Cellular And Molecular Mechanisms Of The Inflammatory Response In Human Lung Tissue Infected With Streptococcus Pneumoniae And Nontypeable Haemophilus Influenzae

Streptococcus pneumoniae (Spn) and nontypeable Haemophilus influenzae (NTHi) are the most common and important pathogens in adults with lower respiratory tract infections such as community-acquired pneumoniae and acute exacerbation of chronic obstructive pulmonary disease (COPD).The pathogenesis of Spn and NTHi is complex and the outcome of these infections depends on bacterial virulence factors and the effectiveness of the host response. The different host cell types, individual host cell receptors and signaling pathways have been shown to be involved in host defense against Spn and NTHi. However, the roles of respiratory epithelial cells and alveolar macrophages in the pulmonary bacterium-induced response and the molecular mechanisms of host cell-bacterium interactions are not fully and clearly demonstrated.Toll-like receptors (TLRs) are a recently identified receptor family. Different TLRs play crucial roles in the immune response by recognizing their distinct pathogen-associated molecular patterns (PAMPs). Among TLRs, TLR2 and TLR4 are implicated in bacterium-associated host immune responses. The downstream mitogen-activated protein kinases (MAPKs) are central signal pathways regulating cellular responses including cell growth, inflammatory cytokine expression, stress reaction and etc. in eukaryocytes.Much of our knowledge about the pathogenesis of Spn and NTHi infections and pulmonary host defense is derived from animal studies on experimental pneumonia, in vitro cell culture models and transaction analysis of clinical specimens, whereas little is known about pathogen-cell interactions and their dynamic development within the human pulmonary compartment. Because lung tissue samples from patients withacute bacterial pneumonia are obviously not available, we developed an acute lung infection model (ALIM), as a bridge to link cell-culture and animal models, to analyze pathogenesis of Spn and NTHi infections within pulmonary compartment, using vital lung specimens from patients undergoing lobectomy due to pulmonary nodules. Histological sections in ALIM which allow for molecular histological and functional studies display a kind of snapshot of the complex mix of events taking place in the course of pulmonary infection diseases. In addition, ALIM compared to chronic persistent infection in COPD patients may help to identify mechanisms of bacterial colonization and inflammation persistence.The rise in antibiotic resistance of pathogens and the limited efficacy of the widely used vaccine urge further efforts to understand the pathogenesis of Spn and NTHi. The demonstration of the cellular and molecular mechanisms in Spn and NTHi infections will provide novel ideas and new therapeutic targets for the prevention and treatment of bacterial infections in the respiratory tract.Part Ⅰ: The establishment of in vitro acute infection models in human lung tissueThe establishment of in vitro acute infection models of Spn and NTHi in human lung tissue was as follows. Vital normal lung tissue at least 5 cm away from pulmonary suspected nodules was used in the experiments. Lung specimens (1cm~3/block) were cocultured with different concentrations of Spn and NTHi. After 4h, 24h and 48h stimulation, supematants were collected and lung specimens were fixed in the newly developed Hepes-Glutamic acid buffer mediated Organic solvent Protection Effect (HOPE)-solution. The lactate dehydrogenase (LDH) levels in the supernatant indicated that the lung cells in ALIM showed time-dependent necrosis in tissue culture and demonstrated that vital lung specimens could be infected with these two pathogens for at least 48 h in our ALIM. Enzyme-linked immunosorbentassay (ELISA) detection showed that Spn and NTHi induced interleukin (IL)-8 release from ALIM in a dose-dependent manner. Total RNA from lung tissue treated by HOPE-solution was intact and not obviously degraded. Reverse transcription-polymerase chain raction (RT-PCR) results showed that IL-8 mRNAexpression in ALIM was upregulated compared to unstimulated lung tissue. β-actin expression in ALIM was easily detected by immunoblot analysis. These results suggested that HOPE-fixative provides an excellent preservation of proteins and antigenic structures. ALIM, as a bridge to link cell-culture and animal models, allows us to analyze a kind of snapshot of the complex mix of events taking place in the course of pulmonary infection diseases on the tissue level. The combination of ALIM and HOPE-fixative provides a new functional model-system for exploring the cellular and molecular pathogenesis of pulmonary diseases.Conclusions:1. In vitro acute infection models of Spn and NTHi in lung tissue were established.2. HOPE-fixative provides an excellent preservation of proteins and antigenic structures and could be applied in the tissue and molecular pathology.Part Ⅱ: Cellular and molecular mechanisms of the inflammatory response in human lung tissue infected with SpnA model of acute Spn infection in lung tissue (ASM) and cell-culture models were used to study the cellular and molecular mechanisms of the pulmonary inflammation induced by Spn. In situ hybridization (ISH) analysis showed that Spn-DNA was detected in 80-90% of alveolar macrophages and 15-30% of alveolar epithelial cells showing the morphologic characteristics of type Ⅱ cells 24-48 h after stimulation. However, bronchial epithelial cells were only sporadically infected (<1%). ASIM was homogenized after extracellular bacteria were killed by antibiotic. Then, Spn was found to grow after tissue homogenates were taken and cultured in sheep blood agar plates for overnight. Immunohistochemistry (IHC) analysis showed that there was no obvious difference in the number of CD68~+ alveolar macrophages between lung tissue treated with Clodronate/liposomes and specimens treated with PBS/liposomes. However, compared with control group, Caspase-3 expression was significantly increased in alveolar macrophages of Clodronate/liposomes-treated lung tissue, followed by the siginificantly decreased release of tumor necrosis factor(TNF)-α from ASIM. These results suggested that Clodronate/liposomes could downregulate Spn-induced pulmonary inflammation by inducing the apoptosis of alveolar macrophages in lung tissue and alveolar macrophages hold an important role in this inflammatory response. Quantitative RT-PCR detection demonstrated that the increased expression of TLR2 and 4 mRN A was observed in infected lung specimens after 24h stimulation. IHC and immunoblot analysis both showed the enhanced phosphorylation of p38 and ERK MAPK in ASIM and infected cell models. TLR-2,4 functional monoclonal antibodies and inhibitors of p38, p44/42 MAPK were adopted to explore the roles of TLRs and MAPKs in Spn-induced inflammatory response in tissue and cell models. ELISA results showed that inhibition of p38 MAPK signaling significantly reduced inflammatory cytokine release (such as IL-8, IL-6, TNF-α) from infected lung tissue in contrast to the blockade of TLR2, TLR4 and ERK MAPK. We further analyzed alveolar macrophages and A549 lung epithelial cells and demonstrated that p38 MAPK is a key element in the inflammatory response to pneumococci.Conclusions:1. Spn may be a facultative intracellular bacterium. Alveolar macrophages and alveolar epithelial cells type Ⅱ may serve as the host cells for acute Spn infection.2. We established in vitro AM-inactivated lung tissue model, which could be used to study the roles of alveolar macrophages in the pulmonary immune response induced by different environmental factors.3. Alveolar macrophages hold an important role in acute inflammatory response in lung tissue infected with Spn.4. Clodronate/liposomes may downregulate Spn-induced pulmonary inflammatory response by inducing the apoptosis of alveolar macrophages via Caspase-3 pathway.5. P38 MAPK is a key signal molecule of pulmonary inflammatory response induced by Spn.Part Ⅲ: Cellular and molecular mechanisms of the inflammatory response in human lung tissue infected with NTHiA model of acute NTHi infection in lung tissue (AHIM) and an A549 cell culture model were used to study the cellular and molecular mechanisms of the inflammatory response in lung tissue infected with NTHi. ISH analysis showed that NTHi-DNA was detected in 70-85% of alveolar macrophages and 15-30% of alveolar epithelial cells showing the morphologic characteristics of type II cells in AHIM 24 h after stimulation. PCR analysis showed that the infection rate of NTHi was 35.7% (15/42) in patients with COPD and ISH results demonstrated that NTHi-DNA was detected in 70-85% of alveolar macrophages and 15-30% of alveolar epithelial cells type II in lung specimens from these COPD patients. The different infection rates of NTHi between acute and chronic infections in lung tissue indicated that lung epithelial cells may serve as the suitable host cells for persistent NTHi colonization. IHC and Immunoblot analysis showed that the phosphorylation of p38 and ERK MAPK was differentially increased in AHIM and A549 cells infected with NTHi. Flow cytometry (FCM) and electrophoretic mobility shift assay (EMSA) detection demonstrated the significantly enhanced the DNA binding activity of NF-kB in AHIM and stimulated A549 cells. Furthermore, the inhibition of p38 MAPK pathway had no effect on nuclear translocation of NF-κB. Using specific inhibitors of p38, p44/42 MAPK and NF-κB, we proved that inhibition of p38 MAPK and NF-κB significantly reduced IL-8 release from infected A549 clles in contrast to blockade of ERK MAPK. Therefore, NTHi induced IL-8 production in A549 cells in a p38 MAPK and NF-κB dependent manner.Conclusions:1. NTHi may be a facultative intracellular bacterium. Alveolar macrophages and alveolar epithelial cells type Ⅱ may serve as the host cells for NTHi infection.2. There are probably different NTHi infection patterns between acute primary infection model and chronic persistent infection in COPD lung tissue. Lung epithelial cells may be the suitable host cells for persistent NTHi colonization in COPD lungtissue.3. NTHi induced the release of inflammatory mediator from A549 cells in a p38 MAPK and NF-κB dependent manner.