Erythropoietin: a potent inducer of peripheral immuno/inflammatory modulation in autoimmune EAE

Abstract

Background: Beneficial effects of short-term erythropoietin (EPO) therapy have been demonstrated in several animal models of acute neurologic injury, including experimental autoimmune encephalomyelitis (EAE)--the animal model of multiple sclerosis. We have found that EPO treatment substantially reduces the acute clinical paralysis seen in EAE mice and this improvement is accompanied by a large reduction in the mononuclear cell infiltration and downregulation of glial MHC class II expression within the inflamed CNS. Other reports have recently indicated that peripherally generated anti-inflammatory CD4(+)Foxp3(+) regulatory T cells (Tregs) and the IL17-producing CD4+ T helper cell (Th17) subpopulations play key antagonistic roles in EAE pathogenesis. However, no information regarding the effects of EPO therapy on the behavior of the general mononuclear-lymphocyte population, Tregs or Th17 cells in EAE has emerged.

Methods and findings: We first determined in vivo that EPO therapy markedly suppressed MOG specific T cell proliferation and sharply reduced the number of reactive dendritic cells (CD11c positive) in EAE lymph nodes during both inductive and later symptomatic phases of MOG(35-55) induced EAE. We then determined the effect in vivo of EPO on numbers of peripheral Treg cells and Th17 cells. We found that EPO treatment modulated immune balance in both the periphery and the inflamed spinal cord by promoting a large expansion in Treg cells, inhibiting Th17 polarization and abrogating proliferation of the antigen presenting dendritic cell population. Finally we utilized tissue culture assays to show that exposure to EPO in vitro similarly downregulated MOG-specific T cell proliferation and also greatly suppressed T cell production of pro-inflammatory cytokines.

Conclusions: Taken together, our findings reveal an important new locus whereby EPO induces substantial long-term tissue protection in the host through signaling to several critical subsets of immune cells that reside in the peripheral lymphatic system.

Figure 1. EPO down-regulates proliferation of MOG-specific T cells and suppresses T cell cytokine production.

a) Single cell suspensions of hyperimmune EAE spleen were prepared 7 days post MOG peptide immunization and quantified by flow cytometry for CD8 and MOG_44–54/H-2Db dimer double positive binding. In normal mice, 0.42% of splenic CD8 cells bound to the MOG_44–54/H-2Db dimer (left side), whereas an 8-fold increase in MOG_44–54/H-2Db dimer positive staining occurred in CD8 T cells obtained from MOG_35–55 immunized EAE animals (middle). In contrast, when a non-specific control peptide was tested, only 0.6% of CD8 cells exhibited non-specific binding to the NP_366–374 dimer (right). b & c) MOG-EAE antigen specific T cells were enriched by exposure to antigen in long term tissue culture, and then exposed to EPO (1 or 10 U/ml) for 72 hrs. Supernatants were collected from the cultured MOG-T cells for cytokine profile analysis and the remaining cells were quantified for number of CD8 and MOG_44–54/H-2Db dimer double binding cells after exposure to EPO or control treatment. EPO induced a marked decrease in pro-inflammatory cytokine production (black bars) when compared to controls (white bars). EPO at 1 U/ml induced a substantial decline in the MOG-specific CD8 T cell population (c-middle panel). Some of the MOG_44–54-peptide/H-2Db dimer positive, non-CD8 T cell (including portion of CD11c positive cells) populations were also reduced by EPO treatment (c-middle panel). Each data point is the mean±SEM of three experiments performed in triplicate. *, p<0.005.

Figure 2. In vivo effect of EPO treatment on MHC expression and number of MOG antigen specific T cells in inguinal lymph nodes.

EPO treatment was started 7 days after mice received MOG_35–55 immunization. Bilateral draining inguinal lymph nodes (DILNs) were obtained from mice on day 11 (after 3 day treatment with either EPO at 5000 U/kg/day or sham treatment with PBS) and single cell suspensions were prepared. a) EPO treatment down-regulated mononuclear cell MHC class I (left) and class II (right) expression in lymph nodes from EAE mice. b) Less than 0.7% of single cells from normal mouse inguinal lymph nodes were double-positive for MOG_44–54 dimer and anti-CD3 antibody. In contrast, about 4% of single cell suspensions from sham treated EAE mice were positive for FITC-CD3 and PE-MOG_40–54/H-2Db dimer double staining (middle), whereas EPO treatment reduced in vivo proliferation of MOG-specific T cells back to 1.5% (right).

Figure 3. Early EPO treatment sharply reduced total numbers of inflammatory cells and limited expansion of the dendritic cell population in DILNs during the EAE induction phase.

C57 mice received EPO or PBS sham treatment starting on the day of MOG-immunization for 6 days and lymph nodes were obtained on day 7. a) Significantly enlarged inguinal lymph nodes were observed in control sham treated MOG-EAE mice (middle), whereas much smaller nodes were found in EPO treated EAE mice (right) similar in size to nodes from normal animals (left). b) Total MNCs were quantified from single cell suspensions of DILNs after lysis of RBCs. Greatly increased inflammatory mononuclear cell numbers were observed in sham treated EAE lymph nodes compared to DILNs from EPO treated EAE mice or normal mice. c) Cells were reacted with fluorophore labeled mAbs specific for surface markers (MHC class II and CD11c) and analyzed by flow cytometry. Note that the large expansion in dendritic cell population found in sham treated EAE mice was dramatically suppressed by EPO therapy. Data represents mean±SEM for 6 individual mice. **, p<0.0001.

Figure 4. EPO induced sustained immuno/inflammatory modulation by expanding peripheral Treg cell numbers and reducing Th17 positive cells.

Mice were immunized with MOG and received daily EPO treatment for 6 days (day1–6). a). EPO therapy for 6 days delayed onset of clinical neurologic signs in animals and reduced the magnitude of clinical deficit in EAE mice (•) compared to sham treated control (○) EAE mice (*, p<0.05). The clinical score was determined as the mean±SEM of 8 mice per group. Data represents the mean±SEM of 8 individual mice. b–c) DILNs were obtained from mice after 6 days treatment with either EPO or PBS. Cells were quantified for number of Tregs (CD4⁺Foxp3⁺) and Th17 by flow cytometry. Panel b shows that about 3.4% Treg cells were detectable in normal mouse inguinal lymph node (left). A reduced number (2.4%) of Treg cells was detected in sham treated EAE control mice nodes (middle), whereas EPO therapy induced a 2-fold increase in Foxp3+ Treg cells on day 16 compared to sham treatment (right). Panel c shows <0.2% of normal healthy lymph node cells stained positive for Th17 (left). Numbers of peripheral Th17 cells greatly increased on day 16 (15-fold) in EAE mice treated with PBS (middle, 3.2%). In marked contrast, EPO therapy (right) sharply reduced the number of peripheral Th17 cells in MOG-immunized animals.

Figure 5. EPO treatment induced peripheral Treg cell expansion while reducing MOG-specific T cells and Th17 positive cells in peripheral lymphoid tissues.

Mice were immunized with MOG and received daily EPO treatment for 6 days (day1–6). DILNs and spinal cords were obtained at three different time points (day 7, day 16 and day 45) from mice treated with EPO or sham treated with PBS. Cells were quantified for number of Tregs (CD4⁺Foxp3⁺), Th17 and MOG-antigen specific T cells by flow cytometry. a) There were reduced numbers of Treg cells in DILNs from sham treated EAE mice at early stages (day 7) as well as at the peak of clinical signs (d16), and during recovery (day 45) compared to normal healthy control animals. b) Numbers of peripheral Th17 cells increased in sham treated EAE mice, whereas EPO therapy sharply reduced the number of peripheral Th17 cells in MOG-immunized animals. c) EPO treatment significantly reduced peripheral MOG_40–54/H-2Db specific T effector cell population compared to sham treated EAE mice. Data represents mean±SEM for 6 individual mice. *, p<0.05; **, p<0.001.

Figure 6. EPO treatment increased numbers of Treg cells and reduced numbers of Th17 cells in MOG-EAE mouse spinal cord.

MOG-immunized C57 mice received daily EPO or PBS treatment for 6 days (day 1–6). Spinal cords were obtained at two different time points (day 16 and day 45) from mice treated with EPO or sham treated with PBS. Cells were quantified for number of Tregs (CD4⁺Foxp3⁺), Th17 and MOG-antigen specific T cells by flow cytometry. a) EPO therapy induced a substantial increase in Foxp3+ Treg cells in EAE spinal cords. The EPO induced expansion of Tregs in the CNS became even more evident in late stages of the disease and correlated with less severe neurologic deficit. b–c) Foxp3⁺ labeled Treg cells in PBS or EPO treated MOG-EAE spinal cord by IHC. Panel b shows a sham treated EAE spinal cord section reacted with Foxp3 antibody containing few labeled cells. By contrast, many more Foxp3+ cells were present in the infiltrates of EPO treated EAE spinal cord (panel c). d) Significantly increased numbers of Th17 cells occurred in sham treated EAE mouse spinal cord and this correlated with more severe clinical neurologic deficits whereas EPO therapy suppressed the number of spinal cord Th17 cells. e) Increased MOG antigen-specific T cells occurred within untreated EAE spinal cord, and EPO induced large reductions in MOG-specific T effector cells within the CNS. Data represents mean±SEM for 6 individual mice. *, p<0.05; **, P<0.001.