IL-12 P40(-/-)小鼠感染呼吸道合胞病毒后肺部的免疫炎症反应

The lung inflammatory and immune responses in IL-12 P40 (-/-) mice infected with RSV

目的探讨白细胞介素_12(IL_12)在呼吸道合胞病毒(RSV)感染中对机体免疫炎症反应的调节作用。方法对IL_12P40基因敲除小鼠[IL_12P40(_/_)]和BALB/c野生型小鼠(WT)气道灌注RSV(2×106PFUs/小鼠)致感染,在感染后第3天和第7天分别检测气道灌洗液中总炎症细胞的数量和分类、肺组织中的CD3+细胞计数、气道粘液分泌水平及Thl、Th2细胞因子的变化。结果IL_12P40(_/_)小鼠感染RSV后尤其在感染后第7天BALF中总炎性细胞数增加达(136.0±8.8)×107/L,显著高于WT小鼠[(88.8±15.2)×107/L],其中以淋巴细胞升高更为明显;肺组织免疫组化染色显示在小气道和小血管周围CD3+细胞浸润在IL_12P40(_/_)小鼠感染7d组为(113.1±12.3)/10个×400视野,显著高于WT感染7d组为(66.8±7.0)/10个×400视野;大气道粘膜粘液分泌在IL_12P40(_/_)小鼠感染7d组AB/PAS染色阳性物质密度为(0.76±0.12)nl/mm2,显著高于WT小鼠感染7d组的(0.45±0.07)nl/mm2。IL_12P40(_/_)小鼠感染RSV后第7天肺组织匀浆上清中Th2细胞因子IL_5为(3.11±1.05)pg/100μg总蛋白,显著高于WT小鼠的(0.87±0.46)pg/100μg总蛋白,P<0.05;IL_13为(0.32±0.08)pg/100μg总蛋白,显著高于WT小鼠的(0.23±0.03)pg/100μg总蛋白,P<0.05;而Thl细胞因子IFN_γ在这两种小鼠均升高,分别为(6.60±1.63)pg/100μg总蛋白和(5.56±0.85)pg/100μg总蛋白,但相互间差异无显著性,P>0.05。IL_18在IL_12P40(_/_)小鼠感染RSV后第3天显著升高。用单克隆抗体阻断IL_18可以进一步提高IL_12P40(_/_)小鼠感染RSV第7天时气道的炎症和粘液分泌水平,同时也进一步促进IL_13的分泌,但不影响IFN_γ的分泌。结论IL_12缺乏可以加重RSV感染肺部炎症、粘液分泌和Th2免疫反应,IL_18的缺乏对此有协同作用,但两者均不影响IFN_γ的分泌水平。

Objective To explore a possible role for IL-12 during acute RSV infection. Methods IL-12 P40 gene knock-out mice [IL-12P40 （-/-）] and wild-type （WT） mice were intratracheally infected with RSV （2 × 10^6 PFUs/ mouse） . The total inflammatory cells and different types of inflammatory cells in BALF stained with HE, CD3^＋ cells in lung tissue stained with immunohistochemical method, mucus in the airway stained with AP/PAS method, and Thl and Th2 cytokines including IL-5, IL-13, IL-18 and IFN-γ in supernatant fluid of lung tissues were detected in both of IL-12 P40（-/-） mice and WT mice on day 3 and day 7 after RSV infection, respectively. Results Total inflammatory cells[（136.0 ± 8.8） × 10^7/L], lymphocytes [ （69.98 ± 6.8） × 10^7/ L], and neutrophils[ （13.1 ± 2.3） × 10^7/L] in BALF were significantly higher in IL-12 P40（-/-） mice than those in WT mice [ （88.8 ± 15.2） × 10^7/L, （32.9 ± 6.1）× 10^7/L and （ 18.9 ± 3.9） × 10^7/L ] on day 7 after RSV infection （P〈 0.01 for all）. CD3^＋ cells [ （113.1 ± 12.3）/× （400 with 10 vision fields] in lung tissue increased obviously in IL-12 P40 （-/-） mice comparing to that in WT mice [ （66.8 ± 7.0）/× 400 with 10 vision fields] on day 7 after RSV infection （P〈 0.01）. Mucus production in the airway was markedly increased in IL-12 P40 （-/-） mice than that in WT mice on day 7 after RSV infection （P〈0.01）. The levels of Th2 cytokines IL-5 [ （3.11 ± 1.05） pg/100μg total protein ] and IL-13 [ （0.32 ± 0.08） pg/100μg total protein ] in the lung tissue were higher in IL-12P40 （-/-） mice than those in WT mice on day 7 after RSV infection （P〈0.05 for all）. The level of IFN-γ in lung tissue increased in either IL-12 P40 （-/-） mice or WT mice on day 7 after RSV infection and without significant difference between them （P 〈0.05）. The levels of IL-18 [ （5.51 ± 0.25） pg/100μg total protein] in the lung tissue were significantly higher in IL-12P40（-/-）mice than those in WT mice [ （3.19±0.37） pg/100μg total protein] on both of day 3 and day 7 after RSV infection （（P 〈0.01 for all）. Furthermore, when IL-18 was blocked with monoclone antibody, the increased level of IL-13 and production of mucus were observed in IL-12P40（-/-） mice on day 7 after RSV infection. However, the level of IFN-7 production was not significantly affected in IL-18 blocked IL-12P40（-/-） mice. Conclusion These findings indicate that IL-12 and/or IL-18 deficiency can increase lung inflammatory and Th2 immune responses to RSV infection, but does not affect the production of IFN-γ.