Volume 84, Issue 4, Pages 745-755 (October 2013) Discrete functions of M2a and M2c macrophage subsets determine their relative efficacy in treating chronic kidney disease  Junyu Lu, Qi Cao, Dong Zheng, Yan Sun, Changqi Wang, Xiao Yu, Ya Wang, Vincent W.S. Lee, Guoping Zheng, Thian K. Tan, Xin Wang, Stephen I Alexander, David C.H. Harris, Yiping Wang  Kidney International  Volume 84, Issue 4, Pages 745-755 (October 2013) DOI: 10.1038/ki.2013.135 Copyright © 2013 International Society of Nephrology Terms and Conditions

Figure 1 Adoptive transfer of M2a and M2c macrophages protects renal functional and structural injury in adriamycin-induced nephropathy (AN) mice. M2c macrophages were more effective than M2a macrophages in reducing proteinuria, glomerular sclerosis, tubular atrophy, and interstitial expansion. (a) Representative periodic acid–Schiff (PAS) sections of renal cortices at day 28 (original magnification × 200). (b–d) Serum creatinine, creatinine clearance, and proteinuria were assessed in normal mice, AN mice, and AN mice treated with M0, M2a, or M2c macrophages at day 28. (e–g) Quantitative analysis of structural changes in kidney. The values are mean±s.e.m. of evaluations for each group (n=6 per group). *P<0.05, **P<0.01, and ***P<0.001 versus AN+M0, #P<0.05 versus AN+M2a. Kidney International 2013 84, 745-755DOI: (10.1038/ki.2013.135) Copyright © 2013 International Society of Nephrology Terms and Conditions

Figure 2 Adoptive transfer of M2a and M2c macrophages reduced renal inflammation. The reduction of CD4+ cell infiltration was greater in adriamycin-induced nephropathy (AN) mice treated with M2c than M2a macrophages. (a) F4/80+, CD4+, and CD8+ T-cell staining in renal cortices at day 28 (original magnification in high power field (hpf) × 200). (b–d) Quantitative analysis of F4/80+ cells, CD4+, and CD8+ T cells in normal mice, AN mice, and AN mice treated with M0, M2a, or M2c macrophages at day 28. The values are mean±s.e.m. of evaluations from each group (n=6 per group). *P<0.05, **P<0.01, and ***P<0.001 versus AN+M0, #P<0.05 versus AN+M2a. Kidney International 2013 84, 745-755DOI: (10.1038/ki.2013.135) Copyright © 2013 International Society of Nephrology Terms and Conditions

Figure 3 Adoptive transfer of M2a and M2c macrophages ameliorated renal fibrosis; the antifibrotic effect of M2c macrophages was significantly superior to that of M2a. (a) Collagen I, α-smooth muscle actin (α-SMA), and fibroblast-specific protein 1 (FSP-1) staining in renal cortices at day 28 (original magnification × 200). (b–d) Quantitative analysis of collagen I-, α-SMA-, and FSP-1-positive staining area in normal mice, adriamycin-induced nephropathy (AN) mice, and AN mice treated with M0, M2a, or M2c macrophages at day 28. The values are mean±s.e.m. of evaluations from each group (n=6 per group). **P<0.01 and ***P<0.001 versus AN+M0; #P<0.05 versus AN+M2a. Kidney International 2013 84, 745-755DOI: (10.1038/ki.2013.135) Copyright © 2013 International Society of Nephrology Terms and Conditions

Figure 4 M2c significantly increased regulatory T cells in renal draining lymph nodes. (a) Adoptive transfer of M2c macrophages increased Foxp3+ cells in local draining lymph nodes (LNs) at day 28 (original magnification × 400). (b–d) Quantitative analysis of Foxp3+ cells staining in LN, spleen, and kidney was performed in normal mice, control adriamycin-induced nephropathy (AN) mice, and AN mice treated with M0, M2a, or M2c macrophages. The values are mean ±s.e.m. of evaluations from each group (n=6 per group). *P<0.05 versus AN+M0 and ##P<0.01 versus AN+M2a. hpf, high power field. Kidney International 2013 84, 745-755DOI: (10.1038/ki.2013.135) Copyright © 2013 International Society of Nephrology Terms and Conditions

Figure 5 Depletion of CD4+CD25+Foxp3+ regulatory T cells (Tregs) in the peripheral blood. (a) BALB/c mice were transfused with M2a or M2c at day 5, following injection with PC61 at days 8 to 13 after adriamycin (ADR) injection. The blood was collected at days 5 and 15 after PC61 administration. (b–d) The fluorescence-activated cell sorting (FACS) result shows that the percentage of Tregs remained relatively stable in normal mice and control adriamycin-induced nephropathy (AN) mice on days 5 and 15, and Tregs were undetectable in AN mice treated with PC61, AN mice treated with PC61 and M2a macrophages, or AN mice treated with PC61 and M2c macrophages. The values are mean±s.e.m. of evaluations from each group (n=6 per group). ***P<0.001 versus AN. Kidney International 2013 84, 745-755DOI: (10.1038/ki.2013.135) Copyright © 2013 International Society of Nephrology Terms and Conditions

Figure 6 Depletion of CD4+CD25+Foxp3+ regulatory T cells (Tregs) in renal draining lymph node (LN), spleen, and kidney. (a) Number of Foxp3+ cells in local renal draining LNs, spleen, and kidney at day 28 (original magnification × 400). (b–d) Quantitative analysis of Foxp3+ cells staining in LN, spleen, and kidney was performed in normal mice, control adriamycin-induced nephropathy (AN) mice, AN mice treated with PC61, AN mice treated with PC61 and M2a macrophages, or AN mice treated with PC61 and M2c macrophages. The values are mean±s.e.m. of evaluations from each group (n=6 per group). ***P<0.001 versus AN. Kidney International 2013 84, 745-755DOI: (10.1038/ki.2013.135) Copyright © 2013 International Society of Nephrology Terms and Conditions

Figure 7 Adoptive transfer of M2a or M2c macrophages provided equal protective effects against renal functional and structural injury after depletion of regulatory T cells (Tregs). (a) Periodic acid–Schiff (PAS) sections of renal cortices at day 28 (original magnification × 200). (b–d) Serum creatinine, creatinine clearance, and proteinuria were assessed in normal mice, adriamycin-induced nephropathy (AN) mice, AN mice treated with PC61, AN mice treated with PC61 and M2a macrophages, or AN mice treated with PC61 and M2c macrophages at day 28. (e–g) Quantitative analysis of structural changes in kidney (PAS). The values are mean±s.e.m. of evaluations from each group (n=6 per group). *P<0.05 and **P<0.01 versus AN+PC61. Kidney International 2013 84, 745-755DOI: (10.1038/ki.2013.135) Copyright © 2013 International Society of Nephrology Terms and Conditions

Figure 8 Adoptive transfer of M2a or M2c macrophages reduced renal inflammation after depletion of regulatory T cells (Tregs). (a) F4/80+, CD4+, and CD8+ cell staining in renal cortices at day 28 (original magnification × 200). (b–d) Quantitative analysis of F4/80+, CD4+, and CD8+ T cells in normal mice, adriamycin-induced nephropathy (AN) mice, AN mice treated with PC61, AN mice treated with PC61 and M2a macrophages, or AN mice treated with PC61 and M2c macrophages at day 28. The values are mean±s.e.m. of evaluations from each group (n=6 per group). *P<0.05 and **P<0.01 versus AN+PC61. hpf, high power field. Kidney International 2013 84, 745-755DOI: (10.1038/ki.2013.135) Copyright © 2013 International Society of Nephrology Terms and Conditions

Figure 9 Adoptive transfer of M2a or M2c macrophages ameliorated renal fibrosis after depletion of regulatory T cells (Tregs). (a) Collagen I, α-smooth muscle actin (α-SMA), and fibroblast-specific protein 1 (FSP-1) staining in renal cortices at day 28 (original magnification × 200). (b–d) Quantitative analysis of collagen I-, α-SMA-, and FSP-1-positive staining area in normal mice, adriamycin-induced nephropathy (AN) mice, AN mice treated with PC61, AN mice treated with PC61 and M2a macrophages, or AN mice treated with PC61 and M2c macrophages at day 28. The values are mean±s.e.m. of evaluations from each group (n=6 per group). *P<0.05, **P<0.01, and ***P<0.001 versus AN+PC61. Kidney International 2013 84, 745-755DOI: (10.1038/ki.2013.135) Copyright © 2013 International Society of Nephrology Terms and Conditions