Immune modulating effects of receptor interacting protein 2 (RIP2) in autoinflammation and immunity.

Receptor-interacting protein 2 (RIP2) is a kinase that is involved in downstream signaling of nuclear oligomerization domain (NOD)-like receptors NOD1 and 2 sensing bacterial peptidoglycans. RIP2-deficiency or targeting of RIP2 by pharmaceutical inhibitors partially ameliorates inflammatory diseases by reducing pro-inflammatory signaling in response to peptidoglycans. However, RIP2 is widely expressed and interacts with several other proteins suggesting additional functions outside the NOD-signaling pathway. In this review, we discuss the immunological functions of RIP2 and its possible role in autoinflammation and immunity.

CASP1 variants influence subcellular caspase-1 localization, pyroptosome formation, pro-inflammatory cell death and macrophage deformability.

CASP1 variants result in reduced enzymatic activity of procaspase-1 and impaired IL-1ß release. Despite this, affected individuals can develop systemic autoinflammatory disease. These seemingly contradictory observations have only partially been explained by increased NF-κB activation through prolonged interaction of variant procaspase-1 with RIP2. To identify further disease underlying pathomechanisms, we established an in vitro model using shRNA-directed knock-down of procaspase-1 followed by viral transduction of human monocytes (THP-1) with plasmids encoding for wild-type procaspase-1, disease-associated CASP1 variants (p.L265S, p.R240Q) or a missense mutation in the active center of procaspase-1 (p.C285A). THP1-derived macrophages carrying CASP1 variants exhibited mutation-specific molecular alterations. We here provide in vitro evidence for abnormal pyroptosome formation (p.C285A, p.240Q, p.L265S), impaired nuclear (pro)caspase-1 localization (p.L265S), reduced pro-inflammatory cell death (p.C285A) and changes in macrophage deformability that may contribute to disease pathophysiology of patients with CASP1 variants. This offers previously unknown molecular pathomechanisms in patients with systemic autoinflammatory disease.

Key Factors Regulating the Interdomain Dynamics May Contribute to the Assembly of ASC.

The canonical ASC domains, PYD and CARD, are interconnected by a lengthy, semi-flexible linker. The molecular basis and purpose of ASC's highly dynamic feature remain elusive. In this study, all-atom molecular dynamics simulations were utilized to examine the role of the linker and the interdomain dynamics of the ASC monomer. As revealed in the principal component analysis (PCA), the flexible linker enables interdomain dynamics and rotation. The stumbling between domains is partially attributed to the helical portion of N-terminal residues in the linker. Additionally, the linker exhibits a certain structural preference due to the turn-type structural inclination of the N-terminal and the presence of several prolines on the linker. Such structural preferences lead to the unavailability of regions for PYD type I interactions to CARDs, as evidenced by the CARD spatial restraint analysis. In conclusion, the semi-flexible linker introduces functionally relevant interdomain dynamics, potentially enhancing PYD self-assembly and the subsequent assembly of the inflammasome complex.

Apoptosis-associated speck-like protein containing a CARD-mediated release of matrix metalloproteinase 10 stimulates a change in microglia phenotype.

Inflammation contributes to amyloid-ß and tau pathology in Alzheimer's disease (AD). Microglia facilitate an altered immune response that includes microgliosis, upregulation of inflammasome proteins, and elevation of matrix-metalloproteinases (MMPs). Studies of cerebrospinal fluid (CSF) and blood in dementia patients show upregulation of two potential biomarkers of inflammation at the cellular level, MMP10 and apoptosis-associated speck-like protein containing a CARD (ASC). However, little is known about their relationship in the context of brain inflammation. Therefore, we stimulated microglia cultures with purified insoluble ASC speck aggregates and MMP10 to elucidate their role. We found that ASC specks altered microglia shape and stimulated the release of MMP3 and MMP10. Furthermore, MMP10 stimulated microglia released additional MMP10 along with the inflammatory cytokines, tumor-necrosis factor-α (TNFα), Interleukin 6 (IL-6), and CXCL1 CXC motif chemokine ligand 1 (CXCL1). A broad-spectrum MMP inhibitor, GM6001, prevented TNFα release. With these results, we conclude that MMP10 and ASC specks act on microglial cells to propagate inflammation.

Alendronate Augments Lipid A-Induced IL-1α Release via Activation of ASC but Not Caspase-11.

Nitrogen-containing bisphosphonates (NBPs), such as alendronate (ALN), are anti-bone-resorptive drugs that have inflammatory side effects. We previously reported that ALN augmented lipid A-induced interleukin (IL)-1ß production and NOD-like receptor pyrin domain-containing-3 (NLRP3)/apoptosis-associated speck-like protein containing a CARD (ASC)-dependent cell death. The present study aimed to examine whether ALN augments lipid A-induced IL-1α release and necroptosis, which is induced by the activation of receptor-interacting protein kinase (RIPK) 3. Treatment of J774.1 cells with ALN augmented lipid A-induced IL-1α release, which was not inhibited by Ac-IETD-CHO, a caspase-8 inhibitor. ALN also activated mixed lineage kinase domain-like (MLKL), a key mediator of the necroptosis pathway, and upregulated the expression of caspase-11, a lipid A receptor. GSK'872, a RIPK3 inhibitor, suppressed the ALN-upregulated expression of caspase-11 and augmented lipid A-induced caspase-8 activation. Moreover, ALN induced the release of NLRP3 and ASC into culture supernatants. GSK'872, but not Ac-IETD-CHO, reduced the ALN-induced release of NLRP3, but not ASC, into culture supernatants, and reduced ALN-induced cell death, but not ALN-induced LDH release. Antibodies against NLRP3 and ASC upregulated caspase-11 expression in the cytosol by inhibiting ALN-induced cell death. However, pretreating cells with an antibody against ASC, but not NLRP3, before ALN addition also inhibited lipid A-induced IL-1α release. Pretreating cells with an antibody against caspase-11 before the addition of ALN or lipid A did not downregulate lipid A-induced production of IL-1α. Taken together, our findings suggest that ALN augments lipid A-induced IL-1α release via activation of ASC, but not caspase-11.

In vivo evidence of inflammasome activation during spontaneous labor at term.

OBJECTIVE: Upon inflammasome activation, the adaptor protein of the inflammasome ASC (apoptosis-associated speck-like protein containing a CARD) forms intracellular specks, which can be released into the extracellular space. The objectives of this study were to investigate whether (1) extracellular ASC is present in the amniotic fluid of women who delivered at term; (2) amniotic fluid ASC concentrations are greater in women who underwent spontaneous labor at term than in those who delivered at term in the absence of labor; and (3) amniotic epithelial and mesenchymal cells can form intracellular ASC specks in vitro. METHODS: This retrospective cross-sectional study included amniotic fluid samples from 41 women who delivered at term in the absence of labor (n = 24) or underwent spontaneous labor at term (n = 17). Amniotic epithelial and mesenchymal cells were also isolated from the chorioamniotic membranes obtained from a separate group of women who delivered at term (n = 3), in which ASC speck formation was assessed by confocal microscopy. Monocytes from healthy individuals were used as positive controls for ASC speck formation (n = 3). RESULTS: (1) The adaptor protein of the inflammasome ASC is detectable in the amniotic fluid of women who delivered at term; (2) amniotic fluid ASC concentration was higher in women who underwent spontaneous labor at term than in those who delivered at term without labor; and (3) amniotic epithelial and mesenchymal cells are capable of forming ASC specks and/or filaments in vitro. CONCLUSION: Amniotic fluid ASC concentrations are increased in women who undergo spontaneous labor at term. Amniotic epithelial and mesenchymal cells are capable of forming ASC specks, suggesting that these cells are a source of extracellular ASC in the amniotic fluid. These findings provide in vivo evidence that there is inflammasome activation in the amniotic cavity during the physiological process of labor at term.

Inflammasome assembly in the chorioamniotic membranes during spontaneous labor at term.

PROBLEM: Inflammasome activation requires two steps: priming and assembly of the multimeric complex. The second step includes assembly of the sensor molecule and adaptor protein ASC (an apoptosis-associated speck-like protein containing a CARD), which results in ASC speck formation and the recruitment of caspase (CASP)-1. Herein, we investigated whether there is inflammasome assembly in the chorioamniotic membranes and choriodecidual leukocytes from women who underwent spontaneous labor at term. METHOD OF STUDY: Using in situ proximity ligation assays, ASC/CASP-1 complexes were determined in the chorioamniotic membranes from women who delivered at term without labor or underwent spontaneous labor at term with or without acute histologic chorioamnionitis (n=10-11 each). Also, ASC speck formation was determined by flow cytometry in the choriodecidual leukocytes isolated from women who delivered at term with or without spontaneous labor (n=9-12 each). RESULTS: (i) ASC/CASP-1 complexes were detected in the chorioamniotic membranes; (ii) ASC/CASP-1 complexes were greater in the chorioamniotic membranes from women who underwent spontaneous labor at term than in those without labor; (iii) ASC/CASP-1 complexes were even more abundant in the chorioamniotic membranes from women who underwent spontaneous labor at term with acute histologic chorioamnionitis than in those without this placental lesion; (iv) ASC speck formation was detected in the choriodecidual leukocytes; and (v) ASC speck formation was greater in the choriodecidual leukocytes isolated from women who underwent spontaneous labor at term than in those without labor. CONCLUSION: There is inflammasome assembly in the chorioamniotic membranes and choriodecidual leukocytes during spontaneous labor at term.

A Role for the Inflammasome in Spontaneous Preterm Labor With Acute Histologic Chorioamnionitis.

Inflammasomes are cytosolic multiprotein complexes that orchestrate inflammation in response to pathogens and endogenous danger signals. Herein, we determined whether the chorioamniotic membranes from women in spontaneous preterm labor with acute histologic chorioamnionitis (1) express major inflammasome components; (2) express caspase (CASP)-1 and CASP-4 as well as their active forms; (3) exhibit apoptosis-associated speck-like protein containing a CARD (ASC)/CASP-1 complex formation; and (4) release the mature forms of interleukin (IL)-1ß and IL-18. We utilized quantitative reverse transcription polymerase chain reaction, enzyme-linked immunosorbent assay, immunoblotting, and immunohistochemistry to determine the messenger RNA (mRNA) and protein expression of major inflammasome components, nucleotide-binding oligomerization domain (NOD) proteins, and the pro- and mature/active forms of CASP-1, CASP-4, IL-1ß, and IL-18. The ASC/CASP-1 complex formation was determined using an in situ proximity ligation assay. When comparing the chorioamniotic membranes from women in spontaneous preterm labor with acute histologic chorioamnionitis to those without this placental lesion, we found that (1) the mRNA of NLR family pyrin domain-containing protein ( NLRP) 1, NLRP3, NLR family CARD domain-containing protein 4 ( NLRC4), and NOD2 were higher; (2) the NLRP3 protein was increased; (3) the mRNA and active form (p10) of CASP-1 were greater; (4) the mRNA and active form of CASP-4 were increased; (5) the mRNA and mature form of IL-1ß were higher; (6) the mature form of IL-18 was elevated; and (7) ASC/CASP-1 complex formation was increased. In conclusion, spontaneous preterm labor with acute histologic chorioamnionitis is characterized by an upregulation of NLRP3 and the active form of CASP-4, as well as increased ASC/CASP-1 complex formation, which may participate in the activation of CASP-1 and the maturation of IL-1ß and IL-18 in the chorioamniotic membranes. These findings provide the first evidence that supports a role for the inflammasome in the pathological inflammation implicated in spontaneous preterm labor with acute histologic chorioamnionitis.