POP1 inhibits MSU-induced inflammasome activation and ameliorates gout.

Canonical inflammasomes are innate immune protein scaffolds that enable the activation of inflammatory caspase-1, and subsequently the processing and release of interleukin (IL)-1ß, IL-18, and danger signals, as well as the induction of pyroptotic cell death. Inflammasome assembly and activation occurs in response to sensing of infectious, sterile and self-derived molecular patterns by cytosolic pattern recognition receptors, including the Nod-like receptor NLRP3. While these responses are essential for host defense, excessive and uncontrolled NLRP3 inflammasome responses cause and contribute to a wide spectrum of inflammatory diseases, including gout. A key step in NLRP3 inflammasome assembly is the sequentially nucleated polymerization of Pyrin domain (PYD)- and caspase recruitment domain (CARD)-containing inflammasome components. NLRP3 triggers polymerization of the adaptor protein ASC through PYD-PYD interactions, but ASC polymerization then proceeds in a self-perpetuating manner and represents a point of no return, which culminates in the activation of caspase-1 by induced proximity. In humans, small PYD-only proteins (POPs) lacking an effector domain regulate this key process through competitive binding, but limited information exists on their physiological role during health and disease. Here we demonstrate that POP1 expression in macrophages is sufficient to dampen MSU crystal-mediated inflammatory responses in animal models of gout. Whether MSU crystals are administered into a subcutaneous airpouch or into the ankle joint, the presence of POP1 significantly reduces neutrophil infiltration. Also, airpouch exudates have much reduced IL-1ß and ASC, which are typical pro-inflammatory indicators that can also be detected in synovial fluids of gout patients. Exogenous expression of POP1 in mouse and human macrophages also blocks MSU crystal-induced NLRP3 inflammasome assembly, resulting in reduced IL-1ß and IL-18 secretion. Conversely, reduced POP1 expression in human macrophages enhances IL-1ß secretion. We further determined that the mechanism for the POP1-mediated inhibition of NLRP3 inflammasome activation is through its interference with the crucial NLRP3 and ASC interaction within the inflammasome complex. Strikingly, administration of an engineered cell permeable version of POP1 was able to ameliorate MSU crystal-mediated inflammation in vivo, as measured by neutrophil infiltration. Overall, we demonstrate that POP1 may play a crucial role in regulating inflammatory responses in gout.

NLRP3 licenses NLRP11 for inflammasome activation in human macrophages.

Intracellular sensing of stress and danger signals initiates inflammatory innate immune responses by triggering inflammasome assembly, caspase-1 activation and pyroptotic cell death as well as the release of interleukin 1ß (IL-1ß), IL-18 and danger signals. NLRP3 broadly senses infectious patterns and sterile danger signals, resulting in the tightly coordinated and regulated assembly of the NLRP3 inflammasome, but the precise mechanisms are incompletely understood. Here, we identified NLRP11 as an essential component of the NLRP3 inflammasome in human macrophages. NLRP11 interacted with NLRP3 and ASC, and deletion of NLRP11 specifically prevented NLRP3 inflammasome activation by preventing inflammasome assembly, NLRP3 and ASC polymerization, caspase-1 activation, pyroptosis and cytokine release but did not affect other inflammasomes. Restored expression of NLRP11, but not NLRP11 lacking the PYRIN domain (PYD), restored inflammasome activation. NLRP11 was also necessary for inflammasome responses driven by NLRP3 mutations that cause cryopyrin-associated periodic syndrome (CAPS). Because NLRP11 is not expressed in mice, our observations emphasize the specific complexity of inflammasome regulation in humans.

Oncostatin M expression induced by bacterial triggers drives airway inflammatory and mucus secretion in severe asthma.

Exacerbations of symptoms represent an unmet need for people with asthma. Bacterial dysbiosis and opportunistic bacterial infections have been observed in, and may contribute to, more severe asthma. However, the molecular mechanisms driving these exacerbations remain unclear. We show here that bacterial lipopolysaccharide (LPS) induces oncostatin M (OSM) and that airway biopsies from patients with severe asthma present with an OSM-driven transcriptional profile. This profile correlates with activation of inflammatory and mucus-producing pathways. Using primary human lung tissue or human epithelial and mesenchymal cells, we demonstrate that OSM is necessary and sufficient to drive pathophysiological features observed in severe asthma after exposure to LPS or Klebsiella pneumoniae. These findings were further supported through blockade of OSM with an OSM-specific antibody. Single-cell RNA sequencing from human lung biopsies identified macrophages as a source of OSM. Additional studies using Osm-deficient murine macrophages demonstrated that macrophage-derived OSM translates LPS signals into asthma-associated pathologies. Together, these data provide rationale for inhibiting OSM to prevent bacterial-associated progression and exacerbation of severe asthma.

Recruitment of pro-IL-1α to mitochondrial cardiolipin, via shared LC3 binding domain, inhibits mitophagy and drives maximal NLRP3 activation.

The balance between NLRP3 inflammasome activation and mitophagy is essential for homeostasis and cellular health, but this relationship remains poorly understood. Here we found that interleukin-1α (IL-1α)-deficient macrophages have reduced caspase-1 activity and diminished IL-1ß release, concurrent with reduced mitochondrial damage, suggesting a role for IL-1α in regulating this balance. LPS priming of macrophages induced pro-IL-1α translocation to mitochondria, where it directly interacted with mitochondrial cardiolipin (CL). Computational modeling revealed a likely CL binding motif in pro-IL-1α, similar to that found in LC3b. Thus, binding of pro-IL-1α to CL in activated macrophages may interrupt CL-LC3b-dependent mitophagy, leading to enhanced Nlrp3 inflammasome activation and more robust IL-1ß production. Mutation of pro-IL-1α residues predicted to be involved in CL binding resulted in reduced pro-IL-1α-CL interaction, a reduction in NLRP3 inflammasome activity, and increased mitophagy. These data identify a function for pro-IL-1α in regulating mitophagy and the potency of NLRP3 inflammasome activation.

The oxidized phospholipid oxPAPC protects from septic shock by targeting the non-canonical inflammasome in macrophages.

Lipopolysaccharide (LPS) of Gram-negative bacteria can elicit a strong immune response. Although extracellular LPS is sensed by TLR4 at the cell surface and triggers a transcriptional response, cytosolic LPS binds and activates non-canonical inflammasome caspases, resulting in pyroptotic cell death, as well as canonical NLRP3 inflammasome-dependent cytokine release. Contrary to the highly regulated multiprotein platform required for caspase-1 activation in the canonical inflammasomes, the non-canonical mouse caspase-11 and the orthologous human caspase-4 function simultaneously as innate sensors and effectors, and their regulation is unclear. Here we show that the oxidized phospholipid 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (oxPAPC) inhibits the non-canonical inflammasome in macrophages, but not in dendritic cells. Aside from a TLR4 antagonistic role, oxPAPC binds directly to caspase-4 and caspase-11, competes with LPS binding, and consequently inhibits LPS-induced pyroptosis, IL-1ß release and septic shock. Therefore, oxPAPC and its derivatives might provide a basis for therapies that target non-canonical inflammasomes during Gram-negative bacterial sepsis.

Identification of an in vivo orally active dual-binding protein-protein interaction inhibitor targeting TNFα through combined in silico/in vitro/in vivo screening.

TNFα is a homotrimeric pro-inflammatory cytokine, whose direct targeting by protein biotherapies has been an undeniable success for the treatment of chronic inflammatory diseases. Despite many efforts, no orally active drug targeting TNFα has been identified so far. In the present work, we identified through combined in silico/in vitro/in vivo approaches a TNFα direct inhibitor, compound 1, displaying nanomolar and micromolar range bindings to TNFα. Compound 1 inhibits the binding of TNFα with both its receptors TNFRI and TNFRII. Compound 1 inhibits the TNFα induced apoptosis on L929 cells and the TNFα induced NF-κB activation in HEK cells. In vivo, oral administration of compound 1 displays a significant protection in a murine TNFα-dependent hepatic shock model. This work illustrates the ability of low-cost combined in silico/in vitro/in vivo screening approaches to identify orally available small-molecules targeting challenging protein-protein interactions such as homotrimeric TNFα.

The PYRIN domain-only protein POP2 inhibits inflammasome priming and activation.

Inflammasomes are protein platforms linking recognition of microbe, pathogen-associated and damage-associated molecular patterns by cytosolic sensory proteins to caspase-1 activation. Caspase-1 promotes pyroptotic cell death and the maturation and secretion of interleukin (IL)-1ß and IL-18, which trigger inflammatory responses to clear infections and initiate wound-healing; however, excessive responses cause inflammatory disease. Inflammasome assembly requires the PYRIN domain (PYD)-containing adaptor ASC, and depends on PYD-PYD interactions. Here we show that the PYD-only protein POP2 inhibits inflammasome assembly by binding to ASC and interfering with the recruitment of ASC to upstream sensors, which prevents caspase-1 activation and cytokine release. POP2 also impairs macrophage priming by inhibiting the activation of non-canonical IκB kinase ɛ and IκBα, and consequently protects from excessive inflammation and acute shock in vivo. Our findings advance our understanding of the complex regulatory mechanisms that maintain a balanced inflammatory response and highlight important differences between individual POP members.

The AIM2 inflammasome is a central regulator of intestinal homeostasis through the IL-18/IL-22/STAT3 pathway.

Inflammasomes are important for maintaining intestinal homeostasis, and dysbiosis contributes to the pathology of inflammatory bowel disease (IBD) and increases the risk for colorectal cancer. Inflammasome defects contribute to chronic intestinal inflammation and increase the susceptibility to colitis in mice. However, the inflammasome sensor absent in melanoma 2 (AIM2) protects against colorectal cancer in an inflammasome-independent manner through DNA-dependent protein kinase and Akt pathways. Yet, the roles of the AIM2 inflammasome in IBD and the early phases of colorectal cancer remain ill-defined. Here we show that the AIM2 inflammasome has a protective role in the intestine. During steady state, Aim2 deletion results in the loss of IL-18 secretion, suppression of the IL-22 binding protein (IL-22BP) in intestinal epithelial cells and consequent loss of the STAT3-dependent antimicrobial peptides (AMPs) Reg3ß and Reg3γ, which promotes dysbiosis-linked colitis. During dextran sulfate sodium-induced colitis, a dysfunctional IL-18/IL-22BP pathway in Aim2-/- mice promotes excessive IL-22 production and elevated STAT3 activation. Aim2-/- mice further exhibit sustained STAT3 and Akt activation during the resolution of colitis fueled by enhanced Reg3b and Reg3g expression. This self-perpetuating mechanism promotes proliferation of intestinal crypt cells and likely contributes to the recently described increase in susceptibility of Aim2-/- mice to colorectal cancer. Collectively, our results demonstrate a central role for the AIM2 inflammasome in preventing dysbiosis and intestinal inflammation through regulation of the IL-18/IL-22BP/IL-22 and STAT3 pathway and expression of select AMPs.

Immunization against an IL-6 peptide induces anti-IL-6 antibodies and modulates the Delayed-Type Hypersensitivity reaction in cynomolgus monkeys.

Interleukin-6 (IL-6) overproduction has been involved in the pathogenesis of several chronic inflammatory diseases and the administration of an anti-IL-6 receptor monoclonal antibody has been proven clinically efficient to treat them. However, the drawbacks of monoclonal antibodies have led our group to develop an innovative anti-IL-6 strategy using a peptide-based active immunization. This approach has previously shown its efficacy in a mouse model of systemic sclerosis. Here the safety, immunogenicity, and efficacy of this strategy was assessed in non human primates. No unscheduled death and clinical signs of toxicity was observed during the study. Furthermore, the cynomolgus monkeys immunized against the IL-6 peptide produced high levels of anti-IL-6 antibodies as well as neutralizing antibodies compared to control groups. They also showed an important decrease of the cumulative inflammatory score following a delayed-type hypersensitivity reaction induced by the Tetanus vaccine compared to control groups (minus 57,9%, P = 0.014). These findings are highly significant because the immunizing IL-6 peptide used in this study is identical in humans and in monkeys and this novel anti-IL-6 strategy could thus represent a promising alternative to monoclonal antibodies.

The PYRIN Domain-only Protein POP1 Inhibits Inflammasome Assembly and Ameliorates Inflammatory Disease.

In response to infections and tissue damage, ASC-containing inflammasome protein complexes are assembled that promote caspase-1 activation, IL-1ß and IL-18 processing and release, pyroptosis, and the release of ASC particles. However, excessive or persistent activation of the inflammasome causes inflammatory diseases. Therefore, a well-balanced inflammasome response is crucial for the maintenance of homeostasis. We show that the PYD-only protein POP1 inhibited ASC-dependent inflammasome assembly by preventing inflammasome nucleation, and consequently interfered with caspase-1 activation, IL-1ß and IL-18 release, pyroptosis, and the release of ASC particles. There is no mouse ortholog for POP1, but transgenic expression of human POP1 in monocytes, macrophages, and dendritic cells protected mice from systemic inflammation triggered by molecular PAMPs, inflammasome component NLRP3 mutation, and ASC danger particles. POP1 expression was regulated by TLR and IL-1R signaling, and we propose that POP1 provides a regulatory feedback loop that shuts down excessive inflammatory responses and thereby prevents systemic inflammation.

Targeting IL-6 by both passive or active immunization strategies prevents bleomycin-induced skin fibrosis.

INTRODUCTION: Interleukin-6 (IL-6) is a pleiotropic cytokine for which preliminary data have suggested that it might contribute to systemic sclerosis (SSc). Our aims were to investigate, firstly, IL-6 expression in patients with SSc and, secondly, the efficacy of both passive and active immunization against IL-6 to reduce skin fibrosis in complementary mouse models of SSc. METHODS: Human serum levels and skin expression of IL-6 were determined by enzyme-linked immunosorbent assay and immunohistochemistry, respectively. We first evaluated the antifibrotic properties of the monoclonal anti-IL-6R antibody, MR16-1, in the bleomycin-induced dermal fibrosis mouse model, reflecting early and inflammatory stages of SSc. Then, we assessed the efficacy of MR16-1 in tight skin-1 (Tsk-1) mice, an inflammation-independent model of skin fibrosis. Additionally, we have developed an innovative strategy using an anti-IL-6 peptide-based active immunization. Infiltrating leukocytes, T cells, and B cells were quantified, and IL-6 levels were measured in the serum and lesional skin of mice after passive or active immunization. RESULTS: Serum and skin levels of IL-6 were significantly increased in patients with early SSc. Treatment with MR16-1 led in the bleomycin mouse model to a 25% (P = 0.02) and 30% (P = 0.007) reduction of dermal thickness and hydroxyproline content, respectively. MR16-1 demonstrated no efficacy in Tsk-1 mice. Thereafter, mice were immunized against a small peptide derived from murine IL-6 and this strategy led in the bleomycin model to a 20% (P = 0.02) and 25% (P = 0.005) decrease of dermal thickness and hydroxyproline content, respectively. Passive and active immunization led to decreased T-cell infiltration in the lesional skin of mice challenged with bleomycin. Upon bleomycin injections, serum and skin IL-6 levels were increased after treatment with MR16-1 and were significantly reduced after anti-IL-6 active immunization. CONCLUSIONS: Our results support the relevance of targeting IL-6 in patients with early SSc since IL-6 is overexpressed in early stages of the disease. Targeting IL-6 by both passive and active immunization strategies prevented the development of bleomycin-induced dermal fibrosis in mice. Our results highlight the therapeutic potential of active immunization against IL-6, which is a seductive alternative to passive immunization.

The PYRIN domain-only protein POP3 inhibits ALR inflammasomes and regulates responses to infection with DNA viruses.

The innate immune system responds to infection and tissue damage by activating cytosolic sensory complexes called 'inflammasomes'. Cytosolic DNA is sensed by AIM2-like receptors (ALRs) during bacterial and viral infections and in autoimmune diseases. Subsequently, recruitment of the inflammasome adaptor ASC links ALRs to the activation of caspase-1. A controlled immune response is crucial for maintaining homeostasis, but the regulation of ALR inflammasomes is poorly understood. Here we identified the PYRIN domain (PYD)-only protein POP3, which competes with ASC for recruitment to ALRs, as an inhibitor of DNA virus-induced activation of ALR inflammasomes in vivo. Data obtained with a mouse model with macrophage-specific POP3 expression emphasize the importance of the regulation of ALR inflammasomes in monocytes and macrophages.

An Update on PYRIN Domain-Containing Pattern Recognition Receptors: From Immunity to Pathology.

Cytosolic pattern recognition receptors (PRRs) sense a wide range of endogenous danger-associated molecular patterns as well as exogenous pathogen-associated molecular patterns. In particular, Nod-like receptors containing a pyrin domain (PYD), called NLRPs, and AIM2-like receptors (ALRs) have been shown to play a critical role in host defense by facilitating clearance of pathogens and maintaining a healthy gut microflora. NLRPs and ALRs both encode a PYD, which is crucial for relaying signals that result in an efficient innate immune response through activation of several key innate immune signaling pathways. However, mutations in these PRRs have been linked to the development of auto-inflammatory and autoimmune diseases. In addition, they have been implicated in metabolic diseases. In this review, we summarize the function of PYD-containing NLRPs and ALRs and address their contribution to innate immunity, host defense, and immune-linked diseases.

Active immunization against IL-23p19 improves experimental arthritis.

INTRODUCTION: IL-23 is a pro-inflammatory cytokine essential for the differentiation of Th17 lymphocytes, a subtype of T lymphocyte implied in auto-immunity. IL-23 shares a subunit with IL-12, IL-12/23p40, and comprises a specific subunit, IL-23p19. We previously demonstrated that active immunization against entire TNF-α and against peptides of IL-1ß was protective in animal models of rheumatoid arthritis. The aim of this study was to evaluate the effect of peptide-based vaccines targeting the IL-23p19 subunit in collagen-induced arthritis (CIA). METHODS: Using bioinformatics, the murine IL-23p19 subunit was modeled and two peptides were defined in the receptor interacting domain. Each peptide was coupled to keyhole limpet hemocyanin (KLH) to obtain two vaccines IL23-K1 and IL23-K2. Both vaccines were used for immunizations in incomplete Freund adjuvant (IFA) in groups of DBA/1 mice. Control groups received KLH or PBS at the same dates. CIA was induced by two subcutaneous injections of bovine type II collagen (CIIb), and the development of disease assessed during the next two months. Anti-CIIb and anti-IL-23 antibody levels were assessed by ELISA. Pro- and anti-inflammatory cytokines mRNA were quantified by qRT-PCR in the spleen and the synovium. T-cell populations in the spleen were evaluated by FACS analysis. RESULTS: The clinical scores showed that mice treated with IL23-K1 developed less arthritis than negative controls (p<0.05). Mice immunized with IL23-K1 produced more anti-IL-23 antibodies than those immunized with IL23-K2 (p<0.001). mRNA quantification showed that the IL23-K1 immunization led to an increase of IL-10 in the spleen (p<0.05 vs. KLH), without any effect on IL-17 level. Histological examination showed that IL23-K1 strongly protected against joint destruction and inflammation (p<0.01 vs. KLH and p<0.001 vs. PBS). T-cell populations in the spleen were not modified by IL-23 modulation. CONCLUSION: These data show that targeting IL-23p19 through a vaccination strategy is protective in CIA. This specific targeting of IL-23 might constitute a promising therapeutic approach to explore in rheumatoid arthritis.

Genomewide association study of a rapid progression cohort identifies new susceptibility alleles for AIDS (ANRS Genomewide Association Study 03).

BACKGROUND: Previous genomewide association studies (GWASs) of AIDS have targeted end points based on the control of viral load and disease nonprogression. The discovery of genetic factors that predispose individuals to rapid progression to AIDS should also reveal new insights into the molecular etiology of the pathology. METHODS: We undertook a case-control GWAS of a unique cohort of 85 human immunodeficiency virus type 1 (HIV-1)-infected patients who experienced rapid disease progression, using Illumina HumanHap300 BeadChips. The case group was compared with a control group of 1352 individuals for the 291,119 autosomal single-nucleotide polymorphisms (SNPs) passing the quality control tests, using the false-discovery rate (FDR) statistical method for multitest correction. RESULTS: Novel associations with rapid progression (FDR, < or = 25%) were identified for PRMT6 (P = 6.1 x 10(-7); odds ratio [OR], 0.24), SOX5 (P = 1.8 x 10(-6); OR, 0.45), RXRG (P = 3.9 x 10(-6); OR, 3.29), and TGFBRAP1 (P = 7 x 10(-6); OR, 0.34). The haplotype analysis identified exonic and promoter SNPs potentially important for PRMT6 and TGFBRAP1 function. CONCLUSIONS: The statistical and biological relevance of these associations and their high ORs underscore the power of extreme phenotypes for GWASs, even with a modest sample size. These genetic results emphasize the role of the transforming growth factor beta pathway in the pathogenesis of HIV-1 disease. Finally, the wealth of information provided by this study should help unravel new diagnostic and therapeutic targets.

Anti-cytokine therapeutics: history and update.

Anti-cytokine therapy has promoted a revolution in the treatment of several inflammatory disorders during the past 10 years. Despite their medical and commercial success, they exhibit several drawbacks: difficulties of production, excessive costs, and a few side-effects. A promising alternative to the passive infusion of monoclonal antibodies or soluble cytokine receptors is the use of the active anti-cytokine immune therapy (ACIT). Surprisingly, clinical studies suggested the interest of this approach during the late 1980's, even before the advent of anti-cytokine passive immunotherapy. In this review, we first explain the involvement of several cytokines in many common diseases involving cytokine overproduction, and identify key targets for anti-cytokine treatments. We then present an update on current advances in preclinical and clinical development of passive anti-cytokine therapeutic approaches. We further discuss progresses in the promising field of active anti-cytokine immunotherapy. Cytokine receptors biologics and small molecules developed using structure/function information, which also constitute important options for treating the cytokine-mediated diseases, are not discussed in this review.

Genomewide association study of an AIDS-nonprogression cohort emphasizes the role played by HLA genes (ANRS Genomewide Association Study 02).

To elucidate the genetic factors predisposing to AIDS progression, we analyzed a unique cohort of 275 human immunodeficiency virus (HIV) type 1-seropositive nonprogressor patients in relation to a control group of 1352 seronegative individuals in a genomewide association study (GWAS). The strongest association was obtained for HCP5 rs2395029 (P=6.79x10(-10); odds ratio, 3.47) and was possibly linked to an effect of sex. Interestingly, this single-nucleotide polymorphism (SNP) was in high linkage disequilibrium with HLA-B, MICB, TNF, and several other HLA locus SNPs and haplotypes. A meta-analysis of our genomic data combined with data from the previously conducted Euro-CHAVI (Center for HIV/AIDS Vaccine Immunology) GWAS confirmed the HCP5 signal (P=3.02x10(-19)) and identified several new associations, all of them involving HLA genes: MICB, TNF, RDBP, BAT1-5, PSORS1C1, and HLA-C. Finally, stratification by HCP5 rs2395029 genotypes emphasized an independent role for ZNRD1, also in the HLA locus, and this finding was confirmed by experimental data. The present study, the first GWAS of HIV-1 nonprogressors, underscores the potential for some HLA genes to control disease progression soon after infection.