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Missense mutations in PLCG2 can cause autoinflammation with phospholipase C gamma 2-associated antibody deficiency and immune dysregulation (APLAID). Here, we generated a mouse model carrying an APLAID mutation (p.Ser707Tyr) and found that inflammatory infiltrates in the skin and lungs were only partially ameliorated by removing inflammasome function via the deletion of caspase-1. Also, deleting interleukin-6 or tumor necrosis factor did not fully prevent APLAID mutant mice from autoinflammation. Overall, these findings are in accordance with the poor response individuals with APLAID have to treatments that block interleukin-1, JAK1/2 or tumor necrosis factor. Cytokine analysis revealed increased granulocyte colony-stimulating factor (G-CSF) levels as the most distinct feature in mice and individuals with APLAID. Remarkably, treatment with a G-CSF antibody completely reversed established disease in APLAID mice. Furthermore, excessive myelopoiesis was normalized and lymphocyte numbers rebounded. APLAID mice were also fully rescued by bone marrow transplantation from healthy donors, associated with reduced G-CSF production, predominantly from non-hematopoietic cells. In summary, we identify APLAID as a G-CSF-driven autoinflammatory disease, for which targeted therapy is feasible.

Coatomer complex I (COPI) mediates retrograde vesicular trafficking from Golgi to the endoplasmic reticulum (ER) and within Golgi compartments. Deficiency in subunit alpha causes COPA syndrome and is associated with type I IFN signalling, although the upstream innate immune sensor involved was unknown. Using in vitro models we find aberrant activation of the STING pathway due to deficient retrograde but probably not intra-Golgi transport. Further we find the upstream cytosolic DNA sensor cGAS as essentially required to drive type I IFN signalling. Genetic deletion of COPI subunits COPG1 or COPD similarly induces type I IFN activation in vitro, which suggests that inflammatory diseases associated with mutations in other COPI subunit genes may exist. Finally, we demonstrate that inflammation in COPA syndrome patient peripheral blood mononuclear cells and COPI-deficient cell lines is ameliorated by treatment with the small molecule STING inhibitor H-151, suggesting targeted inhibition of the cGAS/STING pathway as a promising therapeutic approach.

The inflammasome sensor NLRP1 (nucleotide-binding oligomerization domain-like receptor containing a pyrin domain 1) detects a variety of pathogen-derived molecular patterns to induce an inflammatory immune response by triggering pyroptosis and cytokine release. A number of mutations and polymorphisms of NLRP1 are known to cause autoinflammatory diseases, the functional characterization of which contributes to a better understanding of NLRP1 regulation. Here, we assessed the effect of the common NLRP1 variant M1184V, associated with asthma, inflammatory bowel disease, and diabetes, on the protein level. Our size-exclusion chromatography experiments show that M1184V stabilizes the "function-to-find" domain (FIIND) in a monomeric conformation. This effect is independent of autoproteolysis. In addition, molecular dynamics simulations reveal that the methionine residue increases flexibility within the ZU5 domain, whereas valine decreases flexibility, potentially indirectly stabilizing the catalytic triad responsible for autocleavage. By keeping the FIIND domain monomeric, formation of a multimer of full-length NLRP1 is promoted. We found that the stabilizing effect of the valine further leads to improved dipeptidyl peptidase 9 (DPP9)-binding capacities for the FIIND domain as well as the full-length protein as determined by surface plasmon resonance. Moreover, our immunoprecipitation experiments confirmed increased DPP9 binding for the M1184V protein in cells, consistent with improved formation of an autoinhibited complex with DPP9 in activity assays. Collectively, our study establishes a molecular rationale for the dichotomous involvement of the NLRP1 variant M1184V in autoimmune syndromes.

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The immune system enables organisms to combat infections and to eliminate endogenous challenges. Immune responses can be evoked through diverse inducible pathways. However, various constitutive mechanisms are also required for immunocompetence. The inducible responses of pattern recognition receptors of the innate immune system and antigen-specific receptors of the adaptive immune system are highly effective, but they also have the potential to cause extensive immunopathology and tissue damage, as seen in many infectious and autoinflammatory diseases. By contrast, constitutive innate immune mechanisms, including restriction factors, basal autophagy and proteasomal degradation, tend to limit immune responses, with loss-of-function mutations in these pathways leading to inflammation. Although they function through a broad and heterogeneous set of mechanisms, the constitutive immune responses all function as early barriers to infection and aim to minimize any disruption of homeostasis. Supported by recent human and mouse data, in this Review we compare and contrast the inducible and constitutive mechanisms of immunosurveillance.

BACKGROUND: NLRP1 is an innate immune sensor that can form cytoplasmic inflammasome complexes. Polymorphisms in NLRP1 are linked to asthma; however, there is currently no functional or mechanistic explanation for this. OBJECTIVE: We sought to clarify the role of NLRP1 in asthma pathogenesis. METHODS: Results from the GALA II cohort study were used to identify a link between NLRP1 and asthma in Mexican Americans. In vitro and in vivo models for NLRP1 activation were applied to investigate the role of this inflammasome in asthma at the molecular level. RESULTS: We document the association of an NLRP1 haplotype with asthma for which the single nucleotide polymorphism rs11651270 (M1184V) individually is the most significant. Surprisingly, M1184V increases NLRP1 activation in the context of N-terminal destabilization, but decreases NLRP1 activation on dipeptidyl peptidase 9 inhibition. In vitro studies demonstrate that M1184V increases binding to dipeptidyl peptidase 9, which can account for its inhibitory role in this context. In addition, in vivo data from a mouse model of airway inflammation reveal a protective role for NLRP1 inflammasome activation reducing eosinophilia in this setting. CONCLUSIONS: Linking our in vitro and in vivo results, we found that the NLRP1 variant M1184V reduces inflammasome activation in the context of dipeptidyl peptidase 9 inhibition and could thereby increase asthma severity. Our studies may have implications for the treatment of asthma in patients carrying this variant of NLRP1.

Small extracellular vesicles (SEVs) such as exosomes are released by multiple cell types. Originally believed to be a mechanism for selectively removing unwanted cellular components, SEVs have received increased attention in recent years for their ability to mediate intercellular communication. Apart from proteins and lipids, SEVs contain RNAs, but how RNAs are selectively loaded into SEVs remains poorly understood. To address this question, we profiled SEV RNAs from mouse dendritic cells using RNA-Seq and identified a long noncoding RNA of retroviral origin, VL30, which is highly enriched (>200-fold) in SEVs compared to parental cells. Bioinformatic analysis revealed that exosome-enriched isoforms of VL30 RNA contain a repetitive 26-nucleotide motif. This repeated motif is itself efficiently incorporated into SEVs, suggesting the likelihood that it directly promotes SEV loading. RNA folding analyses indicate that the motif is likely to form a long double-stranded RNA hairpin and, consistent with this, its overexpression was associated with induction of a potent type I interferon response. Taken together, we propose that preferential loading into SEVs of the VL30 RNA containing this immunostimulatory motif enables cells to remove a potentially toxic RNA and avoid autoinflammation. In this way, the original notion of SEVs as a cellular garbage bin should not be entirely discounted.

The signalling receptor for LPS, CD14, is a key marker of, and facilitator for, pro-inflammatory macrophage function. Pro-inflammatory macrophage differentiation remains a process facilitating a broad array of disease pathologies, and has recently emerged as a potential target against cytokine storm in COVID19. Here, we perform a whole-genome CRISPR screen to identify essential nodes regulating CD14 expression in myeloid cells, using the differentiation of THP-1 cells as a starting point. This strategy uncovers many known pathways required for CD14 expression and regulating macrophage differentiation while additionally providing a list of novel targets either promoting or limiting this process. To speed translation of these results, we have then taken the approach of independently validating hits from the screen using well-curated small molecules. In this manner, we identify pharmacologically tractable hits that can either increase CD14 expression on non-differentiated monocytes or prevent CD14 upregulation during macrophage differentiation. An inhibitor for one of these targets, MAP2K3, translates through to studies on primary human monocytes, where it prevents upregulation of CD14 following M-CSF induced differentiation, and pro-inflammatory cytokine production in response to LPS. Therefore, this screening cascade has rapidly identified pharmacologically tractable nodes regulating a critical disease-relevant process.

RIPK1 is a key regulator of innate immune signalling pathways. To ensure an optimal inflammatory response, RIPK1 is regulated post-translationally by well-characterized ubiquitylation and phosphorylation events, as well as by caspase-8-mediated cleavage1-7. The physiological relevance of this cleavage event remains unclear, although it is thought to inhibit activation of RIPK3 and necroptosis8. Here we show that the heterozygous missense mutations D324N, D324H and D324Y prevent caspase cleavage of RIPK1 in humans and result in an early-onset periodic fever syndrome and severe intermittent lymphadenopathy-a condition we term 'cleavage-resistant RIPK1-induced autoinflammatory syndrome'. To define the mechanism for this disease, we generated a cleavage-resistant Ripk1D325A mutant mouse strain. Whereas Ripk1-/- mice died postnatally from systemic inflammation, Ripk1D325A/D325A mice died during embryogenesis. Embryonic lethality was completely prevented by the 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, although 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 also maintains inflammatory homeostasis throughout life.

MLKL is the essential effector of necroptosis, a form of programmed lytic cell death. We have isolated a mouse strain with a single missense mutation, MlklD139V, that alters the two-helix 'brace' that connects the killer four-helix bundle and regulatory pseudokinase domains. This confers constitutive, RIPK3 independent killing activity to MLKL. Homozygous mutant mice develop lethal postnatal inflammation of the salivary glands and mediastinum. The normal embryonic development of MlklD139V homozygotes until birth, and the absence of any overt phenotype in heterozygotes provides important in vivo precedent for the capacity of cells to clear activated MLKL. These observations offer an important insight into the potential disease-modulating roles of three common human MLKL polymorphisms that encode amino acid substitutions within or adjacent to the brace region. Compound heterozygosity of these variants is found at up to 12-fold the expected frequency in patients that suffer from a pediatric autoinflammatory disease, chronic recurrent multifocal osteomyelitis (CRMO).

Despite increasing recognition of the importance of GM-CSF in autoimmune disease, it remains unclear how GM-CSF is regulated at sites of tissue inflammation. Using GM-CSF fate reporter mice, we show that synovial NK cells produce GM-CSF in autoantibody-mediated inflammatory arthritis. Synovial NK cells promote a neutrophilic inflammatory cell infiltrate, and persistent arthritis, via GM-CSF production, as deletion of NK cells, or specific ablation of GM-CSF production in NK cells, abrogated disease. Synovial NK cell production of GM-CSF is IL-18-dependent. Furthermore, we show that cytokine-inducible SH2-containing protein (CIS) is crucial in limiting GM-CSF signaling not only during inflammatory arthritis but also in experimental allergic encephalomyelitis (EAE), a murine model of multiple sclerosis. Thus, a cellular cascade of synovial macrophages, NK cells, and neutrophils mediates persistent joint inflammation via production of IL-18 and GM-CSF. Endogenous CIS provides a key brake on signaling through the GM-CSF receptor. These findings shed new light on GM-CSF biology in sterile tissue inflammation and identify several potential therapeutic targets.

Activation of cyclic GMP-AMP (cGAMP) synthase (cGAS) plays a critical role in antiviral responses to many DNA viruses. Sensing of cytosolic DNA by cGAS results in synthesis of the endogenous second messenger cGAMP that activates stimulator of interferon genes (STING) in infected cells. Critically, cGAMP can also propagate antiviral responses to uninfected cells through intercellular transfer, although the modalities of this transfer between epithelial and immune cells remain poorly defined. We demonstrate here that cGAMP-producing epithelial cells can transactivate STING in cocultured macrophages through direct cGAMP transfer. cGAMP transfer was reliant upon connexin expression by epithelial cells and pharmacological inhibition of connexins blunted STING-dependent transactivation of the macrophage compartment. Macrophage transactivation by cGAMP contributed to a positive-feedback loop amplifying antiviral responses, significantly protecting uninfected epithelial cells against viral infection. Collectively, our findings constitute the first direct evidence of a connexin-dependent cGAMP transfer to macrophages by epithelial cells, to amplify antiviral responses.IMPORTANCE Recent studies suggest that extracellular cGAMP can be taken up by macrophages to engage STING through several mechanisms. Our work demonstrates that connexin-dependent communication between epithelial cells and macrophages plays a significant role in the amplification of antiviral responses mediated by cGAMP and suggests that pharmacological strategies aimed at modulating connexins may have therapeutic applications to control antiviral responses in humans.

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Results

Conclusions

Monogenic autoinflammatory disorders are an increasingly heterogeneous group of conditions characterised by innate immune dysregulation. Improved genetic sequencing in recent years has led not only to the discovery of a plethora of conditions considered to be 'autoinflammatory', but also the broadening of the clinical and immunological phenotypic spectra seen in these disorders. This review outlines the classification strategies that have been employed for monogenic autoinflammatory disorders to date, including the primary innate immune pathway or the dominant cytokine implicated in disease pathogenesis, and highlights some of the advantages of these models. Furthermore, the use of the term 'autoinflammatory' is discussed in relation to disorders that cross the innate and adaptive immune divide. The utilisation of next-generation sequencing (NGS) in this population is examined, as are potential in vivo and in vitro methods of modelling to determine pathogenicity of novel genetic findings. Finally, areas where our understanding can be improved are highlighted, such as phenotypic variability and genotype-phenotype correlations, with the aim of identifying areas of future research.

BACKGROUND: While exome and targeted next-generation DNA sequencing are primarily used for detecting single nucleotide changes and small indels, detection of copy number variants (CNVs) can provide highly valuable additional information from the data. Although there are dozens of exome CNV detection methods available, these are often difficult to use, and accuracy varies unpredictably between and within datasets. FINDINGS: We present Ximmer, a tool that supports an end-to-end process for evaluating, tuning, and running analysis methods for detection of CNVs in germline samples. Ximmer includes a simulation framework, implementations of several commonly used CNV detection methods, and a visualization and curation tool that together enable interactive exploration and quality control of CNV results. Using Ximmer, we comprehensively evaluate CNV detection on four datasets using five different detection methods. We show that application of Ximmer can improve accuracy and aid in quality control of CNV detection results. In addition, Ximmer can be used to run analyses and explore CNV results in exome data. CONCLUSIONS: Ximmer offers a comprehensive tool and method for applying and improving accuracy of CNV detection methods for exome data.

The human pathogen Helicobacter pylori acquires cholesterol from membrane raft domains in eukaryotic cells, commonly known as "lipid rafts." Incorporation of this cholesterol into the H. pylori cell membrane allows the bacterium to avoid clearance by the host immune system and to resist the effects of antibiotics and antimicrobial peptides. The presence of cholesterol in H. pylori bacteria suggested that this pathogen may have cholesterol-enriched domains within its membrane. Consistent with this suggestion, we identified a hypothetical H. pylori protein (HP0248) with homology to the flotillin proteins normally found in the cholesterol-enriched domains of eukaryotic cells. As shown for eukaryotic flotillin proteins, HP0248 was detected in detergent-resistant membrane fractions of H. pylori. Importantly, H. pylori HP0248 mutants contained lower levels of cholesterol than wild-type bacteria (P < 0.01). HP0248 mutant bacteria also exhibited defects in type IV secretion functions, as indicated by reduced IL-8 responses and CagA translocation in epithelial cells (P < 0.05), and were less able to establish a chronic infection in mice than wild-type bacteria (P < 0.05). Thus, we have identified an H. pylori flotillin protein and shown its importance for bacterial virulence. Taken together, the data demonstrate important roles for H. pylori flotillin in host-pathogen interactions. We propose that H. pylori flotillin may be required for the organization of virulence proteins into membrane raft-like structures in this pathogen.

The human pathogen Legionella pneumophila must evade host cell death signaling to enable replication in lung macrophages and to cause disease. After bacterial growth, however, L. pneumophila is thought to induce apoptosis during egress from macrophages. The bacterial effector protein, SidF, has been shown to control host cell survival and death by inhibiting pro-apoptotic BNIP3 and BCL-RAMBO signaling. Using live-cell imaging to follow the L. pneumophila-macrophage interaction, we now demonstrate that L. pneumophila evades host cell apoptosis independent of SidF. In the absence of SidF, L. pneumophila was able to replicate, cause loss of mitochondria membrane potential, kill macrophages, and establish infections in lungs of mice. Consistent with this, deletion of BNIP3 and BCL-RAMBO did not affect intracellular L. pneumophila replication, macrophage death rates, and in vivo bacterial virulence. Abrogating mitochondrial cell death by genetic deletion of the effectors of intrinsic apoptosis, BAX, and BAK, or the regulator of mitochondrial permeability transition pore formation, cyclophilin-D, did not affect bacterial growth or the initial killing of macrophages. Loss of BAX and BAK only marginally limited the ability of L. pneumophila to efficiently kill all macrophages over extended periods. L. pneumophila induced killing of macrophages was delayed in the absence of capsase-11 mediated pyroptosis. Together, our data demonstrate that L. pneumophila evades host cell death responses independently of SidF during replication and can induce pyroptosis to kill macrophages in a timely manner.

Background: Epidemiological studies suggest that compared with the general population, mood disorders are up to 4.7 times more prevalent in substance dependent samples. Comorbid substance use disorder (SUD) and depression has been associated with a more severe and protracted illness course and poorer treatment outcomes. Despite this, the development and assessment of behavioural interventions for treating depression among individuals with SUDs have received little empirical attention. Behavioural Activation Treatment for Depression (BATD-R) is an empirically supported treatment for depression that has shown some efficacy among substance users. This paper describes the study protocol of a parallel, single blind, randomised controlled trial to determine the efficacy and feasibility of a modified version of the BATD-R (Activate) in reducing symptoms of depression and substance dependence among individuals in residential rehabilitation (RR) and opioid substitution therapy (OST). Methods/design: A sample of approximately 200 individuals with depressive symptomatology in treatment for SUD will be recruited from RR and OST services in New South Wales, Australia. Dynamic random allocation following minimisation methodology will be used to assign participants to one of two groups. The control group will receive treatment as usual (TAU), which will be the model of care provided in accordance with standard practice at participating RR and OST services. The intervention group will receive Activate, comprising 10 individual 60-min therapy sessions with a psychologist employed on the research team, in addition to TAU. Data collection will occur at baseline (pre-intervention), and 3-months and 12-months post baseline. Discussion: The association between depression and substance dependence has been well documented, yet practical and effective treatments are scarce. The findings of the present study will contribute significantly to understanding the types of programs that are effect

Inflammatory bowel disease (IBD) is an immunoregulatory disorder, associated with a chronic and inappropriate mucosal immune response to commensal bacteria, underlying disease states such as ulcerative colitis (UC) and Crohn's disease (CD) in humans. Granzyme M (GrzM) is a serine protease expressed by cytotoxic lymphocytes, in particular natural killer (NK) cells. Granzymes are thought to be involved in triggering cell death in eukaryotic target cells; however, some evidence supports their role in inflammation. The role of GrzM in the innate immune response to mucosal inflammation has never been examined. Here, we discover that patients with UC, unlike patients with CD, display high levels of GrzM mRNA expression in the inflamed colon. By taking advantage of well-established models of experimental UC, we revealed that GrzM-deficient mice have greater levels of inflammatory indicators during dextran sulfate sodium (DSS)-induced IBD, including increased weight loss, greater colon length reduction and more severe intestinal histopathology. The absence of GrzM expression also had effects on gut permeability, tissue cytokine/chemokine dynamics, and neutrophil infiltration during disease. These findings demonstrate, for the first time, that GrzM has a critical role during early stages of inflammation in UC, and that in its absence colonic inflammation is enhanced.

Plasmacytoid dendritic cells (pDCs) play an important role in immunity to certain pathogens and immunopathology in some autoimmune diseases. They are thought to have a longer lifespan than conventional DCs (cDCs), largely based on a slower rate of BrdU labeling by splenic pDCs. Here we demonstrated that pDC expansion and therefore BrdU labeling by pDCs occurs in bone marrow (BM). The rate of labeling was similar between BM pDCs and spleen cDCs. Therefore, slower BrdU labeling of spleen pDCs likely reflects the "migration time" (∼2 days) for BrdU labeled pDCs to traffic to the spleen, not necessarily reflecting longer life span. Tracking the decay of differentiated DCs showed that splenic pDCs and cDCs decayed at a similar rate. We suggest that spleen pDCs have a shorter in vivo lifespan than estimated utilizing some of the previous approaches. Nevertheless, pDC lifespan varies between mouse strains. pDCs from lupus-prone NZB mice survived longer than C57BL/6 pDCs. We also demonstrated that activation either positively or negatively impacted on the survival of pDCs via different cell-death mechanisms. Thus, pDCs are also short-lived. However, the pDC lifespan is regulated by genetic and environmental factors that may have pathological consequence.

BACKGROUND: Signals of danger and damage in the cytosol of cells are sensed by NOD-like receptors (NLRs), which are components of multiprotein complexes called inflammasomes. Inflammasomes activate caspase-1, resulting in IL-1-beta and IL-18 secretion and an inflammatory response. To date, the only known activator of rodent Nlrp1 is anthrax lethal toxin (LT), a protease secreted by the bacterial pathogen Bacillus anthracis. Although susceptibility of mouse macrophages to LT has been genetically linked to Nlrp1b, mice harbor two additional Nlrp1 paralogs in their genomes (Nlrp1a and Nlrp1c). However, little is known about their expression profile and sequence in different mouse strains. Furthermore, simultaneous expression of these paralogs may lead to competitional binding of Nlrp1b interaction partners needed for inflammasome activation, thus influencing macrophages susceptibility to LT. To more completely understand the role(s) of Nlrp1 paralogs in mice, we surveyed for their expression in a large set of LT-resistant and sensitive mouse macrophages. In addition, we provide sequence comparisons for Nlrp1a and report on previously unrecognized splice variants of Nlrp1b. RESULTS: Our results show that macrophages from some inbred mouse strains simultaneously express different splice variants of Nlrp1b. In contrast to the highly polymorphic Nlrp1b splice variants, sequencing of expressed Nlrp1a showed the protein to be highly conserved across all mouse strains. We found that Nlrp1a was expressed only in toxin-resistant macrophages, with the sole exception of expression in LT-sensitive CAST/EiJ macrophages. CONCLUSIONS: Our data present a complex picture of Nlrp1 protein variations and provide a basis for elucidating their roles in murine macrophage function. Furthermore, the high conservation of Nlrp1a implies that it might be an important inflammasome sensor in mice.

Pancreatic beta-cell apoptosis is an important feature of islets in type 2 diabetes. Apoptosis can occur through two major pathways, the extrinsic or death receptor mediated pathway, and the intrinsic or Bcl-2-regulated pathway. Hyperglycaemia, hyperlipidaemia and islet amyloid poly-peptide (IAPP) represent important possible causes of increased beta-cell apoptosis. Hyperglycaemia induces islet-cell apoptosis by the intrinsic pathway involving molecules of the Bcl-2 family. High concentrations of palmitate also activate intrinsic apoptosis in islets cells. IAPP oligomers can induce apoptosis by both intrinsic and extrinsic pathways. IL-1b produced through NLRP3 inflammasome activation can also induce islet cell death. Activation of the NLRP3 inflammasome may not be important for glucose or palmitate induced apoptosis in islets but may be important for IAPP mediated cell death. Endoplasmic reticulum (ER) and oxidative stress have been observed in beta cells in type 2 diabetes, and these could be the link between upstream metabolic abnormalities and downstream apoptotic machinery.

Inducible nitric oxide (NO) synthase (iNOS; NOS2) produces NO and related reactive nitrogen species, which are critical effectors of the innate host response and are required for the intracellular killing of pathogens such as Mycobacterium tuberculosis and Leishmania major. We have identified SPRY domain-containing SOCS (suppressor of cytokine signaling) box protein 2 (SPSB2) as a novel negative regulator that recruits an E3 ubiquitin ligase complex to polyubiquitinate iNOS, resulting in its proteasomal degradation. SPSB2 interacts with the N-terminal region of iNOS via a binding interface on SPSB2 that has been mapped by nuclear magnetic resonance spectroscopy and mutational analyses. SPSB2-deficient macrophages showed prolonged iNOS expression, resulting in a corresponding increase in NO production and enhanced killing of L. major parasites. These results lay the foundation for the development of small molecule inhibitors that could disrupt the SPSB-iNOS interaction and thus prolong the intracellular lifetime of iNOS, which may be beneficial in chronic and persistent infections.

PSTPIP1 is a cytoskeleton-associated adaptor protein that links PEST-type phosphatases to their substrates. Mutations in PSTPIP1 cause PAPA syndrome (Pyogenic sterile Arthritis, Pyoderma gangrenosum, and Acne), an autoinflammatory disease. PSTPIP1 binds to pyrin and mutations in pyrin result in familial Mediterranean fever (FMF), a related autoinflammatory disorder. Since disease-associated mutations in PSTPIP1 enhance pyrin binding, PAPA syndrome and FMF are thought to share a common pathoetiology. The studies outlined here describe several new aspects of PSTPIP1 and pyrin biology. We document that PSTPIP1, which has homology to membrane-deforming BAR proteins, forms homodimers and generates membrane-associated filaments in native and transfected cells. An extended FCH (Fes-Cip4 homology) domain in PSTPIP1 is necessary and sufficient for its self-aggregation. We further show that the PSTPIP1 filament network is dependent upon an intact tubulin cytoskeleton and that the distribution of this network can be modulated by pyrin, indicating that this is a dynamic structure. Finally, we demonstrate that pyrin can recruit PSTPIP1 into aggregations (specks) of ASC, another pyrin binding protein. ASC specks are associated with inflammasome activity. PSTPIP1 molecules with PAPA-associated mutations are recruited by pyrin to ASC specks with particularly high efficiency, suggesting a unique mechanism underlying the robust inflammatory phenotype of PAPA syndrome.