Distinct Phenotypes of Inflammation Associated Macrophages and Microglia in the Prefrontal Cortex Schizophrenia Compared to Controls.

Approximately 40% of people with schizophrenia are classified as having "high inflammation." This subgroup has worse neuropathology than patients with "low inflammation." Thus, one would expect the resident microglia and possibly monocyte-derived macrophages infiltrating from the periphery to be "activated" in those with schizophrenia with elevated neuroinflammation. To test whether microglia and/or macrophages are associated with increased inflammatory signaling in schizophrenia, we measured microglia- and macrophage-associated transcripts in the postmortem dorsolateral prefrontal cortex of 69 controls and 72 people with schizophrenia. Both groups were stratified by neuroinflammatory status based on cortical mRNA levels of cytokines and SERPINA3. We found microglial mRNAs levels were either unchanged (IBA1 and Hexb, p > 0.20) or decreased (CD11c, <62% p < 0.001) in high inflammation schizophrenia compared to controls. Conversely, macrophage CD163 mRNA levels were increased in patients, substantially so in the high inflammation schizophrenia subgroup compared to low inflammation subgroup (>250%, p < 0.0001). In contrast, high inflammation controls did not have elevated CD163 mRNA compared to low inflammation controls (p > 0.05). The pro-inflammatory macrophage marker (CD64 mRNA) was elevated (>160%, all p < 0.05) and more related to CD163 mRNA in the high inflammation schizophrenia subgroup compared to high inflammation controls, while anti-inflammatory macrophage and cytokine markers (CD206 and IL-10 mRNAs) were either unchanged or decreased in schizophrenia. Finally, macrophage recruitment chemokine CCL2 mRNA was increased in schizophrenia (>200%, p < 0.0001) and CCL2 mRNA levels positively correlated with CD163 mRNA (r = 0.46, p < 0.0001). Collectively, our findings support the co-existence of quiescent microglia and increased pro-inflammatory macrophages in the cortex of people with schizophrenia.

Inflammation-related transcripts define "high" and "low" subgroups of individuals with schizophrenia and bipolar disorder in the midbrain.

Dopamine dysregulation in schizophrenia may be associated with midbrain inflammation. Previously, we found elevated levels of pro-inflammatory cytokine mRNAs in the post-mortem midbrain of people with schizophrenia (46%) but not from unaffected controls (0%) using a brain cohort from Sydney, Australia. Here, we measured cytokine mRNAs and proteins in the midbrain in the Stanley Medical Research Institute (SMRI) array cohort (N = 105). We tested if the proportions of individuals with schizophrenia and with high inflammation can be replicated, and if individuals with bipolar disorder with elevated midbrain cytokines can be identified. mRNA levels of 7 immune transcripts from post-mortem midbrain tissue were measured via RT-PCR and two-step recursive clustering analysis was performed using 4 immune transcripts to define "high and low" inflammatory subgroups. The clustering predictors used were identical to our earlier midbrain study, and included: IL1B, IL6, TNF, and SERPINA3 mRNA levels. 46% of schizophrenia cases (16/35 SCZ), 6% of controls (2/33 CTRL), and 29% of bipolar disorder cases (10/35 BPD) were identified as belonging to the high inflammation (HI) subgroups [χ2 (2) = 13.54, p < 0.001]. When comparing inflammatory subgroups, all four mRNAs were significantly increased in SCZ-HI and BPD-HI compared to low inflammation controls (CTRL-LI) (p < 0.05). Additionally, protein levels of IL-1ß, IL-6, and IL-18 were elevated in SCZ-HI and BPD-HI compared to all other low inflammatory subgroups (all p < 0.05). Surprisingly, TNF-α protein levels were unchanged according to subgroups. In conclusion, we determined that almost half of the individuals with schizophrenia were defined as having high inflammation in the midbrain, replicating our previous findings. Further, we detected close to one-third of those with bipolar disorder to be classified as having high inflammation. Elevations in some pro-inflammatory cytokine mRNAs (IL-1ß and IL-6) were also found at the protein level, whereas TNF mRNA and protein levels were not concordant.

Peripheral NF-κB dysregulation in people with schizophrenia drives inflammation: putative anti-inflammatory functions of NF-κB kinases.

Elevations in plasma levels of pro-inflammatory cytokines and C-reactive protein (CRP) in patient blood have been associated with impairments in cognitive abilities and more severe psychiatric symptoms in people with schizophrenia. The transcription factor nuclear factor kappa B (NF-κB) regulates the gene expression of pro-inflammatory factors whose protein products trigger CRP release. NF-κB activation pathway mRNAs are increased in the brain in schizophrenia and are strongly related to neuroinflammation. Thus, it is likely that this central immune regulator is also dysregulated in the blood and associated with cytokine and CRP levels. We measured levels of six pro-inflammatory cytokine mRNAs and 18 mRNAs encoding NF-κB pathway members in peripheral blood leukocytes from 87 people with schizophrenia and 83 healthy control subjects. We then assessed the relationships between the alterations in NF-κB pathway genes, pro-inflammatory cytokine and CRP levels, psychiatric symptoms and cognition in people with schizophrenia. IL-1ß and IFN-γ mRNAs were increased in patients compared to controls (both p < 0.001), while IL-6, IL-8, IL-18, and TNF-α mRNAs did not differ. Recursive two-step cluster analysis revealed that high levels of IL-1ß mRNA and high levels of plasma CRP defined 'high inflammation' individuals in our cohort, and a higher proportion of people with schizophrenia were identified as displaying 'high inflammation' compared to controls using this method (p = 0.03). Overall, leukocyte expression of the NF-κB-activating receptors, TLR4 and TNFR2, and the NF-κB subunit, RelB, was increased in people with schizophrenia compared to healthy control subjects (all p < 0.01), while NF-κB-inducing kinase mRNAs IKKß and NIK were downregulated in patients (all p < 0.05). We found that elevations in TLR4 and RelB appear more related to inflammatory status than to a diagnosis of schizophrenia, but changes in TNFR2 occur in both the high and low inflammation patients (but were exaggerated in high inflammation patients). Further, decreased leukocyte expression of IKKß and NIK mRNAs was unique to high inflammation patients, which may represent schizophrenia-specific dysregulation of NF-κB that gives rise to peripheral inflammation in a subset of patients.

Neuroinflammation in schizophrenia: the role of nuclear factor kappa B.

Neuroinflammation, particularly in the dorsolateral prefrontal cortex, is well-established in a subset of people with schizophrenia, with significant increases in inflammatory markers including several cytokines. Yet the cause(s) of cortical inflammation in schizophrenia remains unknown. Clues as to potential microenvironmental triggers and/or intracellular deficits in immunoregulation may be gleaned from looking further upstream of effector immune molecules to transcription factors that control inflammatory gene expression. Here, we focus on the 'master immune regulator' nuclear factor kappa B (NF-κB) and review evidence in support of NF-κB dysregulation causing or contributing to neuroinflammation in patients. We discuss the utility of 'immune biotyping' as a tool to analyse immune-related transcripts and proteins in patient tissue, and the insights into cortical NF-κB in schizophrenia revealed by immune biotyping compared to studies treating patients as a single, homogenous group. Though the ubiquitous nature of NF-κB presents several hurdles for drug development, targeting this key immunoregulator with novel or repurposed therapeutics in schizophrenia is a relatively underexplored area that could aid in reducing symptoms of patients with active neuroinflammation.

Nuclear factor kappa B activation appears weaker in schizophrenia patients with high brain cytokines than in non-schizophrenic controls with high brain cytokines.

BACKGROUND: High inflammation status despite an absence of known infection characterizes a subpopulation of people with schizophrenia who suffer from more severe cognitive deficits, less cortical grey matter, and worse neuropathology. Transcripts encoding factors upstream of nuclear factor kappa B (NF-κB), a major transcriptional activator for the synthesis of pro-inflammatory cytokines, are increased in the frontal cortex in schizophrenia compared to controls. However, the extent to which these changes are disease-specific, restricted to those with schizophrenia and high-neuroinflammatory status, or caused by loss of a key NF-κB inhibitor (HIVEP2) found in schizophrenia brain, has not been tested. METHODS: Post-mortem prefrontal cortex samples were assessed in 141 human brains (69 controls and 72 schizophrenia) and 13 brains of wild-type mice and mice lacking HIVEP2 (6 wild-type, 7 knockout mice). Gene expression of pro-inflammatory cytokines and acute phase protein SERPINA3 was used to categorize high and low neuroinflammation biotype groups in human samples via cluster analysis. Expression of 18 canonical and non-canonical NF-κB pathway genes was assessed by qPCR in human and mouse tissue. RESULTS: In humans, we found non-canonical upstream activators of NF-κB were generally elevated in individuals with neuroinflammation regardless of diagnosis, supporting NF-κB activation in both controls and people with schizophrenia when cytokine mRNAs are high. However, high neuroinflammation schizophrenia patients had weaker (or absent) transcriptional increases of several canonical upstream activators of NF-κB as compared to the high neuroinflammation controls. HIVEP2 mRNA reduction was specific to patients with schizophrenia who also had high neuroinflammatory status, and we also found decreases in NF-κB transcripts typically induced by activated microglia in mice lacking HIVEP2. CONCLUSIONS: Collectively, our results show that high cortical expression of pro-inflammatory cytokines and low cortical expression of HIVEP2 in a subset of people with schizophrenia is associated with a relatively weak NF-κB transcriptional signature compared to non-schizophrenic controls with high cytokine expression. We speculate that this comparatively milder NF-κB induction may reflect schizophrenia-specific suppression possibly related to HIVEP2 deficiency in the cortex.

Regional, cellular and species difference of two key neuroinflammatory genes implicated in schizophrenia.

The transcription factor nuclear factor kappa B (NF-κB) regulates the expression of many inflammatory genes that are overexpressed in a subset of people with schizophrenia. Transcriptional reduction in one NF-κB inhibitor, Human Immunodeficiency Virus Enhancer Binding Protein 2 (HIVEP2), is found in the brain of patients, aligning with evidence of NF-κB over-activity. Cellular co-expression of HIVEP2 and cytokine transcripts is a prerequisite for a direct effect of HIVEP2 on pro-inflammatory transcription, and we do not know if changes in HIVEP2 and markers of neuroinflammation are occurring in the same brain cell type. We performed in situ hybridisation on postmortem dorsolateral prefrontal cortex tissue to map and compare the expression of HIVEP2 and Serpin Family A Member 3 (SERPINA3), one of the most consistently increased inflammatory genes in schizophrenia, between schizophrenia patients and controls. We find that HIVEP2 expression is neuronal and is decreased in almost all grey matter cortical layers in schizophrenia patients with neuroinflammation, and that SERPINA3 is increased in the dorsolateral prefrontal cortex grey matter and white matter in the same group of patients. We are the first to map the upregulation of SERPINA3 to astrocytes and to some neurons, and find evidence to suggest that blood vessel-associated astrocytes are the main cellular source of SERPINA3 in the schizophrenia cortex. We show that a lack of HIVEP2 in mice does not cause astrocytic upregulation of Serpina3n but does induce its transcription in neurons. We speculate that HIVEP2 downregulation is not a direct cause of astrocytic pro-inflammatory cytokine synthesis in schizophrenia but may contribute to neuronally-mediated neuroinflammation.

Genome Sequences of Six Cluster N Mycobacteriophages, Kevin1, Nenae, Parmesanjohn, ShrimpFriedEgg, Smurph, and SpongeBob, Isolated on Mycobacterium smegmatis mc2155.

The annotation of six cluster N Mycobacterium smegmatis phages (Kevin1, Nenae, Parmesanjohn, ShrimpFriedEgg, Smurph, and SpongeBob) reveals regions of genomic diversity, particularly within the central region of the genome. The genome of Kevin1 includes two orphams (genes with no similarity to other phage genes), with one predicted to encode an AAA-ATPase.