Mucosal biopsy shows immunologic changes of the colon in patients with early MS

Abstract

Objective: To investigate immune cells of the colonic mucosa and fecal short-chain fatty acids (SCFAs) in treatment-naive patients with a clinically isolated syndrome (CIS) or early relapsing MS.

Methods: In this cross-sectional proof-of-concept study, we obtained mucosal specimens during ileocolonoscopy from 15 untreated patients with CIS/MS and 10 controls. Mucosal immune cells were analyzed by FACS, and gas chromatography-mass spectrometry measurements of stool samples served to determine SCFA.

Results: The number of total dendritic cells (DCs), CD103+ tolerogenic DCs, and CD4+25+127-regulatory T cells (Tregs) was significantly reduced in the distal colon of patients with CIS/MS compared with controls, whereas we found no differences in the proximal colon. The patients' fecal samples also showed a substantially lower content of SCFA and especially lower levels of butyrate and acetate.

Conclusions: Our findings indicate a disturbed homeostasis of colonic DCs and Tregs in patients with MS which could be associated with colonic SCFA depletion. Although not implying causality, these findings confirm parallel abnormalities of the gut in MS and warrant further research if modulation of the colonic SCFA profile or the colonic Treg pool can serve to modify the course of MS.

Figure 1. FACS analysis of colonic mucosal DCs and Tregs

Colonic specimens were obtained separately from the proximal and distal colon, and lamina propria mononuclear cells were isolated. (A) DCs were identified as live single cells that stain negatively for lin (lineage) 1 markers (CD3, CD14, CD16, CD19, CD20, CD56, and CD34) and positively for HLA-DR. CD103-positive DCs were gated out of the DC population. (B) Tregs were identified as live single cells that stain positively for CD3, CD4, and CD25 markers and negatively for CD127. DC = dendritic cell; FACS = fluorescence-activated cell sorting; Treg = regulatory T cells.

Figure 2. Reduction of mucosal DCs in the distal colon of MS-p

DC numbers from the proximal (A and B) and distal (C and D) colon, see central scheme. (A and B) No significant differences in the number of total and CD103+ DCs were found in the proximal colon of MS-p compared with controls (Ctrl). (C and D) In the distal colon, significant reductions of total and CD103+ DCs were observed in MS-p compared with Ctrl (total DCs: 3.0 [2.2–3.8] vs 5.0 [3.6–6.3], p = 0.019 [‰ of living cells] and 255 [93–381] vs 404 [335–592], p = 0.026 [absolute numbers]; CD103+ DCs: 1.4 [0.9–1.8] vs 2.5 [1.4–3.1], p = 0.036 [‰ of living cells] and 114 [38–174] vs 183 [124–303], p = 0.048 [absolute numbers]) (median [Q1–Q3]; Mann-Whitney U test or t test to compare non-Gaussian and Gaussian variables. Boxplots according to Tukey). DC = dendritic cell; MS-p = patients with MS.

Figure 3. Reduction of mucosal Tregs in the distal colon of MS-p

Treg numbers from the proximal (A and B) and distal (C and D) colon, see central scheme. (A and B) No significant differences in the number of Tregs were found in the proximal colon of MS-p compared with controls (Ctrl). (C and D) In the distal colon, significant reductions of Tregs were observed in MS-p compared with Ctrl (Tregs: 3.1 [1.3–3.7] vs 4.7 [3.4–6.1], p = 0.032 [‰ of living cells] and 153 [83–205] vs 226 [136–510], p = 0.023 [absolute numbers]) (median [Q1–Q3]; Mann-Whitney U test or t test to compare non-Gaussian and Gaussian variables. Boxplots according to Tukey). MS-p = patients with MS; Treg = regulatory T cells.

Figure 4. Decreased SCFA levels in fecal samples of MS-p

(A–C) Fecal samples were analyzed by HPLC. SCFA contents are shown in order of their absolute quantity in μmol/g (total > acetate > propionate > butyrate > valerate > isovalerate > isobutyrate). A significant reduction in MS-p (gray) vs controls (white) was found in total SCFA (252 [148–354] vs 121 [82–162]; p = 0.017), acetate (180 [85–227] vs 70 [45–96]; p = 0.025), and butyrate contents (33 [18–48] vs 14 [8–21]; p = 0.018). Trends were observed in propionate (p = 0.084) and valerate contents (p = 0.075) (median [Q1–Q3]; Mann-Whitney U test or t test to compare non-Gaussian and Gaussian variables. Boxplots according to Tukey). HPLC = high-performance liquid chromatography; MS-p = patients with MS; SCFA = short-chain fatty acid.