IL-6 Trans-Signaling Drives Murine Crescentic GN

Gerald Braun

Yoshikuni Nagayama

Yuichi Maruta

Felix Heymann

David Salant+4 authors

Supplementary Material

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^{Division of Nephrology and Immunology, Rheinisch-Westfälische Technische Hochschule (RWTH) Aachen University, Aachen Germany;}

^{Institute of Biochemistry and Molecular Biology, RWTH Aachen University, Aachen, Germany;}

^{Division of Gastroenterology, Metabolic Diseases and Intensive Care, RWTH Aachen University, Aachen, Germany;}

^{Institute of Pathology, RWTH Aachen University, Aachen, Germany;}

^{Institute of Molecular Biomedicine, Comenius University, Bratislava, Slovakia;}

^{Division of Nephrology, Showa University Fujigaoka Hospital, Yokohama, Japan;}

^{Department of Medicine, Section of Nephrology, Boston University School of Medicine, Boston, MA; and}

^{Institute of Biochemistry, Christian-Albrechts-University, Kiel, Germany}

^{Corresponding author.}

G.S.B. and Y.N. contributed equally to this work.

Correspondence: Dr. Gerald Braun, Medizinische Klinik II, Universitätsklinikum der RWTH, Pauwelsstr. 30, D-52057 Aachen, Germany. Email: ed.nehcaaku@nuarbg

Received 2014 Nov 26; Accepted 2015 Mar 20.

Abstract

The role of IL-6 signaling in renal diseases remains controversial, with data describing both anti-inflammatory and proinflammatory effects. IL-6 can act via classic signaling, engaging its two membrane receptors gp130 and IL-6 receptor (IL-6R). Alternatively, IL-6 trans-signaling requires soluble IL-6R (sIL-6R) to act on IL-6R-negative cells that express gp130. Here, we characterize the role of both pathways in crescentic nephritis. Patients with crescentic nephritis had significantly elevated levels of IL-6 in both serum and urine. Similarly, nephrotoxic serum-induced nephritis (NTN) in BALB/c mice was associated with elevated serum IL-6 levels. Levels of serum sIL-6R and renal downstream signals of IL-6 (phosphorylated signal transducer and activator of transcription 3, suppressor of cytokine signaling 3) increased over time in this model. Simultaneous inhibition of both IL-6 signaling pathways using anti–IL-6 antibody did not have a significant impact on NTN severity. In contrast, specific inhibition of trans-signaling using recombinant sgp130Fc resulted in milder disease. Vice versa, specific activation of trans-signaling using a recombinant IL-6–sIL-6R fusion molecule (Hyper-IL-6) significantly aggravated NTN and led to increased systolic BP in NTN mice. This correlated with increased renal mRNA synthesis of the Th17 cell cytokine IL-17A and decreased synthesis of resistin-like alpha (RELMalpha)-encoding mRNA, a surrogate marker of lesion-mitigating M2 macrophage subtypes. Collectively, our data suggest a central role for IL-6 trans-signaling in crescentic nephritis and offer options for more effective and specific therapeutic interventions in the IL-6 system.

Keywords: glomerular disease, albuminuria, proteinuria, nephritis

Abstract

IL-6 has long been recognized to contribute to inflammatory glomerular diseases.¹–⁵ It is mostly produced by leukocytes but can also be secreted from glomerular endothelial and mesangial cells upon stimulation with, for example, angiotensin II and by podocytes following endotoxin administration in vivo.⁶–⁸ However, the role of IL-6 signaling in glomerular disease, in particular in rapidly progressive glomerulonephritis (RPGN), remains controversial. In rats, IL-6 infusion exerted a beneficial effect in the RPGN model of nephrotoxic nephritis (NTN),⁹ possibly by limiting macrophage activity. Likewise, IL-6 inhibition by either an IL-6- or an IL-6-receptor (IL-6R)-specific antibody aggravated NTN in C57Bl/6 mice leading to increased macrophage activity.¹⁰ However, research in models of systemic lupus erythematosus (SLE) arrived at an opposite conclusion. In one study IL-6 infusion exerted an aggravating effect on GN.¹¹ Although the opposite experiment of IL-6 antagonism did not turn out to be beneficial in this work,¹¹ a second study described disease reduction in another SLE model by an IL-6R-specific antibody.¹²

The IL-6 system consists of IL-6 and two receptors that occur in membrane-bound and soluble forms.¹³ In classic signaling, IL-6 often has anti-inflammatory activity by binding to a membrane-bound heterotetramer of the IL-6R and glycoprotein 130 (gp130), the common signal transducing unit of the IL-6 family of cytokines.¹³ While gp130 is ubiquitous, IL-6R is largely restricted to immune cells and hepatocytes.¹³ In contrast with classic IL-6 signaling, virtually all cells of the body can be stimulated by trans-signaling, in which membrane-bound gp130 transduces a signal upon binding of a complex of IL-6 and the soluble isoform of IL-6R (sIL-6R).¹³ Trans-signaling markedly widens the spectrum of IL-6–susceptible cells and usually exerts proinflammatory actions, especially in the late course of diseases.¹⁴ A circulating soluble isoform of gp130 (sgp130) inhibits trans-signaling and is considered a buffer system limiting trans-signaling in health.¹⁴ Therapeutically, a fusion protein consisting of two sgp130 chains joined by an inactivated Fc receptor portion (sgp130Fc) exerted beneficial anti-inflammatory effects in several non-renal models.¹⁴

Given that IL-6 can exert both anti-inflammatory (usually via classic signaling) and proinflammatory activity (trans-signaling), we hypothesized that this dual mode of action may explain the above controversial findings of previous attempts to target the IL-6 system in nephritis. We therefore first performed a comprehensive assessment of the IL-6 system in human and experimental glomerulonephritis and then either blocked both pathways or selectively inhibited or stimulated trans-signaling in the NTN model.

mc, monoclonal; pc, polyclonal; fc, fluorescent conjugate; IF, immunofluorescence; IHC, immunohistochemistry; WB, Western blot.

Gapdh, glyceraldehyde-3-phosphate dehydrogenase; gp130, glycoprotein 130; Socs3, suppressor of cytokine signaling 3; iNOS, inducible nitric oxide synthase; T-bet, T-box transcription factor 21; Relm-α, resistin-like molecule alpha; GATA-3, GATA binding protein-3; Itgax/CD11c, cluster of differentiation 11c, integrin alpha X.

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Acknowledgments

G.S.B. designed the study and research, performed experiments, analyzed data and prepared the manuscript. Y.N., Y.M., F.H., C.R.V.R., B.M.K., P.B., and L.V. performed experiments and data analysis. D.J.S., U.R., and S.R.J. contributed intellectual input and reagents. T.O. and J.F. conceived, supervised and co-wrote the study.

This study was supported by grants of the Deutsche Forschungsgemeinschaft (SFB TRR 57, projects P17 and P25, and FL 178/4-1) to J.F., P.B., and T.O. and by RO4036/1-1 to C.R.V.R. and BO 3755/2-1 to P.B. Further support came from a grant of the Interdisciplinary Center for Clinical Research (IZKF) at RWTH Aachen University to T.O. and J.F. (E7-2) and from an intramural program to G.S.B. (Rotationsprogramm der Medizinischen Fakultät der RWTH). D.J.S. was supported by research grant DK090029 from the US National Institutes of Health. The work of S.R.J. was supported by the Deutsche Forschungsgemeinschaft (SFB 841, project C1 and the Cluster of Excellence Inflammation at Interfaces). The expert help of Christina Gianussis, Nicole Bataille, Gabriele Dietzel, Gertrud Minnartz, Dagmar Wieland, Esther Stüttgen and Lydia Zimmermanns in performing these studies is gratefully acknowledged.

Acknowledgments

Footnotes

Published online ahead of print. Publication date available at www.jasn.org.

This article contains supplemental material online at http://jasn.asnjournals.org/lookup/suppl/doi:10.1681/ASN.2014111147/-/DCSupplemental.

Footnotes

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