Hyperactivation and altered selection of B cells in patients with paediatric Sjogren's syndrome.

OBJECTIVES: Paediatric Sjögren's syndrome (pSS) is a rare chronic autoimmune disorder, characterised by inflammation of exocrine glands. B cell hyperactivation plays a central role in adult-onset Sjogren. This study was designed to analyse B cell and T cell phenotype, levels of BAFF, and selection of autoreactive B cells in patients with pSS. METHODS: A total of 17 patients diagnosed with pSS and 13 healthy donors (controls) comparable for age were enrolled in the study. B cell and T cell subsets and frequency of autoreactive B cells in peripheral blood were analysed by flow cytometry. Levels of BAFF were analysed by ELISA. RESULTS: The relative frequency of total B cells, transitional, naïve and switched memory B cells was similar between pSS patients and controls. In patients with pSS, we observed a reduction in the frequency of unswitched memory B cells, an increased frequency of atypical memory B cells and an expansion of PD1hi CXCR5- T peripheral helper cells. Levels of BAFF were higher in patients with pSS compared with controls and correlated with serum levels of total IgG and titres of anti-Ro antibodies. The frequency of autoreactive B cells in the transitional, unswitched memory and plasmablast compartment was significantly higher in pSS patients than in controls. CONCLUSIONS: Our results point to a hyperactivation of B cells in pSS. Current therapies do not seem to affect B cell abnormalities, suggesting that novel therapies targeting specifically B cell hyperactivation need to be implemented for paediatric patients.

Pro Nerve Growth Factor and Its Receptor p75NTR Activate Inflammatory Responses in Synovial Fibroblasts: A Novel Targetable Mechanism in Arthritis.

We have recently provided new evidence for a role of p75NTR receptor and its preferential ligand proNGF in amplifying inflammatory responses in synovial mononuclear cells of chronic arthritis patients. In the present study, to better investigate how activation of the p75NTR/proNGF axis impacts synovial inflammation, we have studied the effects of proNGF on fibroblast-like synoviocytes (FLS), which play a central role in modulating local immune responses and in activating pro-inflammatory pathways. Using single cell RNA sequencing in synovial tissues from active and treatment-naïve rheumatoid arthritis (RA) patients, we demonstrated that p75NTR and sortilin, which form a high affinity receptor complex for proNGF, are highly expressed in PRG4pos lining and THY1posCOL1A1pos sublining fibroblast clusters in RA synovia but decreased in RA patients in sustained clinical remission. In ex vivo experiments we found that FLS from rheumatoid arthritis patients (RA-FLS) retained in vitro a markedly higher expression of p75NTR and sortilin than FLS from osteoarthritis patients (OA-FLS). Inflammatory stimuli further up-regulated p75NTR expression and induced endogenous production of proNGF in RA-FLS, leading to an autocrine activation of the proNGF/p75NTR pathway that results in an increased release of pro-inflammatory cytokines. Our data on the inhibition of p75NTR receptor, which reduced the release of IL-1ß, IL-6 and TNF-α, further confirmed the key role of p75NTR activation in regulating inflammatory cytokine production. In a set of ex vivo experiments, we used RA-FLS and cultured them in the presence of synovial fluids obtained from arthritis patients that, as we demonstrated, are characterized by a high concentration of proNGF. Our data show that the high levels of proNGF present in inflamed synovial fluids induced pro-inflammatory cytokine production by RA-FLS. The blocking of NGF binding to p75NTR using specific inhibitors led instead to the disruption of this pro-inflammatory loop, reducing activation of the p38 and JNK intracellular pathways and decreasing inflammatory cytokine production. Overall, our data demonstrate that an active proNGF/p75NTR axis promotes pro-inflammatory responses in synovial fibroblasts, thereby contributing to chronic synovial inflammation, and point to the possible use of p75NTR inhibitors as a novel therapeutic approach in chronic arthritis.

Mutations at the C-terminus of CDC42 cause distinct hematopoietic and autoinflammatory disorders.

BACKGROUND: Pathogenic missense variants in cell division control protein 42 (CDC42) differentially affect protein function, causing a clinically wide phenotypic spectrum variably affecting neurodevelopment, hematopoiesis, and immune response. More recently, 3 variants at the C-terminus of CDC42 were proposed to similarly impact protein function and cause a novel autoinflammatory disorder. OBJECTIVES: We sought to clinically and functionally classify these variants to improve patient management. METHODS: Comparative analysis of the available clinical data and medical history of patients was performed. In vitro and in vivo studies were carried out to functionally characterize individual variants. RESULTS: Differently from what had previously been observed for the p.R186C change causing neonatal-onset cytopenia, autoinflammation, and recurrent hemophagocytic lymphohistiocytosis, p.C188Y and p.∗192Cext∗24 promoted accelerated protein degradation. Unprenylated CDC42C188Y did not behave as a membrane-bound protein, whereas the residual CDC42∗192Cext∗24 mutant replicated the CDC42R186C behavior, being targeted to the Golgi apparatus in a palmitoylation-dependent manner. Assessment of in vitro polarized migration and development in Caenorhabditis elegans documented a loss-of-function behavior of the p.C188Y and p.∗192Cext∗24 variants. Consistently, the 3 pathogenic variants were associated with different clinical presentations, with dysmorphisms, severity, and age of onset of cytopenia and extent of autoinflammation representing major differences. CONCLUSIONS: Pathogenic variants at the CDC42 C-terminus differently impact protein stability, localization, and function, and cause different diseases, with p.R186C specifically associated with neonatal-onset pancytopenia and severe autoinflammation/hemophagocytic lymphohistiocytosis requiring emapalumab and bone marrow transplantation, and p.C188Y and p.∗192Cext∗24 causing anakinra-sensitive autoinflammation.

A novel disorder involving dyshematopoiesis, inflammation, and HLH due to aberrant CDC42 function.

Hemophagocytic lymphohistiocytosis (HLH) is characterized by immune dysregulation due to inadequate restraint of overactivated immune cells and is associated with a variable clinical spectrum having overlap with more common pathophysiologies. HLH is difficult to diagnose and can be part of inflammatory syndromes. Here, we identify a novel hematological/autoinflammatory condition (NOCARH syndrome) in four unrelated patients with superimposable features, including neonatal-onset cytopenia with dyshematopoiesis, autoinflammation, rash, and HLH. Patients shared the same de novo CDC42 mutation (Chr1:22417990C>T, p.R186C) and altered hematopoietic compartment, immune dysregulation, and inflammation. CDC42 mutations had been associated with syndromic neurodevelopmental disorders. In vitro and in vivo assays documented unique effects of p.R186C on CDC42 localization and function, correlating with the distinctiveness of the trait. Emapalumab was critical to the survival of one patient, who underwent successful bone marrow transplantation. Early recognition of the disorder and establishment of treatment followed by bone marrow transplant are important to survival.

Functional Dysregulation of CDC42 Causes Diverse Developmental Phenotypes.

Exome sequencing has markedly enhanced the discovery of genes implicated in Mendelian disorders, particularly for individuals in whom a known clinical entity could not be assigned. This has led to the recognition that phenotypic heterogeneity resulting from allelic mutations occurs more commonly than previously appreciated. Here, we report that missense variants in CDC42, a gene encoding a small GTPase functioning as an intracellular signaling node, underlie a clinically heterogeneous group of phenotypes characterized by variable growth dysregulation, facial dysmorphism, and neurodevelopmental, immunological, and hematological anomalies, including a phenotype resembling Noonan syndrome, a developmental disorder caused by dysregulated RAS signaling. In silico, in vitro, and in vivo analyses demonstrate that mutations variably perturb CDC42 function by altering the switch between the active and inactive states of the GTPase and/or affecting CDC42 interaction with effectors, and differentially disturb cellular and developmental processes. These findings reveal the remarkably variable impact that dominantly acting CDC42 mutations have on cell function and development, creating challenges in syndrome definition, and exemplify the importance of functional profiling for syndrome recognition and delineation.