The Possible Mechanism Of Phagocytic Hypofunction Of Alveolar Macrophages In COPD Rat

Background and objectivesChronic obstructive pulmonary disease (COPD), a common disease, is characterized by persistent airflow limitation which developed progressively and associated with an enhanced chronic inflammation response in the airway and the lung to noxious particles or gases. COPD is characterized by the chronic inflammation of airways, lung parenchyma, pulmonary vascular changes and a variety of inflammatory cells (such as neutrophils, macrophages, etc.) involved in the pathogenesis of COPD.More and more data indicate that patients with severe chronic obstructive pulmonary disease (COPD) occur at a high risk of invasive pulmonary aspergillosis (IPA).Aspergillus is an opportunistic pathogen.For people with normal immune function, Aspergillus spores inhaled can be cleared by the innate immune system; however, for those immunocompromised patients, Aspergillus spores inhaled can propagate in the lungs, which may lead to invasive infection and spread through the blood circulation to other organs under certain conditions, or even lead to death. The research data show that COPD is an important risk factor for ICU patients to be infected with Aspergillus. Some scholars believe that high doses of glucocorticoids, merger of the underlying disease, repeated use of broad-spectrum antibiotics, lung function class III or more, malnutrition and other factors may increase the risk of Aspergillosis in patients with COPD, but this view is lack of a large number of related information. Our laboratory pre-clinical research data show that systemic use of corticosteroids in the stable phase, treatment with three or more antibiotics during hospitalization, and antibiotics treatment longer than10days were independent risk factors associated with IPA. The mechanism of increased susceptibility for Aspergillus in COPD patients are not clear, however, it may be related to decreased immune function.A large number of domestic and foreign studies have shown that long-term smoking may affect AM function and then lead to pulmonary immune dysfunction, consequently, the incidence of bacterial colonization of the respiratory system and lung infections in patients with COPD will increase. The alveolar macrophage is the body’s first line of defense, and plays an important role in the process of removal of conidia of Aspergillus fumigatus. Macrophages clear Aspergillus spores by phagocytosis which is particularly important as the first step of the cleanup process and killing effect. The phagocytosis of macrophages is dependent on the surface PRRs which identificate Aspergillus spores. Among all relevant PRRs, TLRs have a closer relationship with Aspergillus conidia than others. Among TLRs, TLR2is the most closely related to Aspergillus fumigatus.It can identify PAMP of Aspergillus fumigatus, such as beta-glucan, keratin, peptidoglycan, mannose proteins and other cell wall components, and then activate NF-κB and other transcription factors through MyD88adapter protein or non-MyD88myeloid differentiation protein, causing secretion of cytokines, such as IL-1, IL-6, IL-10, TNF-alpha and so on, and promoting the bactericidal and phagocytic activity of macrophages. Meier et al. have confirmed that recognition of the murine macrophage cell toAspergillus spores depends on TLR2. Further in vitro studies suggest that macrophage recognize Aspergillus spores through TLR2etc, and then lead to the production and release of proinflammatory cytokines, while raising the neutrophils to the site of infection.In our study, the rat model of COPD was established with smoke exposure and LPS intrathacheal instillation in the COPD group and evaluated by pathology.Based on the successful replication of COPD rat model, we studied the characteristics of the COPD rat alveolar macrophages to release inflammatory factors and its possible regulatory mechanism; we also research the TLR2expression of alveolar macrophages in COPD rat.Methods1Establishment and identification of chronic obstructive pulmonary disease rat model1.1Establishment of COPD rat model:The animal model of COPD was established with smoke exposure and LPS intrathacheal instillation for one month. Animals were exposed to the smoke45minutes a time and three times a day for30days (except for d9and d19). Additionally,200μL lipopolysaccharide (1mg/mL) was administered intratracheally on days9and19. And control group were injected with an equivalent amount of sterile saline.1.2Identification of COPD rat model:On day30, animals were sacrificed. The differential cell count in the broncho alveolar lavage fluid (BALF) was analyzed. The right lung tissues were taken down and identified by histological detections after being fixed in paraformaldehyde, pathological section and HE staining.1.3The data were statistically analyzed using SPSS13.0statistical software. Measurement data are given as means±SD, two-independent-samples t test analysis was performed if measurement data obeyed normal distribution, if not, Welch approximate t-test was performed. And a=0.05is significant difference test standards.2The roll of Alveolar Macrophages in airway inflammation of COPD rats2.1Establishment of COPD rat model was identical to1.1.2.2Isolation and identification of the alveolar macrophage:The lungs of the rats were lavaged with sterile saline. The cells were collected in the BALF followed by centrifugation. Cells obtained were suspended in1640medium and grown at37℃in5%CO2for2h, the nonadherent cells were removed, the adherent cells were followed by immunofluorescence assay in CD68monoclonal antibody. Alveolar macrophages display green fluorescence.2.3Determination of the inflammatory factors in the culture supernatant: Isolation and identification of the alveolar macrophage was identical to2.2. The supernatant was collected, concentrations of TNF-alpha, MIP-2, IL-10and IL-1βwere measured by ELISA.2.4Determination of phagocytic rate and phagocytic index of Alveolar macrophage phagocytizing Aspergillus spores:The cells were collected in the BALF followed by centrifugation. Cells obtained were seeded in the six-well plates and grown at37℃in5%CO2for2h,then the supernatant were removed, and the six-well plates were added20μL Aspergillus spore suspension and filled with medium up to1mL, and then incubated at37℃,5%CO2for2h. The supernatant was collected, and cells were stained at slide by PAS staining. Finally, we calculated the phagocytic rate and phagocytic index. Phagocytic rate=(number of macrophages phagocytizing spores/the total number of macrophages being counted)×100%, phagocytic index=the total number of spores being phagocytized by macrophages/number of macrophages phagocytizing spores.2.5Determination of NF-κB expression of Alveolar macrophages:Cytoplasmic and nuclear proteins in the alveolar macrophages were extracted from Alveolar macrophages.NF-KB expression was evaluated by Western blot.2.6The data were statistically analyzed using SPSS13.0statistical software. Measurement data are given as means±SD, two-independent-samples t test analysis was performed if measurement data obeyed normal distribution, if not, Welch approximate t-test was performed. And α=0.05is significant difference test standards.3TLR2expression of AMs of COPD rat model3.1Establishment of COPD rat model was identical to1.1.3.2Isolation and treatment of the alveolar macrophage:Cells were collected after centrifugation of BALF, and cultured in RPMI1640medium,37℃,5%CO2for2h. Then nonadherent cells were removed.And then, Pam3CYK4(1ug per well,1mg/mL), Aspergillus spores (2.5×106spores per well) and1mL of saline were respectively added to each group. Finally, cells were collected after4h incubation.3.3TLR2expression of AMs:The total RNA and total protein were respectively extracted from AMs. TLR2expression of AMs was evaluated by qRT-PCR and Western blot.3.4The data were statistically analyzed using statistical software SPSS13.0. Measurement data are expressed as mean±SD. The main effects and interaction effects were analyed by factorial analysis, and single factor was analyzed by one-way ANOVA, And a=0.05was significant difference test standards.Result1Establishment and identification of COPD rat model1.1COPD rat model was successfully established:Serrated proliferation of bronchial cilia cylindrical epithelium, lodging of cilium and defluvium of epithelium were observed under microscope. Bronchial cavity was filled with neutrophils and mucus accumulation.We can also observed desmoplasia and thickening smooth muscle of bronchial wall, also infiltration of Monocytes and lymphocytes. The lymphoid nodules can be seen around the bronchia. Alveolar structure was chaotic, alveolar wall got gauzier than control group and fractured. Alveolar space expanded, and partially fused into bullae. The scores of bronchitis in the COPD group were significantly higher than those in the control group (4.33±1.16vs.1.33±0.58, P=0.016), the same as mean liner intercepts [(168.77±11.35) μm vs.(93.61±4.16) μrn, P=0.000)]. The total cell count in BALF of the COPD group was significantly higher than that in the control group [(2.34±0.15)×108/L vs.(1.72±0.30)×108/L P=0.002], and the AMs and neutrophils were predominant [(72.00±2.22)%and (18.29±8.34)%].2The roll of Alveolar Macrophages in airway inflammation of COPD rats2.1Concentration of inflammatory factors in the culture supernatant: ELISA results showed that the concentrations of TNF-α and MIP-2in culture supernatant of AMs in the COPD group were significantly higher than those in the control group (P=0.000), while the concentrations of IL-10or IL-1βdid not yield any significant difference between the two groups (P>0.05)2.2Phagocytic rate and phagocytic index of Alveolar macrophage phagocytizing Aspergillus spores: Phagocytosis rate of control group was significantly higher than that of COPD group (P=0.000), while the difference of phagocytic index between control group and COPD group was not statistically significant (P=0.053).2.3NF-kB expression of Alveolar macrophages: The cytoplasmic NF-κB p65expression of AMs in the COPD group was significantly lower than that in the control group (P=0.011), while the nuclear NF-κb p65expression was significantly higher.3TLR2expression of AMs in COPD rat 3.1TLR2gene transcription intensity3.1.1The factorial analysis showed that TLR2gene transcription intensity of the COPD group were higher than the control group, the differences were statistically significant (F=680.60, P=0.000). Compared to these three factors, Aspergillus spores affect TLR2gene transcription intensity most deeply, followed by Pam3CYK4, and the last was saline, the difference was statistically significant (F=353.21, P=0.000). Smoke and saline, Aspergillus spores or Pam3CYK4had an interaction effect (F=17.79, P-0.000), namely, the differences of TLR2gene transcription intensity of the COPD group stimulated by saline, Aspergillus spores or Pam3CYK4were significant than the control group; it deems that smoke can affect TLR2gene transcription intensity of rat alveolar macrophage stimulated by saline, Aspergillus spores, or Pam3CYK4.3.1.2In basic state, TLR2gene transcription intensity of alveolar macrophages of the COPD group was significantly lower than the control group (P=0.000), after dropping Aspergillus spores or Pam3CYK4into each group, TLR2gene transcription intensity of alveolar macrophages of the COPD group was lower than the control group (P=0.000).3.1.3TLR2gene transcription intensity of alveolar macrophage added Aspergillus spores or Pam3CYK4of COPD group was significantly higher than the basal state (P=0.000).TLR2gene transcription intensity of alveolar macrophage added Aspergillus spores or Pam3CYK4of control group was significantly higher than the basal state(P=0.000).3.2The protein level of TLR2in AMs3.2.1The factorial analysis showed that TLR2protein level of the COPD group were higher than the control group, the differences were statistically significant (F=349.58, P=0.000). Compared to these three factors, Aspergillus spores affect TLR2protein level most deeply, followed by Pam3CYK4, and the last was saline, the difference was statistically significant (F=77.08, P=0.000). Smoke and saline, Aspergillus spores or Pam3CYK4had an interaction effect (F=18.07, P=0.000), namely, the differences of TLR2protein level of the COPD group stimulated by saline, Aspergillus spores or Pam3CYK4were significant than the control group; it deems that smoke can affect TLR2protein level of rat alveolar macrophage stimulated by saline, Aspergillus spores, or Pam3CYK4.3.2.2In basic state, the protein level of TLR2in AMs was significantly lower than the control group (P=0.000); after dropping Aspergillus spores or Pam3CYK4into each group, The protein level of TLR2in AMs of the COPD group was significantly lower than the control group (P=0.000).3.2.3The protein level of TLR2in AMs added Aspergillus spores or Pam3CYK4of COPD group was significantly higher than the basal state (.P=0.000). The protein level of TLR2in AMs added Aspergillus spores or Pam3CYK4of control group was significantly higher than the basal state(P=0.000).ConclusionIn this study, we have successfully established COPD rat model with smoke exposure and endotoxin, which performed neutrophils and macrophages-dominated chronic inflammation. Activation of NF-κB p65in AMs, the increased expression level of TNF-alpha, the MIP-2and other proinflammatory cytokine and phagocytic dysfunction may be related to that the expression of TLR2in AMs stimulated by long-term smoke cannot be normally up-regulated.