Which inflammatory response with Cytomegalovirus infection?

Which inflammatory response with Cytomegalovirus infection?

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I am thinking about inflammation process with Cytomegalovirus infection. I first thought it is about chronic inflammation, but then changed my mind because of virus infection. I think cytokines must be there in activation.

I am thinking which of these is right mechanism for both of these

  • mononuclear cells and granulocytes (I think this must be chronic inflammation)
  • granulocytes with scar formation (I think no scar)
  • cytopathic cytoproliferation inflammation

I think the cytopathic cytoproliferation inflammation is the right one because cytokines are activating the inflammatory response in virus infection. However, I am not sure if this inflammation is of chronic type.

Which type of inflammation can cytomegalovirus infection lead to?

According to this paper the virus triggers the innate immune system and induces inflammatory cytokines as well as interferon stimulated genes (which is a response to a viral infection). It seems to be recognized by the cells via the Toll-like receptor 2 and CD14, which subsequently trigger the cytokine production. A schematic view comes from the second paper:

These two articles are interesting in this context, the first addresses CMV in special, the second is a pretty recent review about how viruses are recognized and how cytokines are involved:

Seropositivity to Cytomegalovirus, Inflammation, All-Cause and Cardiovascular Disease-Related Mortality in the United States

Affiliation Epidemiology and Biostatistics, School of Public Health, Hunter College, City University of New York (CUNY), CUNY Institute for Demographic Research, New York, New York, United States of America

Affiliation Second Department of Internal Medicine, Center for Medical Research, ZMF, University of Tuebingen Medical School, Tuebingen, Germany

Affiliation Epidemiology and Public Health Group, Peninsula Medical School, Exeter, United Kingdom

Affiliation Epidemiology and Public Health Group, Peninsula Medical School, Exeter, United Kingdom

Affiliation Department of Epidemiology, Center for Social Epidemiology and Population Health, School of Public Health, University of Michigan, Ann Arbor, Michigan, United States of America

Chronic Cytomegalovirus Infection and Inflammation Are Associated with Prevalent Frailty in Community-Dwelling Older Women

From the *Division of Geriatric Medicine, Department of Medicine, University of Calgary, Calgary, Alberta, Canada†Division of Geriatric Medicine and Gerontology, Department of Medicine, and ‡Department of Ophthalmology, School of Medicine, and §Center on Aging and Health, ∥Department of Epidemiology, and ¶Department of Health Policy, Bloomberg School of Public Health, the Johns Hopkins University, Baltimore, Maryland.

From the *Division of Geriatric Medicine, Department of Medicine, University of Calgary, Calgary, Alberta, Canada†Division of Geriatric Medicine and Gerontology, Department of Medicine, and ‡Department of Ophthalmology, School of Medicine, and §Center on Aging and Health, ∥Department of Epidemiology, and ¶Department of Health Policy, Bloomberg School of Public Health, the Johns Hopkins University, Baltimore, Maryland.

From the *Division of Geriatric Medicine, Department of Medicine, University of Calgary, Calgary, Alberta, Canada†Division of Geriatric Medicine and Gerontology, Department of Medicine, and ‡Department of Ophthalmology, School of Medicine, and §Center on Aging and Health, ∥Department of Epidemiology, and ¶Department of Health Policy, Bloomberg School of Public Health, the Johns Hopkins University, Baltimore, Maryland.

From the *Division of Geriatric Medicine, Department of Medicine, University of Calgary, Calgary, Alberta, Canada†Division of Geriatric Medicine and Gerontology, Department of Medicine, and ‡Department of Ophthalmology, School of Medicine, and §Center on Aging and Health, ∥Department of Epidemiology, and ¶Department of Health Policy, Bloomberg School of Public Health, the Johns Hopkins University, Baltimore, Maryland.

From the *Division of Geriatric Medicine, Department of Medicine, University of Calgary, Calgary, Alberta, Canada†Division of Geriatric Medicine and Gerontology, Department of Medicine, and ‡Department of Ophthalmology, School of Medicine, and §Center on Aging and Health, ∥Department of Epidemiology, and ¶Department of Health Policy, Bloomberg School of Public Health, the Johns Hopkins University, Baltimore, Maryland.

From the *Division of Geriatric Medicine, Department of Medicine, University of Calgary, Calgary, Alberta, Canada†Division of Geriatric Medicine and Gerontology, Department of Medicine, and ‡Department of Ophthalmology, School of Medicine, and §Center on Aging and Health, ∥Department of Epidemiology, and ¶Department of Health Policy, Bloomberg School of Public Health, the Johns Hopkins University, Baltimore, Maryland.

From the *Division of Geriatric Medicine, Department of Medicine, University of Calgary, Calgary, Alberta, Canada†Division of Geriatric Medicine and Gerontology, Department of Medicine, and ‡Department of Ophthalmology, School of Medicine, and §Center on Aging and Health, ∥Department of Epidemiology, and ¶Department of Health Policy, Bloomberg School of Public Health, the Johns Hopkins University, Baltimore, Maryland.

From the *Division of Geriatric Medicine, Department of Medicine, University of Calgary, Calgary, Alberta, Canada†Division of Geriatric Medicine and Gerontology, Department of Medicine, and ‡Department of Ophthalmology, School of Medicine, and §Center on Aging and Health, ∥Department of Epidemiology, and ¶Department of Health Policy, Bloomberg School of Public Health, the Johns Hopkins University, Baltimore, Maryland.

From the *Division of Geriatric Medicine, Department of Medicine, University of Calgary, Calgary, Alberta, Canada†Division of Geriatric Medicine and Gerontology, Department of Medicine, and ‡Department of Ophthalmology, School of Medicine, and §Center on Aging and Health, ∥Department of Epidemiology, and ¶Department of Health Policy, Bloomberg School of Public Health, the Johns Hopkins University, Baltimore, Maryland.

From the *Division of Geriatric Medicine, Department of Medicine, University of Calgary, Calgary, Alberta, Canada†Division of Geriatric Medicine and Gerontology, Department of Medicine, and ‡Department of Ophthalmology, School of Medicine, and §Center on Aging and Health, ∥Department of Epidemiology, and ¶Department of Health Policy, Bloomberg School of Public Health, the Johns Hopkins University, Baltimore, Maryland.

From the *Division of Geriatric Medicine, Department of Medicine, University of Calgary, Calgary, Alberta, Canada†Division of Geriatric Medicine and Gerontology, Department of Medicine, and ‡Department of Ophthalmology, School of Medicine, and §Center on Aging and Health, ∥Department of Epidemiology, and ¶Department of Health Policy, Bloomberg School of Public Health, the Johns Hopkins University, Baltimore, Maryland.

From the *Division of Geriatric Medicine, Department of Medicine, University of Calgary, Calgary, Alberta, Canada†Division of Geriatric Medicine and Gerontology, Department of Medicine, and ‡Department of Ophthalmology, School of Medicine, and §Center on Aging and Health, ∥Department of Epidemiology, and ¶Department of Health Policy, Bloomberg School of Public Health, the Johns Hopkins University, Baltimore, Maryland.

Funded by National Institute on Aging (NIA) Grants RO1 AG11703 and R37 AG019905 National Institutes of Health, National Center for Research Resources, Outpatient Department—General Clinical Research Center Grants RR00722, RO1 A141956 and NIA Contract NO1-AG12112.


Objectives: To evaluate the association between asymptomatic chronic cytomegalovirus (CMV) infection and the frailty syndrome and to assess whether inflammation modifies this association.

Design: Cross-sectional analysis.

Setting: Women's Health and Aging Study I & II, Baltimore, Maryland.

Participants: Seven hundred twenty-four community-dwelling women aged 70 to 79 with baseline measures of CMV, interleukin-6 (IL-6), and frailty status.

Measurements: CMV serology and IL-6 concentrations were measured using enzyme-linked immunosorbent assay. Frailty status was based on previously validated criteria: unintentional weight loss, weak grip strength, exhaustion, slow walking speed, and low level of activity. Frail women had three or more of the five components, prefrail women had one or two components, and women who were not frail had none of the components. Multinomial logistic regression adjusted for potential confounders.

Results: Eighty-seven percent of women were CMV seropositive, an indication of chronic infection. CMV was associated with prevalent frailty, adjusting for age, smoking history, elevated body mass index, diabetes mellitus, and congestive heart failure (CMV frail adjusted odds ratio (AOR)=3.2, P=.03 CMV prefrail AOR=1.5, P=.18). IL-6 interacted with CMV, significantly increasing the magnitude of this association (CMV positive and low IL-6 frail AOR=1.5, P=.53 CMV positive and high IL-6 frail AOR=20.3, P=.007 CMV positive and low IL-6 prefrail AOR=0.9, P=.73 CMV positive and high IL-6 prefrail AOR=5.5, P=.001).

Conclusion: Chronic CMV infection is associated with prevalent frailty, a state with increased morbidity and mortality in older adults inflammation enhances this effect. Further prospective studies are needed to establish a causal relationship between CMV, inflammation, and frailty.

Regulation of Inflammation

Cytomegalovirus (CMV) is an important cause of infection in immunocompromised patients (1-5). The two groups that most frequently have clinical infections with CMV are patients infected with the human immunodeficiency virus (HIV) and patients with transplanted organs. CMV can infect many different organ systems, and it is a significant cause of morbidity for patients with HIV infection. CMV infections in patients who have undergone organ transplantation may be an important cause of life-threatening pneumonia and other infections. In addition, CMV has a tendency to infect transplanted organs and trigger rejection. In this regard, CMV infection in a previously seronegative lung transplant recipient is associated with a greater likelihood for the development of severe obliterative bronchiolitis.

There appears to be a greater incidence of CMV infection in patients who have received a transplanted organ compared with patients who are equally immunosuppressed but have not undergone organ transplantation. This clinical observation could be explained by observations that suggest that CMV replicates best in cells that are activated. One by-product of organ transplantation is an intense activation of the immune system in an attempt to reject the transplanted organ. Other cells, like endothelial cells, fibroblasts, and epithelial cells may also be activated by cytokines released by activated inflammatory cells. This cell activation is most intense in the transplanted organ. Thus, the setting of organ transplantation creates an ideal environment for replication of CMV.

Although the control of CMV replication is not entirely understood, it is known that replication is under control of the CMV major immediate early promoter, which controls expression of viral immediate early genes. Expression of the immediate early genes is important for viral replication because the products of these genes are transcription factors necessary for expression of other viral genes. Cell activation is crucial for viral replication because the virus does not express the genes that encode some of the critical transcription factors necessary to activate the major immediate early promoter. Two of the most important of these transcription factors are nuclear factor (NF)κB and cyclic adenosine monophosphate (cAMP)-dependent transcription factors. These transcription factors are expressed at high levels in cells that are activated. Thus, the virus is highly dependent on activation of the infected cell for replication of its genome (Figure 1 ).

Fig. 1. Cell activation is critical for viral replication. Activation of the cell provides critical cell-derived transcription factors (like NFκB and cAMP-derived transcription factors), which are necessary for enhanced expression of the CMV major immediate early promoter. The major immediate early promoter controls viral replication. One function of the virally encoded US28 gene product might be to enhance viral replication because prior studies have shown that it is a signaling receptor that results in cell activation after exposure to chemokines.

The mechanisms by which CMV regulates inflammation are very complex (6-23). There are at least two general ways that CMV can stimulate cells. One mechanism involves attachment of CMV to the cell surface, which results in stimulation of the cell via viral surface glycoproteins. A number of viral coat proteins that mediate attachment to and activation of various types of cells have been identified. This means of cell stimulation probably occurs during active infection. It has also been demonstrated that expression of the viral immediate early genes, in the absence of viral replication, also results in cell activation. Expression of the immediate early genes of CMV occurs during active infection, but it can also occur during latency in the absence of viral replication. The latter observation suggests that latent CMV infection can alter inflammatory responses by cells that contain the viral genome. These observations are also consistent with clinical observations that drugs that inhibit viral replication do not inhibit all of the observed effects of CMV.

There are a number of specific ways that CMV may activate cells (6-23). CMV infection may increase or decrease expression of classes I or II human leukocyte antigen (HLA) antigens. Inhibition of expression of class I antigens by CMV has been proposed as a mechanism used by the virus to escape immune surveillance. By contrast, upregulation of class II antigens has been proposed as mechanisms by which CMV triggers enhanced graft rejection. CMV has also been shown to increase expression of adherence proteins, like intracellular adhesion molecule-1 (ICAM-1) (CD54) and lymphocyte function–associated antigen (LFA)-3 (CD58), and expression of a variety of cytokine genes by monocytes, lymphocytes, epithelial cells, endothelial cells, and fibroblasts. Others studies have shown that epitopes on the CMV IE2 gene product mimic peptides on HLA DR3 antigens (24). This observation suggests that CMV infection might, in some instances, trigger an autoimmune-type disease. Overall, these observations strongly suggest that CMV can augment inflammatory processes. That this can occur in the lung is suggested by an observation that the combination of graft-versus-host disease (GVHD) and CMV can result in interstitial lung disease in animal models. Neither GVHD alone nor CMV alone resulted in lung disease. Although these observations do not eliminate the possibility that GVHD alone can cause lung disease, they do suggest that CMV may increase the expression of this type of lung disorder.

An interesting observation from a number of studies is that the viral genome encodes a number of receptors, including HLA-like antigens that can bind to α-2-microglobulin, Fc receptors, and receptors for various cytokines and chemokines (25-40). In this issue of the Journal, Billstrom and colleagues (41) show that the CMV-encoded chemokine receptor, US28, depletes extracellular regulated upon activation, normal T-cells expressed and secreted (RANTES) during CMV infection. A number of investigators have previously shown that the CMV genome contains four genes (US27, US28, UL33, and UL78) that encode putative homologues of cellular G protein–coupled receptors (25-30). Of these, the US28 gene product has been shown to be a functional receptor for the β-chemokine class of immune modulators. It has also previously been demonstrated that the US28 gene product is a signaling receptor that activates cells and can deplete the extracellular medium of RANTES. The present study by Billstrom and associates extends these observations by providing a mechanism for the depletion of this cytokine. The authors suggest that this may be one mechanism through which CMV might regulate inflammation. Since HIV also utilizes chemokine receptors for infection, the authors also speculate that this might alter the ability of HIV to infect cells. Although these observations are probably the case, the question is why the virus might have evolved to express this receptor. It is unlikely that uptake of RANTES is a protective mechanism for the virus because high levels of RANTES are usually present at sites of infection in spite of this uptake by the cells. More likely, expression of this receptor is important for viral replication, since an interaction of this receptor with its ligand results in cell activation. As noted previously, this is a critical process for viral replication. These observations as a whole are important because they may lead to new, more specific therapies for this important infection.


The present studies demonstrate that, in addition to IL-12, TNF, and IFN-γ, MCMV infections of mice induce detectable IL-1α and IL-6, but not IL-1β, production. The IL-12, TNF, IFN-γ, and IL-1α cytokines all rapidly reach high peak levels in the circulation and decline shortly thereafter. Moreover, cytokine activation of the HPA axis appears to be engaged as endogenous serum corticosterone and ACTH responses also are induced. IL-6 is the critical and required factor leading to peak induction of endogenous glucocorticoids, and IL-1α is a necessary factor for optimal IL-6 production. This pathway to glucocorticoid release through IL-1α and IL-6 is clearly delineated because IL-1β is undetectable in MCMV-infected mice and because IL-1α levels are similar or greater in MCMV- infected IL-6–deficient mice as compared to infected wildtype controls. The IL-6 requirement for glucocorticoid responses is specific to viral infections and virus-type stimuli because, although the synthetic double stranded analogue for viral nucleic acid, poly I:C, the bacterial product, LPS, and restraint stress all induce glucocorticoid responses in normal mice, only peak responses to MCMV and poly I:C are exquisitely dependent upon endogenous IL-6. Taken together, these results identify: (a) the kinetics and magnitudes of early circulating cytokine induction during MCMV infection, (b) a unique IL-6–dependent glucocorticoid response to viral infection and virus-type, non-replicating stimuli, and (c) a specific IL-1α to IL-6 induction pathway. Furthermore, as they present evidence for a distinct cytokine dependency for HPA axis activation, the studies begin to define precise communication pathways between immune and neuroendocrine systems under conditions of different microbial challenges.

IL-12, IFN-γ, and TNF are important for antiviral defense (15, 16) and, as IL-1 and IL-6 have known proinflammatory functions (19, 21), these factors may also promote defense against MCMV. However, at high systemic levels, the cytokines can cause significant and life-threatening pathologies (1, 2, Orange, J.S., T.P Salazar-Mather, and C.A. Biron, manuscript in preparation). As glucocorticoids can decrease production of multiple cytokines, including IL-6, IFN-γ, IL-1, and TNF (8), induction of these endogenous steroid hormones by high virus-induced IL-6 levels may provide feedback inhibition to limit cytokine responses and protect the host from cytokine-mediated disease (22). In support of this hypothesis, another acute viral infection, lymphocytic choriomeningitis virus (LCMV), does not induce detectable systemic cytokines (15 Ruzek, M.C., A.H. Miller, B.D. Pearce, and C.A. Biron, unpublished observation) or significant levels of glucocorticoids (12, Ruzek, M.C., A.H. Miller, B.D. Pearce, and C.A. Biron, data not shown) at early times. As a consequence, immune responses to MCMV are shaped and/or limited by factors in addition to those influencing responses to LCMV. It is noteworthy that in comparison to LCMV, MCMV infections induce relatively weak later T cell responses (14). Thus, glucocorticoids may be specifically elicited during certain viral infections initiating a sequence of events with the potential to result in cytokine-mediated detrimental effects, i.e., inducing extremely high levels of cytokines, but not during viral infections failing to do so, and these hormones may shape additional down-stream immune responses to these viruses.

The IL-1α–induced IL-6 requirement for optimal glucocorticoid stimulation (Fig. 3) is consistent with studies examining recombinant or purified factors in vivo. Administrations of TNF, IL-1, and IL-6 increase circulating ACTH and corticosterone levels (5, 7) with IL-1β being the most potent and rapid inducer (7). However, IL-1α, IL-1β, and TNFα can all stimulate IL-6 production, with IL-1α being a more potent inducer than either IL-1β or TNF (5, 23). In addition, IL-1α and IL-6 have been shown to synergize for release of ACTH (23, 24). This ability of IL-1α to induce and synergize with IL-6 can explain the observation that corticosterone responses after IL-6 administration alone are not as great as those after IL-1 administration (23). As a downstream or cooperative role of IL-6 has not been previously distinguished from direct stimulation, the reported IL-1 requirement for corticosterone responses to pro-inflammatory stimuli may be due to this factor's ability to induce and synergize with IL-6 (23, 24). In addition, given the different potencies of IL-1α and IL-1β for IL-6 induction (5), yet similar activity on the HPA axis (24), IL-1β alone may induce glucocorticoids, but optimal glucocorticoid responses to IL-1α may require IL-6. It is likely that serum IL-1α values reported here are actually an underestimate of overall induced levels, as IL-1α is generally expressed in a membrane-bound form (19).

Although the glucocorticoid response dependency on IL-1α-induced IL-6 is consistent with results from administering cytokines, the IL-6 requirement in response to challenge with particular agents appears to be specific to viral infections. Two additional systems have not found IL-6 essential for glucocorticoid induction these are turpentine and LPS challenges (13). IL-1β is readily detectable after injection of LPS (6), however, it is undetectable in serum from MCMV-infected mice. Therefore, during viral infection, the cytokines leading to glucocorticoid production may be more dependent upon an IL-1α to IL-6 cascade, whereas LPS and/or turpentine may induce other cytokines, including IL-1β, that either alone or in combination induce glucocorticoids independently of IL-6 (3). The poly I:C–induced responses also appear to occur under conditions of minimal IL-1β expression. Thus, the ability to define the IL-1α to IL-6 pathway for glucocorticoid induction, in response to MCMV and/or poly I:C, is most likely possible because of the absence of parallel or overlapping pathways for induction. A model for pathways of glucocorticoid induction, after viral as compared to bacterial stimulation, is presented in Fig. 5.

Our results can be contrasted to others examining virus activation of the HPA axis after exposure to Newcastle disease virus (NDV), influenza virus, and herpes simples type 1 (HSV-1) (25–27). NDV stimulates IL-1–dependent ACTH and corticosterone release (25). However, NDV does not productively infect mice and, similar to poly I:C responses, increases in serum corticosterone are observed within hours of injection (12, 25). Therefore, NDV does not reflect actual events occurring during infection of a permissive host. Influenza virus infections and ocular infections of herpes simplex virus type 1 (HSV-1) also induce increased corticosterone levels, but, in comparison to MCMV infection, these responses peak much later (7 d after infection) and are prolonged (26, 27). As both HSV-1 and influenza virus infections can cause considerable additional physical distress which may itself stimulate the HPA axis, it is likely that other pathways are contributing to induction of glucocorticoid responses at later times after these infections. Interestingly, several other viruses have been shown to stimulate IL-6 mRNA expression in vitro (28). Although a thorough comparison of IL-6 production and corticosterone responses has not been performed during each of these infections, our results suggest that induction of circulating IL-6 may determine whether or not glucocorticoid responses and their consequences are elicited early during viral infections. Taken together, these observations suggest that the kinetics and magnitude of corticosterone production induced by systemic viral infection may be specific to the virus, the extent of virus-induced pathology, and/or the levels of virus-induced IL-6.

In summary, these results demonstrate rapid and dramatic increases in serum cytokines and glucocorticoids early during MCMV infection. An essential role for IL-6 in the induction of peak endogenous glucocorticoid responses is defined, with IL-1 playing an accessory role for production of, and/ or synergism, with IL-6. Taken together with work in other systems, these studies show a stringent regulation of systemic cytokines responses during early MCMV infection, and define a previously uncharacterized cytokine pathway for glucocorticoid induction during a natural infection.

On Evolutionary Biology, Inflammation, Infection, and the Causes of Atherosclerosis

From the Center for Cardiovascular Disease Prevention and the Leducq Center for Cardiovascular Research, Brigham and Women’s Hospital, Harvard Medical School, Boston, Mass.

Often there is no assignable cause for the attack, that is, for the coronary thrombosis itself.

For most of human history, the primary causes of death have included infection and famine. It is thus not surprising that large portions of the human genome are dedicated to 2 interrelated problems: innate immunity and the inflammatory response (how to ward off infection and survive trauma) and cellular metabolism in times of crisis (how to sustain gluconeogenesis during prolonged periods of malnutrition).

From an evolutionary biology standpoint, these interrelated pressures might be expected to select for individuals with relatively enhanced inflammatory function, as well as mild to moderate insulin resistance. 1 However, our ancestors, who lived a demanding hunter-gatherer lifestyle characterized by extended periods of physical activity and a high-protein diet, were also largely free of atherosclerosis and diabetes. Thus, for many investigators interested in the underlying causes of these disorders, a key evolutionary question is now openly being asked: is it possible that the adaptive pattern of an earlier time has resulted in a maladaptive response in our modern environment dominated by increasingly sedentary habits, an abundance of high-carbohydrate foods, and a reduced risk of mortality due to common infections? 1,2 If so, is our current epidemic of atherosclerosis and diabetes predictable on the basis of evolutionary biological principles?

Clinical data supporting this position come from several disparate sources. For one, atherosclerosis is now recognized as a disorder characterized by a chronic alteration of inflammatory function, 3 and key markers of inflammation and the innate immune response, including C reactive protein (CRP), interluekin-6, tumor necrosis factor-α, and several cell adhesion molecules have been linked to the future occurrence of myocardial infarction and stroke in both healthy populations and among those with known coronary disease. 4,5 These data have cemented the need to move beyond cholesterol in our understanding of the causes of atherothrombosis and have lead to the hypothesis that the additional use of inflammatory biomarkers, such as CRP, can improve methods of global cardiovascular risk assessment. 6

It is further recognized that risk factors for atherosclerosis and adult-onset diabetes closely overlap and that the two disorders may derive from similar antecedents. This fact, and the propensity of diabetic patients to have premature atherosclerosis, has lead to a “common soil” hypothesis, which suggests, in part, that both of these disorders share a mutual inflammatory and perhaps genetic basis. 7 In support of this observation are cross-sectional observations linking insulin resistance and diabetes to low-grade inflammation and alterations in the innate immune system, 2,8–10 as well as the fact that adipocytes secrete pro-inflammatory cytokines, thus linking central obesity with both atherogenesis and diabetes. Very recent prospective epidemiological data also support this view. The large-scale Women’s Health Study enrolled apparently healthy individuals with no overt abnormalities of glucose metabolism it discovered that baseline levels of both CRP and interleukin-6, which were previously shown to predict the onset of atherothrombosis, 4,11 were also found to predict the onset of type II diabetes. 12 This finding was true even after adjustment for body mass index and when the analyses were limited to those with hemoglobin A1C levels <6.0 at entry.

A common-cause hypothesis focused on inflammation also helps to explain why pharmacological therapies targeted at reducing atherosclerosis might have efficacy in diabetes prevention. In particular, post hoc evaluation of the West of Scotland Coronary Atherosclerosis Prevention Study found reduced rates of incident diabetes associated with statin use. 13 These data are intriguing because statins not only reduce LDL cholesterol, but also reduce CRP in an LDL-independent manner. 14,15 Similarly, aspirin, an agent known to reduce cardiovascular risk in direct relation to baseline levels of CRP, 16 has very recently been shown at high doses to specifically inhibit the function of I-κ-kinase-β, a key protein involved in the regulation of inflammation that interferes with insulin signaling and contributes to both insulin resistance and diabetes. 17

As intriguing as these observations are, the clinical hypothesis that an enhanced immune response results in increased plaque vulnerability begs the question as to why a population distribution of inflammation exists in the first place and what the underlying determinants of this distribution might be. In the present issue of Circulation, Espinola-Klein and colleagues 18 provide evidence for one potential source of this heightened inflammatory response by evaluating the relationship between infectious burden and the extent and prognosis of patients with atherosclerosis. In brief, among 572 patients undergoing clinical evaluation for coronary disease in the German AtheroGene project, an increasing prevalence of seropositivity directed against herpes simplex virus, cytomegalovirus, Epstein-Barr virus, Hemophilus influenzae, Chlamydia pneumoniae, Mycoplasma pneumoniae, or Helicobacter pylori was found to be associated with an increased prevalence of advanced atherosclerotic lesions and a reduced overall prognosis. These data thus provide confirmatory evidence of a link between total infectious burden and atherosclerotic severity, an observation previously made by others. 19,20

When interpreting these data, investigators must be careful not to confuse association with causation and they need to consider alternative possibilities. Cross-sectional studies cannot establish a temporal relation between exposure and disease, and it is extremely difficult for studies using this (or retrospective) designs to exclude the possibility that the observed associations are due to confounding rather that to any particular causal pathway. Individuals with greater infectious burdens may seem to be at increased vascular risk only because they are older, have increased levels of cigarette consumption, less access to care, or reduced socioeconomic status. All of these factors are associated independently with both infection and atherosclerosis and thus represent alternative (but noncausal) explanations for observed links between infection and coronary disease. In a similar vein, investigators must be careful not to assume that evidence of various infectious organisms residing within atherosclerotic tissue necessarily implies a causal relation. Although suggestive, such studies are prone to selection bias and have difficulty excluding the possibility of an “innocent bystander” effect in which obligate intracellular organisms are present at the lesion site due to an unrelated inflammatory response.

Two alternative approaches to evaluating the association between infection and atherosclerosis that are less likely to be affected by bias and confounding are prospective cohort studies (in which the exposure of interest is ascertained before the onset of disease and in which multiple confounding variables can be simultaneously addressed) and direct randomized trials of antibiotic therapy. Fortunately, great progress is being made on both of these fronts.

With regard to prospective cohort studies evaluating early life exposure to infectious organisms and the subsequent development of cardiovascular disease, results have generally ranged between no observable association to small but nonsignificant effects. In a recent overview analysis of 10 prospective studies of H pylori seropositivity and coronary death or nonfatal myocardial infarction that together evaluated 2916 cases, the pooled odds ratio was only 1.15 (95% confidence intervals, 0.96 to 1.37). 21 Similarly, for C pneumoniae, where retrospective evidence, histological studies, and experimental work are the strongest, a pooled analysis of 3169 case patients from 15 prospective studies found an almost identical overall odds ratio of 1.15 (95% confidence intervals, 0.97 to 1.36). 22 Such large-scale analyses clearly indicate that caution needs to be used when evaluating the infectious hypothesis of atherosclerosis and that investigators need to weigh the value of different study designs carefully. At the same time, it is important to recognize that even large, prospective, epidemiological studies cannot be considered definitive because most did not specifically address the concept of total infectious burden and almost all were limited to the initial development of atherosclerosis rather than to secondary events. Of note, the one prospective study that did evaluate multiple pathogens simultaneously as potential sources of inflammation, as well as determinants of future vascular risk, failed to find significant evidence of association. 23

With regard to antibiotic trials in the secondary prevention of coronary events, published trials have had mixed results, and none have been of adequate size to address the link between infection and atherosclerosis carefully. Of the completed trials, the Azithromycin in Coronary Artery Disease: Elimination of Myocardial Infection with Chlamydia

(ACADEMIC) study 24 has been the most informative, and it indicates that any use of antibiotic therapy to reduce vascular risk is currently inappropriate. However, as Grayston 25 has carefully pointed out, trials of far larger sample size are needed to test this hypothesis fully. At least 3 well-designed, large-scale studies are now well underway employing either azithromycin or gatifloxacin as potent anti-Chlamydial agents in the secondary prevention of acute coronary events.

Clinicians should be aware, however, that even these randomized trials are likely to be only partially informative. If positive, they will provide critical evidence that at least one infectious organism plays a role in the late stages of coronary disease (although an alternative, but less likely, direct anti-inflammatory effect of the agent tested might also be argued). If null, these trials will tell us only that certain antibiotic regimens do not reduce recurrent vascular event rates. Although a null finding in secondary prevention would likely reduce investigator enthusiasm for the antibiotic approach, such a finding should not scientifically be construed to dismiss the possibility of causation, particularly with regard to earlier stages of plaque development. Results of ongoing antibiotic trials will also need to be considered in light of organisms other than Chlamydia. As suggested in the data from Espinola-Klein and colleagues, 18 several alternative bacterial and viral pathogens are capable of triggering a chronic immune response. For example, although again not demonstrating causality, a growing body of literature has found association between periodontal disease and coronary risk. 26

Where then do we stand with regard to inflammation, infection, and atherothrombotic disease and how should we judge the evidence? The answer remains one of uncertainty, and investigators must continue to move forward with critical but open minds. Only a few years ago, there was virtually no clinical evidence that inflammation played a fundamental role in atherothrombosis. It will take several years more to discern what the triggers of that inflammation are and whether infection is a key determinant of that response.

In the meantime, it is hard to imagine that our ancestors, who faced commensal organisms, life threatening plagues, and chronic parasitic infestation, did not have a greater “infectious burden” than what we currently face. Yet, it is only in our modern times that atherosclerosis and diabetes have become epidemic. From a clinical perspective, it is reassuring that diet, exercise, and lifestyle changes can so effectively reduce rates of both atherothrombosis and diabetes. 27,28 If we are, in fact, destined from an evolutionary standpoint to be at risk for these conditions, we should be vigilant in reminding our patients that prevention remains highly effective.

Finally, as cardiologists reflecting on evolutionary determinants of atherogenesis, plaque rupture, platelet aggregation, and acute thrombosis, we may need to step back and recognize how lucky we are to live in an era with a markedly prolonged mean life expectancy. As Fernandez-Real and Ricart 1 have suggested, for our ancestors with a life expectancy of 35 to 40 years, “the advantages of a high cytokine responder (eradication of injury) or moderate insulin resistance (protection from starvation) overcame the possible inconveniences of atherosclerosis.” In our current environment, these inconveniences may prove to be at the root of our ongoing epidemic.

The opinions expressed in this editorial are not necessarily those of the editors or the American Heart Association.

The Second Line of Defense

If you have a cut on your hand, the break in the skin provides a way for pathogens to enter your body. Assume bacteria enter through the cut and infect the wound. These bacteria would then encounter the body&rsquos second line of defense.

Inflammatory Response

The cut on your hand may become red, warm, and swollen. These are signs of aninflammatory response. This is the first reaction of the body to tissue damage or infection. As explained in Figure below, the response is triggered by chemicals called cytokines andhistamines, which are released when tissue is injured or infected. The chemicals communicate with other cells and coordinate the inflammatory response. You can see an animation of the inflammatory response at this link:

This drawing shows what happens during the inflammatory response. Why are changes in capillaries important for this response?


The chemicals that trigger an inflammatory response attract leukocytes to the site of injury or infection. Leukocytes are white blood cells. Their role is to fight infections and get rid of debris. Leukocytes may respond with either a nonspecific or a specific defense.


The SARS-CoV-2 pandemic, which is believed to have originated in Wuhan in 2019, has already led to the deaths of over 340,000 people, a number that is rising steadily at this time [1]. Indeed, the virus represents one of the most important challenges to global health since Spanish Flu in 1918. At this stage, no effective treatment or vaccine is available and the mortality rate is estimated at around 2% [2]. One of the striking features of SARS-CoV-2 infection is that there is a very heterogeneous clinical outcome in different population groups. In particular, mortality risk is greatly increased in older people and also those with underlying health conditions such as cardiovascular disease, hypertension or diabetes. The explanation for these associations is unclear although a dysregulation in immune function with age (‘immune senescence’) is a well-established phenomenon. However, to date, the importance of previous infection history has received little interest as a potential determinant of clinical outcome. In particular, all adults harbour a range of persistent viral infections and this ‘virome’ plays an important role in promoting maturation of immune function and may also impact on the ability to generate immune responses to novel pathogens [3]. As such a primary infection with COVID-19 builds on an established platform of chronic infectious burden and this legacy may act as a determinant of outcome.

The herpesvirus family is one of the best characterized and largest group of persistent viral infections [4]. These eight viruses share a range of features including a relatively mild primary infection in most cases followed by lifelong persistence as a consequence of viral latency and sustained immunological control of viral replication. Cytomegalovirus (CMV) is the largest member of this family with a genome of 235 kb that encodes over 160 proteins. The clinical sequelae of CMV infection include a range of characteristic features and several of these would suggest that this virus, in particular, may act as a important influence on the clinical outcome of SARS infection. In this regard, any such association might be seen in either the extent of SARS-CoV-2 viral replication or the quality of the subsequent immune response. A secondary influence of the acute inflammation leading to enhanced CMV reactivation must also be considered.


Cytomegalovirus is one of the most common persistent infections within the human population and it is likely that over 4 billion people are infected at the current time [5]. Infection is often encountered very early in life but may occur at any age and is usually asymptomatic. The virus then persists in a range of tissues including myeloid cells, vascular endothelium and renal tissue. Of note, the rates of CMV seropositivity (a marker of persistent infection) are very high in populations that have suffered high mortality rates from SARS-CoV-2 infection such as northern Italy, China and Spain [6]. In addition, infection rates are higher in people from lower socio-economic groups, a subset of the population that appears to have higher mortality rates from SARS-CoV-2 infection [7]. A striking feature of Covid-19 is the increased mortality rate in men compared to women and here it may be noteworthy that the influence of CMV on longer term health in women may be less significant than observed for men [8].

One of the characteristic and unique features of cytomegalovirus infection is its influence on the immune response. The virus acts as a hugely important influence on the maturation and long term composition of the immune repertoire [9, 10]. This is seen most clearly in the number and proportion of cytotoxic T and NK lymphocytes within the peripheral circulation which are increased by 30 to 40% in CMV-seropositive individuals [11,12,13]. Importantly this expansion in the number of virus-specific effector and memory cells is associated with a substantial decrease in the relative proportion of naive lymphocytes. Further associations include alterations in systemic inflammatory markers and infection of a proportion of myeloid cells. The significance of these findings in relation to the impact of SARS-CoV-2 infection on immune health are discussed below.

Cytomegalovirus infection of trophoblast cells elicits an inflammatory response: a possible mechanism of placental dysfunction

Objective: We sought to determine whether cytomegalovirus infection of extravillous trophoblast cells induces inflammatory changes that lead to cell death.

Study design: Extravillous trophoblast (HTR-8/SVneo) cells were infected with cytomegalovirus. Cell death assays were performed 8 to 48 hours after infection. The expression and secretion of cytokines (interleukin-6 and -8) preceding cell death was measured by polymerase chain reaction and enzyme-linked immunosorbent assay.

Results: Cell viability (lactate dehydrogenase assay) was reduced approximately 20%, and rates of apoptosis (measured by TdT-mediated dUTP-X nick end labeling [TUNEL] assay) were increased approximately 40% at 16 to 48 hours after infection. Significantly elevated levels of caspase-3 mRNA levels were observed before increased cell death. Interleukin-6 and -8 mRNA expression and protein secretion were up-regulated 8 to 16 hours after cytomegalovirus infection.

Conclusion: Placental exposure to cytomegalovirus induces an inflammatory response that precedes invasive trophoblast cell death. Cytomegalovirus may prevent normal placental invasion, which results in adverse reproductive outcomes that are associated with placental dysfunction.


Background—Positive and negative associations between cytomegalovirus (CMV) infection and coronary artery disease (CAD) have been reported. We postulated that the susceptibility to CMV-induced CAD might relate to patterns of inflammatory and immune responses to CMV infection and that sex might have an effect on these responses.

Methods and Results—In 151 men and 87 women being evaluated for CAD, blood samples were tested for humoral (Ab+) and cellular (Tc+) responses to CMV and for C-reactive protein (CRP). In men, an elevated CRP level was a significant determinant of CAD even after adjustment for CAD risk factors (OR, 3.1 95% CI, 1.21 to 7.97). CMV seropositivity was associated with elevated CRP levels on multivariate analysis (P=0.006). In contrast, in women, CMV seropositivity was independently predictive of CAD (OR, 41.8 95% CI, 4.12 to 423.74). CRP level in women with CAD was >25% higher than those without CAD, but the difference did not reach statistical significance. Importantly, compared with CMV Ab−/Tc− women, CAD prevalence was higher in Ab+/Tc− and Ab+/Tc+ (13% versus 68% and 64%, both P<0.005) but not in Ab−/Tc+ women (25%). There were no differences in age, smoking, diabetes, hypertension, and hypercholesterolemia among women with different types of immune responses to CMV infection.

Conclusions—The mechanisms by which CMV predisposes to CAD in men and women may be different. In men, CMV appears to contribute to CAD risk, insofar as it predisposes to inflammation. In women, other mechanisms, possibly related to the type of immune response generated by the host, appear to be responsible for the proatherogenic effects of CMV.

Various lines of mechanistic evidence imply a possible role of infection in atherosclerosis. Consonant with these investigations are epidemiological studies demonstrating associations between coronary artery disease (CAD) and several pathogens, including cytomegalovirus (CMV), Chlamydia pneumoniae, Helicobacter pylori, and herpes simplex virus. 1 2 3 4 5 6 7 8 Other studies, however, have yielded negative results. 8 9 10 11 12

The link between infection and atherosclerosis has traditionally been assessed by identifying infected individuals through the presence or absence of antibodies directed at the pathogen. We thought the issue might be more complex, because previous studies have shown 13 14 15 that the type of immune response (humoral or cellular) importantly determines whether a given infection leads to pathogen-induced disease. The humoral immune response functions mainly to prevent infection by extracellular agents, whereas the cell-mediated immune response is more critical for elimination and control of intracellular pathogens. The relative intensities of the humoral and cellular immune responses generated by an infectious agent depend on multiple factors, including the specific pathogen and the genetic determinants of the individual host.

Data compatible with the importance of the cellular immune response to control intracellular pathogens come from studies of infectious diseases such as AIDS, 13 14 chronic hepatitis B, 16 and leishmaniasis, 17 18 19 which suggest that a humoral response conveys susceptibility to disease, whereas a cellular response conveys resistance. In addition, our recent studies demonstrated that considerable host variability exists in the inflammatory responses to CMV infection, 20 as reflected by elevated C-reactive protein (CRP) levels.

Because of this heterogeneity and the marked influence of sex on susceptibility to CAD and because of the presumed roles of inflammatory and immune responses in CAD, we postulated that overall susceptibility or resistance to CMV-induced CAD will be determined by sex-related heterogeneity of the inflammatory and immune responses to CMV infection. The purpose of the present investigation, therefore, was to examine the hypotheses that (1) the presence or absence of an inflammatory response to CMV is sex-related and (2) the type of immune response mounted by the host to CMV contributes to susceptibility or resistance to CMV-associated CAD.


Patient Characteristics

This study was approved by the NHLBI Institutional Research Board. Of the 238 individuals, 151 (63%) were men 169 (71%) were white. Ages ranged from 30 to 81 years (mean, 57.2 years median, 57.0 years). Each individual was admitted for evaluation of chest pain or abnormal noninvasive tests and underwent diagnostic coronary angiography. These individuals also formed the basis of another study designed to determine whether the influence of CMV on CAD is modulated by induction of an inflammatory state. 20 For primary analysis, CAD was defined as any angiographic evidence of atherosclerosis, including presence of plaque in any segment of the epicardial coronary arteries. A patient was defined as being free of CAD only if all coronary arteries were angiographically smooth. Approximately 95% of individuals had blood drawn at the time of catheterization, but none had blood drawn >3 years after the diagnostic study. No individual without CAD was admitted to the study unless blood for immunologic testing was drawn within 3 years of coronary angiography. No patient admitted to the study had unstable symptoms, and none had a myocardial infarction within the previous 3 months.

Determination of Risk Factors for CAD

Risk factors for CAD analyzed included age, sex, cigarette smoking, diabetes, hypercholesterolemia, hypertension, and seropositive CMV status. A patient who had stopped smoking >20 years ago and who was <30 years of age when he or she stopped smoking was considered not to have smoking as a risk factor. A patient was considered to have diabetes if he or she was taking insulin or oral hypoglycemic agents or had previously received such treatment and was currently using dietary modification to control the condition. A patient was considered to have hypercholesterolemia if he or she had a serum cholesterol value >240 mg/dL (6.2 mmol/L) or was receiving cholesterol-lowering treatment. A patient was considered to have hypertension if he or she had received the diagnosis or was being treated with antihypertensive medications and/or dietary modification.

Immune Response to CMV Antigens

Blood samples from each individual were tested for (1) anti-CMV IgG antibodies and (2) the proliferation of T lymphocytes from peripheral blood mononuclear cells (PBMCs) in response to CMV antigens. 21

Antibody Status to CMV

Serum collected for detection of antibodies was frozen at −80°C. CMV IgG antibodies were determined by ELISA (Cytomegelisa II, Biowhittaker). Antibody results were calculated from standard curves provided by the manufacturer. A positive result was determined prospectively: an ELISA value <0.25 U was considered negative, and a value of ≥0.25 U was considered positive, indicating prior exposure to CMV. Samples were tested in triplicate and in 2 separate experiments.

Isolation of PBMCs

PBMCs were separated from whole blood on lymphocyte separation medium (Organon Teknika Corp) by centrifugation at 1800 rpm for 25 minutes at room temperature. Separated cells were collected and washed twice in PBS (Gibco Laboratories). The number of viable cells was determined by trypan blue exclusion. PBMCs were cryopreserved in aliquots in liquid nitrogen until used.

CMV Antigen Preparations

Human CMV, Towne strain, was obtained from the American Type Culture Collection and grown in human fibroblasts, HEL299 (ATCC CCL-137), for preparation of the viral antigens. Growth media consisted of MEM (Gibco) supplemented with 2% FBS and antibiotics. Virus titer was measured on HEL299 cells. Protocols for CMV antigen preparations have been published. 21 22 23 CMV antigens were prepared with (1) heat-inactivated CMV (1 hour at 56°C) obtained from supernatants of CMV-infected fibroblasts final viral concentration was 10 5 pfu before inactivation 22 (2) cell lysates of CMV-infected fibroblasts by repeated freezing and thawing 23 and (3) 0.08% glutaraldehyde-fixed CMV-infected fibroblast cells. 21 Both cell lysates and fixed cells were prepared from 2×10 6 cells/mL by infecting a 90% confluent monolayer of human fibroblasts with CMV at a multiplicity of infection of 10. Cells were collected by centrifugation at 50% cytopathic effect. Stocks were divided into aliquots and stored at −70°C. CMV antigen controls were obtained from noninfected fibroblasts (mock-infected cells), prepared exactly as described for CMV-infected cells.

T-Lymphocyte Proliferation

T-lymphocyte proliferative responses were performed in 96-well flat-bottom plates (Costar). PBMCs (100 μL 3×10 6 /mL) were added to each well. PBMCs were cultured at 37°C with 5% CO2 in RPMI 1640 (Gibco) containing 5% human AB serum, 2 mmol/L l -glutamine, 100 U/mL penicillin, 100 μg/mL streptomycin, and HEPES buffer, with or without antigen stimulation. Six days after culture, or 3 days for phytohaemagglutinin stimulation, each well was pulsed with 1 μCi of [ 3 H]thymidine and harvested 18 hours later, and thymidine incorporation was determined. Samples were assayed in triplicate and expressed as mean cpm. Data are presented as stimulation index (cpm of cultures in the presence of CMV antigens divided by cpm of cultures in the absence of antigens). If a sample responded to two thirds CMV antigen preparations (heat-inactivated supernatants of CMV-infected fibroblasts, CMV-infected cell lysates, or fixed CMV-infected fibroblasts) by a stimulation index >4, the response was considered positive. Positive controls included (1) 3 days of stimulation with phytohaemagglutinin (1:200 Gibco) (2) influenza A/Bangkok RX73 (flu grown in embryonated eggs and used as infectious allantoic fluid at an infectivity of 2×10 4 tissue culture infectious dose50/well at a final dilution of 1:1000) (3) Candida antigen (Greer Laboratories, Inc, final dilution of 20 mg/mL) and (4) a pool of irradiated (5000 rad) PBMCs from 3 unrelated healthy donors (2×10 6 /mL). Negative controls included (1) supernatants, cell lysates, and fixed cells from mock-infected fibroblasts prepared exactly as described for CMV-infected cells and (2) RPMI media control.

C-Reactive Protein

Serum CRP was measured by fluorescence polarization immunoassay technology (TDxFLEx analyzer, Abbott Laboratories). CRP levels of 95% and 98% of healthy individuals (n=202) were ≤0.5 mg/dL and ≤1.0 mg/dL, respectively. The between-run coefficients of variation (CVs) of this assay (n=31) were 4.3% and 2.2% at mean levels of 1.10 mg/dL and 2.94 mg/dL, respectively.

Statistical Analysis

Tests were 2-sided. Categorical data were analyzed by Fisher’s exact test. The dichotomous variable indicating presence or absence of CAD was modeled as a function of other factors or variables by multiple logistic regression. The odds ratio was used as a measure of risk of CAD in patients with a given risk factor compared with those without that factor. Covariates considered were age, sex, cigarette smoking, diabetes, hypercholesterolemia, hypertension, and seropositive CMV status. All covariates were individually examined as predictors of CRP and CAD by simple correlation analyses. They were further analyzed as a group for their predictive value for CAD by multiple logistic regression. Multiple linear regression was used to analyze their predictive value for CRP. These analyses were performed with SAS procedures (SAS software system for PC Windows). 24


Of the 238 subjects, 158 (66%) had CAD ranging from presence of plaque to significant stenoses. As previously found, 20 factors significantly associated with CAD were age, male sex, smoking, diabetes, hypercholesterolemia, elevated CRP (>0.5 mg/dL), and CMV seropositivity in univariate analysis. On multivariate analysis, after adjustment for these factors, age (OR, 2.3 P=0.0001), male sex (OR, 6.0 P=0.0005), and hypercholesterolemia (OR, 3.5 P=0.0007) were retained as significant risk factors, whereas diabetes was of borderline significance (OR, 3.0 P=0.0068). 20

Sex Differences in Associations Among CMV Infection, CRP Levels, and CAD

Of 151 men, 113 (75%) had CAD mean age was 57.2 years (median age, 57.0 years). Anti-CMV IgG antibodies were detected in 62% of CAD patients, compared with 61% of those without CAD (Figure 1 ). Mean CRP value was higher in men with CAD than in those without (0.86±0.05 versus 0.59±0.07 mg/dL, P=0.01 Figure 2 ). Of men with CAD, 76% had CRP levels >0.5 mg/dL, versus 50% of those without CAD (P=0.004). When adjusted for traditional CAD risk factors (age, smoking, diabetes, hypercholesterolemia, and hypertension) and CMV seropositivity by multivariate analysis, elevated CRP level was a significant independent predictor of CAD (odds ratio, 3.1 95% CI, 1.21 to 7.97 P=0.02). Although CMV seropositivity was not significantly associated with CAD in men, CMV seropositivity was associated with elevated CRP levels (P=0.03 on univariate analysis) and was an independent determinant of CRP on multivariate analysis after adjustment for CAD and CAD risk factors (β=0.27 95% CI, 0.08 to 0.46, P=0.006).


Of 87 women, 45 (52%) had CAD. Their mean age was 57.0 years (median age, 57.0 years). The frequency of traditional CAD risk factors (except smoking, P<0.05) was similar in men and women (Table 1 ). Of 45 women with CAD, 40 (89%) had anti-CMV IgG antibodies, whereas only 20 of 42 (48%) of those without CAD were CMV-seropositive (P=0.001 Figure 1 ). After adjustment for traditional CAD risk factors, the presence of anti-CMV antibodies was a highly significant predictor of CAD (odds ratio, 41.8 95% CI, 4.12 to 423.74 P=0.0016). In contrast to the findings in men, although mean CRP in women with CAD was >25% higher than CRP in women without CAD, the difference did not reach statistical significance (0.95±0.06 versus 0.75±0.05 mg/dL, P=0.1 Figure 2 ).

Influence of the Type of Immune Responses to CMV on CAD Prevalence

We next determined whether the type of immune response to CMV influenced the prevalence of CAD. Four types of immune response patterns were found. The most common was a humoral response (Ab+/Tc−) the least common was a cellular response (Ab−/Tc+). The types of immune responses to CMV infection generated by the total cohort and by the women and men are shown in Table 2 .

Most importantly, we found that the type of immune response to CMV influences CAD prevalence in women, whereas no relationship between the type of immune response and CAD prevalence was found in men (Figure 3 ). CAD prevalence was 5 times higher in Ab+/Tc− (P=0.0005) and in Ab+/Tc+ women (P=0.003) than in Ab−/Tc− women (those with no immunologic evidence of prior CMV infection). CAD prevalence in women with a cellular response (Ab−/Tc+) was not different from the Ab−/Tc− group but was significantly lower than that of the Ab+/Tc− group (P=0.016). There were no differences in age, smoking, diabetes, hypercholesterolemia, or hypertension among the women with different types of immune response to CMV infection (all P>0.1).


In a previous investigation of the same cohort, 20 we demonstrated that one mechanism by which CMV infection contributes to CAD appears to be through the inflammatory response it evokes, as reflected by elevated CRP levels. We also found that the inflammatory response to CMV infection varied considerably among individuals infected with CMV. In the present investigation, we further explored the variability of host responses to CMV infection and how these might influence susceptibility and resistance to CMV-associated CAD. We focused specifically on sex differences and differences in the type of immune responses: cellular or humoral.

The conclusion presented in our previous paper, that CMV is a risk factor for CAD only insofar as it contributes to an inflammatory response, derived from an analysis of the total cohort. 20 In the present study, however, we found a sex-based heterogeneity in mechanisms and found that the data suggesting a possible link between CMV and CAD via induction of an inflammatory response were driven by the data in men. Women, who constituted a minority of the study cohort (37%), displayed different results, which were masked when data from the population as a whole were assessed.

In the present study, an elevated CRP level was a significant determinant of CAD in men (adjusted odds ratio, 3.1 95% CI, 1.21 to 7.97 P=0.02). Whereas CMV infection was not associated with CAD, it was independently associated with elevated CRP levels (P=0.006). In contrast to the data in men, CMV infection in women was not significantly associated with elevated CRP levels. This finding is compatible with the first hypothesis examined in the present investigation, that the presence or absence of an inflammatory response to CMV is sex-related. Most interestingly, in women, CMV seropositivity was independently predictive of CAD, a relationship that was highly significant (odds ratio, 41.8 95% CI, 4.12 to 423.74 P=0.002). However, the association between elevated CRP levels and CAD was not as strong in women as it was in men. It therefore seems that men, more consistently than women, mount an inflammatory response to CMV infection and that this response appears to predispose to CAD (although the data do not prove a causal relationship between CMV and CAD via a CMV-induced inflammatory response). In women, conversely, the dominant mechanism relating CMV to CAD appears not to relate to inflammation, at least as assessed by CRP levels, but rather to act by some other, as yet undefined mechanisms.

In an attempt to obtain further insights as to how differences in pathogen-host interaction might contribute to host susceptibility versus resistance to CMV-related CAD, we examined our second hypothesis, which proposed that the type of immune response to CMV infection contributes to susceptibility or resistance to CAD. This was stimulated by our recent finding in a group of healthy blood donors that CMV infection evokes diverse immune responses (J.Z. et al, unpublished data, 1998). Some individuals had neither a humoral (antibody) nor T-cell response to CMV antigens (Ab−/Tc− subgroup) these individuals either were never exposed to CMV or were successful in clearing the virus and at the time of testing had no immunologic evidence of prior infection. Others, all of whom demonstrated immunologic evidence of prior infection, had either a humoral phenotype (Ab+/Tc− subgroup), a cellular response (Ab−/Tc+ subgroup), or a combined response (Ab+/Tc+ subgroup). A similar variety of immune responses was found in the present population (Table 2 ).

We found that although there was no influence of immunoresponse patterns on disease susceptibility in men, susceptibility to CMV-related CAD was limited to women with a humoral immune response to CMV infection. Thus, compared with the Ab−/Tc− women, CAD prevalence was higher in the Ab+/Tc− and in the Ab+/Tc+ women (13% versus 68% and 64%, both P<0.005) but not in Ab−/Tc+ women (25%). These differences could not be explained by subgroup-related differences in age, smoking, diabetes, hypercholesterolemia, and hypertension (all P>0.1).

These results indicate that multiple mechanisms exist whereby CMV infection and perhaps infection by other pathogens contribute to CAD. They also indicate that the relative contribution of these mechanisms to atherogenesis is sex-determined and is influenced by whether or not the host mounts an inflammatory response to CMV infection as well as by the nature of the immune response. The data suggest that CMV, at least in men, may contribute to CAD, insofar as it induces an inflammatory response (although it must be emphasized that insofar as an inflammatory response contributes to CAD, CMV can be considered only one possible factor). In women, however, CMV infection is an independent predictor of CAD risk and is not associated with elevated CRP levels.

There are at least 2 possible explanations, not mutually exclusive, to account for the findings in women that susceptibility to CMV-associated CAD occurs in the humoral response subgroups, whereas resistance is observed in the cellular responders. First, it is possible that a cellular response to CMV, an intracellular pathogen, conveys greater control of viral activity than a humoral response. This explanation implies that the cellular response is primary in determining outcome. If this were the sole explanation, however, it might have been expected that greater control of viral activity would be accompanied by lower CRP levels and that women with a combined humoral and cellular response would have a lower prevalence of CAD than women with a humoral response who lacked a cellular response. This was not observed.

The alternative explanation focuses on the humoral immune response as the major player. Thus, it is possible that the humoral response to CMV is a reflection of antibody-induced autoimmune disease. In this regard, there is now growing evidence that autoimmune responses may play a role in atherosclerosis. 25 26 Even more relevant to our concept that the antibody response to CMV infection may predispose to atherosclerosis through autoimmune mechanisms are the many examples of immunopathology triggered by the host’s immune response to viral infection. Perhaps the best-studied potential mechanism for infection-induced immunopathology is that of molecular mimicry, which is based on the invading pathogen having peptides highly homologous to host peptides. The immune response targeted to the infectious pathogen would, through molecular mimicry, effectively result in the development of autoantibodies or autoaggressive T cells to host peptides.

In addition to the information relating to potential mechanisms by which CMV and presumably other pathogens contribute to atherogenesis, our results also help to explain the conflicting epidemiological evidence relating to the possible role of infectious agents in atherosclerosis. Although some studies have found an association between CMV and atherosclerosis or restenosis based on analysis of CMV seropositivity, other studies have questioned such a relationship. This controversy may be due to the paucity of women in these studies in whom a direct association between CMV seropositivity and CAD is observed and to the failure to concomitantly analyze an index of inflammation, such as CRP elevations. Our study also demonstrates the importance of the type of immune response mounted by the host to the infectious agent in determining whether or not the infection predisposes to vascular disease.

Several caveats must be considered relating to our conclusions. First, the study design of this investigation is cross-sectional in nature. Such a design cannot establish causality. It can only establish an association. Hence, any conclusion derived from such a study must be considered preliminary and hypothesis-generating rather than hypothesis-proving. Second, as implied in our discussion about different populations responding differently to a specific infection, it is possible that our conclusions may be limited to the particular population of men and women we studied. Third, our non-CAD control group consisted of individuals who, on clinical evaluation, had some suggestion of CAD. These individuals may not be representative of other individuals without CAD who lack clinical features triggering the decision to perform coronary angiography. In addition, it is unclear why the level of CRP appears to be high in our control group. Fourth, our analysis on the type of immune response is based on single assessments of selected immunologic responses therefore, our proposed hypotheses will have to be evaluated further in the future with more frequent immunologic assessments in larger numbers of men and women. Finally, the number of women in each immune response category is small the results therefore need to be confirmed by larger studies. Nonetheless, we believe it likely that our conclusions will prove to be valid, considering that (1) our hypotheses were formulated prospectively, (2) the results are consistent with those demonstrating the important role of host response in determining susceptibility or resistance to various diseases induced by many other pathogens, and (3) our results help to explain otherwise disparate results in the literature relating to CMV and CAD.

In summary, the results of this investigation provide further evidence that CMV contributes to atherogenesis. The data suggest, however, that the dominant mechanisms by which CMV predisposes to CAD in men and women are different. In men, if CMV contributes to CAD, it would appear to do so insofar as it predisposes to inflammation, whereas in women, CMV is an independent risk factor for CAD. These observations raise the possibility of novel therapeutic strategies for the prevention or treatment of atherosclerosis. Thus, it might be possible to alter disease outcome favorably through the use of vaccines or cytokine-based strategies designed to change an immune response directed against a causally relevant pathogen from one that conveys disease susceptibility to one that enhances resistance. This concept would be especially attractive if similar associations were also demonstrated between CAD and infection with other pathogens, such as C pneumoniae and H pylori, and with the immune response to these pathogens.

Table 1. Patient Characteristics

Data (except age) are number (%) of patients. There were 158 patients with CAD and 80 without CAD in the total. Age, male sex, hypercholesterolemia, diabetes, and smoking, but not hypertension, were significant risk factors for CAD (all P<0.05). There were 113 men with CAD and 38 without CAD. Significant risk factors for CAD in men were similar to in the total. There were 45 women with CAD and 42 without CAD. Significant risk factors, except smoking and hypertension, for CAD in women were similar to in the total. The frequency of CAD risk factors (except smoking, P<0.05) was similar in men and women.

Table 2. Patterns of Humoral and Cellular Immune Responses to CMV Infection

Data presented are number (%) of patients. Ab indicates antibody Tc, T-cell proliferation −, negative and +, positive. P values between sexes were 0.014 in Ab−/Tc−, 0.54 in Ab+/Tc+, 0.62 in Ab+/Tc−, and 0.066 in Ab−/Tc+.

Figure 1. Prevalence of anti-CMV antibodies in men and in women with CAD (CAD+) and without CAD (CAD−).

Figure 2. Mean levels of CRP (mg/dL) in men and in women with CAD (CAD+) and without CAD (CAD−).

Figure 3. Prevalence of CAD in men and in women with different types of immune response to CMV infection. Ab+ and Ab− indicate antibody response positive and negative, respectively Tc+ and Tc−, T-lymphocyte proliferative response positive and negative, respectively.

We thank Rita Mincemoyer for her excellent clinical assistance and data acquisition, Bill Schenke for his help in the preparation of the figures, and Rene Costello for his excellent technical assistance.