using a fluorescent resonance energy transfer probe-based real-time RT-PCR (TaqMan) assay that provides a threshold sensitivity of 125 copy Eq/ml, as previously described [57]. All PCR reactions were run on ABI Prism 7700 Sequence Detection System and the fluorescent signal-based quantitation of viral RNA copy numbers in test samples was determined by ABI sequence detection software (Applied Biosystems, Foster City, CA). Immunohistochemistry, ISH and FISH Immunohistochemical staining and SIV in situ hybridization were performed as previously described [58]. In brief, unselected specimens of tissues of interest were obtained at necropsy, fixed, and paraffin embedded. Immunohistochemistry was performed using a biotin-free polymer approach (MACH-3; Biocare Medical) on 5-µm tissue sections mounted on glass slides, which were dewaxed and rehydrated with double-distilled water. Antigen retrieval was performed by heating sections in 1× DIVA Decloaker reagent (Biocare Medical) in a pressure cooker (Biocare Medical) followed by cooling to room temperature. All slides were stained using the intelliPATH FLX autostaining system (Biocare Medical) according to experimentally determined optimal conditions. This included blocking tissues with Blocking Reagent (Biocare Medical) for 10 min followed by an additional blocking step with TNB (0.1 M Tris-HCL (pH 7.5), 0.15 M NaCl, and 0.5% Blocking Reagent (NEN)) containing 2% Blocking Reagent and 100 µg/mL goat ChromePure IgG (Jackson Immunoresearch) for 10 minutes at room temperature. Endogenous peroxidase was blocked with 1.5% (v/v) H2O2 in TBS (pH 7.4). Primary antibodies were diluted in TNB containing 2% Blocking Reagent and 100 µg/mL goat ChromePure IgG for 1 h at room temperature. Mouse or rabbit MACH-3 secondary polymer systems (Biocare Medical) were applied for 20 minutes each. Double immunohistochemical staining was performed on colon and lymph node sections with either mouse monoclonal anti-LPS-core and rabbit polyclonal anti-IL18 antibodies or mouse monoclonal anti-IFNα and rabbit polyclonal anti-E.coli using the MACH-2 multiplex staining system (Biocare Medical) according to manufacturer's instructions. Sections were developed with ImmPACT DAB (Vector Laboratories) and/or Vulcan Fast Red chromogen (Biocare Medical), counterstained with hematoxylin, and mounted in Permount (Fisher Scientific). All stained slides were scanned at high magnification (400×) using the ScanScope CS System (Aperio Technologies, Inc.) yielding high-resolution data from the entire tissue section. Bacterial PNA FISH was performed using the universal bacterial 16 s ribosomal RNA specific (UniBac) FITC conjugated PNA probe (AdvanDx, Inc.) according to manufacturer's instructions, with the exception that a heat induced epitope retrieval pretreatment step was performed in 1× Diva retrieval buffer (Biocare Medical) for 20 minutes in a 95°C water bath prior to hybridization. FISH samples were examined and imaged using a Nikon 80i upright fluorescent microscope (Nikon Instruments, Inc) equipped with a BrightLine multiband bandpass FITC/Texas Red filter (Semrock; data not shown). Primary antibodies used were: mouse anti-human Interferon-α (clone MMHA-2; PBL InterferonSource), mouse anti-LPS core (clone WN1 222-5; Hycult or provided by Dr. Robin Barclay), mouse anti-macrophage (clone HAM56; Dako), mouse anti-cytokeratin (clone MNF116; Dako), polyclonal rabbit anti-E. coli (Dako), polyclonal rabbit anti-IL-18 (Sigma Prestige Antibodies by Atlas Antibodies) and polyclonal rabbit anti-Claudin-3 (Labvision). Confocal fluorescent microscopy Immunofluorescent confocal microscopy was performed on treated slides as above, but stained overnight with primary antibodies at 4°C, washed, stained with fluorescently conjugated secondary antibodies for 1 h in the dark, counterstained with DAPI (Molecular Probes), mounted in AquaPoly mount (Polysciences, Inc.) and imaged using a Olympus FluoView FV1000. Z-stack images were taken for each high power field that spanned the entire 5 µm tissue section and representative 3-D projections from z-stack images were generated using Imaris 7.0.0 software (Bitplane Inc.). Secondary antibodies used were donkey anti-mouse IgG Alexa Fluor 488 and donkey anti-rabbit IgG Alexa Fluor 555 (all from Molecular Probes). Quantitative image analysis To quantify microbial translocation (LPS) into the LP and the extent of epithelial barrier damage (claudin-3), 5-µm thick sections were cut from paraffin blocks of unselected tissue sections obtained at necropsy and stained with either monoclonal antibody against LPS-core or polyclonal antibody for claudin-3 and counter stained with heamotoxalin. High power (400×) whole tissue scans were obtained using an Aperio ScanScope as described above and imported into Photoshop CS3 (Adobe Systems Inc., Mountain View, California, USA). Images were manually trimmed to remove the submucosae, muscularis and residual luminal content, leaving only the LP mucosae to analyze. The percent area of the LP staining for LPS was determined essentially as previously described using Photoshop CS3 tools with plug-ins from Reindeer Graphics [39], [47]. The percent area of LN staining for LPS was determined from whole LN scans as above but without the need to trim the image. The proportion of the epithelial barrier that was damaged during SIV infection was first determined by manually tracing (in red) the area of the lumen/GI epithelial tract interface that had no claudin-3 staining epithelial cells, using the brush tool in Photoshop CS3. The remaining claudin-3 staining intact epithelial cell regions were then manually traced (in black). The percent damage was calculated by determining the proportion of the image that was red (lack of claudin-3 stain) compared to the total epithelial surface area (red+black) using plug-in tools from Reindeer Graphics. Statistical tests Spearman's rank correlation and Mann-Whitney tests were performed using Prism 4.0 software (Prism, San Diego, CA). Supporting Information Figure S1 Identification of microbial translocation (LPS) in large bowel of chronically SIV+ RMs. Low magnification whole tissue (top panel), 40× (middle panel) and 100× (bottom panel) images from high power whole tissue scans of colon immunohistochemically stained for LPS-core antigen (brown). Rectangles represent regions of the colon magnified in the successive images, while the rectangles displayed in the 100× lower panel images represent the region magnified and displayed in Figure 1. Scale bars = 1 mm. (4.38 MB TIF) Click here for additional data file. Figure S2 Identification of microbial translocation (E. coli) in large bowel of chronically SIV+ RMs. Low magnification whole tissue (top panel), 40× (middle panel) and 100× (bottom panel) images from high power whole tissue scans of colon immunohistochemically stained with a polyclonal antibody against E. coli (brown). Rectangles represent regions of the colon magnified in the successive images, while the rectangles displayed in the 100× lower panel images represent the region magnified and displayed in Figure 2. Scale bars = 1 mm. (4.42 MB TIF) Click here for additional data file. Figure S3 Identification of microbial translocation in gut draining MesLN of chronically SIV+ RMs. Low magnification whole tissue (top panel), 40× (middle panel) and 100× (bottom panel) images from high power whole tissue scans of MesLN immunohistochemically stained for LPS-core antigen (brown). Rectangles represent regions of the colon magnified in the successive images, while the rectangles displayed in the 100× lower panel images represent the region magnified and displayed in Figure 3. Scale bars = 1 mm. (5.35 MB TIF) Click here for additional data file. Figure S4 Identification of microbial translocation in systemic distal AxLN of chronically SIV+ RMs. Low magnification whole tissue (top panel), 40× (middle panel) and 100× (bottom panel) images from high power whole tissue scans of AxLN immunohistochemically stained for LPS-core antigen (brown). Rectangles represent regions of the colon magnified in the successive images, while the rectangles displayed in the 100× lower panel images represent the region magnified and displayed in Figure 4. Scale bars = 1 mm. (5.24 MB TIF) Click here for additional data file. Figure S5 Identification of microbial translocation (E. coli) in the liver of chronically SIV+ RMs. Low magnification whole tissue (top panel), 40× (middle panel) and 100× (bottom panel) images from high power whole tissue scans of liver immunohistochemically stained with a polyclonal antibody against E. coli (brown). Rectangles represent regions of the colon magnified in the successive images, while the rectangles displayed in the 100× lower panel images represent the region magnified and displayed in Figure 5. In 40× and 100× images, arrows point to portal triads, while arrow heads point to central veins. Scale bars = 1 mm. (4.89 MB TIF) Click here for additional data file. Figure S6 Spatial localization of microbial products with type I IFNα+ cells in colon. Images (200×) of colon stained for both E. coli (brown) and IFNα (red) from uninfected and chronically SIV-infected Non-AIDS and AIDS RMs. (8.30 MB TIF) Click here for additional data file. Figure S7 Spatial localization of microbial products with an effector marker of immune activation, IL-18, in MesLN. Images (400×) of MesLN stained for both LPS (brown) and IL-18 (red) from uninfected and SIV-infected Non-AIDS RMs. Note the spatial proximity of LPS+ cells and extracellular LPS with IL-18+ cells in MesLN. Scale bars = 50 µm. (10.20 MB TIF) Click here for additional data file. Figure S8 Damage to the integrity of the epithelial barrier in chronic SIV+ RMs. Low magnification whole tissue (top panel), 40× (middle panel) and 100× (bottom panel) images from high power whole tissue scans of colon from SIV uninfected and chronically SIV+ RMs immunohistochemically stained for the tight junction protein claudin-3 (brown). Rectangles represent regions of the colon magnified in the successive images, while the rectangles displayed in the 100× lower panel images represent the region magnified and displayed in Figure 7. Scale bars = 1 mm. (4.68 MB TIF) Click here for additional data file. Figure S9 GI tract damage results in PMN infiltration within the colon of chronically SIV+ RM. (A) Representative images (200×) of the colon stained for myeloperoxidase (brown) as a marker for PMNs. Note the increasing accumulation of myeloperoxidase+ PMNs adjacent to epithelial lesions in chronically SIV+ RM, reflecting a tissue response to loss of epithelial integrity, but the lack of this association seen in early acute SIV+ RM (4 dpi). (B) Random high power 400× images (10–15) of gut LP were taken and the percent area staining for myeloperoxidase (PMN) were determined in early/acute and chronic SIV+ RM. (3.11 MB TIF) Click here for additional data file. Figure S10 Damage to the integrity of the epithelial barrier is associated with increased enterocyte proliferation. Low magnification whole tissue (top panel), 40× (middle panel) and 100× (bottom panel) images from high power whole tissue scans of colon from SIV uninfected and chronically SIV+ RMs immunohistochemically stained for Ki67 (brown). Rectangles represent regions of the colon magnified in the successive images, while the rectangles displayed in the 100× lower panel images represent the region magnified and displayed in Figure 9. Scale bars = 1 mm. (4.66 MB TIF) Click here for additional data file. Figure S11 Quantitative image analysis of the frequency of HAM56+ macrophages that are E. coli + compared to the proportion of HAM56− E.coli + events in SIV-uninfected and acutely and chronically SIV-infected RMs. Note that throughout the acute phase of infection (1–21 dpi), most microbial products are within or associated with macrophages, however, starting at 28 dpi and extending into the chronic stage macrophages, although still abundant in the GI tract, become progressively inefficient/dysfunction in their ability to bind and/or phagocytose microbial products that cross the epithelial barrier. (0.13 MB TIF) Click here for additional data file.

0 comments Cited 182 times – based on 0 reviews      Review now

Bookmark

All references

Author and article information

Contributors

Daniel C. Douek: Role: Editor

Journal

Journal ID (nlm-ta): PLoS Pathog

Journal ID (iso-abbrev): PLoS Pathog

Journal ID (publisher-id): plos

Journal ID (pmc): plospath

Title: PLoS Pathogens

Publisher: Public Library of Science (San Francisco, USA )

ISSN (Print): 1553-7366

ISSN (Electronic): 1553-7374

Publication date Collection: June 2014

Publication date (Electronic): 26 June 2014

Volume: 10

Issue: 6

Electronic Location Identifier: e1004198

Affiliations

[1 ]Departments of Pharmacology, Case Western Reserve University School of Medicine, Cleveland, Ohio, United States of America

[2 ]Department of Medicine, Case Western Reserve University School of Medicine, Cleveland, Ohio, United States of America

[3 ]The School of Health and Rehabilitation Sciences, Division of Medical Laboratory Science, The Ohio State University College of Medicine, Columbus, Ohio, United States of America

[4 ]Departments of Epidemiology and Biostatistics, Case Western Reserve University School of Medicine, Cleveland, Ohio, United States of America

[5 ]Departments of Pathology, Molecular Biology and Microbiology, Pediatrics, and the Case Comprehensive Cancer Center, Case Western Reserve University School of Medicine, Cleveland, Ohio, United States of America

Vaccine Research Center, United States of America

Author notes

* E-mail: alan.levine@ 123456case.edu

The authors have declared that no competing interests exist.

Conceived and designed the experiments: CYC ADL. Performed the experiments: CYC SLA NTF. Analyzed the data: CYC PF. Wrote the paper: CYC ADL.

Article

Publisher ID: PPATHOGENS-D-13-03109

DOI: 10.1371/journal.ppat.1004198

PMC ID: 4072797

PubMed ID: 24968145

SO-VID: 70bfeff0-ce79-4236-a8bb-18a941ff464d

License:

This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

History

Date received : 24 November 2013

Date accepted : 6 May 2014

Page count

Pages: 18

Funding

This work was supported by grants from the National Institutes of Health (AI 076174 to ADL; T32 GM007250, TL1 TR000441, T32 GM-008803 supported CYC), the Case Western Reserve University Center for AIDS Research (AI 036219), Skin Diseases Research Center (P30 AR-039750), Visual Sciences Research Center Core (P30-EY11373), and School of Dental Medicine (P01 DE-019759). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Progressive Proximal-to-Distal Reduction in Expression of the Tight Junction Complex in Colonic Epithelium of Virally-Suppressed HIV+ Individuals

Read this article at

Abstract

Author Summary

Related collections

HIV Self-Testing

Most cited references 53

Alterations in the gut microbiota associated with HIV-1 infection.

Exposure to HIV-1 Directly Impairs Mucosal Epithelial Barrier Integrity Allowing Microbial Translocation

Damaged Intestinal Epithelial Integrity Linked to Microbial Translocation in Pathogenic Simian Immunodeficiency Virus Infections

Author and article information

Contributors

Journal

Affiliations

Author notes

Article

History

Page count

Funding

Categories

Comments

Comment on this article

Similar content 11

Cited by 31

Most referenced authors 1,582