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Inflammatory responses are crucial for controlling infections and initiating tissue repair. However, excessive and uncontrolled inflammation causes inflammatory disease. Processing and release of the pro-inflammatory cytokines interleukin-1β (IL-1β) and IL-18 depend on caspase-1 activation within inflammasomes. Assembly of inflammasomes is initiated upon activation of cytosolic pattern recognition receptors (PRRs), followed by sequential polymerization of pyrin domain (PYD)-containing and caspase recruitment domain (CARD)-containing proteins mediated by homotypic PYD and CARD interactions. Small PYD- or CARD-only proteins (POPs and COPs, respectively) evolved in higher primates to target these crucial interactions to limit inflammation. Here, we show the ability of COPs to regulate inflammasome activation by modulating homotypic CARD-CARD interactions in vitro and in vivo. CARD16, CARD17, and CARD18 displace crucial CARD interactions between caspase-1 proteins through competitive binding and ameliorate uric acid crystal-mediated NLRP3 inflammasome activation and inflammatory disease. COPs therefore represent an important family of inflammasome regulators and ameliorate inflammatory disease., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Canonical inflammasomes are innate immune protein scaffolds that enable the activation of inflammatory caspase-1, and subsequently the processing and release of interleukin (IL)-1β, 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-1β 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-1β and IL-18 secretion. Conversely, reduced POP1 expression in human macrophages enhances IL-1β 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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 de Almeida, Devi, Indramohan, Huang, Ratsimandresy, Pope, Dorfleutner and Stehlik.)

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-1β 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.

Sensing and responding to pathogens and tissue damage is a core mechanism of innate immune host defense, and inflammasomes represent a central cytosolic pattern recognition receptor pathway leading to the generation of the pro-inflammatory cytokines interleukin-1β and interleukin-18 and pyroptotic cell death that causes the subsequent release of danger signals to propagate and perpetuate inflammatory responses. While inflammasome activation is essential for host defense, deregulated inflammasome responses and excessive release of inflammatory cytokines and danger signals are linked to an increasing spectrum of inflammatory diseases. In this review, we will discuss recent developments in elucidating the role of PYRIN domain-only proteins (POPs) and the related CARD-only proteins (COPs) in regulating inflammasome responses and their impact on inflammatory disease., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

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)-1β 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.

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) Reg3β 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.

Listeria monocytogenes is a Gram-positive intracellular pathogen that causes spontaneous abortion in pregnant women, as well as septicemia, meningitis, and gastroenteritis, primarily in immunocompromised individuals. Although L. monocytogenes can usually be effectively treated with antibiotics, there is still around a 25% mortality rate with individuals who develop clinical listeriosis. Neutrophils are innate immune cells required for the clearance of pathogenic organisms, including L. monocytogenes The diverse roles of neutrophils during both infectious and noninfectious inflammation have recently gained much attention. However, the impact of reactive oxygen species, and the enzymes that control their production, on neutrophil recruitment and function is not well understood. Using congenic mice with varying levels of extracellular superoxide dismutase (ecSOD) activity, we have recently shown that the presence of ecSOD decreases clearance of L. monocytogenes while increasing the recruitment of neutrophils that are not protective in the liver. The data presented here show that ecSOD activity does not lead to a cell-intrinsic increase in neutrophil-homing potential or a decrease in protection against L. monocytogenes Instead, ecSOD activity enhances the production of neutrophil-attracting factors and protects hyaluronic acid (HA) from damage. Furthermore, neutrophils from the livers of ecSOD-expressing mice have decreased intracellular and surface-bound myeloperoxidase, are less capable of killing phagocytosed L. monocytogenes, and have decreased oxidative burst. Collectively, our data reveal that ecSOD activity modulates neutrophil recruitment and function in a cell-extrinsic fashion, highlighting the importance of the enzyme in protecting tissues from oxidative damage., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Listeria monocytogenes is a gram-positive intracellular pathogen that causes meningitis and septicemia in immunocompromised individuals and spontaneous abortion in pregnant women. The innate immune response against L. monocytogenes is primarily mediated by neutrophils and monocytes. Interleukin-23 (IL-23) is an important proinflammatory cytokine well known for its role in neutrophil recruitment in various infectious and autoimmune diseases. We have previously shown that IL-23 is required for host resistance against L. monocytogenes and for neutrophil recruitment to the liver, but not the spleen, during infection. Despite efficient neutrophil recruitment to the spleen, IL-23p19 knockout (KO) mice have an increased bacterial burden in this organ, suggesting that IL-23 may regulate the recruitment/function of another cell type to the spleen. In this study, we show that specific depletion of neutrophils abrogated the differences in bacterial burdens in the livers but not the spleens of C57BL/6 (B6) and IL-23p19 KO mice. Interestingly, L. monocytogenes-infected IL-23p19 KO mice had fewer monocytes in the spleen than B6 mice, as well as a reduction in the monocyte-recruiting chemokines CCL2 and CCL7. Additionally, the overall concentrations of tumor necrosis factor alpha (TNF-α) and nitric oxide (NO(•)), as well as the percentages and total numbers of monocytes producing TNF-α and NO(•), were reduced in IL-23p19 KO mice compared to levels in B6 mice, leading to increased bacterial burdens in the spleens of L. monocytogenes-infected IL-23p19 KO mice. Collectively, our data establish that IL-23 is required for the optimal recruitment of TNF-α- and NO(•)-producing inflammatory monocytes, thus revealing a novel mechanism by which this proinflammatory cytokine provides protection against bacterial infection.

Reactive oxygen species and reactive nitrogen species play important roles during immune responses to bacterial pathogens. Extracellular superoxide dismutase (ecSOD) regulates extracellular concentrations of reactive oxygen species and reactive nitrogen species and contributes to tissue protection during inflammatory insults. The participation of ecSOD in immune responses seems therefore intuitive, yet is poorly understood. In the current study, we used mice with varying levels of ecSOD activity to investigate the involvement of this enzyme in immune responses against Listeria monocytogenes. Surprisingly, our data demonstrate that despite enhanced neutrophil recruitment to the liver, ecSOD activity negatively affected host survival and bacterial clearance. Increased ecSOD activity was accompanied by decreased colocalization of neutrophils with bacteria, as well as increased neutrophil apoptosis, which reduced overall and neutrophil-specific TNF-α production. Liver leukocytes from mice lacking ecSOD produced equivalent NO· compared with liver leukocytes from mice expressing ecSOD. However, during infection, there were higher levels of peroxynitrite (NO(3)·(-)) in livers from mice lacking ecSOD compared with livers from mice expressing ecSOD. Neutrophil depletion studies revealed that high levels of ecSOD activity resulted in neutrophils with limited protective capacity, whereas neutrophils from mice lacking ecSOD provided superior protection compared with neutrophils from wild-type mice. Taken together, our data demonstrate that ecSOD activity reduces innate immune responses during bacterial infection and provides a potential target for therapeutic intervention.

Previous studies have suggested that neutrophils are required for resistance during infection with multiple pathogenic microorganisms. However, the depleting antibody used in those studies binds to both Ly6G and Ly6C (anti-Gr-1; clone RB6-8C5). This antibody has been shown to deplete not only neutrophils but also monocytes and a subset of CD8(+) T cells. Recently, an antibody against Ly6G, which specifically depletes neutrophils, was characterized. In the present study, neutrophils are depleted using the antibody against Ly6G during infection with the intracellular bacterium Listeria monocytogenes (LM). Our data show that neutrophil-depleted mice are much less susceptible to infection than mice depleted with anti-Gr-1. Although neutrophils are required for clearance of LM, their importance is more pronounced in the liver and during a high-dose bacterial challenge. Furthermore, we demonstrate that the protection mediated by neutrophils is due to the production of TNF-α, but not IFN-γ. Additionally, neutrophils are not required for the recruitment of monocytes or the generation of adaptive T-cell responses during LM infection. This study highlights the importance of neutrophils during LM infection, and indicate that depletion of neutrophils is less detrimental to the host than depletion of all Gr-1-expressing cell populations., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Listeria monocytogenes (LM) is a gram-positive bacterium that is a common contaminant of processed meats and dairy products. In humans, ingestion of LM can result in intracellular infection of the spleen and liver, which can ultimately lead to septicemia, meningitis, and spontaneous abortion. Interleukin (IL)-23 is a cytokine that regulates innate and adaptive immune responses by inducing the production of IL-17A, IL-17F, and IL-22. We have recently demonstrated that the IL-23/IL-17 axis is required for optimal recruitment of neutrophils to the liver, but not the spleen, during LM infection. Furthermore, these cytokines are required for the clearance of LM during systemic infection. In other infectious models, IL-22 induces the secretion of anti-microbial peptides and protects tissues from damage by preventing apoptosis. However, the role of IL-22 has not been thoroughly investigated during LM infection. In the present study, we show that LM induces the production of IL-22 in vivo. Interestingly, IL-23 is required for the production of IL-22 during primary, but not secondary, LM infection. Our findings suggest that IL-22 is not required for clearance of LM during primary or secondary infection, using both systemic and mucosal models of infection. IL-22 is also not required for the protection of LM infected spleens and livers from organ damage. Collectively, these data indicate that IL-22 produced during LM infection must play a role other than clearance of LM or protection of tissues from pathogen- or immune-mediated damage.

We determined whether human NK cells could contribute to immune defenses against Mycobacterium tuberculosis through production of IL-22. CD3(-)CD56(+) NK cells produced IL-22 when exposed to autologous monocytes and gamma-irradiated M. tuberculosis, and this depended on the presence of IL-15 and IL-23, but not IL-12 or IL-18. IL-15-stimulated NK cells expressed 10.6 times more DAP10 mRNA compared with control NK cells, and DAP10 siRNA inhibited IL-15-mediated IL-22 production by NK cells. Soluble factors produced by IL-15-activated NK cells inhibited growth of M. tuberculosis in macrophages, and this effect was reversed by anti-IL-22. Addition of rIL-22 to infected macrophages enhanced phagolysosomal fusion and reduced growth of M. tuberculosis. We conclude that NK cells can contribute to immune defenses against M. tuberculosis through production of IL-22, which inhibits intracellular mycobacterial growth by enhancing phagolysosomal fusion. IL-15 and DAP-10 elicit IL-22 production by NK cells in response to M. tuberculosis.

We studied the factors that control IL-17 production in human Mycobacterium tuberculosis infection. CD4(+) cells from healthy tuberculin reactors produced IL-17 in response to autologous M. tuberculosis-stimulated monocytes, and most IL-17(+) cells were Ag experienced, CD4(+)CD62L(-). IL-17 production by CD4(+) cells was inhibited by anti-IL-23, but not by Abs to IL-1, IL-6, or TGF-beta. Anti-NKG2D reduced IL-17 production and the frequency of CD4(+)CD62(-) IL-17(+) cells, suggesting that NKG2D stimulates IL-17 production. CD4(+)NKG2D(+) cells did not produce IL-17. Monocytes and alveolar macrophages from healthy donors produced IL-23 in response to M. tuberculosis. Addition of CD4(+) cells markedly enhanced IL-23 production by M. tuberculosis-stimulated monocytes, and this was inhibited by anti-NKG2D and by Abs to UL-16 binding protein (ULB)1, a ligand for NKG2D on APCs. We conclude that binding of NKG2D to UL-16 binding protein (ULB)1 contributes to IL-23-dependent IL-17 production by CD4(+) cells in human M. tuberculosis infection.