Zheng Tan

Interleukin-33 Ameliorates Experimental Colitis through Promoting Th2/Foxp3+ Regulatory T-Cell Responses in Mice

Crohn’s disease (CD) is characterized by the activation of Th1 and Th17 cells and deficiency of regulatory T cells (Tregs), leading to intestine tissue injury and destruction. As a novel cytokine of the interleukin (IL)-1 family, the role and underlying mechanisms of IL-33 in CD remain poorly understood. Here, we assess the effects and mechanisms of IL-33 on the trinitrobenzene sulfonic acid (TNBS)-induced experimental colitis that mimics human CD. We found that IL-33 levels were increased in the TNBS-treated mice, whereas recombinant IL-33 (rIL-33) administration substantially ameliorated TNBS-mediated colonic tissue injury and clinical symptoms of colitis. The protective effect of rIL-33 was partly associated with the markedly increased induction of Th2-type cytokines. Importantly, rIL-33 treatment resulted in prominently upregulated Foxp3 expression in the TNBS-treated mice, and depletion of Tregs significantly abrogated the impact of IL-33 on reducing the development of colitis. Notably, the level of CD103+ dendritic cells (DCs), which promotes development of Tregs, is also increased in mesenteric lymph node and lamina propria of rIL-33-treated mice. The impact of rIL-33 on CD103+ DC induction was the result of indirectly upregulating intestine epithelial cells that produce thymic stromal lymphopoietin and retinoic acid but do not directly act on DCs. In conclusion, our data provide clear evidence that IL-33 plays a protective role in TNBS-induced colitis, which is closely related to a Th1-to-Th2/Treg switch. Thus, IL-33 is a promising candidate for the development of new treatments for CD.

Gene Silencing of NALP3 Protects Against Liver Ischemia–Reperfusion Injury in Mice

Liver ischemia-reperfusion (I/R) injury is a multifactorial process that affects graft function after liver transplantation. Inflammatory cytokines, such as tumor necrosis factor (TNF)-α, interleukin (IL)-6, IL-1β, and IL-18, have been shown to play key roles in the pathophysiology of liver I/R injury. Studies have indicated that NALP3 (NACHT domain, leucine-rich repeat [LRR] domain, and pyrin domain [PYD]-containing protein-3) inflammasome is pivotal in the processing and releasing of IL-1β and IL-18. The aim of this study was to test whether NALP3 silencing has a protective effect in murine liver I/R injury. Using a partial lobar liver warm ischemia model, mice were hydrodynamically injected with pNALP3shRNA, pshRNANC, or saline 48 hr before ischemia. Those mice pretreated with pNALP3shRNA showed decreased serum alanine aminotransferase levels; inhibited production of proinflammatory cytokines such as IL-1β, IL-18, TNF-α, and IL-6 by downregulation of caspase-1 activation and NF-κB activity; as well as decreased release of HMGB1 (high-mobility group box-1) and inflammatory cell infiltration, leading to the prevention of liver I/R injury, when compared with controls. Histology revealed that pretreatment with pNALP3shRNA significantly ameliorated hepatocellular damage after I/R. Thus, by using a small hairpin RNA approach, our study confirms that NALP3 signaling is involved in liver I/R and that silencing of NALP3 can protect the liver from I/R injury by reducing IL-1β, IL-18, TNF-α, IL-6, and HMGB1 release through downregulation of caspase-1 activation and NF-κB activity.

High-mobility group box 1 promotes early acute allograft rejection by enhancing IL-6-dependent Th17 alloreactive response

Previously, we reported that extracellular high-mobility group box 1 (HMGB1) functions as an innate alarmin implicated in cardiac allograft acute rejection. We now present evidence suggesting that HMGB1 is pivotal in inducing interleukin-17 (IL-17)-producing alloreactive T cells by stimulating dendritic cells secretion of IL-6. Those IL-17+ T cells are likely to be the major effector cells responsible for the early stage of cardiac allograft rejection through mediating an influx of neutrophils into allografts, and therefore, blockade of IL-17A significantly prolonged murine cardiac allograft survival. In contrast to the classical model for a dominant role of IFN-γ+-Th1 cells have in acute allograft rejection, our data suggest that IFN-γ+-Th1 cells are responsible for the late stage of graft destruction by inducing monocyte infiltration when IL-17+ T-cell response recedes. Blockade of HMGB1 significantly decreased splenic alloreactive Th17 cells and IFN-γ-producing CD8+ T cells in the recipients, leading to less infiltration of neutrophils along with lower IL-6 and IL-17 expression levels in the grafts as well as prolongation of cardiac allograft survival. Together, these data support a novel model in which HMGB1 induces IL-17-producing alloreactive T cells to mediate early stage of allograft rejection, whereas IFN-γ-producing alloreactive Th1 cells provoke graft destruction after Th17 response recedes.

Acid-Sensing Ion Channels Contribute to the Effect of Acidosis on the Function of Dendritic Cells

As an H+-gated subgroup of the degenerin/epithelial Na+ channel family, acid-sensing ion channels (ASICs) were reported to be involved in various physiological and pathological processes in neurons. However, little is known about the role of ASICs in the function of dendritic cells (DCs). In this study, we investigated the expression of ASICs in mouse bone marrow-derived DCs and their possible role in the function of DCs. We found that ASIC1, ASIC2, and ASIC3 are expressed in DCs at the mRNA and protein levels, and extracellular acid can evoke ASIC-like currents in DCs. We also demonstrated that acidosis upregulated the expression of CD11c, MHC class II, CD80, and CD86 and enhanced the Ag-presenting ability of DCs via ASICs. Moreover, the effect of acidosis on DCs can be abolished by the nonsteroidal anti-inflammatory drugs ibuprofen and diclofenac. These results suggest that ASICs are involved in the acidosis-mediated effect on DC function.

B7-H4 transfection prolongs β-cell graft survival

B7-H4, a recently discovered member of B7 family, can negatively regulate T cell responses. However, it is not clear whether B7-H4 negatively function in cell transplantation. In this study we investigated the immunosuppressive effect of B7-H4 on beta-cell transplantation. An insulinoma cell line, NIT-1, transfected with B7-H4 (B7-H4-NIT) was established, and transplanted to diabetic C57BL/6 mice by intraperitoneal injection. Proliferation assay of splenocytes in vitro showed that B7-H4-NIT suppressed alloreactive T cell activation. The proportion of IFN-gamma-producing cells in recipient spleen was significantly reduced and the number of Treg cells was upregulated in B7-H4-NIT group compared to the control, EGFP-NIT. The expression of mRNA coding IFN-gamma was lower but that of IL-4 was higher in B7-H4-NIT transplanted recipients than in the control animals. The results of ELISA also revealed the same trends. Diabetic mice reached normalglycemic quickly and gained weight after transplantation of B7-H4-NIT. More importantly, the survival time for recipients transplanted with B7-H4-NIT cells was significantly longer than that with EGFP-NIT cells. These results indicate that B7-H4 transfection prolongs beta-cell graft survival.

Extracellular acidosis modulates the endocytosis and maturation of macrophages.

Extracellular acidosis is involved in various pathological situations of central nervous system and the effects are largely mediated by acid sensing ion channels (ASICs). However, it remains unclear whether extracellular acidosis affects immune cells. Macrophages are immune cells that play important role in immune reactions. In this study we investigated the impact of extracellular acidosis on the function of bone marrow derived macrophages (BMMs). The results showed that extracellular acidosis upregulated the endocytosis, surface molecular expression and interleukin-10 secretion of BMMs, in which the expression of ASIC1 and ASIC3 was detected. Notably, extracellular acidosis stimulated endocytosis and upregulation of surface molecules expression in BMMs could be abolished by amiloride, a blocker of ASICs, and nonsteroid anti-inflammatory drugs. Our findings provide new insight into the role of extracellular acidosis in the regulation of immune function and suggest ASICs as new targets for the modulation of immune response.

CpG ODN pretreatment attenuates concanavalin A-induced hepatitis in mice

T cell-mediated hepatic damage plays a key role in the pathogenesis of liver diseases such as autoimmune hepatitis, viral hepatitis and acute liver failure. CpG-containing oligodeoxynucleotides (CpG ODN), a ligand for toll-like receptor (TLR) 9, is widely used as an immunological adjuvant. In the present study, we investigated the effect of CpG ODN on T cell-mediated liver injury in a murine model of concanavalin A (Con A)-induced hepatitis. We found that the aminotransferase level was significantly decreased in CpG ODN pretreated mice and the survival of the mice was markedly prolonged. CpG ODN pretreatment inhibited NF-kappaB DNA binding activity. As a result, the systemic/liver levels of TNF-alpha and IFN-gamma were significantly suppressed. Furthermore, the activation of inflammatory cells was diminished by CpG ODN pretreatment. These results suggest that CpG ODN pretreatment protects the mice from Con A-induced liver injury via inhibiting hepatocyte apoptosis, inflammation and activation of lymphocytes.

Interleukin-33 is released in spinal cord and suppresses experimental autoimmune encephalomyelitis in mice.

Interleukin-33 (IL-33) is usually expressed in the nucleus as a non-histone chromatin-associated protein. After passively released by necrotic cells, it functions as an IL-1 family member. IL-33 is highly expressed in the central nervous system (CNS), whether IL-33 is actively released in the CNS and involved in experimental autoimmune encephalomyelitis (EAE) remains unclear. In this study, we found that IL-33 and receptor ST2 were expressed in the spinal cord of naïve mice. Compared to naive situation, the intracellular IL-33 was dramatically decreased and extracellular IL-33 was markedly increased in the spinal cord in the pre-onset, onset and peak stage of EAE. In the chronic stage, the reverse happened. The decrease of intracellular IL-33 was related to the activation of astrocytes and the damage of neurons in situ during EAE. Astrocytes secreted IL-33 actively upon inflammatory stimulation in vitro. Furthermore, blockage of the CNS-derived IL-33 exacerbated EAE development. Our data demonstrated that IL-33 was released by activated astrocytes actively, and by damaged neurons during EAE. It plays a suppressive role in EAE development via an autocrine or paracrine manner. Our findings are helpful to understand the release feature and function of the CNS-derived IL-33 and supply a potential therapeutic target for multiple sclerosis.

HMGB1 Is Involved in Chronic Rejection of Cardiac Allograft via Promoting Inflammatory‐Like mDCs

Chronic rejection that leads to diffuse narrowing and occlusion of graft vessels is the most important cause of morbidity and mortality following cardiac transplantation. The role and underlying mechanism of high‐mobility group box 1 (HMGB1), as an established inflammatory mediator in acute rejection, remains poorly understood in chronic rejection. Here, we assessed the effects and mechanisms of HMGB1 on the chronic rejection using single MHC Class II‐mismatched mouse cardiac transplantation model. It was found that HMGB1 was increased accompanying with the development of chronic rejection, while blockade of HMGB1 with specific neutralizing mAb substantially ameliorated chronic rejection‐mediated vasculopathy and fibrosis of allograft, as well as markedly decreased T cell infiltration and production of IL‐17A and interferon‐gamma in allograft and recipients spleen. Further, anti‐HMGB1 antibody treatment significantly declined the number and frequency of mature dendritic cells (DCs) in allograft and recipients spleen, especially CD11b+Ly6Chigh matured DCs that share the phenotypes with inflammatory‐DCs. These findings indicate that HMGB1 contributes to chronic rejection, and HMGB1 blockade may be a novel mean to disrupt the proinflammatory loop after heart transplantation.

Adoptive transfer of FTY720‐treated immature BMDCs significantly prolonged cardiac allograft survival

A sphingosine 1 phosphate receptor modulator, FTY720, has been used to alleviate symptoms in allotransplantation and autoimmune disease models with impressive efficacy, while it only achieved moderate success in clinical trials. Infusion of immature bone marrow‐derived dendritic cell (BMDC) progenitors before transplantation could induce donor specific tolerance. In this study, we investigated the possibility of using FTY720‐DCs (FTY720‐treated immature BMDCs) to prevent severe alloimmune response. Our results indicate that FTY720‐DCs could markedly prolong graft survival compared with Ctrl‐DCs (nonconditioned immature BMDCs) as manifested by reduced inflammatory infiltration into the graft. IFN‐γ production by CD4+ and CD8+ T cells were significantly reduced, while FoxP3+ regulatory T cells among CD4+ T cells were upregulated. Although FTY720 seldom altered the phenotype or the phagocytosis of BMDCs in vitro, it severely hampered their capability to trigger antigen‐specific and allogeneic T‐cell response. When splenic T cells were co‐cultured with FTY720‐DCs, the proportion of regulatory T cells increased, accompanied by elevated IL‐10 production. Consistently, infusion of FTY720‐DCs could preferentially promote Treg proliferation and upregulate PD‐1 expression on conventional T cells in allogeneic mature BMDC priming experiment. These results suggest that infusion of FTY720‐DCs before cardiac transplantation could significantly prolong functional graft survival by acting as a balancer of alloimmune response.