Biology subjects

Gulshan, K.

Publications and source records attributed to Gulshan, K..

9 recordsLinked to original sources

bioRxiv2026

FDA-approved drug library screen identifies antidepressants, antimicrobials, anti-COPD, and anti-CVD agents as blockers of NLRP3 inflammasome and sepsis in a sex-dependent manner.

The NLRP3 inflammasome pathway is central to host defense, but dysregulated activation of inflammasomes promotes diseases associated with metabolic syndrome (diabetes, obesity, CVD, MASLD), neurodegenerative diseases (Alzheimers, Parkinsons), autoinflammatory conditions (CAPS, gout), and respiratory illnesses (asthma/COPD, COVID-19). Therapeutic modulation of NLRP3 is challenging as it requires selective blockade of detrimental inflammasome activation without broadly suppressing innate immunity. Here, we used a phenotypic screen in THP1-ASC-GFP monocytes to identify FDA-approved drugs that can block LPS-induced priming of the NLRP3 inflammasome or inhibit NLRP3 assembly without disrupting upstream priming. Various classes of drugs, such as antidepressants (Fluoxetine, Duloxetine), antihypertensives (Irbesartan, Amlodipine, Nebivolol), antidiabetics (Rosiglitazone), {beta}-adrenergic agonists (Salmeterol), antimalarials (Mefloquine), antifungals (Azoles, Ciclopirox), and antivirals (Saquinavir, Remdesivir), were identified as potent blockers of either priming or assembly of the NLRP3 inflammasome. Secondary validation demonstrated that several compounds suppressed NF-{kappa}B activation, reduced LPS binding to immune cells, decreased pro-inflammatory cytokine production, enhanced efferocytic capacity of macrophages, and enhanced autophagy in vitro and in vivo. Mechanistic analyses further revealed drug-specific effects on lysosomal biogenesis, mitochondrial morphology, and mitochondrial reactive oxygen species. In murine models of acute inflammation and endotoxemia, selected compounds attenuated NLRP3 inflammasome activation, reduced systemic and local inflammatory cytokines, limited tissue injury, and improved survival following LPS-induced sepsis, with efficacy varying by sex. Further studies in primary human cells and in vivo disease models are needed to assess the repurposing and therapeutic relevance of identified drugs.

cell biology↗

bioRxiv2023

Disulfiram reduces atherosclerosis and enhances efferocytosis, autophagy, and atheroprotective gut microbiota in hyperlipidemic mice.

Pyroptosis executor Gasdermin (GsdmD) promotes atherosclerosis in mice and humans. Disulfiram (DSF) was recently shown to potently inhibit GsdmD, but the in-vivo efficacy and mechanism of DSFs anti-atherosclerotic activity is yet to be explored. We used human/mouse macrophages and a hyperlipidemic mouse model of atherosclerosis to determine DSF anti-atherosclerotic efficacy and mechanism. DSF-fed hyperlipidemic apoE-/- mice showed significantly reduced IL-1{beta} release upon in-vivo Nlrp3 inflammasome assembly and showed smaller atherosclerotic lesions ([~]27% and 29% reduction in males and females, respectively). The necrotic core area was also smaller ([~]50% and 46% reduction in DSF-fed males and females, respectively). DSF induced autophagy in macrophages, hepatocytes/liver, and in atherosclerotic plaques. DSF modulated other atheroprotective pathways such as efferocytosis, phagocytosis, and gut microbiota. DSF-treated macrophages showed enhanced phagocytosis/efferocytosis, with a mechanism being a marked increase in cell-surface expression of efferocytic receptor MerTK. Atomic-force microscopy analysis revealed altered biophysical membrane properties of DSF treated macrophages, showing increased ordered-state of the plasma membrane and increased adhesion strength. Furthermore, the 16sRNA sequencing of DSF-fed hyperlipidemic mice showed highly significant enrichment in atheroprotective gut microbiota Akkermansia and a reduction in atherogenic Romboutsia species. Taken together, our data shows that DSF can simultaneously modulate multiple atheroprotective pathways, and thus may serve as novel adjuvant therapeutic to treat atherosclerosis.

cell biology↗

bioRxiv2023

Membrane Bound O-Acyltransferase 7 (MBOAT7) Shapes Lysosomal Lipid Homeostasis and Function to Control Alcohol-Associated Liver Injury

Several recent genome-wide association studies (GWAS) have identified single nucleotide polymorphism (SNPs) near the gene encoding membrane-bound O-acyltransferase 7 (MBOAT7) that is associated with advanced liver diseases. In fact, a common MBOAT7 variant (rs641738), which is associated with reduced MBOAT7 expression, confers increased susceptibility to non-alcoholic fatty liver disease (NAFLD), alcohol-associated liver disease (ALD), and liver fibrosis in those chronically infected with hepatitis viruses B and C. The MBOAT7 gene encodes a lysophosphatidylinositol (LPI) acyltransferase enzyme that produces the most abundant form of phosphatidylinositol 38:4 (PI 18:0/20:4). Although these recent genetic studies clearly implicate MBOAT7 function in liver disease progression, the mechanism(s) by which MBOAT7-driven LPI acylation regulates liver disease is currently unknown. Previously we showed that antisense oligonucleotide (ASO)-mediated knockdown of Mboat7 promoted non-alcoholic fatty liver disease (NAFLD) in mice (Helsley et al., 2019). Here, we provide mechanistic insights into how MBOAT7 loss of function promotes alcohol-associated liver disease (ALD). In agreement with GWAS studies, we find that circulating levels of metabolic product of MBOAT7 (PI 38:4) are significantly reduced in heavy drinkers compared to age-matched healthy controls. Hepatocyte specific genetic deletion (Mboat7HSKO), but not myeloid-specific deletion (Mboat7MSKO), of Mboat7 in mice results in enhanced ethanol-induced hepatic steatosis and high concentrations of plasma alanine aminotransferase (ALT). Given MBOAT7 is a lipid metabolic enzyme, we performed comprehensive lipidomic profiling of the liver and identified a striking reorganization of the hepatic lipidome upon ethanol feeding in Mboat7HSKO mice. Specifically, we observed large increases in the levels of endosomal/lysosomal lipids including bis(monoacylglycero)phosphates (BMP) and phosphatidylglycerols (PGs) in ethanol-exposed Mboat7HSKO mice. In parallel, ethanol-fed Mboat7HSKO mice exhibited marked dysregulation of autophagic flux and lysosomal biogenesis when exposed to ethanol. This was associated with impaired transcription factor EB (TFEB)-mediated lysosomal biogenesis and accumulation of autophagosomes. Collectively, this works provides new molecular insights into how genetic variation in MBOAT7 impacts ALD progression in humans and mice. This work is the first to causally link MBOAT7 loss of function in hepatocytes, but not myeloid cells, to ethanol-induced liver injury via dysregulation of lysosomal biogenesis and autophagic flux.

cell biology↗

bioRxiv2022

Miltefosine attenuates inflammation, reduces atherosclerosis, and alters gut microbiota in hyperlipidemic mice.

Excess cholesterol induces foam cell formation, NLRP3 inflammasome activation, and IL-1{beta} release in atherosclerotic plaques. We have shown previously that Miltefosine increased cholesterol release and dampened NLRP3 inflammasome assembly in macrophages. Here, we show that Miltefosine reduced LPS-induced choline uptake by macrophages and attenuated NLRP3 inflammasome assembly in mice. Miltefosine-fed mice showed reduced plasma IL-1{beta} in a polymicrobial cecal slurry injection model of systemic inflammation. Miltefosine-fed mice showed increased reverse cholesterol transport from macrophages to plasma, liver, and feces. Hyperlipidemic apoE-/- mice fed with Miltefosine showed significantly reduced weight gain and markedly reduced atherosclerotic lesions vs. control mice. 16S rDNA sequencing and analysis showed alterations in the gut microbiota profile of Miltefosine-fed hyperlipidemic apoE-/- vs. control mice, with the most notable changes in Romboutsia and Bacteroidetes species. Taken together, these data indicate that Miltefosine causes pleiotropic effects on lipid metabolism, inflammasome activity, atherosclerosis, and the gut microbiota.

cell biology↗

bioRxiv2022

Myeloid-cell-specific role of Gasdermin D in promoting lung cancer progression in mice.

The activities of the NLRP3 and AIM2 inflammasomes and Gasdermin D (GsdmD), the final executor of inflammasome activity, are implicated in lung cancer pathophysiology but its not clear if their contributions promote or retard lung cancer progression. GsdmD plays a role in release of interleukin-1beta (IL-1 {beta}), and the CANTOS trial and recent studies have highlighted a crucial role of IL-1{beta} in promoting lung cancer. Expression of GsdmD was shown to be upregulated in human non-small cell lung cancer (NSCLC) tissue, but its contribution to in vivo lung cancer metastasis is not known. Using a metastatic Lewis Lung Carcinoma (LLC) cell model, we show that GsdmD knockout (GsdmD-/-) mice form significantly fewer cancer foci in lung, and exhibit markedly decreased lung cancer metastasis. Furthermore, GsdmD-/- mice show a significant ~ 50% increase in median survival rate vs. isogenic WT C57BL6J mice. The cleaved forms of GsdmD and IL-1 {beta} were detected in lung tumor tissue, indicating inflammasome activity in lung tumor microenvironment (TME). Increased migration and growth of LLC cells was observed upon exposure to the conditioned media derived from inflammasome-induced wild type, but not the GsdmD-/-, macrophages. Exposure of human A549 lung cancer cells to the conditioned media derived from inflammasome-induced THP-1 macrophages also resulted in increased cell migration. Using bone marrow transplantation, we show the myeloid-specific contribution of GsdmD in lung cancer metastasis. Taken together, our data show that inflammasome activation in macrophages promotes lung cancer growth and migration, and GsdmD plays a myeloid-specific role in lung cancer progression in mice.

cancer biology↗

bioRxiv2021

Gasdermin D mediates inflammation-induced defects in reverse cholesterol transport and promotes atherosclerosis.

Activation of inflammasomes, such as Nlrp3 and Aim2, can exacerbate atherosclerosis in mice and humans. Gasdermin D (GsdmD) serves as a final executor of inflammasome activity, by generating membrane pores for the release of mature Interleukin-1beta (IL-1{beta}). Inflammation dampens reverse cholesterol transport (RCT) and promotes atherogenesis, while anti-IL-1{beta} antibodies were shown to reduce cardiovascular disease in humans. Though Nlrp3/AIM2 and IL-1{beta} nexus is an emerging atherogenic pathway, the direct role of GsdmD in atherosclerosis is not yet fully clear. Here, we used in-vivo Nlrp3 inflammasome activation to show that the GsdmD-/- mice release ~80% less IL-1{beta} vs WT mice. The GsdmD-/- macrophages were more resistant to Nlrp3 inflammasome mediated reduction in cholesterol efflux, showing ~26% decrease vs. ~60% reduction in WT macrophages. GsdmD expression in macrophages exacerbated foam cell formation in an IL-1{beta} dependent fashion. The GsdmD-/- mice were resistant to Nlrp3 inflammasome mediated defect in RCT, with ~32% reduction in plasma RCT vs. ~ 57% reduction in WT mice, ~ 17% reduction in RCT to liver vs. 42% in WT mice, and ~ 37% decrease in RCT to feces vs. ~ 61% in WT mice. The LDLr anti-sense oligonucleotides (ASO) induced hyperlipidemic mouse model showed the role of GsdmD in promoting atherosclerosis. The GsdmD-/- mice exhibit ~42% decreased atherosclerotic lesion area in females and ~33% decreased lesion area in males vs. WT mice. The atherosclerotic plaque-bearing sections stained positive for the cleaved N-terminal fragment of GsdmD, indicating cleavage of GsdmD in atherosclerotic plaques. Our data show that GsdmD mediates inflammation-induced defects in RCT and promotes atherosclerosis. SummaryGsdmD mediates inflammation-induced defects in RCT and promotes atherosclerosis.

pathology↗

bioRxiv2020

SR-B1 uptake of HDL promotes prostate cancer proliferation and tumor progression

High density lipoprotein (HDL) metabolism, in part, is facilitated by scavenger receptor class B, type 1 (SR-B1) that mediates its uptake into cells. SR-B1 is upregulated in prostate cancer tissue. Here, we report that knockout (KO) of SR-B1 via CRISPR/Cas9 editing led to reduced HDL uptake into prostate cancer cells, and reduced their proliferation in response to HDL. In vivo studies using syngeneic SR-B1 wildtype (SR-B1+/+) and SR-B1 KO (SR-B1-/-) prostate cancer cells in WT and apolipoprotein-AI KO (apoA1-KO) C57BL/6J mice showed that WT hosts, containing higher levels of total and HDL-cholesterol, grew larger tumors than apoA1-KO hosts with lower levels of total and HDL-cholesterol. Furthermore, SR-B1-/- prostate cancer cells formed smaller tumors in WT hosts, than SR-B1+/+ cells in same host model. Tumor volume data was overall similar to survival data. We conclude that tumoral SR-B1 KO reduced HDL-mediated increases in prostate cancer cell proliferation and disease progression.

cancer biology↗

bioRxiv2019

First eight residues of apolipoprotein A-I mediate the C-terminus control of helical bundle unfolding and its lipidation

The crystal structure of a C-terminal deletion of apolipoprotein A-I (apoA1) shows a large helical bundle structure in the amino half of the protein, from residues 8 to 115. Using site directed mutagenesis, guanidine or thermal denaturation, cell free liposome clearance, and cellular ABCA1-mediated cholesterol efflux assays, we demonstrate that apoA1 lipidation can occur when the barrier to this bundle unfolding is lowered. The absence of the C-terminus renders the bundle harder to unfold resulting in loss of apoA1 lipidation that can be reversed by point mutations, such as Trp8Ala, and by truncations as short as 8 residues in the amino terminus, both of which lower the barrier to helical bundle unfolding. Locking the bundle via a disulfide bond leads to loss of apoA1 lipidation. We propose a model in which the C-terminus acts on the N-terminus to destabilize helical bundle. Upon lipid binding to the C-terminus, Trp8 is displaced from its interaction with Phe57, Arg61, Leu64, Val67, Phe71, and Trp72 to destabilize the bundle. However, when the C-terminus is deleted, Trp8 cannot be displaced, the bundle cannot unfold, and apoA1 cannot be lipidated.

biochemistry↗

bioRxiv2018

Miltefosine increased cholesterol efflux, induced autophagy and inhibited NLRP3-inflammasome assembly and IL-1β release.

Miltefosine is an FDA approved oral drug for treating cutaneous and visceral leishmaniasis. Leishmania is a flagellated protozoa, which infects and differentiates in macrophages. Here, we studied the effects of Miltefosine on macrophages lipid homeostasis, autophagy, and NLRP3 inflammasome assembly/activity. Miltefosine treatment conferred multiple effects on macrophage lipid homeostasis leading to increased cholesterol release from cells, increased lipid-raft disruption, decreased phosphatidylserine (PS) flip from the cell-surface, and redistribution of phosphatidylinositol 4,5-bisphosphate (PIP2) from the plasma membrane to actin rich regions in the cells. Enhanced basal autophagy, lipophagy and mitophagy was observed in cells treated with Miltefosine vs. control. Miltefosine treated cells showed marked increased in phosphorylation of kinases involved in autophagy induction such as; Adenosine monophosphate-activated protein kinase (AMPK) and Unc-51 like autophagy activating kinase (ULK1). The Toll like receptor (TLR) signaling pathway was blunted by Miltefosine treatment, resulting in decreased TLR4 recruitment to cell-surface and ~75% reduction in LPS induced pro-IL-1{beta} mRNA levels. Miltefosine reduced endotoxin-mediated mitochondrial reactive oxygen species and protected the mitochondrial membrane potential. Miltefosine treatment induced mitophagy and dampened NLRP3 inflammasome assembly. Collectively, our data shows that Miltefosine induced ABCA1 mediated cholesterol release, induced AMPK phosphorylation and mitophagy, while dampening NLRP3 inflammasome assembly and IL-1{beta} release.\n\nSignificance StatementAtherosclerosis is driven by cholesterol accumulation and inflammation, and the arterial macrophage is a key cell type in both of these processes. The macrophage characteristics that protect against atherosclerosis include increased cholesterol efflux/reverse cholesterol transport, increased autophagy, and deceased inflammatory cytokine production and signaling. Here, we show that one single orally available compound, Miltefosine, can target multiple macrophage pathways involved in lipid homeostasis and inflammation. Miltefosine activated cholesterol release and autophagy while inhibiting pro IL-1{beta} gene expression and NLRP3 inflammasome assembly. Miltefosine activated AMPK signaling pathway and mitophagy, leading to reduced NLRP3 inflammasome assembly and IL-1{beta} release.

cell biology↗