IL-4-secreting CD40L+ MAIT cells support antibody production in the peripheral blood of Heonch-Schönlein purpura patients.

OBJECTIVE: Here, we explored the phenotype and function of MAIT cells in the peripheral blood of patients with HSP. METHODS: Blood samples from HSP patients and HDs were assessed by flow cytometry and single-cell RNA sequencing to analyze the proportion, phenotype, and function of MAIT cells. Th-cytokines in the serum of HSP patients were analyzed by CBA. IgA in cocultured supernatant was detected by CBA to analyze antibody production by B cells. RESULTS: The percentage of MAIT cells in HSP patients was significantly reduced compared with that in HDs. Genes related to T cell activation and effector were up-regulated in HSP MAIT cells, indicating a more activated phenotype. In addition, HSP MAIT cells displayed a Th2-like profile with the capacity to produce more IL-4 and IL-5, and IL-4 was correlated with IgA levels in the serum of HSP patients. Furthermore, CD40L was up-regulated in HSP MAIT cells, and CD40L+ MAIT cells showed an increased ability to produce IL-4 and to enhance IgA production by B cells. CONCLUSION: Our data demonstrate that MAIT cells in HSP patients exhibit an activated phenotype. The enhanced IL-4 production and CD40L expression of MAIT cells in HSP patients could take part in the pathogenesis of HSP.

Regulatory mucosa-associated invariant T cells controlled by ß1 adrenergic receptor signaling contribute to hepatocellular carcinoma progression.

BACKGROUND AND AIMS: The innate-like mucosa-associated invariant T (MAIT) cells are enriched in human liver and have been linked to human HCC. However, their contributions to the progression of HCC are controversial due to the heterogeneity of MAIT cells, and new MAIT cell subsets remain to be explored. APPROACH AND RESULTS: Combining single cell RNA sequencing (scRNA-seq) and flow cytometry analysis, we performed phenotypic and functional studies and found that FOXP3 + CXCR3 + MAIT cells in HCC patients were regulatory MAIT cells (MAITregs) with high immunosuppressive potential. These MAITregs were induced under Treg-inducing condition and predominantly from FOXP3 - CXCR3 + MAIT cells, which displayed mild Treg-related features and represented a pre-MAITreg reservoir. In addition, the induction and function of MAITregs were promoted by ß1 adrenergic receptor signaling in pre-MAITregs and MAITregs, respectively. In HCC patients, high proportion of the intratumoral MAITregs inhibited antitumor immune responses and was associated with poor clinical outcomes. CONCLUSIONS: Together, we reveal an immunosuppressive subset of MAIT cells in HCC patients that contributes to HCC progression, and propose a control through neuroimmune crosstalk.

The CARD8 T60 variant associates with NLRP1 and negatively regulates its activation.

The NLRP1 inflammasome functions as canonical cytosolic sensor in response to intracellular infections and is implicated in auto-inflammatory diseases. But the regulation and signal transduction mechanisms of NLRP1 are incompletely understood. Here, we show that the T60 variant of CARD8, but not the canonical T48 isoform, negatively regulates the NLRP1 inflammasome activation by directly interacting with the receptor molecule NLRP1 and inhibiting inflammasome assembly. Furthermore, our results suggest that different ASC preference in three types of inflammasomes, namely the ASC-indispensable NLRP1 inflammasome, ASC-dispensable mNLRP1b inflammasome and ASC-independent CARD8 inflammasome, is mainly caused by the CARD domain, not the UPA subdomain. Based on the systematic site-directed mutagenesis and structural analysis, we find that signal transduction of the NLRP1 inflammasome relies on multiple interaction surfaces at its CARD domain. Finally, our results partly explain how mutations in NLRP1 lead to its constitutive activation in auto-inflammatory diseases. In conclusion, our study not only reveals how CARD8 downregulates the NLRP1 inflammasome activation, but also provides insights into the assembly mechanisms of CARD-containing inflammasomes.

CD158a+ /CD158b+ NK cell imbalance correlates with hypertension in patients with pre-eclampsia.

PROBLEM: Preeclampsia, a pregnancy complication with hypertension and proteinuria, seriously threats the health and lives of the mother and the baby. The pathogenesis of pre-eclampsia remains incompletely understood. The role of peripheral natural killer cells (NK cells) in the pre-eclampsia is unclear. METHOD OF STUDY: Flow cytometry was performed to detect the expression of CD158a (KIR2DL1) and CD158b (KIR2DL2/3) in peripheral NK cells of healthy pregnant women (HP) and patients with pre-eclampsia (PE). Differentially expressed genes (DEGs) in CD158a+ and CD158b+ NK cells were identified by RNA-sequencing and real-time PCR. Protein array analysis was used to identify altered protein levels in the serum of study participants. RESULTS: CD158a+ NK cell numbers were increased in the peripheral blood of patients while the number of CD158b+ NK cells was reduced. In addition, the percentage of CD158a+ NK cells within the peripheral NK subset was positively correlated with systolic blood pressure while the percentage of CD158b+ NK cells was negatively correlated with systolic blood pressure. RNA-seq and real-time PCR showed that the expression of ERAP2 and GCH1, the genes that regulate blood pressure and angiogenesis, was decreased in CD158a+ compared to CD158b+ NK cells. Consistently, the level of proteins involved in angiogenesis was altered in the serum of pre-eclampsia patients compared to healthy individuals. CONCLUSIONS: CD158a+ NK cells increased while CD158b+ NK cells decreased in the peripheral blood of patients with pre-eclampsia compared to healthy individuals. The change in the frequency of CD158a+ /CD158b+ NK cells is related to the increase in blood pressure.

Homotypic CARD-CARD interaction is critical for the activation of NLRP1 inflammasome.

Cytosolic inflammasomes are supramolecular complexes that are formed in response to intracellular pathogens and danger signals. However, as to date, the detailed description of a homotypic caspase recruitment domain (CARD) interaction between NLRP1 and ASC has not been presented. We found the CARD-CARD interaction between purified NLRP1CARD and ASCCARD experimentally and the filamentous supramolecular complex formation in an in vitro proteins solution. Moreover, we determined a high-resolution crystal structure of the death domain fold of the human ASCCARD. Mutational and structural analysis revealed three conserved interfaces of the death domain superfamily (Type I, II, and III), which mediate the assembly of the NLRP1CARD/ASCCARD complex. In addition, we validated the role of the three major interfaces of CARDs in assembly and activation of NLRP1 inflammasome in vitro. Our findings suggest a Mosaic model of homotypic CARD interactions for the activation of NLRP1 inflammasome. The Mosaic model provides insights into the mechanisms of inflammasome assembly and signal transduction amplification.

Crystal structure of caspase-11 CARD provides insights into caspase-11 activation.

Murine caspase-11 is the centerpiece of the non-canonical inflammasome pathway that can respond to intracellular LPS and induce pyroptosis. Caspase-11 contains two components, an N-terminal caspase recruitment domain (CARD) and a C-terminal catalytic domain. The aggregation of caspase-11 is thought to promote the auto-processing and activation of caspase-11. However, the activation mechanism of caspase-11 remains unclear. In this study, we purified the caspase-11 CARD fused to an MBP tag and found it tetramerizes in solution. Crystallographic analysis reveals an extensive hydrophobic interface formed by the H1-2 helix mediating homotypic CARD interactions. Importantly, mutations of the helix H1-2 hydrophobic residues abolished the tetramerization of MBP-tagged CARD in solution and failed to induce pyroptosis in cells. Our study provides the first evidence of the homotypic interaction mode for an inflammatory caspase by crystal model. This finding demonstrates that the tetramerization of the N-terminal CARD can promote releasing of the catalytic domain auto-inhibition, leading to the caspase-11 activation.

Biochemical characterization of SARS-CoV-2 nucleocapsid protein.

The nucleocapsid (N) protein is an important antigen for coronavirus, which participate in RNA package and virus particle release. In this study, we expressed the N protein of SARS-CoV-2 and characterized its biochemical properties. Static light scattering, size exclusive chromatography, and small-angle X-ray scattering (SAXS) showed that the purified N protein is largely a dimer in solution. CD spectra showed that it has a high percentage of disordered region at room temperature while it was best structured at 55 °C, suggesting its structural dynamics. Fluorescence polarization assay showed it has non-specific nucleic acid binding capability, which raised a concern in using it as a diagnostic marker. Immunoblot assays confirmed the presence of IgA, IgM and IgG antibodies against N antigen in COVID-19 infection patients' sera, proving the importance of this antigen in host immunity and diagnostics.

Biochemical characterization of G64W mutant of acidic beta-crystallin 4.

Crystallins are structural proteins in the lens that last a lifetime with little turnover. Deviant in crystallins can cause rare but severe visual impairment, namely, congenital cataracts. It is reported that several mutations in the acidic ß-crystallin 4 (CRYBA4) are related to congenital cataracts. However, the pathogenesis of these mutants is not well understood at molecular level. Here we evaluate the biochemical properties of wild type CRYBA4 (CRYBA4WT) and a pathogenic G64W mutant (CRYBA4G64W) including protein folding, polymerization state and protein stability. Furthermore, we explore the differences in their interactions with α-crystallin A (CRYAA) and basic ß-crystallin 1 (CRYBB1) via yeast two-hybrid and pull-down assay in vitro, through which we find that G64W mutation leads to protein misfolding, decreases protein stability, blocks its interaction with CRYBB1 but maintains its interaction with CRYAA. Our results deepen our understanding of the pathogenesis of congenital cataracts.

Crystal structure and activation mechanism of DR3 death domain.

Death receptor 3 (DR3) (a.k.a. tumor necrosis factor receptor superfamily 25) plays a key role in the immune system by activating nuclear factor kappa-light-chain-enhancer of activated B cells signaling pathway. Here we present the crystal structures of human and mouse DR3 intracellular death domain (DD) at 2.7 and 2.5 Å resolutions, respectively. The mouse DR3 DD adopts a classical six-helix bundle structure while human DR3 DD displays an extended fold. Though there is one-amino-acid difference in the linker between maltose-binding protein (MBP) tag and DR3 DD, according to our self-interaction analysis, the hydrophobic interface discovered in MBP-hDR3 DD crystal structure is responsible for both hDR3 DD and mDR3 DD homotypic interaction. Furthermore, our biochemical analysis indicates that the sequence variation between human and mouse DR3 DD does not affect its structure and function. Small-angle X-ray scattering analysis shows the averaged solution structures of both human and mouse MBP-DR3 DD are the combination of different conformations with different proportion. Through switching to the open conformation, DR3 DD could improve the interaction with downstream element TNFR-associated death domain (TRADD). Here we propose an activation-dependent structural rearrangement model: the DD region is folded as the six-helix bundles in the resting state, while upon extracellular ligand engagement, it switches to the open conformation, which facilitates its self-association and the recruitment of TRADD. Our results provide detailed insights into the architecture of DR3 DD and the molecular mechanism of activation. DATABASES: All refined structure coordinates as well as the corresponding structure factors have been deposited in the PDB under the accession codes 5YGS, 5YEV, 5YGP, 5ZNY, 5ZNZ.

Chemokine receptor CXCR3 deficiency exacerbates murine autoimmune cholangitis by promoting pathogenic CD8+ T cell activation.

CXC Chemokine Receptor 3 (CXCR3) is functionally pleiotropic and not only plays an important role in chemotaxis, but also participates in T cell differentiation and may play a critical role in inducing and maintaining immune tolerance. These observations are particularly critical for autoimmune cholangitis in which CXCR3 positive T cells are found around the portal areas of both humans and mouse models of primary biliary cholangitis (PBC). Herein, we investigated the role of CXCR3 in the pathogenesis of autoimmune cholangitis. We have taken advantage of a unique CXCR3 knockout dnTGFßRII mouse to focus on the role of CXCR3, both by direct observation of its influence on the natural course of disease, as well as through adoptive transfer studies into Rag-/- mice. We report herein that not only do CXCR3 deficient mice develop an exacerbation of autoimmune cholangitis associated with an expanded effector memory T cell number, but also selective adoptive transfer of CXCR3 deficient CD8+ T cells induces autoimmune cholangitis. In addition, gene microarray analysis of CXCR3 deficient CD8+ T cells reveal an intense pro-inflammatory profile. Our data suggests that the altered gene profiles induced by CXCR3 deficiency promotes autoimmune cholangitis through pathogenic CD8+ T cells. These data have significance for human PBC and other autoimmune liver diseases in which therapeutic intervention might be directed to chemokines and/or their receptors.