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P111 The anti-inflammatory effect of a serine protease inhibitor in a chronic colitis transfer model is mediated by the suppression of Th1 cell differentiation

Van Spaendonk H.*1, Francque S.1,2, De Man J.1, De Winter B.1

1University of Antwerp, Laboratory of Experimental Medicine and Pediatrics, Antwerp, Belgium 2Antwerp University Hospital, Gastroenterology and Hepatology, Antwerp, Belgium

Background

The GI tract is exposed to high levels of proteases. Increasing evidence suggests that a protease/antiprotease dysbalance might play a role in GI diseases such as IBD. In this study, we aimed to investigate the effect of a serine protease inhibitor, nafamostat mesilate (NFM), on chronic colitis in a murine transfer model.

Methods

Colitis was induced in immunodeficient SCID mice by the adoptive transfer of naive T-cells. Animals were treated twice a day with vehicle or NFM (5 mg/kg, i.p.) starting from week 2. Four groups were included: control mice treated with vehicle or NFM and colitis mice treated with vehicle or NFM. Every 2 weeks, colonic inflammation was assessed by clinical outcomes and colonoscopy. After sacrifice at week 4, colonic inflammation was assessed by macroscopy and cytometric bead array (CBA) for IFN-gamma and IL-6. mRNA of T helper (Th) transcription factors such as T-bet (Th1) and protease-activated receptors (PAR) was quantified with RT-qPCR.

Results

Colitis mice significantly lost weight over time whereas CONTROL mice gained weight. Also the clinical disease, colonoscopic and macroscopic score significantly increased at week 4 in the COLITIS group versus the CONTROL group. NFM was able to significantly reduce these signs, resulting in an ameliorated body weight and an improved clinical, endoscopic and macroscopic score. Quantification of colonic cytokines by CBA confirmed these findings: IFN-gamma and IL-6 were significantly upregulated in the COLITIS group versus CONTROL and NFM treatment significantly decreased these levels. RT-qPCR experiments showed an upregulation of the mRNA expression of T-bet and PAR-4 in the COLITIS group (vs CONTROL). Treatment with NFM significantly lowered the mRNA expression. Other transcription factors and PAR receptors showed no statistical differences after NFM treatment.

Table 1. Effect of vehicle or NFM on clinical outcomes

Body weight (%)Clin (0–8)Colo (0–12)Macro (0–12)IFN-gamma (pg/ml)IL-6 (pg/ml)
Week 4Week 4Week 4Week 4Week 4Week 4
CONTROL103.2±1.00.6±0.30.0±0.00.0±0.00.7±0.31.9±0.9
CONTROL+NFM103.9±0.90.7±0.30.1±0.10.0±0.02.3±0.52.3±0.2
COLITIS88.1±1.9#7.1±0.3#8.3±0.5#8.8±0.4#202.1±43.2#133.6±48.5#
COLITIS+NFM95.0±2.8 #,*5.1±0.6#,*6.7±0.6#,*7.0±0.8#,*104.0±20.4#,*35.3±6.5#,*

Data are presented as mean ± SEM and analyzed by two-way ANOVA or one-way ANOVA as appropriate with LSD posthoc testing. #<0.05 versus CONTROL and CONTROL+NFM, *<0.05 versus COLITIS, n=8 in every group.

Table 2. Relative mRNA of Th transcription factors and PARs in colon measured by RTqPCR (vs CONTROL)

T-bet (Th1)GATA-3 (Th2)ROR-gammat (Th17)PAR-2PAR-4
Week 4Week 4Week 4Week 4Week 4
CONTROL1.0±0.11.0±0.11.1±0.21.1±0.11.0±0.1
CONTROL+NFM1.3±0.11.2±0.21.0±0.21.3±0.11.8±0.6
COLITIS3.5±0.4#2.5±0.3#0.2±0.1#1.1±0.12.3±1.1
COLITIS+NFM2.3±0.3 #,*2.5±0.7#0.4±0.1#1.1±0.10.5±0.1£,*

Data are analyzed by two-way ANOVA with LSD posthoc testing. #<0.05 vs CONTROL and CONTROL+NFM, *<0.05 vs COLITIS, £<0.05 vs CONTROL+NFM, n=8 in every group.

Conclusion

Our results show that treatment with NFM ameliorates the course of experimental colitis. The beneficial effect of NFM on the Th1 transcription factor T-bet and major effector cytokine IFN-gamma make us hypothesize that the Th1 T-cell subset plays a pivotal role in the observed anti-inflammatory effect of NFM. We additionally hypothesize that NFM acts through PAR-4 signaling on the crosstalk between innate and adaptive immunity to induce the switch in CD4+ T-cell differentiation.