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. 2010 Jun;21(6):933-42.
doi: 10.1681/ASN.2009060592. Epub 2010 Mar 18.

IL-10/TGF-beta-modified macrophages induce regulatory T cells and protect against adriamycin nephrosis

Qi Cao  1 , Yiping Wang, Dong Zheng, Yan Sun, Ya Wang, Vincent W S Lee, Guoping Zheng, Thian Kui Tan, Jon Ince, Stephen I Alexander, David C H Harris
Affiliations

IL-10/TGF-beta-modified macrophages induce regulatory T cells and protect against adriamycin nephrosis

Qi Cao et al. J Am Soc Nephrol. 2010 Jun.

Abstract

IL-10/TGF-beta-modified macrophages, a subset of activated macrophages, produce anti-inflammatory cytokines, suggesting that they may protect against inflammation-mediated injury. Here, macrophages modified ex vivo by IL-10/TGF-beta (IL-10/TGF-beta Mu2) significantly attenuated renal inflammation, structural injury, and functional decline in murine adriamycin nephrosis (AN). These cells deactivated effector macrophages and inhibited CD4+ T cell proliferation. IL-10/TGF-beta Mu2 expressed high levels of the regulatory co-stimulatory molecule B7-H4, induced regulatory T cells from CD4+CD25- T cells in vitro, and increased the number of regulatory T cells in lymph nodes draining the kidneys in AN. The phenotype of IL-10/TGF-beta Mu2 did not switch to that of effector macrophages in the inflamed kidney, and these cells did not promote fibrosis. Taken together, these data demonstrate that IL-10/TGF-beta-modified macrophages effectively protect against renal injury in AN and may become part of a therapeutic strategy for chronic inflammatory disease.

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Figures

Figure 1.
Figure 1.
Macrophages were polarized into M0, M1, IL-4/13 macrophages (IL-4/13 M2), and IL-10/TGF-β macrophages (IL-10/TGF-β Μ2), as described in the Concise Methods section. (A) MHC-I) and CD86 were assessed by FACS. (B) The mRNA expression of IL-10, TGF-β, TNF-α, iNOS, arginase, MR, CCL17, YM1, and FIZZ1 was measured by quantitative PCR, relative to the control of each experiment. (C) B7 family members were examined using FACS. Data are means ± SEM of four experiments. *P < 0.05, **P < 0.01 versus M0; #P < 0.05 versus IL-4/13 M2.
Figure 2.
Figure 2.
Transfused IL-10/TGF-β M2 provides protective effects on renal structural and functional injury. (A through C) Serum creatinine, creatinine clearance, and urine protein were assessed in normal, AN+vehicle, AN+M0, and AN+IL-10/TGF-β macrophages (IL-10/TGF-β Μ2) at day 28. (D) PAS- and trichrome-stained sections of renal cortices at day 28. (E through I) Kidney injury (PAS) and fibrosis (trichrome) were assessed quantitatively. Data are means ± SEM of evaluations from each group (n = 7 per group). *P < 0.05, **P < 0.01 versus AN+M0. Magnification, ×200.
Figure 3.
Figure 3.
IL-10/TGF-β M2 suppresses M1 in vitro and endogenous renal macrophages in vivo. (A) M1 co-cultured with IL-10/TGF-β Μ2 for 24 hours. The mRNA expression of IL-10, TGF-β, TNF-α, iNOS, arginase, and MR of M1 was examined by quantitative PCR. Data are means ± SEM of four experiments. (B) EM (CD11b+DiI−) were separated from mice that had AN and received a transfusion of IL-10/TGF-β Μ2 by FACS sorting at day 28. (C) Their mRNA expression of IL-10, TGF-β, TNF-α, iNOS, arginase, and MR was assessed by quantitative PCR. Data are means ± SEM of evaluations from each group (n = 7 per group). *P < 0.05, **P < 0.01 versus AN+M0.
Figure 4.
Figure 4.
IL-10/TGF-β M2 inhibits proliferation of CD4+ T cells. (A and B) CD4+ T cells were co-cultured with an equal number of macrophages (Mφ) of various phenotypes (M0, M1, and IL-10/TGF-β M2) in the presence of anti-mouse CD3/CD28, and CD4+ T cell proliferation was assessed using WST-1 at day 2 and day 3 and CFSE at day 3. (C) Inhibition of CD4+ T cell proliferation was examined with various dosages of IL-10/TGF-β M2. (D) Neutralizing anti–IL-10, –TGF-β, and –B7-H4 antibodies were used to block the effect of IL-10/TGF-β Μ2 on CD4+ T cell proliferation. Data are means ± SEM of four experiments. *P < 0.05; **P < 0.01.
Figure 5.
Figure 5.
IL-10/TGF-β Μ2 induces Tregs in vitro and in vivo. (A and B) Macrophages (Mφ) of various phenotypes were co-cultured with CD4+CD25− T cells for 7 days, and the percentage of CD4+Foxp3+ cells was assessed by FACS. Data are means ± SEM of four experiments. **P < 0.01 versus M0+CD4. (C) Neutralizing antibodies against IL-10, TGF-β, B7-H4, or the three antibodies were used to block the effects of IL-10/TGF-β M2. (D) Transwell was used to examine dependence on cell contact. Data are means ± SEM of four experiments. *P < 0.05, **P < 0.01 versus IL-10/TGF-β Μ2+CD4. (E) CD4+Foxp3+ cells were assessed by FACS in RDLN or PBMC from mice that had AN and received a transfusion of M0 or IL-10/TGF-β Μ2. (F) Representative Foxp3 immunostaining of spleen and RDLN. Data are means ± SEM of evaluations from each group (n = 7 per group). *P < 0.05, **P < 0.01 versus AN+M0.
Figure 6.
Figure 6.
Preferential accumulation of IL-10/TGFβ M2 in inflamed kidney and renal draining lymph nodes. (A) DiI-labeled macrophages were seen in kidney, RDLN, and spleen at day 28 after adriamycin administration. (B) Numbers of DiI-labeled macrophages were counted. Data are means ± SEM per high-power field (hpf) from each group (n = 7 per group). *P < 0.05, **P < 0.01 versus AN+M0. Bar = 50 μM.
Figure 7.
Figure 7.
Maintenance of anti-inflammatory phenotyes in vivo of transfused IL-10/TGFβ M2. (A) Transfused IL-10/TGF-β M2 in kidney were separated by FACS sorting. The mRNA expression of IL-10, TGF-β, TNF-α, iNOS, arginase, and MR was examined by real-time PCR. (B) Consecutive frozen sections were stained with iNOS, MR, and B7-H4. Cells fluorescing orange are double positive for DiI and antibody, whereas cells fluorescing red are DiI+ cells and green are iNOS+, MR+, or B7-H4+ cells. Arrows point to the identical DiI+ cells stained by antibodies to iNOS, MR, and B7-H4, respectively. (C) Numbers of double-positive cells were counted. Data are means ± SEM per high-power field from each group (n = 4 per group). *P < 0.05, **P < 0.01 versus AN+M0. Bar = 50 μM.
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