The Immuno-response In Respiratory Infection Following NTHi Challenge And The Expression Of Intracellular Pattern Recognition Receptor TLR9

Higher vertebrates have developed two interactive protective systems:the innate and adaptive immune systems. The innate immune system recognizes invading pathogens by sensing pathogen-associated molecular patterns (PAMPs) proteins. The best understood of the PAMPs are the toll-like receptors (TLRs). Some of the TLRs detect a series of molecules produced by bacteria, fungi and virus. TLR9is different from the other TLRs, it locates intracellularly, sensing intracellular non-methylated bacterial DNA.Respiratory infection has relationship with non-typeable Haemophilus Influenza (NHTi), esp. the lung and middle ear infection, with high morbidity and mortality. NTHi is a non-encapsulated gram-negative pleomorphic rod-shaped bacterium which colonizes the upper respiratory tract of the majority of individuals. It is an opportunistic pathogen most frequently occurs in airway surface that have been compromised by obstruction or loss of mucociliary clearance mechanisms. NTHi is traditionally regarded as an noninvasive extracellular bacterium, however, previous observations suggest that NTHi can internalize to eukaryotic cells. It can invade epithelial cells and reside inside escaping from the activity of antibiotics.We incubated NTHi with epithelial cells and monocytes and observed directly the internalization of NTHi under microscopy by using Antibiotics protecting assay(APA). NTHi could produce extracellular infection and also internalized to intracellular with imflammation production. NTHi can be both extracellular and intracellular bacterium. An acute lung NTHi infection model (ALIM) was built and NTHi could be detected inside the lung compartment and alveolar wall by NTHi-specific DNA probe using in situ hybridization (ISH).By quantitative detection of intracellular cytokine level of human epithelial A549cells and monocytes after NTHi stimulation by APA, the intracellular IL-8and/or TNF-a level was less predominant comparing with thosel released extracellularly. ALIM had cytokines release after NTHi stimulation. There was no significant difference in cytokines production between vialble NTHi and inactivated NHTi. Extracted bacterial DNA could induce cytokines release by monocytes and this action dould be blocked by Chloroquine, an endosomal maturation inhibitor.After the stimulation of human epithelial A549cells, monocytes and ALIM by viable NTHi, inactivated NTHi and bacterial DNA, TLR9mRNA could be up-regulated in lung tissues and monocytes by RT-PCR, but not epithelial A549cells. TLR9protein in monocytes after activated by viable NTHi and bacterial DNA could be up-regulated by Fluorescence-activated cell sorting, and this expression could be blocked by Chlolroquine, however, epithelial A549cells had no expression of TLR9protein under the same stimulation. TLR9protein expression in lung tissue was also up-regulated after the stimulation of viable NTHi, bacterial DNA and synthetic ODN M362in ALIM by Western blotting. The signaling pathway of LTR9protein activation by viable NTHi was involved by MAPK p38and ERK p44/42.The results showed that the varies respiratory tract and cells response to NTHi infection was cell type-specific or bacterial-specific, not fully related to the amount of bacterial. NTHi was traditionally considered as extracellular bacterium, our experiments showed that NTHi could invade inside epithelial A549celles and monocytes. NTHi not only resided intracellularly, escaping the killing effects of antibiotics, but also produced some level of imflammation and had cytokines release. The intracellular cytokine level was about10%to30%of the total cytoking level released by monocytes and epithelial cells after NTHi stimulation. Besides the viable NTHi, heat-inactivated or gentamycin-inactivated NTHi could stimulate IL-8/TNF-a in the epithelial cells, monocytes and ALIM. Viable NTHi, bacterial DNA and synthetic ODN had immune effects to TLR9-mediated inflammation in monocytes and the effects could be modified by Chloroquine. TLR9m RNA could be expressed after sitmulation in lung tissue and monocytes but not epithelial cells by RT-PCR. TLR9could be found up-regulated after viable NTHi and bacterial DNA and be blocked by Chloroquine only in monocytes by FACS. TLR9protein expression in lung tissue was also up-regulated after the stimulation of viable NTHi, bacterial DNA and synthetic ODN M362in ALIM by Western blotting.Conclusion:1. NTHi can invade into epithelial cells and monocytes and produce inflammation intracellularly. The intracellular cytokine level was about10%to30%of the total level released by monocytes and epithelial cells after NTHi stimulation. NTHi can invade and reside in epithelial cells and monocytes, and stimulate respiratory tract. This can be a reasonable explanation for the chronic inflammation and recurrent exacerbation of COPD.2. The inflammation caused by inactivated NTHi has no significant difference comparing with that by viable NTHi, otherwise, both NTHi DNA and ODN M362have weak immune-activities.3. Monocytes and lung tissue express TLR9, but human epithelial cells has no sensitivity to TLR9. Human epithelial cell is not immuno-active to the intracellular bacterial.4. Acute lung NTHi infection model built a good platform connecting in vitro and in vivo for the studying of the interaction between bacterial and the host.