Anti-TNF- α Therapy Suppresses Proinflammatory Activities of Mucosal Neutrophils in Inflammatory Bowel Disease

Abstract

Neutrophils have been found to play an important role in the pathogenesis of inflammatory bowel disease (IBD), and anti-TNF-α mAb (i.e., infliximab) therapy is demonstrated to be effective in the induction of clinical remission and mucosal healing in these patients. However, how anti-TNF-α mAb regulates the functions of neutrophils is still unknown. Herein, we found that anti-TNF-α therapy significantly downregulated infiltration of neutrophils in inflamed mucosa of IBD patients. Importantly, anti-TNF-α mAb could inhibit neutrophils to produce proinflammatory mediators, such as ROS, calprotectin, IL-8, IL-6, and TNF-α. These data indicate that TNF-α plays a critical role in the induction of mucosal inflammatory response, and that blockade of TNF-α modulates intestinal homeostasis through balancing immune responses of neutrophils.

Figure 1

Neutrophils are more activated in the peripheral blood and intestinal mucosa of active IBD patients. (a, b) Peripheral blood samples were collected from healthy donors (HC, n = 24), patients with active CD (A-CD, n = 26), patients with CD in remission (R-CD, n = 22), patients with active UC (A-UC, n = 28), and patients with UC in remission (R-UC, n = 16). The whole blood excluding RBC was analyzed by flow cytometry. ^∗∗∗ P < 0.001 compared with HC. (c) Immunohistochemical staining of MPO in inflamed intestinal mucosa from healthy control (HC, n = 10), patients with A-CD (n = 10), and patients with A-UC (n = 10). The arrows indicate MPO⁺ cells. Original magnification × 200. ^∗ P < 0.05 compared with HC.

Figure 2

The activity of neutrophils is inhibited in the peripheral blood, and the infiltration of neutrophils is decreased in the intestinal mucosa of CD patients after anti-TNF-α treatment. Response group: (a) percentages of CD66b⁺ neutrophils in the peripheral blood of patients with active CD (n = 15) before and after IFX treatment; (b) expression of CD66b mRNA in the peripheral blood neutrophils from patients with active CD (n = 15) before and after IFX treatment by qRT-PCR; (c) expression of CD66b mRNA in intestinal mucosa from patients with active CD (n = 13) before and after IFX treatment by qRT-PCR; (d) immunohistochemical staining of CD66b in the intestinal mucosa of active CD (n = 10) before and after IFX treatment; and (e) expression of S100A8, (f) S100A9, and (g) MPO in the intestinal mucosa of patients with active CD (n = 13) before and after IFX treatment. Failure group: percentage (h) and expression of CD66b (i) of peripheral blood neutrophils from active CD patients (n = 7) before and after IFX. Expression of CD66b (j), S100A8 (k), S100A9 (l), and MPO (m) in intestinal mucosa was detected by qRT-PCR from CD patients in the failure group. ^∗ P < 0.05, ^∗∗ P < 0.01, and ^∗∗∗ P < 0.001.

Figure 3

IFX suppresses the expression of neutrophil-derived S100A8, S100A9, and MPO in intestinal mucosa. Fresh colon biopsies were collected from inflamed mucosa of patients with active CD (n = 12) and active UC (n = 12), and cultured ex vivo with IFX or control human IgG (HIg) (both at 50 μg/ml) for 24 h. Tissues were harvested for detection of expression of S100A8 (a), S100A9 (b), and MPO (c) by qRT-PCR and compared with healthy controls (n = 5). ^∗ P < 0.05 and ^∗∗ P < 0.01.

Figure 4

Anti-TNF-α therapy inhibits peripheral neutrophils to produce proinflammatory cytokines, chemokines, ROS, and MPO. Peripheral neutrophils (5 × 10⁶) from healthy donors (n = 10), patients with active CD (n = 10), and active UC (n = 10) were stimulated by LPS (200 ng/ml) and incubated with or without IFX (50 μg/ml) for 3 h. Cells were collected and expressions of TNF-α (a), IL-8 (b), and IL-6 (c) were detected by qRT-PCR. Peripheral neutrophils (2 × 10⁶) from healthy donors (n = 6), patients with active CD (n = 11), and patients with active UC (n = 4) were simulated with LPS (200 ng/ml) and incubated with or without IFX (50 μg/ml) for 3 h. Culture media were replenished and incubated for another 24 h. Supernatants and protein production of TNF-α (d), IL-8 (e), and IL-6 (f) were measured by ELISA. Peripheral neutrophils (1 × 10⁴) isolated from healthy donors (n = 5), patients with active CD (n = 5), and patients with active UC (n = 5) were measured for ROS (h) and MPO (i) with Amplex Red Hydrogen Peroxide Assay Kit. ^∗ P < 0.05, ^∗∗ P < 0.01, and ^∗∗∗ P < 0.001 compared with medium control and ^# P < 0.05, ^## P < 0.01, and ^### P < 0.001 compared with LPS stimulation.

Figure 5

Anti-TNF-α therapy suppresses neutrophil migration in active CD patients. Peripheral neutrophils (5 × 10⁵) were isolated from patients with active CD (n = 4) and measured with an 8 μm-Transwell plate under attraction with fMLP (10, 30, and 50 nM) for 30 min. The histogram represents the number of migrating neutrophils per high-power field (HPF). ^∗ P < 0.05 and ^∗∗ P < 0.01.

Figure 6

Anti-TNF-α therapy promotes apoptosis of neutrophils in active CD patients. (a–c) Peripheral neutrophils were isolated from healthy donors (n = 8), patients with active CD (n = 10), and patients with active UC (n = 5) and incubated with HIg (50 μg/ml), IFX (50 μg/ml), or TNF-α (20 ng/ml) for 24 h. Cells were collected and detected for apoptosis by flow cytometry. ^∗ P < 0.05.