RESUMO
This study was designed to investigate the analgesic effect of perineural injection of BoNT/A on neuropathic pain induced by sciatic nerve chronic constriction injury (CCI) and possible mechanisms. SD rats were randomly divided into Sham group, CCI group and BoNT/A group. Paw mechanical withdrawal threshold (pMWT) and paw thermal withdrawal latency (pTWL) of each group were detected at different time points after surgery. The expression of myelin markers, autophagy markers and NLRP3 inflammasome-related molecules in injured sciatic nerves were examined at 12 days after surgery. Moreover, C-fiber evoked potential in spinal dorsal horn was recorded. The expression of SNAP-25, neuroinflammation and synaptic plasticity in spinal dorsal horn of each group were examined. Then rats treated with BoNT/A were randomly divided into DMSO group and Wnt agonist group to further explore the regulatory effect of BoNT/A on Wnt pathway. We found that pMWT and pTWL of ipsilateral paw were significantly decreased in CCI group compared with Sham group, which could be improved by perineural injection of BoNT/A at days 7, 9 and 12 after surgery. The peripheral analgesic mechanisms of perineural injection of BoNT/A might be related to the protective effect on myelin sheath by inhibiting NLRP3 inflammasome and promoting autophagy flow, while the central analgesic mechanisms might be associated with inhibition of neuroinflammation and synaptic plasticity in spinal dorsal horn due to inhibiting SNAP-25 and Wnt pathway. As a new route of administration, perineural injection of BoNT/A can relieve CCI induced neuropathic pain probably via both peripheral and central analgesic mechanisms.
Assuntos
Neuralgia , Neuropatia Ciática , Ratos , Animais , Ratos Sprague-Dawley , Doenças Neuroinflamatórias , Constrição , Inflamassomos , Proteína 3 que Contém Domínio de Pirina da Família NLR , Nervo Isquiático/lesões , Analgésicos/farmacologia , Neuropatia Ciática/tratamento farmacológico , Neuropatia Ciática/metabolismo , Neuralgia/tratamento farmacológico , Neuralgia/metabolismo , HiperalgesiaRESUMO
SARS-CoV-2-induced excessive inflammation in brain leads to damage of blood-brain barrier, hypoxic-ischemic injury, and neuron degeneration. The production of inflammatory cytokines by brain microvascular endothelial cells and microglia is reported to be critically associated with the brain pathology of COVID-19 patients. However, the cellular mechanisms for SARS-CoV-2-inducing activation of brain cells and the subsequent neuroinflammation remain to be fully delineated. Our research, along with others', has recently demonstrated that SARS-CoV-2-induced accumulation and activation of mast cells (MCs) in mouse lung could further induce inflammatory cytokines and consequent lung damages. Intracerebral MCs activation and their cross talk with other brain cells could induce neuroinflammation that play important roles in neurodegenerative diseases including virus-induced neuro-pathophysiology. In this study, we investigated the role of MC activation in SARS-CoV-2-induced neuroinflammation. We found that (1) SARS-CoV-2 infection triggered MC accumulation in the cerebrovascular region of mice; (2) spike/RBD (receptor-binding domain) protein-triggered MC activation induced inflammatory factors in human brain microvascular endothelial cells and microglia; (3) MC activation and degranulation destroyed the tight junction proteins in brain microvascular endothelial cells and induced the activation and proliferation of microglia. These findings reveal a cellular mechanism of SARS-CoV-2-induced neuroinflammation.
Assuntos
COVID-19 , SARS-CoV-2 , Humanos , Camundongos , Animais , SARS-CoV-2/metabolismo , COVID-19/metabolismo , Células Endoteliais/metabolismo , Mastócitos/metabolismo , Doenças Neuroinflamatórias , Microglia/metabolismo , Encéfalo/metabolismo , Inflamação/metabolismo , Citocinas/metabolismoRESUMO
SARS-CoV-2 infection-induced hyper-inflammation is a key pathogenic factor of COVID-19. Our research, along with others', has demonstrated that mast cells (MCs) play a vital role in the initiation of hyper-inflammation caused by SARS-CoV-2. In previous study, we observed that SARS-CoV-2 infection induced the accumulation of MCs in the peri-bronchus and bronchioalveolar-duct junction in humanized mice. Additionally, we found that MC degranulation triggered by the spike protein resulted in inflammation in alveolar epithelial cells and capillary endothelial cells, leading to subsequent lung injury. The trachea and bronchus are the routes for SARS-CoV-2 transmission after virus inhalation, and inflammation in these regions could promote viral spread. MCs are widely distributed throughout the respiratory tract. Thus, in this study, we investigated the role of MCs and their degranulation in the development of inflammation in tracheal-bronchial epithelium. Histological analyses showed the accumulation and degranulation of MCs in the peri-trachea of humanized mice infected with SARS-CoV-2. MC degranulation caused lesions in trachea, and the formation of papillary hyperplasia was observed. Through transcriptome analysis in bronchial epithelial cells, we found that MC degranulation significantly altered multiple cellular signaling, particularly, leading to upregulated immune responses and inflammation. The administration of ebastine or loratadine effectively suppressed the induction of inflammatory factors in bronchial epithelial cells and alleviated tracheal injury in mice. Taken together, our findings confirm the essential role of MC degranulation in SARS-CoV-2-induced hyper-inflammation and the subsequent tissue lesions. Furthermore, our results support the use of ebastine or loratadine to inhibit SARS-CoV-2-triggered degranulation, thereby preventing tissue damage caused by hyper-inflammation.
Assuntos
Brônquios , COVID-19 , Degranulação Celular , Mastócitos , SARS-CoV-2 , Traqueia , Animais , Mastócitos/virologia , Mastócitos/imunologia , COVID-19/imunologia , COVID-19/virologia , COVID-19/patologia , Camundongos , Traqueia/virologia , Traqueia/patologia , Brônquios/virologia , Brônquios/patologia , Humanos , Inflamação/virologia , Células Epiteliais/virologia , Modelos Animais de DoençasRESUMO
Mast cells (MCs) are strategically located at the host-environment interface and their non-allergic roles in the immune-surveillance of pathogens have recently gained more attention. However, MC-caused detrimental regulation of immune inflammations can promote viral invasion. Currently, the role of MCs in retroviral infection remains elusive. We have recently proved that human gut MCs could capture and transfer HIV-1 to CD4+ T cells for promoting viral spread; MC-released histamine augments HIV-1-induced functional polarization of dendritic cells to cause immunosuppression via stimulating the differentiation of regulatory T cells. In this study, we used a murine model of MuLV/Friend virus infection to address MC role in acute retroviral infection in vivo. The acute infection of MuLV/Friend virus could be established in C57BL/6 wild type mice, but viral acquisition showed low efficiency in C57BL/6-Kit W - sh/W - sh (Sash) mice which lack MCs. In mechanism, we found that MuLV/Friend virus triggered MC activation for degranulation; MC degranulation subsequently activated the granulocyte-like myeloid derived suppressive cells (G-MDSCs) to inhibit CD8+ T cells- and NK cells-mediated antiviral immune responses. The reconstruction of MCs in Sash mice promoted acute retroviral infection by regulating G-MDSCs functions and antiviral immune responses. Importantly, the administration of MC stabilizers to block cell degranulation elevated antiviral immune response and consequently suppressed retrovirus infection. This study uncovers a specific role of MCs in acute retroviral infection and elucidates the underlying immune-mechanisms. Targeting MCs may provide a novel approach for controlling acute infection by retroviruses.
RESUMO
ABSTRACTHIV-1 latency posts a major obstacle for HIV-1 eradication. Currently, no desirable latency reversing agents (LRAs) have been implicated in the "Shock and Kill" strategy to mobilize the latently infected cells to be susceptible for clearance by immune responses. Identification of key cellular pathways that modulate HIV-1 latency helps to develop efficient LRAs. In this study, we demonstrate that the Wnt downstream ß-catenin/TCF1 pathway is a crucial modulator for HIV-1 latency. The pharmacological activation of the ß-catenin/TCF1 pathway with glycogen synthase kinase-3 (GSK3) inhibitors promoted transcription of HIV-1 proviral DNA and reactivated latency in CD4+ T cells; the GSK3 kinase inhibitor 6-bromoindirubin-3'-oxime (6-BIO)-induced HIV-1 reactivation was subsequently confirmed in resting CD4+ T cells from cART-suppressed patients and SIV-infected rhesus macaques. These findings advance our understanding of the mechanisms responsible for viral latency, and provide the potent LRA that can be further used in conjunction of immunotherapies to eradicate viral reservoirs.
Assuntos
Linfócitos T CD4-Positivos/virologia , Quinase 3 da Glicogênio Sintase/antagonistas & inibidores , HIV-1/crescimento & desenvolvimento , Indóis/farmacologia , Oximas/farmacologia , Ativação Viral/efeitos dos fármacos , Latência Viral/efeitos dos fármacos , Animais , Linfócitos T CD4-Positivos/imunologia , Linhagem Celular Tumoral , HIV-1/efeitos dos fármacos , HIV-1/genética , Células HeLa , Fator 1-alfa Nuclear de Hepatócito/metabolismo , Humanos , Macaca mulatta , Transcrição Gênica/efeitos dos fármacos , Células U937 , Ativação Viral/genética , Latência Viral/genética , Proteínas Wnt/metabolismo , beta Catenina/metabolismoRESUMO
COVID-19 is an immune-mediated inflammatory disease caused by SARS-CoV-2 infection, the combination of anti-inflammatory and antiviral therapy is predicted to provide clinical benefits. We recently demonstrated that mast cells (MCs) are an essential mediator of SARS-CoV-2-initiated hyperinflammation. We also showed that spike protein-induced MC degranulation initiates alveolar epithelial inflammation for barrier disruption and suggested an off-label use of antihistamines as MC stabilizers to block degranulation and consequently suppress inflammation and prevent lung injury. In this study, we emphasized the essential role of MCs in SARS-CoV-2-induced lung lesions in vivo, and demonstrated the benefits of co-administration of antihistamines and antiviral drug remdesivir in SARS-CoV-2-infected mice. Specifically, SARS-CoV-2 spike protein-induced MC degranulation resulted in alveolar-capillary injury, while pretreatment of pulmonary microvascular endothelial cells with antihistamines prevented adhesion junction disruption; predictably, the combination of antiviral drug remdesivir with the antihistamine loratadine, a histamine receptor 1 (HR1) antagonist, dampened viral replication and inflammation, thereby greatly reducing lung injury. Our findings emphasize the crucial role of MCs in SARS-CoV-2-induced inflammation and lung injury and provide a feasible combination antiviral and anti-inflammatory therapy for COVID-19 treatment.
Assuntos
Tratamento Farmacológico da COVID-19 , COVID-19 , Lesão Pulmonar , Doenças dos Roedores , Monofosfato de Adenosina/análogos & derivados , Alanina/análogos & derivados , Animais , Antivirais/farmacologia , Antivirais/uso terapêutico , COVID-19/veterinária , Células Endoteliais , Antagonistas dos Receptores Histamínicos/uso terapêutico , Inflamação/tratamento farmacológico , Inflamação/etiologia , Inflamação/veterinária , Lesão Pulmonar/tratamento farmacológico , Lesão Pulmonar/veterinária , Camundongos , Doenças dos Roedores/tratamento farmacológico , SARS-CoV-2 , Glicoproteína da Espícula de CoronavírusRESUMO
SARS-CoV-2 infection-induced hyper-inflammation links to the acute lung injury and COVID-19 severity. Identifying the primary mediators that initiate the uncontrolled hypercytokinemia is essential for treatments. Mast cells (MCs) are strategically located at the mucosa and beneficially or detrimentally regulate immune inflammations. In this study, we showed that SARS-CoV-2-triggered MC degranulation initiated alveolar epithelial inflammation and lung injury. SARS-CoV-2 challenge induced MC degranulation in ACE-2 humanized mice and rhesus macaques, and a rapid MC degranulation could be recapitulated with Spike-RBD binding to ACE2 in cells; MC degranulation altered various signaling pathways in alveolar epithelial cells, particularly, the induction of pro-inflammatory factors and consequential disruption of tight junctions. Importantly, the administration of clinical MC stabilizers for blocking degranulation dampened SARS-CoV-2-induced production of pro-inflammatory factors and prevented lung injury. These findings uncover a novel mechanism for SARS-CoV-2 initiating lung inflammation, and suggest an off-label use of MC stabilizer as immunomodulators for COVID-19 treatments.
Assuntos
COVID-19/metabolismo , Degranulação Celular , Lesão Pulmonar/metabolismo , Mastócitos/metabolismo , Alvéolos Pulmonares/metabolismo , SARS-CoV-2/metabolismo , Enzima de Conversão de Angiotensina 2/genética , Enzima de Conversão de Angiotensina 2/metabolismo , Animais , COVID-19/genética , Linhagem Celular Tumoral , Feminino , Humanos , Lesão Pulmonar/genética , Lesão Pulmonar/virologia , Macaca mulatta , Masculino , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Alvéolos Pulmonares/virologia , SARS-CoV-2/genética , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/metabolismoRESUMO
Metagenomics next-generation sequencing (mNGS) is increasingly available for the detection of obscure infectious diseases of the central nervous system. However, human DNA contamination from elevated white cells, one of the characteristic cerebrospinal fluid (CSF) features in meningitis patients, greatly reduces the sensitivity of mNGS in the pathogen detection. Currently, effective approaches to selectively reduce host DNA contamination from clinical CSF samples are still lacking. In this study, a total of 20 meningitis patients were enrolled, including 10 definitively diagnosed tuberculous meningitis (TBM) and 10 definite cryptococcal meningitis (CM) cases. To evaluate the effect of reduced human DNA in the sensitivity of mNGS detection, three specimen-processing protocols were performed: (i) To remove human DNA, saponin, a nonionic surfactant, was used to selectively lyse white cells in CSF followed by DNase treatment prior to the extraction of DNA; (ii) to reduce host DNA, CSF was centrifuged to remove human cells, and the supernatant was collected for DNA extraction; and (iii) DNA extraction from the unprocessed specimens was set as the control. We found that saponin processing significantly elevated the NGS unique reads for Cryptococcus (P < 0.01) compared with the control but had no effects for Mycobacterium tuberculosis (P > 0.05). However, detection of centrifuged supernatants improved the NGS unique reads for both TBM and CM compared with controls (P < 0.01). Our results demonstrate that the use of mNGS of centrifuged supernatants from clinical CSF samples in patients with TBM and CM is a simple and effective method to improve the sensitivity of pathogen detection.