Bi-allelic variants in IPO8 cause a connective tissue disorder associated with cardiovascular defects, skeletal abnormalities, and immune dysregulation.

Dysregulated transforming growth factor TGF-ß signaling underlies the pathogenesis of genetic disorders affecting the connective tissue such as Loeys-Dietz syndrome. Here, we report 12 individuals with bi-allelic loss-of-function variants in IPO8 who presented with a syndromic association characterized by cardio-vascular anomalies, joint hyperlaxity, and various degree of dysmorphic features and developmental delay as well as immune dysregulation; the individuals were from nine unrelated families. Importin 8 belongs to the karyopherin family of nuclear transport receptors and was previously shown to mediate TGF-ß-dependent SMADs trafficking to the nucleus in vitro. The important in vivo role of IPO8 in pSMAD nuclear translocation was demonstrated by CRISPR/Cas9-mediated inactivation in zebrafish. Consistent with IPO8's role in BMP/TGF-ß signaling, ipo8-/- zebrafish presented mild to severe dorso-ventral patterning defects during early embryonic development. Moreover, ipo8-/- zebrafish displayed severe cardiovascular and skeletal defects that mirrored the human phenotype. Our work thus provides evidence that IPO8 plays a critical and non-redundant role in TGF-ß signaling during development and reinforces the existing link between TGF-ß signaling and connective tissue defects.

Insights into the expanding intestinal phenotypic spectrum of SOCS1 haploinsufficiency and therapeutic options.

PURPOSE: Hyper activation of the JAK-STAT signaling underlies the pathophysiology of many human immune-mediated diseases. Herein, the study of 2 adult patients with SOCS1 haploinsufficiency illustrates the severe and pleomorphic consequences of its impaired regulation in the intestinal tract. METHODS: Two unrelated adult patients presented with gastrointestinal manifestations, one with Crohn's disease-like ileo-colic inflammation refractory to anti-TNF and the other with lymphocytic leiomyositis causing severe chronic intestinal pseudo-occlusion. Next-generation sequencing was used to identify the underlying monogenic defect. One patient received anti-IL-12/IL-23 treatment while the other received the JAK1 inhibitor, ruxolitinib. Peripheral blood, intestinal tissues, and serum samples were analyzed before-and-after JAK1 inhibitor therapy using mass cytometry, histology, transcriptomic, and Olink assay. RESULTS: Novel germline loss-of-function variants in SOCS1 were identified in both patients. The patient with Crohn-like disease achieved clinical remission with anti-IL-12/IL-23 treatment. In the second patient with lymphocytic leiomyositis, ruxolitinib induced rapid resolution of the obstructive symptoms, significant decrease of the CD8+ T lymphocyte muscular infiltrate, and normalization of serum and intestinal cytokines. Decreased frequencies of circulating Treg cells, MAIT cells, and NK cells, with altered CD56bright:CD16lo:CD16hi NK subtype ratios were not modified by ruxolitinib. CONCLUSION: SOCS1 haploinsufficiency can result in a broad spectrum of intestinal manifestations and need to be considered as differential diagnosis in cases of severe treatment-refractory enteropathies, including the rare condition of lymphocytic leiomyositis. This provides the rationale for genetic screening and considering JAK inhibitors in such cases.

Systemic inflammatory syndrome in children with FARSA deficiency.

Variants in aminoacyl-tRNA synthetases (ARSs) genes are associated to a broad spectrum of human inherited diseases. Patients with defective PheRS, encoded by FARSA and FARSB, display brain abnormalities, interstitial lung disease and facial dysmorphism. We investigated four children from two unrelated consanguineous families carrying two missense homozygous variants in FARSA with significantly reduced PheRS-mediated aminoacylation activity. In addition to the core ARS-phenotype, all patients showed an inflammatory profile associated with autoimmunity and interferon score, a clinical feature not ascribed to PheRS-deficient patients to date. JAK inhibition improved lung disease in one patient. Our findings expand the genetic and clinical spectrum of FARSA-related disease.

Deficiency in Mucosa-associated Lymphoid Tissue Lymphoma Translocation 1: A Novel Cause of IPEX-Like Syndrome.

OBJECTIVE: Early-onset inflammatory bowel diseases can result from a wide spectrum of rare mendelian disorders. Early molecular diagnosis is crucial in defining treatment and in improving life expectancy. Herein we aimed at defining the mechanism of an immunodeficiency-polyendrocrinopathy and enteropathy-X-linked (IPEX)-like disease combined with a severe immunodeficiency in 2 siblings born from distantly related parents. METHODS: Whole exome sequencing was performed on blood-extracted genomic DNA from the 2 affected children and their parents on the genomic platform of Institut IMAGINE. Candidate gene mutation was identified using the in-house software PolyWeb and confirmed by Sanger sequencing. Protein expression was determined by western blot. Flow cytometry was used to assess consequences of the mutation on lymphocyte phenotype and nuclear factor-kappa B (NF-κB) activation at diagnosis and after treatment by hematopoietic stem cell transplantation. RESULTS: We identified a homozygous missense mutation in mucosa-associated lymphoid tissue lymphoma translocation 1 gene (MALT1), which precluded protein expression. In keeping with the known function of MALT1, NF-κB-dependent lymphocyte activation was severely impaired. Moreover, there was a drastic reduction in Forkhead box P3 (FOXP3) regulatory T cells accounting for the IPEX-like phenotype. Following identification of the mutation, both children received hematopoietic stem cell transplantation, which permitted full clinical recovery. Immunological workup at 6 and 12 months after transplantation showed normal NF-κB activation and correction of regulatory T cells frequency. CONCLUSIONS: Along with FOXP3, interleukin 2 receptor alpha chain (IL2RA), and cytotoxic T-lymphocyte protein 4 precursor (CTLA-4) mutations, MALT1 deficiency should now be considered as a possible cause of IPEX-like syndrome associated with immunodeficiency that can be cured by hematopoietic stem cell transplantation.

CD24-triggered caspase-dependent apoptosis via mitochondrial membrane depolarization and reactive oxygen species production of human neutrophils is impaired in sepsis.

Apoptosis is the most common pathway of neutrophil death under both physiological and inflammatory conditions. In this study, we describe an apoptotic pathway in human neutrophils that is triggered via the surface molecule CD24. In normal neutrophils, CD24 ligation induces death through depolarization of the mitochondrial membrane in a manner dependent on caspase-3 and caspase-9 and reactive oxygen species. Proinflammatory cytokines such as TNF-α, IFN-γ, and GM-CSF upregulated the expression of CD24 in vitro, favoring the emergence of a new CD16(high)/CD24(high) subset of cultured neutrophils. We observed that CD24 expression (at both mRNA and protein levels) was significantly downregulated in neutrophils from sepsis patients but not from patients with systemic inflammatory response syndrome. This downregulation was reproduced by incubation of neutrophils from healthy controls with corticosteroids or with plasma collected from sepsis patients, but not with IL-10 or TGF-ß. Decreased CD24 expression observed on sepsis neutrophils was associated with lack of functionality of the molecule, because cross-ligation of CD24 failed to trigger apoptosis in neutrophils from sepsis patients. Our results suggest a novel aspect of CD24-mediated immunoregulation and represent, to our knowledge, the first report showing the role of CD24 in the delayed/defective cell death in sepsis.

Interferon-γ and granulocyte/monocyte colony-stimulating factor production by natural killer cells involves different signaling pathways and the adaptor stimulator of interferon genes (STING).

Natural killer (NK) cells are important for innate immunity in particular through the production of IFN-γ and GM-CSF. Both cytokines are important in restoration of immune function of tolerized leukocytes under inflammatory events. The expression of TLRs in NK cells has been widely studied by analyzing the mRNA of these receptors, rarely seeking their protein expression. We previously showed that murine spleen NK cells express TLR9 intracellularly and respond to CpG oligodeoxynucleotide (CpG-ODN) by producing IFN-γ and GM-CSF. However, to get such production the presence of accessory cytokines (such as IL-15 and IL-18) was required, whereas CpG-ODN or accessory cytokines alone did not induce IFN-γ or GM-CSF. We show here that TLR9 overlaps with the Golgi apparatus in NK cells. Furthermore, CpG-ODN stimulation in the presence of accessory cytokines induces the phosphorylation of c-Jun, STAT3, and IκBα. IFN-γ and GM-CSF production requires NF-κB and STAT3 activation as well as Erk-dependent mechanisms for IFN-γ and p38 signaling for GM-CSF. Using knock-out-mice, we show that UNC93b1 and IL-12 (produced by NK cells themselves) are also necessary for IFN-γ and GM-CSF production. IFN-γ production was found to be MyD88- and TLR9-dependent, whereas GM-CSF was TLR9-independent but dependent on STING (stimulator of interferon genes), a cytosolic adaptor recently described for DNA sensing. Our study thereby allows us to gain insight into the mechanisms of synergy between accessory cytokines and CpG-ODN in NK cells. It also identifies a new and alternative signaling pathway for CpG-ODN in murine NK cells.

NK cell tolerance to TLR agonists mediated by regulatory T cells after polymicrobial sepsis.

As sensors of infection, innate immune cells are able to recognize pathogen-associated molecular patterns by receptors such as TLRs. NK cells present in many tissues contribute to inflammatory processes, particularly through the production of IFN-γ. They may display a protective role during infection but also a detrimental role during sterile or infectious systemic inflammatory response syndrome. Nevertheless, the exact status of NK cells during bacterial sepsis and their capacity directly to respond to TLR agonists remain unclear. The expression of TLRs in NK cells has been widely studied by analyzing the mRNA of these receptors. The aim of this study was to gain insight into TLR2/TLR4/TLR9 expression on/in murine NK cells at the protein level and determine if their agonists were able to induce cytokine production. We show, by flow cytometry, a strong intracellular expression of TLR2 and a low of TLR4 in freshly isolated murine spleen NK cells, similar to that of TLR9. In vitro, purified NK cells respond to TLR2, TLR4, and TLR9 agonists, in synergy with activating cytokines (IL-2, IL-15, and/or IL-18), and produce proinflammatory cytokines (IFN-γ and GM-CSF). Finally, we explored the possible tolerance of NK cells to TLR agonists after a polymicrobial sepsis (experimental peritonitis). For the first time, to our knowledge, NK cells are shown to become tolerant in terms of proinflammatory cytokines production after sepsis. We show that this tolerance is associated with a reduction of the CD27(+)CD11b(-) subset in the spleen related to the presence of regulatory T cells and mainly mediated by TGF-ß.

NOD-like receptors in intestinal homeostasis and epithelial tissue repair.

The intestinal epithelium constitutes a dynamic physical barrier segregating the luminal content from the underlying mucosal tissue. Following injury, the epithelial integrity is restored by rapid migration of intestinal epithelial cells (IECs) across the denuded area in a process known as wound healing. Hence, through a sequence of events involving restitution, proliferation and differentiation of IECs the gap is resealed and homeostasis reestablished. Relapsing damage followed by healing of the inflamed mucosa is a hallmark of several intestinal disorders including inflammatory bowel diseases (IBD). While several regulatory peptides, growth factors and cytokines stimulate restitution of the epithelial layer after injury, recent evidence in the field underscores the contribution of innate immunity in controlling this process. In particular, nucleotide-binding and oligomerization domain-like receptors (NLRs) play critical roles in sensing the commensal microbiota, maintaining homeostasis, and regulating intestinal inflammation. Here, we review the process of intestinal epithelial tissue repair and we specifically focus on the impact of NLR-mediated signaling mechanisms involved in governing epithelial wound healing during disease.

Haploinsufficiency in PTPN2 leads to early-onset systemic autoimmunity from Evans syndrome to lupus.

An exome sequencing strategy employed to identify pathogenic variants in patients with pediatric-onset systemic lupus or Evans syndrome resulted in the discovery of six novel monoallelic mutations in PTPN2. PTPN2 is a phosphatase that acts as an essential negative regulator of the JAK/STAT pathways. All mutations led to a loss of PTPN2 regulatory function as evidenced by in vitro assays and by hyperproliferation of patients' T cells. Furthermore, patients exhibited high serum levels of inflammatory cytokines, mimicking the profile observed in individuals with gain-of-function mutations in STAT factors. Flow cytometry analysis of patients' blood cells revealed typical alterations associated with autoimmunity and all patients presented with autoantibodies. These findings further supported the notion that a loss of function in negative regulators of cytokine pathways can lead to a broad spectrum of autoimmune manifestations and that PTPN2 along with SOCS1 haploinsufficiency constitute a new group of monogenic autoimmune diseases that can benefit from targeted therapy.

Assessment of efficacy of antifungals against Aspergillus fumigatus: value of real-time bioluminescence imaging.

Aspergillus fumigatus causes life-threatening infections, especially in immunocompromised patients. Common drugs for therapy of aspergillosis are polyenes, azoles, and echinocandins. However, despite in vitro efficacy of these antifungals, treatment failure is frequently observed. In this study, we established bioluminescence imaging to monitor drug efficacy under in vitro and in vivo conditions. In vitro assays confirmed the effectiveness of liposomal amphotericin B, voriconazole, and anidulafungin. Liposomal amphotericin B and voriconazole were fungicidal, whereas anidulafungin allowed initial germination of conidia that stopped elongation but allowed the conidia to remain viable. In vivo studies were performed with a leukopenic murine model. Mice were challenged by intranasal instillation with a bioluminescent reporter strain (5 × 10(5) and 2.5 × 10(5) conidia), and therapy efficacies of liposomal amphotericin B, voriconazole, and anidulafungin were monitored. For monotherapy, the highest treatment efficacy was observed with liposomal amphotericin B, whereas the efficacies of voriconazole and anidulafungin were strongly dependent on the infectious dose. When therapy efficacy was studied with different drug combinations, all combinations improved the rate of treatment success compared to that with monotherapy. One hundred percent survival was obtained for treatment with a combination of liposomal amphotericin B and anidulafungin, which prevented not only pulmonary infections but also infections of the sinus. In conclusion, combination therapy increases treatment success, at least in the murine infection model. In addition, our novel approach based on real-time imaging enables in vivo monitoring of drug efficacy in different organs during therapy of invasive aspergillosis.

Toll-like receptors expression and interferon-γ production by NK cells in human sepsis.

INTRODUCTION: During the course of infection, natural killer (NK) cells contribute to innate immunity by producing cytokines, particularly interferon-gamma (IFN-γ). In addition to their beneficial effects against infection, NK cells may play a detrimental role during systemic inflammation, causing lethality during sepsis. Little is known on the immune status of NK cells in patients with systemic inflammatory response syndrome (SIRS) or sepsis in terms of cell surface markers expression and IFN-γ production. METHODS: We investigated 27 sepsis patients and 11 patients with non-infectious SIRS. CD56bright and CD56dim NK cell subsets were identified by flow cytometry and Toll-like receptor (TLR)2, TLR4, TLR9, CX3CR1, CD16 and CD69 expression were analyzed, as well as ex vivo IFN-γ production by NK cells in whole blood samples. RESULTS: We first showed that in NK cells from healthy controls, TLR2 and TLR4 expression is mainly intracellular, similarly to TLR9. Intracellular levels of TLR2 and TLR4, in both CD56bright and CD56dim NK cell subsets from sepsis patients, were increased compared to healthy subjects. In addition, the percentage of CD69+ cells was higher among NK cells of sepsis patients. No difference was observed for TLR9, CX3CR1, and CD16 expression. The ex vivo stimulation by TLR4 or TLR9 agonists, or whole bacteria in synergy with accessory cytokines (IL-15+IL-18), resulted in significant production of IFN-γ by NK cells of healthy controls. In contrast, for SIRS and sepsis patients this response was dramatically reduced. CONCLUSIONS: This study reports for the first time an intracellular expression of TLR2 and TLR4 in human NK cells. Surface TLR4 expression allows discriminating sepsis and SIRS. Furthermore, during these pathologies, NK cells undergo an alteration of their immune status characterized by a profound reduction of their capacity to release IFN-γ.

Dysregulation of the immune response in TGF-ß signalopathies.

The transforming growth factor-ß (TGF-ß) family of cytokines exerts pleiotropic functions during embryonic development, tissue homeostasis and repair as well as within the immune system. Single gene defects in individual component of this signaling machinery cause defined Mendelian diseases associated with aberrant activation of TGF-ß signaling, ultimately leading to impaired development, immune responses or both. Gene defects that affect members of the TGF-ß cytokine family result in more restricted phenotypes, while those affecting downstream components of the signaling machinery induce broader defects. These rare disorders, also known as TGF-ß signalopathies, provide the unique opportunity to improve our understanding of the role and the relevance of the TGF-ß signaling in the human immune system. Here, we summarize this elaborate signaling pathway, review the diverse clinical presentations and immunological phenotypes observed in these patients and discuss the phenotypic overlap between humans and mice genetically deficient for individual components of the TGF-ß signaling cascade.

Structural characterization of a pathogenic mutant of human protein tyrosine phosphatase PTPN2 (Cys216Gly) that causes very early onset autoimmune enteropathy.

PTPN2 is an important protein tyrosine phosphatase (PTP) that plays a key role in cell signaling. Deletions or inactivating mutations of PTPN2 have been described in different pathologies and underline its critical role in hematopoiesis, autoimmunity, and inflammation. Surprisingly, despite the major pathophysiological implications of PTPN2, the structural analysis of this PTP and notably of its pathogenic mutants remains poorly documented. Contrary to other human PTP enzymes, to date, only one structure of PTPN2 (wild-type form) has been reported. Here, we report the first crystal structure of a pathogenic mutant of PTPN2 (Cys216Gly) that causes an autoimmune enteropathy. We show in particular that this mutant adopts a classical PTP fold. More importantly, albeit inactive, the mutant retains its ability to bind substrates and to adopt the characteristic catalytically competent closed form of PTP enzymes. This novel PTPN2 structure may serve as a new tool to better understand PTP structures and the structural impacts of pathogenic mutations. Moreover, the C216G PTPN2 structure could also be helpful to design specific ligands/inhibitors.

Clustering ICU patients with sepsis based on the patterns of their circulating biomarkers: A secondary analysis of the CAPTAIN prospective multicenter cohort study.

BACKGROUND: Although sepsis is a life-threatening condition, its heterogeneous presentation likely explains the negative results of most trials on adjunctive therapy. This study in patients with sepsis aimed to identify subgroups with similar immune profiles and their clinical and outcome correlates. METHODS: A secondary analysis used data of a prospective multicenter cohort that included patients with early assessment of sepsis. They were described using Predisposition, Insult, Response, Organ failure sepsis (PIRO) staging system. Thirty-eight circulating biomarkers (27 proteins, 11 mRNAs) were assessed at sepsis diagnosis, and their patterns were determined through principal component analysis (PCA). Hierarchical clustering was used to group the patients and k-means algorithm was applied to assess the internal validity of the clusters. RESULTS: Two hundred and three patients were assessed, of median age 64.5 [52.0-77.0] years and SAPS2 score 55 [49-61] points. Five main patterns of biomarkers and six clusters of patients (including 42%, 21%, 17%, 9%, 5% and 5% of the patients) were evidenced. Clusters were distinguished according to the certainty of the causal infection, inflammation, use of organ support, pro- and anti-inflammatory activity, and adaptive profile markers. CONCLUSIONS: In this cohort of patients with suspected sepsis, we individualized clusters which may be described with criteria used to stage sepsis. As these clusters are based on the patterns of circulating biomarkers, whether they might help to predict treatment responsiveness should be addressed in further studies. TRIAL REGISTRATION: The CAPTAIN study was registered on clinicaltrials.gov on June 22, 2011, # NCT01378169.

UNC45A deficiency causes microvillus inclusion disease-like phenotype by impairing myosin VB-dependent apical trafficking.

Variants in the UNC45A cochaperone have been recently associated with a syndrome combining diarrhea, cholestasis, deafness, and bone fragility. Yet the mechanism underlying intestinal failure in UNC45A deficiency remains unclear. Here, biallelic variants in UNC45A were identified by next-generation sequencing in 6 patients with congenital diarrhea. Corroborating in silico prediction, variants either abolished UNC45A expression or altered protein conformation. Myosin VB was identified by mass spectrometry as client of the UNC45A chaperone and was found misfolded in UNC45AKO Caco-2 cells. In keeping with impaired myosin VB function, UNC45AKO Caco-2 cells showed abnormal epithelial morphogenesis that was restored by full-length UNC45A, but not by mutant alleles. Patients and UNC45AKO 3D organoids displayed altered luminal development and microvillus inclusions, while 2D cultures revealed Rab11 and apical transporter mislocalization as well as sparse and disorganized microvilli. All those features resembled the subcellular abnormalities observed in duodenal biopsies from patients with microvillus inclusion disease. Finally, microvillus inclusions and shortened microvilli were evidenced in enterocytes from unc45a-deficient zebrafish. Taken together, our results provide evidence that UNC45A plays an essential role in epithelial morphogenesis through its cochaperone function of myosin VB and that UNC45A loss causes a variant of microvillus inclusion disease.

Mechanisms of TNF induction by heat-killed Staphylococcus aureus differ upon the origin of mononuclear phagocytes.

Mononuclear phagocytes are among the first immune cells activated after pathogens invasion. Although they all derive from the same progenitor in the bone marrow, their characteristics differ on the compartment from which they are derived. In this work, we investigated the contribution of phagocytosis for tumor necrosis factor (TNF) production by murine mononuclear phagocytes (monocytes, peritoneal and alveolar macrophages) in response to heat-killed Staphylococcus aureus (HKSA). Mononuclear phagocytes behaved differently, depending on their compartment of residence. Indeed, when bacterial uptake or phagosome maturation was blocked, activation through membrane receptors was sufficient for a maximal production of TNF and interleukin-10 by peritoneal macrophages. In contrast, monocytes, and to a lesser extent alveolar macrophages, required phagocytosis for optimal cytokine production. While investigating the different actors of signalization, we found that p38 kinase and phosphatidylinositol 3-kinase were playing an important role in HKSA phagocytosis and TNF production. Furthermore, blocking the α(5)ß(1)-integrin significantly decreased TNF production in response to HKSA in all three cell types. Finally, using mononuclear phagocytes from NOD2 knockout mice, we observed that TNF production in response to HKSA was dependent on NOD2 for monocytes and peritoneal macrophages. In conclusion, we demonstrate that the mechanisms of activation leading to TNF production in response to HKSA are specific for each mononuclear phagocyte population and involve different recognition processes and signaling pathways. The influence of the compartments on cell properties and behavior should be taken into account, to better understand cell physiology and host-pathogen interaction, and to define efficient strategies to fight infection.