T cell-intrinsic ASC critically promotes T(H)17-mediated experimental autoimmune encephalomyelitis.

Interleukin 1ß (IL-1ß) is critical for the in vivo survival, expansion and effector function of IL-17-producing helper T (T(H)17) cells during autoimmune responses, including experimental autoimmune encephalomyelitis (EAE). However, the spatiotemporal role and cellular source of IL-1ß during EAE pathogenesis are poorly defined. In the present study, we uncovered a T cell-intrinsic inflammasome that drives IL-1ß production during T(H)17-mediated EAE pathogenesis. Activation of T cell antigen receptors induced expression of pro-IL-1ß, whereas ATP stimulation triggered T cell production of IL-1ß via ASC-NLRP3-dependent caspase-8 activation. IL-1R was detected on T(H)17 cells but not on type 1 helper T (T(H)1) cells, and ATP-treated T(H)17 cells showed enhanced survival compared with ATP-treated T(H)1 cells, suggesting autocrine action of T(H)17-derived IL-1ß. Together these data reveal a critical role for IL-1ß produced by a T(H)17 cell-intrinsic ASC-NLRP3-caspase-8 inflammasome during inflammation of the central nervous system.

Regulation of Mesenchymal Cell Fate by Transfer of Active Gasdermin-D via Monocyte-Derived Extracellular Vesicles.

Fibrosis is characterized by inappropriately persistent myofibroblast accumulation and excessive extracellular matrix deposition with the disruption of tissue architecture and organ dysfunction. Regulated death of reparative mesenchymal cells is critical for normal wound repair, but profibrotic signaling promotes myofibroblast resistance to apoptotic stimuli. A complex interplay between immune cells and structural cells underlies lung fibrogenesis. However, there is a paucity of knowledge on how these cell populations interact to orchestrate physiologic and pathologic repair of the injured lung. In this context, gasdermin-D (GsdmD) is a cytoplasmic protein that is activated following cleavage by inflammatory caspases and induces regulated cell death by forming pores in cell membranes. This study was undertaken to evaluate the impact of human (Thp-1) monocyte-derived extracellular vesicles and GsdmD on human lung fibroblast death. Our data show that active GsdmD delivered by monocyte-derived extracellular vesicles induces caspase-independent fibroblast and myofibroblast death. This cell death was partly mediated by GsdmD-independent induction of cellular inhibitor of apoptosis 2 (cIAP-2) in the recipient fibroblast population. Our findings, to our knowledge, define a novel paradigm by which inflammatory monocytes may orchestrate the death of mesenchymal cells in physiologic wound healing, illustrating the potential to leverage this mechanism to eliminate mesenchymal cells and facilitate the resolution of fibrotic repair.

Caspase-1-induced pyroptosis is an innate immune effector mechanism against intracellular bacteria.

Macrophages mediate crucial innate immune responses via caspase-1-dependent processing and secretion of interleukin 1ß (IL-1ß) and IL-18. Although infection with wild-type Salmonella typhimurium is lethal to mice, we show here that a strain that persistently expresses flagellin was cleared by the cytosolic flagellin-detection pathway through the activation of caspase-1 by the NLRC4 inflammasome; however, this clearance was independent of IL-1ß and IL-18. Instead, caspase-1-induced pyroptotic cell death released bacteria from macrophages and exposed the bacteria to uptake and killing by reactive oxygen species in neutrophils. Similarly, activation of caspase-1 cleared unmanipulated Legionella pneumophila and Burkholderia thailandensis by cytokine-independent mechanisms. This demonstrates that activation of caspase-1 clears intracellular bacteria in vivo independently of IL-1ß and IL-18 and establishes pyroptosis as an efficient mechanism of bacterial clearance by the innate immune system.

Activation of the Intracellular Pattern Recognition Receptor NOD2 Promotes Acute Myeloid Leukemia (AML) Cell Apoptosis and Provides a Survival Advantage in an Animal Model of AML.

TLRs, a family of membrane-bound pattern recognition receptors found on innate immune cells, have been well studied in the context of cancer therapy. Activation of these receptors has been shown to induce inflammatory anticancer events, including differentiation and apoptosis, across a wide variety of malignancies. In contrast, intracellular pattern recognition receptors such as NOD-like receptors have been minimally studied. NOD2 is a member of the NOD-like receptor family that initiates inflammatory signaling in response to the bacterial motif muramyl dipeptide. In this study, we examined the influence of NOD2 in human acute myeloid leukemia (AML) cells, demonstrating that IFN-γ treatment upregulated the expression of NOD2 signaling pathway members SLC15A3 and SLC15A4, downstream signaling kinase RIPK2, and the NOD2 receptor itself. This priming allowed for effective induction of caspase-1-dependent cell death upon treatment with muramyl tripeptide phosphatidylethanolamine (MTP-PE), a synthetic ligand for NOD2. Furthermore, the combination of MTP-PE and IFN-γ on AML blasts generated an inflammatory cytokine profile and activated NK cells. In a murine model of AML, dual treatment with MTP-PE and IFN-γ led to a significant increase in mature CD27- CD11b+ NK cells as well as a significant reduction in disease burden and extended survival. These results suggest that NOD2 activation, primed by IFN-γ, may provide a novel therapeutic option for AML.

Microparticulate P2X7 and GSDM-D mediated regulation of functional IL-1ß release.

The pro-inflammatory cytokine IL-1ß is a secreted protein that is cleaved by caspase-1 during inflammasome activation upon recognition of internal and external insults to cells. Purinergic receptor P2X7 has been described to be involved in the release pathway of bioactive mature IL-1ß by activated immune cells. Microparticle (MP) shedding has also been recently recognized as a manner of cytokine IL-1ß release. However, the understanding of purinergic receptor roles in the MP-mediated IL-1ß release process is still rudimentary. Gasdermin-D (GSDM-D), a protein involved in pyroptosis and inflammasome activation, has been recently described to be involved in the release of microparticles by virtue of its pore-forming ability. Hence, our current work is aimed to study the role of P2X7 in regulating GSDM-D-mediated microparticles and thereby bioactive IL-1ß release. We provide evidence that cleaved functional IL-1ß release in microparticles upon LPS stimulation is regulated by GSDM-D and P2X7 in a two-step fashion. GSDM-D activation first regulates release of IL-1ß and P2X7 into microparticles. Then, microparticulate active P2X7 receptor then regulates the release of bioactive IL-1ß encapsulated in microparticles to be able to target other cells inducing IL-8. Using an ATP model of stimulation, we further demonstrated that extracellular ATP stimulation to IL-1ß containing LPS microparticles induces release of its content, which when subjected to epithelial cells induced IL-8. This effect was blocked by P2X7 inhibitor, KN62, as well as by IL-1RA. Taken together, our findings demonstrate for the first time the synergistic critical roles of GSDM-D and purinergic receptors in the regulation of microparticulate bioactive IL-1ß release and induction of target cell responses.

Microparticulate Caspase 1 Regulates Gasdermin D and Pulmonary Vascular Endothelial Cell Injury.

Lung endothelial cell apoptosis and injury occur throughout all stages of acute lung injury/acute respiratory distress syndrome and impact disease progression. Caspases 1, 4, and 5 are essential for completion of the apoptotic program known as pyroptosis that also involves proinflammatory cytokines. Because gasdermin D (GSDMD) mediates pyroptotic death and is essential for pore formation, we hypothesized that it might direct caspase 1-encapsulated microparticle (MP) release and mediate endothelial cell death. Our present work provides evidence that GSDMD is released by LPS-stimulated THP-1 monocytic cells, where it is packaged into microparticles together with active caspase 1. Furthermore, only MP released from stimulated monocytic cells that contain both cleaved GSDMD and active caspase 1 induce endothelial cell apoptosis. MPs pretreated with caspase 1 inhibitor Y-VAD or pan-caspase inhibitor Z-VAD do not contain cleaved GSDMD. MPs from caspase 1-knockout cells are also deficient in p30 active GSDMD, further confirming that caspase 1 regulates GSDMD function. Although control MPs contained cleaved GSDMD without caspase 1, these fractions were unable to induce cell death, suggesting that encapsulation of both caspase 1 and GSDMD is essential for cell death induction. Release of microparticulate active caspase 1 was abrogated in GSDMD knockout cells, although cytosolic caspase 1 activation was not impaired. Last, higher concentrations of microparticulate GSDMD were detected in the plasma of septic patients with acute respiratory distress syndrome than in that of healthy donors. Taken together, these findings suggest that GSDMD regulates the release of microparticulate active caspase 1 from monocytes essential for induction of cell death and thereby may play a critical role in sepsis-induced endothelial cell injury.

Supernatants from stored red blood cell (RBC) units, but not RBC-derived microvesicles, suppress monocyte function in vitro.

BACKGROUND: We have previously shown that critically ill children transfused with red blood cells (RBCs) of longer storage durations have more suppressed monocyte function after transfusion compared to children transfused with fresher RBCs and that older stored RBCs directly suppress monocyte function in vitro, through unknown mechanisms. We hypothesized that RBC-derived microvesicles (MVs) were responsible for monocyte suppression. STUDY DESIGN AND METHODS: To determine the role of stored RBC unit-derived MVs, we cocultured monocytes with supernatants, isolated MVs, or supernatants that had been depleted of MVs from prestorage leukoreduced RBCs that had been stored for either 7 or 30 days. Isolated MVs were characterized by electron microscopy and flow cytometry. Monocyte function after coculture experiments was measured by cytokine production after stimulation with lipopolysaccharide (LPS). RESULTS: Monocyte function was suppressed after exposure to supernatants from 30-day RBC units compared to monocytes cultured in medium alone (LPS-induced tumor necrosis factor-α production, 17,611 ± 3,426 vs. 37,486 ± 5,598 pg/mL; p = 0.02). Monocyte function was not suppressed after exposure to MV fractions. RBC supernatants that had been depleted of MVs remained immunosuppressive. Treating RBC supernatants with heat followed by RNase (to degrade protein-bound RNA) prevented RBC supernatant-induced monocyte suppression. CONCLUSION: Our findings implicate soluble mediators of stored RBC-induced monocyte suppression outside of MV fractions and suggest that extracellular protein-bound RNAs (such as microRNA) may play a role in transfusion-related immunomodulation.

Critical role for phosphoinositide 3-kinase gamma in parasite invasion and disease progression of cutaneous leishmaniasis.

Obligate intracellular pathogens such as Leishmania specifically target host phagocytes for survival and replication. Phosphoinositide 3-kinase γ (PI3Kγ), a member of the class I PI3Ks that is highly expressed by leukocytes, controls cell migration by initiating actin polymerization and cytoskeletal reorganization, which are processes also critical for phagocytosis. In this study, we demonstrate that class IB PI3K, PI3Kγ, plays a critical role in pathogenesis of chronic cutaneous leishmaniasis caused by L. mexicana. Using the isoform-selective PI3Kγ inhibitor, AS-605240 and PI3Kγ gene-deficient mice, we show that selective blockade or deficiency of PI3Kγ significantly enhances resistance against L. mexicana that is associated with a significant suppression of parasite entry into phagocytes and reduction in recruitment of host phagocytes as well as regulatory T cells to the site of infection. Furthermore, we demonstrate that AS-605240 is as effective as the standard antileishmanial drug sodium stibogluconate in treatment of cutaneous leishmaniasis caused by L. mexicana. These findings reveal a unique role for PI3Kγ in Leishmania invasion and establishment of chronic infection, and demonstrate that therapeutic targeting of host pathways involved in establishment of infection may be a viable strategy for treating infections caused by obligate intracellular pathogens such as Leishmania.

Analysis of human bronchial epithelial cell proinflammatory response to Burkholderia cenocepacia infection: inability to secrete il-1ß.

Burkholderia cenocepacia, the causative agent of cepacia syndrome, primarily affects cystic fibrosis patients, often leading to death. In the lung, epithelial cells serve as the initial barrier to airway infections, yet their responses to B. cenocepacia have not been fully investigated. Here, we examined the molecular responses of human airway epithelial cells to B. cenocepacia infection. Infection led to early signaling events such as activation of Erk, Akt, and NF-κB. Further, TNFα, IL-6, IL-8, and IL-1ß were all significantly induced upon infection, but no IL-1ß was detected in the supernatants. Because caspase-1 is required for IL-1ß processing and release, we examined its expression in airway epithelial cells. Interestingly, little to no caspase-1 was detectable in airway epithelial cells. Transfection of caspase-1 into airway epithelial cells restored their ability to secrete IL-1ß following B. cenocepacia infection, suggesting that a deficiency in caspase-1 is responsible, at least in part, for the attenuated IL-1ß secretion.

Inflammasome-dependent release of the alarmin HMGB1 in endotoxemia.

Endotoxin administration recapitulates many of the host responses to sepsis. Inhibitors of the cysteine protease caspase 1 have long been sought as a therapeutic because mice lacking caspase 1 are resistant to LPS-induced endotoxic shock. According to current thinking, caspase 1-mediated shock requires the proinflammatory caspase 1 substrates IL-1ß and IL-18. We show, however, that mice lacking both IL-1ß and IL-18 are normally susceptible to LPS-induced splenocyte apoptosis and endotoxic shock. This finding indicates the existence of another caspase 1-dependent mediator of endotoxemia. Reduced serum high mobility group box 1 (HMGB1) levels in caspase 1-deficient mice correlated with their resistance to LPS. A critical role for HMGB1 in endotoxemia was confirmed when mice deficient for IL-1ß and IL-18 were protected from a lethal dose of LPS by pretreatment with HMGB1-neutralizing Abs. We found that HMGB1 secretion from LPS-primed macrophages required the inflammasome components apoptotic speck protein containing a caspase activation and recruitment domain (ASC), caspase 1 and Nalp3, whereas HMGB1 secretion from macrophages infected in vitro with Salmonella typhimurium was dependent on caspase 1 and Ipaf. Thus, HMGB1 secretion, which is critical for endotoxemia, occurs downstream of inflammasome assembly and caspase 1 activation.

Caspase-7 activation by the Nlrc4/Ipaf inflammasome restricts Legionella pneumophila infection.

Legionella pneumophila (L. pneumophila), the causative agent of a severe form of pneumonia called Legionnaires' disease, replicates in human monocytes and macrophages. Most inbred mouse strains are restrictive to L. pneumophila infection except for the A/J, Nlrc4(-/-) (Ipaf(-/-)), and caspase-1(-/-) derived macrophages. Particularly, caspase-1 activation is detected during L. pneumophila infection of murine macrophages while absent in human cells. Recent in vitro experiments demonstrate that caspase-7 is cleaved by caspase-1. However, the biological role for caspase-7 activation downstream of caspase-1 is not known. Furthermore, whether this reaction is pertinent to the apoptosis or to the inflammation pathway or whether it mediates a yet unidentified effect is unclear. Using the intracellular pathogen L. pneumophila, we show that, upon infection of murine macrophages, caspase-7 was activated downstream of the Nlrc4 inflammasome and required caspase-1 activation. Such activation of caspase-7 was mediated by flagellin and required a functional Naip5. Remarkably, mice lacking caspase-7 and its macrophages allowed substantial L. pneumophila replication. Permissiveness of caspase-7(-/-) macrophages to the intracellular pathogen was due to defective delivery of the organism to the lysosome and to delayed cell death during early stages of infection. These results reveal a new mechanism for caspase-7 activation downstream of the Nlrc4 inflammasome and present a novel biological role for caspase-7 in host defense against an intracellular bacterium.

Brief Report: Increased Cotinine Concentrations are Associated With Reduced Expression of Cathelicidin (LL-37) and NOD-2 in Alveolar Macrophages of PLWH Who Smoke.

There is a strong link between cigarette smoking and pulmonary complications among people living with HIV. However, the effects of smoking on the local lung immune environment in this population remain unclear. Bronchoalveolar lavage and saliva were collected from HIV-infected smokers involved in a prospective study investigating alveolar macrophage expression of host defense molecules. Salivary cotinine concentrations were inversely related to expression of the immune cell receptor nucleotide-binding oligomerization domain-2 and the cathelicidin antimicrobial peptide LL-37. The negative correlation between salivary cotinine and LL-37 was particularly strong. Our study provides insight into how nicotine may adversely affect lung innate immunity in HIV.

Inflammasome Adaptor ASC Is Highly Elevated in Lung Over Plasma and Relates to Inflammation and Lung Diffusion in the Absence of Speck Formation.

Rationale: Caspase-1 is a zymogen whose activation predominantly depends upon the assembly of ASC monomers into insoluble prion-like polymers (specks). ASC polymers support caspase-1 dimer formation inducing a proximity mediated auto-activation of caspase-1. Therefore, the amount and nature of ASC monomers and polymers in lung bronchoalveolar lavage fluid (BALF) might serve as a marker of lung inflammasome activity. Objectives: To determine whether lung ASC concentrations or oligomerization status predicts lung function or activity of lung inflammation. Methods: BALF ASC amount and oligomerization status was studied in three distinct cohorts: (1) young healthy non-smokers, vapers and smokers; (2) healthy HIV+ smokers who underwent detailed lung function studies; and (3) hospitalized patients with suspected pneumonia. We quantified cell free BALF ASC levels by ELISA and immunoblot. Oligomers (i.e., ASC specks) were identified by chemical crosslinking and ability to sediment with centrifugation. Measurement and Main Results: ASC levels are significantly higher in lung lining fluid than in plasma as well as higher in smoker lungs compared to non-smoker lungs. In this context, ASC levels correlate with macrophage numbers, smoking intensity and loss of lung diffusion capacity in a well-characterized cohort of healthy HIV+ smokers. However, only monomeric ASC was found in our BALF samples from all subjects, including patients with lung infections. Conclusions: Even though, most, if not all, extracellular ASC in BALF exists in the soluble, monomeric form, monomeric ASC concentrations still reflect the inflammatory status of the lung microenvironment and correlate with loss of lung function.

Physical activity prevents acute inflammation in a gout model by downregulation of TLR2 on circulating neutrophils as well as inhibition of serum CXCL1 and is associated with decreased pain and inflammation in gout patients.

OBJECTIVES: Gout is the most prevalent inflammatory arthritis. To study the effects of regular physical activity and exercise intensity on inflammation and clinical outcome, we examined inflammatory pathogenesis in an acute model of murine gout and analyzed human gout patient clinical data as a function of physical activity. METHODS: NF-κB-luciferase reporter mice were organized into four groups and exercised at 0 m/min (non-exercise), 8 m/min (low-intensity), 11 m/min (moderate-intensity), and 15 m/min (high-intensity) for two weeks. Mice subsequently received intra-articular monosodium urate (MSU) crystal injections (0.5mg) and the inflammatory response was analyzed 15 hours later. Ankle swelling, NF-κB activity, histopathology, and tissue infiltration by macrophages and neutrophils were measured. Toll-like receptor (TLR)2 was quantified on peripheral monocytes/neutrophils by flow cytometry and both cytokines and chemokines were measured in serum or synovial aspirates. Clinical data and questionnaires accessing overall physical activity levels were collected from gout patients. RESULTS: Injection of MSU crystals produced a robust inflammatory response with increased ankle swelling, NF-κB activity, and synovial infiltration by macrophages and neutrophils. These effects were partially mitigated by low and moderate-intensity exercise. Furthermore, IL-1ß was decreased at the site of MSU crystal injection, TLR2 expression on peripheral neutrophils was downregulated, and expression of CXCL1 in serum was suppressed with low and moderate-intensity exercise. Conversely, the high-intensity exercise group closely resembled the non-exercised control group by nearly all metrics of inflammation measured in this study. Physically active gout patients had significantly less flares/yr, decreased C-reactive protein (CRP) levels, and lower pain scores relative to physically inactive patients. CONCLUSIONS: Regular, moderate physical activity can produce a quantifiable anti-inflammatory effect capable of partially mitigating the pathologic response induced by intra-articular MSU crystals by downregulating TLR2 expression on circulating neutrophils and suppressing systemic CXCL1. Low and moderate-intensity exercise produces this anti-inflammatory effect to varying degrees, while high-intensity exercise provides no significant difference in inflammation compared to non-exercising controls. Consistent with the animal model, gout patients with higher levels of physical activity have more favorable prognostic data. Collectively, these data suggest the need for further research and may be the foundation to a future paradigm-shift in conventional exercise recommendations provided by Rheumatologists to gout patients.

Inflammasome mRNA expression in human monocytes during early septic shock.

RATIONALE: Monocytes are central to the initiation of the inflammatory response in sepsis, with caspase-1 activation playing a key role. Monocyte deactivation during sepsis has been linked to poor outcomes. OBJECTIVES: Given the importance of caspase-1 in the immune response, we investigated whether monocytes from patients early in septic shock demonstrate alterations in mRNAs for caspase-1-related molecules. METHODS: Patients with septic shock (n = 26; age >18 years), critically ill intensive care unit patients (n = 20), and healthy volunteers (n = 22) were enrolled in a prospective cohort study in a university intensive care unit. Demographic, biological, physiologic, and plasma cytokine measurements were obtained. Monocytes were assayed for ex vivo tumor necrosis factor-alpha production, and fresh monocyte mRNA was analyzed by quantitative reverse-transcription polymerase chain reaction for Toll-like receptors, NOD-LRR proteins, cytokines, and nuclear factor-kappaB-related genes. MEASUREMENTS AND MAIN RESULTS: Relative copy numbers for the inflammasome mRNAs for ASC, caspase-1, NALP1, and Pypaf-7 were significantly lower in patients with septic shock compared with critically ill control subjects. NALP1 mRNA levels were linked to survival in patients with sepsis (P = 0.0068) and correlated with SAPS II scores (r = -0.63). CONCLUSIONS: These data suggest that monocyte deactivation occurs during the earliest stages of the systemic inflammatory response and that changes in inflammasome mRNA expression are part of this process.

Circulating Gasdermin-D in Critically Ill Patients.

The key to further improving outcomes in sepsis lies in understanding and abrogating the dysfunctional immune response that leads to organ failure. Activation of gasdermin-D, a pore-forming protein within the inflammasome cascade, has recently been recognized as the critical step in pyroptosis and organ dysfunction. In this study, we sought to investigate the presence of gasdermin-D in critically ill subjects. DESIGN SETTING AND PATIENTS: Prospective pilot study comparing microparticulate active gasdermin-D levels in critically ill patients admitted to the medical ICU at The Ohio State University Medical Center to healthy donors and clinical outcomes. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Plasma was collected from subjects upon consent and microparticles were isolated by ultracentrifugation. Proteins of interest were identified by immunoblot analysis of microparticle lysates. Quantification was accomplished by densitometry using ImageJ software (National Institutes of Health, Bethesda, MD). Investigators were then unblinded and compared microparticulate active gasdermin-D levels to physician adjudicated clinical diagnoses and outcomes. No appreciable levels of active gasdermin-D were observed in microparticles from healthy volunteers and nonseptic critically ill patients. However, elevated levels of gasdermin-D were noted in microparticles from the septic cohort of critically ill patients. Furthermore, a significant positive correlation by linear regression was noted when microparticulate active gasdermin-D levels were compared with microparticulate levels of CD63, an exosomal marker, CD14, a monocyte marker, and CD69, a marker of monocyte activation (R 2 = 0.37, p = 0.0011, R 2 = 0.85, p < 0.0001, and R2 = 0.43, p = 0.0003, respectively). CONCLUSIONS: This is the first study to demonstrate circulating active gasdermin-D in septic patients in the intensive care setting. Our findings also suggest that active gasdermin-D in septic patients is encapsulated in exosomes derived from activated monocytes. Further characterization in the clinical setting is warranted.

Bacterial peptide recognition and immune activation facilitated by human peptide transporter PEPT2.

Microbial detection requires the recognition of pathogen-associated molecular patterns (PAMPs) by pattern recognition receptors (PRRs) that are distributed on the cell surface and within the cytosol. The nucleotide-binding oligomerization domain (NOD)-like receptor (NLR) family functions as an intracellular PRR that triggers the innate immune response. The mechanism by which PAMPs enter the cytosol to interact with NLRs, particularly muropeptides derived from the bacterial proteoglycan cell wall, is poorly understood. PEPT2 is a proton-dependent transporter that mediates the active translocation of di- and tripeptides across epithelial tissues, including the lung. Using computational tools, we initially established that bacterial dipeptides, particularly gamma-D-glutamyl-meso-diaminopimelic acid (gamma-iE-DAP), are suitable substrates for PEPT2. We then determined in primary cultures of human upper airway epithelia and transiently transfected CHO-PEPT2 cell lines that gamma-iE-DAP uptake was mediated by PEPT2 with an affinity constant of approximately 193 microM, whereas muramyl dipeptide was not transported. Exposure to gamma-iE-DAP at the apical surface of differentiated, polarized cultures resulted in activation of the innate immune response in an NOD1- and RIP2-dependent manner, resulting in release of IL-6 and IL-8. Based on these findings we report that PEPT2 plays a vital role in microbial recognition by NLR proteins, particularly with regard to airborne pathogens, thereby participating in host defense in the lung.

A synergistic role for IL-1beta and TNFalpha in monocyte-derived IFNgamma inducing activity.

Although much is known about classic IFNgamma inducers, little is known about the IFNgamma inducing capability of inflammasome-activated monocytes. In this study, supernatants from LPS/ATP-stimulated human monocytes were analyzed for their ability to induce IFNgamma production by KG-1 cells. Unexpectedly, monocyte-derived IFN inducing activity was detected, but it was completely inhibited by IL-1beta, not IL-18 blockade. Moreover, size-fractionation of the monocyte conditioned media dramatically reduced the IFNgamma inducing activity of IL-1beta, suggesting that IL-1beta requires a cofactor to induce IFNgamma production in KG-1 cells. Because TNFalpha is known to synergize with IL-1beta for various gene products, it was studied as the putative IL-1beta synergizing factor. Although recombinant TNFalpha (rTNFalpha) alone had no IFNgamma inducing activity, neutralization of TNFalpha in the monocyte conditioned media inhibited the IFNgamma inducing activity. Furthermore, rTNFalpha restored the IFNgamma inducing activity of the size-fractionated IL-1beta. Finally, rTNFalpha synergized with rIL-1beta, as well as with rIL-1alpha and rIL-18, for KG-1 IFNgamma release. These studies demonstrate a synergistic role between TNFalpha and IL-1 family members in the induction of IFNgamma production and give caution to interpretations of KG-1 functional assays designed to detect functional IL-18.

Francisella induced microparticulate caspase-1/gasdermin-D activation is regulated by NLRP3 independent of Pyrin.

Although the study of pathogen sensing by host defense systems continues to uncover a role for inflammasome components specific to particular pathogens, gaps remain in our knowledge. After internalization, Francisella escapes from the phagosome in mononuclear cells and is thought to be detected by intracellular pathogen-response-receptors pyrin and Aim2 in human and murine models, respectively. However, it remains controversial as to the role of pyrin in detecting Francisella. Our current work aims to study the contribution of inflammasome sensor, Pyrin in regulating microparticulate caspase-1/GSDM-D activation by Francisella. Our findings suggest that NLRP3 is central to the activation/release of active caspase-1/GSDM-D encapsulated in microparticles (MP) by Francisella. We also provide evidence that this regulation is independent of pyrin, implicated in sensing cytosolic Francisella in NLRP3-/- conditions where endogenous Pyrin is present. Absence of NLRP3 completely abrogated Francisella mediated MP caspase-1/GSDM-D activation and release both before and after internalization of the pathogen. However, deletion of pyrin not only enhanced both LPS and Francisella mediated MP active caspase-1/GSDM-D release, but pyrin overexpression resulted in a reduction of inflammasome activation and release; suggesting an inhibitory role of pyrin in LPS and Francisella mediated MP responses. This NLRP3 dependence and inhibitory effect of pyrin correlated with cytokine release as well. These observations also correlated with MPs ability to induce cell death; as LPS and Francisella-induced MPs from pyrin-deficient cells were more potent than wild-type monocytes whereas, NLRP3-/- MPs failed to induce cell death. Taken together, we report that NLPR3 not only mediates Francisella induced cytokine responses, but is also critical for cytokine-independent microparticle-induced inflammasome activation and endothelial cell injury independent of pyrin.