Youhai H. Chen

Negative regulation of TLR4 via targeting of the proinflammatory tumor suppressor PDCD4 by the microRNA miR-21

The tumor suppressor PDCD4 is a proinflammatory protein that promotes activation of the transcription factor NF-κB and suppresses interleukin 10 (IL-10). Here we found that mice deficient in PDCD4 were protected from lipopolysaccharide (LPS)-induced death. The induction of NF-κB and IL-6 by LPS required PDCD4, whereas LPS enhanced IL-10 induction in cells lacking PDCD4. Treatment of human peripheral blood mononuclear cells with LPS resulted in lower PDCD4 expression, which was due to induction of the microRNA miR-21 via the adaptor MyD88 and NF-κB. Transfection of cells with a miR-21 precursor blocked NF-κB activity and promoted IL-10 production in response to LPS, whereas transfection with antisense oligonucleotides to miR-21 or targeted protection of the miR-21 site in Pdcd4 mRNA had the opposite effect. Thus, miR-21 regulates PDCD4 expression after LPS stimulation.

Defective thymocyte apoptosis and accelerated autoimmune diseases in TRAIL-/- mice.

TRAIL, the tumor necrosis factor-related apoptosis-inducing ligand, selectively induces apoptosis of tumor cells, but not most normal cells. Its role in normal, nontransformed tissues is not clear. We report here that mice deficient in TRAIL have a severe defect in thymocyte apoptosis—thus, thymic deletion induced by T cell receptor ligation is severely impaired. TRAIL-deficient mice are also hypersensitive to collagen-induced arthritis and streptozotocin-induced diabetes and develop heightened autoimmune responses. Thus, TRAIL mediates thymocyte apoptosis and is important in the induction of autoimmune diseases.

Development of Foxp3+ Regulatory T Cells Is Driven by the c-Rel Enhanceosome

Regulatory T (Treg) cells are essential for maintaining immune homeostasis. Although Foxp3 expression marks the commitment of progenitors to Treg cell lineage, how Treg cells are generated during lymphocyte development remains enigmatic. We report here that the c-Rel transcription factor controlled development of Treg cells by promoting the formation of a Foxp3-specific enhanceosome. This enhanceosome contained c-Rel, p65, NFAT, Smad, and CREB. Although Smad and CREB first bound to Foxp3 enhancers, they later moved to the promoter to form the c-Rel enhanceosome. c-Rel-deficient mice had up to 90% reductions of Treg cells compared to wild-type mice, and c-Rel-deficient T cells were compromised in Treg cell differentiation. Thus, Treg cell development is controlled by a c-Rel enhanceosome, and strategies targeting Rel-NF-kappaB can be effective for manipulating Treg cell function.

IL‐4 is a differentiation factor for transforming growth factor‐β secreting Th3 cells and oral administration of IL‐4 enhances oral tolerance in experimental allergic encephalomyelitis

We have previously shown that following oral administration of myelin basic protein (MBP), regulatory T cells are generated from gut‐associated lymphoid tissue and that these cells suppress experimental allergic encephalomyelitis (EAE). These regulatory T cells produce transforming growth factor‐β (TGF‐β) with various amounts of IL‐4 and IL‐10 and these TGF‐β‐secreting T cells have been termed Th3 cells. T cells in lymphoid organs drained by mucosal sites secrete IL‐4 as a primary T cell growth factor. In the present study, we examined the role of IL‐4 on oral tolerance and in the generation of TGF‐β secreting cells. Treatment of (PLJ × SJL)F1 mice with intraperitoneal (i.  p.) IL‐4 and low‐dose oral MBP (0.5 mg) given three times reduced the severity of EAE, whereas i.  p. injection of IL‐4 alone or oral MBP alone given in these suboptimal doses, showed no protection. Spleen cells from protected mice produced increased amounts of TGF‐β and reduced IFN‐γ upon stimulation with MBP in vitro. Mucosal MBP‐specific IgA production was significantly increased in IL‐4 plus MBP fed animals. Moreover, oral administration of IL‐4 (1 μg per feeding) also enhanced the suppression of EAE by oral MBP and this protective effect was reversed by administration of anti‐TGF‐β antibody in vivo. Reverse transcription‐PCR showed enhanced suppression of IFN‐γ in Peyers patch in animals fed MBP and IL‐4 versus those fed MBP alone. We then investigated the role of IL‐4 in the generation of TGF‐β‐secreting cells using MBP Ac1‐11 TCR transgenic animals. Cells were cultured with IL‐2, IL‐4, or IFN‐γ in the presence of MBP and limiting dilution analysis for cytokine‐secreting cells performed. We found that IL‐4, but not IL‐2 or IFN‐γ, generated TGF‐β‐secreting T cells from naive splenic T cells and that these cells provided help for IgA production. These findings demonstrate that IL‐4 is a differentiation factor for TGF‐β‐secreting Th3 cells and oral IL‐4 has a synergistic effect on low‐dose oral tolerance that is associated with increased TGF‐β secretion.

Amelioration of collagen-induced arthritis by CD95 (Apo-1/Fas)-ligand gene transfer.

Both rheumatoid arthritis and animal models of autoimmune arthritis are characterized by hyperactivation of synovial cells and hyperplasia of the synovial membrane. The activated synovial cells produce inflammatory cytokines and degradative enzymes that lead to destruction of cartilage and bones. Effective treatment of arthritis may require elimination of most or all activated synovial cells. The death factor Fas/Apo-1 and its ligand (FasL) play pivotal roles in maintaining self-tolerance and immune privilege. Fas is expressed constitutively in most tissues, and is dramatically upregulated at the site of inflammation. In both rheumatoid arthritis and animal models of autoimmune arthritis, high levels of Fas are expressed on activated synovial cells and infiltrating leukocytes in the inflamed joints. Unlike Fas, however, the levels of FasL expressed in the arthritic joints are extremely low, and most activated synovial cells survive despite high levels of Fas expression. To upregulate FasL expression in the arthritic joints, we have generated a recombinant replication-defective adenovirus carrying FasL gene; injection of the FasL virus into inflamed joints conferred high levels of FasL expression, induced apoptosis of synovial cells, and ameliorated collagen-induced arthritis in DBA/1 mice. The Fas-ligand virus also inhibited production of interferon-gamma by collagen-specific T cells. Coadministration of Fas-immunoglobulin fusion protein with the Fas-ligand virus prevented these effects, demonstrating the specificity of the Fas-ligand virus. Thus, FasL gene transfer at the site of inflammation effectively ameliorates autoimmune disease.

The complement inhibitory protein DAF (CD55) suppresses T cell immunity in vivo

Decay-accelerating factor ([DAF] CD55) is a glycosylphosphatidylinositol-anchored membrane inhibitor of complement with broad clinical relevance. Here, we establish an additional and unexpected role for DAF in the suppression of adaptive immune responses in vivo. In both C57BL/6 and BALB/c mice, deficiency of the Daf1 gene, which encodes the murine homologue of human DAF, significantly enhanced T cell responses to active immunization. This phenotype was characterized by hypersecretion of interferon (IFN)-γ and interleukin (IL)-2, as well as down-regulation of the inhibitory cytokine IL-10 during antigen restimulation of lymphocytes in vitro. Compared with wild-type mice, Daf1−/− mice also displayed markedly exacerbated disease progression and pathology in a T cell–dependent experimental autoimmune encephalomyelitis (EAE) model. However, disabling the complement system in Daf1−/− mice normalized T cell secretion of IFN-γ and IL-2 and attenuated disease severity in the EAE model. These findings establish a critical link between complement and T cell immunity and have implications for the role of DAF and complement in organ transplantation, tumor evasion, and vaccine development.

Roles of TNF-Related Apoptosis-Inducing Ligand in Experimental Autoimmune Encephalomyelitis

TRAIL, the TNF-related apoptosis-inducing ligand, induces apoptosis of tumor cells, but not normal cells; the roles of TRAIL in nontransformed tissues are unknown. Using a soluble TRAIL receptor, we examined the consequences of TRAIL blockade in an animal model of multiple sclerosis. We found that chronic TRAIL blockade in mice exacerbated experimental autoimmune encephalomyelitis induced by myelin oligodendrocyte glycoprotein. The exacerbation was evidenced primarily by increases in disease score and degree of inflammation in the CNS. Interestingly, the degree of apoptosis of inflammatory cells in the CNS was not affected by TRAIL blockade, suggesting that TRAIL may not regulate apoptosis of inflammatory cells in experimental autoimmune encephalomyelitis. By contrast, myelin oligodendrocyte glycoprotein-specific Th1 and Th2 cell responses were significantly enhanced in animals treated with the soluble TRAIL receptor. Based on these observations, we conclude that unlike TNF, which promotes autoimmune inflammation, TRAIL inhibits autoimmune encephalomyelitis and prevents activation of autoreactive T cells.

TIPE2, a Negative Regulator of Innate and Adaptive Immunity that Maintains Immune Homeostasis

Immune homeostasis is essential for the normal functioning of the immune system, and its breakdown leads to fatal inflammatory diseases. We report here the identification of a member of the tumor necrosis factor-alpha-induced protein-8 (TNFAIP8) family, designated TIPE2, that is required for maintaining immune homeostasis. TIPE2 is preferentially expressed in lymphoid tissues, and its deletion in mice leads to multiorgan inflammation, splenomegaly, and premature death. TIPE2-deficient animals are hypersensitive to septic shock, and TIPE2-deficient cells are hyper-responsive to Toll-like receptor (TLR) and T cell receptor (TCR) activation. Importantly, TIPE2 binds to caspase-8 and inhibits activating protein-1 and nuclear factor-kappaB activation while promoting Fas-induced apoptosis. Inhibiting caspase-8 significantly blocks the hyper-responsiveness of TIPE2-deficient cells. These results establish that TIPE2 is an essential negative regulator of TLR and TCR function, and its selective expression in the immune system prevents hyperresponsiveness and maintains immune homeostasis.

Critical roles of c-Rel in autoimmune inflammation and helper T cell differentiation

Different members of the Rel/NF-kappaB family may play different roles in immunity and inflammation. We report here that c-Rel-deficient mice are resistant to autoimmune encephalomyelitis and are defective in Th1, but not Th2 responses. The Th1 deficiency appears to be caused by selective blockade of IL-12 production by c-Rel-deficient antigen-presenting cells, as well as by a complete abrogation of IFN-gamma expression in c-Rel-deficient T cells. Interestingly, c-Rel deficiency does not affect T-bet expression, suggesting that c-Rel may act downstream of T-bet during Th1 cell differentiation. Thus, unlike NF-kappaB1, which selectively regulates Th2 cell differentiation, c-Rel is essential for Th1 cell differentiation and Th1 cell-mediated autoimmune inflammation.

Critical roles of TRAIL in hepatic cell death and hepatic inflammation

The TNF-related apoptosis-inducing ligand (TRAIL) induces apoptosis of tumor cells but not most normal cells. Its role in hepatic cell death and hepatic diseases is not clear. In vitro studies suggest that murine hepatocytes are not sensitive to TRAIL-induced apoptosis, indicating that TRAIL may not mediate hepatic cell death. Using two experimental models of hepatitis, we found that hepatic cell death in vivo was dramatically reduced in TRAIL-deficient mice and mice treated with a blocking TRAIL receptor. Although both TRAIL and its death receptor 5 were constitutively expressed in the liver, TRAIL expression by immune cells alone was sufficient to restore the sensitivity of TRAIL-deficient mice to hepatitis. Thus, TRAIL plays a crucial role in hepatic cell death and hepatic inflammation.