Search

Deficiency of adenosine deaminase 2 (DADA2) was first described as a monogenic form of systemic vasculitis that closely resembles polyarteritis nodosa (PAN). The phenotypic spectrum of DADA2 has vastly expanded in recent years and now includes pure red cell aplasia, bone marrow failure syndrome, lymphoproliferative disease, and humoral immunodeficiency. Vasculitis remains the most common presentation of DADA2, and treatment with tumor necrosis factor inhibitors (TNFi) has shown remarkable efficacy in preventing stroke and ameliorating features of systemic inflammation. The precise function of ADA2 has not been elucidated, and how absence of ADA2 ignites inflammation is an active area of research. In this review, we will discuss the current understanding of DADA2 from research and clinical perspectives. We will evaluate several proposed functions of ADA2, including polarization of monocyte phenotype, regulation of neutrophil extracellular trap formation, and modulation of innate immunity. We will also review the role of inflammatory cytokines including TNF and type I interferons. Lastly, we will provide future perspectives on understanding the phenotypic heterogeneity of DADA2 and discuss potential treatment options.

The linear ubiquitin assembly complex (LUBAC) consists of HOIP, HOIL1 and SHARPIN, and is essential for proper immune responses. Patients with HOIP and HOIL1 deficiencies present with severe immunodeficiency, autoinflammation and glycogen storage. In mice, the loss ofSharpinleads to severe dermatitis due to excessive cell death in keratinocytes. Here we report the first patient with SHARPIN deficiency, manifesting fever, arthritis, colitis, chronic otitis media and hepatic glycogenosis but unexpectedly, not associated with dermatologic manifestations. Mechanistically, fibroblasts and B cells from patients with all three LUBAC deficiencies showed attenuated canonical NF-B response and propensity to apoptosis mediated by TNF superfamily members. Furthermore, the SHARPIN deficient patient showed substantial reduction of adenoidal germinal center B cell development. Treatment of the SHARPIN deficient patient with anti-TNF therapies led to complete clinical and transcriptomic resolution of autoinflammation. These findings underscore the critical role of LUBAC as a gatekeeper for apoptosis-mediated immune dysregulation in humans.

Objective Somatic mutations in UBA1 cause a newly defined syndrome known as VEXAS (vacuoles, E1 enzyme, X-linked, autoinflammatory, somatic syndrome). More than 50% of patients currently identified as having VEXAS met diagnostic criteria for relapsing polychondritis (RP), but clinical features that characterize VEXAS within a cohort of patients with RP have not been defined. We undertook this study to define the prevalence of somatic mutations in UBA1 in patients with RP and to create an algorithm to identify patients with genetically confirmed VEXAS among those with RP. Methods Exome and targeted sequencing of UBA1 was performed in a prospective observational cohort of patients with RP. Clinical and immunologic characteristics of patients with RP were compared based on the presence or absence of UBA1 mutations. The random forest method was used to derive a clinical algorithm to identify patients with UBA1 mutations. Results Seven of 92 patients with RP (7.6%) had UBA1 mutations (referred to here as VEXAS-RP). Patients with VEXAS-RP were all male, were on average ≥45 years of age at disease onset, and commonly had fever, ear chondritis, skin involvement, deep vein thrombosis, and pulmonary infiltrates. No patient with VEXAS-RP had chondritis of the airways or costochondritis. Mortality was greater in VEXAS-RP than in RP (23% versus 4%; P = 0.029). Elevated acute-phase reactants and hematologic abnormalities (e.g., macrocytic anemia, thrombocytopenia, lymphopenia, multiple myeloma, myelodysplastic syndrome) were prevalent in VEXAS-RP. A decision tree algorithm based on male sex, a mean corpuscular volume >100 fl, and a platelet count

Monogenic autoinflammatory diseases are a group of rheumatologic disorders caused by dysregulation in the innate immune system. The molecular mechanisms of these disorders are linked to defects in inflammasome-mediated, NF-κB-mediated or interferon-mediated inflammatory signalling pathways, cytokine receptors, the actin cytoskeleton, proteasome complexes and various enzymes. As with other human disorders, disease-causing variants in a single gene can present with variable expressivity and incomplete penetrance. In some cases, pathogenic variants in the same gene can be inherited either in a recessive or dominant manner and can cause distinct and seemingly unrelated phenotypes, although they have a unifying biochemical mechanism. With an enhanced understanding of protein structure and functionality of protein domains, genotype-phenotype correlations are beginning to be unravelled. Many of the mutated proteins are primarily expressed in haematopoietic cells, and their malfunction leads to systemic inflammation. Disease presentation is also defined by a specific effect of the mutant protein in a particular cell type and, therefore, the resulting phenotype might be more deleterious in one tissue than in another. Many patients present with the expanded immunological disease continuum that includes autoinflammation, immunodeficiency, autoimmunity and atopy, which necessitate genetic testing.

ObjectiveMajeed syndrome (MJS) is an autosomal recessive, systemic autoinflammatory disease (SAID) caused by biallelic loss-of-function variants in theLPIN2gene. It is characterized by early-onset chronic recurrent multifocal osteomyelitis (CRMO), dyserythropoietic anemia, and neutrophilic dermatosis. We analyzed a cohort of uncharacterized Indian patients for pathogenic variants inLPIN2and other genes associated with SAIDs.MethodsWe performed whole-exome sequencing (WES) for 1 patient and next-generation sequencing (NGS) targeted gene panel for SAIDs in 3 patients. One patient was a referral from neurology after clinical exome sequencing identified a novel variant inLPIN2. We reviewed the literature for all published studies of mutation-positive MJS patients and have summarized their clinical features and disease-causing variants.ResultsWe describe the largest series of patients with MJS outside of the Middle East. All 5 patients are homozygous for novel, possibly pathogenic variants in theLPIN2gene. Two of these variants are missense substitutions, and 3 are predicted to alter transcript splicing and create a truncated protein. In addition to the classical features of CRMO and anemia, patients exhibited previously unreported features, including abdominal pain, recurrent diarrhea/ear discharge, and erythema nodosum.ConclusionPatients with MJS may present initially to different specialists, and thus it is important to create awareness in the medical community. In India, consanguinity is a common sociocultural factor in many ethnic communities and an abbreviated NGS gene panel for autoinflammatory diseases should include MJS. The unavailability of interleukin 1 inhibitors in some countries poses a treatment challenge.

Background Monogenic autoinflammatory diseases (AID) are caused by mutations in innate immune genes. The effects of these mutations on allergic inflammation are unknown. Objectives We investigated allergic, immunological and clinical phenotypes in FMF (familial Mediterranean fever), CAPS (cryopyrin-associated periodic syndrome), TRAPS (tumour necrosis factor receptor-associated periodic syndrome), HIDS (hyper-IgD syndrome), PAPA (pyogenic arthritis, pyoderma gangrenosum and acne), DADA2 (deficiency of adenosine deaminase 2), HA20 (haploinsufficiency of A20), CANDLE (chronic atypical neutrophilic dermatosis, lipodystrophy, elevated temperature) and SAVI (STING-associated vasculopathy of infancy). Methods In this cross-sectional study, clinical data were assessed in 425 patients with AID using questionnaires and chart reviews. Comparator data were obtained from public databases. Peripheral blood mononuclear cells obtained from 55 patients were stimulated and CD4+ cytokine production assessed. Results Clinical laboratory features of Type 2 immunity were elevated in CAPS but reduced in most AID, particularly DADA2. Physician-diagnosed allergic diseases were prevalent in multiple AID, including CAPS and DADA2. T helper 2 (Th2) cells were expanded in CAPS, TRAPS and HIDS; Th9 cells were expanded in HA20. Conclusions CAPS is characterised by an enhanced Type 2 signature, whereas FMF and CANDLE are associated with reduced Type 2 responses. DADA2 is associated with reduced Type 2 responses but a high rate of physician-diagnosed allergy. Therefore, NLRP3-driven autoinflammation may promote Type 2 immunity, whereas AID like DADA2 may manifest clinical phenotypes that masquerade as allergic disorders. Further investigations are needed to determine the contribution of autoinflammation to allergic clinical and immunological phenotypes, to improve the treatment of patients with AID.

OBJECTIVE. Deficiency of adenosine deaminase 2 (DADA2) is a monogenic form of vasculitis that can resemble polyarteritis nodosa (PAN). This study was undertaken to identify potential disease-causing sequence variants in ADA2 in patients with idiopathic PAN, granulomatosis with polyangiitis (GPA), or microscopic polyangiitis (MPA). METHODS. Patients with idiopathic PAN (n = 118) and patients with GPA or MPA (n = 1,107) were screened for rare nonsynonymous variants in ADA2 using DNA sequencing methods. ADA-2 enzyme activity was assessed in selected serum samples. RESULTS. Nine of 118 patients with PAN (7.6%) were identified as having rare nonsynonymous variants in ADA2. Four patients (3.4%) were biallelic for pathogenic or likely pathogenic variants, and 5 patients (4.2%) were monoallelic carriers for 3 variants of uncertain significance and 2 likely pathogenic variants. Serum samples from 2 patients with PAN with biallelic variants were available and showed markedly reduced ADA-2 enzyme activity. ADA-2 enzyme testing of 86 additional patients revealed 1 individual with strongly reduced ADA-2 activity without detectable pathogenic variants. Patients with PAN and biallelic variants in ADA2 were younger at diagnosis than patients with 1 or no variant in ADA2, with no other clinical differences noted. None of the patients with GPA or MPA carried biallelic variants in ADA2. CONCLUSION. A subset of patients with idiopathic PAN meet genetic criteria for DADA2. Given that tumor necrosis factor inhibition is efficacious in DADA2 but is not conventional therapy for PAN, these findings suggest that ADA-2 testing should strongly be considered in patients with hepatitis B virus–negative idiopathic PAN.

To test the hypothesis that ROSAH (retinal dystrophy, optic nerve oedema, splenomegaly, anhidrosis and headache) syndrome, caused by dominant mutation inThis cohort study systematically evaluated 27 patients with ROSAH syndrome for inflammatory features and investigated the effect ofThe majority of the cohort carried the p.Thr237Met mutation but we also identified a new ROSAH-associated mutation, p.Tyr254Cys.Nearly all patients exhibited at least one feature consistent with inflammation including recurrent fever, headaches with meningeal enhancement and premature basal ganglia/brainstem mineralisation on MRI, deforming arthritis and AA amyloidosis. However, there was significant phenotypic variation, even within families and some adults lacked functional visual deficits. While anti-TNF and anti-IL-1 therapies suppressed systemic inflammation and improved quality of life, anti-IL-6 (tocilizumab) was the only anticytokine therapy that improved intraocular inflammation (two of two patients).Patients' primary samples and in vitro assays with mutated ALPK1 constructs showed immune activation with increased NF-κB signalling, STAT1 phosphorylation and interferon gene expression signature. Knock-in mice with theROSAH syndrome is an autoinflammatory disease caused by gain-of-function mutations in

Background Biallelic loss-of-function variants in NCF1 lead to reactive oxygen species deficiency and chronic granulomatous disease (CGD). Heterozygosity for the p.Arg90His variant in NCF1 has been associated with susceptibility to systemic lupus erythematosus, rheumatoid arthritis, and Sjögren’s syndrome in adult patients. This study demonstrates the association of the homozygous p.Arg90His variant with interferonopathy with features of autoinflammation and autoimmunity in a pediatric patient. Case presentation A 5-year old female of Indian ancestry with early-onset recurrent fever and headache, and persistently elevated antinuclear, anti-Ro, and anti-La antibodies was found to carry the homozygous p.Arg90His variant in NCF1 through exome sequencing. Her unaffected parents and three other siblings were carriers for the mutant allele. Because the presence of two NCF1 pseudogenes, this variant was confirmed by independent genotyping methods. Her intracellular neutrophil oxidative burst and NCF1 expression levels were normal, and no clinical features of CGD were apparent. Gene expression analysis in peripheral blood detected an interferon gene expression signature, which was further supported by cytokine analyses of supernatants of cultured patient’s cells. These findings suggested that her inflammatory disease is at least in part mediated by type I interferons. While her fever episodes responded well to systemic steroids, treatment with the JAK inhibitor tofacitinib resulted in decreased serum ferritin levels and reduced frequency of fevers. Conclusion Homozygosity for p.Arg90His in NCF1 should be considered contributory in young patients with an atypical systemic inflammatory antecedent phenotype that may evolve into autoimmunity later in life. The complex genomic organization of NCF1 poses a difficulty for high-throughput genotyping techniques and variants in this gene should be carefully evaluated when using the next generation and Sanger sequencing technologies. The p.Arg90His variant is found at a variable allele frequency in different populations, and is higher in people of South East Asian ancestry. In complex genetic diseases such as SLE, other rare and common susceptibility alleles might be necessary for the full disease expressivity.

Background Adult-onset inflammatory syndromes often manifest with overlapping clinical features. Variants in ubiquitin-related genes, previously implicated in autoinflammatory disease, may...

Objective Deficiency of adenosine deaminase 2 (DADA2) is a potentially fatal monogenic syndrome characterized by variable manifestations of systemic vasculitis, bone marrow failure, and immunodeficiency. Most cases are diagnosed by pediatric care providers, given the typical early age of disease onset. This study was undertaken to describe the clinical phenotypes and treatment response both in adults and in children with DADA2 in India. Methods A retrospective analysis of pediatric and adult patients with DADA2 diagnosed at various rheumatology centers across India was conducted. Clinical characteristics, diagnostic findings, and treatment responses were analyzed in all subjects. Results In total, 33 cases of DADA2 were confirmed in this cohort between April 2017 and March 2020. Unlike previous studies, nearly one-half of the confirmed cases presented during adulthood. All symptomatic patients exhibited features of vasculitis, whereas constitutional symptoms and anemia were more common in pediatric patients. Cutaneous and neurologic involvement were common, and 18 subjects had experienced at least one stroke. In addition, the clinical spectrum of DADA2 was expanded by recognition of novel features in these patients, including pancreatic infarction, focal myocarditis, and diffuse alveolar hemorrhage. Treatment with tumor necrosis factor inhibitors (TNFi) was initiated in 25 patients. All of the identified disease manifestations showed marked improvement after initiation of TNFi, and disease remission was achieved in 19 patients. Two cases were complicated by tuberculosis infection, and 2 deaths were reported. Conclusion This report presents the first case series of patients with DADA2 from India, diagnosed by adult and pediatric care providers. The findings raise awareness of this syndrome, particularly with regard to its presentation in adults.

NF-κB is a master transcription factor that activates the expression of target genes in response to various stimulatory signals. Activated NF-κB mediates a plethora of diverse functions including innate and adaptive immune responses, inflammation, cell proliferation, and NF-κB is regulated through interactions with IκB inhibitory proteins, which are in turn regulated by the inhibitor of κB kinase (IKK) complex. Together, these 3 components form the core of the NF-κB signalosomes that have cell-specific functions which are dependent on the interactions with other signaling molecules and pathways. The activity of NF-κB pathway is also regulated by a variety of post-translational modifications including phosphorylation and ubiquitination by Lys63, Met1, and Lys48 ubiquitin chains. The physiologic role of NF-κB is best studied in the immune system due to discovery of many human diseases caused by pathogenic variants in various proteins that constitute the NF-κB pathway. These disease-causing variants can act either as gain-of-function (GoF) or loss-of-function (LoF) and depending on the function of mutated protein, can cause either immunodeficiency or systemic inflammation. Typically, pathogenic missense variants act as GoF and they lead to increased activity in the pathway. LoF variants can be inherited as recessive or dominant alleles and can cause either a decrease or an increase in pathway activity. Dominantly inherited LoF variants often result in haploinsufficiency of inhibitory proteins. Here, we review human Mendelian immunologic diseases, which results from mutations in different molecules in the canonical NF-κB pathway and surprisingly present with a continuum of clinical features including immunodeficiency, atopy, autoimmunity, and autoinflammation.

PURPOSE: Deficiency of adenosine deaminase 2 (DADA2) is an autosomal recessive disorder that manifests with fever, early-onset vasculitis, strokes, and hematologic dysfunction. This study aimed to identify disease-causing variants by conventional Sanger and whole exome sequencing in two families suspected to have DADA2 and non-confirmatory genotypes. ADA2 enzymatic assay confirmed the clinical diagnosis of DADA2. Molecular diagnosis was important to accurately identify other family members at risk. METHODS: We used a variety of sequencing technologies, ADA2 enzymatic testing, and molecular methods including qRT-PCR, MLPA. RESULTS: Exome sequencing identified heterozygosity for the known pathogenic variant ADA2: c.1358A>G, p.Y453C in a 14-year-old female with a history of ischemic strokes, livedo, and vasculitis. No second pathogenic variant could be identified. ADA2 enzymatic testing in combination with quantitative RT-PCR suggested a loss-of-function allele. Subsequent genome sequencing identified a canonical splice site variant, c.−47+2T>C, within the 5’UTR of ADA2. Two of her unaffected siblings were found to carry the same two pathogenic variants. A homozygous 800bp duplication comprising exon 7 of ADA2 was identified in a 5-year-old female with features consistent with Diamond-Blackfan anemia (DBA). The duplication was missed by Sanger sequencing of ADA2, chromosomal microarray, and exome sequencing but was detected by MLPA in combination with long-read PCR sequencing. The exon 7 duplication was also identified in her non-symptomatic father and younger sister. CONCLUSIONS: ADA2 pathogenic variants may not be detected by conventional sequencing and genetic testing and may require the incorporation of additional diagnostic methods. A definitive molecular diagnosis is crucial for all family members to make informed treatment decisions.

Familial Mediterranean fever (FMF) is an autoinflammatory disease caused by homozygous or compound heterozygous gain-of-function mutations in MEFV, which encodes pyrin, an inflammasome protein. Heterozygous carrier frequencies for multiple MEFV mutations are high in several Mediterranean populations, suggesting that they confer selective advantage. Among 2,313 Turkish people, we found extended haplotype homozygosity flanking FMF-associated mutations, indicating evolutionarily recent positive selection of FMF-associated mutations. Two pathogenic pyrin variants independently arose >1,800 years ago. Mutant pyrin interacts less avidly with Yersinia pestis virulence factor YopM than with wild-type human pyrin, thereby attenuating YopM-induced interleukin (IL)-1β suppression. Relative to healthy controls, leukocytes from patients with FMF harboring homozygous or compound heterozygous mutations and from asymptomatic heterozygous carriers released heightened IL-1β specifically in response to Y. pestis. Y. pestis-infected MefvM680I/M680I FMF knock-in mice exhibited IL-1-dependent increased survival relative to wild-type knock-in mice. Thus, FMF mutations that were positively selected in Mediterranean populations confer heightened resistance to Y. pestis. Familial Mediterranean fever is an autoinflammatory disease caused by gain-of-function mutations in the pyrin inflammasome. Kastner and colleagues show that mutant pyrin better resists suppression by the plague bacterium Yersinia pestis and may have been positively selected in human Middle Eastern populations.

Ubiquitylation is an essential post-translational modification that regulates intracellular signalling networks by triggering proteasomal substrate degradation, changing the activity of substrates or mediating changes in proteins that interact with substrates. Hundreds of enzymes participate in reversible ubiquitylation of proteins, some acting globally and others targeting specific proteins. Ubiquitylation is essential for innate immune responses, as it facilitates rapid regulation of inflammatory pathways, thereby ensuring sufficient but not excessive responses. A growing number of inborn errors of immunity are attributed to dysregulated ubiquitylation. These genetic disorders exhibit broad clinical manifestations, ranging from susceptibility to infection to autoinflammatory and/or autoimmune features, lymphoproliferation and propensity to malignancy. Many autoinflammatory disorders result from disruption of components of the ubiquitylation machinery and lead to overactivation of innate immune cells. An understanding of the disorders of ubiquitylation in autoinflammatory diseases could enable the development of novel management strategies.

ObjectivesAdenosine deaminase 2 deficiency (DADA2) is a genetic, neurologic, and systemic vasculitis syndrome, which can lead to recurrent strokes, typically lacunar. In the cohort of now 60 patients followed up at the NIH Clinical Center (NIH CC), no patient has had a stroke since starting tumor necrosis factor (TNF) blockade. We present a family with multiple affected children to highlight the importance of TNF blockade not just as secondary stroke prevention but also as primary stroke prevention in genetically affected but clinically asymptomatic patients.MethodsA proband with recurrent cryptogenic strokes was referred for evaluation at the NIH CC. The parents and 3 clinically asymptomatic siblings were also evaluated.ResultsThe proband was diagnosed with DADA2 based on biochemical testing; her antiplatelet therapies were discontinued, and she was started on TNF blockade for secondary stroke prevention. Her 3 asymptomatic siblings were subsequently tested and 2 were found to be biochemically affected. One of them elected to start TNF blockade for primary stroke prevention and the other sibling declined this approach and experienced a stroke. A second genetic sequence variant was subsequently identified in theADA2gene.DiscussionThis family illustrates the importance of testing for DADA2 in young patients with cryptogenic stroke, given the hemorrhagic risks with antiplatelet drugs in these patients and effectiveness of TNF blockade as secondary stroke prevention. In addition, this family highlights the importance of screening all siblings of affected patients because they may be presymptomatic, and we advocate starting TNF blockade for primary stroke prevention in those who are found to be genetically or biochemically affected.

Description, Deubiquitination of proteasome subunits by OTULIN regulates type I IFN production., OTULIN is a linear deubiquitinase that negatively regulates the nuclear factor κB (NF-κB) signaling pathway. Patients with OTULIN deficiency, termed as otulipenia or OTULIN-related autoinflammatory syndrome, present with early onset severe systemic inflammation due to increased NF-κB activation. We aimed to investigate additional disease mechanisms of OTULIN deficiency. Our study found a remarkable activation of type I interferon (IFN-I) signaling in whole blood, peripheral blood mononuclear cells, monocytes, and serum from patients with OTULIN deficiency. We observed similar immunologic findings in OTULIN-deficient cell lines generated by CRISPR. Mechanistically, we identified proteasome subunits as substrates of OTULIN deubiquitinase activity and demonstrated proteasome dysregulation in OTULIN-deficient cells as the cause of IFN-I activation. These results reveal an important role of linear ubiquitination in the regulation of proteasome function and suggest a link in the pathogenesis of proteasome-associated autoinflammatory syndromes and OTULIN deficiency.

Immune responses require a delicate balance: a weak response can cause immunodeficiency, whereas an excessive response can lead to hyperinflammatory disease and hematological malignancy. Because spleen tyrosine kinase has roles in multiple signaling pathways, its gain-of-function alterations in humans cause hypogammaglobulinemia as well as autoinflammation and predisposition to B cell lymphoma.

Dominantly inherited PSTPIP1 mutations cause a spectrum of autoinflammatory manifestations epitomized by PAPA syndrome (pyogenic sterile arthritis, pyoderma gangrenosum, and acne (PAPA) syndrome.). The connections between PSTPIP1 and PAPA syndrome are poorly understood, although evidence suggests involvement of pyrin inflammasome activation. Interleukin-18 (IL-18) is an inflammasome-activated cytokine associated with susceptibility to macrophage activation syndrome (MAS). This study was undertaken to investigate an association of IL-18 with PAPA syndrome.Clinical and genetic data and serum samples were obtained from patients referred to institutions due to symptoms indicative of PAPA syndrome. Serum IL-18, IL-18 binding protein (IL-18BP), and CXCL9 levels were assessed by bead-based assay, and free IL-18 levels were assessed by enzyme-linked immunosorbent assay.The symptoms of PSTPIP1-positive patients with PAPA syndrome overlapped with those of mutation-negative patients with PAPA-like conditions, but mutation-positive patients had earlier onset and a greater proportion had a history of arthritis. We found uniform elevation of total serum IL-18 in treated PAPA syndrome patients at levels nearly as high as those seen in NLRC4-associated autoinflammation with infantile enterocolitis patients, and well above levels found in most familial Mediterranean fever patients. Serum IL-18 elevation in PAPA syndrome patients persisted despite fluctuations in disease activity. Levels of the soluble IL-18 antagonist IL-18BP were modestly elevated, and PAPA syndrome patients had detectable free IL-18. PAPA syndrome was rarely associated with elevation of CXCL9, an indicator of interferon-γ activity, but no PAPA syndrome patients had a history of MAS.PAPA syndrome is a refractory and often disabling monogenic autoinflammatory disease associated with chronic and unopposed elevation of serum IL-18 levels but not with risk of MAS. These findings affect our understanding of the diseases in which IL-18 is overproduced and suggest a link between pyrin inflammasome activation, IL-18, and autoinflammation, without susceptibility to MAS.

Activation of RIPK1 controls TNF-mediated apoptosis, necroptosis and inflammatory pathways1. Cleavage of human and mouse RIPK1 after residues D324 and D325, respectively, by caspase-8 separates the RIPK1 kinase domain from the intermediate and death domains. The D325A mutation in mouse RIPK1 leads to embryonic lethality during mouse development2,3. However, the functional importance of blocking caspase-8-mediated cleavage of RIPK1 on RIPK1 activation in humans is unknown. Here we identify two families with variants in RIPK1 (D324V and D324H) that lead to distinct symptoms of recurrent fevers and lymphadenopathy in an autosomal-dominant manner. Impaired cleavage of RIPK1 D324 variants by caspase-8 sensitized patients' peripheral blood mononuclear cells to RIPK1 activation, apoptosis and necroptosis induced by TNF. The patients showed strong RIPK1-dependent activation of inflammatory signalling pathways and overproduction of inflammatory cytokines and chemokines compared with unaffected controls. Furthermore, we show that expression of the RIPK1 mutants D325V or D325H in mouse embryonic fibroblasts confers not only increased sensitivity to RIPK1 activation-mediated apoptosis and necroptosis, but also induction of pro-inflammatory cytokines such as IL-6 and TNF. By contrast, patient-derived fibroblasts showed reduced expression of RIPK1 and downregulated production of reactive oxygen species, resulting in resistance to necroptosis and ferroptosis. Together, these data suggest that human non-cleavable RIPK1 variants promote activation of RIPK1, and lead to an autoinflammatory disease characterized by hypersensitivity to apoptosis and necroptosis and increased inflammatory response in peripheral blood mononuclear cells, as well as a compensatory mechanism to protect against several pro-death stimuli in fibroblasts.

Receptor Interacting Protein Kinase 1 (RIPK1) is a key regulator of innate immune signalling pathways. To ensure an optimal inflammatory response, RIPK1 is post-translationally regulated by well characterised ubiquitylation and phosphorylation events, as well as caspase-8 mediated cleavage1–7. The physiological relevance of this cleavage remains unclear, though it is believed to inhibit activation of RIPK3 and necroptosis8. Here we show that heterozygous missense mutations p.D324N, p.D324H and p.D324Y prevent caspase cleavage of RIPK1 in humans and result in early-onset periodic fever episodes and severe intermittent lymphadenopathy, a condition we designate ‘Cleavage-resistant RIPK1-Induced Autoinflammatory’ (CRIA) syndrome. To define the mechanism for this disease we generated a cleavage-resistant Ripk1D325A mutant mouse strain. While Ripk1-/- mice die postnatally from systemic inflammation, Ripk1D325A/D325A mice died during embryogenesis. Embryonic lethality was completely prevented by combined loss of Casp8 and Ripk3 but not by loss of Ripk3 or Mlkl alone. Loss of RIPK1 kinase activity also prevented Ripk1D325A/D325A embryonic lethality, however the mice died before weaning from multi organ inflammation in a RIPK3 dependent manner. Consistently, Ripk1D325A/D325A and Ripk1D325A/+ cells were hypersensitive to RIPK3 dependent TNF-induced apoptosis and necroptosis. Heterozygous Ripk1D325A/+ mice were viable and grossly normal, but were hyper-responsive to inflammatory stimuli in vivo. Our results demonstrate the importance of caspase-mediated RIPK1 cleavage during embryonic development and show that caspase cleavage of RIPK1 not only inhibits necroptosis but maintains inflammatory homeostasis throughout life.

Background Biological agents have allowed remarkable improvement in controlling autoimmune arthropathies, although none of the numerous biologics readily available represent a universal treatment standard. Moreover, classical and genetic predictors are currently unsatisfactory to predict individual response to a biologic, and the best treatment selection is still based on a trial-and-error approach. Here, we report a clinical case demonstrating the usefulness of examining the leukocytes’ secretome of patients. We set up and standardized a protocol that examines a patient’s immune responses to establish the secretome of the blood mononuclear leukocytes and personalize the biotherapy. Case presentation A 24-year-old woman was diagnosed with active early rheumatoid arthritis. The initial treatment regimen (prednisone, methotrexate, hydroxychloroquine, naproxen) was inefficient, as well as the anti-TNF adalimumab. The diagnosis was revised as possible rheumatoid arthritis-like psoriatic arthritis and adalimumab was replaced by abatacept (IgG1 Fc-CTLA-4) to no avail. Five years later, abatacept was replaced by the anti-IL-12/IL-23 ustekinumab with no objective control over the symptoms. The patient was thus enrolled in a prospective study based on the quantification of cytokines secreted by peripheral blood leukocytes stimulated with well-known immune activators of pattern recognition receptors and cytokine signalling. The results of this study revealed that plasma concentrations of cytokines were similar between the patient and healthy donors. In comparison to leukocytes from healthy donors, the patient’s secretome showed a unique overproduction of IL-6. The anti-IL-6 receptor tocilizumab was, therefore, administered with a rapid improvement of her active psoriatic arthritis that remained dependent on low prednisone dosage. Clinical parameters progressively returned to normal levels and her quality of life was greatly improved, despite the major delay to begin the present personalized treatment. Conclusions An efficient way to effectively treat patients with complex autoimmune arthropathies, and avoid irreversible disability, is to know their leukocytes’ secretome to identify abnormally secreted cytokines and personalize their biotherapy, as exemplified by this case report.