Leptospiral membrane proteins stimulate pro-inflammatory chemokines secretion by renal tubule epithelial cells through toll-like receptor 2 and p38 mitogen activated protein kinase.
BACKGROUND
Leptospiral membrane proteins extracted from pathogenic Leptospira santarosai serovar Shermani (LMPS) stimulated pro-inflammatory chemokines production in cultured mouse proximal tubule epithelial cells (PTECs) and implicated its role in the pathogenesis of leptospira-induced tubulointerstitial nephritis. PTECs express the functional TLR2 and TLR4, which have been shown to play essential roles in innate immunity. This study investigated the roles of Toll-like receptors (TLRs) and mitogen-activated protein kinases (MAPKs) signalling pathways in the pathogenesis of leptospira-induced tubulointerstitial nephritis.


METHODS
The immortalized mouse PKSV-PR late PTECs were used as the model system. The genes expression and secretion of CCL2/monocyte chemoattractant protein-1 (CCL2/MCP-1) and CXCL2/macrophage inflammatory protein-2 (CXCL2/MIP-2) were measured by reverse transcription-polymerase chain reaction (RT-PCR) and enzyme linked immunosorbent assay (ELISA). We investigated MAPKs signalling pathways by Western blot and their reciprocal roles by specific inhibitors. A specific TLR2 neutralizing antibody was applied to evaluate the crosstalk between TLR2 and MAPKs.


RESULTS
The LMPS stimulated extracellular signal-regulated kinases (ERK1/2), c-Jun N-terminal kinases (JNKs) and p38 mitogen-activated protein kinase (p38 MAPK), initiated the nuclear transcription factor kappaB (NF-kappaB), and enhanced the secretion of CCL2/MCP-1 and CXCL2/MIP-2. The LMPS also unregulated the level of TLR2 mRNA expression in PTECs through time- and dose-dependent effects. The LMPS enhanced the secretion of CCL2/MCP-1 and CXCL8/interleukin-8 (CXCL8/IL-8) in TLR-defective human embryonic kidney (HEK) 293 cells only when transfected with a TLR2 expressing plasmid. The secretions of CCL2/MCP-1 and CXCL2/MIP-2 stimulated by LMPS were significantly reduced by incubating PTECs with SB203580, an inhibitor of p38 MAPK. Furthermore, a neutralizing anti-mouse TLR2 antibody hindered the phosphorylation of p38 and LMPS-stimulated secretion of CCL2/MCP-1 and CXCL2/MIP-2.


CONCLUSIONS
These findings demonstrate that activation of p38 MAPK and release of chemokines by LMPS are mediated by TLR2 in renal proximal tubule cells. These results also implicate the crucial role of innate immunity in leptospira-induced tubulointerstitial nephritis.
Hung
Cheng-Chieh
CC
Kidney Institute, Chang Gung Memorial Hospital, Taipei, Taiwan.
Chang
Chiz-Tzung
CT
Tian
Ya-Chung
YC
Wu
Mai-Szu
MS
Yu
Chun-Chen
CC
Pan
Ming-Jeng
MJ
Vandewalle
Alain
A
Yang
Chih-Wei
CW
D000818:Animals; D001425:Bacterial Outer Membrane Proteins / Q000494:pharmacology*; D002478:Cells, Cultured; D018932:Chemokine CCL2 / Q000235:genetics / Q000378:metabolism; D054426:Chemokine CXCL2; D018925:Chemokines / Q000235:genetics / Q000378:metabolism; D016207:Cytokines / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D006801:Humans; D048031:JNK Mitogen-Activated Protein Kinases / Q000378:metabolism; D007687:Kidney Tubules, Proximal / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D007919:Leptospira; D051379:Mice; D016328:NF-kappa B / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction; D051195:Toll-Like Receptor 2 / Q000235:genetics / Q000378:metabolism*; D014162:Transfection; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism*
D001425:Bacterial Outer Membrane Proteins; C491768:Ccl2 protein, mouse; D018932:Chemokine CCL2; D054426:Chemokine CXCL2; D018925:Chemokines; C404915:Cxcl2 protein, mouse; D016207:Cytokines; D016328:NF-kappa B; D012333:RNA, Messenger; C495342:TLR2 protein, human; C493486:Tlr2 protein, mouse; D051195:Toll-Like Receptor 2; D048049:Extracellular Signal-Regulated MAP Kinases; D048031:JNK Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases