Use of bioluminescence imaging to track neutrophil migration and its inhibition in experimental colitis

Abstract

Inflammatory bowel disease (IBD) is associated with neutrophil infiltration into the mucosa and crypt abscesses. The chemokine interleukin (IL)-8 [murine homologues (KC) and macrophage inflammatory protein (MIP)-2] and its receptor CXCR2 are required for neutrophil recruitment; thus, blocking this engagement is a potential therapeutic strategy. In the present study, we developed a preclinical model of neutrophil migration suitable for investigating the biology of and testing new drugs that target neutrophil trafficking. Peritoneal exudate neutrophils from transgenic β-actin-luciferase mice were isolated 12h after intraperitoneal injection with thioglycollate, and were assessed phenotypically and functionally. Exudate cells were injected intravenously into recipients with dextran sodium sulphate (DSS)-induced colitis followed by bioluminescence imaging of whole-body and ex vivo organs at 2, 4 and 16-22h post-transfer. Anti-KC antibody or an isotype control were administered at 20 µg/mouse 1h before transfer, followed by whole-body and organ imaging 4h post-transfer. The peritoneal exudate consisted of 80% neutrophils, 39% of which were CXCR2(+) . In vitro migration towards KC was inhibited by anti-KC. Ex vivo bioluminescent imaging showed that neutrophil trafficking into the colon of DSS recipients was inhibited by anti-KC 4h post-cell transfer. In conclusion, this study describes a new approach for investigating neutrophil trafficking that can be used in preclinical studies to evaluate potential inhibitors of neutrophil recruitment.

Fig. 1

Phenotypic and functional characterisation of the peritoneal exudate population. Cells were isolated from the peritoneum of FVB/N mice 12 h after intraperitoneal (i.p.) injection with thioglycollate. For the phenotypic analysis, single cell suspensions were stained with fluorochrome-labelled monoclonal antibodies (mAbs) and analysed by flow cytometry in which 20 000 events were recorded. (a) Percentage of neutrophils (i.e. Ly-6G⁺ cells, grey area) present in the peritoneal exudate compared to isotype control antibody (black area). (b) Activation status of the neutrophils using anti-CD69 mAb (grey area) or isotype control antibody (black area). (c) Percentage of the Ly-6G⁺ neutrophils that were CXCR2⁺. Plots shown are representative of five independent experiments. (d) In vitro chemotaxis assay in which migrated cells from triplicate wells of the lower chambers were pooled and counted in trypan blue. For each experiment, the % migration after subtraction of the control (medium alone) was given for keratinocyte-derived chemokine (KC) (alone, no anti-KC) and for two concentrations of anti-KC antibody (0·1 µg/ml and 10 µg/ml), 4 h post-incubation at 37°C. Plots shown represent the mean of three independent experiments using pooled cell populations from three to six mice each time; *P ≤ 0·05, **P < 0·01.

Fig. 4

Effect of anti-mouse KC on neutrophil trafficking. (a). Representative whole body bioluminescence images of wt FVB/N recipients with dextran sodium sulphate (DSS)-induced colitis treated with isotype control rat immunoglobulin (Ig)G2a or anti- keratinocyte-derived chemokine (KC) antibody, 4 h post-adoptive transfer with luc⁺ cells. Non-DSS/naive recipients were also included as a control group. (b) Ex vivo images of colon of (i) non-DSS non-recipient negative control, (ii) non-DSS/naive recipient mouse and DSS recipient mice treated with (iii) IgG control or (iv) anti-KC (20 µg/mouse). Non-DSS/naive recipient, non-DSS-treated and DSS-treated groups; n = 3–5.

Fig. 3

Kinetic analysis of neutrophil trafficking in vivo. Bioluminescent signal in organs dissected from dextran sodium sulphate (DSS) recipient mice at 2, 4 and 16–22 h post-adoptive transfer with luc⁺ exudate cells. Neutrophil trafficking kinetics to (a) colon, (b) mesenteric lymph nodes (MLNs), (c) spleen, (d) liver and (e) lungs are shown. DSS-treated groups; n = 5–8. Plots shown are representative of two independent experiments and the mean values are indicated.

Fig. 2

Inflammation markers in dextran sodium sulphate (DSS)-induced colitis in FVB/N mice. (a) Mean % body weight changes in wild-type FVB/N mice from day 0 of treatment with 4% (w/v) DSS in drinking water for 5 days followed by 1 day of water. (b) Stool consistency and fur texture/posture were used to generate a daily disease activity index (DDAI). DSS-treated groups n = 4 and non-DSS control n = 1. (c) Treatment with DSS significantly increased KC levels and levels of the proinflammatory cytokines interleukin (IL)-1β and IL-6 in the distal colons of the mice; n = 5–6 per group; **P < 0·01, ***P < 0·001. Plots shown are representative of five independent experiments.

Fig. 5

Anti-keratinocyte-derived chemokine (KC) administration markedly reduced neutrophil trafficking to the colons of mice with dextran sodium sulphate (DSS)-induced colitis. Summary of bioluminescent signal in (a) colon and mesenteric lymph nodes (MLNs) and (b) liver, spleen and lungs dissected from naive controls and DSS mice treated with immunoglobulin (Ig)G control or anti-KC antibody, 4 h post-adoptive transfer with luc⁺ cells. Mean signal from the colons of naive recipients was below the background light emission from the non-recipient control colons. P ≤ 0·05; n = 3–5 per group. Plots shown are representative of two independent experiments.