Th2 Conditions Research Articles

Capecitabine mitigates cardiac allograft rejection via inhibition of TYMS-Mediated Th1 differentiation in mice

ObjectivesPrevious studies elucidated that capecitabine (CAP) works as an anti-tumor agent with putative immunosuppressive effects. However, the intricate mechanisms underpinning these effects remain to be elucidated. In this study, we aimed to unravel the molecular pathways by which CAP exerts its immunosuppressive effects to reduce allograft rejection. MethodsHearts were transplanted from male BALB/c donors to male C57BL/6 recipients and treated with CAP for seven days. The rejection of these heart transplants was assessed using a range of techniques, including H&E staining, immunohistochemistry, RNA sequencing, LS-MS/MS, and flow cytometry. In vitro, naïve CD4+ T cells were isolated and cultured under Th1 condition medium with varying treatments, flow cytometry, LS-MS/MS were employed to delineate the role of thymidine synthase (TYMS) during Th1 differentiation. ResultsCAP treatment significantly mitigated acute allograft rejection and enhanced graft survival by reducing graft damage, T cell infiltration, and levels of circulating pro-inflammatory cytokines. Additionally, it curtailed CD4+ T cell proliferation and the presence of Th1 cells in the spleen. RNA-seq showed that TYMS, the target of CAP, was robustly increased post-transplantation in splenocytes. In vitro, TYMS and its metabolic product dTMP were differentially expressed in Th0 and Th1, and were required after activation of CD4+ T cell and Th1 differentiation. TYMS-specific inhibitor, raltitrexed, and the metabolite of capecitabine, 5-fluorouracil, could inhibit the proliferation and differentiation of Th1. Finally, the combined use of CAP and the commonly used immunosuppressant rapamycin can induce long-term survival of allograft. ConclusionCAP undergoes metabolism conversion to interfere pyrimidine metabolism, which targets TYMS-mediated differentiation of Th1, thereby playing a significant role in mitigating acute cardiac allograft rejection in murine models.

Characteristic features of in vitro differentiation of human naïve CD4+ T cells to induced regulatory T cells (iTreg) and T helper (Th) 17 cells: Sharing of lineage-specific markers

In vitro induced regulatory T cells (iTreg) and IL-17 producing T cells (Th17-like cells) can be generated in culture from native CD4+ T cells in peripheral blood by different sets of cytokines. In the presence of transforming growth factor (TGF)-β plus interleukin (IL)-2, cells differentiate into Treg cells with increased expression of the forkhead box P3 (FOXP3). In the presence of TGF-β, IL-6, IL-1β and IL-23, cells differentiate into Th17 cells that produce IL-17A. However, protocols for the generation of human iTreg and Th17 are still controversial. In this study, we characterized the biological features of iTreg and Th17 cells differentiated from peripheral blood naïve CD4+ T cells in vitro using the established protocols. We showed that cells obtained from Treg or Th17 culture conditions shared some phenotypic markers. Cells under Treg conditions had an up-regulated FOXP3 gene and a down-regulated RAR-related orphan receptor C (RORC) gene. Cells derived from the Th17 condition exhibited a down-regulated FOXP3 gene and had significantly higher RORC gene expression than Treg cells. Both resulting cells showed intracellular production of IL-17A and IL-10. Th17 condition-cultured cells exhibited more glycolytic activity and glucose uptake compared to the Treg cells. The findings suggest that cells obtained from established protocols for the differentiation of iTreg and Th17 cells in vitro are possibly in the intermediate stage of differentiation or may be two different types of cells that share a lineage-specific differentiation program.

Daphnetin Alleviates Bleomycin-Induced Pulmonary Fibrosis through Inhibition of Epithelial-to-Mesenchymal Transition and IL-17A.

Idiopathic pulmonary fibrosis (IPF) is a chronic and refractory interstitial lung disease. Although there is no cure for IPF, the development of drugs with improved efficacy in the treatment of IPF is required. Daphnetin, a natural coumarin derivative, has immunosuppressive, anti-inflammatory, and antioxidant activities. However, its antifibrotic effects have not yet been elucidated. In this study, we investigated the antifibrotic effects of daphnetin on pulmonary fibrosis and the associated molecular mechanism. We examined the effects of daphnetin on splenocytes cultured in Th17 conditions, lung epithelial cells, and a mouse model of bleomycin (BLM)-induced pulmonary fibrosis. We identified that daphnetin inhibited IL-17A production in developing Th17 cells. We also found that daphnetin suppressed epithelial-to-mesenchymal transition (EMT) in TGF-β-treated BEAS2B cells through the regulation of AKT phosphorylation. In BLM-treated mice, the oral administration of daphnetin attenuated lung histopathology and improved lung mechanical functions. Our findings clearly demonstrated that daphnetin inhibited IL-17A and EMT both in vitro and in vivo, thereby protecting against BLM-induced pulmonary fibrosis. Taken together, these results suggest that daphnetin has potent therapeutic effects on lung fibrosis by modulating both Th17 differentiation and the TGF-β signaling pathway, and we thus expect daphnetin to be a drug candidate for the treatment of IPF.

Treatment with a red-laser-based wound therapy device exerts positive effects in models of delayed keratinocyte and fibroblast wound healing.

Light therapy is widely used in medicine. Specifically, photobiomodulation has been shown to exert beneficial effects in wound healing disorders, which present a major challenge in health care. The study's aim was providing information on the effect of a novel, red-laser-based wound therapy device (WTD) on keratinocytes and fibroblasts during wound healing under optimal and non-optimal conditions. The scratch wound assay was employed as a wound healing model for mechanical damage with readjustment of specific cell milieus, explicitly chronic TH1 inflammation and TH2-dominant conditions. Furthermore, gene expression analysis of pro-inflammatory cytokines (IL1A, IL6, CXCL8), growth factors (TGFB1, PDGFC), transcription factors (NFKB1, TP53) and heat shock proteins (HSP90AA1, HSPA1A, HSPD1) as well as desmogleins (DSG1, DSG3) in keratinocytes and collagen (COL1A1, COL3A1) in fibroblasts was performed after WTD treatment. It was shown that WTD treatment is biocompatible and supports scratch wound closure under non-optimal conditions. A distinct enhancement of desmoglein and collagen gene expression as well as induction of early growth factor gene expression was observed under chronic inflammatory conditions. Moreover, WTD increased HSPD1 transcript levels in keratinocytes and augmented collagen expression in fibroblasts during wound healing under TH2 conditions. WTD treatment also alleviated the inflammatory response in keratinocytes and induced early growth factor gene expression in fibroblasts under physiological conditions. Positive effects described for wound treatment with WTD could be replicated invitro and seem to be to be conferred by a direct influence on cellular processes taking place in keratinocytes and fibroblasts during wound healing.

Gut microbial metabolite deoxycholic acid facilitates Th17 differentiation through modulating cholesterol biosynthesis and participates in high-fat diet-associated colonic inflammation

BackgroundHigh-fat diet (HFD) is closely associated with the increased prevalence of inflammatory bowel disease (IBD). Excessive gut microbial metabolite deoxycholic acid (DCA) caused by HFD plays significant roles in eliciting intestinal inflammation, however, the mechanism underlining the induction of inflammatory response by DCA has not been fully elucidated. The purpose of this study was to investigate the role of DCA in the triggering of inflammation via affecting CD4+ T cell differentiation.ResultsMurine CD4+T cells were cultured under Th1, Th2 or Th17-polarizing conditions treated with or without different dosage of DCA, and flowcytometry was conducted to detect the effect of DCA on CD4+ T cell differentiation. Alteration of gene expression in CD4+ T cells upon DCA treatment was determined by RNA-sequencing and qRT-PCR. Bioinformatic analysis, cholesterol metabolic profiling, ChIP assay and immuno-fluorescent staining were further applied to explore the DCA-regulated pathway that involved in CD4+T cell differentiation. The results showed that DCA could dose-dependently promote the differentiation of CD4+ T cell into Th17 linage with pathogenic signature. Mechanistically, DCA stimulated the expression of cholesterol biosynthetic enzymes CYP51 and led to the increased generation of endogenous RORγt agonists, including zymosterol and desmosterol, therefore facilitating Th17 differentiation. Up-regulation of CYP51 by DCA was largely mediated via targeting transcription factor SREBP2 and at least partially through bile acid receptor TGR5. In addition, DCA-supplemented diet significantly increased intestinal Th17 cell infiltration and exacerbated TNBS-induced colitis. Administration of cholestyramine to eliminate fecal bile acid obviously alleviated colonic inflammation accompanied by decreased Th17 cells in HFD-fed mice.ConclusionsOur data establish a link between DCA-induced cholesterol biosynthesis in immune cells and gut inflammation. Modulation of bile acid level or targeting cholesterol metabolic pathway may be potential therapeutic measurements for HFD-related colitis.

Icariin restrains NLRP3 inflammasome-mediated Th2 immune responses and ameliorates atopic dermatitis through modulating a novel lncRNA MALAT1/miR-124-3p axis

Context Atopic dermatitis (AD) is a common inflammatory skin disease characterized with hyperactivation of type 2 T helper (Th2) immune responses. Icariin is a flavonoid glucoside with anti-inflammatory activities, which has been used to treat multiple diseases. Objective The present study investigates the underlying mechanisms by which icariin regulates Th2 responses and AD development. Materials and methods BALB/c mice were induced by DNFB to establish AD models, and injected with or without 10 mg/kg icariin for 2 weeks (i.p., daily). CD4+T cells were induced by Th2 condition to simulate AD in vitro, and also treated with or without 100 µM icariin. Results Icariin ameliorated AD-like skin lesion, manifested as a significant decrease in dermatitis scores (from 8.00 ± 1.00 to 3.67 ± 0.58), serum IgE levels (from 3119.15 ± 241.81 to 948.55 ± 182.51 ng/mL), epidermal thickness (from 93.86 ± 4.61 to 42.67 ± 2.48 µm) and infiltration of mast cells (from 60.67 ± 3.21 cells to 36.00 ± 2.65 cells). Also, icariin inactivated NLRP3 inflammasome, inhibited Th2 skewing, reduced lncRNA MALAT1 expression, but elevated miR-124-3p expression in vivo and in vitro. MALAT1 increased NLRP3 expression through targeting miR-124-3p. Knockdown of MALAT1 repressed NLRP3 inflammasome activation and mitigated Th1/Th2 imbalance in Th2-conditioned CD4+T cells, whereas both MALAT1 overexpression and miR-124-3p inhibition ablated the inhibitory effects of icariin on Th2 immune responses. Discussion and conclusions The findings further improve our understanding of the mechanism by which icariin affects AD progression, and highlights the potential of icariin in the treatment of AD.

In vitro differentiated human CD4+ T cells produce hepatocyte growth factor.

Differentiation of naive CD4+ T cells into effector T cells is a dynamic process in which the cells are polarized into T helper (Th) subsets. The subsets largely consist of four fundamental categories: Th1, Th2, Th17, and regulatory T cells. We show that human memory CD4+ T cells can produce hepatocyte growth factor (HGF), a pleiotropic cytokine which can affect several tissue types through signaling by its receptor, c-Met. In vitro differentiation of T cells into Th-like subsets revealed that HGF producing T cells increase under Th1 conditions. Enrichment of HGF producing cells was possible by targeting cells with surface CD30 expression, a marker discovered through single-cell RNA-sequencing. Furthermore, pharmacological inhibition of PI3K or mTOR was found to inhibit HGF mRNA and protein, while an Akt inhibitor was found to increase these levels. The findings suggest that HGF producing T cells could play a role in disease where Th1 are present.

TAp63, a methotrexate target in CD4+ T cells, suppresses Foxp3 expression and exacerbates autoimmune arthritis.

Methotrexate (MTX) is a standard, first-line therapy for rheumatoid arthritis (RA); however, its precise mechanisms of action other than antifolate activity are largely unknown. We performed DNA microarray analyses of CD4+ T cells in patients with RA before and after MTX treatment and found that TP63 was the most significantly downregulated gene after MTX treatment. TAp63, an isoform of TP63, was highly expressed in human IL-17-producing Th (Th17) cells and was suppressed by MTX in vitro. Murine TAp63 was expressed at high levels in Th cells and at lower levels in thymus-derived Treg cells. Importantly, TAp63 knockdown in murine Th17 cells ameliorated the adoptive transfer arthritis model. RNA-Seq analyses of human Th17 cells overexpressing TAp63 and those with TAp63 knockdown identified FOXP3 as a possible TAp63 target gene. TAp63 knockdown in CD4+ T cells cultured under Th17 conditions with low-dose IL-6 increased Foxp3 expression, suggesting that TAp63 balances Th17 cells and Treg cells. Mechanistically, TAp63 knockdown in murine induced Treg (iTreg) cells promoted hypomethylation of conserved noncoding sequence 2 (CNS2) of the Foxp3 gene and enhanced the suppressive function of iTreg cells. Reporter analyses revealed that TAp63 suppressed the activation of the Foxp3 CNS2 enhancer. Collectively, TAp63 suppresses Foxp3 expression and exacerbates autoimmune arthritis.

PI3Kδ shapes Th2 lineage restriction through coordination of IL-2 signaling, Foxo1 inactivation and epigenetic remodeling

Abstract Activated PI3K-delta syndrome (APDS) is a primary immunodeficiency caused by heterozygous activating mutations in PI3Kδ, resulting in recurrent infections as well as allergic diseases. Given that APDS patients show an inability to clear respiratory pathogens (Th1) and development of allergic diseases (Th2), we hypothesized that hyperactivated PI3Kδ (Pik3cd E1020K) may alter CD4 T cell differentiation. Using in vitro polarization of murine naïve CD4 T cells, we observed increased production of IFNγ from Pik3cd E1020Kmice under Th1 inducing conditions and marked increases in IL-4 and IL-13 production under Th2 conditions. However, Pik3cd E1020KTh2 cells also expressed high levels of Tbet and aberrantly produced IFNγ. Strikingly, this dysregulation was also observed in vivo; Pik3cd E1020KCD4 cells show pronounced alterations following house dust mite induced airway inflammation, with impaired Th2 and inappropriate polarization to Th1 responses as visualized by scRNAseq. We linked this unstable Th1 and Th2 differentiation in Pik3cd E1020KCD4 T cells in vitro to hyper-responsiveness to IL-2, associated with increased phosphorylation and inactivation of transcription factor Foxo1. CRISPR-mediated targeting of Foxo1 in naïve WT CD4 cells recapitulated the Pik3cd E1020Kphenotype. Transcriptomic analysis of Pik3cd E1020KCD4 T cells revealed differential expression of genes involved in histone methylation. Accordingly, Pik3cd E1020KCD4 exhibit altered chromatin modifications, including H3K27 and H3K4 trimethylation. Together these data suggest that balanced regulation of PI3Kδ plays a critical role in enforcing Th1 and Th2 lineage identity. This research was supported by the Intramural Research Program of NIAID, NIH

MiRNA-21, which disrupts metabolic reprogramming to facilitate CD4+ T cell polarization toward the Th2 phenotype, accelerates arsenite-induced hepatic fibrosis

Elevated levels of arsenic may be present in groundwater, and long-term exposure to arsenic increases hepatic fibrosis. T helper 2 (Th2) cells are involved in the fibrotic cascade, and cell metabolism is a regulatory factor participating in CD4+ T cell differentiation and function. However, the mechanism for Th2 cell regulation of arsenite-induced hepatic fibrosis is not fully understood. In present study, for arsenite-fed mice, activated hepatic stellate cells may be involved in the infiltration of CD4+ T cells, accompanied by up-regulation of GATA3, a transcription factor, and IL-13, the major Th2 cytokine. Exposed to arsenite, Jurkat cells had increased aerobic glycolysis to promote the cell cycle and cell proliferation. Further, this process elevated levels of marker molecules, including those of the Th2 paradigm characterized by GATA3, IL-4, and IL-13. LX-2 cells were activated when treated with culture medium from Jurkat cells exposed to arsenite. miR-21 may be a therapeutic target for arsenite-induced hepatic fibrosis. In vitro, miR-21 knock-down caused inhibition of the PTEN/PI3K/AKT pathway induced by arsenite. It also reversed the elevated glycolysis and the accelerated cell cycle and cell proliferation. Indeed, this alteration led to diminished expression of GATA3, IL-4, and IL-13 in T cells differentiated under Th2 conditions, which inhibits activation of LX-2 cells. Consistent with the results in vitro, miR-21 knock-out in mice reversed hepatic fibrosis and attenuated the levels of GATA3 and IL-13 induced by arsenite. These findings indicate that miR-21 regulates the glycolysis of CD4+ T cells through the PTEN/PI3K/AKT pathway to accelerate the cell cycle, thereby facilitating CD4+ T cell polarization toward Th2 and releasing the fibrogenic factor IL-13, which participates in arsenite-associated hepatic fibrosis. Inhibition of Th2 polarization of CD4+T cells or miR-21 could be a therapeutic strategy to combat hepatic fibrosis caused by exposure to arsenic.

Association of asthma and herpes zoster, the role of vaccination: A literature review.

Herpes Zoster (HZ) is the reactivation of a previous infection with varicella‐zoster virus (VZV) which shares the same mode of transmission as HZ. It presents with painful erythematous vesicles in a dermatome which is characterized by a burning sensation before and after the rash. Any conditions with suppressed cellular immunity example diabetes mellitus, chronic obstructive pulmonary disease, asthma, cardiovascular diseases, chronic steroid uses, malignancy, etc. causes reactivation of the virus. Impaired immune responses in asthma patients either in any age group may increase their susceptibility to HZ infection owing to skewed Th1/Th2 immunity, resulting in predominant Th2 conditions and an unwarranted Th2 cell response against respiratory allergens. Similarly, many studies have delineated the association of asthma with HZ. However, the relation between steroid use in asthma and HZ is uncertain, its immunosuppressive effect might be responsible for increased susceptibility to the infection. As HZ increases the economic burden and morbidity, its prevention should use vaccines. There are two types of Food and Drug Administration (FDA)‐approved vaccine available against HSV one of which is given as a single dose vaccine called Zostavax, for people 50–59 years but its efficacy falls after 3rd dose and on the subsequent 4th dose and is also contraindicated in human immunodeficiency virus/acquired immunodeficiency syndrome, pregnancy and people taking immunosuppressive drugs. Shingrix is preferred by FDA which is a two doses vaccine that is given 6 months apart for people above 50 years and to immunocompromised people. Hence, proper counseling and education about the risks of herpes should be informed to the patients with timely utilization of the vaccine.

Selenoprotein I deficiency in T cells promotes differentiation into tolerant phenotypes while decreasing Th17 pathology.

Selenoprotein I (SELENOI) is an ethanolamine phospholipid transferase contributing to cellular metabolism and the synthesis of glycosylphosphatidylinositol (GPI) anchors. SELENOI knockout (KO) in T cells has been shown to impair metabolic reprogramming during T cell activation and reduce GPI-anchored Thy-1 levels, which are both crucial for Th17 differentiation. This suggests SELENOI may be important for Th17 differentiation, and we found that SELENOI was indeed up-regulated early during the activation of naïve CD4+ T cells in Th17 conditions. SELENOI KO reduced RORγt mRNA levels by decreasing SOX5 and STAT3 binding to promoter and enhancer regions in the RORC gene encoding this master regulator of Th17 cell differentiation. Differentiation of naïve CD4+ T cells into inflammatory versus tolerogenic Th cell subsets was analyzed and results showed that SELENOI deficiency skewed differentiation away from pathogenic Th17 cells (RORγt+ and IL-17A+ ) while promoting tolerogenic phenotypes (Foxp3+ and IL-10+ ). Wild-type and T cell-specific SELENOI KO mice were subjected to experimental autoimmune encephalitis (EAE), with KO mice exhibiting diminished clinical symptoms, reduced CNS pathology and decreased T cell infiltration. Flow cytometry showed that SELENOI T cell KO mice exhibited lower CD4+ RORγt+ and CD4+ IL-17A+ T cells and higher CD4+ CD25+ FoxP3+ T cells in CNS tissues of mice subjected to EAE. Thus, the metabolic enzyme SELENOI is up-regulated to promote RORγt transcription that drives Th17 differentiation, and SELENOI deficiency shifts differentiation toward tolerogenic phenotypes while protecting against pathogenic Th17 responses.

ITK tunes the Th17/Treg switch response by controlling calcium dependent signaling.

Abstract Naïve CD4+ T cells can differentiate into pro inflammatory T helper type 17 (Th17) and anti inflammatory T regulatory (Treg) lineages following T cell receptor (TCR) activation. The strength of the TCR signal is tuned by Interleukin-2 inducible T cell Kinase (ITK) and ITK can tune the development of Th17 and Treg cells. ITK signals can also tune a Th17/Treg switch under Th17 conditions. How ITK mediated tuning of TCR signal control naïve CD4+ T cell commitment into Th17/Treg lineages is unclear. Here we used ITK inhibitors, conventional ITK and allele specific ITK (ITKas) IL17 GFP/Foxp3 RFP reporter mice, to track naïve CD4+ T cell commitment into Th17/Treg lineages. Under Th17 conditions, reduced TCR signal strength in absence of ITK activity/expression, tuned naïve CD4+ T cell fate by reducing Th17 differentiation and inducing switch to cells resembling Tregs expressing Foxp3. The ITKas model showed this switch is due to specific ITK inhibition. The switched Treg like cells resemble Tregs by expression of Treg markers (CD25, CTLA4, PD1) and suppression of effector T cell proliferation. Transcriptomic analysis of switched Treg like cells by RNA Seq indicate they resemble Tregs, with reduced Th17 (Rorc, Il17) and increased Treg (Foxp3, Nrp1, Ikzf2) gene expression. Analysis of chromatin accessibility by ATAC Seq indicated closed loci for Th17 (Rorc, Il17) genes. Activating intracellular calcium dependent pathways led to a loss of the Th17/Treg switch response in naïve CD4+ T cell commitment, effectively bypassing ITK mediated tuning of the TCR signal critical for the switch response. In conclusion, we show here ITK tunes a Th17/Treg switch response in naïve CD4+ T cell commitment under Th17 conditions by controlling calcium dependent signaling. Supported by grants from NIH (NIH AI129422).

The PGI2 signaling pathway decreases glycolysis and mitochondria respiration in mouse Th2 cells

Abstract Prostaglandin I2 (PGI2) is a lipid molecule produced in the cyclooxygenase (COX) metabolic pathway and regulates T cell function and inflammation. We have previously shown that PGI2 inhibited type 2 cytokine production by Th2 cells in vitro and in vivo. To further investigate the mechanism of the inhibitory effect of PGI2 on Th2 differentiation, we determined the effect of the PGI2 analog cicaprost on T cell metabolism under Th2 conditions. Mouse naïve CD4 T cells from the spleen were activated with antibodies against CD3 and CD28 under Th2 polarization conditions and treated with cicaprost or vehicle for 4 days. Seahorse assays measured cellular glycolysis and mitochondria respiration. We found that cicaprost significantly decreased basal and maximum glycolysis in Th2 cells. Furthermore, when we performed Seahorse assays on the cells rested for 2 days after the initial 4 day T cell activation, we discovered that cicaprost not only suppressed basal glycolysis and glycolytic reserve but also dose-dependently inhibited basal and maximum mitochondria respiration. These results suggest that the PGI2 signaling pathway inhibits Th2 cell metabolism, providing a possible mechanism for PGI2-mediated inhibition of Th2 differentiation and Th2 cell-dependent immune disorders. The Department of Veterans Affairs BX004299 (RSP), NIH AI095227 (RSP), NIH AI111820 (RSP), NIH AI145265 (RSP), NIH AI124456 (RSP), NIH AI145397 (RSP).

TH17 CELLS PROMOTE INTESTINAL FIBROSIS THROUGH THE PRODUCTION OF AMPHIREGULIN

Abstract BACKGROUND & AIMS Intestinal fibrosis is a major serious complication of Crohn’s disease (CD). Gut microbiota-reactive Th17 cells have been established as crucial in the pathogenesis of CD, however, how Th17 cells induce fibrosis is still not completely understood. METHODS Wild-type Th1 cells and Th17 cells, or amphiregulin (Areg)-/- Th17 cells, were transferred into Tcrbxd-/- mice to induce colitis and intestinal fibrosis. CD4+ T cell expression of Areg was determined by qRT-PCR and ELISA. Intestinal fibroblasts were isolated from normal and CD patients and used to determine the effects of Areg on cell proliferation and migration in vitro. Video microscopy and single-cell tracking were used to assess cell migration kinetically. Areg expression was compared in peripheral blood CD4+ T cells, intestinal biopsy tissues, and lamina propria lymphocyte (LPL) from healthy controls and CD patients with or without intestinal fibrosis. RESULTS When transferred into TCRβ/δ-/- mice, although Th1 and Th17 cells induced intestinal inflammation at similar levels, Th17 cells induced more severe intestinal fibrosis than Th1 cells. RNA-seq analysis showed higher levels of AREG expression in Th17 cells compared to Th1 cells, which was further confirmed by qRT-PCR. Transfer of Areg-/- Th17 cells induced less severe fibrosis in TCRβ/δ-/- mice compared with WT Th17 cells. IL-6 but not IL-23 promoted Areg expression in Th17 cells in a time and dose-dependent manner. Treatment with Th17 signature cytokine IL-21, but not IL-17, upregulated T cell expression of Areg. Consistently, IL-21R-/- T cells produced lower levels of Areg. Mechanically, STAT3 mediates Areg expression in Th17 cells, in that IL-6 promoted STAT3 activation, and IL-6 did not induce AREG expression in STAT3-/- T cells. Areg expression was lower in STAT3-/- T cells compared with WT T cells under Th17 conditions. Treatment with RORgt inhibitor, which inhibits Th17 differentiation, suppressed Th17 cell Areg expression, indicating that higher Areg expression requires fully Th17 differentiation. Areg promoted human intestinal fibroblast proliferation and motility, which was mediated by activation of mTOR, and mTOR inhibitors inhibited Areg-induced human intestinal fibroblast proliferation and motility. Finally, Areg expression is increased in peripheral blood CD4+ T cells, intestinal biopsy tissues, and lamina propria lymphocytes of CD patients with fibrosis. CONCLUSION Collectively, these findings reveal that Th17-derived Areg promotes fibrotic responses in both experimental colitis and human CD patients. Thereby, Areg could serve as a potential therapeutic target for fibrosis in CD.

TH17 CELLS PROMOTE INTESTINAL FIBROSIS THROUGH THE PRODUCTION OF AMPHIREGULIN

Intestinal fibrosis is a major serious complication of Crohn’s disease (CD). Gut microbiota-reactive Th17 cells have been established as crucial in the pathogenesis of CD, however, how Th17 cells induce fibrosis is still not completely understood. Wild-type Th1 cells and Th17 cells, or amphiregulin (Areg)-/- Th17 cells, were transferred into Tcrbxd-/- mice to induce colitis and intestinal fibrosis. CD4+ T cell expression of Areg was determined by qRT-PCR and ELISA. Intestinal fibroblasts were isolated from normal and CD patients and used to determine the effects of Areg on cell proliferation and migration in vitro. Video microscopy and single-cell tracking were used to assess cell migration kinetically. Areg expression was compared in peripheral blood CD4+ T cells, intestinal biopsy tissues, and lamina propria lymphocyte (LPL) from healthy controls and CD patients with or without intestinal fibrosis. When transferred into TCRβ/δ-/- mice, although Th1 and Th17 cells induced intestinal inflammation at similar levels, Th17 cells induced more severe intestinal fibrosis than Th1 cells. RNA-seq analysis showed higher levels of AREG expression in Th17 cells compared to Th1 cells, which was further confirmed by qRT-PCR. Transfer of Areg-/- Th17 cells induced less severe fibrosis in TCRβ/δ-/- mice compared with WT Th17 cells. IL-6 but not IL-23 promoted Areg expression in Th17 cells in a time and dose-dependent manner. Treatment with Th17 signature cytokine IL-21, but not IL-17, upregulated T cell expression of Areg. Consistently, IL-21R-/- T cells produced lower levels of Areg. Mechanically, STAT3 mediates Areg expression in Th17 cells, in that IL-6 promoted STAT3 activation, and IL-6 did not induce AREG expression in STAT3-/- T cells. Areg expression was lower in STAT3-/- T cells compared with WT T cells under Th17 conditions. Treatment with RORgt inhibitor, which inhibits Th17 differentiation, suppressed Th17 cell Areg expression, indicating that higher Areg expression requires fully Th17 differentiation. Areg promoted human intestinal fibroblast proliferation and motility, which was mediated by activation of mTOR, and mTOR inhibitors inhibited Areg-induced human intestinal fibroblast proliferation and motility. Finally, Areg expression is increased in peripheral blood CD4+ T cells, intestinal biopsy tissues, and lamina propria lymphocytes of CD patients with fibrosis. Collectively, these findings reveal that Th17-derived Areg promotes fibrotic responses in both experimental colitis and human CD patients. Thereby, Areg could serve as a potential therapeutic target for fibrosis in CD.

Atopic Neutrophils Prevent Postviral Airway Disease.

Respiratory syncytial virus (RSV) infection in infancy is associated with increased risk of asthma, except in those with allergic disease at the time of infection. Using house dust mite allergen, we examined the effect of pre-existing atopy on postviral airway disease using Sendai virus in mice, which models RSV infection in humans. Sendai virus drives postviral airway disease in nonatopic mice; however, pre-existing atopy protected against the development of airway disease. This protection depended upon neutrophils, as depletion of neutrophils at the time of infection restored the susceptibility of atopic mice to postviral airway disease. Associated with development of atopy was an increase in polymorphonuclear neutrophil-dendritic cell hybrid cells that develop in Th2 conditions and demonstrated increased viral uptake. Systemic inhibition of IL-4 reversed atopic protection against postviral airway disease, suggesting that increased virus uptake by neutrophils was IL-4 dependent. Finally, human neutrophils from atopic donors were able to reduce RSV infection of human airway epithelial cells in vitro, suggesting these findings could apply to the human. Collectively our data support the idea that pre-existing atopy derives a protective neutrophil response via potential interaction with IL-4, preventing development of postviral airway disease.

Abstract 1760: Role of Batf-dependant Th17 immune response in PTEN-null mouse model

Abstract Basic leucine zipper transcription factor ATF-like (BATF) belongs to the activator protein 1 (AP-1) family of transcription factors and has been shown to be predominantly expressed in cells of hematopoietic origin, especially in T cells. Mice deficient for BATF were first described to lack T helper cell type 17 (Th17). Th17 cells are a distinct lineage of CD4+ T helper cells that provide protective immunity to infections but are also involved in chronic inflammation and autoimmunity. However, the role of Th17 response in prostate cancer remains controversial. In this study, we took a genetic approach to explore the role of BATF-dependent Th17 cells in prostate cancers by interbreeding BATF-deficient (Batf-/-) mice with mice that are conditionally mutant for PTEN, an established preclinical model for prostate cancer. Batf+/+;Pten-/- and Batf+/-;Pten-/- mice presented similar prostate phenotypes thus were put into one group (named Batf+), which were compared to the Batf-/-;Pten-/- group (named Batf-). We found that Batf- prostates were clearly smaller than Batf+ prostates at 30 weeks. The genitourinary bloc weight was significantly heavier in Batf+ mice than in Batf- mice at 12 and 30 weeks. Batf+ mice developed significantly higher percent of invasive adenocarcinomas in different prostate lobes at 9, 12, and 30 weeks, respectively compared to Batf- mice. Tumors in Batf- mice exhibited lower rates of cellular proliferation and higher apoptosis, as well as reduced inflammatory cell infiltration and angiogenesis in the prostatic stroma. Moreover, Batf- mice exhibited significantly reduced mRNA and protein levels of Th17 cytokines, AP-1 family members and IL-23R, and reduced NF-kB pathway activity in both spleen and prostate tissues. CD4+ T cells from Batf- mice cultured under Th17 conditions for 4 days, expressed reduced AP-1 family-regulated genes compared to Batf+ mice. Prostate cancer cells (LNCaP) with overexpression of BATF in vitro exhibited increased IL-23R expression. Prostate tissues from Batf- mice cultured under IL-6 or Th17 conditions for 4 days had reduced IL-23R expression compared to that from Batf+ mice. Taken together, our results suggest that Batf promotes the initiation and progression of prostate adenocarcinoma via Th17-mediated inflammatory immune responses and that the Batf-IL-23-IL-17 axis is a critical pathway that is needed for prostate adenocarcinoma development and progression. This work was supported by the NIH-National Institute of General Medical Sciences COBRE on Aging and Regenerative Medicine (P20GM103629, PD: S. Michal Jazwinski; PI: Q.Z.) and the Carol Lavin Bernick Faculty Grant from Tulane University (PI: Q.Z.). Citation Format: Qiuyang Zhang, Sen Liu, Bing Zhang. Role of Batf-dependant Th17 immune response in PTEN-null mouse model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1760.

Polyamines polarized Th2/Th9 cell-fate decision by regulating GATA3 expression

Polyamines produced by both prokaryotes and eukaryotes are bioactive substances with pleiotropic effects. Accumulating evidence has demonstrated that polyamines contribute to anti-inflammatory responses by suppressing the expression of proinflammatory cytokines in mononuclear cells and macrophages. However, the effects of polyamines on CD4+ T cell responses remain to be elucidated. Here, we investigated the effect of polyamines on cell fate decisions of naïve CD4+ T cells in vitro. We found that endogenously generated polyamines are essential for the development of T helper 2 (Th2) cells. Treatment with DL-2-difluoromethylornithine (DFMO), an inhibitor of polyamine biosynthesis, diminished GATA3 expression in CD4+ T cells under Th2-skewed conditions. Supplementation of exogenous polyamines rescued GATA3 downregulation caused by DFMO treatment in CD4+ T cells. Transcriptome analysis revealed that deprivation of endogenous polyamines resulted in upregulated Th9-related genes, such as Il9, Irf4, and Batf3, even under the Th2-skewing conditions. Depletion of intracellular polyamines reduced GATA3 expression but increased IL-9-producing CD4+ T cells under both Th2 and Th9-skewing conditions. Furthermore, oral administration of DFMO increased IL-9-producing CD4+ T cells in small intestine in mice. Thus, our data indicate that polyamines play a critical role in the regulation of the Th2/Th9 balance.

Staphylococcus aureus Alpha-Toxin Limits Type 1 While Fostering Type 3 Immune Responses.

Staphylococcus aureus can cause life-threatening diseases, and hospital- as well as community-associated antibiotic-resistant strains are an emerging global public health problem. Therefore, prophylactic vaccines or immune-based therapies are considered as alternative treatment opportunities. To develop such novel treatment approaches, a better understanding of the bacterial virulence and immune evasion mechanisms and their potential effects on immune-based therapies is essential. One important staphylococcal virulence factor is alpha-toxin, which is able to disrupt the epithelial barrier in order to establish infection. In addition, alpha-toxin has been reported to modulate other cell types including immune cells. Since CD4+ T cell-mediated immunity is required for protection against S. aureus infection, we were interested in the ability of alpha-toxin to directly modulate CD4+ T cells. To address this, murine naïve CD4+ T cells were differentiated in vitro into effector T cell subsets in the presence of alpha-toxin. Interestingly, alpha-toxin induced death of Th1-polarized cells, while cells polarized under Th17 conditions showed a high resistance toward increasing concentrations of this toxin. These effects could neither be explained by differential expression of the cellular alpha-toxin receptor ADAM10 nor by differential activation of caspases, but might result from an increased susceptibility of Th1 cells toward Ca2+-mediated activation-induced cell death. In accordance with the in vitro findings, an alpha-toxin-dependent decrease of Th1 and concomitant increase of Th17 cells was observed in vivo during S. aureus bacteremia. Interestingly, corresponding subsets of innate lymphoid cells and γδ T cells were similarly affected, suggesting a more general effect of alpha-toxin on the modulation of type 1 and type 3 immune responses. In conclusion, we have identified a novel alpha-toxin-dependent immunomodulatory strategy of S. aureus, which can directly act on CD4+ T cells and might be exploited for the development of novel immune-based therapeutic approaches to treat infections with antibiotic-resistant S. aureus strains.