Role of collectin-11 in innate defence against uropathogenic Escherichia coli infection

Abstract

Classical collectins (surfactant protein A and D) play a significant role in innate immunity and host defence in uropathogenic Escherichia coli (UPEC)-induced urinary tract infection (UTI). However, the functions of collectin-11 (CL-11) with respect to UPEC and UTI remain largely unexplored. This study aimed to investigate the effect of CL-11 on UPEC and its role in UTI. We further examined its modulatory effect on inflammatory reactions in proximal tubular epithelial cells (PTECs). The present study provides evidence for the effect of CL-11 on the growth, agglutination, binding, epithelial adhesion and invasion of UPEC. We found increased basal levels of phosphorylated p38 MAPK and human cytokine homologue (keratinocyte-derived chemokine) expression in CL-11 knockdown PTECs. Furthermore, signal regulatory protein α blockade reversed the increased basal levels of inflammation associated with CL-11 knockdown in PTECs. Additionally, CL-11 knockdown effectively inhibited UPEC-induced p38 MAPK phosphorylation and cytokine production in PTECs. These were further inhibited by CD91 blockade. We conclude that CL-11 functions as a mediator of innate immunity via direct antibacterial roles as well as dual modulatory roles in UPEC-induced inflammatory responses during UTI. Thus, the study findings suggest a possible function for CL-11 in defence against UTI.

Keywords: Collectin-11; innate immunity; urinary tract infection; uropathogenic Escherichia coli.

Figure 1.

Collectin-11 (CL-11) expression in the kidney and urine of mice. (a) CL-11 proteins were detected in the kidney and urine by Western blot analysis. (b) and (c) Positive staining of CL-11 expression was detected in the tubular epithelial cells of the kidney (original magnification, 200×).

Figure 2.

CL-11 shows bactericidal activity towards uropathogenic E. coli (UPEC) CFT 073. CFT 073 was stained using a LIVE/DEAD BacLight Bacterial Viability Kit. (a) Bacteria were incubated with CL-11 (10 µg/ml). Untreated bacteria served as the control. Bacterial viability was analysed integrating fluorescent changes from viable (green) and dead (red) cells. (b) CFT 073 was incubated without or with CL-11 (10 µg/ml) in the presence of CaCl₂. After incubation, the bacteria were observed by fluorescence microscopy (original magnification, 400×). (c) The percentage of live bacteria was plotted against time. *P < 0.05.

Figure 3.

CL-11 binds to UPEC and inhibits their adhesion and invasion into proximal tubular epithelial cells (PTEC) CFT 073 in vitro. (a) CFT 073 was incubated with or without CL-11 (10 µg/ml) in the presence of CaCl₂ or EDTA. After incubation, the mixture of CFT 073 and protein was washed and centrifuged. The bacterial pellet obtained was subjected to SDS-PAGE, and blotting analysis was performed to detect CL-11 co-sedimented with the bacteria. As a positive control, recombinant CL-11 was loaded. (b) PTECs from mice were isolated and identified as described in the Methods. More than 95% of the isolated cells showed megalin-positive staining. (c) and (d) CFT 073 was pre-treated with CL-11 (10 µg/ml) or vehicle control. PTECs were then incubated with CFT 073. The media was aspirated, and PTECs were washed, treated without or with gentamicin (adhesion or invasion) and finally lysed with TBST to quantitate intracellular bacteria (expressed as % of initial inoculum). *P < 0.05 compared to 0 and 10 µg/ml.

Figure 4.

CL-11 exerts anti-inflammatory effect via SIRPα in uninfected PTECs. (a) CL-11 siRNA effectively reduced CL-11 expression in PTECs. (b) and (c) PTECs were pre-treated with anti-SIPRα Ab or control IgG, and then incubated with or without CL-11 siRNA. Levels of p-p38 MAPK were detected using Western blot analysis. (d) Supernatants were collected and used for measuring keratinocyte-derived chemokine (KC) expression using ELISA. *P < 0.05.

Figure 5.

CD91 is involved in UPEC-induced p-p38 activation. (a) and (b) PTECs were pre-treated with anti-CD91 Ab or control IgG and then incubated with or without UPEC. Levels of phosphorylated p38 MAPK were detected using Western blot analysis. (c) Supernatants were collected and used for measuring KC expression levels using ELISA. *P < 0.05.

Figure 6.

CL-11 exerts pro-inflammatory effect via CD91 in UPEC-infected PTECs. (a) and (b) PTECs were pre-treated with CL-11 siRNA (with or without CD91 Ab) or control siRNA and then incubated with or without UPEC. Levels of p-p38 MAPK were detected in PTECs using Western blot analysis. (c) Supernatants were collected and used for measuring KC expression using ELISA. *P < 0.05.