MicroRNA-21 Deficiency Promotes the Early Th1 Immune Response and Resistance toward Visceral Leishmaniasis.

MicroRNA-21 (miR-21) inhibits IL-12 expression and impairs the Th1 response necessary for control of Leishmania infection. Recent studies have shown that Leishmania infection induces miR-21 expression in dendritic cells and macrophages, and inhibition of miR-21 restores IL-12 expression. Because miR-21 is known to be expressed due to inflammatory stimuli in a wide range of hematopoietic cells, we investigated the role of miR-21 in regulating immune responses during visceral leishmaniasis (VL) caused by Leishmania donovani infection. We found that miR-21 expression was significantly elevated in dendritic cells, macrophages, inflammatory monocytes, polymorphonuclear neutrophils, and in the spleen and liver tissues after L. donovani infection, concomitant with an increased expression of disease exacerbating IL-6 and STAT3. Bone marrow dendritic cells from miR-21 knockout (miR-21KO) mice showed increased IL-12 production and decreased production of IL-10. On L. donovani infection, miR-21KO mice exhibited significantly greater numbers of IFN-γ- and TNF-α-producing CD4+ and CD8+ T cells in their organs that was associated with increased production of Th1-associated IFN-γ, TNF-α, and NO from the splenocytes. Finally, miR-21KO mice displayed significantly more developing and mature hepatic granulomas leading to reduction in organ parasitic loads compared with wild type counterparts. Similar results were noted in L. donovani-infected wild type mice after transient miR-21 depletion. These observations indicate that miR-21 plays a critical role in pathogenesis of VL by suppressing IL-12- and Th1-associated IFN-γ and also inducing disease-promoting induction of the IL-6 and STAT-3 signaling pathway. miR-21 could therefore be used as a potential target for developing host-directed treatment for VL.

MicroRNA155 Plays a Critical Role in the Pathogenesis of Cutaneous Leishmania major Infection by Promoting a Th2 Response and Attenuating Dendritic Cell Activity.

Interferon (IFN)-γ is indispensable in the resolution of cutaneous leishmaniasis (CL), while the Th2 cytokines IL-4, IL-10, and IL-13 mediate susceptibility. A recent study found that miR155, which promotes CD4+ Th1 response and IFN-γ production, is dispensable in the control of Leishmania donovani infection. Here, the role of miR155 in CL caused by L. major was investigated using miR155-deficient (miR155-/-) mice. Infection was controlled significantly quicker in the miR155-/- mice than in their wild-type (WT) counterparts, indicating that miR155 contributes to the pathogenesis of CL. Faster resolution of infection in miR155-/- mice was associated with increased levels of Th1-associated IL-12 and IFN-γ and reduced production of Th2- associated IL-4, IL-10, and IL-13. Concentrations of IFN-γ+CD8+ T cells and natural killer cells in draining lymph nodes were significantly higher in the L. major-infected miR155-/- mice than in the infected WT mice, as indicated by flow-cytometry. After in vitro IFN-γ stimulation, nitric oxide and IL-12 production were increased, IL-10 production was decreased, and parasite clearance was enhanced in L. major-infected miR155-/- DCs compared to those in WT DCs. Furthermore, IFN-γ production from activated miR155-/- T cells was significantly enhanced in L. major-infected miR155-/- DCs. Together, these findings demonstrate that miR155 promotes susceptibility to CL caused by L. major by promoting Th2 response and inhibiting DC function.

Understanding the immune responses involved in mediating protection or immunopathology during leishmaniasis.

Leishmaniasis is a vector-borne Neglected Tropical Disease (NTD) transmitted by the sand fly and is a major public health problem worldwide. Infections caused by Leishmania clinically manifest as a wide range of diseases, such as cutaneous (CL), diffuse cutaneous (DCL), mucosal (MCL) and visceral leishmaniasis (VL). The host innate and adaptative immune responses play critical roles in the defense against leishmaniasis. However, Leishmania parasites also manipulate the host immune response for their survival and replication. In addition, other factors such as sand fly salivary proteins and microbiota also promote disease susceptibility and parasite spread by modulating local immune response. Thus, a complex interplay between parasite, sand fly and the host immunity governs disease severity and outcome. In this review, we discuss the host immune response during Leishmania infection and highlight the factors associated with resistance or susceptibility.

Ibrutinib treatment inhibits breast cancer progression and metastasis by inducing conversion of myeloid-derived suppressor cells to dendritic cells.

BACKGROUND: Ibrutinib is a Bruton's tyrosine kinase (BTK) and interleukin-2-inducible kinase (ITK) inhibitor used for treating chronic lymphocytic leukaemia (CLL) and other cancers. Although ibrutinib is known to inhibit the growth of breast cancer cell growth in vitro, its impact on the treatment and metastasis of breast cancer is unclear. METHODS: Using an orthotopic mouse breast cancer model, we show that ibrutinib inhibits the progression and metastasis of breast cancer. RESULTS: Ibrutinib inhibited proliferation of cancer cells in vitro, and Ibrutinib-treated mice displayed significantly lower tumour burdens and metastasis compared to controls. Furthermore, the spleens and tumours from Ibrutinib-treated mice contained more mature DCs and lower numbers of myeloid-derived suppressor cells (MDSCs), which promote disease progression and are linked to poor prognosis. We also confirmed that ex vivo treatment of MDSCs with ibrutinib switched their phenotype to mature DCs and significantly enhanced MHCII expression. Further, ibrutinib treatment promoted T cell proliferation and effector functions leading to the induction of antitumour TH1 and CTL immune responses. CONCLUSIONS: Ibrutinib inhibits tumour development and metastasis in breast cancer by promoting the development of mature DCs from MDSCs and hence could be a novel therapeutic agent for the treatment of breast cancer.

Deciphering the role of calcium homeostasis in T cells functions during mycobacterial infection.

Calcium plays an important role in regulating cell physiology and immune responses to various pathogens. Our recent work has highlighted the crucial role for calcium homeostasis in dendritic cells and macrophages during various infections. Here we investigated the effect of calcium homeostasis in regulating T cell activation and function during mycobacterial infection. Results show that calcium homeostasis had varied effects in regulating T cell activation and function during mycobacterial infection. This included regulation of the expression of co-stimulatory molecules, cytokine profiles and effector function. A net negative role for Voltage Gated Calcium Channel (VGCC) was observed. Inhibiting VGCC in mycobacteria primed T cells induced increased production of pro-inflammatory cytokines and an increased effector phenotype. Infected macrophages when incubated with VGCC inhibited T cells, induced increased expression of co-stimulatory molecule expression on macrophages, increased the production of pro-inflammatory cytokines and increased autophagy and apoptosis. This collectively led to reduced survival of mycobacteria inside macrophages. The data point towards a fine regulation of protective responses by routes of calcium influx and release that mediate pathogen survival or clearance.

Exploring the potential of drug repurposing for treating depression.

Researchers are interested in drug repurposing or drug repositioning of existing pharmaceuticals because of rising costs and slower rates of new medication development. Other investigations that authorized these treatments used data from experimental research and off-label drug use. More research into the causes of depression could lead to more effective pharmaceutical repurposing efforts. In addition to the loss of neurotransmitters like serotonin and adrenaline, inflammation, inadequate blood flow, and neurotoxins are now thought to be plausible mechanisms. Because of these other mechanisms, repurposing drugs has resulted for treatment-resistant depression. This chapter focuses on therapeutic alternatives and their effectiveness in drug repositioning. Atypical antipsychotics, central nervous system stimulants, and neurotransmitter antagonists have investigated for possible repurposing. Nonetheless, extensive research is required to ensure their formulation, effectiveness, and regulatory compliance.

Genetic Variation in Toll-Like Receptors (TLRs) 2, 4, and 9 Influences HIV Disease Progression Toward Active TB and AIDS.

Background: Toll-like receptors (TLRs) are identified as one of the key components of the innate immune system. The objective of this study was to explore the influence of genetic variability in these TLRs on human immunodeficiency virus (HIV) disease progression with and without tuberculosis (TB) co-infection. Materials and Methods: This prospective, cross-sectional, and longitudinal study included 373 HIV-positive patients without TB infection. This study aimed to examine the genetic variation in TLRs (TLR2, TLR4, and TLR9) between patients with HIV-1 infection and those who progressed to active TB during the two years of follow-up. Results: During the two year follow-up of 373 positive patients, 98 patients progressed to active TB/AIDS (acquired immunodeficiency syndrome). When comparing 98 HIV patients who developed active TB/AIDS to 275 HIV patients who did not, it was discovered that the frequency of the A allele in TLR9 was considerably higher (p <0.001) in HIV patients progressed to active TB/AIDS. Ninety eight HIV individuals who advanced to active TB/AIDS showed a significantly higher frequency of the AA genotype in TLR9 than did in HIV patients who had no TB/AIDS (p <0.001). Conclusion: The increased association of the AA genotype of TLR9 in HIV patients who progressed to active TB during follow-up suggests that HIV-positive patients with the AA genotype of TLR9 have increased susceptibility towards TB during the disease progression.

Butyrate induces oxidative burst mediated apoptosis via Glucose-6-Phosphate Dehydrogenase (G6PDH) in macrophages during mycobacterial infection.

Microorganisms present in the gut modulate host defence responses against infections in order to maintain immune homeostasis. This host-microbe crosstalk is regulated by gut metabolites. Butyrate is one such small chain fatty acid produced by gut microbes upon fermentation that has the potential to influence immune responses. Here we investigated the role of butyrate in macrophages during mycobacterial infection. Results demonstrate that butyrate significantly suppresses the growth kinetics of mycobacteria in culture medium as well as inhibits mycobacterial survival inside macrophages. Interestingly, butyrate alters the pentose phosphate pathway by inducing higher expression of Glucose-6-Phosphate Dehydrogenase (G6PDH) resulting in a higher oxidative burst via decreased Sod-2 and increased Nox-2 (NADPH oxidase-2) expression. Butyrate-induced G6PDH also mediated a decrease in mitochondrial membrane potential. This in turn lead to an induction of apoptosis as measured by lower expression of the anti-apoptotic protein Bcl-2 and a higher release of Cytochrome C as a result of induction of apoptosis. These results indicate that butyrate alters the metabolic status of macrophages and induces protective immune responses against mycobacterial infection.

Epigenetics in renal diseases.

With aging, prevalence of obesity, hypertension, diabetes and renal diseases have increased globally. Over the last two decades, the prevalence of renal diseases has been intensely increasing. Renal disease and renal programming are regulated by epigenetic modifications like DNA methylation and histone modifications. Environmental factors have significant role in the pathophysiology of renal disease progression. Understanding the potential of epigenetic regulation of gene expression may be useful in renal disease prognosis, diagnosis and provides novel therapeutic measures. In a nutshell, this chapter talks about the role of epigenetic mechanisms-DNA methylation, histone modification, and noncoding RNA in different renal diseases. These include diabetic kidney disease, diabetic nephropathy, renal fibrosis, etc.

Receptors of immune cells mediates recognition for tumors.

Over the last few decades, the immune system has been steered toward eradication of cancer cells with the help of cancer immunotherapy. T cells, B cells, monocytes/macrophages, dendritic cells, T-reg cells, and natural killer (NK) cells are some of the numerous immune cell types that play a significant part in cancer cell detection and reduction of inflammation, and the antitumor response. Briefly stated, chimeric antigen receptors, adoptive transfer and immune checkpoint modulators are currently the subjects of research focus for successful immunotherapy-based treatments for a variety of cancers. This chapter discusses ongoing investigations on the mechanisms and recent developments by which receptors of immune cells especially that of lymphocytes and monocytes/macrophages regulate the detection of immune system leading to malignancies. We will also be looking into the treatment strategies based on these mechanisms.

Role of lymphocytes, macrophages and immune receptors in suppression of tumor immunity.

Cancer is now the leading cause of mortality across the world. Inflammatory immune cells are functionally important in the genesis and progression of tumors, as demonstrated by their presence in human tumors. Numerous research has recently been conducted to determine if the innate and adaptive immune systems' cytotoxic cells can inhibit tumor growth and spread. Majority of cancers, when growing into identifiable tumors use multiple strategies to elude immune monitoring by lowering tumor immunity. Immunological suppression in the tumor microenvironment is achieved through interfering with antigen-presenting cells and effector T cells. Treatment of cancer requires managing both the tumor as well as tumor microenvironment (TME). Most patients will not be able to gain benefits from immunotherapy because of the immunosuppressive tumor microenvironment. The actions of many stromal myeloid and lymphoid cells are regulated to suppress tumor-specific T lymphocytes. These frequently exhibit inducible suppressive processes brought on by the same anti-tumor inflammatory response the immunotherapy aims to produce. Therefore, a deeper comprehensive understanding of how the immunosuppressive environment arises and endures is essential. Here in this chapter, we will talk about how immune cells, particularly macrophages and lymphocytes, and their receptors affect the ability of tumors to mount an immune response.

Genetic Analysis of HIBM Myopathy-Specific GNE V727M Hotspot Mutation Identifies a Novel COL6A3 Allied Gene Signature That Is Also Deregulated in Multiple Neuromuscular Diseases and Myopathies.

The GNE-associated V727M mutation is one of the most prevalent ethnic founder mutations in the Asian HIBM cohort; however, its role in inducing disease phenotype remains largely elusive. In this study, the function of this hotspot mutation was profoundly investigated. For this, V727M mutation-specific altered expression profile and potential networks were explored. The relevant muscular disorder-specific in vivo studies and patient data were further analyzed, and the key altered molecular pathways were identified. Our study found that the GNEV727M mutation resulted in a deregulated lincRNA profile, the majority of which (91%) were associated with a down-regulation trend. Further, in silico analysis of associated targets showed their active role in regulating Wnt, TGF-ß, and apoptotic signaling. Interestingly, COL6a3 was found as a key target of these lincRNAs. Further, GSEA analysis showed HIBM patients with variable COL6A3 transcript levels have significant alteration in many critical pathways, including epithelial-mesenchymal-transition, myogenesis, and apoptotic signaling. Interestingly, 12 of the COL6A3 coexpressed genes also showed a similar altered expression profile in HIBM. A similar altered trend in COL6A3 and coexpressed genes were found in in vivo HIBM disease models as well as in multiple other skeletal disorders. Thus, the COL6A3-specific 13 gene signature seems to be altered in multiple muscular disorders. Such deregulation could play a pivotal role in regulating many critical processes such as extracellular matrix organization, cell adhesion, and skeletal muscle development. Thus, investigating this novel COL6A3-specific 13 gene signature provides valuable information for understanding the molecular cause of HIBM and may also pave the way for better diagnosis and effective therapeutic strategies for many muscular disorders.

Cancer Vaccines in the Immunotherapy Era: Promise and Potential.

Therapeutic vaccines are a promising alternative for active immunotherapy for different types of cancers. Therapeutic cancer vaccines aim to prevent immune system responses that are not targeted at the tumors only, but also boost the anti-tumor immunity and promote regression or eradication of the malignancy without, or with minimal, adverse events. Clinical trial data have pushed the development of cancer vaccines forward, and the US Food and Drug Administration authorized the first therapeutic cancer vaccine. In the present review, we discuss the various types of cancer vaccines and different approaches for the development of therapeutic cancer vaccines, along with the current state of knowledge and future prospects. We also discuss how tumor-induced immune suppression limits the effectiveness of therapeutic vaccinations, and strategies to overcome this barrier to design efficacious, long-lasting anti-tumor immune responses in the generation of vaccines.

Field-Deployable Treatments For Leishmaniasis: Intrinsic Challenges, Recent Developments and Next Steps.

Leishmaniasis is a neglected tropical disease endemic primarily to low- and middle-income countries, for which there has been inadequate development of affordable, safe, and efficacious therapies. Clinical manifestations of leishmaniasis range from self-healing skin lesions to lethal visceral infection with chances of relapse. Although treatments are available, secondary effects limit their use outside the clinic and negatively impact the quality of life of patients in endemic areas. Other non-medicinal treatments, such as thermotherapies, are limited to use in patients with cutaneous leishmaniasis but not with visceral infection. Recent studies shed light to mechanisms through which Leishmania can persist by hiding in cellular safe havens, even after chemotherapies. This review focuses on exploring the cellular niches that Leishmania parasites may be leveraging to persist within the host. Also, the cellular, metabolic, and molecular implications of Leishmania infection and how those could be targeted for therapeutic purposes are discussed. Other therapies, such as those developed against cancer or for manipulation of the ferroptosis pathway, are proposed as possible treatments against leishmaniasis due to their mechanisms of action. In particular, treatments that target hematopoietic stem cells and monocytes, which have recently been found to be necessary components to sustain the infection and provide a safe niche for the parasites are discussed in this review as potential field-deployable treatments against leishmaniasis.

Dual-scRNA-seq analysis reveals rare and uncommon parasitized cell populations in chronic L. donovani infection.

Although phagocytic cells are documented targets of Leishmania parasites, it is unclear whether other cell types can be infected. Here, we use unbiased single-cell RNA sequencing (scRNA-seq) to simultaneously analyze host cell and Leishmania donovani transcriptomes to identify and annotate parasitized cells in spleen and bone marrow in chronically infected mice. Our dual-scRNA-seq methodology allows the detection of heterogeneous parasitized populations. In the spleen, monocytes and macrophages are the dominant parasitized cells, while megakaryocytes, basophils, and natural killer (NK) cells are found to be unexpectedly infected. In the bone marrow, the hematopoietic stem cells (HSCs) expressing phagocytic receptors FcγR and CD93 are the main parasitized cells. Additionally, we also detect parasitized cycling basal cells, eosinophils, and macrophages in chronically infected mice. Flow cytometric analysis confirms the presence of parasitized HSCs. Our unbiased dual-scRNA-seq method identifies rare, parasitized cells, potentially implicated in pathogenesis, persistence, and protective immunity, using a non-targeted approach.

Mycobacteria modulate SUMOylation to suppresses protective responses in dendritic cells.

Post translational modifications (PTMs) are exploited by various pathogens in order to escape host immune responses. SUMOylation is one of the PTMs which is involved in regulation of a variety of cellular responses. However, the effects of host SUMOylation on pathogenic bacteria largely remain elusive. We, therefore, investigated the role of SUMOylation in regulating defense responses in dendritic cells (DCs) during mycobacterial infection. Dendritic Cells of female BALB/c mice and THP-1 macrophages were used. Western blotting was performed to measure the expression of level of SUMO1, pSTAT1, pp38, pERK, Beclin-1, LC3, Bax and Cytochrome C. For bacterial burden confocal microscopy and CFU (Colony Forming Unit) were used. Flow cytometry was used for ROS and co-stimulatory molecules measurement. Cytokine level were measured using ELISA. We show that stimulation of Bone Marrow Derived Dendritic Cells (BMDCs) with mycobacterial antigen Rv3416 or live infection with Mycobacterium bovis BCG increases the SUMOylation of host proteins. Inhibition of SUMOylation significantly decreased intracellular bacterial loads in DCs. Additionally, inhibiting SUMOylation, induces protective immune responses by increasing oxidative burst, pro-inflammatory cytokine expression and surface expression of T cell co-stimulatory molecules, and activation of pSTAT1 and Mitogen Activated Protein Kinases (MAPK) proteins- pp38 and pERK. SUMOylation inhibition also increased apoptosis and autophagy in BMDCs. Intriguingly, mycobacteria increased SUMOylation of many of the above molecules. Furthermore, inhibiting SUMOylation in DCs primed T cells that in turn attenuated bacterial burden in infected macrophages. These findings demonstrate that SUMOylation pathway is exploited by mycobacteria to thwart protective host immune responses.

Unlocking therapeutic potential: integration of drug repurposing and immunotherapy for various disease targeting.

Drug repurposing, also known as drug repositioning, entails the application of pre-approved or formerly assessed drugs having potentially functional therapeutic amalgams for curing various disorders or disease conditions distinctive from their original remedial indication. It has surfaced as a substitute for the development of drugs for treating cancer, cardiovascular diseases, neurodegenerative disorders, and various infectious diseases like Covid-19. Although the earlier lines of findings in this area were serendipitous, recent advancements are based on patient centered approaches following systematic, translational, drug targeting practices that explore pathophysiological ailment mechanisms. The presence of definite information and numerous records with respect to beneficial properties, harmfulness, and pharmacologic characteristics of repurposed drugs increase the chances of approval in the clinical trial stages. The last few years have showcased the successful emergence of repurposed drug immunotherapy in treating various diseases. In this light, the present review emphasises on incorporation of drug repositioning with Immunotherapy targeted for several disorders.

Immunization with a Trypanosoma cruzi cyclophilin-19 deletion mutant protects against acute Chagas disease in mice.

Human infection with the protozoan parasite Trypanosoma cruzi causes Chagas disease for which there are no prophylactic vaccines. Cyclophilin 19 is a secreted cis-trans peptidyl isomerase expressed in all life stages of Trypanosoma cruzi. This protein in the insect stage leads to the inactivation of insect anti-parasitic peptides and parasite transformation whereas in the intracellular amastigotes it participates in generating ROS promoting the growth of parasites. We have generated a parasite mutant with depleted expression of Cyp19 by removal of 2 of 3 genes encoding this protein using double allelic homologous recombination. The mutant parasite line failed to replicate when inoculated into host cells in vitro or in mice indicating that Cyp19 is critical for infectivity. The mutant parasite line also fails to replicate in or cause clinical disease in immuno-deficient mice further validating their lack of virulence. Repeated inoculation of mutant parasites into immuno-competent mice elicits parasite-specific trypanolytic antibodies and a Th-1 biased immune response and challenge of mutant immunized mice with virulent wild-type parasites is 100% effective at preventing death from acute disease. These results suggest that parasite Cyp19 may be candidate for small molecule drug targeting and that the mutant parasite line may warrant further immunization studies for prevention of Chagas disease.

Centrin-deficient Leishmania mexicana confers protection against New World cutaneous leishmaniasis.

Leishmaniasis is a neglected protozoan disease affecting over 12 million people globally with no approved vaccines for human use. New World cutaneous leishmaniasis (CL) caused by L. mexicana is characterized by the development of chronic non-healing skin lesions. Using the CRISPR/Cas9 technique, we have generated live attenuated centrin knockout L. mexicana (LmexCen-/-) parasites. Centrin is a cytoskeletal protein important for cellular division in eukaryotes and, in Leishmania, is required only for intracellular amastigote replication. We have investigated the safety and immunogenicity characteristics of LmexCen-/- parasites by evaluating their survival and the cytokine production in bone-marrow-derived macrophages (BMDMs) and dendritic cells (BMDCs) in vitro. Our data shows that LmexCen-/- amastigotes present a growth defect, which results in significantly lower parasitic burdens and increased protective cytokine production in infected BMDMs and BMDCs, compared to the wild type (WT) parasites. We have also determined the safety and efficacy of LmexCen-/- in vivo using experimental murine models of L. mexicana. We demonstrate that LmexCen-/- parasites are safe and do not cause lesions in susceptible mouse models. Immunization with LmexCen-/- is also efficacious against challenge with WT L. mexicana parasites in genetically different BALB/c and C57BL/6 mouse models. Vaccinated mice did not develop cutaneous lesions, displayed protective immunity, and showed significantly lower parasitic burdens at the infection site and draining lymph nodes compared to the control group. Overall, we demonstrate that LmexCen-/- parasites are safe and efficacious against New World cutaneous leishmaniasis in pre-clinical models.

Dissecting role of founder mutation p.V727M in GNE in Indian HIBM cohort.

GNE gene-specific c.2179G>A(p.V727M) is a key alteration reported in patients with hereditary inclusion body myopathy (HIBM) and represents an ethnic founder mutation in the Indian cohort. However, the underlying role of this mutation in pathogenesis remains largely unknown. Thus, in this study, we aimed to access possible mechanisms of V727M mutation that could be leading to myopathy. We evaluated various in silico tools to predict the effect of this mutation on pathogenicity, structural or possible interactions, that could induce myopathy. Our results propose that V727M mutation could induce deleterious effects or pathogenicity and affect the stability of GNE protein. Analysis of differential genes reported in the V727 mutant case suggests that it can affect GNE protein interaction with Myc-proto-oncogene (MYC) transcription factor. Our in silico analysis also suggests a possible interaction between GNE ManNac-kinase domain with MYC protein at the C-terminal DNA-binding domain. MYC targets reported in skeletal muscles via ChIP-seq suggest that it plays a key role in regulating the expression of many genes reported differentially expressed in V727M-mutated HIBMs. We conclude that V727M mutation could alter the interaction of GNE with MYC thereby altering transcription of sialyltransferase and neuromuscular genes, thus understanding these effects could pave the way for developing effective therapies against HIBM.