-Independent Inflammatory Pathways by Activating Distinct TLR4-Dependent and Escherichia coli to Pyelonephritis-Associated Renal Collecting Duct Epithelial Cells React

TLR4 plays a central role in resistance to pyelonephritis caused by uropathogenic Escherichia coli (UPEC). It has been suggested that renal tubule epithelial cells expressing TLRs may play a key role in inﬂammatory disorders and in initiating host defenses. In this study we used an experimental mouse model of ascending urinary tract infection to show that UPEC isolates preferentially adhered to the apical surface of medullary collecting duct (MCD) intercalated cells. UPEC-infected C3H/HeJ ( Lps d ) mice carrying an inactivating mutation of tlr4 failed to clear renal bacteria and exhibited a dramatic slump in proinﬂammatory mediators as compared with infected wild-type C3H/HeOuJ ( Lps n ) mice. However, the level of expression of the leukocyte chemoattractants MIP-2 and TNF- (cid:1) still remained greater in UPEC-infected than in naive C3H/HeJ ( Lps d ) mice. Using primary cultures of microdissected Lps n MCDs that expressed TLR4 and its accessory molecules MD2, MyD88, and CD14, we also show that UPECs stimulated both a TLR4-mediated, MyD88-dependent, TIR domain-containing adaptor-inducing IFN- (cid:2) -independent pathway and a TLR4-independent pathway, leading to bipolarized secretion of MIP-2. Stimulation by UPECs of the TLR4-mediated pathway in Lps n MCDs leads to the activation of NF- (cid:3) B, and MAPK p38, ERK1/2, and JNK. In addition, UPECs stimulated TLR4-independent signaling by activating a TNF receptor-associated factor 2-apoptosis signal-regulatory kinase 1-JNK pathway. These ﬁndings demonstrate that epithelial collecting duct cells are actively involved in the initiation of an immune response via several distinct signaling pathways and suggest that intercalated cells play an active role in the recognition of UPECs colonizing the kidneys. The Journal of Immunology, 2006, 177: 4773–4784. cultures of MCDs microdissected from adult LPS-sensitive C3H/HeOuJ ( Lps n ) and LPS-nonresponsive C3H/HeJ ( Lps d ) mice. Experiments were also conducted with MyD88-deﬁcient ( MyD88 (cid:2) / (cid:2) ) mice (24), which exhibit no inﬂammatory response to the TLR4 ligand LPS (25), and Lps2 mutant ( Lps 2 (cid:2) / (cid:2) ) mice (26), which are deﬁcient for the TIR domain-containing adaptor inducing IFN- (cid:2) (TRIF), an adaptor protein that regulates an MyD88-independent signaling pathway upon stimulation by TLR3 and TLR4 ligands (26, 27 ) . We show that UPEC isolates colonized kidneys after transurethral inoculation and preferentially and constantly adhered to apical membranes of MCD intercalated cells expressing TLR4. UPECs induced the expression of proinﬂammatory mediators in MCD cells and stimulated bipolarized secretion of the chemokine MIP-2 (CXCL2/MIP-2) via both TLR4-mediated MyD88-depen-dent and TLR4-independent pathways requiring distinct MAPK-related signaling molecules. These ﬁndings indicate that MCD cells are involved in UPEC recognition and actively participate to the initiation of immune innate response by triggering rapid stimulation of proinﬂammatory mediators in injured kidneys. (Imgenex), JNK, TRAF6, apo- ptosis regulatory 1 (ASK1), ERK1/2 Biotech-nology), (Sigma-Aldrich) Ags. phosphorylation ASK1 (Cell or