Biology subjects

Chattopadhyay, S.

Publications and source records attributed to Chattopadhyay, S..

At least 19 recordsLinked to original sources

bioRxiv2026

Not all tumors age alike: Bidirectional epigenetic age shifts across 20 solid tumors

Epigenetic ageing and tumor progression have each been studied extensively using DNA methylation, yet their relationship remains poorly understood. Here we integrate DNA methylation age estimation with phyloepigenetic reconstruction across 20 solid cancer spanning 1710 samples from The Cancer Genome Atlas to assess age acceleration in tumor tissue compared to matched normal tissue. MethodsDNA methylation age was estimated for 544 patients with matched normal and tumor samples using Horvaths epigenetic clock, with linear regression used to assess correspondence to chronological age and Wilcoxon signed-rank tests evaluated whether tumor-normal age differences deviated significantly from zero. Tumor evolutionary architecture was reconstructed via UPGMA clustering of genome-wide methylation divergence into phyloepigenetic trees, from which trunk and private methylation events were classified and their chromosomal distribution compared descriptively across cancer types. ResultsApplying Horvaths epigenetic clock to normal tissue yielded a mean absolute error of 17.6 years, substantially exceeding expectations. Contrary to previous reports of pronounced tumor age acceleration any ubiquitous pattern of acceleration did not emerge at the cohort level across all tumor types when tumor age was compared directly against matched normal tissue. Acceleration and deceleration instead varied often consistently by cancer types, with significant positive differences observed in endometrial, lung squamous, head and neck, and prostate cancers, and significant negative differences in renal and thyroid cancers. Phyloepigenetic reconstruction revealed that methylation events arose predominantly through private, subclonal branching rather than early clonal events. The X chromosome was found overrepresented among methylation events across nearly all solid cancers. ConclusionEpigenetic aging in cancer is not uniform or consistently accelerated but reflects tissue-specific and often opposing patterns of change. The predominance of subclonal events suggest ongoing epigenetic diversification throughout tumor evolution, while the consistent overrepresentation of X-chromosome events indicates a distinct chromosome-level vulnerability.

genetics↗

bioRxiv2026

Neuropilin-1 functions as a proviral and immunoregulatory host factor during Chikungunya virus infection

Neuropilin-1 (NRP1) is a transmembrane glycoprotein involved in angiogenesis, neurodevelopment, inflammation, cancer driven immune suppression, and immune homeostasis. However, its contribution to virus-induced immune responses is not explored. Chikungunya virus (CHIKV) is a re-emerging arthritogenic alphavirus that causes severe arthralgia, and myalgia, accompanied by heightened inflammatory cytokine responses. The host factors that drive these inflammatory responses, however, remain poorly defined. Here, in this current study, the role of NRP1 in CHIKV infection was investigated using in vitro, and in vivo model systems. Using genetic manipulation, pharmacological, and antibody blockade-mediated approaches in murine and human cellular infection models, it was demonstrated that NRP1 promotes CHIKV infection while restraining the production of proinflammatory cytokines. Furthermore, NRP1 inhibition selectively increased JNK phosphorylation. Hence, inhibiting JNK reduced the elevated cytokine production caused by NRP1 blockade. Moreover, NRP1 interacted with CHIKV-E1 and is involved in multiple phases of CHIKV infection. In addition, it was demonstrated that NRP1 inhibition using EG00229 trifluoroacetate can reduce viral infection in several CHIKV-susceptible cells, human peripheral blood macrophages, and in the in vivo mice model of infection. Together, these findings indicate that NRP1 is an important host factor and a probable therapeutic target during CHIKV infection. IMPORTANCEChikungunya virus (CHIKV) causes acute febrile illness that can progress to debilitating chronic musculoskeletal and occasional neurological complications. The absence of a globally available effective vaccine and the lack of specific antivirals underscore CHIKV as a major burden, especially in endemic tropical regions. There is a growing need to understand host factors that shape CHIKV-driven immune response. The importance of our study lies in understanding the immunoregulatory role of the host receptor Neuropilin-1 (NRP1) during CHIKV infection. We identify NRP1 as a novel host factor of CHIKV infection that also acts as a rheostat to restrict the inflammatory viral immune response. Moreover, we report anti-viral potential of the NRP1 antagonist EG00229 trifluoroacetate in multiple cell lines, primary cells, and mice model. Our findings indicate NRP1 as a probable therapeutic target in CHIKV pathogenesis.

immunology↗

bioRxiv2026

A human monoclonal antibody from hybrid immunity exhibits exceptional neutralization breadth against antigenically divergent contemporary Omicron JN.1 lineage variants

Hybrid immunity offers stronger and more durable antibody-mediated protection against symptomatic SARS-CoV-2 infection. In the present study, we report the development of a durable antibody response in an individual with hybrid immunity who also received three doses of the prototype COVID-19 vaccine. Polyclonal plasma antibody obtained from this donor also showed extraordinary neutralization breadth against contemporary Omicron variants. One of the functional monoclonal antibodies (ATHS-C30) isolated from this individual, representing the IGHV4-30 lineage-specific B cell with strong binding affinity to JN.1 spike protein, showed extraordinary neutralization breadth, including variants of Omicron lineages that emerged beyond JN.1, such as KP.2, KP.3.1.1, KP.3.2, KP.3.3, LB.1 and XEC. ATHS-C30 was found to bind RBD with high affinity and showed distinct epitope specificity to the other neutralizing mAbs isolated from the same donor through the epitope binning assay. Molecular modelling of the CDHR3 sequence using existing structures indicated that ATHSC-30 belongs to the class 4 antibody, a feature that contributes to breadth, while epitope conservation analysis indicated that the majority of RBD-interacting residues of ATHSC-30 are evolutionarily conserved. Taken together, our study indicate that ATHS-C30 forms the basis of development of a robust and broadly neutralizing antibody response in this individual with hybrid immunity, which overcomes the antigenic variation by targeting highly conserved and cryptic epitopes in destabilizing the spike structure. ImportanceSARS-CoV-2 continues to pose a significant public health threat, particularly to immunocompromised individuals and older adults with underlying comorbidities. Hybrid immunity to SARS-CoV-2 in vaccinated individuals leads to the development of B cells that are qualitatively superior to those that are expected to develop in only vaccinated individuals. In the present study, we found that among individuals with hybrid immunity who developed robust, durable antigen-specific antibody responses, one developed antibody response capable of broadly cross-neutralising contemporary Omicron variants. This was correlated with development of antigen-specific B cell lineage, such as IGHV4-30, that produced antibodies with potent and extraordinary neutralisation breadth against contemporary Omicron lineages, such as KP.2, KP.3.1.1, KP.3.2, KP.3.3, LB.1 and XEC. This is believed to be due to heterologous antigen exposures driving the development of an antigen-specific B cell repertoire that, in turn, facilitates immune imprinting capable of overcoming the ineffectiveness of antibodies to effectively neutralize newly emerging Omicron variants.

immunology↗

bioRxiv2026

A fungal hemophore relay mediates heme transfer via transient protein interactions

The fungal pathobiont Candida albicans acquires heme from host proteins via a set of soluble and anchored extracellular CFEM-type hemophores that can capture the bound heme and exchange it, eventually delivering it to the cell membrane for endocytosis into the yeast cell. Yet the molecular mechanism by which the heme is transferred through this protein cascade across the cell envelope remains unclear. To address this mechanism, we developed a set a fusions of three C. albicans hemophores with fluorescent proteins. Fluorescence of these fusion proteins is strongly quenched when heme is bound to the hemophore moiety, enabling to measure heme transfer instantaneously. Kinetic analysis of the different transfer reactions reveals that heme transfer from the host protein to the CFEM hemophores and between the CFEM hemophores are governed by different regimes. Kinetics of heme transfer from hemoglobin or serum albumin to the CFEM hemophores is mainly first-order, suggesting that heme stochastically released from host proteins is captured by the hemophores. In contrast, transfer of heme between the hemophores is near second-order, consistent with a mechanism requiring protein-protein interactions. To confirm this, we show that CFEM hemophores can interact in homodimeric and heterodimeric complexes. Furthermore, while dimerization-defective mutants of the soluble hemophore Csa2 are proficient in heme binding and extraction, they are defective in heme transfer. This supports a model of heme transfer by direct interaction between the members of the fungal hemophore cascade. SIGNIFICANCEAcquisition of extracellular heme as iron or heme sources is common in microorganisms, and particularly prevalent among pathogenic organisms that must contend with an iron-poor host environment. To extract heme from host proteins, microorganisms deploy various systems that include extracellular soluble and cell-anchored hemophores. Here we describe a new approach for monitoring heme binding and transfer in real time, based on the development of fluorescent derivatives of fungal hemophores. These novel reagents open a new window on the study of a common virulence factor of microbial pathogens.

microbiology↗

bioRxiv2026

CRY1 couples blue light dependent systemic acquired defense through NPR1 interaction and SnRK3.6-mediated translocation

Systemic acquired resistance (SAR) is a whole-plant immune response triggered by localized infection. Cryptochromes (CRY) that are blue light receptors, work together with phytochromes (PHY) to regulate light dependent responses in Arabidopsis. Although the effect of light on plant immunity is fairly well studied, the molecular mechanisms remain unclear. In this study, we show that CRY1 is essential for optimal SAR induction under blue light conditions in Arabidopsis thaliana. The cry1 mutants fail to activate SAR under blue light, as evidenced by reduced expression of defense genes, increased susceptibility to Pseudomonas syringae (PstDC3000), and impaired stomatal defense. While SAR is intact in both wild type and cry1 under white light, blue light selectively reveals critical role of CRY1 in SAR. Interestingly, npr1 mutants activate SAR under blue light, supporting the fact that blue light can trigger SAR through NPR1 independent pathways. Using biochemical and cell-based assays, we show that CRY1 directly interacts with NPR1 in nucleus in a blue-light-enhanced manner. Overexpression of CRY1 induces NPR1 levels and enhances SAR. Expression of each of them individually partly rescued SAR, but their co-expression fully reinstated it, implying that SAR relies on interaction between NPR1 and CRY1. Stomatal movement measurements further show that CRY1 and NPR1 act synergistically to promote stomatal closure to limit pathogen entry. We also identify SnRK3.6, a blue-light-inducible kinase, as a novel regulator of CRY1. SnRK3.6 physically interacts with and phosphorylates CRY1, facilitating its nuclear translocation. Our results revealed a novel role of CRY1-NPR1 complex formation and regulation of CRY1 by SnRK3.6 in inducing SAR.

molecular biology↗

bioRxiv2025

Tretinoin, a Vitamin A derivative, exerts antiviral effects against Chikungunya virus infection through the nuclear retinoid receptor signaling pathways

Chikungunya virus (CHIKV) has reemerged as a global pathogen causing serious public health threat and socio-economic damage, particularly due to the absence of specific antivirals. Retinoids have been reported to exhibit antiviral potentials against multiple viral infections. In the present study, Tretinoin (TR) or all-trans retinoic acid (ATRA) was evaluated for its anti-CHIKV activity using in vitro, in vivo, and ex vivo models. TR was able to impede CHIKV infection efficiently in Vero and physiologically relevant muscle cells, C2C12, with drastic reduction in the viral RNA, proteins and progeny formation. The IC50 of TR against CHIKV was estimated to be 20.05 {micro}M and 19.71 {micro}M in Vero and C2C12 cells, respectively. In addition, the inhibition was effective during entry as well as in the early stages of post CHIKV-infection. Notably, TR showed remarkable virucidal activity. Next, global transcriptome profile demonstrated that the signaling by Retinoic acid pathway was highly upregulated after CHIKV infection and Retinoic acid receptors (RAR), specifically RAR-{beta} are the key mediators for the anti-CHIKV effect of TR. Further, the drug was also capable to modulate CHIKV-induced inflammatory responses by reducing the phosphorylated MAPKs, NF-{kappa}B and proinflammatory cytokines. Interestingly, TR protected C57BL/6 mice from CHIKV challenge by diminishing viral burden leading to reduced clinical scores and better survival. Similar observation was also noticed in the ex vivo model of CHIKV-infected hPBMC-derived monocyte-macrophage populations. In conclusion, this work demonstrated the repurposing potential of TR against CHIKV infection for the first time, encouraging its clinical validation towards future therapeutics. IMPORTANCECHIKV is an arbovirus causing Chikungunya Fever (CHIKF) leading to debilitating arthralgia and myalgia. The unavailability of licensed antivirals or potent vaccines against CHIKV encourages extensive research to find a therapeutic cure. Retinoids, the derivatives of vitamin A, have been repurposed as antiviral agents against a broad range of viruses; however, their efficacy against alphaviruses remains unexplored. Accordingly, Tretinoin (TR), also known as all-trans retinoic acid (ATRA), was validated for anti-CHIKV efficacy in this investigation. Significant perturbation of CHIKV titer through the modulation of Retinoic acid receptors (RAR) was observed upon drug-treatment. Further, TR reduced major inflammatory markers (MAPKs and proinflammatory cytokines) supporting its anti-inflammatory effect. Further, its anti-CHIKV potential was validated in the mouse model and hPBMC-derived monocyte-macrophage cells, indicating the pre-clinical efficacy. Finally, this is the first study to report the anti-CHIKV property of Tretinoin using in vitro, in vivo, and ex vivo approaches suggesting its repurposing potential.

microbiology↗

bioRxiv2025

A genetic screen to identify deubiquitinases as regulators of IRF7

Virus infection rapidly induces the production and secretion of interferons (IFNs), amplifying antiviral responses in infected and neighboring uninfected cells. IFN regulatory factor 7 (IRF7), the master transcription factor, is pivotal in IFN induction, particularly in myeloid cells. Ubiquitination of IRF7 is essential for its transcriptional activation; however, the underlying molecular mechanisms remain poorly understood. We hypothesized that deubiquitinases (DUBs) act as endogenous regulators of IRF7 activity and conducted a genetic screen using a human DUB-targeted siRNA library. This screen identified USP2 as a positive regulator and OTUD5 as a negative regulator of IRF7 activity. OTUD5, an inducible DUB, physically interacted with IRF7 and inhibited its K63-linked ubiquitination, thereby suppressing IRF7 activation. Conversely, USP2 promoted IRF7 activity by binding to IRF7 and removing K27-linked ubiquitin chains, which we found to be inhibitory. Specifically, K27-linked ubiquitination impeded phosphorylation of IRF7, a critical step for its activation. Collectively, our genetic screen and mechanistic studies uncovered USP2 and OTUD5 as novel modulators of IRF7 function, providing new insights into the regulation of antiviral immunity.

immunology↗

bioRxiv2025

Chikungunya virus susceptibility increases in the absence of mk2b and mk3 genes in zebrafish model

Chikungunya virus (CHIKV) infection imposes a significant socio-economic burden due to the absence of effective antiviral treatments and vaccines. Host factors are essential for the CHIKV life cycle, making them promising targets for antiviral therapy. Previous studies have identified Mitogen-activated protein kinase activated protein kinase 2 (MK2) and Mitogen-activated protein kinase activated protein kinase 3 (MK3) as key host factors in CHIKV infection; however, their role in an animal model remain unclear. This study highlights the critical roles of MK2 and MK3 host factors in an animal model following CHIKV infection. To investigate their functions, mk2b (mk2b-/-), mk3 (mk3-/-), and mk2b-mk3 (mk2b-/-mk3-/-) double knockout zebrafish were generated using the CRISPR-Cas9 technique. A significant high viral titer of CHIKV was observed in case of all knockout groups compared to the wild-type (WT) control using plaque assay, RT-qPCR and immunofluorescence assay. Among the knockout groups, mk3-/- displayed the highest susceptibility to CHIKV, followed by mk2b-/-. In contrast, the mk2b-/-mk3-/- double knockout exhibited the lowest susceptibility to CHIKV infection. Additionally, severe symptoms such as bent body, impaired response to physical stimuli, and increased mortality were most pronounced in mk3-/- larvae compared to other knockouts and the WT. The expression levels of inf{phi}1 and rsad2 were also elevated in all knockout groups during the early days of infection indicating higher interferon response in the absence of mk2b and mk3 during CHIKV infection. In conclusion, this study confirms that the mk2b and mk3 host proteins are essential in controlling the CHIKV infection in organism level and subsequently may contribute in designing antiviral therapeutics in future. Furthermore, the knockout model of mk2b and mk3 in zebrafish could serve as a valuable tool for studying their roles in other viral infections. Author SummaryCHIKV, transmitted by Aedes aegypti and Aedes albopictus mosquitoes, causes febrile illness and has spread across Africa, Asia, Europe, and America. Despite extensive research, effective antiviral drugs and vaccines are yet to be commercially available. This study examines the role of mk2b and mk3 host factors following CHIKV infection. For this purpose, mk2b and mk3 single as well as double knockout have been generated in zebrafish using the CRISPR-Cas-9 technique. Findings suggest that zebrafish exhibit high CHIKV susceptibility in the absence of mk2b and mk3, confirming its importance during infection. Moreover, these three knockout models could serve as a valuable platform for examine the role of mk2b and mk3 in the presence of other viral infection.

molecular biology↗

bioRxiv2025

Bayesian inference of cell-to-cell interactions in low-nutrient neuroblastoma cell cultures

Neuroblastoma is characterised by significant intratumoural heterogeneity which complicates treatments. Phenotypic plasticity, i.e., the ability of cells to alter their phenotype without genetic mutations, is a key factor contributing to this heterogeneity. In this study, we quantify cell actions and cell-to-cell interactions that lead to phenotypic adaptation in vitro under stress, specifically low-nutrient conditions. We initially record dynamic cell counts under various nutrient conditions for two neuroblastoma cell lines with known phenotype differences. Subsequently, we compile a list of plausible biological processes that could transpire within the aforementioned systems. We then construct candidate models employing mass-action kinetics. To quantitatively infer processes that lead to phenotypic adaptation, we perform a model selection based on computational Bayesian inference methods.Our results suggest that cell-to-cell interactions promote phenotypic adaptation and that the rate of this adaptation increases with decreasing nutrient concentrations. We perform flow cytometry-based experiments to corroborate changes in phenotype composition in response to both nutrient-deprived and treatmentinduced stress conditions.

cancer biology↗

bioRxiv2025

JNK2 pathway in colonocytes enhances gut barrier integrity in response to microbial acetate.

Weaning involves a nutritional shift from fat-rich milk to carbohydrate-based solid food, reshaping metabolism, microbiota, and gut immune tolerance. While dairy remains a component of the human diet beyond weaning, the impact of continued milk supplementation on gut epithelial homeostasis remains poorly understood. Here, using a mouse model, we show that continued milk-based feeding post-weaning promotes intestinal barrier function by enriching the commensal bacterium Dubosiella newyorkensis, which produces acetate to activate epithelial JNK2 signaling. This pathway enhances barrier integrity and suppresses inflammation induced by mild Dextran Sodium Sulphate (DSS) treatment. In contrast, feeding a lard-based high-fat diet or transient pharmacologic inhibition of JNK2 induces epithelial P38 activation, resulting in barrier disruption and inflammation. Importantly, the beneficial effects of milk were observed only if initiated during the weaning period, when the microbiome is in a metastable transitional state. Initiation of the same intervention two weeks after weaning led to P38 activation and inflammatory responses. These findings highlight the microbiome-dependent, stage-specific effects of diet and underscore the importance of early-life nutritional interventions for long-term gut health.

physiology↗

bioRxiv2024

Telmisartan impedes JEV infection predominately via AT1/PPARγ axis

Japanese encephalitis, caused by Japanese encephalitis virus (JEV) is a vector born disease for which no specific therapeutics are available yet. Binding of angiotensin II (Ang II) to angiotensin II type 1 (AT1) receptor induces release of inflammatory cytokines associated with viral encephalitis. Accordingly, Ang II receptor blockers (ARBs) have been proposed to manage encephalitis. Since Telmisartan (TM, antagonist of AT1 and agonist of PPAR{gamma}) has relatively better brain access than other ARBs, this investigation aims to evaluate its anti-JEV efficacy in vitro and in vivo. TM reduced JEV titer, RNA and protein (NS3) significantly in the BHK-21 cells with IC50 of 24.68M and CC50 of >350 M (Selectivity Index >14.18) indicating its potential of repurposing against JEV. The anti-JEV efficacy of TM was further observed in macrophages (RAW264.7) and neuronal (SH-SY5Y) cells. Interestingly, the viral load was reduced significantly in pre, co and post-treatment conditions of TM, however most efficiently (80%) in post treatment. In presence of GW (PPAR{gamma} antagonist) and AG (AT1 agonist), viral infection was increased remarkably while AT1 was upregulated and PPAR{gamma} was downregulated. Whereas, TM treatment reversed the levels of AT1 and PPAR{gamma} during infection. Further, reduction of inflammatory markers like p-IRF-3, COX-2 and p-NF-{kappa}B was observed after TM treatment in RAW264.7 cells suggesting its immunomodulation through the AT1/PPAR{gamma} axis. Finally, the anti-JEV potential of TM was validated in mice model through the reduction of disease score, viral protein and histological changes. Thus, the preclinical efficacy of TM suggests its suitability for repurposing against JEV.

microbiology↗

bioRxiv2024

Targeting Neuropilin-1 to Enhance Immunotherapy in Melanoma: Reducing Tumour Progression and Peripheral Treg-Mediated Immunosuppression

Melanoma, the most aggressive type of skin cancer with a high mutation rate, is the fifth most common cancer among Caucasians. Despite advancements in treatments like immune checkpoint inhibitors and targeted therapies, over 40% of patients experience immune-related side effects, presenting significant challenges. Neuropilin-1 (NRP1) has become an essential target in cancer therapy due to its overexpression in various cancers, where it enhances regulatory T cell (Treg) function and supports tumor growth, often leading to poor outcomes. This study investigated the effects of NRP1 inhibition in B16-F10 melanoma and its impact on immune responses regulated by Tregs. NRP1 was overexpressed in several cancers, including B16-F10 cells, compared to non-cancerous NIH-3T3 cells. Inhibiting NRP1 selectively caused apoptosis in B16-F10 cells without affecting NIH-3T3 cells. It also reversed the immunosuppression of splenic T cells induced by B16-F10-conditioned media, reducing Treg markers (NRP1, NKG2A, FOXP3), Treg activity, and the production of immunosuppressive cytokines (IL-10, IL-17A). Furthermore, NRP1 inhibition increased T cell proliferation and boosted the release of effector cytokines (TNF, IFN-{gamma}, IL-6, IL-2). NRP1 inhibition also suppressed the STAT, ERK MAPK, and Smad2/3 pathways while activating the PI3K/AKT pathway. In splenic T cells from B16-F10 tumor-bearing mice treated with an NRP1 inhibitor, there was a decrease in Treg markers and activity, along with enhanced T cell proliferation. Additionally, NRP1 inhibitor treatment reduced lung metastasis, decreased tumor size, and improved survival in these mice. This study shows that inhibiting NRP1 may slow B16-F10 melanoma progression and reduce Treg-mediated immunosuppression. This suggests its potential as a promising approach in future cancer immunotherapies, especially in combination with other treatments.

immunology↗

bioRxiv2024

Immunogenicity and protection efficacy of self-amplifying and circular mRNA vaccines for SARS-CoV-2

Recent advances in vaccine technology have positioned messenger RNA (mRNA) vaccines as safe and reliable options for human use. Conventionally, mRNA vaccines were designed using linear or self-amplifying mRNA (SAM), the latter considered to be superior. However, limited success was achieved with SAM vaccines during the COVID-19 pandemic. Further, studies on Circular mRNA (Circ-RNA) vaccines against the SARS-CoV-2, Ebola and monkey pox proved their efficacy. Circ-RNAs are highly stable, neither they induce inflammatory response nor require any extracellular protein for their function. Here, we compared the efficacy of SAM- and Circ-RNA vaccines using the SARS-CoV-2-RBD (receptor binding domain) as the antigen. Both SAM-RBD and Circ-RBD induced a comparable anti-RBD IgG titer and virus-neutralizing antibody titer. However, the latter induced a significantly higher memory T-cell response. Immunization with SAM- and Circ-RBD showed no mortality and improved lung pathophysiology against acute SARS-CoV-2 infection in mice. The Circ-RBD vaccine is stable for 4 weeks at 40C. A bivalent vaccine containing Circ-RBD of both delta and omicron SARS-CoV-2 variants potently neutralized these viruses. These findings demonstrate Circ-RNA-RBD as an excellent vaccine candidate against COVID-19 and also provide a platform for developing bivalent Circ-RNA vaccine candidates against SARS-CoV-2 or other viruses with rapidly emerging variants.

microbiology↗

bioRxiv2024

TLR9 signaling requires ligand-induced phosphorylation of two specific tyrosine residues by EGFR and Syk

Toll-like receptors (TLRs) are transmembrane proteins required for recognizing microbial components or cellular danger signals to activate intracellular signaling pathways leading to induction of anti-microbial and inflammatory genes. Inactive TLRs require ligand-induced activation to recruit adaptor proteins, e.g., MyD88, to trigger the synthesis of cytokines and interferons. TLR9 is an endosomal membrane-bound protein, which binds to CpG-containing microbial DNA or endogenous signals from dead cells or tissue damage. We showed that TLR9 activation requires EGFR, a tyrosine (Tyr) kinase, which interacts with and phosphorylates the cytoplasmic domain of TLR9. Blocking EGFR activity pharmacologically, or knocking out EGFR gene in myeloid cells, suppressed lethal TLR9-induced hepatotoxicity. Here, we reveal that TLR9 required two Src family of kinases, Syk and Lyn, which, together with EGFR, led to phosphorylation and activation of TLR9. Lack of either of these kinases inhibited TLR9-MyD88 interaction, thereby inhibiting TLR9-mediated gene induction. Unlike EGFR, which constitutively binds TLR9, activated Syk interacted with TLR9 in a CpG-dependent manner. Activated Syk interacted with TLR9 and was critical for activating TLR9-bound EGFR. Quantitative mass spectrometric analyses revealed TLR9 was phosphorylated, sequentially, on Tyr870 and Tyr980 by Syk and EGFR, respectively. Mutation of either of these tyrosines led to complete loss of TLR9-induced cytokine production. For activation, Syk was phosphorylated by Lyn, which was activated by CpG-mediated scavenger-receptor A, and surprisingly, independent of TLR9. In summary, our results uncovered the molecular details of TLR9 activation by its Tyr-phosphorylation, which is critical for TLR9-mediated intracellular signaling.

immunology↗

bioRxiv2023

Calcitriol impairs the secretion of IL-4 and IL-13 in Th2 cells via modulating the transcription factors Gata3 and Gfi1

Calcitriol, the bioactive form of vitamin D, exerts its biological functions by binding to its cognate receptor VDR. The indicators of the severity of allergies and asthma have been linked to low vitamin D levels. However, the role of calcitriol in regulating IL-4 and IL-13; two cytokines pivotal to allergic inflammation, remained unclear. Our study observed a decrease in IL-4 and IL-13 secretion in murine and human Th2 cells, treated with calcitriol. In murine Th2 cells, Gata3 expression was attenuated by calcitriol. However, the expression of the transcriptional repressor Gfi1, that prevents Th1 fate of activated CD4+ T cells, too was attenuated in the presence of calcitriol. Ectopic expression of either Gfi1 or VDR impaired the secretion of IL-13 in Th2 cells. Gfi1 significantly impaired Il13 promoter activation; which calcitriol failed to restore. Ecr, a conserved region between the two genes, which enhanced the transactivation of Il4 and Il13 promoters; is essential for calcitriol-mediated suppression of both the genes. In murine Th2 cells, VDR interacted with Gata3 but not Gfi1. Calcitriol augmented the recruitment of VDR to the Il13 promoter and Ecr regions. Gata3 recruitment was significantly impaired at Il13 and Ecr locus in the presence of calcitriol but increased at Il4 promoter. However, in the presence of calcitriol, the overall recruitment of Gfi1 remained unchanged at Il4 but increased at Il13 locus in Th2 cells. Together, our study clearly elucidates that calcitriol modulates VDR, Gata3, and Gfi1 to suppress IL-4 and IL-13 production in Th2 cells. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=81 SRC="FIGDIR/small/569823v3_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@1557aaborg.highwire.dtl.DTLVardef@e8b1dcorg.highwire.dtl.DTLVardef@f505dcorg.highwire.dtl.DTLVardef@53b31c_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2023

Single-cell transcriptome and TCR analysis of human Cytomegalovirus (hCMV)-specific T cell memory reveals effector and pre-effectors of CD8+ and CD4+ cytotoxic T cells

BackgroundLatent human Cytomegalovirus (hCMV) infection can pose a serious threat of reactivation and disease occurrence in immune-compromised individuals. Though, T cells are at the core of the protective immune response to hCMV infection, a detailed characterization of different T cell subsets involved in hCMV immunity is lacking. ResultsHere, in an unbiased manner, we characterized over 8000 hCMV-reactive peripheral memory T cells isolated from seropositive human donors, at a single-cell resolution by analyzing their single-cell transcriptomes paired with the T cell antigen receptor (TCR) repertoires. The hCMV-reactive T cells were highly heterogeneous and consisted of different developmental and functional memory T cell subsets such as, long-term memory precursors and effectors, T helper-17, T regulatory cells (TREGs) and cytotoxic T lymphocytes (CTLs) of both CD4 and CD8 origin. The hCMV-specific TREGs, in addition to being enriched for molecules known for their suppressive functions, showed enrichment for the interferon response signature gene sets. The hCMV-specific CTLs were of two types, the pre-effector and effector-like. The co-clustering of hCMV-specific CD4-CTLs and CD8-CTLs in both pre-effector as well as effector clusters suggest shared transcriptomic signatures between them. The huge TCR clonal expansion of cytotoxic clusters suggest a dominant role in protective immune response to CMV. ConclusionsThe study uncovers the heterogeneity in the hCMV-specific memory T cells reveling many functional subsets with potential implications in better understanding of hCMV-specific T cell immunity. The data presented can serve as a knowledge base for designing vaccines and therapeutics.

immunology↗

bioRxiv2022

A mechanistic insight into Nigella sativa mediated anticancer effect on breast cancer through regulation of various miRNAs: An in vitro & in vivo study

BackgroundCancer continues to threat the mortal alongside scientific community with its burgeoning grasp. Although efforts have been directed to tame cancer by radiotherapy, photodynamic therapy, chemotherapy it came at a cost of fatal side effects. PurposePlant derived bioactive compounds carries an inevitable advantage of being safer, bioavailable & less toxic compared to contemporary chemotherapeutics. This study analyzed anti-cancerous potential of volatile oil, extracted from Nigella sativa, in-vitro against MDA-MB-231, MCF-7 & in-vivo on tumor growth in mice after successful oral administration. Study DesignOur strategic approach employed solvent extraction of black seed oil (BSO) to highlight orchestrated use of its potent integrants - TQ, Carvacrol & TA which in modest amounts show anti-cancerous properties compared to their individual treatment. We attempted to show this cost effective, safe & bioavailable form of dealing with the atrocities of breast cancer by means of MTT, Apoptotic, Western Blot Assays besides Transwell & Wound healing Assay. Reduction in the solid tumour in-vivo & near normalcy restoration of tissue section architecture from the BSO treated tumour sets are indicative of the better anti-tumorigenic potential of BSO. MethodsBSOs Solvent extraction was performed followed by its characterization. MTT aided cytotoxicity study of BSO alongside major components in PBMC & cancer cells while its efficacy was presented by flow cytometric ROS analysis, cell cycle arrest & apoptosis assessment. Anti-migratory potential evaluated by Wound Healing, Transwell Migration & Western Blot while the expression study of a wide range of proteins, miRNAs & the in-vivo studies undertaken climaxed the confirmation of the anti-cancerous potential of BSO. ResultsComparatively reduced concentrations of TQ, TA & Carvacrol in BSO played a synergistic role to enhance apoptotic potential via Caspase 7 & 9, through enhanced ROS & expression of apoptotic family of proteins, miRNAs besides uplifting the anti-migratory perspective by effectively enhancing E-cad & downregulating lamellipodia, filopodia assembly & MMPs in MCF-7 & MDA-MB-231. Similar observations in-vivo outlined the therapeutic potential of BSO. ConclusionThis study culminates isolation & processing of BSO in a simplified procedure, thereby aiming at a more lucrative paradigm to be accepted in contemporary phytomedicine research.

cancer biology↗

bioRxiv2022

Ancestral SARS-CoV-2 driven antibody repertoire diversity in an unvaccinated individual correlates with expanded neutralization breadth

Understanding the quality of immune repertoire triggered during natural infection can provide vital clues that form the basis for development of humoral immune response in some individuals capable of broadly neutralizing pan SARS-CoV-2 variants. We assessed the diversity of neutralizing antibody responses developed in an unvaccinated individual infected with ancestral SARS-CoV-2 by examining the ability of the distinct B cell germline-derived monoclonal antibodies (mAbs) in neutralizing known and currently circulating Omicron variants by pseudovirus and authentic virus neutralization assays. The ability of the antibodies developed post vaccination in neutralizing Omicron variants was compared to that obtained at baseline of the same individual and to those obtained from Omicron breakthrough infected individuals by pseudovirus neutralization assay. Broadly SARS-CoV-2 neutralizing mAbs representing unique B cell lineages with non-overlapping epitope specificities isolated from a single donor varied in their ability to neutralize Omicron variants. Plasma antibodies developed post vaccination from this individual demonstrated neutralization of Omicron BA.1, BA.2 and BA.4 with increased magnitude and found to be comparable with those obtained from other vaccinated individuals who were infected with ancestral SARS-CoV-2. Development of B cell repertoire capable of producing antibodies with distinct affinity and specificities for the antigen immediately after infection capable of eliciting broadly neutralizing antibodies offers highest probability in protecting against evolving SARS-CoV-2 variants. ImportanceDevelopment of robust neutralizing antibodies in SARS-CoV-2 convalescent individuals is known, however varies at population level. We isolated monoclonal antibodies from an individual infected with ancestral SARS-CoV-2 in early 2020 that not only varied in their B cell lineage origin but also varied in their capability and potency to neutralize all the known VOC and currently circulating Omicron variants. This indicated establishment of unique lineages that contributed in forming B cell repertoire in this particular individual immediately following infection giving rise to diverse antibody responses that could compensate each other in providing broadly neutralizing polyclonal antibody response. Individuals who were able to produce such potent polyclonal antibody responses after infection have a higher chance of being protected from evolving SARS-CoV-2 variants.

immunology↗