Novel chemotype NLRP3 inhibitors that target the CRID3-binding pocket with high potency.

The NLRP3 inflammasome plays a central role in various human diseases. Despite significant interest, most clinical-grade NLRP3 inhibitors are derived from sulfonylurea inhibitor CRID3 (also called MCC950). Here, we describe a novel chemical class of NLRP3-inhibiting compounds (NIC) that exhibit potent and selective NLRP3 inflammasome inhibition in human monocytes and mouse macrophages. BRET assays demonstrate that they physically interact with NLRP3. Structural modeling further reveals they occupy the same binding site of CRID3 but in a critically different conformation. Furthermore, we show that NIC-11 and NIC-12 lack the off-target activity of CRID3 against the enzymatic activity of carbonic anhydrases I and II. NIC-12 selectively reduces circulating IL-1ß levels in the LPS-endotoxemia model in mice and inhibits NLRP3 inflammasome activation in CAPS patient monocytes and mouse macrophages with about tenfold increased potency compared with CRID3. Altogether, this study unveils a new chemical class of highly potent and selective NLRP3-targeted inhibitors with a well-defined molecular mechanism to complement existing CRID3-based NLRP3 inhibitors in pharmacological studies and serve as novel chemical leads for the development of NLRP3-targeted therapies.

GSDMD drives canonical inflammasome-induced neutrophil pyroptosis and is dispensable for NETosis.

Neutrophils are the most prevalent immune cells in circulation, but the repertoire of canonical inflammasomes in neutrophils and their respective involvement in neutrophil IL-1ß secretion and neutrophil cell death remain unclear. Here, we show that neutrophil-targeted expression of the disease-associated gain-of-function Nlrp3A350V mutant suffices for systemic autoinflammatory disease and tissue pathology in vivo. We confirm the activity of the canonical NLRP3 and NLRC4 inflammasomes in neutrophils, and further show that the NLRP1b, Pyrin and AIM2 inflammasomes also promote maturation and secretion of interleukin (IL)-1ß in cultured bone marrow neutrophils. Notably, all tested canonical inflammasomes promote GSDMD cleavage in neutrophils, and canonical inflammasome-induced pyroptosis and secretion of mature IL-1ß are blunted in GSDMD-knockout neutrophils. In contrast, GSDMD is dispensable for PMA-induced NETosis. We also show that Salmonella Typhimurium-induced pyroptosis is markedly increased in Nox2/Gp91Phox -deficient neutrophils that lack NADPH oxidase activity and are defective in PMA-induced NETosis. In conclusion, we establish the canonical inflammasome repertoire in neutrophils and identify differential roles for GSDMD and the NADPH complex in canonical inflammasome-induced neutrophil pyroptosis and mitogen-induced NETosis, respectively.

The human inflammasomes.

Two decades of inflammasome research has led to a vast body of knowledge on the complex regulatory mechanisms and pathological roles of canonical and non-canonical inflammasome activation in a plethora of research models of primarily rodent origin. More recently, the field has made notable progress in characterizing human-specific inflammasomes and their regulation mechanisms, including an expansion of inflammasome biology to adaptive immune cells. These exciting developments in basic research have been accompanied by potentially transformative results from large clinical trials and translational efforts to develop inflammasome-targeted small molecule inhibitors for therapeutic use. Here, we will discuss recent findings in the field with a specific emphasis on activation mechanisms of human inflammasomes and their potential role in auto-inflammatory, metabolic and neoplastic diseases.

Delayed development of the protective IL-17A response following a Giardia muris infection in neonatal mice.

Giardia is an intestinal protozoan parasite that has the ability to infect a wide range of hosts, which can result in the clinical condition 'giardiasis'. Over the years, experimental research has shown the crucial involvement of IL-17A to steer the protective immune response against Giardia. The development of the protective response, as reflected by a significant drop in cyst secretion, typically takes around 3 to 4 weeks. However, early-life infections often have a more chronic character lasting for several weeks or months. Therefore, the aim of the current study was to investigate the dynamics of a Giardia muris infection and the subsequent host immune response in neonatal mice infected 4 days after birth. The outcome of the study showed that a G. muris infection in pre-weaned mice failed to trigger a protective IL-17A response, which could explain the prolonged course of infection in comparison to older mice. Only after weaning, a protective intestinal immune response started to develop, characterized by an upregulation of IL-17A and Mbl2 and the secretion of parasite-specific IgA.

Ascaris suum infection was associated with a worm-independent reduction in microbial diversity and altered metabolic potential in the porcine gut microbiome.

The effect of infection of pigs with Ascaris suum on the microbial composition in the proximal colon and fecal matter was investigated using 16S rRNA gene sequencing. The infection significantly decreased various microbial diversity indices including Chao1 richness, but the effect on Chao1 in the colon luminal contents was worm burden-independent. The abundance of 49 genera present in colon contents, such as Prevotella and Faecalibacterium, and 179 operational taxonomic units was significantly changed as a result of infection. Notably, infection was also associated with a significant shift in the metabolic potential of the proximal colon microbiome, where the relative abundance of at least 30 metabolic pathways including carbohydrate metabolism and amino acid metabolism was reduced, while the abundance of 28 pathways was increased by infection. Furthermore, the microbial co-occurrence network in infected pigs was highly modular. Two of 52 modules or subnetworks were negatively correlated with fecal butyrate concentrations (r < -0.7; P < 0.05) while one module with 18 members was negatively correlated with fecal acetate, propionate and total short-chain fatty acids. A partial Mantel test identified a strong positive correlation between node connectivity of the operational taxonomic units assigned to ß-Proteobacteria (especially the family Alcaligenaceae) and fecal acetate and propionate levels (r = 0.82 and 0.74, respectively), while that of the family Porphyromonadaceae was positively correlated with fecal egg counts. Overall, Ascaris infection was associated with a profound change in the gut microbiome, especially in the proximity of the initial site of larval infection, and should facilitate our understanding of the pathophysiological consequence of gastrointestinal nematode infections.

Interleukin-17 receptor A (IL-17RA) as a central regulator of the protective immune response against Giardia.

The protozoan parasite Giardia is a highly prevalent intestinal pathogen with a wide host range. Data obtained in mice, cattle and humans revealed the importance of IL-17A in the development of a protective immune response against Giardia. The aim of this study was to further unravel the protective effector mechanisms triggered by IL-17A following G. muris infection in mice, by an RNA-sequencing approach. C57BL/6 WT and C57BL/6 IL-17RA KO mice were orally infected with G. muris cysts. Three weeks post infection, intestinal tissue samples were collected for RNA-sequencing, with samples from uninfected C57BL/6 WT and C57BL/6 IL-17RA KO animals serving as negative controls. Differential expression analysis showed that G. muris infection evoked the transcriptional upregulation of a wide array of genes, mainly in animals with competent IL-17RA signaling. IL-17RA signaling induced the production of various antimicrobial peptides, such as angiogenin 4 and α- and ß-defensins and regulated complement activation through mannose-binding lectin 2. The expression of the receptor that regulates the secretion of IgA into the intestinal lumen, the polymeric immunoglobulin receptor, was also dependent on IL-17RA signaling. Interestingly, the transcriptome data showed for the first time the involvement of the circadian clock in the host response following Giardia infection.

Host protective ASP-based vaccine against the parasitic nematode Ostertagia ostertagi triggers NK cell activation and mixed IgG1-IgG2 response.

The mucus-dwelling parasite Ostertagia ostertagi is one of the most important gastrointestinal nematodes in cattle. Our group has previously demonstrated the protective capacity of a vaccine against this parasite based on a native activation-associated secreted protein ASP1 (nASP) in combination with the saponin adjuvant QuilA. The aim of the current study was to analyse the effect of both antigen and adjuvant on the cellular and humoral vaccine-induced immune responses by comparing the native ASP to a recombinant version expressed in Pichia pastoris (pASP) and replacing QuilA by Al(OH)3. Immunization of cattle with the protective nASP+QuilA vaccine was associated with antigen-induced proliferation of natural killer (NK) cells combined with IFN-γ secretion and the induction of a mixed IgG1/IgG2 antibody response. ASP-specific activation and proliferation of NK cells was also observed in mice following the same vaccination regime. Replacing QuilA by Al(OH)3 or nASP by pASP significantly decreased the capacity of the vaccines to trigger both NK cell activation and antibody responses and failed to induce protection against a challenge infection. Reduction of the structurally anchoring disulphide bonds of the nASP completely abolished its ability to induce NK cell activation and antibody responses, highlighting the importance of protein conformation for the immunostimulatory activity.

The Intestinal Microbiota Contributes to the Ability of Helminths to Modulate Allergic Inflammation.

Intestinal helminths are potent regulators of their host's immune system and can ameliorate inflammatory diseases such as allergic asthma. In the present study we have assessed whether this anti-inflammatory activity was purely intrinsic to helminths, or whether it also involved crosstalk with the local microbiota. We report that chronic infection with the murine helminth Heligmosomoides polygyrus bakeri (Hpb) altered the intestinal habitat, allowing increased short chain fatty acid (SCFA) production. Transfer of the Hpb-modified microbiota alone was sufficient to mediate protection against allergic asthma. The helminth-induced anti-inflammatory cytokine secretion and regulatory T cell suppressor activity that mediated the protection required the G protein-coupled receptor (GPR)-41. A similar alteration in the metabolic potential of intestinal bacterial communities was observed with diverse parasitic and host species, suggesting that this represents an evolutionary conserved mechanism of host-microbe-helminth interactions.