By measuring molecules enriched in brain endothelial cells involved in both the physical and functional properties of the BBB, we provide evidence supporting an altered BBB in people with schizophrenia. We find the greatest diagnostic change in ICAM1 mRNA, which we confirmed to be localised to the lumen of human brain blood vessels and to be upregulated by cytokines but not antipsychotics in human brain endothelial cells in culture. We have also identified exaggerated transcriptional changes in ABCG2, IFITM, ICAM1, OCLN, CDH5, CD163, CD14, and CD16 mRNAs in the “high inflammatory” subgroup of schizophrenia, demonstrating that some people with schizophrenia have greater molecular changes in the brain endothelial and white blood cell markers compared to others with schizophrenia.

While we focus on the inflammatory effects of cytokines related to the BBB, cytokines can have other neuromodulatory roles. Consistent with this, we have previously published that this “high inflammation” schizophrenia subgroup has greater neuropathology in the DLPFC, such as decreased brain derived neurotrophic factor mRNA, decreased interneuron markers, and evidence for astrogliosis, compared to the “low inflammation” schizophrenia subgroup [14, 41]. Inflammatory clustering was also conducted in the OFC of this cohort with similar overlap, showing neuroinflammation is not necessarily limited to the DLPFC and it is also associated with decreased cortical grey matter and superior frontal gyrus volume [15]. Living people with schizophrenia classified in the “high inflammation” biotype defined using blood-based inflammatory markers have reduced verbal fluency and reduced Broca’s area volume [13]. The mechanisms by which these established inflammatory genes contribute to these pathologies or vice versa remain unclear. A recent clinical trial using an IL-6 receptor antibody failed to demonstrate attenuated schizophrenia symptoms, potentially due to the lack of adequate antibody penetration into the brain [43]. However, there are many other reasons why this relatively small study was not able to demonstrate a beneficial effect, including low statistical power, dosing and time course issues, and the lack of stratifying patients based on inflammatory status prior to administering treatment, which may be necessary to achieve maximum benefit. Assessing neuroinflammation status may be a critical way to stratify people with schizophrenia who display a distinct or exaggerated neuropathology.

One of the most robust changes in this study was an increase in ICAM1 mRNA in schizophrenia compared to controls. Further, the “high inflammation” schizophrenia subgroup had an even greater increase in ICAM1 mRNA consistent with our own and other experimental results showing that IL-1β up-regulates ICAM1 mRNA in cultured endothelial cells [44, 45]. ICAM1 has a role in the attachment of white blood cells to the luminal wall of blood vessels. We confirmed that brain endothelial cells are a cellular source of ICAM1 in the normal adult human brain and in the brains of people with schizophrenia. Infrequent failure to observe ICAM1 signal in the human cortex may be due to low sensitivity of the immunohistochemistry assay and because ICAM1 is normally expressed at low levels and is actively cleaved. ICAM1 also co-localises with astrocytes, an observation more frequent in brains from older individuals. Previous studies found increased extravascular ICAM1 with aging [46, 47], and that GFAP mRNA and protein also increase with age [41, 48, 49]. While ICAM1 can also be expressed in microglia, we did not find ICAM1 to be localised to microglial cells based on morphology. Further, there was no correlation between ICAM1 and IBA1 mRNA. IBA1 mRNA was not changed in our cohort and postmortem studies quantifying microglia density using IBA1 in schizophrenia find no differences [50, 51], though increased microglia density have been observed when using other markers [14]. Our results suggest that beyond endothelial cells, astrocytes and not microglia are the main cellular source of ICAM1 in brain tissue.

Conflicting findings exist as to whether or not peripheral sICAM1 levels are increased, decreased or not changed in schizophrenia relative to controls. Our finding of elevated plasma sICAM1 in chronically ill people with schizophrenia supports studies showing elevated peripheral sICAM1 levels in patients receiving antipsychotic medication [31], and patients with schizophrenia who have a putative disruption of the BBB [28]. While most previous studies have investigated serum, a study in plasma also finds elevated sICAM1 in chronically ill schizophrenia patients [32], consistent with our measures in plasma from chronically ill patients. However, others have reported decreased serum sICAM1 levels in medicated and un-medicated patients with schizophrenia compared to controls [28, 29]. There is also evidence that sICAM1 may vary according to clinical features; for example, lower sICAM1 is linked to better treatment outcomes [30] supporting the hypothesis that higher sICAM1 may be deleterious to brain function.

Reduced sICAM1 was also found in schizophrenia patients after switching to second generation antipsychotics [52]. In our study, we found that peripheral sICAM levels are positively correlated with antipsychotic dose levels at a borderline level of significance. Thus, a higher dose of antipsychotics may elicit an increase in sICAM1, or conversely, elevated sICAM1 (putatively indicative of greater leucocyte extravasation into brain tissue) may require increased doses of antipsychotics. Our experimental results in cultured cells derived from human brain endothelium suggest that antipsychotic drugs may not directly increase ICAM1 expression, but further experiments are needed to determine how they may influence the production and cleavage of the soluble form of ICAM1 in these cells. In summary, the results from studies of sICAM1 in schizophrenia are divergent and may reflect a number of factors. The influence of antipsychotic treatment is unclear and a more systematic approach to research on this topic is required. Our findings support that schizophrenia may have an underlying inflammatory component, but not everyone with schizophrenia is in a high inflammatory state or has clear changes in their brain vasculature or in circulating inflammatory factors like sICAM1.

Membrane bound ICAM1 interacts with lymphocyte function-associated antigen-1 (LFA1) and macrophage associated antigen-1 (MAC1) receptors expressed on leucocytes to promote immune cell infiltration during tissue inflammation [53]. Elevated sICAM1 in the plasma of people with schizophrenia may reflect enhanced cleavage of membrane bound ICAM1 on the endothelium following recruitment of immune cells into the tissue. sICAM1 also interacts with LFA1 and MAC1, thereby interfering with the ability of leucocytes to bind to membrane bound ICAM1. As such, sICAM1 may contribute to the increased circulation of activated monocytes reported in schizophrenia [54, 55] and promote release of pro-inflammatory cytokines from immune cells, further contributing to the inflammatory state.

CD3+ T lymphocytes and CD20+ B lymphocytes have been observed in the hippocampus in a small number of schizophrenia brains [33] and in a broader investigation in the hippocampus, frontal cortex, thalamus, medial temporal lobe and cingulate gyrus in schizophrenia [34]. Inflammation related genes including S100A8, S100A9 and CHI3L1 are elevated in the hippocampus in schizophrenia and perivascular macrophages (CD163+) have been identified in the lumen of blood vessels and surrounding the endothelial cells in at least one schizophrenia hippocampus [21].

In our study, transcript levels of CD16, a marker of natural killer cells and activated macrophages/monocytes, CD163 a marker of perivascular macrophages and CD14, a marker of monocytes were all elevated in schizophrenia cases with high inflammation. CD163+ macrophages were detected in the brain vasculature in all cases irrespective of diagnosis and inflammation. Our study now confirms that monocytes can also be found in the prefrontal cortex of individuals with schizophrenia and for the first time, identifies brain tissue macrophages proximal to neurons in over 40% of individuals with schizophrenia who are in a high inflammatory state.

Our anatomical scope of analysis is restricted as we used 14 µm tissue sections and sampled only a small part (rostro-caudally) of the gyrus rectus microscopically and thus, we cannot comment on the total number of CD163+ cells in the brains of people with schizophrenia. Determining the actual number of cells would require counting using stereological principles, having an entire known volume of the brain area of interest available and systematic sampling of multiple tissue sections. This would require resources (both time and space) that are not currently tractable with the limited amount of human brain tissue typically available for study. We found CD163+ macrophages in the brain parenchyma away from any obvious blood vessels, suggesting that they are capable of infiltrating brain tissue and interacting directly with neurons. Breakdown of the BBB is not a prerequisite for leucocyte infiltration and bone marrow-derived monocytes can access the (rodent) brain without changes to BBB permeability [56]. Therefore, our findings do not necessarily suggest a breakdown or leakiness of the BBB, but rather more potential for circulating immune cells to adhere to the blood vessel endothelium. The presence of immune cells in brain tissue can produce inflammatory factors to further drive the inflammatory cascade by signalling to microglia, astrocytes and back to the endothelium.

Tight junction proteins such as CDH5 and OCLN that contribute to BBB integrity were found to be elevated in relation to inflammation in postmortem tissue in schizophrenia. While this may suggest a tightening of the BBB, it could also be a compensatory response to prevent excessive entry of leucocytes into the perivascular space under inflammatory conditions, or greater utilisation and turnover of these molecules leading to the need for higher synthesis of tight junction proteins in inflammatory states. In addition to cytokine measures, C-reactive protein is a peripheral inflammation marker that could be used to determine inflammatory status and it may be informative for future studies to collect blood for peripheral measures of inflammation along with the brain at time of death. As BBB integrity can be indirectly measured in vivo using novel MRI technology [57], we suggest that MRI could be used with blood cytokine and C-reactive protein measures to determine whether changes in BBB integrity or function and elevated inflammatory status co-exist in living individuals with schizophrenia.

A limitation of our study is that all individuals with schizophrenia were prescribed antipsychotics and were chronically ill. Chlorpromazine equivalent dose positively correlated with ICAM1, IFITM and CDH5 mRNA levels in the postmortem sample, and there was a weak, positive correlation of mean daily chlorpromazine equivalent dose with sICAM1 at a trend level in the living sample. However, exposing cultured endothelial cells to three different antipsychotics did not alter the expression of any genes of interest. Furthermore, all patients were receiving antipsychotics and several mRNAs including ICAM1, IFITM and CDH5 differed significantly between the two inflammatory patient groups. We suggest that antipsychotics alone may not be a primary factor in regulating gene expression of brain endothelial cell transcripts.

Another limitation of our study is the use of tissue homogenates for mRNA experiments, which does not provide cell-specific data. Consulting the Human Protein Atlas indicates that the expressions of brain endothelial markers of interest are predominately found in endothelial cells of the human brain [58]. A previous study using laser microdissection to examine the brain microvasculature finds a down-regulation in inflammation related genes in schizophrenia compared to controls [17] and is in apparent contradiction to our study utilising brain homogenates. However, the status of cytokine or inflammatory mRNAs within the blood endothelial cells was not provided in this previous study and it may be that cytokines are also or alternatively elevated in neuronal or glial cells.

The stress from a lifetime of mental illness may contribute to inflammation in the brain in our chronically ill cohort. While peripheral cortisol levels can be elevated in schizophrenia [59], glucocorticoid receptor mRNA levels are also dysregulated in the brains of people with schizophrenia [60, 61]. When using changes in cortical glucocorticoid and immune pathways to stratify subgroups, we find an overlap in the population of people with schizophrenia in an elevated inflammatory state and in the sample of people with schizophrenia in an elevated stress state [10]. Further research is required to disentangle the reciprocal role of inflammation and stress in the neuropathology of schizophrenia.

In contrast to previous studies [4, 16, 21, 62], we did not detect any diagnostic differences in IFITM mRNA expression in people with schizophrenia compared to controls. This is possibly because our pan-probe measured IFITM1, IFITM2, and IFITM3 transcripts. In contrast, using qPCR Siegel et al [62]. found an increase in IFITM1 expression and with a pan-probe in IFITM2 and IFITM3 expression in schizophrenia. This group also found that IFITM2 mRNA is predominantly expressed in the brain vasculature by in situ hybridisation. IFITM expression can be induced by inflammatory cytokines, IL-1β [63] and IL-6 [64], the same cytokines used to define inflammatory status in our cohort [14]. In support of these findings, we found a significant increase in IFITM expression in the “high inflammation” schizophrenia subgroup compared to the “low inflammation” schizophrenia subgroup.

We are the first to report changes in efflux transporters mRNAs in the brains of people with schizophrenia compared to controls, with greatest decreases in cases with “high inflammation”. The exacerbated decrease in ABCG2 in the “high inflammation” schizophrenia subgroup is consistent with evidence showing that treatment with IL-1β, IL-6 and TNFα reduces both mRNA and protein expression of endothelial ABCG2 in vitro [65]. We did not detect a change in either ABCB1 or ABCC1 expression in people with schizophrenia nor in the context of high inflammation, which is in keeping with the fact that ABCB1 expression is only slightly reduced by IL-6 and increased by TNFα [65]. ABCG2 interacts with substrates in the cell cytoplasm [66] allowing substrates not initially filtered by ABCB1 to be removed. Lower levels of ABCG2 especially in the context of elevated inflammation may result in less capacity to filter out and remove unwanted molecules in the prefrontal cortex for some people with schizophrenia. This dysfunction may delay the brain’s capacity to resolve inflammation and/or could exacerbate existing damage.

In conclusion, we are the first to report molecular alterations in brain endothelial cells and in monocyte/macrophage markers in the PFC from people with schizophrenia that are exaggerated when other signs of inflammation are present. Elevated ICAM1 does not necessarily prove that the BBB is more leaky in individuals with schizophrenia, but instead supports the hypothesis that luminal walls of blood vessels have more potential for increased capture of immune cells in the disease state. Further, we have identified CD163+ macrophages in the parenchyma of some schizophrenia brains. Multiple studies have shown substantial proportions of patients with schizophrenia have peripheral immune system changes and our study supports that these are not necessarily independent of neural changes, but rather that the two are inter-related. Further characterisation of these individuals utilising measurements in both brain and blood will contribute to our understanding of how elevated neuroinflammation contributes to the heterogeneity and pathophysiology of schizophrenia.