16s RNA-based metagenomics reveal previously unreported gut microbiota associated with reactive arthritis and undifferentiated peripheral spondyloarthritis.

OBJECTIVES: Reactive arthritis (ReA) provides a unique opportunity to comprehend how a mucosal infection leads to inflammatory arthritis at a distant site without the apparent invasion of the pathogen. Unfortunately, conventional stool cultures after ReA provide limited information, and there is a dearth of metagenomic studies in ReA. The objective of this study was to identify gut microbiota associated with the development of ReA. METHODS: Patients with ReA or undifferentiated peripheral spondyloarthritis (UpSpA) were included if they presented within 4 weeks of the onset of the current episode of arthritis. Metagenomic DNA was extracted from the stools of these patients and of 36 age- and sex-similar controls. Sequencing and analysis were done using a standard 16S ribosomal pipeline. RESULTS: Of 55 patients, there was no difference between the gut microbiota of postdiarrheal ReA(n = 20) and of upSpA (n = 35). Comparing the gut microbiota of patients vs healthy controls, the patients had significantly higher alpha and beta diversity measures. After stringency filters, Proteobacteria had high abundance while Firmicutes had lesser as compared with the controls. Six families were overexpressed in patients, while another five were overexpressed in controls. Sixteen genera and 18 species were significantly different between patients and controls. At the species level there was strong association of Staphylococcus aureus, Clostridium septicum Klebsiella pneumoniae, Escherichia coli, Empedobacter brevis, Roseburia hominis, Bacillus velezensis, and Crassaminicella with ReA. CONCLUSION: The microbiota of classical gut-associated ReA and upSpA is similar. Patients have higher diversities in their gut microbiota compared with healthy controls. Both known and previously unreported species associated with ReA/upSpA were identified.

RNAseq-based transcriptomics of treatment-naïve multi-inflammatory syndrome in children (MIS-C) demonstrates predominant activation of matrisome, innate and humoral immune pathways.

MIS-C is a rare, highly inflammatory state resembling incomplete Kawasaki disease, temporarily associated with COVID-19. The pathogenesis is not completely known. RNAseq was carried out on whole blood of six treatment-naïve MIS-C patients. This was compared against RNAseq transcriptomics data of five healthy controls (HC), four Kawasaki Disease (KD) and seven systemic Juvenile Idiopathic Arthritis (sJIA). Using PCA, MIS-C clustered separately from HC, KD and sJIA. Amongst the top 50 significant genes in the three comparisons with HC, KD, and sJIA, common genes were: TMCC2, ITGA2B, DMTN, GFI1B, PF4, QSER1, GRAP2, TUBB1. DSEA revealed that maximum number of hits for overexpressed pathways was for NABA matrisome activation when MIS-C was compared against HC. Cytokine stimulated cellular activation pathways, specifically IL-10 were downregulated. MIS-C had more activated pathways of neutrophil degranulation and acquired immune activation but less of coagulation system or heat-shock system involvement as compared to KD. As compared to sJIA, humoral immune response and complements were activated. Matrisome activation was higher, with increased cell-cell interaction and ECM signalling. This analysis revealed novel insights into the pathogenesis of MIS-C, including the potential role of matrisomes, humoral immune system and down-regulated interleukin-10 pathways.

Transgenic mouse models support a protective role of type I IFN response in SARS-CoV-2 infection-related lung immunopathology and neuroinvasion.

Type I interferon (IFN-I) response is the first line of host defense against invading viruses. In the absence of definite mouse models, the role of IFN-I in SARS-CoV-2 infection remains perplexing. Here, we develop two mouse models, one with constitutively high IFN-I response (hACE2; Irgm1-/-) and the other with dampened IFN-I response (hACE2; Ifnar1-/-), to comprehend the role of IFN-I response. We report that hACE2; Irgm1-/- mice are resistant to lethal SARS-CoV-2 infection. In contrast, a severe SARS-CoV-2 infection along with immune cell infiltration, cytokine storm, and enhanced pathology is observed in the lungs and brain of hACE2; Ifnar1-/- mice. The hACE2; Irgm1-/-Ifnar1-/- double-knockout mice display loss of the protective phenotype observed in hACE2; Irgm1-/- mice, suggesting that heightened IFN-I response accounts for the observed immunity. Taking the results together, we demonstrate that IFN-I protects from lethal SARS-CoV-2 infection, and Irgm1 (IRGM) could be an excellent therapeutic target against SARS-CoV-2.

Nimbolide-based nanomedicine inhibits breast cancer stem-like cells by epigenetic reprogramming of DNMTs-SFRP1-Wnt/ß-catenin signaling axis.

Triple-negative breast cancer (TNBC) harbors a high percentage of breast cancer stem-like cells (BCSCs) that significantly contribute to poor prognosis, metastasis, and relapse of the disease. Thus, targeting BCSCs could be a promising approach to combat TNBC. In this context, we investigated nimbolide (Nim), a limonoid triterpenoid that has potent anticancer properties, but poor pharmacokinetics and low bioavailability limit its therapeutic application. So, to enhance the therapeutic potential of Nim, Nim-encapsulated poly(lactic-co-glycolic acid) (PLGA) nanoparticles (Nim NPs) were formulated and the anticancer stem cell (CSC) effects evaluated in vitro and in vivo. In vitro studies suggested that Nim NPs significantly inhibited several inherent characteristics of BCSCs, such as stemness, self-renewability, chemoresistance, epithelial-to-mesenchymal transition (EMT), and migration in comparison to native Nim. Next, the mechanism behind the anti-CSC effect of Nim was explored. Mechanistically, we found that Nim epigenetically restores tumor suppressor gene secreted frizzled-related protein 1 (SFRP1) expression by downregulating DNA methyltransferases (DNMTs), leading to Wnt/ß-catenin signaling inhibition. Further, in vivo results demonstrated that Nim NPs showed enhanced anti-tumor and anti-metastatic effects compared to native Nim in two preclinical models without any systemic toxicity. Overall, these findings provide proof of concept that Nim-based phytonanomedicine can inhibit BCSCs by epigenetic reprogramming of the DNMTs-SFRP1-Wnt/ß-catenin signaling axis.

Biochemical, functional and genomic characterization of a new probiotic Ligilactobacillus salivarius F14 from the gut of tribes of Odisha.

Characterization of new potential probiotics is desirable in the field of research on probiotics for their extensive use in health and disease. Tribes could be an unusual source of probiotics due to their unique food habits and least dependence on medications and consumption of antibiotics. The aim of the present study is to isolate lactic acid bacteria from tribal fecal samples of Odisha, India, and characterize their genetic and probiotic attributes. In this context one of the catalase-negative and Gram-positive isolates, identified using 16S rRNA sequencing as Ligilactobacillus salivarius, was characterized in vitro for its acid and bile tolerance, cell adhesion and antimicrobial properties. The whole genome sequence was obtained and analyzed for strain level identification, presence of genomic determinants for probiotic-specific features, and safety. Genes responsible for its antimicrobial and immunomodulatory functions were detected. The secreted metabolites were analyzed using high resolution mass spectroscopy; the results indicated that the antimicrobial potential could be due to the presence of pyroglutamic acid, propionic acid, lactic acid, 2-hydroxyisocaproic acid, homoserine, and glutathione, and the immuno-modulating activity, contributed by the presence of short chain fatty acids such as acetate, propionate, and butyrate. So, to conclude we have successfully characterized a Ligilactobacillus salivarius species with potential antimicrobial and immunomodulatory ability. The health-promoting effects of this probiotic strain and/or its derivatives will be investigated in future.

Molecular and epigenetic alterations in normal and malignant myelopoiesis in human leukemia 60 (HL60) promyelocytic cell line model.

In vitro cell line model systems are essential in supporting the research community due to their low cost, uniform culturing conditions, homogeneous biological resources, and easy experimental design to study the cause and effect of a gene or a molecule. Human leukemia 60 (HL60) is an in-vitro hematopoietic model system that has been used for decades to study normal myeloid differentiation and leukemia biology. Here, we show that IMDM supplemented with 20% FBS is an optimal culturing condition and induces effective myeloid differentiation compared with RPMI supplemented with 10% FBS when HL60 is induced with 1α,25-dihydroxyvitamin D3 (Vit D3) and all-trans retinoic acid (ATRA). The chromatin organization is compacted, and the repressive epigenetic mark H3K27me3 is enhanced upon HL60-mediated terminal differentiation. Differential gene expression analysis obtained from RNA sequencing in HL60 cells during myeloid differentiation showed the induction of pathways involved in epigenetic regulation, myeloid differentiation, and immune regulation. Using high-throughput transcriptomic data (GSE74246), we show the similarities (genes that did not satisfy |log2FC|>1 and FDR<0.05) and differences (FDR <0.05 and |log2FC|>1) between granulocyte-monocyte progenitor vs HL60 cells, Vit D3 induced monocytes (vMono) in HL60 cells vs primary monocytes (pMono), and HL60 cells vs leukemic blasts at the transcriptomic level. We found striking similarities in biological pathways between these comparisons, suggesting that the HL60 model system can be effectively used for studying myeloid differentiation and leukemic aberrations. The differences obtained could be attributed to the fact that the cellular programs of the leukemic cell line and primary cells are different. We validated several gene expression patterns for different comparisons with CD34+ cells derived from cord blood for myeloid differentiation and AML patients. In addition to the current knowledge, our study further reveals the significance of using HL60 cells as in vitro model system under optimal conditions to understand its potential as normal myeloid differentiation model as well as leukemic model at the molecular level.

Selective autophagy of RIPosomes maintains innate immune homeostasis during bacterial infection.

The NOD1/2-RIPK2 is a key cytosolic signaling complex that activates NF-κB pro-inflammatory response against invading pathogens. However, uncontrolled NF-κB signaling can cause tissue damage leading to chronic diseases. The mechanisms by which the NODs-RIPK2-NF-κB innate immune axis is activated and resolved remain poorly understood. Here, we demonstrate that bacterial infection induces the formation of endogenous RIPK2 oligomers (RIPosomes) that are self-assembling entities that coat the bacteria to induce NF-κB response. Next, we show that autophagy proteins IRGM and p62/SQSTM1 physically interact with NOD1/2, RIPK2 and RIPosomes to promote their selective autophagy and limit NF-κB activation. IRGM suppresses RIPK2-dependent pro-inflammatory programs induced by Shigella and Salmonella. Consistently, the therapeutic inhibition of RIPK2 ameliorates Shigella infection- and DSS-induced gut inflammation in Irgm1 KO mice. This study identifies a unique mechanism where the innate immune proteins and autophagy machinery are recruited together to the bacteria for defense as well as for maintaining immune homeostasis.

An overview of cancer and the human microbiome.

Mutual beneficial associations with the microbial consortia are an essential requisite of human life. Microbial communities have both a symbiotic and a pathogenic standpoint, which portrays a context-dependent scenario of the human microbiome. The symbiotic assemblage works to develop indispensable functions of the human body such as immune system, digestive system, defense against colonization by pathobionts and their toxins, etc. Furthermore, any deviation in the resource utilization by the symbionts due to host factors comprising lifestyle changes, diet, drugs, immunocompromised states, and co-morbidities could perturb beneficial microbes communities and promote the invasion by opportunistic pathogens thus, disrupting the homeostatic state. Microbial infestations have proved to be carcinogenic but this does not spontaneously establish a cancer hallmark, rather they initiate a cascade of events that disturbs the normal cellular activities finally these defective machineries invade distant sites of the body, submitting to a devastative transformed internal milieu. Significant technological and system biology advances have been made in elucidating a lucid but complex basis of such microbe-associated malignancies. This chapter discusses the recent advances, without compromising the concepts of the inception studies, including a brief version of the microbial status in cancer generation, mechanistic approaches adapted, therapeutic interventions, system biology approaches with special mention on the study design gaps, challenges in addressing the drawbacks and finally with a perspective of the future targeted studies, has been a focus of this piece of work.

SHP-1 dephosphorylates histone H2B to facilitate its ubiquitination during transcription.

Dynamic regulation of phosphorylation and dephosphorylation of histones is essential for eukaryotic transcription, but the enzymes engaged in histone dephosphorylation are not fully explored. Here, we show that the tyrosine phosphatase SHP-1 dephosphorylates histone H2B and plays a critical role during transition from the initiation to the elongation stage of transcription. Nuclear-localized SHP-1 is associated with the Paf1 complex at chromatin and dephosphorylates H2B at tyrosine 121. Moreover, knockout of SHP-1, or expression of a mutant mimicking constitutive phosphorylation of H2B Y121, leads to a reduction in genome-wide H2B ubiquitination, which subsequently causes defects in RNA polymerase II-dependent transcription. Mechanistically, we demonstrate that Y121 phosphorylation precludes H2B's interaction with the E2 enzyme, indicating that SHP-1-mediated dephosphorylation of this residue may be a prerequisite for efficient H2B ubiquitination. Functionally, we find that SHP-1-mediated H2B dephosphorylation contributes to maintaining basal autophagic flux in cells through the efficient transcription of autophagy and lysosomal genes. Collectively, our study reveals an important modification of histone H2B regulated by SHP-1 that has a role during eukaryotic transcription.

Isolation and Characterization of Five Severe Acute Respiratory Syndrome Coronavirus 2 Strains of Different Clades and Lineages Circulating in Eastern India.

The emergence of the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) as a serious pandemic has altered the global socioeconomic dynamics. The wide prevalence, high death counts, and rapid emergence of new variants urge for the establishment of research infrastructure to facilitate the rapid development of efficient therapeutic modalities and preventive measures. In agreement with this, SARS-CoV-2 strains were isolated from patient swab samples collected during the first COVID-19 wave in Odisha, India. The viral isolates were adapted to in vitro cultures and further characterized to identify strain-specific variations in viral growth characteristics. The neutralization susceptibility of viral isolates to vaccine-induced antibodies was determined using sera from individuals vaccinated in the Government-run vaccine drive in India. The major goal was to isolate and adapt SARS-CoV-2 viruses in cell culture with minimum modifications to facilitate research activities involved in the understanding of the molecular virology, host-virus interactions, drug discovery, and animal challenge models that eventually contribute toward the development of reliable therapeutics.

MIF confers survival advantage to pancreatic CAFs by suppressing interferon pathway-induced p53-dependent apoptosis.

The presence of activated pancreatic stellate cells (PSCs) in the pancreatic ductal adenocarcinoma (PDAC) microenvironment plays a significant role in cancer progression. Macrophage migration inhibitory factor (MIF) is overexpressed in PDAC tissues and expressed by both cancer and stromal cells. The pathophysiological role of MIF in PDAC-associated fibroblasts or PSCs is yet to be elucidated. Here we report that the PSCs of mouse or cancer-associated fibroblast cells (CAFs) of human expresses MIF and its receptors, whose expression gets upregulated upon LPS or TNF-α stimulation. In vitro functional experiments showed that MIF significantly conferred a survival advantage to CAFs/PSCs upon growth factor deprivation. Genetic or pharmacological inhibition of MIF also corroborated these findings. Further, co-injection of mouse pancreatic cancer cells with PSCs isolated from Mif-/- or Mif+/+ mice confirmed the pro-survival effect of MIF in PSCs and also demonstrated the pro-tumorigenic role of MIF expressed by CAFs in vivo. Differential gene expression analysis and in vitro mechanistic studies indicated that MIF expressed by activated CAFs/PSCs confers a survival advantage to these cells by suppression of interferon pathway induced p53 dependent apoptosis.

Long-read 16S-seq reveals nasopharynx microbial dysbiosis and enrichment of Mycobacterium and Mycoplasma in COVID-19 patients: a potential source of co-infection.

The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a major global health concern. This virus infects the upper respiratory tract and causes pneumonia-like symptoms. So far, few studies have shown alterations in nasopharyngeal (NP) microbial diversity, enrichment of opportunistic pathogens and their role in co-infections during respiratory infections. Therefore, we hypothesized that microbial diversity changes, with increase in the population of opportunistic pathogens, during SARS-CoV2 infection in the nasopharynx, which may be involved in co-infection in COVID-19 patients. The 16S rRNA variable regions, V1-V9, of NP samples of control and COVID-19 (symptomatic and asymptomatic) patients were sequenced using the Oxford Nanopore™ technology. Comprehensive bioinformatics analysis for determining alpha/beta diversities, non-metric multidimensional scaling, correlation studies, canonical correspondence analysis, linear discriminate analysis, and dysbiosis index were used to analyze the control and COVID-19-specific NP microbiomes. We observed significant dysbiosis in the COVID-19 NP microbiome with an increase in the abundance of opportunistic pathogens at genus and species levels in asymptomatic/symptomatic patients. The significant abundance of Mycobacteria spp. and Mycoplasma spp. in symptomatic patients suggests their association and role in co-infections in COVID-19 patients. Furthermore, we found strong correlation of enrichment of Mycobacteria and Mycoplasma with the occurrences of chest pain and fever in symptomatic COVID-19 patients. This is the first study from India to show the abundance of Mycobacteria and Mycoplasma opportunistic pathogens in non-hospitalized COVID-19 patients and their relationship with symptoms, indicating the possibility of co-infections.

Underlying Co-Morbidity Reveals Unique Immune Signatures in Type II Diabetes Patients Infected With SARS-CoV2.

Background: SARS-CoV2 infection in patients with comorbidities, particularly T2DM, has been a major challenge globally and has been shown to be associated with high morbidity and mortality. Here, we did whole blood immunophenotyping along with plasma cytokine, chemokine, antibody isotyping, and viral load from oropharyngeal swab to understand the immune pathology in the T2DM patients infected with SARS-CoV2. Methods: Blood samples from 25 Covid-19 positive patients having T2DM, 10 Covid-19 positive patients not having T2DM, and 10 Covid-19 negative, non-diabetic healthy controls were assessed for various immune cells by analyzing for their signature surface proteins in mass cytometry. Circulating cytokines, chemokines, and antibody isotypes were determined from plasma while viral copy number was determined from oropharyngeal swabs. All our representative data corroborated with laboratory findings. Results: Our observations encompass T2DM patients having elevated levels of both type I and type II cytokines and higher levels of circulating IgA, IgM, IgG1, and IgG2 as compared to NDM and healthy volunteers. They also displayed higher percentages of granulocytes, mDCs, plasmablasts, Th2-like cells, CD4+ EM cells, and CD8+ TE cells as compared to healthy volunteers. T2DM patients also displayed lower percentages of pDCs, lymphocytes, CD8+ TE cells, CD4+, and CD8+ EM. Conclusion: Our study demonstrated that patients with T2DM displayed higher inflammatory markers and a dysregulated anti-viral and anti-inflammatory response when compared to NDM and healthy controls and the dysregulated immune response may be attributed to meta inflammation.

Differential colitis susceptibility of Th1- and Th2-biased mice: A multi-omics approach.

The health and economic burden of colitis is increasing globally. Understanding the role of host genetics and metagenomics is essential to establish the molecular basis of colitis pathogenesis. In the present study, we have used a common composite dose of DSS to compare the differential disease severity response in C57BL/6 (Th1 biased) and BALB/c (Th2 biased) mice with zero mortality rates. We employed multi-omics approaches and developed a newer vector analysis approach to understand the molecular basis of the disease pathogenesis. In the current report, comparative transcriptomics, metabonomics, and metagenomics analyses revealed that the Th1 background of C57BL/6 induced intense inflammatory responses throughout the treatment period. On the contrary, the Th2 background of BALB/c resisted severe inflammatory responses by modulating the host's inflammatory, metabolic, and gut microbial profile. The multi-omics approach also helped us discover some unique metabolic and microbial markers associated with the disease severity. These biomarkers could be used in diagnostics.

Single-Cell Immunogenomic Approach Identified SARS-CoV-2 Protective Immune Signatures in Asymptomatic Direct Contacts of COVID-19 Cases.

The response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is largely impacted by the level of virus exposure and status of the host immunity. The nature of protection shown by direct asymptomatic contacts of coronavirus disease 2019 (COVID-19)-positive patients is quite intriguing. In this study, we have characterized the antibody titer, SARS-CoV-2 surrogate virus neutralization, cytokine levels, single-cell T-cell receptor (TCR), and B-cell receptor (BCR) profiling in asymptomatic direct contacts, infected cases, and controls. We observed significant increase in antibodies with neutralizing amplitude in asymptomatic contacts along with cytokines such as Eotaxin, granulocyte-colony stimulating factor (G-CSF), interleukin 7 (IL-7), migration inhibitory factor (MIF), and macrophage inflammatory protein-1α (MIP-1α). Upon single-cell RNA (scRNA) sequencing, we explored the dynamics of the adaptive immune response in few representative asymptomatic close contacts and COVID-19-infected patients. We reported direct asymptomatic contacts to have decreased CD4+ naive T cells with concomitant increase in CD4+ memory and CD8+ Temra cells along with expanded clonotypes compared to infected patients. Noticeable proportions of class switched memory B cells were also observed in them. Overall, these findings gave an insight into the nature of protection in asymptomatic contacts.

Inhibition of IRGM establishes a robust antiviral immune state to restrict pathogenic viruses.

The type I interferon (IFN) response is the major host arsenal against invading viruses. IRGM is a negative regulator of IFN responses under basal conditions. However, the role of human IRGM during viral infection has remained unclear. In this study, we show that IRGM expression is increased upon viral infection. IFN responses induced by viral PAMPs are negatively regulated by IRGM. Conversely, IRGM depletion results in a robust induction of key viral restriction factors including IFITMs, APOBECs, SAMHD1, tetherin, viperin, and HERC5/6. Additionally, antiviral processes such as MHC-I antigen presentation and stress granule signaling are enhanced in IRGM-deficient cells, indicating a robust cell-intrinsic antiviral immune state. Consistently, IRGM-depleted cells are resistant to the infection with seven viruses from five different families, including Togaviridae, Herpesviridae, Flaviviverdae, Rhabdoviridae, and Coronaviridae. Moreover, we show that Irgm1 knockout mice are highly resistant to chikungunya virus (CHIKV) infection. Altogether, our work highlights IRGM as a broad therapeutic target to promote defense against a large number of human viruses, including SARS-CoV-2, CHIKV, and Zika virus.

Quantitative proteomics of hamster lung tissues infected with SARS-CoV-2 reveal host factors having implication in the disease pathogenesis and severity.

Syrian golden hamsters (Mesocricetus auratus) infected by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) manifests lung pathology. In this study, efforts were made to check the infectivity of a local SARS-CoV-2 isolate in a self-limiting and non-lethal hamster model and evaluate the differential expression of lung proteins during acute infection and convalescence. The findings of this study confirm the infectivity of this isolate in vivo. Analysis of clinical parameters and tissue samples show the pathophysiological manifestation of SARS-CoV-2 infection similar to that reported earlier in COVID-19 patients and hamsters infected with other isolates. However, diffuse alveolar damage (DAD), a common histopathological feature of human COVID-19 was only occasionally noticed. The lung-associated pathological changes were very prominent on the 4th day post-infection (dpi), mostly resolved by 14 dpi. Here, we carried out the quantitative proteomic analysis of the lung tissues from SARS-CoV-2-infected hamsters on day 4 and day 14 post-infection. This resulted in the identification of 1585 proteins of which 68 proteins were significantly altered between both the infected groups. Pathway analysis revealed complement and coagulation cascade, platelet activation, ferroptosis, and focal adhesion as the top enriched pathways. In addition, we also identified altered expression of two pulmonary surfactant-associated proteins (Sftpd and Sftpb), known for their protective role in lung function. Together, these findings will aid in understanding the mechanism(s) involved in SARS-CoV-2 pathogenesis and progression of the disease.