BASIC RESEARCH www.jasn.org

CXCL5 Drives Neutrophil Recruitment in

TH17-Mediated GN

Erik M. Disteldorf,* Christian F. Krebs,* Hans-Joachim Paust,* Jan-Eric Turner,* † ‡ Geraldine Nouailles, André Tittel, Catherine Meyer-Schwesinger,* Gesa Stege,* Silke Brix,* | | Joachim Velden,§ Thorsten Wiech, Udo Helmchen, Oliver M. Steinmetz,* Anett Peters,* ‡ Sabrina B. Bennstein,* Anna Kaffke,* Chrystel Llanto, Sergio A. Lira,¶ Hans-Willi Mittrücker,** ‡ † Rolf A.K. Stahl,* Christian Kurts, Stefan H.E. Kaufmann, and Ulf Panzer*

*III. Medical Clinic and Institutes of |Pathology and **Immunology, Hamburg-Eppendorf University Hospital, Hamburg-Eppendorf, Germany; †Max Planck Institute for Infection Biology, Berlin, Germany; ‡Institute of Molecular Medicine and Experimental Immunology, Bonn, Germany; §Department of Nephropathology, Erlangen University Hospital, Erlangen, Germany; and ¶Immunology Institute, Icahn School of Medicine at Mount Sinai, New York, New York

ABSTRACT Neutrophil trafficking to sites of inflammation is essential for the defense against bacterial and fungal

infections, but also contributes to tissue damage in TH17-mediated autoimmunity. This process is regu- BASIC RESEARCH lated by , which often show an overlapping expression pattern and function in pathogen- and autoimmune-induced inflammatory reactions. Using a murine model of crescentic GN, we show that the pathogenic TH17/IL-17 immune response induces (C-X-C motif) ligand 5 (CXCL5) expression in kidney tubular cells, which recruits destructive neutrophils that contribute to renal tissue injury. By con- trast, CXCL5 was dispensable for neutrophil recruitment and effective bacterial clearance in a murine model of acute bacterial pyelonephritis. In line with these findings, CXCL5 expression was highly upregu- lated in the kidneys of patients with ANCA-associated crescentic GN as opposed to patients with acute bacterial pyelonephritis. Our data therefore identify CXCL5 as a potential therapeutic target for the re- striction of pathogenic neutrophil infiltration in TH17-mediated autoimmune diseases while leaving intact the neutrophil function in protective immunity against invading pathogens.

J Am Soc Nephrol 26: 55–66, 2015. doi: 10.1681/ASN.2013101061

Neutrophils are the most abundant type of leuko- interaction with specific G protein–coupled receptors cytes in the blood and form an indispensable part of that are predominantly expressed on leukocytes. the innate immune system. Their trafficking into Neutrophil infiltration is mainly mediated by chemo- peripheral tissues is pivotal in the defense against kines that have a glutamate-leucine-arginine motif invading bacterial and fungal pathogens.1 To ensure (ELR+ chemokines). In humans, there are seven that neutrophils reach the sites of tissue injury, their ELR chemokine ligands with a C-X-C motif recruitment is regulated by the local expression of (CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, chemoattractants, including chemokines. However, the infiltration of neutrophils also significantly con- Received October 10, 2013. Accepted April 4, 2014. tributes to end-organ damage in autoimmune diseases 2 E.M.D. and C.F.K. contributed equally to this work. mediated by T helper (TH) cell TH17, including 3,4 human and experimental crescentic GN. Published online ahead of print. Publication date available at Chemokines are a large family of small (8–12 kD) www.jasn.org. fi secreted proteins that are identi ed as attractants of Correspondence: Dr. Ulf Panzer, Third Medical Clinic, Hamburg- different types of leukocytes, including neutrophils, Eppendorf University Hospital, Martinistrasse 52, 20246 Hamburg, to sites of infection and inflammation.5 They are Germany. Email: [email protected] produced locally in tissues and act through Copyright © 2015 by the American Society of Nephrology

J Am Soc Nephrol 26: 55–66, 2015 ISSN : 1046-6673/2601-55 55 BASIC RESEARCH www.jasn.org

CXCL7, and CXCL8/IL-8). Their chemotactic effects are me- marker Gr1 (Ly6C/Ly6G) (Figure 1A). In the early stage of diated via binding to the chemokine receptors CXCR1 and nephritis (until day 3), neutrophils were mainly found in the CXCR2. Mice lack complete homologs of the seven human glomerulus (Figure 1, A and B). The infiltration of neutrophils ELR chemokines and have only five members (CXCL1, into the tubulointerstitial area started at day 5, peaked around CXCL2, CXCL3, CXCL5, and CXCL7, which all bind to the day 10, and then declined (Figure 1, A and B). This murine CXCR2).6 Interestingly, previous reports show that IL- demonstrates a previously unknown time- and compart- 17A, the master effector of TH17 cells, induces the ment-specific recruitment of neutrophils into the kidney. expression of the ELR+ chemokines CXCL1, CXCL2, and CXCL57,8 and thereby might drive the recruitment of patho- CXCL1 and CXCL5 Have Unique Functions in the genic neutrophils in autoimmunity. The development of a Recruitment of Neutrophils in Crescentic GN therapeutic strategy targeting ELR+ neutrophil–attracting che- One important prerequisite for the infiltration of neutrophils mokines or their receptors is complicated by an often overlap- into sites of inflammation is the expression of ELR+ chemo- ping expression pattern and function of these molecules in kines, namely CXCL1, CXCL2, CXCL3, CXCL5, and CXCL7, pathogen- and autoimmune-induced inflammatory reac- which act via the CXCR2. Quantitative tions.9 RT-PCR analysis of the renal cortex revealed that CXCL1, Here we describe for the first time a nonredundant function CXCL2, and CXCL3 mRNA expression was strongly upregu- of the chemokines CXCL1 and CXCL5 in murine models of lated in the early stages of nephritis (12 and 24 hours) (Figure crescentic GN and acute bacterial pyelonephritis. CXCL1 1C). Renal CXCL7 expression was low throughout the disease mediated early glomerular neutrophil recruitment in the non- course (data not shown). CXCL1 expression rapidly declined Tcell–dependent initiation phase of GN, whereas CXCL5 to the baseline level by day 3. By contrast, CXCL5 mRNA was responsible for the infiltration of pathogenic neutrophils expression was not increased at early time points but markedly into sites of inflammation in later TH17-dependent phases of increased from day 5 onward, with maximum expression lev- the disease. Of note, CXCL5 did not affect neutrophil infil- els at day 10 (detectable after approximately 25 PCR cycles). tration and bacterial clearance in a murine model of acute Chemokines can be modified post-translationally by proteo- bacterial pyelonephritis, one of the most prevalent kidney lytic cleavage in order to achieve the active form and stored infections in humans. These findingssuggestthatCXCL5 intracellular, therefore, a discrepancy between local mRNA has a unique function in the trafficking of neutrophils in level and protein level/activity might occur. However, our at- TH17 cell–mediated autoimmunity, but not in the innate tempts to quantify renal CXCL5 protein levels in nephritic immune response. CXCL5 therefore represents an attractive mice by ELISA failed because of high background signals therapeutic target for the restriction of pathogenic neutrophil when using CXCL5 knockout (KO) mice as negative controls infiltration in TH17-driven autoimmune diseases without af- (data not shown). fecting the vital functions of neutrophils in the defense against Renal mRNA expression of CXCR2 was characterized by a acute bacterial infections. bimodal course reaching two maxima after 24 hours and 10 days (Figure 1C). In line with this, glomerular and tubuloin- terstitial neutrophil recruitment was reduced in nephritic 2 2 RESULTS CXCR2 / mice at days 3 and 14 (Figure 1D). This showed that CXCR2 drives both the “early” and “late” waves of neu- Time- and Compartment-SpecificInfiltration of trophil recruitment. Neutrophils in Murine Crescentic GN To elucidate whether the expression patterns of CXCL1 and Nephrotoxic nephritis (NTN) is a well characterized model of CXCL5 might result in different functions of these chemokines murine crescentic GN, which is induced by the injection of in the disease course, we performed early (until day 3) and late sheep antiserum raised against kidney cortical components. (days 9 to 14) in vivo neutralization with specific anti-CXCL1 During the early heterologous phase of the disease, the or anti-CXCL514 antibodies. In line with their temporal expression deposited antibodies result in glomerular complement acti- profile, neutralization of CXCL1 resulted in ameliorated glo- vation and neutrophil recruitment, which cause substantial merular neutrophil infiltration at day 3, whereas anti-CXCL5 glomerular injury and renal dysfunction.10 An adaptive im- antibody treatment at day 9 resulted in significantly reduced mune response against the foreign sheep protein develops in tubulointerstitial neutrophil recruitment at day 14 (Figure 1E). the subsequent autologous phase (starting from days 3 to 5), Accordingly, renal FACS analysis revealed a reduction in + + + + 2/2 resulting in the activation of nephritogenic CD4 TH17 and CD45 CD11b Ly6G neutrophils in nephritic CXCL5 TH1 cells in lymphatic organs. First, TH17 cells and, sub- mice at day 14 (Figure 1, F and G), but not at day 3 (Supple- sequently, TH1 cells migrate into the kidney and promote mental Figure 1A). Likewise, tubulointerstitial neutrophil accu- 2 2 renal tissue injury.11–13 The role of neutrophils in the T cell– mulation was significantly decreased in nephritic CXCL5 / mediated phase (starting from day 5) is largely unknown. We mice at day 14 (Figure 1H), but not at day 3 (Supplemental therefore assessed the time course of renal neutrophil infiltra- Figure 1B). Immunofluorescence staining revealed that neutro- 2 2 tion using immunohistochemical staining for the neutrophil phils in nephritic CXCL5 / mice had a reduced capacity to exit

56 Journal of the American Society of Nephrology J Am Soc Nephrol 26: 55–66, 2015 www.jasn.org BASIC RESEARCH

the peritubular capillaries and to enter the tubulointerstitial space (Figure 1I). By comparison, the infiltration of T cells and macrophages/dendritic cells was not significantly affected by CXCL5 deficiency or anti-CXCL5 treatment (Supplemental Figure 2, A–H,J).Furthermore,neutro- phil, monocyte, CD4+,andCD8+ Tcell abundance in the blood, spleen, and kidney under homeostatic and nephritic condi- tions was not significant different between 2 2 wild-type (WT) and CXCL5 / mice (Sup- plemental Figure 3). In addition, kidney morphology and function were not affected by CXCL5 deficiency under basal conditions (Supplemental Figure 4). This indicates that reduced neutrophil abundance in the kidney 2 2 of nephritic CXCL5 / animals was not a consequence of a developmental defect in the hematopoietic system or the kidney, but rather represents reduced recruitment to 2 2 the inflamed tissue in CXCL5 / mice. Toanalyze the role of CXCL1 in the early infiltration of neutrophils into the kidney using a second, independent approach, we induced NTN in WT and CXCL1-deficient mice. In accordance with our anti-CXCL1 neutralization data, neutrophil recruitment 2 2 was reduced in nephritic CXCL1 / mice at day 3, thus underscoring the importance of this chemokine in the early course of nephri- tis (Figure 1, J and K).

5 per time point). (F) FACS analysis of renal 2 2 leukocytes from nephritic WT and CXCL5 / mice stained for the PMN marker Ly6G at day 14. Plots are representative of three in- dependent experiments. (G and H) Quantifi- cation of neutrophils from FACS analysis (G) and quantification of tubulointerstitial PMNs from GR1 stained kidney sections (H) in ne- 2 2 phritic WT and CXCL5 / mice 14 days after nephritis induction. (I) Immunofluorescence Figure 1. CXCL1 and CXCL5 have unique functions in the time- and compartment- staining of nephritic kidney section (day 14) of fi fi 2 2 speci cin ltration of neutrophils in crescentic GN. (A) Immunohistochemistry of kidney WT and CXCL5 / mice using anti-CD31 sections stained for the neutrophil marker GR1 at indicated time points after nephritis (green/endothelial cells), anti-Ly6G (red/ induction. Inserts demonstrate the polymorphonuclear morphology of GR1+ cells PMNs), and collagen type (blue/basement fi fi (neutrophils). (B) Quanti cation of tubulointerstitial (per low-power eld [lpf]) and membrane). (J and K) Quantification of renal fi glomerular (per glomerular cross-section [gcs]) PMN in ltration in the course of ne- neutrophils by FACS analysis (J) and quanti- phritis (n=3–6 per time point). (C) RT-PCR analysis of renal CXCL1, CXCL2, CXCL3, fication of glomerular PMNs from GR1 stained CXCL5, and CXCR2 mRNA expression (n=4–6 per time point). (D) Quantification of 2/2 kidney sections (K) in nephritic WT and 2 2 tubulointerstitial and glomerular PMNs in nephritic WT and CXCR2 mice at day 3 CXCL1 / mice 3 days after nephritis induction. or day 14 after induction of the disease (n=3–5 per time point). (E) Quantification of Symbols/bars represent means6SDs. *P,0.05; tubulointerstitial and glomerular PMNs in nephritic WT mice at day 3 or 14 of nephritis **P,0.01; ***P,0.001. Original magnification, – after administration of anti-CXCL1, anti-CXCL5, or isotype rat-IgG2B antibodies (n=3 3200. Con, control.

J Am Soc Nephrol 26: 55–66, 2015 CXCL5 Recruits Neutrophils in TH17 Response 57 BASIC RESEARCH www.jasn.org

2 2 collected from nephritic CXCL5 / mice (Figure 2B). In line with the CXCL5 mRNA expression pattern, b-Gal–positive cells were detected in the tubular epithe- lium of nephritic mice (Figure 2B). Quantitative RT-PCR analysis of glo- merular and tubulointerstitial tissue sam- ples isolated from nephritic mice using lasermicrodissectionatdays3and14after induction of NTN revealed that CXCL5 mRNA expression was predominantly upregulated at day 14 in the tubulointer- stitial compartment, as indicated by the tubulointerstitial to glomerular expres- sion ratio (Figure 2C). CXCL1 mRNAwas mainly expressed in glomeruli at day 3, whereas glomerular and tubulointersti- tial expression were at control levels at day 14 (Figure 2C). To study whether CXCL5 is mainly pro- duced by renal tissue cells, we generated bone marrow (BM) chimeras using WTand 2 2 CXCL5 / mice as donors (Figure 2D). In- dependent of the BM transplant used, 14 days after induction of nephritis, high CXCL5 mRNA expression levels were only present in animals with WT renal tis- sue cells. An approximately 500-fold in- crease in CXCL5 mRNA expression was found in both WT recipient mice trans- 2 2 planted with WT BM or CXCL5 / BM compared with control mice. By contrast, 2 2 CXCL5 mRNA expression in CXCL5 / 2 2 Figure 2. CXCL5 is expressed by kidney tubular cells. (A) Representative CXCL5 in situ mice receiving WT BM or CXCL5 / BM hybridizations of nephritic mice show that CXCL5 expression colocalizes with kidney b-Gal was not elevated (Figure 2D). As control, tubular cells (arrow) at day 14. (B) Kidney sections of nephritic WT and CXCL5 are we used WT mice carrying the CD45.2 al- stained for b-Gal with specific antibodies (AF488; green), wheat germ agglutinin – lele as recipients. Six weeks after transplan- (TexasRed; red), and nuclear marker draq5 (blue) (arrow, b-Gal positive cells; asterisk, . . tubulus lumen). (C) RT-PCR analysis of compartment-specific CXCL1 and CXCL5 ex- tation, 90% of blood leukocytes, 98% . pression (laser-microdissected glomeruli versus tubulointerstitium) at 24 hours and 14 of BM leukocytes, and 90% of splenic days after induction of nephritis. Data are presented as the ratio of glomerular to leukocytes expressed the phenotypic tubulointerstitial expression and vice versa (n=3 per group). (D) RT-PCR analysis of marker of the transplanted BM (n=6). renal CXCL5 expression in BM chimeric nephritic mice at day 14 (n=4–6 per group). Bars represent means6SDs. The TH17/IL-17 Immune Response Mediates Renal CXCL5 Expression in Crescentic GN Kidney Tubular Cells Produce CXCL5 in Late Crescentic The striking temporal association between CXCL5 expression, GN tubulointerstitial neutrophil infiltration (Figure 1, A–H), and + To localize renal CXCL5 expression, we performed in situ hy- the recruitment of CD4 TH17 cells into the kidney at day 10 bridization experiments. CXCL5 expression was clearly de- (Figure 3A, Supplemental Figure 5) suggest a functional re- tectable in tubular cells 14 days after nephritis induction, lationship. To determine whether IL-17A drives CXCL5 ex- 2 2 but not at day 3 or in control mice (Figure 2A). pression in vivo, we induced nephritis in IL-17A / mice. WT Replacement of the CXCL5 by a b-galactosidase animals showed strong upregulation of CXCL5 mRNA at day 2 2 (b-Gal) gene in CXCL5-deficient mice enabled us to indirectly 10 after nephritis induction, whereas IL-17A / mice dis- 2 2 assess the renal CXCL5 protein expression by b-Gal detection. played reduced upregulation (WT versus IL-17A / , Nuclear b-Gal staining was observed exclusively in tissues P,0.01) (Figure 3B). By contrast, CXCL1 and CXCL2

58 Journal of the American Society of Nephrology J Am Soc Nephrol 26: 55–66, 2015 www.jasn.org BASIC RESEARCH

(Supplemental Figure 6D). Although the in vivo importance of this finding remains to be elucidated, the time kinetic of renal IL-17A (Figure 3A) and TNF-a expression in NTN (Supplemental Figure 6E) is consistent with CXCL5 formation in the inflamed kidney.

Tubulointerstitial Infiltration of CXCR2+ Neutrophils Promotes Renal Tissue Injury To investigate the functional importance of the observed tubulointerstitial neutrophil recruitment, we depleted neutrophils in nephritic mice from days 9 to 14 by using a Ly6G-specificmAb16 (Supplemental Figure 7). Neutrophil depletion significantly reduced tubulointerstitial tissue injury and to a lesser degree glomerular crescent forma- tion, demonstrating the pathogenic role of neutrophils during the TH17 cell–mediated phase of the disease (Figure 4, A and B). Figure 3. IL-17A drives renal CXCL5 expression. (A) RT-PCR analysis of renal IL-17A Levels of BUN, a marker inversely corre- expression in the course of nephritis (n=5–6 per time point). RT-PCR analysis of IL-17A lated with renal function, were slightly but mRNA expression in FACS-sorted renal CD4+ T cells isolated from nephritic kidneys at not significantly reduced (Figure 4B). days 3, 5, 10, and 20. (B) RT-PCR analysis of chemokine mRNA expression in nephritic Next, we showed that CXCR2-deficient 2/2 WT (n=6) and nephritic IL-17A mice (n=6) at day 10. (C) RT-PCR for CXCL5 ex- mice developed less severe nephritis, in pression in murine kidney tubular cells upon stimulation with IL-17A (10 ng/ml), TNF-a termsof renal tissue damage andBUN levels fi (10 ng/ml), and IL-17A+TNF-a for 4 hours. (D) CXCL5-speci c ELISA from supernatant at day 14 (Figure 4C). This is in line with of stimulated tubular cells after 24 hours. Bars represent means6SDs. *P,0.05; reduced neutrophil infiltration in nephritic **P,0.01; ***P,0.001. Con, control. 2 2 CXCR2 / mice (Figure 1D). The recruit- ment of CD3+ T cells and mononuclear phagocytes was not significantly affected 2 2 expression was not significantly altered in nephritic IL-17A / by CXCR2 deficiency (Supplemental Figure 2I). Mouse anti- mice (Figure 3B). sheep globulin–specific IgG, subclass IgG1, subclass IgG2a, 2 2 and IgG2b titers were unaffected in nephritic CXCR2 / IL-17A and TNF-a Synergistically Induce CXCL5 mice compared with the WT group (Supplemental Figure 8F). Expression in Mouse Tubular Cells Given the high renal expression of CXCL5, predominantly in Targeting of CXCL5 Reduces Renal Tissue Damage in tubular cells (Figure 2, A and B), we investigated whether IL-17A the T Cell–Mediated Stage of Crescentic GN drives CXCL5 expression in kidney tubular cells. Because the To investigate whether CXCL5 contributes to renal tissue 2 2 biologic effects of IL-17A are mediated by activation of the injury, nephritis was induced in CXCL5 / and WT mice. At 2 2 IL-17 receptors A and C, we demonstrated the presence of day 14, CXCL5 / mice showed significantly less tubulointer- these receptors in mouse tubular cells by RT-PCR (Supple- stitial and glomerular injury compared with controls (Figure mental Figure 6A). Next, we demonstrated that IL-17A in- 4, D and E). In addition, BUN levels were reduced in nephritic 2 2 duces CXCL5 mRNA and protein production in tubular CXCL5 / mice compared with nephritic WT mice at day 14 cells in a dose-dependent manner (Supplemental Figure (P,0.001; Figure 4E). Albuminuria was upregulated in all 6B), whereas IL-17F and IFN-g had no effect (Supplemental nephritic groups, but was not significantly different between Figure 6C). nephritic WT and CXCL5 KO mice at day 14 (urine albumin/ The signal transduction pathway mediated by IL-17A is still creatinine ratio: WT control [n=6], 0.160.1; WT NTN [n=8], 2 2 incompletely characterized but involves NF-kB and extracellular- 15.864.7; CXCL5 / NTN [n=10], 9.768.9). signal regulated kinase (ERK) activation.15 Therefore, it is of Treatment of nephritic micewith a neutralizinganti-CXCL5 interest that the combination of IL-17A and TNF-a synergis- antibody at day 9 significantly reduced renal tissue injury at day tically amplified the RNA expression and protein secretion 14 (Figure 4F). The more pronounced protection of nephritic 2 2 of CXCL5 (Figure 3, C and D), and synergistically induced CXCL5 / mice compared with anti-CXCL5–treated animals, ERK activation but not NF-kB signaling in tubular cells in terms of BUN levels, is likely the result of a complete

J Am Soc Nephrol 26: 55–66, 2015 CXCL5 Recruits Neutrophils in TH17 Response 59 BASIC RESEARCH www.jasn.org

absence of CXCL5 in the KO mice and the timely restricted CXCL5 neutralization from days 9 to 14. The beneficial effect of neutrophil and CXCL5 targeting on glo- merular injury might reflect reduced renal PMN infiltration. By contrast, the treatment of nephritic mice with a neutralizing anti-CXCL1 anti- body at day 9 (nephritic controls received IgG2a isotype control antibodies) had no effect on the clinical outcome of the disease at day 14 (Figure 4G). Serum titers of anti-sheep IgG, IgG1, IgG2a/c, and IgG2b antibodies directed against the nephritogenic antigen and semi- quantitative scoring of glomerular sheep and mouse IgG deposition revealed no differences between nephritic WT and 2 2 CXCL5 / mice (Supplemental Figure 8, A–E). Moreover, IL-17 and IFN-g produc- tion of sheep IgG-stimulated splenocytes is not impaired in nephritic CXCL5 KO mice compared with nephritic WT mice at day 14 (Supplemental Figure 9), indicating that the absence of CXCL5 did not impair the systemic immune response against the nephritogenic antigen.

CXCL5 Is Dispensable for Neutrophil Recruitment and Bacterial Clearance in a Murine Model of Acute Bacterial Pyelonephritis We next studied the role of CXCL5 in neutrophil trafficking and function in awell established model of acute bacterial pyelo-

renal tissue damage and renal function as- sessed by BUN levels at day 14 in WT and 2 2 CXCR2 / mice. (D) Representative photo- graphs (PAS staining) of the tubulointerstitial compartment of control, nephritic WT, and 2 2 nephritic CXCL5 / mice at day 14. (E) Quantification of glomerular crescent forma- tion, tubulointerstitial damage, BUN levels, and renal neutrophil recruitment 14 days after disease induction. (F and G) Quantification of glomerular crescent formation, tubulointer- stitial damage, BUN levels, and neutrophil infiltration in animals treated with anti-CXCL5 (or IgG2B isotype antibody) (F) and anti- CXCL1 (or IgG2A isotype antibody) (G) 14 Figure 4. The CXCL5/CXCR2 axis promotes renal tissue injury. (A) Representative days after disease induction. Bars represent photographs of PAS-stained kidney sections of mice6neutrophil depletion (from days means6SDs. *P,0.05; **P,0.01; ***P,0.001. 9 to 14) at day 14 after induction of nephritis. (B) Quantification of glomerular crescent Original magnification, 3200 in A; 3400 in D. formation, tubulointerstitial damage, and BUN levels at day 14. (C) Quantification of Con, control; PAS, periodic acid–Schiff.

60 Journal of the American Society of Nephrology J Am Soc Nephrol 26: 55–66, 2015 www.jasn.org BASIC RESEARCH

was detected in WT mice 24 hours after UPEC infection (Figure 5, A and B). Of note, neutrophil recruitment was not af- fected by CXCL5 deficiency, whereas neu- trophils were strongly reduced in infected CXCR2-deficient mice. In line with the pivotal role of neutrophils in bacterial clearance, the numbers of CFUs in the kid- 2 2 neys were significantly higher in CXCR2 / 2 2 mice compared with WTand CXCL5 / an- imals (Figure 5C). Renal expression of CXCL1, and to a lesser degree of CXCL2, was markedly enhanced, whereas CXCL5 was only slightly upregulated 24 hours after infection (Figure 5D).

Renal CXCL5 Expression in Patients with ANCA-Associated GN and Acute Bacterial Pyelonephritis We isolated RNA from renal biopsies and performed real-time PCR to analyze the renal expression of neutrophil-attracting chemokines in patients with acute ANCA- associated GN, the most common form of Figure 5. CXCL5 is dispensable for neutrophil recruitment and bacterial clearance in crescentic GN in humans, and in patients a murine model of acute bacterial pyelonephritis. (A) Representative immunohisto- with acute bacterial pyelonephritis. Al- chemistry for the neutrophil marker GR-1 24 hours after transurethral instillation of + HI 2 though neutrophil recruitment is common UPEC. (B) Quantification of FACS analyses of renal PMNs (CD45 ,GR-1 ,F4/80 , 2 CD11c ), (numbers represent total cells per kidney; n=5–6 per group). (C) CFU per to ANCA-associated GN but even stronger kidney at 24 hours (n=5–6). (D) RT-PCR analysis of renal CXCL1, CXCL2, and CXCL5 in acute pyelonephritis (Figure 6A), upre- expression 24 hours after UPEC infection. Bars/symbols represent means6SDs. gulation of renal tissue CXCL5 expression *P,0.05; **P,0.01; *** P,0.001. Original magnification, 3400. was more profound in ANCA-associated GN (1013-fold; Figure 6B) compared with acute pyelonephritis (16-fold; Figure 6B). Similar results were obtained for CXCL6, which is a close homolog to human CXCL5.6 These results suggest a predomi- nant role of CXCL5 in human autoimmune disease, but not in acute bacterial infection. Table 1 provides the clinical characteristics of the included patients.

DISCUSSION

Figure 6. CXCL5 expression in patients with ANCA-associated GN and acute bacterial pyelonephritis. (A) Representative PMN staining (proteinase 3) in renal biopsies of In recent years, a large number of studies patients with ANCA-associated GN and acute bacterial pyelonephritis. (B) RT-PCR have established the crucial role of TH17 analysis of renal chemokine mRNA expression (n=6–8 per group). Bars represent cells in the pathogenesis of autoimmune – means6SDs. *P,0.05. Original magnification, 3990. diseases,7,18 20 including proliferative and crescentic GN.3,11,21,22 Consequently, the findings from animal models have been nephritis. This model is induced by transurethral instillation of translated into clinical practice, and IL-17A and related cyto- the uropathogenic Escherichia coli strain 536 (UPEC),17 which kines have been successfully targeted for treatment of psoria- invades the kidney and is subsequently cleared by recruited sis23 and Crohn’s disease.24 neutrophils. A massive infiltration of neutrophils into the kid- However, the mechanisms by which the TH17 immunity ney (assessed by immunohistochemistry and flow cytometry) directly contributes to end-organ damage still remain to be

J Am Soc Nephrol 26: 55–66, 2015 CXCL5 Recruits Neutrophils in TH17 Response 61 BASIC RESEARCH www.jasn.org

Table 1. Clinical characteristics of patients with acute ANCA-associated GN and bacterial pyelonephritis PR3/MPO Serum Creatinine Proteinuria Immunosuppression Patient Age (yr) Sex ANCA Type ELISA (U/ml) (mg/dl) (mg/24 h) before Biopsy 1 70 Man pANCA MPO 32.6 2.7 5550 No 2 67 Man pANCA MPO 17.2 1.6 1130 No 3 62 Man pANCA MPO 34 2.5 580 Steroid 4 83 Man cANCA PR3 109 4.8 Anuric Steroid + CyP 5 47 Man cANCA PR3 26.5 1.6 3000 No 6 41 Woman cANCA PR3 .200 1.7 1730 No 7 18 Woman cANCA PR3 .200 0.6 430 No 8 76 Man cANCA PR3 .200 3.2 760 Steroid 9 80 Woman Negative Not assessed 5.5 900 Steroid 10 39 Woman Negative Not assessed 1.2 Not assessed No 11 64 Woman Negative Not assessed 4.8 Not assessed No 12 71 Man Negative Not assessed 5.0 490 No 13 50 Man Negative Not assessed 10.2 .300 No 14 50 Man Negative Not assessed 6.8 .3000 No Clinical characteristics of patients with acute ANCA-associated GN (patients 1–8) and acute pyelonephritis (patients 9–14) at the time of renal biopsy. pANCA, perinuclear ANCA; cANCA, cytoplasmic ANCA; PR3, proteinase 3; MPO, myeloperoxidase; CyP, cypionate.

fully elucidated. TH17 cells produce a variety of proinflamma- before infiltration of the first TH17 cells. By striking contrast, tory , including IL-17A, IL-17F, IL-22, TNF-a,and CXCL5 expression peaked 10 days after induction of the dis- GM-CSF, which drive the recruitment of neutrophils to sites ease and was predominantly expressed by resident tubular of inflammation and thereby promote tissue injury by mech- cells when the renal TH17 immune response had reached its anisms that are thus far incompletely understood.7 Recent maximum level. In line with this, IL-17A resulted in a strong studies indicate that ELR+ chemokines play a pivotal role in upregulation of CXCL5 mRNA and protein expression in mu- this process and might therefore represent an attractive target rine tubular epithelial cells in vitro, which is further enhanced for the prevention of neutrophil-induced tissue injury.25–27 in the presence of TNF-a, potentially as a consequence of This approach is complicated by an often overlapping/redun- synergistic activation of the ERK signaling pathway. This find- dant expression pattern and function of chemokines in auto- ing is in accordance with a recent report by Liu et al. showing immune and infectious disease.5 Targeting of chemokines in that CXCL5 mRNA is induced by IL-17A in alveolar epithelial 26 order to reduce pathogenic neutrophil infiltration in TH17- cells. To further support our hypothesis that IL-17A (pro- mediated autoimmunity might therefore impair defense duced by renal TH17 cells) is directly involved in the induction against microbial pathogens. of CXCL5 expression in tubular cells in vivo, the generation of Recent data by the Chou et al.andMcDonaldet al. suggest mice that are deficient in the IL-17 receptors A and C or in the that neutrophil chemoattractants might have less redundant IL-17 receptor adaptor protein connection to IkBkinaseand functions in the recruitment of neutrophils in vivo than pre- stress-activated protein kinases,34 specifically in renal tubular 28,29 viously thought. To investigate the role of the TH17 im- cells, would be of great interest. mune response for neutrophil recruitment and function in IL-17A promoted renal CXCL5 expression and neutrophil detail, we performed time-kinetic analyses in a well character- infiltration in NTN, whereas CXCL1 and CXCL2 expression 30 ized TH17-dependent model of crescentic GN (NTN). Al- was IL-17A independent. These data further point to non- though NTN does not represent a human disease in all aspects redundant roles of CXCR2 ligands in the attraction of of its pathophysiology (e.g., with respect to immune complex– neutrophils to the kidney, most likely resulting from the GN, anti-glomerular basement membrane nephritis, or temporal and spatial segregation of their expression. CXCL1 ANCA-associated GN), it remains one of the most efficient and CXCL5 in vivo neutralization experiments indeed con- mouse model of crescentic GN.31,32 Neutrophils showed a firmed that early glomerular neutrophil migration is mainly time- and compartment-specific migratory behavior during driven by CXCL1, whereas the second wave of interstitial neu- crescentic GN, suggesting that the interstitial “second wave” of trophil infiltration is strongly dependent on IL-17A–induced neutrophil infiltration is promoted by renal TH17 cells. CXCL5. Our finding is consistent with a recent study in a 2 2 It is generally thought that IL-17A, the major effector model of moderate airway inflammation in CXCL5 / mice cytokine of the TH17 immune response, induces the expres- showing that neutrophil infiltration into the inflamed tissue sion of all neutrophil-attracting ELR+ chemokines in a redun- was dependent on CXCL5.27 In contrast with a study by Mei dant manner.25,26,33 In murine crescentic GN, however, we et al. that indicates that enterocyte-derived CXCL5 in the gut unexpectedly found that the expression of CXCL1 and regulates local IL-17A levels and contributes to CXCR2-dependent CXCL2 was upregulated until day 3 and then declined even neutrophil homeostasis,35 our study did not show any

62 Journal of the American Society of Nephrology J Am Soc Nephrol 26: 55–66, 2015 www.jasn.org BASIC RESEARCH indication of a role for CXCL5 in the kidney under noninflam- Biology (Berlin, Germany)43 and CXCL1-deficient mice were provided matory conditions. The time-dependent role of CXCL5 in GN by S. Lira (Mount Sinai Hospital, New York, NY). All KO mice were 2 2 is further emphasized by the finding that CXCL5 / mice and backcrossed to the C57BL/6 background for at least 10 generations. All anti-CXCL5–treated WTanimals displayed an ameliorated dis- mice were raised under specific pathogen-free conditions. Animal ex- ease course at day 14, but not at day 3. periments were performed according to national animal care and ethical To determine whether the blockade of CXCL5 and CXCR2 guidelines, and were approved by local ethical committees. generally interfereswithneutrophilinfiltration,weinducedacute 2/2 2/2 bacterial pyelonephritis in WT, CXCL5 , and CXCR2 Induction of NTN and Functional Studies mice. In this model, the antibacterial defense relies on the rapid NTN was induced in male mice aged 8 to 10 weeks by intraperitoneal recruitment of neutrophils. As reported, neutrophil trafficking injection of 0.6 ml of nephrotoxic sheep serum per mouse, as and bacterial clearance critically depend on CXCR2, which is previously described.30 Controls were injected intraperitoneally with most likely activated by locally expressed CXCL1 and an equal amount of nonspecific sheep IgG. Urinary albumin excre- CXCL2,17,36–39 making CXCR2 an unfavorable candidate for tion was determined by standard ELISA analysis (Mice-Albumin Kit; therapeutic targeting in autoimmunity. By contrast, CXCL5 Bethyl, Montgomery, TX), whereas urinary creatinine and serum deficiency did not affect early neutrophil infiltration and bac- BUN were measured using standard laboratory methods. For neu- terial clearance in this model of acute bacterial pyelonephritis. tralization/depletion experiments, anti–Ly6G-Ab (clone 1A8),16 A recent study in a murine model of ANCA-associated GN anti-CXCL1 Ab (clone 48415; R&D Systems, Minneapolis, MN),14 demonstrated that IL-17A–producing TH17 effector cells di- or anti-CXCL5 Ab (clone 61905; Leinco Technologies, St. Louis, rectly induce renal inflammation by effector responses involv- MO)14 was used at 100 mg per mouse (intraperitoneally). Nephritic ing neutrophils.40 Moreover, the first studies in human control mice in interventional studies received 100 mgratIgG2A ANCA-associated GN suggest a prominent role for TH17/IL- (clone 54447; R&D Systems) or rat IgG2B (clone 61905; Leinco Tech- 17 responses in this form of crescentic GN.4,41 To examine nologies) antibodies intraperitoneally. Anti-CXCL1 and anti-CXCL5 whether our findings are relevant to human disease, we as- antibodies were given either the day before NTN induction in the case sessed the renal CXCL5 mRNA expression in patients with of early neutralization studies (analysis was performed at day 3) or at acute ANCA-associated GN and acute bacterial pyelonephri- day 9 in the late stages of the interventional experiments (analysis was tis, which are both characterized by neutrophil recruitment. In performed at day 14). line with the results from our animal models, CXCL5 expres- sion was much higher in renal biopsy specimens from patients Acute Bacterial Pyelonephritis Model with autoimmune-mediated ANCA-associated GN compared Female mice aged 8 to 12 weeks were infected by transurethral with those with acute bacterial pyelonephritis. Interestingly, instillation of 13109 UPEC. Three hours later, the procedure was van der Veen et al. recently reported an increased renal CXCL5 repeated to induce pyelonephritis. Twenty-four hours after infection, expression in a murine model of anti-myeloperoxidase IgG/ the number of ascended bacteria was quantified by scoring CFUs after LPS-induced crescentic GN, further supporting the role of overnight culture of kidney collagenase digest as previously de- CXCL5 in autoimmune-mediated ANCA disease.42 The sim- scribed.17 ilar expression pattern of CXCL1, CXCL2, and CXCL5 during the course of the disease (assessed at two time points) might Real-Time RT-PCR Analyses be a consequence of the coapplication of LPS in this model. Total RNA of the renal cortex was prepared according to standard In conclusion, our study provides the first evidence for a laboratory methods. RNA from microdissected tissues was isolated previously unknown nonredundant effector mechanism of the using the RNA Nano prep kit (PALM, Bernried, Germany) as 44 TH17 immune response in experimental crescentic GN involv- previously described. Real-time PCR was performed on a StepO- ing T cell–derived IL-17A–,CXCL5–, and CXCR2–bearing nePlus Real-Time PCR system (Applied Biosystems, Foster City, CA). neutrophils that promote renal tissue injury. By contrast, CXCL5 seems to be dispensable for maintaining neutrophil- In Situ Hybridization mediated innate immune surveillance. However, the potential In situ hybridization was performed as described.45 In brief, the renal functional benefit of an intervention to target CXCL5 in CXCL5 cRNA probe was labeled with a[35S]UTP (1250 Ci/mmol; established crescentic GN remains to be fully elucidated. PerkinElmer) of subcloned cDNA corresponding to nucleotides 208– 532 of cDNA sequence NM_009141.2. In situ hybridization was per- formed on 16-mm cryosections using the 35S-labeled antisense and CONCISE METHODS sense RNA probes. Sections were dipped into Kodak NTB nuclear track emulsion and exposed for 3 weeks; after development, sections Animals were stained with Mayer’shemalaun. 2 2 2 2 CXCR2 / and IFN-g / mice were purchased from The Jackson Laboratory (Bar Harbor, ME). IL-17A–deficient mice were provided Morphologic Analyses by Yoichiro Iwakura (University of Tokyo, Tokyo, Japan), CXCL5- Glomerular crescent formation was assessed in 30 glomeruli per deficient mice were provided by the Max Plank Institute for Infection mouse in a blinded fashion in paraffin sections stained with periodic

J Am Soc Nephrol 26: 55–66, 2015 CXCL5 Recruits Neutrophils in TH17 Response 63 BASIC RESEARCH www.jasn.org acid–Schiff.45 To assess tubulointerstitial injury, photographs of Culture of Mouse Kidney Epithelial Tubular Cells and four nonoverlapping cortical areas from kidney sections stained Stimulation with periodic acid–Schiff were taken per mouse and counted in Mouse kidney tubular cells47 were cultured in DMEM with 3% FCS low-magnification fields (3200). The interstitial area was then de- (Gibco, Eggenstein, Germany) and stimulated with varying concen- termined by superimposing the photographs with a grid containing trations of IL-17A, IL-17F, TNF-a,andIFN-g, as indicated (all from 40 points and by counting the matches of points with interstitial Pepro Tech, Hamburg, Germany). CXCL5 mRNA expression levels tissue (excluding glomeruli, blood vessels, and tubules) in a blinded were analyzed after 4 hours of incubation. Protein levels were deter- fashion. Dividing the positive matches of four photographs by 160 mined after 8 and 24 hours in the supernatants by specificCXCL5 provided the percentage of positive tissue equal to the interstitial ELISA (R&D Systems). Antibodies to detect NF-kB p65 (D14E12) injury score.11,45 and phospho-NFk-B p65 (93H1) as well as ERK and phospho-ERK For immunohistochemical stainings, paraffin-embedded sec- (D13.14.4E) in immunoblots were from Technology tions were deparaffinized, rehydrated, and stained with the follow- (Beverly, MA). ing antibodies: GR-1 (1:50, Ly6 G/C, NIMP-R14; Hycult Biotech, Uden, The Netherlands), CD3 (1:1000, A0452; Dako,Germany), F4/ Laser Microdissection 80 (1:400, BM8; BMA, Germany), MAC-2 (1:1000, M3/38; Cedarlane, Microdissection was carried out on mouse kidney tissue using the ON, Canada), sheep IgG/mouse IgG (both Jackson Immunoresearch PALM MicroBeam IP 230V Z microscope.44 Under direct visual Laboratories), and proteinase 3 to detect PMN in humans. Antigen control, selected tissue compartments (glomeruli or cortical tubu- retrieval was performed by incubation with proteinase type XXIV lointerstitium) were dissected. RNA from microdissected tissue was (5 mg/ml; Sigma-Aldrich, St. Louis, MO) for 15 minutes at 37°C prepared using the PALM RNA extraction kit. (F4/80, GR1, sheep IgG/mouse IgG) or by microwave antigen retriev- al in citrate buffer (DAKO 52367), pH 6.1, for 25 minutes (CD3, BM Transplantation 2/2 MAC-2). Nuclear b-Gal was revealed after protease-mediated antigen Male CXCL5 or WT mice aged 5 to 6 weeks received 9.5 Gy total 2/2 retrieval using a polyclonal antibody (Europa Bioproducts, Cam- body irradiation. Each recipient mouse (WT or CXCL5 ) was in- 6 2/2 bridge, UK). Slides were counterstained with wheat germ agglutinin travenously injected with 5310 BM cells (WTor CXCL5 ) within (Vector, Auckland, New Zealand) and draq 5 (Molecular Probes, Eu- 6 hours of irradiation.48 The efficiency of BM replacement was as- gene, OR). Tissue sections were developed with the Vectastain ABC- sessed using congenic CD45 mouse strains (CD45.1 and CD45.2). AP kit (Vector Laboratories, Burlingame, CA). Glomerular GR1+, After irradiation and transplantation, .90% of circulating leuko- MAC-2+,andCD3+cells in 30 glomerular cross-sections and tubu- cytes, .98% of BM leukocytes, and .90% of splenic leukocytes ex- lointerstitial F4/80+ and CD3+ cells in 30 high-power fields (3400) pressed the phenotypic marker of the transplanted BM (n=6; data not per kidney were counted in a blinded manner. To quantify tubuloin- shown). NTN was induced 4 weeks after BM transplantation. terstitial GR1+ cells, at least 20 low-power fields (3200) were counted. All slides were evaluated under an Axioskop light microscopy (Zeiss, RNA Isolation and RT-PCR Analysis in Human Patients Jena, Germany) and photographed with an Axiocam HRc (Zeiss) or by RNA was isolated with the RNA Micro Kit (Roche) from paraffin- confocal microscopy with a LSM 510 meta microscope using the LSM embedded renal specimens of patients with ANCA-associated GN or software. acute bacterial pyelonephritis. Chemokine expressionwas analyzed by RT-PCR. Baseline kidney allograft biopsies before transplantation (without significant pathology) served as controls. All samples were Isolation of Leukocytes from Kidney and Spleen run in duplicate and were normalized to 18S ribosomal RNA to Previously described methods for leukocyte isolation from murine account for small RNA and cDNA variability. Human analysis was 30 fi kidneys were used. In brief, kidneys were nely minced and diges- approved by the local ethics committee (PV3162). ted for 45 minutes at 37°C with 0.4 mg/ml collagenase D (Roche, Mannheim, Germany). Spleens were minced to isolate splenocytes. Statistical Analyses fi m Both cell suspensions were sequentially ltered through 70- mand Data were expressed as the means6SDs. All statistical analyses were m 2+ 2+ 40- m nylon meshes and washed with HBSS without Ca and Mg performed with the SPSS package and GraphPad Prism 5 software. (Invitrogen). Cell viability was assessed by trypan blue staining be- P,0.05 was considered statistically significant. The t test was used for fl fore ow cytometry or cell transfer experiments. comparison between two groups. In the case of three or more groups, one-way ANOVA was used, followed by a post hoc analysis with ’ Flow Cytometry Bonferroni s test for multiple comparisons. The following antibodies were used for FACS analysis: CD45 (30-F11), CD11b (M1/70), CD11c (HL3), Ly6G (1A8), F4/80 (BM8), MHCII (AF6-120.1), CD3 (506A2), and IL-17A (TC11-18H10) (BD Bio- ACKNOWLEDGMENTS sciences, San Jose, CA; eBioscience, San Diego, CA; or R&D Sys- tems).46 Analyses were performed on a B&D LSRII system using This study was supported by grants from the German Research Diva software and were analyzed using Flowjo software (Treestar, Foundation (KFO 228: PA 754/7-2 to U.P. and C.F.K.; MI 476/4-2 to Ashland, OR). H.-W.M.).

64 Journal of the American Society of Nephrology J Am Soc Nephrol 26: 55–66, 2015 www.jasn.org BASIC RESEARCH

DISCLOSURES 18. Jones SA, Sutton CE, Cua D, Mills KH: Therapeutic potential of tar- – None. geting IL-17. Nat Immunol 13: 1022 1025, 2012 19. Turner JE, Paust HJ, Steinmetz OM, Panzer U: The Th17 immune re- sponse in renal inflammation. Kidney Int 77: 1070–1075, 2010 REFERENCES 20. Dong X, Bachman LA, Miller MN, Nath KA, Griffin MD: Dendritic cells facilitate accumulation of IL-17 T cells in the kidney following acute renal obstruction. Kidney Int 74: 1294–1309, 2008 1. Kolaczkowska E, Kubes P: Neutrophil recruitment and function in health 21. Kitching AR, Holdsworth SR: The emergence of TH17 cells as effectors fl – and in ammation. Nat Rev Immunol 13: 159 175, 2013 of renal injury. JAmSocNephrol22: 235–238, 2011 2. Eyles JL, Hickey MJ, Norman MU, Croker BA, Roberts AW, Drake SF, 22. Tulone C, Giorgini A, Freeley S, Coughlan A, Robson MG: Transferred James WG, Metcalf D, Campbell IK, Wicks IP: A key role for G-CSF- antigen-specific T(H)17 but not T(H)1 cells induce crescentic glomer- fi fl induced neutrophil production and traf cking during in ammatory ar- ulonephritis in mice. Am J Pathol 179: 2683–2690, 2011 – thritis. Blood 112: 5193 5201, 2008 23. Papp KA, Leonardi C, Menter A, Ortonne JP, Krueger JG, Kricorian G, 3. Summers SA, Steinmetz OM, Li M, Kausman JY, Semple T, Edgtton KL, Aras G, Li J, Russell CB, Thompson EH, Baumgartner S: Brodalumab, an Borza DB, Braley H, Holdsworth SR, Kitching AR: Th1 and Th17 cells anti--17-receptor antibody for psoriasis. NEnglJMed366: – induce proliferative glomerulonephritis. JAmSocNephrol20: 2518 1181–1189, 2012 2524, 2009 24. Sandborn WJ, Gasink C, Gao LL, Blank MA, Johanns J, Guzzo C, Sands 4. Velden J, Paust HJ, Hoxha E, Turner JE, Steinmetz OM, Wolf G, Jabs BE, Hanauer SB, Targan S, Rutgeerts P, Ghosh S, de Villiers WJ, WJ, Özcan F, Beige J, Heering PJ, Schröder S, Kneißler U, Disteldorf E, Panaccione R, Greenberg G, Schreiber S, Lichtiger S, Feagan BG; Mittrücker HW, Stahl RA, Helmchen U, Panzer U: Renal IL-17 expression CERTIFI Study Group: Ustekinumab induction and maintenance in human ANCA-associated glomerulonephritis. Am J Physiol Renal therapy in refractory Crohn’sdisease.NEnglJMed367: 1519–1528, Physiol 302: F1663–F1673, 2012 2012 5. Sallusto F, Baggiolini M: Chemokines and leukocyte traffic. Nat Im- 25. Laan M, Cui ZH, Hoshino H, Lötvall J, Sjöstrand M, Gruenert DC, munol 9: 949–952, 2008 Skoogh BE, Lindén A: Neutrophil recruitment by human IL-17 via C-X-C 6. Zlotnik A, Yoshie O: The chemokine superfamily revisited. Immunity 36: chemokine release in the airways. J Immunol 162: 2347–2352, 1999 705–716, 2012 26. Liu Y, Mei J, Gonzales L, Yang G, Dai N, Wang P, Zhang P, Favara M, 7. Korn T, Bettelli E, Oukka M, Kuchroo VK: IL-17 and Th17 cells. Annu Rev Malcolm KC, Guttentag S, Worthen GS: IL-17A and TNF-a exert syn- Immunol 27: 485–517, 2009 ergistic effects on expression of CXCL5 by alveolar type II cells in vivo 8. Fogli LK, Sundrud MS, Goel S, Bajwa S, Jensen K, Derudder E, Sun A, and in vitro. J Immunol 186: 3197–3205, 2011 Coffre M, Uyttenhove C, Van Snick J, Schmidt-Supprian M, Rao A, 27. Mei J, Liu Y, Dai N, Favara M, Greene T, Jeyaseelan S, Poncz M, Lee JS, Grunig G, Durbin J, Casola SS, Rajewsky K, Koralov SB: T cell-derived Worthen GS: CXCL5 regulates chemokine scavenging and pulmonary IL-17 mediates epithelial changes in the airway and drives pulmonary host defense to bacterial infection. Immunity 33: 106–117, 2010 neutrophilia. JImmunol191: 3100–3111, 2013 28. Chou RC, Kim ND, Sadik CD, Seung E, Lan Y, Byrne MH, Haribabu B, 9. Schall TJ, Proudfoot AE: Overcoming hurdles in developing successful Iwakura Y, Luster AD: Lipid-cytokine-chemokine cascade drives neu- drugs targeting chemokine receptors. Nat Rev Immunol 11: 355–363, trophil recruitment in a murine model of inflammatory arthritis. Immu- 2011 nity 33: 266–278, 2010 10. Devi S, Li A, Westhorpe CL, Lo CY, Abeynaike LD, Snelgrove SL, Hall P, 29. McDonald B, Pittman K, Menezes GB, Hirota SA, Slaba I, Waterhouse Ooi JD, Sobey CG, Kitching AR, Hickey MJ: Multiphoton imaging CC, Beck PL, Muruve DA, Kubes P: Intravascular danger signals guide reveals a new leukocyte recruitment paradigm in the glomerulus. Nat fl – Med 19: 107–112, 2013 neutrophils to sites of sterile in ammation. Science 330: 362 366, 2010 11. Paust HJ, Turner JE, Riedel JH, Disteldorf E, Peters A, Schmidt T, Krebs 30. Paust HJ, Turner JE, Steinmetz OM, Peters A, Heymann F, Hölscher C, C, Velden J, Mittrücker HW, Steinmetz OM, Stahl RA, Panzer U: Che- Wolf G, Kurts C, Mittrücker HW, Stahl RA, Panzer U: The IL-23/Th17 axis mokines play a critical role in the cross-regulation of Th1 and Th17 contributes to renal injury in experimental glomerulonephritis. JAm – immune responses in murine crescentic glomerulonephritis. Kidney Int Soc Nephrol 20: 969 979, 2009 82: 72–83, 2012 31. Couser WG: Basic and translational concepts of immune-mediated – 12. Odobasic D, Gan PY, Summers SA, Semple TJ, Muljadi RC, Iwakura Y, glomerular diseases. JAmSocNephrol23: 381 399, 2012 Kitching AR, Holdsworth SR: Interleukin-17A promotes early but at- 32. Kurts C, Panzer U, Anders HJ, Rees AJ: The immune system and kidney tenuates established disease in crescentic glomerulonephritis in mice. disease: Basic concepts and clinical implications. Nat Rev Immunol 13: – Am J Pathol 179: 1188–1198, 2011 738 753, 2013 fi 13. Heymann F, Meyer-Schwesinger C, Hamilton-Williams EE, Hammerich 33. Sadik CD, Kim ND, Alekseeva E, Luster AD: IL-17RA signaling ampli es L, Panzer U, Kaden S, Quaggin SE, Floege J, Gröne HJ, Kurts C: Kid- antibody-induced arthritis. PLoS ONE 6: e26342, 2011 ney dendritic cell activation is required for progression of renal disease 34. Pisitkun P, Ha HL, Wang H, Claudio E, Tivy CC, Zhou H, Mayadas TN, in a mouse model of glomerular injury. J Clin Invest 119: 1286–1297, Illei GG, Siebenlist U: Interleukin-17 cytokines are critical in de- 2009 velopment of fatal lupus glomerulonephritis. Immunity 37: 1104–1115, 14. Pickens SR, Chamberlain ND, Volin MV, Gonzalez M, Pope RM, 2012 Mandelin AM 2nd, Kolls JK, Shahrara S: Anti-CXCL5 therapy amelio- 35. Mei J, Liu Y, Dai N, Hoffmann C, Hudock KM, Zhang P, Guttentag SH, rates IL-17-induced arthritis by decreasing joint vascularization. An- Kolls JK, Oliver PM, Bushman FD, Worthen GS: Cxcr2 and Cxcl5 reg- giogenesis 14: 443–455, 2011 ulate the IL-17/G-CSF axis and neutrophil homeostasis in mice. JClin 15. Gaffen SL: Recent advances in the IL-17 cytokine family. Curr Opin Invest 122: 974–986, 2012 Immunol 23: 613–619, 2011 36. Hang L, Haraoka M, Agace WW, Leffler H, Burdick M, Strieter R, 16. Daley JM, Thomay AA, Connolly MD, Reichner JS, Albina JE: Use of Svanborg C: Macrophage inflammatory protein-2 is required for neu- Ly6G-specific monoclonal antibody to deplete neutrophils in mice. J trophil passage across the epithelial barrier of the infected urinary tract. Leukoc Biol 83: 64–70, 2008 JImmunol162: 3037–3044, 1999 17. Tittel AP, Heuser C, Ohliger C, Knolle PA, Engel DR, Kurts C: Kidney 37. Svensson M, Irjala H, Svanborg C, Godaly G: Effects of epithelial and dendritic cells induce innate immunity against bacterial pyelonephritis. neutrophil CXCR2 on innate immunity and resistance to kidney in- JAmSocNephrol22: 1435–1441, 2011 fection. Kidney Int 74: 81–90, 2008

J Am Soc Nephrol 26: 55–66, 2015 CXCL5 Recruits Neutrophils in TH17 Response 65 BASIC RESEARCH www.jasn.org

38. Svensson M, Yadav M, Holmqvist B, Lutay N, Svanborg C, Godaly G: Compartment-specific expression and function of the chemokine IP- Acute pyelonephritis and renal scarring are caused by dysfunctional 10/CXCL10 in a model of renal endothelial microvascular injury. JAm innate immunity in mCxcr2 heterozygous mice. Kidney Int 80: 1064– Soc Nephrol 17: 454–464, 2006 1072, 2011 45. Riedel JH, Paust HJ, Turner JE, Tittel AP, Krebs C, Disteldorf E, 39. Frendéus B, Godaly G, Hang L, Karpman D, Lundstedt AC, Svanborg C: Wegscheid C, Tiegs G, Velden J, Mittrücker HW, Garbi N, Stahl RA, receptor deficiency confers susceptibility to acute ex- Steinmetz OM, Kurts C, Panzer U: Immature renal dendritic cells recruit perimental pyelonephritis and may have a human counterpart. JExp regulatory CXCR6(+) invariant natural killer T cells to attenuate cres- Med 192: 881–890, 2000 centic GN. JAmSocNephrol23: 1987–2000, 2012 40. Gan PY, Steinmetz OM, Tan DS, O’Sullivan KM, Ooi JD, Iwakura Y, 46. Turner JE, Krebs C, Tittel AP, Paust HJ, Meyer-Schwesinger C, Kitching AR, Holdsworth SR: Th17 cells promote autoimmune anti- Bennstein SB, Steinmetz OM, Prinz I, Magnus T, Korn T, Stahl RA, myeloperoxidase glomerulonephritis. J Am Soc Nephrol 21: 925–931, Kurts C, Panzer U: IL-17A production by renal gd T cells promotes 2010 kidney injury in crescentic GN. JAmSocNephrol23: 1486–1495, 41. van Timmeren MM, Heeringa P: Pathogenesis of ANCA-associated 2012 vasculitis: Recent insights from animal models. Curr Opin Rheumatol 47. Wolf G, Mueller E, Stahl RA, Ziyadeh FN: Angiotensin II-induced hy- 24: 8–14, 2012 pertrophy of cultured murine proximal tubular cells is mediated by 42. van der Veen BS, Petersen AH, Belperio JA, Satchell SC, Mathieson PW, endogenous transforming -beta. J Clin Invest 92: 1366– Molema G, Heeringa P: Spatiotemporal expression of chemokines and 1372, 1993 chemokine receptors in experimental anti-myeloperoxidase antibody- 48. Kluger MA, Zahner G, Paust HJ, Schaper M, Magnus T, Panzer U, Stahl mediated glomerulonephritis. Clin Exp Immunol 158: 143–153, 2009 RA: Leukocyte-derived MMP9 is crucial for the recruitment of proin- 43. Nouailles G, Dorhoi A, Koch M, Zerrahn J, Weiner J 3rd, Faé KC, Arrey flammatory macrophages in experimental glomerulonephritis. Kidney F, Kuhlmann S, Bandermann S, Loewe D, Mollenkopf HJ, Vogelzang A, Int 83: 865–877, 2013 Meyer-Schwesinger C, Mittrücker HW, McEwen G, Kaufmann SH: CXCL5-secreting pulmonary epithelial cells drive destructive neutro- philic inflammation in tuberculosis. J Clin Invest 124: 1268–1282, 2014 44. Panzer U, Steinmetz OM, Reinking RR, Meyer TN, Fehr S, Schneider A, This article contains supplemental material online at http://jasn.asnjournals. Zahner G, Wolf G, Helmchen U, Schaerli P, Stahl RA, Thaiss F: org/lookup/suppl/doi:10.1681/ASN.2013101061/-/DCSupplemental.

66 Journal of the American Society of Nephrology J Am Soc Nephrol 26: 55–66, 2015 ABFACS Tubulointerstitial Glomerular neutrophils neutrophils 2.0 0.8 30 n.s. ) - 1.5 0.6 F4/80

+ 20 cells) + 1.0 0.4 cells/lpf cells/gcs + + 10 Day 3 GR1

0.5 GR1 0.2 (% ofCD45 Wild-type

Neutrophils (Ly6G -/- 0 0.0 0.0 CXCL5 Con NTN Con NTN Con NTN

Supplemental Figure 1: Early neutrophil recruitment in WT and CXCL5-/- mice (A) FACS-based quantification of PMN from the kidney of nephritic wild-type, nephritic CXCL5-/- and wild-type control mice at day 3 after induction of nephritis. (B) Immunohistochemical quantification of renal PMN infiltration into the tubulointerstitial (per low power field, lpf) and glomerular compartment (per glomerular cross section, gcs) of nephritic WT and CXCL5-/- mice (day 3) using the PMN marker GR1. WT Control WT NTN day 14 CXCL5-/- NTN day 14 A B

100 n.s. 80 60 40 20

F4/80+ cells per lpf 0 100 µm Con NTN

C D 2.0 n.s. 1.5

1.0 cells per gcs cells + 0.5

Mac2 0.0 25 µm Con NTN

E F 80 n.s. 60

40 cells perlpf + 20 CD3 0 100 µm Con NTN

G H n.s. 1.5

1.0 cells/gcs

+ 0.5

CD3 Day 14 0.0 25 µm Wild-type Con NTN CXCL5-/-

150 1.0 100 1.5 I 0.8 80 1.0 100 0.6 60 Day 14

0.4 40 cells/gcs cells perlpf cells perlpf + cells per gcs cells

+ 50 0.5 + + Wild-type 0.2 20

CD3 -/- CD3 F4/80 CXCR2 0 Mac2 0.0 0 0.0 ConNTN Con NTN Con NTN Con NTN J 80 2.0 100 1.5 80 60 1.5 1.0 60 40 1.0

40 cells/gcs Day 14 cells perlpf cells perlpf + cells per gcs cells

+ 0.5 + + 20 0.5 20 Isotype CD3 CD3 F4/80 0 Mac2 0.0 0 0.0 Anti-CXCL5 ConNTN Con NTN Con NTN Con NTN Supplemental Figure 2: Renal leukocyte infiltration Paraffin-embedded sections (2 µm) were stained with the following antibodies: F4/80 (BM8, BMA, Germany), MAC-2 (M3/38; Cedaralane, Ontario, Canada) and CD3 (A0452, Dako, Germany). Representative immunohistochemical photographs and quantification of renal leukocyte infiltration in nephritic wild-type, nephritic CXCL5-/- and WT control mice at day 14 after induction of nephritis (lpf = low power field; gcs= glomerular cross section) using the macrophage markers F4/80 (A, B)MAC2(C, D) and the T-cell marker CD3 (E-H). Original magnification x200 for the tubulointersitial compartment (A, E) and X400 for the glomerulus (C, G). F4/80, MAC-2 and CD3 positive cells were also quantified in CXCR2-/- (I) and anti-CXCL5 or IgG2B isotype antibody (J) experiments. A Blood 15 8 20 15 6 15 cells] cells] cells] + + 10 + 10 4 10 T cells T cells + + PMN 5 5 2 5 Monocytes CD4 CD8 [% of CD45 [% of [% cells] of CD45+ [% of CD45 [% of CD45 0 0 0 0 Con NTN ConNTN Con NTN Con NTN

B Spleen 25 30 25 25 20 20 20 cells] cells] cells] cells]

+ 20 + 15 + 15 + 15 T cells T cells + + PMN 10 10 10 10

5 CD4 5 CD8 5 [% ofCD45 [% ofCD45 [% ofCD45 0 [% of CD45 0 0 0 Con NTN Con NTN ConNTN Con NTN C phagocytesMononuclear Kidney Day 14 Wild-type 50 CXCL5-/- 40 cells] + 30 20 10 0 [% of CD11b Con NTN Mononuclear phagocytesMononuclear

Supplemental Figure 3: Immune cell composition in WT and CXCL5-/- mice FACS-based quantification of leukocyte subpopulations in the blood (A), in the spleen (B) and the kidney (C)of WT and CXCL5-/- mice at day 14 after induction of NTN. Untreated WT and CXCL5-/- mice served as controls. PMNs were identified as CD45+,CD11b+,Ly6G+,Ly6cint cells. Blood and bone marrow monocytes were identified as CD45+,CD11b+,Ly6G-,Ly6chigh. Kidney mononuclear phagocytes were identified as CD45+,CD11b+,Ly6G-, F4/80+ cells. CD4+ T cells were identified as CD45+,CD3+,CD4+ cells and CD8+ T cells were identified as CD45+, CD3+,CD8+ cells. (n=4-5 per group, bars represent means ± SD). A WT Control CXCL5-/-

25 µm B

CD0.8 60

0.6 40 0.4

ACR g/g ACR 20 0.2 mg/dl BUN Wild-type CXCL5-/- 0.0 0

Supplemental Figure 4: Renal phenotype of CXCL5-/- mice Renal phenotype and function of wild-type and CXCL5-/- mice under homeostatic conditions. (A)PAS staining revealed no histologic differences between the knockout and wild-type groups. To analyze possible minimal morphologic abnormalities in the glomerular filtration barrier, we performed electron microscopy. As shown in B, no pathologic findings were detectable in both groups in terms of glomerular basement membrane morphology, endothelial cell structure, and podocyte foot process morphology. (C+D) Functional analysis of CXCL5-deficient and wild-type mice demonstrated identical urinary albumin / creatinine ratio (ACR) and blood urea nitrogen (BUN) levels. (n=4 per group, bars represent means ± SD). control NTN day 3 NTN day 10 0.5% 0.4% 7.4% IL-17 CD4

Supplemental Figure 5: Flow cytometry of renal TH17 cells Flow cytometric analyses of renal CD3+ T cells isolated from controls and nephritic kidneys at days 3 and 10. Cells were re-stimulated with PMA/ionomycin and intracellularly stained for IL-17A. IL-17-expression by CD4+ T cells peaked at day 10 and was hardly detectable in controls and at day 3. In contrast CD4-CD3+IL- 17+ cells (such as γδ T cells, see Turner and Krebs et al. JASN, 23: 1486-1495, 2012) were present in control and nephritic mice at day 3. FACS-plots are representative of each point of time as indicated. A B Tubulus cells – CXCL5 (mRNA) Tubulus cells – CXCL5 (protein) 15 1000 O O 2 2 800 IL-17RA H IL-17RC H 10 600 600 bp 400 500 bp 5 pg/ml 200 400 bp

(x-fold of control) 0 0 mRNA expression mRNA Tubulus cells 0 0.1 1 10 100 0 0.1 1 10 100 IL-17A (ng/ml) IL-17A (ng/ml)

C D α α α α CXCL5 - mRNA CXCL5 - protein Control Control IL-17A TNF- IL-17A TNF- IL-17A + TNF- IL-17A + TNF- *** 20 8000 51 64 *** 15 6000 39 51 4000 10 + Basal Phospho-p42/44 Phospho-p65 pg/ml + IL-17A 5 2000 + IFN-γ 51 64 0 0

(x-fold of control) (x-fold + IL-17F mRNA expression mRNA Tubular cells Tubular cells 39 51 p42/44 p65 E 51 51 Renal cortex – TNF-α 39 39 30 β-actin β-actin 20

10

(x-fold of control) 0 mRNA expressionmRNA 0 3 5 10 20 Time (days after NTN induction)

Supplemental Figure 6: IL-17A induces CXCL5 expression in kidney tubular cells (A) Murine epithelial tubular cells express IL-17 receptors IL-17RA and IL-17RC as measured by PCR from cultured cells. (B) IL-17A induces dose-dependent CXCL5 expression in cultured tubular cells. (C) IL-17A but not IFN-γ or IL-17F induced CXCL5 in cultured tubular cells as measured by RT-PCR (after 4 hours of stimulation) and ELISA from the supernatant (after 24 hours). (D) Lysates of stimulated cells were immunoblotted with monoclonal antibodies to phospho-ERK1/2 (upper left) or total ERK1/2 (lower) as well as phospho-p65 and p65 (NF-κB subunit p65, right panel) and β-actin as loading control. (E) Quantitative PCR from renal cortex shows TNF-α expression in the kidney in the course of nephrotoxic nephritis. Bars represent means ±SD (***P < 0.001). A Kidney B Blood

7.84% 0.535% 8.05% 0.042%

Control

83.0% 8.62% 91.7% 0.170%

15.8% 0.853% 24.8% 0.078%

NTN + Isotype (day 14)

74.9% 8.49% 75.0% 0.078%

0.116% 0.534% 0.188% 0.00%

NTN + anti-Ly6G (day 14)

83.1% 16.3% 99.2% 0.657% Ly6G Ly6G

F4/80 CD11b

C Kidney ) - 40 * 40 F4/80 + 30 30 cells) cells] + + 20 20

10 10 (% of CD45 [% of CD45 0 Phagocytes Mono.

Neutrophils (Ly6GNeutrophils 0

D Blood 50 80 *

cells] 40

+ 60 cells] 30 + 40 Day 14 20 Control Monocytes 20 10 NTN + Isotype [% of CD45 NTN + anti-Ly6G

PMN [%PMN of CD45 0 0

Supplemental Figure 7: Anti-Ly6G-antibody depletes neutrophils in vivo Flow cytometric analysis of leukocytes in the kidney (A) and blood (B) of nephritic wild-type mice after administration of PMN depleting rat-anti-Ly6G antibody or IgG isotype. Administration of anti-Ly6G was performed every 48 hours for 3 times, starting at day 9 after induction of the disease. Analysis of leukocytes was performed at day 14. Plots are gated on CD45+ and CD11b+ myelo-monocytic leukocytes and were representative for 3 independent experiments. FACS-based quantification of PMN and myelo-monocytic leukocyte subpopulations from the kidney (C) and the blood (D) of nephritic WT mice after administration of PMN depleting anti-Ly6G antibody or IgG Isotype. A

total IgG IgG 1 IgG 2a/2c IgG 2b 4 4 0.8 4

3 3 0.6 3 Day 14 2 2 0.4 2 Wild-type control (n=3)

OD 450nm 1 OD 450nm 1 OD 450nm 0.2 OD 450nm 1 Wild-type NTN (n=9) CXCL5-/- NTN (n=8) 0 0 0.0 0

0 0 0 0 00 00 20 00 00 10 500 10 500 1: 10 500 1: 1:500 2 1: 1:500 2 1:100 1:50 25 1: 1:500 2 1:25 :1 1:25 :1 1: 1:25 :1 1 1 1

Control WT NTN CXCL5-/- NTN B C 6

4

2 mIgG score mIgG 0 Con NTN D E 6

4

2 sIgG score sIgG 25 µm 0 Con NTN

F total IgG IgG 1 IgG 2a/2c IgG 2b 4 5 2.5 2.5 4 2.0 2.0 3 Day 14 3 1.5 1.5 2 Wild-type control (n=3) 2 1.0 1.0 Wild-type NTN (n=4) OD 450nm 1 OD 450nm 1 OD 450nm 0.5 OD 450nm 0.5 CXCR2-/- NTN (n=4) 0 0 0.0 0.0

0 0 0 0 00 20 00 20 00 20 00 10 500 1: 1: 1: 1: 1:500 2 1:100 1:50 25 1:100 1:50 25 1:100 1:50 25 1:25 :1 1: 1: 1: 1

Supplemental Figure 8: Humoral immune response (A) Quantification of mouse anti-sheep specific IgG subclasses present in the serum of nephritic WT or CXCL5-/- mice and non-nephritic WT control mice measured by ELISA. (B, D) Representative immunohistochemical stainings for glomerular deposition of mouse IgG (B) and sheep IgG (D) in nephritic wild-type and CXCL5-/- mice 14 days after NTN induction (original magnification x400). (C) Quantification of glomerular deposition of mouse IgG (mIgG) and sheep IgG (sIgG) (E) in kidney sections of controls, nephritic wild-type, and nephritic CXCL5-deficient mice. (F) Quantification of mouse anti-sheep specific IgG subclasses present in the serum of nephritic WT or CXCR2-/- mice and non-nephritic WT control mice measured by ELISA. Bars represent means ± SD (* P < 0.05). IL-17A protein IFN-γ protein 80 600

60 400

40

200 IL17 (pg/ml) IL17 20 IFNg in pg/ml Wild-type CXCL5-/- 0 0 ConNTN Con NTN

Supplemental Figure 9: Splenocyte supernatants IL-17A and IFN-γ measurement by ELISA from supernatants of cultured splenocytes from nephritic CXCL5-deficient, nephritic wild-type, and control mice after stimulation with sheep IgG.