Rheumatoid arthritis is an autoimmune disorder that affects the joints and other organs. Pulmonary complications contribute significantly to rheumatoid arthritis mortality. Retinoic acid and its synthetic compound AM80 play roles in immunoregulation but their effect on mucosal autoimmunity remains largely unknown. T follicular helper (Tfh) and Th17 cells are known to promote inflammation and autoantibody production. Using the K/BxN autoimmune arthritis model, we elucidate a novel mechanism whereby oral AM80 administration suppressed lung mucosa–associated Tfh and autoantibody responses by increasing the gut-homing α4β7 integrin expression on Tfh cells. This diverted Tfh cells from systemic (non-gut) inflamed sites such as the lung into the gut-associated lymphoid tissues, Peyer’s patches, and thus reduced the systemic autoantibodies. AM80 also inhibited the lung Th17 response. AM80’s effect in the lungs was readily applied to the joints as AM80 also inhibited Tfh and Th17 responses in the spleen, the major autoantibody producing site known to correlate with K/BxN arthritis severity. Finally, we used anti-β7 treatment as an alternative approach, demonstrating that manipulating T cell migration between the gut and systemic sites alters the systemic disease outcome. The β7 blockade prevented both Tfh and Th17 cells from entering the non-immunopathogenic site, the gut, and retained these T effector cells in the systemic sites, leading to augmented arthritis. These data suggest a dual beneficial effect of AM80, targeting both Tfh and Th17 cells, and warrant strict safety monitoring of gut-homing perturbing agents used in treating intestinal inflammation.

Rheumatoid arthritis (RA) is an autoimmune disease that causes chronic inflammation in the joints as well as in other organs such as the lung. Pulmonary complications are common (19–58%) and rank as the second major cause of death in RA patients (13). Clinical data from RA patients have shown that autoantibodies against citrullinated proteins in the bronchoalveolar lavage fluids are detected in preclinical phase long (5–15 y) before inflammation and destruction of joints. This has led to a long-standing hypothesis that mucosal autoimmunity could predate other systemic development of autoimmune disease in RA (4). These findings suggest that the lung may be an initiating site for RA-related autoimmunity (4). Accordingly, defining the RA-related lung pathogenesis, a poorly understood topic, and identifying the agents that could temper it offers major therapeutic opportunities for both RA-related lung and joint diseases.

K/BxN mice are an autoimmune arthritis model in which transgenic KRN T cells recognize glucose-6-phosphate isomerase, the self-antigen presented by MHC class II I-Ag7 molecules. As in human RA patients, autoantibodies are crucial for disease pathogenesis in K/BxN mice (5). Importantly, K/BxN mice have previously been shown to develop inducible BALT (iBALT)-like structures in their lungs (6), ectopic lymphoid tissues that are known to correlate with lung tissue damage in RA patients (7). T follicular helper (Tfh) cells are a crucial subset of CD4+ T cells that help B cells produce high-affinity and high-titer Abs (810), and an excessive Tfh cell response can lead to many autoimmune conditions including RA (11). Th17 cells, a T effector cell type involved in many autoimmune diseases, promote autoantibody production and inflammation (12). Our previous data have shown that gut microbiota segmented filamentous bacteria (SFB)-induced Tfh and Th17 cells contribute significantly to autoantibody production in K/BxN mice, and a lack of either T effector cell type strongly ameliorates autoantibody production and autoimmune arthritis development (13, 14).

Retinoic acid, a metabolite of vitamin A, has a wide range of biological activity including regulating immune responses (15). AM80 is a synthetic retinoic acid that is characterized by higher stability and fewer potential adverse effects compared with all-trans retinoic acid, one of the most active physiological retinoid metabolites (16, 17). It has been reported that retinoic acid and AM80 ameliorate many autoimmune responses including experimental autoimmune myositis, experimental autoimmune encephalitis, and collagen-induced arthritis (1821). As for retinoic acid’s effects in the lung, retinoic acid treatment has been shown to abrogate pulmonary emphysema (22, 23), but little is known about its effect in autoimmune-related lung diseases. In vitro culture with retinoic acid increases the expression of the gut-homing receptor integrin α4β7 on T cells (2426). The α4β7 integrin receptors are imprinted on lymphocytes by dendritic cells (DCs) from Peyer’s patches (PPs) and mesenteric lymph nodes (MLNs) (26, 27). A recent study discovered that lung DCs could also up-regulate the gut-homing integrin α4β7 in vitro and in vivo (28). A major part of the retinoic acid's anti-inflammatory effects depends on the inhibition of Th17 and promotion of Foxp3+ regulatory T cell (Treg) responses (15, 29). Despite a strong implication of retinoic acid’s involvement in the mucosa, much less is known about its role in mucosal Th17 and Treg responses in vivo such as in the lung and small intestine-lamina propria (SI-LP). Additionally, the role of retinoic acid in the Tfh response remains largely unknown.

In this study, we examine whether the synthetic retinoid AM80 suppresses autoimmune-related lung disease. To elucidate its potential therapeutic mechanism, we compared AM80’s effect on the pathological T effector cells, Tfh and Th17 cells, as well as on Tregs in both mucosal and non-mucosal immune compartments. We determine a novel angle of retinoic acid’s therapeutic mechanism by asking whether AM80 suppresses autoimmune responses by redirecting pathological T effector cells such as Tfh cells and Th17 cells into the intestine and thus away from the systemic (non-gut) inflammatory tissues. It is worth mentioning that despite starting this study with a focus on the effect of retinoic acid on RA-related lung disease, we also included AM80’s effect on joint disease in our analysis. These analyses indicate that our findings in the lung readily apply to joint disease, as we show a strong correlation between the immunoregulatory effects of AM80 in RA-related joint and lung disease.

K/BxN mice were generated by crossing KRN TCR transgenic mice on the C57BL/6 (B6) background with NOD mice. IL17- eGFP reporter mice (C57BL/6-Il17atm1Bcgen/J) from the Jackson Laboratory were crossed with KRN mice to generate IL-17eGFP.KRN mice, which were further crossed with NOD mice to generate IL-17eGFP.K/BxN mice. KikGR.K/BxN mice were generated as previously described (14). Ankle thickness was measured with a caliper (J15 Blet micrometer) as described previously (13). All mice were housed at the animal facility at the University of Arizona. All experiments were conducted according to the guidelines of the Institutional Animal Care and Use Committee at the University of Arizona.

Lungs were perfused with 10 ml PBS to remove blood, then finely minced. Minced lung was placed into 10 ml of digestion buffer containing 1 mg/ml each of Collagenase D (Roche) and MgCl2 and 0.1 mg/ml DNase I (Sigma) in DMEM (HyClone). Lungs were digested for 20–25 min at 37°C with rotation in a shaker (200 rpm) then passed through a 40 μm cell strainer. A plunger from a 5 ml syringe was then used to grind the remaining tissue pieces through the cell strainer. SI-LP cells were isolated as described in our previous report (14). Briefly, PPs were removed from the small intestine. The small intestine was opened longitudinally and excess mucus was removed by scraping gently with forceps along the length of the intestine. The intestine was then thoroughly washed in ice-cold 5 mM EDTA in PBS and cut into 1 cm pieces, which were incubated in 40 ml of 5 mM EDTA and 0.145 mg/ml of DL-DTT in DMEM for 60 min at 37°C at a rotation speed of 100 rpm. After incubation, the epithelial cell layer, containing the intraepithelial lymphocytes, was removed by shaking, and the remaining tissue was cleaned by pressing intestinal pieces over a layer of 100 μm nylon on top of a paper towel. Intestinal pieces were then transferred to an Eppendorf tube with 600 μl of digestion solution containing 1 mg/ml Collagenase D (Roche), 0.15 mg/ml DNase I (Sigma), and 200 ng/ml liberase TL (Roche). The intestine bits were finely minced and transferred to a 50 ml conical tube containing 5 ml of digestion solution. Digestion was performed by incubating the pieces at 37°C for 15 min with rotation at 200 rpm. After digestion, 10 ml of 5 mM EDTA was added and the solution was passed through a 100 μm cell strainer, then through a 40 μm cell strainer. Cells were centrifuged and washed again with EDTA/PBS before being resuspended in 10% FBS DMEM for stimulation.

For surface staining, fluorophore-conjugated mAbs specific for CD4 (RM4-5), CD19 (6D5), CD45 (30-F11), PD-1 (RMP1-30), CTLA-4 (UC10-4B9), CD25 (PC61), and TCRβ (H57-597) were obtained from BioLegend. The mAb recognizing CXCR5 (2G8) was from BD Pharmingen. Anti-integrin α4β7 (DATK32) was from eBioscience. For intracellular cytokine staining, cells were incubated for 4 h with BD GolgiPlug (1:1000 dilution), 50 ng/ml PMA, and 1 μM ionomycin in DMEM (HyClone) supplemented with 10% FCS, 1% nonessential amino acids, penicillin, streptomycin, and glutamine at 37°C. Intracellular cytokine staining was performed with Cytofix/Cytoperm (BD Pharmingen). Abs recognizing IL-17A (TC11-18H10.1) and IgG1 (RMG1-1) were obtained from BioLegend. B cell–expressed IgG1 was stained intracellularly to avoid staining of the serum anti–glucose-6-phosphate isomerase IgG1 that constantly binds to the surface Fc receptor on B cells in the K/BxN model. Surface IgG1 was first blocked by non-fluorophore labeled anti-IgG1 (RMG1-1), then cells were intracellularly stained with fluorophore-conjugated anti-IgG1. For intranuclear staining, buffers from a Foxp3 Staining Buffer Set (eBioscience) were used to stain with Abs recognizing Foxp3 (FJK-16s; eBioscience). Cells were run on an LSR II (BD Biosciences), and analyses were performed with FlowJo (TreeStar) software. Ab recognizing IgG1 was also used for ELISPOT.

Our mouse colony and SFB colonization were described previously (14). For AM80 administration, SFB colonized K/BxN mice (generated from vertical transmission from an SFB gavaged mother) were treated with AM80 (Sigma) at 3 mg/kg body weight in 0.5% carboxymethyl cellulose sodium (CMS; MP Biomedicals) solution or CMS only by orally gavaging, starting from 23 d of age and continuing every other day. To generate SFB positive mice in the β7 blocking experiment, SFB negative mice were weaned at 21 d old and rested for 1 d. Then, mice were orally gavaged with SFB-containing feces collected in house for three consecutive days starting at 23 d old. The SFB negative mice were the ungavaged littermate controls. The SFB colonization status was examined on day 10 after SFB gavage by SFB-specific 16S rRNA quantitative PCR as previously described (13). For the effect of AM80 on SFB colonization, SFB negative mice were gavaged with the same batches of SFB-containing feces and simultaneously treated with AM80 or CMS as described earlier. SFB colonization status was checked on day 10 after the first of 3 d consecutive gavages.

K/BxN mice were i.p. injected with 0.2 mg EdU. Then 12 h after injection, cells were processed and analyzed using the Click-iT Plus EdU Imaging Kit.

ELISA and ELISPOT analyses for glucose-6-phosphate isomerase–specific IgG1 or glucose-6-phosphate isomerase–specific IgG1 autoantibody secreting cells (ASCs), respectively, were performed as described previously (13).

SFB−KRN CD4+ T cells from the spleen were enriched with anti-CD4 MACS beads (Miltenyi), and in vitro cultured for 4 d in Th17 polarization conditions: 2 μg/ml of anti-CD3ε (plate coated), and anti-CD28, IL-6 (50 ng/ml; PeproTech), TGF-β1 (1 ng/ml; PeproTech), and FICZ (6-formylindolo [3,2-b] carbazole, 300 nM; Enzo Life Sciences). AM80 (100 nM) or DMSO vehicle were added during the polarization process.

Survival surgeries were performed under sterile conditions as previously described (14). Anesthetized mice were shaved at the area right around the kidney using electric clippers. The shaved area was wiped with alcohol prep pads. Mice were positioned on their backs and a small incision (∼1.5 cm) was made at the midline, below the costal margin. The PPs were identified and then covered with sterile foil with a ∼4 mm hole to leave one PP exposed to a violet laser at a time (405 nm; peak power <30 mW). A total of four PPs per intestine were exposed to the violet laser. Each PP was exposed to violet light for 30 s and PBS was dripped on the intestine after each PP exposure during surgery to avoid drying. The abdominal muscles were closed using a 4-0 synthetic absorbable suture. The skin incision was closed with clips using a Reflex 9 mm Wound Clip Applier (Robot Surgical Instrument). After surgery, the mice were placed on a heating pad for recovery. Three days after surgery, the spleen and cervical LNs (CLNs) were harvested for analysis.

K/BxN mice were i.p. injected with anti-β7 (0.2 mg per mouse, FIB504; BioXCell), or control Rat IgG (Jackson ImmunoResearch) 1 d after weaning. The mice treated with blocking Abs or control IgG were treated three more times, every 3 d.

Cells from spleen, lung, and SI-LP of SFB+ IL-17eGFP.K/BxN mice (5–6.5 wk old) were cultured with 1 μM ionomycin, 50 ng/ml PMA, and BD GolgiPlug (1:1000 dilution) for 4 h. Cells were stained with Abs recognizing CD4, TCRβ, and CD45 prior to cell sorting (FACSAria). Th17 (CD45+ TCRβ+ CD4+ GFP+) cells from spleen, lung, and SI-LP were sorted. RNA from sorted cells was purified using TriPure (Roche) and further enriched by RNeasy MinElute Cleanup Kit (Qiagen). RNA was frozen and sent to the Boston University MicroArray Core for further processing and analysis using an Affymetrix GeneChip Mouse Gene 1.0 ST Array. Data of Affymetrix GeneChip Mouse Gene 1.0 ST CEL files were normalized to produce gene-level expression values using the implementation of the robust multiarray average. The microarray data have been deposited in the Gene Expression Omnibus repository (www.ncbi.nlm.nih.gov/projects/geo/) under the accession number GSE: 92860.

Lung sections from AM80- or CMS-treated SFB-colonized K/BxN mice were perfused and fixed with 10% (v/v) buffered formalin and stained with H&E for histological evaluation of lymphocyte aggregation. Image analysis was performed using ImageJ software (National Institutes of Health, Bethesda, MD).

Differences were considered significant when p < 0.05 by Student t test (two-tailed, unpaired) or two-way ANOVA (Prism 6; Graph-Pad Software). To compare ankle thickening, the area under the curve was calculated for each mouse within an experimental set followed by the Student t test between groups (Prism 6; Graph-Pad Software). One asterisk (*) indicates p < 0.05, two asterisks (**) indicate p < 0.01, three asterisks (***) indicate p < 0.001, and four asterisks (****) indicate p < 0.0001.

iBALT is a type of ectopic lymphoid tissue found in the lungs of RA patients and is positively correlated with the severity of the patient’s lung disease (7). K/BxN mice develop iBALT-like structures closely resembling the iBALT formations of RA patients (6). We have previously reported that the gut microbiota SFB boost Th17 and Tfh responses in K/BxN mice, which worsens the autoimmune arthritis (13, 14). In this study, unless otherwise mentioned, we intentionally tested the strength of AM80’s immunosuppressive effect in SFB-colonized (SFB+) K/BxN mice, as SFB+ mice display a more severe disease phenotype with stronger pathological Tfh and Th17 cell activity than SFB− K/BxN mice. We first examined the AM80 effect in the lung of SFB+ K/BxN mice. Oral treatment of AM80 ameliorated iBALT-like lesions in the lung of AM80-treated mice compared with vehicle control CMS-treated mice (Fig. 1A, 1B). The decreased lung iBALT-like lesions corresponded with a reduction in the number of self-antigen glucose-6-phosphate isomerase–specific ASCs in the lung draining LNs (LDLNs) (Fig. 1C).

FIGURE 1.

AM80 treatment ameliorates lung autoimmune response in K/BxN mice. (A) SFB+ K/BxN littermate mice were orally gavaged with AM80 or CMS vehicle (control) every other day. Representative images of lung histology (H&E staining) of day 10 (33 d old) AM80- or CMS-treated mice are shown. (B) The combined areas of iBALT-like structure from 10 random, non-overlapped fields of the experiments in (A) were measured using ImageJ software (National Institutes of Health, Bethesda, MD; n = 8 per group, combined from two experiments). (C) The average percentages of anti–glucose-6-phosphate isomerase ASCs of the IgG1 isotype among total B cells + SEM from day 10 AM80-gavaged mice are shown (n = 6 per group, data combined from two assays). **p < 0.01, ***p < 0.001.

FIGURE 1.

AM80 treatment ameliorates lung autoimmune response in K/BxN mice. (A) SFB+ K/BxN littermate mice were orally gavaged with AM80 or CMS vehicle (control) every other day. Representative images of lung histology (H&E staining) of day 10 (33 d old) AM80- or CMS-treated mice are shown. (B) The combined areas of iBALT-like structure from 10 random, non-overlapped fields of the experiments in (A) were measured using ImageJ software (National Institutes of Health, Bethesda, MD; n = 8 per group, combined from two experiments). (C) The average percentages of anti–glucose-6-phosphate isomerase ASCs of the IgG1 isotype among total B cells + SEM from day 10 AM80-gavaged mice are shown (n = 6 per group, data combined from two assays). **p < 0.01, ***p < 0.001.

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Additionally, AM80 also suppressed autoimmune arthritis development (Fig. 2A). Autoantibodies are a hallmark of B cell-mediated autoimmune diseases, including RA. As in RA patients, serum autoantibodies serve as the disease index in the K/BxN model and, indeed, passive transfer of K/BxN arthritic serum (containing anti–glucose-6-phosphate isomerase autoantibodies) into wild type mice is sufficient to induce arthritis development (5). We found that the reduction of arthritis severity in AM80-treated mice corresponded with decreasing levels of serum anti–glucose-6-phosphate isomerase titers (Fig. 2B). To confirm that the effect we observed was due to the direct immunomodulation effect of AM80, not due to AM80 reducing SFB colonization, which would lead to reduced autoimmune activity, we also examined the SFB colonization level after AM80 treatment by quantitative PCR. There was no difference in SFB levels between the AM80 and CMS treatment (Fig. 2C). Therefore, our results suggest that oral AM80 treatment can simultaneously ameliorate diseases at two distinct systemic sites, both at mucosal lung tissue and at the joints.

FIGURE 2.

AM80 administration attenuates autoimmune arthritis development in K/BxN mice. (A) SFB+ K/BxN mice were orally gavaged every other day with AM80 or CMS from the ages of 23–34 d old. Ankle thickening was measured and is shown (n = 9–10 per group, data combined from two independent assays). (B) Serum anti–glucose-6-phosphate isomerase titers from experiments in (A) are shown as mean + SEM. (C) Fecal SFB level from SFB+ K/BxN littermates treated with CMS or AM80 (n = 14 per group; data are combined from three cohorts of littermate mice). **p < 0.01, ****p < 0.0001.

FIGURE 2.

AM80 administration attenuates autoimmune arthritis development in K/BxN mice. (A) SFB+ K/BxN mice were orally gavaged every other day with AM80 or CMS from the ages of 23–34 d old. Ankle thickening was measured and is shown (n = 9–10 per group, data combined from two independent assays). (B) Serum anti–glucose-6-phosphate isomerase titers from experiments in (A) are shown as mean + SEM. (C) Fecal SFB level from SFB+ K/BxN littermates treated with CMS or AM80 (n = 14 per group; data are combined from three cohorts of littermate mice). **p < 0.01, ****p < 0.0001.

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We next set out to systematically determine the mechanism by which AM80 inhibits autoimmune disease in K/BxN mice by examining AM80 effects in a variety of tissues. We compared the immune response with AM80 to CMS treatment in the spleen, which is a systemic lymphoid tissue and the major autoantibody production site corresponding to arthritis development (30). We also examined a systemic mucosal tissue, the lung, as it is the AM80-responsive site that interested us. Finally, we also examined the effect of retinoic acid in the SI-LP because retinoic acid is known to up-regulate the gut-homing α4β7 integrin receptor on lymphocytes. Inhibition of Ag-specific T cell proliferation by retinoic acid has been shown when retinoic acid is delivered by daily i.p. injections. Oral delivery of retinoic acid or AM80 has been adapted by a few groups previously (19, 31, 32). We found that oral AM80 did not suppress T cell proliferation measured in vivo by EdU assay in either systemic tissue (spleen and lung) or gut tissue (SI-LP) (Fig. 3A). Thus, oral treatment of AM80 did not generate an immunosuppressive effect in the general population of CD4+ T cells. Next, we examined AM80’s effect on Foxp3-expressing Tregs. Although induction of Tregs has been proposed as one mechanism of the AM80-mediated anti-inflammatory response, we did not find a difference in the Treg population between the AM80-treated and CMS-treated groups in either systemic sites or the gut (Fig. 3B).

FIGURE 3.

AM80 treatment reduces the systemic Th17 response. (A) Histogram overlays of EdU incorporation in TCRβ+CD4+ T cells from spleen, lung, and SI-LP of AM80- or CMS-treated K/BxN mice are shown. Quantitative data of percentage of EdU+CD4+ T cells are also shown (n = 6 per group, two independent assays). (B) Representative plots and quantitative percentage data of Tregs in spleen, lung, and SI-LP from experiments in (A) are shown. (C) Representative plots and quantitative data of Th17 cell numbers from spleen and lung of AM80- or CMS-treated K/BxN mice (n = 8 per group, two independent assays). (D) Representative plots and quantitative data of Th17 cell number in SI-LP from experiments in (C) are shown. (E) Representative histogram overlays of α4β7 expression on Th17 and non-Th17 cells from spleen and lung from experiments in (C) are shown. Quantitative data are also shown. (F) KRN CD4+ T cells were cultured in vitro with AM80 or DMSO vehicle (control) for 4 d under Th17 polarization conditions. Representative plots and quantitative percentages of Th17 cells are shown (n = 13 replicates from four mice, four independent assays). (G) Histogram overlay of α4β7+ staining on polarized Th17 cells from experiment in (F) is shown. Quantitative percentage of α4β7+ cells in total Th17 cells is also shown. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

FIGURE 3.

AM80 treatment reduces the systemic Th17 response. (A) Histogram overlays of EdU incorporation in TCRβ+CD4+ T cells from spleen, lung, and SI-LP of AM80- or CMS-treated K/BxN mice are shown. Quantitative data of percentage of EdU+CD4+ T cells are also shown (n = 6 per group, two independent assays). (B) Representative plots and quantitative percentage data of Tregs in spleen, lung, and SI-LP from experiments in (A) are shown. (C) Representative plots and quantitative data of Th17 cell numbers from spleen and lung of AM80- or CMS-treated K/BxN mice (n = 8 per group, two independent assays). (D) Representative plots and quantitative data of Th17 cell number in SI-LP from experiments in (C) are shown. (E) Representative histogram overlays of α4β7 expression on Th17 and non-Th17 cells from spleen and lung from experiments in (C) are shown. Quantitative data are also shown. (F) KRN CD4+ T cells were cultured in vitro with AM80 or DMSO vehicle (control) for 4 d under Th17 polarization conditions. Representative plots and quantitative percentages of Th17 cells are shown (n = 13 replicates from four mice, four independent assays). (G) Histogram overlay of α4β7+ staining on polarized Th17 cells from experiment in (F) is shown. Quantitative percentage of α4β7+ cells in total Th17 cells is also shown. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

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Many of the anti-inflammatory functions of AM80 and retinoic acid are attributed to their Th17 inhibitory effect in several autoimmune disease models (1921). In addition, we and others have previously demonstrated that Th17 cells are required to help B cells produce Abs in K/BxN mice and other model systems (13, 33, 34). Therefore, we first examined if AM80 also inhibits the Th17 responses in K/BxN mice at both systemic sites, the spleen and mucosal lung sites. We found that, indeed, AM80 inhibited the Th17 responses in both the spleen and lung (Fig. 3C). Th17 cells have been associated with the pathogenesis of many autoimmune diseases. However, recent studies have shown that, aside from the pathological Th17 effector cells located in the inflammatory tissues, there are also non-pathological types of Th17 cells residing in the intestine and expressing immunoregulatory phenotypes (3537). Indeed, one study has shown that systemic inflammatory Th17 cells can be redirected into the small intestine and be contained there by anti-CD3 treatment in a tolerization model (35). A well-known effect of retinoic acid is to up-regulate the integrin α4β7, a gut-homing receptor that facilitates the return of lymphocytes in systemic sites to the intestine (2426). Putting this information together, we hypothesized a new mechanism whereby AM80 suppresses autoimmune responses by redirecting the Th17 cells into the intestine, to minimize the retention of Th17 cells in systemic inflammatory sites. Most of the gut Th17 cells are located in the SI-LP. To test our hypothesis, we first examined whether there was an increase of Th17 cells in the SI-LP after AM80 treatment; we did not observe such an increase (Fig. 3D). We therefore examined the α4β7 integrin expression on Th17 cells and found there were no differences in α4β7 integrin expression on Th17 cells between the AM80 and CMS-treated groups (Fig. 3E). We were surprised by these findings, as it has been shown that cultured retinoic acid–treated Th17 cells preferentially up-regulate their α4β7 integrin receptors and home to the small intestine after adoptive transfer (24, 25). We therefore tested whether AM80 treatment induces α4β7 integrin expression in vitro in the K/BxN Th17 polarization culture (25). When treating directly in culture, we found a dramatic reduction of the Th17 cell population and an up-regulation of α4β7 expression on Th17 cells (Fig. 3F, 3G). These data suggest that the lack of α4β7 integrin induction in Th17 cells by oral AM80 treatment in K/BxN mice was due to the AM80 exposure route rather than a specific characteristic of K/BxN T cells. Therefore, AM80 inhibits the systemic Th17 response independent of the gut-homing effect induced by AM80.

Little is known about how retinoic acid affects the Tfh response. Thus, we first examined whether AM80 impacted the Tfh response and found that there was a significant drop in number of splenic Tfh cells (Fig. 4A). Because we are interested in the role of AM80 in lung pathogenesis, we also examined the Tfh response in the LDLNs. We did not examine the Tfh response directly in the lung because there is no significant population (if any) of Tfh cells in the lung (C.P. Bradley, F. Teng, D. Naskar, T. Sano, K.M. Felix, N.L. Tran, K. Knox, D. Littman, and H.-J.J. Wu, submitted for publication). Similar to the results in the spleen, there was also a significant decrease of Tfh cells in the LDLNs (Fig. 4A). PPs are a type of gut-associated lymphoid tissue harboring abundant Tfh cells. We recently reported that the gut microbiota SFB drive autoimmune arthritis by promoting the migration of gut Tfh cells from PPs into the spleen to help B cells produce autoantibodies in systemic sites (14). Conversely, because retinoic acid is known to up-regulate α4β7 integrin expression in T cells, we revisited our hypothesis, which was tested on Th17 cells earlier, and asked whether AM80 suppressed the systemic (non-gut) autoimmune response by redirecting systemic Tfh cells into the PPs and minimizing their retention in systemic inflammatory sites. We first addressed this hypothesis by examining the Tfh response in PPs and found that there was indeed an increase of Tfh cells in the PPs of AM80-treated mice (Fig. 4B). We examined whether AM80 increased the PP Tfh population by other mechanisms such as by increasing Tfh cell proliferation in the PPs. We used EdU incorporation to measure PP Tfh cell proliferation in vivo and found there was no difference between the AM80 and CMS groups (Fig. 4C). Bcl-6 is the master transcriptional regulator of Tfh cells and promotes differentiation of non-Tfh CD4+ T cells into Tfh cells (8, 38). Next, we examined whether AM80 increases the PP Tfh population by increasing the expression of Bcl-6 in non-Tfh cells, thus promoting their differentiation into Tfh cells. We did not find that AM80 treatment increased Bcl-6 expression in PP non-Tfh cells, suggesting it did not increase the PP Tfh population by enhancing Tfh cell differentiation (Fig. 4D). In summary, we did not observe that AM80 mediated any boost in Tfh proliferation or differentiation, which strengthens the possibility of our earlier hypothesis, that AM80 could suppress the systemic autoimmune response by redirecting the systemic Tfh cells into the PPs and increasing the Tfh cell population there.

FIGURE 4.

AM80 reduces systemic Tfh while increasing gut Tfh cell population. (A) Representative plots with values indicating the percentage of Tfh cells and quantitative data of Tfh cell numbers in spleen and LDLNs from AM80- or CMS-treated K/BxN mice are shown (n = 12 per group, three independent assays). (B) Representative plots with values indicating the percentage of Tfh cells and quantitative data of Tfh cell numbers in PPs from experiments in (A) are shown. (C) Histograms and quantitative percentage data of PP EdU+Tfh cells from experiments in (A). (D) Histograms and quantitative mean fluorescence intensity (MFI) of Bcl-6 expression in PP-derived non-Tfh cells from experiments in (A). *p < 0.05.

FIGURE 4.

AM80 reduces systemic Tfh while increasing gut Tfh cell population. (A) Representative plots with values indicating the percentage of Tfh cells and quantitative data of Tfh cell numbers in spleen and LDLNs from AM80- or CMS-treated K/BxN mice are shown (n = 12 per group, three independent assays). (B) Representative plots with values indicating the percentage of Tfh cells and quantitative data of Tfh cell numbers in PPs from experiments in (A) are shown. (C) Histograms and quantitative percentage data of PP EdU+Tfh cells from experiments in (A). (D) Histograms and quantitative mean fluorescence intensity (MFI) of Bcl-6 expression in PP-derived non-Tfh cells from experiments in (A). *p < 0.05.

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We next tested whether AM80 redirected Tfh cells to the PPs by increasing the gut-homing α4β7 integrin expression on Tfh cells. Again, we focused on lymphoid tissues, specifically the spleen and LDLNs. We found that, indeed, AM80 increases the α4β7 integrin expression in both splenic and LDLN Tfh cells but not non-Tfh cells (Fig. 5A). To ultimately determine whether AM80 inhibits Tfh cell migration from the PPs to systemic lymphoid tissues, we took advantage of the KikGR transgenic mouse line, which ubiquitously expresses the green-to-red photoconvertible fluorescent protein in its cells (39, 40). We have previously developed a protocol to perform surgery that specifically photoconverts PP cells by treating PPs with violet laser light in KikGR.K/BxN mice that express the KikGR transgene on the K/BxN background (14). Three days later, we monitored the migration of these photoconverted PP cells to the spleen. The control group with sham surgeries displayed very few red fluorescent CD4+ T cells at a background level in the spleen (Fig. 5B). In contrast, a significant number of photoconverted, PP-derived CD4+ T cells were detected in the spleens of the KikGR.K/BxN mice after PP violet light exposure. As expected, we observed an increase of α4β7 integrin expression, an indication of gut origin, in the photoconverted, PP-derived CD4+ T cells compared with the non-photoconverted CD4+ T cells in the spleen. Remarkably, AM80 treatment significantly reduced the percentage of newly arrived, PP-derived, photoconverted Tfh cells (but not non-Tfh cells) in the spleen (Fig. 5C). Because we only carry out a single violet laser exposure on PPs, to collect more photoconverted cells for analysis we also examined another large systemic lymphoid tissue, the CLNs, whose location and function are closely related to the lung, and observed a similar AM80 suppression effect on the migration of PP-derived CD4+ T cells into the CLNs (Fig. 5C) (41). Thus, these results provide direct evidence that AM80 preferentially inhibits Tfh cell, but not non-Tfh cell, migration from PPs to systemic sites. Together, these data support our hypothesis that AM80 suppresses the systemic (non-gut) autoimmune response by up-regulating the α4β7 integrin expression on Tfh cells and redirecting the systemic Tfh cells into the PPs to minimize the retention of Tfh cells in systemic inflammatory sites.

FIGURE 5.

AM80 inhibits the migration of gut Tfh cells to systemic sites. (A) Representative histogram overlays and quantitative data of α4β7 expression on Tfh and non-Tfh cells from spleen and LDLNs of AM80- or CMS-treated K/BxN mice are shown (n = 8 per group, two independent assays). (B) Representative plots of photoconverted, PP-derived (KikR+) CD4+ T cells in spleen of mice 3 d post sham or photoconversion surgeries. Representative plots of α4β7 expression on photoconverted (KikR+) and non-converted (KikG+) cells are also shown. (C) PP-derived (KikR+) CD4+ T cells from spleen and CLNs of AM80- or CMS-treated KikGR.K/BxN mice were further gated on Tfh and non-Tfh cells; representative plots and quantitative data of Tfh and non-Tfh populations within KikR+ CD4+ T cells are shown (n = 4–5 per group, three independent assays). *p < 0.05, **p < 0.01.

FIGURE 5.

AM80 inhibits the migration of gut Tfh cells to systemic sites. (A) Representative histogram overlays and quantitative data of α4β7 expression on Tfh and non-Tfh cells from spleen and LDLNs of AM80- or CMS-treated K/BxN mice are shown (n = 8 per group, two independent assays). (B) Representative plots of photoconverted, PP-derived (KikR+) CD4+ T cells in spleen of mice 3 d post sham or photoconversion surgeries. Representative plots of α4β7 expression on photoconverted (KikR+) and non-converted (KikG+) cells are also shown. (C) PP-derived (KikR+) CD4+ T cells from spleen and CLNs of AM80- or CMS-treated KikGR.K/BxN mice were further gated on Tfh and non-Tfh cells; representative plots and quantitative data of Tfh and non-Tfh populations within KikR+ CD4+ T cells are shown (n = 4–5 per group, three independent assays). *p < 0.05, **p < 0.01.

Close modal

Finally, we used an alternative approach, β7 integrin blockade, to independently provide a proof-of-principle study showing agents that manipulate the migration of T effector cells from systemic (non-gut) sites into gut tissues can alter systemic disease outcome. We have taken advantage of our previous finding that the gut microbiota SFB boost autoimmune arthritis by inducing Th17 and Tfh responses and that SFB colonization also increases the number of α4β7+ Th17 and α4β7+Tfh cells (13, 14). These results suggested that β7 blocking might adversely affect autoimmune arthritis development, given we have previously shown that SFB-mediated migration of PP Tfh cells to systemic sites is essential for disease enhancement. Anti-β7 Ab treatment prevents both gut homing (via α4β7 blocking) and retention (via blocking αEβ7) of lymphocytes (42). Administration of anti-β7 Abs indeed increased the splenic Tfh cell numbers (but not the percentage) but decreased the PP Tfh cell numbers (and percentage) in the gut of SFB+ but not SFB− K/BxN mice (Fig. 6A). This could be due to SFB increasing the percentage of Tfh cells expressing the α4β7 integrin (14), rendering the Tfh cells more susceptible to β7 blockade. There is also an accumulation of Th17 cells in the lung (and a trend of accumulation in the spleen) of the anti-β7 Ab-treated mice, which corresponds to a decrease of Th17 cells in the SI-LP (Fig. 6B). The β7 blockade-mediated increase in the absolute number of Th17 cells in the lung and decrease in the absolute number of Th17 cells in the SI-LP are more obvious in SFB+ compared with SFB− K/BxN mice. Again, this is likely due to a higher baseline number of α4β7+Th17 cells in the SFB+ mice (13).

FIGURE 6.

Integrin β7 blockade exacerbates disease development in K/BxN mice by retaining Tfh and Th17 cells at systemic sites. (A) Representative plots with values indicating the percentage of Tfh cells and quantitative data of Tfh cell numbers from spleen and PPs of anti-β7- or control IgG-treated K/BxN mice are shown (n = 8 per group, combined from four assays). (B) Representative plots with values indicating the percentage of Th17 cells and quantitative data of Th17 cell numbers from spleen, lung, and SI-LP of anti-β7- or control IgG-treated K/BxN mice. (C) Representative histogram overlay and mean fluorescence intensity (MFI) quantitative data of CTLA-4 in Th17 cells from spleen, lung, and SI-LP of SFB+ K/BxN mice are shown (n = 8, two independent assays). (D) We examined the expression of previously reported Th17 pathogenic (red font) and non-pathogenic (blue font) genes by microarray. The fold differences of gene expression in SI-LP Th17 cells over either lung or splenic Th17 cells sorted using a FACSAria are shown for a total of 10 genes indicated on the x-axis. (E) The ankle thickness of SFB− or SFB+ K/BxN mice treated with either anti-β7 Abs or control IgG was measured at 35 d old. Each symbol indicates the average ankle thickness from both ankles of an individual mouse (n = 8 per group, combined from four assays). *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 6.

Integrin β7 blockade exacerbates disease development in K/BxN mice by retaining Tfh and Th17 cells at systemic sites. (A) Representative plots with values indicating the percentage of Tfh cells and quantitative data of Tfh cell numbers from spleen and PPs of anti-β7- or control IgG-treated K/BxN mice are shown (n = 8 per group, combined from four assays). (B) Representative plots with values indicating the percentage of Th17 cells and quantitative data of Th17 cell numbers from spleen, lung, and SI-LP of anti-β7- or control IgG-treated K/BxN mice. (C) Representative histogram overlay and mean fluorescence intensity (MFI) quantitative data of CTLA-4 in Th17 cells from spleen, lung, and SI-LP of SFB+ K/BxN mice are shown (n = 8, two independent assays). (D) We examined the expression of previously reported Th17 pathogenic (red font) and non-pathogenic (blue font) genes by microarray. The fold differences of gene expression in SI-LP Th17 cells over either lung or splenic Th17 cells sorted using a FACSAria are shown for a total of 10 genes indicated on the x-axis. (E) The ankle thickness of SFB− or SFB+ K/BxN mice treated with either anti-β7 Abs or control IgG was measured at 35 d old. Each symbol indicates the average ankle thickness from both ankles of an individual mouse (n = 8 per group, combined from four assays). *p < 0.05, **p < 0.01, ***p < 0.001.

Close modal

Th17 cells are involved in the development of many autoimmune diseases. As mentioned earlier, however, recent studies have shown that in addition to the pathological Th17 effector cells located in inflammatory sites, there are also non-pathological Th17 cell types (3537). One study shows that these non-pathological Th17 cells naturally reside in the intestine and function as gatekeepers against microbial infections (35). These gut Th17 cells express an immunoregulatory phenotype including expression of CTLA-4, a checkpoint receptor that downregulates immune responses. We next tested whether the gut Th17 cells present in the K/BxN model also display a non-pathogenic, immunoregulatory phenotype. We first compared the CTLA-4 expression in the splenic, lung, and SI-LP Th17 cells. The SI-LP Th17 cells indeed express significantly more surface CTLA-4 molecules than those isolated from the spleen and lung (Fig. 6C). We next used microarray assays to examine the previously reported pathogenic and non-pathogenic Th17 transcript signatures. Th17 cells marked by the expression of IL-17-eGFP were isolated from the spleen, lung, and SI-LP of IL-17eGFP.K/BxN mice. We compared the fold differences of gene expression in SI-LP Th17 cells to those from spleen or lung for genes associated with pathogenic (red) and non-pathogenic (blue) Th17 cell signatures reported previously (3537). The SI-LP Th17 cells in K/BxN mice indeed had an increased expression of non-pathogenic and a decreased expression of pathogenic genes compared with those from the lung and spleen (Fig. 6D). Therefore, both subsets of pathogenic effector T cells, Tfh and Th17 cells, in the SFB+ K/BxN model were retained by β7 blockade in systemic inflammatory sites away from the non-pathogenic gut area (PPs for Tfh cells and SI-LP for Th17 cells). These results suggest that β7 blockade could cause an adverse effect on autoimmune arthritis development in the SFB+ group. Indeed, we found that SFB enhanced arthritis development as reported previously (13, 14) and β7 blockade further augmented the disease severity as shown by an increase of ankle thickness in SFB+ but not SFB− K/BxN mice (Fig. 6E).

We demonstrate that oral AM80 treatment ameliorates lung autoimmune response in K/BxN mice by reducing the populations of both lung Th17 and LDLN Tfh cells. Autoantibodies are a hallmark of B cell-mediated autoimmune disease (43, 44). We and others have demonstrated that both Tfh and Th17 cells contribute to germinal center B cell differentiation and Ab production (13, 14, 33, 34). Thus, there is a dual benefit of AM80 treatment for autoantibody mediated autoimmune diseases as it strikes down both Tfh and Th17 responses when compared with a Tfh- or Th17-only targeted therapy. Regarding iBALT formation, our unpublished observations (C.P. Bradley et al., submitted for publication) show that Th17 cells are essential for iBALT formation in K/BxN mice. We do not think Tfh cells have a significant role in iBALT formation because we did not observe a significant induction (if any) of the Tfh cell population in the lung of SFB+ mice (C.P. Bradley et al., submitted for publication). Rather, we think Tfh cells in LDLNs contribute to the lung autoimmune response by providing help to LDLN B cells to generate autoantibodies that are likely dispersed into the lung due to LDLNs’ close proximity. We have previously demonstrated that both Tfh and Th17 cells contribute significantly to autoantibody and arthritis development in K/BxN mice (14). The oral administration of AM80 also reduces autoimmune arthritis development as Tfh and Th17 cell counts also decrease in the spleen, which is the major systemic autoantibody producing site corresponding to arthritis development (30). Therefore, we are able to demonstrate AM80 has a global autoimmune suppression effect in a systemic autoimmune condition involving multiple organs.

In our previous study, we asked whether SFB-induced Tfh cells in PPs could directly help B cells in PPs and/or exit to systemic sites, helping B cells there (14). We found that, despite spleen and PPs having similar percentages of anti–glucose-6-phosphate isomerase–specific B cells, the spleen produces a much higher absolute number of anti–glucose-6-phosphate isomerase ASCs compared with PPs because it contains a much larger B cell pool. We also showed that PP-derived Tfh cells are required to migrate to the systemic lymphoid tissues to contribute to the systemic autoimmune response there. PP depletion in SFB+ K/BxN mice reduces systemic Tfh cells in the spleen as well as the popliteal LNs (foot draining LNs) subsequently leading to amelioration of serum autoantibody titers and arthritis (14). In this study, we have elucidated a novel mechanism whereby AM80 suppresses the systemic Tfh responses in both mucosal (LDLN) and non-mucosal (spleen) sites by specifically increasing the expression of the gut-homing α4β7 integrin on Tfh cells. This diverts the pathologic Tfh cells into the gut associated lymphoid tissues (PPs) and, as a result, keeps the pathologic effector Tfh cells away from the systemic inflammatory sites. The AM80 effect is a mirror image of the SFB effect, in that AM80 prevents the migration of PP Tfh cells to the systemic lymphoid tissues, which minimizes Tfh help to B cells in systemic sites. AM80 specifically up-regulates the expression of α4β7 integrin on Tfh cells but not non-Tfh or Th17 cells. In vitro, retinoic acid or AM80 significantly promotes α4β7 expression on Th17 cells [(25), Fig. 3G], but during oral AM80 treatment of in vivo autoimmune arthritis models, there appears to be no induction of α4β7 on Th17 cells in the AM80-treated group. Rather, AM80’s inhibitory effect on systemic Th17 populations in K/BxN mice could rely on a direct AM80-mediated suppression of Th17 differentiation as reported previously (45). It is peculiar that oral AM80 treatment has different effects on Th17 and Tfh cells in vivo in terms of α4β7 imprinting and up-regulation. We speculate that this difference may result from the differential locations of Tfh and Th17 cells in the gut, i.e., Tfh cells are mostly located in PPs and Th17 cells are mostly located in SI-LP. Studies have shown that DCs from PPs and MLNs can naturally convert Vitamin A to retinoic acid, which helps DCs to imprint α4β7 on lymphocytes (26, 27). Little is known about the molecular mechanism by which PP or MLN DC imprint α4β7 on lymphocytes after retinoic acid exposure. However, because PPs are one of the natural sites where α4β7 imprinting takes place, one can picture a likely scenario that Tfh cells located in the PP have a more direct access to the machinery required for α4β7 up-regulation upon AM80 exposure than Th17 cells located at the SI-LP.

Th17 cells are a pathological T effector cell type involved in many autoimmune diseases (12). Moreover, Th17 cells are present and play a pathological role in the lungs during many chronic inflammatory conditions such as asthma and fibrotic lung diseases (46). The effect of retinoic acid in the lung Th17 response during autoimmune conditions has not been studied previously. We found that AM80 treatment inhibits the Th17 response in the lung similar to what we and others have observed in the spleen and other effector sites (20, 21). However, AM80 treatment did not significantly reduce the Th17 population in another mucosal tissue that we examined, the SI-LP. This different outcome suggests that retinoic acid’s effect on Th17 inhibition depends on the environmental condition. A previous study has shown that a Vitamin A–deficient diet surprisingly downregulates the Th17 response in the SI-LP (47), also suggesting a different role of Vitamin A and its derivative products in regulating the Th17 population between systemic sites and the gut. Our results showing that AM80 did not promote Treg responses in K/BxN mice are consistent with previous reports showing retinoic acid promotes Treg differentiation only in vitro but not in vivo during the development of experimental autoimmune encephalomyelitis (EAE) and Listeria monocytogenes infection (21, 45). The lack of a Treg promotion effect by retinoic acid or AM80 may be due to a limitation of TGF-β, as the ability of retinoic acid to promote the generation of Tregs requires a synergistic effect with TGF-β (24, 45, 48).

Previously, pathologic Th17 signatures have been found in Th17 cells from the CNS of EAE models whereas non-pathologic Th17 signatures have been observed in the intestinal Th17 cells of an anti-CD3 Ab-mediated tolerization model (35). The intestinal Th17 cells induced by anti-CD3 treatment even display an immunoregulatory phenotype and express a high level of CTLA4 compared with lung and splenic Th17 cells. However, little is known about whether intestinal Th17 cells will acquire an inflammatory phenotype in a systemic autoimmune disease. Our results suggest that at least in the K/BxN model, intestinal Th17 cells still display an immunoregulatory phenotype despite the systemic autoimmune condition. In contrast, Th17 cells isolated from another mucosal organ, the lung, behaved more like those from the spleen, expressing a stronger pathogenic signature and weaker non-pathogenic Th17 signature than SI-LP. These data imply that in a systemic autoimmune condition, diverting the Th17 cells from systemic inflammatory sites to the gut could improve the disease. Conversely, agents that block the entry of Th17 cells into the gut could have detrimental effects, worsening the disease. Our anti-β7 treatment experiment supports this hypothesis. Aside from the AM80 data, the β7 blockade experiments independently provide another proof-of-principle study showing agents that alter the migration of T effector cells between the systemic (non-gut) sites and the gut could alter the outcome of disease at the systemic sites. In this case, blocking β7 augmented the autoimmune arthritis development, but only when SFB was present in the host. A combination of β7 blockade and SFB colonization was particularly detrimental for disease development because the gut microbiota SFB increase the homeostatic level of α4β7+Tfh and α4β7+Th17 cells (13, 14), which were further elevated at the systemic site by the β7 blockade, as they were unable to re-enter the gut, ultimately leading to augmented autoimmune arthritis. As dysbiosis (imbalance of gut microbiota) is highly prevalent in western societies, urgent attention is required for patients undergoing anti-β7 or other treatment that manipulates immune cell gut-homing (4951).

Many anti-adhesion drugs targeting α4β1, α4β7, and αEβ7 integrins, or MadCAM-1, the ligands for α4β7, are already on the market or have been undergoing development with a focus on treating intestinal diseases such as inflammatory bowel disease (IBD) (52). In addition to affecting the gastrointestinal tract, IBD has several systemic (non-gut/extraintestinal) manifestations including arthritis, which occurs in ∼30% of IBD patients (53, 54). Our results suggest strong precautions should be taken when using anti-adhesive drugs in IBD patients with systemic diseases such as autoimmune arthritis. Thus far, because these drugs aim to provide therapies to treat IBD by blocking gut-homing of T lymphocytes, the studies associated with them have largely focused on the intestinal but not the systemic disease. However, we did find a study involving treating an animal EAE model with Abs against the α4β1 integrin, which governs the migration of T cells into the CNS, that also applied anti-α4β7 Abs as a control (55). In that study, only α4β1 blockage prevented inflammation in CNS. What is interesting to us is that not only did the α4β7 blockade provide no benefit, the survival rate in the anti-α4β7 treated group actually fell to just 21 d compared with the placebo group, which was 25 d. Although this was not significant in the number of animals used in the study (number of placebo versus anti-α4β7 was 4:7), it raises a concern that anti-adhesive drugs could augment another systemic autoimmune disease, in this case EAE.

Taken together, we have supplied two models that display a mirror image with regard to how preventing (e.g., by β7 blockade) or promoting (e.g., by AM80) gut homing of T effector cells augments or ameliorates a systemic autoimmune disease. Our results deliver two critical messages. One is that agents that modify gut homing, such as AM80, could act as another layer of treatment for systemic inflammatory or autoimmune diseases. In contrast, our β7 blockade data show that retaining the pathologic effector cells in the systemic site, and a subsequent enhancement of the autoimmune arthritis, warrants strict safety monitoring for these anti-adhesive drugs in IBD patients. The results of this study thus elucidate that AM80 ameliorates systemic autoimmune pathology by targeting Tfh and Th17 cells, while identifying potential side effects of gut-homing perturbing agents used in treating intestinal inflammation.

This work was supported by grants from the National Institutes of Health (R56AI107117 and R01AI107117) and by the Southwest Clinic and Research Institute Fund to H.-J.J.W.

The microarray data presented in this article have been submitted to the Gene Expression Omnibus repository (www.ncbi.nlm.nih.gov/projects/geo/) under accession number GSE92860.

Abbreviations used in this article:

ASC

autoantibody secreting cell

CLN

cervical LN

CMS

carboxymethyl cellulose sodium

DC

dendritic cell

EAE

autoimmune encephalomyelitis

iBALT

inducible BALT

IBD

inflammatory bowel disease

LDLN

lung draining LN

LN

lymph node

MLN

mesenteric lymph node

PP

Peyer’s patch

RA

rheumatoid arthritis

SFB

segmented filamentous bacteria

SI-LP

small intestine-lamina propria

Tfh

T follicular helper

Treg

regulatory T cell.

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The authors have no financial conflicts of interest.