Recirculating Intestinal IgA-Producing Cells Regulate Neuroinflammation via IL-10

Abstract

Plasma cells (PC) are found in the CNS of multiple sclerosis (MS) patients, yet their source and role in MS remains unclear. We find that some PC in the CNS of mice with experimental autoimmune encephalomyelitis (EAE) originate in the gut and produce immunoglobulin A (IgA). Moreover, we show that IgA⁺ PC are dramatically reduced in the gut during EAE, and likewise, a reduction in IgA-bound fecal bacteria is seen in MS patients during disease relapse. Removal of plasmablast (PB) plus PC resulted in exacerbated EAE that was normalized by the introduction of gut-derived IgA⁺ PC. Furthermore, mice with an over-abundance of IgA⁺ PB and/or PC were specifically resistant to the effector stage of EAE, and expression of interleukin (IL)-10 by PB plus PC was necessary and sufficient to confer resistance. Our data show that IgA⁺ PB and/or PC mobilized from the gut play an unexpected role in suppressing neuroinflammation.

Keywords: B cells; EAE; IgA; MS; experimental autoimmune encephalomyelitis; microbiota; multiple sclerosis; plasma cells; small intestinal lamina propria.

Figure 1.. IgA⁺ PB and/or PC Are Detected in the Brain and Spinal Cord during EAE

(A) Expression of indicated markers on Prdm1-YFP⁺ cells in the Br, Sc, BM, and LN during the chronic phase of EAE. Grey filled histograms represent the FMO for each stain and empty histograms represent specific Ab staining as determined by flow cytometry. (B) Absolute numbers of Prdm1⁻YFP⁺B220^−/dim cells in the Br and Sc at different time points of EAE determined by flow cytometry. (C) Representative contour plots of intracellular IgA and IgG expression by Prdm1⁻YFP⁺B220^−/dim cells determined by flow cytometry. (D) Summary of frequency data obtained by flow cytometry. (E) Number of IgA or IgG ASC in the Sc and Br determined by two-color ELISPOT of unimmunized (UI) or EAE WT mice (chronic phase). (F) Number of IgA or IgG ASC in the Br determined by two-color ELISPOT of UI mice or IgA^−/− chimeric EAE mice. (A–E) Experiments were repeated 3 times with at least 5 mice per group. (F) Two experiments were performed with at least 6 mice per group. *p < 0.05, ^**p < 0.01, NS (not significant) by Mann-Whitney test with mean and SD depicted. Corresponding EAE data for IgA^−/− chimeric EAE mice are found in Table S1 and Figure S6A. See also Figure S1.

Figure 2.. PB and/or PC Are Diminished in the Gut during EAE, and Transfer of Gut-Derived Blimp⁺ Cells to PB and/or PC-Deficient Mice Reduces Disease Severity

(A) Representative immunofluorescence images of Aicd-YFP small intestines stained with DAPI, anti-CD8α, and anti-CD138 and visualized for YFP at the pre-onset or chronic phase of EAE. (B) Quantification of CD138⁺ cells using the frequency of CD8⁺ cells as an unchanged denominator. (C) Quantification of YFP⁺ cells using the frequency of CD8⁺ cells as an unchanged denominator. (D and E) EAE clinical scores of PB and/or PC adoptive transfer in (D) CD19^crePrdm1^fl/fl recipients or (E) Jht^−/− recipients, with adoptive transfer time-points indicated by arrows. (F) Detection of adoptively transferred Prdm1-YFP⁺ cells in the Br, BM, and LN of PB and/or PC-deficient recipient mice. Experiments in (A) were repeated 3 times with 3–8 mice per group. Experiments in (D) were repeated 5 times with 3–4 mice per group. Experiments in (E) were repeated 3 times with 3–5 mice per group. *p < 0.05, ^**p < 0.01 using two-way ANOVA test or Mann-Whitney test with mean and SD depicted. Scale bar depicted in (A) is 200 μm. See also Figure S2 and Table S1.

Figure 3.. IgA⁺ B Cells from the SILP Recirculate to Distal Tissues

(A) RV-specific IgA and IgG ASC in the SILP of uninfected (UI) mice or mice infected with RV and/or Flu. (B) As in (A) but assessing RV-specific IgA and IgG ASC in the BM. (C) As in (A) but assessing RV-specific IgA and IgG ASC in the lungs. These experiments were repeated 5 times, and the graphs depicted contain all pooled data (n = 7–15 mice per group). *p < 0.05, ^**p < 0.01, ^***p < 0.001 (Mann-Whitney test with mean and SD depicted. (D) Schematic of parabiosis experimental design. Parabiosis experiments were conducted over 60 days with RV-only (“A”), and UI (“B”) mice. A and B mice were subsequently paired together. (E) Fecal samples from each mouse were collected and tested for RV antigen via ELISA. Mice infected with RV cleared the virus by D12 and were subsequently paired with their parabiont partner on D15 post-infection (p.i.). (F) Sample ELISPOTs from each collected tissue. Except for the first vertical panel, Parabiont 1 was RV-infected and Parabiont 2 was UI. Both pairs in the first vertical panel were uninfected. (G) Total RV-specific IgA ASC/1×10⁶ cells in the SILP of parabionts. (H) Total RV IgA ASC/1×10⁶ cells in BM of parabionts. Note that due to the highly damaged state of the gut at the peak of influenza infection at D6 post-flu infection, only limited numbers of cells could be isolated from the gut thus D6 was not included in our analysis. These experiments were repeated 2 times, and the graphs depicted contain all pooled data (n = 4 pairs of mice per group). *p < 0.05 using Mann-Whitney test with mean and SD depicted. See also Figures S3 and S4.

Figure 4.. RV-Specific IgA⁺ ASC Migrate to the CNS during MOG_35-55-Induced EAE

(A) Schematic representation of RV-EAE experiments. (B) RV-specific IgA ASC in the Br of unimmunized (UI) mice or mice infected with RV and/or immunized with MOG_35–35. (C) As in (B) but assessing RV-specific IgA ASC in the Sc. (D) As in (B) but assessing RV-specific IgA ASC in the SILP. (E) As in (B) but assessing RV-specific IgA ASC in the BM. (F) Representative RV-specific IgA ASC ELISPOT from Br, Sc, SILP, and BM of RV-EAE mice at the chronic stage of the disease. (G) Representative images of commensal-reactive IgA ASC derived from the SILP of naive WT, Germ Free, and Cd19^crePrdm1^fl/fl mice. PBS-coated wells were used as a negative control for the assay. (H) Quantitative summary of the results shown in (G). (I) Quantification of commensal-reactive IgA ASC derived from sorted Prdm1-YFP⁺ B220⁻ or Prdm1-YFP⁻ B220⁺ cells from the SILP of Prdm1^YFP mice. (J) Representative images of commensal-reactive IgA ASC from results depicted in (I). (K) Quantification of commensal-reactive IgA ASC in the BM (top) and brain (bottom) from UI and EAE (chronic) WT mice. (L) Representative images of commensal-reactive IgA ASC from results depicted in (K). These experiments were repeated 3 times. The number of mice depicted in (B)–(E) are as follows: UI (n = 7), RV (n = 6), EAE (n = 6), RV EAE peak (n = 4), and RV EAE chronic (n = 5). The number of mice depicted in (H), (I), and (K) are n = 4–7 for each group. ^*p < 0.05, ^**p < 0.01, ^***p < 0.001, ^****p < 0.0001 using Mann-Whitney test with mean and SD depicted. See also Figure S4.

Figure 5.. IgA-Binding of Bacteria in the Gut Is Reduced in MS Patients during an Acute Relapse

(A) Study overview: fresh-frozen fecal samples of 33 patients with clinically Isolated syndrome (CIS) or relapslng-remlttlng multiple sclerosis (RRMS) (n = 22 during disease remission, n = 11 during an acute relapse) and 32 healthy controls were collected. Fecal bacteria were isolated and IgA-binding of autologous gut bacteria was quantified by bacterial flow cytometry (BUGFlow) and ELISA. (B) BUGFlow: bacteria were identified based on forward and side scatter and the IgA-positive population was defined based on an isotype control. Shown is an example of staining of fecal bacteria from an RRMS patient with an isotype control (left) as well as with anti-IgA during remission (middle) and during relapse (right). (C) Percentage of fecal-bound IgA by BUG Flow: 1 × 10⁶ fecal bacteria were stained with anti-IgA and % of IgA binding was assessed by flow cytometry. Values shown are replicates from 2 experiments. (D) Quantification of fecal-bound IgA by ELISA: 1 × 10⁶ fecal bacteria were assessed for IgA binding using a commercially available quantitative IgA ELISA assay. Values shown are mean values of duplicate measurements. *p < 0.05, t test with Welch correction with mean and SEM depicted. See also Table S2.

Figure 6.. PC-Derived Factors that Are Involved in EAE Suppression

(A) Representative immunofluorescence images of WT and IL-10^−/− small intestines stained with DAPI, anti-IgA, and anti-IL-10. (B) Mixed BM chimeras whereby PB and/or PC-deficient mice were reconstituted with and 80/20 mixture of PB and/or PC-deficient + IL-10^−/− BM and control chimeras reconstituted with an 80/20 mixture of WT + IL-10^−/− BM were subjected to EAE and clinical score was measured overtime. Cumulative score is shown on the right. (C) Quantitative analysis of H&E (left) and LFB (right) staining of thoracic spinal cord sections derived from mixed BM chimeras. (D and E) Representative images of H&E (D) and LFB (E) stains from thoracic spinal cord sections derived from mixed BM chimeras acquired at4× magnification using a light microscope to visualize inflammation and demyelination. (F) Representative images of commensal-reactive IgA ASC pre-sorted as Dump⁻ (CD4, CD8, F4/80 negative) and subsequently sorted as Thy1.1⁺ or Thy1.1^- cells from the brain (7,500 cells/well) or BM (150 cells/well) during the chronic phase of EAE. Note that the pre-sort step results in an enrichment in these number of spots detected. (G) Quantification of commensal-reactive IgA ASC from (F). Experiments in (A)were repeated two times with at least 5 mice per group, with IL10^−/− staining control being performed once. Experiments in (B) were performed three times. Experiments in (C)–(E) were performed three times with 4–8 mice per group. Experiments in (F) and (G) were performed three times with 5 mice per group. *p < 0.05, ^**p < 0.01, ^***p < 0.001 using two-way ANOVA test for(B) and Mann-Whitney test for(C) and (G) with mean and SD depicted, (C) mean and SEM depicted. Scale bar depicted in (A) and (D) is 200 μm. See also Figures S5 and S6 and Table S1.

Figure 7.. BAFF-Tg Mice Are Resistant to MOG_35–55-Induced EAE via IL-10

(A) Representative immunofluorescence images of the SILP of BAFF-Tg^+/+ mice before (UI) and during the chronic phase of MOG_35–55-induced EAE stained with DAPI, anti-CD138, and anti-IgA. (B) Quantification of CD138⁺ cells in the SILP using CD8⁺ cells as an unchanged denominator. (C) Quantification of IgA⁺ cells in the SILP using CD8⁺ cells as an unchanged denominator. (D and E) Number of IgA or IgG ASC enumerated by ELISPOT analysis in the (D) Br and (E) Sc of BAFF-Tg^+/+ mice during MOG35_–55-induced EAE (chronic phase) compared with UI BAFF^+/+-Tg mice. (F) Clinical scores of BAFF^+/+-Tg, BAFF^+/−-Tg, and WT littermates after immunization with MOG_35–55. (G) Clinical scores of BAFF^+/+ and WT mice after immunization with hrMOG. (H) Frequency of MOG_35–55 IFNγ⁺ CD4⁺ T cells (left) or IL-17 CD4⁺ T cells (right) from WT or BAFF^+/−-Tg littermates with chronic EAE. (I) EAE Clinical scores of adoptive transfer EAE whereby pre-primed T cells were transferred into WT or BAFF^+/−-Tg recipient mice. (J) Representative immunofluorescence images of BAFF^+/+-Tg and BAFF^+/−-Tg x IL-10^−/− small intestines stained with DAPI, anti-IgA, and anti-IL-10. (K) Clinical scores of BAFF^+/−-Tg, BAFF^+/−-Tg x IL-10^−/−, and WT littermates after immunization with MOG_35–55. (L) EAE Clinical scores of Baff^+/−-Tg IL-10^−/− recipients, after transfer of Prdm1^YFP-IL-10^−/− gut PC or Prdm1^YFP gut PC. Experiments in (A)–(F) and (K)–(L) were repeated 3 times with at least 4 mice per group. The experiment in (G) was repeated two times with at least 4–5 mice per group. ^*p < 0.05, ^**p < 0.01, ^***p < 0.001 using two-way ANOVA test for (F), (G), (I), (K), and (L) and Mann-Whitney test for (B)–(E) and (H) with mean and SD depicted. Scale bar depicted in (A) is 200 μm. See also Figure S7 and Table S1.