P-199 Lipocalin2 Affecting Bacterial Clearance in Activated Macrophages Contributes the Onset of Intestinal Inflammation

Abstract

Lipocalin2 (Lcn2) is an extracellular transport protein expressed in neutrophil granules, activated macrophages, and epithelial cells of the respiratory and gastrointestinal tract in response to inflammatory signals. It is well known that Lcn2 exerts bacteriostatic effect by sequestrating iron-containing siderophores because iron is essential for the growth and virulence of most bacterial species. In this regards, Lcn2 is considered to be involved in innate immune response. However, little is known about the exact role of Lcn2 in the intestinal inflammation. The aim of this study is to investigate the role of Lcn2 in the intestinal inflammation using IL-10 knock out (KO) mice. (1) Time-dependent expression of Lcn2 in the colonic tissues of IL-10 KO mice was compared with that of C57BL/6 wild type (WT) mice by quantitative PCR (qPCR) and immunohistochemistry (IHC). (2) We compared intestinal inflammation between IL-10 KO and Lcn2/IL-10 double knock out (DKO) mice. Inflammation was quantified by histologic scores, TNF-alpha and IL-12p40 secretion from cultured colonic explants, and IFN-gamma and IL-17A secretion from unseparated mesenteric lymph node (MLN) cells stimulated with cecal bacterial lysate. (3) Thioglycollate-elicited peritoneal macrophages were obtained from IL-10 KO (IL-10 KO-derived TEPM) and Lcn2/IL-10 DKO mice (DKO-derived TEPM), and infected with Escherichia coli. We examined the bacteria-killing function of macrophages by gentamicin protection assay. Also, TNF-alpha secretion from infected macrophages was determined by enzyme-linked immune-sorbent assay (ELISA). (1) qPCR and IHC demonstrated that Lcn2 expression in the colonic tissues of IL-10 KO mice was apparently up-regulated compared to that of WT mice from early 4 weeks after birth, and further increased over time. (2) Histologic scores in DKO mice were significantly higher than those in IL-10 KO mice at 4 weeks after birth (DKO versus IL-10 KO mice, 15.0 ± 0.0 versus 0.6 ± 0.5). Gene expressions of IL-1beta, TNF-alpha, IFN-gamma, and IL-17A in the colonic tissues of DKO mice were significantly up-regulated compared to those in IL-10 KO mice. TNF-alpha and IL-12p40 secretion from colonic explants of DKO mice were significantly higher than those of IL-10 KO mice (DKO versus IL-10 KO mice, TNF-alpha; 6.5 ± 0.7 versus 3.6 ± 1.2 ng/mL, IL-12p40; 30.6 ± 9.2 versus 5.3 ± 1.9 ng/mL). IFN-gamma and IL-17A production from MLN cells of DKO mice were higher than those of IL-10 KO mice (DKO versus IL-10 KO mice, IFN-gamma; 28.6 ± 33.7 versus 21.3 ± 32.7, P = 0.29, IL-17A; 2.5 ± 3.8 versus 0.6 ± 0.8 ng/mL, P < 0.05). (3) Uptake of bacteria was significantly enhanced in DKO-derived TEPM compared to that in IL-10 KO-derived TEPM (colony forming unit (CFU) at 2 hours after infection; DKO versus IL-10 KO-derived TEPM, 1461.7 ± 201.0 versus 868.3 ± 300.0 cfu/mL). In addition, intracellular survival of phagocytosed bacteria in DKO-derived TEPM persisted compared to that in IL-10 KO-derived TEPM (CFU at 8 hours after infection; DKO versus IL-10 KO-derived TEPM, 30.0 ± 17.9 versus 8.3 ± 13.3 cfu/mL, P < 0.05). Consequently, TNF-alpha production from DKO-derived TEPM significantly increased compared to IL-10 KO-derived TEPM (DKO versus IL-10 KO-derived TEPM, 14.6 ± 1.2 versus 4.0 ± 0.9 pg/mL at 16 hours after infection). Lcn2 affecting bacterial clearance in activated macrophages contributes to the onset of intestinal inflammation.