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. 2023 Nov 7:14:1212167.
doi: 10.3389/fimmu.2023.1212167. eCollection 2023.

Hepatocyte-specific regulation of autophagy and inflammasome activation via MyD88 during lethal Ehrlichia infection

Omid Teymournejad  1 , Aditya Kumar Sharma  1 , Mohammed Abdelwahed  1   2 , Muhamuda Kader  3 , Ibrahim Ahmed  1 , Hoda Elkafas  1 , Nahed Ismail  1
Affiliations

Hepatocyte-specific regulation of autophagy and inflammasome activation via MyD88 during lethal Ehrlichia infection

Omid Teymournejad et al. Front Immunol. .

Abstract

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.

Keywords: Ehrlichia spp.; HMGB1; MyD88; autophagy; inflammasome.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Ehrlichia replication in hepatocytes is regulated by MyD88. (A) Absolute copy number of intracellular bacteria in primary E japonica-infected WT and MyD88-/- HCs at 12 and 24 hr p.i. via amplification of dsb gene. (B) Relative expression of E japonica 16s rRNA normalized to GAPDH to determine live replicative Ehrlichia in primary WT and MyD88-/- HCs in the presence/absence of E japonica infection at 12 and 24 hr p.i. (C) Relative expression of 16s rRNA, normalized to GAPDH, in uninfected and E japonica-infected HCs in the presence/absence of MyD88 inhibitor at 24 hr p.i. Data are presented as Mean ± SD from three different experiments. (*P<0.05, **P<0.01, ***P<0.001).
Figure 2
Figure 2
CASP1, in HCs, is negatively regulated by MyD88 in fatal infection. (A, B) Immunoblot of pro and cleaved CASP1 in uninfected and E japonica-infected primary WT and MyD88-/- HCs at 24 hr p.i. (C) Relative mRNA expression of Casp1 in uninfected and infected primary WT and MyD88-/- HCs at 24 hr p.i. (D) Relative mRNA expression of IL-1β in uninfected and infected primary WT and MyD88-inhibited HCs at 24 hr p.i. Data is from three independent experiments represented as Mean ± SD (**P<0.01, ***P<0.001).
Figure 3
Figure 3
CASP 3 and CASP11, in HCs, is positively regulated by MyD88 in fatal infection. (A, B) Immunoblot analysis of cleaved CASP3 protein at 24 hr p.i., normalized to β-actin, in uninfected and E japonica-infected primary WT and MyD88-/- HCs cultured in the presence or absence of CASP1 inhibitor. (C) Relative mRNA expression of active CASP11 production in uninfected and E japonica-infected primary WT and MyD88-inhibited HCs at 24 hr p.i. (D, E) Immunoblot of pro and cleaved CASP11 in uninfected and E japonica-infected primary WT and MyD88-/- HCs at 24 hr p.i. (F, G) Immunoblot analysis of pro and cleaved Gasdermin (GSDM) D protein normalized to β-actin in uninfected and E japonica-infected primary WT and MyD88-/- HCs in the presence/absence of CASP1 and 11 inhibitor at 24 hr p.i. (H) LDH percentage release in uninfected and E japonica-infected HCs in the presence/absence of MyD88 inhibitor at 24 hr p.i. All results are presented as mean ± SD (*P<0.05, **P<0.01, ***P<0.001) from three independent experiments. ns, not significant.
Figure 4
Figure 4
Effects of MyD88 signaling on HMGB1 nuclear translocation and extracellular secretion. (A) Immunofluorescence staining showing HMGB1 accumulation in uninfected and E japonica-infected primary WT and MyD88-/- HCs at 24 hr p.i. (B) Quantitative analysis showing the percentage of HMGB1 in the nucleus in uninfected and E japonica-infected primary WT and MyD88-/- HCs (C) Western blot of HMGB1 proteins levels in uninfected and E japonica-infected primary WT and MyD88-/- HCs at 24 hr p.i. (D) Normalized level of total HMGB1 in relation to GAPDH (E) ELISA of HMGB1 levels in the culture supernatant of uninfected and E japonica-infected HCs in the presence/absence of MyD88 inhibitor at 24 hr p.i. (F) Relative mRNA expression of RAGE receptor in uninfected and E japonica-infected HCs in the presence/absence of MyD88 inhibitor at 24 hr p.i. All in vitro experiments are done thrice and are independent, represented as Mean ± SD (*P<0.05, **P<0.01). ns, not significant.
Figure 5
Figure 5
MyD88 mediates the induction of autophagy in HCs. (A) Immunoblot of LC3I and LC3II in uninfected and E japonica-infected primary WT and MyD88-/- HCs at 24 hr p.i. (B) Analyzing LC3II/LC3I ratio in uninfected and E japonica-infected WT and MyD88-/- HCs at 24 hr p.i. (C) Immunoblot of p62 in uninfected and E japonica-infected HCs WT and MyD88-/- HCs at 24 hr p.i. (D) Relative mRNA expression of p62 in uninfected and E japonica-infected HCs in WT and MyD88-/- HCs at 24 hr p.i. Data is from three independent experiments represented as Mean ± SD (*P<0.05, **P<0.01, ****P<0.0001). ns, not significant.
Figure 6
Figure 6
MyD88 compensates for the inhibition of autophagy and mTORC1 activation in HCs. (A) Immunoblot of pS6 in uninfected and E japonica-infected HCs in the presence/absence of MyD88 inhibitor at 24 hr p.i. (B) Immunoblot of pS6 activation in uninfected and E japonica-infected HCs with or without MyD88 inhibitor and with or without rapamycin treatment at 24 hr p.i. (C, D) Immunoblot of LC3I and LC3II in uninfected and E.japonica-infected HCs with/without MyD88 inhibitor and rapamycin at 24 hr p.i. Data is from three independent experiments represented as Mean ± SD (*P<0.05, **P<0.01, ***P<0.001).
Figure 7
Figure 7
Proposed model of the role of MyD88 in the pathogenesis of Ehrlichia-induced liver injury (1). E. japonica enters host cells (HCs) and residing within the Phagosome (2); E. japonica entry leads to the induction of TLR9 and MyD88 (3); This activation of TLR9 and MyD88 triggers the activation of NF-kB, resulting in the upregulation of inflammatory genes like IL-6, IL-1β, NLRP3, and HMGB1 (4); E. japonica triggers mitochondrial damage as suggested by our prior studies. Such mitochondrial dysfunction may result in the release of mitochondrial DAMPS that triggers activation of NLRP3 inflammasome complex. NLRP3 activation trigger cleavage/activation of CASP1 (5); On the other hand, MyD88 signals negatively regulate CASP1 activation, which accounts for lack of secretion of biologically active IL-1β by infected HCs (6); MyD88 activates CASP11 and CASP3. CASP3 causes apoptotic cell death, while activated CASP11 induces Gasdermin D, leading to pyroptosis and release of HMGB1 (7); MyD88 also activates mTORC1 (8); MyD88 also promotes an early induction of mTORC1-independent non-canonical autophagy (9); MyD88 also blocks autophagy flux via mechanisms that may include activation of mTORC1; and (10) The above downstream signaling pathways mediated by MyD88 in HC following infection with virulent E. japonica result in host-pathogenic response including inflammation and apoptotic and pyroptotic cell death. Solid arrows indicate established mechanisms by data in this study as well as our previous studies. The dotted arrows indicate hypothetical mechanisms.
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NI: College of Medicine, UIC, Department of Pathology Start-up funds.