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Angiotensin IV is Induced in Experimental Autoimmune Encephalomyelitis but Fails to Influence the Disease

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Abstract

In multiple sclerosis (MS) and its corresponding animal models, over-activity of the renin-angiotensin system (RAS) has been reported and pharmacological RAS blockade exerts beneficial effects. The RAS generates a number of bioactive angiotensins, thereby primarily regulating the body’s sodium homeostasis and blood pressure. In this regard, angiotensin IV (AngIV), a metabolite of the RAS has been shown to modulate inflammatory responses. Here we studied potential implications of AngIV signalling in myelin oligodendrocyte glycoprotein (MOG) peptide induced murine experimental autoimmune encephalomyelitis (EAE), a close-to-MS animal model. Mass spectrometry revealed elevated plasma levels of AngIV in EAE. Expression of cognate AT4 receptors was detected in macrophages and T cells as major drivers of pathology in EAE. Yet, AngIV did not modulate macrophage or T cell functions in vitro or displayed detectable effects on neuroantigen specific immune responses in vivo. The data argue against a major contribution of AngIV signalling in the immunopathogenesis of MOG-EAE.

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Acknowledgments

This work was supported by the ELAN fonds of the Friedrich-Alexander University Erlangen-Nuremberg. We wish to thank Silvia Seubert for technical assistance.

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Authors and Affiliations

  1. Department of Neurology, University of Erlangen-Nuremberg, Erlangen, Germany

    Arndt Manzel, Agnes Kovacs & Ralf A. Linker

  2. Department of Internal Medicine III, General Hospital Vienna, Vienna, Austria

    Oliver Domenig

  3. Department of Neurology, Ruhr-University Bochum, Bochum, Germany

    Bjoern Ambrosius & Ralf Gold

  4. Department of Nephrology, Heinrich-Heine University, Düsseldorf, Germany

    Johannes Stegbauer

  5. Attoquant Diagnostics, Vienna, Austria

    Marko Poglitsch

  6. Experimental and Clinical Research Center, a joint cooperation between the Charitè, Medical Faculty and the Max-Delbruck Center for Molecular Medicine, Berlin, Germany

    Dominik N. Mueller

  7. Department of Neurology, University Hospital Erlangen, Schwabachanlage 6, 91054, Erlangen, Germany

    Ralf A. Linker

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  1. Arndt Manzel
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  2. Oliver Domenig
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  3. Bjoern Ambrosius
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  4. Agnes Kovacs
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  5. Johannes Stegbauer
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  9. Ralf A. Linker
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Correspondence to Ralf A. Linker.

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Supplementary Figure 1

Inflammation increases plasma angiotensins Mice were immunized with MOG/CFA or left untreated (naïve), plasma was collected on day 10 and 16 after immunization and analysed by the RAS-Fingerprint technique. Angiotensin peptides are identified by bracketed amino acid numbers. (Ang(2-8) = Ang III, Bars represent mean +/- SEM; n = 6, values below quantification limit included as “0“). Plasma levels of detectable downstream peptide metabolites are increased in MOG/CFA immunized mice. (GIF 32 kb)

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Supplementary Figure 2

Lnpep and c-Met proteins are expressed by macrophages. Peritoneal exudate cells were obtained by lavage from naïve C57BL/6 mice and cultured for 48 h in the presence or absence of 100 ng/ml LPS. Cells were fixed, permeabilised and stained with primary antibodies directed against Lnpep and c-Met before analysis on a flow cytometer. [a] Cells in the pM gate were further analysed (LC = Lymphocytes, GC = Granulocytes, pM = peritoneal macrophages). [b] Representative histograms of Lnpep and c-Met expression in peritoneal macrophages (iso = primary AB omitted). [c, d] Lnpep and c-Met protein expression quantified by mean fluorescence intensity. Lnpep and c-Met proteins are present in peritoneal macrophages, but not regulated upon LPS stimulation (bars represent mean +/- SEM, n = 3 replicates, pooled cells from 4-6 mice). (GIF 80 kb)

High Resolution Image (TIFF 19587 kb)

Supplementary Figure 3

AngIV does not influence T cell differentiation. [a-c] Naïve T cells were isolated from spleens of C57BL/6 mice and cultured in the presence of anti-CD3, anti-CD28 and polarizing cytokines under addition of AngIV. After 4 days, differentiated T helper cells were quantified by intracellular cytokine staining/flow cytometry. Addition of AngIV had no effect on T helper cell polarization (n = 6 replicates of pooled cells, bars represent mean +/- SEM, n = 3 p.g.). (GIF 21 kb)

High Resolution Image (TIFF 8358 kb)

Supplementary Figure 4

Cytokine production after MOG restimulation in vitro is not affected by AngIV. [a, b] Mice received continuous AngIV or saline infusion and underwent EAE induction. On day 10 post EAE induction, splenocytes were restimulated in vitro with 20 μg/ml MOG peptide for 48 h and cytokines secreted into supernatants were assayed by ELISA. [a] Production of typically T helper cell derived cytokines was not affected in AngIV infused mice. [b] Production of cytokines typically derived from antigen presenting cells or macrophages was not affected in AngIV infused mice (bars in a,b represent mean +/- SEM; n = 4 mice p.g.). (GIF 35 kb)

High Resolution Image (TIFF 12482 kb)

Supplementary Figure 5

AngIV does not impact on macrophage effector functions. [a] Peritoneal exudate cells were obtained by PBS lavage from naïve C57BL/6 mice and cultured for 24 h in the presence or absence of 100 ng/ml LPS. Cells were analysed for Ccl2/MCP-1 mRNA expression by semi-quantitative realtime PCR. AngIV had no effect on Ccl2 expression (n = 6, pool of 2 experiments). [b] Bone marrow precursor cells were differentiated into M0 macrophages, then polarised into M1 macrophages for 24h in the presence of IFN-γ, LPS and AngIV. AngIV did not influence M1 polarisation (bars represent mean +/- SEM, n = 3 p.g.). (GIF 34 kb)

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Manzel, A., Domenig, O., Ambrosius, B. et al. Angiotensin IV is Induced in Experimental Autoimmune Encephalomyelitis but Fails to Influence the Disease. J Neuroimmune Pharmacol 9, 533–543 (2014). https://doi.org/10.1007/s11481-014-9548-y

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