Peripheral and intestinal T lymphocyte subsets in dogs with chronic inflammatory enteropathy

Abstract

Background: Dysregulated T lymphocyte response is thought to play a key role in chronic intestinal inflammation (CIE).

Objectives: To evaluate the presence of changes in peripheral and intestinal T lymphocyte subsets and to describe potential immune and inflammatory biomarkers in dogs with CIE.

Animals: Sixteen healthy dogs and 26 dogs were diagnosed with CIE.

Methods: Prospective case-control study evaluating peripheral and intestinal T lymphocytes using flow cytometry and inflammatory markers obtained from complete blood cell counts.

Results: Dogs with CIE had higher peripheral activated T helper (Th) lymphocytes (87/μL [18-273] CIE, 44/μL [16-162] healthy control (HC, P = .013) and regulatory T cells (Treg; 108/μL [2-257] CIE, 34/μL [1-114] HC, P = .004). In the intestinal epithelium, CIE dogs presented lower percentages of Th (4.55% [1.75-18.67] CIE, 8.77% [3.79-25.03] HC, P = .002), activated Th cells (0.16% [0.02-0.83] CIE, 0.33% [0.05-0.57] HC, P = .03) and CD4/CD8 ratio (0.08 [0.02-0.39] CIE, 0.21 [0.07-0.85] HC, P = .003). Conversely, higher percentage of activated T cytotoxic cells (20.24% [3.12-77.12] CIE, 12.32% [1.21-39.22] HC, P = .04) and interferon-gamma (IFN-γ) producing T lymphocytes (7.36% [0.63-55.83] CIE, 1.44% [0.00-10.56] HC, P = .01) within the epithelium was observed. In the lamina propria the percentage of Treg lymphocytes was higher (6.02% [1.00-21.48] CIE, 3.52% [0.18-10.52] HC, P = .02).

Conclusions and clinical importance: Systemic and intestinal immune alterations occur in dogs with CIE suggesting that blood IFN-γ producing T lymphocytes and the systemic immune-inflamation index (SII) could potentially serve as biomarkers for the disease.

Keywords: IBD; T lymphocytes; flow cytometry; intraepithelial lymphocytes; lamina propria lymphocytes; peripheral blood lymphocytes.

FIGURE 1

Example of flow cytometry characterization. Phenotypes of PBL, IEL, and LPL Th (CD3+ CD4+), Tc (CD3+ CD8+), double positive (CD3+ CD4+ CD8+) and double negative (CD3+ CD4− CD8−) T cells. Data are presented as histograms showing the expression of 1 parameter and as dot plots showing percentages of cells with various properties. (A‐C) Representative dot plot showing the forward/side scatter (FCS/SSC) properties of the analyzed PBL (A), IEL (B), and LPL (C) gating the lymphocyte population. (D‐F) Representative histograms of CD3 expression in PBL (D), IEL (E), and LPL (F), with negative cells on the left (gray) and positive cells on the right side of the graphs (green, blue, and orange, respectively). All cells on the right of where the negative cells end are considered positive. Numbers in the right upper corners of the graphs indicates the percentages of the respective positive populations. (G‐I) Representative dot plots of CD3+ gated cells labeled with mAbs against CD4 and CD8 in PBL (G), IEL (H), and LPL (I). CD4 positive cells are displayed in the upper left corner while CD8 cells are displayed in the lower right corner of the graphs. CD4+ CD8+ cells are displayed in the upper right and CD4− CD8− cells in the lower left corner of the graphs. The adjacent numbers indicate the percentage of the respective positive populations.

FIGURE 2

Significantly different T lymphocyte subpopulations between the HC group and CIE group in the 3 immune compartments evaluated. (A) Percentages of different T PBL subpopulations. (B) Absolute value of different T PBL subpopulations. (C,D) Percentages of different T IEL subpopulations. (E) Ratio CD4/CD8 from duodenal epithelium. (F) Percentages of different T LPL subpopulations. The lines correspond to the median, the whiskers to the range and the bars to the confidence interval. Significant P‐values: ns, no significant; <.05*; <.01**.