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Pathogenic effects of inhibition of mTORC1/STAT3 axis facilitates Staphylococcus aureus-induced pyroptosis in human macrophages.

ABSTRACT: BACKGROUND:Pyroptosis is a recently identified pathway of caspase-mediated cell death in response to microbes, lipopolysaccharide, or chemotherapy in certain types of cells. However, the mechanism of how pyroptosis is regulated is not well-established. METHODS:Herein, the intracellular bacteria were detected by staining and laser confocal microscopy and TEM. Live/dead cell imaging assay was used to examine macrophage death. Western blot and immunohistochemical staining were used to examine the protein changes. IFA was used to identify typical budding vesicles of pyroptosis and the STAT3 nuclear localization. SEM was used to observe the morphological characteristics of pyroptosis. ELISA was used to detect the level of inflammatory cytokines. Pyroptosis was filmed in macrophages by LSCM. RESULTS:S. aureus was internalized by human macrophages. Intracellular S. aureus induced macrophage death. S. aureus invasion increased the expression of NLRP3, Caspase1 (Casp-1 p20) and the accumulation of GSDMD-NT, GSDMD-NT pore structures, and the release of IL-1? and IL-18 in macrophages. Macrophages pyroptosis induced by S. aureus can be abrogated by blockage of S. aureus phagocytosis. The pyroptosic effect by S. aureus infection was promoted by either rapamycin or Stattic, a specific inhibitor for mTORC1 or STAT3. Inhibition of mTORC1 or STAT3 induced pyroptosis. mTORC1 regulated the pyroptosic gene expression through governing the nuclear localization of STAT3. mTORC1/STAT3 axis may play a regulatory role in pyroptosis within macrophages. CONCLUSIONS:S. aureus infection induces human macrophage pyroptosis, inhibition of mTORC1/STAT3 axis facilitates S. aureus-induced pyroptosis. mTORC1 and STAT3 are associated with pyroptosis. Our findings demonstrate a regulatory function of the mTORC1/STAT3 axis in macrophage pyroptosis, constituting a novel mechanism by which pyroptosis is regulated in macrophages. Video Abstract Macrophages were infected with S. aureus for 3 h (MOI 25:1), and pyroptosis was filmed in macrophages by laser confocal microscopy. A representative field was recorded. Arrow indicates lysing dead cell.

SUBMITTER: Yao R

PROVIDER: S-EPMC7706204 | biostudies-literature | 2020 Nov

REPOSITORIES: biostudies-literature

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Pathogenic effects of inhibition of mTORC1/STAT3 axis facilitates Staphylococcus aureus-induced pyroptosis in human macrophages.

Yao Ruiyuan R Chen Yuhao Y Hao Huifang H Guo Zhixin Z Cheng Xiaoou X Ma Yuze Y Ji Qiang Q Yang Xiaoru X Wang Yanfeng Y Li Xihe X Wang Zhigang Z

Cell communication and signaling : CCS 20201130 1

<h4>Background</h4>Pyroptosis is a recently identified pathway of caspase-mediated cell death in response to microbes, lipopolysaccharide, or chemotherapy in certain types of cells. However, the mechanism of how pyroptosis is regulated is not well-established.<h4>Methods</h4>Herein, the intracellular bacteria were detected by staining and laser confocal microscopy and TEM. Live/dead cell imaging assay was used to examine macrophage death. Western blot and immunohistochemical staining were used t ...[more]

PMID: 33256738

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Project description:Macrophages are active contributors to the innate immune defense system. As macrophage activation is clearly affected by the surrounding microenvironment, the present study investigated the effect of sulforaphane (SFN) on the bactericidal activity of macrophages and the underlying molecular mechanisms involved in this process. Human THP‑1‑derived macrophages, primary human peripheral blood mononuclear cell‑derived macrophages, and primary mouse bone marrow derived‑macrophages (BMDMs) pretreated with SFN or DMSO were utilized in a model of Staphylococcus aureus infection. The results suggested that SFN pretreatment of macrophages effectively repressed the intracellular survival of S. aureus through modulation of p38/JNK signaling and decreased S. aureus‑induced caspases‑3/7‑dependent cell apoptosis, potentially through downregulation of microRNA (miR)‑142‑5p and miR‑146a‑5p. As SFN is a well‑known activator of nuclear factor erythroid 2‑related factor 2 (Nrf2), Nrf2‑/‑ BMDMs were used to demonstrate that the SFN‑mediated inhibitory effect was independent of Nrf2. Nevertheless, an increase in intracellular bacterial survival in Nrf2‑deficient macrophages was observed. In addition, SFN pretreatment suppressed S. aureus‑induced transcriptional expression of genes coding for the proinflammatory cytokines interleukin (IL)‑1β, IL‑6, and tumor necrosis factor‑α (TNF‑α), as well as for the M1 markers C‑C motif chemokine receptor 7, IL‑23 and inducible nitric oxide synthase (iNOS). Western blot analysis indicated that S. aureus challenge activated p38 mitogen‑activated protein kinase (MAPK) (p38) and c‑Jun N‑terminal kinase (JNK) MAPK signaling pathways, while SFN pretreatment prevented p38 and JNK phosphorylation. Pretreatment with 2 specific inhibitors of p38 and JNK, SB203580 and SP600125, respectively, resulted in a decrease in S. aureus‑induced proinflammatory gene expression levels compared with those observed in the SFN‑pretreated macrophages. Furthermore, THP‑1‑derived macrophages pretreated with SB203580 or SP600125 prior to bacterial infection exhibited a significant inhibition in intracellular S. aureus survival. In conclusion, we hypothesize that concomitant targeting of the p38/JNK‑inflammatory response and the S. aureus‑induced apoptosis with SFN may be a promising therapeutic approach in S. aureus infection.

Dataset Information

Pathogenic effects of inhibition of mTORC1/STAT3 axis facilitates Staphylococcus aureus-induced pyroptosis in human macrophages.

Publications

Pathogenic effects of inhibition of mTORC1/STAT3 axis facilitates Staphylococcus aureus-induced pyroptosis in human macrophages.

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