Kristin L. Gressitt

Gastrointestinal inflammation and associated immune activation in schizophrenia

Immune factors are implicated in normal brain development and in brain disorder pathogenesis. Pathogen infection and food antigen penetration across gastrointestinal barriers are means by which environmental factors might affect immune-related neurodevelopment. Here, we test if gastrointestinal inflammation is associated with schizophrenia and therefore, might contribute to bloodstream entry of potentially neurotropic milk and gluten exorphins and/or immune activation by food antigens. IgG antibodies to Saccharomyces cerevisiae (ASCA, a marker of intestinal inflammation), bovine milk casein, wheat-derived gluten, and 6 infectious agents were assayed. Cohort 1 included 193 with non-recent onset schizophrenia, 67 with recent onset schizophrenia and 207 non-psychiatric controls. Cohort 2 included 103 with first episode schizophrenia, 40 of whom were antipsychotic-naïve. ASCA markers were significantly elevated and correlated with food antigen antibodies in recent onset and non-recent onset schizophrenia compared to controls (p≤0.00001-0.004) and in unmedicated individuals with first episode schizophrenia compared to those receiving antipsychotics (p≤0.05-0.01). Elevated ASCA levels were especially evident in non-recent onset females (p≤0.009), recent onset males (p≤0.01) and in antipsychotic-naïve males (p≤0.03). Anti-food antigen antibodies were correlated to antibodies against Toxoplasma gondii, an intestinally-infectious pathogen, particularly in males with recent onset schizophrenia (p≤0.002). In conclusion, gastrointestinal inflammation is a relevant pathology in schizophrenia, appears to occur in the absence of but may be modified by antipsychotics, and may link food antigen sensitivity and microbial infection as sources of immune activation in mental illness.

Discordant patterns of bacterial translocation markers and implications for innate immune imbalances in schizophrenia

The origin of inflammation in psychiatric disorders is not well understood. The translocation of commensal microbiota across the gastrointestinal barrier can result in a persistent state of low-grade immune activation and/or inflammation. We measured serological surrogate markers of bacterial translocation (soluble CD14 (sCD14) and lipopolysaccharide binding protein (LBP)) in two psychiatric cohorts and compared these levels to C-reactive protein (CRP), body mass index (BMI), and food-related and autoimmune antibodies. The two cohorts were composed of the following: (1) n=141 schizophrenia, n=75 bipolar disorder, n=78 controls; (2) n=78 antipsychotic-naïve first-episode schizophrenia, n=38 medicated first-episode schizophrenia. sCD14 seropositivity conferred a 3.1-fold increased odds of association with schizophrenia (multivariate regressions, OR=3.09, p<0.0001) compared to controls. Case-control differences in sCD14 were not matched by LBP. Quantitative levels of LBP, but not sCD14, correlated with BMI in schizophrenia (R(2)=0.21, p<0.0001). sCD14 and LBP also exhibited some congruency in schizophrenia with both significantly correlated with CRP (R(2)=0.26-0.27, p<0.0001) and elevated in females compared to males (p<0.01). Antipsychotic treatment generally did not impact sCD14 or LBP levels except for significant correlations, especially sCD14, with gluten antibodies in antipsychotic-naïve schizophrenia (R(2)=0.27, p<0.0001). In bipolar disorder, sCD14 levels were significantly correlated with anti-tissue transglutaminase IgG (R(2)=0.37, p<0.001). In conclusion, these bacterial translocation markers produced discordant and complex patterns of activity, a finding that may reflect an imbalanced, activated innate immune state. Whereas both markers may upregulate following systemic exposure to Gram-negative bacteria, non-lipopolysaccharide-based monocyte activation, autoimmunity and metabolic dysfunction may also contribute to the observed marker profiles.

Chlorovirus ATCV-1 is part of the human oropharyngeal virome and is associated with changes in cognitive functions in humans and mice

Significance Human mucosal surfaces contain a wide range of microorganisms. The biological effects of these organisms are largely unknown. Large-scale metagenomic sequencing is emerging as a method to identify novel microbes. Unexpectedly, we identified DNA sequences homologous to virus ATCV-1, an algal virus not previously known to infect humans, in oropharyngeal samples obtained from healthy adults. The presence of ATCV-1 was associated with a modest but measurable decrease in cognitive functioning. A relationship between ATCV-1 and cognitive functioning was confirmed in a mouse model, which also indicated that exposure to ATCV-1 resulted in changes in gene expression within the brain. Our study indicates that viruses in the environment not thought to infect humans can have biological effects. Chloroviruses (family Phycodnaviridae) are large DNA viruses known to infect certain eukaryotic green algae and have not been previously shown to infect humans or to be part of the human virome. We unexpectedly found sequences homologous to the chlorovirus Acanthocystis turfacea chlorella virus 1 (ATCV-1) in a metagenomic analysis of DNA extracted from human oropharyngeal samples. These samples were obtained by throat swabs of adults without a psychiatric disorder or serious physical illness who were participating in a study that included measures of cognitive functioning. The presence of ATCV-1 DNA was confirmed by quantitative PCR with ATCV-1 DNA being documented in oropharyngeal samples obtained from 40 (43.5%) of 92 individuals. The presence of ATCV-1 DNA was not associated with demographic variables but was associated with a modest but statistically significant decrease in the performance on cognitive assessments of visual processing and visual motor speed. We further explored the effects of ATCV-1 in a mouse model. The inoculation of ATCV-1 into the intestinal tract of 9–11-wk-old mice resulted in a subsequent decrease in performance in several cognitive domains, including ones involving recognition memory and sensory-motor gating. ATCV-1 exposure in mice also resulted in the altered expression of genes within the hippocampus. These genes comprised pathways related to synaptic plasticity, learning, memory formation, and the immune response to viral exposure.

Inflammatory Molecular Signature Associated With Infectious Agents in Psychosis

Schizophrenia (SZ) is a devastating mental condition with onset in young adulthood. The identification of molecular biomarkers that reflect illness pathology is crucial. Recent evidence suggested immune and inflammatory cascades in conjunction with infection may play a role in the pathology. To address this question, we investigated molecular changes in cerebrospinal fluid (CSF) from antipsychotic-naïve patients with SZ and at risk mental status for psychosis (ARMS), in comparison with healthy controls (HCs). We measured 90 analytes using a broad multiplex platform focusing on immune and inflammatory cascades then selected 35 with our quality reporting criteria for further analysis. We also examined Toxoplasma gondii (TG) and herpes simplex virus 1 antibody levels in CSF. We report that expression of 15 molecules was significantly altered in the patient groups (SZ and ARMS) compared with HCs. The majority of these molecular changes (alpha-2-macroglobulin [α2M], fibrinogen, interleukin-6 receptor [IL-6R], stem cell factor [SCF], transforming growth factor alpha [TGFα], tumor necrosis factor receptor 2 [TNFR2], IL-8, monocyte chemotactic protein 2 [MCP-2/CCL8], testosterone [for males], angiotensin converting enzyme [ACE], and epidermal growth factor receptor) were consistent between SZ and ARMS patients, suggesting these may represent trait changes associated with psychotic conditions in general. Interestingly, many of these analytes (α2M, fibrinogen, IL-6R, SCF, TGFα, TNFR2, IL-8, MCP-2/CCL8, and testosterone [for males]) were exacerbated in subjects with ARMS compared with subjects with SZ. Although further studies are needed, we optimistically propose that these molecules may be good candidates for predictive markers for psychosis from an early stage. Lastly, reduction of IL-6R, TGFα, and ACE was correlated with positivity of TG antibody in the CSF, suggesting possible involvement of TG infection in the pathology.

Anti-Gluten Immune Response following Toxoplasma gondii Infection in Mice

Gluten sensitivity may affect disease pathogenesis in a subset of individuals who have schizophrenia, bipolar disorder or autism. Exposure to Toxoplasma gondii is a known risk factor for the development of schizophrenia, presumably through a direct pathological effect of the parasite on brain and behavior. A co-association of antibodies to wheat gluten and to T. gondii in individuals with schizophrenia was recently uncovered, suggesting a coordinated gastrointestinal means by which T. gondii and dietary gluten might generate an immune response. Here, we evaluated the connection between these infectious- and food-based antigens in mouse models. BALB/c mice receiving a standard wheat-based rodent chow were infected with T. gondii via intraperitoneal, peroral and prenatal exposure methods. Significant increases in the levels of anti-gluten IgG were documented in all infected mice and in offspring from chronically infected dams compared to uninfected controls (repetitive measures ANOVAs, two-tailed t-tests, all p≤0.00001). Activation of the complement system accompanied this immune response (p≤0.002–0.00001). Perorally-infected females showed higher levels of anti-gluten IgG than males (p≤0.009) indicating that T. gondii-generated gastrointestinal infection led to a significant anti-gluten immune response in a sex-dependent manner. These findings support a gastrointestinal basis by which two risk factors for schizophrenia, T. gondii infection and sensitivity to dietary gluten, might be connected to produce the immune activation that is becoming an increasingly recognized pathology of psychiatric disorders.

Seroreactive marker for inflammatory bowel disease and associations with antibodies to dietary proteins in bipolar disorder

Immune sensitivity to wheat glutens and bovine milk caseins may affect a subset of individuals with bipolar disorder. Digested byproducts of these foods are exorphins that have the potential to impact brain physiology through action at opioid receptors. Inflammation in the gastrointestinal (GI) tract might accelerate exposure of food antigens to systemic circulation and help explain elevated gluten and casein antibody levels in individuals with bipolar disorder.

Complement C1q formation of immune complexes with milk caseins and wheat glutens in schizophrenia

Immune system factors including complement pathway activation are increasingly linked to the etiology and pathophysiology of schizophrenia. Complement protein, C1q, binds to and helps to clear immune complexes composed of immunoglobulins coupled to antigens. The antigenic stimuli for C1q activation in schizophrenia are not known. Food sensitivities characterized by elevated IgG antibodies to bovine milk caseins and wheat glutens have been reported in individuals with schizophrenia. Here, we examined the extent to which these food products might comprise the antigen component of complement C1q immune complexes in individuals with recent onset schizophrenia (n=38), non-recent onset schizophrenia (n=61) and non-psychiatric controls (n=63). C1q seropositivity was significantly associated with both schizophrenia groups (recent onset, odds ratio (OR)=8.02, p≤0.008; non-recent onset, OR=3.15, p≤0.03) compared to controls (logistic regression models corrected for age, sex, race and smoking status). Casein- and/or gluten-IgG binding to C1q was significantly elevated in the non-recent onset group compared to controls (OR=4.36, p≤0.01). Significant amounts of C1q-casein/gluten-related immune complexes and C1q correlations with a marker for gastrointestinal inflammation in non-recent onset schizophrenia suggests a heightened rate of food antigens in the systemic circulation, perhaps via a disease-associated altered intestinal permeability. In individuals who are in the early stages of disease onset, C1q activation may reflect the formation of immune complexes with non-casein- or non-gluten-related antigens, the presence of C1q autoantibodies, and/or a dissociated state of immune complex components. In conclusion, complement activation may be a useful biomarker to diagnose schizophrenia early during the course of the disease. Future prospective studies should evaluate the impacts of casein- and gluten-free diets on C1q activation in schizophrenia.

Metagenomic Sequencing Indicates That the Oropharyngeal Phageome of Individuals With Schizophrenia Differs From That of Controls

Mucosal sites such as the oropharynx contain a wide range of microorganisms, collectively designated as the microbiome. The microbiome can affect behavior through a number of neurobiological and immunological mechanisms. Most previous studies have focused on the bacterial components of the microbiome. However, the microbiome also includes viruses such as bacteriophages, which are viruses that infect bacteria and alter their metabolism and replication. We employed metagenomic analysis to characterize bacteriophage genomes in the oral pharynx of 41 individuals with schizophrenia and 33 control individuals without a psychiatric disorder. This analysis was performed by the generation of more than 100,000,000 sequence reads from each sample and the mapping of these reads to databases. We identified 79 distinct bacteriophage sequences in the oropharyngeal samples. Of these, one bacteriophage genome, Lactobacillus phage phiadh, was found to be significantly different in individuals with schizophrenia (P < .00037, q < 0.03 adjusted for multiple comparisons). The differential levels of Lactobacillus phage phiadh remained significant when controlling for age, gender, race, socioeconomic status, or cigarette smoking (P < .006). Within the group of individuals with schizophrenia, the level of Lactobacillus phage phiadh correlated with the prevalence of immunological disorders as well as with the administration of valproate, which has been shown in animal models to alter the microbiome. The bacteriophage composition of the oropharynx in individuals with schizophrenia differs from that of controls. The biological consequences of this difference and the potential effects of altering bacteriophage levels through therapeutic interventions are worthy of further investigation.

Candida albicans exposures, sex specificity and cognitive deficits in schizophrenia and bipolar disorder

Immune aberrations in schizophrenia and bipolar disorder have led to the hypotheses that infectious agents or corresponding immune responses might contribute to psychiatric etiopathogeneses. We investigated case–control differences in exposure to the opportunistic fungal pathogen, Candida albicans, and examined associations with cognition, medication, lifestyle, and somatic conditions. We quantified C. albicans IgG antibodies in two cohorts totaling 947 individuals and evaluated odds ratios (OR) of exposure with psychiatric disorder using multivariate regressions. The case–control cohort included 261 with schizophrenia, 270 with bipolar disorder, and 277 non-psychiatric controls; the second included 139 with first-episode schizophrenia, 78 of whom were antipsychotic naive. No differences in C. albicans exposures were found until diagnostic groups were stratified by sex. In males, C. albicans seropositivity conferred increased odds for a schizophrenia diagnosis (OR 2.04–9.53, P⩽0.0001). In females, C. albicans seropositivity conferred increased odds for lower cognitive scores on Repeatable Battery for the Assessment of Neuropsychological Status (RBANS) in schizophrenia (OR 1.12, P⩽0.004), with significant decreases on memory modules for both disorders (P⩽0.0007–0.03). C. albicans IgG levels were not impacted by antipsychotic medications. Gastrointestinal (GI) disturbances were associated with elevated C. albicans in males with schizophrenia and females with bipolar disorder (P⩽0.009–0.02). C. albicans exposure was associated with homelessness in bipolar males (P⩽0.0015). In conclusion, sex-specific C. albicans immune responses were evident in psychiatric disorder subsets. Inquiry regarding C. albicans infection or symptoms may expedite amelioration of this treatable comorbid condition. Yeast exposure as a risk factor for schizophrenia and its associated cognitive and GI effects require further investigation including the possible contribution of gut–brain mechanisms.

Maternal complement C1q and increased odds for psychosis in adult offspring

The presence of maternal antibodies to food and infectious antigens may confer an increased risk of developing schizophrenia and psychosis in adult offspring. Complement factor C1q is an immune molecule with multiple functions including clearance of antigen-antibody complexes from circulation and mediation of synaptic pruning during fetal brain development. To determine if maternal C1q was associated with offspring schizophrenia and psychosis, we evaluated 55 matched case-control maternal serum pairs from the National Collaborative Perinatal Project. Sample pairs were composed of mothers whose offspring developed psychoses as adults and those whose offspring were free from psychiatric disease. Matching criteria for offspring included birth date, delivery hospital, race, and gender, with further matching based on mothers age. IgG markers of C1q, bovine milk casein, egg ovalbumin, and wheat gluten were measured with enzyme-linked immunosorbent assays. C1q levels were compared to food antigen IgG and to previously generated data for C-reactive protein, adenovirus, herpes simplex viruses, influenza viruses, measles virus, and Toxoplasma gondii. C1q was significantly elevated in case mothers with odds ratios of 2.66-6.31 (conditional logistic regressions, p ≤ 0.008-0.05). In case mothers only, C1q was significantly correlated with antibodies to both food and infectious antigens: gluten (R(2)=0.26, p ≤ 0.004), herpes simplex virus type 2 (R(2)=0.21, p ≤ 0.02), and adenovirus (R(2)=0.25, p ≤ 0.006). In conclusion, exposure to maternal C1q activity during pregnancy may be a risk factor for the development of schizophrenia and psychosis in offspring. Prenatal measurement of maternal C1q may be an important and convergent screening tool to identify potentially deleterious immune activation from multiple sources.