RGS10 deficiency ameliorates the severity of disease in experimental autoimmune encephalomyelitis

Generation of RGS10-null mice (C57BL/6) has been described previously [27]. The 2D2 TCR mice C57BL/6J mice were purchased from The Jackson Laboratory (Bar Harbor, ME). Eight to 12-week-old male mice were used for experiments, except where indicated. Experimental procedures involving use of animal tissue were performed in accordance with the NIH Guidelines for Animal Care and Use and approved by the Institutional Animal Care and Use Committee at Emory University School of Medicine in Atlanta, GA. Unless noted, mice were euthanized by intraperitoneal Euthasol injection.

RGS10-null mice and wild-type (WT) littermates (C57BL/6 background) were actively immunized with myelin oligodendrocyte protein (MOG)_35–55 as described to initiate EAE [29, 30]. Briefly, mice were injected subcutaneously (s.c.) with 100 μg of MOG_35–55 peptide emulsified in complete Freund’s adjuvant (CFA) supplemented with 250 μg of heat-inactivated Mycobacterium tuberculosis H37 RA. In addition, mice received an intraperitoneal (i.p.) pertussis toxin injection (250 ng) at the time of sensitization and 48 h later. Mice were monitored daily for clinical signs, and disease was evaluated as described [31]. For induction of EAE by adoptive transfer, mice were injected with MOG_35–55/CFA and pertussis toxin as described above. Splenocytes and draining lymph node cells were harvested on day 9 after immunization and expanded in vitro with 10 μg/ml of MOG_35–55 and recombinant mouse (rm) IL-12 (10 ng/ml, R&D Systems) to induce Th1 cells or rmIL-23 (10 ng/ml, R&D Systems) to induce Th17 cells for additional 72 h. Cells were then harvested, washed once with saline, counted and injected i.p. into 5- to 6-week-old WT naïve recipient mice (10 million cells per mouse) i.p. Mice were followed clinically up to at least day 30 post-transfer.

At the time of sacrifice, the spinal cords were removed and fixed in 4 % paraformaldehyde for 24 h and then embedded in paraffin. Sections were cut at 20 μm on a microtome and stained by Luxol fast blue (LFB) to reveal demyelinated areas. For LFB staining, the sections were fixed in 4 % PFA for 10 min, followed by washing in 1× PBS for 5 min. The sections were cleaned by xylene for 10 min and then was hydrated in 100 % EtOH for 5 min and 95 % EtOH for 5 min. The sections were stained in Luxol fast blue solution for 1 h and 45 min, followed by a rise of 95 % EtOH for 5 min and Milli-Q water for 3 min. The sections were differentiated for 10 s in lithium carbonate solution, then 10 s in 70 % EtOH, 10 s in milli-H₂O, and 5 s in lithium carbonate again, and 5 s in 70 % EtOH. Images of RGS10 EAE sections were captured under ×20 objective lens on a Nikon 90i microscope using thresholding analysis on image J software by an investigator blinded to treatment history.

Mononuclear cells from the spinal cord were isolated by mechanical and enzymatic dissociation methods followed by Percoll gradient (70/30 %) centrifugation [32]. T cells (CD45+CD3+), neutrophils (CD11b+Ly6G+), B cells (CD19+), myeloid cells (Ly6G-CD11b+), Th1 (CD4+T-bet+), or Th17 (CD4+RORγt+) were analyzed by flow cytometry. Flow cytometry data were acquired using an LSRII instrument and analyzed using FlowJo Software.

Spleen and lymph nodes were collected from RGS10-null or WT mice at day 10 post-MOG_35–55/CFA immunization. Single-cell suspensions were prepared by mechanical disruption in RPMI-1640 medium supplemented with 10 % FBS, 100 IU/ml penicillin, 100 μg/ml streptomycin, 1× non-essential amino acids, 1 μM sodium pyruvate, 50 mM 2-ME, and 2 mM L-glutamine; 2 × 10⁵ cells per well in a 96-well plate were activated by different concentrations of MOG_35–55 or plate-bound anti-CD3 (5 μg/ml, 145-2C11, eBioscience) plus soluble anti-CD28 (5 μg/ml, 37.51, eBioscience) for 72 h and proliferation was assessed via MTS assay (Promega). Supernatants were collected after 72 h of culture, and cytokine levels were measured by mouse multiplexed Meso Scale Discovery ELISAs (Meso Scale Discovery) [27].

Dendritic cells (DCs) were isolated from the spleens and lymph nodes of RGS10-null or WT mice. Tissues were incubated with CD90.2 beads to deplete T cells followed by positive selection using CD11c beads (Miltenyl Biotech). CD4+ T cells were isolated from the spleens of 6-week-old 2D2 TCR mice using the CD4+ T cell isolation kit II (Miltenyl Biotech); 2 × 10⁴ DCs were incubated with 1 × 10⁵ CD4+ T cells in the presence of different concentrations of MOG_35–55 for 72 h, and T cell proliferation was assessed via MTS assay.

A BD transwell system with a pore size of 5 μm (Corning, Lowell, MA, USA) was used for the migration assay. In the bottom compartment, CXCL-12 (10 nM, R&D System) was added in chemotaxis buffer (0.5 % BSA)/RPMI-1640. As a chemokinesis control, we included CXCL-12 (10 nM) in both the bottom and top compartments. CD4⁺ T cells (1 × 10⁵) were seeded in the top compartment, and after a 2-h incubation (37 °C, 5 % CO₂), the inserts were removed, and the cells that had migrated through the filter to the lower chamber were counted by flow cytometry. Polystyrene beads (15.0 μm diameter, Polysciences, Warrington, PA, USA) were added to each well to allow the cell count to be normalized. A ratio was generated and percent input migration was calculated.

Naïve T cells (CD4+CD25−) from 6–8-week-old RGS10-null or WT male mice were isolated from the spleen using Miltenyi beads. T cells (2 × 10⁵ cells/well) were incubated in flat-bottomed 96-well plates at 37 °C for 3 days with plate-bound anti-CD3 (1 μg/ml) plus soluble anti-CD28 (1 μg/ml) in the presence of (i) anti-IL-4 neutralizing antibody (10 μg/ml, 11B11, eBioscience) and rm IL-12 (10 ng/ml) for Th1 differentiation or (ii) anti-IL-4 (10 μg/ml) and anti-IFN-γ neutralizing antibodies (10 μg/ml, R4-6A2, BD Bioscience), rmIL-6 (20 ng/ml, R&D System), and human TGF-β1 (3 ng/ml, R&D System) for Th17 differentiation. Phorbol myristate acetate (PMA) (50 ng/ml) and ionomycin (1 μg/ml) plus protein transport inhibitor (eBioscience) were added for the last 5 h of the culture period. The percentages of IFNγ+ and IL-17A+ CD4+ T cells were analyzed by flow cytometry.

Power analyses, outlined in the 3rd Edition of Design and Analysis, by Geoffrey Keppel, were used to estimate group sizes. The number of animals was based on our published work [33], and our studies and estimates per group for physiologic outcome measures yielded power values >0.80 with α = 0.05. Animal EAE scoring data were analyzed using the non-parametric Mann-Whitney t test. Comparison of quantitative data between two groups was assessed using the Student’s t test. Comparison between two groups for in vitro experiments was analyzed by two-way ANOVA followed by the Bonferroni post hoc test for p values. p < 0.05 was considered statistically significant. Specific statistical tests used for each experiment are indicated in the figure legend.

We hypothesized that RGS10 modulates CNS autoimmunity by regulating T lymphocyte infiltration and/or effector functions. To test this possibility, we induced EAE in WT and RGS10-null mice by active immunization with MOG_35–55. We found that RGS10-null mice induced to develop EAE by active immunization exhibited less severe clinical signs in the acute phase of disease (approximately day 9 through day 15 post-immunization (p.i.). Clinical symptoms remained mild through day 35 p.i. (Fig. 1).

Consistent with the clinical findings, LFB staining analysis and blinded measures of myelination area revealed that RGS-null mice displayed less demyelination at day 32 p.i.) (Fig. 1b, c). LFB staining in naïve CNS tissues and at day 12 p.i. showed no differences between genotypes (data not shown). In summary, RGS10-null mice had significantly lower EAE incidence and delayed onset of disease, as well as lower mean maximum clinical scores (Table 1). Potential causes for the phenotype of RGS10-null mice in EAE include, but are not limited to (1) functional defects of antigen presenting cells (APCs), such as DCs, (2) a defect in T cell proliferation or effector functions such as or cytokine secretion, (3) a defect in the expansion of autoreactive T cells, and/or (4) a defect in infiltration of effector T cells into the CNS.

We asked if the milder EAE in RGS10-null mice was due to impaired APC function. To test this possibility, we evaluated the ability of RGS10-null or WT CD11c+ DCs to activate MOG_35–55-specific 2D2 CD4+ T cells. We found that RGS10-null DCs stimulated proliferation of MOG_35–55-specific 2D2 CD4+ T cells as well as their WT counterparts (Fig. 2). This suggests that peripheral RGS10-null DCs are competent in their ability to activate autoreactive CD4+ T cells.

We have previously shown that young (3–7-month old) RGS10-null mice do not display any abnormalities in immune cell distribution within the CNS and peripheral lymphoid tissues [34]. We found no differences between naive WT and RGS10-null mice with respect to absolute numbers of CD4+ and CD8+ T cells, B cells, and monocytes within the spleens, blood, and brain [34]. To determine whether CD4+ T cells from RGS10-null mice have defects in their ability to respond to mitogenic stimuli, we stimulated CD4+ cells from RGS10-null vs. WT mice with either (i) anti-CD3/CD28 antibodies or (ii) a combination of PMA and ionomycin (Fig. 3). In response to these mitogens, CD4+ T cells from RGS10-null mice proliferated to the same extent as cells from WT animals. In fact, CD4+ T cells from RGS10-null animals secreted higher levels of IFN-γ, IL-2, and IL-5 compared to WT cells. Thus, RGS10-null CD4+ T cells do not exhibit defects in proliferation or cytokine secretion when stimulated non-specifically through the T cell receptor (anti-CD3/anti-CD28) or when the T cell receptor is bypassed via activation with phorbol ester and calcium ionophore (PMA/ionomycin). Collectively, our data suggest that RGS10-null T cells do not have inherent, global defects in TCR signaling and that RGS10-null DCs are competent in their ability to present antigen to T cells.

Next, we asked whether RGS10-null T cells were capable of differentiating into Th1 or Th17 effector cells. For this, naive T cells from RGS10-null and WT mice were isolated and then differentiated into Th1 or Th17 cells under in vitro polarization conditions [35]. We found similar proportions of IFNγ+ and IL-17+ cells among RGS10-null vs. WT CD4+T cells that were differentiated under Th1 or Th17 conditions, respectively. Thus, CD4+ T cell-expressed RGS10 is dispensable for Th1 and Th17 differentiation in vitro (Fig. 3c).

To further characterize T cell proliferation and effector functions in RGS10-null animals, we examined autoantigen-specific recall responses. RGS10-null or WT mice were immunized with MOG_35–55. Ten days later, draining (inguinal) lymph node cells and splenocytes were isolated and re-stimulated with MOG_35–55 for 3 days. Upon re-stimulation with MOG_35–55 in vitro, draining lymph node cells and splenocytes from RGS10-null mice proliferated less and produced cytokines at levels significantly lower than their WT counterparts (Fig. 4a, b). Lymph node cells and splenocytes from RGS10-null mice produced cytokines including IFN-γ, IL-17, and IL-10 at levels significantly lower than WT cells upon re-stimulation with MOG_35–55 in vitro (Fig. 4c). These data indicate that RGS10-null mice might have an impairment in the generation or maintenance of autoreactive T cell populations after initial activation, which may contribute to the attenuated EAE phenotype in RGS10-null mice.

Next, we examined cellular infiltration within the CNS and the peripheral lymphoid tissues of RGS10-null and WT mice. We explored the possibility that milder EAE in RGS10-null mice could be due to reduced leukocytic infiltration into the CNS. We found no significant difference in the percentages and numbers of Th1 or Th17 cells in draining the lymph node and spleen at day 15 p.i. between genotypes, as determined by flow cytometry (data not shown). However, there were significantly fewer infiltrating leukocytes (CD45+), specifically, CD3+ T cells and CD11b+ myeloid cells in CNS tissues of RGS10-null mice. Conversely, the spinal cords from RGS10-null and WT mice contained comparable numbers of B cells and neutrophils (Fig. 5a). We also found that the fraction of CD45^high myeloid cells among CD11b+ gated cells in RGS10-null EAE mice is reduced relative to that in WT EAE mice, which suggests that there are relatively fewer activated microglia or infiltrating macrophages in the CNS of RGS10-null EAE mice compared to those of WT EAE mice (Fig. 5b). We also examined the composition of T lymphocytes within the CNS. Consistent with our observations of fewer CD3+ CD4+ T cells in RGS10-null CNS tissues (Fig. 5a), we found that RGS10-null CNS tissues contained significantly fewer Th1 (T-bet+) cells and Th17 (RORγt+) cells than their WT counterparts (Fig. 5c). Notably, the relative proportion of T-bet+ and RORγt+ cells within the CD4+ T lymphocyte population did not significantly differ between WT and RGS10-null mice, suggesting that RGS10 does not preferentially impact Th1 vs. Th17 differentiation within the CNS (data not shown). Collectively, our results demonstrate that there is significantly reduced inflammation in the CNS of RGS10-null mice induced to develop EAE, which is consistent with the reduced demyelination observed in RGS10-null mice with chronic EAE (day 32 p.i.) (Fig. 1b, c).

The resistance of RGS10-null mice to EAE could be explained by a defect in generation or maintenance of autoreactive T cell populations; however, given the reduced frequency of T cells in the spinal cords of RGS10-null mice with EAE (Fig. 5), it is also possible that RGS10-null CD4+ T cells have defects in their ability to traffic into the CNS. Thus, we next examined whether RGS10 regulates CD4+ T cell migration to chemokines in vitro. We found that, compared to WT cells, RGS10-null CD4+ T cells displayed attenuated migration in response to CXCL12 (a ligand for CXCR4) (Fig. 6b) and CCL19 (aligand for CCR7) (data not shown). Notably, CXCR4 and CCR7 protein were expressed at similar levels in WT vs. RGS10-null CD4+ T cells, suggesting that attenuated migration is not due to abnormal receptor expression (Fig. 6a and data not shown). Rather, RGS10 may regulate signaling pathways downstream of chemokine receptors on CD4+ T cells.

To determine whether the decreased EAE disease severity in the RGS10-null mice was due to autoreactive T cell-intrinsic defects, we performed adoptive transfer experiments. Encephalitogenic, MOG_35–55-reactive Th1 or Th17 cells from WT or RGS10-null mice were transferred to WT recipient mice. Our results showed that the recipients of Th1 cells from RGS10-null EAE mice developed attenuated disease compared to mice that received WT Th1 cells. Conversely, the recipients of Th17 cells from RGS10-null mice developed clinical EAE that was statistically indistinguishable from recipients of WT Th17 cells. These findings suggest that a key determinant of the EAE phenotype in RGS10-null mice is impaired Th1 cell function and/or trafficking (Fig. 7).