pubmed.ncbi.nlm.nih.gov

Genistein Promotes M2 Macrophage Polarization via Aryl Hydrocarbon Receptor and Alleviates Intestinal Inflammation in Broilers with Necrotic Enteritis - PubMed

  • ️Mon Jan 01 2024

Genistein Promotes M2 Macrophage Polarization via Aryl Hydrocarbon Receptor and Alleviates Intestinal Inflammation in Broilers with Necrotic Enteritis

Shuli Quan et al. Int J Mol Sci. 2024.

Abstract

The aryl hydrocarbon receptor (AhR) is a transcription factor that regulates the immune system through complicated transcriptional programs. Genistein, an AhR ligand, exhibits anti-inflammatory properties. However, its role in modulating immune responses via the AhR signaling pathway remains unclear. In this study, 360 male Arbor Acre broilers (1-day-old) were fed a basal diet supplemented with 40 or 80 mg/kg genistein and infected with or without Clostridium perfringens (Cp). Our results demonstrated that genistein ameliorated Cp-induced intestinal damage, as reflected by the reduced intestinal lesion scores and improved intestinal morphology and feed-to-gain ratio. Moreover, genistein increased intestinal sIgA, TGF-β, and IL-10, along with elevated serum IgG, IgA, and lysozyme levels. Genistein improved intestinal AhR and cytochrome P450 family 1 subfamily A member 1 (CYP1A1) protein levels and AhR+ cell numbers in Cp-challenged broilers. The increased number of AhR+CD163+ cells in the jejunum suggested a potential association between genistein-induced AhR activation and anti-inflammatory effects mediated through M2 macrophage polarization. In IL-4-treated RAW264.7 cells, genistein increased the levels of AhR, CYP1A1, CD163, and arginase (Arg)-1 proteins, as well as IL-10 mRNA levels. This increase was attenuated by the AhR antagonist CH223191. In summary, genistein activated the AhR signaling pathway in M2 macrophages, which enhanced the secretion of anti-inflammatory cytokines and attenuated intestinal damage in Cp-infected broilers Cp.

Keywords: M2 macrophages; aryl hydrocarbon receptor; genistein; intestinal inflammation; necrotic enteritis.

PubMed Disclaimer

Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1

Genistein alleviates intestinal damage in broilers infected with Clostridium perfringens. (A) Hematoxylin and eosin (HE) staining of duodenum of broilers (×40 magnification, scale bar = 200 μm). (B) The intestinal lesion scores of broilers. (C) The villus height (VH), crypt depth (CD), and the ratio of VH to CD (VH/CD) in the duodenum of broilers. Con, basal diet; Gen40, basal diet supplemented with 40 mg/kg genistein; Gen80, Gen80 diet; Cp, basal diet and Cp infection; Cp+Gen40, Gen40 diet and Cp infection; Cp+Gen80, Gen80 diet and Cp infection. The differences among groups were determined by ANOVA using Duncan’s test. The results are presented as mean ± SEM, n = 12. Letter a indicates p < 0.05 vs. Con group; letter b indicates p < 0.05 vs. Cp group.

Figure 1
Figure 1

Genistein alleviates intestinal damage in broilers infected with Clostridium perfringens. (A) Hematoxylin and eosin (HE) staining of duodenum of broilers (×40 magnification, scale bar = 200 μm). (B) The intestinal lesion scores of broilers. (C) The villus height (VH), crypt depth (CD), and the ratio of VH to CD (VH/CD) in the duodenum of broilers. Con, basal diet; Gen40, basal diet supplemented with 40 mg/kg genistein; Gen80, Gen80 diet; Cp, basal diet and Cp infection; Cp+Gen40, Gen40 diet and Cp infection; Cp+Gen80, Gen80 diet and Cp infection. The differences among groups were determined by ANOVA using Duncan’s test. The results are presented as mean ± SEM, n = 12. Letter a indicates p < 0.05 vs. Con group; letter b indicates p < 0.05 vs. Cp group.

Figure 2
Figure 2

Genistein enhances immune responses in the intestine and serum of broilers. (A) The content of sIgA, TGF-β, and IL-10 in the jejunal mucosa of broilers was measured by ELISA kits. (B) The concentration of serum IgG and IgA of broilers was determined by ELISA kits. The lysozyme activity was measured by lysozyme assay kits. Con, basal diet; Gen40, basal diet supplemented with 40 mg/kg genistein; Gen80, Gen80 diet; Cp, basal diet and Cp infection; Cp+Gen40, Gen40 diet and Cp infection; Cp+Gen80, Gen80 diet and Cp infection. The differences among groups were determined by ANOVA using Duncan’s test. The results are presented as mean ± SEM, n = 12. Letter a indicates p < 0.05 vs. Con group; letter b indicates p < 0.05 vs. Cp group.

Figure 3
Figure 3

Genistein activates the intestinal AhR pathway in broilers challenged with NE. (A) Western blot of AhR and CYP1A1 in the jejunum of broilers. (B) Quantification of AhR and CYP1A1 protein levels, n = 3. Con, basal diet; Gen40, basal diet supplemented with 40 mg/kg genistein; Gen80, Gen80 diet; Cp, basal diet and Cp infection; Cp+Gen40, Gen40 diet and Cp infection; Cp+Gen80, Gen80 diet and Cp infection. The differences among groups were determined by ANOVA using Duncan’s test. The results are presented as mean ± SEM. Letter a indicates p < 0.05 vs. Con group; letter b indicates p < 0.05 vs. Cp group.

Figure 4
Figure 4

Genistein promotes AhR expression in intestinal M2 macrophages of broilers. (A) Sections of jejunum tissues were immunostained with DAPI (blue), AhR (green), and CD163 (red) (×400 magnification, scale bar = 50 μm). (B) The quantified number of AhR+ and AhR+CD163+ cells in the jejunum of broilers, n = 3. Con, basal diet; Gen40, basal diet supplemented with 40 mg/kg genistein; Gen80, Gen80 diet; Cp, basal diet and Cp infection; Cp+Gen40, Gen40 diet and Cp infection; Cp+Gen80, Gen80 diet and Cp infection. The differences among groups were determined by ANOVA using Duncan’s test. The results are presented as mean ± SEM. Letter b indicates p < 0.05 vs. Cp group.

Figure 5
Figure 5

Genistein promotes M2 macrophage polarization depending on AhR in RAW264.7 cells. (A) Cells were treated with genistein (0, 25, 50, 100, 200 μM) for 48 h. The effect of genistein on cell viability was determined by the CCK-8 assay, n = 10. (B) Cells were prestimulated with IL-4 (20 ng/mL) for 24 h and then treated with genistein (0, 25, 50, 100, 200 μM) and IL-4 (20 ng/mL) for 24 h. The relative mRNA expression levels of AhR and Arg-1 were measured by qRT-PCR. (C) Cells were prestimulated with IL-4 (20 ng/mL) for 24 h and then treated with genistein (50 μM), FICZ (100 nM), CH223191 (5 μM), genistein + CH223191 in the presence of IL-4 for 24 h. The relative mRNA expression levels of IL-10 were measured by qRT-PCR. (D) The protein levels of AhR, CYP1A1, CD163, and Arg-1 were determined by Western blot. The differences among groups were determined by ANOVA using Duncan’s test. The results are presented as mean ± SEM, n = 3. Letter a indicates p < 0.05 vs. Con group; letter b indicates p < 0.05 vs. IL-4-treatment group; letter c indicates p < 0.05 vs. Gen-treatment group.

Figure 5
Figure 5

Genistein promotes M2 macrophage polarization depending on AhR in RAW264.7 cells. (A) Cells were treated with genistein (0, 25, 50, 100, 200 μM) for 48 h. The effect of genistein on cell viability was determined by the CCK-8 assay, n = 10. (B) Cells were prestimulated with IL-4 (20 ng/mL) for 24 h and then treated with genistein (0, 25, 50, 100, 200 μM) and IL-4 (20 ng/mL) for 24 h. The relative mRNA expression levels of AhR and Arg-1 were measured by qRT-PCR. (C) Cells were prestimulated with IL-4 (20 ng/mL) for 24 h and then treated with genistein (50 μM), FICZ (100 nM), CH223191 (5 μM), genistein + CH223191 in the presence of IL-4 for 24 h. The relative mRNA expression levels of IL-10 were measured by qRT-PCR. (D) The protein levels of AhR, CYP1A1, CD163, and Arg-1 were determined by Western blot. The differences among groups were determined by ANOVA using Duncan’s test. The results are presented as mean ± SEM, n = 3. Letter a indicates p < 0.05 vs. Con group; letter b indicates p < 0.05 vs. IL-4-treatment group; letter c indicates p < 0.05 vs. Gen-treatment group.

Similar articles

Cited by

References

    1. Rothhammer V., Quintana F.J. The aryl hydrocarbon receptor: An environmental sensor integrating immune responses in health and disease. Nat. Rev. Immunol. 2019;19:184–197. doi: 10.1038/s41577-019-0125-8. - DOI - PubMed
    1. Schmidt J.V., Bradfield C.A. Ah receptor signaling pathways. Annu. Rev. Cell Dev. Biol. 1996;12:55–89. doi: 10.1146/annurev.cellbio.12.1.55. - DOI - PubMed
    1. Gronke K., Hernández P.P., Zimmermann J., Klose C.S.N., Kofoed-Branzk M., Guendel F., Witkowski M., Tizian C., Amann L., Schumacher F., et al. Interleukin-22 protects intestinal stem cells against genotoxic stress. Nature. 2019;566:249–253. doi: 10.1038/s41586-019-0899-7. - DOI - PMC - PubMed
    1. Climaco-Arvizu S., Dominguez-Acosta O., Cabanas-Cortes M.A., Rodriguez-Sosa M., Gonzalez F.J., Vega L., Elizondo G. Aryl hydrocarbon receptor influences nitric oxide and arginine production and alters M1/M2 macrophage polarization. Life Sci. 2016;155:76–84. doi: 10.1016/j.lfs.2016.05.001. - DOI - PMC - PubMed
    1. Cui X., Ye Z., Wang D., Yang Y., Jiao C., Ma J., Tang N., Zhang H. Aryl hydrocarbon receptor activation ameliorates experimental colitis by modulating the tolerogenic dendritic and regulatory T cell formation. Cell Biosci. 2022;12:46. doi: 10.1186/s13578-022-00780-z. - DOI - PMC - PubMed

MeSH terms

Substances