| Background and Purpose: Granuloma formation is the main pathological hallmark caused by Mycobacterium tuberculosis. Granuloma formation enables M.tuberculosis infection becomes localized, and this is conducive to immune cells to clear them. Macrophage recruitment activation, lymphocyte infiltration is a major cellular basis for granuloma formation. Cytokines and chemokines and their receptors involved in the formation of granulomas by modulating macrophage and lymphocyte function, but the specific mechanism is not yet clear. As the body’s defense microbial invasion outside, complement plays an important role in the infection process, but the current relationship between granuloma formation and complement has not been reported. Our experiment was to study the role of Mannan binding lectin-associated serine protease 2(MASP-2) in the formation of granuloma. Predicting the functional domains on MASP-2 and selecting CCP1-CCP2-SP domain to further study its effection on tuberculous granuloma formation, providing new ideas for TB,s immune theropy. Method:1. Using rabbit skin tuberculosis granuloma model to study the influence of r Ad-h MASP-2 on granuloma formation and liquefaction process. Rabbits were divided into three groups with four animals in each group: BCG+PBS, BCG+r Ad and BCG+r Ad-h MASP-2. All animals were injected intradermally at two sites on each flank with 100ml BCG containing 5×106 CFU. BCG+r Ad and BCG+r Ad-h MASP-2 groups were injected subcutaneously 1×108 PFU(200μl) r Ad or r Ad-h MASP-2 on day 4 and day-3, BCG+PBS group subcutaneously 200μl PBS on day 4 and day-3. The lesions were observed every day on their sizes and the liquefaction and healing process. We recorded the time when the lesions formed granulomas, time of ulceration, time of liquefaction peak, onset of healing and time of the lesions healed, and blooding respectively, determine IL-2 and IFN-γ in the serum by ELISA. When the lesions at the stage of liquefaction peak, the number of tubercle bacilli within liquefied caseum was determined. The lesions and normal skin tissues nearby were collected when the lesions healed and were made for pathological sections and subjected to HE staining.2. Download the MASP-2 domain CCP1/2SP, CCP2 SP, SP and C4 protein structures in Protein Data Bank, predicting the functional domains of MASP-2 about cleavage of C4 through Aut Dock. CCP1/2SP, CCP2 SP, SP structure were docking simulation with C4, respectively. Calculate the docking energy(E). Docking mode divided into situ and free docking.3. Constructing eukaryotic expression vector p Sec Tag-2B-CCP1/2SP, p Sec Tag-2B-CCP2 SP, p Sec Tag 2B-SP. All animals were inoculated intranasally with 60ml BCG containing 1×109 CFU to establish granuloma lung infection model. Kunming mice were divided into five groups with five animals in each group:1BCG+p Sec Tag2B-CCP1/2SP; 2BCG+p Sec Tag2B-CCP2SP; 3BCG+p Sec Tag2B-SP; 4 BCG+p Sec Tag2B; 5 BCG+PBS. BCG+p Sec Tag2B-CCP1/2SP, BCG+p Sec Tag2B-CCP2 SP, BCG+p Sec Tag2B-SP and BCG+p Sec Tag2 B were injected with 25 ug vector by tail vein twice a week. BCG+PBS injected equal volume of PBS(200μl). 3 weeks later, mice were sacrificed and detection: 1 Lungs stained with hematoxylin-eosin, and the figures were collected from the optical microscopic imaging system. We calculated granuloma area by Image-Pro Plus 6.0, and analyzed the percentage through IBM SPSS Statistics 18.0 software. The acid-fast stain of Ziehl-Neelsen was used to define BCG. 2 Lungs, homogente of mice cultured on 7H10 solid medium, counted the single colony of BCG. 3 We detected CD4+ T cells, CD8+ T cells, CD4+PD1+ T cells, CD8+PD1+ T cells, CD4+Tim3+ T cells, CD8+Tim3+ T cells by the Flow cytometry. Result:1. Compared with the BCG+r Ad group, the volume of skin granuloma formation in BCG+r Ad-h MASP-2 group(449.15±117.20 mm3) increased significantly(P=0.001), the amount of viable BCG(3.61×104±1.44×104 CFU/g) was significantly reduced(P <0.001).2. Compared with BCG+r Ad group, serum IL-2(granulomatous stage: 9.90±1.41 pg/ml; liquefaction peak: 12.76±1.52 pg/ml) and IFN-γ(granulomatous stage: 78.81±6.01 pg/ml; liquefaction peak: 109.37±20.52 pg/ml) levels were significantly higher in BCG+r Ad-h MASP-2 group(P <0.05).3. If docking mode is situ docking, CCP1/2SP domain and C4 bind more solid, and with the binding energy E is-260.8; If docking mode is free dockingf, SP domain and C4 bind more solid, the energy E is-440.8, but its docking site changes.4. Comparison with BCG+p Sec Tag2 B group, the percentage of granuloma area in BCG+p Sec Tag2B-CCP1/2SP group(35.21±21.91) was significantly increased(P=0.036), but the number of viable BCG bacilli(1.71×104±1.91×104 CFU/g) did not significantly decreased(P=0.912).5. Comparison with BCG+p Sec Tag2 B group, in BCG+p Sec Tag2B-CCP1/2SP group, the total ltmphocytes, CD4+ T cells(4.58×104±6.15×104), CD8+ T cells(2.61×104±5.50×103), CD4+PD1+ T cells(1.10×103±3.24×102), CD4+Tim3+ T cells(2.18×103±1.08×103), CD8+PD1+ T cells(3.66×102±2.05×102), CD8+Tim3+ number T cells(5.43×103±1.93×103) were obviously increased(P <0.05). Conclusion:1. MASP-2 can promote the formation of tuberculous granulomas on rabbit skin, and reduce the TB load in liquefied material.2. MASP-2 can promote the secretion of IFN-γ and IL-2 in BCG-infected rabbit, suggesting that MASP-2 can induce BCG infection Rabbit T cell immunity to Th1-type cell development.3. By the molecular docking, we predict that the domains which MASP-2 binding and functional with C4 is CCP1/2SP segment.4. MASP-2-CCP1/2SP can promote the formation of the granuloma in the lung of BCG injected mice.5. MASP-2-CCP1/2SP can increase the number of CD4+ T cells and CD8+ T cells, promote the accumulation and activation of lymphocytes, and participate in granuloma occurrence and development.6. MASP-2-CCP1/2SP can increase the number of CD4+PD1+ T cells, CD4+Tim3+ T cells, CD8+PD1+ T cells, CD8+Tim3+ T cells in BCG infected mice lung tissue, suggesting CCP1/2SP play an important role in protective immunity responses, while in order to balance of lung immune, suppression immune response also has been induced. |
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