Ro60-Roles in RNA Processing, Inflammation, and Rheumatic Autoimmune Diseases.

The Ro60/SSA2 autoantigen is an RNA-binding protein and a core component of nucleocytoplasmic ribonucleoprotein (RNP) complexes. Ro60 is essential in RNA metabolism, cell stress response pathways, and cellular homeostasis. It stabilises and mediates the quality control and cellular distribution of small RNAs, including YRNAs (for the 'y' in 'cytoplasmic'), retroelement transcripts, and misfolded RNAs. Ro60 transcriptional dysregulation or loss of function can result in the generation and release of RNA fragments from YRNAs and other small RNAs. Small RNA fragments can instigate an inflammatory cascade through endosomal toll-like receptors (TLRs) and cytoplasmic RNA sensors, which typically sense pathogen-associated molecular patterns, and mount the first line of defence against invading pathogens. However, the recognition of host-originating RNA moieties from Ro60 RNP complexes can activate inflammatory response pathways and compromise self-tolerance. Autoreactive B cells may produce antibodies targeting extracellular Ro60 RNP complexes. Ro60 autoantibodies serve as diagnostic markers for various autoimmune diseases, including Sjögren's disease (SjD) and systemic lupus erythematosus (SLE), and they may also act as predictive markers for anti-drug antibody responses among rheumatic patients. Understanding Ro60's structure, function, and role in self-tolerance can enhance our understanding of the underlying molecular mechanisms of autoimmune conditions.

The role of TLR9 polymorphism in susceptibility to pulmonary tuberculosis.

Mycobacterium tuberculosis (MTB) is the causative agent of pulmonary tuberculosis (PTB), a major health problem that leads to 1.5 million deaths annually. Host genetic factors play a significant role in disease resistance/susceptibility by altering immunity against MTB. Toll-like receptor (TLR) sensors such as TLR2, TLR4, TLR8, and TLR9 are known to play a pivotal role in PTB via modulating sensor expression and/or effector responses. Single-nucleotide polymorphism (SNP) rs187084 (T-1486C) of the TLR9 promoter is associated with various autoimmune disorders and cancers. A recent bioinformatic analysis predicted that the T-1486C SNP is involved in PTB, although its potential role is unclear. To investigate the role of T-1486C in PTB, we stimulated PBMCs with the H37Rv whole cell lysate. We found that the presence of the "C" allele increases the transcriptional activity of the TLR9, which in turn induces high levels of Interferon gamma-induced protein 10 (IP-10), a biomarker for PTB. However, the expression of protective cytokines such as IFNγ and TNFα was observed significantly less with "C" allele in comparison to "T" allele. We further selected three different tribe populations showing differential susceptibility to PTB and performed genotypic analyses for the TLR9 promoter. We found a significantly lower minor allele frequency (MAF) of T-1486C in the Baiga tribe, wherein fewer PTB cases were reported, than that in the Gond and Korku tribes. Collectively, these data suggest that the minor "C" allele at rs187084 locus may be associated with susceptibility to PTB, which may explain the relatively lower PTB rates observed in Baiga tribe members.

Comparison of different methods for isolating CD8+ T lymphocyte-derived extracellular vesicles and supramolecular attack particles.

CD8+ T lymphocytes play vital roles in killing infected or deranged host cells, recruiting innate immune cells, and regulating other aspects of immune responses. Like any other cell, CD8+ T cells also produce extracellular particles. These include extracellular vesicles (EVs) and non-vesicular extracellular particles (NVEPs). T cell-derived EVs are proposed to mediate cell-to-cell signalling, especially in the context of inflammatory responses, autoimmunity, and infectious diseases. CD8+ T cells also produce supramolecular attack particles (SMAPs), which are in the same size range as EVs and mediate a component of T cell mediated killing. The isolation technique selected will have a profound effect on yield, purity, biochemical properties and function of T cell-derived particles; making it important to directly compare different approaches. In this study, we compared commonly used techniques (membrane spin filtration, ultracentrifugation, or size exclusion liquid chromatography) to isolate particles from activated human CD8+ T cells and validated our results by single-particle methods, including nanoparticle tracking analysis, flow cytometry, electron microscopy and super-resolution microscopy of the purified sample as well as bulk proteomics and lipidomics analyses to evaluate the quality and nature of enriched T cell-derived particles. Our results show that there is a trade-off between the yield and the quality of T cell-derived particles. Furthermore, the protein and lipid composition of the particles is dramatically impacted by the isolation technique applied. We conclude that from the techniques evaluated, size exclusion liquid chromatography offers the highest quality of T cell derived EVs and SMAPs with acceptable yields for compositional and functional studies.

Gonadal status of male recipient mice influences germ cell development in immature buffalo testis tissue xenograft.

Growth and development of immature testis xenograft from various domestic mammals has been shown in mouse recipients; however, buffalo testis xenografts have not been reported to date. In this study, small fragments of testis tissue from 8-week-old buffalo calves were implanted subcutaneously onto the back of immunodeficient male mouse recipients, which were either castrated or left intact (non-castrated). The xenografts were retrieved and analyzed 12 and 24 weeks later. The grafted tissue survived and grew in both types of recipient with a significant increase in weight and seminiferous tubule diameter. Recovery of grafts from intact recipients 24 weeks post-grafting was significantly lower than that from the castrated recipients. Seminal vesicle indices and serum testosterone levels were lower in castrated recipients at both collection time points in comparison to the intact recipients and non-grafted intact mouse controls. Pachytene spermatocytes were the most advanced germ cells observed in grafts recovered from castrated recipients 24 weeks post-grafting. Complete spermatogenesis, as indicated by the presence of elongated spermatids, was present only in grafts from intact recipients collected 24 weeks post-grafting. However, significant number of germ cells with DNA damage was also detected in these grafts as indicated by TUNEL assay. The complete germ cell differentiation in xenografts from intact recipients may be attributed to efficient Sertoli cell maturation. These results suggest that germ cell differentiation in buffalo testis xenograft can be completed by altering the recipient gonadal status.

Lessons from a large-scale COVID-19 vaccine trial.

Global vaccination coverage remains indispensable in combatting the ongoing SARS-CoV-2 pandemic. Safety, efficacy, and durability of immune protection are the key parameters of randomized controlled trials (RCTs) and are essential for vaccine approvals, global distribution, and comprehensive population-vaccination programs. Immune protection from either vaccination or natural infection decreases over time, further challenged by rapid viral evolution. In this issue of the JCI, Sobieszczyk and colleagues report an update on the safety, efficacy, and durability of immune protection of AZD12222 in a large-scale, multinational, Phase III RCT. They report that protection lasted through 6 months, with immunity waning after 180 days. The study also highlights challenges facing vaccine trials, including the need for early unblinding for vulnerable participants, which may affect outcome measurements. Another challenge is to ensure fair representation of marginalized and minority ethnic groups in vaccine safety and efficacy studies worldwide.

Searching for escape-resistant anti-SARS-CoV-2 neutralizing antibodies.

A major goal of SARS-CoV-2 vaccination is the induction of neutralizing antibodies (nAbs) capable of blocking infection by preventing interaction of the SARS-CoV-2 Spike protein with ACE2 on target cells. Cocktails of monoclonal nAbs can reduce the risk of severe disease if administered early in infection. However, multiple variants of concern (VOCs) have arisen during the pandemic that may escape from nAbs. In this issue of the JCI, Jia Zou, Li Li, and colleagues used yeast display libraries to identify mAbs that bind to Spike proteins with a vast array of single amino acid substitutions. The authors identified mutation-resistant monoclonal nAbs for potential use as therapeutics. Multimerization further improved the potency of selected nAbs. These findings suggest a way forward in development of better nAb cocktails. However, the emergence of the highly mutated omicron (B.1.1.529) variant heightens the importance of finding effective anti-SARS-CoV-2 nAb therapeutics despite rapid viral evolution.

NIX-mediated mitophagy regulate metabolic reprogramming in phagocytic cells during mycobacterial infection.

RNASeq analysis of PBMCs from treatment naïve TB patients and healthy controls revealed that M. tuberculosis (Mtb) infection dysregulates several metabolic pathways and upregulates BNIP3L/NIX receptor mediated mitophagy. Analysis of publicly available transcriptomic data from the NCBI-GEO database indicated that M. bovis (BCG) infection also induces similar rewiring of metabolic and mitophagy pathways. Mtb chronic infection and BCG in-vitro infection both downregulated oxidative phosphorylation and upregulated glycolysis and mitophagy; therefore, we used non-pathogenic mycobacterial species BCG as a model for Mtb infection to gain molecular insights and outcomes of this phenomenon. BCG infection in PBMCs and THP-1 macrophages induce mitophagy and glycolysis, leading to differentiation of naïve macrophage to M1 phenotype. Glucose consumption and lactate production were quantified by NMR, while the mitochondrial mass assessment was performed by mitotracker red uptake assay. Infected macrophages predominantly exhibit M1-phenotype, which is indicated by an increase in M1 specific cytokines (IL-6, TNF-α, and IL-1ß) and increased NOS2/ARG1, CD86/CD206 ratio. NIX knockdown abrogates this upregulation of glycolysis, mitophagy, and secretion of pro-inflammatory cytokines in BCG infected cells, indicating that mycobacterial infection-induced immunometabolic changes are executed via NIX mediated mitophagy and are essential for macrophage differentiation and resolution of infection.