Toll-like receptor 2 mediates early inflammation by leptospiral outer membrane proteins in proximal tubule cells.
Tubulointerstitial nephritis is a cardinal renal manifestation in leptospirosis and LipL32, the major lipoprotein component of leptospiral outer membrane proteins (OMPs), induces a robust inflammatory response in cultured renal proximal tubule cells through a nuclear factor-kappaB-related pathway. Here, we investigated whether Toll-like receptor (TLR), known to play a pivotal role in innate immunity, could mediate the inflammatory response induced by leptospiral OMPs in renal proximal tubule cells. TLR expression was analyzed by flow cytometry and indirect immunofluorescence in cultured mouse proximal tubule (pyruvate kinase simian virus 40-proximal straight (PKSV-PR)) cells. Reverse transcription-competitive polymerase chain reaction and enzyme-linked immunosorbent assay were undertaken to analyze the inducible effects of inducible nitric oxide synthase (iNOS) and monocyte chemoattractant protein-1 (MCP-1 also termed CCL2) by pathogenic and non-pathogenic leptospiral OMPs and recombinant lipoproteins in either PKSV-PR cells or TLR-transfected human embryonic kidney (HEK) 293 cells. Anti-TLR antibodies were used for blocking experiments. Leptospira santarosai serovar Shermani OMPs and LipL32 induced a significant increase in TLR2 but not TLR4 expression in PKSV-PR cells. The increase in iNOS and CCL2/MCP-1 mRNA expressions could be prevented by an anti-TLR2 antibody, but not by an anti-TLR4 antibody. Furthermore, leptospiral OMPs stimulated both CCL2/MCP-1 mRNA and secreted protein in transfected HEK 293 cells with a TLR2-expressing plasmid, but had no effect in cells with a TLR4-expressing plasmid. In conclusion, these findings indicate that the stimulation of iNOS and CCL2/MCP-1 caused by pathogenic leptospiral OMPs, in particular LipL32, in proximal tubule cells requires TLR2 for the early inflammatory response.
Yang
C-W
CW
Department of Nephrology, Kidney Institute, Chang Gung Memorial Hospital, 199 Tun-Hwa North Road, Taipei, 105 Taiwan. cwyang@adm.cgmh.org.tw
Hung
C-C
CC
Wu
M-S
MS
Tian
Y-C
YC
Chang
C-T
CT
Pan
M-J
MJ
Vandewalle
A
A
D000818:Animals; D000942:Antigens, Bacterial / Q000008:administration & dosage; D001425:Bacterial Outer Membrane Proteins / Q000276:immunology; D001483:Base Sequence; D002478:Cells, Cultured; D018932:Chemokine CCL2 / Q000378:metabolism; D004247:DNA / Q000235:genetics; D006801:Humans; D018836:Inflammation Mediators / Q000378:metabolism; D007687:Kidney Tubules, Proximal / Q000276:immunology* / Q000469:parasitology*; D007919:Leptospira / Q000276:immunology*; D007922:Leptospirosis / Q000276:immunology; D008074:Lipoproteins / Q000276:immunology; D051379:Mice; D052247:Nitric Oxide Synthase Type II / Q000378:metabolism; D011994:Recombinant Proteins / Q000008:administration & dosage / Q000276:immunology; D051195:Toll-Like Receptor 2 / Q000235:genetics / Q000378:metabolism*; D051197:Toll-Like Receptor 4 / Q000235:genetics; D014162:Transfection
D000942:Antigens, Bacterial; D001425:Bacterial Outer Membrane Proteins; C491768:Ccl2 protein, mouse; D018932:Chemokine CCL2; D018836:Inflammation Mediators; C406589:LipL32 protein, Leptospira; D008074:Lipoproteins; D011994:Recombinant Proteins; C495342:TLR2 protein, human; C495345:TLR4 protein, human; C493486:Tlr2 protein, mouse; D051195:Toll-Like Receptor 2; D051197:Toll-Like Receptor 4; D004247:DNA; D052247:Nitric Oxide Synthase Type II; C496317:Nos2 protein, mouse