Infliximab therapy increases the frequency of circulating CD16(+) monocytes and modifies macrophage cytokine response to bacterial infection

Abstract

Crohn's disease (CD) has been correlated with altered macrophage response to microorganisms. Considering the efficacy of infliximab treatment on CD remission, we investigated infliximab effects on circulating monocyte subsets and on macrophage cytokine response to bacteria. Human peripheral blood monocyte-derived macrophages were obtained from CD patients, treated or not with infliximab. Macrophages were infected with Escherichia coli, Enterococcus faecalis, Mycobacterium avium subsp. paratuberculosis (MAP) or M. avium subsp avium, and cytokine levels [tumour necrosis factor (TNF) and interleukin (IL)-10] were evaluated at different time-points. To evaluate infliximab-dependent effects on monocyte subsets, we studied CD14 and CD16 expression by peripheral blood monocytes before and after different infliximab administrations. We also investigated TNF secretion by macrophages obtained from CD16(+) and CD16(-) monocytes and the frequency of TNF(+) cells among CD16(+) and CD16(-) monocyte-derived macrophages from CD patients. Infliximab treatment resulted in elevated TNF and IL-10 macrophage response to bacteria. An infliximab-dependent increase in the frequency of circulating CD16(+) monocytes (particularly the CD14(++) CD16(+) subset) was also observed (before infliximab: 4·65 ± 0·58%; after three administrations: 10·68 ± 2·23%). In response to MAP infection, macrophages obtained from CD16(+) monocytes were higher TNF producers and CD16(+) macrophages from infliximab-treated CD patients showed increased frequency of TNF(+) cells. In conclusion, infliximab treatment increased the TNF production of CD macrophages in response to bacteria, which seemed to depend upon enrichment of CD16(+) circulating monocytes, particularly of the CD14(++) CD16(+) subset. Infliximab treatment of CD patients also resulted in increased macrophage IL-10 production in response to bacteria, suggesting an infliximab-induced shift to M2 macrophages.

Keywords: cytokine secretion; inflammatory bowel diseases; infliximab therapy; macrophages; monocyte subsets.

Figure 1

Bacterial-stimulation index (bacteria-induced cytokine secretion) by macrophages from Crohn's disease (CD) and CD- infliximab (IFX) patients after infection with Escherichia coli (EC), E. faecalis (EC), Mycobacterium avium subsp. paratuberculosis (MAP) or M. avium subsp. avium (MA) at 3 h (T3h), 3 days (T3d) and 7 days (T7d) after infection. Bacterial-stimulation index was calculated as the difference between cytokine levels measured in infected versus uninfected macrophage cultures obtained from the same subject. Significant differences between CD-IFX and CD macrophages were calculated using Student's t-test (*P < 0·005).

Figure 2

Dot-plot of peripheral blood monocytes from a representative CD patient, according to CD14 and CD16 staining. Monocytes were divided into three subsets.

Figure 3

(a) Percentage of CD16⁻ (CD14⁺⁺CD16⁻) (left panel) and CD16⁺ (CD14⁺CD16⁺⁺ and CD14⁺⁺CD16⁺) (right panel) monocytes from healthy controls and CD patients before and after treatment with different doses of infliximab (IFX). (b) Distribution of the CD16⁺ monocytes by the two subpopulations. Asterisks not associated with a line indicate a significant difference with control (*P < 0·050; **P < 0·010; ***P < 0·001; ****P < 0·0001).

Figure 4

Tumour necrosis factor (TNF) production by macrophages obtained from CD14⁺CD16⁻ and CD14⁺CD16⁺ peripheral blood monocytes from healthy donors, in response to challenge with Mycobacterium avium subsp. paratuberculosis (MAP) for 3 h, at a multiplicity of infection (MOI0 of 10:1. Monocyte differentiation into macrophages was accomplished by culturing the isolated monocyte populations for 7 days. *P < 0·050; ***P < 0·001.

Figure 5

Mycobacterium avium subsp. paratuberculosis (MAP)-induced increment in the frequency of tumour necrosis factor (TNF)⁺ cells, as calculated by the ratio of the percentage of TNF⁺ cells in MAP-infected versus uninfected CD16⁻ (left) or CD16⁺ (right) macrophages. *P < 0·050.