Fig 2: Chronic morphine induced NLRX1-mediated immunosuppression in brain. A Confocal microscopy analysis of NLRX1 (green) and NeuN (red), CD31 (red) or GFAP (red). Bar = 50 µm. B, C, D The mRNA expression of inflammatory cytokine genes were analyzed in cortex (B), striatum (C) and cerebellum (D) in mice (n = 3–5). Quantitative graphs are shown by mean ± SEM. Data represent the mean ± SEM. Student's t-test or Mann–Whitney U test were used to measure significance between two groups (* p < 0.05, ** p < 0.01, *** p < 0.001 and ns p > 0.05)

Fig 3: NLRX1-mediated mitophagy occurred in microglia independent of PINK1–Parkin pathway, but not astrocyte (A, B) Quantitative graphs of NLRX1 mRNA (A, n = 6) and representative Western blots of mitophagy (B, n = 3) in primary astrocyte. Quantitative graphs of NLRX1 mRNA (C, n = 6) and representative Western blots of mitophagy (D, n = 3) in MA cells. E Co-staining of HSP60 or Tim23 and LC3B in primary astrocyte. Bar = 10 µm. F The quantitative graphs of JC-1 assays as measured by flow cytometry (n = 4). G The NLRX1 mRNA expression of BV2 cells with or without CCCP treatment (n = 6). Data represent the mean ± SEM. Two-sided Student’s t tests were used to measure significance between two groups (* p < 0.05, ** p < 0.01, *** p < 0.001 and ns p > 0.05)

Fig 4: NLRX1-mediated incomplete mitophagy facilitated morphine induced microglial immunosuppression and susceptibility to infection. A NLRX1 regulated the mitophagy-induced immunosuppression in morphine-treated BV2 cells (n = 4–8), as measured by qPCR analysis. B Torin1 treatment partly rescued the inhibition of proinflammatory cytokines including IL-1ß, TNF-a and iNOS (n = 4). C After treatment with CCCP or Bafilomycin A1 at the absent or present of morphine, the expressions of proinflammatory cytokines were measured by qPCR analysis (n = 4). D, E The cell viability was detected in BV2 cells with 100 ng/ml LPS treatment (D, n = 3) or 1ug/ml LPS treatment (E, n = 3). F LPS challenge was performed in scrambled-shRNA or NLRX1-shRNA primary microglial cells. The cell viability was measured by CCK8 (n = 3). Data represent the mean ± SEM. Student's t-test or Mann–Whitney U test were used to measure significance between two groups (* p < 0.05, ** p < 0.01, *** p < 0.001 and ns p > 0.05)

Fig 5: Schematic model of NLRX1-mediated insufficient mitophagy in morphine-treated microglial cells. Morphine enhanced the ability of NLRX1-mediated mitophagy in a LC3-dependent manner, while impairing lysosomal function including generation, mitophagosome–lysosome fusion and acidification, thus leading to disturbance of mitochondrial–lysosomal homeostasis. NLRX1-mediated incomplete mitophagy in morphine-treated microglial cells ultimately contributed to microglial immunosuppressive condition