We have determined that clustering by inflammatory genes as well as clustering by stress genes results in groups enriched in individuals with a diagnosis of psychosis. Furthermore, it would seem that inflammation has a greater association with schizophrenia while stress signaling has a greater association with bipolar disorder. However, when the gene lists are combined together there are a greater than expected number of individuals with schizophrenia and a trend toward a greater number of bipolar disorder individuals categorized as in the high inflammation/stress group. Our study builds on the mounting evidence suggesting that a subset of individuals with psychotic illness have increased expression of immune-related genes in the brain,14 cerebrospinal fluid,45 and blood.46, 47 By showing that the immune changes are often concomitant with alterations in the stress signaling system, we have expanded the understanding of possible molecular mechanisms involved in the pathophysiology and of the therapies targeting these systems.

We examined the cohort demographics for evidence that the inflammatory/stress cluster could be a reflection of some external factors other than psychiatric disease state. We found decreased pH in the high inflammatory/stress group, which may be a response to the increased metabolic demands of an inflammatory immune response; however, this lowered pH could be a sign of tissue damage and in itself may exacerbate inflammation.48 This damage may be reflected in our observation of decreased brain weight in the high inflammatory/stress group, which remains significantly different even after co-varying for age and sex (data not shown). This brain mass decrease may be related to the observed volumetric and brain thickness decreases found in schizophrenia and bipolar disorder, but the link between inflammation and brain volume requires further exploration.49 It is also possible that the decrease in brain mass is related to the stress of the disease states as individuals undergoing chronic stress in adolescence and adulthood have decreased brain volumes.50, 51 Some of the observed brain inflammatory changes may be related to peripheral inflammation, which were identified in individuals found in both high and low inflammatory/stress groups. Interactions between peripheral IL-1 activation and the central nervous system have been demonstrated in animal models.52 Although obesity could be a possible cause of peripheral inflammation in some humans, it also may be directly related to psychiatric disease or to treatment.53 This is a key area that requires further research to understand the interactions.

The most robust mRNA finding from this study, and one that has been consistently replicated in other studies, was the elevation in SERPINA3 mRNA present in the DLPFC of individuals with schizophrenia. We found an almost 200% increase in the expression of SERPINA3 in schizophrenia patients compared to controls (note results on log scale) in the Stanley Array Cohort, which is now the fourth separate post-mortem cohort studied where this elevation is found.14, 54, 55 This suggests that upregulation of SERPINA3 mRNA is a highly reproducible finding in the frontal cortex in schizophrenia but its more specific involvement in schizophrenia as compared with bipolar disorder remains unexplored. SERPINA3 is known to code for a protein with an anti-chymotripsin action (SERPINA3 is also known as alpha 1-antichymotripsin), although this may not be the only protease that SERPINA3 inhibits.56 The precise cellular source of the SERPINA3 mRNA elevations in the brains of individuals with schizophrenia is unknown, but macrophages, reactive astrocytes and activated microglia have been shown to express the SERPINA3 protein in pathological conditions.57 In Alzheimer’s disease, SERPINA3 forms complexes with amyloid beta protein and also alters the expression of astrocytic and inflammatory genes.58, 59 Transcription of SERPINA3 is associated with inflammatory activation and the protein itself has multiple roles including prevention of excessive tissue damage from the secretion of chymotrypsin originating in phagocytes (microglia are the equivalent central nervous system phagocytes) as well as an unidentified DNA-binding role thought to modulate chymotrypsin-like chromatin enzymes.60, 61 Recently, it has been shown that glucocorticoids, working synergistically with inflammatory molecules (specifically TNF) increase the transcription of SERPINA3.62 Although the levels of GR are lower in individuals with schizophrenia, which may be a consequence of increased stress, the GR signaling system may remain functional, and in the face of elevated TNF this may result in the elevations in SERPINA3 present in our cohorts as well as others. The evidence for TNF being elevated in schizophrenia and bipolar disorder is mixed, but nevertheless a number of studies have demonstrated TNF increases in the brain and blood.63, 64, 65 The coactivation and upregulation of SERPINA3 suggests a possible compensatory role in the disease in an attempt to attenuate a chronic inflammatory response. It is perplexing that the elevation in SERPINA3 only becomes apparent in bipolar disorder after clustering by other inflammatory/stress factors, implying that although inflammation is common between the two diseases, specific elements of the response are more prevalent or exaggerated in schizophrenia. Further research is required to elucidate the role of SERPINA3 in the brain, and particularly in schizophrenia.

We were unable to replicate the overall group increases found in the cytokines IL-6 and IL-8 mRNAs previously identified in the schizophrenia patients from our Sydney TRC cohort.14 However, the subgroup of individuals with high inflammation/stress in the current study did display elevated IL1RL1, IL-6, TNF and IL-1β mRNA compared with the majority of controls. We were also unable to replicate a previous finding of overall increased IL-1β in individuals with bipolar disorder.18 It does seem that the generalized activation of the immune/inflammatory system is a common factor between all of these studies; however, the precise factors and the percentage of individuals demonstrating these changes at the time of death appear to be variable. Alternatively, as we suspect that the number of individuals with psychosis who have active inflammation may be only 40–50% of the total, nonrandom sampling may be responsible for variable research outcomes.

Our data suggest an interrelationship between stress signaling and immune function in the frontal cortex of a portion of individuals, primarily those with bipolar disorder and schizophrenia. Although data on cortisol levels of individuals in this cohort are not currently available, it is plausible that elevated cortisol levels in individuals with schizophrenia and bipolar disorder may drive the observed changes in both stress and inflammatory gene mRNAs. Elevated glucocorticoid levels, which have been demonstrated in individuals with schizophrenia and bipolar disorder,66, 67 have been shown to decrease GR mRNA expression and protein abundance in the rodent and primate frontal cortex and hippocampus.68, 69, 70, 71 Glucocorticoids also suppress immune function and immune gene expression,34, 35, 36, 37, 72 resulting in inflammatory resurgence after chronic administration.73 In schizophrenia and bipolar disorder it is possible that chronic hypercortisolemia, in a portion of patients with greater negative symptoms,74 may cause a chronic decrease in the expression and function of GR protein leading to attenuation of the natural inhibition of the immune system by stress.

Our idea of stress-related gene expression changes being more primary originates from the greater contribution of stress-related genes to the combined inflammatory/stress clustering. Our pathway analysis expands upon the limited number of genes involved in our mRNA findings and provides an opportunity for integration of our results with other schizophrenia and bipolar disorder findings. The high inflammation/stress group shows a downregulation of a number of gene groups in the diagnosis of schizophrenia and bipolar disorder. Key among these are the growth factor-related genes (NRTN (neurturin), BDNF and GDNF family receptor α2 (GFRA2)) and the inhibitory and excitatory signaling (GABA A receptor subunit γ2, α1 and δ, glutamic acid decarboxylase 1, somatostatin and glutamate receptor ionotropic AMPA 1, respectively). NRTN works through the GDNF receptor pathway, including GFRA2, and has been shown to be neuroprotective in the cortex of animal models.75 BDNF mRNA has been previously found decreased in the DLPFC of schizophrenia and BDNF can inhibit major histocompatibility complex-II inducibility in microglia.76, 77, 78 We, as well as others, have previously shown that major histocompatibility complex II-labeled microglia are increased in individuals with schizophrenia, and this may provide a possible link to decreased BDNF.14 The health of inhibitory interneurons and their signaling has been the focus of extensive research efforts in both schizophrenia and bipolar disorder with BDNF thought to be an important survival factor for inhibitory interneurons. Knockout of BDNF in mice resulted in a decrease density of cortical interneurons of the somatostatin subtype.79 Decreased amounts of BDNF have further been shown to correlate with decreased expression of glutamic acid decarboxylase 1 (GAD67) in subjects with schizophrenia.2, 80 Decreased amounts of somatostatin and glutamic acid decarboxylase 1 mRNA in particular, have been widely replicated in schizophrenia, with glutamic acid decarboxylase 1 also found decreased in bipolar disorder.81, 82, 83, 84 These mRNA decreases are exacerbated in the high inflammation/stress group of both diseases in our current study as well as previously in the high inflammation group of the TRC schizophrenia cohort.14 The involvement of the GABA A receptor subunits with stress and inflammation, particularly the significant decreases in the almost ubiquitous γ2 and α1 encoding subunits, together with the extrasynapic δ subunit, may explain some of the contradictory findings reported in both diseases.83, 85, 86 A number of immune-related genes are also found in this pathway including TNF, IL8 and TNFSF10 all of which as well as a number of other inflammatory-related genes have been implicated in schizophrenia and bipolar disorder.14, 87, 88, 89, 90 IL-8 was significantly elevated only in bipolar, similar to our qPCR data, when using the microarray generated data. However, microarrays, owing to their sensitivity issues are not optimal for measuring cytokine mRNA.91 TNFSF10 is also known as TNF-related apoptosis-inducing ligand and works through a caspase cascade including caspase 9 to cause apoptosis.92 The decrease in TNF-related apoptosis-inducing ligand is in direct contrast to our previously observed TNFSF13 (APRIL) increases in both disease states implying abnormal apoptotic-related process may be present.88 Cytokines, including IL-1β and TNF also work through mitogen-activated protein kinases to phosphorylate ATF2, leading to increased c-Jun and anti-apoptotic activity.92 A decrease in the latter part of this pathway may indicate a reduced ability to counter the pro-apoptotic activity of elevated cytokines found in the high inflammation/stress group, although the presence of apoptosis in these diseases is disputed.

It is noteworthy that we observed a significant number of controls (18% of the group) that also displayed the elevated markers of both inflammation and stress in the brain. This may suggest that some individuals have a biological resiliency to the negative effects of chronic inflammation, the mechanisms of which have not yet been identified, or that we are observing a mix of individuals in both chronic and acute inflammatory states. Individuals identified in the high inflammation/stress control group may be in the midst of an acute inflammatory response to a stressor that may have resolved without significant or ongoing neuronal damage.31 In schizophrenia, the often observed peripheral cytokine elevations over a variety of ages would lead us to believe that the higher than normal inflammatory response may be more chronic, rather than acute, possibly leading to cumulative damage.93

In summary, we have established that our previously observed post-mortem changes in cytokine expression are not consistently detectable across all cohorts.14 However, a generalized pattern of immune activation/inflammatory processes seems to be occurring in under half of the individuals diagnosed with schizophrenia and bipolar disorder. This immune activation may also co-occur with previously observed GR signaling abnormalities found in both diseases.28 In combination, the inflammatory/stress dysfunction may integrate previously independent observations about the pathophysiology of the disorders and provide additional targets for novel, individually or biological subgroup-targeted treatment interventions.