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MyD88-dependent inflammasome activation and autophagy inhibition contributes to Ehrlichia-induced liver injury and toxic shock.

ABSTRACT: Severe hepatic inflammation is a common cause of acute liver injury following systemic infection with Ehrlichia, obligate Gram-negative intracellular bacteria that lack lipopolysaccharide (LPS). We have previously shown that type I IFN (IFN-I) and inflammasome activation are key host-pathogenic mediators that promote excessive inflammation and liver damage following fatal Ehrlichia infection. However, the underlying signals and mechanisms that regulate protective immunity and immunopathology during Ehrlichia infection are not well understood. To address this issue, we compared susceptibility to lethal Ixodes ovatus Ehrlichia (IOE) infection between wild type (WT) and MyD88-deficient (MyD88-/-) mice. We show here that MyD88-/- mice exhibited decreased inflammasome activation, attenuated liver injury, and were more resistant to lethal infection than WT mice, despite suppressed protective immunity and increased bacterial burden in the liver. MyD88-dependent inflammasome activation was also dependent on activation of the metabolic checkpoint kinase mammalian target of rapamycin complex 1 (mTORC1), inhibition of autophagic flux, and defective mitophagy in macrophages. Blocking mTORC1 signaling in infected WT mice and primary macrophages enhanced bacterial replication and attenuated inflammasome activation, suggesting autophagy promotes bacterial replication while inhibiting inflammasome activation. Finally, our data suggest TLR9 and IFN-I are upstream signaling mechanisms triggering MyD88-mediated mTORC1 and inflammasome activation in macrophages following Ehrlichia infection. This study reveals that Ehrlichia-induced liver injury and toxic shock are mediated by MyD88-dependent inflammasome activation and autophagy inhibition.

SUBMITTER: Kader M

PROVIDER: S-EPMC5663626 | biostudies-literature | 2017 Oct

REPOSITORIES: biostudies-literature

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MyD88-dependent inflammasome activation and autophagy inhibition contributes to Ehrlichia-induced liver injury and toxic shock.

Kader Muhamuda M Alaoui-El-Azher Mounia M Vorhauer Jennie J Kode Bhushan B BB Wells Jakob Z JZ Stolz Donna D Michalopoulos George G Wells Alan A Scott Melanie M Ismail Nahed N

PLoS pathogens 20171019 10

Severe hepatic inflammation is a common cause of acute liver injury following systemic infection with Ehrlichia, obligate Gram-negative intracellular bacteria that lack lipopolysaccharide (LPS). We have previously shown that type I IFN (IFN-I) and inflammasome activation are key host-pathogenic mediators that promote excessive inflammation and liver damage following fatal Ehrlichia infection. However, the underlying signals and mechanisms that regulate protective immunity and immunopathology dur ...[more]

PMID: 29049365

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Project description:Hepatocytes play a crucial role in host response to infection. Ehrlichia is an obligate intracellular bacterium that causes potentially life-threatening human monocytic ehrlichiosis (HME) characterized by an initial liver injury followed by sepsis and multi-organ failure. We previously showed that infection with highly virulent Ehrlichia japonica (E. japonica) induces liver damage and fatal ehrlichiosis in mice via deleterious MyD88-dependent activation of CASP11 and inhibition of autophagy in macrophage. While macrophages are major target cells for Ehrlichia, the role of hepatocytes (HCs) in ehrlichiosis remains unclear. We investigated here the role of MyD88 signaling in HCs during infection with E. japonica using primary cells from wild-type (WT) and MyD88-/- mice, along with pharmacologic inhibitors of MyD88 in a murine HC cell line. Similar to macrophages, MyD88 signaling in infected HCs led to deleterious CASP11 activation, cleavage of Gasdermin D, secretion of high mobility group box 1, IL-6 production, and inflammatory cell death, while controlling bacterial replication. Unlike macrophages, MyD88 signaling in Ehrlichia-infected HCs attenuated CASP1 activation but activated CASP3. Mechanistically, active CASP1/canonical inflammasome pathway negatively regulated the activation of CASP3 in infected MyD88-/- HCs. Further, MyD88 promoted autophagy induction in HCs, which was surprisingly associated with the activation of the mammalian target of rapamycin complex 1 (mTORC1), a known negative regulator of autophagy. Pharmacologic blocking mTORC1 activation in E. japonica-infected WT, but not infected MyD88-/- HCs, resulted in significant induction of autophagy, suggesting that MyD88 promotes autophagy during Ehrlichia infection not only in an mTORC1-indpenedent manner, but also abrogates mTORC1-mediated inhibition of autophagy in HCs. In conclusion, this study demonstrates that hepatocyte-specific regulation of autophagy and inflammasome pathway via MyD88 is distinct than MyD88 signaling in macrophages during fatal ehrlichiosis. Understanding hepatocyte-specific signaling is critical for the development of new therapeutics against liver-targeting pathogens such as Ehrlichia.

Project description:Although the detrimental effects of diabetes mellitus/hyperglycemia have been observed in many liver disease models, the function and mechanism of hyperglycemia regulating liver-resident macrophages, Kupffer cells (KCs), in thioacetamide (TAA)-induced liver injury remain largely unknown. In this study, we evaluated the role of hyperglycemia in regulating NOD-like receptor family pyrin domain-containing 3 protein (NLRP3) inflammasome activation by inhibiting autophagy induction in KCs in the TAA-induced liver injury model. Type I diabetes/hyperglycemia was induced by streptozotocin treatment. Compared with the control group, hyperglycemic mice exhibited a significant increase in intrahepatic inflammation and liver injury. Enhanced NLRP3 inflammasome activation was detected in KCs from hyperglycemic mice, as shown by increased gene induction and protein levels of NLRP3, cleaved caspase-1, apoptosis-associated speck-like protein containing a caspase recruitment domain and interleukin-1?, compared with control mice. NLRP3 inhibition by its antagonist CY-09 effectively suppressed inflammasome activation in KCs and attenuated liver injury in hyperglycemic mice. Furthermore, inhibited autophagy activation was revealed by transmission electron microscope detection, decreased LC3B protein expression and p-62 protein degradation in KCs isolated from TAA-stressed hyperglycemic mice. Interestingly, inhibited 5' AMP-activated protein kinase (AMPK) but enhanced mammalian target of rapamycin (mTOR) activation was found in KCs from TAA-stressed hyperglycemic mice. AMPK activation by its agonist 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) or mTOR signaling knockdown by small interfering RNA restored autophagy activation, and subsequently, inhibited NLRP3 inflammasome activation in KCs, leading to ultimately reduced TAA-induced liver injury in the hyperglycemic mice. Our findings demonstrated that hyperglycemia aggravated TAA-induced acute liver injury by promoting liver-resident macrophage NLRP3 inflammasome activation via inhibiting AMPK/mTOR-mediated autophagy. This study provided a novel target for prevention of toxin-induced acute liver injury under hyperglycemia.

Project description:IFN?I signaling exacerbate liver pathology and contribute to sepsis following fatal Ehrlichia infection by promoting Ehrlichia replication and mediating detrimental inflammasome activation. Inflammasomes are an important innate immune host defense against intracellular microbial infection. Activation of inflammasomes by microbial or host ligands results in cleavage of caspase?1 (canonical pathway) or caspase?11 (noncanonical pathway), release of interleukin (IL)?1?, IL?18, high mobility group box 1 (HMGB1), and inflammatory cell death known as pyroptosis. Ehrlichia are obligate, intracellular, gram?negative bacteria that lack lipopolysaccharide but cause potentially life?threatening monocytic ehrlichiosis in humans and mice that is characterized by liver injury followed by sepsis and multiorgan failure. Employing murine models of mild and fatal ehrlichiosis caused by infection with mildly and highly virulent Ehrlichia muris (EM) and Ixodes ovatus Ehrlichia (IOE), respectively, we have previously shown that IOE infection triggers type I interferon (IFN?I) response and deleterious caspase?11 activation in liver tissues, which promotes liver injury and sepsis. In this study, we examined the contribution of IFN?I signaling in hepatocytes (HCs) to Ehrlichia?induced liver injury. Compared to EM infection, we found that IOE enter and replicate in vitro cultured primary murine HCs and induce secretion of IFN? and several chemokines, including regulated upon activation, normal T?cell expressed, and secreted (RANTES), monocyte chemoattractant protein 1 (MCP1), monokine induced by gamma (MIG)/chemokine (C?X?C motif) ligand 9 (CXCL9), macrophage inflammatory protein 1 alpha (MIP1?), keratinocyte?derived chemokine (KC), and granulocyte?macrophage colony?stimulating factor (GM?CSF). Notably, in vitro stimulation of uninfected and Ehrlichia?infected HCs with recombinant IFN? triggered activation of caspase?1/11, cytosolic translocation of HMGB1, and enhanced autophagy and intracellular bacterial replication. Secretion of HMGB1 by IOE?infected HCs was dependent on caspase?11. Primary HCs from IOE? but not EM?infected mice also expressed active caspase?1/11. Conclusion: HC?specific IFN?I signaling may exacerbate liver pathology during infection with obligate intracellular Ehrlichia by promoting bacterial replication and detrimental caspase?11?mediated inflammasome activation.

Project description:BackgroundThe NLRP3 inflammasome is a multiprotein complex that regulates the innate immune inflammatory response by activating caspase-1 and subsequent IL-1β and IL-18. Fluoxetine has been shown to have the anti-inflammatory properties in many disease models. However, the effects and mechanisms of these effects of fluoxetine in early brain injury after subarachnoid hemorrhage (SAH) have not been defined.MethodsThe SAH model was induced by an endovascular perforation in adult male Sprague-Dawley (SD) rats weighing 300-320 g. N-Ac-Tyr-Val-Ala-Asp-chloromethyl ketone (AC-YVAD-CMK) was injected intraperitoneally (5 mg/kg) 1 h after SAH. Fluoxetine was administered via intravenous route 6 h after SAH. 3-Methyladenine (3-MA) was intracerebroventricularly injected 20 min before SAH. SAH grade, neurological function, brain water content, propidium iodide (PI) staining, western blot, double immunostaining, and transmission electron microscopy were performed.ResultsExpression of caspase-1 increased and peaked at 24 h after SAH. Caspase activation was along with the increased necrotic cells, which occurred mainly in neurons. Necrotic cell death of microglia and astrocyte were also found. Administration of AC-YVAD-CMK, a caspase-1 inhibitor, reduced the expression of IL-1β and IL-18 and the number of PI-positive cells, attenuated brain edema, and improved neurological function, which was also observed in fluoxetine-treated rats. Furthermore, fluoxetine treatment significantly decreased the expression of NLRP3 and cleaved caspase-1 and upregulated the expression of beclin-1, a marker for autophagy. Finally, the effects of fluoxetine in NLRP3 inflammasome activation were reversed by additional 3-MA administration.ConclusionsTogether, our present study indicated that NLRP3 inflammasome and caspase-1 activation play a deleterious role in early brain injury and fluoxetine mitigates NLRP3 inflammasome and caspase-1 activation through autophagy activation after SAH, providing a potential therapeutic agent for SAH treatment.

Dataset Information

MyD88-dependent inflammasome activation and autophagy inhibition contributes to Ehrlichia-induced liver injury and toxic shock.

Publications

MyD88-dependent inflammasome activation and autophagy inhibition contributes to Ehrlichia-induced liver injury and toxic shock.

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