Activation of Peripheral Blood CD4+ T-Cells in IBS is not Associated with Gastrointestinal or Psychological Symptoms

Abstract

Immune activation may underlie the pathogenesis of irritable bowel syndrome (IBS), but the evidence is conflicting. We examined whether peripheral CD4+ T-cells from IBS patients demonstrated immune activation and changes in cytokine production. To gain mechanistic insight, we examined whether immune activation correlated with psychological stress and changing symptoms over time. IBS patients (n = 29) and healthy volunteers (HV; n = 29) completed symptom and psychological questionnaires. IBS patients had a significant increase in CD4+ T-cells expressing the gut homing marker integrin β7 (p = 0.023) and lymphoid marker CD62L (p = 0.026) compared to HV. Furthermore, phytohaemagglutinin stimulated CD4+ T-cells from IBS-D patients demonstrated increased TNFα secretion when compared to HV (p = 0.044). Increased psychological scores in IBS did not correlate with TNFα production, while stress hormones inhibited cytokine secretion from CD4+ T-cells of HV in vitro. IBS symptoms, but not markers of immune activation, decreased over time. CD4+ T-cells from IBS-D patients exhibit immune activation, but this did not appear to correlate with psychological stress measurements or changing symptoms over time. This could suggest that immune activation is a surrogate of an initial trigger and/or ongoing parallel peripheral mechanisms.

Figure 1

Flow cytometry for integrin β7 and CD62L in IBS patients and HV. (A) Flow cytometry revealed a significant increase in the % of CD4+ T-cells expressing the gut homing marker, integrin β7, in patients with IBS (38.3% [34.8–43.1], n = 12) when compared to HV (30.2% [22.5–35.6], n = 12; p = 0.023 vs. IBS). A significant increase in the % of CD4+ T-cells expressing the lymph node homing marker, CD62L was also seen in IBS (B) (IBS: 85.2% [83.3–88.9], n = 12; HV: 80.1% [73.5–84.4], n = 12; p = 0.026), as well as in the % of CD4+ T-cells co-expressing CD62L and integrin β7 (C) (IBS: 35.6% [30.2–42.2], n = 12; HV: 27.3% [17.4–32.5], n = 12; p = 0.040, when compared to HV).

Figure 2

CD4+ T-cell stimulated cytokines in IBS patients and HV. No differences in CD4+ T-cell stimulated TNFα (A), IL-6 (B) or IL-10 (C) were seen when comparing HV (n = 29) to IBS patients (n = 29). CD4+ T-cell derived TNFα concentrations were significantly increased in IBS-D (315.3 pg/mL [184.3–710.3], n = 10) patients (D) when compared to HV (65.2 pg/mL [28.6–313.6], n = 29. p = 0.044 Kruskal Wallis Test, p < 0.05 HV vs. IBS-D). No significant differences were seen in CD4+ T-cell derived IL-6 (E) or IL-10 (F) concentrations when comparing IBS subgroups.

Figure 3

Psychological scores in IBS patients and HV. Anxiety and depression scores were assessed using the anxiety and depression components of the HADS score, while somatization was assessed using the PHQ-15 score. The anxiety (A) (HV: 4.0 [1.5–6.0], n = 29; IBS-D: 12.5 [9.5–16.0], n = 10; IBS-C: 7.0 [5.0–13.0], n = 11; IBS-M: 10.5 [5.5–13.8], n = 8; Kruskal Wallis Test p < 0.0001; HV vs. IBS-D p < 0.001, HV vs. IBS-M p < 0.05) and depression (B) (HV: 1.0 [0–2.5], n = 29; IBS-D: 6.5 [2.0–7.3], n = 10; IBS-C: 2.0 [1.0–11.0], n = 11; IBS-M: 5/8 [3.5–8.8], n = 8; Kruskal Wallis Test p < 0.0001; HV vs. IBS-D p < 0.01, HV vs. IBS-M p < 0.01) scores for both IBS-D and IBS-M patients were significantly different from HV; no differences between subgroups was noted. Similarly, while all IBS subtypes exhibited increased somatization scores when compared to HV (C) (HV: 3.0 [1.0–5.0], n = 29; IBS-D: 18.0 [10.8–19.3], n = 10; IBS-C: 12.0 [10.0–16.0], n = 11; IBS-M: 13.0 [9.5–14.8], n = 8; Kruskal Wallis Test p < 0.0001; HV vs. IBS-D p < 0.001, HV vs. IBS-C p < 0.001, HV vs. IBS-M p < 0.01), no differences between subgroups were noted.

Figure 4

The effect of stress hormones on CD4+ T-cell stimulated cytokine production. CD4+ T-cells were incubated with media alone, PHA 5 μM and/or the stress hormones epinephrine 1 nM + corticosterone 1 μM. Epinephrine and corticosterone significantly inhibited CD4+ T-cell stimulated production of TNFα (A) [Unstimulated: 0 pg/mL (0–0); Epinephrine/corticosterone alone: 0 pg/mL (0–0); PHA: 356.6 pg/mL (65.6–780.6); PHA + Epinephrine/corticosterone: 34.6 pg/mL (8.8–51.3)], IL-6 (B) [(Unstimulated: 0.4 pg/mL (0–3.5); Epinephrine/corticosterone alone: 0.7 pg/mL (0.2–7.7); PHA: 533.8 pg/mL (250.9–830.1); PHA + Epinephrine/corticosterone: 250.3 pg/mL (98.5–571.6)] and IL-10 (C) [(Unstimulated: 1.1 pg/mL (0.7–5.3); Epinephrine/corticosterone alone: 1.0 pg/mL (0.02–3.8); PHA: 481.0 pg/mL (192.9–703.3); PHA + Epinephrine/corticosterone: 158.3 pg/mL (86.7–419.1)]. Individual responses from each patient are shown graphically. *p = 0.031 vs. PHA response; n = 6 donors in all cases.