Recruitment of activated neutrophils correlates with disease severity in adult Crohn's disease

Abstract

Neutrophils are detected in inflamed colon in Crohn's disease (CD). However, whether the frequency and/or activation of circulating or gut tissue neutrophils correlate with endoscopic severity remains to be investigated. A cohort of 73 CD patients was prospectively enrolled according to endoscopic severity and treatment history. Individuals with active disease were stratified using the Montreal classification. Harvey-Bradshaw Index (HBI) and Simple Endoscopic Score for Crohn's Disease (SES-CD) were performed at the time of ileocolonoscopy. Frequency of neutrophils and their expression of CD66b and CD64 were assessed in paired blood and colonic biopsies using flow cytometry. The percentage of neutrophils increased in inflamed colon and correlated with SES-CD in the entire cohort of patients examined, as well as in the subgroup with inflammatory (B1) active disease. SES-CD further correlated with neutrophil CD66b expression in mucosa but not blood and, conversely, with neutrophil CD64 expression in blood but not mucosa. However, the evaluation of neutrophil activation in mucosa when compared to blood reflected disease activity more clearly. Finally, a neutrophil activation power index (CD66b in mucosa X CD64 in blood) that correlated with SES-CD discriminated between patients with mild and severe disease. In conclusion, the frequency and activation of colonic neutrophils correlated with SES-CD, highlighting that mucosal neutrophils are associated with disease severity in CD.

Keywords: CD64; CD66b; Crohn’s disease; SES-CD; neutrophil.

Figure 1

Gating strategy for identification of neutrophils and eosinophils in the mucosa and the blood of Crohn’s disease (CD) patients and cellular frequencies. (a) Representative gating strategy, cell size and morphology by Giemsa–Wright staining for identification of eosinophils and neutrophils in freshly isolated hematopoietic cells from inflamed colon mucosa and peripheral blood of CD patients. Scale bar: 20 μm. (b) Frequencies of colonic and peripheral blood eosinophils and neutrophils among patients with active (n = 57) versus inactive disease (n = 16) (Mann–Whitney U‐test). (c) Frequencies of colonic neutrophils among subgroups according to disease behaviour (Kruskal–Wallis test followed by Dunn’s test. (d) Histological score of active CD patients (n = 18) with high and low mucosal neutrophil frequencies according to the median of the distribution of neutrophils (median = 3.15%) assessed by flow cytometry left panel (Mann–Whitney U‐test), correlation between the frequencies of colonic neutrophils and histological score (middle panel) (Spearman’s rank correlation coefficient) and comparison between histological subscores relative to polymorphonuclear infiltration in active (n =18) or inactive disease (n = 5) (right panel) (Mann–Whitney U‐test); B1 *P < 0·05; **P < 0·01; ****P < 0·0001.

Figure 2

Frequency of colonic neutrophils and the expression of CD66b correlate with Simple Endoscopic Score for Crohn’s Disease (SES‐CD). (a) Correlations between mucosal neutrophil frequencies and SES‐CD in the entire cohort (n = 73), exclusive colonic disease ‘L2’ (n = 42), B1 disease (n = 52) (Pearson’s correlation coefficient). (b) CD66b mean fluorescence intensity (MFI) on neutrophils in colon of patients with active or inactive disease (Mann–Whitney U‐test). (c) Histological score of active CD patients (n = 18) with high and low CD66b MFI according to median MFI (= 5179.5) of CD66b expression on colonic neutrophils (Mann–Whitney U‐test) (left panel) and correlation between the MFI of CD66b on colonic neutrophils and histological score (n = 18, Spearman’s rank correlation coefficient). (d) CD66b MFI on neutrophils in colon and blood of individuals with B1 or non‐B1 disease, complete mucosal healing (n = 12) and healthy controls (healthy controls: colon samples n = 10, blood samples n = 16) (Kruskal–Wallis test followed by Dunn’s test (*), Wilcoxon’s matched‐pairs signed‐rank test for colonic versus circulating neutrophils (+) (healthy controls n = 10 paired samples). (e) Correlations between CD66b MFI on colonic neutrophils and SES‐CD among the entire cohort (n = 73), exclusive colonic disease (L2, n = 42), B1 disease (n = 52) (Pearson’s correlation coefficient). *+P < 0·05; **++P <  0·01; ***+++P < 0·001; ****++++P < 0·0001.

Figure 3

CD64 expression on circulating neutrophils correlates with Simple Endoscopic Score for Crohn’s Disease (SES‐CD). CD64 mean fluorescence intensity (MFI) on neutrophils (a) in blood of patients with active and inactive disease (Mann–Whitney U‐test), (b) in colon and blood of individuals with B1 versus non‐B1 disease, complete mucosal healing (n = 12) and healthy controls (healthy controls: colon samples: n = 10, blood samples n = 16) (Kruskal–Wallis followed by Dunn’s test between B1, B2 or B3, and healthy control groups (*), Wilcoxon matched‐pairs signed‐rank test for colonic versus circulating neutrophils (+) (healthy controls n = 10 paired samples). (c) Dot‐plot representing CD64 and CD66b co‐expression after gating on purified circulating and colonic neutrophils. (d) Correlations between CD64 MFI on circulating neutrophils and SES‐CD among the entire cohort (n = 73), exclusive colonic disease ‘L2’ (n = 42), B1 disease (n = 52) (Pearson’s correlation coefficient) *P < 0·05; **++P < 0·01; ***+++P < 0·001; ****++++P < 0·0001.

Figure 4

Expression of activation markers on mucosal and circulating neutrophils discriminates disease activity and severity in Crohn’s disease (CD) patients with B1 disease. (a) CD66b mean fluorescence intensity (MFI) on colonic neutrophils (left panel) and CD64 MFI on peripheral blood neutrophils (middle panel) according to the presence of complete mucosal healing [Simple Endoscopic Score for Crohn’s Disease (SES‐CD) ≥ 1: n = 43, SES‐CD = 0: n =12) (Mann–Whitney U‐test)]. Performance of each parameter to discriminate complete mucosal healing from individuals with SES‐CD ≥ 1 area under the curve (AUC) and their exact binomial confidence interval are presented as well as pairwise comparison of ROC curves (DeLong’s test) (right panel). (b) CD66b MFI on colonic neutrophils (left panel), CD64 MFI on circulating neutrophils (middle panel) and neutrophil activation power index (right panel) according to endoscopic disease severity in the subgroup with B1 disease (n = 40) (Kruskal–Wallis followed by Dunn’s test). *P < 0·05; **P < 0·01; ****P < 0·0001.