Fig 2: nCB activates the HIF1a pathway to increase lactate in macrophages.(A) Representative Western blots showing expression of the mTORC1 downstream targets p-S6K (T389) and p-4E-BP1 (T37/46). n = 3 biological replicates per group. (B and C) Effects of rapamycin on nCB-treated BMDMs. Cells were exposed to nCB (50 µg/ml) for 24 hours, followed by 100 nM rapamycin (rap.) for another 24 hours. n = 4 biological replicates per group. (D) Heatmap of selected HIF1a-regulated genes in CD11c+ lung APCs from PBS-exposed or nCB-exposed WT mice. n = 3 per group. (E and F) Representative Western blots showing HIF1a in nCB-treated macrophages. n = 2 or 3 biological replicates per group. (G) Detection of hypoxia by pimonidazole in PBS- or nCB-exposed lungs. Scale bars, 100 µm. n = 5 biological replicates per group. (H) Lactate production in supernatants of nCB-treated BMDMs. Supernatants were collected 24 hours after nCB treatment. n = 4 per group. Representative (I) lactate production in the lungs and quantification of total live (J) FOXP3+ Treg cells; (K) PD-1+ LAG3+ CD4 T cells, (L) PD-1+ LAG3+ CD8 T cells, (M) PD-L1+ interstitial macrophages, and (N) PD-L1+ alveolar macrophages. n = 3 to 5 per group. All experiments have been repeated for two to three times and shown as representatives. Significance was determined by Student’s t test, one-way ANOVA, and two-way ANOVA with Holm’s correction. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Fig 3: Loss of alveolar macrophages exaggerates inflammation and tumors in response to nCB.(A) Top five pathways altered in CD11c+ APCs from nCB-exposed lungs. (B) Heatmap of the IL-10–regulated genes shown in (A). n = 3 per group. (C) IL-10 and TGFß in lung CD11c+ APCs. n = 3 to 15 per group. (D) Representative IL-10 expression in lung macrophages. n = 3 per condition. (E) Schematic diagram of alveolar macrophage depletion in Pts4d/d mice. Three-month-old Pts4d/d mice received five doses of nCB intranasally in the first 1.5 week. Clodronate or control liposomes were intranasally delivered to the mice three times per week for 3 weeks after the third dose of nCB. Mice were euthanized at 4 months of age. (F) Total live alveolar macrophages. (G) Whole-lung lactate levels and total cell numbers of interstitial macrophages and Ly6C+ monocytes. (H) Total cell numbers of live PD-1+ LAG3+ CD4 or CD8 T cells and FOXP3+ Treg cells. (I) IL-1ß, IL-6, and IL-10 expression in lungs. n = 7 or 8 per group. (J) Representative H&E stains and summary statistics of tumor foci found in liposome-treated nCB lungs. All experiments have been repeated for two to three times and shown as representatives. Significance was determined by Student’s t test and Fisher’s tests. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.