Complement Receptor 1 and 2 Deficiency Increases Coxsackievirus B3-Induced , Dilated Cardiomyopathy, and Failure by Increasing Macrophages, This information is current as IL-1β, and Immune Complex Deposition in of September 27, 2021. the Heart DeLisa Fairweather, Sylvia Frisancho-Kiss, Dolores B. Njoku, Jennifer F. Nyland, Ziya Kaya, Susy A. Yusung,

Sarah E. Davis, J. Augusto Frisancho, Masheka A. Barrett Downloaded from and Noel R. Rose J Immunol 2006; 176:3516-3524; ; doi: 10.4049/jimmunol.176.6.3516 http://www.jimmunol.org/content/176/6/3516 http://www.jimmunol.org/

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2006 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Complement Receptor 1 and 2 Deficiency Increases Coxsackievirus B3-Induced Myocarditis, Dilated Cardiomyopathy, and by Increasing Macrophages, IL-1␤, and Immune Complex Deposition in the Heart1

DeLisa Fairweather,2*† Sylvia Frisancho-Kiss,† Dolores B. Njoku,†‡ Jennifer F. Nyland,*† Ziya Kaya,†§ Susy A. Yusung,† Sarah E. Davis,*† J. Augusto Frisancho,† Masheka A. Barrett,† and Noel R. Rose†¶ Downloaded from Complement and complement receptors (CR) play a central role in immune defense by initiating the rapid destruction of invading microorganisms, amplifying the innate and adaptive immune responses, and mediating solubilization and clearance of immune complexes. Defects in the expression of C or CR have been associated with loss of tolerance to self proteins and the development of immune complex-mediated autoimmune diseases such as systemic lupus erythematosus. In this study, we examined the role of CR on coxsackievirus B3 (CVB3)-induced myocarditis using mice deficient in CR1/2. We found that CR1/2 deficiency significantly increased acute CVB3 myocarditis and pericardial fibrosis resulting in early progression to dilated cardiomyopathy and heart http://www.jimmunol.org/ failure. The increase in inflammation was not due to increased viral replication, which was not significantly altered in the of CR1/2-deficient mice, but was associated with increased numbers of macrophages, IL-1␤ levels, and immune complex deposition in the heart. The complement regulatory protein, CR1-related gene/protein Y (Crry), was increased on cardiac macrophage populations, while immature B220low B cells were increased in the spleen of CR1/2-deficient mice during acute CVB3-induced myocarditis. These results show that expression of CR1/2 is not necessary for effective clearance of CVB3 infection, but prevents immune-mediated damage to the heart. The Journal of Immunology, 2006, 176: 3516–3524.

he consists of 12 soluble plasma pro- products of C3, including iC3b, C3dg, and C3d. CR2 expression by guest on September 27, 2021 teins that interact with one another in three distinct enzy- on B cells plays a major role in the induction of Ab responses and T matic activation cascades, the classical, alternative, and has been proposed to be a primary mediator of the immune-en- lectin pathways, and in a nonenzymatic cytolytic membrane attack hancing role of C (3–5). CR1 (CD35) has dual roles as a receptor pathway (1). Complement components activate the innate immune for C3b, C4b, C1q, and mannan-binding lectin, is important in response by responding to bound Ab and to molecular patterns clearing immune complexes (IC), and is a C regulator with decay found on pathogens. Activation leads to the formation of C split accelerating and membrane cofactor activities (1, 3). Although products that have potent proinflammatory properties amplifying CR1 and 2 are encoded on separate genes in humans, in the mouse both the innate and adaptive immune responses (2, 3). Comple- they are derived from the same gene, Cr2, so that mice deficient in 3 ment receptor 2 (CR2) (CD21) is the receptor for the cleavage one receptor are also deficient in the other (6, 7). Deficiencies in C can enhance disease by allowing increased susceptibility to infec-

*Department of Environmental Health Sciences, †Department of Pathology, ‡Depart- tion (8) or by increasing some autoimmune diseases, such as rheu- ment of Anesthesiology and Critical Care Medicine, Johns Hopkins School of Med- matoid arthritis (RA) and systemic lupus erythematosus (SLE), by icine and Bloomberg School of Public Health, Baltimore, MD 21205; §Department of failing to clear IC and/or by failing to regulate the immune re- Internal Medicine III-Cardiology, University of Heidelberg, Heidelberg, Germany; and ¶W. Harry Feinstone Department of Molecular Microbiology and Immunology, sponse (4, 5, 9, 10). Johns Hopkins Bloomberg School of Public Health, Baltimore, MD 21205 Myocarditis is a principal cause of heart disease in young adults Received for publication June 30, 2005. Accepted for publication January 3, 2006. and is often a precursor of heart failure due to the progression to The costs of publication of this article were defrayed in part by the payment of page dilated cardiomyopathy (DCM) (11, 12). Coxsackievirus B3 charges. This article must therefore be hereby marked advertisement in accordance (CVB3) infection of susceptible A/J or BALB/c mice results in a with 18 U.S.C. Section 1734 solely to indicate this fact. disease similar to that observed in humans, with the development 1 This work was supported by National Institutes of Health Grants HL67290, HL70729, ES03819, and AI51835, and by the Deutsche Forschungsgemeinschaft KA of acute myocarditis from days 7 to 14 after infection that 179712-1 (to Z.K.). progresses to chronic myocarditis and DCM from day 28 to at least 2 Address correspondence and reprint requests to Dr. DeLisa Fairweather, Department day 58 after infection (13, 14). The pathogenesis of myocarditis of Environmental Health Sciences, Johns Hopkins University, Bloomberg School of involves both -mediated and Ab-mediated mechanisms (15– Public Health, 615 North Wolfe Street, Room E7628, Baltimore, MD 21205. E-mail address: [email protected] 17), with IgG Ab and C deposition in the hearts of patients with 3 Abbreviations used in this paper: CR, complement receptor; IC, immune complex; DCM or A/J mice inoculated with cardiac myosin and adjuvant to RA, rheumatoid arthritis; SLE, systemic lupus erythematosus; DCM, dilated cardio- induce experimental autoimmune myocarditis (EAM) (18–21). myopathy; CVB3, coxsackievirus B3; EAM, experimental autoimmune myocarditis; p.i., postinfection; MZ, marginal zone; Crry, CR1-related gene/protein Y; T reg, reg- Proinflammatory cytokines are critical for the development of ulatory T cell. myocarditis. Increased IL-1␤ or IL-18 levels in the heart directly

Copyright © 2006 by The American Association of Immunologists, Inc. 0022-1767/06/$02.00 The Journal of Immunology 3517 correlate with increased acute CVB3-induced myocarditis in sus- Plaque assay ␤ ceptible strains of mice (22–24), while administration of IL-1 The level of infectious virus was determined in individual homogenates by during the innate immune response to CVB3 infection induces plaque assay according to standard procedures (22). Samples were pro- autoimmune myocarditis in genetically resistant strains of mice cessed in the same manner as for cytokine analysis. Dilutions of tissue (25). However, the role of CR1/2 in the development of CVB3- supernatants were incubated on Vero cell monolayers (ATCC) for1hat 37°C and 5% CO to allow viral attachment and then incubated for 3 days induced myocarditis has not been previously examined. 2 to allow plaque formation. Virus titers were expressed as the mean PFU per To investigate the effect of CR1/2 on the development of CVB3- gram of tissue Ϯ SEM and the limit of detection was 10 PFU/g tissue. induced myocarditis, we used A/J mice deficient in CR1/2. We hypothesized that CR1/2 deficiency could 1) increase myocarditis Heart digestion and FACS analysis by increasing viral replication, 2) decrease myocarditis due to the “adjuvant effect” of C, or 3) increase myocarditis by preventing The heart was perfused at a constant flow of 14 ml/min with cold PBS clearance of IC. We found that CR1/2 deficiency did not increase (Biofluids) for 2 min, as described previously (29, 30). Briefly, immune cells were released from the myocardium by digestion with collagenase II viral replication in the heart but significantly increased myocardi- (1 mg/ml; Sigma-Aldrich) and protease XIV (0.5 mg/ml; Sigma-Aldrich) tis, pericardial fibrosis, and was associated with increased IC dep- in PBS for 7 min at 37°C, and single-cell separation was completed using osition in the heart, resulting in early progression to DCM and razor blades to dislodge immune cells from the tissue. Individual cell sus- heart failure during the acute phase of disease. CR1/2 deficiency pensions from seven mice were pooled by group and immune cells were separated from heart cells using a magnetic column and anti-CD45 para- increased the number of macrophages, CR1-related gene/protein Y magnetic beads (30F11.1; Miltenyi Biotec). (Crry) expression on macrophages, and proinflammatory IL-1␤ Splenocytes were isolated, RBC were lysed with ACK lysing buffer, and levels in the heart during acute myocarditis. These results show samples were pooled by group. Immune cells from the heart and spleen Downloaded from that CR1/2 expression is not necessary for effective clearance of were stained with the following mAbs (BD Pharmingen) diluted in 1% CVB3 infection, but reduces inflammation and fibrosis in the heart FBS in PBS (Invitrogen Life Technologies): anti-CD3 (total T cells, clone 17A2), anti-CD4 (Th cells, GK1.5), anti-CD8 (CTLs, clone 53-6.7), anti- and the progression to severe heart disease. B220 (B cells, clone RA3-6B2), and anti-GR-1 (granulocytes, clone RB6- 8C5). Anti-F4/80 to measure macrophages was purchased from eBio- Materials and Methods science (clone BM8). Cell fluorescence was measured using a FACSCalibur flow cytometer (BD Biosciences) and data were analyzed Mice http://www.jimmunol.org/ using CellQuest software (BD Biosciences). The same results were ob- Male A/J mice (6–8 wk of age) were obtained from The Jackson Labo- tained in three separate experiments using seven mice per group. ratory. CR1/2-deficient mice were backcrossed from the C57BL/6 mouse strain to the A/J strain for five generations, as described previously (26). Immunofluorescence The murine Cr2 gene encodes for both CR1 and CR2 transcripts by alter- native splicing (6). Furthermore, A/J mice have a 2-bp deletion in the gene For analysis of IC deposition on the heart during acute CVB3-induced encoding C5 that renders mice deficient in functional C5 and unable to myocarditis, 5-␮m cryosections of OCT preserved A/J- or CR1/2-deficient form the membrane attack complex C5b-C9 (26, 27). This deficiency al- hearts were fixed in acetone and stained at room temperature for 30 min lowed us to separate complement effects on the inductive phase of myo- with fluorescence-labeled Abs against FITC-labeled C3 (ICN Biomedi- from the effector mechanisms related to the membrane attack com- cals), Texas Red-labeled IgM or FITC-labeled IgG (Vector Laboratories). plex. Mice were maintained under pathogen-free conditions in the animal Sections were coverslipped with Vectashield mounting medium (Vector by guest on September 27, 2021 facility at Johns Hopkins School of Medicine, and approval was obtained Laboratories), viewed with an Olympus fluorescence microscope, and an- from the Animal Care and Use Committee of the Johns Hopkins University alyzed using Image-Pro Plus version 3.1 software. for all procedures. Myocarditis Statistical analysis Individual experiments were conducted at least three times with 7–14 mice/ Normally distributed data were analyzed by the Student t test. The Mann- group. Mice, 6–8 wk of age, were inoculated i.p. with a tissue culture- Whitney U test was used to evaluate nonparametric data. Significant dif- derived stock of CVB3 (Nancy strain) originally obtained from American ferences were obtained by comparing CVB3-infected knockout mice with Type Culture Collection (ATCC) and grown in Vero cells (ATCC). CVB3 infected A/J controls. Test values with a p Ͻ 0.05 were considered signif- p Ͻ ,ءءء ;p Ͻ 0.01 ,ءء ;p Յ 0.05 ,ء ;was diluted in sterile saline and 103 PFU were injected i.p. on day 0 and icantly different from control values tissues were collected on day 2 (innate response) or on day 10 or 12 (acute 0.001. myocarditis) postinfection (p.i.). Mice inoculated i.p. with PBS or unin- fected tissue homogenate did not develop myocarditis (data not shown). Hearts were cut longitudinally and fixed in 10% phosphate-buffered for- Results malin and embedded in paraffin. Sections, 5-␮m thick, were cut at various CR1/2 deficiency increases acute CVB3-induced myocarditis and depths in the tissue section and stained with H&E to determine the level of fibrosis inflammation and DCM and with Masson’s trichrome to detect collagen deposition. Sections were examined by two independent investigators in a CR1/2 deficiency is associated with certain autoimmune diseases blinded manner, and myocarditis, fibrosis or pericardial fibrosis was as- such as SLE (4, 5, 9, 10). To investigate the role of CR1/2 in sessed as the percentage of the heart section with inflammation or fibrosis compared with the overall size of the heart section, with the aid of a mi- CVB3-induced myocarditis, A/J mice deficient in CR1/2 were ex- croscope eyepiece grid. The development of DCM was assessed by gross amined for the development of acute myocarditis at day 10 or 12 observation of histology sections at low magnification, as described pre- after infection with 103 PFU of CVB3 i.p. We found, by histolog- viously (28). ical examination of H&E sections, that CR1/2 deficiency signifi- Cytokine measurement cantly increased the level of inflammation in the heart at day 10 (data not shown) or 12 p.i. compared with infected wild-type A/J Ϫ Hearts were frozen in dry ice immediately and stored at 80°C until ho- controls (Fig. 1A). Fibrosis usually develops during the progres- mogenized. Tissues were homogenized at 10% w/v in 2% MEM and su- pernatants were stored at Ϫ80°C until used in ELISA or plaque assays. sion to chronic myocarditis following CVB3 infection (28). In this Cytokines were measured in heart supernatants using Quantikine cytokine study, we found a significant increase in myocardial fibrosis in ELISA kits (R&D Systems), according to manufacturer’s instructions. The CR1/2-deficient hearts (Fig. 1, B and D) compared with wild-type limits of detection for the cytokine kits were as follows: TNF-␣, 5.1 pg/ml; A/J hearts (Fig. 1, B and C). Areas of myocardial fibrosis were IL-1␤, 3 pg/ml; IL-12, 2.5 pg/ml; IL-18, 25 pg/ml; IFN-␥, 2 pg/ml; and IL-4, 2 pg/ml. Cytokines were below detectable levels in the 2% MEM closely associated with inflammation. Thus, we show here that used to homogenate samples (data not shown). Cytokines were expressed CR1/2 deficiency increases acute CVB3-induced myocarditis and as picograms per gram of heart tissue Ϯ SEM. fibrosis. 3518 CR1/2 PROTECTS AGAINST MYOCARDITIS

FIGURE 2. CR1/2 deficiency increases DCM and reduces survival dur- Downloaded from ing acute CVB3-induced myocarditis. CR1/2-deficient (CR1/2Ϫ/Ϫ)(A and FIGURE 1. CR1/2 deficiency increases acute CVB3-induced myocar- C) and wild-type A/J control (A and B) mice received 103 PFU of CVB3 Ϫ Ϫ ditis and fibrosis. Mice deficient in CR1/2 (CR1/2 / )(A, B, and D) were i.p. on day 0 and mice were observed daily for survival (A) until day 12 p.i. compared with wild-type A/J controls (A–C) for the development of acute when hearts were cut longitudinally and assessed histologically for dilation myocarditis (A) or fibrosis (B–D). Mice received 103 PFU of CVB3 i.p. on (B and C) at low power. A/J control mice did not develop DCM during day 0 and hearts were collected on day 12 p.i. Myocardial sections were acute CVB3 myocarditis (B) whereas ϳ50% of surviving CR1/2-deficient stained with H&E to detect inflammation or Masson’s trichrome to detect mice developed DCM (C). Individual experiments were conducted four http://www.jimmunol.org/ collagen deposition (stains bright blue, C and D) and assessed as the per- times with 7–14 mice per group, with one representative heart shown for centage of the heart section with inflammation or fibrosis compared with each group (B and C). Magnification, ϫ2.5. the overall size of the heart section. Individual experiments were conducted four times with 7–14 mice per group. Data are presented as the mean Ϯ During viral infections, C3 and C4 directly coat virions, including .p Ͻ 0.01. Magnification, ϫ2.5 ,ءء ;p Ͻ 0.05 ,ء .SEM CVB3, preventing or neutralizing viral infection of target cells and facilitating lysis of virus-infected host tissues (34, 35). Comple- CR1/2 deficiency reduces survival following CVB3 infection ment deficiencies are often associated with reduced resistance to Because adults rarely die from acute viral myocarditis (11), we bacterial and viral infections (8, 36, 37). Hence, increased myo- developed a model of CVB3-induced myocarditis where 100% of carditis (Fig. 1) and deaths (Fig. 2A) in CR1/2-deficient mice could by guest on September 27, 2021 mice survive acute myocarditis (from days 7 to 14 p.i.) and be due to increased viral replication in the heart. However, when progress to develop chronic autoimmune myocarditis and DCM we examined the level of infectious CVB3 in the heart by plaque (days 35 to 56 p.i.) (13, 14). In this study, we found survival was assay, we did not find a significant difference in viral replication at reduced by ϳ50% on days 7–8 p.i. in CR1/2-deficient mice (Fig. day 2 (Fig. 3A), day 10 (Fig. 3B), or day 12 (Fig. 3C) p.i. By day 2A), indicating that CR1/2 deficiency results in premature death 14 p.i., CVB3 could no longer be detected in the heart of wild-type during acute myocarditis. A/J or CR1/2-deficient mice by plaque assay (data not shown). Thus, CVB3 infection is effectively cleared from the heart inde- CR1/2 deficiency increases DCM pendent of CR1/2 expression. DCM is characterized by progressive myocardial hypertrophy and dilation, as well as contractile (systolic) dysfunction. It often de- CR1/2 deficiency increases pericardial fibrosis velops following myocarditis (12) and is a major cause of heart We have recently shown that an adhesive, fibrotic is failure in patients (12, 31) and mice (28, 32, 33). DCM in mice can associated with increased DCM and reduced survival in IFN-␥- be determined in heart cross-sections as dilation (28) and by as- deficient mice following CVB3 infection (28). The development of sessing cardiac function (32, 33). DCM is not observed during fibrosis is an important feature in a number of pathological con- acute CVB3-induced myocarditis (days 7 to 12 p.i.), but develops ditions including myocarditis, and is a key determinant in the clin- in susceptible strains of mice (i.e., BALB/c and A/J) by day 35 p.i. ical outcome of chronic heart disease (28, 31). Fibrosis involves during chronic myocarditis (14, 28). We previously reported that proliferation of fibroblasts and deposition of extracellular matrix DCM is more severe in mice deficient in IFN-␥ during chronic proteins like collagen (38). Fibroblasts are a major component of CVB3-induced myocarditis or EAM, and is associated with in- cardiac tissue and so it is not surprising that fibrosis is an important creased heart failure and death (28, 32, 33). In this study, we found contributor to the development of DCM and congestive heart fail- that wild-type A/J control mice did not develop DCM (0%) during ure (38, 39). In our model of CVB3-induced myocarditis, fibrosis acute myocarditis (Fig. 2B), whereas ϳ50% of surviving CR1/2- is not observed during acute myocarditis but develops with the deficient mice (Fig. 2A) developed DCM, by histological analysis, progression to chronic disease (14, 28, 40). In this study, we ob- during the acute phase of CVB3-induced myocarditis (Fig. 2C). served increased fibrotic pericarditis during acute CVB3 myocar- Thus, CR1/2-deficient mice rapidly develop DCM following ditis (day 10 or 12 p.i.) in CR1/2-deficient hearts (Fig. 4, A and C) CVB3 infection. compared with wild-type A/J controls (Fig. 4, A and B). Pericardial fibrosis was indicated by bright blue staining for collagen deposi- CR1/2 deficiency does not increase viral replication in the heart tion (Fig. 4, B and C) and was associated with pericardial inflam- Complement is a critical component of the innate immune re- mation. Pericarditis was observed in A/J mice in some experi- sponse that protects the host against invading microorganisms. ments, and was always increased in CR1/2-deficient mice, but was The Journal of Immunology 3519 Downloaded from

FIGURE 4. Pericarditis is increased in CR1/2-deficient mice during acute CVB3 myocarditis. CR1/2-deficient mice (CR1/2Ϫ/Ϫ)(A and C) were compared with wild-type A/J controls (A and B) for the level of pericarditis. Mice received 103 PFU of CVB3 i.p. on day 0 and hearts were collected on day 10 p.i. Pericarditis (A) was assessed as the percentage of http://www.jimmunol.org/ the heart with pericarditis and pericardial fibrosis compared with the over- all size of the heart section. Pericardial fibrosis (B and C) was assessed FIGURE 3. CR1/2 deficiency does not alter CVB3 replication in the using Masson’s trichrome stain, which stains areas with collagen deposi- Ϫ/Ϫ heart. Mice deficient in CR1/2 (CR1/2 ) were compared with wild-type tion as bright blue. Individual experiments were conducted four times and p Ͻ ,ءءء .A/J controls for the level of viral replication in the heart during the innate data presented as the mean Ϯ SEM of 7–14 mice per group immune response at day 2 (A), or during acute myocarditis at day 10 (B) 0.001. Magnification, ϫ64. or 12 (C) p.i. Mice received 103 PFU of CVB3 i.p. on day 0 and hearts were collected on day 2, 10, or 12 p.i. for analysis by plaque assay. Data are presented as the mean PFU/g Ϯ SEM of 7–14 mice per group.

(Fig. 5A). The percentage of B cells was also decreased in the by guest on September 27, 2021 hearts of CR1/2-deficient mice (A/J 20.7% vs CR1/2Ϫ/Ϫ 13.6%) (Fig. 5B). The reduced percentage of T and B cells in the hearts of not observed in A/J mice in the experiment shown in Fig. 4. Thus, CR1/2-deficient mice following CVB3 infection is consistent with CR1/2 deficiency rapidly induces fibrotic pericarditis following previous reports of reduced B cell and T cell activation in virally CVB3 infection, which is associated with increased dilation of the infected or adjuvant immunized C or CR-deficient mice (26, 41– heart and reduced survival. 43). Thus, CR1/2 deficiency reduces T and B cells in the heart during acute CVB3-induced myocarditis. CR1/2 deficiency reduces T and B cells in the heart low Attachment of C3 and Ag or IC to CR2 promotes Ag processing by CR1/2 deficiency increases B220 B cell populations in the macrophages, dendritic cells, and B cells (4). Coligation of the spleen BCR with CR2 and CD19 increases the activity of B cells leading Splenic B cells can be divided into mature B2 cells that express to increased Ab production. CR1/2-deficient mice have been high levels of B220 (B220high) and immature or marginal zone shown to have defective T and B cell responses to infection or (MZ) B cells that express lower levels of B220 (B220low). MZ B immunization (5, 8, 26, 41–43). Expression of CR1/2 on murine cells function very early in the immune response to infection or CD4ϩ and CD8ϩ T cells has recently been shown to modulate the immunization by binding IgM-IC via C and CR2 and transporting activation of T cells (26, 43). To better understand the mechanisms IC to the follicles for Ag presentation to dendritic cells (3, 4, 44, involved in the increased inflammation in the hearts of CR1/2- 45). Increased percentages of MZ B cells have been reported in deficient mice, we examined specific cell populations in the in- various immune deficiencies and are believed to account for the flammatory infiltrate of the heart during acute CVB3 myocarditis poor immune responses in these individuals or mice (44). The MZ to determine whether they were altered by CR1/2 deficiency. We B cell compartment has also been shown to contain higher levels isolated CD45ϩ immune cells from the hearts of CR1/2-deficient of clones with autoreactivity, and is associated with certain auto- mice and compared the relative proportions of macrophages (F4/ immune diseases such as SLE (46, 47), but has not been previously 80), granulocytes (GR-1), eosinophils (GR-1low), Th cells (CD4), examined during CVB3-induced myocarditis. In this study, we CTLs (CD8), and B cells (B220) to wild-type A/J controls at day found no differences in T cell or macrophage percentages in the 12 p.i. by FACS analysis. Although the overall percentage of in- spleen of CR1/2-deficient mice compared with A/J control mice by flammation in the heart by histological examination was increased FACS analysis during acute myocarditis (data not shown). Total B in CR1/2-deficient mice (Fig. 1A), the cardiac infiltrate of CR1/2- cell (B220) numbers were also unaltered between CR1/2-deficient deficient mice contained fewer CD4ϩ T cells (A/J 21.8% vs CR1/ and A/J control splenocytes (A/J 16.9% vs CR1/2Ϫ/Ϫ 18.3%) (Fig. 2Ϫ/Ϫ 10.6%) and fewer CD8ϩ T cells (A/J 8.7% vs CR1/2Ϫ/Ϫ 6, R3). However, there was an increase in the percentage of B cells 3.2%) than CVB3 infected wild-type A/J mice by FACS analysis with a B220low phenotype in CR1/2- deficient mice (A/J 5.1% vs 3520 CR1/2 PROTECTS AGAINST MYOCARDITIS Downloaded from FIGURE 5. CR1/2 deficiency reduces T cell and B cell numbers in the heart. Flow cytometry dot plots depict the percentage of T cells (A) and B cells (B) isolated from the hearts of A/J control (left) and CR1/2-deficient (right) mice during acute myocarditis. Mice received 103 PFU of CVB3 i.p. ϩ FIGURE 6. CR1/2 deficiency increases immature B cell numbers in the on day 0 and CD45 immune cells were isolated from the heart on day spleen. Flow cytometry dot plots depict the percentage of immature 12 p.i. after enzymatic digestion. Individual immune cells were separated B220low B cells (R2) and total B cells (R3) from the spleen of control A/J from heart cells using magnetic beads, and the relative proportions of each 3 http://www.jimmunol.org/ ϩ ϩ (A) and CR1/2-deficient (B) mice. Mice received 10 PFU of CVB3 i.p. on of the following cell types were evaluated by FACS: CD4 T cells, CD8 day 0 and splenocytes were analyzed by FACS at day 12 p.i. for the per- T cells, and B cells (B220). Individual experiments were conducted three centage of B220ϩ B cells. Individual experiments were conducted three times using 7–14 mice per group. times using 7–14 mice per group.

CR1/2Ϫ/Ϫ 10.6%) (Fig. 6, R2), which may represent immature or MZ B cells. These results indicate that CR1/2 deficiency reduces cells isolated from the heart at day 12 p.i. for Crry expression by the maturation of B cells in the spleen following CVB3 infection, FACS, we found that Crry levels were unchanged on T and B cells by guest on September 27, 2021 similar to other infections. (data not shown) but were increased on macrophages by ϳ30% (A/J 43.2% vs CR1/2Ϫ/Ϫ 56.8%) over the isotype control (Fig. CR1/2 deficiency increases macrophages and Crry expression in 7B). Not only were absolute numbers of macrophages increased in the heart the hearts of CR1/2-deficient mice compared with A/J controls Macrophages play an important role in initiating and regulating (Fig. 7A), but relative Crry levels on CR1/2-deficient macrophages immune and inflammatory responses (30, 48). Complement en- were increased when equal numbers of macrophages were com- hances the immunogenicity of various viruses by promoting Ag pared (Fig. 7B). Thus, in the absence of CR1/2, macrophage num- trapping to CR1 expressed on macrophages in the MZ of second- bers and Crry expression on macrophages is increased during acute ary lymphoid organs, leading to the activation of B cells (37). C3 CVB3-induced myocarditis. is also important for recruiting inflammatory cells, including mac- rophages, to the heart following immunization with cardiac myosin ␤ and adjuvant in EAM (26, 49). However, the effect of CR1/2 de- CR1/2 deficiency increases IL-1 levels in the heart ficiency on macrophages following CVB3 infection has not been For some time proinflammatory cytokines have been known to previously examined. We found, in contrast to T and B cells, that contribute to the development of chronic myocarditis in mice (22, macrophage numbers were consistently increased by ϳ40% in the 23, 25, 28). We have previously shown that IL-1␤ and IL-18 levels hearts of CR1/2-deficient mice (A/J 34.6% vs CR1/2Ϫ/Ϫ 49.3%) in the heart directly correlate with the severity of acute CVB3- (Fig. 7A) by FACS analysis. Overall granulocytes, which make up induced myocarditis (22). Proinflammatory cytokines are likely to approximately one-third of the inflammatory infiltrate (30), were be produced by macrophages and during acute myo- not significantly increased in CR1/2-deficient hearts (A/J 34.5% vs carditis, as these cells constitute over 60% of the inflammatory CR1/2Ϫ/Ϫ 37.9%). Likewise, eosinophils (GR-1low) were not sig- infiltrate (29, 30). In this study, we examined the levels of TNF-␣, nificantly altered (A/J 8.0% vs CR1/2Ϫ/Ϫ 10.0%). Thus, CR1/2 IL-1␤, IL-12, IL-18, IFN-␥, and IL-4 in heart or spleen homoge- deficiency preferentially increases the number of macrophages in nates from CR1/2-deficient or wild-type A/J control mice that had the heart during acute CVB3-induced myocarditis. been infected with CVB3 10 days earlier. We found that CR1/2 Crry is a complement regulatory protein that possesses both de- deficient mice had significantly decreased levels of TNF-␣ and cay accelerating and membrane cofactor activities like CR1 (7, significantly increased levels of IL-1␤ in the heart during acute 50). However, Crry does not bind C3b or C4b and so does not myocarditis (Fig. 8). There were no significant changes in the lev- function as a receptor for C or facilitate clearance of IC (50). Crry els of IL-12, IL-18, IFN-␥, or IL-4 in the hearts of these mice (Fig. is not present in humans, but has been suggested to function as a 8). We did not observe significant differences in any of the cyto- costimulatory molecule on T cells and to favor Th2 (IL-4-produc- kines we tested in spleen homogenates at this time point (data not ing) responses in mice (51). When we examined inflammatory shown). Our results indicate that CR1/2 deficiency reduces TNF-␣ The Journal of Immunology 3521

FIGURE 7. CR1/2 deficiency increases macrophage numbers and Crry expression on macrophages from the heart. Flow cytometry dot plots depict the percentage of macrophages (A) and expression of Crry on macro- phages (B) isolated from the hearts of A/J control (A, left; B, middle) and CR1/2 deficient (A, right; B, right) mice during acute myocarditis. Gating was based on the FITC control (B, left). Mice received 103 PFU of CVB3 i.p. on day 0 and CD45ϩ immune cells were isolated from the heart on day 12 p.i. after enzymatic digestion. Individual immune cells were separated from heart cells using magnetic beads, and the percentage of immune cells analyzed by FACS. Individual experiments were conducted three times using 7–14 mice per group. Downloaded from

and increases IL-1␤ levels in the heart during acute CVB3-induced rapidly succumb to heart failure during the acute phase of CVB3 myocarditis. myocarditis due to increased inflammation, pericardial fibrosis, and DCM. Increased inflammation is associated with increased CR1/2 deficiency increases IC deposition in the heart numbers of macrophages, IL-1␤ levels, and IC deposition in the http://www.jimmunol.org/ Deposition of IC in the heart contributes to the pathogenesis of heart, whereas viral replication is unchanged. Overall, these find- chronic myocarditis in patients with DCM or A/J mice with EAM ings provide evidence that CR1/2 expression reduces immune-me- (18–21). CR1 in humans and CR1/2 in mice is known to be critical diated damage in the heart during acute CVB3-induced myocar- for disposing of IC as well as the products of inflammatory injury ditis by reducing IC deposition and fibrosis. such as apoptotic cells (3). However, the role of CR1/2 deficiency Complement deficiencies are often associated with reduced in IC deposition in the heart during CVB3-induced myocarditis has resistance to bacterial or viral infections (36, 37). However, a not been investigated. We report here that IC deposition occurs in previous study showed that depletion of C3 with cobra venom both A/J- and CR1/2-deficient hearts during acute CVB3 myocar-

factor did not alter viral replication or myocarditis in CVB3- by guest on September 27, 2021 ditis (Fig. 9). Specific fluorescence staining for IC was observed on infected BALB/c mice (17). In the present study, CVB3 repli- the myocardium and pericardium of all mice examined. Areas cated in the heart at similar levels in wild-type A/J and CR1/ of the heart that showed increased pericarditis and/or fibrosis 2-deficient mice (Fig. 3). These results indicate that CR1/2 also had increased deposition of IgM, IgG, and C3, with the highest intensity of staining along the pericardium (Fig. 9). Al- expression is not required for effective clearance of CVB3 in- though staining was fairly similar between A/J- and CR1/2- fection, and show that increased myocarditis, fibrosis, and deficient hearts for IgM and C3 deposition, IgG deposition was deaths in CR1/2-deficient mice are not due to increased viral greater in CR1/2-deficient hearts (Fig. 9). Thus, CR1/2 defi- replication. Because CVB3 infection is effectively cleared with- ciency is associated with increased IC deposition in the heart out expression of CR1/2, other factors must be necessary for during acute CVB3-induced myocarditis. viral clearance. We recently demonstrated that IL-12-induced IFN-␥ reduces CVB3 replication during myocarditis (22, 28, Discussion 30). In this study, IL-12 and IFN-␥ levels were unaltered in the We report here, for the first time, that CR1/2 deficiency increases heart (Fig. 8) or spleen (data not shown) of CR1/2-deficient CVB3-induced inflammatory heart disease. CR1/2-deficient mice mice, indicating that IFN-␥ could protect CR1/2-deficient mice

FIGURE 8. CR1/2 deficiency reduces TNF-␣ and in- creases IL-1␤ in the heart during acute CVB3 myocar- ditis. Mice deficient in CR1/2 (CR1/2Ϫ/Ϫ) were com- pared with wild-type A/J controls for the level of cytokines in the heart. Mice received 103 PFU of CVB3 i.p. on day 0 and hearts were collected on day 10 p.i. and analyzed for TNF-␣, IL-1␤, IL-12, IL-18, IFN-␥, and IL-4 levels by ELISA. Individual experiments were con- ducted at least three times and data presented as the .p Ͻ 0.05 ,ء .mean Ϯ SEM of 7–14 mice per group 3522 CR1/2 PROTECTS AGAINST MYOCARDITIS

ated autoimmune diseases like SLE (44, 46, 47). In this study, we observed increased numbers of B220low B cells in the spleen of CR1/2-deficient mice (Fig. 6). Bone marrow-derived B2 cells characteristically have high levels of B220, whereas B1 cells dis- play low to intermediate levels of B220 but reside predominantly in the pleural or peritoneal cavities of the adult mouse (42, 44, 45). Immature B or MZ B cells also have low levels of B220 but reside in the spleen. Hence, the population of B220low B cells increased in CR1/2-deficient splenocytes in this study may be immature or MZ B cells (Fig. 6). This population of immature B cells may contribute to the development of CVB3-induced heart disease by increasing levels and IC-mediated pathology. In sup- port of this, we found increased deposition of IgG associated with areas of increased inflammation and fibrosis in the heart of CR1/ FIGURE 9. CR1/2 deficiency increases IgG deposition in the heart dur- 2-deficient mice (Fig. 9). ing acute CVB3 myocarditis. Deposition of IgM, C3, and IgG was ob- served in the myocardium and pericardium of wild-type A/J and CR1/2- A body of circumstantial evidence links the development of deficient (CR1/2Ϫ/Ϫ) mice compared with isotype controls (Control) by many autoimmune diseases with preceding infections, such as immunofluorescent staining. Frozen sections of A/J or CR1/2-deficient SLE with EBV and myocarditis with CVB3 (24). Environmen- hearts were collected on day 12 p.i. with 103 PFU of CVB3 i.p. Analysis tal factors like infections are thought to trigger autoimmune Downloaded from of IC deposition was conducted in two separate experiments with seven disease in susceptible individuals, although the precise mecha- mice per group, with one representative heart shown for each group. Mag- nisms remain unclear (24). CR1 levels are significantly lower in nification, ϫ20. SLE patients, and genetically susceptible CR1/2-deficient mice develop increased lupus-like disease (5, 9, 10, 54). Molecular genetics studies and analysis of twin pairs discordant for the

presence of SLE suggest that reduced levels of CR1 are an http://www.jimmunol.org/ against viral infection. So, what mechanisms are responsible for acquired phenomenon (55, 56). Thus, it is possible that viral or increasing heart disease in CR1/2-deficient mice? bacterial infections could reduce CR1 expression resulting in Increased levels of proinflammatory cytokines have been as- increased autoimmune disease in susceptible individuals. It has sociated with increased heart disease and heart failure following been shown that LPS or bacterial infection in mice results in CVB3 infection of mice (22, 23, 25, 28, 40). Proinflammatory reduced CR2 transcript levels (57). It is important to keep in cytokines are released from a number of inflammatory cells in mind that not all results obtained from mice may be directly the heart, including T cells and macrophages, as well as by applicable to humans due to the fact that humans encode two resident myocytes and mast cells (22, 23). Reduced levels of different genes for CR1 and CR2 while mice use alternatively TNF-␣ in the heart of CR1/2-deficient mice in this study may be spliced variants of the same gene, which differ in their extra- by guest on September 27, 2021 due to reduced numbers of T cells (Figs. 5 and 8), and suggest cellular and intracellular signaling domains (6, 58). Thus, it is a protective role for TNF-␣ during acute CVB3 myocarditis. difficult to separate the individual roles of CR1 and CR2 in the CD4ϩ T cells can be an important source of local TNF-␣ pro- mouse. duction during inflammation (52). Increased levels of IL-1␤ in A major function of CR1 is to remove IC from the circulation the heart may reflect greater numbers of macrophages in the (1, 4, 5). Pathology in systemic autoimmune diseases like SLE hearts of CR1/2-deficient mice. Increased IL-1␤ levels in the is believed to involve increased autoantibody-IC deposition and heart directly correlate with increased inflammation during vasculitis. IC-mediated cardiac involvement in SLE occurs in acute CVB3-induced myocarditis (22), and are known to alter Ͼ50% of cases, with myocarditis and pericarditis occurring myocyte function (53). Furthermore, we have found that in- most frequently (40, 59). IgG specific for cardiac creased IL-1␤ levels, a strongly profibrotic cytokine, are asso- myosin are found in myocarditis and DCM patients (60), and ciated with increased pericardial fibrosis and DCM in chronic IgG1 is deposited in the myocardium and pericardium of A/J CVB3-induced myocarditis (28). Thus, increased IL-1␤ levels mice during EAM (20). An explanation for the increased peri- in the heart of CR1/2-deficient mice may contribute to increased cardial fibrosis and DCM observed in CR1/2-deficient mice in myocarditis, fibrosis, and DCM. this study (Figs. 2C and 4) is damage to the heart due to IC CR1/2-deficient mice have reduced numbers of T and B cells in deposition and reduced clearance of IC by CR1 (Fig. 9). The the heart following CVB3 infection (Fig. 5). Because T and B cells increased levels of Crry on macrophages in CR1/2-deficient are important in the pathogenesis of myocarditis (13–19), the re- mice may be an attempt by the immune system to compensate duced numbers of T and B cells in CR1/2-deficient mice would be for the loss of CR1. However, Crry does not bind C3b and C4b expected to reduce disease. However, other pathogenic factors and does not facilitate clearance of IC (50). Thus, CR1/2 is may outweigh the effects of reduced T and B cell inflammation in important in reducing IC-mediated damage to the heart follow- the heart. For example, macrophage infiltration is increased (Fig. ing CVB3 infection in A/J mice. 7). Another possibility is that a protective population of T cells, In this study, we show that CR1/2 deficiency hastens the such as CD25ϩ T regulatory (Treg) cells, may be reduced in the development of chronic, fibrotic perimyocarditis and DCM fol- heart resulting in increased myocarditis. In support of this idea, we lowing CVB3 infection. CR1/2 deficiency results in increased found that LPS-stimulated T cells from the spleen of CR1/2-defi- macrophages, IL-1␤, and IC deposition in the heart–factors that cient mice have reduced CD25ϩ expression (43), a molecule ex- all contribute to fibrosis, pericarditis, and DCM. Dysfunctional pressed on Treg cell populations that is important in reducing au- or absent CR1 expression promotes the development of IC-me- toimmune disease. diated diseases like SLE and RA in humans, while IC-mediated Increased numbers of immature or MZ B (B220low) cells are damage is considered to be an important pathological mecha- associated with increased autoantibodies and increased IC-medi- nism in the development of DCM and heart failure in patients. The Journal of Immunology 3523

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