The Analysis Of Expression Spectrum Of Intrapulmonary TREMs Family And Protective Effects Of TREM-2and Regulatory Effect Of Vasoactive Intestinal Peptide In ALI

Acute lung injury (ALI) is a common pathway for many respiratory diseases. The progress of ALI can be divided into three phases:(Ⅰ) exudative phase characterized by cytokine production and increased inflammation (Ⅱ) proliferative phase and (Ⅲ) fibrotic phase. Infections caused by Gram-negative bacteria is a major risk factor for ALI, the lipopolysaccharide (LPS) of external membrane can activate a variety of inflammatory cells such as macrophages and neutrophils, which released a large number of inflammatory factors, and inflammatory self-limiting mechanism was broken, inducing the excessive inflammatory response, and thus resulting in respiratory distress, non-cardiogenic pulmonary edema and hypoxemia, etc. Therefore, it is important to search the therapeutic target which can cut off the inflammation start and automatic waterfall response in ALI early, and promote lung repair.Alveolar macrophages (AM) is one of the oldest and largest cells which expose to antigen in lungs, and it play an important role in starting and maintaining of early inflammation. AM as phagocytes phagocytosis which can kill and digest microorganisms, and involving in multiple stages of the adaptive immune response, its functional status can affect the occurrence and development of ALI. A variety of pathogen recognition receptors such as Toll-like receptors (TLR), chemotactic factor receptor and the scavenger receptor are express on the AM surface, and these receptors can transmit immunomodulatory signals into intracellular, and they are one of the main factors for inflammation started in ALI early. Lung fibroblast can secrete the precursor of extracellular matrix, is one of the important effector cells involving in tissue repair of ALI.Triggering receptors expressed on myeloid cells (TREMs) is IgG-like receptors as natural killer cell receptor. TREMs family has six recognized members, and TREM-1, TREM-2, TREM-3, TREM like receptor-1(TLT-1), TLT-2and TLT-4are the membrane-type receptor, and sTREM-1, sTREM-2and sTLT-1are soluble receptor. TREM-2is a immune inhibitory receptors, which can induce chemokine expression in dendritic cells around and ultimately play a role in regulating the function of dendritic cells, and inhibit the TLR ligand activation of macrophages and promote phagocytosis of bacteria by bone marrow-derived macrophage, it suggest that TREM-2can regulate the severe sepsis and chronic inflammation.We have observed that TREM-2express in AM and lung fibroblasts. Therefore, in-depth study of effect of TREM-2on the inflammation startup and apoptosis of AM and fibroblast, it will clear the role and status of TREM-2in the early stage of ALI, and elucidate the pathogenesis of ALI and screen the new therapeutic targets.Vasoactive intestinal peptide (VIP) is one of the most abundant nerve regulatory peptides in lungs, which can enhance migration of bronchial epithelial cells and dilate of blood vessels, and relax of airway smooth muscle, and scavenge oxygen free radicals and anti-apoptosis. Whether pulmonary VIP regulates the expression of TREM-2, and inhibits inflammatory response and promote damage repair in ALI.Object:To analyze the changes of pulmonary TREMs family expression profile, to explore TREM-2function and the VIP on the regulation of TREM-2expression of the lungs in ALI.Methods:(1) Kunming mice were sensitized to LPS by inmmunization. LPS was administrated into the peritoneal cavity of mouse (15mg/kg). Buxco system was used to monitor the changes in respiratory function parameters of rats in ALI. HE staining of mouse lung was observed to judge the pathological changes. TREM-1, TREM-2, TREM-3, TLT-1, TLT-2, and TLT-4expressions in lung were detected by RT-PCR and Western Blot. Expressions of TREM-1, TREM-2, TREM-3, TLT-1, TLT-2, and TLT-4in AM or fibroblast or bronchial epithelial cells or alveolar type II epithelial cells were detected by RT-PCR and flow cytometry.(2) pEGFP-N1/TREM-2recombinant plasmid was transfected into AM, and the transfection and expression of TREM-2recombinant plasmid were detected by fluorescence microscopy and RT-PCR and flow cytometry. Effects of TREM-2on the TNF-a and IL-10expressions in LPS-stimulated AM were tested by RT-PCR and ELISA. Effect of TREM-2on the NF-κB expression in LPS-stimulated AM was detected by RT-PCR, and nuclear translocation of NF-κB was tested by immunofluorescence.(3) Flow cytometry surgery and PI staining detect the effect of TREM-2on the fibroblast apoptosis, and the expressions and activity caspase3, caspase8and caspase9were tested by RT-PCR and apoptotic protein activity assay kit.(4) Construction of the pGC-FU-recombinant lentivirus of the VIP and infected Kunming mice, effect of VIP on the TREM-2expression in mouse lungs of ALI was analyzed by RT-PCR and Western Blot, RT-PCR and flow cytometry detect the effect of VIP on the TREM-2expression in LPS-actived macrophages and its mechanism.Results:1. Profiling analysis of TREMs family in ALI(1) Buxco detection shows that the respiratory function of LPS-stimulated mice was depressed, such as respiratory frequency (RF) and tidal volume (TV) and dynamic compliance (Cdyn) were dropped, but total pulmonary resistance (TPR) were increased (P<0.05). The alveolar space of mice was filled with white blood cells and alveolar macrophages, and the alveolar septum of lung was destructed.(2) TREM-1, TREM-2, TREM-3, TLT-1, TLT-2and TLT-4express in the lung of normal mice. Compared with normal group, expressions of TREM-1, TREM-3, TLT-1, TLT-2and TLT-4were increased in ALI group, but TREM-2mRNA expression was decreased (P<0.05). The analytical results of Western Blot were same as the RT-PCR results.(3) RT-PCR detection results showed that:TREM-2express in resting fibroblasts, alveolar type Ⅱ epithelial cells and bronchial epithelial cell, but no TREM-1, TREM-3, TLT-1, TLT-2and TLT-4mRNA expressions. The resting AMs express TREM-1, TREM-3, TREM-2, TLT-1, TLT-2and TLT-4mRNA, and TREM-1, TREM-3, TLT-1, TLT-2and TLT-4mRNA expressions of LPS-activated AM were elevated, but TREM-2mRNA expression decreased (P<0.05). The results of Flow cytometry detection were consistent with the above RT-PCR. 2. The build of pEGFP-Nl/TREM-2recombinant plasmid and effects of TREM-2on inflammation start-up and zoom effects of AM(1) Successfully build pEGFP-N1/TREM-2recombinant plasmid and it was transfected into AM. Fluorescence microscopy analysis showed that the transfection efficiency was about40%. Real-time PCR analysis and flow cytometry results show that TREM-2mRNA expression of AM in pEGFP-N1/TREM-2recombinant plasmid group was significantly more than pEGFP-N1plasmid group (P<0.01).(2) RT-PCR analysis showed that TNF-a mRNA expression significantly increased in LPS-stimulated AM. Compared with LPS+pEGFP-N1plasmid group, TNF-a mRNA expression of AM in LPS+pEGFP-Nl/TREM-2plasmid group was significantly reduced. ELISA results showed that TNF-a protein content of AM (260.64±47.58) pg/mL in LPS+pEGFP-N1/TREM-2plasmid group was lower than the LPS+pEGFP-N1plasmid group (352.16±43.82) pg/mL (P<0.05).(3) RT-PCR detection results showed that:Compared with LPS+pEGFP-Nl plasmid group, IL-10mRNA expression of AM in LPS+pEGFP-Nl/TREM-2plasmid group was increased. ELISA results showed that IL-10protein content of AM (242.96±63.23) pg/mL in LPS+pEGFP-Nl/TREM-2plasmid group was higher than that in LPS+pEGFP-N1plasmid group (184.38±34.33) pg/mL (P<0.05).(4) NF-κB mRNA expression of AM in LPS group was significantly higher than Control group, and NF-κB mRNA expression of AM in LPS+pEGFP-N1plasmid group was significantly higher than that in pEGFP-Nl plasmid group, but NF-κB mRNA expression of AM in LPS+pEGFP-N1/TREM-2plasmid group was lower than that in LPS+pEGFP-N1plasmid group (P<0.05). Compared with Control group, NF-κB nuclear translocation of AM in LPS-stimulated AM increased significantly, and NF-κB nuclear translocation of AM in LPS+pEGFP-N1/TREM-2plasmid group was less than that in LPS+pEGFP-N1plasmid group.3. Effects of TREM-2on the apoptosis of mouse fibroblasts (1) Real-time PCR analysis showed that TREM-2mRNA expression of fibroblast in pEGFP-Nl/TREM-2recombinant plasmid group was significantly higher than that of pEGFP-Nl plasmid group after plasmid transfected for24h (P<0.01).(2) Flow cytometry results showed that the Gl percent of fibroblast in pEGFP-N1plasmid group (70.88%±1.17%) was higher than that in pEGFP-N1/TREM-2recombinant plasmid transfection group (67.13%±1.35%), and apoptosis rate of fibroblast in pEGFP-N1plasmid group (0.96%±0.16%) more than pEGFP-N1/TREM-2recombinant plasmid transfection group (0.66%±0.05%). PI staining results showed that the apoptosis rate of fibroblast in LPS group was significantly increased, and the apoptosis rate of fibroblast in pEGFP-Nl/TREM-2recombinant plasmid group (23.67%±8.08%) was significantly less than that in LPS+pEGFP-N1plasmid group (59.33%±17.34%)(P<0.01)(3) Compared with Control group, the caspase8, Caspase3and caspase9mRNA expressions of fibroblast in LPS group were significantly elevated, and the caspase8, Caspase3and caspase9mRNA expressions of fibroblast in LPS+pEGFP-N1/TREM-2plasmid group were lower than that in LPS+pEGFP-Nl plasmid group (P<0.05). The activity of caspase8, Caspase3and caspase9of fibroblast in LPS group were more than that of Control group, but the activity of caspase8, Caspase3and caspase9of fibroblast in pEGFP-N1/TREM-2recombinant plasmid group were weak than that in pEGFP-N1plasmid group (P<0.05).4. Regulation of VIP on the TREM-2expression of lungs in ALI(1) pGC-FU-VIP recombinant lenti viral was successfully constructed, and the fluorescence microscopy result show that pulmonary green fluorescence intensity is positively correlated with the titer of pGC-FU-VIP recombinant lenti viral. VIP mRNA expression in ALI group was significantly increased, and VIP mRNA expression of pGC-FU-VIP+ALI group was higher than ALI group and pGC-FU+ALI group (P<0.01).(2) Compared with normal group, TREM-2mRNA expression of lungs in ALI group was decreased, and VIP increased the TREM-2mRNA expression of lungs in ALI group (P<0.01). Different concentrations (10-10,10-9,10-8,10-7and10-6mol/L) of VIP has no significant effect on the resting macrophages, and TREM-2mRNA expression of macrophages was no significant change at different time points (at0,2,4,6,12and24h) after VIP pretreatmentand. VIP increased the TREM-2mRNA expression of the LPS-stimulated macrophages in dose-and time-dependent. Flow cytometry showed that VIP has no effect on TREM-2expression of resting macrophages, but upregulate the TREM-2expression of LPS-stimulated macrophage(P<0.01).(3) RT-PCR analysis showed that VIP receptor antagonist against the regulation of VIP on TREM-2mRNA expression in the LPS-stimulated macrophages (P<0.05). Flow cytometry showed that:compared with LPS+VIP group, TREM-2protein expression of macrophages in LPS+VIP+VIP receptor antagonist group was decreased (P<0.05). Flow cytometry showed that NF-κB blockers can elevated the mean fluorescence intensity of TREM-2in LPS-activated macrophages. Compared with LPS+VIP group, the signal blocking agents H-89and PD98059can reduce the TREM-2expression of macrophages (P<0.01).(4) JASPAR and IFTI databases show that API and NF-κB may be the core transcription regulation factor of TREM-2expression, and Real-time PCR analysis showed that VIP can increase API mRNA expressions in LPS-stimulated macrophages. Flow cytometry showed that NF-κB blockers can elevated the mean fluorescence intensity of TREM-2in LPS-activated macrophages.Conclusions:1. TREM-1, TREM-3, TLT-1, TLT-2and TLT-4express in AM, and their expressions of lungs were increased in ALI. TREM-2mainly expressed in AM, fibroblasts, bronchial epithelial cells and alveolar type II epithelial cells, and its expression was decreased in ALI.2. TREM-2can inhibit NF-κB activation of LPS-stimulated AM, affect the activation of AM and then cut off the start and amplification of the early inflammatory.3. TREM-2can inhibit the death receptor and mitochondria-mediated apoptosis pathway, and reduce apoptosis of LPS-stimulated lung fibroblasts, to maintain the dynamic equilibrium of fiber cell number and function, and in favor of a timely repair for injury lungs.4. VIP through PKA and ERK signaling pathways up-regulate the expression of the core transcription factor API, and restrain activation of NF-κB, thereby increasing the generation of TREM-2, participate in the lung tissue injury and repair.