Journal of Experimental Medicine

The IKAROS family transcription factors regulate lymphocyte development. Loss-of-function variants in IKZF1 cause primary immunodeficiency, but IKAROS family members IKZF2 and IKZF3 have not yet been associated with immunodeficiency yet. Here, we describe a pedigree with a heterozygous truncating variant in IKZF2, encoding the translational activator and repressor HELIOS which is highly expressed in regulatory T cells and effector T cells, particularly of the CD8+ T cell lineage. Protein-protein interaction analysis revealed that the variant abolished HELIOS dimerizations as well as binding to members of the Mi-2/NuRD chromatin remodeling complex. Patients carrying the IKZF2 variant presented with a combined immunodeficiency phenotype characterized by recurrent upper respiratory infections, thrush and mucosal ulcers, as well as chronic lymphadenopathy. With extensive immunophenotyping, functional assays, and transcriptional analysis we show that reduced HELIOS expression was associated with chronic T cell activation and increased production of pro-inflammatory cytokines both in effector and regulatory T cells. Lymph node histology from patients indicated dysregulated germinal center reactions. Moreover, affected individuals displayed profoundly reduced circulating MAIT cell numbers. In summary, we show that this novel loss-of-function variant in HELIOS leads to an immunodeficiency with signs of immune overactivation. One sentence summaryTruncating variant of HELIOS causes immunodeficiency with signs of immune overactivation.

The linear ubiquitin assembly complex (LUBAC) consists of HOIP, HOIL1 and SHARPIN, and is essential for proper immune responses. Patients with HOIP and HOIL1 deficiencies present with severe immunodeficiency, autoinflammation and glycogen storage. In mice, the loss of Sharpin leads to severe dermatitis due to excessive cell death in keratinocytes. Here we report the first patient with SHARPIN deficiency, manifesting fever, arthritis, colitis, chronic otitis media and hepatic glycogenosis but unexpectedly, not associated with dermatologic manifestations. Mechanistically, fibroblasts and B cells from patients with all three LUBAC deficiencies showed attenuated canonical NF-B response and propensity to apoptosis mediated by TNF superfamily members. Furthermore, the SHARPIN deficient patient showed substantial reduction of adenoidal germinal center B cell development. Treatment of the SHARPIN deficient patient with anti-TNF therapies led to complete clinical and transcriptomic resolution of autoinflammation. These findings underscore the critical role of LUBAC as a gatekeeper for apoptosis-mediated immune dysregulation in humans.

ATR (Ataxia Telangiectasia and Rad3-related) kinase and its interacting protein ATRIP orchestrate the replication stress response. Two patients of independent ancestry with microcephaly, primordial dwarfism, and recurring infections were found to be homozygous for splice donor site variants of ATRIP exon 5, resulting in ATRIP deficiency. The c.829+5G>T patient exhibited autoimmune hemolytic anemia, lymphopenia, poor vaccine response, and intermittent neutropenia. Immunophenotyping revealed reduced CD16+ NK cells and absent naive T cells, mucosal-associated invariant T cells (MAITs), and invariant natural killer T cells (iNKTs). Lymphocytic defects were characterized by T cell receptor (TCR) oligoclonality, abnormal class switch recombination (CSR), and impaired T cell proliferation. ATRIP deficiency resulted in low-grade ATR activation but impaired CHK1 phosphorylation upon genotoxic stress. Consequently, ATRIP deficient cells inadequately regulated DNA replication, leading to chromosomal instability, compromised cell cycle control, and impaired cell viability. CRISPR-SelectTIME confirmed reduced cell fitness induced by both variants. This study establishes ATRIP deficiency as a monogenic cause of microcephalic primordial dwarfism, highlights ATRIPs critical role in protecting immune cells from replication stress, and brings a renewed perspective to the canonical functions of ATRIP.

Homozygous OTU deubiquitinase with linear linkage specificity (OTULIN) variants cause OTULIN-Related Autoinflammatory Syndrome (ORAS). This disease is characterized by early-onset autoinflammation, fever, panniculitis, diarrhea, and arthritis. In contrast, heterozygous and compound-heterozygous OTULIN variants have been associated with a phenotype defined by abscess development in different organs. Whether homozygous OTULIN variants can cause abscessing in affected patients is currently unknown. Here, we report a juvenile female patient harboring a novel homozygous OTULIN variant (Chr5:14687605G>T, p.V185F; referred to as OTULINV185F), presenting with autoinflammation and sterile abscesses in lung and skin. Through in silico analysis and functional assays, we show that OTULINV185F impairs OTULIN function, leading to compromised degradation of linear ubiquitin linkages. Notably, the patient clinically improved on anti-TNF therapy. Our findings underscore the diverse clinical manifestations of OTULIN dysfunction and call for a new classification of the disease that includes abscess formation as potential ORAS symptom.

Acquired mutations in the UBA1 gene, occurring in myeloid cells and resulting in expression of a catalytically impaired isoform of the enzyme E1, were recently identified in patients with severe adult-onset auto-inflammatory syndrome called VEXAS (vacuoles, E1 enzyme, X-linked, autoinflammatory, somatic). The precise physiological and clinical impact of these mutations remains poorly defined. Here, we studied a unique prospective cohort of individuals with severe autoinflammatory disease with (VEXAS) or without (VEXAS-like) UBA1 somatic mutations and compared with low-risk myelodysplastic syndromes (MDS) and aged gender-matched healthy controls. We performed an integrated immune analysis including multiparameter phenotyping of peripheral blood leukocytes, cytokines profiling, bulk and single-cell gene expression analyses and skin tissue imaging mass cytometry. Focusing on myeloid cells, we show that monocytes from UBA1-mutated individuals were quantitatively and qualitatively impaired and displayed features of exhaustion with aberrant expression of chemokine receptors. Within affected tissues, pathological skin biopsies from VEXAS patients showed an abundant enrichment of CD16+ CD163+ monocytes adjacent to blood vessels and M1 macrophages, possibly promoting local inflammation in part through STAT3 activation. In peripheral blood from VEXAS patients, we identified a significant increase in circulating levels of many proinflammatory cytokines, including IL-1{beta} and IL-18 which reflect inflammasome activation and markers of myeloid cells dysregulation. Gene expression analysis of whole blood confirmed the role of circulating cells in the IL-1{beta} and IL-18 dysregulation in VEXAS patients and revealed a significant enrichment of TNF- and NF{kappa}B signaling pathways that could mediate cell death and inflammation. Single-cell analysis confirmed the inflammatory state of monocytes from VEXAS patients and allowed us to identify specific molecular pathways that could explain monocytopenia, especially the activation of PANoptosis and a deficiency in the TYROBP/DAP12 axis and {beta}-catenin signaling pathway. Together, these findings on monocytes from patients with UBA1 mutations provide important insights into the molecular mechanisms involving the mature myeloid commitment in VEXAS syndrome and suggest that the control of the undescribed inflammasome activation and PANoptosis could be novel therapeutic targets in this condition. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=198 SRC="FIGDIR/small/22281005v1_ufig1.gif" ALT="Figure 1"> View larger version (56K): org.highwire.dtl.DTLVardef@10a14e4org.highwire.dtl.DTLVardef@e1b88eorg.highwire.dtl.DTLVardef@11bb0a2org.highwire.dtl.DTLVardef@e971ca_HPS_FORMAT_FIGEXP M_FIG C_FIG

Human endosomal Toll-like receptors TLR7 and TLR8 recognize self and non-self RNA ligands, and are important mediators of innate immunity and autoimmune pathogenesis. TLR7 and TLR8 are encoded by the adjacent X-linked genes, TLR7 and TLR8. We previously established that TLR7 evades X chromosome inactivation in female immune cells, and that mononuclear blood cells express more TLR7 protein in women than in men. Using RNA fluorescence in situ hybridization, we now show that TLR8 likewise evades X chromosome inactivation in CD14+ monocytes and CD4+ T lymphocytes, and that cells harboring TLR7 or TLR8 transcript foci are more frequent in women than in men. In parallel, we found TLR7 and TLR8 simultaneous transcription to be disproportionally frequent in female monocytes and T cells, and disproportionally scarce in the male cells, resulting in a 7-fold difference in frequency. These transcriptional biases were again observable when comparing the single X of XY males with the active X of female cells. Among (47,XXY) Klinefelter syndrome males, both TLR7 and TLR8 escape X chromosome inactivation, and co-transcription frequencies on the active X of monocytes were intermediate overall between those for XY males and XX females, and encompassed both male- and female-like individual patterns. These findings indicate that the TLR7 and TLR8 genes form a co-regulated gene cluster, which we have called the X-linked Toll-like receptor locus, with different sex- and sexual karyotype-dependent modes of transcription. Interestingly, TLR8 protein expression was significantly higher in female mononuclear blood cells, including all monocyte subsets, than in the male cells. Thus, co-dependent transcription from the active X chromosome and escape from inactivation could both contribute to higher TLR8 protein abundance in female cells, which may have implications for the response to viruses and bacteria, and the risk of developing inflammatory and autoimmune diseases. HighlightsO_LITLR8, like TLR7, escapes X chromosome inactivation in immune cells from women and 47,XXY Klinefelter syndrome (KS) men. C_LIO_LIThe frequency of cells double-positive for TLR7 and TLR8 primary transcripts is 7-fold higher in women than in men. C_LIO_LITLR7 and TLR8 form a co-regulated gene cluster on the human X chromosome, with sex-specific, divergent transcriptional patterns observable in monocytes and CD4+ T lymphocytes. C_LIO_LICo-dependent transcription of the TLR7 and TLR8 genes on the active X was observed in women and KS men, contrasting with mutually exclusive transcription in euploid men. C_LIO_LIBlood mononuclear cells, including monocyte subsets, expressed higher levels of TLR8 protein in females than in males. C_LI

Initial symptoms of COVID-19 infection depend on viral replication, while hyperinflammation is a hallmark of critical illness and may drive severe pneumonia and death. Among the mechanisms potentially involved in the hyperinflammatory state, we focused on the unfolded protein response, because the IRE1-XBP1 branch can be activated as result of the endoplasmic reticulum stress produced by the overwhelming synthesis of viral components and synergizes with Toll-like receptor signaling to induce cytokine expression. Viral RNA may trigger the IRE1-XBP1 branch via TLR7/8 activation and like TLR2 and TLR4 may underpin cytokine expression trough XBP1 splicing (sXBP1). The expression of IL1B, IL6, and TNF mRNA in bronchoalveolar aspirates (BAAs) were higher in COVID-19 patients under mechanical ventilation and intubation who showed sXBP1. The scrutiny of monocytic/macrophagic markers during active infection showed a reduction of those involved in antigen presentation and survival, as well as the IFN stimulated gene MX1. These changes reverted after infection tests turned negative. In contrast, the expression of the mRNA of the serine protease TMPRSS2 involved in S protein priming showed a high expression during active infection. TLR8 mRNA showed an overwhelming expression as compared to TLR7 mRNA, which suggests the presence of monocyte-derived dendritic cells (MDDCs). In vitro experiments in MDDCs activated with ssRNA40, a positive-sense, single-stranded RNA (+ssRNA) like SARS-CoV-2 RNA, induced sXBP1 and the expression of IL-1{beta}, IL-6, and TNF at mRNA and protein levels. These responses were blunted by the IRE1 ribonuclease inhibitor MKC8866. Given the analogies between the results observed in BAAs and the effects induced by +ssRNA in MDDCs, IRE1 ribonuclease inhibition might be a druggable target in severe COVID-19 disease. O_FIG O_LINKSMALLFIG WIDTH=180 HEIGHT=200 SRC="FIGDIR/small/22269752v1_ufig1.gif" ALT="Figure 1"> View larger version (53K): org.highwire.dtl.DTLVardef@13b04b3org.highwire.dtl.DTLVardef@1b1af7corg.highwire.dtl.DTLVardef@780104org.highwire.dtl.DTLVardef@8ad0ba_HPS_FORMAT_FIGEXP M_FIG C_FIG Author summaryCOVID-19 pandemics put an unprecedented pressure on health systems. The need of new therapies urged research on the mechanisms triggered by the interaction of SARS-CoV-2 virus with host cells and the ensuing pathophysiology driving pneumonia and multiorgan failure. Hyperinflammation soon appeared as a mechanism involved in mortality that could even proceed after viral infection comes to an end. Hyperinflammation is supported by an inappropriate production of cytokines, and this explains the use of the term cytokine storm to refer to this phase of the disease. Given that insight into the molecular mechanisms driving cytokine storm should focus on the interaction of viral components with immune cells, experiments addressing the effect of viral components on its cognate receptors were carried out. It was observed that viral RNA induces a cytokine pattern like the one observed in bronchoalveolar aspirates of COVID-19 patients with critical disease. Overall, the study revealed that both cell organelle overload and receptors involved in the recognition of viral RNA may team up to induce proinflammatory cytokines. This mechanism can be exploited to develop new treatments for COVID-19 disease.

Cystic fibrosis (CF) is a life-shortening genetic disorder, caused by mutations in the gene that encodes Cystic Fibrosis Transmembrane-conductance Regulator (CFTR), a cAMP-activated chloride and bicarbonate channel. Although multiple organ systems can be affected, CF lung disease claims the most morbidity and mortality due to chronic bacterial infection, persistent neutrophilic inflammation, and mucopurulent airway obstruction. Despite the clear predominance of neutrophils in these pathologies, how CFTR loss-of-function affects these cells per se remains incompletely understood. Here, we report the profiling and comparing of transcriptional signatures of peripheral blood neutrophils from CF participants and healthy human controls (HC) at the single-cell level. Circulating CF neutrophils had an aberrant basal state with significantly higher scores for activation, chemotaxis, immune signaling, and pattern recognition, suggesting that CF neutrophils in blood are prematurely primed. Such an abnormal basal state was also observed in neutrophils derived from an F508del-CF HL-60 cell line, indicating an innate characteristic of the phenotype. LPS stimulation drastically shifted the transcriptional landscape of HC circulating neutrophils towards a robust immune response, however, CF neutrophils were immune-exhausted. Moreover, CF blood neutrophils differed significantly from CF sputum neutrophils in gene programming with respect to neutrophil activation and aging, as well as inflammatory signaling, highlighting additional environmental influences on the neutrophils in CF lungs. Taken together, loss of CFTR function has intrinsic effects on neutrophil immune programming that leads to premature priming and dysregulated response to challenge.

RIG-I is a cytosolic immune receptor that provides the first line of defense by detecting viral RNA and triggering antiviral responses. Its physiological role in humans remains unclear, as no patients with complete RIG-I deficiency have yet been reported. We identified a critically ill COVID-19 patient with severe RIG-I deficiency caused by heterozygous RIG-I G731R, a novel dominant loss-of-function variant. The G731R mutation in helicase motif VI disrupts the arginine finger, impairing the ATPase activity of RIG-I, but not its RNA-binding ability. However, viral RNA binding fails to expose the signaling domains, thereby impairing the IFN-{beta} response of G731R. Instead, G731R competes with wild-type RIG-I, exerting a dominant negative effect. The loss-of-function is caused by bulky-charged substitutions at G731, as alanine or leucine substitution results in an unexpected gain-of-function phenotype. These findings highlight the importance of uncompromised RIG-I function for human antiviral immunity and the pleiotropic effects of single mutations.

Calcium signaling is essential for lymphocyte activation, with genetic disruptions resulting in severe immunodeficiency. The inositol 1,4,5-trisphosphate receptor (IP3R), formed from homo- or hetero-tetramers of the IP3R isoforms 1-3, amplifies lymphocyte signaling by releasing Ca2+ from ER stores into the cytosol following antigen-stimulation. While knockout of all 3 IP3R isoforms results in immunodeficiency in mice, the seeming redundancy of subunits was thought to explain the absence of IP3R mutation as a cause of human immunodeficiency. Here, we identify compound heterozygous variants in ITPR3 in two unrelated Caucasian patients presenting with combined immunodeficiency, in one case requiring hematopoietic stem cell transplantation. We observed disrupted Calcium homeostasis in patient-derived fibroblasts and immune cells, with abnormal proliferation and activation responses following B and T cell receptor stimulation. Reconstitution of IP3R knockout cell lines identified the variants as functional hypomorphs with reduced discrimination between homeostatic and induced states, validating a link between genotype and phenotype. These results demonstrate a functional linkage between defective ER Ca2+ channels and immunodeficiency, and identify IP3Rs as diagnostic targets for patients with specific inborn errors of immunity.

BackgroundCytokine storm syndromes (CSS), including hemophagocytic lymphohistiocytosis (HLH), are increasingly recognized as hyper-inflammatory states leading to multi-organ failure and death. Familial HLH (FHL) in infancy results from homozygous genetic defects in perforin-mediated cytolysis by CD8 T-lymphocytes and natural killer (NK) cells. Later onset CSS are frequently associated with heterozygous defects in FHL genes, but genetic etiologies for most are unknown. We identified rare DOCK8 variants in CSS patients. ObjectiveWe explore the role of CSS patient derived DOCK8 mutations on cytolytic activity in NK cells. We further study effects of Dock8-/- in murine models of CSS. MethodsDOCK8 cDNA from 2 unrelated CSS patients with different missense mutations were introduced into human NK-92 NK cells by foamy virus transduction. NK cell degranulation (CD107a), cytolytic activity against K562 target cells, and interferon-gamma (IFN{gamma}) production were explored by flow cytometry (FCM). A third CSS patient DOCK8 mRNA splice acceptor site variant was explored by exon trapping. Dock8-/- mice were assessed for features of CSS (weight loss, splenomegaly, hepatic inflammation, cytopenias, and IFN{gamma} levels) upon challenge with lymphochoriomeningitic virus (LCMV) and excess IL-18. ResultsBoth patient DOCK8 missense mutations decreased cytolytic function in NK cells in a partial dominant-negative fashion in vitro. The patient DOCK8 splice variant disrupted mRNA splicing in vitro. Dock8-/- mice tolerated excess IL-18 but developed features of CSS upon LCMV infection. ConclusionMutations in DOCK8 may contribute to CSS-like hyper-inflammatory states by altering cytolytic function in a threshold model of disease. Key MessagesO_LIHeterozygous missense mutations in DOCK8 may contribute to decreased NK cell function via partial dominant-negative effects on perforin-mediated cytolysis. C_LIO_LIHeterozygous mutations in DOCK8 may contribute to hyper-inflammatory syndromes in a threshold model of disease. C_LIO_LILCMV infection of Dock8-/- mice recapitulates features of murine FHL. C_LI Capsule SummaryHeterozygous missense and splice site mutations in DOCK8 may contribute to hyper-inflammation in patients with CSS. DOCK8 is important for optimal NK cell cytolytic function, and LCMV infection of Dock8-/- mice resembles murine FHL.

T cells play key protective but also pathogenic roles in COVID-19. We studied expression of long non-coding RNAs (lncRNAs) in COVID-19 T cell transcriptomes by integrating previously published single-cell RNA sequencing datasets. The long intergenic non-coding RNA MALAT1 was the most highly transcribed lncRNA in T cells, with Th1 cells demonstrating the lowest and CD8+ resident memory cells the highest MALAT1 expression, amongst CD4+ and CD8+ T cells populations, respectively. We then identified gene signatures that covaried with MALAT1 in single T cells. A significantly higher number of transcripts correlated negatively with MALAT1 than those that correlated. Enriched functional annotations of the MALAT1-anti-correlating gene signature included processes associated with T cell activation such as cell division, oxidative phosphorylation and response to cytokine. The MALAT1 anti-correlating gene signature shared by both CD4+ and CD8+ T cells marked dividing T cells in both lung and blood of COVID-19 patients. Focussing on the tissue, we used an independent patient cohort of post-mortem COVID-19 lung samples and demonstrated that MALAT1 suppression was indeed a marker of MKI67+ proliferating CD8+ T cells. Our results reveal MALAT1 suppression and its associated gene signature are a hallmark of human proliferating T cells.

Dysfunctions in nucleic acid-sensing Toll-like receptors (TLRs) disrupt nucleic acid tolerance, leading to systemic lupus erythematosus (SLE). Here, we report a novel homozygous gain-of-function p.R95L mutation in the TLR chaperone protein UNC93B1 in an SLE patient. Bulk and single-cell transcriptional analysis of the patients peripheral blood mononuclear cells (PBMCs) revealed significantly elevated inflammation in T cells and myeloid cells due to enhanced dendritic cells function. The UNC93B1 R95L mutation leads to hyperactivation of TLR7/8, but not TLR3/9, upon stimulation with specific agonists in vitro. Transgenic Unc93b1R95L/R95L mice develop inflammatory and autoimmune phenotypes, and the upregulation of inflammatory signaling differs among organs, with a specific contribution of malfunctioned T cells and B cells. In human and mouse cell lines, the UNC93B1 R95L mutation promotes TLR7/8 signaling by enhancing its binding to ssRNA, without affecting TLR7/8 translocation. Overall, our results elucidate the pathology of organs and the immunological profiles of immune cell populations in the development of SLE caused by the UNC93B1 R95L mutation through the TLR7/8 axis.

BackgroundChronic rhinosinusitis with nasal polyps (CRSwNP) is a complex disease characterized by multiple inflammatory endotypes. Although recent progress has been made in endotype-based classification, developing tailored therapeutic strategies for CRSwNP remains challenging. This study aimed to optimize therapeutic outcomes in CRSwNP by identifying potential molecular markers. MethodsWe utilized an integrated approach that combined bulk and single-cell RNA sequencing (scRNA-seq) to delineate the molecular signatures inherent to the cellular components of nasal polyp (NP) tissue. The levels of C11-BODIPY (as a marker of lipid peroxidation) and SLC27A2/FATP2 were assessed using quantitative PCR and immunofluorescence (IF) staining. The effects of lipofermata, a FATP2 inhibitor, were examined in air-liquid interface (ALI) cultured epithelial cells derived from CRSwNP patients and healthy controls. ResultsDeconvolution analysis of NP tissue revealed an upregulation of genes associated with lipid metabolism in the NP epithelium. In CRSwNP patients, we observed a significant increase in lipid peroxidation and SLC27A2/FATP2 expression in the NP epithelium. A marked expression of genes critical to metabolic pathways involved in lipid peroxidation was identified in SLC27A2-positive epithelial cells. Additionally, FATP2 and lipid peroxidation staining patterns exhibited a positive correlation in their respective % Area levels. Elevated SLC27A2 expression was associated with disease pathogenesis and correlated with disease severity. Treatment with lipofermata resulted in decreased mRNA levels of ALOX15, a key mediator of inflammation and lipid peroxidation, and FOXJ1, a marker of abnormal ciliogenesis. ConclusionElevated SLC27A2 expression in the NP epithelium correlates with the severity of CRSwNP, highlighting its potential as a therapeutic target for managing advanced CRSwNP cases.

Birdshot chorioretinopathy uveitis (BCR-UV) is strongly associated with HLA-A29 which implicates MHC-I pathway mediated perturbation of natural killer (NK) cells as a potential disease mechanism. We profiled blood NK cells at single-cell resolution in a cohort of patients and healthy controls and investigated the links between NK cell subpopulations and disease activity. Flow cytometry analysis of major immune cell lineages revealed substantial expansion of the CD56dim CD16+ NK cells in BCR-UV compared to healthy controls and to other types of non-infectious uveitis. Ex vivo restimulation showed that NK cells from BCR-UV patients exhibit increased secretion of TNF-alpha, a cytokine considered central to the pathogenesis of BCR-UV. Unbiased transcriptomic characterization at single-cell resolution established that the expanded CD16+ (i.e., FCGR3A+) NK cells also co-express high levels of CD8A and CD244, indicating expansion of a subset of CD56dim CD16+ CD8+ NK cells in patients. Confirmation of these results by high-dimensional flow cytometry further showed that the BCR-UV-associated CD8bright CD244bright NK cells displayed activation receptors including CD314 (NKG2D), and cytotoxicity receptor CD337 (NKp30). Finally, longitudinal monitoring of patients showed that clinical remission after systemic immunomodulatory treatment correlated with a significant decrease in CD8bright CD244bright NK cells. In conclusion, there is an expansion of CD8bright CD244bright NK cells during active disease in BCR-UV patients which decrease upon successful systemic immunomodulatory treatment, suggesting that CD8bright/CD244bright NK cells may be a pro-inflammatory NK subset involved in the underlying disease mechanism.

Viral respiratory infections are the most common childhood infection worldwide. However, even common pathogens can have significant consequences in the context of patients with primary immunodeficiency diseases. More than half or viral infections annually are due to rhinovirus/enterovirus strains. Most clinical manifestations of viral infection are mild. However 3% of cases result in hospitalization in patients who have no other known risk factors. These patients may have an inborn error of immunity, a genetic susceptibility to viral infections. Here we present the case of an adult male who suffered respiratory viral infections his whole life and developed chronic, inflammatory damage to sinuses and lungs as a consequence. Genomic sequencing identified compound heterozygous variants in the IFIH1 gene, encoding the protein Melanoma Differentiation Association Protein 5 (MDA5), a RIG-I-like cytoplasmic sensor of RNA intracellular infections. We show a dominant negative effect on these variants on the level of interferon-induced expression of MDA5 protein. This work supports that loss-of-function variants in IFIH1 affect the sensing of viral infections. Underlying genomic variants may dictate the point at which recurrent, respiratory viral infections leave commonplace experience and incur lasting damage.

Cryopyrin-associated periodic syndromes (CAPS) also known as NLRP3-associated auto-inflammatory diseases, are a spectrum of rare auto-inflammatory diseases caused by gain-of-function mutations in the NLRP3 gene, resulting in inflammasome hyper-activation and dysregulated release of Interleukin-1{beta} (IL-1{beta}). Many patients with CAPS develop progressive sensorineural hearing loss (SNHL) due to cochlear auto-inflammation which, in rare cases, may be the sole manifestation. This study was undertaken to establish the suspected diagnosis of CAPS in a family presenting autosomal dominant progressive/acute SNHL and a novel missense variant in the NLRP3 gene of unknown significance (NM_001079821:c.1790G>A, p.Ser597Asn). To do so, we conducted an ex vivo functional assessment of the NLRP3 inflammasome in carries (n=10) and healthy family members (n=5). The assay revealed hyper-activation of the inflammasome among carriers, supporting the hypothesis that this missense variant is a pathogenic gain-of-function mutation. Administration of anti-IL-1 therapy resulted in a substantial clinical improvement among pediatric patients, who exhibited near resolution of hearing impairment within 1-3 months of treatment. Our findings highlight the crucial role of early diagnosis and treatment of hearing loss due to hyperactivation of the inflammasome with an anti-IL-1 agent in reversing cochlear damage. Furthermore, our results suggest that high and ultrahigh frequency ranges need to be included in the auditory assessment to enable early detection of subclinical SNHL. Finally, incorporating functional inflammasome assessment as part of the clinical evaluation could establish the diagnosis in inconclusive cases.

RationaleInvestigations on the mechanisms of airway smooth muscle remodeling, a prominent asthma feature contributing to its clinical manifestations and severity, have largely focused on its hyperplastic growth. Conversely, limited data and virtually no translational research have been produced on a plausible role of apoptosis in the homeostasis and remodeling of airway smooth muscle. ObjectivesWe aimed at demonstrating an involvement of apoptosis, an essential regulator of organ structure and cell turnover, in the pathophysiology of airway smooth muscle remodeling in asthma. MethodsMurine experimental asthma was modeled to analyze airway hyperresponsiveness, contractile tissue remodeling and apoptosis detection outcomes at early and late cutoffs, and under pharmacological inhibition of apoptosis by employing a caspase blocker. Clinical investigation followed through analyses on human bronchial biopsies. ResultsAirway hyperresponsiveness and contractile tissue remodeling were already established in early experimental asthma, and a subsequent upregulation of apoptosis limited the airway contractile tissue growth. Caspase inhibition elicited chaotic pulmonary mechanics and an unusual growth of airway smooth muscle that was structurally disorganized. In bronchial biopsies, airway smooth muscle increased from controls through subjects with intermittent and persistent moderate and severe asthma. Cleaved poly-ADP ribose polymerase (c-PARP, a byproduct of caspase activity) was increased in severe asthma. ConclusionsApoptosis is involved in airway contractile cell turnover and in shaping the size, structure and proper function of the airway smooth muscle layer. Apoptosis inhibitors may complicate concomitant asthma, whereas agents favouring airway contractile cell apoptosis may provide a novel pipeline of therapeutic development. Key messagesO_LIHow normal airway smooth muscle structure is preserved, and whether counteracting responses to remodeling are elicited in asthma, are outstanding questions not probed in vivo nor in the clinical setting. C_LIO_LIIn this work, combined investigations on murine experimental asthma and human bronchial biopsies show that airway contractile cell apoptosis is involved in the homeostasis of airway smooth muscle, and apoptotic activity is upregulated as part of the remodeling process of this tissue in asthma. C_LIO_LIApoptosis arises as a key regulator of the size and structure of the airway smooth muscle layer. This concept draws implications for clinical practice and drug development. C_LI

PGAP3 is a glycosylphosphatidylinositol (GPI) phospholipase gene localized within chromosome 17q12-21, a region highly linked to asthma. Although much is known about the function of other chromosome 17q12-21 genes expressed at increased levels in bronchial epithelium such as ORMDL3 and GSDMB, little is known about the function of increased PGAP3 expression in bronchial epithelium in the context of asthma. The aim of this study was therefore to determine whether increased PGAP3 expression in human bronchial epithelial cells regulated expression of mRNA pathways important to the pathogenesis of asthma by utilizing RNA-sequencing and bioinformatic analysis. We performed RNA-sequencing on normal human bronchial epithelial cells transfected with PGAP3 for 24 and 48 hours. PGAP3 regulated genes were compared to asthma and respiratory virus (influenza A, rhinovirus, respiratory syncytial virus) reference data sets to identify PGAP3 target genes and pathways. Approximately 9% of the upregulated PGAP3-induced genes were found in an asthma reference data set, 41% in a rhinovirus reference data set, 33% in an influenza A reference data set, and 3% in a respiratory syncytial virus reference data set. PGAP3 significantly upregulated the expression of several genes associated with the innate immune response and viral signatures of respiratory viruses associated with asthma exacerbations. Two of the highest expressed genes induced by PGAP3 are RSAD2, OASL, and IFN-{lambda}, which are anti-viral genes associated with asthma. PGAP3 also upregulated the antiviral gene BST2, which like PGAP3 is a GPI-anchored protein. We conclude that PGAP3 expression in human bronchial epithelial cells regulates expression of genes known to be linked to asthma, and also regulates the bronchial epithelial expression of genes pertinent to the pathogenesis of respiratory viral triggered asthma exacerbations.

BackgroundTrefoil factor family cytokines (TFF1-3) have documented reparative and immunoregulatory effects on mucosal epithelial cells that include: blocking apoptosis, directed cell migration, and regulation of pro-inflammatory cytokine production. TFF1 and TFF3 have been shown to undergo altered expression within sinonasal tissues of patients with various forms of chronic rhinosinusitis including those with or wthout polyp associated disease, but the cellular source(s) of TFF members in CRS remains unclear. To further explore the role of TFF3 in inflammatory sinonasal disease, we sought to determine its expression pattern in immune and non-immune cell types in CRS disease. MethodsPolyp and inferior turbinate tissues were isolated from patients undergoing surgical resection for treatment of CRS. Tissue homogenates and single cell suspensions were subjected to enzyme linked immunosorbent assay (ELISA) and single-cell RNA sequencing. Air liquid interface cultures of sinonasal epithelia were subjected to immunofluorescence (IF) microscopy. Interleukin 25, TFF2 and TFF3 protein levels were compared to sinonasal outcome test scores (SNOT-22) to determine whether levels associated with post-operative outcomes. ResultsTFF3 was broadly expressed in goblet cells, ciliated cells, Tuft cells and T lymphocytes. TFF3 protein levels positively associated with IL-25 in CRSwNP patients. Lastly, TFF3 protein levels positively associated with clinical improvement post-surgery. ConclusionsTFF3 is broadly expressed within multiple epithelial and immune cell lineages in patients with CRSwNP. Protein levels of TFF3 correlate with IL-25 and clinical signs of disease, however TFF3 levels associate with clinical improvement following surgical intervention indicating a potential beneficial role for this reparative cytokine in CRS patients.