Lihua Duan

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.

Protective Effect of Antagonist of High‐mobility Group Box 1 on Lipopolysaccharide‐Induced Acute Lung Injury in Mice

We explored the effects of recombinant A‐box (rA‐box), a specific blockade for endogenous high mobility group box 1 (HMGB1) protein, on acute lung inflammation induced by lipopolysaccharide (LPS) in vivo. Acute lung injury (ALI) was produced successfully by intratracheal administration of LPS (10 μg/mouse) in male BALB/c mice. rA‐box (0.3, 0.6 mg/mouse, i.p.) was administered 30 min prior to or 2 h after LPS exposure. Bronchoalveolar lavage fluid (BALF) was obtained to measure chemokines, proinflammatory cytokines, total cell counts and proteins at the indicated time points. It was found that rA‐box caused a significant reduction in the total cells and neutrophils in BALF, a significant reduction in the W/D ratio and protein leakage at 24 h after LPS challenge. In addition, rA‐box was also believed to have downregulated the expression of LPS‐induced chemokines (keratinocyte‐derived chemokine) and proinflammatory cytokines, including early mediator TNF‐a and late mediator HMGB1. These findings confirm the significant protection of rA‐box against LPS‐induced ALI, and the effect mechanism of rA‐box was associated with decreasing the expression of chemokines and proinflammatory cytokines.

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.

Gene delivery of indoleamine 2,3-dioxygenase prolongs cardiac allograft survival by shaping the types of T-cell responses.

We investigated the hypothesis that overexpression of indoleamine 2,3‐dioxygenase (IDO) by a cardiac allograft may result in a survival advantage of the allograft by creating a tolerogenic microenvironment.

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.

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.

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.

Flt3L combined with rapamycin promotes cardiac allograft tolerance by inducing regulatory dendritic cells and allograft autophagy in mice.

The induction of immune tolerance is still a formidable challenge in organ transplantation. Dendritic cells (DCs) play an important role in orchestrating immune responses by either mediating protective immune responses or inducing antigen specific tolerance. Previous studies demonstrated that the fms-like tyrosine kinase 3 receptor (Flt3) and its ligand (Flt3L) play an essential role in the regulation of DC commitment and development. Here, we report a synergic effect between Flt3L and low-dose rapamycin (Rapa) in the protection of allograft rejction. It was found that Flt3L combined with Rapa significantly prolonged murine cardiac allograft survival time as compared with that of untreated recipients or recipients treated with Rapa or Flt3L alone. Mechanistic studies revealed that Flt3L combined with low-dose of Rapa induced the generation of tolerogenic DCs along with the production of CD25+ Foxp3+ regulatory T cells and IL-10 secretion. We also observed enhanced autophagy in the cardiac allograft, which could be another asset contributing to the enhanced allograft survival. All together, these data suggest that Flt3L combined with low-dose of Rapa could be an effective therapeutic approach to induce tolerance in clinical setting of transplantation.

High‐mobility group box 1 accelerates early acute allograft rejection via enhancing IL‐17+ γδ T‐cell response

Th17 and γδ T cells are the dominant IL‐17‐producing cell. We previously reported that high‐mobility group box 1 (HMGB1) is critical in inducing IL‐17‐producing alloreactive T cells during early stage of acute allograft rejection. However, the role of γδ T cells during this process and its implication in HMGB1–mediated allograft rejection are not fully understood. Here, we use a murine model of cardiac allograft transplantation to further study the role of HMGB1 and IL‐17‐producing γδ T cells in acute allograft rejection. It was found that the expression of HMGB1 was increased in allograft, while blockade of HMGB1 suppressed IL‐17+ γδ T‐cell response and inhibited the gene transcription of IL‐23 and IL‐1β. Furthermore, in vitro HMGB1 indirectly promoted the development of IL‐17+ γδ T cells by stimulating dendritic cells to produce IL‐23 and IL‐1β, meanwhile depletion of γδ T cells in vivo prolonged allograft survival and reduced the level of IL‐17 in serum. In conclusion, our findings inferred that increased HMGB1 expression could enhance IL‐17+ γδ T‐cell response by promoting the secretion of IL‐23 and IL‐1β, while IL‐17+ γδ T cells contribute to the early stage of acute allograft rejection.