Double stranded RNA drives anti-viral innate immune responses, sickness behavior and cognitive dysfunction dependent on dsRNA length, IFNAR1 expression and age.

Double stranded RNA is generated during viral replication. The synthetic analogue poly I:C is frequently used to mimic anti-viral innate immune responses in models of psychiatric and neurodegenerative disorders including schizophrenia, autism, Parkinson's disease and Alzheimer's disease. Many studies perform limited analysis of innate immunity despite these responses potentially differing as a function of dsRNA molecular weight and age. Therefore fundamental questions relevant to impacts of systemic viral infection on brain function and integrity remain. Here, we studied innate immune-inducing properties of poly I:C preparations of different lengths and responses in adult and aged mice. High molecular weight (HMW) poly I:C (1-6 kb, 12 mg/kg) produced more robust sickness behavior and more robust IL-6, IFN-I and TNF-α responses than poly I:C of < 500 bases (low MW) preparations. This was partly overcome with higher doses of LMW (up to 80 mg/kg), but neither circulating IFNß nor brain transcription of Irf7 were significantly induced by LMW poly I:C, despite brain Ifnb transcription, suggesting that brain IFN-dependent gene expression is predominantly triggered by circulating IFNß binding of IFNAR1. In aged animals, poly I:C induced exaggerated IL-6, IL-1ß and IFN-I in the plasma and similar exaggerated brain cytokine responses. This was associated with acute working memory deficits selectively in aged mice. Thus, we demonstrate dsRNA length-, IFNAR1- and age-dependent effects on anti-viral inflammation and cognitive function. The data have implications for CNS symptoms of acute systemic viral infection such as those with SARS-CoV-2 and for models of maternal immune activation.

Acute systemic inflammation exacerbates neuroinflammation in Alzheimer's disease: IL-1ß drives amplified responses in primed astrocytes and neuronal network dysfunction.

Neuroinflammation contributes to Alzheimer's disease (AD) progression. Secondary inflammatory insults trigger delirium and can accelerate cognitive decline. Individual cellular contributors to this vulnerability require elucidation. Using APP/PS1 mice and AD brain, we studied secondary inflammatory insults to investigate hypersensitive responses in microglia, astrocytes, neurons, and human brain tissue. The NLRP3 inflammasome was assembled surrounding amyloid beta, and microglia were primed, facilitating exaggerated interleukin-1ß (IL-1ß) responses to subsequent LPS stimulation. Astrocytes were primed to produce exaggerated chemokine responses to intrahippocampal IL-1ß. Systemic LPS triggered microglial IL-1ß, astrocytic chemokines, IL-6, and acute cognitive dysfunction, whereas IL-1ß disrupted hippocampal gamma rhythm, all selectively in APP/PS1 mice. Brains from AD patients with infection showed elevated IL-1ß and IL-6 levels. Therefore, amyloid leaves the brain vulnerable to secondary inflammation at microglial, astrocytic, neuronal, and cognitive levels, and infection amplifies neuroinflammatory cytokine synthesis in humans. Exacerbation of neuroinflammation to produce deleterious outcomes like delirium and accelerated disease progression merits careful investigation in humans.

Acute transient cognitive dysfunction and acute brain injury induced by systemic inflammation occur by dissociable IL-1-dependent mechanisms.

Systemic inflammation can impair cognition with relevance to dementia, delirium and post-operative cognitive dysfunction. Episodes of delirium also contribute to rates of long-term cognitive decline, implying that these acute events induce injury. Whether systemic inflammation-induced acute dysfunction and acute brain injury occur by overlapping or discrete mechanisms remains unexplored. Here we show that systemic inflammation, induced by bacterial LPS, produces both working-memory deficits and acute brain injury in the degenerating brain and that these occur by dissociable IL-1-dependent processes. In normal C57BL/6 mice, LPS (100 µg/kg) did not affect working memory but impaired long-term memory consolidation. However prior hippocampal synaptic loss left mice selectively vulnerable to LPS-induced working memory deficits. Systemically administered IL-1 receptor antagonist (IL-1RA) was protective against, and systemic IL-1ß replicated, these working memory deficits. Dexamethasone abolished systemic cytokine synthesis and was protective against working memory deficits, without blocking brain IL-1ß synthesis. Direct application of IL-1ß to ex vivo hippocampal slices induced non-synaptic depolarisation and irreversible loss of membrane potential in CA1 neurons from diseased animals and systemic LPS increased apoptosis in the degenerating brain, in an IL-1RI-dependent fashion. The data suggest that LPS induces working memory dysfunction via circulating IL-1ß but direct hippocampal action of IL-1ß causes neuronal dysfunction and may drive neuronal death. The data suggest that acute systemic inflammation produces both reversible cognitive deficits, resembling delirium, and acute brain injury contributing to long-term cognitive impairment but that these events are mechanistically dissociable. These data have significant implications for management of cognitive dysfunction during acute illness.

Correction: Acute transient cognitive dysfunction and acute brain injury induced by systemic inflammation occur by dissociable IL-1-dependent mechanisms.

Following publication of this article, the authors noticed an error in the abstract, where they incorrectly stated that: "Direct application of IL-1ß to ex vivo hippocampal slices induced non-synaptic depolarisation and irreversible loss of membrane potential in CA1 neurons from diseased animals and systemic LPS increased apoptosis in the degenerating brain, in an IL-1RI-/--dependent fashion". This has now been corrected to: "Direct application of IL-1ß to ex vivo hippocampal slices induced non-synaptic depolarisation and irreversible loss of membrane potential in CA1 neurons from diseased animals and systemic LPS increased apoptosis in the degenerating brain, in an IL-1RI-dependent fashion". The authors would like to apologise for this error. This has been corrected in both the PDF and HTML versions of the article.

Chronic neurodegeneration induces type I interferon synthesis via STING, shaping microglial phenotype and accelerating disease progression.

Type I interferons (IFN-I) are the principal antiviral molecules of the innate immune system and can be made by most cell types, including central nervous system cells. IFN-I has been implicated in neuroinflammation during neurodegeneration, but its mechanism of induction and its consequences remain unclear. In the current study, we assessed expression of IFN-I in murine prion disease (ME7) and examined the contribution of the IFN-I receptor IFNAR1 to disease progression. The data indicate a robust IFNß response, specifically in microglia, with evidence of IFN-dependent genes in both microglia and astrocytes. This IFN-I response was absent in stimulator of interferon genes (STING-/- ) mice. Microglia showed increased numbers and activated morphology independent of genotype, but transcriptional signatures indicated an IFNAR1-dependent neuroinflammatory phenotype. Isolation of microglia and astrocytes demonstrated disease-associated microglial induction of Tnfα, Tgfb1, and of phagolysosomal system transcripts including those for cathepsins, Cd68, C1qa, C3, and Trem2, which were diminished in IFNAR1 and STING deficient mice. Microglial increases in activated cathepsin D, and CD68 were significantly reduced in IFNAR1-/- mice, particularly in white matter, and increases in COX-1 expression, and prostaglandin synthesis were significantly mitigated. Disease progressed more slowly in IFNAR1-/- mice, with diminished synaptic and neuronal loss and delayed onset of neurological signs and death but without effect on proteinase K-resistant PrP levels. Therefore, STING-dependent IFN-I influences microglial phenotype and influences neurodegenerative progression despite occurring secondary to initial degenerative changes. These data expand our mechanistic understanding of IFN-I induction and its impact on microglial function during chronic neurodegeneration.

Cyclooxygenase-1-dependent prostaglandins mediate susceptibility to systemic inflammation-induced acute cognitive dysfunction.

Systemic inflammatory events often precipitate acute cognitive dysfunction in elderly and demented populations. Delirium is a highly prevalent neuropsychiatric syndrome that is characterized by acute inattention and cognitive dysfunction, for which prior dementia is the major predisposing factor and systemic inflammation is a frequent trigger. Inflammatory mechanisms of delirium remain unclear. We have modeled aspects of delirium during dementia by exploiting progressive neurodegeneration in the ME7 mouse model of prion disease and by superimposing systemic inflammation induced by the bacterial endotoxin lipopolysaccharide (LPS). Here, we have used this model to demonstrate that the progression of underlying disease increases the incidence, severity, and duration of acute cognitive dysfunction. This increasing susceptibility is associated with increased CNS expression of cyclooxygenase (COX)-1 in microglia and perivascular macrophages. The COX-1-specific inhibitor SC-560 provided significant protection against LPS-induced cognitive deficits, and attenuated the disease-induced increase in hippocampal and thalamic prostaglandin E2, while the COX-2-specific inhibitor NS-398 was ineffective. SC-560 treatment did not alter levels of the proinflammatory cytokines interleukin (IL)-1ß, tumor necrosis factor-α, IL-6, or C-X-C chemokine ligand 1 in blood or brain, but systemic IL-1RA blocked LPS-induced cognitive deficits, and systemic IL-1ß was sufficient to induce similar deficits in the absence of LPS. Furthermore, the well tolerated COX inhibitor ibuprofen was protective against IL-1ß-induced deficits. These data demonstrate that progressive microglial COX-1 expression and prostaglandin synthesis can underpin susceptibility to cognitive deficits, which can be triggered by systemic LPS-induced IL-1ß. These data contribute to our understanding of how systemic inflammation and ongoing neurodegeneration interact to induce cognitive dysfunction and episodes of delirium.

Hyperthermia elevates brain temperature and improves behavioural signs in animal models of autism spectrum disorder.

BACKGROUND: Autism spectrum disorders (ASD) are predominantly neurodevelopmental and largely genetically determined. However, there are human data supporting the idea that fever can improve symptoms in some individuals, but those data are limited and there are almost no data to support this from animal models. We aimed to test the hypothesis that elevated body temperature would improve function in two animal models of ASD. METHODS: We used a 4 h whole-body hyperthermia (WBH) protocol and, separately, systemic inflammation induced by bacterial endotoxin (LPS) at 250 µg/kg, to dissociate temperature and inflammatory elements of fever in two ASD animal models: C58/J and Shank3B- mice. We used one- or two-way ANOVA and t-tests with normally distributed data and Kruskal-Wallis or Mann-Whitney with nonparametric data. Post hoc comparisons were made with a level of significance set at p < 0.05. For correlation analyses, data were adjusted by a linear regression model. RESULTS: Only LPS induced inflammatory signatures in the brain while only WBH produced fever-range hyperthermia. WBH reduced repetitive behaviours and improved social interaction in C58/J mice and significantly reduced compulsive grooming in Shank3B- mice. LPS significantly suppressed most activities over 5-48 h. LIMITATIONS: We show behavioural, cellular and molecular changes, but provide no specific mechanistic explanation for the observed behavioural improvements. CONCLUSIONS: The data are the first, to our knowledge, to demonstrate that elevated body temperature can improve behavioural signs in 2 distinct ASD models. Given the developmental nature of ASD, evidence that symptoms may be improved by environmental perturbations indicates possibilities for improving function in these individuals. Since experimental hyperthermia in patients would carry significant risks, it is now essential to pursue molecular mechanisms through which hyperthermia might bring about the observed benefits.

Exacerbation of CNS inflammation and neurodegeneration by systemic LPS treatment is independent of circulating IL-1ß and IL-6.

BACKGROUND: Chronic neurodegeneration comprises an inflammatory response but its contribution to the progression of disease remains unclear. We have previously shown that microglial cells are primed by chronic neurodegeneration, induced by the ME7 strain of prion disease, to synthesize limited pro-inflammatory cytokines but to produce exaggerated responses to subsequent systemic inflammatory insults. The consequences of this primed response include exaggerated hypothermic and sickness behavioural responses, acute neuronal death and accelerated progression of disease. Here we investigated whether inhibition of systemic cytokine synthesis using the anti-inflammatory steroid dexamethasone-21-phosphate was sufficient to block any or all of these responses. METHODS: ME7 animals, at 18-19 weeks post-inoculation, were challenged with LPS (500 µg/kg) in the presence or absence of dexamethasone-21-phosphate (2 mg/kg) and effects on core-body temperature and systemic and CNS cytokine production and apoptosis were examined. RESULTS: LPS induced hypothermia and decreased exploratory activity. Dexamethasone-21-phosphate prevented this hypothermia, markedly suppressed systemic IL-1ß and IL-6 secretion but did not prevent decreased exploration. Furthermore, robust transcription of cytokine mRNA occurred in the hippocampus of both ME7 and NBH (normal brain homogenate) control animals despite the effective blocking of systemic cytokine synthesis. Microglia primed by neurodegeneration were not blocked from the robust synthesis of IL-1ß protein and endothelial COX-2 was also robustly synthesized. We injected biotinylated LPS at 100 µg/kg and even at this lower dose this could be detected in blood plasma. Apoptosis was acutely induced by LPS, despite the inhibition of the systemic cytokine response. CONCLUSIONS: These data suggest that LPS can directly activate the brain endothelium even at relatively low doses, obviating the need for systemic cytokine stimulation to transduce systemic inflammatory signals into the brain or to exacerbate existing pathology.

Double stranded RNA drives innate immune responses, sickness behavior and cognitive impairment dependent on dsRNA length, IFNAR1 expression and age.

Double stranded RNA is generated during viral replication. The synthetic analog poly I:C is frequently used to mimic anti-viral innate immune responses in models of psychiatric and neurodegenerative disease including autism, schizophrenia, Parkinsons disease and Alzheimers disease. Many studies perform limited analysis of innate immunity despite these responses potentially differing as a function of dsRNA molecular weight and age. Therefore fundamental questions relevant to impacts of systemic viral infection on brain function and integrity remain. Here, we studied innate immune-inducing properties of poly I:C preparations of different lengths and responses in adult and aged mice. High molecular weight (HMW) poly I:C (1 to 6 kb, 12 mg/kg) produced more robust sickness behavior and more robust IL-6, IFN-I and TNF alpha responses than poly I:C of less than 500 bases (low MW) preparations. This was partly overcome with higher doses of LMW (up to 80 mg/kg), but neither circulating IFN beta nor brain transcription of Irf7 were significantly induced by LMW poly I:C, despite brain Ifnb transcription, suggesting that brain IFN-dependent gene expression is predominantly triggered by circulating IFN beta binding of IFNAR1. In aged animals, poly I:C induced exaggerated IL-6, IL-1beta and IFN-I in the plasma and similar exaggerated brain cytokine responses. This was associated with acute working memory deficits selectively in aged mice. Thus, we demonstrate dsRNA length, IFNAR1 and age-dependent effects on antiviral inflammation and cognitive function. The data have implications for CNS symptoms of acute systemic viral infection such as those with SARS-CoV-2 and for models of maternal immune activation.

Microglia in the degenerating brain are capable of phagocytosis of beads and of apoptotic cells, but do not efficiently remove PrPSc, even upon LPS stimulation.

Despite the phagocytic machinery available to microglia the aberrant amyloid proteins produced during Alzheimer's and prion disease, amyloid-ß and PrP(Sc), are inefficiently cleared. We have shown that microglia in the ME7 model of prion disease show morphological evidence of activation, synthesize low levels of pro-inflammatory cytokines and are primed to produce exaggerated responses to subsequent inflammatory challenges. Whether these microglia engage in significant phagocytic activity in the disease per se, or upon subsequent inflammatory challenge is not clear. In the present study we show transcriptional activation of a large number of scavenger receptors (SRs), matrix metalloproteinases (MMPs), oxidative enzymes, and cathepsins in ME7 animals. Hippocampally-injected inert latex beads (6 µm) are efficiently phagocytosed by microglia of ME7 prion-diseased animals, but not by microglia in normal animals. Stimulation of ME7 animals with systemic bacterial endotoxin (lipopolysaccharide, LPS) induced further increases in SR-A2, MMP3, and urokinase plasminogen activator receptor (uPAR) but decreased, or did not alter, transcription of most phagocytosis-related genes examined and did not enhance clearance of deposited PrP(Sc). Furthermore, intracerebral injection with LPS (0.5 µg) induced marked microglial production of IL-1ß, robust cellular infiltration and marked apoptosis but also did not induce further clearance of PrP(Sc). These data indicate that microglia in the prion-diseased brain are capable of phagocytosis per se, but show limited efficacy in removing PrP(Sc) even upon marked escalation of CNS inflammation. Furthermore, microglia/macrophages remain IL-1ß-negative during phagocytosis of apoptotic cells. The data demonstrate that phagocytic activity and pro-inflammatory microglial phenotype do not necessarily correlate.

Endogenous IL-1 in cognitive function and anxiety: a study in IL-1RI-/- mice.

Interleukin-1 (IL-1) is a key pro-inflammatory cytokine, produced predominantly by peripheral immune cells but also by glia and some neuronal populations within the brain. Its signalling is mediated via the binding of IL-1α or IL-1ß to the interleukin-1 type one receptor (IL-1RI). IL-1 plays a key role in inflammation-induced sickness behaviour, resulting in depressed locomotor activity, decreased exploration, reduced food and water intake and acute cognitive deficits. Conversely, IL-1 has also been suggested to facilitate hippocampal-dependent learning and memory: IL-1RI(-/-) mice have been reported to show deficits on tasks of visuospatial learning and memory. We sought to investigate whether there is a generalised hippocampal deficit in IL-1RI(-/-) animals. Therefore, in the current study we compared wildtype (WT) mice to IL-1RI(-/-) mice using a variety of hippocampal-dependent learning and memory tasks, as well as tests of anxiety and locomotor activity. We found no difference in performance of the IL-1RI(-/-) mice compared to WT mice in a T-maze working memory task. In addition, the IL-1RI(-/-) mice showed normal learning in various spatial reference memory tasks including the Y-maze and Morris mater maze, although there was a subtle deficit in choice behaviour in a spatial discrimination, beacon watermaze task. IL-1RI(-/-) mice also showed normal memory for visuospatial context in the contextual fear conditioning paradigm. In the open field, IL-1RI(-/-) mice showed a significant increase in distance travelled and rearing behaviour compared to the WT mice and in the elevated plus-maze spent more time in the open arms than did the WT animals. The data suggest that, contrary to prior studies, IL-1RI(-/-) mice are not robustly impaired on hippocampal-dependent memory and learning but do display open field hyperactivity and decreased anxiety compared to WT mice. The results argue for a careful evaluation of the roles of endogenous IL-1 in hippocampal and limbic system function.

Systemic inflammation induces acute behavioral and cognitive changes and accelerates neurodegenerative disease.

BACKGROUND: Chronic neurodegeneration results in microglial activation, but the contribution of inflammation to the progress of neurodegeneration remains unclear. We have shown that microglia express low levels of proinflammatory cytokines during chronic neurodegeneration but are "primed" to produce a more proinflammatory profile after systemic challenge with bacterial endotoxin (lipopolysaccharide [LPS]). METHODS: Here, we investigated whether intraperitoneal (IP) challenge with LPS, to mimic systemic infection, in the early stages of prion disease can 1) produce exaggerated acute behavioral (n = 9) and central nervous system (CNS) inflammatory (n = 4) responses in diseased animals compared with control animals, and 2) whether a single LPS challenge can accelerate disease progression (n = 34-35). RESULTS: Injection of LPS (100 microg/kg), at 12 weeks postinoculation (PI), resulted in heightened CNS interleukin-1 beta (IL-1beta), tumor necrosis factor-alpha (TNF-alpha), and interferon-beta (IFN-beta) transcription and microglial IL-1beta translation in prion-diseased animals relative to control animals. This inflammation caused exaggerated impairments in burrowing and locomotor activity, and induced hypothermia and cognitive changes in prion-diseased animals that were absent in LPS-treated control animals. At 15 weeks PI, LPS (500 microg/kg) acutely impaired motor coordination and muscle strength in prion-diseased but not in control animals. After recovery, these animals also showed earlier onset of disease-associated impairments on these parameters. CONCLUSIONS: These data demonstrate that transient systemic inflammation superimposed on neurodegenerative disease acutely exacerbates cognitive and motor symptoms of disease and accelerates disease progression. These deleterious effects of systemic inflammation have implications for the treatment of chronic neurodegeneration and associated delirium.