BASIC RESEARCH www.jasn.org IL-34–Dependent Intrarenal and Systemic Mechanisms Promote Lupus Nephritis in MRL-Faslpr Mice Yukihiro Wada,1 Hilda M. Gonzalez-Sanchez,1 Julia Weinmann-Menke,2 Yasunori Iwata,1 Amrendra K. Ajay,1 Myriam Meineck,2 and Vicki R. Kelley1 1Renal Division, Department of Medicine, Brigham and Women’s Hospital, Boston, Massachusetts; and 2Department of Nephrology and Rheumatology, University Medical Center of the Johannes Gutenberg University Mainz, Mainz, Germany ABSTRACT lpr Background In people with SLE and in the MRL-Fas lupus mouse model, macrophages and autoanti- bodies are central to lupus nephritis. IL-34 mediates macrophage survival and proliferation, is expressed by tubular epithelial cells (TECs), and binds to the cFMS receptor on macrophages and to a newly identified second receptor, PTPRZ. Methods To investigate whether IL-34–dependent intrarenal and systemic mechanisms promote lupus lpr nephritis, we compared lupus nephritis and systemic illness in MRL-Fas mice expressing IL-34 and IL-34 lpr knockout (KO) MRL-Fas mice. We also assessed expression of IL-34 and the cFMS and PTPRZ receptors in patients with lupus nephritis. lpr Results Intrarenal IL-34 and its two receptors increase during lupus nephritis in MRL-Fas mice. In knock- out mice lacking IL-34, nephritis and systemic illness are suppressed. IL-34 fosters intrarenal macrophage accumulation via monocyte proliferation in bone marrow (which increases circulating monocytes that are recruited by chemokines into the kidney) and via intrarenal macrophage proliferation. This accumulation leads to macrophage-mediated TEC apoptosis. We also found suppression of circulating autoantibodies and glomerular antibody deposits in the knockout mice. This is consistent with fewer activated and pro- liferating intrarenal and splenic B cells in mice lacking IL-34, and with our novel discovery that PTPRZ is expressed by macrophages, B and T cells. These findings appear translatable to human patients with lupus lpr nephritis, whose expression of IL-34, cFMS, and PTPRZ is similar to that seen in the MRL-Fas lupus mouse model. Moreover, expression of IL-34 in TECs correlates with disease activity. Conclusions IL-34 is a promising novel therapeutic target for patients with lupus nephritis. J Am Soc Nephrol 30: 244–259, 2019. doi: https://doi.org/10.1681/ASN.2018090901 Nephritisiscommoninpatientswithlupus.1,2 Even mice, Mø in lupus-prone MRL-Faslpr mice are defec- with optimal therapy, up to 25% of these patients tive in shifting from M1 to M2 and hyperproliferate progress to ESRD.2–4 Moreover, a new therapeutic to Mø growth factors,8 thereby promoting an for lupus nephritis has not been approved in over five decades. Therefore, the need for a novel thera- Received September 6, 2018. Accepted November 16, 2018. peutic target for lupus nephritis is pressing and timely. Myeloid cells, most notably Mø, regulate the in- Y.W.,H.M.G.-S.,andJ.W.-M.contributed equally to this work. flammatory response to kidney injury and repair. Published online ahead of print. Publication date available at Activated Mø are broadly conceptually divided into www.jasn.org. “ ” “ ” M1 destroyers and M2 healers. Mø are integral Correspondence: Dr. Vicki R. Kelley, Harvard Institutes of Med- in AKI that resolves in normal mice,5–7 but trigger icine, 4 Blackfan Circle, Boston, MA 02115. Email: vkelley@rics. CKD in lupus-prone mice.7 For example, after a bwh.harvard.edu transient kidney insult (ischemia), unlike normal Copyright © 2019 by the American Society of Nephrology 244 ISSN : 1046-6673/3002-244 J Am Soc Nephrol 30: 244–259, 2019 www.jasn.org BASIC RESEARCH accumulation of Mø that escalate inflammation in the renal Significance Statement tubular-interstitium. Moreover, MRL-Faslpr Mø are defective in removing apoptotic cells, leading to the induction of Macrophages and autoantibodies play a central role in the pathol- lpr autoantibodies that circulate, lodge in glomeruli, and thereby ogy of lupus nephritis in patients with lupus and in the MRL-Fas compromise glomerular filtration.7 Thus, the accumulation of mouse model. The authors demonstrate that IL-34 and its two re- ceptors, cFMS and PTPRZ, are upregulated in the kidney with ad- lpr Mø in lupus-prone mice is central to initiating and driving vancing nephritis in MRL-Fas mice. Genetically deleting IL-34 in lupus nephritis. these mice suppresses nephritis and the systemic illness via mac- IL-34 and colony stimulating factor 1 (CSF-1) are the prin- rophage- and autoantibody-mediated mechanisms within and ciple Mø growth factors that regulate the accumulation of outside of the kidney. The authors also found that patients with Mø in inflamed tissues. CSF-1 functions by engaging a high- lupus nephritis have elevated IL-34 in serum and urine; intrarenal and systemic expression of IL-34, cFMS, and PTPRZ similar to that fi lpr af nity receptor tyrosine kinase encoded by the cFMS proto- displayed in MRL-Fas mice; and IL-34 expression that correlates oncogene, CSF-1R (cFMS, CD115).9,10 cFMS is principally with histopathologic index of disease activity. These findings sug- expressed on mononuclear phagocytes, including progenitor gest that IL-34 is a promising novel therapeutic target for patients cells,11 monoblasts, promonocytes, monocytes,12 Mø,den- with lupus nephritis. dritic cells,13 and some epithelial cells.14,15 The discovery – fi that cFMS null mice were not viable, and that CSF-1 de cient (Fms-eGFP) mice expressing eGFP under the control of Fms fi mice survived, led to the identi cation of a second cFMS promoter and first intron, referred to as MacGreen, provided 16 ligand, known as IL-34. by David Hume (Roslin Institute, University of Edinburgh, IL-34 and CSF-1 have shared and differing properties. Both Edinburgh, Scotland)20;(2) (TgN[Csf1-Z]Ers7/+) mice ex- cytokines promote the growth and survival of monocytes and pressing lacZ under the control of Csf1 promoter and the first 17 formation of Mø colonies from BM, but differ in spatiotem- intron,21 referred to as TgZ mice, provided by Richard Stanley 17 poral expression in some adult and developing tissues, as (Albert Einstein College of Medicine, New York, NY); and (3) well as during disease. Although IL-34 and CSF-1 both signal tm1Mom fi B6.129S7-Rag1 /J (JAX). IL-34 KO mice deleted of Il34 through cFMS, a second IL-34 receptor, PTPRZ, was identi ed exons 3–5 and intercrossed with Il34 LacZ/+ offspring,22 pro- in brain.18 We elucidated a role for IL-34 using ischemia/ vided by Marco Colonna (Washington University, St. Louis, reperfusion renal injury (I/R),19 an acute model of tubular MO), were backcrossed onto the MRL-Faslpr background injury. However, unlike I/R, lupus nephritis is a systemic illness (backcrossed eight generations, then using “speed congenics” involving cell- and antibody-mediated mechanisms driving [JAX] at generations 4 and 6 we selected breeders with max- chronic tubulointerstitial and glomerular disease. Given the imal MRL-Faslpr genes). We bred and housed mice in the an- dissimilarities between IL-34 and CSF-1, and I/R and lupus imal facility at Harvard Medical School, Boston, MA. Use of nephritis, it is unclear whether IL-34–mediated mechanisms mice in this study was reviewed and approved by the Standing lead to lupus nephritis. Committee on Animals in the Harvard Medical School (Pro- To test the hypothesis that IL-34 is a potential therapeutic tocol #2016N000161), in adherence to standards set in the target for lupus nephritis, we compared IL-34 KO, wild-type lpr Guide for the Care and Use of Laboratory Animals (eighth (WT), and heterozygous (+/-) mice on the MRL-Fas back- ground during age-related advancing lupus nephritis. The edition, The National Academies Press, revised 2011). central questions are: (1) Do IL-34 and IL-34 receptors in- crease with progressive lupus nephritis in MRL-Faslpr mice? Serum and Renal Biopsy Specimens (2) Does deleting IL-34 suppress renal disease along with the Human renal biopsy specimens with the diagnosis of lupus fi fi systemic illness in MRL-Faslpr mice? (3) Is the accumulation of nephritis (as de ned by the ISN/RPS 2004 classi cation and Mø in lupus nephritis a result of IL-34–mediated mechanisms histopathology activity and chronicity indices) and of healthy within or outside of the kidney? (4) Are the IL-34 receptors, controls (patients with normal serum creatinine, no protein- cFMS and PTPRZ, expressed by different cells within and out- uria but dysmorphic erythrocytes in the urine, and no evidence side of the kidney? And (5) are the IL-34–dependent findings of kidneydisease in the kidney biopsy specimen) were provided in lupus-prone mice translatable to patients with lupus by the Department of Pathology, Friedrich-Alexander Univer- nephritis? sity Erlangen-Nuermberg, Germany.23 Renal pathologists, without access to the patient’s clinical data, evaluated these biopsy specimens. After informed consent, serum specimens METHODS were taken from patients who fulfilled the classification of SLE and diagnosis of lupus nephritis.24 Volunteers (age range, Mice 18–70 years) were screened for health by exclusion of any prior MRL-Faslpr, C57BL/6J, and B6.129S7-Rag1tm1Mom/J mice were kidney diseases, diabetes, hypertension, and autoimmune dis- purchased from The Jackson Laboratory (JAX), Bar Harbor, eases. Healthy controls had normal serum creatinine levels and ME. The following mutant mice were backcrossed onto the no proteinuria in spot urine (assessed by proteinuria and MRL-Faslpr background for more than ten generations: (1) albuminuria strip). Freshly voided urine and drawn blood J Am Soc Nephrol 30: 244–259, 2019 IL-34 Promotes Lupus Nephritis 245 BASIC RESEARCH www.jasn.org samples were collected, centrifuged, aliquoted, and stored the numbers of: glomerular IgG and C3 deposits,7,32 intrarenal at 280°C before analysis as previously described.23 proliferating Mø and B cells,19 Mø, CD4 T cells, B220 unique double-negative T cells,29 apoptotic tubular epithelial cells Survival (TECs),7 and cells expressing PTPRZ.19 We assessed survival in IL-34 KO and WT MRL-Faslpr mice dying with renal disease (proteinuria).