- Open Access
The innate immune response to coxsackievirus B3 predicts progression to cardiovascular disease and heart failure in male mice
Biology of Sex Differencesvolume 2, Article number: 2 (2011)
Men are at an increased risk of dying from heart failure caused by inflammatory heart diseases such as atherosclerosis, myocarditis and dilated cardiomyopathy (DCM). We previously showed that macrophages in the spleen are phenotypically distinct in male compared to female mice at 12 h after infection. This innate immune profile mirrors and predicts the cardiac immune response during acute myocarditis.
In order to study sex differences in the innate immune response, five male and female BALB/c mice were infected intraperitoneally with coxsackievirus B3 (CVB3) or phosphate buffered saline and their spleens were harvested 12 h later for microarray analysis. Gene expression was determined using an Affymetrix Mouse Gene 1.0 ST Array. Significant gene changes were verified by quantitative real-time polymerase chain reaction or ELISA.
During the innate immune response to CVB3 infection, infected males had higher splenic expression of genes which are important in regulating the influx of cholesterol into macrophages, such as phospholipase A2 (PLA2) and the macrophage scavenger receptor compared to the infected females. We also observed a higher expression in infected males compared to infected females of squalene synthase, an enzyme used to generate cholesterol within cells, and Cyp2e1, an enzyme important in metabolizing cholesterol and steroids. Infected males also had decreased levels of the translocator protein 18 kDa (TSPO), which binds PLA2 and is the rate-limiting step for steroidogenesis, as well as decreased expression of the androgen receptor (AR), which indicates receptor activation. Gene differences were not due to increased viral replication, which was unaltered between sexes.
We found that, compared to females, male mice had a greater splenic expression of genes which are important for cholesterol metabolism and activation of the AR at 12 h after infection. Activation of the AR has been linked to increased cardiac hypertrophy, atherosclerosis, myocarditis/DCM and heart failure in male mice and humans.
Cardiovascular disease (CVD) is the leading cause of death in the USA . Heart failure can result from a number of cardiovascular conditions, including coronary artery disease, myocarditis and dilated cardiomyopathy (DCM), and men have a higher incidence and severity of these diseases than women [1–5]. Infections, such as coxsackievirus B3 (CVB3), toxins and hypersensitivity drug reactions are known to induce myocarditis [6, 7]. Although myocarditis occurs more often in men, the rates of CVB3 infection are similar between men and women worldwide [8–10]. The factors that predict progression to myocarditis and DCM remain unknown.
We have developed a mouse model of autoimmune myocarditis and DCM using a heart-passaged strain of CVB3 that includes infectious virus and heart antigens in the inoculum . All infected mouse strains develop acute myocarditis from day 8 to 12 post infection (pi), but only certain strains such as A/J and BALB/c develop DCM by day 35 pi . Male BALB/c mice infected with heart-passaged CVB3 develop more severe acute myocarditis than females. More severe disease in males is associated with increased Toll-like receptor (TLR)4 expression on mast cells and macrophages in the heart during acute myocarditis and in the spleen at 12 h pi [12–14]. TLR4 signalling following infection increases cardiac interleukin (IL)-1β and IL-18 resulting in a more prominent T helper type 1 (Th1) immune response in males [12, 13, 15]. Although males have more severe CVB3 myocarditis, the virus replicates to the same level in the hearts of both male and female mice [12, 13]. Gonadectomy of male BALB/c mice reduces myocarditis, making males appear immunologically like females with increased IL-4 and more alternatively activated macrophages and regulatory T cells in the heart . Furthermore, as early as 6 h-12 h after infection mast cells and macrophages obtained from the spleen or peritoneum of males express more TLR4 than females, displaying the same innate immunological profile in the spleen as observed in the heart during acute myocarditis .
As innate immunity directs the adaptive immune response [13, 16], we hypothesized that sex differences in innate immune genes in the spleen would uncover pathways that are important in the susceptibility to acute myocarditis and DCM. We found that the primary gene networks elevated in males compared to females at 12 h after infection involved cholesterol metabolism and activation of the androgen receptor (AR) in immune cells.
BALB/cJ (BALB/c) mice were obtained from The Jackson Laboratory (ME, USA). Mice were maintained under pathogen-free conditions in the animal facility at the Johns Hopkins School of Medicine and approval was obtained from the Animal Care and Use Committee of the Johns Hopkins University for all procedures.
Male and female BALB/c mice (8 to 10 weeks old) were inoculated intraperitoneally (ip) with 103 plaque forming units (PFU) of heart-passaged CVB3 (contains infectious virus and heart tissue) diluted in sterile phosphate buffered saline (PBS) and the spleen, pancreas and sera harvested at 12 h and 48 h pi. Age matched control males and females received PBS only. In separate experiments, mice received CVB3 on day 0 and echocardiography was conducted at day 10 pi and hearts were collected for histology at day 12 and 35 pi. CVB3 (Nancy strain) was obtained from the American Type Culture Collection (ATCC, VA, USA), grown in Vero cells (ATCC) and passaged through the heart as described . Mice inoculated ip with uninfected cardiac tissue supernatant diluted in PBS, or PBS alone, do not develop myocarditis [12–14]. For this reason PBS alone injections were used as uninfected controls for the innate experiments.
Myocarditis and DCM
Hearts were fixed in 10% buffered formalin, stained with haematoxylin and eosin (H&E) and myocarditis was assessed as the percentage of the heart section with inflammation compared to the overall size of the heart section using a microscope eyepiece grid. The sections were examined by two independent investigators. The development of DCM was assessed by gross observation of histology sections at low magnification and functionally by echocardiography. Individual experiments were conducted three times with 7 - 10 mice per group.
Cardiac function was examined by trans-thoracic echocardiography in conscious mice using the Sequoia Acuson C256 ultrasound machine (PA, USA) equipped with a 15 MHz linear transducer, as previously described . The heart was imaged in a two-dimensional mode in the parasternal short axis view. From the M mode, the left ventricular (LV) wall thickness and chamber dimensions were determined. Ejection fraction represents stroke volume (the volume of blood ejected with each beat) divided by end diastolic LV volume.
The spleen and pancreas from individual mice were homogenized at 10% weight/volume in 2% minimal essential medium (MEM) and individual supernatants were used in plaque assays to determine the level of infectious virus . Virus titres are expressed as the mean plaque-forming unit (PFU)/g tissue ± standard error of mean (SEM) and the limit of detection is 10 PFU/g of tissue. Individual experiments were conducted three times with 5 - 7 mice per group.
Spleens were homogenized at 10% weight/volume in 2% MEM and individual supernatants used in ELISA (seven/group). Secreted PLA2 levels were determined in homogenized supernatants using USCN Life Science kits (Wuhan, China), according to the manufacturer's instructions. PLA2 levels were expressed as pg/g of spleen tissue ±SEM. The kit did not specify sPLA2 subtype.
RNA extraction and microarray
Individual spleens from uninfected PBS-inoculated control and heart-passaged CVB3-infected male and female BALB/c mice were used for microarray analysis (five/group, not pooled) or in a separate experiment to verify microarray data by real-time polymerase chain reaction (RT-PCR; seven/group, not pooled). Spleens were harvested at 12 h pi, flash frozen in liquid nitrogen and stored at -80°C. The spleens were homogenized in 2 mL TRIzol (Invitrogen, CA, USA) according to the manufacturer's protocol. The PureLink Micro-to-Midi Total RNA Purification System (Invitrogen) was used for extraction and purification of RNA. RNA was quantified using a NanoDrop spectrophotometer and quality assessed by RNA Nano LabChip analysis on an Agilent BioAnalyzer 2100 (Agilent Technologies, CA, USA). Processing and GeneChip analysis for microarray were performed on five samples for each treatment group. We processed 100 ng of the total RNA for hybridization to Affymetrix Mouse Gene ST 1.0 microarrays using the Affymetrix GeneChip Whole Transcript Sense Target Labeling Assay, according to the manufacturer's protocol and previously published methods (Affymetrix, CA, USA) [18, 19]. The Affymetrix Mouse GeneChip Gene 1.0 ST Array interrogates 28,853 well-annotated genes with 764,885 distinct probes. The expertise, facilities and instrumentation for Affymetrix GeneChip experimentation and analyses were provided and supported by the Johns Hopkins Malaria Research Institute.
Analysis of microarray data was performed with Partek Genomics Suite (GS) Version 6.4 (Partek, MO, USA). Gene expression patterns for each gene were normalized to the median array intensity for all chips and data from infected animals was normalized to uninfected PBS controls . Microarray data were analysed with Partek GS software by 2-way ANOVA in order to look for significant differences between conditions (with sex and infection as factors) and then P values and fold changes were generated using Fisher's least significant difference (LSD) post hoc analysis for comparisons of sex. False discovery rate (FDR) corrections for multiple comparisons (Benjamini-Hochberg) were applied to reduce the total number of false-positives. Genes were considered significant if they had a P value less than 0.05. Ingenuity Pathway Analysis (IPA; Ingenuity Systems, CA, USA) was used to generate IPA network data by inputting microarray data analysed with Partek GS. Ingenuity generates networks by identifying published gene relationships and provides an IPA score (P value) indicating the likelihood that the significantly up or down-regulated genes found in our microarray would be present in a given network. The P value is calculated as the -log of the Fisher's exact test.
Microarray data accession number
The Affymetrix gene expression data were deposited to the GEO http://www.ncbi.nlm.nih.gov/geo with accession number GSE26630.
Validation of microarray data by quantitative RT-PCR
Results obtained from microarray data were verified in a separate experiment using seven mice/group. Total RNA from spleens was validated by qRT-PCR using Assay-on-Demand primers and probe sets and the ABI 7000 Taqman System from Applied Biosystems (CA, USA), according to Rangasamy et al. . Hypoxanthine phosphoribosyltransferase 1 (HPRT) was used as a normalization control. There was no significant difference in HPRT expression in the spleen between males and females before or after infection. The mRNA data are presented as a relative gene expression (RGE). RGE is calculated as the ratio of target gene expression [fold change of messenger RNA (mRNA) of interest) to the normalization control gene expression (fold change of normalization control mRNA). Data is expressed as the mean of seven mice/group ± SEM. Normally distributed data were analysed by the Student's t test. The Mann-Whitney U test was used to evaluate nonparametric data. A value of P < 0.05 was considered significant.
Statistical analysis of the microarray data is described in the 'RNA Extraction and Microarray' section of the Methods. All other data were analysed by the Student's t test for normally distributed data and the Mann-Whitney U test for nonparametric data. A value of P < 0.05 was considered significant.
Males develop increased myocarditis and progress to heart failure and DCM
We showed previously that male BALB/c mice with acute CVB3 myocarditis develop significantly increased inflammation in the heart compared to females, while the virus replicates in the heart at the same level in both sexes [12, 13]. Increased acute CVB3 myocarditis in males- compared to females (Figure 1A) was associated with reduced heart function with a lower ejection fraction by echocardiography at day 10 pi (Figure 1B). An ejection fraction ≤45% is indicative of heart failure . By day 35 pi male hearts became dilated while female hearts appeared normal (Figure 1C).
Genes altered in male versus female spleens prior to and following infection
Previously, we showed that immune factors that determine sex differences in CVB3 myocarditis, such as increased TLR4 expression on mast cells and macrophages following CVB3 infection, are upregulated as early as 12 h pi in the spleen of male compared to female mice [13, 22]. As the innate immune response in the spleen is important in driving the adaptive immune response, we examined sex differences in gene expression by microarray at 12 h in the spleens of male and female BALB/c mice inoculated with 103 PFU CVB3 or PBS ip on day 0. Although we conducted a two-way ANOVA analysis of data from the four groups (males versus females, PBS versus CVB3), in this paper we report only the effect of sex (we do not report gene differences between PBS versus CVB3 infected groups).
After the two-way ANOVA, we performed a post-hoc Fisher's LSD analysis in order to identify gene changes between uninfected males and uninfected females or infected males and infected females with a P < 0.05 (Table 1). FDR corrections were applied in order to reduce the total number of false-positives. The main category of genes that was identified following FDR analysis in infected or uninfected male versus female spleens was × and Y-linked genes (Table 1). These × and Y-linked genes are routinely observed in microarray studies of sex differences in hearts from humans and mice .
Genes increased in male versus female spleens prior to infection
As our previous research has shown that distinct immune differences exist between infected males and infected females at 12 h pi in the spleen that relate to heart disease, we analysed the microarray data without correcting for multiple comparisons. The primary problem with this type of analysis is that it is likely to generate false positives. Thus, findings must be verified with other methods such as RT-PCR or ELISA. We suspect that small gene changes will be important indicators of disease susceptibility because the innate immune response to CVB3 between males and females is very similar, differing mainly in amplitude (severity) [12–14]. For example, both male and female splenocytes upregulate TLR4 following CVB3 infection but more immune cells express TLR4 in males than in females . Genes found to be increased in uninfected males versus uninfected females are listed in Table 2.
Using mRNA obtained in a separate experiment from the one used for the microarray analysis, we verified by qRT-PCR that the top five genes listed in Table 2 were more highly expressed in uninfected males compared to uninfected females (Table 2, Figure 2). Several of these genes are important in regulating oxidative stress to infection including Cyp2e1, aldehyde dehydrogenase (Aldh1a7) and sulphotransferase 1e1 (Sult1e1; Figure 2). The cytochrome P450 Cyp2e1 is known to be elevated in men and is important in metabolizing cholesterol and steroids. Cyp2e1 also induces the production of reactive oxygen species (ROS) that activate proinflammatory responses and reduce viral replication [24, 25]. Several other genes reported to be important in promoting heart disease and regulating lipid metabolism were elevated in the spleen of uninfected males compared to uninfected females (Table 2). These genes included: carbonic anhydrase 3 (Ca3), an enzyme known to contribute to cardiac hypertrophy and heart failure when expressed in the heart ; haptoglobin (Hp), a biomarker of inflammation and cardiovascular disease (CVD) ; and heat shock protein 90 (Hsp90aa1; Table 2). We verified that these genes had a higher expression in uninfected males by qRT-PCR even for Hsp90, which had a fold change of only 1.19 (Table 2, Figure 2). We did not verify all of the genes identified in Table 2 and so other genes in this list could include false positives. Our findings suggest that uninfected males have an underlying immune profile in the spleen that may predispose them to a proinflammatory response and inflammatory heart disease following infection.
Endocrine system development and function genes increase in CVB3 infected males compared to infected females at 12 h pi
Analysis of the microarray data, without correcting for multiple comparisons, revealed a number of genes expressed more highly in CVB3 infected males than in infected females (data not shown). IPA of the data found that the gene network with the most genes present, and thus the highest IPA score, from our study of the spleen of infected males compared to infected females involved endocrine system development and function with an IPA score of 42 (P = 1 × 10-42; Figure 3). The IPA score (P value) indicates the likelihood that the significantly up or down-regulated genes found in our microarray would be present in a given gene network. Using mRNA obtained in a separate experiment from the one used for the microarray analysis, we verified by qRT-PCR several of the genes shown in Figure 3. Cyp2e1 was the most highly expressed gene in this network with a fold increase of 1.8 (P = 9.4 × 10-4; Figure 3). Cytochrome P450 s such as aromatase play a critical role in the metabolism of steroids . We were surprised to find that the AR was downregulated in the spleen of CVB3 infected males compared to infected females (Figure 3). However, it has been reported that elevated levels of testosterone decrease mRNA expression of the AR but increase AR protein and indicate activation or the use of the receptor [28, 29]. Hsp90 acts as a chaperone preventing movement of the AR to the nucleus. We found that Hsp90 mRNA was more highly expressed in males prior to infection (Table 2, Figure 2). We verified that Cyp2e1 (Figure 3 and 4A) and the macrophage scavenger receptor (Msr1; Figures 3 and 4B), which transports oxidized lipids into macrophages, had greater expression in CVB3 infected males than infected females at 12 h pi. Reduced mRNA expression of the AR in CVB3 infected males compared to infected females was also verified by qRT-PCR (Figure 4C). TSPO is the rate-limiting step for steroid synthesis and is expressed in mast cells, macrophages, heart tissue and the spleen, for example . We found that, similar to the AR, TSPO mRNA levels were also decreased in infected males compared to infected females at 12 h pi in the spleen (Figure 4D). We did not verify other genes from this network and it is likely that some of the genes shown in Figure 3 are false positives. However, we verified that Msr1, TSPO, AR and Cyp2e1, genes critical for cholesterol and steroid synthesis, are more elevated or 'activated' in the spleen of infected males compared to infected females at 12 h pi.
CVB3 replicates at the same level in females and males during the innate immune response
In order to examine whether sex differences in gene expression in CVB3 infected males and infected females were related to viral replication, we inoculated male and female BALB/c mice with 103 PFU CVB3 or PBS ip on day 0 and examined the level of viral replication in the spleen and pancreas at 12 and 48 h pi (Figure 5). No viral replication was detected in uninfected PBS controls (data not shown) or at 12 h in the spleen of infected mice (Figure 5A), confirming earlier findings . There was no significant difference in viral replication between CVB3 infected males and infected females at 12 h pi in the pancreas or at 48 h pi in the spleen and pancreas (the pancreas is a target organ for CVB3 infection; Figure 5A and 5B). Thus, altered gene expression that occurs between males and females at 12 h pi in the spleen by microarray is not simply due to differences in viral replication.
Genes associated with cardiovascular disease increase in CVB3 infected males compared to infected females at 12 h pi
The network with the second highest number of genes, and thus second highest IPA score, identified by IPA of data that had not been corrected for multiple comparisons in infected males compared to infected females was DNA replication, recombination and repair, with an IPA score of 41 (P = 1 × 10-41; data not shown). Remember, the IPA score (P value) indicates the likelihood that the significantly up or down-regulated genes found in our microarray would be present in a given gene network. This gene network also received the highest IPA score for data that had not been corrected for multiple comparisons between uninfected males and uninfected females, with an IPA score of 46 (P = 1 × 10-46; data not shown). None of the other networks comparing uninfected males to uninfected females corresponded to those revealed for CVB3 infected males versus infected females, which indicates that infection activated a distinct immune profile in the spleen of males that was not simply due to baseline sex differences.
Unexpectedly, we found that the networks with the 3rd and 5th highest IPA scores in the spleen of CVB3 infected males compared to those in the infected females from data that had not been corrected for multiple comparisons included cardiovascular system development and function with an IPA score of 38 (P = 1 × 10-38; data not shown) and cardiovascular and metabolic disease with an IPA score of 29 (P = 1 × 10-29; Figure 6, Table 3). These CVD gene networks were not observed in uninfected males versus uninfected females. Genes with well known roles in promoting CVD that were more highly expressed in the spleen of CVB3 infected males compared to infected females at 12 h pi included carbonic anhydrase (Ca1-Ca3; Figure 6, Table 2 and 3) and PLA2 (Cpla2, Pla2g4c, Pla2g12a; Figure 6, Table 3) . Elevated lipoprotein-associated (Lp)-PLA2 is associated with the development of atherosclerosis and stroke . Other key proteins in this network included: farnesyl-diphosphate faresyltranserferase 1 (Fdft1), also called squalene synthase, which increases intracellular cholesterol levels ; MAP2 kinase 3 (Map2k3) which has been shown to contribute to sex-based differences in myocardial remodelling and heart failure [33, 34]; and the chemokine CXCL12 which regulates neutrophil and macrophage function (Figure 6, Table 3) .
Using separate experiments from the one used for the microarray, we verified by qRT-PCR or ELISA several of the genes shown in Table 3. We confirmed that PLA2 was increased in the spleen of CVB3 infected males versus infected females using ELISA (Figure 7A) and that infected males had greater mRNA expression of carbonic anhydrase 1 (Ca1; Figure 7B) and squalene synthase (Fdft1; Figure 7C) by qRT-PCR. Although we were able to confirm that carbonic anhydrase 3 (Ca3) expression was greater in uninfected males than uninfected females (Figure 2), we were unable to confirm that Ca3 was elevated in CVB3 infected males by RT-PCR. We did not verify other genes in Table 3 and so the list could include other false positives. However, we were able to verify that Ca1, PLA2 and squalene synthase had higher expression in the spleen of CVB3 infected males than in infected females at 12 h pi (Figure 7). Thus, a number of genes associated with the development of CVD and heart failure, and genes that regulate cholesterol metabolism, show greater expression in the spleen of CVB3 infected males during innate immunity. Our findings indicate that these genes may be involved in initiating the immune response that results in inflammatory heart disease. Further studies are necessary to confirm their role.
Recent evidence indicates that the innate immune response is critical in determining the adaptive response to infection or vaccination. We previously showed that as early as 12 h pi males respond to CVB3 infection with an elevated proinflammatory response . Elevated TLR4 expression on mast cells and macrophages in the spleen and heart of CVB3 infected males not only increases acute myocarditis but induces expression of the profibrotic cytokine IL-1β resulting in cardiac dilatation and heart failure later [13–15, 36]. In this study, we examined the sex differences in gene expression in the spleen of BALB/c mice prior to, and during, the innate immune response to CVB3 infection. We found that FDR analysis of sex differences following infection revealed mainly differences in × and Y-linked genes. However, by analysing the microarray data without correcting for multiple comparisons we were able to identify by Partek and Ingenuity software and to verify by qRT-PCR and ELISA a number of other gene changes between sexes. Similar to our findings, several studies in rodents and humans examining sex differences in heart disease using FDR analysis yielded few gene changes aside from × and Y-linked genes [4, 23, 34, 37]. This is an important issue for the study of sex differences in heart disease and other chronic inflammatory conditions. These findings suggest that small gene changes are likely to be important in driving sex differences in inflammatory diseases. Most of the low-fold gene changes we verified from the IPA networks involved genes critical to the regulation of cholesterol and steroid synthesis in immune cells and in macrophages in particular (Figure 8).
For many years it has been known that underlying sex differences exist in the expression of hepatic enzymes such as cytochrome P450 s, sulphotransferases and glutathione S-transferases [24, 38]. We verified that male spleens have a higher expression of Cyp2e1 and Sult1e1 (Table 2, Figures 2 and 4). These enzymes are important in metabolizing drugs, steroids, fatty acids and environmental chemicals, as well as amplifying a protective immune response against infection. Thus, elevated levels of these enzymes in the spleen of males may predispose them to increased inflammation following CVB3 infection.
In addition, we found that males respond to CVB3 infection by upregulating genes specifically involved in cholesterol metabolism (Figure 8). Squalene synthase (Fdft1) regulates intracellular cholesterol levels particularly the endogenous production of steroid hormones, vitamin D, bile acids and lipoprotein particles (Figures 6, 7 and 8, Table 3). Squalene synthase inhibitors have been found to reduce plasma levels of total- and low-density lipoprotein (LDL)-cholesterol in clinical studies . Another source of cholesterol for macrophages comes from extracellular LDL-cholesterol. LDL is recruited from the circulation to the cell wall where it becomes oxidized by secreted PLA2 (sPLA2) among other factors . The superfamily of PLA2 enzymes includes 15 distinct groups that fall into four main categories: secreted (sPLA2); cytosolic (cPLA2); calcium-independent PLA2; and Lp-PLA2 . Approximately 80% of Lp-PLA2 is associated with LDL in the sera, and elevated levels of Lp-PLA2 and sPLA2s occur in patients with atherosclerosis [31, 40, 41]. PLA2 catalyzes the removal of fatty acids from the sn- 2 position of membrane phospholipids releasing arachidonic acid for the generation of lipid mediators that are crucial for many inflammatory processes such as leukotrienes, prostaglandins and thromboxanes [42, 43]. Increased sPLA2 levels have been associated with a number of inflammatory conditions including: rheumatoid arthritis; sepsis; psoriasis; pancreatitis; and cancer [43, 44]. cPLA2 levels increase in phagocytic cells, such as mast cells, macrophages and neutrophils, following their activation where cPLA2 has been found to be essential for phagocytic function . Similarly, sPLA2 is secreted from mast cells and macrophages following activation. In this study we were able to verify increased sPLA2 in the spleen of CVB3 infected males compared to infected females (Figures 6 and 7A, Table 3). PLA2 is also a ligand for TSPO and so elevated levels of PLA2 in the spleen of males could facilitate AR-mediated activation of immune cells during the innate immune response to CVB3 infection (Figure 8). Oxidized-LDL becomes a ligand for LDL receptors (LDLR) and macrophage scavenger receptors (Msr1) that transport cholesterol into macrophages to be metabolized to steroids and other lipid mediators that increase proinflammatory responses (Figure 8) . Our observation of greater expression of sPLA2 and Msr1 in the spleen of CVB3 infected males may allow oxidation and uptake of LDL-cholesterol and activation of TSPO for the production of steroids in splenic macrophages (Figures 4 and 7). Interestingly, PLA2 expression in the heart was found to be a sex-related gene that predicted the progression to heart failure in CVD patients . Haptoglobin was also more highly expressed in the spleen of males prior to infection (Table 2, Figure 2). By binding Apo-AI, haptoglobin inhibits the transfer of lipids from pro-inflammatory LDL particles to anti-inflammatory HDL thereby potentially increasing LDL levels (Figure 8) [39, 45].
Findings from our study that examined sex differences in the spleen correspond closely to clinical studies of sex differences in gene expression in CVD and heart failure patients. Sex studies of normal and diseased hearts in rodents and humans have found sex differences in carbonic anhydrase (Table 2 and 3), PLA2 (Table 3), Map2k3 (Table 3) and the AR (Figure 3 and 4; see, for example, [4, 23, 34, 37, 46]. With an ever increasing world population developing CVD a better understanding of the genes that predispose men to coronary heart disease and DCM is critical. Our findings show that the gene changes that contribute to an increased pro-inflammatory response in males are initiated within hours of infection in the spleen.
Men are at an increased risk of developing atherosclerosis, myocarditis, DCM and heart failure compared to women but the early immunological factors that drive the proinflammatory response remain unclear. In this study we found that the spleens of CVB3 infected male mice had a greater expression of genes associated with cholesterol and steroid influx and metabolism such as PLA2, Msr1 and squalene synthase compared to infected females. TSPO, the rate-limiting step for steroid synthesis and the AR were decreased in CVB3 infected males compared to infected females consistent with activation of the receptors. Thus, increased cholesterol metabolism in the spleen of males during the innate immune response to CVB3 infection may drive the testosterone-mediated proinflammatory response that we observe during acute CVB3 myocarditis, including mast cell and macrophage proliferation, NFκB activation and TLR4 signalling. These findings have important implications for other inflammatory CVDs that are increased in males.
myocarditis and dilated cardiomyopathy
false discover rate
hypoxanthine phosphoribosyl transferase
Ingenuity Pathway Analysis
lest significant difference
phosphate buffered saline
plaque forming units
- PLA2 :
- q RT-PCR:
quantitative real-time polymerase chain reaction
relative gene expression
translocator protein 18 kDa.
WRITING GROUP MEMBERS, Lloyd-Jones D, Adams RJ, Brown TM, Carnethon M, Dai S, De Simone G, Ferguson TB, Ford E, Furie K, Gillespie C, American Heart Association Statistics Committee and Stroke Statistics Subcommittee, et al.: Heart disease and stroke statistics- 2010 update: a report from the American Heart Association. Circulation 2010, 121: e46-e215. [Erratum in Circulation. 2010, 121: e260] 10.1161/CIRCULATIONAHA.109.192667
Wexler RK, Elton T, Pleister A, Feldman D: Cardiomyopathy: an overview. Am Fam Physician 2009, 79: 778–784.
Dec GW: Introduction to clinical myocarditis. In Myocarditis: From Bench to Bedside. Edited by: Cooper LT Jr, Totowa N. NY: Humana Press; 2003:257–281.
Regitz-Zagrosek V, Oertelt-Prigione S, Seeland U, Hetzer R: Sex and gender differences in myocardial hypertrophy and heart failure. Circ J 2010, 74: 1265–1273. 10.1253/circj.CJ-10-0196
Fairweather D, Frisancho-Kiss S, Rose NR: Sex differences in autoimmune disease from a pathologic perspective. Am J Pathol 2008, 173: 600–609. 10.2353/ajpath.2008.071008
Cooper LT Jr: Myocarditis. N Engl J Med 2009, 360: 1526–1538. 10.1056/NEJMra0800028
Cihakova D, Rose NR: Pathogenesis of myocarditis and dilated cardiomyopathy. Adv Immunol 2008, 99: 95–114. full_text
Schoub BD, Johnson S, McAnerney JM, Dos Santos IL, Klaassen KL: Epidemic Coxsackie B virus infection in Johannesburg, South Africa. J Hyg (Lond) 1985, 95: 447–455. 10.1017/S0022172400062872
Dechkum N, Pangsawan Y, Jayavasu C, Saguanwongse S: Coxsackie B virus infection and myopericarditis in Thailand, 1987–1989. Southeast Asian J Trop Med Public Health 1998, 29: 273–276.
Khetsuriani N, LaMonte-Fowlkes A, Oberste MS, Pallansch MA: Enterovirus surveillance- United States, 1970–2005. MMWR Surveill Summ 2006, 55: 1–20.
Fairweather D, Rose NR: Coxsackievirus-induced myocarditis in mice: a model of autoimmune disease for studying immunotoxicity. Methods 2007, 41: 118–122. 10.1016/j.ymeth.2006.07.009
Frisancho-Kiss S, Nyland JF, Davis SE, Frisancho JA, Barrett MA, Rose NR, Fairweather D: Sex differences in coxsackievirus B3-induced myocarditis: IL-12Rβ1 signaling and IFN-β increase inflammation in males independent from STAT4. Brain Res 2006, 1126: 139–147. 10.1016/j.brainres.2006.08.003
Frisancho-Kiss S, Davis SE, Nyland JF, Frisancho JA, Cihakova D, Rose NR, Fairweather D: Cutting Edge: Cross-regulation by TLR4 and T cell Ig mucin-3 determines sex differences in inflammatory heart disease. J Immunol 2007, 178: 6710–6714.
Frisancho-Kiss S, Coronado MJ, Frisancho JA, Lau VM, Rose NR, Klein SL, Fairweather D: Gonadectomy of male BALB/c mice increases Tim-3+ alternatively activated M2 macrophages, Tim-3+ T cells, Th2 cells and Treg in the heart during acute coxsackievirus-induced myocarditis. Brain Behav Immun 2009, 23: 649–657. 10.1016/j.bbi.2008.12.002
Fairweather D, Yusung S, Frisancho-Kiss S, Barrett M, Gatewood S, Steele R, Rose NR: IL-12Rβ1 and TLR4 increase IL-1β and IL-18-associated myocarditis and coxsackievirus replication. J Immunol 2003, 170: 4731–4737.
Frisancho-Kiss S, Nyland JF, Davis SE, Barrett MA, Gatewood SJL, Njoku DB, Cihakova D, Silbergeld EK, Rose NR, Fairweather D: Cutting Edge: T cell Ig mucin-3 reduces inflammatory heart disease by increasing CTLA-4 during innate immunity. J Immunol 2006, 176: 6411–6415.
Cihakova D, Barin JG, Afanasyeva M, Kimura M, Fairweather D, Berg M, Talor MV, Baldeviano GC, Frisancho-Kiss S, Gabrielson K, et al.: Interleukin-13 protects against experimental autoimmune myocarditis by regulating macrophage differentiation. Am J Pathol 2008, 172: 1195–1208. 10.2353/ajpath.2008.070207
Page KR, Jedlicka AE, Fakheri B, Noland GS, Kesavan AK, Scott AL, Kumar N, Manabe YC: Myocabacterium-induced potentiation of type I immune responses and protection against malaria are host specific. Infect Immun 2005, 73: 8369–8380. 10.1128/IAI.73.12.8369-8380.2005
Irizarry RA, Warren D, Spencer F, Kim IF, Biswal S, Frank BC, Gabrielson E, Garcia JGN, Geoghegan J, Germino G, et al.: Multiple-laboratory comparison of microarray platforms. Nat Methods 2005, 2: 345–350. 10.1038/nmeth756
Rangasamy T, Misra V, Zhen L, Tankersley GC, Tuder RM, Biswal S: Cigarette smoke-induced emphysema in A/J mice associated with pulmonary oxidative stress, apoptosis of lung cells and global alterations in gene expression. Am J Physiol Lung Cell Mol Physiol 2009, 296: L888-L900. 10.1152/ajplung.90369.2008
Kass DA, Kitzman DW, Alvarez GE: The restoration of chronotropic competence in heart failure patients with normal ejection fraction (RESET) study: rationale and design. J Cardiac Fail 2010, 16: 17–24. 10.1016/j.cardfail.2009.08.008
Fairweather D, Frisancho-Kiss S, Gatewood S, Njoku D, Steele R, Barrett M, Rose NR: Mast cells and innate cytokines are associated with susceptibility to autoimmune heart disease following coxsackievirus B3 infection. Autoimmunity 2004, 37: 131–145. 10.1080/0891693042000196200
Isensee J, Witt H, Pregla R, Hetzer R, Regitz-Zagrosek V, Noppinger PR: Sexually dimorphic gene expression in the heart of mice and men. J Mol Med 2008, 86: 61–74. 10.1007/s00109-007-0240-z
Waxman DJ, Holloway MG: Sex differences in the expression of hepatic drug metabolizing enzymes. Mol Pharmacol 2009, 76: 215–228. 10.1124/mol.109.056705
Cederbaum A: Nrf2 and antioxidant defense against CYP2E1 toxicity. Expert Opin Drug Metab Toxicol 2009, 5: 1223–1244. 10.1517/17425250903143769
Alvarez BV, Johnson DE, Sowah D, Soliman D, Light PE, Xia Y, Karmazyn M, Casey JR: Carbonic anhydrase inhibition prevents and reverts cardiomyocyte hypertrophy. J Physiol 2007, 579: 127–145. 10.1113/jphysiol.2006.123638
Quaye IK: Haptoglobin, inflammation and disease. Trans R Soc Trop Med Hyg 2008, 102: 735–742. 10.1016/j.trstmh.2008.04.010
Thum T, Borlak J: Testosterone, cytochrome P450, and cardiac hypertrophy. FASEB J 2002, 16: 1537–1549. 10.1096/fj.02-0138com
Yeap BB, Krueger RG, Leedman PJ: Differential posttranscriptional regulation of androgen receptor gene expression by androgen in prostate and breast cancer cells. Endocrinology 1999, 140: 3282–3291. 10.1210/en.140.7.3282
Veenman L, Gavish M: The peripheral-type benzodiazepine receptor and the cardiovascular system. Implications for drug development. Pharmacol Ther 2006, 110: 503–524. 10.1016/j.pharmthera.2005.09.007
Packard CJ: Lipoprotein-associated phospholipase A 2 as a biomarker of coronary heart disease and a therapeutic target. Curr Opin Cardiol 2009, 24: 358–363. 10.1097/HCO.0b013e32832bcb22
Do R, Kiss RS, Gaudet D, Engert JC: Squalene synthase: a critical enzyme in the cholesterol biosynthesis pathway. Clin Genet 2009, 75: 19–29. 10.1111/j.1399-0004.2008.01099.x
Liao P, Georgakopoulos D, Kovacs A, Zheng M, Lerner D, Pu H, Saffitz J, Chien K, Xiao R-P, Kass DA, Wang Y: The in vivo role of p38 MAP kinases in cardiac remodeling and restrictive cardiomyopathy. Proc Nat Acad Sci USA 2001, 98: 12283–12288. 10.1073/pnas.211086598
Boheler KR, Volkova M, Morrell C, Garg R, Zhu Y, Margulies K, Seymour A-M, Lakatta EG: Sex- and age-dependent human transcriptome variability: Implications for chronic heart failure. Proc Nat Acad Sci USA 2003, 100: 2754–2759. 10.1073/pnas.0436564100
Karin N: The multiple faces of CXCL12 (SDF-1β) in the regulation of immunity during health and disease. J Leukoc Biol 2010, 88: 463–473. 10.1189/jlb.0909602
Fairweather D, Frisancho-Kiss S, Yusung SA, Barrett MA, Gatewood SJL, Davis SE, Njoku DB, Rose NR: IFN-β protects against chronic viral myocarditis by reducing mast cell degranulation, fibrosis, and the profibrotic cytokines TGF-β 1 , IL-1β and IL-4 in the heart. Am J Pathol 2004, 165: 1883–1894. 10.1016/S0002-9440(10)63241-5
Diedrich M, Tadic J, Mao L, Wacker MA, Nebrich G, Hetzer R, Regitz-Zagrosek V, Klose J: Heart protein expression related to age and sex in mice and humans. Int J Mol Med 2007, 20: 865–874.
Zordoky BN, El-Kadi AO: Modulation of cardiac and hepatic cytochrome P450 enzymes during heart failure. Curr Drug Metab 2008, 9: 122–128. 10.2174/138920008783571792
Tabet F, Rye K-A: High-density lipoproteins, inflammation and oxidative stress. Clin Sci 2009, 116: 87–98. 10.1042/CS20080106
Burke JE, Dennis EA: Phospholipase A 2 structure/function, mechanism, and signaling. J Lipid Res 2009, 50: S237-S242. 10.1194/jlr.R800033-JLR200
White H: Why inhibition of lipoprotein-associated phospholipase A 2 has the potential to improve patient outcomes. Curr Opin Cardiol 2010, 25: 299–301. 10.1097/HCO.0b013e32833aaa94
Levy R: The role of cytosolic phospholipase A 2 -alpha in regulation of phagocytic functions. Biochim Biophys Acta 2006, 1761: 1323–1334.
Biolard E, Lai Y, Larabee K, Balestrieri B, Ghomashchi F, Fujioka D, Gobezie R, Coblyn JS, Weinblatt ME, Massarotti EM, et al.: A novel anti-inflammatory role for secretory phospholipase A 2 in immune complex-mediated arthritis. EMBO Mol Med 2010, 2: 172–187. 10.1002/emmm.201000072
Mounier CM, Wendum D, Greenspan E, Flejou J-F, Rosenberg DW, Lambeau G: Distinct expression pattern of the full set of secreted phospholipases A 2 in human colorectal adenocarcinomas: sPLA 2 -III as a biomarker candidate. Br J Cancer 2008, 98: 587–595. 10.1038/sj.bjc.6604184
Henderson RJ, Wasan KM, Leon CG: Haptoglobin inhibits phospholipid transfer protein activity in hyperlipidemic human plasma. Lip Health Dis 2009, 8: 27. 10.1186/1476-511X-8-27
Von Haehling S, Schefold JC, Lainscak M, Doehner W, Anker SD: Inflammatory biomarkers in heart failure revisited: much more than innocent bystanders. Heart Failure Clin 2009, 5: 549–560. 10.1016/j.hfc.2009.04.001
The authors thank J Augusto Frisancho and Jaewook Shin for their technical assistance with the studies. This work was supported by National Institutes of Health Grants HL087033 (DF) and ES007062 (TRG).
The authors declare that they have no competing interests.
JAO and MJC contributed equally to this study, and are joint first authors. JAO carried out the microarray studies and analysis, participated in qRT-PCR assays and drafted the manuscript. MJC carried out the microarray studies and analysis and the qRT-PCR assays and analysis and helped draft the manuscript. AEG participated in the microarray studies and analysis and in qRT-PCR analysis and conducted plaque assays. JBK conducted plaque assays. AB conducted ELISAs. DB conducted echocardiography and interpreted the results. KLG interpreted the echocardiography results and helped to draft the manuscript. TRG participated in the design of the study and the interpretation of data. DF conceived the study, participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.
Jennifer A Onyimba, Michael J Coronado contributed equally to this work.
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