Fig 1.
Deficiency in OGR1 results in the attenuation of EAE.
(A) EAE was induced in wild type (WT) and OGR1-KO mice by injection of MOG35-55/CFA and pertussis toxin and mice were observed daily for clinical signs of EAE for 28 days. Data shown are means + SEM of clinical scores obtained from 3 independent experiments that each used N = 5 mice/group. (B-D) In one experiment, cross sections of WT and OGR1-KO spinal cords (N = 10 sections/mouse) were prepared from N = 8 mice/group and were stained with Luxol Fast Blue and hematoxylin and eosin. Sections were scored blindly for meningeal and perivascular inflammation and demyelination. (B) shows a representative area of a spinal cord cross section from one WT and OGR1-KO mouse. The arrow indicates perivascular infiltration and dashed outlined section indicates area of demyelination. (C) shows the percent of spinal cord quadrants that were affected by meningeal inflammation or that contained perivascular cuffs. (D) shows the percent of the spinal cord white matter that was demyelinated. In C and D, values are means + SEM of values obtained in the N = 8 mice/group. *p<0.05, **p<0.01, ***p<0.001 by two-tailed t-test.
Table 1.
Clinical features of disease in OGR1-KO and WT mice.
Fig 2.
The frequency and total number of Th1 and Th17 cells were significantly reduced in OGR1-KO CNS during EAE.
WT and OGR1-KO mice were immunized with MOG35-55/CFA and were provided pertussis toxin and at 28 days post-immunization brains and spinal cords were removed and CNS mononuclear cells were isolated by Percoll gradient centrifugation. Lymphocytes were counted, restimulated in vitro with PMA and ionomycin in the presence of brefeldin A, and stained with antibodies specific for CD4, IFN-γ and IL-17. (A-B) The percentages of IFN-γ+ and IL-17+ cells within the CD4+ gate were measured by flow cytometry and (C) the total cell number of each population was calculated. Data are means + SEM of individual mice (N = 3/group) and are representative of 3 independent experiments. **p<0.01, ***p<0.001 by two-tailed t-test. Similar studies were conducted at day 10 post-immunization with similar results.
Fig 3.
OGR1 deficiency impairs the generation of antigen-specific T cells.
(A-B) Mice (N = 6/group) were immunized with MOG35-55/CFA and at 10 days post-immunization draining lymph nodes (dLN) were removed and were pooled together. Lymph node mononuclear cells were labeled with CFSE and cultured with MOG35-55 peptide (0–10 μg/mL) for 3 days. Cells were collected and stained with anti-CD4 antibody and the percentage of divided CD4+ cells was analyzed by CFSE dilution. (C-D) Supernatants from the cultures were used to measure the levels of IL-17 (C) or IFN-γ (D) by ELISA. Data are means + SEM of values obtained from triplicate cultures and are representative of 3 independent experiments. *p<0.05, **p<0.01, ***p<0.001 by t-test (two-tailed).
Table 2.
Total cell numbers x 106 in Naïve WT and OGR1-KO mice.
Table 3.
Percent of Live Cells in Naïve WT and OGR1-KO mice.
Fig 4.
OGR1-KO T cells show no inherent defects in their capacity to proliferate and differentiate.
(A) CD4+ T cells were isolated from WT and OGR1-KO spleen and lymph nodes (N = 4 mice/group), were pooled and labeled with CFSE and stimulated with anti-CD3 and anti-CD28 (2 μg/ml) antibodies. After 3 days of culture, cells were collected and stained with anti-CD4 antibody and cell proliferation was measured by CFSE dilution. Data are means + SEM of triplicate cultures and are representative of 4 independent experiments. (B) WT or OGR1-KO CD4+ T cells were cultured with soluble anti-CD28 antibody (2 μg/ml) on anti-CD3-coated (2 μg/ml) plates for 5 days and then were re-stimulated with PMA and ionomycin and IL-17 and IFN-γ were measured by ELISA. Data are means + SEM of triplicate cultures and are representative of 3 independent experiments. (C-E) CD4+ T cells from WT or OGR1-KO mice were stimulated with anti-CD3 and anti-CD28 (both at 2 μg/ml) under Th1 or Th17 polarizing conditions. After a 5-day incubation, cells were re-stimulated with PMA and ionomycin in the presence of brefeldin A for an additional 4 hours. Cells were then collected and stained with antibodies for CD4, IFN-γ or IL-17 and the percentage of IFN-γ+ and IL-17+ CD4+ cells was calculated. Results in C show representative FACs plots while data in D and E are means + SEM of triplicate cultures. Data shown are from one of three experiments that were performed. ns = not significant, by Student’s t-test (two-tailed).
Fig 5.
The frequency and total number of macrophages and DCs are reduced in OGR1-KO dLN after immunization.
(A-B) Draining lymph nodes (dLN) were removed from WT and OGR1-KO mice at 10 days post-immunization with MOG35-55/CFA. The frequencies (A) and total number (B) of CD11c+ and F4/80+ cells were determined by flow cytometry. Data in A are representative flow plots, while data in B are means + SEM of values obtained from N = 6 mice/group. Data shown are representative of 2 independent experiments.
Fig 6.
APC from OGR1-KO mice have a decreased ability to support T cell proliferation and cytokine production.
Splenocytes were isolated from WT and OGR1-KO mice and T cells were depleted by MACs separation as described in the Methods. T cell-depleted splenocytes (APC) were stimulated with LPS overnight, washed and co-cultured (1:1 ratio) with CFSE-labeled WT CD4+ T cells that were plated onto anti-CD3 antibody-coated plates. The percentage of divided CD4+ cells (A-B) was examined after 3 days of culture. Data shown are from 4 individual experiments. (C-D) Cells were stimulated with PMA, ionomycin in the presence of brefeldin A for an additional 4 hours and intracellular staining for IFN-γ was performed. (C) shows representative FACs plots of IFN-γ expression versus side scatter of CD4+ cells and (D) shows the means + SEM of values obtained from 3 individual experiments. *p<0.05, **p<0.01 by t-test (two-tailed).
Fig 7.
OGR1-KO macrophages have a greater ability than WT macrophages to inhibit the proliferation and cytokine production by CD4+ T cells.
CFSE-labeled WT CD4+ T cells were plated onto anti-CD3 antibody-coated plates and co-cultured with either WT or OGR1-KO macrophages (mac) that had been pre-stimulated with 0.1 μg/mL LPS. The percentage of divided CD4+ cells (A-B) was examined after a 3-day incubation period. (A) shows representative data while (B) shows means + SEM of values obtained from 6 individual experiments. *p< 0.05 by Student’s t-test (two-tailed). (C-D) WT CD4+ T cells were co-cultured with either WT or OGR1-KO macrophages, at a ratio of 1:0.2, for 5 days (C) or 4 days (D) and supernatants were used to measure the levels of IFN-γor IL-17 by ELISA. Data are means + SEM of triplicate cultures and are representative of 3 independent experiments. *p<0.05, **p<0.01 Student’s t-test (two-tailed).
Fig 8.
Differential NO Production Accounts for the Differential Ability of OGR1-KO and WT Macrophages to Support T Cell Proliferation.
(A) WT and OGR1-KO macrophages were stimulated with 0.1 μg/mL LPS overnight and supernatants were collected and analyzed for nitrite concentration using the Griess assay. Data are means + SEM of values obtained from N = 5 mice/group. (B) The expression of iNOS was examined by flow cytometry in WT (solid line) and OGR1-KO (dashed line) macrophages after stimulation with 0.1 μg/mL LPS overnight. Histograms of cells positive for iNOS, within the CD11b+F4/80+ gate, are shown. Staining with the isotype control antibody (dotted line) is also shown. This panel is representative of 3 experiments that were performed on N = 3 mice/group. (C-E) WT CD4+ T cells and either WT or OGR1-KO macrophages (mac) were co-cultured in the presence or absence of 10 μM L-NIL. (C) The percent of divided CD4+ cells was determined by staining CFSE-labeled T cells in the co-culture with anti-CD4 antibody after a 3-day incubation. (D-E) Supernatants were collected and analyzed for IFN-γ and IL-17 by ELISA. Values in C-E are means + SEM of results from triplicate cultures and are representative of 3 independent experiments that were performed. *p<0.05, **p<0.01, ***p<0.001 by Student’s t-test (two-tailed) or one-way ANOVA.