Retinoic Acid Induces Functionally Suppressive Foxp3+RORγt+ T Cells In Vitro.

Introduction: CD4+ T cells with regulatory function co-expressing Foxp3 and RORγt are linked to the development of oral tolerance towards innocuous food antigens in mice. This study aimed to discern the role played by IL-6 and retinoic acid (RA) in the in vitro generation of Foxp3+RORγt+ T cells and to investigate whether such cells have suppressive properties. Methods: CD4+CD25- T cells isolated from the spleen of BALB/c mice, were stimulated in the presence of IL-2 alone or together with TFG-ß and different concentrations of IL-6 and/or RA. Percentage of Foxp3+, RORγt+, IL-17+, Foxp3+RORγt-, Foxp3+RORγt+, and Foxp3-RORγt+ T cells within the total CD4+ T cell population, production of cytokines (IL-10 and IL-17A) and gene expression (Foxp3, Rorc, Tgfb1, Il6, Il10, and Il17) were assessed at different time points. The phenotype and ability of cells generated from CD4+CD44-CD62L+ cells in the presence of RA to suppress effector T cell proliferation was assessed. Results: TGF-ß plus IL-6 induced the generation of Foxp3+ and double positive Foxp3+RORγt+ T cells to a higher extent than TGF-ß alone at the beginning of the incubation period, although expression of Foxp3 subsequently declined. RA, added to TGF-ß, increased Foxp3 and Rorc expression and Foxp3 and RORγt transcription and promoted the differentiation of Foxp3+RORγt- and Foxp3+RORγt+ cells that expressed and secreted IL-17. Foxp3+ T cells generated in vitro in presence of RA were functionally suppressive. Conclusions: Under the influence of IL-2 and TGF-ß, suppressive Foxp3+RORγt+ T cells that express and secrete IL-17 can be produced in vitro and RA further contributes to stabilize this phenotype.

Correlations of Helicobacter pylori with liver function, inflammatory factors and serum levels of FoxP3 and RORγt in patients with hepatitis B cirrhosis.

OBJECTIVE: To investigate the correlations of Helicobacter pylori (HP) with liver function, inflammatory factors and serum levels of forkhead box P3 (FoxP3) and retinoic acid receptor-related orphan receptor gamma-t (RORγt) in patients with hepatitis B cirrhosis (HBC). PATIENTS AND METHODS: A total of 60 HBC patients were divided into HBC group (n=30) and HP-infected HBC group (HP&HBC group, n=30). QRT-PCR was conducted to determine the messenger ribonucleic acid (mRNA) levels of FoxP3 and RORγt in serum samples. ELISA was applied to measure the levels of relevant inflammatory factors. Besides, immunohistochemical staining was conducted to detect positive expressions of FoxP3 and RORγt in liver tissues of patients in the two groups. RESULTS: No significant differences in gender, drinking, smoking, diabetes and age were found between HBC group and HP&HBC group (p>0.05). Globulin and albumin levels were comparable between the two groups (p>0.05). Liver function indexes, including ALT, AST and TBIL were higher in HP&HBC group than those in HBC group (p<0.05). The HBV-DNA level was lower in HBC group in comparison with that in HP&HBC group. The interferon-gamma (IFN-γ) level was remarkably higher in HBC group than that in HP&HBC group (p<0.01), and the levels of interleukin (IL)-6, IL-10, IL-17 and transforming growth factor (TGF)-ß1 were notably lower in HBC group in comparison with those in HP&HBC group (p<0.01). Additionally, the mRNA levels of FoxP3 and RORγt in HBC group were distinctly lower than those in HP&HBC group (p<0.01). The mRNA levels of FoxP3 and RORγt were positively related to those of IL-6, IL-10, IL-17, and TGF-ß1, and negatively associated with IFN-γ level. Immunohistochemical results indicated that positive expression rates of FoxP3 and RORγt in the liver tissues were approximately 50% in HP&HBC group and B. Zhao, Q.-J. Sheng, Y. Qin, X.-L. Wang, H. Zhao, N. Zhaowere 15% in HBC group, and the difference was statistically significant (p<0.05). CONCLUSIONS: Expression levels of FoxP3 and RORγt in serum and liver tissues are elevated in HP-infected HBC patients, and inflammatory factors are correlated with their expressions, suggesting the aggravated liver damage.

c-MAF, a Swiss Army Knife for Tolerance in Lymphocytes.

Beyond its well-admitted role in development and organogenesis, it is now clear that the transcription factor c-Maf has owned its place in the realm of immune-related transcription factors. Formerly introduced solely as a Th2 transcription factor, the role attributed to c-Maf has gradually broadened over the years and has extended to most, if not all, known immune cell types. The influence of c-Maf is particularly prominent among T cell subsets, where c-Maf regulates the differentiation as well as the function of multiple subsets of CD4 and CD8 T cells, lending it a crucial position in adaptive immunity and anti-tumoral responsiveness. Recent research has also revealed the role of c-Maf in controlling Th17 responses in the intestine, positioning it as an essential factor in intestinal homeostasis. This review aims to present and discuss the recent advances highlighting the particular role played by c-Maf in T lymphocyte differentiation, function, and homeostasis.

Maf deficiency in T cells dysregulates Treg - TH17 balance leading to spontaneous colitis.

The maintenance of homeostasis in the gut is a major challenge for the immune system. Here we demonstrate that the transcription factor MAF plays a central role in T cells for the prevention of gastro-intestinal inflammation. Conditional knock out mice lacking Maf in all T cells developed spontaneous late-onset colitis, correlating with a decrease of FOXP3+RORγt+ T cells proportion, dampened IL-10 production in the colon and an increase of inflammatory TH17 cells. Strikingly, FOXP3+ specific conditional knock out mice for MAF did not develop colitis and demonstrated normal levels of IL-10 in their colon, despite the incapacity of regulatory T cells lacking MAF to suppress colon inflammation in Rag1-/- mice transferred with naïve CD4+ T cells. We showed that one of the cellular sources of IL-10 in the colon of these mice are TH17 cells. Thus, MAF is critically involved in the maintenance of the gut homeostasis by regulating the balance between Treg and TH17 cells either at the level of their differentiation or through the modulation of their functions.

Clinicopathological analysis of immunohistochemical expression of retinoic acid-related orphan receptor-γt in peripheral T-cell lymphoma, not otherwise specified.

Peripheral T-cell lymphoma, not otherwise specified (PTCL-NOS) is cytologically and phenotypically heterogeneous. Retinoic acid-related orphan receptor-γt (RORγt) is a transcription factor that regulates the differentiation of naïve CD4+ helper T cells to Th17 cells. In the present study, we immunohistochemically confirmed the expression of RORγt in PTCL-NOS. Pathological and clinical investigations were performed for 170 cases of PTCL-NOS. RORγt-positive cases accounted for 17.6% (30/170) of the total cases, and they showed a significantly higher frequency of CD8 positivity (P = .033), lower counts of white blood cells (P = .030) and neutrophils (P = .039) in the peripheral blood, higher levels of hypergammaglobulinemia (P = .031), a higher frequency of a complete response (P = .009), and a tendency for a lower International Prognostic Index (P = .061) and better overall survival (P = .0806). These results suggest that RORγt-positive PTCL-NOS could be a subpopulation of PTCL-NOS. Further research associated with this genomic abnormality at the transcriptional level is needed to confirm the results of this study.

The expression change of RORγt, BATF, and IL-17 in Chinese vitiligo patients with 308 nanometers excimer laser treatment.

This study aims to explore the expression of RORγt, BATF, and IL-17 in Chinese vitiligo patients with 308 nm excimer laser treatment. One hundred and sixty-four vitiligo patients treated with 308 nm excimer laser were enrolled as Case group and 137 health examiners as Control group. Quantitative real-time polymerase chain reaction and immunohistochemistry were conducted to detect the expressions of RORγt, BATF, and IL-17. Expression of RORγt, BATF, IL-17A, and IL-17F were higher in Case group than Control group, with the diagnostic accuracy of 88.04, 87.38, 97.34, and 89.04%, respectively. Pearson correlation analysis showed a positive correlation in RORγt, BATF, IL-17A, and IL-17F mRNAs in vitiligo patients. Moreover, their expressions were higher in active vitiligo patients than stable ones. Besides, the expressions of RORγt, BATF, IL-17A, and IL-17F in vitiligo skin were significantly higher than those in non lesional skin and normal controls. After treatment, their expressions were significantly decreased. Active vitiligo and the high expressions of RORγt, BATF, and IL-17F were the independent risk factors for the ineffectiveness of 308 nm excimer laser treatment. The expressions of RORγt, BATF, IL-17 were significantly enhanced in vitiligo patients, which were correlated with the activity of vitiligo and 308 nm excimer laser therapeutic effects.

The differentiation of ROR-γt expressing iNKT17 cells is orchestrated by Runx1.

iNKT cells are a unique lineage of T cells that recognize glycolipid presented by CD1d. In the thymus, they differentiate into iNKT1, iNKT2 and iNKT17 effector subsets, characterized by preferential expression of Tbet, Gata3 and ROR-γt and production of IFN-γ, IL-4 and IL-17, respectively. We demonstrate that the transcriptional regulator Runx1 is essential for the generation of ROR-γt expressing iNKT17 cells. PLZF-cre Runx1 cKO mice lack iNKT17 cells in the thymus, spleen and liver. Runx1-deficient iNKT cells have altered expression of several genes important for iNKT17 differentiation, including decreased expression of IL-7Rα, BATF and c-Maf and increased expression of Bcl11b and Lef1. However, reduction of Lef1 expression or introduction of an IL-7Rα transgene is not sufficient to correct the defect in iNKT17 differentiation, demonstrating that Runx1 is a key regulator of several genes required for iNKT17 differentiation. Loss of Runx1 leads to a severe decrease in iNKT cell numbers in the thymus, spleen and liver. The decrease in cell number is due to a combined decrease in proliferation at Stage 1 during thymic development and increased apoptosis. Thus, we describe a novel role of Runx1 in iNKT cell development and differentiation, particularly in orchestrating iNKT17 differentiation.

Pharmacological Inhibition of Bromodomain Proteins Suppresses Retinal Inflammatory Disease and Downregulates Retinal Th17 Cells.

Experimental autoimmune uveitis (EAU), in which CD4+ Th1 and/or Th17 cells are immunopathogenic, mimics various clinical features of noninfectious uveitis in humans. The impact of bromodomain extraterminal (BET) inhibitors on Th17 cell function was studied in a mouse model of EAU in vivo and in mouse and human Th17 cells in vitro. Two BET inhibitors (GSK151 and JQ1) were able to ameliorate the progression of inflammation in EAU and in mouse CD4+ T cells in vitro, downregulating levels of Th17 cells. Additionally, the uveitogenic capacity of Th17 cells to transfer EAU was abrogated by BET inhibitors in an adoptive transfer model. In human CD4+ T cells, a 5-d exposure to BET inhibitors was accompanied by a significant downregulation of Th17-associated genes IL-17A, IL-22, and retinoic acid-related orphan receptor γt. However, in vitro, the inhibitors had no effect on already polarized Th17 cells. The key finding is that, in response to BET inhibitors, Th17-enriched cultures developed a regulatory phenotype, upregulated FOXP3 expression and IL-10 secretion, and lost pathogenicity in vivo. We conclude that BET targeting of Th17 cells is a potential therapeutic opportunity for a wide range of inflammatory and autoimmune diseases, including uveitis.

Vitamin A Controls the Presence of RORγ+ Innate Lymphoid Cells and Lymphoid Tissue in the Small Intestine.

Changes in diet and microbiota have determining effects on the function of the mucosal immune system. For example, the active metabolite of vitamin A, retinoic acid (RA), has been described to maintain homeostasis in the intestine by its influence on both lymphocytes and myeloid cells. Additionally, innate lymphoid cells (ILCs), important producers of cytokines necessary for intestinal homeostasis, are also influenced by vitamin A in the small intestines. In this study, we show a reduction of both NCR(-) and NCR(+) ILC3 subsets in the small intestine of mice raised on a vitamin A-deficient diet. Additionally, the percentages of IL-22-producing ILCs were reduced in the absence of dietary vitamin A. Conversely, mice receiving additional RA had a specific increase in the NCR(-) ILC3 subset, which contains the lymphoid tissue inducer cells. The dependence of lymphoid tissue inducer cells on vitamin A was furthermore illustrated by impaired development of enteric lymphoid tissues in vitamin A-deficient mice. These effects were a direct consequence of ILC-intrinsic RA signaling, because retinoic acid-related orphan receptor γt-Cre × RARα-DN mice had reduced numbers of NCR(-) and NCR(+) ILC3 subsets within the small intestine. However, lymphoid tissue inducer cells were not affected in these mice nor was the formation of enteric lymphoid tissue, demonstrating that the onset of RA signaling might take place before retinoic acid-related orphan receptor γt is expressed on lymphoid tissue inducer cells. Taken together, our data show an important role for vitamin A in controlling innate lymphoid cells and, consequently, postnatal formed lymphoid tissues within the small intestines.

RORγt(+) IL-22-producing NKp46(+) cells protect from hepatic ischemia reperfusion injury in mice.

BACKGROUND & AIMS: NKp46(+) cells are major effector cells in the pathogenesis of hepatic ischemia reperfusion injury (IRI). Nevertheless, the precise role of unconventional subsets like the IL-22-producing NKp46(+) cells (NK22) remains unknown. The purpose of this study was to examine the role of NK22 cells in IRI in transplantation, particularly with respect to regulation by the transcription factor ROR-gamma-t (RORγt). METHODS: To explore the role of NK22 cells in IRI in the absence of adaptive immunity, B6.RORγt-(gfp/wt)-reporter and B6.RORγt-(gfp/gfp)-knockout (KO) mice on a Rag KO background underwent 90min partial warm ischemia, followed by 24h of reperfusion. RESULTS: Rag KO mice that possess fully functional NKp46(+) cells, and Rag-common-γ-chain-double-KO (Rag-γc-DKO) mice that lack T, B and NKp46(+) cells, were used as controls. We found that Rag-γc-DKO mice lacking NK22 cells show more severe levels of hepatocellular damage (GPT, histological injury) when compared to both Rag-RORγt-reporter and Rag KO mice that possess NK22 cells. Importantly, Rag-RORγt-reporter and Rag KO mice undergoing IRI expressed high protein levels of both IL-22 and GFP (RORγt), suggesting a protective role for RORγt(+) NK22 cells in IRI. Therefore, we tested the hypothesis that RORγt critically protects from IRI through the induction of hepatic NK22 cells by studying Rag-Rorγt-DKO mice under IRI conditions. We found that the lack of RORγt(+) NK22 cells in Rag-Rorγt-DKO mice significantly enhanced IR-induced hepatocellular injury, a phenotype that could be reversed upon adoptive transfer of Rag-Rorγt-reporter NK22 cells into DKO mice. CONCLUSIONS: RORγt(+) NK22 cells play an important protective role in IRI in mice.

Mucosal-associated invariant T cell-rich congenic mouse strain allows functional evaluation.

Mucosal-associated invariant T cells (MAITs) have potent antimicrobial activity and are abundant in humans (5%-10% in blood). Despite strong evolutionary conservation of the invariant TCR-α chain and restricting molecule MR1, this population is rare in laboratory mouse strains (≈0.1% in lymphoid organs), and lack of an appropriate mouse model has hampered the study of MAIT biology. Herein, we show that MAITs are 20 times more frequent in clean wild-derived inbred CAST/EiJ mice than in C57BL/6J mice. Increased MAIT frequency was linked to one CAST genetic trait that mapped to the TCR-α locus and led to higher usage of the distal Vα segments, including Vα19. We generated a MAIThi congenic strain that was then crossed to a transgenic Rorcgt-GFP reporter strain. Using this tool, we characterized polyclonal mouse MAITs as memory (CD44+) CD4-CD8lo/neg T cells with tissue-homing properties (CCR6+CCR7-). Similar to human MAITs, mouse MAITs expressed the cytokine receptors IL-7R, IL-18Rα, and IL-12Rß and the transcription factors promyelocytic leukemia zinc finger (PLZF) and RAR-related orphan receptor γ (RORγt). Mouse MAITs produced Th1/2/17 cytokines upon TCR stimulation and recognized a bacterial compound in an MR1-dependent manner. During experimental urinary tract infection, MAITs migrated to the bladder and decreased bacterial load. Our study demonstrates that the MAIThi congenic strain allows phenotypic and functional characterization of naturally occurring mouse MAITs in health and disease.

[The level of RORγt increases in rat lung tissues of bronchiolitis caused by respiratory syncytial virus].

OBJECTIVE: To study the level of retinoic acid receptor-related orphan receptor γt (RORγt) in rat lung tissues of bronchiolitis caused by respiratory syncytial virus (RSV) and its implication. METHODS: The rats were randomly divided into normal group and bronchiolitis group. After the model of bronchiolitis was established successfully by nasal dripping, the pathological changes of lung tissues were detected by HE staining; the plasma levels of interleukin 23 (IL-23), IL-17 were detected by ELISA; the level of RORγt mRNA in lung tissues and peripheral blood mononuclear cells (PBMCs) were detected by real-time quantitative PCR; the level of RORγt protein in lung tissues was examined by Western blotting. RESULTS: Compared with the normal group, the rats with bronchiolitis presented with pulmonary interstitial hyperemia and edema, more inflammatory cell infiltration, wider alveolar septa and bronchial collapse and deformation. Compared with the normal group, the level of RORγt mRNA in the lung tissues and PBMCs increased in rats with bronchiolitis. The level of RORγt protein in lung tissues and the plasma levels of IL-23 and IL -17 were higher in rats with bronchiolitis than in normal rats. CONCLUSION: The level of RORγt was elevated in the lung tissues of rats with RSV-induced bronchiolitis.

IL-17 Producing Cells and RORγt mRNA Transcriptional Factor in Cirrhotic and HCC Egyptian Patients.

Retinoic acid-related orphan receptor gT (RORγT) is the orphan nuclear receptor that regulates the development of Th17 cells and the expression of IL-17. The differentiation of Th17 cells is associated with the upregulation of RORγT mRNA, and the mechanisms regulating that process in human cells are not well understood. RORγt as transcription factor was selectively expressed in Th17 cells and is regulated by STAT3. The relationship between Th17 cells and tumor immunopathology has been controversial. Aim of the study is to evaluate Th17 cells and RORγt transcriptional factor in cirrhotic, early and advanced HCC patients. Ninety patients were studied (30 cirrhotic, 30 early stage and 30 advanced stage HCC patients). They were recruited from the National Liver Institute, Menoufia University, and subjected to full clinical examination, investigations to detect liver cirrhosis, portal vein thrombosis and tumor staging. Peripheral blood mononuclear cells (PBMCs) were stimulated with cytosim, ionomycin and monensin, and surface (CD4-PE)as well as intracellular staining for Th17 (IL-17 FITC) was performed. Analysis of cells was carried out using FACS Calibur. RORγt mRNA expression in PBMCs was measured by real time RT- PCR. Cirrhotic patients showed increased Th17% cells, without significant change in RORγt mRNA as compared to early stage HCC. Advanced stage HCC patients showed significant increase of Th17 cells% and RORγt mRNA compared to studied patients group. Positive correlation of Th17 and RORγtmRNA was found with aminotransferases and bilirubin levels while, negative with serum albumin in advanced stage patients group (P < 0.001). Both markers were significantly increased with tumor size; RORγt mRNA increased with multiple tumor foci. In conclusion, Th17 cells and RORγt may be useful prognostic markers for advanced liver cirrhosis and HCC.

Expression of interleukin-22 and its significance in the pathogenesis of chronic rhinosinusitis.

In order to explore the role of IL-22 in the pathogenesis of CRS, we observed the expression of IL-22 and associated factors in chronic rhinosinusitis with nasal polyps (CRSwNP) and chronic rhinosinusitis without nasal polyps (CRSsNP). Immunohistochemical staining was applied to detect the expression of IL-22, IL-22R, STAT3, retinoic acid orphan receptor C (RORC) and aryl hydrocarbon recptor (AhR). There was significantly higher expression of IL-22 in CRSsNP than in controls (P<0.05). But the expression of IL-22 had no significant difference when comparing CRSwNP with CRSsNP and controls. The expression of IL-22R was significantly lower in CRSwNP compared to controls and CRSsNP (P<0.05). The expression of AhR was lower in CRSwNP than in CRSsNP (P<0.05). There was no significant difference of RORC and STAT3 among CRSwNP, CRSsNP and controls. IL-22 plays the important role in the pathogenesis of CRS, and further research is needed to understand the complex interactions with other cytokines and the exact mechanism of transcriptional regulation for IL-22.

Activation of RANKL-induced osteoclasts and memory T lymphocytes by Porphyromonas gingivalis is serotype dependant.

AIM: Destructive periodontitis is associated with a Th1-Th17 immune response and activation of RANKL-induced osteoclasts. In addition, Porphyromonas gingivalis K1 and K2 serotypes induce a strong Th1-Th17 response. This study aimed to investigate whether these P. gingivalis serotypes induce higher osteoclasts activation, by increased Th17-associated RANKL production, and an antigen-specific memory T-lymphocyte response. MATERIAL AND METHODS: The RANKL production and TRAP(+) osteoclast induction were quantified on naïve T lymphocytes stimulated with dendritic cells primed with the P. gingivalis serotypes. The T-bet, GATA-3, RORC2 and Foxp3 expression was correlated with RANKL production. The frequency of proliferating memory T lymphocytes in response to P. gingivalis serotypes was determined in both periodontitis and healthy subjects. RESULTS: T lymphocytes stimulated by K1 or K2-primed dendritic cells elicited higher levels of RANKL and TRAP(+) osteoclasts than cells stimulated with the other serotypes. RANKL positively correlated with RORC2. Whereas periodontitis patients had a higher frequency of memory T lymphocytes responding to K1 or K2, healthy subjects had a higher frequency of memory T lymphocytes responding to K4 or K(-) . CONCLUSIONS: P. gingivalis serotypes K1 and K2, but not others, are associated with an increased production of the osteoclastogenesis-related factor RANKL. This important information suggests that these serotypes could elicit a greater bone resorption in vivo and have a role in the periodontitis pathogenesis.

Distinct human T-lymphocyte responses triggered by Porphyromonas gingivalis capsular serotypes.

AIM: Porphyromonas gingivalis can synthesize an extracellular capsule and different serotypes have been described based on capsular antigenicity. On dendritic cells (DCs), the type of capsule present plays a role on the strength of the developed immune response. This study aimed to investigate the T-lymphocyte responses when stimulated with autologous mature DCs exposed to different P. gingivalis K-serotypes. MATERIALS AND METHODS: Naïve CD4(+) T-lymphocytes were obtained from healthy subjects and stimulated with autologous DCs primed with increasing multiplicity of infections of the different P. gingivalis K-serotypes. The Th1, Th2, Th17 and T-regulatory cytokines and transcription factor levels were quantified. RESULTS: Distinct types of response were detected when T-lymphocytes were stimulated by DCs primed with the different P. gingivalis K-serotypes. T-lymphocytes stimulated by K1 or K2-primed DCs elicited higher levels of Th1 and Th17-associated cytokines, T-bet and RORC2 than T-lymphocytes stimulated with DCs primed with the other serotypes. Conversely, the serotypes K3-K5 induced higher levels of Th2-associated cytokines and GATA-3 than the others. CONCLUSIONS: These results demonstrate that DCs primed with the different P. gingivalis K-serotypes elicited distinct T-cell responses. Strains K1 (W83) and K2 (HG184) induced a Th1/Th17 pattern of immune response and K3 (A7A1-28), K4 (ATCC(®49417™) ), and K5 (HG1690) a Th2 response.

Vascular cell adhesion molecule 1 expression by biliary epithelium promotes persistence of inflammation by inhibiting effector T-cell apoptosis.

UNLABELLED: Chronic hepatitis occurs when effector lymphocytes are recruited to the liver from blood and retained in tissue to interact with target cells, such as hepatocytes or bile ducts (BDs). Vascular cell adhesion molecule 1 (VCAM-1; CD106), a member of the immunoglobulin superfamily, supports leukocyte adhesion by binding α4ß1 integrins and is critical for the recruitment of monocytes and lymphocytes during inflammation. We detected VCAM-1 on cholangiocytes in chronic liver disease (CLD) and hypothesized that biliary expression of VCAM-1 contributes to the persistence of liver inflammation. Hence, in this study, we examined whether cholangiocyte expression of VCAM-1 promotes the survival of intrahepatic α4ß1 expressing effector T cells. We examined interactions between primary human cholangiocytes and isolated intrahepatic T cells ex vivo and in vivo using the Ova-bil antigen-driven murine model of biliary inflammation. VCAM-1 was detected on BDs in CLDs (primary biliary cirrhosis, primary sclerosing cholangitis, alcoholic liver disease, and chronic hepatitis C), and human cholangiocytes expressed VCAM-1 in response to tumor necrosis factor alpha alone or in combination with CD40L or interleukin-17. Liver-derived T cells adhered to cholangiocytes in vitro by α4ß1, which resulted in signaling through nuclear factor kappa B p65, protein kinase B1, and p38 mitogen-activated protein kinase phosphorylation. This led to increased mitochondrial B-cell lymphoma 2 accumulation and decreased activation of caspase 3, causing increased cell survival. We confirmed our findings in a murine model of hepatobiliary inflammation where inhibition of VCAM-1 decreased liver inflammation by reducing lymphocyte recruitment and increasing CD8 and T helper 17 CD4 T-cell survival. CONCLUSIONS: VCAM-1 expression by cholangiocytes contributes to persistent inflammation by conferring a survival signal to α4ß1 expressing proinflammatory T lymphocytes in CLD.

A low-molecular-weight dialysable leukocyte extract selectively enhances development of CD4⁺RORγt⁺ T cells and IL-17 production.

A low-molecular-weight (under 10 kDa) dialysable leukocyte extract (called transfer factor, TF) has been shown to be a prospective substance to improve or modulate immune response in autoimmunity, inflammation, infectious diseases or cancers. However, the use of TF has been limited by the absence of any data on the mechanism of its action. Here we show that TF prepared from peripheral blood leukocytes of healthy human donors displays multiple regulatory effects on individual parameters of the immune system. TF decreases proliferation of T and B lymphocytes and partially alters the production of cytokines and nitric oxide by activated macrophages. TF also inhibits production of T helper 1 (Th1) cytokines interleukin 2 (IL-2) and interferon γ, slightly stimulates production of Th2 cytokine IL-10 and considerably enhances the secretion of IL-17 by activated mouse spleen T cells. At the molecular level, TF enhances expression of genes for transcription factor RORγt and for IL-17. The enhanced expression of the RORgt gene corresponds with an increase in the number of RORγt⁺CD4⁺ Th17 cells and with enhanced IL-17 production. In contrast, the expression of the Foxp3 gene and the proportion of CD4⁺CD25⁺Foxp3⁺ regulatory T cells are not significantly changed in the presence of TF. These results suggest that the activation of pro-inflammatory Th17 cells, which have multiple immunoregulatory properties, could be the main mechanism of the immunomodulatory action of a low-molecular-weight leukocyte extract.

IL-17A plays a critical role in the pathogenesis of liver fibrosis through hepatic stellate cell activation.

Liver fibrosis is a severe, life-threatening clinical condition resulting from nonresolving hepatitis of different origins. IL-17A is critical in inflammation, but its relation to liver fibrosis remains elusive. We find increased IL-17A expression in fibrotic livers from HBV-infected patients undergoing partial hepatectomy because of cirrhosis-related early-stage hepatocellular carcinoma in comparison with control nonfibrotic livers from uninfected patients with hepatic hemangioma. In fibrotic livers, IL-17A immunoreactivity localizes to the inflammatory infiltrate. In experimental carbon tetrachloride-induced liver fibrosis of IL-17RA-deficient mice, we observe reduced neutrophil influx, proinflammatory cytokines, hepatocellular necrosis, inflammation, and fibrosis as compared with control C57BL/6 mice. IL-17A is produced by neutrophils and T lymphocytes expressing the Th17 lineage-specific transcription factor Retinoic acid receptor-related orphan receptor γt. Furthermore, hepatic stellate cells (HSCs) isolated from naive C57BL/6 mice respond to IL-17A with increased IL-6, α-smooth muscle actin, collagen, and TGF-ß mRNA expression, suggesting an IL-17A-driven fibrotic process. Pharmacologic ERK1/2 or p38 inhibition significantly attenuated IL-17A-induced HSC activation and collagen expression. In conclusion, IL-17A(+) Retinoic acid receptor-related orphan receptor γt(+) neutrophils and T cells are recruited into the injured liver driving a chronic, fibrotic hepatitis. IL-17A-dependent HSC activation may be critical for liver fibrosis. Thus, blockade of IL-17A could potentially benefit patients with chronic hepatitis and liver fibrosis.

Imbalance of placental regulatory T cell and Th17 cell population dynamics in the FIV-infected pregnant cat.

BACKGROUND: An appropriate balance in placental regulatory T cells (Tregs), an immunosuppressive cell population, and Th17 cells, a pro-inflammatory cell population, is essential in allowing tolerance of the semi-allogeneic fetus. TGF-ß and IL-6 are cytokines that promote differentiation of Tregs and Th17 cells from a common progenitor; aberrant expression of the cytokines may perturb the balance in the two cell populations. We previously reported a pro-inflammatory placental environment with decreased levels of FoxP3, a Treg marker, and increased levels of IL-6 in the placentas of FIV-infected cats at early pregnancy. Thus, we hypothesized that FIV infection in the pregnant cat causes altered placental Treg and Th17 cell populations, possibly resulting in placental inflammation. METHODS: We examined the effect of FIV infection on Treg and Th17 populations in placentas at early pregnancy using quantitative confocal microscopy to measure FoxP3 or RORγ, a Th17 marker, and qPCR to quantify expression of the key cytokines TGF-ß and IL-6. RESULTS: FoxP3 and RORγ were positively correlated in FIV-infected placentas at early pregnancy, but not placentas from normal cats, indicating virus-induced alteration in the balance of these cell populations. In control cats the expression of IL-6 and RORγ was positively correlated as predicted, but this relationship was disrupted in infected animals. TGF-ß was reduced in infected queens, an occurrence that could dysregulate both Treg and Th17 cell populations. Co-expression analyses revealed a highly significant positive correlation between IL-6 and TGF-ß expression in control animals that did not occur in infected animals. CONCLUSION: Collectively, these data point toward potential disruption in the balance of Treg and Th17 cell populations that may contribute to FIV-induced inflammation in the feline placenta.