A Translational Investigation of IFN-α and STAT1 Signaling in Endothelial Cells during Septic Shock Provides Therapeutic Perspectives.

Septic shock and disseminated intravascular coagulation (DIC) are known to be characterized by an endothelial cell dysfunction. The molecular mechanisms underlying this relationship are, however, poorly understood. In this work, we aimed to investigate human circulating IFN-α in patients with septic shock-induced DIC and tested the potential role of endothelial Stat1 (signal transducer and activator of transcription 1) as a therapeutic target in a mouse model of sepsis. For this, circulating type I, type II, and type III IFNs and procoagulant microvesicles were quantified in a prospective cohort of patients with septic shock. Next, we used a septic shock model induced by cecal ligation and puncture in wild-type mice, in Ifnar1 (type I IFN receptor subunit 1)-knockout mice, and in Stat1 conditional knockout mice. In human samples, we observed higher concentrations of circulating IFN-α and IFN-α1 in patients with DIC compared with patients without DIC, whereas concentrations of IFN-ß, IFN-γ, IFN-λ1, IFN-λ2, and IFN-λ3 were not different. IFN-α concentration was positively correlated with CD105 microvesicle concentrations, reflecting endothelial injury. In Ifnar1-/- mice, cecal ligation and puncture did not induce septic shock and was characterized by lesser endothelial cell injury, with lower aortic inflammatory cytokine expression, endothelial inflammatory-related gene expression, and fibrinolysis. In mice in which Stat1 was specifically ablated in endothelial cells, a marked protection against sepsis was also observed, suggesting the relevance of an endothelium-targeted strategy. Our work highlights the key roles of type I IFNs as pathogenic players in septic shock-induced DIC and the potential pertinence of endothelial STAT1 as a therapeutic target.

NKG2D ligands in inflammatory joint diseases: analysis in human samples and mouse models.

OBJECTIVES: NKG2D ligands (NKG2DLs) are stress-inducible molecules involved in multiple inflammatory settings. In this work, we quantified MICA, an NKG2DL, in the synovial fluid of patients suffering various arthritides and measured Nkg2dLs gene expression in murine models of acute joint inflammation. METHODS: Soluble MICA (sMICA) was quantified by ELISA is synovial fluids harvested from patients suffering osteoarthritis, rheumatoid arthritis, psoriatic arthritis, calcium pyrophosphate crystal arthritis, urate crystal arthritis and reactive arthritis. Transcripts encoding murine NKG2DLs were quantified by RT-qPCR in the joints of mouse models of rheumatoid arthritis, urate crystal arthritis and osteoarthritis. RESULTS: Marked overproduction of sMICA was observed in the synovial fluid of RA patients. Mouse studies highlighted the complex transcriptional regulation of Nkg2d ligands encoding genes depending on the inflammatory setting and microenvironment CONCLUSIONS: sMICA quantification could be an interesting biomarker to identify acute inflammation in RA patients in whom classical markers (i.e. anti-citrullinated protein antibodies, ACPA) are undetectable.

Matching for the nonconventional MHC-I MICA gene significantly reduces the incidence of acute and chronic GVHD.

Graft-versus-host disease (GVHD) is among the most challenging complications in unrelated donor hematopoietic cell transplantation (HCT). The highly polymorphic MHC class I chain-related gene A, MICA, encodes a stress-induced glycoprotein expressed primarily on epithelia. MICA interacts with the invariant activating receptor NKG2D, expressed by cytotoxic lymphocytes, and is located in the MHC, next to HLA-B Hence, MICA has the requisite attributes of a bona fide transplantation antigen. Using high-resolution sequence-based genotyping of MICA, we retrospectively analyzed the clinical effect of MICA mismatches in a multicenter cohort of 922 unrelated donor HLA-A, HLA-B, HLA-C, HLA-DRB1, and HLA-DQB1 10/10 allele-matched HCT pairs. Among the 922 pairs, 113 (12.3%) were mismatched in MICA MICA mismatches were significantly associated with an increased incidence of grade III-IV acute GVHD (hazard ratio [HR], 1.83; 95% confidence interval [CI], 1.50-2.23; P < .001), chronic GVHD (HR, 1.50; 95% CI, 1.45-1.55; P < .001), and nonelapse mortality (HR, 1.35; 95% CI, 1.24-1.46; P < .001). The increased risk for GVHD was mirrored by a lower risk for relapse (HR, 0.50; 95% CI, 0.43-0.59; P < .001), indicating a possible graft-versus-leukemia effect. In conclusion, when possible, selecting a MICA-matched donor significantly influences key clinical outcomes of HCT in which a marked reduction of GVHD is paramount. The tight linkage disequilibrium between MICA and HLA-B renders identifying a MICA-matched donor readily feasible in clinical practice.

Identification of driver genes for critical forms of COVID-19 in a deeply phenotyped young patient cohort.

The drivers of critical coronavirus disease 2019 (COVID-19) remain unknown. Given major confounding factors such as age and comorbidities, true mediators of this condition have remained elusive. We used a multi-omics analysis combined with artificial intelligence in a young patient cohort where major comorbidities were excluded at the onset. The cohort included 47 "critical" (in the intensive care unit under mechanical ventilation) and 25 "non-critical" (in a non-critical care ward) patients with COVID-19 and 22 healthy individuals. The analyses included whole-genome sequencing, whole-blood RNA sequencing, plasma and blood mononuclear cell proteomics, cytokine profiling, and high-throughput immunophenotyping. An ensemble of machine learning, deep learning, quantum annealing, and structural causal modeling were used. Patients with critical COVID-19 were characterized by exacerbated inflammation, perturbed lymphoid and myeloid compartments, increased coagulation, and viral cell biology. Among differentially expressed genes, we observed up-regulation of the metalloprotease ADAM9. This gene signature was validated in a second independent cohort of 81 critical and 73 recovered patients with COVID-19 and was further confirmed at the transcriptional and protein level and by proteolytic activity. Ex vivo ADAM9 inhibition decreased severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) uptake and replication in human lung epithelial cells. In conclusion, within a young, otherwise healthy, cohort of individuals with COVID-19, we provide the landscape of biological perturbations in vivo where a unique gene signature differentiated critical from non-critical patients. We further identified ADAM9 as a driver of disease severity and a candidate therapeutic target.

Contrasting role of NLRP12 in autoinflammation: evidence from a case report and mouse models.

OBJECTIVE: To explore at the molecular level the phenotype of a patient suffering an autoinflammatory syndrome which was diagnosed as familial cold autoinflammatory syndrome type 2 (FCAS-2). To explore the functions of Nlrp12 in inflammation using mouse models. METHODS: Whole exome sequencing and Nlrp12 targeted resequencing were performed on DNA isolated from the patient and her family members. In vivo and ex vivo models of inflammation (urate crystals-dependent acute joint inflammation and urate crystals-induced peritonitis) were analysed in Nlrp12-deficient and Nlrp12-competent mice. RESULTS: A rare missense NLRP12 variant (c.857C>T, p.P286L) was identified in the patient and her healthy relatives. Nlrp12-deficient mice exhibit reduced systemic inflammation and neutrophilic infiltration. CONCLUSION: Nlrp12 mediates proinflammatory functions in mice. In humans, the identification of Nlrp12 variants must be cautiously interpreted depending on clinical and paraclinical data to diagnose FCAS-2.

A mouse model of MSU-induced acute inflammation in vivo suggests imiquimod-dependent targeting of Il-1ß as relevant therapy for gout patients.

Rationale: The role of Monosodium Urate (MSU) crystals in gout pathophysiology is well described, as is the major impact of IL-1ß in the inflammatory reaction that constitutes the hallmark of the disease. However, despite the discovery of the NLRP3 inflammasome and its role as a Pattern Recognition Receptor linking the detection of a danger signal (MSU) to IL-1ß secretion in vitro, the precise mechanisms leading to joint inflammation in gout patients are still poorly understood. Methods: Acute urate crystal inflammation was obtained by subcutaneous injections of MSU crystals in mice. Symptoms were followed by scoring, cytokine quantification by ELISA and western blot, gene expression by RT-qPCR and RNAseq; Magnetic Resonance Imaging was also used to assess inflammation. Results: We provide an extensive clinical, biological and molecular characterization of an acute uratic inflammation mouse model which accurately mimics human gout. We report the efficacy of topical imiquimod treatment and its impact on Interferon-dependent down modulation of Il-1ß gene expression in this experimental model. Conclusion: Our work reveals several key features of MSU-dependent inflammation and identifies novel therapeutic opportunities for gout patients.

Compatibility at amino acid position 98 of MICB reduces the incidence of graft-versus-host disease in conjunction with the CMV status.

Graft-versus-host disease (GVHD) and cytomegalovirus (CMV)-related complications are leading causes of mortality after unrelated-donor hematopoietic cell transplantation (UD-HCT). The non-conventional MHC class I gene MICB, alike MICA, encodes a stress-induced polymorphic NKG2D ligand. However, unlike MICA, MICB interacts with the CMV-encoded UL16, which sequestrates MICB intracellularly, leading to immune evasion. Here, we retrospectively analyzed the impact of mismatches in MICB amino acid position 98 (MICB98), a key polymorphic residue involved in UL16 binding, in 943 UD-HCT pairs who were allele-matched at HLA-A, -B, -C, -DRB1, -DQB1 and MICA loci. HLA-DP typing was further available. MICB98 mismatches were significantly associated with an increased incidence of acute (grade II-IV: HR, 1.20; 95% CI, 1.15 to 1.24; P < 0.001; grade III-IV: HR, 2.28; 95% CI, 1.56 to 3.34; P < 0.001) and chronic GVHD (HR, 1.21; 95% CI, 1.10 to 1.33; P < 0.001). MICB98 matching significantly reduced the effect of CMV status on overall mortality from a hazard ratio of 1.77 to 1.16. MICB98 mismatches showed a GVHD-independent association with a higher incidence of CMV infection/reactivation (HR, 1.84; 95% CI, 1.34 to 2.51; P < 0.001). Hence selecting a MICB98-matched donor significantly reduces the GVHD incidence and lowers the impact of CMV status on overall survival.

Lipolysis is altered in MHC class I zinc-alpha(2)-glycoprotein deficient mice.

Non-conventional major histocompatibility complex class I molecules are involved in a variety of physiological functions, most at the periphery of the immune system per se. Zinc-alpha(2)-glycoprotein (ZAG), the sole soluble member of this superfamily has been implicated in cachexia, a poorly understood yet life-threatening, severe wasting syndrome. To further ascertain the role of ZAG in lipid metabolism and perhaps the immune system, we inactivated both ZAG alleles by gene targeting in mice. Subjecting these ZAG deficient animals to standard or lipid rich food regimens led to increased body weight in comparison to identically treated wild-type mice. This phenotype appeared to correlate with a significant decrease in adipocytic lipolysis that could not be rescued by several pharmacological agents including beta(3)-adrenoreceptor agonists. Furthermore, in contrast to previously reported data, ZAG was found to be ubiquitously and constitutively expressed, with an especially high level in the mouse liver. No overt immunological phenotype was identified in these animals.

Rapid evolution of major histocompatibility complex class I genes in primates generates new disease alleles in humans via hitchhiking diversity.

A plausible explanation for many MHC-linked diseases is lacking. Sequencing of the MHC class I region (coding units or full contigs) in several human and nonhuman primate haplotypes allowed an analysis of single nucleotide variations (SNV) across this entire segment. This diversity was not evenly distributed. It was rather concentrated within two gene-rich clusters. These were each centered, but importantly not limited to, the antigen-presenting HLA-A and HLA-B/-C loci. Rapid evolution of MHC-I alleles, as evidenced by an unusually high number of haplotype-specific (hs) and hypervariable (hv) (which could not be traced to a single species or haplotype) SNVs within the classical MHC-I, seems to have not only hitchhiked alleles within nearby genes, but also hitchhiked deleterious mutations in these same unrelated loci. The overrepresentation of a fraction of these hvSNV (hv1SNV) along with hsSNV, as compared to those that appear to have been maintained throughout primate evolution (trans-species diversity; tsSNV; included within hv2SNV) tends to establish that the majority of the MHC polymorphism is de novo (species specific). This is most likely reminiscent of the fact that these hsSNV and hv1SNV have been selected in adaptation to the constantly evolving microbial antigenic repertoire.

Mutations in signal recognition particle SRP54 cause syndromic neutropenia with Shwachman-Diamond-like features.

Shwachman-Diamond syndrome (SDS) (OMIM #260400) is a rare inherited bone marrow failure syndrome (IBMFS) that is primarily characterized by neutropenia and exocrine pancreatic insufficiency. Seventy-five to ninety percent of patients have compound heterozygous loss-of-function mutations in the Shwachman-Bodian-Diamond syndrome (sbds) gene. Using trio whole-exome sequencing (WES) in an sbds-negative SDS family and candidate gene sequencing in additional SBDS-negative SDS cases or molecularly undiagnosed IBMFS cases, we identified 3 independent patients, each of whom carried a de novo missense variant in srp54 (encoding signal recognition particle 54 kDa). These 3 patients shared congenital neutropenia linked with various other SDS phenotypes. 3D protein modeling revealed that the 3 variants affect highly conserved amino acids within the GTPase domain of the protein that are critical for GTP and receptor binding. Indeed, we observed that the GTPase activity of the mutated proteins was impaired. The level of SRP54 mRNA in the bone marrow was 3.6-fold lower in patients with SRP54-mutations than in healthy controls. Profound reductions in neutrophil counts and chemotaxis as well as a diminished exocrine pancreas size in a SRP54-knockdown zebrafish model faithfully recapitulated the human phenotype. In conclusion, autosomal dominant mutations in SRP54, a key member of the cotranslation protein-targeting pathway, lead to syndromic neutropenia with a Shwachman-Diamond-like phenotype.

Increased Viral Dissemination in the Brain and Lethality in MCMV-Infected, Dicer-Deficient Neonates.

Among Herpesviruses, Human Cytomegalovirus (HCMV or HHV-5) represents a major threat during congenital or neonatal infections, which may lead to encephalitis with serious neurological consequences. However, as opposed to other less prevalent pathogens, the mechanisms and genetic susceptibility factors for CMV encephalitis are poorly understood. This lack of information considerably reduces the prognostic and/or therapeutic possibilities. To easily monitor the effects of genetic defects on brain dissemination following CMV infection we used a recently developed in vivo mouse model based on the neonatal inoculation of a MCMV genetically engineered to express Luciferase. Here, we further validate this protocol for live imaging, and demonstrate increased lethality associated with viral infection and encephalitis in mutant mice lacking Dicer activity. Our data indicate that miRNAs are important players in the control of MCMV pathogenesis and suggest that miRNA-based endothelial functions and integrity are crucial for CMV encephalitis.

Use of in vivo imaging to monitor the progression of experimental mouse cytomegalovirus infection in neonates.

Human Cytomegalovirus (HCMV or HHV-5) is a life-threatening pathogen in immune-compromised individuals. Upon congenital or neonatal infection, the virus can infect and replicate in the developing brain, which may induce severe neurological damage, including deafness and mental retardation. Despite the potential severity of the symptoms, the therapeutic options are limited by the unavailability of a vaccine and the absence of a specific antiviral therapy. Furthermore, a precise description of the molecular events occurring during infection of the central nervous system (CNS) is still lacking since observations mostly derive from the autopsy of infected children. Several animal models, such as rhesus macaque CMV, have been developed and provided important insights into CMV pathogenesis in the CNS. However, despite its evolutionary proximity with humans, this model was limited by the intracranial inoculation procedure used to infect the animals and consistently induce CNS infection. Furthermore, ethical considerations have promoted the development of alternative models, among which neonatal infection of newborn mice with mouse cytomegalovirus (MCMV) has recently led to significant advances. For instance, it was reported that intraperitoneal injection of MCMV to Balb/c neonates leads to infection of neurons and glial cells in specific areas of the brain. These findings suggested that experimental inoculation of mice might recapitulate the deficits induced by HCMV infection in children. Nevertheless, a dynamic analysis of MCMV infection of neonates is difficult to perform because classical methodology requires the sacrifice of a significant number of animals at different time points to analyze the viral burden and/or immune-related parameters. To circumvent this bottleneck and to enable future investigations of rare mutant animals, we applied in vivo imaging technology to perform a time-course analysis of the viral dissemination in the brain upon peripheral injection of a recombinant MCMV expressing luciferase to C57Bl/6 neonates.

Deregulation of type I IFN-dependent genes correlates with increased susceptibility to cytomegalovirus acute infection of dicer mutant mice.

Regulation of gene expression by microRNAs (miRNAs) is now considered as an essential mechanism for cell development and homeostasis. Indeed, numerous studies have reported that modulating their expression, maturation, or activity can affect cell survival, identity or activation. In particular, miRNAs are key players in the tight regulation of signaling cascades, and as such, they appear as perfectly suited immunomodulators. Several immune-related processes, including inflammation, have recently been demonstrated to require specific miRNAs. In addition, the discovery of herpesvirus-encoded miRNAs has reinforced this assumption. To decipher the potential roles of miRNAs in innate antiviral immune response, we developed an in vivo model based on the inoculation of mouse cytomegalovirus (MCMV) in mice. Furthermore, we exploited a mouse line carrying a hypomorphic mutation in the Dicer gene to visualize the impact of impaired miRNA biogenesis upon the anti-MCMV response. Our data indicate that miRNAs are important actors in mounting an efficient response against herpesviruses. We suggest that a rapid and transient interferon response following viral infection requires miRNA-dependent repressor release. In addition, our in vivo efforts identified several miRNA targets, thus providing a conceptual framework for future analyzes on the regulation of specific actors involved in the Type I interferon pathway.

The non-conventional MHC class I MR1 molecule controls infection by Klebsiella pneumoniae in mice.

As opposed to the well established role of MHC-linked, polymorphic, class I (MHC-I) genes in adaptive immunity, a universal role for non-conventional MHC-I is unknown, thus requiring a case-by-case study. The MHC unlinked, monomorphic, but ß2microglobulin (ß2m)-associated "MHC class I related" MR1 molecule interacts with a semi-invariant TCR. The pathophysiology of this interaction or more importantly of this peculiar MHC-I remains mostly unknown. Recently it was shown that ß2m deficient mice were more susceptible to infection by Klebsiella pneumoniae, a widely spread Gram-negative bacteria that causes diverse and often severe ailments in man. Here we demonstrate, using both an in vivo imaging system and survival tests, the increased susceptibility to K. pneumoniae (but not to several other Gram negative bacteria) of MR1 deficient mice. This is accompanied by a consequent change in body temperature and systemic cytokine profile. Hence MR1 controls K. pneumoniae infection in vivo.

The heterogeneous allelic repertoire of human toll-like receptor (TLR) genes.

Toll-Like Receptors (TLR) are critical elements of the innate arm of the vertebrate immune system. They constitute a multigenic family of receptors which collectively bind a diverse array of--exogeneous as well as endogeneous--ligands. An exponential burst of knowledge has defined their biological role in fight against infections and generation/modulation of auto-immune disorders. Hence, they could at least be conceptually recognized--despite being structurally unrelated - as innate counterparts to Major Histocompatibility Complex (MHC) molecules--equally recognizing antigenic ligands (albeit structurally more homogeneous i.e., peptides), again derived from self and/or non-self sources--preeminent this time in adaptive immunity. Our great disparities in face of infections and/or susceptibility to auto-immune diseases have provoked an intense search for genetic explanations, in part satisfied by the extraordinary MHC allelic repertoire. An equally in-depth and systematic analysis of TLR diversity is lacking despite numerous independent reports of a growing number of SNPs within these loci. The work described here aims at providing a preliminary picture of the allelic repertoire--and not purely SNPs--of all 10 human TLR coding sequences (with exception of TLR3) within a single cohort of up to 100 individuals. It appears from our work that TLR are unequally polymorphic: TLR2 (DNA alleles: 7/protein alleles: 3), 4 (4/3), 7 (6/3), 8 (9/2) and 9 (8/3) being comparatively least diverse whereas TLR1 (11/10), 5 (14/12), 6 (10/8) and 10 (15/10) show a substantial number of alleles. In addition to allelic assignment of a large number of SNPs, 10 new polymorphic positions were hereby identified. Hence this work depicts a first overview of the diversity of almost all human TLR genes, a prelude for large-scale population genetics as well as genetic association studies.