Fig 2: Myeloid-specific GP96 deficiency prevents chronic alcohol-induced endotoxin and pro-inflammatory cytokine production. (A) Endotoxin level was measured in serum (n = 8-12). (B) CyP2e1 was detected in liver microsomal fraction by western blot, and calnexin was used as an internal loading control (n = 3). Total RNA from liver tissue was subjected to quantitative RT-PCR for analysis of pro-inflammatory cytokines TNF-a, IL-6, MCP-1, and IL-1ß; NLRP3; anti-inflammatory markers IL-10, TGF-ß, and ATF3 (C); and macrophage markers (n = 6-10) (F). (D) Total liver protein level for TNF-a was measured in tissue extracts of pair-fed and alcohol-fed mice by ELISA (n = 6-10). (E) ATF3 protein level was analyzed by western blot in whole-liver extracts using tubulin as an internal control (n = 3). The mRNA expression profile of (G) pro-inflammatory, and (H) anti-inflammatory and restorative macrophage markers, Trem2 and ATF3, were analyzed in liver macrophages of pair-fed and alcohol-fed mice (n = 5). (I) The mRNA level of pro-inflammatory cytokines was analyzed in BMDMs isolated from WT and M-GP96KO mice and stimulated with 100 ng/mL LPS for 2 hours (n = 9). Data are represented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig 3: Activating transcription factor 3 (ATF3)/CCL2 regulates microglia activation in rats. (a) mRNA expression of pro-inflammatory factors in rat brain tissues by reverse transcriptase quantitative polymerase chain reaction (RT-qPCR). (b) mRNA expression of anti-inflammatory factors in rat brain tissues by RT-qPCR. (c) Immunofluorescence validation of ATF3 and CCL2 localization in microglia. (d) Immunohistochemical detection of IBA1 expression in rat brain tissues. (e) Assessment of microglia activation by immunofluorescence detection of BrdU/IBA1. Data are expressed as mean ± SD, and statistical significance was determined using one-way analysis of variance (ANOVA) (a–e), followed by Tukey multiple comparison test. *#&p < .05

Fig 4: Activating transcription factor 3 (ATF3) represses CCL2 transcription in rats with cerebral ischemia. (a) Analysis of differentially expressed genes (DEGs) in brain tissues of rats with cerebral ischemia by mRNA microarray. (b–d) The biological process, cellular components and molecular functions of DEGs, respectively analyzed by gene ontology (GO) enrichment analysis. (e) Pathways distributed by DEGs analyzed by KEGG pathway enrichment analysis. (f) Interactions between DEGs analyzed by protein–protein interactions (PPI) network. (g) The binding site between ATF3 and the upstream promoter of CCL2 predicted by a bioinformatics website. (h) the binding relation between ATF3 and CCL2 examined by chromatin immunoprecipitation (ChIP). Data are expressed as mean ± SD, and statistical significance was determined using two-way analysis of variance (ANOVA) (h), followed by Tukey multiple comparison test. * p < .05

Fig 5: ATF3 immunostaining in nuclei of neurons in cervical level 7 (C7) spinal cord ventral horns, showing the highest presence in HRHF-untreated rats. A-D, ATF3 immunoexpression in motor neurons located in spinal cord ventral horns, at segmental level C7. NeuN was used as a neuronal counterstain and DAPI is used as a nuclear counterstain in panels A, B, and D. Asterisks in the panels indicate areas enlarged in their insets. Insets in panels A and B show the presence of ATF3 only in neuronal cell bodies of HRHF-untreated and HRHF-Rest/IgG rats. E, Quantification of the percent of neurons (NeuN+ cells) in cervical ventral horns that were also immunopositive for ATF3. Same number of limbs/gp as in the legend of Figure 3. *P < .05 and **P < .01 compared with FRC rats; ## P < .01 compared with HRHF-untreated or HRHF-Rest/IgG rats as shown. Scale bar = 50 µm. FRC, food restricted control; HRHF, high repetition high force [Color figure can be viewed at wileyonlinelibrary.com]