Alveolar cytokines and interferon autoantibodies in COVID-19 ARDS.

Background: Type I interferon (IFN-I) and IFN autoantibodies play a crucial role in controlling SARS-CoV-2 infection. The levels of these mediators have only rarely been studied in the alveolar compartment in patients with COVID-19 acute respiratory distress syndrome (CARDS) but have not been compared across different ARDS etiologies, and the potential effect of dexamethasone (DXM) on these mediators is not known. Methods: We assessed the integrity of the alveolo-capillary membrane, interleukins, type I, II, and III IFNs, and IFN autoantibodies by studying the epithelial lining fluid (ELF) volumes, alveolar concentration of protein, and ELF-corrected concentrations of cytokines in two patient subgroups and controls. Results: A total of 16 patients with CARDS (four without and 12 with DXM treatment), eight with non-CARDS, and 15 healthy controls were included. The highest ELF volumes and protein levels were observed in CARDS. Systemic and ELF-corrected alveolar concentrations of interleukin (IL)-6 appeared to be particularly low in patients with CARDS receiving DXM, whereas alveolar levels of IL-8 were high regardless of DXM treatment. Alveolar levels of IFNs were similar between CARDS and non-CARDS patients, and IFNα and IFNω autoantibody levels were higher in patients with CARDS and non-CARDS than in healthy controls. Conclusions: Patients with CARDS exhibited greater alveolo-capillary barrier disruption with compartmentalization of IL-8, regardless of DXM treatment, whereas systemic and alveolar levels of IL-6 were lower in the DXM-treated subgroup. IFN-I autoantibodies were higher in the BALF of CARDS patients, independent of DXM, whereas IFN autoantibodies in plasma were similar to those in controls.

A Novel CDC42 Variant with Impaired Thymopoiesis, IL-7R Signaling, PAK1 Binding, and TCR Repertoire Diversity.

Genetic variants in cell division cycle 42 (CDC42) can manifest with dysmorphic features, autoinflammation, hemophagocytic lymphohistiocytosis, and thrombocytopenia, whereas defective thymopoiesis is a rare disease manifestation. We report a novel CDC42 missense variant (c.46A > G, p.Lys16Glu) resulting in infection and HPV-driven carcinogenesis in the mosaic mother and impaired thymopoiesis and profound T cell lymphopenia in the heterozygous daughter identified through newborn screening for SCID. We found that surface expression of IL-7Rα (CD127) was decreased, consistent with reduced IL-7-induced STAT5 phosphorylation and accelerated apoptotic T cell death. Consistent with the vital role of IL-7 in regulating thymopoiesis, both patients displayed reduced T cell receptor CDR3 repertoires. Moreover, the CDC42 variant prevented binding to the downstream effector, p21-activated kinase (PAK)1, suggesting this impaired interaction to underlie reduced IL-7Rα expression and signaling. Here, we provide the first report of severely compromised thymopoiesis and perturbed IL-7Rα signaling caused by a novel CDC42 variant and presenting with diverging clinical and immunological phenotypes in patients.

A Distinct Dexamethasone-Dependent Gene Expression Profile in the Lungs of COVID-19 Patients.

The effects of dexamethasone (DXM) treatment on pulmonary immunity in COVID-19-associated acute respiratory distress syndrome (CARDS) remain insufficiently understood. We performed transcriptomic RNA-seq analysis of bronchoalveolar lavage fluid from 20 mechanically ventilated patients: 12 with CARDS (with or without DXM) and 8 non-COVID-19 critically ill controls. CARDS with DXM was characterized by upregulation of genes related to B-cell and complement pathway activation, antigen presentation, phagocytosis, and FC-γ receptor signaling. Most interferon-stimulated genes were upregulated in CARDS, particularly in CARDS without DXM. In conclusion, DXM treatment was not associated with regulation of proinflammatory pathways in CARDS but with regulation of other local immune responses. Clinical Trials Registration. NCT04354584.

Life-threatening viral disease in a novel form of autosomal recessive IFNAR2 deficiency in the Arctic.

Type I interferons (IFN-I) play a critical role in human antiviral immunity, as demonstrated by the exceptionally rare deleterious variants of IFNAR1 or IFNAR2. We investigated five children from Greenland, Canada, and Alaska presenting with viral diseases, including life-threatening COVID-19 or influenza, in addition to meningoencephalitis and/or hemophagocytic lymphohistiocytosis following live-attenuated viral vaccination. The affected individuals bore the same homozygous IFNAR2 c.157T>C, p.Ser53Pro missense variant. Although absent from reference databases, p.Ser53Pro occurred with a minor allele frequency of 0.034 in their Inuit ancestry. The serine to proline substitution prevented cell surface expression of IFNAR2 protein, small amounts of which persisted intracellularly in an aberrantly glycosylated state. Cells exclusively expressing the p.Ser53Pro variant lacked responses to recombinant IFN-I and displayed heightened vulnerability to multiple viruses in vitro-a phenotype rescued by wild-type IFNAR2 complementation. This novel form of autosomal recessive IFNAR2 deficiency reinforces the essential role of IFN-I in viral immunity. Further studies are warranted to assess the need for population screening.

Fulminant H1N1 and severe acute respiratory syndrome coronavirus-2 infections with a 4-year interval without an identifiable underlying cause: a case report.

BACKGROUND: The clinical presentation of severe acute respiratory syndrome coronavirus-2 infection is highly variable from asymptomatic infection to fulminant disease. The reasons for the variation are only starting to unravel, with risk factors including age and certain comorbidities as well as genetic defects causing immunological perturbations in the interferon pathways. CASE PRESENTATION: We report the case of an otherwise healthy Caucasian man, who at ages 60 and 64 years suffered from severe H1N1 influenza virus infection and severe acute respiratory syndrome coronavirus-2 infections, respectively. In both cases, there were acute kidney impairment and the need for intensive care unit admission as well as mechanical ventilation. Fortunately, after both infections there was full clinical recovery. The severity of the infections indicates an underlying impairment in the ability to control these kinds of infections. Challenge of patient peripheral blood mononuclear cells showed impaired type I and III antiviral interferon responses and reduced interferon-stimulated gene expression. However, despite investigation of patient samples by whole exome sequencing and enzyme-linked immunosorbent assay, no known disease-causing genetic variants related to interferon pathways were found, nor were interferon autoantibodies demonstrated. Thus, any underlying immunological cause of this unusual susceptibility to severe viral infections remains unresolved. CONCLUSION: The patient experienced very similar severe clinical pictures triggered by H1N1 and severe acute respiratory syndrome coronavirus-2 infections, indicating an underlying inability to contain these infections. We were unable to show that the patient had any of the currently known types of immune incompetence but identified genetic changes possibly contributing to the severe course of both infections. Further analyses to delineate contribution factors are needed.

Host Genetics and Antiviral Immune Responses in Adult Patients With Multisystem Inflammatory Syndrome.

COVID-19 associated multisystem inflammatory syndrome (MIS) is a rare condition mostly affecting children but also adults (MIS-A). Although severe systemic inflammation and multiorgan dysfunction are hallmarks of the syndrome, the underlying pathogenesis is unclear. We aimed to provide novel immunological and genetic descriptions of MIS-A patients. Cytokine responses (IL-6, IL-1ß, TNFα, CXCL10, type I, II and III interferons) following SARS-CoV-2 infection of peripheral blood mononuclear cells in vitro were analyzed as well as antibodies against IFNα and IFNω (by ELISA) in patients and healthy controls. We also performed whole exome sequencing (WES) of patient DNA. A total of five patients (ages 19, 23, 33, 38, 50 years) were included. The patients shared characteristic features, although organ involvement and the time course of disease varied slightly. SARS-CoV-2 in vitro infection of patient PBMCs revealed impaired type I and III interferon responses and reduced CXCL10 expression, whereas production of proinflammatory cytokines were less affected, compared to healthy controls. Presence of interferon autoantibodies was not detected. Whole exome sequencing analysis of patient DNA revealed 12 rare potentially disease-causing variants in genes related to autophagy, classical Kawasaki disease, restriction factors and immune responses. In conclusion, we observed an impaired production of type I and III interferons in response to SARS-CoV-2 infection and detected several rare potentially disease-causing gene variants potentially contributing to MIS-A.

STK4 Deficiency Impairs Innate Immunity and Interferon Production Through Negative Regulation of TBK1-IRF3 Signaling.

BACKGROUND: STK4 deficiency due to homozygous mutations in the STK4 gene encoding the STK4/MST1 kinase was first described in 2012. STK4/MST1 kinase regulates cell proliferation, survival, differentiation, and immune responses through canonical and non-canonical Hippo signaling pathways. OBJECTIVE: We describe an 11-year-old girl with a clinical presentation consisting of severe recurrent herpes zoster, chronic warts, and recurrent pneumonias, as well as a somatic phenotype with hypothyroidism and low stature. Whole exome sequencing revealed STK4 deficiency due to homozygosity for a novel frameshift variant in STK4, c.523dupA, p.(L174fsTer45), resulting in a premature stop codon within the kinase domain. METHODS: We performed a thorough investigation of the genetics and innate and adaptive immunological abnormalities in STK4 deficiency. RESULTS: We show significantly impaired type I, II, and III interferon (IFN) responses and partly reduced proinflammatory cytokine responses to ligands of Toll-like receptor (TLR)3, TLR9, and the cytosolic RNA and DNA sensors as well as to microorganisms. Impaired IFN responses could be attributed to reduced phosphorylation of TBK1 and IRF3. Moreover, virus infection induced enhanced cell death by apoptosis. Importantly, autophagy pathways were slightly disturbed, with enhanced LC3B-Ito LCB3-II conversion at the single cell level but normal overall formation of LCB3 punctae. Finally, the patient displayed some indicators of impaired adaptive immunity in the form of insufficient vaccination responses, T cell lymphopenia, and reduced Treg fractions, although with largely normal T cell proliferation and normal IFNg production. CONCLUSION: Here, we demonstrate disturbances in various immune cell populations and pathways involved in innate immune responses, cell death, autophagy, and adaptive immunity in a patient homozygous for a novel STK4 frameshift mutation.

SARS-CoV2-mediated suppression of NRF2-signaling reveals potent antiviral and anti-inflammatory activity of 4-octyl-itaconate and dimethyl fumarate.

Antiviral strategies to inhibit Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV2) and the pathogenic consequences of COVID-19 are urgently required. Here, we demonstrate that the NRF2 antioxidant gene expression pathway is suppressed in biopsies obtained from COVID-19 patients. Further, we uncover that NRF2 agonists 4-octyl-itaconate (4-OI) and the clinically approved dimethyl fumarate (DMF) induce a cellular antiviral program that potently inhibits replication of SARS-CoV2 across cell lines. The inhibitory effect of 4-OI and DMF extends to the replication of several other pathogenic viruses including Herpes Simplex Virus-1 and-2, Vaccinia virus, and Zika virus through a type I interferon (IFN)-independent mechanism. In addition, 4-OI and DMF limit host inflammatory responses to SARS-CoV2 infection associated with airway COVID-19 pathology. In conclusion, NRF2 agonists 4-OI and DMF induce a distinct IFN-independent antiviral program that is broadly effective in limiting virus replication and in suppressing the pro-inflammatory responses of human pathogenic viruses, including SARS-CoV2.

Author Correction: SARS-CoV2-mediated suppression of NRF2-signaling reveals potent antiviral and anti-inflammatory activity of 4-octyl-itaconate and dimethyl fumarate.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Systemic juvenile idiopathic arthritis and recurrent macrophage activation syndrome due to a CASP1 variant causing inflammasome hyperactivation.

OBJECTIVES: We investigated a patient with systemic juvenile idiopathic arthritis (sJIA) and recurrent macrophage activation syndrome (MAS) to discover genetic and immunological contributing factors. METHODS: Severe recurrent MAS motivated whole exome sequencing (WES) to identify genetic variants potentially involved in disease pathogenesis. In vitro peripheral blood mononuclear cell (PBMC) stimulations for cytokine expression and caspase-1 activity assays as well as NF-κB reporter luciferase assays were performed to functionally characterize variants. RESULTS: WES revealed an extremely rare heterozygous missense variant, c.482G>A, p.R161H in the CASP1 gene encoding pro-caspase-1. Lipopolysaccharide (LPS) stimulation of patient PBMCs induced high levels of IL-6 compared to controls, and activation of the NLRP3 inflammasome resulted in increased production of IL-1ß and IL-18 as well as significantly elevated caspase-1 activity. Constitutive and inducible levels of IL-18 and IFNγ in whole blood were markedly elevated. Expression of the CASP1 variant in an NF-κB reporter luciferase assay induced increased NF-κB activation in a RIP2-dependent manner. The disease course of the patient was complicated by severe recurrent MAS. However, dual IL-1 and IL-6 blockade caused disease remission. CONCLUSION: For the first time, we demonstrate the involvement of a CASP1 variant in sJIA and recurrent MAS. This variant is gain-of-function for both inflammasome and NF-κB activation leading to increased production of IL-6, IL-1ß and IL-18. Although dual IL-1 and IL-6 blockade may be beneficial in patients, in whom single treatment is not sufficient to control MAS, caution should be practiced, since interstitial lung disease may progress despite apparent clinical and biochemical remission.

Autoinflammatory disease with corneal and mucosal dyskeratosis caused by a novel NLRP1 variant.

OBJECTIVES: Here we investigated a patient with inflammatory corneal intraepithelial dyskeratosis, mucosal inflammation, tooth abnormalities and, eczema to uncover the genetic and immunological basis of the disease. METHODS: On suspicion of an autoinflammatory condition, Sanger sequencing of nucleotide-binding oligomerization domain-like, leucine-rich repeat pyrin domain containing 1 (NLRP1) was performed and combined with an in vitro inflammasome reconstitution assay to measure caspase-1-mediated IL-1ß cleavage, stimulation of patient peripheral blood mononuclear cells (PBMCs) and whole blood to measure IL-1ß, IL-18 production and quantification of apoptosis-associated speck-like protein containing CARD (ASC) speck formation as a measure of inflammasome activation by flow cytometry. RESULTS: Sanger sequencing revealed a novel mutation (c.175G>C, p.A59P; NM_33004.4) in the inflammasome molecule NLRP1 segregating with disease, although with incomplete penetrance, in three generations. We found that patient PBMCs produced increased IL-1ß in response to inflammatory stimuli, as well as increased constitutive levels of IL-18. Moreover, we demonstrate that expression of the identified NLRP1 A59P variant caused spontaneous IL-1ß cleavage to mature IL-1ß. In addition, patient PBMCs responded to NLRP1 stimulation with increased ASC speck formation as a reflection of elevated inflammasome activity. CONCLUSION: We demonstrate that this novel NLRP1 A59P variant caused increased activation of the NLRP1 inflammasome, resulting in constitutively and inducibly elevated IL-1ß and IL-18 synthesis. We suggest the NLRP1 mutation underlies the pathogenesis of this rare autoinflammatory dyskeratotic disease inherited in an autosomal dominant manner with incomplete penetrance in the patient and within the family for several generations.

Host Genetics, Innate Immune Responses, and Cellular Death Pathways in Poliomyelitis Patients.

PURPOSE: Poliovirus (PV) is one of the most studied viruses. Despite efforts to understand PV infection within the host, fundamental questions remain unanswered. These include the mechanisms determining the progression to viremia, the pathogenesis of neuronal infection and paralysis in only a minority of patients. Because of the rare disease phenotype of paralytic poliomyelitis (PPM), we hypothesize that a genetic etiology may contribute to the disease course and outcome. METHODS: We used whole-exome sequencing (WES) to investigate the genetic profile of 18 patients with PPM. Functional analyses were performed on peripheral blood mononuclear cells (PBMCs) and monocyte-derived macrophages (MdMs). RESULTS: We identified rare variants in host genes involved in interferon signaling, viral replication, apoptosis, and autophagy. Upon PV infection of MdMs, we observed a tendency toward increased viral burden in patients compared to controls, suggesting reduced control of PV infection. In MdMs from patients, the IFNß response correlated with the viral burden. CONCLUSION: We suggest that genetic variants in innate immune defenses and cell death pathways contribute to the clinical presentation of PV infection. Importantly, this study is the first to uncover the genetic profile in patients with PPM combined with investigations of immune responses and viral burden in primary cells.

Defective interferon priming and impaired antiviral responses in a patient with an IRF7 variant and severe influenza.

Influenza infection is common worldwide with many individuals affected each year during epidemics and occasionally pandemics. Previous studies in animal models and a few human cases have established an important role of innate type I and III interferon (IFN) for viral elimination and mounting of antiviral responses. However, genetic and immunological determinants of very severe disseminated influenza virus infection in humans remain incompletely understood. Here, we describe an adult patient with severe influenza virus A (IAV) infection, in whom we identified a rare variant E331V in IFN regulatory factor (IRF)7 by whole-exome sequencing. Examination of patient cells demonstrated a cellular phenotype suggesting functional IRF7 impairment, since priming with IFN was almost abolished and IFN responses to IAV were significantly impaired in patient cells. Moreover, IAV replication was significantly higher in patient cells than in controls. Finally, expression of IRF7 E331V in HEK293 cells demonstrated significantly reduced activation of both IFNA7 and IFNB promoters in a luciferase reporter gene expression assay compared to IRF7 wild type. These findings provide further support for the essential role of IRF7 in amplifying antiviral IFN responses to ensure potent and sustained IFN responses during influenza virus infection in humans.

Interferon signature in patients with STAT1 gain-of-function mutation is epigenetically determined.

STAT1 gain-of-function (GOF) variants lead to defective Th17 cell development and chronic mucocutaneous candidiasis (CMC), but frequently also to autoimmunity. Stimulation of cells with STAT1 inducing cytokines like interferons (IFN) result in hyperphosphorylation and delayed dephosphorylation of GOF STAT1. However, the mechanism how the delayed dephosphorylation exactly causes the increased expression of STAT1-dependent genes, and how the intracellular signal transduction from cytokine receptors is affected, remains unknown. In this study we show that the circulating levels of IFN-α were not persistently elevated in STAT1 GOF patients. Nevertheless, the expression of interferon signature genes was evident even in the patient with low or undetectable serum IFN-α levels. Chromatin immunoprecipitation (ChIP) experiments revealed that the active chromatin mark trimethylation of lysine 4 of histone 3 (H3K4me3), was significantly enriched in areas associated with interferon-stimulated genes in STAT1 GOF cells in comparison to cells from healthy donors. This suggests that the chromatin binding of GOF STAT1 variant promotes epigenetic changes compatible with higher gene expression and elevated reactivity to type I interferons, and possibly predisposes for interferon-related autoimmunity. The results also suggest that epigenetic rewiring may be responsible for treatment failure of Janus kinase 1/2 (JAK1/2) inhibitors in certain patients.

Functional IRF3 deficiency in a patient with herpes simplex encephalitis.

Herpes simplex encephalitis (HSE) in children has previously been linked to defects in type I interferon (IFN) production downstream of Toll-like receptor 3. Here, we describe a novel genetic etiology of HSE by identifying a heterozygous loss-of-function mutation in the IFN regulatory factor 3 (IRF3) gene, leading to autosomal dominant (AD) IRF3 deficiency by haploinsufficiency, in an adolescent female patient with HSE. IRF3 is activated by most pattern recognition receptors recognizing viral infections and plays an essential role in induction of type I IFN. The identified IRF3 R285Q amino acid substitution results in impaired IFN responses to HSV-1 infection and particularly impairs signaling through the TLR3-TRIF pathway. In addition, the R285Q mutant of IRF3 fails to become phosphorylated at S386 and undergo dimerization, and thus has impaired ability to activate transcription. Finally, transduction with WT IRF3 rescues the ability of patient fibroblasts to express IFN in response to HSV-1 infection. The identification of IRF3 deficiency in HSE provides the first description of a defect in an IFN-regulating transcription factor conferring increased susceptibility to a viral infection in the CNS in humans.

Listeria monocytogenes induces IFNß expression through an IFI16-, cGAS- and STING-dependent pathway.

Listeria monocytogenes is a gram-positive facultative intracellular bacterium, which replicates in the cytoplasm of myeloid cells. Interferon ß (IFNß) has been reported to play an important role in the mechanisms underlying Listeria disease. Although studies in murine cells have proposed the bacteria-derived cyclic-di-AMP to be the key bacterial immunostimulatory molecule, the mechanism for IFNß expression during L. monocytogenes infection in human myeloid cells remains unknown. Here we report that in human macrophages, Listeria DNA rather than cyclic-di-AMP is stimulating the IFN response via a pathway dependent on the DNA sensors IFI16 and cGAS as well as the signalling adaptor molecule STING. Thus, Listeria DNA is a major trigger of IFNß expression in human myeloid cells and is sensed to activate a pathway dependent on IFI16, cGAS and STING.

Inflammatory cytokines break down intrinsic immunological tolerance of human primary keratinocytes to cytosolic DNA.

Keratinocytes are involved in protecting the body from infections and environmental challenges, but also in inflammatory conditions like psoriasis. DNA has emerged as a potent stimulator of innate immune responses, but there is largely no information of how keratinocytes respond to cytosolic DNA. In this study, we report that human keratinocytes are tolerant to cytoplasmic DNA. However, if treated with inflammatory cytokines, keratinocytes gained the capacity to respond to DNA through a mechanism antagonized by the antimicrobial peptide LL37, proposed to be involved in activation and regulation of skin inflammation. The DNA sensor IFN-inducible protein 16 (IFI16) colocalized with DNA and the signaling molecule stimulator of IFN genes (STING) in the cytoplasm only in cytokine-stimulated cells, correlating with recruitment of the essential kinase TANK-binding kinase 1. Moreover, IFI16 was essential for DNA-driven innate immune responses in keratinocytes. Finally, IFI16 was upregulated in psoriasis skin lesions and localized to the cytoplasm in a subpopulation of cells. Collectively, this work suggests that inflammatory environments in the skin can lead to breakdown of tolerance for DNA in keratinocytes, which could contribute to the development of inflammatory diseases.