The deficiency of DNASE1L3 does not affect systemic sclerosis pathogenesis in two inducible murine models of the disease.

We induced systemic sclerosis (SSc)-like disease in both wild-type and Dnase1l3-deficient mice using two distinct approaches involving bleomycin and hypochlorous acid injections. Our observations revealed that the deficiency in DNASE1L3 did not affect tissue fibrosis or inflammation caused by these treatments. Despite the association of single nucleotide polymorphisms in humans with SSc pathogenesis, our study demonstrates that DNASE1L3 is dispensable in two inducible murine models of SSc-like pathogenesis.

Grb2-Mediated Recruitment of USP9X to LAT Enhances Themis Stability following Thymic Selection.

Themis is a new component of the TCR signaling machinery that plays a critical role during T cell development. The positive selection of immature CD4+CD8+ double-positive thymocytes and their commitment to the CD4+CD8- single-positive stage are impaired in Themis-/- mice, suggesting that Themis might be important to sustain TCR signals during these key developmental processes. However, the analysis of Themis mRNA levels revealed that Themis gene expression is rapidly extinguished during positive selection. We show in this article that Themis protein expression is increased in double-positive thymocytes undergoing positive selection and is sustained in immature single-positive thymocytes, despite the strong decrease in Themis mRNA levels in these subsets. We found that Themis stability is controlled by the ubiquitin-specific protease USP9X, which removes ubiquitin K48-linked chains on Themis following TCR engagement. Biochemical analyses indicate that USP9X binds directly to the N-terminal CABIT domain of Themis and indirectly to the adaptor protein Grb2, with the latter interaction enabling recruitment of Themis/USP9X complexes to LAT, thereby sustaining Themis expression following positive selection. Together, these data suggest that TCR-mediated signals enhance Themis stability upon T cell development and identify USP9X as a key regulator of Themis protein turnover.

Enrichment of Large Numbers of Splenic Mouse Dendritic Cells After Injection of Flt3L-Producing Tumor Cells.

Dendritic cells (DCs) are antigen-presenting cells (APCs) that shape innate and adaptive immunity. There are multiple subsets of DCs distinguished according to their phenotype and functional specialization. DCs are present in lymphoid organs and across multiple tissues. However, their frequency and numbers at these sites are very low making their functional study difficult. Multiple protocols have been developed to generate DCs in vitro from bone marrow progenitors, but they do not fully recapitulate DC complexity found in vivo. Therefore, directly amplifying endogenous DCs in vivo appears as an option to overcome this specific caveat. In this chapter, we describe a protocol to amplify murine DCs in vivo by the injection of a B16 melanoma cell line expressing the trophic factor FMS-like tyrosine kinase 3 ligand (Flt3L). We have also compared two methods of magnetic sorting of amplified DCs, both giving high yields of total murine DCs, but different representation of the main DC subsets found in vivo.

Corrigendum: The Role of Nucleases and Nucleic Acid Editing Enzymes in the Regulation of Self-Nucleic Acid Sensing.

[This corrects the article DOI: 10.3389/fimmu.2021.629922.].

The Role of Nucleases and Nucleic Acid Editing Enzymes in the Regulation of Self-Nucleic Acid Sensing.

Detection of microbial nucleic acids by the innate immune system is mediated by numerous intracellular nucleic acids sensors. Upon the detection of nucleic acids these sensors induce the production of inflammatory cytokines, and thus play a crucial role in the activation of anti-microbial immunity. In addition to microbial genetic material, nucleic acid sensors can also recognize self-nucleic acids exposed extracellularly during turn-over of cells, inefficient efferocytosis, or intracellularly upon mislocalization. Safeguard mechanisms have evolved to dispose of such self-nucleic acids to impede the development of autoinflammatory and autoimmune responses. These safeguard mechanisms involve nucleases that are either specific to DNA (DNases) or RNA (RNases) as well as nucleic acid editing enzymes, whose biochemical properties, expression profiles, functions and mechanisms of action will be detailed in this review. Fully elucidating the role of these enzymes in degrading and/or processing of self-nucleic acids to thwart their immunostimulatory potential is of utmost importance to develop novel therapeutic strategies for patients affected by inflammatory and autoimmune diseases.

Selectins impair regulatory T cell function and contribute to systemic lupus erythematosus pathogenesis.

Systemic lupus erythematosus (SLE) is a systemic autoimmune disease characterized by a loss of tolerance toward self-nucleic acids, autoantibody production, interferon expression and signaling, and a defect in the regulatory T (Treg) cell compartment. In this work, we identified that platelets from patients with active SLE preferentially interacted with Treg cells via the P-selectin/P-selectin glycoprotein ligand-1 (PSGL-1) axis. Selectin interaction with PSGL-1 blocked the regulatory and suppressive properties of Treg cells and particularly follicular Treg cells by triggering Syk phosphorylation and an increase in intracytosolic calcium. Mechanistically, P-selectin engagement on Treg cells induced a down-regulation of the transforming growth factor-ß axis, altering the phenotype of Treg cells and limiting their immunosuppressive responses. In patients with SLE, we found an up-regulation of P- and E-selectin both on microparticles and in their soluble forms that correlated with disease activity. Last, blocking P-selectin in a mouse model of SLE improved cardinal features of the disease, such as anti-dsDNA antibody concentrations and kidney pathology. Overall, our results identify a P-selectin-dependent pathway that is active in patients with SLE and validate it as a potential therapeutic avenue.

Self-Nucleic Acid Sensing: A Novel Crucial Pathway Involved in Obesity-Mediated Metaflammation and Metabolic Syndrome.

Obesity and overweight are a global health problem affecting almost one third of the world population. There are multiple complications associated with obesity including metabolic syndrome that commonly lead to development of type II diabetes and non-alcoholic fatty liver disease. The development of metabolic syndrome and severe complications associated with obesity is attributed to the chronic low-grade inflammation that occurs in metabolic tissues such as the liver and the white adipose tissue. In recent years, nucleic acids (mostly DNA), which accumulate systemically in obese individuals, were shown to aberrantly activate innate immune responses and thus to contribute to metabolic tissue inflammation. This minireview will focus on (i) the main sources and forms of nucleic acids that accumulate during obesity, (ii) the sensing pathways required for their detection, and (iii) the key cellular players involved in this process. Fully elucidating the role of nucleic acids in the induction of inflammation induced by obesity would promote the identification of new and long-awaited therapeutic approaches to limit obesity-mediated complications.

Pooled Analysis of the Cow's Milk-related-Symptom-Score (CoMiSS™) as a Predictor for Cow's Milk Related Symptoms.

PURPOSE: The diagnosis of cow's milk (CM) allergy is a challenge. The Cow's Milk-related-Symptom-Score (CoMiSS™) was developed to offer primary health care providers a reliable diagnostic tool for CM related symptoms. The predictive prospective value of the CoMiSS™ was evaluated in three clinical trials. METHODS: Pooled analyses of the three studies were conducted based on regressing the results of the month-1 challenge test on the month-1 CoMiSS™, adjusting for baseline CoMiSS™ using a logistic regression model. In addition a logistic regression model was also fitted to the month-1 challenge test result with the change in CoMiSS™ from baseline as a predictor. RESULTS: Results suggest that infants having a low CoMiSS™ (median, 5) after 1 month dietary treatment free from intact CM protein have a significant risk of having a positive challenge test (odds ratio, 0.83; 95% confidence interval, 0.75-0.93; p=0.002). Pooled data suggest that the change in CoMiSS™ from baseline to month-1 can predict CM related symptoms as a confirmed diagnosis according to the challenge test at month-1. However, in order to validate such a tool, infants without CM related symptoms would also need to be enrolled in a validation trial. A concern is that it may not be ethical to expose healthy infants to a therapeutic formula and a challenge test. CONCLUSION: Pooled data analysis emphasizes that the CoMiSS™ has the potential to be of interest in infants suspected to have CM-related-symptoms. A prospective validation trial is needed.

Themis1 enhances T cell receptor signaling during thymocyte development by promoting Vav1 activity and Grb2 stability.

The T cell signaling protein Themis1 is essential for the positive and negative selection of thymocytes in the thymus. Although the developmental defect that results from the loss of Themis1 suggests that it enhances T cell receptor (TCR) signaling, Themis1 also recruits Src homology 2 domain-containing phosphatase-1 (SHP-1) to the vicinity of TCR signaling complexes, suggesting that it has an inhibitory role in TCR signaling. We used TCR signaling reporter mice and quantitative proteomics to explore the role of Themis1 in developing T cells. We found that Themis1 acted mostly as a positive regulator of TCR signaling in vivo when receptors were activated by positively selecting ligands. Proteomic analysis of the Themis1 interactome identified SHP-1, the TCR-associated adaptor protein Grb2, and the guanine nucleotide exchange factor Vav1 as the principal interacting partners of Themis1 in isolated mouse thymocytes. Analysis of TCR signaling in Themis1-deficient and Themis1-overexpressing mouse thymocytes demonstrated that Themis1 promoted Vav1 activity both in vitro and in vivo. The reduced activity of Vav1 and the impaired T cell development in Themis1(-/-) mice were due in part to increased degradation of Grb2, which suggests that Themis1 is required to maintain the steady-state abundance of Grb2 in thymocytes. Together, these data suggest that Themis1 acts as a positive regulator of TCR signaling in developing T cells, and identify a mechanism by which Themis1 regulates thymic selection.