SARS-CoV-2-encoded small RNAs are able to repress the host expression of SERINC5 to facilitate viral replication.

Serine incorporator protein 5 (SERINC5) is a key innate immunity factor that operates in the cell to restrict the infectivity of certain viruses. Different viruses have developed strategies to antagonize SERINC5 function but, how SERINC5 is controlled during viral infection is poorly understood. Here, we report that SERINC5 levels are reduced in COVID-19 patients during the infection by SARS-CoV-2 and, since no viral protein capable of repressing the expression of SERINC5 has been identified, we hypothesized that SARS-CoV-2 non-coding small viral RNAs (svRNAs) could be responsible for this repression. Two newly identified svRNAs with predicted binding sites in the 3'-untranslated region (3'-UTR) of the SERINC5 gene were characterized and we found that the expression of both svRNAs during the infection was not dependent on the miRNA pathway proteins Dicer and Argonaute-2. By using svRNAs mimic oligonucleotides, we demonstrated that both viral svRNAs can bind the 3'UTR of SERINC5 mRNA, reducing SERINC5 expression in vitro. Moreover, we found that an anti-svRNA treatment to Vero E6 cells before SARS-CoV-2 infection recovered the levels of SERINC5 and reduced the levels of N and S viral proteins. Finally, we showed that SERINC5 positively controls the levels of Mitochondrial Antiviral Signalling (MAVS) protein in Vero E6. These results highlight the therapeutic potential of targeting svRNAs based on their action on key proteins of the innate immune response during SARS-CoV-2 viral infection.

Roles of Bone Morphogenetic Protein Receptor 1A in Germinal Centers and Long-Lived Humoral Immunity.

In response to T-dependent Ag, germinal centers (GC) generate bone marrow-resident plasma cells (BMPC) and memory B cells (MBC). In this study, we demonstrate that the bone morphogenetic protein receptor 1A (BMPR1A) signaling pathway, which regulates differentiation and self-renewal in multiple stem cell populations, regulates GC dynamics and resultant establishment of BMPC and MBC. Expression studies using quantitative PCR and novel Bmpr1aIRES.EGFP reporter mice demonstrated that Bmpr1a expression is upregulated among GC B cells (GCBC) and subsets of MBC, bone marrow plasmablasts, and BMPC. In immunized mice carrying B cell-targeted Bmpr1a gene deletions, the GC response was initially diminished. Subsequently, the GCBC compartment recovered in size, concurrent with accumulation of GCBC that carried unmodified rather than deleted Bmpr1a alleles. Similarly, the resulting class-switched MBC and BMPC carried retained non-recombined alleles. Despite the strong selective pressure for "leaky" B cells that retained Bmpr1a, there was a permanent marked reduction in switched bone marrow Ab-forming cells (plasmablasts + plasma cells), BMPC, MBC, and Ag-specific serum IgM in mice carrying B cell-targeted Bmpr1a gene deletions. These findings demonstrate a novel role for BMPR1A in the modulation of the B cell response and in the establishment of long-term memory.

Human Extrafollicular CD4+ Th Cells Help Memory B Cells Produce Igs.

Follicular helper T (Tfh) cells are necessary for germinal center B cell maturation during primary immune responses; however, the T cells that promote humoral recall responses via memory B cells are less well defined. In this article, we characterize a human tonsillar CD4+ T cell subset with this function. These cells are similar to Tfh cells in terms of expression of the chemokine receptor CXCR5 and the inhibitory receptor PD-1, IL-21 secretion, and expression of the transcription factor BCL6; however, unlike Tfh cells that are located within the B cell follicle and germinal center, they reside at the border of the T cell zone and the B cell follicle in proximity to memory B cells, a position dictated by their unique chemokine receptor expression. They promote memory B cells to produce Abs via CD40L, IL-10, and IL-21. Our results reveal a unique extrafollicular CD4+ T cell subset in human tonsils, which specialize in promoting T cell-dependent humoral recall responses.

TFH cells progressively differentiate to regulate the germinal center response.

Germinal center (GC) B cells undergo affinity selection, which depends on interactions with CD4(+) follicular helper T cells (TFH cells). We found that TFH cells progressed through transcriptionally and functionally distinct stages and provided differential signals for GC regulation. They initially localized proximally to mutating B cells, secreted interleukin 21 (IL-21), induced expression of the transcription factor Bcl-6 and selected high-affinity B cell clones. As the GC response evolved, TFH cells extinguished IL-21 production and switched to IL-4 production, showed robust expression of the co-stimulatory molecule CD40L, and promoted the development of antibody-secreting B cells via upregulation of the transcription factor Blimp-1. Thus, TFH cells in the B cell follicle progressively differentiate through stages of localization, cytokine production and surface ligand expression to 'fine tune' the GC reaction.

A Critical Role of IL-21-Induced BATF in Sustaining CD8-T-Cell-Mediated Chronic Viral Control.

Control of chronic viral infections by CD8 T cells is critically dependent on CD4 help. In particular, helper-derived IL-21 plays a key role in sustaining the CD8 T cell response; however, the molecular pathways by which IL-21 sustains CD8 T cell immunity remain unclear. We demonstrate that IL-21 causes a phenotypic switch of transcription factor expression in CD8 T cells during chronic viral infection characterized by sustained BATF expression. Importantly, BATF expression during chronic infection is both required for optimal CD8 T cell persistence and anti-viral effector function and sufficient to rescue "unhelped" CD8 T cells. Mechanistically, BATF sustains the response by cooperating with IRF4, an antigen-induced transcription factor that is also critically required for CD8 T cell maintenance, to preserve Blimp-1 expression and thereby sustain CD8 T cell effector function. Collectively, these data suggest that CD4 T cells "help" the CD8 response during chronic infection via IL-21-induced BATF expression.

Dynamic signaling by T follicular helper cells during germinal center B cell selection.

T follicular helper (T(FH)) cells select high-affinity, antibody-producing B cells for clonal expansion in germinal centers (GCs), but the nature of their interaction is not well defined. Using intravital imaging, we found that selection is mediated by large but transient contacts between T(FH) and GC B cells presenting the highest levels of cognate peptide bound to major histocompatibility complex II. These interactions elicited transient and sustained increases in T(FH) intracellular free calcium (Ca(2+)) that were associated with T(FH) cell coexpression of the cytokines interleukin-4 and -21. However, increased intracellular Ca(2+) did not arrest TFH cell migration. Instead, T(FH) cells remained motile and continually scanned the surface of many GC B cells, forming short-lived contacts that induced selection through further repeated transient elevations in intracellular Ca(2+).

Clinical significance of high levels of soluble tumour necrosis factor-α receptor-2 produced by alternative splicing in rheumatoid arthritis: a longitudinal prospective cohort study.

OBJECTIVES: We investigated whether serum levels of an alternatively spliced soluble (s)TNF receptor-2 (DS-TNFR2) affected the clinical response to anti-TNF-α therapy, classical DMARDs or radiological evidence of disease progression in patients with RA. METHODS: We included 116 patients with RA. Cohort 1: 52 DMARD-naïve early RA patients [mean (s.d.) disease duration 8.5 (6.2) months] who started gold salts and MTX therapies. Cohort 2: 64 MTX-resistant established RA patients [144 (107) months] who started infliximab therapy. We evaluated the European League Against Rheumatism (EULAR) response to therapy and the serum levels of DS-TNFR2, sTNFR2 and ACPAs at baseline and at 12 months. In Cohort 1, radiological progression and levels of MMP-1 were also determined. RESULTS: In Cohort 1, 40% of patients had high baseline levels (HL > 50 ng/ml) of DS-TNFR2 with significantly higher RF and ACPA levels than patients with normal levels (NL ≤ 50 ng/ml) of DS-TNFR2. The EULAR response to DMARDs was similar in HL and NL patients. Radiographic progression was observed in 23.5% of all patients after 12 months. In Cohort 2, 26.6% of patients had HL of DS-TNFR2 with significantly higher RF and ACPA levels than patients with NLs. The EULAR response from 6 to 30 weeks was prolonged in the HL group compared with the NL group. CONCLUSIONS: Patients with HL of DS-TNFR2 maintained a prolonged therapeutic response to anti-TNF-α therapy and had proportionally less radiographic progression compared with patients with NLs.

Soluble TNF-alpha receptor 2 produced by alternative splicing is paradoxically associated with markers of liver injury.

Tumor necrosis factor-alpha and insulin resistance play central roles in the pathogenesis of abnormal hepatocellular function. We evaluate the relationship between a novel serum DS-TNFR2 (an alternatively spliced soluble TNF-alpha receptor 2) isoform and parameters of liver health. Serum ALT, AST and GGT, insulin resistance, adiponectin and DS-TNFR2 isoform concentrations were measured in 492 subjects from two different Caucasian Spanish populations. We found a significant negative association between serum ALT and DS-TNFR2 levels in both populations (r=-0.269; p=0.002 and r=-0.152; p=0.01, respectively). DS-TNFR2 levels also correlated negatively with serum AST (r=-0.142; p=0.042) and GGT (r=-0.206; p=0.003) in population 1 and with AST (r=-0.127; p=0.038) in population 2. In multiple regression analysis models, serum DS-TNFR2 was shown to be an independent modulator of serum ALT activity after adjusting for sex, age, BMI, HOMA and adiponectin in both populations. These results suggest potential anti-inflammatory properties of this TNF-alpha receptor 2 isoform at the hepatic level.

An alternatively spliced soluble TNF-alpha receptor is associated with metabolic disorders: a replication study.

In a previous study, we identified a biologically active form of tumor necrosis factor-alpha receptor 2 (sTNFR2) produced by differential splicing (DS-TNFR2) which antagonized TNF-alpha biological activity. Obesity, insulin resistance and type 2 diabetes are linked to increased TNF-alpha action. We hypothesized that subjects with detectable DS-TNFR2 would be protected from developing obesity and related metabolic disorders. Thus, we investigated if circulating DS-TNFR2 concentration was associated with components of the so-called metabolic syndrome among 269 consecutive subjects from the general population. DS-TNFR2 was measured using a monoclonal antibody against an epitope present in TNFR2 (first 14 residues of the juxtamembrane region) but predicted to be absent in soluble proteolytic cleavage-produced TNFR2. Plasma DS-TNFR2 concentration was significantly decreased among patients with glucose intolerance or type 2 diabetes mellitus (p=0.026). DS-TNFR2 tended to be associated with fasting and post-load glucose (both r=-0.11, p=0.054), and with diastolic blood pressure in men (r=-0.16, p=0.07). Serum DS-TNFR2 concentration was significantly associated with LDL cholesterol (r=-0.28, p=0.002), uric acid (r=-0.13, p=0.04) and with blood glycated hemoglobin (r=-0.13, p=0.04). DS-TNFR2 declined with increased number of components of the metabolic syndrome (p=0.03). Those subjects with 2 or more components had significantly decreased circulating DS-TNFR2 levels (0.96+/-2.2 versus 1.7+/-3.2, p=0.033). In summary, the circulating concentration of DS-TNFR2 seems to be inversely linked to metabolic disorders, hinting at a possible anti-inflammatory role.

An alternative spliced variant of circulating soluble tumor necrosis factor-alpha receptor-2 is paradoxically associated with insulin action.

OBJECTIVE: Serum concentrations of soluble tumor necrosis factor-alpha (TNF-alpha) receptor 2 (sTNFR2) are associated with insulin resistance. In a recent study, we provided evidence for the existence of a biologically active form of sTNFR2 produced by alternative splicing (DS-TNFR2). We aimed to evaluate whether this circulating DS-TNFR2 is associated with insulin action in humans. DESIGN AND METHODS: Real time PCR (light cycler technology) evaluated DS-TNFR2 expression in monocytes. DS-TNFR2 was measured using a monoclonal antibody against an epitope present in TNFR2 (first 14 residues of the juxtamembrane region) but predicted to be absent in soluble proteolytic cleavage-produced TNFR2. Insulin sensitivity was measured using euglycemic hyperinsulinemic clamp (n = 76) and homeostatic model of assessment (HOMA) value in a replication study of 223 subjects. RESULTS: Real time PCR confirmed gene expression of DS-TNFR2 in monocytes from healthy subjects. A significant and positive association was found between serum DS-TNFR2 concentration and insulin sensitivity (P = 0.032, n = 76). This association was most significant in subjects with normal glucose tolerance (r = 0.44, P = 0.002). The subjects in whom DS-TNFR2 was detectable were more insulin sensitive than those with undetectable DS-TNFR2 (42.12+/-22.08 vs 31.71+/- 16.95 micromol x kg(-1) x min(-1), P = 0.039). DS-TNFR2 was inversely associated with body mass index, waist-to-hip ratio, systolic and diastolic blood pressure, fasting serum glucose, serum triglycerides and serum uric acid concentration and with the HOMA value (P = 0.03) in the replication study. Circulating DS-TNFR2 declined with increased number of components of the metabolic syndrome. CONCLUSION: Native sTNFR2 and DS-TNFR2 show opposite associations with insulin action. DS-TNFR2 might play a role as a counterpart of the proinflammatory environment associated with insulin resistance.

A polymorphism in the 3' untranslated region of the gene for tumor necrosis factor receptor 2 modulates reporter gene expression.

The gene encoding the human TNF alpha receptor (TNFR) 2 contains polymorphisms in the 3' untranslated region (UTR). Previous studies have shown that some variant alleles in this region are associated with obesity and insulin resistance. However, the effect of these polymorphisms on the expression of TNFR2 has not been studied to date. To examine the role played by different haplotypes in the control of TNFR2 expression (haplotypes A1-A5, referring to nucleotides 1663 G/A, 1668 T/G, and 1690 T/C), we introduced these sequences into the 3'-UTR of a heterologous reporter gene and expressed the corresponding constructs in a human T-cell line. We demonstrate that a 485-nt fragment of the TNFR2 3'-UTR that contains a U-rich region decreases reporter expression and that haplotypes A1-A4 exert a stronger effect than A5. Furthermore, time-course assays of mRNA stability using actinomycin D revealed that haplotypes A1-A4 destabilize the mRNA. The proximal TNFR2 3'-UTR, independently of haplotype differences, responded to T-cell activation by increasing mRNA decay. Electromobility shift analysis demonstrated that protein(s) found in T-cell extracts bind to the 485-nt fragment. We suggest that an increased rate of TNFR2 mRNA decay protects cells from unrestrained TNF alpha effects and that this protection is weakened in A5 subjects. These findings may explain the association of this haplotype with obesity and increased leptin levels.

Identification and characterization of a novel spliced variant that encodes human soluble tumor necrosis factor receptor 2.

Tumor necrosis factor (TNF)-alpha is a pleiotropic cytokine involved in a broad spectrum of inflammatory and immune responses including proliferation, differentiation and cell death induction in several cell types. The biological effects of TNF-alpha are mediated via the cell-surface TNF receptors TNFR1 and TNFR2. Soluble forms of these two receptors, which contain the extracellular ectodomains, are proteolytically cleaved from the membrane. High levels of soluble (s) TNFR2 in serum have been documented in multiple inflammatory pathologies. We describe here a new differential spliced isoform of human TNFR2 missing exons 7 and 8, DS-TNFR2(Delta7,8). This novel isoform lacks the transmembrane and cytoplasmic domains. Expression studies with DS-TNFR2(Delta7,8) cDNA transiently transfected COS cells showed that it encodes a sTNFR2 receptor of approximately 42 kDa. Soluble DS-TNFR2(Delta7,8) blocked TNF-alpha-induced apoptosis, which suggests that it regulates TNF-alpha function by antagonizing its biological activity. An ELISA was developed that quantifies sTNFR2 generated by alternative splicing. Our data show that sTNFR2 generated by alternative splicing can be found in sera of healthy individuals, at increased levels in patients with sepsis and at high concentrations in rheumatoid arthritis patients.

Shedding of TNF-alpha receptors, blood pressure, and insulin sensitivity in type 2 diabetes mellitus.

Tumor necrosis factor-alpha (TNF-alpha) is increasingly recognized as a key component in the development of insulin resistance and increased blood pressure. In a sample of 368 individuals, the ratio of soluble TNF-alpha receptors (sTNFR2/sTNFR1) correlated positively with systolic and diastolic blood pressure (P < 0.01). This ratio was significantly greater in type 2 diabetic subjects (DM-2) than in type 1 diabetic patients and was greater than in control nondiabetic subjects (P < 0.00001). The TNF-alpha receptor 1 (TNFR1) density in peripheral blood monocytes was similar in DM-2 patients and in nondiabetic subjects. After phorbol 12-myristate 13-acetate, TNFR1 shedding was significantly decreased in DM-2 compared with control subjects, and it was directly associated with insulin sensitivity (r = 0.54, P = 0.03). Serum sTNFR1 concentration was also linked to the vasodilatory response to glyceryltrinitrate (P = 0.01). Conversely, TNF-alpha receptor 2 shedding was negatively associated with insulin sensitivity (r = -0.54, P = 0.03), whereas shedding of L-selectin showed no significant association. After exercise-induced lowering of blood pressure, a parallel decrease in sTNFR2/sTNFR1 was observed in DM-2 patients. Our findings suggest that insulin resistance and blood pressure are linked to altered shedding of TNF-alpha receptors in DM-2. The latter seems reversible and is not genetically determined.