Journal: Journal of Cellular Physiology

Article Title: Interleukin‐37 inhibits osteoclastogenesis and alleviates inflammatory bone destruction

doi: 10.1002/jcp.27526

Figure Lengend Snippet: IL‐37b inhibited the expression of specific mRNAs and proteins at different stages of osteoclast differentiation. The MyD88 dimerization inhibitor (ST 2825) reversed the effects of IL‐37b on osteoclastogenesis. (a) Cells were treated with RANKL (50 ng/ml), M‐CSF (50 ng/ml), and IL‐37b (200 ng/ml) for 24 or 72 hr, and then lysed for Western blot analyses using antibodies against NFATc1 and GAPDH. (b,c) The expression of genes associated with osteoclast differentiation, fusion, and function were detected by qRT‐PCR. * p < 0.05 and ** p < 0.01 compared with the RANKL‐treated control. (d) TRAP staining showed that 10 μg/ml ST 2825 partially counteracted the inhibitory effects of IL‐37b on osteoclast formation. Scale bar = 100 μm. (e) Western blot showing the level of NFATc1 in cells stimulated with RANKL in the presence or absence of ST 2825. The results were normalized to GAPDH levels. (f) Co‐IP analysis of the interaction between SIGIRR and MyD88 in RAW264.7 cells treated with RANKL and IL‐37b. Co‐IP: coimmunoprecipitation; GAPDH: glyceraldehyde 3‐phosphate dehydrogenase; IL‐37b: Interleukin‐37b; mRNA: messenger RNA; MyD88: myeloid differentiation factor 88; M‐CSF: macrophage colony stimulating factor; NFATc1: nuclear factor of activated T cells 1; qRT‐PCR: quantitative real‐time polymerase chain reaction; RANKL: receptor activator of nuclear factor‐κB ligand; SIGIRR: single Ig IL‐1‐related receptor; TRAP: tartrate‐resistant acid phosphatase [Color figure can be viewed at wileyonlinelibrary.com]

Article Snippet: MyD88 pharmacologic inhibitor (ST 2825) was purchased from Medchemexpress (Princeton, NJ).

Techniques: Expressing, Western Blot, Quantitative RT-PCR, Control, Staining, Co-Immunoprecipitation Assay, Real-time Polymerase Chain Reaction

Journal: Journal of Cellular Physiology

Article Title: Interleukin‐37 inhibits osteoclastogenesis and alleviates inflammatory bone destruction

doi: 10.1002/jcp.27526

Figure Lengend Snippet: Interleukin 37 downregulates osteoclastogenesis through direct and indirect actions. IL: interleukin; MyD88: myeloid differentiation factor 88; NF‐kB: nuclear factor‐κB; RANKL: receptor activator of nuclear factor‐κB ligand; TNF‐α: tumor necrosis factor‐α [Color figure can be viewed at wileyonlinelibrary.com]

Article Snippet: MyD88 pharmacologic inhibitor (ST 2825) was purchased from Medchemexpress (Princeton, NJ).

Techniques:

Journal: Frontiers in Immunology

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

doi: 10.3389/fimmu.2023.1212167

Figure Lengend Snippet: 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).

Article Snippet: E. japonica was added to cultured primary murine HCs or AML2 HCs at a multiplicity of infection (MOI) of 5 before infection, cells were pretreated for 1 hr. with the MyD88 inhibitor ST 2825 (APExBIO Technology LLC, Houston, TX) at a 15 μM to block MyD88 signaling.

Techniques: Bacteria, Infection, Amplification, Expressing

Journal: Frontiers in Immunology

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

doi: 10.3389/fimmu.2023.1212167

Figure Lengend Snippet: 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).

Article Snippet: E. japonica was added to cultured primary murine HCs or AML2 HCs at a multiplicity of infection (MOI) of 5 before infection, cells were pretreated for 1 hr. with the MyD88 inhibitor ST 2825 (APExBIO Technology LLC, Houston, TX) at a 15 μM to block MyD88 signaling.

Techniques: Infection, Western Blot, Expressing

Journal: Frontiers in Immunology

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

doi: 10.3389/fimmu.2023.1212167

Figure Lengend Snippet: 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.

Article Snippet: E. japonica was added to cultured primary murine HCs or AML2 HCs at a multiplicity of infection (MOI) of 5 before infection, cells were pretreated for 1 hr. with the MyD88 inhibitor ST 2825 (APExBIO Technology LLC, Houston, TX) at a 15 μM to block MyD88 signaling.

Techniques: Infection, Western Blot, Cell Culture, Expressing

Journal: Frontiers in Immunology

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

doi: 10.3389/fimmu.2023.1212167

Figure Lengend Snippet: 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.

Article Snippet: E. japonica was added to cultured primary murine HCs or AML2 HCs at a multiplicity of infection (MOI) of 5 before infection, cells were pretreated for 1 hr. with the MyD88 inhibitor ST 2825 (APExBIO Technology LLC, Houston, TX) at a 15 μM to block MyD88 signaling.

Techniques: Translocation Assay, Immunofluorescence, Staining, Infection, Western Blot, Enzyme-linked Immunosorbent Assay, Expressing, In Vitro

Journal: Frontiers in Immunology

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

doi: 10.3389/fimmu.2023.1212167

Figure Lengend Snippet: 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.

Article Snippet: E. japonica was added to cultured primary murine HCs or AML2 HCs at a multiplicity of infection (MOI) of 5 before infection, cells were pretreated for 1 hr. with the MyD88 inhibitor ST 2825 (APExBIO Technology LLC, Houston, TX) at a 15 μM to block MyD88 signaling.

Techniques: Western Blot, Infection, Expressing

Journal: Frontiers in Immunology

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

doi: 10.3389/fimmu.2023.1212167

Figure Lengend Snippet: 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).

Article Snippet: E. japonica was added to cultured primary murine HCs or AML2 HCs at a multiplicity of infection (MOI) of 5 before infection, cells were pretreated for 1 hr. with the MyD88 inhibitor ST 2825 (APExBIO Technology LLC, Houston, TX) at a 15 μM to block MyD88 signaling.

Techniques: Inhibition, Activation Assay, Western Blot, Infection

Journal: Frontiers in Immunology

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

doi: 10.3389/fimmu.2023.1212167

Figure Lengend Snippet: 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.

Article Snippet: E. japonica was added to cultured primary murine HCs or AML2 HCs at a multiplicity of infection (MOI) of 5 before infection, cells were pretreated for 1 hr. with the MyD88 inhibitor ST 2825 (APExBIO Technology LLC, Houston, TX) at a 15 μM to block MyD88 signaling.

Techniques: Activation Assay, Infection, Protein-Protein interactions