Fig. 7.

Effect of dietary αKG on hypoxia-immune response in mice. The C57/BL6 mice (5-6 weeks old) were administered with 1% αKG via drinking water, 12 hrs prior to hypoxia exposure (11% oxygen for 48 hrs), experimental design is presented as a schematic in Fig. S14a. (a-b) After treatment, plasma and PBMC-granulocyte cell pellet were used for measuring αKG in (a) plasma and (b) PBMCs (1 × 10⁶ cells) lysate using colorimetry based assay. Unpaired t-test was used for comparison between groups (n=7 in each group), **p<0.01, *p<0.05 and ns=non-significant. (c-d) Monocytes and neutrophils counts were measured from whole blood using hematology analyzer. (e-f) Plasma level of cytokines IL1β and IL6 were assessed using CBA assay. (g-h) Monocyte and neutrophil counts were measured from bronchoalveolar lavage fluid (BALF) using hematology analyzer. The counts of both cell types in whole blood and BALF were confirmed using staining with CD45, CD11b, Ly6C and Ly6G and analyzed using flow cytometry analysis, mentioned in Fig. S13. Each dot represents a mouse from three independent experiments. Unpaired t-test was used for comparison between the groups as in Fig. 7c-h, ***p<0.001 **p<0.01, and *p<0.05. (i) The H & E staining of lung section of mice showed the infiltration of immune cells into airway space under hypoxia treatment (magnification view shows large numbers of leukocytes inside airway space), which was rescued by 48 hrs αKG supplementation; arrows indicate the cell infiltration, bars in all images ~ 50 μm. (J) The infiltration score was calculated in case of each treatment group based on H & E lung sections from independent experiment. Additional images for quantification has been depicted in Fig. S14g. (k) The expression of Cxcr4 gene in leukocytes was assessed using real-time PCR and normalized with housekeeping gene Hprt. Unpaired t-test was used for comparison between groups, **p<0.01, and *p<0.05.(l) Expression of P-p65 and HIF2α in leukocytes pellet was evaluated by western blot to ascertain the enzymatic function of PHD2 after αKG administration under hypoxia. Representative blot shows data of 2 mice from each group. Densitometry showed data from 5 mice for P-p65/p65 and 8 mice for HIF2α from independent experiments as mentioned in Fig. S15u-w. The αKG treatment for 48 hrs rescued more effectively than 24 hrs treatment for all above parameters in mice exposed for 48 hrs under 11% hypoxia.