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      NK-, NKT- and CD8-Derived IFNγ Drives Myeloid Cell Activation and Erythrophagocytosis, Resulting in Trypanosomosis-Associated Acute Anemia

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          African trypanosomes are the causative agents of Human African Trypanosomosis (HAT/Sleeping Sickness) and Animal African Trypanosomosis (AAT/Nagana). A common hallmark of African trypanosome infections is inflammation. In murine trypanosomosis, the onset of inflammation occurs rapidly after infection and is manifested by an influx of myeloid cells in both liver and spleen, accompanied by a burst of serum pro-inflammatory cytokines. Within 48 hours after reaching peak parasitemia, acute anemia develops and the percentage of red blood cells drops by 50%. Using a newly developed in vivo erythrophagocytosis assay, we recently demonstrated that activated cells of the myeloid phagocytic system display enhanced erythrophagocytosis causing acute anemia. Here, we aimed to elucidate the mechanism and immune pathway behind this phenomenon in a murine model for trypanosomosis. Results indicate that IFNγ plays a crucial role in the recruitment and activation of erythrophagocytic myeloid cells, as mice lacking the IFNγ receptor were partially protected against trypanosomosis-associated inflammation and acute anemia. NK and NKT cells were the earliest source of IFNγ during T. b. brucei infection. Later in infection, CD8+ and to a lesser extent CD4+ T cells become the main IFNγ producers. Cell depletion and transfer experiments indicated that during infection the absence of NK, NKT and CD8+ T cells, but not CD4+ T cells, resulted in a reduced anemic phenotype similar to trypanosome infected IFNγR-/- mice. Collectively, this study shows that NK, NKT and CD8+ T cell-derived IFNγ is a critical mediator in trypanosomosis-associated pathology, driving enhanced erythrophagocytosis by myeloid phagocytic cells and the induction of acute inflammation-associated anemia.

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          African trypanosomes are the causative agents of Human and Animal African Trypanosomosis, impairing economic development and causing death throughout the African continent. Anemia and inflammation are hallmark features of virtually every type of trypanosome infection. During experimental murine trypanosomosis, early inflammation causes enhanced red blood cell phagocytosis by cells of the myeloid phagocytic system, leading to severe anemia within 48 hours past peak parasitemia. Here, we identify the pro-inflammatory cytokine IFNγ as the main driver of the early inflammatory reaction and enhanced red blood cell phagocytosis. This IFNγ is derived consecutively by NK, NKT and CD8+ T cells, hence these cells all play a crucial role in the induction of inflammation and anemia.

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          Coordinated and distinct roles for IFN-alpha beta, IL-12, and IL-15 regulation of NK cell responses to viral infection.

          NK cell cytotoxicity, IFN-gamma expression, proliferation, and accumulation are rapidly induced after murine CMV infections. Under these conditions, the responses were shown to be elicited in overlapping populations. Nevertheless, there were distinct signaling molecule requirements for induction of functions within the subsets. IL-12/STAT4 was critical for NK cell IFN-gamma expression, whereas IFN-alphabeta/STAT1 were required for induction of cytotoxicity. The accumulation/survival of proliferating NK cells was STAT4-independent but required IFN-alphabeta/STAT1 induction of IL-15. Taken together, the results define the coordinated interactions between the cytokines IFN-alphabeta, IL-12, and IL-15 for activation of protective NK cell responses during viral infections, and emphasize these factors' nonredundant functions under in vivo physiological conditions.
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            Innate and cytokine-driven signals, rather than microbial antigens, dominate in natural killer T cell activation during microbial infection

            Invariant NK T cells (iNKT cells) recognize microbial and endogenous cellular lipid antigens presented by CD1d molecules (Brigl and Brenner, 2004; Kronenberg, 2005; Bendelac et al., 2007). iNKT cells express an invariant TCR-α chain (Vα14Jα18 in mice and Vα24Jα18 in humans) paired with a limited set of TCR-β chains, display surface receptors typically found on NK cells, and have a memory/effector phenotype in the absence of prior stimulation (Godfrey et al., 2004). iNKT cells constitutively express mRNA, but not protein, for IFN-γ, poising them for rapid effector function (Stetson et al., 2003). Together, these features distinguish iNKT cells from MHC-restricted T cells and suggest distinct modalities of activation. A growing body of evidence documents a critical role for iNKT cells during bacterial, viral, fungal, and protozoan infections (Tupin et al., 2007; Cohen et al., 2009). The protective, and in some instances detrimental, functions of iNKT cells during infection are often the result of their ability to rapidly produce copious amounts of IFN-γ and to contribute to the recruitment and activation of other cell types, including neutrophils, macrophages, DCs, NK cells, and B cells. These properties enable iNKT cells to orchestrate and amplify the protective immune response to infection (Brigl and Brenner, 2004; Tupin et al., 2007; Cohen et al., 2009). Yet how CD1d-restricted iNKT cells, with their limited TCR diversity, become activated rapidly in response to vastly diverse microbial infections remains incompletely understood. Two distinct pathways have been described for iNKT cell activation during microbial infection. The microbial antigen-driven pathway involves direct recognition of CD1d-presented microbial lipid antigens by iNKT cells. Glycosphingolipids (GSLs) found in Sphingomonas spp. (Kinjo et al., 2005; Mattner et al., 2005; Sriram et al., 2005) and diacylglycerols isolated from both Borrelia burgdorferi (Kinjo et al., 2006) and Streptococcus pneumoniae (Burrows et al., 2009) can be presented by CD1d molecules, and these microbial lipid antigens have been proposed to drive iNKT cell activation in a TCR-dependent manner during infection (Kinjo et al., 2005, 2006; Mattner et al., 2005). After exposure to these microbial antigens, iNKT cells produce both IFN-γ and IL-4 within hours (Kinjo et al., 2005, 2006; Mattner et al., 2005). The recognition of microbial glycosylceramides by iNKT cells has been proposed to fill a gap in the innate recognition of Gram-negative LPS-negative α-proteobacteria such as Sphingomonas spp. and Ehrlichia spp. (Kinjo et al., 2005; Mattner et al., 2005). The production of IFN-γ by iNKT cells in response to antigen stimulation does not require IL-12 signaling; however, IL-12 is known to play a critical role in the trans-activation of NK cells and the systemic release of IFN-γ after iNKT cell stimulation (Kitamura et al., 1999; Kawakami et al., 2001; Matsuda et al., 2003). In contrast to the TCR-mediated recognition of microbial lipid antigens, iNKT cells can be activated fully in response to microbial products by an innate cytokine- and self-antigen–driven pathway. In this scenario, iNKT cell activation results from combined stimulation with a weak TCR-mediated signal from recognition of endogenous CD1d-presented lipids, together with cytokine-mediated co-stimulation by IL-12, released by DCs after toll-like receptor (TLR)–mediated activation (Brigl et al., 2003; Mattner et al., 2005; Nagarajan and Kronenberg, 2007). iNKT cell activation after stimulation of DCs with TLR agonists can be modulated by alterations in CD1d-presented self-lipids and changes in CD1d expression levels (Sköld et al., 2005; Raghuraman et al., 2006; Paget et al., 2007; Salio et al., 2007). In some cases, such as stimulation with LPS from Escherichia coli or during viral infection, iNKT cell activation can be so dominantly driven by IL-12 and IL-18 that very little or no TCR-mediated stimulation by CD1d-presented self-lipids is needed (Nagarajan and Kronenberg, 2007; Tyznik et al., 2008; Wesley et al., 2008). This innate cytokine-driven pathway of activation allows iNKT cell recognition of pathogens that express TLR ligands but appear to lack CD1d-presented lipid antigens, such as viruses or the Gram-negative bacterium Salmonella typhimurium (Brigl et al., 2003; Mattner et al., 2005; Tyznik et al., 2008; Wesley et al., 2008). The current model suggests that, dependent on the expression of antigens by the microbe, iNKT cell activation during microbial infection is cognate, foreign antigen driven, or innate cytokine driven (Mattner et al., 2005; Tupin et al., 2007; Brigl and Brenner, 2010). In this paper, we investigated the relative contributions of microbial antigen– versus cytokine-driven pathways in iNKT cell activation using a large panel of diverse bacterial pathogens, several of which are known to express iNKT cell antigens and/or have been shown to require iNKT cells for protective immunity. Unexpectedly, we found that iNKT cell IFN-γ production was dominantly dependent on innate mechanisms with TLR-mediated signaling and the production of IL-12 by APCs, irrespective of whether or not bacteria express CD1d-presented iNKT cell antigens. Furthermore, high levels of IL-12 receptor were expressed by iNKT cells, readying them for rapid cytokine-mediated stimulation. Thus, our data suggest that innate signals, together with cytokine-driven activation, are the dominant pathway enabling rapid iNKT cell responses to diverse microbial infections. RESULTS Antigen- and cytokine-driven pathways of iNKT cell activation Studies using only NKT cell hybridomas do not adequately model the NKT cell activation mechanism that may occur in vivo because such systems lack the potential to respond to both antigen and cytokine signals. To investigate the mechanisms of iNKT cell activation by microbes, we used a system with primary mouse iNKT cell lines and BM-derived DCs that is able to respond to a variety of stimuli (Chiba et al., 2009). iNKT cell lines incubated with DCs and stimulated with the CD1d-presented microbial GSL antigen GSL-1, which is found in Sphingomonas spp. (Kinjo et al., 2005; Mattner et al., 2005; Sriram et al., 2005), produced large amounts of IFN-γ (Fig. 1 A, left). This IFN-γ response was dependent on recognition of CD1d by iNKT cell lines, as use of CD1d-deficient DCs resulted in markedly reduced IFN-γ secretion (Fig. 1 A). To determine the requirement for TLR- and cytokine-mediated stimulation for the activation of iNKT cell lines, we performed experiments using DCs deficient in the adaptor protein MyD88 or the production of IL-12, as well as blocking antibodies against IL-12. Stimulation of iNKT cell lines with GSL-1 was not altered when DCs deficient in MyD88 were used (Fig. 1 B) and was only marginally reduced when IL-12–deficient DCs were used or when mAbs to IL-12 were added to the cultures (Fig. 1 C). Thus, iNKT cell activation by a microbial lipid antigen required CD1d expression and was essentially independent of TLR signaling or IL-12 production by APCs. The slightly reduced iNKT cell IFN-γ response to GSL-1 in the presence of IL-12–deficient DCs or after addition of mAb against IL-12 was likely the result of a lack of IL-12–mediated amplification of iNKT cell IFN-γ production that has been noted after CD40L-mediated stimulation of DCs by antigen-activated iNKT cells (Tomura et al., 1999). Figure 1. Antigen- versus cytokine-driven pathways of iNKT cell activation. (A–C) iNKT cell lines were cultured with WT (filled squares) and CD1d-deficient (A), MyD88-deficient (B), or IL-12p35–deficient (C; all open squares) BM-derived DCs and stimulated with various concentrations of LPS, CpG, or GSL-1 for 16–24 h. Cytokine concentrations in culture supernatants were measured by ELISA. Data are presented as means of duplicate values ± SD and are representative of at least three independent experiments. Bar graphs show percent inhibition of IFN-γ secretion in culture supernatants comparing CD1d-deficient (A), MyD88-deficient (B), or IL-12p35-deficient (C) DCs (filled bars) to WT DCs after stimulation with 2 ng/ml LPS, 2 µg/ml CpG, or 10 µg/ml GSL-1, and data are summarized from four independent experiments (mean ± SD). Open bars in C represent the percentage of IFN-γ secretion in the presence of 10 µg/ml of blocking anti–IL-12 antibodies. Data are summarized from two independent experiments. No significant inhibition was observed with control antibodies (not depicted). (D and E) Experiments showing IL-4 production in response to GSL-1, LPS, or CpG using CD1d- (D) and MyD88- and IL-12–deficient (E) BM DCs were performed as in A–C. IL-4 concentrations in culture supernatants were measured by ELISA. n.a. indicates not applicable because IL-4 levels with WT DCs were very low. Data are presented as means of duplicate values ± SD and are representative of at least three independent experiments. In contrast to their direct stimulation by CD1d-presented microbial lipid antigens, iNKT cells can also be stimulated with TLR agonists in the presence of DCs by a self-antigen– and cytokine-driven pathway that does not require the cognate recognition of microbial antigens by iNKT cells (Brigl et al., 2003; Nagarajan and Kronenberg, 2007; Paget et al., 2007; Salio et al., 2007). Indeed, iNKT cell lines incubated with WT DCs and stimulated with the TLR agonists LPS (TLR4) or CpG (TLR9) produced copious amounts of IFN-γ (Fig. 1 A, second and third panel). To determine the requirements for a CD1d–TCR interaction, TLR signaling, and IL-12 stimulation after activation of iNKT cells with TLR agonists, we used DCs from CD1d-, MyD88-, or IL-12–deficient mice and blocking antibodies against IL-12. Using CD1d-deficient DCs, TLR agonist–induced IFN-γ production by iNKT cell lines was significantly reduced in response to LPS or CpG, respectively, compared with stimulation in the presence of WT DCs (Fig. 1 A). Because no exogenous microbial antigens were present under these conditions, recognition of CD1d-presented cellular self-antigens appeared to be required for iNKT cell activation, as has been previously noted (Brigl et al., 2003). When MyD88-deficient DCs were used during stimulation of iNKT cell lines, IFN-γ production in response to LPS or CpG was markedly reduced (Fig. 1 B). Furthermore, iNKT cell activation in response to LPS or CpG was strikingly reduced when DCs deficient in IL-12 production were used and when mAb to IL-12 was added to the cultures (Fig. 1 C). In response to antigens, iNKT cells are also known to secrete IL-4. After stimulation with GSL-1, iNKT cell lines produced IL-4 in a CD1d-dependent and MyD88- and IL-12–independent manner (Fig. 1, D and E). In response to LPS or CpG, no significant amounts of IL-4 were detected (Fig. 1, D and E). Thus, TLR agonist–induced IFN-γ secretion by iNKT cell lines in the presence of DCs was driven by recognition of CD1d-presented self-antigens and IL-12, which are produced by APCs after TLR-mediated activation, whereas antigen-driven activation results in TLR- and IL-12–independent IFN-γ and IL-4 secretion. Expression of microbial iNKT cell antigens To examine the mechanism of iNKT cell activation in response to bacterial infection, we assembled a panel of 11 bacteria, 5 of which are known to express CD1d-presented iNKT cell antigens (Table I). The diverse panel of bacteria was selected to include clinically important pathogens such as the Gram-positive bacteria S. pneumoniae, Listeria monocytogenes and Staphylococcus aureus, the Gram-negative bacteria Pseudomonas aeruginosa, S. typhimurium, and E. coli, the spirochete B. burgdorferi, the mycobacterium Mycobacterium tuberculosis, and the α-proteobacteria Sphingomonas capsulata, Novosphingobium aromaticivorans, and Sphingomonas yanoikuyae. Members of the class of α-proteobacteria are known to cause opportunistic infections in humans and have been associated with the induction of autoimmunity (Mohammed and Mattner, 2009; Ryan and Adley, 2010). Furthermore, iNKT cells are critical in mice for protective immunity to infection with S. pneumoniae, B. burgdorferi, P. aeruginosa, S. capsulata, and L. monocytogenes, and iNKT cells have been implicated in the development of primary biliary cirrhosis in a mouse model after infection with N. aromaticivorans (see Table I for summary and references). Table I. Bacteria used in this study Organism Strain Antigens Role of iNKT in infection Reference Gram-positive S. pneumoniae URF918 GlcDAG, GalGlcDAG Protective Burrows et al., 2009; Kawakami et al., 2003 L. monocytogenes 10403S N.d. Protective Arrunategui-Correa and Kim, 2004 S. aureus 25923a N.d. N.d. Brigl et al., 2003 Gram-negative P. aeruginosa D4 N.d. Protective Nieuwenhuis et al., 2002 S. typhimurium MT110 N.d. Not protective Berntman et al., 2005; Brigl et al., 2003 E. coli 25922a N.d. N.d. α-Proteobacteria S. capsulata 14666a GSL-1 (GlcAGSL) Protective Mattner et al., 2005 N. aromaticivorans 700278a GSL-1 Induction of PBC Mattner et al., 2008 S. yanoikuyae 51230a GSL-1 Protective Kinjo et al., 2005 Spirochetes B. burgdorferi N40 BbGL-II (GalDAG) Protective Kinjo et al., 2006; Kumar et al., 2000; Tupin et al., 2008 Mycobacteria M. tuberculosis H37Rv PIM4 b Not protective Behar et al., 1999; Fischer et al., 2004 Description of bacteria used in this study, microbial lipid antigens, if any, described for the respective bacterium, and role of iNKT cells during infection. GlcAGSL, glucuronic acid containing GSL; BbGL-II, B. burgdorferi glycolipid II; GalDAG, galactosyl diacylglycerol; PIM4, phosphatidylinositol mannoside 4; N.d., not described; PBC, primary biliary cirrhosis. a American Type Culture Collection number. b Antigenic activity of this lipid could not be confirmed by some investigators (Kinjo et al., 2006). From among the panel of bacteria, microbial CD1d-presented lipid antigens that are capable of stimulating iNKT cells have been described for S. capsulata, N. aromaticivorans, and S. yanoikuyae (all three GSL-1), B. burgdorferi (BbGL-II), and S. pneumoniae (monoglycosyl- and diglycosyldiacylglycerol; for details and references see Table I). To confirm that bacteria used in this study indeed expressed iNKT cell antigens, we isolated polar lipids from S. capsulata, N. aromaticivorans, S. yanoikuyae, B. burgdorferi, and S. pneumoniae and subjected the lipids to electrospray mass spectrometry analysis. Lipids from S. capsulata, N. aromaticivorans, and S. yanoikuyae yielded ions representing the [M – H]− ions of the antigenic α-glucuronosylceramide GSL-1 (Fig. 2, A–C; and Table S1). For B. burgdorferi lipids, ions corresponding to the [M + CH3COO]− adduct ions of α-galactosyldiacylglycerol, the BbGL-II antigen, were detected (Fig. 2 D and Table S1). In positive-ion mode, the lipids from S. pneumoniae gave rise to [M + Na]+ ions corresponding to the α-glucosyldiacylglycerol (GlcDAG), together with ions corresponding to the α–galactosyl GlcDAG (GalGlcDAG; Fig. 2 E and Table S1). The structural assignments for individual lipid species were supported by multiple-stage ion-trap tandem mass spectrometry (Table S1 and not depicted). Further analysis of the bacterial lipids by liquid chromatography, light scattering detection, and mass spectrometry analysis confirmed the presence of the respective antigenic lipid species in these five bacteria and suggested that all bacteria expressed significant amounts of the respective antigens (Fig. S1). Thus, 5 of the 11 bacteria used in this study expressed known iNKT cell lipid antigen species as expected. No lipids capable of stimulating iNKT cells directly have so far been described for the other 6 bacteria. Figure 2. Detection of microbial lipid antigens expressed by bacteria. Lipids were extracted from bacteria and analyzed by ESI mass spectrometry. (A–C) [M – H]− adducts of the GSL-1 (GlcAGSL) antigen in S. capsulata, N. aromaticivorans, and S. yanoikuyae. (D) [M + CH3COO]− adducts of the GalDAG (BbGL-II) antigen in B. burgdorferi. (E) [M + Na]+ adducts of the GlcDAG and GalGlcDAG antigens in S. pneumoniae. For annotation of sphingosine and acyl chain composition see Table S1. For corresponding LC and MS data of lipid antigens expressed by bacteria see SI Fig. 1. Mechanism of bacteria-induced iNKT cell activation We next examined the mechanism of iNKT cell activation in response to the 11 bacteria described in the previous section. iNKT cell lines incubated with WT DCs and stimulated with any of the 11 heat-killed bacteria produced substantial amounts of IFN-γ (Fig. 3 A). iNKT cell activation induced by heat-killed bacteria required a CD1d-TCR interaction, as IFN-γ production by iNKT cell lines in the presence of CD1d-deficient DCs and any of the 11 heat-killed bacteria was reduced by a mean of 50–80%, compared with stimulation in the presence of WT DCs (Fig. 3 A). Little or no IL-4 was generated by iNKT cells after stimulation with most of the heat-killed bacteria, whereas stimulation with B. burgdorferi, S. capsulata, N. aromaticivorans, and S. yanoikuyae resulted in production of low amounts of IL-4 (Fig. 3 B and not depicted). The amounts of IL-4 detectable in culture supernatants varied substantially between experiments and were generally reduced after repeated stimulation of iNKT cell lines, and no significant amounts of IL-4 were detected with iNKT cells isolated directly from Vα14 TCR transgenic mice (unpublished data). Figure 3. Cytokine responses of iNKT cells to diverse bacteria. iNKT cell lines were cultured with WT (filled squares) or CD1d-deficient (open squares) DCs in the presence of heat-inactivated bacteria. (A and B) IFN-γ (A) and IL-4 (B) concentrations were measured in culture supernatants by ELISA after 16–24 h. Data represent means of duplicate values ± SD and are representative of at least three independent experiments. Next, we determined if iNKT cell activation by heat-killed bacteria required TLR-mediated signaling by the APCs. iNKT cell lines incubated with MyD88-deficient DCs plus any of the 11 heat-killed bacteria resulted in reduced IFN-γ secretion when compared with WT DCs (Fig. 4 A and Fig. S2). Thus, whether the bacteria used for stimulation expressed known microbial lipid antigens, iNKT cell IFN-γ secretion was critically dependent on TLR-mediated signaling by DCs. This result suggested that iNKT cell activation in response to bacteria expressing microbial iNKT cell antigens might not be mediated by TCR recognition of the microbial lipid antigens and instead might be cytokine-driven. Figure 4. Mechanism of iNKT cell activation in response to diverse bacteria (A) iNKT cell lines were cultured with WT or MyD88-deficient DCs in the presence of heat-inactivated bacteria. Data for IFN-γ secretion in culture supernatants are shown as percent inhibition comparing MyD88-deficient to WT DCs after stimulation with 10 bacteria per DC for E. coli (Ec), P. aeruginosa (Pa), and S. typhimurium (St) and with 100 bacteria per DC for S. pneumoniae (Sp), L. monocytogenes (Lm), S. aureus (Sa), B. burgdorferi (Bb), S. capsulata (Sc), N. aromoticivorans (Na), S.yanoikuyae (Sy), and M. tuberculosis (Mt). Data are summarized from four independent experiments (mean ± SD). (B) Concentration of IL-12p40 (ng/ml, filled bars) and IL-12p70 (pg/ml, open bars) measured by ELISA in culture supernatants of DCs stimulated with microbial products or heat-killed bacteria as in Fig. 1 and Fig. 4 A, respectively (mean ± SD). No IL-18 was detected in culture supernatants and IFN-β concentrations were not different between unstimulated and stimulated conditions (not depicted). (C) iNKT cell lines were cultured with WT or IL-12p35–deficient DCs in the presence of stimuli as described in A. Data are shown as percentage of IFN-γ secretion in culture supernatants comparing IL-12–deficient to WT DCs after stimulation (filled bars; mean ± SD). Open bars represent the percentage of IFN-γ secretion in the presence of 10 µg/ml blocking mAb against IL-12. No significant inhibition was observed with isotype-matched control antibodies (not depicted). Data are summarized from two independent experiments. (D) Expression of the early activation marker CD25 by iNKT cells after 24 h of co-culture with WT or IL-12–deficient DCs in the presence of stimuli as in A. GSL-1 was used at 2 µg/ml, LPS at 10 ng/ml, and CpG at 2 µg/ml. (E) IL-4 production by iNKT cells in response to heat-inactivated bacteria using WT (filled squares), MyD88-deficient (open squares), or IL-12–deficient (open circles) DCs. Experiments were performed as in Fig. 1 (D and E). IL-4 concentrations in culture supernatants were measured by ELISA. Data are presented as means of duplicate values ± SD and are representative of at least three independent experiments. IL-12, IL-18, and type I IFNs have been implicated in activating iNKT cells after TLR-mediated stimulation of DCs in the absence of iNKT cell recognition of CD1d-presented microbial lipid antigens (Brigl et al., 2003; Mattner et al., 2005; Nagarajan and Kronenberg, 2007; Paget et al., 2007; Salio et al., 2007). We determined if any of these cytokines were generated by DCs after stimulation with the panel of heat-killed bacteria by measuring the concentration of IL-12, IL-18, and IFN-β in supernatants from DCs cultured with TLR agonist, GSL-1, or any of the 11 heat-killed bacteria. Significant quantities of IL-12p40 and IL-12p70, but not of IL-18 or IFN-β, were detected in supernatants of DCs exposed to LPS, CpG, or any of the 11 heat-killed bacteria (Fig. 4 B and not depicted). In contrast, no IL-12p40 or IL-12p70 was detected in supernatants of DCs cultured in the presence of the GSL-1 antigen. Then, we determined whether IL-12 was required for IFN-γ secretion by iNKT cell lines in response to stimulation with heat-killed bacteria. Indeed, incubation of iNKT cell lines with IL-12–deficient DCs in the presence of any of the 11 heat-killed bacteria resulted in substantially reduced IFN-γ production compared with stimulation in the presence of WT DCs (Fig. 4 C and Fig. S2). Similarly, addition of blocking mAb against IL-12 reduced iNKT cell IFN-γ secretion after stimulation with any of the 11 heat-killed bacteria (Fig. 4 C). In contrast, stimulation of iNKT cell lines with IL-18–deficient DCs or after stimulation of iNKT cell/DC co-cultures in the presence of antibodies blocking type I IFN signaling did not show a requirement for IL-18 or type I IFNs, respectively, in response to any of the 11 heat-killed bacteria (Fig. S3, A and B). As described, MyD88- and IL-12–deficient DCs were capable of presenting the purified GSL-1 antigen to stimulate iNKT cell lines but were not capable of eliciting IFN-γ secretion by iNKT cell lines in response to the TLR agonists LPS and CpG (Fig. 1). We next determined if expression of the early activation marker CD25 on iNKT cells after stimulation was dependent on IL-12. Expression levels of CD25 were increased after stimulation with GSL-1, TLR agonists, or heat-killed bacteria (Fig. 4 D and Fig. S4). Stimulation with TLR agonists or heat-killed bacteria, but not with GSL-1, resulted in decreased expression on CD25 when DCs deficient in IL-12 were used. Similar results were obtained for the early activation marker CD69 (unpublished data). iNKT cell IFN-γ production and CD25 expression were also both reduced on day 2 after stimulation with antigen, TLR agonists, or heat-killed bacteria, suggesting that iNKT cell activation in the absence of IL-12 was not delayed (Fig. S5, A and B). The weak IL-4 responses observed after stimulation with Borrelia and Sphingomonas spp. showed a trend toward being reduced when MyD88-deficient DCs were used and toward being increased when IL-12–deficient DCs were used (Fig. 4 E). iNKT cells freshly isolated from WT mice by tetramer sorting also showed a strong dependence of the iNKT cell IFN-γ production on MyD88 and IL-12, whereas little or no IL-4 was generated (Fig. S6, A and B). The CD1d-dependence of TLR agonist- and bacteria-induced activation of freshly isolated iNKT cell was reduced compared with what was observed using iNKT cell lines, which is likely the result of stimulation of iNKT cells using TCR-β antibodies and tetramer for their purification by cell sorting, as has been observed previously (Matsuda et al., 2003; Nagarajan and Kronenberg, 2007). To determine if alterations in CD1d expression levels by the APCs may contribute to the observed iNKT cell activation, CD1d expression levels were determined on WT DCs after exposure to heat-killed bacteria. Stimulation with the TLR agonists LPS and CpG and all 11 of the heat-killed bacteria, but not GSL-1, resulted in increased expression of CD1d by DCs (Fig. S7, A and B). The increased expression of CD1d after stimulation was absent in the presence of MyD88-deficient DCs, suggesting that TLR-mediated signaling was required. Thus, iNKT cell activation by a wide variety of heat-killed bacterial pathogens in vitro, including bacteria which expressed CD1d-presented microbial lipid antigens, such as Sphingomonas spp., B. burgdorferi, M. tuberculosis, or S. pneumoniae, was dominantly dependent on TLR signaling and IL-12–driven stimulation. iNKT cell activation after live in vitro infection To further investigate the mechanism of iNKT cell activation in response to DCs infected with live bacteria, we used an in vitro live infection model with three microorganisms shown to express iNKT cell antigens (B. burgdorferi, S. capsulata, and N. aromaticivorans; Table I) and three microorganisms for which iNKT cell antigens have not been described (P. aeruginosa, S. typhimurium, and L. monocytogenes). DCs were infected with bacteria for 3 h and infected DCs were then co-cultured with iNKT cell lines, resulting in production of substantial amounts of IFN-γ for all six bacteria tested (Fig. 5 A). To determine the relative requirements for a CD1d–TCR interaction, TLR signaling, and IL-12 stimulation after activation of iNKT cells with infected DCs, we used DCs from CD1d-, MyD88-, or IL-12–deficient mice. Infection of DCs deficient in CD1d, MyD88, or IL-12 production with P. aeruginosa, S. typhimurium, L. monocytogenes, B. burgdorferi, S. capsulata, or N. aromaticivorans resulted in reduced IFN-γ production by iNKT cell lines, compared with infection of WT DCs (Fig. 5 A). Live in vitro infection with B. burgdorferi, and to a lesser extent S. capsulata, resulted in the production of IL-4 (Fig. 5 B). This IL-4 production was largely MyD88 dependent but IL-12 independent. Thus, similar to the results obtained after stimulation with heat-killed bacteria, iNKT cell IFN-γ production in response to DCs infected with live bacteria was dependent on CD1d recognition, TLR-signaling, and IL-12 stimulation, irrespective of the expression of iNKT cell antigens by the bacteria. This suggested that iNKT cell activation in response to infected DCs was dominantly cytokine driven and that TLR-mediated signals were required, whereas CD1d-mediated signals played a variable role. Figure 5. iNKT cell response to live in vitro infection. DCs were incubated with 2 µg/ml CpG, 10 µg/ml GSL-1, P. aeruginosas, S. typhimurium (both 10 live bacteria per DC), L. monocytogenes, B. burgdorferi, S. capsulata, or N. aromaticivorans (all 100 live bacteria per DC) for 3 h, followed by washing and plating with iNKT cell lines. (A and B) IFN-γ (A) or IL-4 (B) concentrations were measured in culture supernatants by ELISA after 16–24 h. Data are displayed as means of triplicate measurements ± SD and are representative of at least two independent experiments. In vivo activation of iNKT cells during Sphingomonas infection Next, we analyzed iNKT cell activation in a mouse model of Sphingomonas infection. Mononuclear cells were isolated from WT mice 18 h after intravenous infection with S. yanoikuyae and iNKT cells were identified using CD1d tetramers. CD1d tetramer–positive iNKT cells from livers of uninfected mice stained at intermediate intensity for the early activation marker CD69 (mean fluorescence intensity [MFI], 465 ± 13) and, 18 h after infection, expression of CD69 increased to an MFI of 2,990 ± 639 (Fig. 6 A). IFN-γ secretion by iNKT cells showed that a mean of 41 ± 14% of the CD1d tetramer–positive iNKT cells were positive 18 h after infection, compared with a mean of 2 ± 0.1% in uninfected controls (Fig. 6 B). Thus, iNKT cells became rapidly activated after S. yanoikuyae infection. To assess whether IL-12 was required for the in vivo activation of iNKT cells during S. yanoikuyae infection, as suggested by our in vitro experiments, we compared IFN-γ secretion and CD69 expression by liver iNKT cells in WT versus IL-12–deficient mice after infection. 18 h after infection, IL-12 deficiency resulted in reduced IFN-γ secretion and CD69 expression (Fig. 6, C and D). Figure 6. In vivo activation of iNKT cells during S. yanoikuyae infection. (A) CD69 surface staining on CD1d tetramer–positive iNKT cells isolated from the livers of uninfected WT mice (dotted line) or 18 h after i.v. infection with S. yanoikuyae (bold line). (B) IFN-γ secretion by CD1d tetramer–positive liver iNKT cells from uninfected animals (left, bold line) or 18 h after i.v. infection with S. yanoikuyae (right, bold line) in comparison with staining with isotype control antibodies (dotted line). (C and D) Comparison of IFN-γ secretion (C) and CD69 expression (D) by CD1d tetramer–positive liver iNKT cells in WT and IL-12–deficient mice 18 h after i.v. infection with S. yanoikuyae (mean ± SD). (E) WT or IL-12-deficient mice were injected i.v. with 20, 5, or 1.25 µg GSL-1, and IFN-γ secretion by CD1d tetramer–positive liver iNKT cells was determined after 80 min. Means ± SD are shown. Data are pooled from three independent experiments and represent four to six mice per condition. (F) IL-4 secretion by liver iNKT cells 45 min after injection of 2 µg α-GalCer. Similar results were observed with GSL-1 (not depicted). (G) IL-4 secretion by liver iNKT cells from uninfected animals or 18 h after i.v. infection with S. yanoikuyae in comparison with staining with isotype control antibodies. Data are representative of two independent experiments using three to four mice per condition. To compare the requirement for IL-12 in iNKT cell IFN-γ secretion after infection with S. yanoikuyae bacteria that express the GSL-1 antigen with iNKT cell activation induced by the GSL-1 antigen, we analyzed iNKT cell activation after stimulation with purified GSL-1 antigen. After injection of a range of doses of GSL-1 antigen, IFN-γ secretion by CD1d tetramer–positive liver iNKT cells was similar in WT or IL-12–deficient mice (Fig. 6 E). IL-4 production by iNKT cell in vivo was readily observed after injection of the α-galactosylceramide (GalCer) antigen; however, during infection with S. yanoikuyae, S. capsulata, or N. aromaticivorans no significant amounts of IL-4 were detected in either WT or IL-12–deficient mice (Fig. 6, F and G; and not depicted). Thus, iNKT cell activation after infection with a GSL-1–expressing microbe is dependent on IL-12, whereas iNKT cell activation with a microbial antigen alone does not require IL-12. iNKT cell activation during S. pneumoniae infection Next, we examined the role of iNKT cells after infection with S. pneumoniae. Infection of WT and iNKT cell–deficient Jα18−/− mice with S. pneumoniae showed reduced survival of iNKT cell–deficient Jα18−/− mice (Fig. 7 A). Between days 6 and 12 after infection, >50% of Jα18−/− mice succumbed to infection, whereas none of the WT animals died. Similar results were obtained in CD1d-deficient animals (Fig. S8 A). Bacterial burden in lungs of infected WT or Jα18−/− animals were comparable on day 3 after infection. However, on day 6 after infection, bacterial burden in lungs of Jα18−/− were ∼1,000-fold higher when compared with WT animals (Fig. 7 B). Thus, iNKT cells were critically important for protective immunity after pulmonary infection with S. pneumoniae. Figure 7. In vivo activation of iNKT cells during S. pneumoniae infection. WT or Jα18-deficient mice were infected intranasally with S. pneumoniae. (A) Survival was recorded daily for 2 wk for WT (filled squares; n = 9) and Jα18−/− (open squares; n = 9) mice. Results are representative of two independent experiments. (B) The number of CFU was determined in lung tissues of WT (open bars; n = 6) or Jα18−/− (filled bars; n = 6) on days 3 and day 6 after infection. Data are pooled from two independent experiments (mean ± SD). (C and D) Lymphocytes were isolated from lungs of mice infected intratracheally with S. pneumoniae or from uninfected mice and analyzed by flow cytometry. Staining for surface expression of CD69 (C) or secreted IFN-γ or IL-4 (D) on CD1d tetramer–positive lymphocytes are shown. Data represent means ± SD (n = 6) and are pooled from two independent experiments. (E and F) WT or IL-12p35–deficient mice were infected intratracheally with S. pneumoniae and secretion of IFN-γ (E) or expression of CD69 by iNKT cells (F) was determined as described in C and D on day 2 after infection. Data represent means ± SD for three to four mice per group and one experiment of two similar experiments is shown. To examine iNKT cell activation after pulmonary infection with S. pneumoniae, lymphocytes were isolated from lungs of infected and uninfected animals and iNKT cells were analyzed by flow cytometry for surface expression of the activation marker CD69 and for secretion of IFN-γ. Expression of CD69 was intermediate on iNKT cells from uninfected mice and increased on days 2, 4, and 7 after infection (Fig. 7 C). Thus, iNKT cells became activated rapidly after S. pneumoniae infection. Cytokine secretion assays showed significant IFN-γ secretion by iNKT cells on days 2 and 4 after infection that returned to baseline by day 7 after infection (Fig. 7 D). No significant production of IL-4 by iNKT cells was observed in either WT or IL-12–deficient mice during infection at any of the time points examined (Fig. 7 D and not depicted). Thus, iNKT cells became activated rapidly and expressed IFN-γ early during pulmonary infection with S. pneumoniae. Next, we determined whether IL-12 was required for the activation of iNKT cells during S. pneumoniae infection, as was the case in the in vitro experiments described in the previous paragraphs. IL-12 deficiency resulted in reduced IFN-γ secretion and CD69 expression on days 2 and 4 after infection (Fig. 7, E and F; and Fig. S8, B and C). Thus, most of the iNKT cell activation and IFN-γ production during S. pneumoniae infection in vivo was critically dependent on IL-12. Expression and function of IL-12 receptor on iNKT cells Responsiveness to IL-12 correlates with expression of IL-12 receptor (IL-12R) on NK and activated T cells (Presky et al., 1996; Trinchieri, 2003). To determine the expression of IL-12R by iNKT cells, we took complementary approaches. First, expression of mRNA for the two IL-12R subunits, IL-12Rβ1 and IL-12Rβ2, was determined by microarray analysis in iNKT cells and other lymphocyte subsets. IL-12Rβ1, which is responsible for binding IL-12, was expressed at very low levels by naive CD4 and CD8 T cells, memory CD8 T cells, γδT cells, and NK cells, whereas slightly higher levels were detected in memory CD4 T cells (Fig. 8 A). In contrast, much higher levels of IL-12Rβ1 mRNA were detected in both CD4+ and CD4− iNKT cells isolated from spleen or liver. IL-12Rβ2, which is required for IL-12R–mediated signaling, was expressed at very low levels by naive CD4 and CD8 T cells, memory CD8 T cells, and γδT cells, whereas slightly higher levels were expressed by memory CD4 T cells (Fig. 8 A). In comparison, much higher levels of IL-12Rβ2 mRNA were detected in NK cells. Intermediate levels of IL-12Rβ2 were expressed by CD4+ and CD4− iNKT cells isolated from spleen, and high levels, similar to those detected on NK cells, were detected in CD4+ and CD4− liver iNKT cells. Second, we determined if the expression levels of IL-12R mRNA correlated with protein expression. Using CD1d tetramers to detect iNKT cells, we found that expression of IL-12Rβ1 could be detected exclusively on tetramer+/CD3+ lymphocytes (iNKT cells) but not on tetramer−/CD3− lymphocytes (mainly NK cells) or tetramer−/CD3+ (containing MHC-restricted CD4 and CD8 αβT cells and γδT cells; Fig. 8 B). Figure 8. Expression and function of IL-12 receptor on iNKT cells. (A) IL-12Rβ1 (left) and IL-12Rβ2 (right) mRNA expression in purified splenocyte subsets or iNKT cell subsets purified from spleen or liver was assessed by microarray. Data are representative of two to four independent experiments and are shown as mean ± SD. (B) Flow cytometry staining of CD19− splenocytes with CD1d tetramers and anti-CD3 antibodies (left). The tetramer−/CD3− gate contains NK cells, the tetramer−/CD3+ gate contains MHC-restricted CD4 and CD8 αβT cells and γδT cells, and the tetramer+/CD3+ gate contains iNKT cells. Surface expression of IL-12Rβ1 was determined by flow cytometry on gated lymphocyte subpopulations. (C) Naive and memory CD4 T cells, NK cells, and iNKT cells were isolated from spleens of WT mice and purified by cell sorting. Cells were stimulated in the presence of 1 ng/ml recombinant IL-12 for 1 h and subsequently stained with antibodies against the phosphorylated form of STAT4 (solid lines) or isotype control antibodies (dotted lines). (D) iNKT cell lines were incubated with WT (filled squares) or CD1d−/− (open squares) DCs in the presence of various concentrations of recombinant IL-12. Data are presented as means of duplicate values ± SD and are representative of at least three independent experiments. (E) iNKT cell lines were incubated with WT DCs and various concentrations of the microbial lipid antigen GSL-1 in the absence or presence of various concentrations of recombinant IL-12. IFN-γ concentrations were measured in culture supernatants by ELISA after 16–24 h. Data are presented as means of triplicate values ± SD and are representative of two independent experiments. The specific cellular effects of IL-12 are mainly a result of its ability to induce STAT4 activation. To determine if the constitutive expression of high levels of IL-12 receptor enabled prompt STAT4 activation in iNKT cells after IL-12 stimulation, we purified naive and memory CD4 T cells, NK cells, and iNKT cells by cell sorting and stimulated the purified cell populations with IL-12 for 1 h. Intracellular phosphorylation of STAT4 was assessed by antibody staining and flow cytometry. No STAT4 phosphorylation was detected in naive and memory CD4 T cells (Fig. 8 C). In contrast, phosphorylation of STAT4 was detected in a large number of NK cells and iNKT cells. Thus, unique among T cells but similar to NK cells, high constitutive expression of functional IL-12 receptor appears to provide a mechanistic explanation for the ability of iNKT cells to rapidly secrete IFN-γ in response to IL-12–mediated stimulation. Last, we sought to address how IL-12–mediated co-stimulation alters iNKT cell activation by CD1d-presented self- and microbial lipids. The addition of recombinant IL-12 to iNKT cell lines cultured in the presence of WT DCs resulted in notably increased IFN-γ secretion, as compared with the addition of IL-12 to iNKT cell lines co-cultured in the presence of CD1d-deficient DCs (Fig. 8 D). As no exogenous antigens were added in this system, the stimulation provided by CD1d recognition was likely a result of recognition of CD1d-presented self-lipids. Next, we determined how co-stimulation with IL-12 altered iNKT cell responses to microbial antigens. A cross-titration of GSL-1 and IL-12 revealed that in particular low concentrations of GLS-1 and low concentrations of IL-12 resulted in a synergistic stimulation of IFN-γ by iNKT cells (Fig. 8 E). Thus, weak TCR-mediated stimulation of iNKT cells with CD1d-present self- or microbial antigens was significantly amplified by IL-12, resulting in potent secretion of IFN-γ by iNKT cells. DISCUSSION iNKT cells have been shown to play important protective roles during several bacterial, parasitic, and viral infections. However, the central question of how they are activated by microbes is not fully explained. Because viruses lack lipid antigens and antigens have only been described for a few pathogens so far, it is important to determine how many diverse pathogens can activate iNKT cells. To investigate the mechanism of iNKT cell activation in response to infection, we used a large panel of diverse bacteria, including several that express known CD1d-presented iNKT cell lipid antigens. Unexpectedly, we found that, irrespective of the expression of CD1d-presented lipid antigens by the microbes, microbe-induced iNKT cell activation and IFN-γ production in vitro and in vivo was critically dependent on IL-12 released by DCs after TLR-mediated activation. Thus, our data do not support a recently proposed model in which iNKT cell activation during infection is driven by TCR-mediated recognition of CD1d-presented microbial antigens (Mattner et al., 2005; Tupin et al., 2007). Instead, our data suggest that innate cytokine-driven activation is the dominant pathway for iNKT cell activation in response to virtually all infectious agents examined to date that induce the production of IL-12 by APCs after TLR-mediated activation (Fig. 9). Figure 9. Innate and cytokine-driven iNKT cell activation during microbial infection. iNKT cell activation during microbial infection is dominantly driven by innate TLR-mediated signals and IL-12, which is released by DCs after stimulation with microbial products. In addition, TCR-mediated stimulation contributes to iNKT cell activation. However, the TCR-mediated signal alone, provided either by recognition of CD1d-presented self- or microbial antigens, is not sufficient to result in iNKT cell IFN-γ production in the absence of IL-12 stimulation. The constitutive expression of high levels of IL-12 receptor endows iNKT cells with the ability to respond rapidly to cytokine-mediated stimulation and ensures immediate iNKT cell activation in response to virtually any infectious agent that induces the production of IL-12, irrespective of the expression of microbial lipid antigens, thus allowing iNKT cells to overcome their restricted TCR specificity. In addition to IL-12 as a critical mediator for stimulating iNKT cell responses to microbes, other studies have suggested a role for IL-18 and type I IFN in the iNKT cell response to E. coli LPS or the TLR9 agonist CpG, respectively (Nagarajan and Kronenberg, 2007; Paget et al., 2007). However, our in vitro experiments did not show a significant role for IL-18 or type I IFNs in response to any of the 11 bacterial pathogens tested. Instead, our in vitro and in vivo experiments repeatedly implicated IL-12 as the critical cytokine required for iNKT cell activation and IFN-γ secretion in response to all microbes tested. Underscoring the critical and dominant role of IL-12 for rapid activation of iNKT cells, we detected that constitutive expression of high levels of IL-12Rβ1 and IL-12Rβ2 chains by iNKT cells and IL-12 stimulation rapidly led to STAT4 activation in iNKT cells. In contrast, MHC-restricted T cells lack constitutive expression of components of the IL-12 receptor and are only known to express functional IL-12 receptor after activation and Th1 differentiation (Presky et al., 1996; Trinchieri, 2003). In agreement with previous studies, we found that naive and memory CD4+ T cells were not able to activate STAT4 after IL-12 stimulation in the absence of additional stimulation or differentiation. Recently, MAIT (mucosal-associated invariant T) lymphocytes, another lymphocyte population characterized by expression of an invariant TCR-α chain, have been shown to become activated by microbes independently of TLR- and cytokine-mediated stimulation, suggesting that these innate T cells require recognition of cognate microbial antigens for their activation (Le Bourhis et al., 2010). Thus, among T cells, iNKT cells appear to be uniquely equipped to promptly secrete IFN-γ upon primary encounter of an inflammatory condition that results in the production of IL-12. Our in vitro experiments showed that full iNKT cell activation in response to all pathogens tested also required CD1d-dependent signals and that increased expression of CD1d by bacteria-stimulated APCs may contribute to iNKT cell activation. However, in the absence of co-stimulation by IL-12, the CD1d-mediated signals were not sufficient to induce IFN-γ secretion by iNKT cells. Our in vitro studies showed that iNKT cell responses to weak self-antigens or to low concentrations of microbial antigen were amplified by exogenously added IL-12. Therefore, although not able to activate iNKT cells through TCR-mediated signaling alone, it seems likely that even small numbers of CD1d–microbial antigen complexes or presentation of more potent self-antigens may contribute to or modulate iNKT cell activation during infection in the presence of IL-12–mediated co-stimulation. Furthermore, the requirement for CD1d-TCR–mediated signals may ensure that IL-12–amplified iNKT cell activation occurs only in close contact with CD1d-expressing APCs such as monocytes, DCs, macrophages, and B cells. The lack of dependence on specific recognition of CD1d-presented microbial antigens found in this study suggests that the exceptional potency of the pharmacological iNKT cell agonist α-GalCer, which is much greater than that of any naturally occurring iNKT cell antigen found to date, may have led to overestimation of the importance of microbial antigens in iNKT cell activation. iNKT cell responses to all of the microbial antigens described so far result in the production of both IFN-γ and IL-4 by iNKT cells both in vitro and in vivo. Most of the bacteria tested in our studies resulted in a bias toward production of IFN-γ, using iNKT cell lines, freshly isolated iNKT cells, and during in vivo infection. Interestingly, the occasionally observed IL-4 induced by Sphingomonas spp. in vitro was increased in the absence of IL-12, suggesting that IL-12 may play an important role in polarizing iNKT cell responses to infection. However, in vivo infection with Sphingomonas spp. in IL-12–deficient mice did not result in a notable increase in the number of iNKT cells producing IL-4 (unpublished data). Stimulation of iNKT cells with B. burgdorferi resulted in IL-4 production by iNKT cells in our studies, which is consistent with previous studies detecting IL-4 production by a small number of iNKT cells in spleen after B. burgdorferi infection (Tupin et al., 2008). The in vitro IL-4 production observed in our studies appeared to be IL-12 independent but MyD88 dependent, suggesting that innate signals, rather than microbial antigens, are responsible for the iNKT cell IL-4 production in response to this bacterium. Further studies will be required to understand the nature of the signals responsible for bacteria-induced IL-4 production by iNKT cells. Activation of iNKT cells during infection driven by innate signals and cytokines, rather than microbial antigens, provides several advantages. As an amplification loop for innate signals, cytokine-mediated activation enables the activation of iNKT cells in response to virtually any microorganism that induces IL-12 production, irrespective of the expression of iNKT cell ligands, and thus allows iNKT cells to overcome their restricted TCR repertoire and conserved mode of antigen-recognition. Such a mechanism of activation may be critical for iNKT cell responses to viral infection, because viruses are not known to encode enzymes for the production of unique microbial lipids, and for responses to other microorganisms that lack expression of CD1d-presented iNKT cell ligands. For the organisms that contain iNKT cell lipid antigens, synthesis, uptake, and loading of antigens into CD1d molecules, and subsequent transport of antigen–CD1d complexes to the surface of APCs, may be inefficient and slower than the IL-12–driven iNKT cell response. The rapid innate cytokine-driven activation of iNKT cells provides the evolving immune response with T cell effector functions early during the course of infection that otherwise would only be available after the expansion and differentiation of antigen-specific MHC-restricted T cells (Brigl et al., 2003; Chiba et al., 2008). During infection, iNKT cell–secreted IFN-γ appears to serve several critically important functions. This includes a critical role in activation of macrophages and in neutrophil-mediated clearance of pathogens (Nieuwenhuis et al., 2002; Nakamatsu et al., 2007). In addition, the early IFN-γ provided by iNKT cells has been shown to be critical for the development of adaptive Th1 immune responses and may affect the magnitude and quality of MHC-restricted CD4 and CD8 T cell responses during infection (Tupin et al., 2007; Cohen et al., 2009). Together, iNKT cells, and the IFN-γ they produce, have emerged as critical regulators of the early immune response during infection. In summary, our findings indicate that during infection with a variety of bacterial pathogens, including bacteria which express known iNKT cell antigens, iNKT cell activation is driven predominantly by IL-12, rather than by TCR-mediated recognition of CD1d-presented microbial antigens (Fig. 9). In contrast to the dominant role observed for foreign antigen recognition in the activation of MHC-restricted T cells, the lack of dependence on specific recognition of cognate microbial antigen defines a different role for the TCR in innate T cell activation. For the activation of iNKT cells during infection, CD1d–TCR interactions instead appear to determine the cell–cell interaction between iNKT cells and APCs and allow T cell activation to become manifest. This may control such innate cytokine-driven activation so that it is localized to sites of APC-produced IL-12. The constitutive expression of high levels of IL-12 receptor uniquely equips iNKT cell for immediate cytokine-driven responses. Our results suggest a unified dominantly innate cytokine-driven model of iNKT cell activation that explains how these cells become activated efficiently and rapidly in response to highly diverse microbial pathogens. MATERIALS AND METHODS Mice. C57BL/6, IL-12p40−/−, IL-12p35−/−, and IL-18−/− mice were obtained from The Jackson Laboratory. Jα18−/− (Cui et al., 1997) and CD1d−/− mice (Exley et al., 2003) were provided by M. Exley (Harvard Medical School, Boston, MA), Vα14Jα18 TCR transgenic mice (Bendelac et al., 1996) by A. Bendelac (University of Chicago, Chicago, IL), and MyD88−/− (Adachi et al., 1998) by K. Kobayashi (Harvard Medical School, Boston, MA). All mice were on the C57BL/6 background, were housed in specific pathogen-free conditions or a biosafety level 2 animal facility after in vivo infections, and female mice at 6–14 wk of age were used for experiments. All animal studies were approved by the Dana-Faber Cancer Institute Animal Care and Use Committee. In vitro iNKT cell assay. iNKT cell lines were generated and maintained in complete RPMI medium (RPMI supplemented with 10% FBS [Gemini], Hepes [Invitrogen], L-glutamine, penicillin/streptomycin, and 2-ME) supplemented with 20 U/ml of recombinant mouse IL-2 (PeproTech) and 10 ng/ml IL-7 as previously described (Chiba et al., 2009). DCs were generated from BM of mice cultured in complete RPMI supplemented with 10 ng/ml of recombinant mouse GM-CSF and 1 ng/ml IL-4 for 5–8 d and purified with CD11c magnetic beads (Miltenyi Biotec). iNKT cell lines (105/well) were cultured with DCs (2 × 104/well) in 96-well plates (Costar; Corning) in complete RPMI. Freshly isolated iNKT cells were obtained from spleens of WT B6 animals after depletion of CD19+ and CD8a+ cells using magnetic beads (Miltenyi Biotec), followed by cell sorting using CD1d tetramers and TCR-β staining on a FACSAria IIu instrument (BD). GSL-1 (National Institutes of Health [NIH] Tetramer Core Facility), LPS (Salmonella enterica serotype Typhimurium; Sigma-Aldrich), CpG (ODN1826; InvivoGen), recombinant mouse IL-12 (PeproTech), or bacteria were added and cultures were incubated for 16–48 h at 37°C. Culture supernatants were analyzed by ELISA with matched antibody pairs (IFN-γ and IL-4 [BD], sensitivity 10 and 2 pg/ml, respectively; IL-18 and IFN-β [R&D Systems]). Blocking anti–IL-12 (BD) and anti–mouse IFN-α/β R1 antibodies (R&D Systems) were added to cultures at 5–10 µg/ml. Expression of CD25 was determined on iNKT cells by flow cytometry (gated by FSC/SSC). Surface expression of CD1d on CD11c+ BM DCs was determined using CD1d Abs (BD) after incubation with various stimuli. Bacteria. S. pneumoniae (for strains and references see Table I; provided by K. Kawakami (University of the Ryukyus, Nishihara, Okinawa, Japan), cultured in THY [BD] or BHI [Oxoid] medium), L. monocytogenes (BHI medium), S. aureus (LB medium; BD), P. aeruginosa (LB medium, provided by R. Blumberg, Harvard Medical School, Boston, MA), S. typhimurium (LB medium), E. coli (LB medium), S. capsulata (MH medium, 30°C; Oxoid), N. aromaticivorans (MH medium), S. yanoikuyae (TSB), and B. burgdorferi (BSK-H medium, 33°C; provided by L. Bockenstedt, Yale School of Medicine, New Haven, CT; Sigma-Aldrich) were grown in culture medium at 37°C, unless otherwise noted, until mid- to late-log phase, washed 3× in LPS-free PBS (Invitrogen), and heat inactivated at 65°C for 45 min. B. burgdorferi were inactivated at 48°C for 30 min. M. tuberculosis were inactivated by γ radiation (Colorado State University, Fort Collins, CO). All bacterial preparations, except gram-negative bacteria, tested negative for LPS by Limulus amebocyte lysate test (limit of detection 0.03 EU/ml; Associates of Cape Cod). Lipids, lipid extraction, and MS analysis. Lipids were extracted and analyzed by thin layer chromatography, as described elsewhere (Tatituri et al., 2007), or analyzed by high performance liquid chromatography (HPLC) using a AutoPurification HPLC system coupled to a mass detector with both ES and APCI capabilities, evaporative light scattering detector, and photodiode array detector (Waters). MS analysis, including low-energy CAD MSn experiments, were conducted on a linear ion-trap (LIT) mass spectrometer (Finnigan; Thermo Fisher Scientific) with Xcalibur operating system. Lipid extracts dissolved in chloroform/methanol (1/2) were continuously infused (2 µl/min) to the ESI source, where the skimmer was set at ground potential, the electrospray needle was set at 4.5 kV, and temperature of the heated capillary was 300°C. The automatic gain control of the ion trap was set to 2 × 104, with a maximum injection time of 100 ms. Helium was used as the buffer and collision gas at a pressure of 10−3 mbar (0.75 mTorr). The MSn experiments were performed with an optimized relative collision energy ranging from 18–25%, an activation q value at 0.25, and the activation time at 30–50 ms to leave a minimal residual abundance of precursor ion (around 10%). Mass spectra were accumulated in the profile mode, typically for 3–10 min for MSn (n = 2, 3, and 4) spectra. The mass resolution of the instrument was tuned to 0.6 D at half peak height. Synthetic GSL-1 was provided by the NIH tetramer facility and P.B. Savage (Brigham Young University, Provo, UT; Mattner et al., 2005). Synthesis of α-GalCer has been previously described (Veerapen et al., 2009). In vitro infections. Bacteria were grown to mid-log phase, as described in Bacteria, and added to DCs cultured in antibiotic-free complete RPMI. After a 3-h incubation at 37°C, infected DCs were washed 3× in complete RPMI. Infected DCs (5 × 104/well) were co-cultured with iNKT cell lines (105/well) in complete RPMI medium in 96-well plates for 16–24 h. In vivo infection and flow cytometry. S. pneumoniae was grown as described in Bacteria and mice were anesthetized with i.p. ketamine/xylazine or by inhalation isoflurane and inoculated intranasally or intratracheally, respectively, with 2–3 × 106 bacteria in PBS. CFUs were determined on blood agar plates. For flow cytometric analysis, mononuclear cells were isolated from lungs perfused with PBS followed by digestion with collagenase/DNase. S. yanoikuyae was grown in TSB supplemented with 5% sheep blood and passaged in SCID mice. Mice were injected i.v. with 4 × 109 bacteria in 200 µl PBS. Cells were stained with antibodies against CD45, CD19, TCR-β, PBS57-loaded CD1d tetramers (NIH Tetramer Core Facility), CD69, or isotype-matched control antibodies. Secretion of IFN-γ or IL-4 was determined by cytokine secretion assay (Miltenyi Biotec) according to the manufacturer’s instructions. Flow cytometric data were collected on a LSR II flow cytometer (BD) and analyzed with FlowJo software (Tree Star). IL-12 receptor expression. iNKT cells were purified from spleen or liver on a MoFlo cell sorter (Dako) gating on CD19−, B220−, Ter119−, CD11b−, CD11c−, LyG/Gr-1−, CD45+, TCRint, CD1d-tetramer+, CD4+, or CD4− iNKT cells, followed by RNA extraction with Trizol (Invitrogen), RNA amplified, and hybridized to Affymetrix 1.0 ST MuGene arrays within the NIAID/NIH ImmGen project (protocols and raw data available at http://www.immgen.org; Heng and Painter, 2008). For flow cytometric analysis, cells were stained with CD1d tetramers and antibodies against IL-12Rβ1 (BD). STAT4 phosphorylation. Naive (CD62LhiCD44lo) and memory (CD62LloCD44hi) TCRβ+CD4+ T cells, NK cells (NK1.1+TCR-β−), and iNKT cells (as in the previous section) were isolated by negative selection and cell sorting. 1–5 × 105 purified cells were cultured in completed RPMI and stimulated with recombinant IL-12 for 1 h at 37°C and subsequently stained with antibodies against pSTAT4 (BD) according to manufacturer’s protocols. Online supplemental material. Fig. S1 shows mass spectrometry analysis of microbial lipids separated by liquid chromatography. Fig. S2 shows representative dose titrations for bar graphs depicted in Fig. 4 (A and C). Fig. S3 shows IL-18 and type I IFN independence of iNKT cell activation in response to microbes. Fig. S4 shows representative FACS plots for bar graphs depicted in Fig. 4 D. Fig. S5 shows IL-12 dependence of IFN-γ secretion and CD25 expression by iNKT cells on day 2 after activation with microbial products. Fig. S6 shows cytokine responses to microbial stimulation of freshly isolated iNKT cells. Fig. S7 shows MyD88-dependent CD1d expression by DCs after stimulation with microbial products. Fig. S8 shows survival of WT and CD1d-deficient mice after S. pneumoniae infection and in vivo activation of iNKT cells on day 4 after S. pneumoniae infection. Online supplemental material is available at http://www.jem.org/cgi/content/full/jem.20102555/DC1.
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              Tip-DC Development during Parasitic Infection Is Regulated by IL-10 and Requires CCL2/CCR2, IFN-γ and MyD88 Signaling

              Introduction Inflammatory immune responses against invading pathogens require the recruitment of immune cells to the site of infection. However, the infiltration of infected tissue by activated immune cells may result in tissue injury justifying a profound understanding of the mechanisms underlying the recruitment and activation of inflammatory cells. Experimental infections with African trypanosomes, extracellular blood-borne parasites that cause sleeping sickness in humans and Nagana disease in cattle in sub-Saharan Africa [1], [2], are used as model systems to study infection-associated liver pathogenicity [3]. In murine models, the control of parasitemia is mostly mediated in the liver by IFN-γ- and MyD88-dependent generation of classically activated monocytic cells (M1) that secrete trypanotoxic molecules TNF and NO and exert phagocytic activity [4], [5], [6], [7]. Within M1, CD11b+Ly6C+CD11c+ inflammatory DCs have been identified as the main population producing TNF and NO during Trypanosoma brucei infection [8]. These TNF and iNOS producing DCs (Tip-DCs) originated from bone marrow CD11b+Ly6C+ monocytic cells recruited to the liver, spleen and lymph nodes of infected mice. On the other hand, the production of TNF and NO contributes to infection-associated pathogenicity including liver cell apoptosis/necrosis, resulting in organ failure and thus negatively affecting survival of the T. brucei infected host [3]. In this respect, IL-10 was shown to limit liver inflammation/injury and prolong survival by dampening the IFN-γ producing activity of T cells [9], [10] and by directly limiting the differentiation of CD11b+Ly6C+ monocytic cells to functional Tip-DCs [8]. Furthermore, IL-10 triggers the expression of genes associated with alternative activation of monocytic cells (M2) that could contribute to anti-inflammatory and wound-healing processes during African trypanosome infection [10], [11]. Since the recruitment of bone marrow derived CD11b+Ly6C+ monocytic cells to inflamed tissue and their subsequent differentiation in M1-type, TNF and NO producing CD11b+Ly6C+CD11c+ Tip-DCs may have a negative impact on the outcome of T. brucei infection, interfering with their recruitment to inflamed tissue could lead to new anti-disease treatments. Therefore we scrutinized the potential pathways governing the recruitment of inflammatory CD11b+Ly6C+ monocytic cells to the liver as well as their subsequent differentiation to functional Tip-DCs during T. brucei infection. Results Induction of chemokines in the liver of T. brucei infected mice T. brucei infection in C57Bl/6 mice is characterized by a massive expansion of CD11b+Ly6C+ monocytic cells in the liver [8]. These cells are closely related to CD11b+Ly6C+CCR2+ inflammatory monocytes that are recruited in infected tissues during microbial infection [12]. To identify pathway(s) possibly involved in the recruitment of liver CD11b+Ly6C+ monocytic cells, a custom in-house developed mRNA array was used to screen for the expression of chemokine genes in total liver extracts at day 6 post infection, i.e. when the first most important peak of parasitemia and the highest expansion of CD11b+Ly6C+ monocytic cells in the liver (reaching 27.8±2.5% within liver non-parenchymal cells in infected animals versus 2.1±0.3% in non-infected mice) occur [8]. Seven chemokine genes were found to be significantly induced (more than 2-fold as compared to gene expression in total liver extracts from non-infected mice) and their expression in total liver extracts was subsequently confirmed by RT-PCR (Table 1). The identified genes included (i) T cell-attracting chemokine genes; Cxcl10 (IP-10) and Cxcl9 (Mig) acting through the CXCR3 receptor and (ii) monocyte-attracting chemokine genes; Ccl3 (MIP-1α), Ccl4 (MIP-1β) and Ccl5 (RANTES), all ligands for the CCR5 chemokine receptor; Ccl2 (MCP-1), the preferential ligand for chemokine receptor CCR2; and Mif that interacts predominantly with the CD74 receptor but also potentially with CXCR2 and CXCR4. These data raised the possibility that signaling through CCR5 or CCR2 chemokine receptor or through Mif may be involved in the recruitment of liver associated CD11b+Ly6C+ monocytic cells during T. brucei infection. This hypothesis was supported by the observation that liver CD11b+Ly6C+ monocytic cells expressed CCR2, CCR5 and CD74 on their surface, although at different levels (Fig. 1A). 10.1371/journal.ppat.1001045.g001 Figure 1 CD11b + Ly6C + monocytic cells are affected by CCR2 but not CCR5 or Mif signaling during T. brucei infection. A) Inflammatory monocytic cells gated based on their expression of CD11b and Ly6C from non-parenchymal cells isolated from the liver of WT mice on day 6 of T. brucei infection were stained for CCR2, CCR5 or CD74 expression (filled grey curves compared to dotted line curves representing specific isotype controls) FACS profiles are representative of one of four animals tested in two independent experiments. B) Percentage and D) Number of CD11b+Ly6C+ monocytic cells in liver non-parenchymal, blood and bone marrow of CCR2 KO, CCR5 KO and Mif KO mice on day 6 post infection. Data are shown as mean ± SEM of three individual mice of one of three independent experiments performed. C) Non-parenchymal cells isolated from liver, blood and bone marrow of WT and CCR2 KO mice on day 6 post infection were assayed for co-expression of CD11b and Ly6C. Percentages of CD11b+Ly6C+ monocytic cells within the indicated gate are shown. FACS profiles are representative of one of nine animals tested in three independent experiments. *, significantly lower (p 75%) as compared to infected WT mice in both the liver and the blood, and coincided with an increase of the percentage of CD11b+Ly6C+ monocytic cells in the bone marrow (Figure 1B, 1C). Similar modulations were observed for absolute numbers of CD11b+Ly6C+ monocytic cells in the liver, blood and bone marrow of the respective mouse strains during infection (Figure 1D). These data suggest that the recruitment of CD11b+Ly6C+ monocytic cells to the liver of T. brucei infected mice consists of a two-step process comprising a CCR2-dependent, yet CCR5- and Mif-independent egression step from bone marrow to blood, followed by an extravasation step from blood to liver. Since in CCR2 KO mice CD11b+Ly6C+ monocytic cells accumulated in the bone marrow, it was unclear whether CCR2 also played a role in blood to liver extravasation during infection. To address this question, CD11b+Ly6C+ monocytic cells were purified from the bone marrow of T. brucei infected WT and CCR2 KO mice, labeled (using PKH26 and CellVue labeling kits respectively), and co-injected at a 1-1 ratio in infected WT recipient mice. Twenty four hours later, liver non-parenchymal cells from recipient mice were analyzed for the presence of labeled cells. As shown in figure 2, the 1-1 ratio of transferred WT to CCR2 KO CD11b+Ly6C+ monocytic cells could be traced back in infected WT recipient mice, demonstrating that the absence of CCR2 signaling in monocytic cells had no effect on blood to liver migration of CD11b+Ly6C+ monocytic cells during T. brucei infection. To verify whether expression of CCR2 by cells other than CD11b+Ly6C+ monocytic cells affected the recruitment of the latter cells from the blood to the liver, CD11b+Ly6C+ monocytic cells isolated from the bone marrow of T. brucei infected WT mice and labeled with PKH26 were transferred into infected CCR2 KO or WT recipient mice. The percentage of transferred CD11b+Ly6C+ cells found back in the liver after 24 hours was not significantly different between CCR2 KO or WT recipients (0.4±0.2% or 0.3±0.1%, respectively). Thus, these data indicate that the CCR2 chemokine receptor signaling in CD11b+Ly6C+ monocytic cells is crucial for their bone marrow emigration but dispensable for their blood to liver extravasation during T. brucei infection. 10.1371/journal.ppat.1001045.g002 Figure 2 CCR2 does not contribute to liver extravasation of CD11b + Ly6C + monocytic cells during T. brucei infection. WT and CCR2 KO CD11b+Ly6C+ monocytic cells were isolated from bone marrow of mice on day 6 of infection, labeled with PKH26 and CellVue kits, respectively, and injected at a 1-1 ratio in the blood of recipient WT mice on day 6 of T. brucei infection. Left panel shows percentages of gated, labeled WT and CCR2 KO cells before injection. 24 hours after transfer liver non-parenchymal were harvested from recipient mice and analysed for the presence of labeled cells. Percentages of gated, labeled WT and CCR2 KO cells are shown (right panel). FACS profiles are representative of one of six animals tested in two independent experiments. Impaired CCR2 signaling reduces liver Tip-DC percentage and pathogenicity during T. brucei infection During T. brucei infection, up to 70% of the CD11b+Ly6C+ liver monocytic cells can co-express CD11c, classifying them as inflammatory CD11b+Ly6C+CD11c+ dendritic cells [8]. The percentage of CD11c+ cells within the CD11b+Ly6C+ monocytic cell population in the liver of T. brucei infected mice was not affected by the absence of CCR2 (Figure 3). Also, the percentage of TNF+ and iNOS+ cells within CD11b+Ly6C+ monocytic cells (Figure 3) and their expression levels of co-stimulatory molecules CD80/CD86 and MHC class II (not shown) was similar in WT and CCR2 KO mice, indicating that CCR2 signaling is not involved in the differentiation of CD11b+Ly6C+ monocytic cells to functional Tip-DCs during T. brucei infection. However, in agreement with the reduced percentage of CD11b+Ly6C+ monocytic cells in the liver of CCR2 KO mice (Figure 1), we observed a significantly reduced percentage of TNF+ and iNOS+ Tip-DCs within the total liver non-parenchymal cell population of CCR2 KO mice compared to WT mice (Figure 3). The reduced percentage of Tip-DCs in the liver of infected CCR2 KO mice associated with (i) a significant reduction of TNF, NO and IFN-γ secretion in liver non-parenchymal cell cultures and ii) a reduced percentage of liver IFN-γ+CD4+ and IFN-γ+CD8+ T cells compared to infected WT mice (Figure 4). 10.1371/journal.ppat.1001045.g003 Figure 3 CCR2, IL-10R, MyD88 and IFN-γ signaling differentially contribute to Tip-DC differentiation/maturation during T. brucei infection. A) Liver non-parenchymal cells were isolated from WT, CCR2 KO, anti-IL-10R treated WT, MyD88 KO and IFN-γ KO mice on day 6 of infection. CD11b+Ly6C+ monocytic cells were gated (as indicated in figure 1C) and assayed for co-expression of CD11b and CD11c (top panels), TNF (middle panels) or iNOS (bottom panels). Percentages of CD11c+, TNF+ or iNOS+ cells (rectangular gate) within CD11b+Ly6C+ monocytic cells are indicated. Corresponding percentages of CD11c+, TNF+ or iNOS+ cells within the total number of living non-parenchymal cells recovered from the liver are indicated between brackets. Data are shown as mean ± SEM of three individual mice of one of three independent experiments performed. FACS profiles are representative of one of nine animals tested in three independent experiments performed. B) CD11b+Ly6C+ monocytic cells were isolated from bone marrow of WT and MyD88 KO or IFN-γR KO mice on day 6 of T. brucei infection, labeled with PKH26 (WT) and CellVue kits (KO), and injected at a 1-1 ratio in the blood of recipient WT mice on day 6 of T. brucei infection. Twenty four hours after transfer, liver non-parenchymal were harvested from recipient mice and analyzed for the presence of labeled cells. Percentages of gated, labeled WT and MyD88 or IFN-γR KO cells after transfer are shown (left panels). The expression of MHC-II molecules on the gated labeled cells is shown in the right panels. Data are shown as mean ± SEM of three individual mice of one of three independent experiments performed. FACS profiles are representative of one of four animals tested in three independent experiments. Note that due to the low numbers of transferred cells recovered from the liver, we were unable to assess iNOS or TNF expression on transferred cells using intracellular staining assays. *, significantly lower (p 75% of T. brucei are removed from circulation by resident liver Kupffer cells arguing for a major role of these cells in parasite control [36]. On the other hand, a more pro-pathogenic function for recruited CD11b+Ly6C+ monocytic cells/Tip-DCs is supported by the observation that in CCR2 KO mice (this study) or in T. brucei infected mice treated with IL-10 [8], reduced percentage of recruited CD11b+Ly6C+ monocytic cells/Tip-DCs associated with reduced pathogenicity and increased survival without affecting parasitemia. In the same vein, drastic shortened survival time of T. brucei infected IL-10 KO mice due to excessive tissue injury coincided with increased levels of CD11b+Ly6C+ monocytic cells/Tip-DC in the liver but normal parasite clearance capacity [8], [37]. Finally, our observation that the transfer of CD11b+Ly6C+ monocytic cells from infected WT mice that can maturate to Tip-DCs [8], but not from TNF KO mice, increased TNF concentration and ALT activity in serum of infected CCR2 KO recipients supports a role for these cells in the induction of liver injury during T. brucei infection. In conclusion, we have unraveled the pathways involved in the recruitment of a major pathogenic Tip-DC population in the liver of T. brucei infected mice (Figure 8). The development of Tip-DCs is a multi-step process including (i) a CCR2-dependent egression of CD11b+Ly6C+ inflammatory monocytic cells from bone marrow, followed by (ii) a differentiation step to immature inflammatory DCs (CD11c+ but CD80/CD86/MHC-IIlow) which is IFN-γ and MyD88 signaling independent and (iii) a maturation step of inflammatory DCs to functional (CD80/CD86/MHC-IIhigh) TNF and NO producing Tip-DCs which is IFN-γ and MyD88 signaling dependent. IL-10 could inhibit the CCL2/CCR2-mediated egression of CD11b+Ly6C+ monocytic cells from the bone marrow as well as their differentiation and maturation to Tip-DCs in the liver. Liver injury in various etiologies can result from uncontrolled activation of monocyte-derived cells recruited to the liver via CCR2 signaling and can be modulated by IL-10 [38], [39], [40], [41], [42]. African trypanosome infections thus represent a useful model to unravel these activation mechanisms and hereby to identify new tools, including monocyte associated, IL-10 inducible genes we have previously identified [10], [11], to treat hepatic inflammation. 10.1371/journal.ppat.1001045.g008 Figure 8 Model for migration, differentiation and maturation of CD11b + Ly6C + monocytes to Tip-DCs during T. brucei infection. Materials and Methods Ethics statement The experiments, maintenance and care of mice complied with the guidelines of the European Convention for the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes (CETS n° 123). The experiments for this study were approved by the Ethical Committee for Animal Experiments of the Vrije Universiteit Brussel, VUB, Brussel, Belgium. Parasites, mice and infections Trypanosoma brucei AnTat 1.1E parasites were a kind gift from N. Van Meirvenne (Institute for Tropical Medicine, Belgium). Female mice kept under filter barrier were used at 8–12 weeks of age. Wild type C57BL/6 mice were from Harlan, The Netherlands. CCR2 KO, CCR5 KO and Mif KO C57BL/6 mice were kindly provided by Dr. D. Engel, University Clinic of Bonn, Germany, Dr. F. Tacke, RWTH-University Hospital Aachen, Germany and Prof. R. Bucala, Yale University School of Medicine, New Haven, USA). IFN-γ KO, TNF KO and MyD88 KO C57BL/6 mice were bred in our animal facility. Parasites stored at −80°C were used to infect cyclophosphamide treated F1 (C57BL/6× BALB/c) mice bred in-house by i.p. inoculation. At day 4 post infection, mice were bled, and parasites were purified by diethyl-aminoethyl-cellulose chromatography. C57BL/6 mice were then infected i.p. with 2000 purified parasites. Parasitemia was monitored by tail blood puncture. When required, mice were injected i.v. with 100 µl of a 1 mg/ml PBS solution of anti-IL-10R antibody (1B1.3a, kindly provided by Dr. M. Moser and C. Coquerelle, ULB, Gosselies, Belgium with the agreement of Schering-Plough Biopharma) or control antibody (rat IgG1, BD Biosciences) on days 7 and 9 of infection and sacrificed on day 10. Isolation of liver, blood and bone marrow immune cells Liver non-parenchymal cells were isolated as follows. Animals were euthanized (CO2) and livers were perfused through the portal vein with 10 ml of 100 U/ml collagenase type III (Worthington Biochemical Corporation) in HBSS. Then, the liver was minced and incubated in 10 ml of a 100 U/ml collagenase III solution in HBSS (20 min, 37°C). The resulting cell suspension was passed through a 70 µm nylon mesh filter and then washed by adding 30 ml of HBSS supplemented with 2 mM EDTA and 10% FCS followed by centrifugation (300 g, 10 min, 4°C). After erythrocyte lysis, pellet was resuspended in 10 ml of Lymphoprep (Lucron Bioproducts) and overlayed with 10 ml of HBSS supplemented with 2 mM EDTA and 10% FCS. After centrifugation (430 g, 30 min, 17°C), the layer of low-density cells at the interface containing non-parenchymal cells was harvested. CD11b+Ly6C+Ly6G+ liver granulocytes were not retained within the non-parenchymal fractions due to their high density characteristic. Bone marrow cells were isolated from hind leg bone of CO2 euthanized animals by perfusion with 20 ml of HBSS supplemented with 10% FCS (HBSS/FCS). The resulting cell suspension was passed through a 100 µm nylon mesh filter, centrifuged (300 g, 10 min, 4°C) and resuspended in HBSS/FCS. Blood was isolated on EDTA by heart puncture of euthanized mice, followed by thorough erythrocyte lysis, centrifugation (300 g, 10 min, 4°C) and resuspension of blood immune cells in HBSS/FCS. Immune cells and used buffers were kept on ice during isolation protocols and subsequent analysis. Transfer of monocytes CD11b+Ly6C+ monocytes were isolated from the bone marrow of infected WT, CCR2 KO, TNF KO, MyD88 KO or IFN-γR KO mice by MACS purification on day 6 post infection. CD11c+ and Ly6G+ cells were first depleted via negative MACS selection. CD11b+Ly6C+ monocytes were then isolated via positive CD11b selection on magnetic separation columns according to the manufacturer's protocol (Miltenyi Biotec) resulting in purity ranging from 90–95%. WT and CCR2 KO or MyD88 KO or IFN-γR KO bone marrow cells were then fluorescently labeled using respectively PKH26 (Sigma-Aldrich) and CellVue membrane labeling kits (Polysciences) according to the manufacturer's protocol. Labeled cells were adoptively transferred at a 1-1 ratio (total of 4×106 cells/mouse) through tail vein injection in infected recipient mice on day 6 post infection. Alternatively, CellVue labeled WT or TNF KO monocytes from infected mice were injected in CCR2 KO mice on day 6 post infection (8×106 cells/mouse). Twenty four hours after transfer the recipient mice were sacrificed and presence of labeled cells was analyzed in the liver non-parenchymal cell fraction. Quantification of cytokines and NO2 Liver cells were resuspended at 2×106/ml in RPMI 1640 (Gibco) supplemented with 10% FCS, 100 U/ml penicillin, 100 µg/ml streptomycin, 0.1 mM non-essential amino acids, 2 mM L-glutamine, and 5×10−5 M 2-mercaptoethanol (all from Invitrogen Life Technologies) and cultured in vitro (37°C). Cytokines were quantified in culture supernatants collected after 2 days with specific sandwich ELISAs for IFN-γ (PharMingen) or TNF (R&D Systems), in accordance to the manufacturers' protocols. NO2 quantification was assayed by a standard Griess reaction as described [37]. FACS analysis For surface markers, cells were stained for 30 min at 4°C using conventional protocols. Cells were pre-incubated with anti-FcγR Ab (clone 2.4G2) before adding (1 µg/106 cells): FITC-conjugated or APC-conjugated MHC-II (clone M5/114.15.2), PerCP-Cy5.5-conjugated or PE-Cy7 conjugated CD11b (M1/70), FITC-conjugated CD80 (16-10A1), FITC-conjugated CD86 (Gl-1), FITC-conjugated Ly6C (AL-21), PE-conjugated or APC-conjugated CD11c (HL3), PE-conjugated CCR5 (C34-3448), unconjugated CCR2 (MC-21, a gift of Dr Matthias Mack, University of Regensburg, Regensburg, Germany), FITC-conjugated CD74 (In-1). Dead cells were excluded by 7-AAD staining. For intracellular TNF staining, cells were cultured 6 hours in the presence of brefeldin-A (BD Bioscience). For intracellular IFN-γ staining, cells were cultured 2 hours with anti-CD3 before adding brefeldin-A (BD Bioscience). Four hours later, cells were fixed, permeabilised (Fix/Perm kit, eBioscience) and analyzed. Antibodies used for intracellular staining were APC-conjugated TNF (clone MP6-XT22), unlabeled rabbit iNOS (M19) and APC-conjugated anti-rabbit IgG. Cells were analyzed on a FACSCanto II and analysis was performed using FlowJo. Antibodies were purchased from BD Biosciences, eBioscience or R&D Systems. Gene expression analysis CD11b+ Ly6C+ cells were isolated from liver non-parenchymal cells by negative selection for CD4, CD8 and CD19 followed by positive Ly6C selection, using a two-step labeling with Ly6C-PE (BD Bioscience) and anti-PE beads (Miltenyi Biotec) on magnetic separation columns according to the manufacturer's protocol (Miltenyi Biotec) with a purity ranging from 90–95%. Three ×106 purified CD11b+Ly6C+ liver cells were put in Trizol (Invitrogen) and stored at −80°C. For total liver extracts, pieces of liver (0.5×0.5 cm) were minced, washed with HBSS and after centrifugation put in Trizol. Gene expression in liver extracts or isolated populations was analyzed by quantitative real time PCR using the conditions described [43]. Results of the PCR analyses were normalized against the house-keeping gene S12. Primers used were: Cxcl-9 (Forward: TCCTTTTGGGCATCATCTTC, Reverse: TTCCCCCTCTTTTGCTTTTT), Cxcl-10 (Forward: GGATGGCTGTCCTAGCTCTG, Reverse: ATAACCCCTTGGGAAGATGG), Ccl3 (Forward: CGGAAGATTCCACGCCAATTC, Reverse: GGTGAGGAACGTGTCCTGAAG), Ccl4 (Forward: GCCCTCTCTCTCCTCTTGCT, Reverse: GTCTGCCTCTTTTGGTCAGG), Ccl5 (Forward: ACAGGTCAAACTACAACTCCA, Reverse: TCAGCTCTTAGCAGACATTGG), Ccl2 (Forward: CACTCACCTGCTGCTACTCATTCAC, Reverse: GGATTCACAGAGAGGGAAAAATGG), Mif (Forward: CTTTTAGCGGCACGAACGAT, Reverse: AAGAACAGCGGTGCAGGTAA). Microscopy, ALT and AST levels Liver were fixed in Bouin solution (Sigma). Histological sections embedded in paraffin were stained with Hematoxylin-eosin-saffron for microscopic evaluations. Liver alanine aminotransferase (ALT) was measured in serum samples, using commercially available kits (Boehringer Mannheim, Mannheim, Germany). Statistical analysis All comparisons were tested for statistical significance using the unpaired t test with Welch's correction from GraphPad Prism 4.0 software.
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                Contributors
                Role: Editor
                Journal
                PLoS Pathog
                PLoS Pathog
                plos
                plospath
                PLoS Pathogens
                Public Library of Science (San Francisco, CA USA )
                1553-7366
                1553-7374
                12 June 2015
                June 2015
                : 11
                : 6
                : e1004964
                Affiliations
                [1 ]Laboratory of Cellular and Molecular Immunology, Vrije Universiteit Brussel, Brussels, Belgium
                [2 ]Department of Structural Biology, Vlaams Instituut voor Biotechnologie (VIB), Brussels, Belgium
                [3 ]Department of Myeloid Cell Immunology, VIB, Brussels, Belgium
                [4 ]International Centre for Genetic Engineering and Biotechnology, Cape Town Component, Cape Town, South Africa
                [5 ]Faculty of Health Sciences (IDM, Division Immunology), University of Cape Town, Cape Town, South Africa
                [6 ]Bioceros, Utrecht, The Netherlands
                National Institute of Health, UNITED STATES
                Author notes

                The authors have declared that no competing interests exist.

                Conceived and designed the experiments: JC CDT BS JK FK MB RK FB SM. Performed the experiments: JC CDT BS JK FK MB RK SM. Analyzed the data: JC JK SM. Contributed reagents/materials/analysis tools: FB SM LB. Wrote the paper: JC SM FB.

                Article
                PPATHOGENS-D-15-00202
                10.1371/journal.ppat.1004964
                4466398
                26070118
                f252076e-427e-45e9-acd9-1e6cb0bf9d87
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                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
                : 26 January 2015
                : 20 May 2015
                Page count
                Figures: 7, Tables: 0, Pages: 19
                Funding
                This work was supported by the Fonds voor Wetenschappelijk Onderzoek (FWO) Project fwoTM597, the FWO-NRF bilateral Flanders-Shout Africa grant n° GA10410N, the Interuniversity Attraction Pole Program (PAI-IAP N. P7/41) and the South African Medical Research Council (SAMRC), the Research Chair initiative (SARCHi) by DST and NRF. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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                Infectious disease & Microbiology
                Infectious disease & Microbiology

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