Volume 85, Issue 4, Pages 794-806 (April 2014) Failed renoprotection by alternatively activated bone marrow macrophages is due to a proliferation-dependent phenotype switch in vivo  Qi Cao, Yiping Wang, Dong Zheng, Yan Sun, Changqi Wang, Xin M. Wang, Vincent W.S. Lee, Ya Wang, Guoping Zheng, Thian K. Tan, Yuan M. Wang, Stephen I. Alexander, David C.H. Harris  Kidney International  Volume 85, Issue 4, Pages 794-806 (April 2014) DOI: 10.1038/ki.2013.341 Copyright © 2014 International Society of Nephrology Terms and Conditions

Figure 1 Similar phenotypic features of bone marrow (BM) and splenic (SP) M2 macrophages. Macrophages were polarized into M2 macrophages as described in Methods. (a) CD80, CD86, B7-H1, Gr-1, and CD115 were assessed by fluorescence-activated cell sorting. (b, c) The mRNA expression of arginase, mannose receptor (MR), YM1, FIZZ1, interleukin (IL)-10, transforming growth factor (TGF)-β, tumor necrosis factor (TNF)-α, and IL-6 was measured by quantitative PCR. (d) Inhibition of CD4+ T-cell proliferation was examined with various dosages of BM-M2 and SP-M2 macrophages. Data represent the mean±standard error of mean of four experiments. *P<0.05 vs. BM-M0, and #P<0.05 vs. SP-M0. Kidney International 2014 85, 794-806DOI: (10.1038/ki.2013.341) Copyright © 2014 International Society of Nephrology Terms and Conditions

Figure 2 Failed protection by bone marrow (BM)-M2 macrophages of renal structural and functional injury in adriamycin nephropathy (AN) mice. (a–c) Serum creatinine, creatinine clearance, and proteinuria were assessed in normal, AN+vehicle, AN+BM-M0, AN+BM-M2, AN+Splenic (SP)-M0, AN+SP-M2 at day 28 after adriamycin injection. (d) Periodic acid–Schiff (PAS)-stained sections of renal cortices at day 28 (× 200). (e–g) Kidney injury (glomerulosclerosis, damaged tubules, and interstitial volume) was assessed quantitatively. The values represent the mean±standard error of mean of evaluations from each group (n=7 per group). *P<0.05 and **P<0.01 vs. AN+SP-M0. Kidney International 2014 85, 794-806DOI: (10.1038/ki.2013.341) Copyright © 2014 International Society of Nephrology Terms and Conditions

Figure 3 In vivo tracking of transfused M2 macrophages. Carboxyfluorescein diacetate succinimidyl ester (CFSE)-labeled bone marrow (BM)-M2 and splenic (SP)-M2 macrophages were transfused into BALB/c mice at day 5 after adriamycin injection. 2 days and 23 days later, kidney and kidney draining lymph node (KDLN) were harvested for analysis. BM-M2 (a) and SP-M2 (b) macrophages were seen in the kidney and KDLN at day 2 and day 23 after macrophage transfusion. Bar=50μM. (c) Numbers of CFSE-labeled BM-M2 and SP-M2 macrophages were counted. The values represent the mean±standard error of mean of evaluations from each group (n=6 per group). **P<0.01 vs. day 2. (d) Chemokine receptors (CCR1, CCR2, CCR3, CCR5, CCR7, and CX3CR1) were assessed by quantitative PCR in fresh cultured BM-M2 and SP-M2 macrophages. Data represent the mean±s.e.m. of four experiments. **P<0.01 vs. SP-M2. (e) Chemokines (CCL2, CCL3, CCL4, CCL5, CX3CL1, and IP-10) were assessed by quantitative PCR in the kidney of normal (Nor) and adriamycin nephropathy (week (wk)1, wk2, wk4) mice. The values represent the mean±s.e.m. evaluations from each group (n=6 per group). *P<0.05 vs. normal. Kidney International 2014 85, 794-806DOI: (10.1038/ki.2013.341) Copyright © 2014 International Society of Nephrology Terms and Conditions

Figure 4 Loss of protective phenotype of bone marrow (BM)-M2 macrophage in diseased kidney. Carboxyfluorescein diacetate succinimidyl ester (CFSE)-positive cells were sorted from kidneys of adriamycin (AN) mice at days 2, 7, and 23 after BM-M2 and splenic (SP)-M2 macrophage transfusion. The mRNA expression of arginase, mannose receptor (MR), YM1, FIZZ1, interleukin (IL)-10, transforming growth factor (TGF)-β, iNOS, tumor necrosis factor (TNF)-α, IL-6, and CCL2 of transfused BM-M2 macrophages (a, b) and SP-M2 macrophages (c, d) was examined by quantitative PCR. The values represent the mean±standard error of mean of evaluations from each group (n=6 per group). *P<0.05, **P<0.01, and ***P<0.001 vs. day 0. Kidney International 2014 85, 794-806DOI: (10.1038/ki.2013.341) Copyright © 2014 International Society of Nephrology Terms and Conditions

Figure 5 Proliferation of bone marrow (BM)-M2 macrophages in diseased kidney. Carboxyfluorescein diacetate succinimidyl ester (CFSE)-labeled BM-M2 and splenic (SP)-M2 macrophages were transfused into BALB/c mice at day 5 after adriamycin (ADR) injection. Total kidney cells were separated from kidneys of adriamycin nephropathy (AN) mice at days 2, 7, and 23 after M2 macrophage transfusion. Proliferation of transfused macrophages in kidney was measured by calculating the percentage and mean fluorescence intensity. Transfused BM-M2 (a) and SP-M2 (b) macrophages in the kidney were counted by flow cytometry at days 2, 7, and 23 after macrophage transfusion. (c) The percentage of transfused BM-M2 and SP-M2 macrophages in kidney was assessed. (d) Mean fluorescence intensity (MFI) of CFSE-labeled BM-M2 and SP-M2 macrophages in the kidney was assessed quantitatively. The values represent the mean±standard error of mean of evaluations from each group (n=6 per group). **P<0.01 vs. day 2. Kidney International 2014 85, 794-806DOI: (10.1038/ki.2013.341) Copyright © 2014 International Society of Nephrology Terms and Conditions

Figure 6 Loss of M2 phenotype of proliferated bone marrow (BM)-M2 macrophages in vitro. (a) Carboxyfluorescein diacetate succinimidyl ester (CFSE)-labeled BM-M2 and splenic (SP)-M2 macrophages were cultured in vitro for 2 weeks, and CFSE-low cells regarded as proliferated (P) and CFSE-high cells regarded as nonproliferated (NP). These populations of BM-M2 macrophages were separated by flow cytometry at week 1 and week 2. (b) Separated nonproliferated (NP) and proliferated (P) BM-M2 macrophages after 1 week of culture, proliferated BM-M2 macrophages after 2 weeks of culture, and SP-M2 macrophages after 1 or 2 weeks of culture were co-cultured with splenic CD4+ T cells for 72h. CD4+ T-cell proliferation was examined by 3H thymidine incorporation assay. (c, d) The mRNA expression of arginase, mannose receptor (MR), interleukin (IL)-10, and transforming growth factor (TGF)-β in BM-M2 and SP-M2 macrophages was examined by quantitative PCR. Data represent the mean±standard error of mean of four experiments. *P<0.05, **P<0.01, ***P<0.001 vs. BM-M2, and #P<0.05 vs. SP-M2. Kidney International 2014 85, 794-806DOI: (10.1038/ki.2013.341) Copyright © 2014 International Society of Nephrology Terms and Conditions

Figure 7 Proliferation of bone marrow (BM) and splenic (SP) M2 macrophages in vitro. BM-M2 and SP-M2 macrophages were cultured in six-well plates (1 × 104 per well) with macrophage colony-stimulating factor (M-CSF) (2ng/ml), or M-CSF plus neutralizing M-CSF receptor antibody for 14 days. (a) Photos of BM-M2 and SP-M2 macrophages were taken by phase-contrast microscopy at days 0, 4, 7, and 14. (b) Quantitative analysis of numbers of BM-M2 and SP-M2 macrophages at days 0, 4, 7, and 14. (c) Quantitative analysis of numbers of BM-M2 and SP-M2 macrophages at day 7 after culture with various doses of M-CSF (2ng/ml, 10ng/ml, 50ng/ml). Data represent the mean±standard error of mean of four experiments. **P<0.01 vs. BM-M2+antibodies (Abs). Kidney International 2014 85, 794-806DOI: (10.1038/ki.2013.341) Copyright © 2014 International Society of Nephrology Terms and Conditions

Figure 8 Correlation of proliferation of transfused bone marrow (BM) M2 macrophages with macrophage colony-stimulating factor (M-CSF) expression on tubule cells. (a)Carboxyfluorescein diacetate succinimidyl ester (CSFE)-labeled transfused BM-M2 and splenic (SP)-M2 macrophages (red) and M-CSF-positive tubule cells (green) were seen in kidney at days 2, 7, and 23 after M2 macrophage transfusion. Bar=50μM. (b) The percentage of M-CSF-positive area in kidney section was quantified at days 2, 7, and 23 after M2 macrophage transfusion. (c) Numbers of CFSE-labeled BM-M2 and SP-M2 macrophages in kidney sections were counted. The values represent the mean±standard error of mean per high-power field (h.p.f.) from each group (n=6 per group). **P<0.01, and ***P<0.001 vs. SP-M2. (d–g) Correlation between numbers of BM-M2 and SP-M2 macrophages with the percentage of M-CSF-positive area in kidney sections was assessed at day 7 and day 23 after M2 macrophage transfusion. Kidney International 2014 85, 794-806DOI: (10.1038/ki.2013.341) Copyright © 2014 International Society of Nephrology Terms and Conditions

Figure 9 c-fms blockade of bone marrow (BM)-M2 macrophage proliferation and phenotype change. BM-M2 macrophages were cultured with macrophage colony-stimulating factor (M-CSF) (10ng/ml) and with various doses of c-fms inhibitor GW2580 (0, 0.01, 0.1, 1, 10μM/ml) for 2 weeks. The mRNA expression of arginase, mannose receptor (MR), interleukin (IL)-10, and transforming growth factor (TGF)-β in BM-M2 macrophages was examined by quantitative PCR. Data represent the mean±standard error of mean of four experiments. *P<0.05, **P<0.01, ***P<0.001 vs. M2. Kidney International 2014 85, 794-806DOI: (10.1038/ki.2013.341) Copyright © 2014 International Society of Nephrology Terms and Conditions