IL-12 Protects against Coxsackievirus B3-Induced by Increasing IFN- γ and Macrophage and Populations in the This information is current as of September 24, 2021. DeLisa Fairweather, Sylvia Frisancho-Kiss, Susy A. Yusung, Masheka A. Barrett, Sarah E. Davis, Ronelle A. Steele, Shannon J. L. Gatewood and Noel R. Rose J Immunol 2005; 174:261-269; ; doi: 10.4049/jimmunol.174.1.261 Downloaded from http://www.jimmunol.org/content/174/1/261

References This article cites 67 articles, 31 of which you can access for free at: http://www.jimmunol.org/ http://www.jimmunol.org/content/174/1/261.full#ref-list-1

Why The JI? Submit online.

• Rapid Reviews! 30 days* from submission to initial decision

• No Triage! Every submission reviewed by practicing scientists by guest on September 24, 2021 • Fast Publication! 4 weeks from acceptance to publication

*average

Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts

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 © 2005 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

IL-12 Protects against Coxsackievirus B3-Induced Myocarditis by Increasing IFN-␥ and Macrophage and Neutrophil Populations in the Heart1

DeLisa Fairweather,* Sylvia Frisancho-Kiss,* Susy A. Yusung,* Masheka A. Barrett,* Sarah E. Davis,* Ronelle A. Steele,* Shannon J. L. Gatewood,* and Noel R. Rose2*†

Th1-type immune responses, mediated by IL-12-induced IFN-␥, are believed to exacerbate certain autoimmune diseases. We recently found that signaling via IL-12R␤1 increases coxsackievirus B3 (CVB3)-induced myocarditis. In this study, we examined the role of IL-12 on the development of CVB3-induced myocarditis using mice deficient in IL-12p35 that lack IL-12p70. We found that IL-12 deficiency did not prevent myocarditis, but viral replication was significantly increased. Although there were no changes in the total percentage of inflammatory cells in IL-12-deficient compared with wild-type BALB/c controls by FACS analysis, Downloaded from macrophage and neutrophil populations were decreased. This decrease corresponded to reduced TNF-␣ and IFN-␥ levels in the heart, suggesting that macrophage and/or neutrophil populations may be a primary source of TNF-␣ and IFN-␥ during acute CVB3 myocarditis. Increased viral replication in IL-12-deficient mice was not mediated by reduced TNFRp55 signaling, because viral replication was unaltered in TNFRp55-deficient mice. However, STAT4 or IFN-␥ deficiency resulted in significantly in- creased viral replication and significantly reduced TNF-␣ and IFN-␥ levels in the heart, similar to IL-12 deficiency, indicating that http://www.jimmunol.org/ the IL-12/STAT4 pathway of IFN-␥ production is important in limiting CVB3 replication. Furthermore, STAT4 or IFN-␥ defi- ciency also increased chronic CVB3 myocarditis, indicating that therapeutic strategies aimed at reducing Th1-mediated autoim- mune diseases may exacerbate common viral infections such as CVB3 and increase chronic inflammatory heart disease. The Journal of Immunology, 2005, 174: 261–269.

iral myocarditis is a frequent cause of sudden death in ␥-mediated mechanisms (6). These findings suggest that IL-12- young adults and can progress to chronic dilated cardio- mediated Th1 responses increase myocarditis. This study further V myopathy (DCM),3 a major cause of asso- examines the role of IL-12 on the development of CVB3-induced ciated with an autoimmune response (1, 2). Although most people myocarditis. by guest on September 24, 2021 recover from acute viral myocarditis, susceptible individuals de- IL-12 is potent at inducing IFN-␥ production from NK and T velop chronic myocarditis and DCM. Coxsackievirus B3 (CVB3) cells and promotes the differentiation of T cells to a Th1 phenotype infection of susceptible mice results in a disease pattern similar to (7). Th1-mediated immune responses have been implicated in a that observed in humans. Acute myocarditis occurs from days 7 to number of autoimmune diseases, including some forms of colitis, 14 postinfection (p.i.) in all strains of mice, while susceptible mice type I diabetes, multiple sclerosis, rheumatoid arthritis, and myo- (100% prevalence) progress to a chronic phase of disease from day (6, 8, 9). IL-12 is a heterodimer composed of IL-12p35 and 28 to at least 58 p.i. that is associated with the development of IL-12p40 subunits and is secreted as a biologically active IL- DCM (3). Susceptible BALB/c mice respond to CVB3 infection 12p70 molecule primarily by macrophages, , and den- with elevated levels of TNF-␣, IL-1␤, and IL-12p70 in the heart dritic cells. Signaling by IL-12 requires coexpression of the IL- during the innate response (4). We have shown that increased ␤ ␤ IL-1␤ and IL-18 levels in the heart correlate with increased acute 12R 1 and IL-12R 2 chains for high affinity IL-12p70 binding ␥ CVB3 myocarditis, and are mediated by TLR4 and IL-12R␤1 sig- and maximal IFN- production. In the mouse, IL-12R signaling naling (5). Recently, CD4ϩ Th1 cells were shown to promote activates STAT1, 3, 4, and 5, with STAT4 believed to be respon- CVB3-induced myocarditis in susceptible BALB/c mice by IFN- sible for most of the biological activities of IL-12p70 through the production of IFN-␥ (7, 10, 11). Similar to IL-12, IL-23 activates STAT1, 3, and 4; induces IFN-␥ production; and exacerbates au- *Department of Pathology and †W. Harry Feinstone Department of Molecular Mi- crobiology and Immunology, Johns Hopkins Medical Institutions, Baltimore, MD toimmune disease (12–15). However, the role of IL-12 and IL-23 21205 is functionally distinct, with IL-12 preferentially affecting naive T Received for publication July 8, 2004. Accepted for publication October 22, 2004. cells, while IL-23 induces proliferation of memory T cells (7). The costs of publication of this article were defrayed in part by the payment of page IL-12 is also required for resistance to certain bacterial, viral, charges. This article must therefore be hereby marked advertisement in accordance and intracellular parasitic infections (16–19). IL-12 exerts its in- with 18 U.S.C. Section 1734 solely to indicate this fact. fluence over many infectious agents by stimulating the production 1 This work was supported by National Institutes of Health Grants HL67290, HL70729, and AI51835. of TNF-␣ and IFN-␥, which act synergistically to control infec- 2 Address correspondence and reprint requests to Dr. Noel R. Rose, Department of tions by recruiting and activating macrophages, NK cells, and T Pathology, Johns Hopkins Medical Institutions, Ross Building, Room 659, 720 Rut- cells to perform their effector functions (17, 20–23). IL-12-in- land Avenue, Baltimore, MD 21205. E-mail address: [email protected] duced IFN-␥ appears to act on different effector cells in different 3 Abbreviations used in this paper: DCM, dilated cardiomyopathy; CVB3, coxsack- ievirus B3; EAM, experimental autoimmune myocarditis; LCMV, lymphocytic cho- viral infections. For example, CD8 CTLs have been shown to re- riomeningitis virus; p.i., postinfection. duce lymphocytic choriomeningitis virus (LCMV) infection (18),

Copyright © 2005 by The American Association of Immunologists, Inc. 0022-1767/05/$02.00 262 IL-12 REDUCES MYOCARDITIS while NK cells play a major role in defense against murine cyto- Cytokine measurement megalovirus infection (3, 24). Although clearance of viral infec- Hearts were frozen in dry ice immediately and stored at Ϫ80°C until ho- tions is usually thought to be mediated by destruction of infected mogenized. Tissues were homogenized at 10% weight/volume in 2% cells by effector immune cells, it is now becoming clear that re- MEM, and supernatants were stored at Ϫ80°C until used in ELISAs or lease of IFNs and TNF at the site of infection is also important in plaque assays. Cytokines were measured in heart supernatants using Quan- purging virus from infected cells noncytopathically (22, 25, 26). tikine cytokine ELISA kits purchased from R&D Systems, according to manufacturer’s instructions. The limits of detection for the cytokine kits Cytokines can also control viral infections indirectly by modulat- were as follows: TNF-␣, 5.1 pg/ml; IL-1␤, 3 pg/ml; IL-12, 2.5 pg/ml; ing the immune response and by up-regulating Ag processing and IL-18, 25 pg/ml; and IFN-␥, 2 pg/ml. Cytokines were below detectable display of viral epitopes on the surface of infected cells (22). How- levels in the 2% MEM used to homogenate samples (data not shown). Ϯ ever, the role of IL-12 in the development of CVB3-induced myo- Cytokines were expressed as pg/g heart tissue SEM. carditis has not been previously investigated. Plaque assay In the present study, we examine the role of IL-12 on the de- The level of infectious virus was determined in individual homogenates by velopment of CVB3-induced myocarditis using mice deficient in plaque assay according to standard procedures (5, 28). Samples were pro- IL-12p35, STAT4, or IFN-␥. We found that IL-12 deficiency did cessed in the same manner as for cytokine analysis. Dilutions of tissue not prevent the development of acute myocarditis, but allowed a supernatants were incubated on confluent Vero cell (ATCC) monolayers significant increase in viral replication in the heart. STAT4 or for1hat37°C and 5% CO2 to allow viral attachment, and then incubated ␥ for 3 days to allow plaque formation. Virus titers were expressed as the IFN- deficiency also resulted in significantly increased levels of mean PFU/g tissue Ϯ SEM, and the limit of detection was 10 PFU/g tissue. viral replication, indicating that IFN-␥ produced by IL-12 and STAT4 transcription is important in reducing CVB3 levels in the Heart digestion and FACS analysis Downloaded from heart. IL-12- or IFN-␥-deficient hearts had reduced levels of The mice were anesthetized, the chest cavity was opened, and the root of TNF-␣ and IFN-␥ during acute CVB3 myocarditis, and reduced the aorta was exposed and cannulated with a 27-gauge needle connected to numbers of macrophages and neutrophils. However, increased vi- a Dynamax peristaltic pump (Rainin Instrument). The heart was perfused at a constant flow of 14 ml/min with cold PBS (Biofluids) for 2 min, and ral replication was not mediated by TNFRp55 signaling, because then digested with collagenase II (1 mg/ml; Sigma-Aldrich) and protease viral replication was unaltered in TNFRp55-deficient mice. These XIV (0.5 mg/ml; Sigma-Aldrich) in PBS for 7 min at 37°C (29). The heart results demonstrate that the IL-12/STAT4 pathway of IFN-␥ pro- was then removed from the chest cavity, and single cell separation was http://www.jimmunol.org/ duction is important in limiting CVB3 replication during acute completed using razor blades to dislodge immune cells from the tissue. myocarditis, but does not exacerbate myocarditis. Furthermore, Individual cell suspensions from seven mice were pooled by group, and ␥ leukocytes were separated from heart cells using anti-CD45 paramagnetic STAT4 or IFN- was found to protect against the development of beads (30F11.1; Miltenyi Biotec) on a magnetic column (Miltenyi Biotec). chronic CVB3 myocarditis, indicating that therapeutic strategies Leukocytes were stained with the following mAbs (BD Pharmingen) di- aimed at reducing Th1-mediated autoimmune diseases may exac- luted in 1% FBS (Invitrogen Life Technologies) in PBS: FITC anti-CD3 erbate common viral infections such as CVB3 and increase chronic (total T cells, clone 17A2), PE anti-CD4 (Th cells, GK1.5), CyChrome anti-CD8 (CTLs, clone 53-6.7), CyChrome anti-B220 (B cells, clone RA3- inflammatory heart disease. 6B2), PE anti-CD49b (DX5, NK, and NKT cells), and PE anti-Gr1 (gran- ulocytes, clone RB6-8C5). FITC anti-F4/80 to measure macrophages was by guest on September 24, 2021 purchased from eBioScience (clone BM8). Cell fluorescence was measured using a FACSCalibur flow cytometer (BD Biosciences), and data were Materials and Methods analyzed using CellQuest software (BD Biosciences). The same results Mice were obtained in three separate experiments. ␥ IL-12p35-, STAT4-, and IFN- -deficient mice and wild-type BALB/cJ Statistical analysis (BALB/c) controls were obtained from The Jackson Laboratory. We gen- erated TNFRp55-deficient mice on a BALB/c genetic background by back- Normally distributed data were analyzed by Student’s t test; otherwise, the crossing C57BL/6 TNFRp55-deficient mice to susceptible BALB/cJ mice Mann-Whitney U test was used. Test values with a p Ͻ 0.05 were con- p Ͻ ,ءء ;p Ͻ 0.05 ,ء .for 10 generations. TNFRp55-deficient mice on a C57BL/6 genetic back- sidered significantly different from control values .p Ͻ 0.001 ,ءءء ;ground were a gift from T. Mak (University of Toronto, Toronto, Canada) 0.01 (27). TNFRp55 heterozygotes were interbred to generate wild-type and TNFRp55 homozygotes in the Johns Hopkins Animal Facility. Mice were Results confirmed as deficient for TNFRp55 using a previously published PCR IL-12p35-deficient mice develop acute myocarditis similar to protocol (27). All mice used in these studies were on a BALB/c genetic background. All mice were maintained under specific pathogen-free con- wild-type BALB/c mice ditions, and approval was obtained from the Animal Care and Use Com- CVB3-induced myocarditis in BALB/c mice is believed to be me- mittee of the Johns Hopkins University for all procedures. diated by pathogenic mechanisms involving a Th1 response (6). Previously, we found that signaling via IL-12R␤1 increases both Myocarditis CVB3-induced myocarditis and cardiac myosin-induced experi- mental autoimmune myocarditis (EAM) (5, 30). To investigate the Individual experiments were conducted at least three times with 7–10 mice role of IL-12 during the acute phase of CVB3-induced myocardi- per group. Mice, 6–8 wk of age, were inoculated i.p. with a heart-passaged stock of CVB3 (Nancy strain) originally obtained from the American Type tis, BALB/c mice deficient in IL-12p35 (therefore lacking IL- Culture Collection (ATCC). CVB3 was diluted in sterile saline; 103 PFU 12p70) were examined for the development of acute myocarditis was injected i.p. on day 0; and tissues were collected on day 12 (acute 12 days after i.p. infection with 103 PFU of CVB3. Day 12 was myocarditis) or day 35 (chronic myocarditis) p.i. This model of myocar- chosen because this time point represents the peak of acute in- ditis is subacute, and thus no deaths occurred during the acute or chronic flammation following CVB3 infection in BALB/c mice and is sim- phase of disease (data not shown). Mice inoculated i.p. with PBS or un- infected heart homogenate did not develop acute or chronic myocarditis ilar to the acute myocarditis observed at day 21 in the EAM model (data not shown). Hearts were cut longitudinally, fixed in 10% phosphate- (3, 5, 30). We found, by histological examination, that IL-12 de- buffered formalin, and embedded in paraffin. Sections 5-␮m thick were cut ficiency did not reduce the level of inflammation in the heart at this at various depths in the section and stained with H&E. Sections were ex- time point compared with wild-type BALB/c controls (Fig. 1). amined by two independent investigators in a blinded manner, and myo- ␤ carditis was assessed as the percentage of the heart section with inflam- Thus, although IL-12R 1 signaling increases acute CVB3 myo- mation compared with the overall size of the heart section, with the aid of carditis, IL-12 is not required for the development of myocardial a microscope eyepiece grid. inflammation. The Journal of Immunology 263

FIGURE 2. IL-12p35 deficiency increases CVB3 replication in the heart. Mice deficient in IL-12p35 (IL-12p35Ϫ/Ϫ) were compared with wild- type BALB/c controls for the level of viral replication in the heart 12 days

after CB3 infection. Mice received 103 PFU of CVB3 i.p. on day 0, and Downloaded from hearts were collected on day 12 p.i. for analysis by plaque assay. Data are presented as the mean Ϯ SEM of seven mice per group from one exper- .p Ͻ 0.01 ,ءء .iment of three

specific cell populations in the inflammatory infiltrate of IL-12p35- deficient mice to determine whether they were altered following http://www.jimmunol.org/ infection. To investigate this question, we isolated immune cells (contained in the CD45ϩ fraction) from the hearts of IL-12p35- deficient mice and compared the relative proportions of macro- phages (F4/80), neutrophils (Gr1 low), eosinophils (Gr1 high), NK FIGURE 1. IL-12p35 deficiency does not prevent acute CVB3 myocar- cells (DX5), Th cells (CD4), CTLs (CD8), and B cells (B220) with Ϫ/Ϫ ditis. Mice deficient in IL-12p35 (IL12p35 )(A and C) were compared wild-type BALB/c controls at day 12 p.i. (Fig. 3). We found that with wild-type BALB/c (A and B) controls for the development of myo- ϩ the total numbers of CD45 cells in the heart at day 12 were carditis. Mice received 103 PFU of CVB3 i.p. on day 0, and hearts were collected on day 12 p.i. Myocarditis was assessed as the percentage of the similar between IL-12p35-deficient mice and wild-type controls, by guest on September 24, 2021 heart section with inflammation compared with the overall size of the heart confirming the histological analysis of the percentage of inflam- section stained with H&E, with the aid of a microscope eyepiece grid. mation in the heart (see Fig. 1). However, the inflammatory infil- Individual experiments were conducted three times with seven mice per trate in the heart of IL-12p35-deficient mice consistently contained group, with one representative heart shown for each group (B and C; orig- ϳ12% fewer macrophages and ϳ10% fewer neutrophils than inal magnification ϫ400). Data are presented as the mean Ϯ SEM. CVB3-infected wild-type BALB/c mice (Fig. 3). All other cell

IL-12p35 deficiency increases viral replication in the heart Because virus infection initiates myocarditis (31), we were inter- ested in the effect of IL-12p35 deficiency on viral replication in the heart during acute CVB3 myocarditis. We found that IL-12p35 deficiency (or the lack of IL-12p70) significantly increased the amount of infectious virus in the heart compared with wild-type BALB/c controls at day 12 p.i. (Fig. 2). Previously, we showed a strong correlation between the level of inflammation during acute CVB3 myocarditis and IL-1␤/IL-18 levels in the heart (r ϭ 0.80) (5). When the severity of inflammation (Fig. 1) was compared with the level of infectious virus (Fig. 2) in this study, we found no significant correlation in either mouse strain (wild-type BALB/c, FIGURE 3. Macrophage and neutrophil populations decrease in IL- Ϫ/Ϫ r ϭ 0.20; IL-12p35 , r ϭ 0.16). Because increased infectious 12p35-deficient hearts. The composition of the inflammatory infiltrate dur- virus in the heart did not directly result in increased myocardial ing acute CVB3 myocarditis was compared between wild-type BALB/c inflammation, factors other than viral replication are decisive in mice and mice deficient in IL-12p35 (IL-12p35Ϫ/Ϫ). Mice received 103 ϩ attracting inflammatory cells to the heart (5). These results show PFU of CVB3 i.p. on day 0, and CD45 immune cells were isolated from that IL-12 reduces CVB3 replication in the heart during acute the heart on day 12 p.i. by enzymatic digestion. Individual cell types were myocarditis. separated using magnetic beads, and the percentage of cells was analyzed by FACS. The relative proportion of each of the following cell types was IL-12p35 deficiency reduces macrophage and neutrophil evaluated: macrophages (F4/80), neutrophils (NEU; Gr1 low), eosinophils populations in the heart (EOS; Gr1 high), NK cells (DX5), Th cells (CD4), CTLs (CD8), and B cells (B220). Similar results were obtained in three separate experiments To understand the mechanisms involved in the increase in CVB3 using seven mice per group. Data are presented as the mean Ϯ SEM of replication in the hearts of IL-12p35-deficient mice, we examined three experiments. 264 IL-12 REDUCES MYOCARDITIS populations that we examined either remained the same (EOS and are currently investigating the role of IL-23 on the pathogenesis of DX5) or increased in number in the heart infiltrate (Fig. 3). Over- CVB3-induced myocarditis. Thus, IL-12 significantly alters the all, the percentage of inflammatory cells in the heart remained production of TNF-␣ and IFN-␥ in the heart during acute CVB3 similar between the two strains. Our results suggest that in the myocarditis. absence of IL-12, fewer macrophages and neutrophils are recruited to the heart following CVB3 infection. Because neutrophils and macrophages are important in reducing viral replication (22), re- TNFRp55 deficiency does not increase viral replication or duced numbers of these cells could result in increased viral repli- myocarditis cation in the heart. Thus, IL-12 is important in inducing and/or Several clinical studies have found increased TNF-␣ levels in the maintaining appropriate macrophage and neutrophil function fol- sera of patients with congestive heart failure (32–34). We also lowing CVB3 infection. found that increased TNF-␣ levels in the heart during the innate response to viral infection are associated with the development of ␣ ␥ IL-12p35 deficiency reduces TNF- and IFN- levels in the chronic myocarditis (4, 35–37). In contrast, TNF-␣ has been found heart to play a critical role in the control of certain viral infections such We previously reported that IL-12R␤1-deficient BALB/c mice as murine cytomegalovirus (38, 39), but not for other viruses such have significantly reduced acute myocarditis that correlates with as LCMV (40). However, the role of TNF on the replication of reduced levels of IL-1␤ and IL-18 in the heart following CVB3 CVB3 during acute myocarditis is not known. Because IL-12 de- infection (5). However, TNF-␣, IL-12, and IFN-␥ levels are not ficiency led to significantly decreased levels of TNF-␣ in the heart significantly altered by IL-12R␤1 signaling (5). In this study, we during acute CVB3 myocarditis (Fig. 4), we wanted to determine Downloaded from examined the levels of TNF-␣, IL-1␤, IL-12, IL-18, and IFN-␥ in whether reduced TNF signaling was responsible for the increase in the hearts of IL-12p35-deficient and wild-type BALB/c control viral replication (see Fig. 2). The biological activities of TNF are mice that had been infected i.p. with 103 PFU of CVB3 12 days mediated through two distinct receptors, TNFRp55 and TNFRp75, earlier. We found that IL-12p35-deficient mice had significantly that are expressed on many cell types (41). Most of the immune decreased levels of TNF-␣, IL-12, and IFN-␥ in the heart com- responses classically associated with TNF function are mediated pared with wild-type controls (Fig. 4). Significant changes in by TNFRp55 (41). We backcrossed TNFRp55-deficient C57BL/6 http://www.jimmunol.org/ IL-1␤ and IL-18 levels were not observed in IL-12p35-deficient mice to a susceptible BALB/c genetic background for 10 genera- mice (Fig. 4). These findings were in stark contrast to those ob- tions. BALB/c mice homozygous for TNFRp55 deficiency Ϫ Ϫ tained in IL-12R␤1-deficient hearts (5). Thus, the effect of IL-12 (TNFR / ) were infected i.p. with 103 PFU of CVB3 and com- on cytokine production in the heart following CVB3 infection op- pared with wild-type BALB/c littermates for the level of myocar- erates primarily via a signaling pathway other than IL-12R␤1. Be- ditis and viral replication in the heart. We found that TNFRp55 cause IL-12R␤1 binds the IL-12p40 component of IL-12 or IL-23 deficiency did not significantly alter the level of viral replication in and IL-12R␤2 binds only IL-12p35 (7), our findings suggest that the heart at day 12 p.i. (Fig. 5A). Furthermore, TNFRp55 defi- IL-23 (or another IL-12p40-related cytokine) leads to increased ciency did not significantly reduce the level of inflammation in the IL-1␤ and IL-18 levels (5), while IL-12 increases TNF-␣ and heart during acute myocarditis (Fig. 5B), similar to the results ob- by guest on September 24, 2021 IFN-␥ in the heart during acute CVB3 myocarditis. Because IL- tained with IL-12-deficient mice (Fig. 1). These results show that 12R␤2-deficient mice on a BALB/c genetic background are not the increased viral replication observed in IL-12p35-deficient currently available, we are unable to determine whether the IL- hearts was not due to a requirement for TNFRp55 signaling. How- 12p35 effect on TNF-␣ and IFN-␥ occurs via IL-12R␤2 signaling. ever, we did not examine the effect of TNFRp75 signaling on In preliminary results, we have detected IL-23 transcripts by RT- CVB3 viral replication or myocarditis because this strain is not yet PCR in the hearts of CVB3-infected mice at day 12 p.i. (S. Fri- available on a BALB/c background. Thus, the actions of TNF-␣ sancho-Kiss and D. Fairweather, unpublished observations). We that are mediated by TNFRp55 signaling do not directly influence viral replication or inflammation during acute CVB3 myocarditis in BALB/c mice.

STAT4 or IFN-␥ deficiency increases viral replication without increasing myocarditis Because the biological activities of IL-12 influencing IFN-␥ pro- duction are primarily mediated via STAT4 (7, 10, 11), we were interested in determining whether STAT4 was also involved in protecting mice from CVB3 infection. We previously found that IFN-␥ deficiency did not alter acute myocarditis after CVB3 in- fection, but significantly increased viral replication in the heart at day 12 p.i (5), similar to the effect of IL-12p35 deficiency reported in this study (see Figs. 1 and 2). In this study, we further evaluated the role of STAT4 and IFN-␥ on the development of acute myo- carditis following CVB3 infection using STAT4- and IFN-␥-defi- ␣ ␥ FIGURE 4. IL-12p35 deficiency reduces TNF- and IFN- in the heart. cient mice. We found that STAT4 or IFN-␥ deficiency signifi- Mice deficient in IL-12p35 (IL-12p35Ϫ/Ϫ) were compared with wild-type cantly increased viral replication in the heart 12 days after CVB3 BALB/c controls for the level of cytokines in the heart 12 days after CVB3 infection. Mice received 103 PFU of CVB3 i.p. on day 0, and hearts were infection compared with BALB/c controls (Fig. 6A), but that myo- collected on day 12 p.i. and analyzed for TNF-␣, IL-1␤, IL-12, IL-18, and carditis was not significantly altered (Fig. 6B). These results were IFN-␥ levels by ELISA. Individual experiments were conducted three similar to the effects on myocarditis and viral replication observed times, and data were presented as the mean Ϯ SEM of seven mice per in IL-12p35-deficient mice (Figs. 1 and 2). These findings indicate -␥-p Ͻ 0.05. that activation of STAT4 transcription is important for IFN ,ء .group The Journal of Immunology 265 Downloaded from http://www.jimmunol.org/

FIGURE 5. TNFRp55 deficiency does not increase viral replication or by guest on September 24, 2021 myocarditis. Mice deficient in TNFRp55 (TNFRϪ/Ϫ) were compared with wild-type BALB/c controls for the level of viral replication (A) or myo- FIGURE 6. ␥ carditis (B) in the heart 12 days after CVB3 infection. Mice received 103 STAT4 or IFN- deficiency increases viral replication with- PFU of CVB3 i.p. on day 0, and hearts were collected on day 12 p.i. out increasing myocardial inflammation. Mice deficient in STAT4 Ϫ/Ϫ ␥ ␥Ϫ/Ϫ Individual experiments were conducted three times, and data were pre- (STAT4 ) or IFN- (IFN- ) were compared with wild-type BALB/c A B sented as the mean Ϯ SEM of 7–10 mice per group. controls for the level of viral replication ( ) or myocarditis ( ) in the heart 12 days after CVB3 infection. Mice received 103 PFU of CVB3 i.p. on day 0, and hearts were collected on day 12 p.i. Individual experiments were conducted at least three times, and data were presented as the mean Ϯ SEM .p Ͻ 0.01 ,ءء ;p Ͻ 0.05 ,ء .of 7–10 mice per group mediated reduction of viral replication in the heart during acute CVB3-induced myocarditis.

STAT4 or IFN-␥ deficiency reduces TNF-␣ and IFN-␥ in the IFN-␥ deficiency results in reduced macrophages and heart neutrophils in the heart Because TNF-␣ and IFN-␥ were significantly reduced in IL- To determine whether reduced IFN-␥ levels were responsible for 12p35-deficient hearts (Fig. 4) and associated with increased viral the reduced macrophages and neutrophils we observed in the replication (Fig. 2), we next determined the cytokine environment hearts of IL-12p35-deficient mice (see Fig. 4), we examined the in STAT4- and IFN-␥-deficient hearts following CVB3 infection. percentages of individual cell populations within the infiltrate of Mice were infected with 103 PFU of CB3 i.p., and hearts were IFN-␥-deficient hearts, as before. We found that macrophage and analyzed 12 days later for TNF-␣, IL-1␤, IL-12, IL-18, or IFN-␥ neutrophil populations were decreased in IFN-␥-deficient hearts at levels, as before. STAT4 (Fig. 7A) or IFN-␥ (Fig. 7B) deficiency day 12 p.i. (data not shown), similar to IL-12p35-deficient mice significantly reduced the level of TNF-␣ and IFN-␥ in the heart, (see Fig. 3). The primary difference between mice deficient in IL- like IL-12p35-deficient mice (see Fig. 4). In addition, STAT4- 12p35 or IFN-␥ compared with wild-type BALB/c mice was that deficient mice had significantly lower levels of IL-12 (Fig 7A), IFN-␥-deficient mice had increased numbers of eosinophils in the similar to IL-12p35-deficient mice (Fig. 4). IL-1␤ was somewhat acute infiltrate (wild-type BALB/c 4.4% compared with IFN-␥- reduced in IFN-␥-deficient hearts at day 12 p.i., but not signifi- deficient mice with 12.7%). The increase in eosinophils may re- cantly ( p ϭ 0.06) (Fig. 7B). Thus, the significant reduction in flect the skewing of the immune response to an IL-4-mediated Th2 TNF-␣ and IFN-␥ in IL-12p35-, STAT4-, and IFN-␥-deficient phenotype, which is known to occur in the absence of IFN-␥ (42, hearts further supports the view that this cytokine pathway is in- 43). Thus, IL-12-induced IFN-␥ results in increased numbers of volved in reducing viral replication in the heart during acute CVB3 macrophages and neutrophils in the heart during acute CVB3-in- myocarditis. duced myocarditis. 266 IL-12 REDUCES MYOCARDITIS

FIGURE 8. STAT4 or IFN-␥ deficiency increases chronic, autoimmune myocarditis. Mice deficient in STAT4 (STAT4Ϫ/Ϫ) or IFN-␥ (IFN-␥Ϫ/Ϫ) Downloaded from were compared with wild-type BALB/c controls for the development of chronic myocarditis. Mice received 103 PFU of CVB3 i.p. on day 0, and hearts were collected on day 35 p.i. Myocarditis was assessed as the per- centage of the heart section with inflammation and necrosis compared with the overall size of the heart section stained with H&E. Individual experi- ments were conducted more than three times with seven mice per group.

Data are presented as the mean Ϯ SEM. http://www.jimmunol.org/

replication during acute myocarditis, but this signaling pathway also reduces the severity of chronic inflammatory heart disease. Thus, strategies aimed at reducing IFN-␥-induced Th1 responses to prevent autoimmune diseases may actually aggravate viral rep- FIGURE 7. STAT4 or IFN-␥ deficiency reduces TNF-␣ and IFN-␥ in lication and chronic myocarditis. the heart. Mice deficient in STAT4 (STAT4Ϫ/Ϫ)(A) or IFN-␥ (IFN-␥Ϫ/Ϫ)

(B) were compared with wild-type BALB/c controls for the level of cyto- by guest on September 24, 2021 3 Discussion kines in the heart 12 days after CVB3 infection. Mice received 10 PFU of IL-12 is believed to exacerbate many autoimmune diseases by CVB3 i.p. on day 0, and hearts were collected on day 12 p.i. and analyzed ␥ ␣ ␤ ␥ increasing IFN- levels and Th1 responses. In this study, IL-12 for TNF- , IL-1 , IL-12, IL-18, and IFN- levels by ELISA. Individual ␥ experiments were conducted at least three times, and data were presented deficiency reduced IFN- levels in the heart during acute viral p Ͻ 0.01. CVB3 myocarditis. However, myocardial inflammation was not ,ءء ;p Ͻ 0.05 ,ء .as the mean Ϯ SEM of 7–10 mice per group decreased, suggesting that IL-12 is not necessary for the develop- ment of acute myocarditis. Analysis of the cellular composition of the infiltrate revealed that macrophage and neutrophil populations ␥ STAT4 or IFN- deficiency increases chronic, autoimmune were decreased. These populations were also decreased in IFN-␥- CVB3-induced myocarditis deficient hearts during acute CVB3 myocarditis, indicating that Because we had established that STAT4-induced IFN-␥ produc- IL-12 may influence both viral replication and inflammation via tion in the heart was important in reducing CVB3 replication dur- IFN-␥. Thus, IL-12-induced IFN-␥ is important in inducing and/or ing acute myocarditis, we next investigated the effect of this path- maintaining macrophage and neutrophil function in the heart dur- way on the development of the chronic, autoimmune phase of ing acute CVB3 myocarditis. Reduction of these cell populations disease. We have previously shown that IFN-␥ protects against the has important ramifications for the development of myocarditis development of EAM by reducing activated T cells (30). Recently, due to their role in preventing viral replication. Indeed, mice de- we found that IFN-␥ also protects against the development of ficient in IL-12, STAT4, or IFN-␥ had significantly increased lev- chronic CVB3 myocarditis by reducing inflammation, fibrosis, and els of CVB3 in the heart during acute myocarditis compared with the profibrotic cytokines TGF-␤1, IL-1␤, and IL-4 in the heart (44, wild-type controls. We conclude that IL-12/STAT4-mediated 45). In contrast, IL-12 signaling does not influence the develop- IFN-␥ production increases macrophage and neutrophil numbers ment of chronic CVB3 myocarditis (44). Because we have not in the heart and the ability to reduce viral replication during acute previously examined the role of STAT4 on the development of CVB3 myocarditis (Fig. 9). chronic myocarditis, in this study we infected STAT4- or IFN-␥- Studies examining the role of IL-12 on the development of au- deficient mice with 103 PFU of CVB3 i.p. and analyzed hearts for toimmunity have provided conflicting reports (15, 30, 46–50). IL- the development of myocarditis at day 35 p.i., during the peak of 12-induced/IFN-␥-mediated Th1 responses have been shown to be chronic disease (3). CVB3 is cleared from the heart by day 14 p.i. necessary for the development of many autoimmune diseases and does not reactivate due to the lack of IFN-␥ throughout the (6–9, 50–53). With the recent understanding that IL-12 is part of chronic phase of disease (day 21 through 35 p.i.) (44). We found a family of heterodimeric cytokines that include IL-23 and IL-27, that STAT4 or IFN-␥ deficiency resulted in significantly increased a dichotomy between IL-12R␤1 and IL-12R␤2 signaling has levels of inflammation in the heart at day 35 p.i. (Fig. 8). Thus, not emerged (5, 7, 15, 49). Previous studies in experimental animals only does the lack of STAT4-induced IFN-␥ protect against CVB3 that concluded that IL-12 exacerbates autoimmune disease, using The Journal of Immunology 267

function by reducing immune cell activation and chronic inflam- mation (30, 44, 45, 57, 60). The apparent role of IFN-␥ in exac- erbating autoimmune diseases may be explained, at least in part, by the fact that proinflammatory responses involving TNF-␣ and/or IL-1␤ often lead to greater IFN-␥ production (22, 61–63). Rather than reducing CVB3 infection, TNF may be more impor- tant in inducing apoptosis of lymphocytes, thereby preventing a chronic accumulation of inflammatory cells in the heart. We have not yet examined the effect of TNFRp55 deficiency on the devel- opment of chronic CVB3 myocarditis. It is not surprising that IFN-␥ protects against viral infections. IFNs, including IFN-␣, IFN-␤, and IFN-␥, are essential for effec- tive clearance of many viral infections (21, 22, 25, 26, 40, 64). IFN-␥ reduces viral replication directly, by inhibiting protein syn- thesis and degrading RNA (65, 66), and indirectly, by inducing NO production and apoptosis (67, 68). TNF-␣ is also an important antiviral agent, providing an essential function by amplifying the activity of IFN-␥ (23, 63). Results from the present study show

that IL-12 is important in stimulating production in the heart of Downloaded from TNF-␣ in addition to IFN-␥ during acute CVB3 myocarditis (Fig. 4). However, TNF-␣ levels were also reduced in STAT4- and IFN- ␥-deficient hearts (Fig. 7), indicating that IFN-␥ also modulates TNF-␣ levels. IFN-␥ does not directly induce apoptosis, but in- fluences apoptosis by increasing TNF levels (67, 68). Clearance of

FIGURE 9. Possible mechanisms of IL-12-mediated protection against LCMV infection was found to require IFN-␥, but not TNF-␣ (40). http://www.jimmunol.org/ CVB3 replication in the heart. IL-12p35, STAT4, or IFN-␥ deficiency re- Thus, it is possible that even though TNF levels are influenced by sults in increased viral replication in the heart during acute CVB3 myo- IL-12, STAT4, and IFN-␥ deficiency, TNF is not involved in the carditis, but no overall increase in inflammation. However, macrophage clearance of CVB3 from the heart. However, we have not yet ␥ and neutrophil populations are decreased in IL-12p35- or IFN- -deficient examined the role of TNFRp75 signaling in CVB3 replication or hearts relative to other immune cell populations, suggesting that macro- myocarditis. TNFRp75 signaling has been shown to protect against phages and neutrophils are important in clearing CVB3 from the heart. the development of experimental autoimmune encephalomyelitis by reducing Th1 cytokine production and infiltration in the CNS (69). Thus, it is possible that TNFRp75 signaling could be in- neutralizing Abs or genetically deficient mice for the various com- volved in reducing IFN-␥ levels and infiltration in the heart of by guest on September 24, 2021 ponents of IL-12 or IL-12R signaling, need to be reinterpreted in IL-12- or IFN-␥-deficient mice in this study. light of our current understanding of the divergent roles of these Our results also suggest that macrophages and neutrophils may cytokines (54, 55). For example, IL-23, which signals via IL- be a primary source of TNF and IFN-␥ in the heart, because cell 12R␤1, stimulates a strong proinflammatory response that has numbers and cytokine levels were reduced in IL-12- and IFN-␥- been shown to exacerbate experimental autoimmune encephalo- deficient mice (Figs. 3, 4, and 7). Traditionally, NK and T cells are myelitis (12–15). Likewise, we have found that signaling via IL- considered to be the major producers of IFN-␥ (22). However, 12R␤1 increases both EAM- and CVB3-induced acute myocarditis recent studies have shown that TNF-␣ and IFN-␥ are also pro- (5, 30, 45). In this study, we found that IL-12 did not exacerbate duced by macrophages and neutrophils (23, 62, 70). Macrophages acute CVB3 myocarditis. That is, although certain immune cell and neutrophils are rapidly recruited to sites of virus infection, populations were increased in the heart, there was no overall in- where they can kill virus-infected cells by releasing cytokines and crease in the percentage of inflammation (Figs. 4 and 8). It is likely NO and by recruiting other inflammatory cells to the infected tis- that IL-12 influences macrophage and neutrophil populations via sues (22, 70–72). Neutrophils are known to clear various bacterial IFN-␥ because these populations were also reduced in IFN-␥-de- infections, but their contribution to the antiviral response has been ficient mice. Thus, this study further separates the role of IL-12 unclear (22). There is evidence that neutrophils contribute to the from IL-12R␤1 signaling, showing that IL-12 protects against control of HSV infection of the cornea (71) and respiratory syn- CVB3 replication, but does not increase the severity of acute cytial virus infection of the lung (72). Our results suggest that myocarditis. neutrophils may also be important in clearing CVB3 infection Not only are there conflicting reports on the effect of IL-12 on from the heart. the development of autoimmune disease, but the role of IFN-␥ has The results of this study highlight the difficulty in delineating the also been controversial. After investigating the role of IFN-␥ in contribution of inflammation and viral replication to the develop- experimental models of autoimmune disease, many researchers, ment of viral myocarditis. Clinicians struggle with determining including us, have found a protective role for IFN-␥ (30, 43, 47, whether patient treatment should be aimed at reducing viral rep- 48, 56–58). In this study, we also found that STAT4 and IFN-␥ lication in the heart or reducing inflammation and proinflammatory reduced the severity of the chronic, autoimmune phase of CVB3- cytokines. In our model of CVB3-induced myocarditis, immune induced myocarditis (see Fig. 8). Protection against chronic CVB3 mechanisms appear to be more important than viral replication in myocarditis is not due to IL-12R␤1 signaling because there is no determining the development of chronic myocarditis (5, 31, 35, 36, significant difference in the level of chronic CVB3 myocarditis in 44, 45). In this study, we demonstrate for the first time that the IL-12R␤1-deficient mice (44). Thus, even though increased IFN-␥ IL-12/STAT4-induced IFN-␥ pathway is not responsible for acute has sometimes been associated with autoimmune diseases (59), it CVB3-induced myocarditis, because mice deficient in this path- may be that IL-12-induced IFN-␥ serves a primarily protective way developed myocarditis at the same level as wild-type controls. 268 IL-12 REDUCES MYOCARDITIS

But rather, elevated levels of the proinflammatory cytokines 23. Ishida, Y., T. Maegawa, T. Kondo, A. Kimura, Y Iwakura, S. Nakamura, and ␥ TNF-␣ and IL-1␤ are associated with susceptibility to disease (4, N. Makaida. 2004. Essential involvement of IFN- in Clostridium difficile toxin A-induced enteritis. J. Immunol. 172:3018. 5, 31), and can induce disease in resistant strains of mice (35, 36). 24. Shellam, G. R., J. E. Allan, J. M. Papadimitriou, and G. J. Bancroft. 1981. In- Because treatments to reduce Th1 responses in patients with au- creased susceptibility to cytomegalovirus infection in beige mutant mice. Proc. toimmune diseases are being considered (52), it is important to Natl. Acad. Sci. USA 78:5104. 25. Horwitz, M. S., T. Krahl, C. Fine, J. Lee, and N. Sarvetnick. 1999. Protection clarify the role of IL-12 and IFN-␥ in viral myocarditis. The pos- from lethal coxsackievirus-induced pancreatitis by expression of ␥ interferon. sibility exists that reduction of Th1 responses may exacerbate J. Virol. 73:1756. common viral infections such as CVB3 and increase chronic in- 26. Fairweather, D., and N. R. Rose. 2002. Type I diabetes: virus infection or auto- immune disease? Nat. Immunol. 3:338. flammatory heart disease. 27. Pfeffer, K., T. Matsuyama, T. M. Kundig, A. Wakeham, K. Kishihara, A. Shahinian, K. Wiegmann, P. S. Ohashi, M. Kronke, and T. W. Mak. 1993. Mice deficient for the 55 kd tumor necrosis factor receptor are resistant to en- Acknowledgments dotoxic shock, yet succumb to L. monocytogenes infection. Cell 73:457. We thank Mehmet Guler, Dolores Njoku, William Baldwin, 28. Lenzo, J. C., D. Fairweather, V. Cull, G. R. Shellam, and C. M. Lawson. 2002. Barbara Wasowska, and Millie Samaniego for a critical reading of the Characterization of murine cytomegalovirus myocarditis: cellular infiltration of the heart and virus persistence. J. Mol. Cell. Cardiol. 34:629. manuscript and helpful discussions. 29. Afanasyeva, M., D. Georgakopoulos, D. F. Belardi, A. C. Ramsundar, J. G. Barin, D. A. Kass, and N. R. Rose. 2004. Quantitative analysis of myocar- dial inflammation by flow cytometry in murine autoimmune myocarditis. References Am. J. Pathol. 164:807. 1. Feldman, A. M., and D. McNamara. 2000. Myocarditis. N. Engl. J. Med. 30. Afanasyeva, M., Y. Wang, Z. Kaya, E. A. Stafford, K. M. Dohmen, 343:1388. A. A. Sadighi Akha, and N. R. Rose. 2001. Interleukin-12 receptor/STAT4 sig-

2. Dec, G. W. 2003. Introduction to clinical myocarditis. In Myocarditis: From naling is required for the development of autoimmune myocarditis in mice by an Downloaded from Bench to Bedside. L. T. Cooper, Jr., ed. Humana Press, Totowa, p. 157. interferon-␥-independent pathway. Circulation 104:3145. 3. Fairweather, D., Z. Kaya, G. R. Shellam, C. M. Lawson (James), and N. R. Rose. 31. Fairweather, D., S. Frisancho-Kiss, and N. R. Rose. 2004. Viruses as adjuvants 2001. From infection to . J. Autoimmun. 16:175. for autoimmunity: evidence from coxsackieviruses. Rev. Med. Virol. In press. 4. Fairweather, D., S. Frisancho-Kiss, S. Gatewood, D. Njoku, R. Steele, M. Barrett, 32. Levine, B., J. Kalman, L. Mayer, H. M. Fillit, and M. Packer. 1990. Elevated and N. R. Rose. 2004. Mast cells and innate cytokines are associated with sus- circulating levels of tumor necrosis factor in severe chronic heart failure. N. Engl. ceptibility to autoimmune heart disease following coxsackievirus B3 infection. J. Med. 323:236. Autoimmunity 37:131. 33. Katz, S. D., R. Rao, J. W. Berman, M. Schwarz, L. Demopoulos, R. Bijou, and 5. Fairweather, D., S. Yusung, S. Frisancho-Kiss, M. Barrett, S. Gatewood, T. H. LeJemtel. 1994. Pathophysiological correlates of increased serum tumor http://www.jimmunol.org/ ␤ ␤ R. Steele, and N. R. Rose. 2003. IL-12R 1 and TLR4 increase IL-1 and IL- necrosis factor in patients with congestive heart failure. Circulation 90:12. 18-associated myocarditis and coxsackievirus replication. J. Immunol. 170:4731. 34. Matsumori, A. 2003. Cytokines in experimental myocarditis. In Myocarditis: ␥␦ ϩ 6. Huber, S. A., D. Sartini, and M. Exley. 2002. V 4 T cells promote autoim- From Bench to Bedside. L. T. Cooper, Jr., ed. Humana Press, Totowa, p. 109. mune CD8ϩ cytolytic T lymphocyte activation in coxsackievirus B3-induced ϩ 35. Lane, J. R., D. A. Neumann, A. LaFond-Walker, A. Herskowitz, and N. R. Rose. myocarditis in mice: role for CD4 Th1 cells. J. Virol. 76:10785. 1992. Interleukin 1 or tumor necrosis factor can promote coxsackievirus B3- 7. Trinchieri, G., S. Pflanz, and R. A. Kastelein. 2003. The IL-12 family of het- induced myocarditis in resistant B10.A mice. J. Exp. Med. 175:1123. erodimeric cytokines: new players in the regulation of responses. 36. Lane, J. R., D. A. Neumann, A. LaFond-Walker, A. Herskowitz, and N. R. Rose. 19:641. 1993. Role of IL-1 and tumor necrosis factor in coxsackievirus-induced autoim- 8. Segal, B. M., D. M. Klinman, and E. M. Shevach. 1997. Cutting edge: microbial mune myocarditis. J. Immunol. 151:1682. products induce autoimmune disease by an IL-12-dependent pathway. J. Immu- nol. 158:5087. 37. Lenzo, J. C., D. Fairweather, G. R. Shellam, and C. M. Lawson. 2001. Immu- nomodulation of murine cytomegalovirus-induced myocarditis in mice treated 9. Caspi, R. R. 1998. IL-12 in autoimmunity. Clin. Immunol. Immunopathol. 88:4. by guest on September 24, 2021 with lipopolysaccharide and tumor necrosis factor. Cell. Immunol. 213:52. 10. O’Garra, A. 1998. Cytokines induce the development of functionally heteroge- neous T helper cell subsets. Immunity 8:275. 38. Sambhi, S. K., M. R. J. Kohonen-Corish, and I. A. Ramshaw. 1991. Local pro- duction of tumor necrosis factor encoded by recombinant vaccinia virus is ef- 11. Moser, M., and K. M. Murphy. 2000. Dendritic cell regulation of Th1-Th2 de- fective in controlling viral replication in vivo. Proc. Natl. Acad. Sci. USA velopment. Nat. Immunol. 1:199. 88:4025. 12. Oppmann, B., R. Lesley, B. Blom, J. C. Timans, Y. Xu, B. Hunte, F. Vega, 39. Pavic, I., B. Polic, I. Crnkovic, P. Lucin, S. Jonjic, and U. H. Koszinowski. 1993. N. Yu, J. Wang, K. Singh, et al. 2000. Novel p19 protein engages IL-12p40 to ␣ form a cytokine, IL-23, with biological activities similar as well as distinct from Participation of endogenous tumor necrosis factor ( ) in host resistance to cyto- J. Gen. Virol. 74:2215 IL-12. Immunity 13:715. megalovirus infection. . 13. Parham, C., M. Chirica, J. Timans, E. Vaisberg, M. Travis, J. Cheung, S. Pflanz, 40. Klavinskis, L. S., R. Geckeler, and M. B. A. Oldstone. 1989. Cytotoxic T lym- phocyte control of acute lymphocytic choriomeningitis virus infection: interferon R. Zhang, K. P. Singh, F. Vega, et al. 2002. A receptor for the heterodimeric ␥ ␣ cytokine IL-23 is composed of IL-12R␤1 and a novel cytokine receptor subunit, , but not tumor necrosis factor , displays antiviral activity in vivo. J. Gen. IL-23R. J. Immunol. 168:5699. Virol. 70:3317. 14. Cua, D. J., J. Sherlock, Y. Chen, C. A. Murphy, B. Joyce, B. Seymour, L. Lucian, 41. Peschon, J. J., D. S. Torrance, K. L. Stocking, M. B. Glaccum, C. Otten, W. To, S. Kwan, T. Churakova, et al. 2003. Interleukin-23 rather than interleu- C. R. Willis, K. Charrier, P. J. Morrissey, C. B. Ware, and K. M. Mohler. 1998. kin-12 is the critical cytokine for autoimmune inflammation of the brain. Nature TNF receptor-deficient mice reveal divergent roles for p55 and p75 in several 421:744. models of inflammation. J. Immunol. 160:943. 15. Zhang, G.-X., B. Gran, S. Yu, J. Li, I. Siglienti, X. Chen, M. Kamoun, and 42. Afanasyeva, M., Y. Wang, Z. Kaya, S. Park, M. J. Zilliox, B. H. Schofield, A. Rostami. 2003. Induction of experimental autoimmune encephalomyelitis in S. L. Hill, and N. R. Rose. 2001. Experimental autoimmune myocarditis in A/J IL-12 receptor-␤2-deficient mice: IL-12 responsiveness is not required in the mice is an interleukin-4-dependent disease with a Th2 phenotype. Am. J. Pathol. pathogenesis of inflammatory demyelination in the central nervous system. J. Im- 159:193. munol. 170:2153. 43. Tang, H., G. C. Sharp, K. P. Peterson, and H. Braley-Mullen. 1998. IFN-␥- 16. Mattner, F., J. Magram, J. Ferrante, P. Launois, K. Di Padova, R. Behin, deficient mice develop severe granulomatous experimental autoimmune thyroid- M. K. Gately, J. A. Louis, and G. Alber. 1996. Genetically resistant mice lacking itis with eosinophil infiltration of the thyroids. J. Immunol. 160:5105. interleukin-12 are susceptible to infection with Leishmania major and mount a 44. Fairweather, D., S. Frisancho-Kiss, S. A. Yusung, M. A. Barrett, S. J. L. Gatewood, polarized Th2 cell response. Eur. J. Immunol. 26:1553. S. E. Davis, D. B. Njoku, and N. R. Rose. IFN-␥ protects against chronic viral 17. Hunter, C. A., T. Slifer, and F. Araujo. 1996. Interleukin-12-mediated resistance myocarditis by reducing mast cell degranulation, fibrosis, and the profibrotic cyto- ␤ ␤ to Trypanosoma cruzi is dependent on tumor necrosis factor ␣ and ␥ interferon. kines TGF- 1, IL-1 , and IL-4 in the heart. Am. J. Pathol. In press. Infect. Immun. 64:2381. 45. Fairweather D., M. Afanasyeva, and N. R. Rose. 2004. Cellular immunity: a role 18. Orange, J. S., S. F. Wolf, and C. A. Biron. 1994. Effects of IL-12 on the response for cytokines. In The Handbook of Systemic Autoimmune Diseases: The Heart in and susceptibility to experimental viral infections. J. Immunol. 152:1253. Systemic Autoimmune Diseases. A. Doria and P. Pauletto, eds. Elsevier Science, 19. Wysocka, M., M. Kubin, L. Q. Vieira, L. Ozmen, G. Garotta, P. Scott, and Amsterdam, p. 3. G. Trinchieri. 1995. Interleukin-12 is required for interferon-␥ production and 46. Leonard, J. P., K. E. Waldburger, and S. J. Goldman. 1995. Prevention of ex- lethality in lipopolysaccharide-induced shock in mice. Eur. J. Immunol. 25:672. perimental autoimmune encephalomyelitis by against interleukin 12. 20. Ozmen, L., M. Aguet, G. Trinchieri, and G. Garotta. 1995. The in vivo antiviral J. Exp. Med. 181:381. activity of interleukin-12 is mediated by ␥ interferon. J. Virol. 69:8147. 47. Tarrant, T. K., P. B. Silver, J. L. Wahlsten, L. V. Rizzo, C.-C. Chan, B. Wiggert, 21. Orange, J. S., B. Wang, C. Terhorst, and C. A. Biron. 1995. Requirement for and R. R. Caspi. 1999. Interleukin 12 protects from a T helper type 1-mediated natural killer cell-produced interferon ␥ in defense against murine cytomegalo- autoimmune disease, experimental autoimmune uveitis, through mechanisms in- virus infection and enhancement of this defense pathway by interleukin 12 ad- volving interferon-␥, nitric oxide, and apoptosis. J. Exp. Med. 189:219. ministration. J. Exp. Med. 182:1045. 48. Eriksson, U., M. O. Kurrer, W. Sebald, F. Brombacher, and M. Kopf. 2001. Dual 22. Guidotti, L. G., and F. V. Chisari. 2001. Noncytolytic control of viral infections role of the IL-12/IFN-␥ axis in the development of autoimmune myocarditis: by the innate and adaptive immune response. Annu. Rev. Immunol. 19:65. induction by IL-12 and protection by IFN-␥. J. Immunol. 167:5464. The Journal of Immunology 269

49. Camoglio, L., N. P. Juffermans, M. Peppelenbosch, A. A. te Velde, F. J. ten Kate, 60. Chu, C. Q., S. Wittmer, and D. K. Dalton. 2000. Failure to suppress the expansion S. J. H. van Deventer, and M. Kopf. 2002. Contrasting roles of IL-12p40 and of the activated T cell population in interferon ␥-deficient mice leads to exacer- IL-12p35 in the development of hapten-induced colitis. Eur. J. Immunol. 32:261. bation of experimental autoimmune encephalomyelitis. J. Exp. Med. 192:123. 50. Grabie, N., M. W. Delfs, J. R. Westrich, V. A. Love, G. Stavrakis, F. Ahmad, 61. Hodge-Dufour, J., M. W. Marino, M. R. Horton, A. Jungbluth, M. D. Burdick, C. E. Seidman, J. G. Seidman, and A. H. Lichtman. 2003. IL-12 is required for R. M. Strieter, P. W. Noble, C. A. Hunter, and E. Pure. 1998. Inhibition of differentiation of pathogenic CD8ϩ T cell effectors that cause myocarditis. interferon ␥ induced interleukin 12 production: a potential mechanism for the J. Clin. Invest. 111:671. anti-inflammatory activities of tumor necrosis factor. Proc. Natl. Acad. Sci. USA 51. Okura, Y., K. Takeda, S. Honda, H. Hanawa, H. Watanabe, M. Kodama, 95:13806. T. Izumi, Y. Aizawa, S. Seki, and T. Abo. 1998. Recombinant murine interleu- 62. Yeaman, G. R., J. E. Collins, J. K. Currie, P. M. Guyre, C. R. Wira, and kin-12 facilitates induction of cardiac myosin-specific type 1 helper T cells in M. W. Fanger. 1998. IFN-␥ is produced by polymorphonuclear neutrophils in rats. Circ. Res. 82:1035. human uterine endometrium and by cultured peripheral blood polymorphonuclear 52. Chang, J. T., B. J. Segal, and E. M. Shevach. 2000. Role of costimulation in the neutrophils. J. Immunol. 160:5145. induction of the IL-12/IL-12 receptor pathway and the development of autoim- 63. Nguyen, K. B., and C. A. Biron. 1999. Synergism for cytokine-mediated disease munity. J. Immunol. 164:100. during concurrent endotoxin and viral challenges: roles for NK and T cell IFN-␥ 53. Trinchieri, G. 2003. Interleukin-12 and the regulation of innate resistance and production. J. Immunol. 162:5238. adaptive immunity. Nat. Rev. Immunol. 3:133. 64. Lucin, P., I. Pavic, B. Polic, S. Jonjic, and U. H. Koszinowski. 1992. ␥ Interferon- 54. Gor, D. O., N. R. Rose, and N. S. Greenspan. 2003. Th1-Th2: a Procrustean dependent clearance of cytomegalovirus infection in salivary glands. J. Virol. paradigm. Nat. Immunol. 4:503. 66:1977. 55. Watford, W. T., and J. J. O’Shea. 2003. A case of mistaken identity. Nature 65. Landolfo, S., G. Gribaudo, A. Angeretti, and M. Gariglio. 1995. Mechanisms of 421:706. viral inhibition by interferons. Pharmacol. Ther. 65:415. 56. Camoglio, L., A. A. te Velde, A. de Boer, F. J. ten Kate, M. Kopf, and 66. Boehm, U., T. Klanp, M. Groot, and J. C. Howard. 1997. Cellular responses to S. J. van Deventer. 2000. Hapten-induced colitis associated with maintained Th1 interferon-␥. Annu. Rev. Immunol. 15:749. and inflammatory responses in IFN-␥ receptor-deficient mice. Eur. J. Immunol. 67. MacMicking, J., Q. Xie, and C. Nathan. 1997. Nitric oxide and macrophage 30:1486. function. Annu. Rev. Immunol. 15:323. 57. Furlan, R., E. Brambilla, F. Ruffini, P. L. Poliani, A. Bergami, P. C. Marconi, 68. Bogdan, C. 2001. Nitric oxide and the immune response. Nat. Immunol. 2:907. D. M. Franciotta, G. Penna, G. Comi, L. Adorini, and G. Martino. 2001. Intra- 69. Suvannavejh, G. C., H. O. Lee, J. Padilla, M. C. Dal Canto, T. A. Barrett, and Downloaded from thecal delivery of IFN-␥ protects C57BL/6 mice from chronic-progressive ex- S. D. Miller. 2000. Divergent roles for p55 and p75 tumor necrosis factor recep- perimental autoimmune encephalomyelitis by increasing apoptosis of central ner- tors in the pathogenesis of MOG(35–55)-induced experimental autoimmune en- vous system-infiltrating lymphocytes. J. Immunol. 167:1821. cephalomyelitis. Cell. Immunol. 205:24. 58. Jones, L. S., L. V. Rizzo, R. K. Agarwal, T. K. Tarrant, C. C. Chan, B. Wiggert, 70. Ellis, T. N., and B. L. Beaman. 2004. Interferon-␥ activation of polymorphonu- and R. R. Caspi. 1997. IFN-␥-deficient mice develop experimental autoimmune clear neutrophil function. Immunology 112:2. uveitis in the context of a deviant effector response. J. Immunol. 158:5997. 71. Daheshia, M., S. Kanangat, and B. T. Rouse. 1998. Production of key molecules 59. Abel, L. C. J., L. V. Rizzo, B. Ianni, F. Albuquerque, F. Bacal, D. Carrara, by ocular neutrophils early after herpetic infection of the cornea. Exp. Eye Res.

E. A. Bocchi, H. C. Teixeira, C. Mady, J. Kalil, and E. Cunha-Neto. 2001. 67:619. http://www.jimmunol.org/ Chronic Chagas disease cardiomyopathy patients display an increased IFN-␥ re- 72. Wang, S. Z., and K. D. Forsyth. 2000. The interaction of neutrophils with respi- sponse to Trypanosoma cruzi infection. J. Autoimmun. 17:99. ratory epithelial cells in viral infection. Respirology 5:1. by guest on September 24, 2021