Histamine H3R antagonist counteracts the impaired hippocampal neurogenesis in Lipopolysaccharide-induced neuroinflammation.

Adult neurogenesis in hippocampus dentate gyrus (DG) is associated with numerous neurodegenerative diseases such as aging and Alzheimer's disease (AD). Overactivation of microglia induced neuroinflammation is well acknowledged to contribute to the impaired neurogenesis in pathologies of these diseases and then leading to cognitive dysfunction. Histamine H3 receptor (H3R) is a presynaptic autoreceptor regulating histamine release via negative feedback way. Recently, studies show that H3R are highly expressed not only in neurons but also in microglia to modulate inflammatory response. However, whether inhibition of H3R is responsible for the neurogenesis and cognition in chronic neuroinflammation induced injury and the mechanism remains unclear. In this study, we found that inhibition of H3R by thioperamide reduced the microglia activity and promoted a phenotypical switch from pro-inflammatory M1 to anti-inflammatory M2 in microglia, and ultimately attenuated lipopolysaccharide (LPS) induced neuroinflammation in mice. Additionally, thioperamide rescued the neuroinflammation induced impairments of neurogenesis and cognitive function. Mechanically, the neuroprotection of thioperamide was involved in histamine dependent H2 receptor (H2R) activation, because cimetidine, an H2R antagonist but not pyrilamine, an H1R antagonist reversed the above effects of thioperamide. Moreover, thioperamide activated the H2R downstream phosphorylated protein kinase A (PKA)/cyclic AMP response element-binding protein (CREB) pathway but inhibited nuclear factor kappa-B (NF-κB) signaling. Activation of CREB by thioperamide promoted interaction of CREB-CREB Binding Protein (CBP) to increase anti-inflammatory cytokines (Interleukin-4 and Interleukin-10) and brain-derived neurotrophic factor (BDNF) release but inhibited NF-κB-CBP interaction to decrease pro-inflammatory cytokines (Interleukin-1ß, Interleukin-6 and Tumor necrosis factor α) release. H89, an inhibitor of PKA/CREB signaling, abolished effects of thioperamide on neuroinflammation and neurogenesis. Taken together, these results suggested under LPS induced neuroinflammation, the H3R antagonist thioperamide inhibited microglia activity and inflammatory response, and ameliorated impairment of neurogenesis and cognitive dysfunction via enhancing histamine release. Histamine activated H2R and reinforced CREB-CBP interaction but weakened NF-κB-CBP interaction to exert anti-inflammatory effects. This study uncovered a novel histamine dependent mechanism behind the therapeutic effect of thioperamide on neuroinflammation.

CCPG1 involved in corneal Aspergillus fumigatus infection.

AIM: To investigate whether non-canonical autophagy transport receptor cell cycle progression 1 (CCPG1) is involved in the corneal antifungal immune response. METHODS: Human corneal epithelial cells (HCECs) and human myeloid leukemia mononuclear cells (THP-1) macrophages stimulated by Aspergillus fumigatus (A. fumigatus) were used as cell models. The expression of CCPG1 mRNA was detected by qRT-PCR. Western blot was used to determine the protein expression of CCPG1 and interleukin-1ß (IL-1ß). The dectin-1 neutralizing antibody was used to detect the association between dectin-1 and CCPG1. Immunofluorescence was used to observe the colocalization of CCPG1 and C-type lectin-like receptor-1 (CLEC-1) in THP-1 macrophages. RESULTS: The expression of CCPG1 started to increase at 4h after infection and increased in a time-dependent manner in HCECs and THP-1 macrophages. With dectin-1 neutralizing antibody pretreatment, the expression of IL-1ß was down-regulated. CCPG1 up-regulation in response to A. fumigatus infection was independent of dectin-1. Immunofluorescence showed the colocalization of CCPG1 and CLEC-1 in THP-1 macrophages. CONCLUSION: As a specific autophagy protein of non-canonical autophagy pathway, CCPG1 is involved in corneal infection with A. fumigatus.

Thioperamide attenuates neuroinflammation and cognitive impairments in Alzheimer's disease via inhibiting gliosis.

Alzheimer's disease (AD) is an age-related neurodegenerative disease, which characterized by deposition of amyloid-ß (Aß) plaques, neurofibrillary tangles, neuronal loss, and accompanied by neuroinflammation. Neuroinflammatory processes are well acknowledged to contribute to the progression of AD pathology. Histamine H3 receptor (H3R) is a presynaptic autoreceptor regulating histamine release via negative feedback way. Recently, studies show that H3R are highly expressed not only in neurons but also in microglia and astrocytes. H3R antagonist has been reported to have anti-inflammatory efficacy. However, whether inhibition of H3R is responsible for the anti-neuroinflammation in glial cells and neuroprotection on APPswe, PSEN1dE9 (APP/PS1 Tg) mice remain unclear. In this study, we found that inhibition of H3R by thioperamide reduced the gliosis and induced a phenotypical switch from A1 to A2 in astrocytes, and ultimately attenuated neuroinflammation in APP/PS1 Tg mice. Additionally, thioperamide rescued the decrease of cyclic AMP response element-binding protein (CREB) phosphorylation and suppressed the phosphorylated P65 nuclear factor kappa B (p-P65 NF-κB) in APP/PS1 Tg mice. H89, an inhibitor of CREB signaling, abolished these effects of thioperamide to suppress gliosis and proinflammatory cytokine release. Lastly, thioperamide alleviated the deposition of amyloid-ß (Aß) and cognitive dysfunction in APP/PS1 mice, which were both reversed by administration of H89. Taken together, these results suggested the H3R antagonist thioperamide improved cognitive impairment in APP/PS1 Tg mice via modulation of the CREB-mediated gliosis and inflammation inhibiting, which contributed to Aß clearance. This study uncovered a novel mechanism involving inflammatory regulating behind the therapeutic effect of thioperamide in AD.

SARS­CoV­2 spike protein­induced host inflammatory response signature in human corneal epithelial cells.

Coronavirus disease 2019 (COVID­19), caused by the severe acute respiratory syndrome coronavirus­2 (SARS­CoV­2), led to an outbreak of viral pneumonia in December 2019. The present study aimed to investigate the host inflammatory response signature­caused by SARS­CoV­2 in human corneal epithelial cells (HCECs). The expression level of angiotensin­converting enzyme 2 (ACE2) in the human cornea was determined via immunofluorescence. In vitro experiments were performed in HCECs stimulated with the SARS­CoV­2 spike protein. Moreover, the expression levels of ACE2, IL­8, TNF­α, IL­6, gasdermin D (GSDMD) and IL­1ß in HCECs were detected using reverse transcription­quantitative PCR and/or western blotting. It was identified that ACE2 was expressed in normal human corneal epithelium and HCECs cultured in vitro. Furthermore, the expression levels of IL­8, TNF­α and IL­6 in HCECs were decreased following SARS­CoV­2 spike protein stimulation, while the expression levels of GSDMD and IL­1ß were increased. In conclusion, the present results demonstrated that the SARS­CoV­2 spike protein suppressed the host inflammatory response and induced pyroptosis in HCECs. Therefore, blocking the ACE2 receptor in HCECs may reduce the infection rate of COVID­19.

CLEC-1 Acts as a Negative Regulator of Dectin-1 Induced Host Inflammatory Response Signature in Aspergillus fumigatus Keratitis.

Purpose: C-type lectin-like receptor-1 (CLEC-1) is a member of the Dectin-1 cluster of pattern recognition receptors (PRRs). It is involved in host immunity, has immunoregulatory function, and supports allograft tolerance. Our study aimed to describe the role of CLEC-1 in response to fungal keratitis, in situ, in vivo, and in vitro. Methods: Quantitative polymerase chain reaction (qRT-PCR) and immunofluorescence were used to detect the expression of CLEC-1 in corneas of patients with Aspergillus fumigatus (A. fumigatus) keratitis. In vitro and in vivo experiments were designed in THP-1 macrophages and C57BL/6 mouse models, respectively. The expression of CLEC-1 in corneas of mice model was determined by qRT-PCR, Western blot, and immunofluorescence. CLEC-1 overexpression in mouse corneas was achieved by intrastromal injection of adeno-associated virus (AAV) vectors. Disease response was evaluated by slit-lamp photography, clinical score, and colony forming unit (CFU). Bioluminescence imaging system image acquisition, myeloperoxidase (MPO) assays, immunofluorescence staining, qRT-PCR, and Western blot were used to investigate the role of CLEC-1. To further define the role of CLEC-1, we used lentivirus vectors to overexpress CLEC-1 or/and Dectin-1 in THP-1 macrophages. Results: The expression of CLEC-1 was increased in corneas of patients with A. fumigatus keratitis. In corneas of mice from the A. fumigatus keratitis model, the expression of CLEC-1 was decreased in the acute inflammatory stage and increased during convalescence. Following Natamycin treatment, CLEC-1 was upregulated in A. fumigatus keratitis mice. Compared with normal C57BL/6 mice, overexpression of CLEC-1 converted the characteristic susceptible response to resistance, as demonstrated by slit-lamp photography and clinical score. In vivo studies revealed decreased MPO levels and neutrophils recruitment and higher fungal load after the upregulation of CLEC-1. Compared with control corneas, CLEC-1 overexpression impaired corneal pro-inflammatory cytokine IL-1ß production. Conclusions: These findings demonstrate that CLEC-1 may act as a negative regulator of Dectin-1 induced host inflammatory response via suppressing neutrophils recruitment and production of pro-inflammatory cytokine IL-1ß production in response to A. fumigatus keratitis.

Nerolidol inhibits the LOX-1 / IL-1ß signaling to protect against the Aspergillus fumigatus keratitis inflammation damage to the cornea.

PURPOSE: Nerolidol, a naturally occurring sesquiterpene has both anti-microbial and anti-inflammatory properties. The current study aims to investigate the antifungal and the anti-inflammatory effects of nerolidol against mouse Aspergillus fumigatus (A. fumigatus) keratitis. METHODS: The minimum inhibitory concentration (MIC) and cytotoxicity tests were used to study the antifungal ability. For in vivo and in vitro studies, the mouse corneas and the human corneal epithelial cells (HCECs) infected with A. fumigatus spores were intervented with nerolidol or phosphate buffer saline (PBS). Thereafter, the effect of the nerolidol on the response against inflammation was analyzed using the following parameters: recruitment of the neutrophils or macrophages and the expression of the lectin-type oxidized low density lipoprotein receptor-1 (LOX-1) and interleukin 1ß (IL-1ß). Techniques used were the slit lamp, immunofluorescence, myeloperoxidase (MPO) detection, quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot. RESULTS: Nerolidol directly inhibits the growth of A. fumigatus. The administration of nerolidol reduced the severity of fungal keratitis with infiltration of fewer inflammatory cells and reduced levels of the LOX-1, as well the anti-inflammatory cytokines such as IL-1ß were reduced compared with the PBS group. Additionally, in vitro studies showed that treatment with nerolidol inhibited the production of the LOX-1 / IL-1ß levels in A. fumigatus stimulated HCECs. CONCLUSION: Nerolidol attenuated the A. fumigatus keratitis inflammatory response by inhibiting the growth of A. fumigatus, reducing the recruitment of the neutrophils and the macrophages, and inhibiting the LOX-1/ IL-1ß signaling.

Protective Effects of Baicalin on Lipopolysaccharide-Induced Injury in Caenorhabditis elegans.

OBJECTIVES: Sepsis-induced inflammation injury and oxidative stress are well known causes of mortality. The anti-inflammatory effects of baicalin have been proposed in a mouse model of experimental sepsis. Here, we investigated its protective effects and associated mechanisms with respect to lipopolysaccharide (LPS)-induced injury in Caenorhabditis elegans. METHODS: Worms were stimulated by LPS (100 µg/mL), with baicalin (1, 10, 100 µmol/L), for 24 h. Animal survival rates and behaviors (reversal and omega turn) were then determined. Further, levels of the inflammatory cytokines interleukin 6 (IL-6), IL-1, and tumor necrosis factor (TNF)-α were detected by enzyme-linked immunosorbent assay. Western blotting was also performed to determine the protein expression levels of Toll-like receptor 4 (TLR4), nuclear factor-κB (NF-κB), Bax, and Bcl-2. The activities of malondialdehyde (MDA) and superoxide dismutase (SOD) contents were determined using corresponding kits. RESULTS: Baicalin (10, 100 µmol/L) improved LPS-stimulated C. elegans survival and rescued behavioral phenotypes. It also suppressed the oxidative stress related to LPS injury by decreasing MDA levels and increasing SOD activity. Moreover, the inflammatory response was inhibited as evidenced by decreased levels of cytokines including IL-6, IL-1, and TNF-α. In addition, baicalin treatment significantly decreased cleaved Bax levels and increased Bcl-2 expression in C. elegans treated with LPS. Simultaneously, the expression of NF-κB and TLR4 was significantly decreased. CONCLUSION: Baicalin treatment protects against LPS-induced injury by decreasing oxidative stress, repressing the inflammatory cascade, and inhibiting apoptosis.

Carnosine protects against permanent cerebral ischemia in histidine decarboxylase knockout mice by reducing glutamate excitotoxicity.

Recently, we showed that carnosine protects against NMDA-induced excitotoxicity in differentiated PC12 cells through a histaminergic pathway. However, whether the protective effect of the carnosine metabolic pathway also occurs in ischemic brain is unknown. Utilizing the model of permanent middle cerebral artery occlusion (pMCAO) in mice, we found that carnosine significantly improved neurological function and decreased infarct size in both histidine decarboxylase knockout and the corresponding wild-type mice to the same extent. Carnosine decreased the glutamate levels and preserved the expression of glutamate transporter-1 (GLT-1) but not the glutamate/aspartate transporter in astrocytes exposed to ischemia in vivo and in vitro. It suppressed the dissipation of Delta Psi(m) and generation of mitochondrial reactive oxygen species (ROS) induced by oxygen-glucose deprivation in astrocytes. Furthermore, carnosine also decreased the mitochondrial ROS and reversed the decrease in GLT-1 induced by rotenone. These findings are the first to demonstrate that the mechanism of carnosine action in pMCAO may not be mediated by the histaminergic pathway, but by reducing glutamate excitotoxicity through the effective regulation of the expression of GLT-1 in astrocytes due to improved mitochondrial function. Thus, our study reveals a novel antiexcitotoxic agent in ischemic injury.