Latent Toxoplasma gondii infections are associated with elevated biomarkers of inflammation and vascular injury

Toxoplasma gondii is a protozoan parasite that infects felines as definitive hosts and other warm-blooded animals as intermediate hosts, including humans as accidental or dead-end hosts [38]. Feces of infected cats contain T. gondii oocysts with infectious sporozoites that can remain viable in soil for years [16, 34]. Intermediate hosts including livestock can become infected through ingestion of oocyst contaminated soil. The parasite forms a life-long infection in the hosts persisting in bradyzoite-containing infectious tissue cysts in muscles, the central nervous system, and other tissues. The parasite completes its life cycle when cats ingest tissues of infected intermediate hosts. Humans become infected mainly through consumption of raw or undercooked meat or accidental ingestion of environmental oocysts [30].

Serum IgM response appears for a short period after initial infection. Serum IgG antibodies to this parasite usually peak in 2 to 3 months and then gradually decline to a lower but still detectable level characteristic of a chronic infection [46]. Therefore, serum IgM and IgG tests are typically used for detecting and differentiating acute and latent T. gondii infections [31, 32]. The latest available national US surveillance shows 13.2% IgG seroprevalence in individuals older than 5 years of age [31].

Clinical symptoms of new infections in humans include ocular disease, lymphadenitis, encephalitis, and myocarditis [24]. However, about three-quarters of new infections in healthy individuals are asymptomatic [58]. T. gondii infection during pregnancy is especially dangerous as the parasite can cause spontaneous abortion or severe neurological abnormalities in a newborn [30]. T. gondii infections in rodents and primates have been associated with behavioral modifications that make them more vulnerable to predation by felines [29, 42, 57]. Behavioral abnormalities in infected animals have been associated with chronic neuroinflammation [10, 27].

Latent T. gondii infections in humans have been associated with various adverse neuropsychiatric outcomes including suicide and increased risk of traffic accidents [50], schizophrenia and bipolar disorder [11, 13], obsessive compulsive disorder [39], and increased aggression and impulsivity [14]. Other studies linked latent infections with an increased risk of type 2 diabetes [36], rheumatoid arthritis [28] and Alzheimer’s disease [40].

Latent T. gondii infections have also been linked with immune activation and subtle neurophysiological changes [53, 55]. Previous research demonstrated associations between T. gondii infection and elevated serum levels of markers of inflammation, dyslipidemia, and cardiovascular events, specifically endothelial adhesion molecules, ICAM-1, VCAM-1, as well as pro-inflammatory cytokines [21, 23, 55].

Our previous epidemiological study in 206 adults in North Carolina demonstrated that T. gondii seropositivity was associated with an elevated allostatic load – a composite measure of physiological dysregulation comprised of 15 biomarkers of neuroendocrine, metabolic, immune and endothelial function including ICAM-1, VCAM-1, CRP and SAA [21]. While associations with many individual biomarkers were positive, only a few of these effects including increased levels of myeloperoxidase (the enzyme involved in immune response to the parasite), proinflammatory cytokine IL-6, and VCAM-1 were statistically significant.

The objective of the present study was to test associations of latent T. gondii infections with individual biomarkers of inflammation and vascular injury in a larger sample of adult individuals. The study involved analysis of four biomarkers that have been linked to T. gondii in previous in vivo or in vitro studies and that are known predictors of adverse health outcomes in humans: ICAM-1, VCAM-1, CRP and SAA. Adhesion molecules ICAM-1 and VCAM-1 are released into circulation in response to inflammation by vascular endothelial cells. They mediate leukocyte adherence to the vascular endothelium and transmigration. Previous research suggested that T. gondii exploits these natural cell trafficking pathways to cross cellular barriers including the blood-brain barrier [3, 25]. CRP and SAA are biomarkers of inflammation. Elevated levels of ICAM-1, VCAM-1, CRP and SAA have been associated with coronary artery disease, cancer and psychiatric disorders including schizophrenia [4, 9, 33, 41, 55, 60].

This cross-sectional epidemiological study demonstrated that T. gondii IgG seropositivity (a marker of latent infection) was associated with elevated serum levels of three vascular injury and inflammation biomarkers: VCAM-1, ICAM-1 and CRP (the latter two associations were only marginally significant). The study involved 694 adult individuals, 67 of whom were seropositive to T. gondii. Compared to our previous study in the same area [21], this new study had almost three-and-a-half times bigger sample size and four times as many seropositive individuals.

The observed 9.7% seroprevalence of T. gondii in this study of adults was comparable with the 13.2% national estimate of unadjusted seroprevalence in the 2009-2010 NHANES study which involved individuals of at least 5 years of age [31]. As previous studies showed a declining T. gondii seroprevalence in the US, it is likely that the current national seroprevalence rate is below the 2009-2010 estimate.

This study involved two subsets of participants recruited at EPA and NIEHS facilities located in the Raleigh-Durham-Chapel Hill, NC metropolitan area approximately 17 km apart. Participants were recruited using convenience sampling approaches. As a result, the study population was different from the source population due to the greater willingness of certain categories of people, such as women, to participate. The EPA recruitment site was located on the campus of University of North Carolina in Chapel Hill, a city of about 60,000 residents with a high proportion of university graduates. The NIEHS recruitment site was located closer to the city of Raleigh (the capital of North Carolina, population 250,000); it is likely that the NIEHS subset included more residents of Raleigh, but the residence data were not available for this analysis. The observed difference between T. gondii seropositivity rates in NIEHS and EPA subsets was largely explained by their sociodemographic compositions and life histories. Although the pooled study population was more representative of the diverse sociodemographic conditions in central North Carolina than each subset, the seroprevalence estimate in this study might not accurately represent T. gondii seroprevalence in the general population of this area.

In the present study, history of living on a farm was a predictor of T. gondii seropositivity. This finding is consistent with previously published results [44]. Other common risk factors for T. gondii infections such as eating undercooked meat, contacts with soil and owning a cat were detected in our previous analysis [21], but could not be confirmed in the present study given discrepancies in how questions in EPA and NIEHS questionnaires regarding cat ownership, eating raw or undercooked meat and handling soil were formulated. It should also be noted that data on consumption of lamb and game meat, which may be important sources of T. gondii infections, were not collected.

In this study, smoking and T. gondii seropositivity were positively associated. Previous analysis of National Health and Nutrition Examination Survey (NHANES) data in the US demonstrated that latent T. gondii infections were associated with a slightly reduced likelihood of self-reported tobacco usage as well as significantly reduced usage of heroin and methamphetamine [5]. The effects on tobacco usage, however, were inconsistent between different rounds of NHANES survey and across income and education categories with T. gondii being a risk factor for tobacco usage in subgroups with high income and high education. Further research is needed to elucidate potential relationships between T. gondii and smoking.

The effects of latent T. gondii infections on vascular injury and inflammation biomarkers observed in this study reflect underlying pathophysiological processes, which may be triggered by a periodic rupture of the tissue cysts and release of bradyzoites that happens spontaneously in intermediate hosts [20]. In immunocompetent individuals, this triggers immune system activation that clears the released bradyzoites. Adhesion molecules VCAM-1 and ICAM-1 are released into circulation by vascular endothelial cells in response to inflammation. They mediate leukocyte adherence to the vascular endothelium and transmigration. In vitro experiments also demonstrated that expressions of ICAM-1 and VCAM-1 are upregulated in T. gondii infected bovine endothelial cells [52]. The parasite has been shown to use ICAM-1 to cross the blood-brain barrier and other endothelial barriers during the acute phase of infection [3, 25]. The role of ICAM-1 during the latent infection stage remains to be characterized. VCAM-1 is essential for controlling T. gondii infection [17, 47] and infections are associated with elevated serum levels of VCAM-1 in rodents [55]. Chronically elevated levels of these adhesion molecules are linked with the development of atherosclerosis [9, 35, 60]. Previous research has also associated elevated levels of VCAM-1 and ICAM-1 with schizophrenia [41]. CRP is a marker of acute inflammation which is elevated in T. gondii-infected animals [55]. Chronically elevated CRP is also associated with cardiovascular disease [33, 60], major depressive disorder [26], generalized anxiety disorder [15], and schizophrenia [37], as well as greater severity of symptoms in patients with schizophrenia [12].

Previous epidemiological studies have demonstrated associations between T. gondii seropositivity and ICAM-1 [23], VCAM-1 [21] and CRP [8, 51], consistent with our new findings. The positive association between latent T. gondii infection and serum levels of VCAM-1 in humans was demonstrated for the first time in our previous publication [21] and confirmed in the present analysis.

Our analysis of data on the same set of individuals [49] also demonstrated that CMV IgG seropositivity or the intensity of anti-CMV IgG responses were associated with elevated levels of the same vascular injury biomarkers, VCAM-1, ICAM-1 and CRP. The present study showed that the effects of T. gondii on these biomarkers were largely independent from the effects of CMV. Adjusting for CMV serostatus did not have substantial impacts on effects estimates for T. gondii seropositivity (Table 5, models 2 and 3): effect estimates for VCAM-1, ICAM-1, and CRP were reduced from 11.4 to 11.2% (2% reduction of the effect size), from 8.6 to 8.2% (5% reduction), and from 35.8 to 34.2% (4% reduction), respectively. It should be noted, however, that the latter adjusted effects were only marginally significant.

Both CMV and T. gondii infections have been linked with psychiatric disorders suggesting potential overlapping biological pathways to detrimental health effects [11]. Chronic inflammation plays an important role in pathophysiology of psychiatric disorders but specific cause-effect mechanisms underlying this association remain to be elucidated [45]. Previous research demonstrated that T. gondii infection causes neural damage and reactive tissue repair in mice similar to those observed in the brain of schizophrenia patients, and that both T. gondii infections in mice and schizophrenia are associated with elevated levels of CRP and VCAM-1 [55]. While there is accumulating evidence of immune response activation sustaining inflammation in T. gondii-infected individuals as a pathway to behavioral changes, neurological deficit and psychiatric disorders, specific molecular mechanisms of these effects remain to be characterized [56].

Furthermore, previous studies also demonstrated that exposure to common air pollutants is associated with increased serum levels of CRP, ICAM-1 and VCAM-1 in humans [6, 7], suggesting that T. gondii-infected individuals may be more susceptible to the effects of air pollution. Schizophrenia patients have elevated levels of these biomarkers [41], and exposure to common ambient air pollutants is associated with increased hospital admissions for schizophrenia and other psychiatric disorders [2, 19, 43] as well as atherosclerosis, thrombosis, and increased mortality in chronically exposed individuals [1, 54]. Further investigations of the interaction effects of T. gondii infections and exposure to environmental hazards on the risk of neuropsychiatric and systemic diseases are warranted.

This cross-sectional observational study could only demonstrate statistical associations; it was not designed to establish a cause-effect relationship. Some of the observed associations, especially those that were marginally significant, could be due to random effects. The observed associations need to be further validated in different populations and countries, particularly in South America and other areas where more virulent strains are circulating [48]. Another important limitation of this study is that only a limited set of sociodemographic and socioeconomic variables was available for statistical analysis in the combined dataset due to inconsistently formulated questions in EPA and NIEHS survey forms. Most importantly, individual and household level education and income data were not available in this study. As residential addresses were not available for the NIEHS subset, we could not use area level socioeconomic indicators as a proxy for individual socioeconomic status. However, our previous analysis did not find an association between education level and T. gondii seropositivity [21] suggesting that socioeconomic status was unlikely to confound the observed associations between seropositivity and biomarkers of inflammation and vascular injury in this study population. Due to the small sample size, this analysis was limited to contrasting biomarker levels in seropositive and seronegative individuals. A bigger study with a greater number of seropositive individuals would be needed to explore potential associations between the intensity of anti-T. gondii IgG responses in infected individuals and levels of biomarkers of inflammation and vascular injury.

In this study T. gondii IgG seropositivity was significantly associated with an increased level of soluble VCAM-1 in serum and marginally significantly with two more serum biomarkers of vascular injury and inflammation, ICAM-1 and CRP. Exposure to air pollution is known to affect the same biomarkers leading to various adverse health outcomes. Further research is needed to assess potential interaction effects of latent T. gondii infections and exposure to environmental hazards on morbidity and mortality.