Activation of the Absent in Melanoma 2 Inflammasome in Peripheral Blood Mononuclear Cells From Idiopathic Pulmonary Fibrosis Patients Leads to the Release of Pro-Fibrotic Mediators.
ABSTRACT: Idiopathic pulmonary fibrosis (IPF) is a chronic fibro-proliferative disease characterized by poor prognosis, with a mean survival of ~2-3?years after definite diagnosis. The cause of IPF is still unknown but it is a heterogeneous condition in which the aberrant deposition of extracellular matrix leads to extensive lung remodeling. This remodeling is a consequence of inflammatory responses, but the mechanisms involved are poorly understood. In this study, we first analyzed a bleomycin-induced mouse model, which showed that higher expression of IL-1?, but not IL-18, was correlated to pulmonary cell infiltration and fibrosis. Then, we found that peripheral blood mononuclear cells (PBMCs) from IPF patients released IL-1? and IL-18 in a NLRP3- and calpain-independent manner after LPS?±?ATP stimulation. Instead, the activation of the absent in melanoma 2 (AIM2) inflammasome induced the release of IL-1? in a caspase-1-/caspase-8-independent manner; whereas IL-18 release was caspase-1 dependent. These effects correlated with the release of the pro-fibrotic TGF-?, which was induced by AIM2 activation in a caspase-1- and TLR4-independent manner, but dependent on IL-1?. In this context, the activation of AIM2 induced the release of caspase-4 from IPF-derived PBMCs, which correlated with the mRNA levels of this caspase that was higher in IPF than in healthy PBMCs. In conclusion, our findings identify a novel molecular mechanism whereby the activation of AIM2 could lead to the activation of the non-canonical inflammasome (caspase-4 dependent) that induces the release of IL-1? responsible for the release of TGF-? from PBMCs of IPF patients.
Project description:Idiopathic pulmonary fibrosis (IPF) has been linked to chronic lung inflammation. Drosha ribonuclease III (DROSHA), a class 2 ribonuclease III enzyme, plays a key role in microRNA (miRNA) biogenesis. However, the mechanisms by which DROSHA affects the lung inflammation during idiopathic pulmonary fibrosis (IPF) remain unclear. Here, we demonstrate that DROSHA regulates the absent in melanoma 2 (AIM2) inflammasome activation during idiopathic pulmonary fibrosis (IPF). Both DROSHA and AIM2 protein expression were elevated in alveolar macrophages of patients with IPF. We also found that DROSHA and AIM2 protein expression were increased in alveolar macrophages of lung tissues in a mouse model of bleomycin-induced pulmonary fibrosis. DROSHA deficiency suppressed AIM2 inflammasome-dependent caspase-1 activation and interleukin (IL)-1β and IL-18 secretion in primary mouse alveolar macrophages and bone marrow-derived macrophages (BMDMs). Transduction of microRNA (miRNA) increased the formation of the adaptor apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) specks, which is required for AIM2 inflammasome activation in BMDMs. Our results suggest that DROSHA promotes AIM2 inflammasome activation-dependent lung inflammation during IPF.
Project description:Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive interstitial lung disease. Chronic lung inflammation is linked to the pathogenesis of IPF. DROSHA, a class 2 ribonuclease III enzyme, has an important role in the biogenesis of microRNA (miRNA). The function of miRNAs has been identified in the regulation of the target gene or protein related to inflammatory responses via degradation of mRNA or inhibition of translation. The absent-in-melanoma-2 (AIM2) inflammasome is critical for inflammatory responses against cytosolic double stranded DNA (dsDNA) from pathogen-associated molecular patterns (PAMPs) and self-DNA from danger-associated molecular patterns (DAMPs). The AIM2 inflammasome senses double strand DNA (dsDNA) and interacts with the adaptor apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), which recruits pro-caspase-1 and regulates the maturation and secretion of interleukin (IL)-1? and IL-18. A recent study showed that inflammasome activation contributes to lung inflammation and fibrogenesis during IPF. In the current review, we discuss recent advances in our understanding of the DROSHA-miRNA-AIM2 inflammasome axis in the pathogenesis of IPF.
Project description:Chronic obstructive pulmonary disease (COPD) is now the fourth-leading cause of death worldwide and its prevalence is increasing. The progressive decline of lung function and airway remodelling are a consequence of chronic inflammatory responses. It was recently postulated the involvement of the inflammasome in COPD, although the underlying mechanism/s still need to be elucidated. Therefore, we isolated peripheral blood mononuclear cells (PBMCs) from exacerbated/unstable COPD patients. The stimulation of PBMCs with an AIM2 inflammasome activator, Poly dA:dT, led to IL-1?, but not IL-1?, release. The release of this cytokine was caspase-1- and caspase-4-dependent and correlated to higher levels of 8-OH-dG in COPD compared to non-smoker and smoker-derived PBMCs. Interestingly, AIM2-depedent IL-1? release was responsible for higher TGF-? levels, crucial mediator during pro-fibrotic processes associated to COPD progression. In conclusion, our data highlight the involvement of AIM2/caspase-1/caspase-4 in IL-1?-induced TGF-? release in unstable COPD-derived PBMCs, opening new therapeutic perspectives for unstable COPD patients.
Project description:BACKGROUND:Human immunodeficiency virus (HIV) promotes an inflammatory process, leading to the progressive loss of the functional capacity of the immune system. The HIV infection induces alterations in several tissues, but mainly in the gut-associated lymphoid tissue (GALT). However, the degree of GALT deterioration varies among infected individuals. In fact, it has been shown that HIV-controllers, who spontaneously control viral replication, exhibit a lower inflammatory response, and a relative normal frequency and function of most of the immune cells. Inflammasomes are molecular complexes involved in the inflammatory response, being NLRP1, NLRP3, NLRC4, AIM2 and Pyrin inflammasomes, the best characterized so far. These complexes regulate the maturation of cytokines of the IL-1 family, including IL-1? and IL-18. These cytokines have been associated with immune activation and expansion of HIV target cells, promoting viral replication. Interesting, some reports indicate that HIV induces the activation of the NLRP3 inflammasome, but the role of this, and other inflammasomes during HIV infection, especially in GALT, remains unclear. OBJECTIVE:To compare the relative expression of inflammasome components and the proinflammatory response related to their activity, between HIV-progressors and HIV-controllers. METHODS:GALT biopsies and peripheral blood mononuclear cells (PBMCs) from 15 HIV-controllers and 15 HIV-progressors were obtained. The relative expression of the following inflammasome components were evaluated by RT-PCR: NLRP3, NLRC4, NLRP1, AIM2, ASC, Caspase-1, IL-1? and IL-18. In addition, plasma concentration of IL-18 was evaluated as an indicator of baseline proinflammatory status. Finally, in supernatants of PBMCs in vitro stimulated with inflammasome agonists, the concentrations of IL-1? and IL-18 were quantified by ELISA. RESULTS:HIV-progressors exhibited higher expression of IL-1?, IL-18 and caspase-1 genes in GALT and PBMCs compared with HIV-controllers. In addition, HIV-progressors had also increased expression of ASC in PBMCs. When plasma levels were evaluated, IL-18 was increased in HIV-progressors. Interesting, these patients also showed an increased production of IL-1? in supernatants of PBMCs stimulated in vitro with the agonists of AIM2, NLRP1 and NLRC4 inflammasomes. Finally, the expression of caspase-1, NLRP1, IL-1? and IL-18 in GALT or peripheral blood was correlated with CD4+ T-cell count and viral load. CONCLUSION:Our results suggest that during HIV-infection, the required signals to induce the expression of different components of the inflammasomes are produced, both in GALT and in periphery. The activation of these molecular complexes could increase the number of target cells, favoring HIV replication and cell death, promoting the disease progression.
Project description:BACKGROUND:HIV-1 induces an uncontrolled inflammatory response of several immune components, such as inflammasomes. These molecular complexes, associated with Toll-like receptor (TLR) activity, induce the maturation and release of IL-1β and IL-18 and eventually induce pyroptosis. It has been previously demonstrated that HIV induces inflammasome activation, which is significantly lower in the gastrointestinal tissue and blood from people living with HIV-1 with spontaneous control of viral replication. Therefore, immunomodulatory agents could be useful in improving HIV prognosis. OBJECTIVE:To evaluate the potential inhibitory effect of sulfasalazine (SSZ) on inflammasomes and TLRs in peripheral blood mononuclear cells (PBMCs) from people living with HIV and healthy donors. METHODS:PBMCs were obtained from 15 people living with HIV and 15 healthy donors. Cells were stimulated with agonists of TLRs and inflammasomes and subsequently treated with SSZ. The concentration of IL-1β and the relative expression of NLRP3, NLRC4, NLRP1, AIM2, ASC, Caspase-1, IL-1β, and IL-18 were quantified. RESULTS:Cells treated with SSZ exhibited a decreased IL-1β production after inflammasome and TLR stimulation, as well as regulation of inflammasome-related genes, in both people with HIV and healthy individuals. The concentration of IL-1β was positively correlated with the CD4+ T-cell count and negatively with the viral load. CONCLUSION:Our results suggest that SSZ has an immunomodulatory effect on inflammasome and TLR activation that depends on the clinical HIV status.
Project description:The inflammasome is an intracellular multi-protein complex that orchestrates the release of the pro-inflammatory cytokines IL-1? and IL-18, and a form of cell death known as pyroptosis. Tyrosine phosphorylation of the inflammasome sensors NLRP3, AIM2, NLRC4, and the adaptor protein, apoptosis-associated speck-like protein (ASC) has previously been demonstrated to be essential in the regulation of the inflammasome. By using the pharmacological protein tyrosine phosphatase (PTPase) inhibitor, phenylarsine oxide (PAO), we have demonstrated that tyrosine dephosphorylation is an essential step for the activation of the NLRP3 and AIM2 inflammasomes in human and murine macrophages. We have also shown that PTPase activity is required for ASC nucleation leading to caspase-1 activation, IL-1?, and IL-18 processing and release, and cell death. Furthermore, by site-directed mutagenesis of ASC tyrosine residues, we have identified the phosphorylation of tyrosine Y60 and Y137 of ASC as critical for inflammasome assembly and function. Therefore, we report that ASC tyrosine dephosphorylation and phosphorylation are crucial events for inflammasome activation.
Project description:The inflammasome activates caspase-1 and the release of interleukin-1? (IL-1?) and IL-18, and several inflammasomes protect against intestinal inflammation and colitis-associated colon cancer (CAC) in animal models. The absent in melanoma 2 (AIM2) inflammasome is activated by double-stranded DNA, and AIM2 expression is reduced in several types of cancer, but the mechanism by which AIM2 restricts tumor growth remains unclear. We found that Aim2-deficient mice had greater tumor load than Asc-deficient mice in the azoxymethane/dextran sodium sulfate (AOM/DSS) model of colorectal cancer. Tumor burden was also higher in Aim2(-/-)/Apc(Min/+) than in APC(Min/+) mice. The effects of AIM2 on CAC were independent of inflammasome activation and IL-1? and were primarily mediated by a non-bone marrow source of AIM2. In resting cells, AIM2 physically interacted with and limited activation of DNA-dependent protein kinase (DNA-PK), a PI3K-related family member that promotes Akt phosphorylation, whereas loss of AIM2 promoted DNA-PK-mediated Akt activation. AIM2 reduced Akt activation and tumor burden in colorectal cancer models, while an Akt inhibitor reduced tumor load in Aim2(-/-) mice. These findings suggest that Akt inhibitors could be used to treat AIM2-deficient human cancers.
Project description:Inflammasomes regulate the activity of caspase-1 and the maturation of interleukin 1beta (IL-1beta) and IL-18. AIM2 has been shown to bind DNA and engage the caspase-1-activating adaptor protein ASC to form a caspase-1-activating inflammasome. Using Aim2-deficient mice, we identify a central role for AIM2 in regulating caspase-1-dependent maturation of IL-1beta and IL-18, as well as pyroptosis, in response to synthetic double-stranded DNA. AIM2 was essential for inflammasome activation in response to Francisella tularensis, vaccinia virus and mouse cytomegalovirus and had a partial role in the sensing of Listeria monocytogenes. Moreover, production of IL-18 and natural killer cell-dependent production of interferon-gamma, events critical in the early control of virus replication, were dependent on AIM2 during mouse cytomegalovirus infection in vivo. Collectively, our observations demonstrate the importance of AIM2 in the sensing of both bacterial and viral pathogens and in triggering innate immunity.
Project description:The DNA sensor absent in melanoma 2 (AIM2) forms an inflammasome complex with ASC and caspase-1 in response to Francisella tularensis subspecies novicida infection, leading to maturation of IL-1? and IL-18 and pyroptosis. AIM2 is critical for host protection against F. novicida infection in vivo; however, the role of pyroptosis downstream of the AIM2 inflammasome is unknown. Recent studies have identified gasdermin D (GSDMD) as the molecule executing pyroptosis by forming pores on the plasma membrane following activation by inflammatory caspase-1 and -11. In this study, we report that GSDMD-deficient mice were susceptible to F. novicida infection compared with wild type mice. Interestingly, we observed that GSDMD is required for optimal caspase-1 activation and pyroptotic cell death in F. novicida-infected bone marrow-derived macrophages. Furthermore, caspase-1 activation was compromised in bone marrow-derived macrophages lacking GSDMD stimulated with other AIM2 inflammasome triggers, including poly(dA:dT) transfection and mouse CMV infection. Overall, our study highlights a function, to our knowledge previously unknown, for GSDMD in promoting caspase-1 activation by AIM2 inflammasome.
Project description:Macrophages respond to cytosolic nucleic acids by activating cysteine protease caspase-1 within a complex called the inflammasome. Subsequent cleavage and secretion of proinflammatory cytokines IL-1beta and IL-18 are critical for innate immunity. Here, we show that macrophages from mice lacking absent in melanoma 2 (AIM2) cannot sense cytosolic double-stranded DNA and fail to trigger inflammasome assembly. Caspase-1 activation in response to intracellular pathogen Francisella tularensis also required AIM2. Immunofluorescence microscopy of macrophages infected with F. tularensis revealed striking colocalization of bacterial DNA with endogenous AIM2 and inflammasome adaptor ASC. By contrast, type I IFN (IFN-alpha and -beta) secretion in response to F. tularensis did not require AIM2. IFN-I did, however, boost AIM2-dependent caspase-1 activation by increasing AIM2 protein levels. Thus, inflammasome activation was reduced in infected macrophages lacking either the IFN-I receptor or stimulator of interferon genes (STING). Finally, AIM2-deficient mice displayed increased susceptibility to F. tularensis infection compared with wild-type mice. Their increased bacterial burden in vivo confirmed that AIM2 is essential for an effective innate immune response.