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      The Application of Drugs and Nano-Therapies Targeting Immune Cells in Hypoxic Inflammation

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          Abstract

          Immune cells are pivotal in the dynamic interplay between hypoxia and inflammation. During hypoxic conditions, HIF-1α, a crucial transcription factor, facilitates the adaptation of immune cells to the hypoxic micro-environment. This adaptation includes regulating immune cell metabolism, significantly impacting inflammation development. Strategies for anti-inflammatory and hypoxic relief have been proposed, aiming to disrupt the hypoxia-inflammation nexus. Research extensively focuses on anti-inflammatory agents and materials that target immune cells. These primarily mitigate hypoxic inflammation by encouraging M2-macrophage polarization, restraining neutrophil proliferation and infiltration, and maintaining Treg/TH17 balance. Additionally, oxygen-releasing nano-materials play a significant role. By alleviating hypoxia and clearing reactive oxygen species (ROS), these nano-materials indirectly influence immune cell functions. This paper delves into the response of immune cells under hypoxic conditions and the resultant effects on inflammation. It provides a comprehensive overview of various therapies targeting specific immune cells for anti-inflammatory purposes and explores nano-materials that either carry or generate oxygen to alleviate anoxic micro-environments.

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          PD-L1 is a novel direct target of HIF-1α, and its blockade under hypoxia enhanced MDSC-mediated T cell activation

          Hypoxia is a common feature of solid tumors (Semenza, 2011). Hypoxic zones in tumors attract immunosuppressive cells such as myeloid-derived suppressor cells (MDSCs; Corzo et al., 2010), tumor-associated macrophages (TAMs; Doedens et al., 2010; Imtiyaz et al., 2010), and regulatory T cells (T reg cells; Clambey et al., 2012). MDSCs are a heterogeneous group of relatively immature myeloid cells and several studies have described mechanisms of MDSC-mediated immune suppression (Gabrilovich et al., 2012). A large body of preclinical and clinical data indicates that antibody blockade of immune checkpoints can significantly enhance antitumor immunity (Pardoll, 2012; West et al., 2013). Recently, antibody-mediated blockade of preprogrammed death 1 (PD-1; Topalian et al., 2012) and its ligand, PD-L1 (Brahmer et al., 2012), was shown to result in durable tumor regression and prolonged stabilization of disease in patients with advanced cancers. PD-1, a cell surface glycoprotein with a structure similar to cytotoxic T lymphocyte antigen 4 (CTLA-4), belongs to the B7 family of co-stimulatory/co-inhibitory molecules and plays a key part in immune regulation (Greenwald et al., 2005). PD-1 has two known ligands, PD-L1 (B7-H1) and PD-L2 (B7-DC). Although hypoxia has been shown to regulate the function and differentiation of MDSCs (Corzo et al., 2010), several major questions remain unresolved. The influence of hypoxia on the regulation of immune checkpoint receptors (PD-1 and CTLA-4) and their respective ligands (PD-L1, PD-L2, CD80, and CD86) on MDSCs remains largely obscure. Furthermore, the potential contribution of these immune checkpoint receptors and their respective ligands on MDSC function under hypoxia is still unknown. In the present study, we showed that hypoxia via hypoxia-inducible factor-1α (HIF-1α) selectively up-regulated PD-L1 on MDSCs, but not other B7 family members, by binding directly to the HRE in the PD-L1 proximal promoter. Blockade of PD-L1 under hypoxia abrogated MDSC-mediated T cell suppression by modulating MDSCs cytokine production. RESULTS AND DISCUSSION Differential expression of PD-L1 on tumor-infiltrating MDSCs versus splenic MDSCs and selective up-regulation of PD-L1 in splenic MDSCs under hypoxic stress We first compared the level of expression of PD-L1 and PD-L2 between splenic MDSCs and tumor-infiltrating MDSCs from tumor-bearing mice. We found that the percentage of PD-L1+ cells was significantly higher on tumor-infiltrating MDSCs as compared with splenic MDSC in B16-F10, LLC (Fig. 1 A), CT26, and 4T1 (Fig. 1 B) tumor models. No significant difference was found in the percentage of PD-L2+ cells in splenic MDSCs as compared with tumor-infiltrating MDSCs in four tumor models tested (Fig. 1 C). We did not observe any significant difference in the expression levels of other members of the B7 family such as CD80, CD86, PD-1, and CTLA-4 on MDSCs from spleen and tumor (unpublished data). Youn et al. (2008) previously observed no significant differences in the percentage of PD-L1+ or CD80+ cells within the splenic MDSCs from tumor-bearing mice and immature myeloid cells from naive tumor-free mice. However, by comparing the expression of immune checkpoint inhibitors between splenic and tumor-infiltrating MDSCs, we showed that there is a differential expression of PD-L1 on tumor-infiltrating MDSCs. Figure 1. Tumor-infiltrating MDSCs differentially express PD-L1 as compared with splenic MDSCs, and hypoxia selectively up-regulates PD-L1 on splenic MDSCs in tumor-bearing mice. Surface expression level of PD-L1 and PD-L2 on Gr1+ CD11b+ cells (MDSCs) from (B16-F10 and LLC; A; CT26 and 4T1; B) in spleens (black dotted line histogram) and tumor (black line histogram) as compared with isotype control (gray-shaded histogram) was analyzed by flow cytometry. (C) Statistically significant differences (indicated by asterisks) between tumor-infiltrating MDSCs and splenic MDSCs are shown (*, P 20 fold for HRE-4), comparable to their binding to an established HRE in VEGF, LDHA, and Glut1 genes. To determine whether this HIF-1α site (HRE-4) was a transcriptionally active HRE, MSC-1 cells were co-transfected with pGL4-hRluc/SV40 vector and pGL3 EV, pGL3 HRE-4, or pGL3 HRE-4 MUT vectors (Fig. 3 M) and grown under normoxia or hypoxia. After 48 h, firefly and renilla luciferase activities were measured. As shown in Fig. 3 N, hypoxia significantly increased the luciferase activity of HRE-4 reporter by more than threefold as compared with normoxia. More interestingly, the luciferase activity of HRE-4 MUT was significantly decreased (>50%) as compared with HRE-4 under hypoxia (Fig. 3 N). The results presented in Figs. 3 (H–N) demonstrate that PD-L1 is a direct HIF-1α target gene in MSC-1 cells. Thus, we provide evidence here that HIF-1α is a major regulator of PD-L1 mRNA and protein expression, and that HIF-1α regulates the expression of PD-L1 by binding directly to the HRE-4 in the PD-L1 proximal promoter. Blocking PD-L1 decreases MDSC-mediated T cell suppression under hypoxia by down-regulating MDSC IL-6 and IL-10 To directly test the functional consequences of hypoxia-induced up-regulation of PD-L1 in MDSC-mediated T cell suppression, the expression of PD-L1 was blocked on ex vivo MDSCs by using anti–PD-L1 monoclonal antibody. Hypoxia increased the ability of MDSCs to suppress both specific and nonspecific stimuli-mediated T cell proliferation (Fig. 4, A and B). Interestingly, blockade of PD-L1 under hypoxia significantly abrogated the suppressive activity of MDSCs in response to both nonspecific stimuli (anti-CD3/CD28 antibody; Fig. 4 A) and specific stimuli (TRP-2(180–88) peptide; Fig. 4 B). Under hypoxia, MDSCs acquired the ability to inhibit T cell function (Fig. 4, C and D) by decreasing the percentage of IFN-γ+ CD8+ and CD4+ T cells; whereas the percentage of IFN-γ+ CD8+ (Fig. 4 C) and IFN-γ+ CD4+ T cells (Fig. 4 D) significantly increased after PD-L1 blockade under hypoxic conditions. Thus, the immune suppressive function of MDSCs enhanced under hypoxia was abrogated after blocking PD-L1, and hypoxic up-regulation of PD-L1 on MDSCs is involved in mediating the suppressive action of MDSCs, at least in part, as we were not able to completely restore T cell proliferation and function after PD-L1 blockade on MDSCs under hypoxia. Figure 4. Blockade of PD-L1 under hypoxia down-regulates MDSC IL-6 and IL-10 and enhances T cell proliferation and function. MDSCs isolated from spleens of B16-F10 tumor-bearing mice were pretreated for 30 min on ice with 5 µg/ml control antibody (IgG) or antibody against PD-L1 (PDL1 Block) and co-cultured with splenocytes under normoxia and hypoxia for 72 h. (A and B) Effect of MDSC on proliferation of splenocytes stimulated with (A) anti-CD3/CD28 coated beads or (B) TRP-2(180–88) peptide under the indicated conditions. Cell proliferation was measured in triplicates by [3H]thymidine incorporation and expressed as counts per minute (CPM). (C and D) MDSCs were cultured with splenocytes from B16-F10 mice stimulated with anti-CD3/CD28. Intracellular IFN-γ production was evaluated by flow cytometry by gating on (C) CD3+CD8+ IFN-γ+ and (D) CD3+CD4+ IFN-γ+ populations. Statistically significant differences (indicated by asterisks) are shown (**, P 95% as evaluated by FACS analysis. MDSC functional assays. For evaluation of T cell proliferation, splenocytes from B16-F10 mice were plated into U-bottom 96-well plates along with MDSCs at different ratios (50,000 MDSC:200,000 splenocytes/well). Plates were stimulated with either anti-CD3/CD28 beads (Miltenyi Biotec) or TRP-2 180–88 peptide for 72 h at 37°C. Co-cultures were pulsed with thymidine (1 µCi/well; Promega) for 16–18 h before harvesting, and [3H]thymidine uptake was counted using Packard’s TopCount NXT liquid scintillation counter and expressed as counts per minute (CPM). For assessment of T cell functions, MDSCs co-cultured with splenocytes from B16-F10 mice were stimulated with anti-CD3/CD28 beads. After 72 h, intracellular IFN-γ production was evaluated by flow cytometry by gating on CD3+CD8+ IFN-γ+ and CD3+CD4+ IFN-γ+ populations. MDSCs cytokine production (ELISA). MDSCs isolated from spleens of B16-F10 tumor-bearing mice were pretreated for 30 min on ice with 5 μg/ml control antibody (IgG) or Anti-Mouse PD-L1 (B7-H1) Functional Grade Purified antibody 5 µg/ml (clone MIH5; eBioscience; PDL1 Block) and cultured under normoxia and hypoxia for 72 h. Supernatants were collected and the secretion of IL-6, IL-10, and IL-12p70 (eBioscience) was determined by ELISA. ChIP assay. ChIP was performed with lysates prepared from MSC-1 by using SimpleChIP Enzymatic Chromatin IP kit (Cell Signaling Technology). SYBR Green RT-qPCR was performed using the primers detailed in Table S1. Arginase enzymatic activity and NO (nitric oxide) production. Arginase activity was measured in MDSC cell lysates, and for NO production, culture supernatants were mixed with Greiss reagent and nitrite concentrations were determined as described earlier (Youn et al., 2008). Luciferase reporter assay. A 653-bp section corresponding to mouse PD-L1 promoter containing HRE4 sequence was inserted into the NheI–XhoI sites of pGL3-Basic vector (Promega). Mutation of HRE4 was performed by site-directed mutagenesis and verified by sequencing. A 56-bp mouse PD-L1 gene sequence was inserted into the Bgl II site of pGL3-Promoter (Promega). MSC-1 cells were co-transfected with 0.2 µg of pGL4-hRluc/SV40 vector (which contains renilla luciferase sequences downstream of the SV40 promoter) and 1 µg of pGL3 empty vector, pGL3 HRE-4, or pGL3 HRE-4 MUT vectors in 6-well plates with Lipofectamine 2000 (Invitrogen) in OPTIMEM (Invitrogen) medium and grown under normoxia or hypoxia. After 48 h, firefly and Renilla luciferase activities were measured using the Dual-Luciferase Reporter assay (Promega) and the ratio of firefly/Renilla luciferase was determined. Statistics. Data were analyzed with GraphPad Prism. Student’s t test was used for single comparisons. Online supplemental material. Table S1 shows genomic oligonucleotide primers used for amplification of immunoprecipitated DNA samples from ChIP assays. Online supplemental material is available at http://www.jem.org/cgi/content/full/jem.20131916/DC1. Supplementary Material Supplemental Material
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            Functional polarization of tumour-associated macrophages by tumour-derived lactic acid.

            Macrophages have an important role in the maintenance of tissue homeostasis. To perform this function, macrophages must have the capacity to monitor the functional states of their 'client cells': namely, the parenchymal cells in the various tissues in which macrophages reside. Tumours exhibit many features of abnormally developed organs, including tissue architecture and cellular composition. Similarly to macrophages in normal tissues and organs, macrophages in tumours (tumour-associated macrophages) perform some key homeostatic functions that allow tumour maintenance and growth. However, the signals involved in communication between tumours and macrophages are poorly defined. Here we show that lactic acid produced by tumour cells, as a by-product of aerobic or anaerobic glycolysis, has a critical function in signalling, through inducing the expression of vascular endothelial growth factor and the M2-like polarization of tumour-associated macrophages. Furthermore, we demonstrate that this effect of lactic acid is mediated by hypoxia-inducible factor 1α (HIF1α). Finally, we show that the lactate-induced expression of arginase 1 by macrophages has an important role in tumour growth. Collectively, these findings identify a mechanism of communication between macrophages and their client cells, including tumour cells. This communication most probably evolved to promote homeostasis in normal tissues but can also be engaged in tumours to promote their growth.
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              Trained immunity: A program of innate immune memory in health and disease.

              The general view that only adaptive immunity can build immunological memory has recently been challenged. In organisms lacking adaptive immunity, as well as in mammals, the innate immune system can mount resistance to reinfection, a phenomenon termed "trained immunity" or "innate immune memory." Trained immunity is orchestrated by epigenetic reprogramming, broadly defined as sustained changes in gene expression and cell physiology that do not involve permanent genetic changes such as mutations and recombination, which are essential for adaptive immunity. The discovery of trained immunity may open the door for novel vaccine approaches, new therapeutic strategies for the treatment of immune deficiency states, and modulation of exaggerated inflammation in autoinflammatory diseases.
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                Author and article information

                Journal
                Int J Nanomedicine
                Int J Nanomedicine
                ijn
                International Journal of Nanomedicine
                Dove
                1176-9114
                1178-2013
                09 April 2024
                2024
                : 19
                : 3441-3459
                Affiliations
                [1 ]Jilin Provincial Key Laboratory of Tooth Development and Bone Remodeling, Hospital of Stomatology, Jilin University , Changchun, 130021, People’s Republic of China
                [2 ]Department of Oral Implantology, Hospital of Stomatology, Jilin University , Changchun, 130021, People’s Republic of China
                Author notes
                Correspondence: Yidi Zhang; Yanmin Zhou, Department of Oral Implantology, Hospital of Stomatology, Jilin University, Changchun, 130021, People’s Republic of China, Email zhangyidi@jlu.edu.cn; zhouym@jlu.edu.cn
                [*]

                These authors contributed equally to this work

                Article
                456533
                10.2147/IJN.S456533
                11015843
                38617798
                ff421ee0-3245-49f6-b009-dd6081cc31aa
                © 2024 Luo et al.

                This work is published and licensed by Dove Medical Press Limited. The full terms of this license are available at https://www.dovepress.com/terms.php and incorporate the Creative Commons Attribution – Non Commercial (unported, v3.0) License ( http://creativecommons.org/licenses/by-nc/3.0/). By accessing the work you hereby accept the Terms. Non-commercial uses of the work are permitted without any further permission from Dove Medical Press Limited, provided the work is properly attributed. For permission for commercial use of this work, please see paragraphs 4.2 and 5 of our Terms ( https://www.dovepress.com/terms.php).

                History
                : 09 January 2024
                : 29 March 2024
                Page count
                Figures: 5, Tables: 2, References: 159, Pages: 19
                Categories
                Review

                Molecular medicine
                hypoxia,inflammation,immunometabolism,immunotherapy,nanotherapy
                Molecular medicine
                hypoxia, inflammation, immunometabolism, immunotherapy, nanotherapy

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