CSF-1-dependant donor-derived macrophages mediate chronic graft-versus-host disease

Abstract

Chronic GVHD (cGVHD) is the major cause of late, nonrelapse death following stem cell transplantation and characteristically develops in organs such as skin and lung. Here, we used multiple murine models of cGVHD to investigate the contribution of macrophage populations in the development of cGVHD. Using an established IL-17-dependent sclerodermatous cGVHD model, we confirmed that macrophages infiltrating the skin are derived from donor bone marrow (F4/80+CSF-1R+CD206+iNOS-). Cutaneous cGVHD developed in a CSF-1/CSF-1R-dependent manner, as treatment of recipients after transplantation with CSF-1 exacerbated macrophage infiltration and cutaneous pathology. Additionally, recipients of grafts from Csf1r-/- mice had substantially less macrophage infiltration and cutaneous pathology as compared with those receiving wild-type grafts. Neither CCL2/CCR2 nor GM-CSF/GM-CSFR signaling pathways were required for macrophage infiltration or development of cGVHD. In a different cGVHD model, in which bronchiolitis obliterans is a prominent manifestation, F4/80+ macrophage infiltration was similarly noted in the lungs of recipients after transplantation, and lung cGVHD was also IL-17 and CSF-1/CSF-1R dependent. Importantly, depletion of macrophages using an anti-CSF-1R mAb markedly reduced cutaneous and pulmonary cGVHD. Taken together, these data indicate that donor macrophages mediate the development of cGVHD and suggest that targeting CSF-1 signaling after transplantation may prevent and treat cGVHD.

Figure 10. Lung GVHD develops in an IL-17– and CSF-1R–dependent manner.

(A) B10.BR recipients treated with 120 mg/kg/day cyclophosphamide (days –3 and –2) and lethally irradiated (day –1; 850 cGy) were transplanted with B6 BM with either B6 or Rorc^–/– Sp (n = 4/all groups). On day 60 after transplantation, pulmonary function measures were performed. R, resistance; E, elastance; C, compliance. Recipients of Rorc^–/– grafts exhibited significantly improved pulmonary function compared with that of WT graft recipients (R: *P = 0.02, *P = 0.03; E: *P = 0.01, *P = 0.03; C: **P = 0.005,*P = 0.01). (B) Similarly, recipients of Csf1r^–/– BM plus Csf1r^–/– Sp grafts had significantly improved pulmonary function compared with that of mice receiving WT BM plus Csf1r^–/– Sp. (R: *P = 0.024,*P = 0.042,*P = 0.0431; E: *P = 0.013,*P = 0.031,*P = 0.01; C: **P = 0.001, **P = 0.0031, *P = 0.033). n = 4. (C) Trichrome staining illustrates reduced collagen deposition in mice that received Csf1r^–/– grafts compared with those that received WT grafts, quantified in D as trichrome area/total area (**P = 0.0037; ***P = 0.0005; **P = 0.0023). (E) IHC for F4/80 expression in recipients of WT B6 plus Sp or BM-only grafts that were given control IgG or M279 mAb after transplantation from days 0 to 28 after transplantation. Minimal F4/80⁺ cells were noted in recipients treated with M279 mAb compared with those detected in control IgG–treated recipients. (F) Lung function parameters confirmed an improvement in lung function after M279 mAb treatment (R: **P = 0.001,**P = 0.008; E: **P = 0.003, *P = 0.034; C: **P = 0.004,*P = 0.02). n = 4. (G) Trichrome staining confirmed that M279 mAb treatment significantly reduced collagen deposition, quantified in H (**P = 0.017; ***P = 0.0003). Statistically significant differences were calculated using unpaired t tests. Data represent the mean ± SEM. Original magnification, ×20.

Figure 9. Anti–CSF-1R Ab treatment after transplantation attenuates cutaneous GVHD.

Lethally irradiated B6 mice received G-CSF–mobilized WT BALB/c grafts and were treated with rat IgG control or anti–CSF-1R mAb (M279; 400 μg/3 times week) from days 7 to 33 after transplantation. (A) IHC to detect F4/80 expression 34 days after transplantation (n = 8–10/group for all groups; n = 3/group for TCD), confirming that M279 treatment resulted in a significant depletion of F4/80⁺ cells, quantified in B as positive/pixels/mm² (***P = 0.002). (C and D) Lethally irradiated B6D2F1 mice received B6 BM and T cell grafts and were treated with either rat IgG or M279 from days 7 to 48 after transplantation (n = 7–8/group; n = 3/group for TCD). (C and D) H&E-stained images and semiquantitative histopathology confirmed that M279 treatment resulted in a significant reduction in cutaneous pathology and cutaneous fibrosis (*P = 0.01; ***P = 0.0006). (E) Representative dot plots of PB monocyte and macrophage analysis of recipients 48 days after transplantation. Numbers in each dot plot indicate the percentage of Ly6C^hi cells (top 2 quadrants) and Ly6C^lo cells (bottom 2 quadrants) and their expression of CCR2. Results show a significant increase in the frequency of Ly6C^hi cells and a significant decrease in Ly6C^lo cell frequencies in M279-treated recipients (**P = 0.0012; *P = 0.026). n = 7/group. Statistically significant differences were calculated using 2-tailed Mann-Whitney U tests. Data represent the mean ± SEM. Original magnification, ×5.

Figure 8. F4/80⁺ macrophage infiltration and cutaneous fibrosis post BMT is CCL2/CCR2 independent.

(A) Serum CCL2 (MCP-1; pg/ml) in lethally irradiated B6D2F1 mice that received B6 BM plus T cell (Allo) or TCD grafts 7, 14, 21, and 28 days after transplantation (days 7 and 14, *P = 0.015; day 21, *P = 0.035; day 28, **P = 0.009). n = 4–6/group. (B) Lethally irradiated B6D2F1 mice received BM plus T cell grafts from B6 or Ccr2^–/– donors. IHC to detect F4/80 expression at day 28 shows that recipients of Ccr2^–/– grafts had F4/80⁺ macrophage infiltration levels similar to those in mice that received WT grafts, quantified in C as positive/pixels/mm² (P = 0.207) (n = 9 WT, n = 8 KO, n = 5 TCD). (D and E) H&E-stained images and semiquantitative histopathology (n = 15 WT, n = 14 KO, n = 6 TCD) illustrating that recipients of Ccr2^–/– grafts had cutaneous pathology similar to that of recipients of WT grafts (P = 0.720). (F and G) Lethally irradiated B6 mice received G-CSF–mobilized BALB/c or TCD grafts and were treated with hamster IgG or anti-CCL2 from days 7 to 33 (n = 15 hamster IgG, n = 14 anti-CCL2, n = 3 TCD). Similar levels of F4/80⁺ infiltration were noted in both groups, quantified in G as positive/pixels/mm² (P = 0.2727). (H) Semiquantitative histopathology score illustrating that anti-CCL2 administration had no effect on cutaneous pathology (P = 0.922). (I) Representative dot plots of recipient PB monocyte and macrophage analysis at day 21. Numbers in each dot plot indicate the percentage of Ly6C^hi cells (top 2 quadrants) and Ly6C^lo cells (bottom 2 quadrants) and their expression of CCR2. Results show no significant changes in Ly6C^hi or Ly6C^lo cell frequencies (n = 4/group). Statistically significant differences were calculated using 2-tailed Mann-Whitney U tests. Data represent the mean ± SEM. Original magnification, ×5.

Figure 7. F4/80⁺ macrophage infiltration and cutaneous fibrosis after BMT is GM-CSF/GM-CSFR independent.

(A) GM-CSF serum levels (pg/ml) in lethally irradiated B6D2F1 mice that received either B6 BM plus T cell (Allo) or TCD grafts 7, 14, 21,and 28 days after transplantation. (B) Representative images of IHC to detect F4/80 expression illustrate that recipients of common β chain^–/– grafts still acquired F4/80⁺ macrophage infiltration after transplantation (n = 6 WT, n = 4 KO, n = 3 TCD). (C) Lethally irradiated B6 mice received either G-CSF–mobilized BALB/c grafts or TCD grafts and were treated with anti–GM-CSF (400 μg every 2 weeks) from days 7 to 33 after transplantation. Representative IHC images of F4/80 expression at day 34 after transplantation (n = 4/group for all groups, except 3/TCD group), confirming that M250 treatment did not reduce F4/80⁺ macrophage infiltration, quantified in D as positive/pixels/mm². (E) Representative dot plots of PB monocyte/macrophage analysis of recipients 21 days after transplantation. Numbers in each dot plot indicate the percentage of Ly6C^hi cells (top 2 quadrants) and Ly6C^lo cells (bottom 2 quadrants) and their expression of CCR2. Results illustrate no significant changes in either Ly6C^hi or Ly6C^lo cell frequencies between groups (n = 4/group). Statistically significant differences were calculated using 2-tailed Mann-Whitney U tests. Data represent the mean ± SEM. Original magnification, ×5.

Figure 6. F4/80⁺ macrophage infiltration and cutaneous fibrosis following BMT are CSF-1/CSF-1R dependent.

Lethally irradiated B6D2F1 mice received BM plus T cells from WT FLCs or Csf1r^–/– FLCs (KO FLCs). (A) Mice that received KO FLC grafts had significantly lower GVHD clinical scores (day 7, ***P ≤ 0.0001; day 14, **P = 0.007; day 21, ***P = 0.0001; day 28, ***P = 0.0003; day 35, *P = 0.043). (B) IHC for F4/80 expression from skin 21 days after transplantation illustrates that recipients of KO FLC grafts had less F4/80⁺ macrophage infiltration than did mice that received WT FLC grafts. Minimal F4/80⁺ macrophage infiltrate was noted in mice that received TCD grafts. (C) Quantification of F4/80⁺ staining as positive/pixels/mm² (**P = 0.003). (D) Masson’s trichrome images and semiquantitative histopathology (n = 19 WT, n = 23 KO, n = 6 TCD) scores for cutaneous pathology and total cutaneous fibrosis on day 48 after transplantation confirmed that recipients of KO FLC grafts had significantly lower fibrosis compared with those that received WT FLC grafts (*P = 0.045; **P = 0.008). (E) Representative dot plots of PB monocyte/macrophage analysis of recipients 21 days after transplantation. Numbers in each dot plot indicate the percentage of Ly6C^hi cells (top 2 quadrants) and Ly6C^lo cells (bottom 2 quadrants) and their expression of CCR2. Results illustrate a significant increase in the frequency of Ly6C^hi cells and a significant decrease in Ly6C^lo cell frequency in recipients of KO FLC grafts (*P = 0.0286, *P = 0.0286). n = 4/group. Statistically significant differences were calculated using 2-tailed Mann-Whitney U tests. Data represent the mean ± SEM. Original magnification, ×5.

Figure 5. Analysis of PB and cutaneous monocyte and macrophage populations after transplantation and CSF-1 treatment.

(A) Representative dot plots of PB monocyte and macrophage analysis of recipients 19 days after transplantation. Numbers in each dot plot indicate the percentage of Ly6C^hi cells (top 2 quadrants) and Ly6C^lo cells (bottom 2 quadrants) and their expression of CCR2. Results illustrate a trend toward increased frequencies of Ly6C^lo cells after CSF-1 treatment. Representative dot plot shows frequency of Ly6C^hi cells (saline, 96.4% and CSF-1, 89.4%) and Ly6C^lo cells (saline, 3.57% and CSF-1, 10.66%). (B) Representative dot plots illustrate the gating strategy for cutaneous monocyte and macrophage analysis of recipients 19 days after transplantation. Numbers in each dot plot indicate the percentage of positive cells in each gate. Results illustrate a significant increase in the absolute numbers of CD45⁺ monocytes (Ly6C^loCD11b⁺F4/80⁺) and CD45⁺ macrophages (Ly6C^loF4/80⁺). CD45⁺Ly6C^loCD11b⁺F4/80⁺ monocyte absolute numbers: **P = 0.0087; CD45⁺Ly6C^loF4/80⁺ macrophage absolute numbers: *P = 0.026 (n = 6/group, from 3 mice/group; 2 ears/mouse). Statistically significant differences were calculated using 2-tailed Mann-Whitney U tests. Data represent the mean ± SEM.

Figure 4. CSF-1 treatment after SCT exacerbates cutaneous cGVHD.

Lethally irradiated B6 mice received G-CSF–mobilized WT BALB/c (CD45.1) or TCD grafts and were treated with either saline or CSF-1 (10 μg/daily) from days 14 to 19 after transplantation. (A) Representative IHC images of F4/80 expression in the skin of saline- and CSF-1–treated mice. CSF-1 treatment resulted in a significant increase in F4/80⁺ cells within the skin compared with cells detected in saline-treated control mice, quantified in B as positive/pixels/mm² (**P = 0.007). (C) CSF-1 treatment preferentially expanded donor M2 macrophages in the skin (CD45.1⁺ [donor], F4/80⁺, CD206⁺, and iNOS^–) compared with levels detected in saline control mice. (D) Representative H&E-stained images and semiquantitative histopathology (n = 8/group for all groups, except 6/group for TCD) illustrating that CSF-1 treatment resulted in a significantly higher cutaneous pathology score compared with that for saline-treated mice (**P = 0.006). Statistically significant differences were calculated using 2-tailed Mann-Whitney U tests. Data represent the mean ± SEM. Original magnification, ×5 (A and B) and ×8 (C).

Figure 3. Cytokine/chemokine dependency of PB monocyte populations in naive mice.

(A) Representative dot plots illustrate the gating strategy for PB monocytes and macrophages. Numbers in each dot plot indicate the percentage of positive cells in each gate. (B) Analysis of PB monocytes and macrophages from B6, Ccr2^–/–, and common β chain^–/– mice as well as from WT and Csf1r^–/– FLCs. Numbers in each dot plot indicate the percentage of Ly6C^hi cells (top 2 quadrants) and Ly6C^lo cells (bottom 2 quadrants) and their expression of CCR2. (C) Frequency (percentage) and absolute numbers (×10⁶/ml) of Ly6C^hi versus Ly6C^lo monocyte and macrophage populations in naive mice (frequency of Ly6C^hi cells: **P = 0.004, ***P = 0.0002; frequency of Ly6C^lo cells: **P = 0.004, ***P = 0.0002; absolute numbers of Ly6C^hi cells: **P = 0.0081; absolute numbers of Ly6C^lo cells: *P = 0.0286). n = 8 for all groups. Statistically significant differences were calculated using 2-tailed Mann-Whitney U tests. Data represent the mean ± SEM.

Figure 2. F4/80⁺ macrophages infiltrating the skin after transplantation are donor M2–like macrophages that express CSF-1R.

(A) Representative IHC images from lethally irradiated B6 mice that received G-CSF–mobilized BALB/c (CD45.1) or G-CSF–mobilized TCD grafts. IHC to analyze F4/80, CD45.1, CD206, and iNOS expression illustrates that at day 21 after transplantation, donor M2–like macrophages were present within the skin (F4/80⁺, CD45.1⁺, CD206⁺, and iNOS^–). Minimal donor M2–like macrophage infiltrate was detected in mice that received TCD grafts. Original magnification, ×5. (B) Representative IF images 28 days after transplantation from skin of lethally irradiated B6D2F1 mice that received BM plus T cell grafts from MacGreen transgenic mice (Csf1r promoter driving EGFP). n = 3/time point. Original magnification, ×20. IF confirmed that CSF-1R⁺ cells (green) were also F4/80⁺ (red); white arrows.

Figure 1. F4/80⁺ macrophages infiltrate the skin of mice receiving either mobilized or nonmobilized grafts.

(A–H) IHC to detect F4/80 expression in recipients of mobilized and nonmobilized grafts. Representative images of skin from lethally irradiated B6 mice that received G-CSF–mobilized BALB/c grafts. IHC was performed on days 7 (A), 14 (B), and 21 (C) after transplantation (n = 3/time point). F4/80⁺ macrophages were present in the dermis by day 7 after transplantation (A), with robust infiltration throughout the dermis at day 14 (B) and subcutaneous fat from day 21 (C). (D) Minimal F4/80⁺ macrophage infiltrate was noted in the skin of mice that received TCD grafts. (E–H) Representative images of F4/80 IHC in lethally irradiated B6D2F1 mice that received B6 BM plus T cell grafts. Skin was examined on days 21 (E) , 28 (F), and 35 (G) after transplantation (n = 3/time point). F4/80⁺ macrophage infiltrate was present in the dermis from day 21 after transplantation (E) and throughout the subcutaneous fat layer by day 28 (F and G). There was minimal F4/80⁺ macrophage infiltrate in mice that received TCD grafts (H). Original magnification, ×5.