TWEAK-Fn14 Signaling Activates Myofibroblasts to Drive Progression

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TWEAK-Fn14 Signaling Activates Myoﬁbroblasts to Drive Progression of Fibrotic Kidney Disease

† † † Ivan G. Gomez,* Allie M. Roach,* Naoki Nakagawa, Aldo Amatucci,* † ‡ ‡ Bryce G. Johnson,* Kadeshia Dunn, Mark C. Kelly, Gamze Karaca,* Timothy S. Zheng,* ‡ † Suzanne Szak,* Claire M. Peppiatt-Wildman, Linda C. Burkly,* and Jeremy S. Dufﬁeld*

*Research & Development, Biogen, Cambridge, Massachusetts; †Division of Nephrology, Departments of Medicine & Pathology, and Institute of Stem Cell & Regenerative Medicine, University of Washington, Seattle, Washington; and ‡Medway School of Pharmacy, University of Kent, Chatham, Kent, United Kingdom

ABSTRACT The identification of the cellular origins of myofibroblasts has led to the discovery of novel pathways that potentially drive myofibroblast perpetuation in disease. Here, we further investigated the role of innate immune signaling pathways in this process. In mice, renal injury-induced activation of pericytes, which are myofibroblast precursors attached to endothelial cells, led to upregulated expression of TNF receptor superfamily member 12a, also known as fibroblast growth factor-inducible 14 (Fn14), by these cells. In live rat kidney slices, administration of the Fn14 ligand, TNF-related weak inducer of apoptosis (TWEAK), promoted pericyte-dependent vasoconstriction followed by pericyte detachment from capillaries. In vitro, administration of TWEAK activated and differentiated pericytes into cytokine-producing myofibroblasts, and further activated established myofibroblasts in a manner requiring canonical and noncanonical NF-kB signaling pathways. Deficiency of Fn14 protected mouse kidneys from fibrogenesis, inflammation, and associated vascular instability after in vivo injury, and was associated with loss of NF-kB signaling. In a genetic model of spontaneous CKD, therapeutic delivery of anti-TWEAK blocking antibodies attenuated disease progression, pre- served organ function, and increased survival. These results identify the TWEAK-Fn14 signaling pathway as an important factor in myofibroblast perpetuation, fibrogenesis, and chronic disease progression.

J Am Soc Nephrol 27: 3639–3652, 2016. doi: 10.1681/ASN.2015111227

Fibrosis is a central problem in many forms of including types VI, VIII, and XII, and other matrix chronic disease affecting internal organs including proteins including fibrillin, hyaluronan, and fibro- the liver, lung, heart, central nervous system, and nectin.2–4 Recently, genetic fate-mapping studies kidney. The deposition of pathologic fibrotic ma- have demonstrated that in virtually all organs, dis- terial between and within organ units, as well as the crete populations of resident mesenchymal cells cellular activation required for such deposition, form the cellular link between initial tissue injury have been strongly implicated in the perpetuation and excessive, pathologic matrix production by mi- and progression of chronic diseases. In the kidney, grating from their niche and depositing pathologic fibrosis manifests histologically as interstitial fibro- matrix as (myo)fibroblasts.2,5–12 In the kidney, sis, glomerulosclerosis, and arteriosclerosis.1 The numerical expansion and activation of myofibroblasts is central to fibrosis. Although a number of Received November 13, 2015. Accepted February 16, 2016. cell types have the potential to produce and secrete Published online ahead of print. Publication date available at extracellular matrix (ECM) proteins, including ep- www.jasn.org. ithelial cells, endothelial cells, and leukocytes, it is Correspondence: Dr. Jeremy S. Duffield or Dr. Linda C. Burkly, myofibroblasts that produce the pathogenic, fibril- Biogen, Building 6, 115 Broadway, Cambridge, MA, 02142. lar collagenous matrix in vivo, especially collagen Email: jeremy.duffi[email protected] or [email protected] types I, III, V, VII, and XI, microfibril collagens Copyright © 2016 by the American Society of Nephrology

J Am Soc Nephrol 27: 3639–3652, 2016 ISSN : 1046-6673/2712-3639 3639 BASIC RESEARCH www.jasn.org many of these mesenchymal cells are closely associated with Furthermore, release of TWEAK by increased blood flow capillaries, perform vascular functions, have mesenchymal in the kidney may be an early feature of the development stem cell characteristics, and may be referred to as pericytes of kidney disease.34 Since such microvascular problems are or perivascular cells.1,3,9,13,14 In the mouse kidney, many of recognized to drive kidney disease,14 and since pericytes are these perivascular cells lack expression of NG2 but express directly implicated in the fibrogenic process, we hypothe- PDGFRb, whereas in rat and human kidneys the expression sized that Fn14 may contribute to fibrogenesis by regulating of NG2 is more variable.15–17 pericyte functions directly. Here, we evaluate the role of The activation of pericytes results in migration from the Fn14 in the activation of kidney myofibroblast precursors, capillary to the interstitial space. This detachment from the as well as in the persistence of established myofibroblasts, capillary is associated with vascular instability, which may and determined its contribution to renal fibrogenesis and result in pathologic angiogenesis or capillary loss.3,13,18 Studies end organ failure. suggest that vascular instability and pericyte activation are directly linked due the loss of survival signals, quiescence signals, and basement membrane maintenance. Failure to resolve RESULTS these responses, either as a result of repetitive injury or loss of normal restorative mechanisms, appears to be central to the Fn14 is Expressed by Pericytes and Myofibroblasts in progression to chronic disease. the Kidney Although several secreted factors that contribute to cellular Tnfrsf12 (Fn14) and Tnfsf12 (TWEAK) were highly upregu- crosstalk and perpetuation of the fibrotic response have been lated in the setting of kidney injury with fibrosis caused by identified, including TGFb,VEGF,CTGF,WNTligands, surgical unilateral ureteral obstruction (UUO) (Figure 1, A Hedgehog ligands, and PDGFs, additional factors likely play and B). Flow cytometry-sorted pericytes from normal kid- critical roles in fibrogenesis. In particular, myofibroblasts neys, and activated pericytes (which progressively differentiate show activation of innate immune signaling pathways, sug- into myofibroblasts) sorted from diseased kidneys were eval- gesting that additional cytokine/receptor interactions contrib- uated for expression of Tnfrsf12 by quantitative PCR (qPCR). ute to their appearance and maintenance in a pathologic Tnfrsf12 was present in healthy pericytes and expression pro- state.3,4,11,19,20 gressively increased with disease (Figure 1B). In keeping with TNFreceptor and TNF superfamily receptor–signaling have high expression of Fn14 by myofibroblasts, kidney tissue been identified as potentially contributing to the pathogenesis sections from the UUO model were stained with anti-Fn14 of kidney diseases, including diabetic nephropathy, IgA ne- antibodies and costained with markers for myofibroblasts. phropathy, and lupus nephritis.21–25 TNF-related weak in- Fn14 was detected at high levels in interstitial myofibro- ducer of apoptosis (TWEAK) and its receptor fibroblast blasts, but was also detected in endothelial cells (Figure growth factor-inducible 14 (Fn14) are TNF superfamily 1C). A minority of injured epithelial cells also expressed a (TNFSF) members and have been implicated in models of low level of Fn14. Notably, only a subpopulation of myofi- kidney injury and disease, including models featuring renal broblasts appeared to express Fn14 and these were in the fibrosis, particularly where autoimmunity is thought to drive areas of greatest myofibroblast accumulation. Fn14 staining the pathogenesis. Such links with autoimmunity may derive in kidneys lacking Fn14 did not detect any specificsignal, from reports that TWEAK overexpression in vivo leads to the highlighting the specificity of this method for detecting pro- development of autoimmunity. However, the receptor and tein expression (Figure 1C, Supplemental Figure 1). A sim- ligand are upregulated in human chronic fibrosing diseases ilar pattern of staining was detected in biopsies from human of the kidney, such as FSGS and diabetic nephropathy, where CKD, where the majority of staining was detected in the there is no autoimmunity26,27; yet a role for this receptor and interstitium in myofibroblasts and endothelium (Supple- ligand directly in fibrogenesis remains undetermined.28–30 mental Figure 2). Fn14 expression was not detected in hu- Fn14 signaling via TNF receptor-associated factors has been man glomeruli. Further validation of these observations was reported to stabilize NF-kB–inducing kinase (NIK), thereby made in primary cultures of mouse kidney cells. Pericytes activating the P52/RelB NF-kB complex and causing down- expressed Fn14, seen as a single band by Western blotting. stream activation of NF-kB target genes, which have been Myofibroblasts, purified from chronically diseased and fi- suggested to be important factors in human kidney dis- brotic kidneys, expressed the receptor at significantly higher ease.31 Recently, we identified Fn14 (TNFRSF12)asanupre- levels. Epithelial and endothelial cells weakly expressed the gulated receptor on activated fibroblasts precursors, known receptor in vitro. Cultured macrophages did not express the as pericytes, and their pathologic counterparts in diseased receptor (Figure 1D). A similar pattern of expression was kidney, known as myofibroblasts. Transcripts for Fn14 were seen in primary cultures of human kidney cells (Supple- found to be enriched in myofibroblasts relative to other mental Figure 2). In contrast to this pattern of expression, cell types in the diseased kidney.3,4,20 Fn14 was reported transcripts for Tnfsf12 were not detected in epithelial cells to contribute to microvascular permeability in cerebral is- but were detected at high levels in cultured macrophages chemia, as well as pathologic angiogenesis in tumors.32,33 (Figure 1E).

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Fn14 Receptor Activation Converts Pericytes into Inflammatory Myofibroblasts In Vitro TWEAK induced dose-dependent proliferation of pericytes at higher concentrations, an effect blocked by antibodies against Fn14 (Figure 2A). An important component of myofibroblast function is the acquisition of migratory characteristics. Pericytes were interrogated for their capacity to migrate across 50 mm transwell membranes in response to the chemoattractant PDGF-BB or TWEAK (Figure 2B). At higher concentrations, TWEAK induced migration similar to PDGF. Next, we evaluated the effect of TWEAK on expression of the interme- diate filament aSMA, often used as a marker of myofibroblast differentiation and contractile capacity. TWEAK upregulated Acta2 transcripts within 6 hours (Figure 2C) and increased protein synthesis within 24 hours (Figure 2, D and E). Consistent with myofibroblast differentiation, TWEAK stimulated stress fiber formation, as detected by phalloidin staining of cells in culture (Figure 2, F and G). The extent of stress fiber formation was similar to the effect of TGFb1 stimulation, but was selectively blocked by anti-Fn14 antibodies. Therefore, TWEAK triggers myofibroblast transition in vitro. In addition, TWEAK-activated pericytes released significant amounts of the monocyte chemoattractant MCP1, as well as the inflammatory cytokine IL6 (Figure 2, H and I), though not as robustly as TLR4 stimulation Figure 1. Fn14 is highly expressed by kidney myofibroblasts and TWEAK is highly by highly pure LPS. expressed by macrophages. (A) Quantitative PCR (qPCR) results showing changes in Tnfrsf12 (Fn14) and Tnfsf12 (TWEAK) transcript levels in whole tissue from the UUO Fn14 Activation Results in Sustained model of kidney disease in mice versus sham. (B) qPCR showing changes in Tnfrsf12 Pericyte-Mediated Vasoconstriction transcript levels in FACS-sorted collagen I-producing cells in the UUO model of kidney followed by Pericyte Detachment fl disease in mice versus sham. (C) Immuno uorescence images showing the localization from Capillaries of Fn14 in the UUO kidney disease model at day 10. Note a predominantly interstitial Given the pronouncedeffectsof TWEAKon pattern of expression and predominant colocalization with a subpopulation of myofibroblasts, particularly in areas of intense interstitial cell accumulation (arrowheads), pericyte biology, we tested the functional whereas some myofibroblasts do not coexpress Fn14. Weak expression in the me- consequences of pericyte exposure to sangium (*) of glomeruli (g) can be seen. Expression of Fn14 on endothelium is also TWEAK in situ in the medulla of the kid- detected (thick arrows) and while almost all tubular epithelium (T) does not express ney. Medullary pericytes regulate blood Fn14, a minority of injured cells can be seen to express Fn14 (thin arrows) (a, arteriole). flow into the medulla of the kidney.35 (D) Western blot showing Fn14 protein expression in primary cultures of cells. Live rat kidney slices were initially Quantitation is based on three independent experiments. (E) qPCR showing relative immunostained for Fn14, which was found expression of Tnfsf12 transcripts in primary cell cultures. Bar, 25 mm; *P,0.05; to be expressed strongly by medullary , , **P 0.01; ***P 0.001; n=5/group unless otherwise stated. pericytes (Figure 3A). We applied TWEAK or Angiotensin II (AngII) to the slice. Within 30 seconds, TWEAK induced pericyte contraction around the capillaries (Figure 3, B

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assay (Figure 3, D–H). TWEAK stimulated sustained contraction of the capillaries (Figure 3D) and caused a shape change to pericytes, characterized by increased pericyte longitudinal process length and reduced radial process length (Figure 3, E and F). This was associated with increased separation of pericytes and reduced pericyte number attached to capillaries (Figure 3, G and H). The combination of shape change characterized by retraction of radial processes and reduced pericyte density on the capillary suggested pericytes may be de- taching in response to sustained TWEAK exposure. Such detachment could be observed over the 4-hour period, by separation of cell processes from the capillary, spreading of cell bodies away from the capillary and by quantifying the separation of pericytes from the inner endothelial sur- face (Figure 3, I and J). Sustained contraction followed by detachment is indicative of capillary dysfunction that may lead to abnormal salt and water homeostasis, and the subsequent detachment of pericytes is consistent with the early stages of their transition to interstitial myoﬁbroblasts.

TWEAK Enhances Myofibroblast Activation and Increases Inflammatory Cytokine Production We next studied the effect of TWEAK on established myofibroblasts that are charac- teristic of chronic disease. Myofibroblasts, purified from kidneys 3 weeks following Figure 2. Kidney pericyte cultures are activated by TWEAK. (A, B) Graphs showing the (A) chronic ischemic injury when severe fibrosis proliferative response and (B) migratory response of pericytes across a transwell barrier in response to TWEAK. (C–E) The effect of TWEAK on pericyte expression of Acta2 tran- has become established, exhibited enhanced script levels and its protein, the myofibroblast marker aSMA (C) quantitative PCR, (D) proliferation, migration, and spontaneous Western blot, and (E) normalized densitometry of Western blots. (F) Fluorescence images cytokine production compared with pri- and (G) quantification of stress fiber formation as detected by phalloidin-Cy3 in pericytes mary pericyteculturesinidenticalconditions in response (24 hours) to TWEAK or TGFb1. Note, anti-Fn14 (A-Fn14) blocking antibodies (Figures 2 and 4). TWEAK exposure en- inhibit TWEAK-mediated stress fiber formation, but not TGFb-mediated stress fibers. hanced proliferation and migration of myo- (H, I) Graphs showing release of (H) MCP1 or (I) IL6 into the supernatant by primary fibroblasts to a similar level as those achieved pericyte cultures. Note, at higher concentrations pericytes release substantial amounts of by serum or PDGF-BB, respectively (Figure m , , these cytokines. Bar, 25 m; *P 0.05; **P 0.01; n=4/group. 4, A and B). The TWEAK effect was blocked by anti-Fn14 blocking antibodies, confirm- ing TWEAK selectivity for Fn14. In addition and C), which was clearly detectable and was similar to the to these characteristics, TWEAK enhanced MCP-1 and IL6 re- effect of AngII. The capillary contraction was predominantly lease by myofibroblasts to similar levels achieved by LPS (Figure at the site of the pericyte cell body. To test whether TWEAK 4, C and D), indicating TWEAK was a potent proinflammatory exerted a tonic effect on capillaries, we applied anti-TWEAK cytokine for myofibroblasts and that established myofibroblasts antibodies (Figure 3B), but observed no dilation of capillaries, had greater sensitivity to Fn14 signaling than pericytes. Similar indicating TWEAK does not tonically regulate capillary diam- to pericytes, however, myofibroblasts generated a repertoire of eter in healthy conditions. Next, we determined the effect of chemokines and cytokines in response to TWEAK (Supplemen- chronic TWEAK exposure (4 hours) in the live kidney slice tal Table 1).

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Bioinformatic Analysis Identifies TWEAK Signaling Pathways in Myofibroblasts The mechanism by which TWEAK acts on myofibroblasts and the significance in driving fibrogenic responses in myofibroblasts was dissected by comparing transcriptional responses of myofibroblasts to cytokines implicated in fibrogenesis, including TWEAK, using hierarchical clustering analysis. TWEAK prominently activated pathways in myofibroblasts that promote inflammation and fibrosis (Figure 5A, Sup- plemental Figure 3), including the pathway of hepatic fibrosis mediated by hepatic stellate cells. Notably, TWEAK increases collagen V, collagen VI, laminin, and fibrillin matrix genes in myofibroblasts (Figure 5C, Supplemental Figure 3D, Supplemental Table 1). TWEAK activates intracellular signaling pathways including canonical and noncanonical NF-kB, JNK, AKT, and MAPK, as annotated in type 1 diabetes mellitus signaling, NF-kB signaling, and TNF receptor 2 signaling pathways (Figure 5A). Strikingly, TWEAK promotes greater enrichment of genes involved in fibrotic pathways than other known fibrogenic cytokines, including TGFb.Whenthe myofibroblast responsive genes were re-evaluated to enrich specifically for Figure 3. TWEAK stimulates acute microvascular vasoconstriction mediated by pericytes TWEAK-responsive genes, additional in situ, followed by pericyte detachment. (A) Fluorescence micrograph showing isolectin pathways IFN signaling, MIF regulation (IB4) labeled endothelium and immunodetection of Fn14 in rat kidney slice. Note of innate immunity, and iNOS signaling m prominent expression of Fn14 by perivascular cells (arrowheads) (bar, 25 m). (B) Phase were identified, collectively resulting in en- contrast images of medullary capillaries (vasa recta) in live kidney slice showing the effect hanced biologic functions in myofibro- of TWEAK on capillary diameter after 270 seconds of exposure (blue: pericyte body, red: blasts including immune cell movement, diameter at pericyte body, yellow: diameter at nonpericyte area). Note washout of TWEAK (after 6 minutes) results in relaxation. Application of anti-TWEAK antibodies has cell proliferation, and cell-to-cell signaling no effect on diameter. (C) Quantification of change in capillary diameter 300 seconds (Figure 5B, Supplemental Figure 3A). after application of AngII or TWEAK in pericyte or nonpericyte areas of the capillary (n=8/ Genes regulated by TWEAK within the group). (D) Graph of capillary diameter after treatment of kidney slice with TWEAK or cell movement module were those whereby AngII for 4 hours. (E, F) Graphs showing the length of processes of NG2-labeled pericytes myofibroblasts promote leukocyte recruit- where (E) longitudinal processes increase in length but (F) radial processes retract. (G, H) ment, including chemokines CXCL-1,-2,-10, Morphometric measurements of pericyte density long the vasa recta capillary 4 hours and -16, (also known as KC, MIP2, and after treatment with TWEAK or AngII compared with control. (I) Fluorescent images of IP10 respectively) and CCL-2,-7, -9, and pericytes labeled with NG2 and capillary lumen labeled with IB4. In control settings (PSS), -20 (also known as MCP1, MCP3, MIP1, long pericyte processes remain longitudinal with the capillary and associated with cell and MIP3A respectively), and cell adhesion bodies (shown by arrowheads), whereas after 4 hours of TWEAK, pericyte processes molecules (ICAM and VCAM), many of without associated cell bodies are seen (arrows) indicative of separation, and in other instances cell bodies show enlargement and spreading (arrowhead) (bar, 10 mm). (J) which are implicated in myeloid cell re- fl Morphometry of pericyte endothelial distance between pericyte basal membrane and cruitment and in ammation (Figure 5B, – capillary wall. *P,0.05; n=12/group. PSS, physiological saline solution; RBC, red blood Supplemental Figure 3, B D, Supplemental cell. Table 1). Altered cell movement genes included Rho/RAC family members GRK5, RND1, and ARHGEF6, troponins (Supple- mental Figure 3, Supplemental Table 1),

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than for other downstream signaling pathways, with evidence of NIK accumulation, increased phosphorylation of p100, and p100 cleavage to yield the p52 subunit (Fig- ure 5E). As suggested by the bioinformatic analysis, TWEAK activates IFN regulatory factors (IRF), which may be responsible for IFN-b production and MAPK activation (Figure 5F). Also, TWEAK activates the ERK signaling pathway, whereas AKTsig- naling, constitutively active in myofibroblasts, is downregulated by TWEAK (Figure5G).Toevaluatetheimportance of these regulated pathways in myofibroblast responses, canonical NF-kBsignal- ing was blocked with the IkKb inhibitor, ACHP, resulting in an 80% reduction in migration. A NIK inhibitor, which blocks noncanonical pathway signaling, similarly inhibited migration, as did an inhib- Figure 4. Established kidney myofibroblasts are highly sensitive to TWEAK mediated itor of ERK phosphorylation (Figure activation. (A, B) Graphs showing the proliferative response at (A) 16 hours and (B) 5H). Overall, these results are consistent fi migratory response (24 hours) across a transwell barrier of myo broblasts in response to with the in vitro functional studies re- TWEAK. (C, D) Graphs showing release of (C) MCP1 or (D) IL6 into the supernatant by ported above, whereby TWEAK pro- myofibroblast cultures. Note, at higher concentrations myofibroblasts release large motesmorepathologicmyofibroblast amounts of these cytokines. *P,0.05; **P,0.01; n=4/group. characteristics. and ACTA2, (Figure 2) all of which are involved in contraction, Fn14 Deficiency Attenuates Fibrogenesis and Vascular migration, and cell signaling in response to changes in the and Tubular Injury in the UUO Model of Kidney Fibrosis stiffness of the ECM microenvironment. The cell proliferation The studies above, strongly implicate Fn14 as an important module regulated by TWEAK includes cell cycle regulators, receptor in vascular reactivity, in differentiation of perivascu- such as cyclin D1, as well as cytokines such as PDGF-B and lar cells to myofibroblasts, myofibroblast persistence, and leu- CSF-1, whereby myofibroblasts can exert autocrine and para- kocyte recruitment to tissues. To test the contribution of Fn14 crine effects on stromal cells and macrophages. The cell-to-cell and TWEAK in vivo,westudiedfibrogenesis in the UUO signaling module includes the most highly upregulated gene, model, a robust model for the study of early fibrogenesis. 2 2 the IL33 receptor, known as IL1LR1 (ST2), as well as IL-1R1 Mice deficient in Fn14 (Tnfrsf12 / ) (Supplemental Figure 1) and TNF receptor-2, which may serve to amplify innate im- were subjected to surgical UUO, compared with wild-type mune signaling (Supplemental Figure 3, B–D, Supplemental littermates, and evaluated at days 5, 7, and 10. 2 2 Table 1). In addition, canonical and noncanonical NF-kB Tnfrsf12 / mice have normal appearing and functioning transcriptional regulators NFKB2 (P100), NFKBIA (IkBa), kidneys. At day 10 of disease, there was substantially less fi- 2 2 NFKBIE,andRELBareupregulated.JUNB,NRF3,and brosis in the kidney tissue of Tnfrsf12 / mice compared with FOXS1, regulators of inflammatory signaling pathways, littermate controls (Figure 6A, Supplemental Figure 4). The such as AKT, are enhanced (Figure 5A, Supplemental Figure appearance of aSMA+ cells was markedly reduced, as was the 3A, Supplemental Table 1). Also notable is the upregulation of recruitment of leukocytes (Figure 6A, Supplemental Figure 4). multiple vascular permeability regulators, including S1PR3, Consistent with reduced activation of myofibroblasts and re- S1PR2,VCAM,ICAM1,ECSCR,VEGF-C,VEGFR1,and duced leukocyte infiltration, the tubular epithelial cells of the RGS5 (Supplemental Figure 3, B–D, Supplemental Table 1). nephron were less injured and there was significant preserva- Since the promotion of vascular permeability, reflecting the tion of the microvasculature (Figure 6, B and C). Whole tissue destabilization of vessels, is associated with pathologic fibrosis transcriptional analysis was performed in healthy kidneys and when chronically maintained, these findings further implicate diseased kidneys at day 10. A total of 977 probe sets were 2 2 TWEAK in the process of vascular instability. differentially expressed between Tnfrsf12 / and littermate Consistent with the bioinformatic analysis, TWEAK-treated controls, with the majority (74%, n=720) being more highly 2 2 myofibroblasts phosphorylated IkBa and p65, indicative of ca- expressed in Tnfrsf12 / mice. Reduced expression genes nonical NF-kB activation (Figure 5D). The noncanonical path- were analyzed by KEGG annotated ontology pathways (Sup- way was also activated, with kinetics slower and more sustained plemental Figure 4E, Supplemental Table 2). The most

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reduced through absence of Fn14. The proteins regulated by Fn14 signaling were evaluated by String protein-protein interaction analysis to identify nodes or modules (Fig- ure 6D).36 Strikingly, eight distinct nodes emerged controlling inflammatory signaling, cytokine and chemokine generation, angiogenesis, contraction, fibrogenesis, and chromatin modification. Prominent among matrix genes regulated by Fn14 in vivo were collagen VI and collagen V, as had been identified in the myofibroblast cultures (Figure 5). In fact, many of the signaling nodes identified in the myofibroblast and pericyte cultures overlapped with the whole tissue analysis, pointing to a key role for Fn14 in regulating myofibroblast behavior in vivo. Consistent with these findings, collagen VI and laminin were deposited in response to kidney injury, and the increase in these proteins was almost completely abated by the absence of Tnfrsf12 (Figure 6, A and E, Supplemental Figure 4). Next, we dissected the signaling pathways downstream of Fn14 that we had identified in myofibroblasts (Figure 5) and found significant reductions in canonical NF-kB component p65 and noncanonical NF-kB pathway activation, indicated by reduced NIK and P-p100 levels in the Tnfrsf12-deficient kidneys. In addition, Tnfrsf12 deficiency led to reduced P-IRF-3 Figure 5. Transcriptional analysis of cytokine-stimulated myofibroblasts identifies and IRF-4 signaling (Figure 6, F–I). The TWEAK-induced fibrogenic signaling via canonical and noncanonical NF-kB, IRF, and reduction of P65 activity correlated closely ERK. (A) Ingenuity IPA software was used to identify pathways that are enriched in with total levels of P65, whereas the reduc- fi b b myo broblasts treated with TWEAK, IL1 ,TGF , or PDGF-BB compared with vehicle after tion in P52 activity was directly regulated by 16 hours. The top ten pathways for each cytokine based on P values were pooled, and upstream accumulation of NIK, suggesting the heatmap shows z-scores for these pathways across all stimuli. (B) The heatmap shows the intensity of genes differentially expressed with TWEAK treatment at 16 hours for each that loss of Fn14 results primarily in the re- of the biologic processes shown. Vehicle and TWEAK treatments were performed in duction of noncanonical pathway activation triplicate. (C) Normalized gene expression for matrix proteins upregulated by TWEAK in vivo. In addition, the ERK, MAPK, and treatment (log base 2). (D) Blots showing canonical NF-kB activation by the increase in AKT signaling pathways were also less acti- P-IkBa, P-p65 (RELA) with time in myofibroblasts in response to TWEAK. (E) Blots showing vated when Tnfrsf12 was absent. noncanonical NF-kB activation by accumulation of NIK and activation of P-p100 to generate the cleaved and active p52 subunit. (F) Blots showing the effect of TWEAK on Anti-TWEAK Blocking Antibodies active P-IRF-3 accumulation and IRF-4 accumulation. (G) The effect of TWEAK on active Slow Progression of Chronic Fibrotic k k b P-ERK and P-AKT signaling adaptors. (H) The effect of canonical NF- B inhibitor (I K Disease of the Kidney and Enhance k inhibitor), noncanonical NF- B inhibitor (NIK inhibitor), and ERK inhibitor on TWEAK Survival stimulated myofibroblast migration. *P,0.05. The studies above provided compelling evidence that Fn14 signaling and its down- prominent pathways included amoebiasis, which encapsulates stream pathways might be important drivers of chronic disease cell movement, followed by pathways involving focal adhe- progression in the kidney through actions on myofibroblasts, sion, ECM deposition, leukocyte transendothelial migration, potentially mediated by the secretion of TWEAK from recruited chemokine signaling, and NF-kB signaling annotated in B cell macrophages. To evaluate the importance of this pathway in signaling. In addition, enrichment for genes in Notch and chronic disease triggered by microvascular injury, we delivered VEGF signaling pathways implicated in angiogenesis was anti-TWEAK antibodies that block its interaction with Fn1437

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2 2 Figure 6. Fn14 deficiency (Tnfrsf12 / )reducesfibrogenesis in the UUO model of kidney disease. (A) Photomicrographic images showing the extent of fibrosis (day 10), aSMA+ myofibroblasts (day 7), collagen type VI (day 7), and CD45+ leukocytes (day 7) in the UUO model. (B, C) Graphs quantifying tubular injury and vascular density. (D) String analysis showing known and predicted protein- protein interactions of 200 genes most enriched as a result of loss of Fn14 in the diseased kidney. Different types of evidence denoted by the color of the connecting lines, where green is neighborhood, red is gene fusion, blue is co-occurrence, black is coexpression, purple is experiments, cyan is databases, yellow is text mining, and lilac is homology. Proteins are colored according to nodes of interaction. (E) Blots showing the effect of Fn14 deficiency on matrix protein accumulation in whole tissue. (F, G) Blots showing the

3646 Journal of the American Society of Nephrology J Am Soc Nephrol 27: 3639–3652, 2016 www.jasn.org BASIC RESEARCH therapeutically to mice with a mutation in the capillary base- critical to kidney function only in areas of pericyte coverage. ment membrane gene, Col4a3, which causes microvascular in- Sustained microvascular contraction gives way to pericyte de- stability in the kidney, leading to chronic fibrotic kidney tachment, followed by differentiation to myofibroblasts. disease analogous to human Alport nephropathy (Figure 7).38 TWEAK is not only sufficient to cause pericyte differentiation 2 2 Col4a3 / mice on the 129sv background develop albuminuria to myofibroblasts, but also causes inflammatory cytokine and by 3 weeks and develop progressive glomerulosclerosis, tubu- chemokine release by pericytes that causes leukocyte recruit- lointerstitial kidney disease, and loss of organ function, followed ment. TWEAK strongly activates established myofibroblasts, by death from kidney failure by 60–80 days, with a median sur- which derive from perivascular cells1,9,10,20 (Figure 8). Impor- vival of 70 days. Mice received either an isotype-matched control tantly, comparative studies showed that TWEAK also activates Ig or anti-TWEAK antibody (10 mg/kg) twice weekly by intra- fibrogenic and inflammatory signaling in established myofi- peritoneal (i.p.) injection from 24 days onward, once albumin- broblasts more strongly than other well established fibrogenic uria had developed (Figure 7A). In one cohort, kidneys were cytokines. Moreover, TWEAK stimulates myofibroblast pro- analyzed at 56 days to assess kidney function and pathology. liferation, suggesting that in chronic disease, TNF superfamily Anti-TWEAK antibody treatment reduced levels of BUN, a signaling may be important in sustaining pathologic tissue marker of kidney function, by approximately 50%, and attenu- responses. Although TWEAK is found in several cell types, ated albumin leak, another marker of kidney function (Figure 7, macrophages are shown here to produce high levels; therefore, B and C). A second cohort of mice (n=14/group) was treated TWEAK may be one of the paracrine factors used by macro- from 24 days onward with anti-TWEAK antibody (10 mg/kg i.p. phages to promote fibrogenic responses (Figure 8). The stud- injection twice a week) to evaluate the effect on longevity. At this ies also demonstrate TWEAK or its receptor Fn14 to be concentration, anti-TWEAK antibodies specifically enhanced potential targets in the treatment of chronic fibrosing diseases, survival, with a median improvement of 9.5 days (equivalent such as those initiated by mutations in the basement mem- to a 13.5% increase in survival) (Figure 7D). Standard histologic brane proteins, including Alport nephropathy, thereby iden- evaluation revealed a marked reduction in interstitial fibrosis tifying TWEAK and Fn14 as important amplifiers of the and interstitial myofibroblasts (Figure 7, E–G),aswellasasub- chronic fibrogenic process. Evidence of activation of the path- stantial reduction in matrix proteins, including collagen VI and way in chronic human fibrosing diseases, including FSGS and laminin (Figure 7H). Such a reduction in myofibroblasts was diabetic kidney disease, point to a possible role of this pathway closely associated with a reduction in leukocytes (Figure 7E, in driving many chronic fibrosing diseases.26,27 In addition, Supplemental Figure 5), preservation of the microvasculature the findings here further support the notion that the blockade (Figure 7I), and a reduction in injury to the epithelium (Figure of myofibroblast activation can attenuate tissue injury, as 7J). Moreover, the loss of glomerular capillaries and glomerular well as the deposition of ECM, and therefore improve tissue scarring that occurs with this disease was also attenuated by anti- function. TWEAK antibodies (Figure 7, I–K, Supplemental Figure 5). The Factors that drive fibrogenic signaling in vivo include sev- major signaling pathways blocked by chronic anti-TWEAK an- eral developmental morphogenic receptor-signaling path- tibody treatment were assessed. Marked reduction in both ca- ways, including TGFb, WNT, Hedgehog, PDGF, and VEGF. nonical and noncanonical NF-kB signaling was detected, with However, there has been relatively little attention given to in- the most striking changes in the noncanonical pathway (Figure nate immune inflammatory signaling pathways directly in the 7L). In addition, IRF signaling was attenuated (Figure 7M), fibrogenic process. The findings reported here suggest TNF whereas ERK and AKT activation were not reduced in this superfamily member TWEAK, signaling through Fn14, may chronic disease model (Supplemental Figure 5). be a critical fibrogenic signaling pathway. It is interesting to note that pirfenidone, a small molecule recently shown to be effective in treating human lung fibrosis,aswellaskidney DISCUSSION fibrosis,39,40 has significant impact in the generation of inflammatory cytokines, including the TNF family,41 but its pre- These results presented here identify a novel role for the TNF cise mechanism of action is poorly understood. Unlike TNFa superfamily receptor signaling pathway, TWEAK-Fn14, as signaling, Fn14 is known to activate both canonical and non- an important inflammatory signaling cascade that regulates canonical NF-kB signaling pathways in cells, but does not pericyte and myofibroblast biology, and as a consequence, activate a cell death pathway. We show here that in established microvascular responses. In acute settings, TWEAK triggers myofibroblasts, Fn14 activation does signal through both sustained contraction of kidney capillaries, whose flow is NF-kB pathways, as well as ERK, but that inhibition of the

effect of disease and Fn14 deficiency on activation of factors in the (F) canonical and (G) noncanonical NF-kB signaling pathways. (H) Blots showing the effect of disease and Fn14 deficiency on activation of P-IRF-3 and IRF-4. (I) Blots showing the effect of Fn14 deficiency on active P-ERK and P-AKT. *P,0.05; **P,0.01; n=7/group; bar, 50 mm.

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noncanonical pathway alone is sufficient to attenuate downstream effects of TWEAK in myofibroblasts, implicating noncanonical signaling as well as canonical signaling in fibrogenesis. In the disease models, it was the noncanonical pathway that was most suppressed by loss of Fn14. Fn14 activation in myofibroblasts triggers an IFN response, particularly activating IRF-3 and IRF-4, both of which have recently been implicated in the fibrogenic process in other tissues.42,43 These findings suggest that myofibroblasts may be an important, un- appreciated source of IFNb. Whereas these IFN signaling pathways have been shown to play key roles in immune cell response to infections, their role in pathologic fibrogenic responses to tissue injury has not been explored. One of the conundrums in fibrogenesis is that the process is widely considered an aberrant wound healing response. Whereas many signaling pathways that coordinately regulate tissue genesis or regeneration become activated in pathologic disease, the reason they are pathologic is unclear. One explanation for this is the requirement for the coactivation of pathways associated with innate immune responses in stromal cells as a prerequisite for pathologic responses to tissue injury. The bioinformatic studies highlight several important aspects of Fn14 signaling in myofibroblasts that are instructive. Firstly, Fn14 directly promotes matrix synthesis, but the major collagens promoted by Fn14 are collagens V and VI, as well as laminin and fibrillin. In vivo, fibrillar collagens I and III are also reduced by absence of Fn14. Recent evidence indicates that many organs, including the kidney, generate significant levels of fibrillar collagens when healthy, yet these are not assembled into a complex matrix but are degraded. In fact, chronic disease is a state of low matrix Figure 7. Anti-TWEAK antibodies block fibrogenesis and the progression of CKD in turnover.44 It is striking, therefore, that col- 2 2 Col4a3 / mice. (A) Schema showing the experimental approach. (B) BUN levels at 8 lagens V and VI, and microfibrillar 2 2 weeks showing the effect of anti-TWEAK in Col4a3 / mice. (C) Time course of al- 2 2 buminuria in Col4a3 / mice (D) Kaplein–Meier survival curve showing the effect of 2 2 anti-TWEAK in Col4a3 / mice. (E) Photomicrographs showing the extent of interstitial fibrosis (Sirius red stain), CD45+ leukocytes, tubular epithelial injury (periodic sclerosis, 4=75%–100% sclerosis). (L) Repre- 2 2 acid–Schiff stain), and glomerular scarring (silver stain) at 8 weeks of age in Col4a3 / sentative blots showing the effect of anti-TWEAK kidneys. (F–H) Quantification of Sirius red+ fibrosis, aSMA+ myofibroblasts, and matrix antibodies on canonical and noncanonical NF-kB proteins. (I, J) Quantification of vascular density and tubular epithelial injury. (K) signaling factors. (M) Representative blots show- Glomerulosclerosis scores showing percentage of glomeruli with each of the disease ing the effect of anti-TWEAK antibodies on P-IRF- scores (0=no sclerosis, 1=1%–25% sclerosis, 2=25%–50% sclerosis 3=50%–75% 3 and IRF-4. *P,0.05; ** P,0.01; n=14/group; bar, 50 mm.

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activated in a wide range of settings, particularly in response to infection. However, we increasingly recognize chronic diseases as states of inflammation, and NF-kBacti- vation has been reported in these settings. The role of noncanonical NF-kBhasbeen thought to be restricted to regulation by receptors such as CD40, BAFF, APRIL, LTbR, RANK, and Fn14. Several of these receptors have been implicated in fibrosis, and all these receptors have the capacity to induce canonical as well as noncanonical NF-kB signaling. But, the relative contribution of canonical versus noncanonical NF-kBinmyofibroblasts from TNFSF receptors has not been determined previously. We report distinct differences between IL1b-mediated signaling in fi Figure 8. Tweak triggers vasoconstriction, pericyte activation and myofibroblast myo broblasts, which activates P65 persistence in the kidney. Schema summarizing the effect of TWEAK on the fibroblast predominantly, compared with TWEAK. lineage in kidney disease. TWEAK promotes a stronger tendency to- ward noncanonical NF-kB signaling and stimulates chemokine production and collagens are highly regulated in myofibroblasts by Fn14 sig- genes associated with contraction/migration. Therefore, the naling. Collagen VI has been implicated in liver and cardiac noncanonical NF-kB pathway appears to be an important fibrosis, and recent human studies of CKD identify collagens V new signaling pathway in myofibroblasts that may contribute and VI as important discriminators of pathologic fibrosis.45,46 to their pathologic functions. Collagen V is important in assembly of collagens I and III, and Blocking antibodies against TWEAK or Fn14 deficiency are collagen VI is widely believed to be important in organizing protective in a rodent model of autoimmunity with features of fibrils with proteoglycans and anchoring them to basement lupus nephritis25; a progressive kidney disease characterized membranes in collagens I and III47,48. One implication from by autoimmune B cell activation, immune complex deposi- these observations is that by regulating collagens V and VI, tion, as well as leukocyte recruitment in the kidney, where the deposition of stable crosslinked fibrillar matrix can be immune complex deposition is thought to play a major role controlled. in the early pathogenesis. However, lupus nephritis progresses In addition to an important role in matrix production and as a microvascular fibrosing kidney disease, similar to many stabilization, these new studies highlight the high level of expres- other forms of CKD. Our studies show that anti-TWEAK an- sion of Fn14 in activated pericytes and their descendants, tibodies not only inhibit fibrosis in an acute model of kidney myofibroblasts. Combined with significant endothelial ex- injury, but also protect against the progression of a CKD trig- pression, the receptor is therefore implicated in vascular gered by a mutation in a kidney capillary basement membrane responsiveness. In acute settings, TWEAK behaves similarly protein, Col4a3. This disease model closely replicates human to AngII, having the capacity to vasoconstrict kidney capillar- Alport syndrome and has close similarities to a number of ies. Therefore, TWEAK may regulate BP and sodium balance other human CKDs characterized by inflammation and fi- in the setting of tissue injury. Furthermore, pericyte detach- brosis. Therefore, our studies suggest anti-TWEAK anti- ment from capillaries, stimulated by TWEAK, is believed to bodies may be effective at retarding other forms of CKD, contribute to vascular instability and eventual capillary rare- including Alport nephropathy, IgA nephropathy, and other faction, thereby further promoting fibrogenesis through forms of ischemic CKD, by directly targeting the fibrogenic hypoxia-induced signals. In addition, TWEAK stimulates process. myofibroblasts to produce a number of factors that may adversely regulate vascular homeostasis, including S1P receptors and VEGF-C.49 CONCISE METHODS The downstream signaling pathways activated by Fn14 in pericytes and myofibroblasts are identified in these studies. In Animals, Human Tissue, and Models of Kidney Disease addition to noncanonical and canonical NF-kB signaling, C57BL/6J mice were purchased from Jackson ImmunoResearch TWEAK activates ERK and IRF signaling, both of which Laboratories (West Grove, PA). Col1a1-GFPTg mice on the C57BL6 2 2 may play roles in the fibrogenic process. NF-kBsignalingis background have been previously described.2 Tnfrsf12 / (also

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2 2 known as Fn14 / ) mice on the C57BL6 background have been pre- ACKNOWLEDGMENTS viously described and genotyping was performed as reported.50 Lit- 2 termate control colonies were maintained as controls. Col4a3+/ We are grateful to Nicol Hutchison (University of Washington) for mice were purchased from Jackson ImmunoResearch Laboratories assisting with experiments, to Bill Stallcup for generating anti- (129-Col4a3tm1Dec/J) and are on the 129 background. Mice were PDGFRb antibodies, to the Lynne and Mike Garvey Microscopy Suite bred as heterozygotes and genotyping was performed at 3 weeks as at University of Washington, to Catherine Quigley and Michelle Ols 2 2 described.51 Col4a3 / mice were weighed at 3 weeks and random- (Biogen) for assistance with experiments, and to G. Mabbutt, ized in a sex-matched manner to vehicle or specific antibody. Anti- K. Saromi, and Carol Crawford (University of Kent) for assistance TWEAK antibodies or isotype-matched control Ig (10 mg/kg) were with live kidney slice experiments and confocal imaging of fixed subcutaneously administered in 200 ml of saline twice weekly until tissue. week 8 or until week 12. Mice (n=12/group) were euthanized at week These studies were funded by Biogen, National Institutes of Health 8.5 for analysis of blood, urine, and tissue, or studied for survival grants (DK087389, DK093493, DK094768, HL122895, and TR000504), (n=14/group). Timed urine collections were performed at weeks 4, 6, an American Heart Association grant 12040023 (to J.S.D.), and a Core and 8. Adult (12–16 week old) mice (C57BL/6 littermate controls or Fulbright Visiting Scholarship (to Nicol Hutchison). 2 2 Tnfrsf12 / ) were anesthetized with ketamine/xylazine (100/10 mg/kg, i.p. injection) before surgery. UUO or sham surgery was performed as previously described2 and kidneys were collected on DISCLOSURES Tg days 0, 2, 5, 7, and 10. In other experiments, Col1a1-GFP male EmployeesofBiogen,Cambridge,MA(I.G.G.,A.M.R.,A.A.,B.G.J.,G.K., mice were anesthetized and unilateral ischemia reperfusion injury T.S.Z., S.S., L.C.B., J.S.D.) have stock in the company. was performed by clamping the left renal artery for 30 minutes, as described.52 All animal studies were performed under protocols ap- proved by Department of Comparative Medicine, University of REFERENCES Washington (Protocol 4244–01) or the Institutional Animal Care and Use Committee at Biogen (Protocol 0489–2013). Human kidney 1. Duffield JS: Cellular and molecular mechanisms in kidney fibrosis. J Clin tissue was obtained from discarded kidneys during nephrectomy Invest 124: 2299–2306, 2014 fi procedures performed at Tufts New England Medical Center 2. 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JK: Urinary collagen fragments are significantly altered in diabetes: a link TWEAK, via its receptor Fn14, is a novel regulator of mesenchymal to pathophysiology. PLoS One 5: e13051, 2010 progenitor cells and skeletal muscle regeneration. EMBO J 25: 45. Veidal SS, Karsdal MA, Vassiliadis E, Nawrocki A, Larsen MR, Nguyen 5826–5839, 2006 QH, Hägglund P, Luo Y, Zheng Q, Vainer B, Leeming DJ: MMP medi- 51. Gomez IG, MacKenna DA, Johnson BG, Kaimal V, Roach AM, Ren S, ated degradation of type VI collagen is highly associated with liver Nakagawa N, Xin C, Newitt R, Pandya S, Xia TH, Liu X, Borza DB, Grafals fibrosis–identification and validation of a novel biochemical marker M, Shankland SJ, Himmelfarb J, Portilla D, Liu S, Chau BN, Duffield JS: assay. PLoS One 6: e24753, 2011 Anti-microRNA-21 oligonucleotides prevent Alport nephropathy pro- 46. Naugle JE, Olson ER, Zhang X, Mase SE, Pilati CF, Maron MB, gression by stimulating metabolic pathways. JClinInvest125: 141– Folkesson HG, Horne WI, Doane KJ, Meszaros JG: Type VI collagen 156, 2015 induces cardiac myofibroblast differentiation: implications for post- 52. Lin SL, Li B, Rao S, Yeo EJ, Hudson TE, Nowlin BT, Pei H, Chen L, Zheng infarction remodeling. Am J Physiol Heart Circ Physiol 290: H323– JJ, Carroll TJ, Pollard JW, McMahon AP, Lang RA, Duffield JS: Mac- H330, 2006 rophage Wnt7b is critical for kidney repair and regeneration. Proc Natl 47. Keene DR, Engvall E, Glanville RW: Ultrastructure of type VI collagen in Acad Sci U S A 107: 4194–4199, 2010 human skin and cartilage suggests an anchoring function for this fila- 53. Crawford C, Kennedy-Lydon T, Sprott C, Desai T, Sawbridge L, Munday mentous network. JCellBiol107: 1995–2006, 1988 J, Unwin RJ, Wildman SS, Peppiatt-Wildman CM: An intact kidney slice 48. Kielty CM, Whittaker SP, Grant ME, Shuttleworth CA: Type VI collagen model to investigate vasa recta properties and function in situ. Neph- microfibrils: evidence for a structural association with hyaluronan. JCell ron, Physiol 120: 17–31, 2012 Biol 118: 979–990, 1992 49. Shea BS, Brooks SF, Fontaine BA, Chun J, Luster AD, Tager AM: Pro- longed exposure to sphingosine 1-phosphate receptor-1 agonists ex- acerbates vascular leak, fibrosis, and mortality after lung injury. Am J See related editorial, “Beyond EMT: Epithelial STAT3 as a Central Regulator of – Respir Cell Mol Biol 43: 662 673, 2010 Fibrogenesis,” on pages 3502–3504. 50. Girgenrath M, Weng S, Kostek CA, Browning B, Wang M, Brown SA, Winkles JA, Michaelson JS, Allaire N, Schneider P, Scott ML, This article contains supplemental material online at http://jasn.asnjournals. Hsu YM, Yagita H, Flavell RA, Miller JB, Burkly LC, Zheng TS: org/lookup/suppl/doi:10.1681/ASN.2015111227/-/DCSupplemental.

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