Direction of leukocyte polarization and migration by the phosphoinositide-transfer protein TIPE2.

The polarization of leukocytes toward chemoattractants is essential for the directed migration (chemotaxis) of leukocytes. How leukocytes acquire polarity after encountering chemical gradients is not well understood. We found here that leukocyte polarity was generated by TIPE2 (TNFAIP8L2), a transfer protein for phosphoinositide second messengers. TIPE2 functioned as a local enhancer of phosphoinositide-dependent signaling and cytoskeleton remodeling, which promoted leading-edge formation. Conversely, TIPE2 acted as an inhibitor of the GTPase Rac, which promoted trailing-edge polarization. Consequently, TIPE2-deficient leukocytes were defective in polarization and chemotaxis, and TIPE2-deficient mice were resistant to leukocyte-mediated neural inflammation. Thus, the leukocyte polarizer is a dual-role phosphoinositide-transfer protein and represents a potential therapeutic target for the treatment of inflammatory diseases.

Epithelial TIPE1 Protein Guards against Colitis by Inhibiting TNF-α-Mediated Inflammation.

Intestinal epithelial cells (IECs) at the internal/external interface orchestrate the mucosal immune response, and IEC dysfunction has been linked to multiple inflammatory diseases, including inflammatory bowel disease. In this study, we found that a member of the TNF-α-induced protein 8 (TNFAIP8 or TIPE) family called TIPE1 is indispensable for maintaining epithelial cell barrier integrity and homeostasis under inflammatory conditions. TIPE1-deficient mice, or chimeric mice that were deficient in TIPE1 in their nonhematopoietic cells, were more sensitive to dextran sulfate sodium-induced experimental colitis; however, TIPE1 deficiency had no impact on the development of inflammation-associated and sporadic colorectal cancers. Mechanistically, TIPE1 prevented experimental colitis through modulation of TNF-α-dependent inflammatory response in IECs. Importantly, genetic deletion of both TIPE1 and its related protein TNFAIP8 in mice led to the development of spontaneous chronic colitis, indicating that both of these two TIPE family members play crucial roles in maintaining intestinal homeostasis. Collectively, our findings highlight an important mechanism by which TIPE family proteins maintain intestinal homeostasis and prevent inflammatory disorders in the gut.

Exosomal PD-L1 contributes to immunosuppression and is associated with anti-PD-1 response.

Tumour cells evade immune surveillance by upregulating the surface expression of programmed death-ligand 1 (PD-L1), which interacts with programmed death-1 (PD-1) receptor on T cells to elicit the immune checkpoint response1,2. Anti-PD-1 antibodies have shown remarkable promise in treating tumours, including metastatic melanoma2-4. However, the patient response rate is low4,5. A better understanding of PD-L1-mediated immune evasion is needed to predict patient response and improve treatment efficacy. Here we report that metastatic melanomas release extracellular vesicles, mostly in the form of exosomes, that carry PD-L1 on their surface. Stimulation with interferon-γ (IFN-γ) increases the amount of PD-L1 on these vesicles, which suppresses the function of CD8 T cells and facilitates tumour growth. In patients with metastatic melanoma, the level of circulating exosomal PD-L1 positively correlates with that of IFN-γ, and varies during the course of anti-PD-1 therapy. The magnitudes of the increase in circulating exosomal PD-L1 during early stages of treatment, as an indicator of the adaptive response of the tumour cells to T cell reinvigoration, stratifies clinical responders from non-responders. Our study unveils a mechanism by which tumour cells systemically suppress the immune system, and provides a rationale for the application of exosomal PD-L1 as a predictor for anti-PD-1 therapy.

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.

The SCFß-TrCP E3 Ubiquitin Ligase Regulates Immune Receptor Signaling by Targeting the Negative Regulatory Protein TIPE2.

TNFAIP8-like 2 (TIPE2) is a negative regulator of immune receptor signaling that maintains immune homeostasis. Dysregulated TIPE2 expression has been observed in several types of human immunological disorders. However, how TIPE2 expression is regulated remains to be determined. We report in this study that the SCFß-TrCP E3 ubiquitin ligase regulates TIPE2 protein abundance by targeting it for ubiquitination and subsequent degradation via the 26S proteasome. Silencing of either cullin-1 or ß-TrCP1 resulted in increased levels of TIPE2 in immune cells. TAK1 phosphorylated the Ser3 in the noncanonical degron motif of TIPE2 to trigger its interaction with ß-TrCP for subsequent ubiquitination and degradation. Importantly, the amount of TIPE2 protein in immune cells determined the strength of TLR 4-induced signaling and downstream gene expression. Thus, our study has uncovered a mechanism by which SCFß-TrCP E3 ubiquitin ligase regulates TLR responses.

Caspase-8 promotes c-Rel-dependent inflammatory cytokine expression and resistance against Toxoplasma gondii.

Caspase-8 is a key integrator of cell survival and cell death decisions during infection and inflammation. Following engagement of tumor necrosis factor superfamily receptors or certain Toll-like receptors (TLRs), caspase-8 initiates cell-extrinsic apoptosis while inhibiting RIPK3-dependent programmed necrosis. In addition, caspase-8 has an important, albeit less well understood, role in cell-intrinsic inflammatory gene expression. Macrophages lacking caspase-8 or the adaptor FADD have defective inflammatory cytokine expression and inflammasome priming in response to bacterial infection or TLR stimulation. How caspase-8 regulates cytokine gene expression, and whether caspase-8-mediated gene regulation has a physiological role during infection, remain poorly defined. Here we demonstrate that both caspase-8 enzymatic activity and scaffolding functions contribute to inflammatory cytokine gene expression. Caspase-8 enzymatic activity was necessary for maximal expression of Il1b and Il12b, but caspase-8 deficient cells exhibited a further decrease in expression of these genes. Furthermore, the ability of TLR stimuli to induce optimal IκB kinase phosphorylation and nuclear translocation of the nuclear factor kappa light chain enhancer of activated B cells family member c-Rel required caspase activity. Interestingly, overexpression of c-Rel was sufficient to restore expression of IL-12 and IL-1ß in caspase-8-deficient cells. Moreover, Ripk3-/-Casp8-/- mice were unable to control infection by the intracellular parasite Toxoplasma gondii, which corresponded to defects in monocyte recruitment to the peritoneal cavity, and exogenous IL-12 restored monocyte recruitment and protection of caspase-8-deficient mice during acute toxoplasmosis. These findings provide insight into how caspase-8 controls inflammatory gene expression and identify a critical role for caspase-8 in host defense against eukaryotic pathogens.

The anti-inflammatory TIPE2 is an inhibitor of the oncogenic Ras.

The connection between cancer and inflammation is widely recognized, yet the underlying molecular mechanisms are poorly understood. We report here that TIPE2 provides a molecular bridge from inflammation to cancer by targeting the Ras signaling pathway. TIPE2 binds the Ras-interacting domain of the RalGDS family of proteins, which are essential effectors of activated Ras. This binding prevented Ras from forming an active complex, thereby inhibiting the activation of the downstream signaling molecules Ral and AKT. Consequently, TIPE2 deficiency led to heightened activation of Ral and AKT, resistance to cell death, increased migration, and dysregulation of exocyst complex formation. Conversely, TIPE2 overexpression induced cell death and significantly inhibited Ras-induced tumorigenesis in mice. Importantly, TIPE2 expression was either completely lost or significantly downregulated in human hepatic cancer. Thus, TIPE2 is an inhibitor of both inflammation and cancer, and a potential drug target for inflammatory and neoplastic 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.

Silencing c-Rel in macrophages dampens Th1 and Th17 immune responses and alleviates experimental autoimmune encephalomyelitis in mice.

Autoimmune Th1 and Th17 responses are critical for the development of central nervous system (CNS) pathology in experimental autoimmune encephalomyelitis (EAE), an animal model for human multiple sclerosis. Although macrophages play important roles in the development of Th1 and Th17 responses, whether modulating macrophage gene transcription can diminish the Th1- and Th17 cell-induced CNS pathology is unclear. In this study, we successfully silenced the expression of the transcription factor c-Rel in macrophages of mice with EAE (including those infiltrating the CNS) using chemically modified c-Rel-specific siRNAs delivered by nanoparticles. Knocking down c-Rel in macrophages in vitro inhibited expression of NF-κB targets, such as pro-inflammatory cytokines interleukin 1ß (IL-1ß) and p40 of interleukin 12 (IL-12)/interleukin 23 (IL-23), in macrophages, leading to reduced interferon γ (IFN-γ) and interleukin 17A (IL-17A) production by co-cultured MOG-specific T cells from EAE mice. Such effects correlated with diminished T-cell infiltration in the CNS, reduced clinical symptoms, as well as downregulated pathogenic Th1 and Th17 responses in EAE mice. Taken together, our findings indicate that targeting c-Rel in macrophages dampens CNS-specific Th1 and Th17 immune responses, and can be effective for treating autoimmune diseases of the CNS.

Exacerbated experimental colitis in TNFAIP8-deficient mice.

The TNF-α-induced protein 8 (TNFAIP8 or TIPE) is a risk factor for cancer and bacterial infection, and its expression is upregulated in a number of human cancers. However, its physiologic and pathologic functions are unclear. In this study, we describe the generation of TIPE-deficient mice and their increased sensitivity to colonic inflammation. TIPE-deficient mice were generated by germ line gene targeting and were born without noticeable developmental abnormalities. Their major organs, including lymphoid organs and intestines, were macroscopically and microscopically normal. However, after drinking dextran sodium sulfate-containing water, TIPE-deficient mice developed more severe colitis than wild type mice did, as demonstrated by decreased survival rates, increased body weight loss, and enhanced leukocyte infiltration, bacterial invasion, and inflammatory cytokine production in the colon. Bone marrow chimeric experiments revealed that TIPE deficiency in nonhematopoietic cells was responsible for the exacerbated colitis in TIPE-deficient mice. Consistent with this result, TIPE-deficient intestinal epithelial cells had increased rate of cell death and decreased rate of proliferation as compared with wild type controls. These findings indicate that TIPE plays an important role in maintaining colon homeostasis and in protecting against colitis.

Crucial roles of TNFAIP8 protein in regulating apoptosis and Listeria infection.

TNF-α-induced protein 8 (TNFAIP8 or TIPE) is a newly described regulator of cancer and infection. However, its precise roles and mechanisms of actions are not well understood. We report in this article that TNFAIP8 regulates Listeria monocytogenes infection by controlling pathogen invasion and host cell apoptosis in a RAC1 GTPase-dependent manner. TNFAIP8-knockout mice were resistant to lethal L. monocytogenes infection and had reduced bacterial load in the liver and spleen. TNFAIP8 knockdown in murine liver HEPA1-6 cells increased apoptosis, reduced bacterial invasion into cells, and resulted in dysregulated RAC1 activation. TNFAIP8 could translocate to plasma membrane and preferentially associate with activated RAC1-GTP. The combined effect of reduced bacterial invasion and increased sensitivity to TNF-α-induced clearance likely protected the TNFAIP8-knockout mice from lethal listeriosis. Thus, by controlling bacterial invasion and the death of infected cells through RAC1, TNFAIP8 regulates the pathogenesis of L. monocytogenes infection.

TIPE2 protein prevents injury-induced restenosis in mice.

Proliferation of vascular smooth muscle cells (VSMCs) plays an important role in restenosis, a disease characterized by smooth muscle cell hyperplasia and neointimal formation. How proliferation signals are controlled to avoid restenosis is not fully understood. Here we report that TIPE2, the tumor necrosis factor (TNF) α-induced protein 8-like 2 (TNFAIP8L2), suppresses injury-induced restenosis by inhibiting VSMCs proliferation. TIPE2 was significantly upregulated in VSMCs in response to PDGF-BB stimuli and injury. Enforced TIPE2 expression significantly suppressed VSMCs proliferation and cell cycle progression, whereas TIPE2 deficiency in VSMCs promoted cell proliferation and upregulated the expression of Cyclins D1 and D3. TIPE2 likely regulated VSMC proliferation via Rac1-STAT3 and ERK1/2 signaling pathways. It blocked STAT3 activation and nuclear translocation in a Rac1-dependent manner. As a result, TIPE2-deficient VSMCs exhibited enhanced proliferation whereas TIPE2-deficient mice developed more severe restenosis in response to vascular injury. Conversely, adenovirus-mediated gene transfer of TIPE2 significantly reduced injury-induced restenosis in mice. These results indicate that TIPE2 plays a suppressive role in injury-induced restenosis and may serve as a new therapeutic target for treating the disease.

Treating psoriasis by targeting its susceptibility gene Rel.

Psoriasis is a chronic inflammatory disorder of the skin. Accumulating evidence indicates that the Rel gene, a member of the NF-κB family, is a risk factor for the disease. We sought to investigate whether psoriasis can be prevented by directly targeting the Rel gene transcript, i.e., the c-Rel mRNA. Using chemically-modified c-Rel specific siRNA (siRel) and poly(ethylene glycol)-b-poly(l-lysine)-b-poly(l-leucine) (PEG-PLL-PLLeu) micelles, we successfully knocked down the expression of c-Rel, and showed that the expression of cytokine IL-23, a direct target of c-Rel that can drive the development of IL-17-producing T cells, was markedly inhibited. More importantly, treating mice with siRel not only prevented but also ameliorated imiquimod (IMQ)-induced psoriasis. Mechanistic studies showed that siRel treatment down-regulated the expression of multiple inflammatory cytokines. Taken together, these results indicate that the susceptibility gene Rel can be targeted to treat and prevent psoriasis.

Hepatitis C virus promotes hepatocellular carcinogenesis by targeting TIPE2, a new regulator of DNA damage response.

Infection of hepatitis C virus (HCV) is associated with primary hepatocellular carcinoma (HCC). However, its underlying molecular mechanisms remain enigmatic. Tumor necrosis factor-α-induced protein 8-like 2 (TIPE2), a new negative regulator of immunity, plays significant roles in modulating inflammation and tumorigenesis. We hypothesized that TIPE2 might be involved in the development of HCV-induced HCC. To test this hypothesis, the expression of TIPE2 was determined by Western blot in the tumor and pericarcinomatous tissues collected from ten HCV-positive HCC patients; the interaction between TIPE2 and HCV-encoded non-structural proteins was analyzed by immunoprecipitation and immunofluorescence assays, and tumorigenesis and its mechanisms were studied in cell models and nude mice. Our results demonstrated that the expression of TIPE2 was significantly reduced in HCC tissues compared to that in the paracarcinoma tissues. HCV-encoded non-structural protein NS5A could specifically interact with TIPE2 and induce its degradation. Downregulation of TIPE2 by shRNA in cell lines increased genomic DNA damage and promoted cell colony formation in vitro and tumorigenesis in nude mice. In contrast, overexpression of TIPE2 had an opposite effect. Downregulation of TIPE2 by NS5A is associated with genomic DNA instability and HCV-induced HCC development. Thus, TIPE2 may be a new therapeutic target for the treatment of HCV-associated HCC.

c-Rel is Required for the Induction of pTregs in the Eye but Not in the Gut Mucosa.

Regulatory T (Treg) cells play an integral role in maintaining immune homeostasis and preventing autoimmune diseases. Forkhead box P3 expression marks the commitment of progenitor cells to the Treg lineage. Although the essential function of the nuclear factor (NF)-κB family transcription factor c-Rel in the regulation of natural Treg cells has been firmly established, little is known about whether c-Rel is involved in the in vivo generation of peripheral Treg cells (pTregs), which develop from mature CD4+ conventional T cells outside of the thymus. We sought to answer this question through the induction of pTregs in the eye and gut mucosa using ovalbumin-specific T cell receptor transgenic mice that do or do not express c-Rel. Our results showed that Tregs can be induced in the eye in a c-Rel-dependent manner when immune-mediated inflammation occurs. However, c-Rel is dispensable for the induction of pTregs in the gut mucosa after oral antigen administration. Thus, c-Rel may play distinct roles in regulating the development of pTregs in different organs. Abbreviations ACAID: Anterior Chamber-Associated Immune Deviation; ATF: activating transcription factor; CREB: cAMP responsive element-binding protein; DMEM: Dulbecco minimum essential medium; HBSS: Hanks Balanced Salt Solution; NFAT: Nuclear Factor of Activated T cells; PBS: Phosphate-buffered saline; PE: Phycoerythrin; WT: wild type.

Critical roles of TIPE2 protein in murine experimental colitis.

Both commensal bacteria and infiltrating inflammatory cells play essential roles in the pathogenesis of inflammatory bowel disease. The molecular mechanisms whereby these pathogenic factors are regulated during the disease are not fully understood. We report in this article that a member of the TNF-α-induced protein 8 (TNFAIP8) family called TIPE2 (TNFAIP8-like 2) plays a crucial role in regulating commensal bacteria dissemination and inflammatory cell function in experimental colitis induced by dextran sodium sulfate (DSS). Following DSS treatment, TIPE2-deficient mice, or chimeric mice that are deficient in TIPE2 only in their hematopoietic cells, lost less body weight and survived longer than wild-type controls. Consistent with this clinical observation, TIPE2-deficient mice exhibited significantly less severe colitis and colonic damage. This was associated with a marked reduction in the colonic expression of inflammatory cytokines, such as TNF-α, IL-6, and IL-12. Importantly, the ameliorated DSS-induced colitis in TIPE2(-/-) mice also was associated with reduced local dissemination of commensal bacteria and a weaker systemic inflammatory response. Combined with our previous report that TIPE2 is a negative regulator of antibacterial immunity, these results indicate that TIPE2 promotes colitis by inhibiting mucosal immunity to commensal bacteria.

Enhanced atherosclerosis in TIPE2-deficient mice is associated with increased macrophage responses to oxidized low-density lipoprotein.

Atherosclerosis has been widely recognized as an inflammatory disease of the arterial wall in which macrophages play a major role. Yet, how macrophage-mediated pathology is regulated during atherosclerosis is poorly understood. TNF-α-induced protein 8-like 2 (TIPE2, also known as TNFAIP8L2) is highly expressed in resting macrophages and can negatively regulate inflammation through inhibiting immune receptor signaling. We report in this article that TIPE2 plays a crucial atheroprotective role likely by regulating macrophage responses to oxidized low-density lipoprotein (ox-LDL). TIPE2-deficient macrophages treated with ox-LDL produced more oxidative stress and proinflammatory cytokines, and exhibited heightened activation of the JNK, NF-κB, and p38 signaling pathways. As a consequence, TIPE2 deficiency in bone marrow-derived cells exacerbated atherosclerosis development in Ldlr(-/-) mice fed a high-fat diet. Importantly, ox-LDL markedly downregulated TIPE2 mRNA and protein levels in macrophages, suggesting that ox-LDL mediates atherosclerosis by TIPE2 inhibition. These results indicate that TIPE2 is a new inhibitor of atherosclerosis and a potential drug target for treating the disease.

TIPE2 protein serves as a negative regulator of phagocytosis and oxidative burst during infection.

Phagocytosis and oxidative burst are two major effector arms of innate immunity. Although it is known that both are activated by Toll-like receptors (TLRs) and Rac GTPases, how their strengths are controlled in quiescent and TLR-activated cells is not clear. We report here that TIPE2 (TNFAIP8L2) serves as a negative regulator of innate immunity by linking TLRs to Rac. TLRs control the expression levels of TIPE2, which in turn dictates the strengths of phagocytosis and oxidative burst by binding to and blocking Rac GTPases. Consequently, TIPE2 knockout cells have enhanced phagocytic and bactericidal activities and TIPE2 knockout mice are resistant to bacterial infection. Thus, TIPE2 sets the strengths of phagocytosis and oxidative burst and may be targeted to effectively control infections.

Nuclear factor-κB binding motifs specify Toll-like receptor-induced gene repression through an inducible repressosome.

Sustained Toll-like receptor (TLR) stimulation continuously activates antimicrobial genes but paradoxically represses inflammatory genes. This phenomenon, termed TLR tolerance, is essential for preventing fatal inflammatory conditions such as sepsis, but its underlying mechanisms are unclear. We report here that NF-κB binding nucleic acids of gene promoters are tolerogenic motifs, which selectively recruit an NcoR-Hdac3-deacetylated-p50 repressosome to inflammatory genes. Genome-wide analyses of TLR4-induced genes revealed that NF-κB motifs were the only regulatory elements significantly enriched in tolerizable genes. Mutating the NF-κB motifs of tolerizable genes converted them into nontolerizable ones, whereas inserting NF-κB binding motifs into nontolerizable genes conferred the tolerance. Although NF-κB p50 was essential for assembling the repressosome, genetic disruption of the NcoR-Hdac3 interaction alone was sufficient to completely abolish TLR4 tolerance and to render mice vulnerable to sepsis. Thus, the specificity of TLR tolerance is dictated by evolutionally conserved nucleic acid motifs that bound by NF-κB and the NcoR repressosome.

TIPE2 controls innate immunity to RNA by targeting the phosphatidylinositol 3-kinase-Rac pathway.

RNA receptors such as TLR3 and retinoid acid-inducible gene I/melanoma differentiation-associated gene 5 play essential roles in innate immunity to RNA viruses. However, how innate immunity to RNAs is controlled at the molecular level is not well understood. We describe in this study a new regulatory pathway of anti-RNA immunity that is composed of PI3K, its target GTPase Rac, and the newly described immune regulator TNF-α-induced protein 8 like-2 (TIPE2, or TNFAIP8L2). Polyinosinic-polycytidylic acid [Poly (I:C)], a dsRNA receptor ligand, activates Rac via its guanine nucleotide exchange factor Tiam; this leads to the activation of cytokine genes and, paradoxically, downregulation of the Tipe2 gene. TIPE2 is a negative regulator of immunity; its deficiency leads to hyperactivation of the PI3K-Rac pathway as exemplified by enhanced AKT, Rac, P21-activated kinase, and IFN regulatory factor 3 activities. As a consequence, TIPE2 knockout myeloid cells are hyperreactive to Poly (I:C) stimulation, and TIPE2 knockout mice are hypersensitive to Poly (I:C)-induced lethality. These results indicate that TIPE2 controls innate immunity to RNA by targeting the PI3K-Rac pathway. Therefore, manipulating TIPE2 or Rac functions can be effective for controlling RNA viral infections.