Xueqiang Zhao

C-type Lectin Receptor Dectin-3 Mediates Trehalose 6,6′-Dimycolate (TDM)-induced Mincle Expression through CARD9/Bcl10/MALT1-dependent Nuclear Factor (NF)-κB Activation

Background: Recent studies suggest that Mincle expression is induced by Dectin-3-mediated signaling in response to TDM stimulation. Results: Deficiency in Dectin-3 and the CARD9-Bcl10-Malt1 complex are defective for TDM-induced NF-κB activation and Mincle. Conclusion: Dectin-3- and CARD9/Bcl10/Malt1-dependent NF-κB activation plays an essential role for TDM-induced Mincle expression. Significance: This study provides the molecular insight for designing adjuvants that stimulate the immune system. Previous studies indicate that both Dectin-3 (also called MCL or Clec4d) and Mincle (also called Clec4e), two C-type lectin receptors, can recognize trehalose 6,6′-dimycolate (TDM), a cell wall component from mycobacteria, and induce potent innate immune responses. Interestingly, stimulation of Dectin-3 by TDM can also induce Mincle expression, which may enhance the host innate immune system to sense Mycobacterium infection. However, the mechanism by which Dectin-3 induces Mincle expression is not fully defined. Here, we show that TDM-induced Mincle expression is dependent on Dectin-3-mediated NF-κB, but not nuclear factor of activated T-cells (NFAT), activation, and Dectin-3 induces NF-κB activation through the CARD9-BCL10-MALT1 complex. We found that bone marrow-derived macrophages from Dectin-3-deficient mice were severely defective in the induction of Mincle expression in response to TDM stimulation. This defect is correlated with the failure of TDM-induced NF-κB activation in Dectin-3-deficient bone marrow-derived macrophages. Consistently, inhibition of NF-κB, but not NFAT, impaired TDM-induced Mincle expression, whereas NF-κB, but not NFAT, binds to the Mincle promoter. Dectin-3-mediated NF-κB activation is dependent on the CARD9-Bcl10-MALT1 complex. Finally, mice deficient for Dectin-3 or CARD9 produced much less proinflammatory cytokines and keyhole limpet hemocyanin (KLH)-specific antibodies after immunization with an adjuvant containing TDM. Overall, this study provides the mechanism by which Dectin-3 induces Mincle expression in response to Mycobacterium infection, which will have significant impact to improve adjuvant and design vaccine for antimicrobial infection.

CARD9 mediates Dectin-1–induced ERK activation by linking Ras-GRF1 to H-Ras for antifungal immunity

CARD9 is dispensable for NF-κB activation induced by Dectin-1 ligands in mice. However, Dectin-1–induced H-Ras activation is mediated by a complex with CARD9, which leads to ERK activation for host innate immune responses to Candida albicans infection.

JNK1 negatively controls antifungal innate immunity by suppressing CD23 expression

Opportunistic fungal infections are a leading cause of death among immune-compromised patients, and there is a pressing need to develop new antifungal therapeutic agents because of toxicity and resistance to the antifungal drugs currently in use. Although C-type lectin receptor– and Toll-like receptor–induced signaling pathways are key activators of host antifungal immunity, little is known about the mechanisms that negatively regulate host immune responses to a fungal infection. Here we found that JNK1 activation suppresses antifungal immunity in mice. We showed that JNK1-deficient mice had a significantly higher survival rate than wild-type control mice in response to Candida albicans infection, and the expression of JNK1 in hematopoietic innate immune cells was critical for this effect. JNK1 deficiency leads to significantly higher induction of CD23, a novel C-type lectin receptor, through NFATc1-mediated regulation of the CD23 gene promoter. Blocking either CD23 upregulation or CD23-dependent nitric oxide production eliminated the enhanced antifungal response found in JNK1-deficient mice. Notably, JNK inhibitors exerted potent antifungal therapeutic effects in both mouse and human cells infected with C. albicans, indicating that JNK1 may be a therapeutic target for treating fungal infection.

Dectin-3 Deficiency Promotes Colitis Development due to Impaired Antifungal Innate Immune Responses in the Gut

Interactions between commensal fungi and gut immune system are critical for establishing colonic homeostasis. Here we found that mice deficient in Dectin-3 (Clec4d -/-), a C-type lectin receptor that senses fungal infection, were more susceptible to dextran sodium sulfate (DSS)-induced colitis compared with wild-type mice. The specific fungal burden of Candida (C.) tropicalis was markedly increased in the gut after DSS treatment in Clec4d -/- mice, and supplementation with C. tropicalis aggravated colitis only in Clec4d -/- mice, but not in wild-type controls. Mechanistically, Dectin-3 deficiency impairs phagocytic and fungicidal abilities of macrophages, and C. tropicalis-induced NF-κB activation and cytokine production. The conditioned media derived from Dectin-3-deficient macrophages were defective in promoting tissue repairing in colonic epithelial cells. Finally, anti-fungal therapy was effective in treating colitis in Clec4d -/- mice. These studies identified the role of Dectin-3 and its functional interaction with commensal fungi in intestinal immune system and regulation of colonic homeostasis.

Differential Regulation of c-Jun Protein Plays an Instrumental Role in Chemoresistance of Cancer Cells

Background: The mechanism underlying intrinsic tumor resistance to the widely used drug cisplatin remains elusive. Results: Cisplatin induced c-Jun down-regulation via cleavage and ubiquitylation-dependent degradation of c-Jun in CDDP-sensitive cancer cells and c-Jun up-regulation via activation of JNK2 in CDDP-resistant cancer cells. Conclusion: c-Jun plays an instrumental role in chemoresistance of cancer cells. Significance: c-Jun is a molecular target for improving cancer therapy. The chemotherapeutic drug cisplatin (cis-diamminedichloroplatinum(II) (CDDP)) is widely used in the treatment of human cancers. However, the mechanism underlying intrinsic tumor resistance to CDDP remains elusive. Here, we demonstrate that treatment with CDDP resulted in down-regulation of c-Jun expression via caspase-9-dependent cleavage of c-Jun at Asp-65 and MEKK1-mediated ubiquitylation and degradation of c-Jun in CDDP-sensitive cancer cells. In contrast, activation of JNK2 (but not JNK1) phosphorylated and up-regulated the expression of c-Jun in CDDP-resistant cells. Activated c-Jun bound to the promoter regions of the MDR1 gene and promoted the expression of MDR1. Expression of a cleavage-resistant c-Jun mutant (D65A) suppressed CDDP-induced apoptosis of CDDP-sensitive cells, whereas depletion of JNK2, c-Jun, or MDR1 in CDDP-resistant cancer cells promoted apoptosis upon CDDP treatment. In addition, mammary gland tumors induced by polyomavirus middle T antigen in JNK2−/− mice were more sensitive to CDDP compared with those in JNK2+/+ mice. These findings highlight the instrumental role of c-Jun in the resistance of tumors to treatment with CDDP and indicate that c-Jun is a molecular target for improving cancer therapy.

E3 ubiquitin ligase Cbl-b negatively regulates C-type lectin receptor–mediated antifungal innate immunity

Innate immune responses mediated by C-type lectin receptors Dectin-2 and Dectin-3 against fungal infections are negatively regulated by Cbl-b ubiquitination.

Regulation of linear ubiquitin chain assembly complex by Caspase-mediated cleavage of RNF31

ABSTRACT Cell death and survival signaling pathways have opposed but fundamental functions for various cellular processes and maintain cell homeostasis through cross talk. Here we report a novel mechanism of interaction between these two pathways through the cleavage of RNF31 by caspases. RNF31, a component of the linear ubiquitin chain assembly complex (LUBAC), regulates cell survival by inducing linear ubiquitination of NF-κB signaling components. We found that RNF31 is cleaved under apoptosis conditions through various stimulations. The effector caspases caspase 3 and caspase 6 are responsible for this event, and aspartates 348, 387, and 390 were identified as target sites for this cleavage. Cleavage of RNF31 suppressed its ability to activate NF-κB signaling; thus, mutation of cleavage sites inhibited the induction of apoptosis by treatment with tumor necrosis factor alpha (TNF-α). Our findings elucidate a novel regulatory loop between cell death and the survival signal and may provide guidance for the development of therapeutic strategies for cancers through the sensitization of tumor cells to death-inducing drugs.

CARD9S12N facilitates the production of IL-5 by alveolar macrophages for the induction of type 2 immune responses

The adaptor CARD9 functions downstream of C-type lectin receptors (CLRs) for the sensing of microbial infection, which leads to responses by the TH1 and TH17 subsets of helper T cells. The single-nucleotide polymorphism rs4077515 at CARD9 in the human genome, which results in the substitution S12N (CARD9S12N), is associated with several autoimmune diseases. However, the function of CARD9S12N has remained unknown. Here we generated CARD9S12N knock-in mice and found that CARD9S12N facilitated the induction of type 2 immune responses after engagement of CLRs. Mechanistically, CARD9S12N mediated CLR-induced activation of the non-canonical transcription factor NF-κB subunit RelB, which initiated production of the cytokine IL-5 in alveolar macrophages for the recruitment of eosinophils to drive TH2 cell–mediated allergic responses. We identified the homozygous CARD9 mutation encoding S12N in patients with allergic bronchopulmonary aspergillosis and revealed activation of RelB and production of IL-5 in peripheral blood mononuclear cells from these patients. Our study provides genetic and functional evidence demonstrating that CARD9S12N can turn alveolar macrophages into IL-5-producing cells and facilitates TH2 cell–mediated pathologic responses.CARD9 serves as an adaptor for C-type lectin receptor signaling. Xin Lin and colleagues show that CARD9 inhibits RelB-mediated IL-5 expression. The CARD9S12N mutant, prevalent in humans, cannot interact with RelB and promotes enhanced allergic responses to fungal pathogens.

Gain-of-Function Mutation of Card14 Leads to Spontaneous Psoriasis-like Skin Inflammation through Enhanced Keratinocyte Response to IL-17A

Summary Genetic mutations of CARD14 (encoding CARMA2) are observed in psoriasis patients. Here we showed that Card14E138A/+ and Card14&Dgr;Q136/+ mice developed spontaneous psoriasis‐like skin inflammation, which resulted from constitutively activated CARMA2 via self‐aggregation leading to the enhanced activation of the IL‐23‐IL‐17A cytokine axis. Card14−/− mice displayed attenuated skin inflammation in the imiquimod‐induced psoriasis model due to impaired IL‐17A signaling in keratinocytes. CARMA2, mainly expressed in keratinocytes, associates with the ACT1‐TRAF6 signaling complex and mediates IL‐17A‐induced NF‐&kgr;B and MAPK signaling pathway activation, which leads to expression of pro‐inflammatory factors. Thus, CARMA2 serves as a key mediator of IL‐17A signaling and its constitutive activation in keratinocytes leads to the onset of psoriasis, which indicates an important role of NF‐&kgr;B activation in keratinocytes in psoriatic initiation. Graphical Abstract Figure. No caption available. HighlightsCard14E138A/+ and Card14&Dgr;Q136/+ mice develop spontaneous psoriasis‐like phenotype&agr;&bgr; T cells, not &ggr;&dgr; T cells, are the main source of IL‐17A in Card14E138A/+ miceCard14 E138A mutation leads to CARMA2 aggregation and activationCARMA2 mediates IL‐17A signaling by interacting with the ACT1‐TRAF6 complex &NA; Mutations in the CARMA2 gene (CARD14) are identified in psoriasis patients, but whether and how these mutations initiate psoriasis is unclear. Wang et al. reveal that Card14E138A/+ mice develop spontaneous psoriasis via CARMA2 self‐aggregation and activation, and the deficiency or mutation of Card14 affects the activation of IL‐17A signaling in keratinocytes.

CARMA3 Is a Host Factor Regulating the Balance of Inflammatory and Antiviral Responses against Viral Infection

Host response to RNA virus infection is sensed by RNA sensors such as RIG-I, which induces MAVS-mediated NF-κB and IRF3 activation to promote inflammatory and antiviral responses, respectively. Here, we have found that CARMA3, a scaffold protein previously shown to mediate NF-κB activation induced by GPCR and EGFR, positively regulates MAVS-induced NF-κB activation. However, our data suggest that CARMA3 sequesters MAVS from forming high-molecular-weight aggregates, thereby suppressing TBK1/IRF3 activation. Interestingly, following NF-κB activation upon virus infection, CARMA3 is targeted for proteasome-dependent degradation, which releases MAVS to activate IRF3. When challenged with vesicular stomatitis virus or influenza A virus, CARMA3-deficient mice showed reduced disease symptoms compared to those of wild-type mice as a result of less inflammation and a stronger ability to clear infected virus. Altogether, our results reveal the role of CARMA3 in regulating the balance of host antiviral and pro-inflammatory responses against RNA virus infection.