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Bhushan, A.

Publications and source records attributed to Bhushan, A..

9 recordsLinked to original sources
bioRxiv2026

Osteoprotegerin-Enabled Immune Evasion by Pathological Adipose Stromal Cells Drives Metabolic Dysfunction in Obesity

Diet-induced obesity (DIO) promotes the accumulation of stromal cells with senescent characteristics in the adipose tissue (AT). Selectively clearing these cells--either through chemical senolytics or activation of invariant natural killer T (iNKT) cells--improves glucose homeostasis in obese mice, however the identity of the responsible stromal population remains unknown. Here, we use transcriptional profiling of AT stromal cells coupled with C12FDG-based enrichment of senescent populations in mice with DIO and healthy controls to identify a distinct subset of adipose progenitor cells (APCs) with robust senescence signatures that accumulate in DIO across multiple AT depots. We show that these cells, which we term senescent APCs (sAPCs), are not merely passive markers of metabolic stress but are instead active stromal organizers, accumulating in parallel with the emergence of lipid-associated macrophages (LAMs) and the diminution of multipotent mesenchymal progenitors. sAPCs promoted CCR2-dependent macrophage chemotaxis, directly linking stromal senescence to chemokine-mediated remodeling of the AT immune niche. Comparative transcriptional analysis revealed a remarkable similarity between sAPCs and inflammatory cancer-associated fibroblasts (iCAFs), including the strong induction of periostin (POSTN) and the production of osteoprotegerin (OPG), a decoy receptor for RANKL and TRAIL that enables tumoral immune evasion. Indeed, OPG production by AT stromal cells was induced by DIO across AT depots. Exogenous OPG inhibited the ability of iNKT cells to kill senescent APCs in vitro, whereas antibody-mediated OPG neutralization reciprocally enhanced such cytotoxic killing. In vivo, systemic OPG neutralization both reduced sAPC accumulation in AT and normalized glucose homeostasis in obese mice. Together, these findings identify sAPCs as a pathological stromal population that expands in obesity through elaboration of immunomodulatory factors. In particular, secreted OPG enables sAPCs to evade iNKT-mediated immune surveillance and contributes to metabolic dysfunction, highlighting OPG and sAPCs as promising therapeutic targets for restoring AT immune and metabolic homeostasis.

immunology↗
bioRxiv2025

Bile Salts and Bacterial Bile Salt Hydrolase Activity Differentially Influence Escherichia coli Colonization and FXR Signaling in Colorectal Cancer Organoids

Bile salts are key regulators of host-microbe interactions and epithelial signaling in the intestine. Gut bacteria can deconjugate primary bile salts, which can be leveraged to engineer targeted therapies through bile salt hydrolase (BSH) activity. However, the interplay between bile salt signaling, microbial metabolism, and tumor-associated colonization remains poorly understood. Here, we establish an in vitro colon organoid platform to dissect bile salt-driven epithelial-microbe dynamics at cellular resolution. Using this system, we found that bile salts markedly modulated colonization of native Escherichia coli (EcAZ-2) and a BSH-expressing strain (EcAZ-2LsBSH) in healthy versus tumor murine organoids. Under bile salt exposure, EcAZ-2LsBSH preferentially colonized tumor organoids, though overall colonization was lower than that of the non-BSH strain. Beyond effects on bacterial colonization, bile salts also directly activated host signaling pathways. In presence of bile salts, EcAZ-2LsBSH significantly enhanced activation of bile acid receptors such as farnesoid X receptor (FXR) by 2-fold in healthy organoids. These findings reveal a mechanistic bridge between in vivo microbial function and bile salt interactions, offering potential microbiota and bile acid targeted therapeutic strategies.

cancer biology↗
bioRxiv2025

Blocking Osteoprotegerin Reprograms Cancer Associated Fibroblast to Promotes Immune Infiltration into the Tumor Microenvironment

AbstractThe stromal compartment of many solid tumors plays a critical role in shaping an immunosuppressive microenvironment that limits the effectiveness of immune-based therapies1. Among stromal constituents, cancer-associated fibroblasts (CAFs) have emerged as key regulators of antitumor immunity2-5. Here, we identify a distinct subset of CAFs in both murine and human stroma-rich cancers that secrete osteoprotegerin (OPG)-- a soluble decoy receptor that neutralizes receptor activator of nuclear factor kappa-B ligand (RANKL) and TNF-related apoptosis-inducing ligand (TRAIL), both of which are involved in T cell function. In vitro, OPG directly impairs CD8 T cell-mediated killing of target cells. In murine models of pancreatic and breast cancer, antibody-mediated blockade of OPG promotes robust immune infiltration into the tumor microenvironment, leading to significant tumor regression. Stromal profiling revealed that OPG blockade induces a shift in CAF cells--reducing immunosuppressive OPG fibroblasts while expanding interferon-responsive fibroblasts, thus recalibrating the tumor stroma toward a pro-immunogenic landscape. These findings uncover a previously unrecognized mechanism of stromal immune suppression and highlight OPG as a stromal immune checkpoint controlling CD8 T cell infiltration. Targeting OPG may offer a novel therapeutic strategy to convert immunologically "cold" tumors into T cell-infiltrated, tumor microenvironment.

cancer biology↗
bioRxiv2024

Epithelial cytokeratin 6a restricts secretory autophagy of proinflammatory cytokines by interacting with Sec16A

Epithelial cells form a crucial barrier against harmful microbes and inflammatory stimuli. Restraining inflammatory responses at the corneal barrier is necessary for avoiding sight-threatening tissue damage. Yet, epithelial cell-intrinsic mechanisms that dampen inflammation are largely unexplored. Keratin 6a (K6a) is a common type II cytokeratin highly expressed in corneal and other stratified epithelial cells. In a mouse model of sterile corneal inflammation, K6a knockout mice exhibit disease exacerbation. Here, we investigated cell-intrinsic mechanisms by which cytoplasmic K6a curbs corneal inflammation. We stimulated wild-type (WT) and K6a siRNA-knockdown (K6a-KD) human corneal epithelial (hTCEpi) cells with inflammatory P. aeruginosa culture supernatant. Our results showed that, under both basal and inflammatory conditions, K6a-KD cells secreted higher levels of cytokines and chemokines (IL-1, IL-6, IL-8, CXCL1, CCL20) as compared to WT cells. K6a-KD cells also had increased level of LC3-II, a marker for autophagosomes, while autophagic degradation of SQSTM1/p62 remained unchanged. In K6a-KD cells, the majority of LC3-II puncta were associated with non-acidified autophagosomes rather than acidified autolysosomes. Upon stimulation, IL-8 was found to co-localize with LC3-II by confocal microscopy. Mechanistically, mass spectrometric analysis of K6a immunoprecipitates identified Sec16A, a protein involved in secretory autophagy, as an interacting partner of K6a. Further experiments showed that knocking down key proteins involved in autophagosome formation (ATG5) and the secretory autophagy process (Sec16A, GRASP55, Rab8) abolished the augmentative effect of K6a-KD on cytokine and chemokine secretion. These findings reveal a novel repressive role of K6a in secretory autophagy-mediated proinflammatory cytokine secretion and provide new insights into cell-intrinsic mechanisms of inflammation control at epithelial barriers.

cell biology↗
bioRxiv2023

Keratin 6a attenuates Toll-like receptor-triggered proinflammatory response in corneal epithelial cells by suppressing ELKS/IKKϵ-dependent activation of NF-κB

The corneal epithelium at the ocular surface is constantly exposed to the environment and represents the first line of defense against infection, mechanical injury or chemical irritation. Through TLR-mediated recognition of pathogen- and damage-associated molecular patterns, it engages in direct antimicrobial responses and alerts the immune system on intruder and tissue damage by secreting pro-inflammatory and chemotactic cytokines that promote immune cell infiltration. How the corneal epithelium downregulates TLR signaling is unclear, yet it highly expresses keratin 6a (K6a), a cytoskeletal protein that has emerged to play essential regulatory roles in corneal innate immune response. Here we report that mice harboring genetic deletion of K6a are more susceptible to developing bacterial keratitis with unresolved corneal opacification and higher bacterial load. Such disease phenotype is caused by the increased pro-inflammatory cytokine and chemokine secretions from the K6a-null corneal epithelium, which further promotes the infiltration of immune cells and their associated pro-inflammatory response. Using human corneal epithelial cells immortalized by telomerase reverse transcriptase (hTCEpi cells), we demonstrated that knocking down K6a enhances NF-{kappa}B/ RelA-dependent cytokine and chemokine expression. Moreover, proteomic screen reveals that K6a interacts with ELKS, a critical NEMO-binding scaffold that links between canonical IKK/{beta} and the principal cytoplasmic inhibitor of RelA, i.e. I{kappa}B., to promote its phosphorylation and degradation. Surprisingly, K6a does not antagonize any of these canonical NF-{kappa}B signaling events. Instead, we found that ELKS in addition to canonical IKKs interacts with the atypical IKK member IKK{varepsilon}. Furthermore, knockdown of K6a in hTCEpi cells promotes ELKS-dependent phosphoactivation of IKK{varepsilon}, which in turn phosphorylates and activates RelA. Our study thus demonstrated an unexpected role of cytosolic K6a as a novel negative regulator of TLR/NF-{kappa}B signaling in preventing excess proinflammatory cytokine and chemokine expressions. It further highlighted the functional importance of ELKS as a common signaling scaffold for both canonical and atypical IKK-dependent activation of NF-{kappa}B in corneal epithelial cells. Using both IKK classes other than only canonical IKKs for TLR/NF-{kappa}B induction as in other cell types including myeloid immune cells suggest that the cornea epithelium is more flexible in modulating its inflammatory response, which could greatly minimize corneal damage while preserving its essential functions for barrier protection and light refraction.

immunology↗
bioRxiv2023

Trilliumosides A-B, two novel steroidal saponins isolated from Rhizomes of Trillium govanianum as potent anticancer agents targeting apoptosis in A-549 cancer cell line.

Two novel steroidal saponins, Trilliumosides A (1) and B (2) were isolated from the rhizomes of Trillium govanianum by bioactivity-guided phytochemical investigation along with seven known compounds protodioscin (3) govanoside B (4), borassoside E (5), 20-hydroxyecdysone (6), 5-20-hydroxyecdysone (7), govanic acid (8), and diosgenin (9). The structure of novel compounds 1-2 were established using spectroscopic methods such as 1D, 2D NMR data and HR-ESI-MS. The isolated compounds were evaluated for in-vitro cytotoxic activity against a panel of human cancer cell lines. Compound 1 showed significant cytotoxic activity against A-549 (Lung) and SW-620 (Colon) cell lines with IC50 values of 1.83 and 1.85 {micro}M, whereas compound (2) IC50 value against A-549 cell line was found to be 1.79 {micro}M. Among previously known compounds (3), (5) and (9) their cytotoxic IC50 value was found to be in the range of 5-10 {micro}M. In detailed anticancer analysis compound (2) was seen inhibiting colony forming potential and in-vitro migration in the A-549 cell line. Furthermore, the mechanistic study of compound (2) on the A-549 cell line revealed characteristic changes including nuclear morphology, increased ROS generation, and reduced levels of MMP. Above mentioned events eventually induce apoptosis, a key hallmark in cancer studies, by upregulating the pro-apoptotic protein BAX and downregulating the anti-apoptotic protein BCL-2 thereby activating Caspase-3. Our study reports the first mechanistic anticancer evaluation of the compounds isolated from the rhizomes of Trillium govanianumwith remarkable activity in the desired micro molar range. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=108 SRC="FIGDIR/small/537170v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@14d9769org.highwire.dtl.DTLVardef@10f7658org.highwire.dtl.DTLVardef@22a38borg.highwire.dtl.DTLVardef@1f0b28_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗
bioRxiv2020

A Novel Standalone Microfluidic Device for Local Control of Oxygen Tension for Intestinal-Bacteria Interactions

The intestinal environment is unique because it supports the intestinal epithelial cells under a normal oxygen environment and the microbiota under an anoxic environment. Due to importance of understanding the interactions between the epithelium and the microbiota, there is a strong need for developing representative and simple experimental models. Current approaches do not capture the dual-oxygen environment, require external anaerobic chambers, or are complex. Another major limitation is that in the solutions that can mimic the dual-oxygen environment, the oxygenation level of the epithelial cells is not known, raising the question whether the cells are hypoxic. We report standalone microfluidic devices that form a dual-oxygen environment without the use of an external anaerobic chamber or oxygen scavengers to coculture intestinal epithelial and bacterial cells. By changing the thickness of the device cover, the oxygen tension in the chamber could be modulated. We verified the oxygen levels using several tests: microscale oxygen sensitive sensors incorporated within the devices, hypoxic immunostaining of Caco-2 cells, and genetically encoded bacteria. Collectively, these methods monitored oxygen concentrations in devices more comprehensively than previous reports and allowed for control of oxygen tension to match the requirements of both intestinal cells and anaerobic bacteria. Our experimental model is supported by the mathematical model that considers diffusion of oxygen into the top chamber and the cellular oxygen consumption rate. This allowed us to experimentally determine the oxygen consumption rate of the epithelial cells more precisely.

bioengineering↗
bioRxiv2019

BET proteins are required for transcriptional activation of the senescent beta cell secretome in Type 1 Diabetes

OBJECTIVEType 1 Diabetes (T1D) results from progressive loss of pancreatic beta cells due to autoimmune destruction. We recently reported that during the natural history of T1D in humans and the female nonobese diabetic (NOD) mouse model, beta cells acquire a senescence-associated secretory phenotype (SASP) that is a major driver of disease onset and progression, but the mechanisms that activate SASP in beta cells were not explored. The objective of this study was to identify transcriptional mechanisms of SASP activation in beta cells.\n\nMETHODSWe used the female NOD mouse model of spontaneous autoimmune T1D and ex vivo experiments on NOD mouse and human islets to test the hypothesis that Bromodomain Extra-Terminal domain (BET) proteins activate the beta cell SASP transcriptional program.\n\nRESULTSHere we show that beta cell SASP is transcriptionally controlled by BET proteins, including BRD4. Chromatin analysis of key beta cell SASP genes in NOD islets revealed binding of BRD4 at active regulatory regions. BET protein inhibition in NOD islets diminished not only the transcriptional activation and secretion of SASP factors but also the non-cell autonomous activity. BET protein inhibition also decreased the extent of SASP induction in human islets exposed to DNA damage. The BET protein inhibitor iBET-762 prevented diabetes in NOD mice and also attenuated SASP in beta cells in vivo.\n\nCONCLUSIONSTaken together, our findings support a crucial role for BET proteins in the activation of the beta cell SASP transcriptional program. These studies suggest avenues for preventing T1D by transcriptional inhibition of SASP.

physiology↗