Fig 2: Creatine promotes IL-4 induced-M2 polarization in vitro. (A) Heatmap analysis of RNA-seq dataset of PMs showing subsets of genes in M1 and M2 macrophages relative to controls treated with LPS/IFN-? or IL-4 for 24 hours. (B) Transcription levels of GATM, GAMT and Slc6a8 in BMDMs were detected by quantitative PCR (n=3). (C) Arg1 and GATM expression levels in BMDMs were detected using western blotting (n=3). (D) WT BMDMs were pretreated with creatine overnight and subsequently activated with IL-4 for 24 hours. Expression of Arg1, Retnla and Ccl17 mRNA were determined by quantitative PCR (n=4). (E) siRNA blocks STAT6 expression in IL-4 activated (24 hours) AMs. STAT6, GATM, Arg1 and Mrc1 expression was determined by quantitative PCR and (F), western blotting. (G) Heatmap analysis of RNA-seq dataset of PMs showing subsets of genes in WT and GATM-/- (n=3). (H) Transcription levels of Scd1 in AMs were detected by quantitative PCR (n=3). Data are expressed as mean ± SEM. Statistical significance was determined by one-way ANOVAs with Tukey’s multiple comparisons test; n.s., not significant *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

Fig 3: GATM promoted M2 polarization in BMDMs but had only a small effect on M1 polarization. (A, B), GATM-/- BMDMs were activated with LPS and IFN-? for 24 hours. Level of NO and Nos2, IL1b and IL12b mRNA expression was determined by quantitative PCR (n=3). (C) GATM-/- BMDMs were treated with IL-4 activation for 24 hours. Arg1, Mrc1, Ccl17 and Retnla mRNA expression was determined by quantitative PCR (n=3). (D, E), GATM-/- AMs were treated with LPS and IFN-? or IL-4 activation for 24 hours. iNOS and Arg1 protein expression was determined by western blotting (n=3). Data are expressed as mean ± SEM. Statistical significance was determined using one-way ANOVAs with Tukey’s multiple comparisons test; n.s., not significant; *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

Fig 4: GATM is involved in the polarization of M2 type macrophages in asthma. (A) Immunofluorescence staining of CD68, Arg1 and GATM in lung tissue. Original magnifications x 200 left and x 630 right panel (n=4). Scale bar=20 uM. (B) Morphology of AMs under the microscope. Original magnification x 100 (n=5). Scale bar=200 uM. (C) Transcription levels of Arg1 and Retnla in AMs were detected by quantitative PCR (n=5). (D) Transcription levels of GATM, GAMT and Slc6a8 in AMs were detected by quantitative PCR (n=5). (E) Arg1 and GATM expression levels in AMs were detected by western blotting (n=4). Data are expressed as mean ± SEM. Statistical significance was determined using t-tests; *P<0.05; **P<0.01, ***P<0.001, ****P<0.0001.

Fig 5: Pulmonary expression of GATM and creatine in HDM-induced asthma model. (A) Experimental timeline. (B) AHR as measured by lung resistance (RL; n=7). (C) Serum level of IgE and BALF levels of IL-4 (n=6–8). (D) Representative H&E-stained lung sections showing different groups. Original magnification x 200 (n=6). Scale bar=100 uM. (E) Representative PAS-stained lung sections showing goblet cell metaplasia in different groups. Original magnification x 200 (n=4). Scale bar=100 uM. (F) Representative Masson-stained lung sections showing submucosal collagen deposition in different groups. Original magnification x 200 (n=4). Scale bar=100 uM. (G) Total cells, eosinophils and lymphocytes in BALF (n=6). (H) The expression of IL-4, IL-5, IL-13 in asthma. (I) Creatine concentration in asthma. (J) western blotting detection of GATM protein expression in the lung (n=4). (K) Immunofluorescence detection of GATM protein expression in the lung (n=4). Scale bar=20 uM. Data expressed as mean ± SEM. Statistical significance was determined using t-tests; *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.