Basic Res Cardiol (2014) 109:408 DOI 10.1007/s00395-014-0408-y

ORIGINAL CONTRIBUTION

Adiponectin promotes coxsackievirus B3 myocarditis by suppression of acute anti-viral immune responses A. Jenke • L. Holzhauser • M. Lo¨bel • K. Savvatis • S. Wilk • A. Weitha¨user S. Pinkert • C. Tscho¨pe • K. Klingel • W. Poller • C. Scheibenbogen • H. P. Schultheiss • C. Skurk

•

Received: 9 July 2013 / Accepted: 14 March 2014 / Published online: 2 April 2014 Ó Springer-Verlag Berlin Heidelberg 2014

Abstract Adiponectin (APN) is an immunomodulatory adipocytokine that improves outcome in patients with virus-negative inflammatory cardiomyopathy and mice with autoimmune myocarditis. Here, we investigated whether APN modulates cardiac inflammation and injury in coxsackievirus B3 (CVB3) myocarditis. Myocarditis was induced by CVB3 infection of APN-KO and WT mice. APN reconstitution was performed by adenoviral gene transfer. Expression analyses were performed by qRT-PCR and immunoblot. Cardiac histology was analyzed by H&Estain and immunohistochemistry. APN-KO mice exhibited diminished subacute myocarditis with reduced viral load, attenuated inflammatory infiltrates determined by NKp46, F4/80 and CD3/CD4/CD8 expression and reduced IFNb, IFNc, TNFa, IL-1b and IL-12 levels. Moreover, Electronic supplementary material The online version of this article (doi:10.1007/s00395-014-0408-y) contains supplementary material, which is available to authorized users. A. Jenke
L. Holzhauser
K. Savvatis
A. Weitha¨user
C. Tscho¨pe
W. Poller
H. P. Schultheiss
C. Skurk (&) Department of Cardiology and Pneumology, Charite´ University Medicine Berlin, Campus Benjamin Franklin, Hindenburgdamm 30, 12200 Berlin, Germany e-mail: [email protected] M. Lo¨bel
S. Wilk
C. Scheibenbogen Institute of Medical Immunology, Charite´ University Medicine Berlin, Campus Virchow-Klinikum, Augustenburger Platz 1, 13353 Berlin, Germany S. Pinkert Biotechnological Institute, Technical University Berlin, Gustav-Meyer-Allee 25, 13355 Berlin, Germany K. Klingel Department of Molecular Pathology, University of Tu¨bingen, Liebermeisterstrasse 8, 72076 Tu¨bingen, Germany

myocardial injury assessed by necrotic lesions and troponin I release was attenuated resulting in preserved left ventricular function. Those changes were reversed by APN reconstitution. APN had no influence on adhesion, uptake or replication of CVB3 in cardiac myocytes. In acute CVB3 myocarditis, cardiac viral load did not differ between APN-KO and WT mice. However, APN-KO mice displayed an enhanced acute immune response, i.e. increased expression of myocardial CD14, IFNb, IFNc, IL12, and TNFa resulting in increased cardiac infiltration with pro-inflammatory M1 macrophages and activated NK cells. Up-regulation of cardiac CD14 expression, type I and II IFNs and inflammatory cell accumulation in APN-KO mice was inhibited by APN reconstitution. Our observations indicate that APN promotes CVB3 myocarditis by suppression of toll-like receptor-dependent innate immune responses, polarization of anti-inflammatory M2 macrophages and reduction of number and activation of NK cells resulting in attenuated acute anti-viral immune responses. Keywords Adiponectin
Coxsackievirus B3
Myocarditis
Toll-like receptor
Innate immunity

Introduction Coxsackievirus B3 (CVB3) is a positive, single-strand RNA virus of the Picornaviridae family inducing viral myocarditis. Clinical outcome in CVB3 myocarditis is determined by the interplay between pathogen virulence and host response ranging from subclinical disease to acute heart failure and death or dilated cardiomyopathy (DCM) due to incomplete virus clearance or autoimmune disease [6, 22, 21]. Immediately after CVB3 infection of the heart, innate and adaptive immune responses are induced that

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lead to virus clearance and sessation of the inflammatory response that can also affect the myocardium [11], because infiltrated immune cells have been shown to damage cardiac tissue either directly through secretion of cytolytic proteins (e.g. perforin) or indirectly via overexpression of pro-inflammatory cytokines or induction of fibrosis [8]. The initiation of a robust inflammatory response in the acute phase of CVB3 myocarditis represents a crucial factor to achieve early virus clearance, to avoid direct cardiac damage and detrimental persistent accumulation of infiltrated immune cells within the heart. The acute immune response is characterized by the up-regulation of type I and II interferons (IFNs) associated with infiltration of activated macrophages and natural killer (NK) cells to sites of CVB3 infection. It precedes a second wave of infiltrating antigen-specific T and B cells in the subacute phase of myocarditis [8]. A prolonged and exaggerated reaction of the immune system due to CVB3 persistence in the heart induces myocardial cell death and adverse extracellular matrix (ECM) remodeling resulting in cardiac dysfunction, ventricular dilatation and heart failure [18]. Adiponectin (APN) is an abundant plasma cytokine, that is primarily expressed in adipocytes [36]. Moreover, APN expression has also been shown for endothelial cells, skeletal myocytes and cardiac cells [36]. APN exists as full-length protein in several oligomeric forms or as proteolytic cleavage fragment in high concentrations of 3–30 lg/mL in human plasma [36]. APN functions as important modulator of immune reactions exerting a wide spectrum of anti-inflammatory effects [36]. Results of in vitro experiments demonstrate that APN inhibits activation of the pro-inflammatory transcription factor nuclear factor (NF)-jB in cardiac myocytes and fibroblasts [3] as well as expression of tumor necrosis factor (TNF)a in endothelial cells [28]. Moreover, APN suppresses immune cell activation [27], adhesion to target cells [28] and controls antigen-specific expansion of T cells [41]. Accordingly, results from in vivo studies of chronic inflammation show that APN mediates cardioprotection by modulation of pro-inflammatory responses in non-viral heart disease [16]. Moreover, APN confers resistance against cardiac injury and fibrosis following ischemia [29, 33, 45], attenuates left ventricular hyperthrophy after pressure overload and favors positive outcome in patients with virus-negative inflammatory cardiomyopathy [3]. Therefore, APN has been characterized as anti-inflammatory, cardioprotective cytokine. Early descriptive studies investigating APN effects in virus myocarditis showed protective effects regarding myocardial inflammation and function [35, 34, 17]. However, inhibition of innate immune responses, i.e. amelioration of Toll-like receptor (TLR) signaling [47] as well as attenuation of antigen-specific T cell proliferation [41] while protective against chronic inflammation might be

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deleterious in viral infection, since viral clearance is important for prevention of direct viral and inflammationinduced tissue damage [1]. Moreover, APN has been shown to enhance hepatitis B virus (HBV) replication [46]. Accordingly, serum APN levels are associated with HBV virus load in HBV carriers [4]. Here, we comparatively investigated the course of CVB3-induced myocardial inflammation and injury in wild-type (WT) and APN-knockout (KO) mice. We could show that in the absence of APN viral infection was cleared more rapidly with less myocardial inflammation and injury at day 7 post-infection (p.i.).

Methods Cell culture and reagents Neonatal cardiac myocytes and fibroblasts were prepared from hearts of 1–3-day old Wistar Unilever rats. Mouse splenocytes were isolated from spleens of 6–8 weeks old male WT and APN-KO mice. Splenic NK cells were enriched using the NK cell Isolation Kit II (Miltenyi Biotech). Recombinant human full-length APN produced in a mammalian expression system was purchased from R&D Systems; measured endotoxin contamination (KineticQCL, Lonza) was below 10 pg/lg protein. TLR ligands LPS, P(I:C) and R-848 were purchased from Enzo Life Sciences. Animals and mouse model of CVB3 myocarditis APN-knockout (APN-KO) mice and corresponding C57BL/6 WT mice were purchased from Jackson Laboratories. The investigation was approved by the institutional ethics committee and the respective authorities in Berlin (Germany), it conformed to the NIH Guide for the Care and Use of Laboratory Animals (8th edition, published 2011) and has been performed in accordance with the Declaration of Helsinki (6th revision, published 2008). Before injections and euthanasiation animals were anaesthized by inhalation of 2.0 vol % isoflurane for 5 min using an automatic delivery system. Male mice were infected by intraperitoneal injection of 200 lL phosphate-buffered saline (PBS) containing 5 9 105 plaque forming units (PFU) of transfection-derived purified CVB3 (Nancy strain; ATCC Number: VR-30). 3 and 7 days p.i. infected APN-KO mice and WT mice were compared with PBStreated mice of both groups. For APN reconstitution experiments, APN-KO mice were injected with 1 9 109 PFU of Ad-APN vector 7 days before CVB3 infection. APN gene transfer leads to transduction of stable expression of APN in the liver that can be detected in serum as

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long as 4 weeks [16]. As shown in Suppl. Fig. 1c reconstitution of APN-KO mice resulted in systemic APN levels comparable to WT mice. Systemic APN levels were measured by ELISA (R&D Systems) for all animals included in the study using duplicates of serum samples. A total number of 52 WT mice and 73 APN-KO mice were originally used for this study. 3 and 7 days p.i. infected APN-KO mice and WT mice were compared with PBS-treated mice of both groups. Moreover, APN-KO mice reconstituted by APN gene transfer were examined in CVB3 myocarditis. Therefore, APN-KO mice were injected with 1 9 109 PFU of Ad-APN vector 7 days before CVB3 infection. All animals were randomly assigned to the different study groups. A minimum CVB3 positive strand copy number of 1 9 103 per ng cDNA in left ventricular tissue specimens as determined by qRT-PCR was used as threshold to include infected animals into the study. The CVB3 positive strand copy number was measured for all CVB3-infected animals using triplicates of left ventricular cDNA samples. These criteria finally resulted in the following number of animals per group:

Group

Number of animals Infected

Tissue sections of frozen heart samples were used for detection of viral positive strand RNA with a S35-labeled enterovirus-specific RNA probe. Sections were exposed for 3 weeks and counterstained with hematoxylin and eosin (H&E). Quantification of CVB3 RNA following in situ hybridization was done using the following score obtained in high power fields (2009): 0 = no positive cells; 1 = a few small foci with positive cells; 2 = a few foci with [100 positive cells; 3 = B10 % of the tissue sections contain positive cells; and 4 = 10–30 % of the tissue sections contain positive cells (subacute CVB3 myocarditis: n = 15 animals per group, acute CVB3 myocarditis: n = 5 animals per group, scoring of four sections per animal). Plaque assay

WT

–

15

APN-KO

–

15

WT-CVB3

19

15

APN-KO CVB3

16

15

APN-KO ? Ad-APN CVB3

15

13

Acute CVB3 myocarditis (day 3 p.i.) – –

In situ hybridization

included in study

Subacute CVB3 myocarditis (day 7 p.i.)

WT APN-KO

pressure was measured. Cardiac hemodynamics were measured for n = 6–8 animals per group. Animals were euthanasiazed immediately after hemodynamic measurements. Hearts were removed, transversely dissected in 3-mm-thick slices and snap frozen for later analysis. Serum levels of troponin I were measured by ELISA (Life Diagnostics) using duplicates of serum samples for n = 10 animals per group.

8 8

WT-CVB3

10

8

APN-KO CVB3

10

8

APN-KO ? Ad-APN CVB3

9

8

For certain analyses such as immunohistochemical measurements of cardiac protein expression smaller numbers of animals were randomly chosen from the different study groups to allow an efficient but representative comparison of all groups of mice. Cardiac hemodynamics and troponin I measurements Parameters of left ventricular function were recorded via a microconductance catheter (1.2F) system in closed-chest animals. Moreover, heart rate as well as mean blood

HeLa cells (Wisconsin strain) were maintained in monolayer culture in minimal essential medium supplemented with 5 % FBS, 1 % gentamicin and non-essential amino acids. For plaque assay analysis, confluent HeLa cell layers in 24 well plates were infected with serially diluted CVB3 containing tissue extracts for an inoculation time of 30 min. HeLa cells were overlaid with agar containing Eagle’s MEM and incubated for 3 days at 37 °C. To visualize plaques, cells were stained with 0,025 % (w/v) neutral red and virus titer was determined by counting plaques. The assay was measured for all animals included in the study and was repeated at least twice for each mouse. In vitro replication of CVB3 in cardiac myocytes Transfection-derived and purified CVB3 (Nancy strain; ATCC Number: VR-30) was used for experiments with cardiac myocytes. To analyze adhesion, uptake and replication of CVB3 in vitro cardiac myocytes were inoculated with CVB3 at a multiplicity of infection (MOI) of 1 for 30 min in serum-free medium in presence or absence of APN. After inoculation CVB3-containing medium was removed and cells washed twice with PBS to eliminate unattached virus. For measurement of CVB3 adhesion, RNA was isolated immediately from PBS-washed cardiac

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myocytes. For measurement of CVB3 internalization, PBSwashed cardiac myocytes were incubated in DMEM containing 10 % FBS for 30 min at 37 °C. To remove noninternalized CVB3 from the cell surface, cardiac myocytes were trypsinized and pelletized by centrifugation before RNA was isolated. For measurement of viral replication PBS-washed cardiac myocytes were cultured in DMEM containing 10 % FBS for 12 h. To quantify APN expression in cardiac myocytes following CVB3 infection in vitro cardiac myocytes were inoculated with CVB3 at a MOI of 1 for 30 min in a serum-free medium. After inoculation CVB3-containing medium was removed and cells washed twice with PBS. The PBS-washed cardiac myocytes were cultured in DMEM containing 10 % FBS for 6 h. RNA was isolated using TRIzol (Invitrogen). Copy numbers of CVB3 RNA were determined by qRT-PCR. Quantitative real-time polymerase chain reaction (qRTPCR) RNA from tissues and cultured cells was extracted using TRIzol (Invitrogen) and the RNeasy Mini Kit (Qiagen). RNA integrity was checked by 2100 Bioanalyzer (Agilent Technologies). qRT-PCR was performed using High capacity cDNA Reverse Transcription Kit, TaqMan Universal PCR Master Mix and TaqMan gene expression assays (Applied Biosystems). Triplicates of cDNA samples were used for all measurements. Histological measurements Tissue Tec—embedded frozen left ventricular tissue sections were used for histological measurements. Inflammatory infiltrates and associated cardiac myocyte necrosis were quantified in H&E-stained section high power fields (2009). The inflammatory score was calculated by multiplication of the share of section area marked by immune cell infiltrates with the average degree of inflammatory. The degree of inflammatory foci was discriminated as follows: 0 = no immune cell infiltrates; 1 = small inflammatory foci with few cells; 2 = medium grade foci with B100 immune cells; 3 = severe inflammatory foci with C100 immune cells foci (n = 15 animals per group, scoring of four sections per animal). The extent of cardiac myocyte necrosis in areas of inflammatory infiltrates was quantified in H&E-stained section high power fields (4009). The necrotic score was calculated by multiplicating the share of section area affected by cardiac myocyte necrosis with the average degree of necrotic foci. The degree of necrotic foci was discriminated as follows: 0 = no necrotic cardiac myocytes; 1 = share of necrotic cardiac myocytes B20 %; 2 = 20 % \ share of necrotic cardiac myocytes B50 %; 3 = share of necrotic cardiac

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myocytes C50 % (n = 6 animals per group, scoring of four sections per animal). Immunohistochemistry was performed using primary antibodies for CD11b (BD Pharmingen; subacute CVB3 myocarditis: n = 6–8 animals per group, acute CVB3 myocarditis: n = 5 animals per group), collagen type 1 (Millipore; n = 6 animals per group) and CD14 (Santa Cruz; n = 6–8 animals per group) followed by the Envision horseradish peroxidase technique (DAKO). For CD14 immunohistochemistry sections were pre-treated with a permeabilisation solution containing 0.1 % Triton X-100. Results were quantified by digital image analyses of four sections per animal. Immunoblot Cardiac protein expression of CD14 was quantified by immunoblot using anti-CD14 (Santa Cruz) primary and anti-rabbit (Cell Signaling Technologies) secondary antibodies (n = 6–8 animals per group). Protein bands were visualized using a chemoluminescence system (Thermo Scientific). Statistical analysis SPSS 20 was used for statistical data analysis. Results are expressed as box plots with median. Statistical differences were assessed using the Kruskal–Wallis test followed by post-hoc testing via Mann–Whitney U test for pair-wise comparisons between individual groups. Differences were considered statistically significant at a two-sided value of P \ 0.05. No Bonferroni adjustment has been performed.

Results APN regulation in CVB3 myocarditis APN expression following CVB3 infection was studied in WT mice. CVB3 infection induced a significant up-regulation of cardiac APN expression as early as day 3 p.i. (Suppl. Fig. 1a) that was due to the inflammatory process since CVB3-dependent up-regulation of APN in cardiac myocytes was absent in vitro (Suppl. Fig. 1b). However, systemic plasma concentrations of APN did not differ between WT and CVB3-infected WT mice on day 3 p.i. (Suppl. Fig. 1c). APN-KO mice show reduced virus load in subacute myocarditis Cardiac virus load in subacute myocarditis, i.e. at day 7 p.i., measured by plaque assay (Fig. 1a) or CVB3 copy number (Suppl. Fig. 2a) was significantly reduced in APN-

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KO mice (P \ 0.01). These results were corroborated by in situ hybridization (ISH, Fig. 1b), as APN-KO mice showed significantly lower levels of CVB3 RNA in cardiac myocytes and immune cells when compared to WT littermates (P \ 0.01). Taken together, those data implicate APN in the regulation of cardiac CVB3 infection by facilitating viral replication. APN-KO mice exhibit diminished inflammatory infiltrates and pro-inflammatory cytokine expression in subacute myocarditis APN-KO and WT mice exhibit low numbers of inflammatory cells in cardiac tissue under baseline conditions (Fig. 1c). At day 7 p.i., there was a significant increase in cardiac mononuclear cell accumulation in WT animals. In APN-KO mice, however, increased accumulation of inflammatory cells was significantly blunted following CVB3 infection. The score of inflammatory infiltrates in hearts of APN-KO mice infected with CVB3 was less than half of that calculated for WT animals (P \ 0.01) (Fig. 1c). In line with these data, gene expression of immune cell markers cluster of differentiation (CD)3z (Fig. 1d), CD4, CD8a and perforin (T cells) (Suppl. Fig. 2b), protein expression of the immune cell marker CD11b abundantly expressed on macrophages and NK cells (Fig. 1e) as well as gene expression of F4/80 (macrophages) and NKp46 (NK cells) (Fig. 1f) was significantly increased 7 days p.i. compared with baseline in WT animals (P \ 0.01), whereas up-regulation of those markers was significantly diminished in APN-KO mice (P \ 0.01 vs. WT-CVB3, respectively). Taken together our data indicate lower grades of inflammatory cell accumulation in hearts of APN-KO mice in subacute myocarditis. Accordingly, WT as well as APN-KO mice showed an increase in cardiac gene expression of pro-inflammatory cytokines at day 7 post-CVB3 infection compared to baseline conditions (Fig. 2). However, gene expression of IFNb, IFNc, TNFa, interleukin (IL)-1b, IL-6 and IL-12 was significantly diminished in APN-KO mice when compared to WT littermates 7 days p.i. (P \ 0.01, respectively). APN-KO mice show attenuated myocardial injury and improved left ventricular function in subacute CVB3 myocarditis Chronic inflammation and ongoing virus replication have been shown to trigger myocardial injury in CVB3 myocarditis [21]. In subacute CVB3 myocarditis on day 7 p.i. WT mice exhibited substantial myocardial necrosis located primarily around inflammatory infiltrates (Fig. 3a) which was accompanied by increased serum levels of troponin I, a specific marker for cardiac myocyte injury (Fig. 3b).

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Correlating with decreased myocardial virus load and attenuated cardiac inflammation following CVB3 infection, APN-KO mice showed significantly diminished necrotic lesions (P \ 0.01, Fig. 3a) and troponin I serum levels (P \ 0.05, Fig. 3b) compared to their WT littermates. Moreover, APN reconstitution by gene transfer in APN-KO mice resulting in systemic APN levels comparable to WT animals reversed the observed reduction of troponin I serum levels in APN-deficient mice (P \ 0.05, Fig. 3b). Persistent myocardial inflammation and associated tissue injury induce pro-fibrotic ECM remodeling in CVB3 myocarditis. Therefore, mRNA expression of Col1a1 and Col3a1 (Suppl. Fig. 2c) as well as protein expression of collagen type 1 (Fig. 3c) was determined in hearts of mice. CVB3 myocarditis resulted in increased collagen mRNA and protein expression primarily located around inflammatory infiltrates which was significantly attenuated in APN-deficient mice. Taken together, our data implicate that diminished virus load and attenuated cardiac inflammation in APN-KO mice are associated with reduced myocardial tissue injury. In accordance with these findings, hemodynamic parameters were less impaired in APN— deficient mice compared to their WT littermates 1 week following CVB3 infection (Table 1). APN reconstitution by gene transfer in APN-KO mice resulting in systemic APN levels comparable to WT animals reversed the observed improvement of left ventricular function in APNdeficient mice implicating APN in adverse outcome of CVB3 myocarditis. APN has no effect on CVB3 replication in cardiac myocytes The observed anti-viral effect of APN deficiency could be due to (1) inhibition of myocardial CVB3 adhesion, entry or replication (i.e., direct effects of APN on viral life cycle in target cells), or (2) immunosuppressive actions of APN. To exclude that APN promotes viral entry, expression of CAR, the coxsackievirus-and-adenovirus-receptor mediating cellular entry that was determined in hearts of animals on day 3 post-CVB3 infection. CAR expression was not different between the groups of mice studied (Suppl. Fig. 3a). Moreover, cardiac myocytes were infected with CVB3 in vitro in the presence or absence of APN and viral adhesion, entry and replication were determined. As shown in Fig. 4a, APN had neither any inherent effect on infection nor on replication of CVB3 virus particles in cardiac myocytes. Therefore, inhibition of viral replication in APN-KO mice observed 7 days p.i. is most likely due to the immunoregulatory role of APN. Therefore, pancreatic (data not shown) and cardiac CVB3 virus load as determined by plaque assay and ISH was assessed 3 days following infection with CVB3 in APN-KO and WT animals.

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Basic Res Cardiol (2014) 109:408 b Fig. 1 APN-KO mice exhibit reduced virus load and inflammatory

cell infiltration in subacute CVB3 myocarditis. CVB3 virus load in hearts of WT and APN-KO mice on day 7 p.i. was determined by a plaque assay and by b in situ hybridization (n = 15 animals per group). c Inflammatory cell infiltration in hearts of WT and APN-KO mice on day 7 p.i. was scored by H&E staining (n = 15 animals per group). d mRNA expression of immune cell marker CD3z in hearts of WT and APN-KO mice on day 7 p.i. was determined by qRT-PCR (n = 15 animals in per group). e Protein expression of CD11b in hearts of WT and APN-KO mice on day 7 p.i. was quantified by immunohistochemistry. Left panel: reddish colouring in representative pictures indicates CD11b? cells. Right panel: box plot indicating number of CD11b? cells per mm2 (n = 6–8 animals per group). f mRNA expression of F4/80 and NKp46 in hearts of WT and APNKO mice on day 7 p.i. was determined by qRT-PCR (n = 15 animals per group)

In accordance with our in vitro data, the copy number of CVB3 genome was not different in pancreas and hearts of infected APN-KO and WT mice (Suppl. Fig. 3b). Those data were corroborated by plaque assay and ISH (Fig. 4b), further implicating altered immune responses rather than reduced in vivo CVB3 infection for the higher virus load observed in hearts of WT mice at day 7 p.i. APN-KO mice show increased CD14 expression in myocardial tissue in acute CVB3 myocarditis TLRs are key recognition components of the innate immune system that recognize pathogen-associated molecular

Fig. 2 APN-KO mice show reduced expression of interferons and pro-inflammatory cytokines in subacute CVB3 myocarditis. mRNA expression of IFNb, IFNc, TNFa, IL-1b, IL-6 and IL-12(p40) in

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patterns (PAMPs) and up-regulate pro-inflammatory gene expression. CD14 acts as co-receptor in the TLR signal transduction pathway. It binds to TLR3, TLR4 and TLR7/8 and amplifies pro-inflammatory signaling. CVB3 triggers an innate immune response by binding to the CD14-TLR complex as the first line of defense. TLR expression was not different on day 3 following CVB3 infection in WT and APN-KO mice (data not shown). However, CVB3 myocarditis induced an up-regulation of CD14 expression on cardiac myocytes that was significantly enhanced in APNKO mice (Fig. 4c). Immunohistochemical CD14 staining was apparent on the surface of cardiac myocytes and immune cells as well as intracellularly (Fig. 4d). Moreover, reconstitution of APN by gene transfer reversed the observed up-regulation of CD14 expression in hearts of APN-deficient animals following CVB3 infection (Fig. 4c, d). To investigate regulation of CD14 expression more precisely, cardiac myocytes were incubated with polyinosine-polycytidylic acid (P(I:C)), R-848 or lipopolysaccharide (LPS), known activators for TLR3, TLR7/8 and TLR4, in the presence or absence of APN in vitro. Stimulation with respective TLR ligands led to an up-regulation of CD14 expression. However, APN incubation did not affect CD14 expression in cardiac myocytes in vitro (Suppl. Fig. 4a-b). Instead, APN significantly inhibited TLR3mediated up-regulation of IFN expression following stimulation of cardiac and immune cells with P(I:C) (Fig. 4e). In

hearts of WT and APN-KO mice on day 7 p.i. was determined by qRT-PCR (n = 15 animals per group)

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Basic Res Cardiol (2014) 109:408

Fig. 3 APN-KO mice show attenuated myocardial injury and fibrosis in subacute CVB3 myocarditis. a Extent of cardiac myocyte necrosis in hearts of WT and APN-KO mice on day 7 p.i. was scored in H&Estained tissue sections. Left panel: arrowheads in representative pictures indicate necrotic cardiac myocytes. Right panel: box plot indicating calculated necrosis scores (n = 6 animals per group). b Troponin I as specific marker for cardiac myocyte injury was

Table 1 Hemodynamic function in subacute myocarditis

* Significantly different vs. WT # Significantly different vs. WT CVB3 §

Significantly different vs. APN-KO CVB3

WT

APN-KO

WT-CVB3

APN-KO CVB3

APN-KO CVB3 ? Ad-APN

LV maximal pressure (mm Hg)

79.5 ± 2.8

75.2 ± 2.7

45.2 ± 1.9*

60.3 ± 3.7#

46.2 ± 2.6§

LV end systolic pressure (mm Hg)

69.4 ± 3.0

63.9 ± 2.9

44.8 ± 1.2*

55.7 ± 4.2#

43.9 ± 1.2§

LV end diastolic pressure (mm Hg)

3.4 ± 0.6

2.7 ± 0.6

7.9 ± 0.9*

4.3 ± 1.1#

7.5 ± 0.4§

Stroke volume (lL)

19.9 ± 2.7

18.0 ± 1.1

9.5 ± 0.4*

13.1 ± 0.7#

Cardiac output (mL/min) Ejection fraction (%)

15,710 ± 1,968 64.9 ± 4.6

accordance with attenuation of TLR/CD14 signaling by APN, we determined a significant increase of IFNb and IFNc expression in hearts of APN-KO mice compared to

123

measured in serum of WT and APN-KO mice on day 7 p.i. (n = 10 animals per group). c Protein expression of collagen type 1 in hearts of WT and APN-KO mice on day 7 p.i. was quantified by immunohistochemistry. Left panel: reddish colouring in representative pictures indicates collagen type 1. Right panel: box plot indicating area fraction of collagen type 1-positive tissue (n = 6 animals per group)

9.7 ± 0.2§ #

12,569 ± 850

4,197 ±266*

6,602 ± 1,295

66.3 ± 4.4

51.0 ± 1.6*

63.6 ± 2.0#

4,240 ± 99§ 48.0 ± 2.4§

WT animals at day 3 p.i. that was reversed by APN reconstitution via gene transfer (Fig. 4f). Taken together, our data implicate APN in downregulation of cardiac myocyte

Basic Res Cardiol (2014) 109:408

CD14 expression in vivo and inhibition of TLR3 signaling resulting in enhanced cardiac cytokine expression and increased recruitment/activation of immune cells in APNKO mice in acute CVB3 myocarditis. APN-KO mice exhibit increased number and activation of macrophages and NK cells in acute CVB3 myocarditis In acute CVB3 myocarditis 3 days p.i. the initial anti-viral inflammatory response is carried out by activated immune cells, i.e. macrophages and NK cells [8]. The immune cell marker CD11b is predominantly expressed on these cell types. At day 3 p.i. we observed an increased number of CD11b? cells in the myocardium of WT and APN-KO mice compared to baseline conditions (P \ 0.01, Fig. 5a). However, the increase in macrophage and NK cell accumulation following CVB3 infection was significantly enhanced in APN-KO mice compared to their WT littermates (P \ 0.05). Importantly, CD3? T cell accumulation was not significantly different in all examined groups of mice (Suppl. Fig. 3c). Detailed expression analysis of specific macrophage and NK cell activation markers by qRT-PCR at day 3 p.i. revealed a significant increase in cardiac expression of F4/80, CD69 and perforin in APNKO mice (P \ 0.05 APN-KO vs. WT mice, respectively) (Fig. 5b, c). Moreover, the increased number and activation of macrophages and NK cells in APN-KO animals in acute CVB3 myocarditis was reversed by APN reconstitution via gene transfer. In summary, our data implicate a different time course of viral clearance in WT and APNKO mice due to differential activation of immune cells in viral myocarditis. APN-KO mice show increased macrophage polarization towards the pro-inflammatory M1 phenotype and enhanced anti-viral NK cell activity in acute CVB3 myocarditis Macrophages display two distinct polarization states, M1 and M2. The ‘‘classical’’ M1 macrophage has a proinflammatory and anti-viral phenotype. In contrast, the ‘‘alternative’’ M2 macrophage exerts predominantly antiinflammatory effects. Macrophage galactose N-acetylgalactosamine specific lectin-1 (Mgl1) represents a specific marker to distinguish progressive M1 or M2 polarization [27]. Macrophages are typically polarized to the M1 state in response to PAMPs or alternatively by released IFNs following viral infections [27, 30]. Interestingly, although APN-KO mice exhibited an increased accumulation of mononuclear cells (i.e., macrophages and NK cells) at day 3 post-CVB3 infection, a significantly diminished Mgl1/ F4/80 expression ratio indicating a shift to the pro-

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inflammatory, anti-viral M1 phenotype was determined compared to WT animals (P \ 0.01, Fig. 5c). Moreover, the observed shift of cardiac macrophage polarization in APN-KO mice was completely reversed by APN reconstitution via gene transfer (P \ 0.01). Activated M1 macrophages typically up-regulate expression of proinflammatory cytokines (i.e. TNFa and IL-12) and chemokines (i.e. CC chemokine ligand (CCL)5) [27]. Accordingly, significantly up-regulated expression levels of TNFa, IL-12, CCL5 and CXC chemokine ligand (CXCL)10 were determined in hearts of APN-KO mice compared to their WT littermates at day 3 p.i. (Suppl. Fig. 5). Besides macrophages NK cells define the first line of anti-viral defense. As shown in Fig. 5d splenic NK cells isolated from APN-KO mice expressed significantly higher quantities of activation marker CD69 (P \ 0.05), IFNc (P \ 0.05, data not shown) and perforin (Fig. 5d, (P \ 0.01)) compared to their WT littermates at day 3 post-CVB3 infection. Taken together, those data indicate increased polarization of resident cardiac macrophages to the pro-inflammatory anti-viral M1 phenotype as well as enhanced cardiac infiltration of activated, perforin-releasing NK cells in APN-KO mice that might be responsible for accelerated virus clearance compared to WT mice in acute CVB3 myocarditis (Fig. 6).

Discussion In the present study we show for the first time that APN deficiency is beneficial in acute viral infection in the model of CVB3-induced myocarditis. In contrast to other models of inflammatory heart disease APN-KO mice more rapidly clear viral infection with less inflammation and myocardial damage following CVB3 infection. APN deficiency was associated with an improved outcome in CVB3 myocarditis characterized by reduced CVB3 load, enhanced CD14 and cytokine gene expression, diminished extent of inflammatory lesions and attenuated myocardial necrosis resulting in improved left ventricular function. Our observations indicate that APN promotes CVB3 myocarditis by modulating polarization of activated macrophages towards the anti-inflammatory M2 phenotype and attenuating the number of activated NK cells resulting in suppression of acute anti-viral innate immune responses. Those data are in stark contrast to other models of chronic inflammation, in which APN protects against inflammation and tissue injury [32, 33]. Cardiac injury in CVB3 myocarditis is caused by viral and inflammatory mechanisms. First, viral replication directly damages cardiac myocytes in severely immunocompromised mice (SCID) [5] and insufficient virus clearance is associated with the development of DCM [22,

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Basic Res Cardiol (2014) 109:408 b Fig. 4 APN-KO mice exhibit similar virus load, increased CD14

expression and TLR signaling in acute CVB3 myocarditis. a Cardiac myocytes were infected in vitro with CVB3 (MOI = 1 PFU) in presence or absence of APN (10 lg/mL). CVB3 adhesion, uptake and replication were determined by qRT-PCR using a probe specific for CVB3 positive strand RNA. Results are presented as Mean ± SEM (n = 6). b CVB3 virus load in hearts of WT and APN-KO mice on day 3 p.i. was determined by in situ hybridization (n = 5 animals per group). c Cardiac CD14 mRNA expression on day 3 post-CVB3 infection was determined by qRT-PCR (n = 8 animals per group). d Cardiac CD14 protein expression is increased on day 3 post-CVB3 infection and APN reconstitution inhibits CD14 up-regulation. Upper panel: representative images indicating immunhistochemical detection of CD14 (reddish colouring) on cardiac myocyte surface and intracellularly. Lower panel: immunoblots indicating cardiac expression of CD14 relative to GAPDH e Left panel: cardiac myocytes were incubated with APN (10 lg/mL) or vehicle (Albumin 10 lg/mL) for 18 h before stimulation with TLR ligands P(I:C) (50 lg/mL) and R-848 (5 lg/mL) for 6 h. mRNA expression of IFNß was determined by qRT-PCR (n = 4). Right panel: splenocytes from WT and APNKO mice were stimulated with P(I:C) (50 lg/mL) for 6 h. mRNA expression of IFNb was determined by qRT-PCR. (n = 5). f mRNA expression of IFNb and IFNc in hearts of WT and APN-KO mice on day 3 p.i. was determined by qRT-PCR (n = 8 animals per group)

21]. Picornavirus protease 2A cleaves important host proteins, such as eukaryotic initiation factor-4G (eIF-4G) resulting in inhibition of the host cell protein synthesis machinery [24] or dystrophin thereby disrupting the sarcolemma membrane [1]. Accordingly, cardiospecific overexpression of CVB protease 2A is sufficient to induce cardiomyopathy [43]. In this regard, results of our plaque assay and ISH experiments show that cardiac CVB3 virus load was significantly reduced in APN-KO mice compared to WT animals 7 days p.i. indicating a protective effect of APN deficiency. The reduced virus load in hearts of APNKO mice observed in subacute CVB3 myocarditis might result from (1) delayed dissemination of CVB3 from the gastrointestinal tract or diminished entry into the heart, (2) attenuated CVB3 replication in cardiac myocytes or (3) an enhanced anti-viral innate immune response within the heart. The pancreas constitutes one of the first target organs that is readily attacked by CVB3 following intraperitoneal injection. However, we observed no significant difference in pancreatic CVB3 virus load between APN-KO and WT mice on day 3 p.i., indicating equal dissimination of CVB3 from the gastrointestinal tract in both groups of mice. Moreover, CAR expression and CVB3 virus load in hearts of APN-KO and WT mice were not significantly different in acute myocarditis. In fact, APN had no influence on CVB3 adhesion, uptake or replication in cardiac myocytes in vitro. Thus, the reduced viral load in APN-deficient mice in subacute myocarditis results from an increased anti-viral immune response. Recognition of virus-associated molecular patterns by TLRs and their co-receptor CD14 induces the expression of cytokines (TNFa, IL-12) and type I and II IFNs in cardiac

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resident cells that act in concert to limit viral replication, to activate endothelial cells and to trigger the recruitment and activation of immune cells. CVB3 is recognized by and activates TLR3-, TLR4- and TLR7/8-CD14 signaling complexes [44]. In this regard, TLR3-deficient mice are characterized by increased viral replication and enhanced cardiac damage following CVB infection [26] and transgenic overexpression of TLR3 decreases mortality in CVB3 myocarditis. Accordingly, mice lacking the TLR3 adaptor protein TIR-domain-containing adapter-inducing interferon-b (TRIF) have impaired IFN type I responses, aggravated myocarditis and enhanced viral replication resulting in high mortality [31]. Moreover, loss of function single nucleotid polymorphisms in the TLR3 gene is associated with reduced viral clearance and a blunted immune response against enterovirus in humans [14]. Taken together, TLR virus sensing mechanisms play an important role in the pathogenesis of CVB3 myocarditis. CD14 expression, a co-receptor and enhancer of TLR signaling [10] was regulated by APN in vivo. Moreover, APN inhibited TLR3 signaling in cardiac and immune cells in vitro. In accordance with these findings, we recently have shown that APN attenuates TLR4 signaling by disengaging APN receptor 1 from the CD14-TLR4 complex resulting in attenuation of TLR4 downstream signaling [16]. Therefore, inhibition of TLR signaling by APN diminishes viral clearance. In line with these observations an increased expression of immune response mediators downstream of TLRs such as IFNb, IFNc, IL-12, TNFa, CCL5 and CXCL10 was determined within the hearts of APN-deficient mice on day 3 post-CVB3 infection. IFNs are essential for an effective anti-viral response. IFNb- [7] or IFN type I receptor-[39] deficient mice show attenuated survival in CVB3 myocarditis and IFNb application for 6 months in patients with viral DCMi resulted in complete viral clearance and improved left ventricular function [23]. Similarly, TNFa and IL-12 have been shown to protect from virus myocarditis in a dose- and time-dependent manner. TNFa-deficient mice show enhanced cardiac injury and increased mortality following encephalomyocarditis virus (EMCV) infection and can be rescued by systemic application of exogenous TNFa in the acute phase of disease [38]. Moreover, IL-12 protects against development of CVB3 myocarditis by increasing IFNc expression and macrophage populations in the heart [9]. Taken together, TLR-dependent up-regulation of pro-inflammatory cytokines in acute myocarditis might help eliminating virus load and up-regulation of chemokines (i.e., CCL5, CXCL10) might facilitate immune cell recruitment. Our immune cell data support this interpretation. In acute CVB3 myocarditis we observed a significantly increased formation of CD11b? inflammatory infiltrates and enhanced expression levels of the macrophage

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Fig. 5 APN-KO mice show increased macrophage and NK cell activation in acute CVB3 myocarditis. a Protein expression of CD11b in hearts of WT and APN-KO mice on day 3 p.i. was quantified by immunohistochemistry. Left panel: reddish colouring in representative pictures indicates CD11b? cells. Right panel: box plot indicating number of CD11b? cells per mm2 (n = 5 animals per group). b mRNA expression of CD69 and perforin in hearts of WT and APN-KO mice on day 3 p.i. was determined by qRT-PCR (n = 8 animals per group). c mRNA expression of F4/80 and Mgl1 in hearts of WT and APN-KO mice on day 3 p.i. was determined by qRT-PCR (n = 8 animals per group). d mRNA expression of CD69 and IFNc in splenic NK cells of WT and APN-KO mice on day 3 p.i. was determined by qRT-PCR (n = 5 animals per group)

activation marker F4/80 in hearts of WT mice. Cardiac expression of the ‘‘alternative’’ macrophage polarization marker Mgl1 was reduced in CVB3-infected APN-KO

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mice, indicating an increased macrophage polarization to the anti-viral, pro-inflammatory M1 phenotype. Promotion of the anti-inflammatory M2 phenotype in peritoneal and

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Fig. 6 Working model for effects of APN in CVB3 myocarditis. CVB3 infection of cardiac cells induces up-regulation of CD14 and activates TLR signaling via PAMP recognition. In response, cardiac resident cells up-regulate cytokines and chemokines and attract immune cells such as macrophages and NK cells. APN inhibits upregulation of CD14 thereby suppressing TLR signaling. Moreover, differentiation (i.e., ‘‘classical’’ M1 macrophage phenotype) as well

as activation of innate immune cells within the myocardium that is mandatory for virus control is diminished by APN. Attenuated virus clearance induces chronic inflammation that is characterized by upregulation of cytokine expression and accumulation of immune cells within the myocardium resulting in tissue injury, fibrosis and heart failure

stromal macrophages of adipose tissue by APN has been reported before [27]. However, pathogen-induced cardiac differentiation of M1 macrophages in acute virus myocarditis might attenuate virus replication and dissemination and facilitate CVB3 clearance in APN-KO mice. Moreover, the number of activated, IFNc- and perforin-producing NK cells important for viral clearance was significantly increased in hearts of CVB3-infected APNKO mice. NK cells are important for cardioprotection in acute CVB3 myocarditis since they limit dissemination of CVB3 by killing infected cardiac myocytes. In this regard, depletion of NK cells was shown to increase viral titers and to aggravate myocyte degeneration in CVB3 myocarditis [13]. Recently, our group has shown that the number and function of NK cells are controlled by APN [40]. Besides virus load, a persistent and uncontrolled inflammatory response that has been triggered to control viral replication will result in myocardial injury [21]. Especially persistently activated T cells have been shown to exert detrimental effects in CVB3 myocarditis [42]. Accordingly, CD4- and CD8-KO mice as well as CD4 CD8 double-KO mice are protected from myocardial injury in CVB3 myocarditis and display improved survival. In the present study we observed significantly reduced numbers of infiltrated CD4? and CD8? T cells in APN-KO mice in

the subacute phase of CVB3 myocarditis. In line with attenuated inflammatory cell infiltration a reduced cardiac expression of inflammatory cytokines such as TNFa, IL1b, IL-6 and IL-12 in APN-KO mice was observed in subacute CVB3 myocarditis. In this regard, increased cardiac inflammation has been shown to impair outcome of patients with viral myocarditis [20]. Taken together, the ability of the host to limit viral replication while minimizing tissue injury attributable to detrimental proinflammatory responses is a pre-requisite of favorable outcome. Reduced virus load and inflammation in hearts of APNKO mice in subacute CVB3 myocarditis was associated with diminished tissue injury as assessed by attenuated cardiac myocyte necrosis and decreased serum levels of troponin I. Mechanistically, large scale release of perforin by cytotoxic CD8? T cells has been identified as main trigger of immune cell-induced death of cardiac myocytes in CVB3 myocarditis [12]. Cardiac myocytes exposed to high concentrations of perforin die from osmotic lysis, a form of necrotic cell death [37]. Accordingly, attenuated cardiac myocyte necrosis in hearts of APN-KO mice during subacute CVB3 myocarditis is accompanied by reduced infiltration of CD8? T cells and diminished perforin expression. Abundant release of pro-inflammatory

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cytokines in CVB3 myocarditis associated with virus- and immune cell-dependent tissue injury induces pathological ECM remodeling resulting in cardiac fibrosis, which is characterized by excess collagen deposition within the heart [19]. In this regard, alternatively activated M2 macrophages have been implicated in enhanced pro-fibrotic remodeling of the heart [25]. Accordingly, attenuated virus load, reduced inflammation, enhanced M1 macrophage polarization and diminished tissue injury in hearts of APNKO mice during subacute CVB3 myocarditis was associated with reduced cardiac expression of collagen type 1 and 3. IL-1b is a major regulator of collagen expression in cardiac fibroblasts and has been shown to control profibrotic ECM remodeling in myocarditis [2]. Compared with their WT littermates APN-KO mice displayed significantly attenuated cardiac expression of IL-1b at day 7 p.i., thereby contributing to attenuated cardiac fibrosis in subacute CVB3 myocarditis. Taken together, attenuated virus replication in cardiac myocytes along with reduced TLR-dependent expression of pro-inflammatory cytokines such as TNFa and IL-2 as well as diminished release of perforin by cytotoxic T cells might explain the decreased extent of tissue injury in hearts of APN-KO mice in subacute CVB3 myocarditis. Our data are in stark contrast to other models of inflammatory heart disease such as experimental autoimmune myocarditis (EAM) [16], myocardial infarction [32, 33, 45] and EMCV myocarditis [17, 34, 35], in which APN inhibits cardiac inflammation, tissue injury and fibrosis. In fact, several descriptive studies to date have clearly shown a protective effect of APN in virus myocarditis. The opposing findings might be related to different animal and virus strains employed (i.e., leptin-deficient mice and encephalomyocarditis virus) inducing different kinetics and quality of immune responses. Specifically, we employed APN-deficient mice and a human pathogenic cardiotropic virus. In support of our hypothesis, preliminary data in a small cohort of CVB3-positive DCMi patients show higher cardiac APN expression in CVB3-positive patients compared to virus-negative DCMi patients and lower APN expression in responders, i.e. patients spontaneously clearing the virus (C. Skurk, unpublished data). The difference in outcome in pathogen- vs. autoimmune-induced injury models might be related to the fact that if an effective early clearance of virus can be achieved an enhanced immune response in the absence of APN is beneficial, whereas in the setting of a more prolonged and pathogenic (auto)-immune response the immunosuppressive effect of APN is warranted. We have shown previously that APN attenuates the innate immune response by diminishing TLR signaling [16]. Moreover, APN inhibited antigen-specific expansion of T cells in response to stimulation by viral antigens, among them CVB3 [3, 41].

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Furthermore, APN attenuated differentiation and activation of NK cells, a cell type important for the immune response against viral antigens [40]. Therefore, inhibition of innate immunity, i.e. TLR signaling, macrophage differentiation and NK cell activation, as well as suppression of the adaptive immune response, e.g. antigen-specific T cell activation, by APN attenuates virus clearance leading to enhanced cardiac tissue injury and fibrosis that results in impaired left ventricular function. In this regard, immunosuppressive therapy in the absence of virus together with symptomatic heart failure medication is the mainstay of clinical therapy and has been shown to protect cardiac tissue from inflammatory injury in prospective clinical trials [15]. However, proven viral replication is a contraindication for immunosuppression, since viral clearance is mandatory for immunosuppression to show beneficial effects [6]. Therefore, care should be taken in future APN modulating therapies in virus-positive myocarditis patients. In summary, contrary to its beneficial role in murine EAM [16] and human virus-negative DCMi [3] APN exerts deleterious effects in CVB3-induced myocarditis. While protecting against chronic autoimmune inflammation associated with EAM and post-viral DCMi, APN prevents early virus clearance by suppressing the acute anti-viral immune response in CVB3 myocarditis resulting in increased pathogen- and inflammation-induced myocardial injury and fibrosis. Acknowledgments This work was supported by a Grant of Deutsche Forschungsgemeinschaft (SFB TR19, TP B7 to CS and CS as well as TP Z4 to KK). Conflict of interest On behalf of all authors, the corresponding author states that there is no conflict of interest.

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