Expression of the cathelicidin LL-37 is modulated by short chain fatty acids in colonocytes: relevance of signalling pathways

Abstract

Background and aims: Short chain fatty acids (SCFA) exert profound effects on the colonic mucosa. In particular, SCFA modulate mucosal immune functions. The antimicrobial cathelicidin LL-37 is expressed by colon epithelial cells. In the present study the effect of SCFA on LL-37 expression was investigated.

Methods: LL-37 expression in vivo was assessed by immunohistochemistry. Real time quantitative reverse transcription-polymerase chain reaction was employed to determine LL-37 expression in colonocytes in vitro after treatment with various cytokines, SCFA, or flavone. LL-37 levels were correlated to cell differentiation which was determined by alkaline phosphatase (AP) activity. In addition, intracellular signalling pathways such as MEK-ERK (mitogen/extracellular signal protein kinase (MEK)/extracellular signal regulated protein kinase (ERK)) and p38/mitogen activated protein (MAP) kinase were explored.

Results: In vivo, LL-37 expression in healthy mucosa was restricted to differentiated epithelial cells in human colon and ileum. In colonocytes, increased LL-37 expression associated with cell differentiation was detected in vitro following treatment with butyrate, isobutyrate, propionate, and trichostatin A. Flavone induced LL-37 transcription but did not affect AP activity while cytokines had no effect. To dissect pathways mediating differentiation and LL-37 expression, specific inhibitors were applied. Inhibition of the protein kinase MEK enhanced butyrate induced AP activity while LL-37 expression in colon epithelial cells was blocked. In contrast, inhibition of p38/MAP kinase blocked cell differentiation without inhibiting LL-37 expression.

Conclusions: Expression of the cathelicidin LL-37 in colonocytes and cellular differentiation are separately modulated by SCFA via distinct signalling pathways. These data may provide a rationale for dietary modulation of mucosal defence mechanisms.

Figure 1

Expression of LL-37 in SW620 colon cells detected by reverse transcription-polymerase chain reaction (RT-PCR) and Southern hybridisation during butyrate (2 mmol/l) treatment (A). Quantitative real time RT-PCR analyses of LL-37 expression in SW620 colon cells (B). Cells were incubated with butyrate (2 mmol/l) and real time RT-PCR analyses using a TaqMan system were performed. No LL-37 transcripts were detected in medium control treated SW620 cells at any time point. LL-37 expression levels are therefore displayed as “fold induction” relative to induction after incubation with butyrate 2 mmol/l for 24 hours. **p<0.01; ***p<0.001. Similar results were obtained in SW480 cells (data not shown). Induction of LL-37 transcription in Geki2 (left) and HT-29 (right) colon cells incubated with butyrate 2 or 4 mmol/l is displayed as “fold induction” relative to medium treated control Geki2 cells and HT-29 cells, respectively (C). *p<0.05; ***p<0.001.

Figure 2

Effect of butyrate on alkaline phosphatase (AP) activity of SW620, HT-29, and Geki2 colon epithelial cells. Cells were incubated with butyrate 2 mmol/l or medium alone and AP activity was measured. Values are expressed as mU of AP activity per mg of cellular protein and are means (SD). **p<0.01; ***p<0.001. Similar results were obtained in SW480 cells (data not shown).

Figure 3

(A) Comparison of LL-37 induction by different short chain fatty acids, lactate, flavone, and trichostatin A in SW620 colon cells. LL-37 expression was analysed by quantitative real time reverse transcription-polymerase chain reaction analyses, as described. **p<0.01; ***p<0.001. (B) Effect of assorted luminal factors on alkaline phosphatase (AP) activity as a surrogate marker of colon epithelial cell differentiation. SW620 cells were incubated with the indicated substrates and AP activity was measured. Values are expressed as mU of AP activity per mg cellular protein and are means (SD). *p<0.05; ***p<0.001.

Figure 4

Expression of LL-37 peptide in epithelial cells in human colon and ileum. LL-37 expression was detected in colorectal (A) and ileal (B) biopsy specimens from healthy individuals by staining serial sections with the polyclonal antibody for LL-37. Sections are shown at 100× and 250× magnifications. As a negative control, the primary antibody was preabsorbed with the synthetic LL-37 peptide (displayed on the right panel).

Figure 5

Effect of specific kinase inhibitors on butyrate induced alkaline phosphatase (AP) activity and LL-37 expression. SW620 colon cells were incubated with butyrate or trichostatin A (TSA) after preincubation with or without the MEK inhibitor U0126 or the p38/MAP kinase inhibitor SB203580, and LL-37 expression was analysed by quantitative reverse transcription-polymerase chain reaction (A). (B) Influence of the inhibitors on butyrate or trichostatin A induced AP activity as a surrogate marker for differentiation. *p<0.05; ***p<0.001.