MyD88-dependent inflammasome activation and autophagy inhibition contributes to <i>Ehrlichia</i>-induced liver injury and toxic shock

<div><p>Severe hepatic inflammation is a common cause of acute liver injury following systemic infection with <i>Ehrlichia</i>, 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 <i>Ehrlichia</i> infection. However, the underlying signals and mechanisms that regulate protective immunity and immunopathology during <i>Ehrlichia</i> infection are not well understood. To address this issue, we compared susceptibility to lethal <i>Ixodes ovatus Ehrlichia</i> (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 <i>Ehrlichia</i> infection. This study reveals that <i>Ehrlichia</i>-induced liver injury and toxic shock are mediated by MyD88-dependent inflammasome activation and autophagy inhibition.</p></div

MyD88 inhibits mitophagy in macrophages.

<p><b>(A)</b> Representative confocal immunofluorescence colocalization (yellow) of LC3 (green) and mitochondria (MitoTracker; red), and DAPI nuclear stain (blue) in uninfected (media) and IOE-infected WT and MyD88^-/- BMM at 24h p.i. Cells stimulated with LPS (200ng/mL) were used as positive controls. (B) Quantification of LC3 colocalization with MitoTracker per cell. Data are presented as mean ± SEM from three different experiments. *P<0.05.</p

Schematic representation of MyD88-mediated inflammasome activation and immunopathology during fatal <i>Ehrlichia</i> infection.

<p>Infection of macrophages (main target cells) with virulent LPS-negative <i>Ehrlichia</i> triggers TLR9/MyD88 signaling and increased expression of pro-inflammatory cytokines (e.g.TNF-α) and upregulation of pro-IL-1β and inflammasome components (e.g. NLRP3), as well as induction of IRF7 and production of IFN-I (A-C). Recognition of PAMPs triggers TLR9 signaling and downstream MyD88 adaptor molecule activation, which in turn blocks autophagy induction in macrophages via mTORC1 activation. MyD88-induced block of autophagy can result in defective mitophagy and accumulation of mitochondrial DAMPs secondary to IOE-induced mitochondrial damage (1–4). These PAMPs and mitochondrial DAMPs can further induce TLR9 signaling, in a positive feedback loop (5), as well as activation of canonical (caspase 1 dependent) and non-canonical (caspase 11 dependent) inflammasome pathways (4). In addition, IFN-I produced by infected macrophages binds in an autocrine manner to IFNAR, which in turn induces activation of caspase 11 (D). This causes secretion of IL-1β/IL-1α and pyroptosis of infected macrophages (6). Defective autophagy can negatively influence MHC-II antigen presentation, which would lead to reduced proliferation of CD4⁺ Th1 cells (7). In addition, excessive inflammasome activation and host cell death promote expansion and activation of pathogenic CD8⁺ T cells (7), causing further liver damage.</p

<i>Ehrlichia</i>-induced inflammasome activation is MyD88 dependent.

<p>(A) Western blot analysis of pro- and active/cleaved caspase-1 (p20) in whole liver lysates from uninfected and IOE-infected WT mice, compared with MyD88^-/- mice on day 7 p.i. GAPDH used as loading control. The density of bands in each group quantified and normalized to GAPDH expression. Levels of IL-1β (B) and IL-1α (C) in sera of naïve/uninfected and infected WT or MyD88^-/- mice on day 7 p.i. (D) mRNA expression of caspase-1, IL-1β and caspase-11 in liver tissues at day 7 p.i. in uninfected and IOE-infected WT and MyD88^-/- mice. (E) mRNA expression of NLRP3, NLRC4 and AIM2 in liver at day 7 p.i. WT and MyD88^-/- BMM were infected with IOE and the levels of IL-1β (F) and IL-1α (G) and TNF-α (H) in culture supernatants were measured at 0, 8, 12, and 24h post infection. (I) Expression of pro-caspase-1 and active/cleaved caspase-1 in uninfected and infected WT and MyD88^-/- BMM measured by immunoblotting at 24h p.i. (J) Expression of pro-caspase-11 and active/cleaved caspase-11 in uninfected and IOE-infected WT and MyD88^-/- BMM measured by immunoblotting at 24h p.i. (K) Level of LDH at 24h p.i. in uninfected or IOE-infected WT-BMM cultured in the presence/absence of caspase-1 inhibitor (Inh) or caspase-11 inhibitor (Inh). LPS+ATP were used as positive control. Data from <i>in vivo</i> experiments are from 3 mice/group and representative of three independent experiments. Data from <i>in vitro</i> experiments are representative of three independent experiments. All results presented as mean ± SD (* P<0.05, **P<0.01, ***P<0.001).</p

MyD88 is required for type I IFN-mediated inflammasome activation.

<p>(A) mRNA expression of IRF-7 and IFNβ in uninfected and IOE-infected WT and MyD88^-/- BMM at 24h p.i. (B) Level of IL-1β in cell culture supernatants of uninfected and IOE-infected WT and MyD88^-/- BMM in the presence/absence of IFNβ (500 IU/ml) at 24hr p.i. LPS + ATP is used as positive control. (C) The relative mRNA expression of IFNAR in uninfected and IOE-infected WT and MyD88^-/- BMM normalized to GAPDH at 24h p.i. All results are presented as mean ± SD (* P<0.05, ***P<0.001) from three independent <i>in vitro</i> experiments. ns = not significant.</p

MyD88 inhibits autophagy induction and activates inflammasome via activation of mTORC1 pathway in macrophages.

<p>(A) Representative immunoblots of phosphorylation of S6 (pS6) and total S6 (S6) in whole liver lysates from uninfected and IOE-infected WT or MyD88^-/- mice on day 7 p.i. (B) Representative immunoblots showing expression of 4E-BP1, phospho- S6 (pS6), total S6 (S6), phospho-AKT (pAKT), and total AKT in uninfected and IOE-infected WT and MyD88^-/- BMM. (C) Representative Immunoblots showing phosphorylation of S6 (pS6) in WT-BMM infected with IOE and treated with/without Dynasore (Dyn). pS6 normalized to GAPDH at 24h p.i. (D) Transmission electron microscopy images of WT-BMM infected with IOE and treated with/without Dynasore at 24h p.i. Left two panels shows low magnification (scale bar 2μm), and right two panels show high magnification (scale bar 500nM) magnification, respectively. Arrow in no Dynasore-treated cells indicating <i>Ehrlichia</i> morula. Arrow on Dynasore-treated cell indicates normal mitochondrial morphology. (E) Immunoblotting analysis showing the expression of LC3I and LC3II in untreated and rapamycin-treated, uninfected and IOE infected WT-BMM at 24h p.i. The density of LC3II:LC3I bands were quantified and normalized to GAPDH loading control, and the ratio of normalized LC3II:I band is shown. (F) Transmission electron microscopy of uninfected (left two panels) and IOE-infected (right two panels) WT-BMM at 24h p.i., in the presence or absence of rapamycin showing higher formation of multiple double membrane autophagosomes in both uninfected and infected cells upon rapamycin treatment (Arrows). All panels are of high magnification (scale bar 500nM) (G) Concentrations of IL-1α and IL-1β in culture supernatant from uninfected and IOE-infected WT-BMM in the presence/absence of rapamycin at 24h p.i. LPS + ATP used as positive control. (H) mRNA expression of Caspase-11 and immunoblot of pro- and active/cleaved caspase-1 in uninfected and IOE-infected WT and MyD88^-/- BMMs in the presence/absence of rapamycin at 24h p.i. (I) TNF-α in culture supernatant from uninfected and IOE-infected WT-BMM infected with/without rapamycin treatment at 24h p.i. Negative controls included uninfected cells with or without rapamycin, while positive control included WT-BMM stimulated with LPS and ATP that activates NLRP3 inflammasome. All results are presented as mean ± SD (* P<0.05, **P<0.01, ***P<0.001) from three independent <i>in vitro</i> experiments. ns = not significant. Quantification of immunoblot bands is presented as mean pixel density of bands/GAPDH from three mice/group and is representative of three independent experiments.</p

TLR9-dependent inflammasome and mTORC1 activation in IOE infected macrophages.

<p>A) IL-1β in cell culture supernatants from uninfected and IOE infected (MOI 5) WT, TLR7^-/-, and TLR9^-/- BMM at 24h p.i. (B) Representative Immunoblot analyzing phosphorylation of phospho S6 (pS6) in WT, TLR7^-/-, and TLR9^-/- BMM at 24h p.i. GAPDH used as loading control. (C) Immunoblot analysis of Caspase-1 in WT and TLR9^-/- BMM at 24h p.i. β-actin used as loading control. mRNA expression of Caspase-11 in infected WT and TLR9^-/- BMM and controls at 24h p.i. (D) Survival of uninfected and IOE-infected WT and TLR9^-/- mice. Data showing 85% survival of IOE infected TLR9^-/- mice compared to WT mice till day 60 p.i. (n = 6/group). (E) Representative H&E and TUNEL staining of liver sections from naïve/uninfected and IOE-infected WT and TLR9^-/- mice on day 7 p.i. The insert in the H&E staining of liver section from IOE-infected TLR9^-/- mice demonstrates an enhanced cellular infiltration consistent with regenerative changes. (F) Quantification of TUNEL positive Kupffer cells and hepatocytes per high power field (HPF) in TLR9^-/- and WT mice. Data from <i>in vitro</i> experiments are representative of three independent experiments. All results are presented as mean ± SD (* P<0.05, ***P<0.001).</p