Role of Gut Commensal Microflora in the Development of Experimental Autoimmune Encephalomyelitis

Mice procedures

Female 6-wk-old SJL mice were obtained from The Jackson Laboratory. All mice were maintained in accordance with institutional policies for animal health and well-being at Dartmouth College Animal Resources Center under pathogen-free conditions in individual ventilated cages under HEPA-filtered barrier conditions and were fed sterile food and water ad libitum. Mice were treated with the following antibiotics dissolved in drinking water or dissolved in water: ampicillin (1 g/ml), vancomycin (0.5 g/ml), neomycin sulfate (1 g/ml), and metronidazole (1 g/ml) (15). When required, dissolved antibiotics were administered by oral gavages or i.p. injections at daily single 200-μl doses of 1 g/ml. Mice treated i.p. or by oral gavages received normal drinking water during the length of the experiments. Serial dilutions of intestinal and fecal samples were cultured in general bacteriological agar plates (CDC blood agar; BD Biosciences) for 48 h at 37°C. Plates were cultured in aerobic and anaerobic conditions. Total bacteria per gram of sample was calculated based on the CFU counted in each serial dilution.

EAE induction by proteolipid protein (PLP)_139–151 or myelin oligodendrocyte glycoprotein (MOG)_35–55 challenge

Female SJL/J or C57BL/6 mice (four per group) were challenged s.c. with 200 μg of PLP_139–151 or 250 μg of MOG_35–55 (Peptides International), respectively, in 200 μl of CFA (Sigma-Aldrich). Mice received i.p. 200 ng (SJL/J) or 400 ng (C57BL/6) of Bordetella pertussis toxin (PT; List Biological Laboratories) (days 0 and 2) (12). Spinal cords were harvested 12 days after challenge and fixed with neutral buffered formalin (VWR International), embedded into paraffin, sectioned at 3 μm, and stained with H&E and Luxol fast blue for pathological changes and inflammatory cell infiltration (12).

Cytokine detection by Luminex, ELISA, and PCR

Spleen and CLN cell suspensions were costimulated with PLP_139–151 (30 μg/ml) or with anti-CD3 mAb (coated well; 10 μg/ml; BD Pharingen) and soluble anti-CD28 mAb (5.0 μg/ml; BD Pharmingen) for 3 days (12). Luminex was employed to quantify triplicate sets of samples to measure cytokines (IFN-γ, TNF-α, MIP-1α, MIP-1β, MCP-1, IL-6, IL-17, IL-10, IL-4, and IL-13). Capture ELISA was used to quantify IFN-γ, IL-17, IL-10, and IL-13 from supernatants of cells cultured after FACS sort, as previously described (13). For PCR detection of IL-13 mRNA (r-GGTCCTGTAGATGGCATTGCA; l-GGAGCTGAGCAACATCACACA; Invitrogen) a total of 1.0 μg of RNeasy-purified mRNA (Qiagen) was reverse transcribed using MultiScribe RT (Amersham Biosciences). cDNA (200 ng) was amplified using the 2× SYBR Green mix (Applied Biosystems) on a Bio-Rad iCycler.

FACS analysis

Single-cell preparations were prepared as described above (12). Cell subsets were analyzed using fluorochrome-conjugated mAbs (BD Pharmingen). For the analysis of T cell subpopulations, CD3, CD4, CD8, CD25, and CD45Rb (BD Pharmingen) were used. Intracellular staining for FoxP3 was done using flourochrome-labeled anti-FoxP3 mAb (clone FJK-16S; eBioscience). For NK cells, DX5, B220, and CD11b were used. For B cells, CD19 and B220 (BD Pharmingen) were used. For macrophages and DC subpopulations, CD11b, CD11c, CD103, B220, CD8, Gr-1, and F4/80 mAb were used (BD Pharmingen). For DC activation, MHC class II (I-Ad; clone AMS-32.1) and CD80 (BD Pharmingen) were used. Fluorescence was analyzed with a FACSCanto (BD Biosciences).

Cell purifications

CD4⁺ T cells and CD8⁺ T cells were obtained with magnetic beads (Dynal Biotec). The enriched CD4⁺ T cells were cell-sorted for FITC-anti-CD4 and allophycocyanin-anti-CD25 mAbs (BD Pharmingen). CD11c⁺ cells were obtained with magnetic beads (StemCell Technologies). The enriched CD11c⁺ cells were cell-sorted (FACSVantage with Turbo-Sort; BD Biosciences) following staining with FITC-anti-CD103 into CD11c^highCD103⁺ cells.

In vitro T cell assays

To assess T_reg cell suppressor activity, 1.5 × 10⁵ responder CD25⁻CD4⁺ T cells were labeled with CFSE and subsequently cocultured in triplicate with CD25⁺CD4⁺ T cells at 1:1, 1:0.1, 1:0.01, and 1:0.001 CD25⁻/CD25⁺ T cell ratios. Feeder cells (T cell-depleted mitomycin C-treated) splenocytes prepared from naive mice (16) were added at 1.5 × 10⁵ cells per well. Cells were incubated at 37°C in 5% CO₂ for 72 h. CD4⁺ T cell proliferation was compared by FACS.

To asses cytokine production by CD25⁻CD4⁺ T cells and CD25⁺CD4⁺ T cells, sorted cells (2 × 10⁵) were stimulated in vitro with PLP_139–151 (30 μg/ml) or with anti-CD3 mAb-coated wells (10 μg/ml; BD Pharmingen) plus soluble anti-CD28 mAb (5 μg/ml; BD Pharmingen) (13). Capture ELISA was used to quantify IFN-γ, IL-17, IL-10, and IL-13 production.

To assess the role of CD11c^highCD103⁺ and CD11c^highCD103⁻ DCs in the in vitro conversion of splenic CD4⁺ T cells sorted from naive SJL/J mice into FoxP3⁺ T_reg cells, 1.5 × 10⁵ naive CD25⁻CD4⁺ T cells were cocultured in triplicate with CD11c^highCD103⁻ or CD11c^highCD103⁺ DCs. Anti-CD3 mAb (10 μg/ml; BD Pharmingen) and IL-2 (20 U/well) were added. Retinoic acid (4 nM) and TGF-β (1 ng/ml) were added to some cultures to enhance the T_reg cell conversion rates. Cells were incubated at 37°C in 5% CO₂ for 72 h. Conversion of naive CD25⁻CD4⁺ T cells into FoxP3⁺ T_reg cells was compared by FACS.

Adoptive transfer experiments

CD25⁺CD4⁺ T cells (4 × 10⁵) or CD25⁻CD4⁺ T cells (4 × 10⁵) were i.v. injected into naive recipients. In different experiments, 1 × 10⁶ CD4⁺ T cells and CD8⁺ T cells were adoptively transferred. One day after the adoptive transfer of T cells, mice were challenged with PLP_139–151 to induce EAE.

In vivo inactivation of CD25⁺CD4⁺ T cells

Mice were orally treated with antibiotics 7 days before EAE challenge with PLP_139–151 and PT. To inactivate CD25⁺CD4⁺ T cells, the same mice were given 0.3 mg of anti-CD25 mAb (American Type Culture Collection no. TIB-222, clone PC 61.5.3) on days 4 and 2 before EAE challenge (12). As a control group, treated and naive mice received 0.3 mg of purified rat IgG Ab on the same days before EAE challenge. CD25 depletion was confirmed by FACS analysis of peripheral blood samples obtained 2 days after the administration of the second dose of anti-CD25 or rat IgG Abs. A separate control group was immunized with PBS 7 days before EAE challenge.

Statistical analysis

Student’s t test was applied to show differences of combined experiments in clinical scores, body, spleen, and cecum weights, Luminex detection of cytokines, as well as in the flow cytometry of T_reg cell and DC experiments. ANOVA followed by post hoc Tukey’s test was applied to show differences in EAE clinical scores. Values of p <0.05 and <0.01 are indicated.

Oral treatment with antibiotics protects mice against EAE

Studies in germ-free animals have demonstrated that microbial populations of the gut are essential for the complete development of a normal immune response. To assess whether modification of normal gut commensal microflora would influence the induction and development of an acute demyelinating process in the CNS, mice were treated with an antibiotic cocktail that has been shown to effectively reduce the gut intestinal bacterial burden (15). Drinking water was supplemented with ampicillin (1 g/L; Sigma-Aldrich), neomycin sulfate (1 g/L; Sigma- Aldrich), metronidazole (1 g/L; Sigma-Aldrich), and vancomycin (0.5 g/L; Sigma-Aldrich) for 7 days (15). EAE was induced with PLP_139–151 in SJL and MOG_35–55 in C57BL/6 mice previously treated with antibiotics (Fig. 1⇓). Control mice were treated with PBS and i.p. with the same antibiotics. There have been different reports implicating a direct neurological effect by injections of minocycline, a second generation type of tetracycline. Minocycline provides partial protection against EAE when combined with glatiramer acetate and IFN-β (17, 18), provoking a down-regulation in the Ag presentation capability of blood monocyte-derived DC Ag presentation in mice and activation capability in MS patients (17).

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FIGURE 1.

Oral treatment with antibiotics reduces EAE clinical scores in PLP_139–151- and MOG_35–55-challenged mice. SJL/J and C57BL/6 mice were treated with antibiotics for 7 days and subsequently EAE challenged with PLP_139–151 (A) or MOG_35–55 (B). At the end of the treatments, body weights were measured and small intestines and colons were aseptically removed and the tissues were combined with fecal contents and cultured in blood general agar media in aerobic and anaerobic conditions at 37°C for 48 h. Serial dilutions were used to calculate the bacterial CFU, represented as total bacteria per gram of tissue and fecal content. SJL/J mice were treated orally with antibiotics dissolved in drinking water, by i.p. daily injections, or by daily oral gavages. Mice treated with antibiotics supplied in the drinking water were protected against EAE and suffered body weight reductions. Mice supplied with normal water but treated with the same antibiotics were protected against EAE; however, their body weights were not altered when compared with untreated control mice. Both groups had significant reductions in the bacterial CFU per gram, as opposed to untreated and i.p. treated mice. Depicted is a representative experiment (n = 4) of the three separate experiments for a total of 14 mice per group: ∗, p < 0.01 for oral drinking treatment vs naive and i.p. treatment. ^‡, p < 0.01 for oral gavage treatment vs naive and i.p. treatment (A). C57BL/6 mice treated orally with antibiotics were protected against EAE. The gut microflora was reduced, but treatment with antibiotics did not alter body weights (B). Depicted is a representative experiment of the three separate experiments for a total of 12 mice per group: ∗, p < 0.01 for oral drinking treatment vs naive and i.p. treatment.

Oral treatment with antibiotics previous to challenge with PLP_139–151 significantly reduced the severity of EAE when compared with PBS control and i.p. treated animals (Fig. 1⇑A). Continuous oral antibiotic treatment in the drinking water resulted in body weight reduction of SJL/J mice. However, treatment of SJL/J mice with the same combination of antibiotics administered by once daily oral gavages (200-μl doses of 1 g/ml) and free access to untreated drinking water had no effect on body weight (Fig. 1⇑A). These mice were protected against EAE compared with control mice that had free access to normal drinking water and were not treated with antibiotics. Both PBS- and i.p. treated mice developed significant clinical disease compared with mice treated with oral antibiotics. Differences were observed in the onset of the disease and the cumulative scores of PBS vs i.p. compared with orally treated mice (supplemental Table SI).⁴

Similar studies were done with oral antibiotic treatment in C57BL/6 mice in which EAE was induced following immunization with MOG_35–55 (Fig. 1⇑B). The antibiotic-treated mice were protected against disease and there was no significant alteration in body weight (Fig. 1⇑B). The EAE cumulative scores of mice treated orally with antibiotics were significantly reduced (p < 0.01) when compared with PBS-treated (13.1 ± 1.2 vs 52.0 ± 2.1) and i.p. treated mice (13.1 ± 1.2 vs 49.7 ± 1.1) (supplemental Table SII). No significant differences in the EAE clinical scores were observed between C57BL/6 mice treated with PBS and mice treated i.p. with antibiotics. These results suggest that the protection observed was independent of any physiologic stress induced by continuous oral antibiotic treatment and more importantly the genetic background of the animals used in the studies.

Oral treatment with antibiotics significantly reduced the bacterial counts present in fecal and intestinal samples of SJL/J (Fig. 1⇑A) and C57BL/6 mice (Fig. 1⇑B). Aerobic and anaerobic conditions were examined and, in both cases, a significant reduction of bacterial counts was found after 1 wk of treatment. No bacterial CFU were detected in fecal samples of mice treated orally with antibiotics as opposed to the culture of fecal intestinal contents, suggesting that fresh pellets might be insufficient to compare total bacterial loads. When fresh fecal pellets and intestinal fractions of mice were combined and cultured, we observed that only oral treatment by drinking water or by oral gavages but not i.p. treatment with antibiotics reduced gut commensal microflora. However, antimicrobial treatment did not completely deplete bacterial presence, showing that certain bacterial populations remain viable despite antibiotic treatment. When animals were subsequently provided with normal drinking water, intestinal recolonization was observed 1 wk later (supplemental Fig. 2A). The treatment with antibiotics does not render the gut sterile but rather substantially reduces the bacterial load and perhaps alters the composition of the normal gut microflora. No significant differences in the splenic and cecum weights were observed in mice treated i.p. with antibiotics when compared with naive mice (supplemental Fig. 2B).

Our results show that daily administration of an antibiotic cocktail by single oral gavages or continuous administration in the drinking water rendered SJL/J mice resistant to EAE. Antibiotic treatment by oral gavages did not affect mouse weight that remained stable compared with baseline (no treatment). As weight loss did not appear to mediate resistance, SJL/J mice were treated with antibiotics by continuous administration in the drinking water in all further studies.

Oral treatment with antibiotics reduces proinflammatory responses

To study the cytokine pattern of mice treated with antibiotics, Peyer’s patches (PP), mesenteric lymph nodes (MLN), and splenic and cervical lymph nodes (CLN) lymphocytes were harvested from naive and mice treated orally with antibiotics and costimulated with αCD3/αCD28 Abs (Table I⇓). Results show that the reduction of gut commensal microflora significantly diminished the production of MIP-1α, MIP-1β, and IL-6 in PP. MLN of animals treated with antibiotics produced lesser amounts of IFN-γ, MIP-1α, MIP-1β, and IL-6 and significantly increased levels of IL-13. Splenic and CLN cells derived from these mice produced reduced IFN-γ, MIP-1α, MIP-1β, MCP-1, IL-17, and IL-6 levels, whereas IL-13 and IL-10 (in CLN) were significantly enhanced when compared with untreated control mice.

Oral treatment with antibiotics enhances the frequency of FoxP3⁺ T_reg cells in the MLN and CLN

Flow cytometry was used to compare the populations of T cells, B cells, DCs, macrophages, NK cells, and NKT cells (supplemental Fig. 1). A significant reduction in CD4⁺ T cells and an enhanced CD8⁺ T cell response was observed in mice treated orally with antibiotics when compared with naive and i.p. treated mice. Phenotypic analysis of the various immune compartments within the PP of animals treated orally with antibiotics showed a significant reduction in T cell, B cell, and CD11c⁺CD11b⁻ percentages. Conversely, there was a significant increase in CD11c⁺CD11b⁺ DCs when compared with either naive or mice treated i.p. with the same antibiotic cocktail. Percentages of CD11b⁺F4/80⁺ monocytes, NK cells, and NKT cells of treated mice failed to show any significant difference when compared with untreated control mice. The MLN of mice treated with oral antibiotics showed a significant reduction in total T cells, but no change in B cells, CD11b⁺F4/80⁺ monocytes, NK cells, NKT cells, or CD11c⁺CD11b⁺ or CD11b⁻ DC populations. The percentage of splenic T cells was significantly higher in orally treated than in naive and i.p. treated mice. No alterations were observed in CD11c⁺CD11b⁺, CD11c⁺CD11b⁻, and CD11c⁺Gr-1⁺ DCs or in CD11b⁺F4/80⁺ monocytes. Interestingly, a significant reduction in NK and NKT cell percentages in the spleen was observed in mice after oral treatment with antibiotics. Finally, analysis of CLN showed that percentages of T cells were reduced significantly in mice treated orally with antibiotics, with no modifications in the rest of cellular populations compared.

Oral treatment with antibiotics altered significantly CD4⁺ T cell subpopulations (Fig. 2⇓ and supplemental Fig. 5, A and B). FACS analysis revealed that the frequency of CD4⁺CD25⁺ T cells was reduced in PP of mice orally treated with antibiotics, but significantly increased (p < 0.01) in MLN, spleens, and CLN when compared with naive and i.p. treated mice (Fig. 2⇓A). Lymph nodes of treated mice showed reciprocal reduction and enhancement of activated CD45Rb^lowCD4⁺ T cells in MLN and CLN of CD25⁺ T cell populations when compared with naive and mice treated i.p. with antibiotics (data not shown). Interestingly, FACS analysis showed that oral treatment with antibiotics provoked a significant reduction (p < 0.01) in the frequency of FoxP3⁺CD25⁺/total CD4⁺ T cells in spleens but was otherwise unchanged from control values (Fig. 2⇓B). When total numbers of FoxP3⁺ T_reg cells were compared, significant reductions (p < 0.01) were measured in PP and spleens of mice subjected to oral treatment with antibiotics. However, gut flora alterations enhanced FoxP3⁺T_reg cell numbers significantly (p < 0.01) in MLN and CLN when compared with naive and mice treated i.p. (Fig. 2⇓C).

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FIGURE 2.

Oral treatment with antibiotics significantly modifies CD4⁺CD25⁺ and FoxP3⁺ T_reg cell populations in the GALT and distal peripheral lymph nodes. SJL mice were treated orally or i.p. with antibiotics for 7 days and cell suspensions were obtained from PP, MLN, spleens, and CLN of naive and treated mice. CD4⁺CD25⁺ T cells (A) and the frequency of FoxP3⁺CD25⁺ T cells (B) were analyzed by flow cytometry (previously gated in CD4⁺ cells). Oral treatment with antibiotics provoked a reduction in the percentages of CD4⁺CD25⁺ T cells in PP, but significant enhancements (p < 0.01) in MLN, spleens, and CLN (A). A significant reduction in the CD25⁺FoxP3⁺ percentage of total CD4⁺ T cells was observed in PP of i.p. treated mice and MLN and spleens of orally treated mice (B). When total cell numbers were compared, a significant increase of FoxP3⁺ T_reg cells in MLN and CLN of mice treated orally was observed when compared with naive or i.p. treated mice (C) (p < 0.01). Depicted are the means ± SEM from three separate experiments for a total of 12 mice per group. ∗, p < 0.05 between naive vs orally treated and orally vs i.p. treated.

The role of CD11c^highCD103⁺ DCs in the conversion of naive CD4⁺ T cells into Foxp3⁺ T_reg cells has been demonstrated (19), and the potential role for commensal bacteria in this conversion was hypothesized (19, 20). It has been suggested that CD103⁺ DCs might migrate from the intestine to the MLN, where they could generate T_reg cells (21). We investigated whether the enhancement of FoxP3⁺ T_reg cells observed in the lymph nodes of mice treated with antibiotics could be driven by this specific mucosal DC subpopulation. We observed a significant increase in the percentages and total numbers of CD11c^highCD103⁺ and CD11c^highCD103⁻ DCs in PP and MLN of mice orally treated with antibiotics when compared with naive and i.p. treated mice (Fig. 3⇓A; gating strategy and surface characterization of CD11c^highCD103⁺ and CD11c^highCD103⁻ DCs are shown in supplemental Fig. 4A).

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FIGURE 3.

PP and MLN CD11c^highCD103⁺ DCs of mice treated with antibiotics are increased and enhance the conversion in vitro of naive CD4⁺ T cells into FoxP3⁺ T_reg cells. The percentages of CD103⁺CD11c^high and CD103⁻CD11c^high DCs were compared (A; the gating strategy used and characterization of CD103⁺CD11c^high and CD103⁻CD11c^high DCs are shown in supplemental Fig. 4A). The percentages and total cell numbers of CD103⁻ and CD103⁺CD11^high DCs were significantly enhanced in PP and MLN when the microflora was altered. CD11c⁺ cells from MLN of naive mice and mice treated orally with antibiotics were cell-sorted into CD103⁻ and CD103⁺ subpopulations (B; sort is shown in Supplemental Fig. 4B). Naive CD25⁻CD4⁺ T cells were cocultured with CD11c^highCD103⁻ or CD11c^highCD103⁺ DCs. Conversion rates of naive CD4⁺ T cells into FoxP3⁺T_reg cells were significantly enhanced when cocultured with CD11c^highCD103⁺ DCs from mice treated with antibiotics. No differences were observed in cultures with CD11c^highCD103⁻ DCs. ∗, p < 0.01 between naive mice and i.p. treated vs orally treated mice.

We compared the effect of CD11c^highCD103⁺ and CD11c^highCD103⁻ DCs purified from naive and mice treated with antibiotics in the conversion of splenic naive CD4⁺ T cells into FoxP3⁺ T_reg cells (Fig. 3⇑B). The acquisition of FoxP3 by T cells was significantly augmented when cells were cocultured with CD11c^highCD103⁺ DCs of mice treated orally with antibiotics when compared with naive and i.p. treated mice (Fig. 3⇑B). The effect of CD11c^highCD103⁻ DCs in the conversion of naive CD4⁺ T cells into FoxP3⁺ T_reg cells was significantly lower, and no substantial differences in the conversion rates were observed between naive mice treated orally or mice treated i.p. with antibiotics (Fig. 3⇑B). No differences in cell proliferation were observed in cultures with CD11c^highCD103⁻ DCs or CD11c^highCD103⁺ DCs (data not shown). The role of TGF-β and retinoic acid (RA) in the enhancement of T_reg cell conversion by MLN CD103⁺ DCs has been described before (19). We compared the effect of TGF-β and RA in our experimental groups (supplemental Fig. 4B). As described before, rates of conversion by CD11c^highCD103⁺ DCs were increased for all groups tested. The conversion rates induced by CD11c^highCD103⁻ DCs were significantly reduced even in the culture with RA and TGF-β (supplemental Fig. 4B and Ref. 19).

Protection against EAE is associated with increased IL-10-producing FoxP3⁺ T_reg cell percentages and Th2-type immune responses

The reduction of proinflammatory responses in the GALT and other peripheral lymph nodes after oral treatment with antibiotics was shown previously (Table I⇑) (14). We tested whether the changes in gut commensal microflora would protect against EAE by alterations in the cytokine patterns (Fig. 4⇓). Total lymphocytes were harvested from CLN 13 days after EAE induction and cytokines were quantified by capture ELISA after restimulation with PLP_139–151. Lymphocytes obtained from the oral treatment protected group of mice released to the media reduced levels of IL-17 and IFN-γ, whereas IL-10 and IL-13 were enhanced when compared with PBS- and i.p. treated mice lymphocytes (Fig. 4⇓A). IL-13 was enhanced in lymphocytes of i.p. treated compared with PBS-treated mice. However, the level of cytokine production was significantly lower when compared with mice treated orally with antibiotics.

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FIGURE 4.

Oral treatment with antibiotics induces EAE protection by immune deviation toward Th2-type responses. Total lymphocytes from CLN were harvested from mice on day 13 post-EAE induction and restimulated in the presence of PLP_139–151 and cultured for 72 h. CD4⁺ T cells were enriched with magnetic beads and FACS sorted in CD25⁻ and CD25⁺CD4⁺ T cells. Total lymphocytes (A) and sorted CD4⁺CD25⁻ or CD4⁺CD25⁺ T cells (B) were stimulated with PLP_139–151 and cultured for 72 h. Supernatants of the cultures were analyzed by cytokine ELISA. Animals orally treated with antibiotics showed significantly enhanced IL-13 and IL-10 levels and reduced IL-17 and IFN-γ when compared with PBS- and i.p. treated mice. Depicted are the combined results from three separate experiments for a total of 15 mice per group for A and a total of 9 mice per group for B: ∗, p < 0.01 for PBS vs oral treatment and orally vs i.p. treated mice. τ, p < 0.01 for i.p. vs PBS-treated mice.

To identify the CD4⁺ T cell population responsible for the switching of the cytokine profiles of mice protected against EAE, CLN lymphocytes were sorted in CD4⁺CD25⁺ and CD4⁺CD25⁻ T cells and pulsed with PLP_139–151. CD4⁺CD25⁻ T cells from orally treated mice produced significantly reduced levels of IFN-γ and IL-17 when compared with PBS- and i.p. treated mice (Fig. 4⇑B). However, IL-10 and IL-13 were significantly increased in these orally treated mice. When the cytokine profile of CD4⁺CD25⁺ T cells was compared, cells sorted from mice treated orally with antibiotics produced significantly higher levels of IL-10 when compared with PBS- and i.p. treated mice. No significant differences were observed in IFN-γ, IL-17, or IL-13 cytokines in the CD4⁺CD25⁺ T cell populations compared (Fig. 4⇑B). Our results show that oral treatment with antibiotics caused immune deviation in mice characterized by a reduction in proinflammatory responses and significant enhancement in Th2-type immunity.

PCR analysis showed enhanced levels of IL-13 expression in the brains of animals protected against EAE by oral treatment with antibiotics when compared with PBS-treated mice and animals treated i.p. with antibiotics (supplemental Fig. 3). No significant differences in IL-13 production were observed in brains of mice treated i.p. and control PBS-treated mice. Demyelination and nucleated cell infiltration levels were reduced in orally treated mice. No significant differences were observed between PBS- and i.p. treated mice (supplemental Table SIII and supplemental Fig. 3). Interestingly, when mice were treated with the antibiotics during the entire length of the experiment, mice were fully protected with no evidence of disease development as determined by clinical score (supplemental Fig. 3B). These data suggest that intestinal colonization with certain bacterial populations can evoke clinical disease consistent with EAE.

To assess whether protection against EAE was associated with an increase in the T_reg cell populations, lymphocytes derived from PP, MLN, spleen, and CLN were analyzed for presence of FoxP3⁺ T_reg cells (Fig. 5⇓ and supplemental Fig. 5, C and D). A significantly higher (p < 0.01) percentage of FoxP3⁺T_reg cells was observed in spleen and MLN in animals treated with oral doses of antibiotics and protected against EAE when compared with untreated mice and animals treated i.p. with antibiotics. A nonsignificant increase in the frequency of FoxP3⁺ T_reg cells in CLN of orally treated mice was observed; however, when total numbers were compared a significant increase of FoxP3⁺ T_reg cells in CLN, MLN, and spleens of orally treated mice was observed when compared with PBS- and i.p. treated mice (Fig. 5⇓B and supplemental Fig. 5, C and D).

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FIGURE 5.

FoxP3⁺ T_reg cells are enhanced in mice protected against EAE. PP, MLN, spleens, and CLN of mice orally or i.p. treated with antibiotics and PBS-treated mice and subsequently EAE induced were harvested on day 13 after EAE induction and cells were analyzed by FACS. Animals treated orally with antibiotics showed significant enhancement of CD4⁺CD25⁺ T cell populations in MLN and spleens and not a significant increase in PP and CLN when compared with PBS- and i.p. treated mice. FoxP3 frequencies on these populations were significantly enhanced (A). Total cell numbers of mice treated orally with antibiotics and protected against EAE were significantly higher in spleens and CLN when compared with naive or i.p. treated mice (B). Depicted are the combined results from three separate experiments for a total of 15 mice per group: ∗, p < 0.05 or p < 0.01 for naive vs oral treatment and orally vs i.p. treated mice.

Our results suggest that a combination of Th2-type immune responses and the induction of regulatory T cell subpopulations may provide an important framework that can offer protection against EAE when bacterial communities of the gut are challenged with antibiotics.

Oral antibiotic treatment induces protective T_reg cells

Adoptive transfer experiments were performed to determine whether the elicited T cell response following oral antibiotic treatment was responsible for the reduced susceptibility to EAE. In the first experiment, the protective role of CD4⁺ or CD8⁺ T cells was compared (Fig. 6⇓, A and B). SJL/J mice were treated for 7 days with ampicillin, vancomycin, neomycin sulfate, and metronidazole dissolved in drinking water, or with normal drinking water (naive control group). After the treatment, CLN were harvested and CD4⁺ or CD8⁺ T cell populations were enriched by selection with magnetic microbeads. Adoptive transfer of 1 × 10⁶ cells per mouse (≥96% pure) was performed 1 day before EAE induction with PLP_139–151. CD4⁺ T cells isolated from CLN of mice treated with antibiotics significantly reduced the EAE clinical scores of SJL mice when compared with CD4⁺ T cells obtained from naive mice (Fig. 6⇓A). In contrast, no significant differences were observed in the clinical outcome of the disease after adoptive transfer of CD8⁺ T cell-enriched populations from CLN of mice treated with antibiotics when compared with PBS-treated mice or mice treated with naive CD8⁺ T cells (Fig. 6⇓B). These results suggest that CD8⁺ T cells of mice treated with antibiotics do not play a role in the protection against EAE observed previously.

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FIGURE 6.

CD4⁺CD25⁺ T cells from CLN of mice treated with antibiotics confer protection against EAE. CD8⁺ T cells do not mediate in protection conferred by modulation of commensal microflora. CD4⁺ (A) and CD8⁺ T cells (B) were purified with microbeads from combined peripheral lymph nodes of naive and mice orally treated with antibiotics by negative selection using coated microbeads. Cells (1 × 10⁶) were adoptively transferred into naive SJL recipient mice and EAE was induced with PLP_139–151 1 day later. Adoptive transfer of CD4⁺ T cells from mice treated with antibiotics reduced significantly EAE clinical scores when compared with PBS treatment and adoptive transfer of CD4⁺ T cells obtained from naive mice (A). No significant differences were observed in the clinical scores of mice that received CD8⁺ T cells from naive or mice previously treated with antibiotics (B). CD4⁺ T cells from CLN of naive and mice treated with antibiotics were enriched by negative selection and CD4⁺CD25⁻ and CD4⁺CD25⁺ T cells were sorted by flow cytometry. Sorted cells (4 × 10⁵) were adoptively transferred into naive recipient SJL mice and EAE was induced 1 day later (C). Transfer of CD4⁺CD25⁺ T cells obtained from mice treated with antibiotics significantly reduced EAE clinical scores when compared with adoptively transferred naive CD4⁺CD25⁺ T cells and CD4⁺CD25⁻ T cells from naive or animals treated with antibiotics. Sorted cells used for the adoptive transfer experiments were cultured in the presence of anti-CD3 and anti-CD28 mAbs for 72 h. Cytokines were quantified by specific ELISA (D). Depicted are the combined results from two separate experiments for a total of 10 mice per group: ∗, p < 0.01 for naive vs oral treatment and orally vs i.p. treated mice.

We next evaluated whether CD25⁺CD4⁺ or CD25⁻CD4⁺ T cells obtained from CLN of mice treated with antibiotics would be suppressive in vitro and would confer protection against EAE after adoptive transfer. The suppressive capacity of antibiotic-treated, FoxP3-enriched CD25⁺CD4⁺ T cells was significantly enhanced at a 1:10 suppressor T-to-effector T cell ratio (supplemental Fig. 6). To analyze a potential protective role of these cell populations, naive recipient SJL mice were adoptively transferred with 4 × 10⁵ cells per mouse of CD25⁺CD4⁺ or CD25⁻CD4⁺ T cells obtained from CLN of naive mice or mice previously treated with antibiotics 1 day prior to EAE induction with PLP_139–151. When CD25⁺CD4⁺ T cells (>75% FoxP3⁺) purified from CLN of SJL mice treated with antibiotics were transferred, a significant reduction of the EAE clinical scores was observed (Fig. 6⇑C). No protection was observed after adoptive transfer of the control arms, including CD25⁻CD4⁺ T cells purified from mice treated with antibiotics and CD25⁺CD4⁺ and CD25⁻CD4⁺ T cells obtained from naive mice.

Analysis of the cytokine profile of adoptively transferred CD25⁺CD4⁺ and CD25⁻CD4⁺ T cells showed that protective CD4⁺CD25⁺ T cells (>75% FoxP3⁺) sorted from mice treated orally with antibiotics produced significantly enhanced levels of IL-10 (p < 0.01) and IL-13 (not significant) when compared with naive CD4⁺CD25⁺ T cells. When CD25⁻CD4⁺T cells were compared, those obtained from orally treated mice showed significant reductions in IFN-γ and IL-17 and not significant differences in IL-10 and IL-13 when compared with naive levels (Fig. 6⇑D).

To confirm the protective capacity of the T_reg cells from oral antibiotic-treated mice, in vivo neutralization of CD25-expressing cells was performed using a depleting anti-CD25 mAb (clone PC-61). Two doses of 300 μg/mouse on days 3 and 5 after the initiation of oral antibiotic treatment reduced the CD25⁺ in CD4⁺ T cells of naive mice as well as mice treated with either orally or i.p. with antibiotics when compared with control treatment with rat IgG isotype control (Fig. 7⇓A). Partial reversion of protection was observed by depletion of CD25⁺ T cells in mice treated with oral antibiotics (Fig. 7⇓B). The onset of clinical disease occurred earlier (p < 0.05) in all groups treated with anti-CD25 mAb when compared with rat IgG-treated mice (supplemental Table SIV). The cumulative scores and mortality of mice treated orally with antibiotics and subsequently with anti-CD25 mAb were significantly more severe (p < 0.05) when compared with mice treated orally with antibiotics and injected with rat IgG. Interestingly, EAE clinical scores were also significantly reduced in CD25-neutralized mice previously treated with antibiotics when compared with either naive (p < 0.05) or i.p. treated (p < 0.05) mice.

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FIGURE 7.

CD25⁺ cell inactivation restores EAE susceptibility in mice treated orally with antibiotics. Mice were orally or i.p. treated with antibiotics for 7 days before EAE challenge with PLP_139–151 and PT. To inactivate CD25⁺CD4⁺ T cells, the same mice were given 0.3 mg of anti-CD25 mAb (clone PC 61.5.3) on days 4 and 2 before EAE induction. As a control group, treated and naive mice received 0.3 mg of purified rat IgG Ab on the same days before EAE challenge. FACS analysis of peripheral blood samples showed that treatment with anti-CD25 mAb reduced very significantly the CD25⁺ percentages in CD4⁺ T cells of naive mice and treated orally and i.p. with antibiotics when compared with treatment with rat IgG isotype control (A). When EAE was induced, protection observed in mice treated orally with antibiotics was partially lost (B). Depicted are the combined results from two separate experiments for a total of 12 mice per group: ∗, p < 0.01 for naive vs oral treatment and orally vs i.p. treated mice.