Browsing by Author "Hiral Thacker"

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Autoimmunity and clinical pathology amelioration in SLE by dexamethasone primed mesenchymal stem cell derived conditioned media
(BioMed Central Ltd, 2025) Khushbu Priya; Sonali Rawat; Doli Das; Manaswi Chaubey; Hiral Thacker; Kiran Rajendra Giri; Shambhavi Singh; Madhukar Rai; Sujata P. Mohanty; Geeta Rai
Background: This study aimed to investigate the therapeutic potential of cell-free Dexamethasone (Dex) primed Wharton’s jelly Mesenchymal stem cells derived conditioned media (DW) in addressing complications associated with systemic lupus erythematosus (SLE), focusing on its immunomodulatory effects. Methods: Peripheral blood mononuclear cells from 74 SLE patients were stimulated and treated with Dex, DW and W. Culture supernatant were evaluated for autoantibody levels, IL-10 and TGF-β by ELISA, Treg subtypes, Breg subtypes, TH17 cells Double negative T cells and inflammatory neutrophils by flow cytometry, IL-10 and IL-17A by qPCR. In vivo studies were performed on 60 pristane induced female BALB/c mice. Dex and DW treatments were evaluated for autoantibody production, proteinuria, immunomodulation of immune cells, organ function, and histopathology. In vivo imaging of internal organs was done using VevoLAZR-X photoacoustic imaging system. Results: DW treatment significantly expanded different Treg and Bregs subtypes. DW suppressed pathogenic TH17, Double negative T cells and inflammatory neutrophils. Comparative analyses with hydroxychloroquine showed similar effects, with combined treatment enhancing efficacy. Inhibition studies implicated the TGF-β pathway in DW's mechanism. In vivo studies using the PIL mouse model showed that DW treatment reduced mortality, prevented proteinuria, and ameliorated symptoms such as limb inflammation, seizures, and alopecia. Detailed organ-specific evaluations through live imaging and histopathological analyses revealed DW’s protective effects on kidneys, liver, lungs, heart, and spleen. Conclusion: DW shows promise as a cell-free biological therapy for SLE and related autoimmune disorders, capable of modulating immune responses effectively without the adverse effects of glucocorticoids. © The Author(s) 2025.
Clinical Outcomes of Defective Hematopoiesis
(Apple Academic Press, 2024) Geeta Rai; Doli Das; Khushbu Priya; Hiral Thacker
The process of production of immune cells through hematopoiesis is an interesting paradigm in stem cell research providing an array of researchable components. Hematopoietic stem cells can differentiate into multiple committed lineages, ensuring the generation of cells with specific functions and also retaining the self-renewing capability. HSCs play a major role in disease biology as irregularities in HSCs generation and differentiation lead to life-threatening diseases. In-depth studies of these defective mechanisms not only reveal the pathophysiology of hematological diseases, including malignancies, immunodeficiencies, bone marrow (BM) failure syndromes, and hemoglobinopathies, but also helps to understand the survival and function of HSCs, thus opening a new door of translational research. © 2024 Apple Academic Press, Inc.
Complement component 5a receptor 1 and leukotriene B4 receptor 1 regulate neutrophil extracellular trap (NET) formation through Rap1a/B-Raf/ERK signaling pathway and their deficiency in term low birth weight newborns leads to deficient NETosis
(Elsevier B.V., 2024) Doli Das; Hiral Thacker; Khushbu Priya; Madhu Jain; Shambhavi Singh; Geeta Rai
Background: Neutrophil extracellular traps (NETs) being one of the predominant activities of neutrophils has become its key defense mechanism owing to its extensive role in inflammation and infection. However, the mechanisms regulating NET formation or NETosis still remains to be better understood. Our earlier whole genome transcriptomic data revealed two G-protein couple receptors (GPCRs) − complement component 5a receptor 1 (C5aR1) and leukotriene B4 receptor 1 (LTB4R1) were downregulated in term low birth weight (tLBW) newborns with deficient NET formation abilities. Neutrophils employ C5aR1 and LTB4R1 for mediating their immune responses, inflammation and antimicrobial activity. Hence, this study was aimed to explore the role of two GPCRs, C5aR1 and LTB4R1 including their downstream signaling molecules in NETs induction and regulation. Methods: The validation of the transcriptomic data for C5aR1 and LTB4R1 was done using quantitative real time PCR. Pharmacological inhibition of C5aR1 and LTB4R1 using W-54011 and LY223982 on neutrophils of adults and newborns’ was done to study their impact on NETosis. Extracellular DNA release, Reactive oxygen species (ROS) generation, expression of NET proteins, and signaling molecules downstream to C5aR1 and LTB4R1 were quantified using plate reader based assay, immunofluorescence, and western blotting. Myeloperoxidase (MPO)-DNA quantified by flow cytometry. Knockdown studies using siRNA against C5aR1 and LTB4R1 were done in HL-60 cells derived surrogate neutrophils and expression of downstream molecules of the two GPCRs, C5aR1 and LTB4R1 signaling axis along with NET proteins was quantified by western blotting. Results: The expression of C5aR1 and LTB4R1, extracellular DNA, ROS and NET associated proteins (NE, CitH3, PAD4 and MPO) was notably increased upon NET induction in healthy adults and normal birth weight (NBW) newborns’ neutrophils. Pharmacological inhibition of these two GPCRs led to substantial reduction in NETosis, extracellular DNA, ROS generation, and expression of NET associated proteins like CitH3, NE, PAD4, MPO along with downstream signaling molecules Rap1a, B-Raf and pERK. Our observations suggest a precise role of C5aR1 and LTB4R1 on induction of NETs via Rap1a/B-Raf/ERK signaling axis. Conclusion: The C5aR1 and LTB4R1 signaling via Rap1a/B-Raf/ERK axis acts as a signal-relay mechanism to regulate NET formation in neutrophils. Further, C5aR1 and LTB4R1 signaling cascade along with NET-associated proteins are remarkably downregulated in tLBW newborns’ neutrophils leading to impaired NETosis in them. Therefore, C5aR1 and LTB4R1 and their signaling molecules could provide an effective therapeutic target for compromised NETosis like tLBW newborns. © 2024
Dexamethasone and IFN-γ primed mesenchymal stem cells conditioned media immunomodulates aberrant NETosis in SLE via PGE2 and IDO
(Frontiers Media SA, 2024) Khushbu Priya; Hiral Thacker; Manaswi Chaubey; Madhukar Rai; Shambhavi Singh; Sonali Rawat; Kiran Giri; Sujata Mohanty; Geeta Rai
Background: Systemic Lupus Erythematosus (SLE) is characterized by dysregulated immune responses, with neutrophil extracellular traps (NETs) playing a significant role. NETs are recognized by autoantibodies in SLE patients, exacerbating pathology. Both excessive NET formation and impaired degradation contribute to SLE pathophysiology. Objective: To investigate the immunomodulatory effects of Dexamethasone-primed Wharton’s jelly (WJ) derived MSCs CM (DW) and IFN-γ-primed WJ-MSCs-CM (IW) on NETosis and associated protein markers in SLE patients’ LPS or ribonucleoprotein immune complexes (RNP ICs) induced neutrophils and in pristane induced lupus (PIL) model. And to elucidate the mechanism involved therein. Methods: We investigated the immunomodulatory effects of DW and IW on NETosis in SLE. Utilizing ex vivo and in vivo models, we assessed the impact of preconditioned media on NET formation and associated protein markers neutrophil elastase (NE), citrullinated histone (citH3), myeloperoxidase (MPO), cytoplasmic and mitochondrial ROS production. We also examined the involvement of key immunomodulatory factors present in DW and IW, including prostaglandin E2 (PGE2), indoleamine 2,3-dioxygenase (IDO), and transforming growth factor-beta (TGF-β). Results: Preconditioned media effectively suppressed NETosis and reduced ROS generation in SLE neutrophils, indicating their immunomodulatory potential. Inhibition studies implicated IDO and PGE2 in mediating this effect. Combined treatment with DW or IW together with hydroxychloroquine (HCQ) demonstrated superior efficacy over HCQ alone, a standard SLE medication. In PIL mouse model, DW and IW treatments reduced NETosis, ROS generation, as evidenced by decreased NET-associated protein expression in vital organs. Conclusion: Our study highlights the multifaceted impact of IW and DW on NETosis, ROS dynamics, and lupus severity in SLE. These findings underscore the potential of preconditioned media for the development of targeted, personalized approaches for SLE treatment. Copyright © 2024 Priya, Thacker, Chaubey, Rai, Singh, Rawat, Giri, Mohanty and Rai.
Immunogenetic perspective of inflammatory disorders
(Elsevier, 2022) Geeta Rai; Hiral Thacker; Doli Das; Khushbu Priya
Understanding of the genetic basis underlying inflammatory disorders has progressed in recent years. Contribution of proinflammatory cytokines, human leukocyte antigen (HLA), and non-HLA polymorphisms in the pathogenesis of several autoimmune and immune-mediated inflammatory disorder is critical. HLA plays a central role in disease pathology. Harmful stimuli triggering the signaling mechanisms including nuclear factor-kappa B pathway, Janus kinase-signal transducer and activator of transcription pathway, and mitogen-activated protein kinase pathway results in the release of inflammatory mediators. From acute to chronic inflammation, the etiology of various inflammatory disorders is poorly understood. Inflammatory disorder such as COVID 19 is a devastating havoc to the world. As we reach the end of 2020, >1 million people have succumbed to death worldwide. Disease-manifesting clinical features include mild to severe pneumonia, loss of respiratory function progressing to acute respiratory distress syndrome with occasional multiorgan failure. Cytokine storm, decreased T cell count, and insufficient immune response are conducive issues to COVID 19 pandemic. Varied immune responses to the same antigen across different individuals determine the genetic perspective of disease susceptibility. Through genome-wide association studies, next-generation sequencing and other genetic techniques, several genetic risk loci associated with various inflammatory diseases such as inflammatory bowel disease, psoriasis, sclerosis, and systemic lupus erythematosus (SLE) have been identified. Dysregulated inflammatory pathways, gene mutation, or elevated cytokine level may lead to the disease progression. However, the production of autoantibodies against the nuclear antigens is a hallmark of diseases like SLE and rheumatoid arthritis. Moreover, environmental factors like smoking also increase the risk of inflammatory disorders. Understanding the functional aspects of casual genetic factors underlying the disease pathogenesis greatly facilitates the ability to identify the therapeutic targets relevant to disease. The current chapter deals with the idea of genetic perspective associated with various inflammatory disorders and their potential therapeutic targets along with the factors contributing to disease susceptibility. © 2022 Elsevier Inc. All rights reserved.