Immunomodulation of Murine Chronic DSS-Induced Colitis by Tuftsin-Phosphorylcholine

Abstract

Helminths or their products can immunomodulate the host immune system, and this phenomenon may be applied as the basis of new anti-inflammatory treatments. Previously, we have shown the efficacy of tuftsin-phosphorylcholine (TPC), based on a helminth product, in four animal models of autoimmune diseases: arthritis, colitis, systemic lupus erythematosus, and experimental autoimmune encephalomyelitis. We demonstrated that TPC reduced inflammatory process ex vivo in peripheral blood lymphocytes (PBLs) and in biopsies from giant-cell arteritis. In the present study, we assessed the therapeutic potential of TPC treatment on a chronic colitis murine model. C57BL/6 mice with chronic colitis were treated with TPC after the third cycle of 2% dextran sodium sulfate (DSS). Oral TPC treatment resulted in amelioration of the colitis clinical manifestations exemplified by reduced disease activity index (DAI) score, expansion of mesenteric lymph nodes (MLN) T regulatory cells (shown by Fluorescence Activated Cell Sorting (FACS)), significant reduction in the expression of pro-inflammatory cytokines (IL-1β, IL17, IL-6, TNFα), and elevation in the expression of anti-inflammatory cytokine IL-10 (shown by RT-PCR). This study demonstrated the potential immunomodulatory effects of oral administration of TPC in a chronic colitis murine model. Further clinical trials are needed in order to evaluate this novel approach for the treatment of patients with inflammatory bowel disease.

Keywords: colitis; inflammatory bowel disease; phosphorylcholine; tuftsin.

Figure 1

Chronic experimental colitis model and Tuftsin–Phosphorylcholine (TPC) administration.

Figure 2

The effect of TPC on disease activity in a chronic colitis model. Day 0 in this graph presents day 30 of chronic colitis. DAI score: The levels of DAI score are presented as an average number, in each group of mice ±standard error (n = 20 per group), treated with tuftsin–phosphorylcholine (TPC) or phosphate-buffered saline (PBS). DSS mice that were treated with TPC had significantly lower average DAI score in comparison to mice treated with PBS (p = 0.001).

Figure 3

The effect of tuftsin–phosphorylcholine (TPC) on colon length. Colon length measurements represented in centimeters, on Day 45 of experiment, after three cycles of 2% DSS and 15 days of treatment with TPC or phosphate-buffered saline (PBS). (A) TPC-treated mice demonstrated significantly less shortening of the colon (p < 0.002). (B) Comparison of the colon length between TPC treated mice and PBS subjected mice.

Figure 4

Histological analysis of distal colon sections in chronic colitis mice. Histological analysis: H&E staining in representative distal colon section from each studied group of chronic colitis mice magnification of ×200 and ×400. (A) PBS control mice: massive infiltration of lymphocytes, destruction of the normal structure, and cryptitis. Histological score for DSS = 12 points (over 10% loss of epithelium = 3 points, over 20% loss of crypts = 3 points, severe depletion of goblet cells = 3 points, severe infiltration of inflammatory cells = 3 points). (B) TPC-treated mice: mild pathology of cryptitis. Histological score for DSS = 6 points (over 10% loss of epithelium = 3 points, 10–20% loss of crypts = 2 points, moderate depletion of goblet cells = 2 points, mild infiltration of inflammatory cells = 1 points). (C) TPC-treated mice: No significant infiltration of lymphocytes with no cryptitis. Histological score for DSS = 0 points (no loss of epithelium = 0 points, no loss of crypts = 0 points, no depletion of goblet cells = 0 points, no infiltration of inflammatory cells = 0 points). (D) Normal healthy column.

Figure 5

T regulatory cells expansion in isolated mesenteric lymph nodes of TPC-treated chronic colitis mice. (A). The data is presented as a percentage of Tregs CD4⁺CD25⁺FOXP3⁺ expansion in isolated mesenteric lymph nodes of TPC and PBS-treated mice (n = 20). Values are the mean ± SD, p < 0.0001. (B) Representative flow cytometry analyses of Tregs CD4⁺CD25⁺FOXP3⁺ (gated on CD4⁺) in mesenteric lymph nodes derived from the TPC and PBS-treated mice. TPC—5.63% PBS—2.58%. (C) Percentage of T regulatory upon treatment with TPC in comparison to treatment with PBS.

Figure 6

Cytokines gene expression of IL-1β, IL-17, IL-6, TNFα, IL-10 by mesenteric lymph nodes (MLN) from oral administration of tuftsin–phosphorylcholine (TPC) in comparison to phosphate-buffered saline (PBS) subjected mice. The data was normalized with β-Actin expression. One Asterix p < 0.001, two Asterix p < 0.02-0.03, three Asterix p < 0.001.