Fig 2: Circulating parvalbumin suppresses M2 macrophage polarization in mice.a Parvalbumin protein expression in muscle and serum from WT and parvalbumin KO mice. b Representative M2 macrophage marker gene expression in scWAT and BAT of WT and parvalbumin KO mice. WT, n = 10; PVALB KO, n = 10. c Representative flow cytometry plots and (d) quantification demonstrate the numbers of M2 macrophages (CD206+/CD11C−) in the scWAT of WT and parvalbumin KO mice. WT, n = 13; PVALB KO, n = 13. e Male parvalbumin KO mice were intravenously injected with Ad-parvalbumin or Ad-GFP through the tail vein. Parvalbumin protein expression in the liver and serum were examined. f Representative M2 macrophage marker gene expression in scWAT and BAT of parvalbumin KO mice injected with Ad-GFP or Ad-parvalbumin. Adv-GFP, n = 8; adv-PVALB, n = 8. g Representative flow cytometry plots and (h) quantification demonstrate the numbers of M2 macrophages (CD206+/CD11C−) in the scWAT of parvalbumin KO mice injected with Ad-GFP and Ad-parvalbumin. Adv-GFP, n = 14; Ad-parvalbumin, n = 14. i Macrophage parvalbumin deficiency had no effect on M2 activation. Cd301, Cd206, and Tgm2 mRNA expression in WT and parvalbumin KO BMDM treated with IL4 and parvalbumin (100 ng/ml) (n = 3 per group). Data are represented as mean ± SEM. P values were determined by unpaired two-tailed Student’s t test (b, d, f, h, and i). Source data are provided as a Source Data file.

Fig 3: MnJ promotes antigen presentation and specific T-cell responses. a, b WT mice were immunized subcutaneously with PBS, phycoerythrin (PE, 10 μg), PE (10 μg) + MnJ (20 μg), or PE (10 μg) + Imject Alum (200 μg). Inguinal lymph nodes were collected 12 or 24 h later (n = 3). The ratio and number of PE+ APCs (a) and Mo-DCs (b) among dLN cells were analyzed by FACS. Live cells were identified by DAPI staining. Among live singlet cells, APCs were identified as the cell subset single or double positive for CD11c and F4/80. Among APCs, Mo-DCs were identified as CD8a+ CD64+ cells. c BMDCs were treated with MnCl2 (20 μg/ml), MnJ (20 μg/ml), or Imject Alum (20 μg/ml) for 20 h. CD86 expression was analyzed by FACS. d CD45.1+ OT-I CD8+ or OT-II CD4+ T cells were labeled with CFSE and transferred to CD45.2+ WT mice. These mice were then immunized with OVA (1 μg), OVA (1 μg) + MnJ (10 μg), or OVA (1 μg) + Imject Alum (1320 μg). After 3 days, T-cell proliferation was analyzed by FACS (n = 3). e WT mice were immunized intramuscularly with OVA (10 μg), OVA (10 μg) + MnJ (10 μg), or OVA (10 μg) + Imject Alum (1320 μg) on days 0, 7, and 14. Splenocytes were collected on day 21 and stimulated with OVA (100 μg/ml). IL-2, IL-4, IFNγ, and IL-10 secretion by T cells was measured by ELISA (n = 3). f OVA-specific cytotoxicity was measured on day 21 in an in vivo killing assay (mice were immunized on days 0, 7, and 14, n = 4). g Numbers of Tfh or GC B cells in dLNs from WT mice were analyzed by FACS. Live cells were identified by DAPI staining. Among live singlet cells, CD4+ T cells were identified as the cell subset double positive for CD3 and CD4. Among CD4+ T cells, Tfh cells were identified as PD1+ CXCR5+ cells. B cells were identified as the cell subset double positive for CD45 and B220. Among B cells, GC B cells were identified as Fas+ GL7+ cells (n = 3). One representative experiment of at least three independent experiments is shown, and each was performed in triplicate. Error bars represent the SEM; data were analyzed by an unpaired t-test. ns not significant; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001

Fig 4: Arf1 inhibition triggers ER stress and induces DAMPs and DC infiltration.a–e Huh-7 cells were treated with control DMSO or GCA and then injected into athymic nude mice. After 3 weeks, the tumors from the treated cells were excised and stained for molecular markers. Calr, HMGB1, ERp46, and the lysosome protein LAMP1 were dramatically induced in the GCA-treated tumors (a and b). Nuclear HMGB1 moved to the extracellular space (b). The ER stress marker peIF2a and the ERp46 receptor PEAR1 were also significantly increased in GCA-treated tumors (c and d). Cleaved and active Caspase 1 p20 but not cleaved Caspase 3 was also dramatically induced in the GCA-treated tumor (e). f, g Direct treatment of Huh-7 cells with GCA induced the expression and translocation of HMGB1 (f) and the translocation of Calr (g) from the nucleus to the extracellular space. h Treatment of Huh-7 cells with the Arf1 inhibitor BFA or GCA induced ATP secretion from control but not Arf1 (sh-Arf1) knockdown cells (n = 5 wells cell each group, *p < 0.05, t-test; repeat three independent experiments). i Treatment of mice with Arf1 inhibitor GCA increased ATP release in vivo. j, k LRP1 and CD11c expression in the intestine of Lgr5/Apc or Lgr5/Arf1/Apc mice. l LRP1 and CD11c expression in the liver of MYC-ON liver tumor mice treated with the indicated reagents. Each experiment replicates three times. Data are shown as the mean ± /SEM. *p < 0.05, **p < 0.01 by Student’s t-test. Scale bars are as indicated.

Fig 5: ATG5 is required in CD11c+ cells to regulate the early influx of neutrophils during M. tuberculosis infection in vivo. (A) Representative flow cytometry plots of neutrophils (CD45+Ly6G+CD11b+) at 14 dpi from Atg5fl/fl, Atg5fl/fl-LysM-Cre, Atg5fl/fl-CD11c-Cre, and Atg5fl/fl-Mrp8-Cre mice. (B) Proportion of CD45+ cells that are neutrophils in the lung at 14 dpi in Mtb-GFP infected Atg5fl/fl (n = 15), Atg5fl/fl-LysM-Cre (n = 8), Atg5fl/fl-CD11c-Cre (n = 10), and Atg5fl/fl-Mrp8-Cre (n = 6) mice. Neutrophil frequency is reported as a ratio relative to the average neutrophil frequency in Atg5fl/fl control mice at 14 dpi within a given experiment. (C) Lung burden from the right lung at 14 dpi in Mtb-GFP infected Atg5fl/fl (n = 15), Atg5fl/fl-LysM-Cre (n = 8), Atg5fl/fl-CD11c-Cre (n = 10), and Atg5fl/fl-Mrp8-Cre (n = 6) mice. (D) Proportion of CD45+ cells that are neutrophils in the lung at 21 dpi in Mtb-GFP infected Atg5fl/fl (n = 19), Atg5fl/fl-LysM-Cre (n = 6), Atg5fl/fl-CD11c-Cre (n = 6), and Atg5fl/fl-Mrp8-Cre (n = 7) mice. Neutrophil frequency is reported as a ratio relative to the average neutrophil frequency in Atg5fl/fl control mice at 21 dpi within a given experiment. Susceptible and healthy Atg5fl/fl-Mrp8-Cre mice are defined as done previously where susceptible Atg5fl/fl-Mrp8-Cre mice have lost more than 5% of their pre-infection body weight by 20 dpi and healthy Atg5fl/fl-Mrp8-Cre mice have lost less than 5% of their pre-infection body weight at 20 dpi [3]. (E) Lung burden from the right lung at 21 dpi in Mtb-GFP infected Atg5fl/fl (n = 19), Atg5fl/fl-LysM-Cre (n = 6), Atg5fl/fl-CD11c-Cre (n = 6), and Atg5fl/fl-Mrp8-Cre (n = 7) mice. (F) Kaplan–Meier curve of survival proportions during Mtb-GFP infection of Atg5fl/fl (n = 9) and Atg5fl/fl-CD11c-Cre (n = 9) mice. Statistical differences were determined by a log-rank Mantel–Cox test (F) or one-way ANOVA and Šídák multiple comparison test (B–E). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Differences that are not statistically significant are designated as ns. Pooled data from at least 2 separate experiments is graphed where each data point is from 1 biological replicate. The individual numerical values used to generate the graphed data in Fig 1, the statistical analyses performed to analyze these data, and the p values from these statistical tests are in S1 Data. dpi, days postinfection.