original article http://www.kidney-international.org & 2007 International Society of Nephrology Different roles of TIR8/SIGIRR on toll-like receptor signaling in intrarenal antigen-presenting cells and tubular epithelial cells M Lech1, C Garlanda2, A Mantovani2, CJ Kirschning3, D Schlo¨ndorff1 and H-J Anders1 1Nephrological Center, Medical Policlinic, University of Munich, Munich, Germany; 2Istituto Clinico Humanitas and Fondazione Humanitas per la Ricerca, Rozzano, Italy and 3Institute of Medical Microbiology, Immunology, and Hygiene, Technical University of Munich, Munich, Germany Toll-like receptors (TLRs) exist on both myeloid and intrinsic The toll-like receptor (TLR)/interleukin 1 receptor (IL-1R) renal cells contributing to the initiation of innate immunity superfamily has a central role for initiating innate anti- during renal infection with uropathogenic Escherichia coli. microbial immunity and, hence, may contribute to renal Toll–interleukin 1 receptor (IL-1R) (TIR)8/SIGIRR is an orphan pathology in infective pyelonephritis.1,2 In fact, renal tubular receptor of the TLR/IL-1R family, which suppresses TLR epithelial cells express TLR1-4 and -11 and can produce signaling of immune cells and is highly expressed in the proinflammatory cytokines and chemokines in response to kidney. Lack of TIR8/SIGIRR is associated with enhanced renal respective TLR ligands.3–6 Furthermore, renal dendritic cells chemokine signaling upon exposure to lipopolysaccharide contribute to innate immunity in the kidney as they are (LPS). This was because of TIR8/SIGIRR expression on resident activated via TLRs either directly by pathogens or by intrarenal myeloid cells rather than tubular epithelial cells endogenous molecules such as Tamm–Horsfall protein.7 which express it on basolateral and luminal membranes. The Uropathogenic Escherichia coli (UPEC) is the most common lack of TIR8/SIGIRR does not enhance TLR/IL-1R signaling in pathogen involved in infective pyelonephritis and UPEC tubular epithelial cells as was observed in monocytes. TIR8/ lipopolysaccharide (LPS) ligates TLR4 as part of the LPS SIGIRR is induced in monocytes treated with LPS or tumor receptor complex.8 We have recently reported experiments necrosis factor and interferon-c in a dose-dependent manner with TLR4 bone marrow chimeric mice which showed that but was downregulated in treated tubule epithelial cells. This TLR4 on intrinsic renal cells as well as on myeloid cells cell type-specific regulation and function did not relate to contributes to renal chemokine signaling and subsequent mRNA splice variants but was associated with N- and O- renal abcess formation in infective pyelonephritis with glycosylation of the receptor in renal cells of myeloid and UPEC.9 nonmyeloid origin. Our studies show that resident myeloid What are the molecular mechanisms that control renal cells contribute to TLR-mediated antimicrobial immunity in TLR signaling? Excessive TLR signaling may lead to severe the kidney and that this function is controlled by TIR8/SIGIRR. inflammation and inappropriate immunity-related tissue TIR8/SIGIRR does not suppress TLR signaling in tubular damage.10 In fact, a number of negative regulators of TLRs epithelial cells, which supports their role as sensors of have been identified, which include splice variants of microbial infection in the kidney. signaling molecules, soluble TLRs, cleavage enzymes, and Kidney International (2007) 72, 182–192; doi:10.1038/sj.ki.5002293; receptors of the TLR/IL-1R family, T1/ST2 and toll–IL- published online 2 May 2007 1R(TIR)8/SIGIRR.11–13 The Tir8/Sigirr gene was identified by KEYWORDS: SIGIRR; toll-like receptor; lipopolysaccharide; infection searching EST databases for TIR domain-containing se- quences of yet unknown members of the TLR/IL-1R family.14,15 TIR8/SIGIRR is the only TIR domain-containing member of the TLR/IL-1R superfamily that has a single extracellular immunoglobulin (Ig) domain.14,15 The intracel- lular domain of TIR8/SIGIRR differs from the typical C terminus of TLR/IL-1Rs and is similar to that of the Drosophila toll protein.14 Neither IL-1 nor other ligands bind to TIR8/SIGIRR. TIR8/SIGIRR does not activate Correspondence: H-J Anders, Medizinische Poliklinik, Universita¨tMu¨nchen, 14,16 Pettenkoferstr. 8a, Mu¨nchen 80336, Germany. nuclear factor-kB (NF-kB), most probably because it E-mail: [email protected] does not retain two amino acids (Ser447 and Tyr536) in the 14 Received 12 October 2006; revised 2 February 2007; accepted 8 March highly conserved TIR domain. TIR8/SIGIRR was proposed 2007; published online 2 May 2007 as an endogenous inhibitor of TLR signaling, because 182 Kidney International (2007) 72, 182–192 M Lech et al.: TIR8/SIGIRR in renal TLR signaling original article overexpression of TIR8/SIGIRR in Jurkat or HepG2 cells tubular epithelial cells. Obviously, the regulation and substantially reduced IL-1- or IL-18-induced activation of function of TIR8/Sigirr is different in immune and NF-kB.15,17,18 Tir8/Sigirr-deficient mice are more susceptible nonimmune cells in the kidney. to dextran-induced inflammatory bowel disease and a TIR8/ Sigirr-blocking antibody was shown to aggravate Pseudomo- RESULTS 19,20 nas aeruginosa keratitis in Balb/c mice. This was referred TIR8/Sigirr is expressed in renal antigen-presenting cells and to TIR8/Sigirr-mediated suppression of TLR signaling in renal tubular epithelial cells of 6-week-old C57BL/6 mice dendritic cells, which express TIR8/Sigirr constitutively. TLR function has been reported to be age-dependent;21 Interestingly, TIR8/Sigirr mRNA is expressed at high levels hence, we analyzed TIR8/Sigirr mRNA levels by real-time in the murine and human kidney in comparison to other reverse transcription (RT)–PCR in C57BL/6 mice, either 10 solid organs including lymphoid tissues.15,17 This may either days, 6 weeks, or 1 year of age. At 6 weeks of age, high levels relate to TIR8/Sigirr expression in intrarenal myeloid cells or of TIR8/Sigirr mRNA were found in kidneys, threefold tubular cells. We hypothesized a role for TIR8/Sigirr in compared to respective TIR8/Sigirr levels in spleen (Figure regulating TLR signaling in both cell types during exposure 1a). By contrast, brain, heart, lung, liver, small intestine, to UPEC LPS. However, we discovered that renal TLR colon, skin, and muscle expressed TIR8/Sigirr mRNA at signaling is independent of TIR8/Sigirr expression in renal lower levels as in spleen. In most organs, TIR8/Sigirr mRNA a 0.00020 1.5 weeks * 6.0 weeks 0.00015 52 weeks 0.00010 0.00005 * * # * Tir8/Sigirr mRNA/18s rRNA # * * * * * # 0 * * * Spleen Thymus Brain Lung Liver Kidney Small Skin intestine b c 2×10−5 Kidney Tir8/Sigirr rRNA 1×10−5 +/+ −/− Tir8/Sigirr n.d. 0 Intrarenal Tubular Mesangial CD11b+ epithelial cells cells cells d Figure 1 | TIR8/Sigirr expression in C57BL/6 mice.(a) mRNA was extracted from organs of C57BL/6 mice of different age as indicated (n ¼ 3–6). TIR8/Sigirr mRNA expression levels were determined by real-time RT–PCR and expressed as mean of the ratio TIR8/Sigirr /18s- rRNA7s.e.m.; *Po0.05 vs 1.5 weeks, #Po0.05 vs 6 weeks. (b) TIR8/Sigirr protein expression was determined by Western blot analysis. Proteins were prepared from kidneys of 6–week-old C57BL/6 wild-type mice or TIR8/Sigirr-deficient mice as indicated. (c) Primary cells were isolated from of 6-week-old C57BL/6 mice as described in Materials and Methods. TIR8/Sigirr mRNA levels were determined by real-time RT–PCR and expressed as mean of the ratio TIR8/Sigirr/18s-rRNA7s.e.m. N.d. ¼ not detected. (d) F4/80 immunostaining for intrarenal antigen-presenting cells (black) in kidney cortex (left) and medulla (right). Original magnification  200. Kidney International (2007) 72, 182–192 183 original article M Lech et al.: TIR8/SIGIRR in renal TLR signaling levels declined from young to old age. Interestingly, TIR8/ originates from tubular epithelial cells and intrarenal Sigirr mRNA levels in 6-week-old C57BL/6 mice were seven- immune cells, resident antigen-presenting cells. to eightfold higher as compared to 10-day- or 1-year-old mice. The prominent expression of TIR8/Sigirr in kidneys of TIR8/Sigirr localizes to luminal and basolateral membranes of 6-week-old mice was confirmed on the protein level by tubular epithelial cells Western blot (Figure 1b). Cortex and medulla from kidneys The transmembrane molecule TIR8/Sigirr has been reported of 6-week-old C57BL/6 mice expressed equal levels of TIR8/ to suppress LPS signaling in Jurkat cells by interacting with Sigirr mRNA, suggesting a tubular or interstitial origin rather the intracellular domain of TLR4 and both extracellular Ig than glomerular cells, which predominantely locate to the domain and the intracellular TIR domain of IL-1R.18 Thus, renal cortex (data not shown). In fact, unlike primary TIR8/Sigirr should localize to outer membranes of these cells. mesangial cells tubular epithelial cells and resident CD11b/ In fact, flow cytometry of primary tubular epithelial cells F4/80-positive renal myeloid cells both expressed TIR8/Sigirr using a TIR8/Sigirr-specific antibody revealed a robust signal mRNA (Figure 1c). The latter cells localize to the interstitium on the cell surface (Figure 2a). The cellular distribution of of the renal cortex and medulla of mice (Figure 1d). These TIR8/Sigirr was confirmed by the immunostaining on data indicate that the profound renal TIR8/Sigirr expression primary tubular epithelial cells. Positive staining signals were 100 100 abTir8/Sigirr +/+ Tir8/Sigirr −/− 80 80 60 60 Counts Counts 40 40 20 20 0 0 100 101 102 103 104 100 101 102 103 104 FL2-H FL2-H cd * ef* * * ** * * Figure 2 | TIR8/Sigirr expression in tubular epithelial cells. (a and b) Flow cytometry for TIR8/Sigirr was performed using primary tubular epithelial cells prepared from (a) wild-type mice and (b) Tir8/Sigirr-deficient mice as indicated.