Characterization of antifungal C-type lectin receptor expression on murine epithelial and endothelial cells in mucosal tissues.
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ABSTRACT:
SUBMITTER: Stappers MHT
PROVIDER: S-EPMC8593890 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:B cell linker protein (BLNK) is a pivotal adaptor protein that interfaces the B cell receptor (BCR)-associated spleen tyrosine kinase (Syk) to regulate B cell function and development. However, it is still not well understood whether BLNK is involved in Syk-coupled C-type lectin receptor (CLR) signaling in myeloid cells, particularly in the context of antifungal immunity. Here, we show that stimulation with fungi-derived β-glucans or α-mannans induced the phosphorylation of BLNK in macrophages, which was significantly impaired due to the deficiency of CLRs including Dectin-1 and Dectin-2, as well as their downstream mediators Syk and Fc-receptor gamma-chain (FcRγ). Furthermore, BLNK is induced to be interacted with casitas B-lineage lymphoma (c-Cbl), which is completely dependent on the engagement of Dectin-1 and Dectin-2. Notably, BLNK deficiency facilitates CLR-mediated recruitment of c-Cbl/phosphatidylinositol 3-kinase (PI3K) complex to F-actin cytoskeleton, thereby promoting macrophage migration. Consequently, mice with monocyte-specific deficiency of BLNK are highly resistant to the infection with Candida albicans, a major human fungal pathogen, through increasing the infiltration of Ly6C+macrophages into kidneys. Together, our data indicate that BLNK functions downstream of Syk-coupled CLRs to negatively regulate antifungal immunity against C. albicans infection. These findings unravel a previously unidentified role of BLNK that negatively regulates macrophage migration through inhibiting CLR-mediated recruitment of c-Cbl/PI3K complex to the cytoskeleton.
2023-11-07 | GSE246891 | GEO
Project description:Human CARD9 deficiency is an autosomal recessive primary immunodeficiency disorder caused by biallelic mutations in the gene CARD9, which encodes a signaling protein that is found downstream of many C-type lectin receptors (CLRs). CLRs encompass a large family of innate recognition receptors, expressed predominantly by myeloid and epithelial cells, which bind fungal carbohydrates and initiate antifungal immune responses. Accordingly, human CARD9 deficiency is associated with the spontaneous development of persistent and severe fungal infections that primarily localize to the skin and subcutaneous tissue, mucosal surfaces and/or central nervous system (CNS). In the last 3 years, more than 15 missense and nonsense CARD9 mutations have been reported which associate with the development of a wide spectrum of fungal infections caused by a variety of fungal organisms. The mechanisms by which CARD9 provides organ-specific protection against these fungal infections are now emerging. In this review, we summarize recent immunological and clinical advances that have provided significant mechanistic insights into the pathogenesis of human CARD9 deficiency. We also discuss how genetic mutations in CARD9-coupled receptors (Dectin-1, Dectin-2) and CARD9-binding partners (MALT1, BCL10) affect human antifungal immunity relative to CARD9 deficiency, and we highlight major understudied research questions which merit future investigation.
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Project description:BLNK negatively regulates C-type lectin receptor-mediated antifungal immunity through inhibiting macrophage migration
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Project description:Membrane glycoproteins play vital roles in many fundamental physiological and pathophysiological processes in the central nervous system and represent important targets for pharmaceuticals and biomarker discovery. However, their isolation and characterization have been greatly limited. Lectin affinity chromatography (LAC) has evolved as a powerful method to enrich glycoproteins in biofluid and cell/tissue lysate. However, its use in the hydrophobic fraction of the samples has rarely been explored. In this study, we have conducted a systematic investigation on the lectin binding efficiency in the presence of four commonly used detergents. We have found that, under certain concentrations, detergents can minimize nonspecific binding and facilitate the elution of hydrophobic glycoproteins. With the detergent assisted lectin affinity chromatography (DALAC), a total of 1491 proteins were identified with low numbers of false positives from two lectins. Proteins (699) were identified with at least two unique peptides, of which 219 are membrane glycoproteins. Compared to the traditional methods, the DALAC approach significantly increased the recovery of plasma membrane and glycoproteins. NP-40 is recommended as a well rounded detergent for DALAC, but the conditions for enriching certain target proteins need to be empirically determined. This study represents the first global identification of the murine brain glycoproteome.
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Project description:CD47 is a ubiquitously expressed transmembrane glycoprotein that regulates inflammatory responses and tissue repair. Here, we show that normal mice treated with anti-CD47 antibodies, and Cd47-null mice have impaired intestinal mucosal wound healing. Furthermore, intestinal epithelial cell (IEC)-specific loss of CD47 does not induce spontaneous immune-mediated intestinal barrier disruption but results in defective mucosal repair after biopsy-induced colonic wounding or Dextran Sulfate Sodium (DSS)-induced mucosal damage. In vitro analyses using primary cultures of CD47-deficient murine colonic IEC or human colonoid-derived IEC treated with CD47-blocking antibodies demonstrate impaired epithelial cell migration in wound healing assays. Defective wound repair after CD47 loss is linked to decreased epithelial ?1 integrin and focal adhesion signaling, as well as reduced thrombospondin-1 and TGF-?1. These results demonstrate a critical role for IEC-expressed CD47 in regulating mucosal repair and raise important considerations for possible alterations in wound healing secondary to therapeutic targeting of CD47.
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Project description:Numerous bacterial pathogens infect the mammalian host by initially associating with epithelial cells that line the intestinal lumen. Recent work has revealed that commensal bacteria that reside in the intestine promote defense against pathogenic infection, however whether the microbiota direct host pathways that alter pathogen adherence is not well-understood. Here, by comparing germ-free mice, we identify that the microbiota decrease bacterial pathogen adherence and dampen epithelial expression of the cell surface glycoprotein C-type lectin 2e (Clec2e). Functional studies revealed that overexpression of this lectin promotes adherence of intestinal bacterial pathogens to mammalian cells. Interestingly, microbiota-sensitive downregulation of Clec2e corresponds with decreased histone acetylation of the Clec2e gene in intestinal epithelial cells. Histone deacetylation and transcriptional regulation of Clec2e depends on expression and recruitment of the histone deacetylase HDAC3. Thus, commensal bacteria epigenetically instruct epithelial cells to decrease expression of a C-type lectin that promotes pathogen adherence, revealing a novel mechanism for how the microbiota promote innate defense against infection.
| S-EPMC6514056 | biostudies-literature
Project description:The C-type lectin-like receptors (CTLRs) play important roles in innate immunity as one type of pattern recognition receptors. Here, we cloned and characterized a C-type lectin-like receptor (LycCTLR) from large yellow croaker Larimichthys crocea. The full-length cDNA of LycCTLR is 880 nucleotides long, encoding a protein of 215 amino acids. The deduced LycCTLR contains a C-terminal C-type lectin-like domain (CTLD), an N-terminal cytoplasmic tail, and a transmembrane region. The CTLD of LycCTLR possesses six highly conserved cysteine residues (C1-C6), a conserved WI/MGL motif, and two sugar binding motifs, EPD (Glu-Pro-Asp) and WYD (Trp-Tyr-Asp). Ca(2+) binding site 1 and 2 were also found in the CTLD. The LycCTLR gene consists of five exons and four introns, showing the same genomic organization as tilapia (Oreochromis niloticus) and guppy (Poecilia retitculata) CTLRs. LycCTLR was constitutively expressed in various tissues tested, and its transcripts significantly increased in the head kidney and spleen after stimulation with inactivated trivalent bacterial vaccine. Recombinant LycCTLR (rLycCTLR) protein produced in Escherichia coli BL21 exhibited not only the hemagglutinating activity and a preference for galactose, but also the agglutinating activity against two food-borne pathogenic bacteria E. coli and Bacillus cereus in a Ca(2+)-dependent manner. These results indicate that LycCTLR is a potential galactose-binding C-type lectin that may play a role in the antibacterial immunity in fish.
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Project description:BackgroundCryopreservation of leukocytes isolated from the cervicovaginal and colorectal mucosa is useful for the study of cellular immunity (see Hughes SM et al. PLOS ONE 2016). However, some questions about mucosal biology and sexually transmitted infections are better addressed with intact mucosal tissue, for which there is no standard cryopreservation protocol.Methods and findingsTo find an optimal preservation protocol for mucosal tissues, we tested slow cooling (1°C/min) with 10% dimethylsulfoxide (designated "cryopreservation") and fast cooling (plunge in liquid nitrogen) with 20% dimethylsulfoxide and 20% ethylene glycol ("vitrification"). We compared fresh and preserved human cervicovaginal and colorectal tissues in a range of assays, including metabolic activity, human immunodeficiency virus infection, cell phenotype, tissue structure by hematoxylin-and-eosin staining, cell number and viability, production of cytokines, and microbicide drug concentrations. Metabolic activity, HIV infectability, and tissue structure were similar in cryopreserved and vitrified vaginal tissues. However, vitrification led to poor cell recovery from the colorectal mucosa, with 90% fewer cells recovered after isolation from vitrified colorectal tissues than from cryopreserved. HIV infection rates were similar for fresh and cryopreserved ectocervical tissues, whereas cryopreserved colorectal tissues were less easily infected than fresh tissues (hazard ratio 0.7 [95% confidence interval 0.4, 1.2]). Finally, we compared isolation of cells before and after cryopreservation. Cell recoveries were higher when cells were isolated after freezing and thawing (71% [59-84%]) than before (50% [38-62%]). Cellular function was similar to fresh tissue in both cases. Microbicide drug concentrations were lower in cryopreserved explants compared to fresh ones.ConclusionsCryopreservation of intact cervicovaginal and colorectal tissues with dimethylsulfoxide works well in a range of assays, while the utility of vitrification is more limited. Cell yields are higher from cryopreserved intact tissue pieces than from thawed cryopreserved single cell suspensions isolated before freezing, but T cell functions are similar.
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Project description:TNF plays an integral role in inflammatory bowel disease (IBD), as evidenced by the dramatic therapeutic responses in Crohn's disease (CD) patients induced by chimeric anti-TNF mAbs. However, treatment of CD patients with etanercept, a decoy receptor that binds soluble TNF, fails to improve disease. To explore this discrepancy, we investigated the role of TNF signaling in Wnt/?-catenin-mediated intestinal stem cell and progenitor cell expansion in CD patients, human cells, and preclinical mouse models. We hypothesized that TNF exerts beneficial effects on intestinal epithelial cell (IEC) responses to injury. In CD patients, intestinal stem cell and progenitor cell Wnt/?-catenin signaling correlates with inflammation status. TNF-deficient (Tnf-/-) mice exhibited increased apoptosis, less IEC proliferation, and less Wnt signaling when stimulated with anti-CD3 mAb. Bone marrow (BM) chimera mice revealed that mucosal repair depended on TNF production by BM-derived cells and TNFR expression by radioresistant IECs. Wild-type?Tnfr1/2-/- BM chimera mice with chronic dextran sodium sulfate colitis exhibited delayed ulcer healing, more mucosal inflammation, and impaired Wnt/?-catenin signaling, consistent with the hypothesis that epithelial TNFR signaling participates in mucosal healing. The direct effect of TNF on stem cells was demonstrated by studies of TNF-induced Wnt/?-catenin target gene expression in murine enteroids and colonoid cultures and TNF-induced ?-catenin activation in nontransformed human NCM460 cells (TOPFlash) and mice (TOP-GAL). Together, these data support the hypothesis that TNF plays a beneficial role in enhancing Wnt/?-catenin signaling during ulcer healing in IBD. These novel findings will inform clinicians and therapeutic chemists alike as they strive to develop novel therapies for IBD patients.
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Project description:Fibrinogen C domain containing 1 (FIBCD1) is a transmembrane receptor that binds chitin and other acetylated compounds with high affinity. FIBCD1 has previously been shown to be present in the epithelium of the gastrointestinal tract. In the present study, we performed a detailed analysis of normally structured human tissues for the expression of FIBCD1 by quantitative PCR and immunohistochemistry. We find that FIBCD1 is expressed in epithelial cells derived from all three germ layers. Endodermal-derived epithelial cells throughout the gastrointestinal tract and the respiratory system showed high expression of FIBCD1 and also mesodermal-derived cells in the genitourinary system and ectodermal-derived epidermis and sebaceous glands cells expressed FIBCD1. In some columnar epithelial cells, for example, in the salivary gland and gall bladder, the FIBCD1 expression was clearly polarized with strong apical reaction, while other columnar cells, for example, in small and large intestine and in bronchi, the staining was equally strong apically and basolaterally. In keratinocytes in skin, tongue, and oral cavity, the FIBCD1 staining was granular. This expression pattern together with the known binding properties supports that FIBCD1 plays a role in innate immunity in the skin and at mucosal surfaces.
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