Bruton's tyrosine kinase regulates TLR9 but not TLR7 signaling in human plasmacytoid dendritic cells

Plasmacytoid dendritic cells (PDCs) represent a key cell type for both innate and adaptive immunity. PDCs express both TLR7 and TLR9 and the recognition of nucleic acids by these two receptors triggers the production of a large amount of type‐I IFN and the induction of PDC maturation into APCs. This unique feature of PDCs is at the basis of clinical development of both TLR7 and TLR9 agonists for infectious diseases, allergy, cancer, and asthma. However, TLR7 and TLR9 recognition of self‐nucleic acids is linked to many autoimmune diseases including lupus, and a better understanding of the signaling pathways of these two receptors in PDCs is thus important. We have identified Bruton's tyrosine kinase (Btk) as an important player for TLR9 but not TLR7 signaling in human PDCs. Blocking Btk using a specific inhibitor leads to the reduction of all TLR9‐induced responses in PDCs, including cytokine production and expression of costimulatory molecules, while this has no impact on the TLR7 response. This identifies Btk as a key molecule in TLR9 signaling in PDCs and is the first demonstration that the TLR7 and TLR9 pathways can be dissociated in human PDCs.     

Clonal lineage tracing reveals shared origin of conventional and plasmacytoid dendritic cells

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干细胞旁分泌效应在ALI/ARDS治疗中的研究进展

急性肺损伤(acute lung injury,ALI)以及它的严重形式——急性呼吸窘迫综合征(acute respiratorydistress syndrome,ARDS)是危重病人发病和死亡的重要原因之一,最近2个世纪以来,死亡率仍在36%～44%左右。ALI/ARDS的病因众多,发病机制十分复杂,涉及的环节多,受损的靶细胞多,主要涉及的环节有:炎症反应失控、细胞损伤与修复、细胞凋     

PI3K/Akt信号通路上调急性肺损伤大鼠肺泡上皮钠通道表达

目的 探讨PI3K/Akt信号通路对急性肺损伤(ALI)大鼠肺泡上皮钠通道(ENaC)α、β和γ亚基表达的影响.方法 成年SD大鼠随机分为对照组、ALI组(脂多糖)、胰岛素组及渥曼青霉素组,每组5只.观察肺组织病理改变,收集支气管肺泡灌洗液(BALF),测量总肺水含量,RT-PCR和Western blot测定ENaC mRNA和蛋白、p-Akt表达.结果 胰岛素组BALF蛋白含量、髓过氧化物酶(MPO)活性、总肺水含量较ALI组显著减少(P＜0.05),渥曼青霉素组BALF蛋白含量、MPO活性及总肺水含量较胰岛素组显著增加(P＜0.05).ALI组α-、β-和γ-ENaC蛋白表达显著低于对照组(0.33 ±0.06 vs 1.27 ±0.07,0.18±0.04 vs 0.72±0.04,0.37±0.04 vs0.69±0.05)(P＜0.05).胰岛素组蛋白表达α-ENaC(2.19 ±0.04)、β-ENaC(1.18 ±0.07)和γ-ENaC(1.18 ±0.08)显著高于ALI组(P＜0.05).渥曼青霉素组蛋白表达α-ENaC(0.86 ±0.09)、β-ENaC (0.58±0.05)和γ-ENaC (0.59±0.02)显著低于胰岛素组(P＜ 0.05).胰岛素组ENaC mRNA和p-Akt较ALI组显著升高(P＜0.05).渥曼青霉素组ENaC mRNA和p-Akt较胰岛素组显著降低(P＜0.05).结论 激活H3K/Akt通路上调3种ENaC亚基表达,从而清除肺水肿液.     

PI3K/Akt和JAK2/STAT3信号转导通路在SO_2抗大鼠肢体缺血再灌注致急性肺损伤中的作用

目的:探讨PI3K/Akt和JAK2/STAT3信号转导通路在二氧化硫(SO2)抗肢体缺血再灌注(I/R)致急性肺损伤中的作用。方法:应用双大腿根部绑扎止血带复制大鼠双后肢缺血再灌注肺损伤模型。在再灌注前20 min腹腔注射Na2SO3/Na HSO3;在再灌注前1 h静脉注射Stattic或LY294002。应用TUNEL、ELISA、Western blot等方法检测细胞凋亡、细胞因子表达及相关信号通路蛋白表达的情况。结果:与对照组相比,I/R组的MDA及MPO含量、肺系数、细胞凋亡指数、细胞因子表达以及p-STAT3、p-Akt蛋白的水平均显著增高;当应用Na2SO3/Na HSO3后,上述反映肺损伤的各项指标均下降。Western blot检测结果显示I/R后,肺组织中p-STAT3和p-Akt蛋白的水平均明显增加。而应用Na2SO3/Na HSO3后,p-Akt蛋白的水平继续增加,但p-STAT3蛋白的水平却减少(P0.05)。结论:JAK2/STAT3和PI3K/Akt信号通路都参与了SO2抗肢体缺血再灌注致急性肺损伤的作用。JAK2/STAT3通路的活化,能够使I/R损伤加重;相反,PI3K/Akt信号通路的活化,可以使I/R损伤减弱。此外,JAK2/STAT3和PI3K/Akt信号通路之间存在交互作用。     

FcgammaRII expression on follicular dendritic cells and immunoreceptor tyrosine-based inhibition motif signaling in B cells

Immune complexes (IC) initiate immunoreceptor tyrosine-based inhibition motif (ITIM) signaling and inhibit B cell activation by coligating B cell receptor for antigen (BCR) and FcgammaRII. Nevertheless, IC on follicular dendritic cells (FDC) stimulate rapid germinal center (GC) B cell proliferation suggesting that interactions between IC and FDC render IC capable of B cell activation. To understand this, we studied the kinetics of FDC FcgammaRII and complement receptors 1 and 2 (CR1&2) expressions during the GC reaction and determined whether FDC FcgammaRII could bind Fc in IC and block ITIM signaling. Mice were immunized with sheep red blood cells (SRBC), and CR1&2 and FcgammaRII levels in FDC reticula were monitored. The role of FDC FcgammaRII was studied using anti-BCR-stimulated A20 cells. Levels of FDC FcgammaRII in spleens of SRBC-injected mice increased within 24 h and were dramatically increased (approximately 50-fold) on days 3 and 5. In contrast, CR1&2 levels increased less than twofold. Addition of normal FDC, but not FDC lacking FcgammaRII, reduced and reversed anti-BCR-induced SH2 domain-containing inositol phosphatase (SHIP)-1 phosphorylation in A20 cells. FDC were able to induce normal recall responses even after overnight incubation of the lymphocytes with IC to stimulate ITIM signaling. Engagement of Ig Fc with numerous FcgammaRII on FDC appears to minimize IC-induced ITIM signaling. Thus, rapid up-regulation of FDC FcgammaRII may explain why poorly immunogenic IC are rendered highly immunogenic when presented by FDC in GC.     

p38 MAPK-HSP27信号通路在内毒素致大鼠肺损伤中的作用

 目的：观察p38丝裂原激活蛋白激酶（p38 MAPK)-热休克蛋白27（HSP27）信号通路在急性肺损伤病理过程中的变化规律。方法：健康雄性Wistar大鼠（300~320 g）随机分成正常对照组（A组）、急性肺损伤组（B组）及急性肺损伤+SB203580组（C组）。通过腹腔注射内毒素建立急性肺损伤大鼠模型,分别于实验开始后的0、2、4、6、8 h处死各组大鼠。检测支气管肺泡灌洗液（BALF）白细胞介素6（IL-6）、肿瘤坏死因子α（TNF-α）及BALF中蛋白含量。苏木素-伊红（HE）染色检查肺组织病理变化及免疫荧光方法检测内皮细胞内F-actin和G-actin,计算肺湿干重比值（W/D）。检测肺组织中磷酸化p38 MAPK（p-p38 MAPK）及磷酸化HSP27（p-HSP27）的含量。 结果：B组在实验后2 h BALF中蛋白水平和肺W/D开始明显增加,给予内毒素后8 h肺泡上皮肿胀,肺泡壁增宽,肺泡间质和肺泡腔水肿明显,肺泡内炎症细胞、红细胞和蛋白渗出明显增多,表现出急性肺损伤的病理改变。在给予了p38 MAPK抑制剂SB203580后的C组BALF中蛋白水平及肺W/D分别比B组明显减少,肺泡内炎症细胞、红细胞和蛋白渗出、间质与肺泡水肿均较B组减轻。B组均在实验后2 h血清及BALF中TNF-α和IL-6的浓度开始增加,p-p38 MAPK及p-HSP27的肺内表达开始增加,与A组相比有显著差异。B组实验后8 h的F-actin的表达明显比A组实验后0 h及8 h的增加,给予p38 MAPK抑制剂SB203580的C组肺p-HSP27 和F-actin的表达分别比B组明显减少。结论：内毒素可以通过激活p38 MAPK-HSP27信号通路引起急性肺损伤;阻断该信号通路可以减轻肺损伤。     

Axl/Gas6 pathway positively regulates FLT3 activation in human natural killer cell development

Activation of the fibromyalgia syndrome‐like tyrosine kinase 3 (FLT3) by its ligand, FLT3 ligand (FL), strongly augments the development of natural killer (NK) cells from human CD34⁺ hematopoietic progenitor cells (HPCs) in the presence of IL‐15, compared with NK‐cell development in the presence of IL‐15 alone. In this study, we observed that blocking the receptor tyrosine kinase Axl/Gas6 pathway with a soluble Axl‐IgG1 Fc fusion protein (Axl‐Fc) in the presence of FL significantly diminished the absolute number of CD3^?CD56⁺ NK cells derived from human CD34⁺ HPCs. Axl‐Fc reduced the expression levels of the IL‐2/15 receptor β chain (CD122) and γ chain (CD132) induced by activation of FLT3 and consequently reduced the frequency of NK precursor cells responding to IL‐15. Furthermore, Axl‐Fc diminished FL‐induced FLT3 phosphorylation and impeded the physical interaction between Axl and FLT3 in CD34⁺ HPCs. Collectively, our data suggest that the Axl/Gas6 pathway contributes to normal human NK‐cell development at least in part via its positive regulatory effect on FLT3 signaling in CD34⁺ HPCs.     

MiR-24 inhibits inflammatory responses in LPS-induced acute lung injury of neonatal rats through targeting NLRP3

Inflammation plays an important role in the development of acute lung injury (ALI) in preterm infants. Despite the critical role of microRNA in inflammatory response, little is known about its function in ALI. In this study, we investigate the role of MicroRNA-24 (miR-24) in lipopolysaccharide (LPS) induced neonatal rats ALI and its potential mechanism. LPS was used to induce ALI neonatal animal model. miR-24 expression in the lung tissues of LPS-challenged neonatal rats was detected by qPCR. Proinflammatory factors, including tumor necrosis factor-alpha (TNF-α), IL-1β, IL-18 in the bronchoalveolar lavage fluid and lung tissues of LPS-challenged neonatal rats were measured by qRT-PCR and western blot, respectively. The mRNA levels of surfactant protein A (SP-A) and D (SP-D) was measured by qRT-PCR. Direct binding of miR-24 and pyrin domain-containing 3(NLRP3) were determined by dual luciferase assay. The levels of NLRP3, apoptosis-associated speck-like protein containing a C?terminal caspase recruitment domain (ASC) and caspase-1 protein expression were detected by immunohistochemistry (IHC) staining and western blot, respectively. Our data indicated that LPS-induced lung injury in neonatal rats and resulted in significant downregulated of miR-24 expression. Overexpression of miR-24 significantly reduced LPS-induced lung damage and decreased the release of proinflammatory cytokine TNF-α, IL-6, IL-1β and SP-A, SP-D expression induced by LPS. In addition, miR-24 inhibited the expression of NLRP3 by directly targeting to the CDS region of NLRP3 mRNA. Furthermore, miR-24 overexpression attenuated lung inflammation and deactivated the NLRP3/caspase-1/IL-1β pathway in LPS-challenged neonatal rats. These data show that miR-24 alleviated inflammatory responses in LPS-induced ALI via targeting NLRP3.     

Lack of FLT3-ITD in Tunisian childhood acute lymphoblastic leukemia

BackgroundThe fms-like tyrosine kinase 3 (FLT3) gene belong to the class III receptor tyrosine kinases witch is predominantly expressed on hematopoietic progenitor cells, and plays an important role in haematopoiesis. Targeting the FMS-like tyrosine kinase receptor-3 (FLT3) in acute leukemia is mainly important. Therefore, activating mutations in FLT3, primarily the FLT3-internal tandem duplication (FLT3-ITD), was used as a prognostic marker especially in myeloid leukemia; however, in ALL, the prognostic relevance of FLT3 mutations is less clear.ObjectivesThis study was conducted to evaluate the frequency of FLT3-ITD mutation in Tunisian childhood acute lymphoblastic leukemia, and to correlate this mutation with prognostic parameters.MethodsGenomic DNA was extracted from EDTA-anticoagulant blood samples from a total of 25 children suffering from acute lymphoblastic leukemia (ALL). After DNA extraction, the polymerase chain reaction using specific primers was conducted to screen the FLT3-ITD.ResultsIn acute lymphoblastic leukemia (ALL), 9 cases with LAL-B were detected and the median age is 13 years. Chromosome abnormalities were detected in 5 with ALL and are correlated with worse prognosis (very high risk and relapse). At molecular lever, never FLT3-ITD was detected.ConclusionsOur findings suggest that FLT3 mutations are not common in Tunisian childhood ALL and thus do not affect clinical outcome.     

Toll-like receptor ligand activation of murine bone marrow-derived dendritic cells

Dendritic cells (DCs) are required for the initiation of primary immune responses. The pattern of Toll-like receptor (TLR) expression on various subsets of these cells has been shown to differ, suggestive of distinct roles in influencing immune responses. We have examined here the responses of immature DCs derived from murine bone marrow (BMDCs) to a range of TLR ligands. BMDCs cultured for 6 days in the presence of granulocyte-macrophage colony-stimulating factor were stimulated for 24 hr with ligands to TLR1-2 [Pam(3)Cys-Ser-(Lys)(4) (PAM)], TLR2-6 (macrophage-activating lipopeptide-2 (MALP-2); zymosan or peptidoglycan (PG)], TLR3 (polyinosinic-polycytidylic acid), TLR4 [lipopolysaccharide R515 (LPS)], TLR5 (flagellin), TLR7 (polyuridylic acid) and TLR9 [CpG ODN2395 (CpG)]. DC activation was monitored using membrane marker expression and analysis of culture supernatants for cytokine/chemokine release. Ligands to TLR3 and TLR7 failed to activate BMDCs. All other TLR ligands caused elevated expression of membrane markers. PAM, MALP-2 and LPS induced high-level expression of proinflammatory cytokines and chemokines. Treatment with CpG was associated with a preferential type 1 cytokine and chemokine profile. Zymosan and PG were proinflammatory but also skewed towards a type 2 pattern of cytokines and chemokines. In contrast, flagellin did not cause marked secretion by BMDCs of cytokines or chemokines. These data for BMDCs are largely consistent with the reported TLR repertoire of freshly isolated murine Langerhans cells. In addition, murine BMDCs show selective responses to TLR ligands with respect to general activation, with differentiated cytokine patterns suggestive of potential priming for divergent immune responses.     

骨髓来源间充质干细胞培养上清液减轻脂多糖诱导的小鼠急性肺损伤

 目的:观察经尾静脉输注骨髓间充质干细胞培养上清液（MSCs CdM）对脂多糖(LPS)诱导的小鼠急性肺损伤的治疗作用及其机制。方法:采用全骨髓培养法分离纯化骨髓间充质干细胞,传至第3代时观察细胞形态,流式细胞术检测细胞表面标志,并且收集上清液用超滤离心管进行离心。30只BALB/c小鼠随机分为对照组、 LPS模型组和MSCs CdM治疗组。对照组腹腔内注射生理盐水（0.01 mL/g）,LPS组和MSCs CdM治疗组腹腔内注射LPS（5 mg/kg,0.01 mL/g）制备急性肺损伤模型。造模1 h后经尾静脉输注MSCs CdM（MSCs  CdM治疗组）或生理盐水 （LPS组或对照组）300 μL。6 h后处死小鼠,留取标本检测肺组织病理形态学、肺组织湿干重比（W/D）、支气管肺泡灌洗液（BALF）中蛋白含量、血清及BALF中细胞因子水平和肺组织中髓过氧化物酶(MPO)的活性。结果:与对照组比较,LPS处理后肺组织病理损伤严重,BALF中蛋白、血清肿瘤坏死因子α(TNF-α)和白细胞介素6(IL-6)含量、肺组织中MPO活性及肺组织湿干重比均显著升高。与LPS组比较,MSCs CdM治疗组肺组织病理损伤程度减轻,BALF中蛋白、血清TNF-α和IL-6含量、肺组织中MPO活性及肺组织湿干重比均显著降低,而BALF中白细胞介素10（IL-10）和角质细胞生长因子（KGF）水平显著高于LPS组和对照组。结论:骨髓间充质干细胞培养上清液可有效减轻LPS诱导的急性肺损伤,其作用机制可能与其调节肺部TNF-α、IL-6、IL-10和KGF的水平有关。     

部分液体通气治疗家猪急性肺损伤的抗炎效应

目的：探讨部分液体通气治疗肺灌洗诱导的 急性肺损伤家猪模型时，是否具有抗炎作用。方法：16只健康家猪，采 用生理盐水肺内灌洗复制急性肺损伤模型，随机分为部分液体通气组及机械通气组给予不同 治疗，观察其肺脏湿/干比值及肺通透指数，观察其血浆、支气管肺泡灌洗液及肺组织匀浆 中TNF-α、MDA含量及SOD、MPO活性。结果：（1）部分液体通气组家猪 肺脏湿/干比值、肺通透指数及支气管肺泡灌洗液中白细胞计数明显低于机械通气组。（2） 肺组织MDA、MPO含量部分液体通气组明显低于机械通气组，但两组间SOD活性无明显差别。 （3）部分液体通气组支气管肺泡灌洗液及肺组织匀浆中TNF-α含量明显低于机械通气组。 结论：部分液体通气改善动物肺损伤指标，提示以氟碳化合物为呼吸媒 介的部分液体通气对肺灌洗诱导的急性肺损伤家猪具有抗炎效应。     

Role of natural interferon-producing cells and T lymphocytes in porcine monocyte-derived dendritic cell maturation

Maturation of dendritic cells (DC) is a key immunological process regulating immune responses to pathogens and vaccines, as well as tolerance and autoimmune processes. Consequently, the regulation of DC maturation should reflect these multifaceted immunological processes. In the present study, we have defined the role of particular cytokines, Toll-like receptor (TLR) ligands and T lymphocytes in the porcine monocyte-derived DC (MoDC). Interferon-alpha (IFN-alpha) alone was a poor inducer of MoDC maturation, but in association with tumour necrosis factor-alpha (TNF-alpha), or TLR ligands such as lipopolysaccharide and polyinosinic-polycytidylic acid I:C, an up-regulation of major histocompatibility complex II and CD80/86 expression was noted, along with reduced endocytic activity. In contrast, TNF-alpha alone or in combination with the TLR ligands was a poor inducer of DC maturation, but co-operated with T-lymphocytes in the presence of antigen to induce DC maturation. Natural interferon producing cells (NIPC, or plasmacytoid DCs) represent a danger-recognition system of the immune defences, and can respond to viruses not otherwise recognized as posing a danger. Indeed, MoDC did not respond to transmissible gastroenteritis virus (TGEV), whereas NIPC produced high levels of IFN-alpha and TNF-alpha after TGEV stimulation. Moreover, supernatants from the stimulated NIPC induced maturation in MoDCs. These matured MoDCs displayed an enhanced ability to present antigen to and thus stimulate T cells. Taken together, the present work demonstrates that maturation of MoDC not only results from TLR signalling, but can require co-operation with various cell types--principally NIPC and activated T cells--which would reflect the particular immunological situation.     

CCN1在脂多糖诱导急性肺损伤中的表达调控和在炎症反应中的作用

目的:体内观察富半胱氨酸蛋白61(CYR61/CCN1)在正常肺组织中的表达定位和在脂多糖(LPS)诱导小鼠急性肺损伤中的表达变化,体外研究LPS调控CCN1表达的分子机制和CCN1在LPS诱导炎症介质表达中的作用。方法:分别通过免疫组化(IHC)染色及免疫荧光法观察CCN1在小鼠肺组织和气道上皮细胞16HBE中的表达定位;气道滴注LPS建立小鼠急性肺损伤模型,IHC观察CCN1在肺组织中的表达变化;体外研究中,分别予气道上皮16HBE细胞以ERK1/2、JNK、P38和PI3K信号通路特异性抑制剂预处理2 h后加入LPS刺激,通过RT-qPCR和Western blot检测CCN1的mRNA和蛋白表达变化;分别通过CCN1-siRNA和重组CCN1蛋白刺激16HBE细胞,qPCR检测炎症介质白细胞介素6(IL-6)、IL-8、转化生长因子β(TGF-β)和血管内皮生长因子(VEGF)的mRNA水平。结果:CCN1在正常肺组织中以气道上皮表达为主,在LPS诱导的急性肺损伤小鼠气道上皮细胞中CCN1表达升高;LPS可刺激16HBE细胞中CCN1的表达水平升高,其中ERK1/2、JNK、P...