Welcome to the ESID 2022 Meeting Interactive Programme

Background and Aims

STAT3-hyper IgE syndrome (STAT3-HIES) patients suffer from recurrent lung infections, leading to tissue destructive changes with pneumatocele formation and severe lung defects. To improve the pulmonary therapy of STAT3-HIES patients, we aim to better understand the pathophysiology underlying the destructive lung disease.

Methods

Using a transgenic mouse model (mutStat3) with a STAT3-HIES like immunologic phenotype carrying the dominant negative mutation Stat3-ΔV463 (Steward-Tharp et al. Blood 2014), we model lung infections by inducing acute lung injury with intratracheal instillation of lipopolysaccharide (LPS). Inflammatory responses and tissue injury were analyzed by quantification of bronchoalveolar lavage (BAL) cells, ELISA, and protein quantification of BAL fluid. Lung tissue was collected for histology and expression analysis.

Results

Instillation of LPS induced lung injury in a dosage-dependent manner in wildtype (wt) and mutStat3 mice as shown by an overall increase in BAL protein concentrations with higher levels in mutStat3 compared to wt samples. We found increased immune cell infiltration predominantly neutrophils and increased TNFalpha release into the air space significant higher in mutStat3 compared to control animals. Quantification of immunohistologically stained lung tissue with pro surfactant protein C (pro-SpC) as a marker for alveolar type II (AT2) cells, showed reduced positive pro-SpC cells in lung tissue in mutStat3 compared to wt mice after LPS challenge.

Conclusions

Our in vivo mutStat3 mouse model of lung injury indicates a higher susceptibility to pulmonary tissue damage with elevated lung inflammation and deficient epithelial recovery in STAT3-HIES after pulmonary injury.

Background and Aims

Germline, monoallelic, gain-of-function (GOF) mutations in STAT1 cause an ultra-rare form of primary immunodeficiency (PID) through overactivation of the Janus-associated kinase STAT1 signalling pathway. Heightened basal levels of STAT1 protein have previously been shown to be a key driver of disease^{1 2} but the exact mechanism of STAT1 GOF still remains unclear.

Methods

A nanoBRET system was developed to investigate the conformations adopted by STAT1 dimers pre- and post-IFNγ stimulation in STAT1 (^-/-) cell lines lentivirally transduced with wild-type or GOF STAT1. Nuclear localisation and transcriptomics were assessed in the same cell lines pre- and post-IFN stimulation, and in response to Ruxolitinib.

Results

We show that the majority of GOF mutations destabilise the inactive dimeric conformations of STAT1 and promote the adoption of an active-like conformation at baseline. In contrast, some mutations stabilise the active conformation and induce stronger DNA binding, suggesting that discreet GOF mutations function through different mechanisms. We show that the nuclear localisation sequence is exposed in the active orientation, and that consequently, GOF mutants had greater nuclear presence at baseline. Transcriptomic analysis detailed that STAT1-mediated gene expression was induced under inactive status for GOF mutants and that each mode of pathogenesis has unique gene expression signatures. We compared the clinical phenotypes described for different for STAT1 GOF mutations and identified that patients with mutations that stabilise the active conformation are more likely to experience autoimmune complications.

Conclusions

This work has identified alternative modes of pathogenesis for STAT1 GOF which may explain clinical heterogeneity.

Background and Aims

Disseminated coccidioidomycosis (DCM) is caused by Coccidioides, pathogenic fungi endemic to Western United States and Mexico. While exposure is common, pneumonia is thought to occur in ~3% of residents annually with <1% of those developoing disseminated disease.

Methods

We enrolled DCM patients, performed whole-exome sequencing and assessed cytokine production in peripheral blood mononuclear cells. Confocal microscopy co-localized DECTIN-1 and fungal endospores. Transfection demonstrated DECTIN-1, DUOX1 and DUOXA1 roles in β-glucan-stimulated H₂O₂ production. RNA was sequenced from STAT3-mutated, autosomal-dominant Hyper-IgE syndrome (AD-HIES) patients' respiratory tissues. Duox1^-/- mice were infected with Coccidioides.

Results

In an exploratory set of 67 DCM patients, two had haploinsufficient STAT3 mutations. Defects in β-glucan sensing and response were seen in 34/67 (50.7%) cases. Damaging CLEC7A (n=14) and PLCG2 (n=11) variants were found and PBMC from patients with these variants produced less β-glucan-stimulated TNF-α than healthy controls (P<0.005). Using ancestry matched controls, damaging variants in CLEC7A and PLCG2 were over-represented in DCM (P=0.0206, P=0.015, respectively) including CLEC7A Y238* (P=0.0105) and PLCG2 R268W (P=0.0025). In a validation cohort of 112 DCM patients PLCG2 R268W (P=0.0276), CLEC7A I223S (P=0.044) and CLEC7a Y238* (P=0.0656) were confirmed. Fifteen discovery cohort patients had heterozygous DUOX1 or DUOXA1 variants which impaired H₂O₂ production in transfected cells. AD-HIES patient airway epithelial cells had decreased DUOX1/DUOXA1 transcripts. Duox1^-/- mice had increaed morbidity and mortaligy following Coccidoiodes infection.

Conclusions

Patients with DCM have impaired β-glucan sensing or responsiveness affecting H₂O₂ production. Genetically impaired Coccidioides recognition and cellular response decrease inflammatory cytokine production and underlie susceptibility to disseminated coccidioidomycosis.

Background and Aims

Chronic granulomatous disease (CGD) is characterized by recurrent life-threatening infections and hyperinflammatory complications.It is caused by mutations in the NADPH oxidase complex and the consequent loss of reactive oxygen species (ROS) production. Recombinant human interferon gamma (rIFNγ) is used as prophylaxis to reduce the risk of severe infections, but the mechanisms behind its efficacy in CGD are still unknown.

Methods

We compared the immune and metabolic profile of immmune cells from CGD patients with healthy controls using scRNAseq analysis, ATACseq profiling, ELISA, proteomics, Seahorse and metabolomics. Finally, we investigated the effect of in vitro rIFNγ treatment on some of those parameters.

Results

We found that innate immune myeloid cells from CGD patients are epigenetically and functionally reprogrammed to have a hyperactivated immune status. In parallel, they present an impaired in vitro induction of trained immunity. CGD monocytes have deficient intracellular amino acids levels and have profound functional metabolic defects, both at the glycolytic and mitochondrial level. In vitro treatment with rIFNγ restored these myeloid metabolic defects and reduced abnormal IL-1β and IL-6 production in response to fungal stimuli in CGD monocytes, suggesting that prophylactic rIFNγ efficacy in CGD patients has a metabolic basis.

Conclusions

Learning more about the immunometabolic defects underlying diseases will not only give new insight into their pathogenesis but, beyond CGD, might open doors for efficient and targeted immunotherapy aimed at correcting these defects. In conclusion, our findings address a new avenue of research exploring the consequences of a defective NADPH oxidase complex in the metabolic rewiring of immune cells.

Background and Aims

Chronic Granulomatous Disease is a mendelian disorder caused by loss-of-function mutations genes encoding subunits of the NADPH oxidase complex 2. Increasing reports have underlined the role of the NLRP3 inflammasome in the pathophysiology of inflammation in CGD. However, the precise mechanism provoking a disproportionate inflammatory response in CGD patients remains elusive. Interestingly, the NOX2-deficiency model – characterized by defective production of cytosolic Reactive Oxygen Species (ROS) is at odds with the classical concept that chronic inflammation is caused by prolonged and sustained ROS production. These opposite views suggest that efficient regulation of the inflammatory response requires a well-balanced ROS signaling.

The objective of our study is to refine the precise role of cytosolic ROS in regulating inflammation and discover alternative strategies for treating chronic inflammatory diseases.

Methods

We assessed the dynamics of the inflammatory response in NOX2-deficient patients’ primary monocytes and a CRISPR-engineered NOX2-deficient phagocytic THP-1 cell line.

Results

We show that the defective redox signaling in CGD phagocytes is responsible for post-translational priming of the pyroptosome as evidenced by an enhanced oligomerization of its principal component ASC. NOX2 deficiency also increases the phosphorylation of GasderminD (GSDMD) at Serine 252, an amino acid that seems critical for its pyroptotic activity. Interestingly, GSDMD cleavage further activates the NLRP3 inflammasome by facilitating the release of mitochondrial DNA in the cytosol and by lowering the intracellular K+ concentration through GSDMD membrane pores.

Conclusions

We unveil the pivotal role of GSDMD in the amplification of the inflammatory response in CGD, paving the way for targeted therapies.

Background and Aims

Hyperactive or constitutive STAT6 signalling is associated to increased Th2 differentiation, responsible of allergic inflammation. STAT6 variants have been associated to food allergies¹ and STAT6 somatic mutations have been identified in 11% of follicular lymphoma.² Recently, STAT6 variants have been detected in two patients with profound allergic immunedysregulation.³

Methods

We describe a clinical case of allergic immunedysregulation. Diagnostic work-up included immunologic investigations using FACS analysis, clinical exome sequencing (CES) and proteomic approach through Olink assay.

Results

A 21yo-girl presented early-onset refractory atopic dermatitis, recurrent respiratory infections, severe asthma, multiple inhalants, drugs and food severe allergies, HPV-negative laryngeal papillary hyperplasia, gastrointestinal disorders with not-specific mucosal infiltrate and chronic EBV infection.

Diagnostic work-up showed hyper-eosinophilia, hyper-IgE (699-12550 kU/L), positive RAST and ISAC test for multiple allergens, normal Ig levels, normal frequency of Tcells and Tregs, reduced frequency of Th17cells, normal circulating Tfhcells, slightly reduced Bcells frequency with normal Bcell maturation, reduced in vitro Tcell proliferation and Bcell antibodies production.

CES disclosed a de novo heterozygous mutation in STAT6 gene (c.1255G>A; p.D419N), predicted pathogenic by ACGM (CADD score 31) not reported in gnomAD, but described in follicular lymphoma. Proteomic analysis showed a baseline higher percent-change in IL-4 level compared to healthy control.

Conclusions

We suggest that the mutation identified in our patient could be responsible of her clinical phenotype and could be stated as a novel Primary Atopic Disorder. More functional studies are ongoing to confirm its pathogenic role and to suggest a targeted therapeutical choice.

¹Allergy. 2018 Jun;73(6):1337-1341.

²Blood. 2015 Jan 22;125(4):668-79.

³medRxiv preprint doi:10.1101/2022.04.25.22274265

Background and Aims

Monogenic defects in type I interferon (IFN-I) signaling components have been identified in patients with herpes simplex encephalitis (HSE), emphasizing inborn errors of immunity underlying pathogenesis. In vitro studies demonstrate that the cytoplasmic RNA sensor RIG-I, which is well known to restrict RNA viruses, also critically contributes to the innate immune responses to DNA viruses including HSV-1.

Methods

We identified compound heterozygous mutations in the gene GTF3A in a family afflicted by HSE in early childhood. We studied primary patient cells as well as several CRISPR/Cas9-edited GTF3A mutant cells.

Results

GTF3A encodes for the transcription factor TFIIIA, which is part of the Pol III complex. We confirmed that the patient TFIIIA mutants have an impaired promoter-binding ability. We tested HSV-1 replication in the patient fibroblasts and GTF3A mutant cells and observed enhanced viral replication. To understand the underlying mechanism, we took an unbiased approach and searched for novel transcriptional targets of TFIIIA by ChIP-seq analysis and identified the pseudogene RNA5SP141, previously described as a RIG-I agonist. We found that RNA5SP141 is upregulated following HSV-1 infection and that this induction is abrogated in primary patient cells, in GTF3A gene-edited mutant cells, and upon targeted knockdown using siRNA. Finally, we explored the downstream effects of impaired RNA5SP141 expression on anti-herpesviral immunity and found abrogated RIG-I activation and markedly diminished induction of antiviral genes during HSV-1 infection.

Conclusions

Our work unveils a novel role for TFIIIA, acting as a moonlighting protein that regulates innate immune responses to HSV-1 by transcriptional regulation of the host-derived RNA5SP141 RIG-I ligand.