Coordinated local mucosal and systemic immune responses following severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection either protect against coronavirus disease 2019 (COVID-19) ...pathologies or fail, leading to severe clinical outcomes. To understand this process, we performed an integrated analysis of SARS-CoV-2 spike-specific antibodies, cytokines, viral load and bacterial communities in paired nasopharyngeal swabs and plasma samples from a cohort of clinically distinct patients with COVID-19 during acute infection. Plasma viral load was associated with systemic inflammatory cytokines that were elevated in severe COVID-19, and also with spike-specific neutralizing antibodies. By contrast, nasopharyngeal viral load correlated with SARS-CoV-2 humoral responses but inversely with interferon responses, the latter associating with protective microbial communities. Potential pathogenic microorganisms, often implicated in secondary respiratory infections, were associated with mucosal inflammation and elevated in severe COVID-19. Our results demonstrate distinct tissue compartmentalization of SARS-CoV-2 immune responses and highlight a role for the nasopharyngeal microbiome in regulating local and systemic immunity that determines COVID-19 clinical outcomes.
Type I interferons (IFNs) are essential mediators of antiviral responses. These cytokines have been implicated in the pathogenesis of autoimmunity, most notably systemic lupus erythematosus (SLE), ...diabetes mellitus, and dermatomyositis, as well as monogenic type I interferonopathies. Despite a fundamental role in health and disease, the direct quantification of type I IFNs has been challenging. Using single-molecule array (Simoa) digital ELISA technology, we recorded attomolar concentrations of IFNα in healthy donors, viral infection, and complex and monogenic interferonopathies. IFNα protein correlated well with functional activity and IFN-stimulated gene expression. High circulating IFNα levels were associated with increased clinical severity in SLE patients, and a study of the cellular source of IFNα protein indicated disease-specific mechanisms. Measurement of IFNα attomolar concentrations by digital ELISA will enhance our understanding of IFN biology and potentially improve the diagnosis and stratification of pathologies associated with IFN dysregulation.
Coronavirus disease 2019 (COVID-19) is characterized by distinct patterns of disease progression that suggest diverse host immune responses. We performed an integrated immune analysis on a cohort of ...50 COVID-19 patients with various disease severity. A distinct phenotype was observed in severe and critical patients, consisting of a highly impaired interferon (IFN) type I response (characterized by no IFN-β and low IFN-α production and activity), which was associated with a persistent blood viral load and an exacerbated inflammatory response. Inflammation was partially driven by the transcriptional factor nuclear factor-κB and characterized by increased tumor necrosis factor-α and interleukin-6 production and signaling. These data suggest that type I IFN deficiency in the blood could be a hallmark of severe COVID-19 and provide a rationale for combined therapeutic approaches.
The cholinergic system has been proposed as a potential regulator of COVID-19-induced hypercytokinemia. We investigated whole-blood expression of cholinergic system members and correlated it with ...COVID-19 severity. Patients with confirmed SARS-CoV-2 infection and healthy aged-matched controls were included in this non-interventional study. A whole blood sample was drawn between 9-11 days after symptoms onset, and peripheral leukocyte phenotyping, cytokines measurement, RNA expression and plasma viral load were determined. Additionally, whole-blood expression of native alpha-7 nicotinic subunit and its negative dominant duplicate (CHRFAM7A), choline acetyltransferase and acetylcholine esterase (AchE) were determined. Thirty-seven patients with COVID-19 (10 moderate, 11 severe and 16 with critical disease) and 14 controls were included. Expression of CHRFAM7A was significantly lower in critical COVID-19 patients compared to controls. COVID-19 patients not expressing CHRFAM7A had higher levels of CRP, more extended pulmonary lesions and displayed more pronounced lymphopenia. COVID-19 patients without CHRFAM7A expression also showed increased TNF pathway expression in whole blood. AchE was also expressed in 30 COVID-19 patients and in all controls. COVID-19-induced hypercytokinemia is associated with decreased expression of the pro-inflammatory dominant negative duplicate CHRFAM7A. Expression of this duplicate might be considered before targeting the cholinergic system in COVID-19 with nicotine.
Heterozygous missense mutations in coatomer protein subunit α, COPA, cause a syndrome overlapping clinically with type I IFN-mediated disease due to gain-of-function in STING, a key adaptor of IFN ...signaling. Recently, increased levels of IFN-stimulated genes (ISGs) were described in COPA syndrome. However, the link between COPA mutations and IFN signaling is unknown. We observed elevated levels of ISGs and IFN-α in blood of symptomatic COPA patients. In vitro, both overexpression of mutant COPA and silencing of COPA induced STING-dependent IFN signaling. We detected an interaction between COPA and STING, and mutant COPA was associated with an accumulation of ER-resident STING at the Golgi. Given the known role of the coatomer protein complex I, we speculate that loss of COPA function leads to enhanced type I IFN signaling due to a failure of Golgi-to-ER STING retrieval. These data highlight the importance of the ER-Golgi axis in the control of autoinflammation and inform therapeutic strategies in COPA syndrome.
Microbial nucleic acid recognition serves as the major stimulus to an antiviral response, implying a requirement to limit the misrepresentation of self nucleic acids as non-self and the induction of ...autoinflammation. By systematic screening using a panel of interferon-stimulated genes we identify two siblings and a singleton variably demonstrating severe neonatal anemia, membranoproliferative glomerulonephritis, liver fibrosis, deforming arthropathy and increased anti-DNA antibodies. In both families we identify biallelic mutations in DNASE2, associated with a loss of DNase II endonuclease activity. We record increased interferon alpha protein levels using digital ELISA, enhanced interferon signaling by RNA-Seq analysis and constitutive upregulation of phosphorylated STAT1 and STAT3 in patient lymphocytes and monocytes. A hematological disease transcriptomic signature and increased numbers of erythroblasts are recorded in patient peripheral blood, suggesting that interferon might have a particular effect on hematopoiesis. These data define a type I interferonopathy due to DNase II deficiency in humans.
Host immunity to infection with SARS-CoV-2 is highly variable, dictating diverse clinical outcomes ranging from asymptomatic to severe disease and death. We previously reported reduced type I ...interferon in severe COVID-19 patients preceded clinical worsening. Further studies identified genetic mutations in loci of the TLR3- or TLR7-dependent interferon-I pathways, or neutralizing interferon-I autoantibodies as risk factors for development of COVID-19 pneumonia. Here we show in patient cohorts with different severities of COVID-19, that baseline plasma interferon α measures differ according to the immunoassay used, timing of sampling, the interferon α subtype measured, and the presence of autoantibodies. We also show a consistently reduced induction of interferon-I proteins in hospitalized COVID-19 patients upon immune stimulation, that is not associated with detectable neutralizing autoantibodies against interferon α or interferon ω. Intracellular proteomic analysis shows increased monocyte numbers in hospitalized COVID-19 patients but impaired interferon-I response after stimulation. We confirm this by ex vivo whole blood stimulation with interferon-I which induces transcriptomic responses associated with inflammation in hospitalized COVID-19 patients, that is not seen in controls or non-hospitalized moderate cases. These results may explain the dichotomy of the poor clinical response to interferon-I based treatments in late stage COVID-19, despite the importance of interferon-I in early acute infection and may guide alternative therapeutic strategies.
NMOSD is not formally recognised as a side-effect of therapy with recombinant interferon alpha. ...we submitted requests for data on adverse events to spontaneous reporting schemes (the UK Medicines ...and Healthcare products Regulatory Agency's Yellow Card Scheme and VigiBase, the WHO global database of individual case safety reports) and reviewed published drug safety reports. Interferon beta therapy is widely used and moderately effective for the treatment of multiple sclerosis, but its use in people with NMOSD is associated with disease worsening8 and increasing aquaporin-4 antibody titres.9 Therefore, our findings are consistent with a growing body of evidence that suggests that activation of the type I IFN pathway causes opposing therapeutic responses in individuals with NMOSD and multiple sclerosis. Supplementary Material Supplementary appendix Recombinant interferon alpha therapy Increased endogenous IFNα Patient 1 Patient 2 Patient 3 Patient 4 Underlying disease Systemic mastocytosis Hepatitis C Monogenic interferonopathy Systemic lupus erythematosus Interferon exposure Interferon alpha (10 years) Interferon alpha (1 year) 872 fg/mL (normal 0–8 fg/mL) 815 fg/mL (normal 0–8 fg/mL) Clinical presentation Bilateral leg numbness and weakness Bilateral leg numbness and weakness Unilateral arm weakness Bilateral arm and leg numbness and weakness MRI findings LETM thoracic LETM thoracic LETM high cervical LETM high cervical Aquaporin-4 antibodies by cell-based assay Positive Positive Positive Positive Diagnostic criteria for NMOSD met Yes Yes Yes Yes Treatment Corticosteroids, azathioprine, and rituximab Corticosteroids and azathioprine Corticosteroids and ruxolitinib Corticosteroids, azathioprine, and rituximab Clinical course Relapsing transverse myelitis Relapsing transverse myelitis Single LETM Single LETM Table Patient characteristics
NLRP3 inflammasome is suggested to contribute to the complex pathogenesis of systemic lupus erythematosus, but its role in cutaneous lupus erythematosus has not been addressed. This study ...investigated the expression of NLRP3 inflammasome components and levels of type I interferons in the skin of 20 patients with cutaneous lupus erythematosus. Expression of NLRP1/3, adaptor protein ASC (apoptosis-associated speck-like protein), caspase-1, interferon-α (IFN-α), myxovirus resistance protein (MxA), and interferon-induced proteins 1 and 2 (IFIT 1/2) in the skin was assessed using reverse transcription quantitative real-time PCR (RT-qPCR), western blotting and immunohistochemistry. Serum interferon-α protein levels from 12 patients were measured using digital enzyme-linked immunoassay (ELISA). Interleukin-1β expression was significantly upregulated in the lesional skin of patients with cutaneous lupus erythematosus compared with their uninvolved skin. However, NLRP1/3, ASC and caspase-1 were not significantly upregulated compared with the skin of control persons. IFN-α and IFN-induced proteins MxA and IFIT1/2 were strongly expressed in cutaneous lupus erythematosus skin. Variability in the expression of NLRP3 inflammasome components among patients suggests heterogeneity of pathological pathways in cutaneous lupus erythematosus.
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