Nafamostat reduces systemic inflammation in TLR7-mediated virus-like illness.

BACKGROUND: The serine protease inhibitor nafamostat has been proposed as a treatment for COVID-19, by inhibiting TMPRSS2-mediated viral cell entry. Nafamostat has been shown to have other, immunomodulatory effects, which may be beneficial for treatment, however animal models of ssRNA virus infection are lacking. In this study, we examined the potential of the dual TLR7/8 agonist R848 to mimic the host response to an ssRNA virus infection and the associated behavioural response. In addition, we evaluated the anti-inflammatory effects of nafamostat in this model. METHODS: CD-1 mice received an intraperitoneal injection of R848 (200 µg, prepared in DMSO, diluted 1:10 in saline) or diluted DMSO alone, and an intravenous injection of either nafamostat (100 µL, 3 mg/kg in 5% dextrose) or 5% dextrose alone. Sickness behaviour was determined by temperature, food intake, sucrose preference test, open field and forced swim test. Blood and fresh liver, lung and brain were collected 6 h post-challenge to measure markers of peripheral and central inflammation by blood analysis, immunohistochemistry and qPCR. RESULTS: R848 induced a robust inflammatory response, as evidenced by increased expression of TNF, IFN-γ, CXCL1 and CXCL10 in the liver, lung and brain, as well as a sickness behaviour phenotype. Exogenous administration of nafamostat suppressed the hepatic inflammatory response, significantly reducing TNF and IFN-γ expression, but had no effect on lung or brain cytokine production. R848 administration depleted circulating leukocytes, which was restored by nafamostat treatment. CONCLUSIONS: Our data indicate that R848 administration provides a useful model of ssRNA virus infection, which induces inflammation in the periphery and CNS, and virus infection-like illness. In turn, we show that nafamostat has a systemic anti-inflammatory effect in the presence of the TLR7/8 agonist. Therefore, the results indicate that nafamostat has anti-inflammatory actions, beyond its ability to inhibit TMPRSS2, that might potentiate its anti-viral actions in pathologies such as COVID-19.

Unique pathways downstream of TLR-4 and TLR-7 activation: sex-dependent behavioural, cytokine, and metabolic consequences.

Introduction: Post-infection syndromes are characterised by fatigue, muscle pain, anhedonia, and cognitive impairment; mechanistic studies exploring these syndromes have focussed on pathways downstream of Toll-like receptor (TLR) 4 activation. Here, we investigated the mechanistic interplay between behaviour, metabolism, and inflammation downstream of TLR-7 activation compared to TLR-4 activation in male and female CD1 mice. Methods: Animals received either a TLR-4 (LPS; 0.83 mg/kg) or TLR-7 (R848, 5 mg/kg) agonist, or saline, and behaviour was analysed in an Open Field (OF) at 24 h (n = 20/group). Plasma, liver, and prefrontal cortex (PFC) were collected for gene expression analysis at 24 h and 1H-NMR metabolomics. Results: TLR-4 and TLR-7 activation decreased distance travelled and rearing in the OF, but activation of each receptor induced distinct cytokine responses and metabolome profiles. LPS increased IL-1ß expression and CXCL1 in the PFC, but TLR7 activation did not and strongly induced PFC CXCL10 expression. Thus, TLR7 induced sickness behaviour is independent of IL-1ß expression. In both cases, the behavioural response to TLR activation was sexually dimorphic: females were more resilient. However, dissociation was observed between the resilient female mice behaviour and the levels of gene cytokine expression, which was, in general, higher in the female mice. However, the metabolic shifts induced by immune activation were better correlated with the sex-dependent behavioural dimorphisms; increased levels of antioxidant potential in the female brain are intrinsic male/female metabolome differences. A common feature of both TLR4 and TLR7 activation was an increase in N-acetyl aspartate (NAA) in the PFC, which is likely be an allostatic response to the challenges as sickness behaviour is inversely correlated with NAA levels. Discussion: The results highlight how the cytokine profile induced by one PAMP cannot be extrapolated to another, but they do reveal how the manipulation of the conserved metabolome response might afford a more generic approach to the treatment of post-infection syndromes.

Omega-3 alleviates behavioral and molecular changes in a mouse model of stress-induced juvenile depression.

Introduction: Depression is increasingly diagnosed in adolescence, necessitating specific prevention and treatment methods. However, there is a lack of animal models mimicking juvenile depression. This study explores a novel model using ultrasound (US) stress in juvenile mice. Methods: We employed the US stress model in one-month-old C57/BL6 mice, exposing them to alternating ultrasound frequencies (20-25 kHz and 25-45 kHz) for three weeks. These frequencies correspond to negative and neutral emotional states in rodents and can induce a depressive-like syndrome. Concurrently, mice received either an omega-3 food supplement (FS) containing eicosapentaenoic acid (EPA; 0.55 mg/kg/day) and docosahexaenoic acid (DHA; 0.55 mg/kg/day) or a vehicle. Post-stress, we evaluated anxiety- and depressive-like behaviors, blood corticosterone levels, brain expression of pro-inflammatory cytokines, and conducted metabolome analysis of brain, liver and blood plasma. Results: US-exposed mice treated with vehicle exhibited decreased sucrose preference, a sign of anhedonia, a key feature of depression, increased anxiety-like behavior, elevated corticosterone levels, and enhanced TNF and IL-1ß gene expression in the brain. In contrast, US-FS mice did not display these changes. Omega-3 supplementation also reduced anxiety-like behavior in non-stressed mice. Metabolomic analysis revealed US-induced changes in brain energy metabolism, with FS increasing brain sphingomyelin. Liver metabolism was affected by both US and FS, while plasma metabolome changes were exclusive to FS. Brain glucose levels correlated positively with activity in anxiety tests. Conclusion: Chronic omega-3 intake counteracted depressive- and anxiety-like behaviors in a US model of juvenile depression in mice. These effects likely stem from the anti-inflammatory properties of the supplement, suggesting potential therapeutic applications in juvenile depression.