| Nontypeable Haemophilus influenzae(NTHi) is the nonencapsulated Haemophilus influenzae. It is a major opportunistic pathogen causing lower respiratory tract infections such as otitis media in children and chronic obstructive pulmonary disease(COPD) in adults. The lower respiratory tract infected by NTHi is associated with acute exacerbations of COPD. The infections induced by NTHi are related to the deficiencies of innate immune function, airway barrier destruction and lung damage. The previous studies showed that inflammatory response in airway after NTHi infection was triggered by the activation of pro-inflammatory cytokines including IL-1β, CXCL-2 and TNF-α, which was regulated by mitogen-activated protein kinases(MAPK) and NF-κB signals through Toll-like receptors(TLRs). Recently, NOD-like receptors(NLRs) are reported to play an important role in innate immune response and are essential to defend against pathogen infection. NLRP3 inflammasome can activate Caspase-1 and promote the maturity and release of the downstream pro-inflammatory cytokines such as IL-1β and IL-18 so that it participants in the inflammatory response. In order to induce the pro-inflammatory response and antibacterial reactions, Caspase-1 is one important component of NLRP3 inflammasome which activates Caspase-1 and cleaves pro-IL-1β and pro-IL-18 to be the mature form after the inflammasome automated assembly of inflammasome. Although the activation of NLRP3 inflammasome and Caspase-1 in innate responses induced by Listeria monocytogenes and Staphylococcus aureus, the mechanism of NTHi and Caspase-1 in respiratory tract infections has less characterized. Therefore, to investigate the expression of Caspase-1 in the host infected by NTHi, a model in vitro of airway infection was firstly built by the human alveolar epithelial cells A549 infected with different concentrations of NTHi and the survival and apoptosis of A549 infected by NTHi were assessed. The expression of the downstream signals Caspase-1 and IL-1 family members of NLRP3 inflammasome were analysed by molecular biology. At the same time, the expression of cytokines IL-6 and TNF-α were tested. Next, the mouse were stimulated with different concentrations of NTHi to build a model in vivo. And the damage of airways and lungs were assessed by HE and the expression of Caspase-1 were analysed by IHC. Moreover, the expressions of Caspase-1, IL-1β and NF-κB m RNA were tested though RT-PCR. The results showed that the periods of NTHi mid-growth were between 9 and 13 h. The linear relationship between the concentrations of NTHi and OD600 was Y=0.586x+8.015. The results in vitro showed the infection of NTHi decreased the survival rates of cells, increased the numbers of apoptosis and cell death also upregulate the expression of the inflammatory cytokines IL-6 and TNF-α. In addition, the expression of IL-1β and IL-18 of the A549 cells infected by NTHi increased in a dose-dependent manner. However, when the number of NTHi was 1×108 CFU/m L, the expression of Caspase-1 was inhibited and the expressions of IL-1β and IL-18 continued increasing. The results in vivo demonstrated that alveolar septurn of infected mice were broken and plenty more of inflammatory cells were recruited. The pathologic change of tissues was associated with the concentrations of NTHi. Moreover, the protein and m RNA expression of Caspase-1, NF-κB and IL-1β in the airways and lungs of mice infected by NTHi were significantly upregulated. The overall results indicated that the infection of NTHi decreased the survival rates of A549 cells and increased the apoptosis even cell death. The infection of NTHi enhanced the inflammatory responses and activated the expression of Caspase-1 and the downstream signals IL-1β and IL-18. Additonally, the results of the lungs and airways of the mice infected by NTHi showed the significant pathological change. The expression of Caspase-1 may be associated with the severity of respiratory mucosa damage. |
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