Hepatic Treg Research Articles

Pretransplant targeting of TNFRSF25 and CD25 stimulates recipient Tregs in target tissues ameliorating GVHD post-HSCT.

The current approach to minimize transplant-associated complications, including graft-versus-host disease (GVHD) includes long-term pharmacological immune suppression frequently accompanied by unwanted side effects. Advances in targeted immunotherapies regulating alloantigen responses in the recipient continue to reduce the need for pan-immunosuppression. Here, in vivo targeting of the TNF superfamily receptor 25 (TNFRSF25) and the high affinity IL-2 receptor with a TL1A-Ig fusion protein and low dose IL-2, respectively, was used to pretreat recipient mice prior to allogeneic-HSCT (aHSCT). Pretreatment induced Treg expansion persisting early post-aHSCT leading to diminished GVHD and improved transplant outcomes. Expansion was accompanied by an increase in frequency of stable and functionally active Tregs as evidenced by in vitro assays using cells from major GVHD target tissues including colon, liver, and eye. Importantly, pretreatment supported epithelial cell function/integrity, a diverse microbiome including reduction of pathologic bacteria overgrowth and promotion of butyrate producing bacteria, while maintaining physiologic levels of obligate/facultative anaerobes. Notably, using a sphingosine 1-phosphate receptor agonist to sequester T cells in lymphoid tissues, we found that the increased tissue Treg frequency included resident CD69 + CD103 + FoxP3 + hepatic Tregs. In contrast to infusion of donor Treg cells, the strategy developed here resulted in the presence of immunosuppressive target tissue environments in the recipient prior to the receipt of donor allo-reactive T cells and successful perseveration of GVL responses. We posit strategies that circumvent the need of producing large numbers of ex-vivo manipulated Tregs, may be accomplished through in vivo recipient Treg expansion, providing translational approaches to improve aHSCT outcomes.

Ileal microbial microbiome and its secondary bile acids modulate susceptibility to nonalcoholic steatohepatitis in dairy goats

BackgroundLiver damage from nonalcoholic steatohepatitis (NASH) presents a significant challenge to the health and productivity of ruminants. However, the regulatory mechanisms behind variations in NASH susceptibility remain unclear. The gut‒liver axis, particularly the enterohepatic circulation of bile acids (BAs), plays a crucial role in regulating the liver diseases. Since the ileum is the primary site for BAs reabsorption and return to the liver, we analysed the ileal metagenome and metabolome, liver and serum metabolome, and liver single-nuclei transcriptome of NASH-resistant and susceptible goats together with a mice validation model to explore how ileal microbial BAs metabolism affects liver metabolism and immunity, uncovering the key mechanisms behind varied NASH pathogenesis in dairy goats.ResultsIn NASH goats, increased total cholesterol (TC), triglyceride (TG), and primary BAs and decreased secondary BAs in the liver and serum promoted hepatic fat accumulation. Increased ileal Escherichia coli, Erysipelotrichaceae bacterium and Streptococcus pneumoniae as well as proinflammatory compounds damaged ileal histological morphology, and increased ileal permeability contributes to liver inflammation. In NASH-tolerance (NASH-T) goats, increased ursodeoxycholic acid (UDCA), isodeoxycholic acid (isoDCA) and isolithocholic acid (isoLCA) in the liver, serum and ileal contents were attributed to ileal secondary BAs-producing bacteria (Clostridium, Bifidobacterium and Lactobacillus) and key microbial genes encoding enzymes. Meanwhile, decreased T-helper 17 (TH17) cells and increased regulatory T (Treg) cells proportion were identified in both liver and ileum of NASH-T goats. To further validate whether these key BAs affected the progression of NASH by regulating the proliferation of TH17 and Treg cells, the oral administration of bacterial UDCA, isoDCA and isoLCA to a high-fat diet-induced NASH mouse model confirmed the amelioration of NASH through the TH17 cell differentiation/IL-17 signalling/PPAR signalling pathway by these bacterial secondary BAs.ConclusionThis study revealed the roles of ileal microbiome and its secondary BAs in resilience and susceptibility to NASH by affecting the hepatic Treg and TH17 cells proportion in dairy goats. Bacterial UDCA, isoDCA and isoLCA were demonstrated to alleviate NASH and could be novel postbiotics to modulate and improve the liver health in ruminants.A8SM95maBgMZc1-WqV_tnDVideo

β-Catenin disruption decreases macrophage exosomal α-SNAP and impedes Treg differentiation in acute liver injury.

Hepatic macrophages and regulatory T cells (Tregs) play an important role in the maintenance of liver immune homeostasis, but the mechanism by which hepatic macrophages regulate Tregs in acute liver injury remains largely unknown. Here, we found that the hepatic Treg proportion and β-catenin expression in hepatic macrophages were associated with acetaminophen- and d-galactosamine/LPS-induced acute liver injury. Interestingly, β-catenin was markedly upregulated only in infiltrating macrophages but not in resident Kupffer cells. Myeloid-specific β-catenin-knockout mice showed an increased inflammatory cell infiltration and hepatocyte apoptosis. Moreover, myeloid β-catenin deficiency decreased the hepatic Treg proportion in the injured liver. Mechanistically, in vitro coculture experiments revealed that macrophage β-catenin modulated its exosome composition and influenced Treg differentiation. Using mass spectrometry-based proteomics, we identified that macrophage β-catenin activation increased the level of exosomal alpha soluble NSF attachment protein (α-SNAP), which in turn promoted Treg differentiation. Overall, our findings demonstrated a molecular mechanism that macrophage β-catenin regulated the Treg proportion in the liver by enhancing the expression of exosomal α-SNAP, providing insights into the pathophysiology of acute liver injury.

Macrophage Notch1 signaling modulates regulatory T cells via the TGFB axis in early MASLD

Background & aimsHepatic immune imbalance is critical in driving metabolic dysfunction-associated steatotic liver disease (MASLD) progression. The role of hepatic regulatory T cells (Tregs) in MASLD initiation and the mechanisms responsible for their change are not completely understood. MethodsA mouse model subjected to a short-term high-fat diet (HFD) to mimic early steatosis, along with liver biopsy samples from patients with simple steatosis and macrophage-specific Notch1 knockout mice (Notch1M-KO)， was used to investigate the role of Tregs in early MASLD and the effect of hepatic macrophage Notch1 signaling on Treg frequency. Using Exos-miRNA-sequencing, we analyzed the miRNA correlated with Treg differentiation. ResultsHere we showed that a decrease in Tregs contributed to HFD-induced hepatic steatosis and insulin resistance (n=5/group/time point, p<0.001). Remarkably, the frequency of Tregs exhibited a negative correlation with Notch1 activation in hepatic macrophages during hepatic steatosis (n=38/group, r=-0.735, p<0.001). Furthermore, we found that Notch1 deficiency attenuated hepatic lipid deposition and reversed Treg levels (n=5 per group, p<0.01, p<0.05). Moreover, Treg depletion in Notch1M-KO mice greatly diminished the ameliorative effect of macrophagic Notch1 deletion on hepatic steatosis. Mechanistically, macrophage Notch1 activation increased its exosomal miR-142a-3p level (1-2-fold), impairing Treg differentiation by targeting TGFBR1 on T cells. Consistently, HFD-fed Notch1M-KO mice exhibited reduced miR-142a-3p levels, elevated TGFBR1 expression on T cells, and increased Tregs frequency in the liver. ConclusionsThese findings highlight the critical role of hepatic Tregs in the early stage of MASLD and add a novel, non-negligible pathway for macrophage involvement in hepatic steatosis. We identify a previously unrecognized molecular mechanism of macrophage Notch1/exosomal miR-142a-3p/TGFBR1 pathway in regulating Treg differentiation, providing the rationale for refined therapeutic strategies for MASLD. Impact and implicationsThe immune mechanisms driving MASLD progression, particularly in the early stages, are not fully understood, which limits the development of effective interventions. This study elucidated a novel mechanism by which hepatic macrophage Notch1 signaling modulated Tregs through the exosomal miR-142a-3p/TGFBR1 axis, contributing to the progression of MASLD. These findings provide a rationale for a potential immunological approach to treat MASLD in the future.

Regulatory T cells in inflamed liver are dysfunctional in murine primary biliary cholangitis.

Primary biliary cholangitis (PBC) is a chronic autoimmune disease characterized by immune-mediated destruction of intrahepatic small bile ducts. CD8 T cells play a critical role in biliary destruction. However, regulatory T cells (Tregs) have also been identified in the portal tracts of PBC patients. This study tested the hypothesis that hepatic Tregs in PBC were dysfunctional in suppressing immune responses in disease by using our human PBC-like autoimmune cholangitis (AIC) mouse model induced by 2-octynoic acid-conjugated ovalbumin (2-OA-OVA). Our results showed that female and male mice immunized with 2-OA-OVA developed AIC; however, female AIC mice had more severe liver inflammation and fibrosis than male AIC mice. Levels of functional effector CD8 T cells and their chemoattractants, CXCL9 and CXCL10, in the liver were markedly elevated in female AIC mice than in male AIC mice. These results reinforce that CD8 T cells are the primary effector cells in PBC. The number of hepatic Tregs in AIC mice was also higher than in saline-treated mice, but there was no difference between male and female AIC mice. The suppressive function of AIC Tregs was evident despite a discrepancy in the changes in their co-inhibitory receptors and inhibitory cytokines. However, the expansion of hepatic Tregs by low-dose IL-2 treatment did not reduce immune responses to AIC, which may be due to the dysfunction of Tregs in inhibiting T cells. In conclusion, the function of Tregs in the inflamed liver of PBC was insufficient, and low-dose IL-2 treatment could not restore their function to suppress pathological immune responses. Transferring normal Tregs or directly targeting effector CD8 T cells may be beneficial for treating PBC.

Immunosuppressive CD29+ Treg accumulation in the liver in mice on checkpoint inhibitor therapy

ObjectiveLiver metastases are often resistant to immune checkpoint inhibitor therapy (ICI) and portend a worse prognosis compared with metastases to other locations. Regulatory T cells (Tregs) are one of several...

Abstract 3808: MicroRNA-206 drives antitumor immunity by disrupting the communication between Kupffer cells and Tregs

Abstract Kupffer cells (KCs) account for 15% of the total liver cells. However, their roles in hepatocellular carcinoma (HCC) is poorly described. Clec4fCre-tdTomato knock-in mice expressing Cre recombinase and tdTomato-NLS under the KC-specific Clec4f promoter were hydrodynamically injected with oncogenic AKT and Nras (AKT/Ras) to induce HCC. A LoxP-stop-LoxP (LSL) system was used to express miR-206 in KCs of mice. AKT activation was observed in hepatocytes and KCs of HCC patients. Hydrodynamic injection (HDI) of AKT/Ras led to activation of AKT signaling in KCs and hepatocytes. AKT/Ras mice developed lethal HCC within 6-8 weeks post injection. Activation of AKT signaling led to M2 polarization of KCs, increased hepatic Tregs and exhaustion of CTLs. MicroRNA (miR) profiling revealed that AKT/Ras impaired miR-206 biogenesis in KCs by driving translation of Yin Yang 1 (YY1), a transcription repressor of miR-206. HDI of miR-206 into AKT/Ras mice fully prevented HCC, while all control mice died of HCC 6-8 weeks post injection. However, HDI of miR-206 only transfected ~ 20% of hepatocytes, indicating that it is unreasonable to conclude that the full prevention of HCC in AKT/Ras/miR-206 mice is caused by inhibited proliferation of HCC cells. We, therefore, posited that miR-206-mediated recovery of immune surveillance, at least in part, accounted for the full prevention of HCC in AKT/Ras/miR-206 mice. We next used the LSL system to overexpress miR-206 in KCs of Clec4fCretdTomato mice injected with AKT/Ras. KC-specific expression of miR-206 fully prevented HCC in AKT/Ras mice, while 100% control mice died of HCC. Mechanistically, AKT signaling in KCs drove CCL22 (C-C motif chemokine ligand 22) production by phosphorylating β-catenin that activates transcription of Ccl22. In contrast, miR-206 disrupted β-catenin signaling by targeting LEF1 (lymphoid enhancer-binding factor 1), an “architectural” transcription factor of β-catenin. CCL22 recruits Tregs. Indeed, KC-specific expression of miR-206 led to elevated CCL22, M2 to M1 transition of KCs, reduced hepatic Tregs, and elevated CTLs. Depletion of CTLs in AKT/Ras mice fully offset the ability of miR-206 to prevent HCC. Conclusions: AKT-educated KCs initiate Treg recruitment via a regulatory loop consisting of YY1, β-catenin/LEF1, miR-206, and CCL22; once this loop is activated, AKT activates β-catenin axis, which drives CCL22 production, Treg recruitment and HCC growth; while KC-specific expression of miR-206 reversed this process by blocking β-catenin axis in KCs. miR-206 represents a novel immunotherapy against HCC. Citation Format: Guisheng Song, Ningning Liu. MicroRNA-206 drives antitumor immunity by disrupting the communication between Kupffer cells and Tregs. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3808.

FGF15 Protects Septic Mice by Inhibiting Inflammation and Modulating Treg Responses.

Fibroblast growth factor 15 (FGF15) through its FGF-receptor (FGFR)-4 inhibits hepatic inflammation. The current study aimed at investigating whether FGF15 could inhibit septic inflammation and its compensative regulatory T cell (Treg) responses in a mouse sepsis model of cecal ligation and puncture (CLP) and in vitro transwell co-culture. Following the sham or CLP procedure, male CLP C57BL/6 mice were intravenously injected with vehicle saline or FGF15 beginning at 2 h post the procedure every 12 h for three days. Some mice were euthanized and their serum and liver samples were collected for examination of cytokines and Tregs by enzyme-linked immunosorbent assay (ELISA), Western blot and flow cytometry. The remaining mice were monitored for their survival up to 14 days post procedure. Moreover, the purified hepatic CD4+ T cells were co-cultured in transwell plates with unmanipulated NCTC 1469 cells or the cells that had been transfected with the control or FGFR4-specific siRNA and treated with, or without, Lipopolysaccharides (LPS) for 24 h, followed by treatment with vehicle PBS or FGF15 for 48 h. Compared with the CLP group of mice, treatment with FGF15 significantly prolonged the mean survival days of mice (12 vs 1.17 in the CLP group, P = 0.022), mitigated hepatic inflammation and reduced the frequency of apoptotic cells in the liver of mice. FGF15 treatment decreased the percentages of hepatic Tregs, hepatic IL-2, TGF-β and FOXP3 expression in septic mice, accompanied by decreasing serum IL-1β, TNF-α, IL-6 and IL-10 levels. Similarly, FGF15 treatment also attenuated the LPS-increased frequency of Tregs, FOXP3 and IL-2 expression and IL-1β, TNF-α, IL-6 and IL-10 secretion in vitro after co-culture with NCTC 1469 cells, but not co-cultured FGFR4-silenced NCTC 1649 cells. FGF15 treatment through FGFR4 ameliorated hepatic inflammation and its compensative Treg responses, which were associated with protecting from septic death in mice.

Potential correlation of allograft infiltrating group 2 innate lymphoid cells with acute rejection after liver transplantation.

Accumulating evidence indicates the critical roles of group 2 innate lymphoid cells (ILC2s) in immunoregulation. However, the role of ILC2s in acute rejection after liver transplantation (LT) remains elusive. In this study, we analyzed the frequency, counts, and signature cytokines of ILC2s in liver transplant recipients by flow cytometric analysis and multiplex immunofluorescence assay. We also assessed the spatial distribution and correlation between hepatic ILC2s and Treg cells. The changes of ILC2s were dynamically monitored in the mouse LT model. We found that the frequencies of circulating ILC2s were comparable in liver transplant recipients with either rejection or non-rejection compared with the control group. The hepatic ILC2s counts were significantly increased in the rejection group than in the non-rejection and control groups, and a similar trend was observed for Treg cells. In the mouse LT model, allograft infiltrating ILC2s dramatically increased within 14 days post-transplant. The frequency of ILC2s in bone marrow significantly increased at 7 days post-transplant and rapidly decreased at 14 days after LT. Similarly, there was a significant increase in the frequency of splenic ILC2s within two weeks post-transplant. Multiplex immunofluorescence assay showed a close correlation between hepatic ILC2s and Treg cells by analyzing their spatial distribution and distance. In conclusion, the number of allograft infiltrating ILC2s was closely related to rejection after LT. Allograft infiltrating ILC2s may play inhibitory roles in posttransplant immune homeostasis, favoring resolution of liver allograft rejection by interacting with Treg cells or promoting the migration of Tregs cells into the liver allograft.

Abstract 2582: Regulatory T cells restrain efficacy of immunotherapy in murine liver tumors

Abstract Background: Checkpoint blockade immunotherapy (CBI) can induce a durable response for some patients with hepatic tumors, but many derive no or incomplete oncologic benefit for unclear reasons. Regulatory T cells (Tregs) in the liver play a central role in maintaining the tolerogenic local immune environment, both in homeostasis and disease. Here we sought to explore the impact of CBI on hepatic Tregs to determine if they play a role in CBI resistance of liver tumors in mice. Methods: High-dimensional flow cytometry was used to examine the ex vivo characteristics of lymphocytes from both tumor-free and orthotopic tumor-bearing livers of mice treated with CBI or other immunomodulatory agents. Results: All hepatic T cell subsets examined displayed higher proliferative and apoptotic indices compared with those of splenic T lymphocytes. We found that hepatic Tregs intensely proliferate and undergo apoptosis compared with splenic Tregs under homeostatic conditions. Hepatic Treg proliferation was enhanced after administration of CBI treatment. This effect was abrogated by co-treatment with sirolimus. CD8, macrophages, and the gut microbiome were found to be dispensable for the in vivo in response to αPD1. Co-treatment of mice with αPD1 and αCD25 sensitized MC38-bearing liver tumors. Conclusion: Murine liver Tregs naturally proliferate and undergo apoptosis due to the mTOR rheostat at homeostasis making them highly responsive to CBI. This behavior potentially explains liver-specific CBI-resistance in tumors. Citation Format: Benjamin L. Green, Chi Ma, Qianfei Zhang, Benjamin Ruf, Umberto Rosato, Jonathan Qi, Simon Wabitsch, Kylynda Bauer, Yuta Myojin, Justin McCallen, John C. McVey, Varun Subramanyam, Vanessa Catania, Amran Nur, Firouzeh Korangy, Changqing Xie, Tim F. Greten. Regulatory T cells restrain efficacy of immunotherapy in murine liver tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2582.

MicroRNA-206 enhances antitumor immunity by disrupting the communication between malignant hepatocytes and regulatory T cells in c-Myc mice.

Intertumoral accumulation of regulatory T cells (Tregs) has been implicated in the pathogenesis of HCC. Because of poor understanding of the immunosuppression mechanism(s) in HCC, immunotherapy is largely unsuccessful for the treatment of HCC. Hydrodynamic injection (HDI) of c-Myc into mice resulted in enlarged spleens and lethal HCC associated with an increase in hepatic Tregs and depletion of CTLs (cytotoxic T lymphocytes). Malignant hepatocytes in c-Myc mice overproduced TGFβ1, which enhanced the suppressor function of Tregs and impaired the proliferation and cytotoxicity of CTLs. In addition to activating TGFβ signaling, c-Myc synergized with Yin Yang 1 to impair microRNA-206 (miR-206) biogenesis. HDI of miR-206 fully prevented HCC and the associated enlargement of the spleen, whereas 100% of control mice died from HCC within 5-9 weeks postinjection. Mechanistically, by directly targeting errant kirsten ras oncogene (KRAS) signaling, miR-206 impeded the mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK)axis that drives expression of Tgfb1. By blocking the KRAS/MEK/ERK axis, miR-206 prevented TGFβ1 overproduction, thereby impairing the suppressor function and expansion of Tregs, but enhancing the expansion and cytotoxic program of CTLs. Disrupting the interaction between miR-206 and Kras offset the roles of miR-206 in inhibiting immunosuppression and HCC. Depletion of CD8+ T cells impaired the ability of miR-206 to inhibit HCC. c-Myc-educated hepatocytes promoted immunosuppression by overproducing TGFβ1, which promoted HCC development. miR-206, by attenuating TGFβ1 overproduction, disrupted the communication of malignant hepatocytes with CTLs and Tregs, which prevented HCC. miR-206 represents a potential immunotherapeutic agent against HCC.

MicroRNA-15a/16-1 Prevents Hepatocellular Carcinoma by Disrupting the Communication Between Kupffer Cells and Regulatory T Cells

Hepatocellular carcinoma (HCC) is characterized by intratumoral accumulation of regulatory T cells (Tregs), which suppresses antitumor immunity. This study was designed to investigate how microRNAs regulate immunosuppression in HCC. FVB/NJ mice were hydrodynamically injected with AKT/Ras or c-Myc and Sleeping Beauty transposon to induce HCC. The Sleeping Beauty system was used to deliver microRNA-15a/16-1 into livers of mice. Flow cytometry and immunostaining were used to determine changes in the immune system. Hydrodynamic injection of AKT/Ras or c-Myc into mice resulted in hepatic enrichment of Tregs and reduced cytotoxic T cells (CTLs) and HCC development. HCC impaired microRNA-15a/16-1 biogenesis in Kupffer cells (KCs) of AKT/Ras and c-Myc mice. Hydrodynamic injection of microRNA-15a/16-1 fully prevented HCC in AKT/Ras and c-Myc mice, while 100% of control mice died of HCC. Therapeutically, microRNA-15a/16-1 promoted a regression of HCC in both mouse models, impaired hepatic enrichment of Tregs, and increased hepatic CTLs. Mechanistically, a significant increase was observed in serum C-C motif chemokine 22 (CCL22) and transcription of Ccl22 in KCs of AKT/Ras and c-Myc mice. MicroRNA-15a/16-1 prevented KCs from overproducing CCL22 by inhibiting nuclear factor-κB that activates transcription of Ccl22. By reducing CCL22 binding to C-C chemokine receptor type 4 on Tregs, microRNA-15a/16-1 impaired Treg chemotaxis. Disrupting the interaction between microRNA-15a/16-1 and nuclear factor-κB impaired the ability of microRNA-15a/16-1 to prevent hepatic Treg accumulation and HCC. Depletion of cluster of differentiation 8+ T cells and additional treatment of CCL22 recovered growth of HCC that was fully prevented by microRNA-15a/16. MicroRNA-15a/16-1 attenuates immunosuppression by disrupting CCL22-mediated communication between KCs and Tregs. MicroRNA-15a/16-1 represents a potential immunotherapy against HCC.

HIF-1α modulates sex-specific Th17/Treg responses during hepatic amoebiasis

An invasive form of intestinal Entamoeba (E.) histolytica infection, which causes amoebic liver abscess, is more common in men than in women. Immunopathological mechanisms are responsible for the more severe outcome in males. Here, we used a mouse model of hepatic amoebiasis to investigate the contribution of hepatic hypoxia-inducible factor (HIF)-1α to T helper 17 (Th17)/regulatory T cell (Treg) responses in the context of the sex-specific outcome of liver damage. C57BL/6J mice were infected intrahepatically with E.histolytica trophozoites. HIF-1α expression was determined by qPCR, flow cytometry and immunohistochemistry. Tregs and Th17 cells were analysed by immunohistochemistry and flow cytometry. Finally, male and female hepatocyte-specific Hif1α knockout mice were generated, and the effect of HIF-1α on abscess development, the cytokine milieu, and Th17/Treg differentiation was examined. E.histolytica infection increased hepatic HIF-1α levels, along with the elevated frequencies of hepatic Th17 and Treg cells. While the Th17 cell population was larger in male mice, Tregs characterised by increased expression of Foxp3 in female mice. Male mice displayed increased IL-6 expression, contributing to immunopathology; this increase in IL-6 expression declined upon deletion of hepatic HIF-1α. In both sexes, hepatic deletion of HIF-1α reduced the Th17 cell frequency; however, the percentage of Tregs was reduced in female mice only. Hepatic HIF-1α modulates the sex-specific outcome of murine E.histolytica infection. Our results suggest that in male mice, Th17 cells can be modulated by hepatic HIF-1α via IL-6, indicating marked involvement in the immunopathology underlying abscess development. Strong expression of Foxp3 by hepatic Tregs from female mice suggests a potent immunosuppressive function, leading to initiation of liver regeneration. Infection with the parasite Entamoeba histolytica activates immunopathological mechanisms in male mice, which lead to liver abscesses that are larger than those in female mice. In the absence of the protein HIF-1α in hepatocytes, abscess formation is reduced; moreover, the sex difference in abscess size is abolished. These results suggest that HIF-1α modulates the immune response involved in the induction of immunopathology, resulting in differential disease susceptibility in males and females.

Regulatory T-cell and neutrophil extracellular trap interaction contributes to carcinogenesis in non-alcoholic steatohepatitis

Regulatory T-cells (Tregs) impair cancer immunosurveillance by creating an immunosuppressive environment that fosters tumor cell survival. Our previous findings demonstrated that neutrophil extracellular traps (NETs), which are involved both in innate and adaptive immunity, are abundant in livers affected by non-alcoholic steatohepatitis (NASH). However, how NETs interact with Tregs in the development of NASH-associated hepatocellular carcinoma (NASH-HCC) is not known. A choline-deficient, high-fat diet+diethylnitrosamine mouse model and the stelic animal model were utilized for NASH-HCC and a western diet mouse model was used for NASH development. Treg depletion was achieved using FoxP3-DTR mice. RNA sequencing was used to explore the mechanism by which NETs could regulate Treg differentiation. Bioenergetic analyses of naïve CD4+ T-cells were assessed by Seahorse. Although the absolute number of CD4+ T-cells is lower in NASH livers, the Treg subpopulation is selectively increased. Depleting Tregs dramatically inhibits HCC initiation and progression in NASH. There is a positive correlation between increased NET and hepatic Treg levels. RNA sequencing data reveals that NETs impact gene expression profiles in naïve CD4+ T-cells, with the most differentially expressed genes being those involved in mitochondrial oxidative phosphorylation. By facilitating mitochondrial respiration, NETs can promote Treg differentiation. Metabolic reprogramming of naïve CD4+ T-cells by NETs requires toll-like receptor 4. Blockade of NETs invivo using Pad4-/- mice or DNase I treatment reduces the activity of Tregs. Tregs can suppress immunosurveillance in the premalignant stages of NASH. NETs facilitate the crosstalk between innate and adaptive immunity in NASH by promoting Treg activity through metabolic reprogramming. Therapies targeting NETs and Treg interactions could offer a potential strategy for preventing HCC in patients with NASH. Regulatory T-cells (Tregs) can promote tumor development by suppressing cancer immunosurveillance, but their role in carcinogenesis during non-alcoholic steatohepatitis (NASH) progression is unknown. Herein, we discovered that selectively increased intrahepatic Tregs can promote an immunosuppressive environment in NASH livers. Neutrophil extracellular traps (NETs) link innate and adaptive immunity by promoting Treg differentiation via metabolic reprogramming of naïve CD4+ T-cells. This mechanism could be targeted to prevent liver cancer in patients with NASH.

Foxp3+ Regulatory T Cells Inhibit CCl4-Induced Liver Inflammation and Fibrosis by Regulating Tissue Cellular Immunity.

Foxp3+ regulatory T (Treg) cells are pivotal in maintaining immunological self-tolerance and tissue homeostasis; however, it remains unclear how tissue Treg cells respond to liver injury and regulate chronic inflammation, which can cause liver fibrosis. We report here that hepatic Treg cells play a critical role in preventing liver pathology by suppressing inflammatory cellular immunity that can promote liver damage and fibrosis. Chronic liver inflammation induced by injections of carbon tetrachloride (CCl4) led to preferential expansion of hepatic Treg cells that prevented liver fibrosis. In contrast, depletion of Treg cells in the CCl4-induced liver fibrosis model exacerbated the severity of liver pathology. Treg depletion unleashed tissue cellular immunity and drove the activation and expansion of the pro-fibrotic IL-4-producing T helper 2 cells, as well as CCR2high Ly-6Chigh inflammatory monocytes/macrophages in the inflamed liver. Although Treg expression of amphiregulin plays a key role in tissue remodeling and repair in various inflammation models, amphiregulin from hepatic Treg cells, the largest producer among liver immune cells, was dispensable for maintaining liver homeostasis and preventing liver fibrosis during CCl4-induced chronic inflammation. Our results indicate that Treg cells control chronic liver inflammation and fibrosis by regulating the aberrant activation and functions of immune effector cells. Harnessing Treg functions, which effectively regulate tissue cellular immunity, may be a therapeutic strategy for preventing and treating liver fibrosis.

ApoE deficiency promotes hepatic pathology by aggravating Th17/Treg imbalance in murine schistosomiasis japonica.

The Schistosoma japonicum (Sjaponicum)-infected ApoE gene deficiency (ApoE-/- ) mice were used to determine effect of ApoE on hepatic immunopathology. Murine activities and appetite, body weight, and ratio of liver weight to its body weight (Hepatic mass index, HMI) were observed. Worm load and liver egg burden were evaluated as the infection intensity. Number and size of liver egg granulomas and serum levels of alanine aminotransferase (ALT) were investigated. We analysed hepatic fibrosis by markers of fibrosis in tissue, detected hepatic Th17 and Treg frequency by flow cytometry, and measured hepatic expressions of RORγt, Foxp3, IL-17A and TGF-β1 via qPCR. Lipid metabolism was determined by serum levels of cholesterol (TC) and triglyceride (TG) as well as hepatic Oil red O staining. In the infected ApoE-/- mice, the increased infection intensity aggravated the hepatic immunopathology (evidenced by increased HMI, elevated egg granulomas and increased ALT levels) and fibrosis (increased hepatic collagen deposition). ApoE deficiency resulted in significantly elevated ratio of hepatic Th17/Treg and higher serum levels of TC and TG, along with higher level of hepatic Oil red O staining. ApoE deficiency promotes hepatic pathology and fibrosis by exacerbating Th17/Treg imbalance and altering lipid metabolism in murine schistosomiasis japonica.

Hepatocytes paradoxically affect intrahepatic IFN-γ production in autoimmune hepatitis due to Gal-9 expression and TLR2/4 ligand release

Hepatocytes are the targets in autoimmune hepatitis (AIH) that results in T cell-dependent liver injury. However, hepatocytes may also affect the hepatic T cells in AIH, but the underlying mechanisms are not fully understood. Here we report that hepatocytes could secrete galectin-9 (Gal-9) to suppress the intrahepatic production of Th1 cytokine IFN-γ and restrict AIH development, but hepatocyte damage resulted in opposite effects due to release of TLR2/4 ligands that promoted the intrahepatic production of IL-1β, IL-6, and IL-12. Through Tim-3, Gal-9 could efficiently suppress the intrahepatic T cell activation despite presence of TLR2/4 ligands, thus attenuating Th1 response in AIH. Intriguingly, intrahepatic IL-6/IL-12 suppressed the effect of TGF-β on Treg cells. Therefore, in AIH, Gal-9 promoted Foxp3 expression and function of hepatic Treg cells through TL1A signaling, although Treg function was still impaired, compared with that in naive state. Due to its promoting effect on Treg function, together with its effect on T effector cells in a Tim-3-independent way, Gal-9 could attenuate intrahepatic IFN-γ production by hindering the increase of hepatic CD4+CD43+ T cells resulting from extrahepatic T cell activation. TLR2/4 ligands attenuated the effects of Gal-9 on Treg cells and CD4+CD43+ T cells by increasing intrahepatic IL-6 and IL-12. Blocking TLR2/4 ligands could efficiently suppress intrahepatic IFN-γ production, liver injury, and hepatic fibrosis. These findings suggest that hepatocytes paradoxically affect Th1 response in AIH due to Gal-9 expression and TLR2/4 ligands release, and that targeting TLR2/4 signaling may provide an important approach in the therapeutic strategy for AIH.

Interleukin 2 Promotes Hepatic Regulatory T Cell Responses and Protects From Biliary Fibrosis in Murine Sclerosing Cholangitis.

In the multidrug resistance protein 2 (Mdr2)-/- mouse model, low phospholipid bile instigates biliary epithelial injury, sterile inflammation, and fibrosis, thereby recapitulating disease mechanisms implicated in biliary atresia (BA) and primary sclerosing cholangitis. We hypothesize that T lymphocytes contribute to the biliary injury and fibrosis in murine sclerosing cholangitis (SC) and that they are susceptible to suppression by regulatory T cells (Tregs). In juvenile Mdr2-/- mice, intrahepatic CD8+ lymphocytes were expanded, and contraction of intrahepatic Tregs coincided with rising serum alanine transferase and alkaline phosphatase (ALP) levels between days 14-30 of life. Antibody-mediated depletion of intrahepatic CD8+ lymphocytes during that time reduced ALP levels and the expression of osteopontin (Opn), a pro-fibrogenic cytokine. Depletion of intrahepatic Tregs with anti-CD25 antibody between days 7-30 increased intrahepatic CD8+ T cells, Opn expression, and fibrosis. Conversely, expansion of intrahepatic Tregs with interleukin 2/anti-interleukin 2 immune complexes (IL-2c) downregulated hepatic expression of Opn and Tnf, reduced frequency of intrahepatic CD8+ lymphocytes, and diminished biliary injury and fibrosis. Treatment with IL-2c upregulated hepatic Treg expression of CD39, an ectonucleotidase capable of hydrolyzing pro-inflammatory adenosine triphosphate. In vitro, Tregs expressing CD39 suppressed the proliferation of hepatic CD8+ lymphocytes from Mdr2-/- mice more efficiently than those lacking CD39. In infants with BA, infiltration of interlobular bile ducts with CD8+ cells was associated with biliary expression of Opn and its transcription was negatively correlated with mRNA expression of Treg-associated genes. Conclusion: Hepatic CD8+ T lymphocytes drive biliary injury and fibrosis in murine SC. Their proliferation is controlled by hepatic Tregs through the purinergic pathway, which is responsive to IL-2c, suggesting that Treg-directed low-dose Il-2 treatment may be considered as therapy for SC.

The IL‐33‐ST2‐MyD88 axis promotes regulatory T cell proliferation in the murine liver

Hepatic Foxp3+ Treg cells are crucial for maintaining local immune homeostasis in the liver. However, the environmental cues required for hepatic Treg cell homeostasis are unclear. In this study, we showed that the IL-33 receptor ST2 was preferentially expressed on Treg cells in the mouse liver, but it was more lowly expressed in the spleen, mesenteric lymph nodes, and blood. More importantly, we found that IL-33 promoted the proliferation of hepatic Treg cells through myeloid differentiation factor MyD88 signaling concomitant with increased cyclin-dependent kinase 4 and cyclin D1 expression. These results suggest that IL-33 is a potential tissue-specific factor controlling Treg cell homeostasis via increased Treg proliferation in the liver.

Increased hepatic Th2 and Treg subsets are associated with biliary fibrosis in different strains of mice caused by Clonorchis sinensis

Previous studies showed that CD4+T cells responses might be involved in the process of biliary fibrosis. However, the underlying mechanism resulting in biliary fibrosis caused by Clonorchis sinensis remains not yet fully elucidated. The objectives of the present study were to investigate the different profiles of hepatic CD4+T cell subsets (Th1, Th2, Th17 and Treg cells) and their possible roles in the biliary fibrosis of different strains of mice (C57BL/6, BALB/c and FVB mice) induced by C. sinensis infection. C57BL/6, BALB/c and FVB mice were orally gavaged with 45 metacercariae. All mice were sacrificed on 28 days post infection in deep anesthesia conditions. The leukocytes in the liver were separated to examine CD4+T cell subsets by flow cytometry and the left lobe of liver was used to observe pathological changes, collagen depositions and the concentrations of hydroxyproline. The most serious cystic and fibrotic changes appeared in FVB infected mice indicated by gross observation, Masson’s trichrome staining and hydroxyproline content detection. In contrast to C57BL/6 infected mice, diffuse nodules and more intensive fibrosis were observed in the BALB/c infected mice. No differences of the hepatic Th1 subset and Th17 subset were found among the three strains, but the hepatic Th2 and Treg cells and their relative cytokines were dramatically increased in the BALB/c and FVB infected groups compared with the C57BL/6 infected group (P<0.01). Importantly, increased Th2 subset and Treg subset all positively correlated with hydroxyproline contents (P<0.01). This result for the first time implied that the increased hepatic Th2 and Treg cell subsets were likely to play potential roles in the formation of biliary fibrosis in C. sinensis-infected mice.