Biology subjects

Girish, N.

Publications and source records attributed to Girish, N..

5 recordsLinked to original sources

bioRxiv2025

TNF signaling drives expansion of Reg4+ epithelial cells in colitis.

Reg4+ secretory cells are upregulated in colitis and promote epithelial repair, but their regulation is poorly understood. We show that TNF-TNFR1 signaling controls Reg4+ cell numbers in mouse and human colonoids and in vivo, with TNFR1 deficiency reducing these cells and TNF restoring them dose-dependently. In UC patients and DSS-colitis, REG4+ cells mark regenerating crypts, and persistent REG4, DUOX2, and TNFR1 expression identifies non-responders to anti-TNF therapy. These findings reveal a TNFR1-dependent mechanism regulating Reg4+ cells and suggest potential strategies to improve therapeutic responses in IBD.

cell biology↗

bioRxiv2025

TNF Receptor 1 regulates colonic mesenchymal cell diversity and the epithelial stem cell niche.

BACKGROUND & AIMSAnti-tumor necrosis factor (anti-TNF) is a mainstay of inflammatory bowel disease (IBD) therapy but fails in many patients. Although TNF has pro-inflammatory effects, depletion of TNF receptor 1 (TNFR1) paradoxically exacerbates chronic colitis. Because colitis induces remodeling of mesenchymal cell populations, which provide a niche for epithelial stem cells involved in mucosal healing, we hypothesized that TNFR1 promotes colonic mesenchymal cell diversity and stem cell niche function. METHODSMesenchymal TNFR1 function was studied using TNFR1-/-, platelet derived growth factor receptor alpha (PDGFR)-Cre;TNFR1fl/fl mice, and mixed-genotype mesenchymal-epithelial co-cultures. Mesenchymal cell diversity and gene function were assessed using single-cell RNA-Seq of primary colonic myofibroblasts (CMFs) and via anti-integrin A6 (ITGA6) antibody treatment and exogenous R-spondin 3 (RSPO3) supplementation. RESULTSTNFR1-/- mesenchyme exhibits reduced cell diversity, with specific depletion of specialized TNF- and interferon-signaling pericryptal cell-type. Deletion of TNFR1 in the pericryptal mesenchyme diminished the (PDGFR)+ CMF population and reduced RSPO3 expression, but increased ITGA6 expression relative to controls (TNFR1+/-). Moreover, inhibition of ITGA6 reversed the proliferative and migratory phenotype of TNFR1-/- CMFs and restored expression of PDGFR and RSPO3. Co-cultures of colonoids with TNFR1-/- CMFs resulted in downregulation of stem cell marker expression; this was rescued by supplementation with RSPO3. Supporting the role for mesenchymal TNFR1 in regulating colonic epithelial stem cells, mice deficient for TNFR1 in PDGFR+ cells showed a 40% loss of Lgr5+ stem cells, consistent with the global TNFR1-deficient mouse. CONCLUSIONTNFR1-mediated signaling regulates specification and function of colonic mesenchyme, performing an integral role in the maintenance of the crypt stem cell population.

cell biology↗

bioRxiv2021

Anal skin-like epithelium mediates colonic wound healing

Intestinal epithelial wound healing, which is essential for health, is compromised and represents a therapeutic target in inflammatory bowel disease (IBD). While studies have elucidated important subpopulations of intestinal epithelial cells in repair, these have yet to translate to therapies. Here, in mouse models of acute colitis, we demonstrate a distinct and essential source of wound-healing cells that re-epithelialize the distal colon. Using 3-d imaging, lineage tracing, and single-cell transcriptomics, we show that neighboring skin-like (squamous) cells of the anus rapidly migrate into the injured colon and establish a permanent epithelium of crypt-like morphology. These squamous cells derive from a small unique transition zone, at the boundary of colonic and anal epithelium, that resists colitis. The cells of this zone have a pre-loaded program of colonic differentiation and further upregulate key aspects of colonic epithelium during repair. Thus, heterologous cell-types at tissue junctions represent unique reserve cells capable of repair and plasticity.

developmental biology↗

bioRxiv2021

A highly thermotolerant, trimeric SARS-CoV-2 receptor binding domain derivative elicits high titers of neutralizing antibodies

The Receptor Binding Domain (RBD) of SARS-CoV-2 is the primary target of neutralizing antibodies. We designed a trimeric, highly thermotolerant glycan engineered RBD by fusion to a heterologous, poorly immunogenic disulfide linked trimerization domain derived from cartilage matrix protein. The protein expressed at a yield of [~]80-100 mg/liter in transiently transfected Expi293 cells, as well as CHO and HEK293 stable cell lines and formed homogeneous disulfide-linked trimers. When lyophilized, these possessed remarkable functional stability to transient thermal stress of upto 100 {degrees}C and were stable to long term storage of over 4 weeks at 37 {degrees}C unlike an alternative RBD-trimer with a different trimerization domain. Two intramuscular immunizations with a human-compatible SWE adjuvanted formulation, elicited antibodies with pseudoviral neutralizing titers in guinea pigs and mice that were 25-250 fold higher than corresponding values in human convalescent sera. Against the beta (B.1.351) variant of concern (VOC), pseudoviral neutralization titers for RBD trimer were [~] three-fold lower than against wildtype B.1 virus. RBD was also displayed on a designed ferritin-like Msdps2 nanoparticle. This showed decreased yield and immunogenicity relative to trimeric RBD. Replicative virus neutralization assays using mouse sera demonstrated that antibodies induced by the trimers neutralized all four VOC to date, namely B.1.1.7, B.1.351, P.1 and B.1.617.2 without significant differences. Trimeric RBD immunized hamsters were protected from viral challenge. The excellent immunogenicity, thermotolerance, and high yield of these immunogens suggest that they are a promising modality to combat COVID-19, including all SARS-CoV-2 VOC to date.

microbiology↗

bioRxiv2020

Design of a highly thermotolerant, immunogenic SARS-CoV-2 spike fragment

Virtually all SARS-CoV-2 vaccines currently in clinical testing are stored in a refrigerated or frozen state prior to use. This is a major impediment to deployment in resource-poor settings. Several use viral vectors or mRNA. In contrast to protein subunit vaccines, there is limited manufacturing expertise for these novel, nucleic acid based modalities, especially in the developing world. Neutralizing antibodies, the clearest known correlate of protection against SARS-CoV-2, are primarily directed against the Receptor Binding Domain (RBD) of the viral spike protein. We describe a monomeric, glycan engineered RBD protein fragment that is expressed at a purified yield of 214mg/L in unoptimized, mammalian cell culture and in contrast to a stabilized spike ectodomain, is tolerant of exposure to temperatures as high as 100{degrees}C when lyophilized, upto 70{degrees}C in solution and stable for over four weeks at 37{degrees}C. In prime:boost guinea pig immunizations, when formulated with the MF59 like adjuvant AddaVax, the RBD derivative elicited neutralizing antibodies with an endpoint geometric mean titer of ~415 against replicative virus, comparing favourably with several vaccine formulations currently in the clinic. These features of high yield, extreme thermotolerance and satisfactory immunogenicity suggest that such RBD subunit vaccine formulations hold great promise to combat COVID-19.

biophysics↗