Kohler JB et al. (2019), Microenvironmental stimuli induce different mac...

ECB-ART-47128

Biol Open 2019 Apr 26;84:. doi: 10.1242/bio.040808.

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Microenvironmental stimuli induce different macrophage polarizations in experimental models of emphysema.

Kohler JB , Cervilha DAB , Riani Moreira A , Santana FR , Farias TM , Alonso Vale MIC , Martins MA , Prado CM , Tibério IC , Ito JT .

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Macrophages play a pivotal role in the development of emphysema and depending on the microenvironment stimuli can be polarized into M1- or M2-like macrophage phenotypes. We compared macrophage polarizations in cigarette smoke (CS)- and porcine pancreatic elastase (PPE)-induced emphysema models. C57BL/6 mice were subdivided into four experimental groups. In the PPE group, animals received an intranasal instillation of PPE (0.677 IU); in the saline group, animals received an intranasal instillation of saline (0.9%). Animals from both groups were euthanized on day 28. In the CS group, animals were exposed to CS for 30 min, twice a day, 5 days per week for 12 weeks. In the control group, animals received filtered air. We observed an increase in total macrophages for both experimental models. For M1-like macrophage markers, we observed an increase in TNF-α+ and IFN-γ+ cells, Cxcl-9 and Cxcl-10 expressions in PPE and CS groups. Only in the CS group, we detected an increased expression of IL-12b For M2-like macrophages markers we observed a down regulation in IL-10, IL-4, IL-13, Arg1 and Fizz1 and an increase of TGF-β+ cells in the PPE group, while for the CS group there was an increase in TGF-β+ cells and IL-10 expression. All exposure groups were compared to their respective controls. In summary, we demonstrated that CS- and PPE-induced models resulted in different microenvironmental stimuli. CS exposure induced an environmental stimulus related to M1- and M2-like macrophage phenotypes similar to previous results described in COPD patients, whereas the elastase-induced model provided an environmental stimulus related only to the M1 phenotype.

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Species referenced: Echinodermata
Genes referenced: irf4 irfl LOC574780 LOC586250

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	Fig. 1. Lm (A,B) values measured in experimental groups. (A) P=0.009, (B) P=0.001. Results were expressed as the mean±s.e. Saline n=10; PPE n=8; control n=8; CS n=9.
	Fig. 2. Photomicrographs of Lm in the distal airspaces of saline, PPE, control and CS groups. 200× magnification, scale bars: 100 μm.
	Fig. 3. The density of positive cells for MAC-2 (A,B). (A) P=0.001, (B) P=0.001 compared to their respective controls. Values are expressed as the mean±s.e. Saline n=11; PPE n=10; control n=11; CS n=9.
	Fig. 4. Photomicrographs of positive cells for MAC-2 and their respective negative controls in lung parenchyma in saline, PPE, control and CS groups. The arrows show the MAC-2+ cells in lung parenchyma. 400× magnification, scale bars: 50 μm.
	Fig. 5. The density of positive cells for IFN- γ (A,B) and TNF-α (C,D), M1-like macrophages markers. (A) P=0.004, (B) P=0.001, (C) P=0.037, (D) P=0.001, compared to their respective controls. Values are expressed as the mean±s.e. IFN-γ: saline n=8; PPE n=10; control n=9; CS n=9. TNF-α: saline n=8; PPE n=10; control n=9; CS n=9.
	Fig. 6. The density of positive cells for TGF-β (A,B) and IL-10 (C,D), M2-like macrophages markers. (A) P=0.001, (B) P=0.001, (C) P=0.041, (D) P=0.029, compared to their respective controls. Values are expressed as the mean±s.e. TGF-β: saline n=9; PPE n=8; control n=8; CS n=9. IL-10: saline n=8; PPE n=8; control n=7; CS n=7.
	Fig. 7. Photomicrographs of positive cells for IFN-γ, TNF-α, TGF-β and IL-10 in lung parenchyma in saline, PPE, control and CS groups. The arrows show IFN-γ+, TNF-α+, TGF-β+ and IL-10+ cells in lung parenchyma. 400× magnification, scale bars: 50 μm.
	Fig. 8. Photomicrographs of negative control images for IFN-γ, TNF-α, TGF-β and IL-10 in lung parenchyma in saline, PPE, control and CS groups. 400× magnification, scale bars: 50 μm.
	Fig. 9. Protein quantification of IL-4 (A,B), IL-10 (C,D), and IL-13 (E,F) by ELISA. (A) P=0.016, (C) P=0.046, (D) P=0.028, (E) P=0.007, compared to their respective controls. Values are expressed as the mean±s.e. IL-4: saline n=10; PPE n=9; control n=8; CS n=6. IL-10: saline n=7; PPE n=8; control n=6; CS n=6. IL-13: saline n=6; PPE n=9; control n=6; CS n=7.
	Fig. 10. Gene expression for Cxcl9 (A,B), Cxcl10 (C,D), Il-12b (E,F). (A) P=0.004, (B) P=0.017, compared to their respective controls. (C) P=0.001, (D) P=0.036, compared to their respective controls. (F) *P=0.026, compared to the control group. We did not observe any significant results following iNos (G,H) and Irf-5 (I,J) analysis. Values are expressed as the mean±s.e. CXCL9: saline n=14; PPE n=13; control n=11; CS n=13. CXCL10: saline n=14; PPE n=14; control n=11; CS n=16. IL-12b: saline n=13; PPE n=13; control n=10; CS n=11. iNOS: saline n=7; PPE n=4; control n=4; CS n=3. Irf5: saline n=14; PPE n=15; control n=12; CS n=17.
	Fig. 11. Gene expression for Arg1 (A,B), Fizz1 (C,D). (A)P=0.005 compared to the saline group. (C) P=0.001 compared to the saline group. No differences in Ym1 and (E,F) Irf4 (G,H) gene expression were detected. Values are expressed as the mean±s.e. Arg1: saline n=14; PPE n=15; control n=12; CS n=17. Fizz1: saline n=13; PPE n=14; control n=12; CS n=16. Ym1: saline n=13; PPE n=14; control n=9; CS n=16. Irf4: saline n=14; PPE n=14; control n=10; CS n=14.

References [+] :

Barnes, Alveolar macrophages as orchestrators of COPD. 2004, Pubmed

	Fig. 1. Lm (A,B) values measured in experimental groups. (A) P=0.009, (B) P=0.001. Results were expressed as the mean±s.e. Saline n=10; PPE n=8; control n=8; CS n=9.
	Fig. 2. Photomicrographs of Lm in the distal airspaces of saline, PPE, control and CS groups. 200× magnification, scale bars: 100 μm.
	Fig. 3. The density of positive cells for MAC-2 (A,B). (A) P=0.001, (B) P=0.001 compared to their respective controls. Values are expressed as the mean±s.e. Saline n=11; PPE n=10; control n=11; CS n=9.
	Fig. 4. Photomicrographs of positive cells for MAC-2 and their respective negative controls in lung parenchyma in saline, PPE, control and CS groups. The arrows show the MAC-2+ cells in lung parenchyma. 400× magnification, scale bars: 50 μm.
	Fig. 5. The density of positive cells for IFN- γ (A,B) and TNF-α (C,D), M1-like macrophages markers. (A) P=0.004, (B) P=0.001, (C) P=0.037, (D) P=0.001, compared to their respective controls. Values are expressed as the mean±s.e. IFN-γ: saline n=8; PPE n=10; control n=9; CS n=9. TNF-α: saline n=8; PPE n=10; control n=9; CS n=9.
	Fig. 6. The density of positive cells for TGF-β (A,B) and IL-10 (C,D), M2-like macrophages markers. (A) P=0.001, (B) P=0.001, (C) P=0.041, (D) P=0.029, compared to their respective controls. Values are expressed as the mean±s.e. TGF-β: saline n=9; PPE n=8; control n=8; CS n=9. IL-10: saline n=8; PPE n=8; control n=7; CS n=7.
	Fig. 7. Photomicrographs of positive cells for IFN-γ, TNF-α, TGF-β and IL-10 in lung parenchyma in saline, PPE, control and CS groups. The arrows show IFN-γ+, TNF-α+, TGF-β+ and IL-10+ cells in lung parenchyma. 400× magnification, scale bars: 50 μm.
	Fig. 8. Photomicrographs of negative control images for IFN-γ, TNF-α, TGF-β and IL-10 in lung parenchyma in saline, PPE, control and CS groups. 400× magnification, scale bars: 50 μm.
	Fig. 9. Protein quantification of IL-4 (A,B), IL-10 (C,D), and IL-13 (E,F) by ELISA. (A) P=0.016, (C) P=0.046, (D) P=0.028, (E) P=0.007, compared to their respective controls. Values are expressed as the mean±s.e. IL-4: saline n=10; PPE n=9; control n=8; CS n=6. IL-10: saline n=7; PPE n=8; control n=6; CS n=6. IL-13: saline n=6; PPE n=9; control n=6; CS n=7.
	Fig. 10. Gene expression for Cxcl9 (A,B), Cxcl10 (C,D), Il-12b (E,F). (A) P=0.004, (B) P=0.017, compared to their respective controls. (C) P=0.001, (D) P=0.036, compared to their respective controls. (F) *P=0.026, compared to the control group. We did not observe any significant results following iNos (G,H) and Irf-5 (I,J) analysis. Values are expressed as the mean±s.e. CXCL9: saline n=14; PPE n=13; control n=11; CS n=13. CXCL10: saline n=14; PPE n=14; control n=11; CS n=16. IL-12b: saline n=13; PPE n=13; control n=10; CS n=11. iNOS: saline n=7; PPE n=4; control n=4; CS n=3. Irf5: saline n=14; PPE n=15; control n=12; CS n=17.
	Fig. 11. Gene expression for Arg1 (A,B), Fizz1 (C,D). (A)P=0.005 compared to the saline group. (C) P=0.001 compared to the saline group. No differences in Ym1 and (E,F) Irf4 (G,H) gene expression were detected. Values are expressed as the mean±s.e. Arg1: saline n=14; PPE n=15; control n=12; CS n=17. Fizz1: saline n=13; PPE n=14; control n=12; CS n=16. Ym1: saline n=13; PPE n=14; control n=9; CS n=16. Irf4: saline n=14; PPE n=14; control n=10; CS n=14.