Fiona Moghaddas

Familial autoinflammation with neutrophilic dermatosis reveals a regulatory mechanism of pyrin activation

A mutation in pyrin that disrupts regulation leads to autoinflammatory disease. Guarding inflammation The innate immune system is hard-wired to protect people from infection. However, mutations in these protective genes can lead to uncontrolled inflammation, resulting in autoinflammatory disease. Now, Masters et al. describe a family with an autoinflammatory disease caused by a previously unreported mutation in pyrin. This mutation disrupts pyrin regulation and mimics the effect of pathogen sensing by pyrin, leading to proinflammatory interleukin-1β (IL-1β) production. Indeed, targeting IL-1β resolved disease in one patient. These data suggest that pyrin is regulated through a guard-like mechanism, which guards against autoinflammation in humans. Pyrin responds to pathogen signals and loss of cellular homeostasis by forming an inflammasome complex that drives the cleavage and secretion of interleukin-1β (IL-1β). Mutations in the B30.2/SPRY domain cause pathogen-independent activation of pyrin and are responsible for the autoinflammatory disease familial Mediterranean fever (FMF). We studied a family with a dominantly inherited autoinflammatory disease, distinct from FMF, characterized by childhood-onset recurrent episodes of neutrophilic dermatosis, fever, elevated acute-phase reactants, arthralgia, and myalgia/myositis. The disease was caused by a mutation in MEFV, the gene encoding pyrin (S242R). The mutation results in the loss of a 14-3-3 binding motif at phosphorylated S242, which was not perturbed by FMF mutations in the B30.2/SPRY domain. However, loss of both S242 phosphorylation and 14-3-3 binding was observed for bacterial effectors that activate the pyrin inflammasome, such as Clostridium difficile toxin B (TcdB). The S242R mutation thus recapitulated the effect of pathogen sensing, triggering inflammasome activation and IL-1β production. Successful therapy targeting IL-1β has been initiated in one patient, resolving pyrin-associated autoinflammation with neutrophilic dermatosis. This disease provides evidence that a guard-like mechanism of pyrin regulation, originally identified for Nod-like receptors in plant innate immunity, also exists in humans.

Monogenic autoinflammatory diseases: Cytokinopathies.

Rapid advances in genetics are providing unprecedented insight into functions of the innate immune system with identification of the mutations that cause monogenic autoinflammatory disease. Cytokine antagonism is profoundly effective in a subset of these conditions, particularly those associated with increased interleukin-1 (IL-1) activity, the inflammasomopathies. These include syndromes where the production of IL-1 is increased by mutation of innate immune sensors such as NLRP3, upstream signalling molecules such as PSTPIP1 and receptors or downstream signalling molecules, such as IL-1Ra. Another example of this is interferon (IFN) and the interferonopathies, with mutations in the sensors STING and MDA5, the upstream signalling regulator AP1S3, and a downstream inhibitor of IFN signalling, ISG15. We propose that this can be extended to cytokines such as IL-36, with mutations in IL-36Ra, and IL-10, with mutations in IL-10RA and IL-10RB, however mutations in sensors or upstream signalling molecules are yet to be described in these instances. Additionally, autoinflammatory diseases can be caused by multiple cytokines, for example with the activation of NF-κB/Rel, for which we propose the term Relopathies. This nosology is limited in that some cytokine pathways may be degenerate in their generation or execution, however provides insight into likely autoinflammatory disease candidates and the cytokines with which newly identified mutations may be associated, and therefore targeted.

A novel Pyrin-Associated Autoinflammation with Neutrophilic Dermatosis mutation further defines 14-3-3 binding of pyrin and distinction to Familial Mediterranean Fever

Objective Pyrin-Associated Autoinflammation with Neutrophilic Dermatosis (PAAND) is a recently described monogenic autoinflammatory disease. The causal p.S242R MEFV mutation disrupts a binding motif of the regulatory 14-3-3 proteins within pyrin. Here, we investigate a family with clinical features consistent with PAAND in whom the novel p.E244K MEFV mutation, located in the +2 site of the 14-3-3 binding motif in pyrin, has been found. Methods Multiplex cytokine analyses were performed on p.E244K patient and control serum. Peripheral blood mononuclear cells were stimulated ex vivo with lipopolysaccharide (LPS). In vitro, inflammasome complex formation was evaluated by flow cytometry of Apoptosis-associated Speck-like protein containing a Caspase recruitment domain (ASC) specks. Interleukin-1β (IL-1β) and IL-18 production was quantified by ELISA. The ability of the p.E244K pyrin mutation to interact with 14-3-3 was assessed by immunoprecipitation. Results PAAND p.E244K patient serum displayed a different cytokine profile compared with patients with Familial Mediterranean Fever (FMF). In overexpression models, p.E244K pyrin was associated with decreased 14-3-3 binding and increased ASC speck formation. THP-1 monocytes expressing PAAND pyrin mutations demonstrated spontaneous caspase-1-dependent IL-1β and IL-18 secretion, as well as cell death, which were significantly greater than those of wild-type and the FMF-associated mutation p.M694V. Conclusion In PAAND, disruption of the +2 position of a 14-3-3 binding motif in pyrin results in its constitutive activation, with spontaneous production of IL-1β and IL-18, associated with inflammatory cell death. The altered serum cytokine profile may explain the different clinical features exhibited by PAAND patients compared with those with FMF.

Autoinflammatory mutation in NLRC4 reveals a leucine-rich repeat (LRR)–LRR oligomerization interface

Background: Monogenic autoinflammatory disorders are characterized by dysregulation of the innate immune system, for example by gain‐of‐function mutations in inflammasome‐forming proteins, such as NOD‐like receptor family CARD‐containing 4 protein (NLRC4). Objective: Here we investigate the mechanism by which a novel mutation in the leucine‐rich repeat (LRR) domain of NLRC4 (c.G1965C, p.W655C) contributes to autoinflammatory disease. Methods: We studied 2 unrelated patients with early‐onset macrophage activation syndrome harboring the same de novo mutation in NLRC4. In vitro inflammasome complex formation was quantified by using flow cytometric analysis of apoptosis‐associated speck‐like protein containing a caspase recruitment domain (ASC) specks. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 techniques and lentiviral transduction were used to generate THP‐1 cells with either wild‐type or mutant NLRC4 cDNA. Cell death and release of IL‐1&bgr;/IL‐18 were quantified by using flow cytometry and ELISA, respectively. Results: The p.W655C NLRC4 mutation caused increased ASC speck formation, caspase‐1–dependent cell death, and IL‐1&bgr;/IL‐18 production. ASC contributed to p.W655C NLRC4–mediated cytokine release but not cell death. Mutation of p.W655 activated the NLRC4 inflammasome complex by engaging with 2 interfaces on the opposing LRR domain of the oligomer. One key set of residues (p.D1010, p.D1011, p.L1012, and p.I1015) participated in LRR‐LRR oligomerization when triggered by mutant NLRC4 or type 3 secretion system effector (PrgI) stimulation of the NLRC4 inflammasome complex. Conclusion: This is the first report of a mutation in the LRR domain of NLRC4 causing autoinflammatory disease. c.G1965C/p.W655C NLRC4 increased inflammasome activation in vitro. Data generated from various NLRC4 mutations provides evidence that the LRR‐LRR interface has an important and previously unrecognized role in oligomerization of the NLRC4 inflammasome complex. GRAPHICAL ABSTRACT Figure. No caption available.

A Mutation Outside the Dimerization Domain Causing Atypical STING-Associated Vasculopathy With Onset in Infancy

Background Mutations in the gene encoding stimulator of interferon genes (STING) underlie a type I interferon (IFN) associated disease, STING-associated vasculopathy with onset in infancy (SAVI). Patients suffer cutaneous vasculopathy and interstitial lung disease, but are not known to suffer life-threatening infection. Case We describe a child who presented with Pneumocystis jirovecii pneumonia in early life, from which he recovered. He went on to suffer failure to thrive, developmental delay, livedo reticularis, and vesicular rash, but without cutaneous vasculitis, and with normal C-reactive protein and erythrocyte sedimentation rates. At 3 years of age, he developed life-threatening pulmonary hypertension. Methods Whole genome sequencing (WGS) was performed using the Illumina HiSeqX10 platform and the Seave platform was used for bioinformatic analysis. mRNA expression of IFN-stimulated genes and inflammatory cytokines from peripheral blood mononuclear cells was determined by quantitative polymerase chain reaction. Luciferase assay was used to model IFNβ and NF-κB activity in vitro. Results WGS revealed a de novo mutation p.Arg284Ser in STING at an amino acid previously associated with SAVI. Although this mutation did not fall in the dimerization domain (DD), mRNA analysis revealed constitutive IFN-gene activation consistent with an interferonopathy, which correlated to STING activation in vitro. The patient was treated with corticosteroids and the JAK inhibitor Ruxolitinib, resulting in a rapid improvement of pulmonary hypertension, general well-being, and resolution of the IFN gene signature. However, he did go on to evolve a nasal septal erosion suggesting incomplete control of disease. Conclusion This case provides molecular evidence to support the p.Arg284Ser variant in STING exerting pathogenicity through a gain-of-function mechanism. The lack of cutaneous vasculitis or elevated systemic inflammatory markers, and the occurrence of an opportunistic infection are notable, and raise the possibility that variants outside the STING DD may potentially manifest with an atypical SAVI phenotype. Nevertheless, there was an objective clinical improvement in response to JAK inhibition.

Autoantibodies directed to centromere protein F in a patient with BRCA1 gene mutation.

BackgroundAutoantibodies directed to centromere protein F were first reported in 1993 and their association with malignancy has been well documented.CaseWe present the case of a 48-year-old Caucasian female with a BRCA1 gene mutation associated with bilateral breast cancer. Antinuclear autoantibody immunofluorescence performed for workup of possible inflammatory arthropathy showed a high titre cell cycle related nuclear speckled pattern, with subsequent confirmation by addressable laser bead immunoassay of the target antigen as an immunodominant epitope at the C-terminus of centromere protein F.ConclusionHere we review the current literature on centromere protein F, its association with breast cancer and present the first case of this antibody being identified in a person with a BRCA1 gene mutation.

Whole exome sequencing in systemic juvenile idiopathic arthritis.

Systemic juvenile idiopathic arthritis (sJIA) shares clinical features with classic monogenic autoinflammatory diseases, characterised by fevers, arthritis and evanescent rashes. Disease exacerbations are associated with elevated serum cytokine levels including 1L-1β, IL-6, and IL-18; and clinical response to anakinra, canakinumab and tocilizumab suggests that cytokine dysregulation is a key pathophysiological mechanism. Macrophage activation syndrome (MAS) may complicate sJIA, rendering individuals clinically indistinguishable from their familial haemophagocytic lymphohistiocytosis (fHLH) counterparts; with NK and CD8+ cell dysfunction leading to sustained immune cell activation and cytokine storm. Whilst there have been HLA associations and polymorphisms noted on sJIA genome-wide association studies, in rare cases mutations have been found in genes encoding key components of the inflammatory response, which may contribute to disease pathogenesis.

Incidental antinuclear autoantibody to sp100 and gp210 in subjects with normal liver function tests

Aim: Primary biliary cirrhosis (PBC) specific antinuclear antibodies (ANA) are found in about 50% of patients with PBC. They are largely directed to sp100 and gp210 and are associated with ANA immunofluorescence (IIF) staining patterns of multiple nuclear dots (MND) and nuclear pore complex (NPC) respectively. They have not previously been reported in subjects with normal liver function tests (LFTs). We review the incidental incidence of these autoantibodies in our test population and their association with normal and cholestatic LFTs. Method: Retrospective analysis of lineblots performed based on ANA IIF pattern of MND and NPC. LFTs were recorded from date of ANA testing and at one and two years of follow up. Results: 187 lineblots were positive for autoantibody to sp100 and/or gp210 detected on the basis of ANA pattern to MND and NPC. Twenty-nine patients with anti-gp210 antibodies and 51 patients with anti-sp100 antibodies detected incidentally on the basis of ANA pattern remained biochemically and symptomatically stable during a follow up period of one year. Discussion: The population identified here could serve as a basis for long term studies, contributing to our knowledge as the predictive value of these specificities detected incidentally is currently unknown.

Autoantibodies directed to centromere protein F in a patient with BRCA1 gene mutation

Aim: Autoantibodies directed to centromere protein F (CENP-F) were first reported in 1993 and their association with malignancy has been well documented. We present a case of this autoantibody detected in a 47-year-old female with BRCA1 gene mutation associated with bilateral breast cancer and ovarian cancer. Method and results: Antinuclear autoantibody immunofluorescence carried out for possible inflammatory arthropathy showed high titre nuclear speckled-II (NSII) pattern consistent with CENP-F that was confirmed by addressable laser bead immunoassay (ALBIA) for the C-terminal p-F4, an immunodominant CENP-F peptide. Discussion: We review the current literature on CENP-F, its association with breast cancer and present the first documented case of this antibody being identified in a person with BRCA1 gene mutation.