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Primary immunodeficiencies (PIDs) are a large and growing group of disorders commonly associated with recurrent infections. However, nowadays, we know that PIDs often carry with them consequences related to organ or hematologic autoimmunity, autoinflammation, and lymphoproliferation in addition to simple susceptibility to pathogens. Alongside this conceptual development, there has been technical advancement, given by the new but already established diagnostic possibilities offered by new genetic testing (e.g., next-generation sequencing). Nevertheless, there is also the need to understand the large number of gene variants detected with these powerful methods. That means advancing beyond genetic results and resorting to the clinical phenotype and to immunological or alternative molecular tests that allow us to prove the causative role of a genetic variant of uncertain significance and/or better define the underlying pathophysiological mechanism. Furthermore, because of the rapid availability of results, laboratory immunoassays are still critical to diagnosing many PIDs, even in screening settings. Fundamental is the integration between different specialties and the development of multidisciplinary and flexible diagnostic workflows. This paper aims to tell these evolving aspects of immunodeficiencies, which are summarized in five key messages, through introducing and exemplifying five clinical cases, focusing on diseases that could benefit targeted therapy.

Background Systemic lupus erythematosus (SLE) is a complex multi-system disease, characterized by both autoimmune and autoinflammatory clinical and laboratory features. The role of type I interferon (IFN) in SLE has been demonstrated from the 2000s, by gene expression analyses showing significant over-expression of genes related to type I IFN signalling pathway (IFN signature). However, several studies questioned the role of measuring the intensity of IFN signature (IFN score) to chase SLE activity. We would assess if the IFN signature can help the clinical and therapeutic stratification of patients with pediatric SLE. Methods We measured the IFN score in peripheral whole blood from a series of subjects with childhood-onset SLE and correlated the results with clinical and laboratory parameters. Results Thirty-one subjects were included in the study, among which the 87% displayed a positive IFN score. The only significant relation was found for high IFN score in subjects with normocomplementemia. No correlation was observed between IFN score and SLEDAI-2K, BILAG-2004 and SLICC. Patients with high IFN score and normal complement levels also presented lower anti-dsDNA antibodies. Conclusions The integration between IFN signature analysis and complement levels may easily distinguish two groups of subjects, in which the autoimmune or autoinflammatory component of the disease seems to be prevalent.

Off-label use of medications is still a common practice in pediatric rheumatology. JAK inhibitors are authorized in adults in the treatment of rheumatoid arthritis, psoriatic arthritis and ulcerative colitis. Although their use is not authorized yet in children, JAK inhibitors, based on their mechanism of action and on clinical experiences in small series, have been suggested to be useful in the treatment of pediatric interferon-mediated inflammation. Accordingly, an increased interferon score may help to identify those patients who might benefit of JAK inhibitors. We describe the clinical experience with JAK inhibitors in seven children affected with severe inflammatory conditions and we discuss the correlation between clinical features and transcriptomic data. Clinical improvements were recorded in all cases. A reduction of interferon signaling was recorded in three out of seven subjects at last follow-up, irrespectively from clinical improvements. Other signal pathways with significant differences between patients and controls included upregulation of DNA repair pathway and downregulation of extracellular collagen homeostasis. Two patients developed drug-related adverse events, which were considered serious in one case. In conclusion, JAK inhibitors may offer a valuable option for children with severe interferon-mediated inflammatory disorders reducing the interferon score as well as influencing other signal pathways that deserve future studies.

Background Chronic Atypical Neutrophilic Dermatosis with Lipodystrophy and Elevated Temperature (CANDLE) syndrome is a complex autoinflammatory disorders arising from inborn defects in immunoproteasome. Several genes can be involved and cases with digenic inheritance have been described. However, many cases remain without the identification of a specific genetic defect. A positive interferon signature is typically found in patients and may serve as a diagnostic clue. Objectives To describe clinical and genetic features in a girl with CANDLE and in her relatives with a variety of different rheumatologic complaints. Methods We performed Whole Exome Sequencing (WES) on 10 family members. Moreover, we assessed RNA-seq on three sample from the proband, collected during acute phases of disease (samples positive to class I interferon signature (IS)) (1), and her parents. Results of RNA sequencing (RNA-seq) were compared with specimens from healthy controls. Differentially expressed genes (DEGs) were filtered by fold change > 2 and padj Results We describe the case of a 20 years old girl with clinical and biological data supportive of CANDLE syndrome. At the age of 3 years, she started presenting a clinical picture reminiscent of amyopathic dermatomyositis, with skin rash, lipodystrophy, subcutaneous panniculitis nodules, and more recently with chilblains, skin ulcerations, polyarticular arthritis and alopecia. Her pedigree includes several relatives with rheumatic disorders, but none has a clinical picture as complex and severe as our patient. This girl was found to have a strongly positive class I IS in peripheral blood cells. After several unsuccessful therapeutic attempts with antirheumatic drugs and biologics, the girl showed a dramatic clinical response to the JAK inhibitors tofacitinbib. IS resulted positive also in 4 of her relatives, three of whom presented also rheumatologic symptoms. Conversely, one uncle of the girl was affected with rheumatologic symptoms but had negative IS. The pedigree may suggest a complex pattern of inheritance, likely with a major dominant disorder, whose expression can be modulated by multigenic and/or environmental factors. WES failed to detect significant genetic variants in proteasome components. However, RNA-seq revealed a profile of differentially expressed IFN-regulated genes similar to that reported by Anja Brehm et al. in subjects with CANDLE/PRAAS (2). Conclusion Our results suggest that our family may present a multigenic form of CANDLE, with a complete clinical picture only in the proband, whilst other relatives may only present partial or incomplete forms of the disease. References [1] Rice GI, et al. Assessment of Type I Interferon Signaling in Pediatric Inflammatory Disease. J Clin Immunol. 2017 Feb;37(2):123- 132. doi: 10.1007/s10875-016-0359-1. Epub 2016 Dec 9. [2] Brehm A, et al. Additive loss-of- function proteasome subunit mutations in patients with CANDLE/PRAAS promote type I IFN production. J Clin Invest. 2016 Feb;126(2):795. doi: 10.1172/JCI86020. Epub 2016 Feb 1. Disclosure of Interests None declared

Background and aimsMultiple monogenic disorders present as very early onset inflammatory bowel disease (VEO-IBD) or as IBD with severe and atypical features. Establishing a genetic diagnosis may change patients’ management and prognosis. In this study, we describe the diagnostic approach to suspected monogenic IBD in a real clinical setting, discussing genetic and phenotypic findings and therapeutic implications of molecular diagnosis.MethodsInformation of patients with VEO-IBD and early onset IBD with severe/atypical phenotypes (EO-IBD s/a) managed between 2008–2017 who underwent a genetic workup were collected.ResultsNinety-three patients were included, and 12 (13%) reached a genetic diagnosis. Candidate sequencing (CS) was performed in 47 patients (50%), and next generation sequencing (NGS) was performed in 84 patients (90%). Candidate sequencing had a good diagnostic performance only when guided by clinical features specific for known monogenic diseases, whereas NGS helped finding new causative genetic variants and would have anticipated one monogenic diagnosis (XIAP) and consequent bone marrow transplant (BMT). Patients with monogenic IBD more frequently were male (92% vs 54%; P = 0.02), had extraintestinal findings (100% vs 34%; P < 0.001), and had disease onset ≤1 month of life (25% vs 1%; P = 0.006). Genetic diagnosis impacted patient management in 11 patients (92%), 7 of whom underwent BMT.ConclusionA genetic diagnosis can be established in a significant proportion of suspected monogenic IBD and has an impact on patients’ management. Candidate sequencing may be deployed when clinical findings orientate toward a specific diagnosis. Next generation sequencing should be preferred in patients with nonspecific phenotypes.

More and more findings suggest that neurological disorders could have an immunopathological cause. Thus, immune-targeted therapies are increasingly proposed in neurology (even if often controversial), as anakinra, inhibiting IL-1 for febrile inflammatory illnesses, and JAK inhibitors for anti-interferons treatment. Precision medicine in neurology could be fostered by a better understanding of the disease machinery, to develop a rational use of immuno-modulators in clinical trials. In this review, we focus on monogenic disorders with neurological hyper-inflammation/autoimmunity as simplified “models” to correlate immune pathology and targeted treatments. The study of monogenic models yields great advantages for the elucidation of the pathogenic mechanisms that can be reproduced in cellular/animal models, overcoming the limitations of biological samples to study. Moreover, monogenic disorders provide a unique tool to study the mechanisms of neuroinflammatory and autoimmune brain damage, in all their manifestations. The insight of clinical, pathological, and therapeutic aspects of the considered monogenic models can impact knowledge about brain inflammation and can provide useful hints to better understand and cure some neurologic multifactorial disorders.

To evaluate how mucosal bacteria impact on the spontaneous and muramyl dipeptide (MDP)-induced inflammation in Crohn’s disease (CD) and ulcerative colitis (UC). METHODS Colonic mucosal biopsies were collected from children with active or remissive CD, UC and controls. Twotissue samples were taken from inflamed mucosal segments (in patients with active disease) or fromnoninflamed mucosa [in patients in remission or in healthy controls (HC)]. Experiments were performed in the presence or absence of antibiotics, to assess whether the disease-associated microbiota can modulate the cytokine response ex vivo . For this purpose, each specimen was half-cut to compare spontaneous and MDP-induced inflammation in the presence of live bacteria (LB) or antibiotics. After 24 h of culture, an array of 17 cytokines was assessed in supernatants. Statistical analyses were performed to find significant differences in single cytokines or in patterns of cytokine response in the different groups. RESULTS We demonstrated that subjects with CD display aspontaneous production of inflammatory cytokines including granulocyte-colony stimulating factor (G-CSF), interleukin (IL) 6, IL8, IL10 and IL12, that was not significantly influenced by the addition of antibiotics. UC specimens also displayed a trend of increased spontaneous secretion of several cytokines, which however was not significant due to broader variability among patients. After the addition of antibiotics, spontaneous IL8 secretion was significantly higher in UC than in controls. In HC, a trend towards the weakening of spontaneous IL8 production was observed in the presence of live mucosal bacteria with respect to the presence of antibiotics. In contrast, in the presence of LB UC showed an increasing trend of spontaneous IL8 production, while MDP stimulation resulted in lower IL8 production in the presence of antibiotics. We also showed that subjects with CD seem to have a lowered production of IL8 in response to MDP in the presence of LB. Only with the addition of antibiotics, likely reducing the contribution of LB, multivariate statistical analysis could identify the combination of measures of G-CSF, tumor necrosis factor alpha, IL4 and IL17 as a good discriminator between CD and UC. CONCLUSION We showed that the presence of LB or antibiotics can significantly influence the inflammatory response ex vivo in inflammatory bowel diseases.

AIM: To evaluate the inflammatory state in Crohn's disease (CD) patients and correlate it with genetic background and microbial spreading. METHODS: By means of flow cytometry, production of tumor necrosis factor-alpha (TNF-α) was measured in peripheral blood monocytes from patients suffering from CD, ulcerative colitis (UC) and in healthy subjects after stimulation of the NOD2 and TLR pathways. CD patients were genotyped for the three most common NOD2 variants (R702W, G908R and L1007Pfs*2) and basal production of TNF-α was correlated to NOD2 genotype. Also, production of TNF-α was correlated to plasmatic levels of LPS Binding Protein (LBP), soluble (s) CD14 and to the activity state of the disease. RESULTS: The patients with CD were characterized by a significantly higher monocyte basal expression of TNF-α compared with healthy subjects and UC patients, and after stimulation with Pam3CSK4 (ligand of TLR2/1) and MDP-L18 (ligand of NOD2) this difference was maintained, while other microbial stimuli (LPS, ligand of TLR4 and PolyI:C, ligand of TLR3) induced massive activation in CD monocytes as well as in UC and in healthy control cells. There was no significant difference in the production of TNF-α between patients who carried CD-associated heterozygous or homozygous variants in NOD2 and patients with wild type NOD2 genotype. Although serum LBP levels have been shown to correlate positively with the state of activity of the disease, TNF-α production did not show a clear correlation with either LBP or sCD14 levels in plasma. Moreover, no clear correlation was seen between TNF-α production and activity indices in either CD or UC. CONCLUSION: Peripheral monocytes from CD express higher basal and stimulated TNF-α than controls, regardless of NOD2 genotype and without a clear correlation with disease activity.