Interaction of Leishmania donovani promastigotes with human monocyte-derived macrophages: parasite entry, intracellular survival, and multiplication.

Leishmania donovani promastigotes were incubated with human monocyte-derived macrophages in vitro to assess the role of macrophages in the early stage of visceral leishmaniasis. Adherent mononuclear cells, obtained from nonimmune human donors, were cultivated on glass cover slips for 5 days and then incubated with axenically grown promastigotes in the presence of heat-inactivated autologous serum. Promastigotes attached to macrophages with either their flagellar or aflagellar ends, and macrophage pseudopodia formed around them. Intracellular parasites were identified within phagocytic vacuoles by electron microscopy, and the parasites assumed a form similar to that of amastigotes obtained from infected hamster spleens. Initially, 67 +/- 5% of the macrophages were infected with a mean of 4.2 +/- 0.7 parasites per infected cell. After 6 days of incubation, 79 +/- 7% of the macrophages were infected with 15.9 +/- 3.2 parasites per infected cell. The total number of parasites per monolayer increased from 4.8 +/- 0.8 x 10(5) to 1.8 +/- 0.4 x 10(6) (P less than 0.05). Dividing parasites were identified in macrophage vacuoles by electron microscopy. Human monocyte-derived macrophage vacuoles by electron microscopy. Human monocyte-derived macrophages can phagocytize promastigotes, allow the conversion of promastigotes to an amastigote-like state, and support intracellular multiplication.     

Tumor suppressor p53 induces apoptosis of host lymphocytes experimentally infected by Leishmania major,by activation of Bax and caspase-3: a possible survival mechanism for the parasite

Apoptosis of infected host macrophages by Leishmania spp. is mainly addressed as one of the survival mechanisms of the parasite. However, there is no eligible data about whether tumor suppressor p53 could induce the apoptosis of host lymphocytes-treated Leishmania major via the mitochondrial intrinsic pathway. In this study, the amastigotes of L. major obtained from ten cutaneous leishmaniases (CL) patients were separately isolated and cultured in N.N.N and RPMI 1640 media. L. major was definitely confirmed by targeting Cyt b gene following sequencing. Subsequently, 2–3 × 10⁶ lymphocytes obtained from ten healthy individuals were isolated and co-cultured with 1–2 × 10⁶ L. major promastigotes. Following 6 h of exposure time, the enzymatic activity of caspase-3 was determined by fluorometric assay in each L. major-treated lymphocytes and cell control (only lymphocyte). The mRNA expressions of Bax, Bcl-2, p53, and caspase-3 genes were assessed by quantitative real-time-PCR analysis following 6 to 9 h of exposure times. The Bcl-2 mRNA expression in L. major-treated lymphocytes was 100-fold down-regulated relative to cell control. The mRNA expressions of p53 and caspase-3 were over-expressed 1.8- and 3.2-fold up-regulated relative to control lymphocytes, respectively. The Bax/Bcl-2 ratio and caspase-3 activity were higher than the control group (Pv <0.05). The current new findings indicate that the apoptotic effects of L. major-treated host lymphocytes dependent on p53 tumor suppressor via mitochondrial pathway may probably address as an auxiliary survival mechanism of L. major in CL patients. However, here is much work ahead to figure out the multiple functions played by apoptosis in the evasion of L. major.

     

Molecular mechanisms involved in interleukin-4-induced human neutrophils: expression and regulation of suppressor of cytokine signaling

Interleukin-4 (IL-4) is a CD132-dependent cytokine known to activate the Jak-STAT pathway in different cells and cell lines. Although IL-4 has been demonstrated previously to be an agonist in human neutrophils, its capacity to activate different cell signaling pathways in these cells has never been investigated. Two types of IL-4 receptor (IL-4R) exist: the Type I (CD132/IL-4Ralpha heterodimer) and the Type II (IL-4Ralpha/IL-13Ralpha1 heterodimer). In a previous study, we demonstrated that neutrophils express the Type I receptor. Herein, using flow cytometry, we demonstrated that neutrophils, unlike U-937 cells, do not express IL-13Ralpha1 and IL-13Ralpha2 and confirmed the expression of CD132 and IL-4Ralpha on their surface. We also demonstrated that IL-4 induced phosphorylation of Syk, p38, Erk-1/2, JNK, Jak-1, Jak-2, STAT6, and STAT1 and that treatment of cells with the inhibitors piceatannol, SB203580, PD98059, or AG490 reversed the ability of IL-4 to delay neutrophil apoptosis. Using RT-PCR, we demonstrated for the first time that neutrophils express mRNA for all suppressor of cytokine signaling (SOCS) members, namely SOCS1-7 and cytokine-inducible Src homology 2 protein. It is interesting that IL-4 increased expression of SOCS3 at the mRNA and protein levels. The effect of IL-4 on SOCS3 protein expression was increased markedly when the proteasome inhibitor MG132 was added to the cultures, but this was inhibited by cycloheximide, suggesting that SOCS3 is de novo-synthesized in response to IL-4. We conclude that neutrophils express only the Type I IL-4R on their surface and that IL-4 signals via different cell signaling pathways, including the Jak/STAT/SOCS pathway.     

Dynamin inhibition interferes with inflammasome activation and cytokine gene expression in Streptococcus pyogenes-infected human macrophages

In the present study, we have analysed the ability of Streptococcus pyogenes [Group A streptococcus (GAS)] to activate the NACHT-domain-, leucine-rich repeat- and PYD-containing protein 3 (NALP3) inflammasome complex in human monocyte-derived macrophages and the molecules and signalling pathways involved in GAS-induced inflammatory responses. We focused upon analysing the impact of dynamin-dependent endocytosis and the role of major streptococcal virulence factors streptolysin O (SLO) and streptolysin S (SLS) in the immune responses induced by GAS. These virulence factors are involved in immune evasion by forming pores in host cell membranes, and aid the bacteria to escape from the endosome–lysosome pathway. We analysed cytokine gene expression in human primary macrophages after stimulation with live or inactivated wild-type GAS as well as with live SLO and SLS defective bacteria. Interleukin (IL)-1β, IL-10, tumour necrosis factor (TNF)-α and chemokine (C-X-C motif) ligand (CXCL)-10 cytokines were produced after bacterial stimulation in a dose-dependent manner and no differences in cytokine levels were seen between live, inactivated or mutant bacteria. These data suggest that streptolysins or other secreted bacterial products are not required for the inflammatory responses induced by GAS. Our data indicate that inhibition of dynamin-dependent endocytosis in macrophages attenuates the induction of IL-1β, TNF-α, interferon (IFN)-β and CXCL-10 mRNAs. We also observed that pro-IL-1β protein was expressed and efficiently cleaved into mature-IL-1β via inflammasome activation after bacterial stimulation. Furthermore, we demonstrate that multiple signalling pathways are involved in GAS-stimulated inflammatory responses in human macrophages.     

Leishmania RAB7: characterisation of terminal endocytic stages in an intracellular parasite

Leishmania species are intracellular parasites that inhabit a parasitophorous vacuole (PV) within host macrophages and engage with the host endo-membrane network to avoid clearance from the cell. Intracellular Leishmania amastigotes exhibit a high degree of proteolytic/lysosomal activity that may assist degradation of MHC class II molecules and subsequent interruption of antigen presentation. As an aid to further analysis of the endosomal/lysosomal events that could facilitate this process, we have characterised a Leishmania homologue of the late endosomal marker, Rab7, thought to be involved in the terminal steps of endocytosis and lysosomal delivery. The Leishmania major Rab7 (LmRAB7) protein is expressed throughout the life-cycle, shows 73 and 64% identity to Trypanosoma cruzi and Trypanosoma brucei Rab7s (TcRAB7 and TbRAB7), respectively, and includes a kinetoplastid-specific insertion. The recombinant protein binds GTP and polyclonal antibodies raised against this antigen recognise structures in the region of the cell between the nucleus and kinetoplast. By immunoelectron microscopy of axenic amastigotes, Leishmania mexicana Rab7 (LmexRAB7) is found juxtaposed to and overlapping membrane structures labelled for the megasomal marker, cysteine proteinase B, confirming a late-endosomal/lysosomal localisation.     

Leishmania donovani-induced ceramide as the key mediator of Akt dephosphorylation in murine macrophages: role of protein kinase Czeta and phosphatase

Leishmania donovani is an intracellular protozoan parasite that impairs the host macrophage immune response to render it suitable for its survival and establishment. L. donovani-induced immunosuppression and alteration of host cell signaling is mediated by ceramide, a pleiotropic second messenger playing an important role in regulation of several kinases, including mitogen-activated protein kinase and phosphatases. We observed that the endogenous ceramide generated during leishmanial infection led to the dephosphorylation of protein kinase B (PKB) (Akt) in infected cells. The study of ceramide-mediated Akt phosphorylation revealed that Akt was dephosphorylated at both Thr308 and Ser473 sites in infected cells. Further investigation demonstrated that ceramide was also responsible for the induction of PKCzeta, an atypical Ca-independent stress kinase, as well as the ceramide-activated protein phosphatases (e.g., protein phosphatase 2A [PP2A]). We found that Akt dephosphorylation was mediated by ceramide-induced PKCzeta-Akt association and PP2A activation. In addition, treatment of L. donovani-infected macrophages with PKCzeta-specific inhibitor peptide could restore the translocation of phosphorylated Akt to the cell membrane. This study also revealed that ceramide is involved in the inhibition of proinflammatory cytokine tumor necrosis factor alpha release by infected macrophages. These observations strongly suggest the importance of ceramide in the alteration of normal cellular functions, impairment of the kinase/phosphatase balance, and thereby establishment of leishmaniasis in the hostile macrophage environment.     

Leishmania major abrogates gamma interferon-induced gene expression in human macrophages from a global perspective

Infection with Leishmania major triggers several pathways in the host cell that are crucial to initial infection as well as those that are used by Leishmania to enhance its replication and virulence. To identify the molecular events of the host cell in response to Leishmania, the global gene expression of the human monocytic cell line THP-1 either infected with Leishmania major in the presence and absence of gamma interferon (IFN-gamma) or in the presence of IFN-gamma alone was analyzed using high-density human oligonucleotide microarrays, followed by statistical analysis. The persistence of the parasite despite an extensive response to IFN-gamma, added 24 h after infection with L. major, suggests that L. major can survive in an IFN-gamma-enriched environment in vitro. Results demonstrate that L. major counteracts the IFN-gamma response in macrophages on a large scale. Expression of genes involved in the innate immune response, cell adhesion, proteasomal degradation, Toll-like receptor expression, a variety of signaling molecules, and matrix metalloproteinases was significantly modulated.     

Targeting intracellular signaling: a novel approach to vaccination

Vaccination is well known to control many current infectious diseases. However, the development of cellular (Th1) immunity to control viral pathogens, among others, requires the development of new vaccine adjuvants. The use of Toll-like receptor ligands or cytokines has shown much promise, although specificity and toxicity are issues with these strategies. Targeting intracellular signaling pathways may allow for greater specificity of the adjuvant, as well as reducing systemic toxicity. Studies targeting these pathways are discussed, as well as their potential applications in the future.     

细胞因子信号转导抑制因子3在脓毒症小鼠肝脏和脾脏中的表达

目的 探讨细胞因子信号转导抑制因子3(SOCS3)在脓毒症小鼠肝脏和脾脏中的表达情况及其可能的作用机制.方法 采用盲肠结扎并穿刺术(CLP)制作小鼠脓毒症模型.检测肝脏和脾脏组织的SOCS3 mRNA及蛋门表达,采用RT-PCR检测组织中SOCS3 mRNA的相对含量,用免疫印迹方法测定组织中SOCS3相对蛋白含量.用SPSS统计软件对上述指标间的变化关系进行分析.结果 脓毒症手术后SOCS3在肝脏内基因和蛋白表达量有升高趋势,但与对照组比较筹异无统计学意义(P>0.05).SOCS3在脾脏内的基因和蛋白表达在术后2 h迅速升高.至12 h达峰值.在肝脏和脾脏中SOCS3的基因表达和蛋白表达均正相关(r=0.353、0.731,P均<0.05).结论 CLP导致的脓毒症可以诱导SOCS3在小鼠肝脏和脾脏中表达增多,提示SOCS3在脓毒症后的免疫变化中有重要作用.     

细胞因子信号抑制因子3在小鼠肾炎进程中的表达

目的: 探索细胞因子信号抑制因子3(SOCS3)在小鼠肾炎进程中的表达变化规律和意义。方法: 采用含兔抗小鼠肾小球基底膜抗体的肾毒素血清(NTS)制备小鼠肾炎模型。在第10、15、20和25 d分别收集小鼠尿液并摘取肾脏。以蛋白尿水平、肾脏组织HE和Masson染色评价肾炎进程;采用免疫组化和Western blotting 法检测SOCS3蛋白表达变化,以及Janus激酶2(JAK2)和信号转导及转录激活因子3(STAT3)在小鼠肾炎进程中磷酸化情况。结果: 小鼠肾炎模型表现出典型的发病、进展与转归;SOCS3在肾炎进程中未出现立即随JAK2和STAT3磷酸化增多而表达增加的现象,仅在肾炎严重期(第20 d)表达增加(P<0.05),在恢复期(第25 d)表达明显增加(P<0.01)。恢复期SOCS3表达显著增加,表现出抑制JAK2和STAT3磷酸化的作用。结论: 机体自身条件下,SOCS3的表达未立即随JAK2/STAT3磷酸化增多而增加,SOCS3对免疫性肾炎的保护作用可能主要在肾炎恢复期。     

Lactobacillus rhamnosus GG and Streptococcus thermophilus induce suppressor of cytokine signalling 3 (SOCS3) gene expression directly and indirectly via interleukin-10 in human primary macrophages

In the present study we have characterized T helper type 2 (Th2) [interleukin (IL)-10]/Th1 (IL-12) cytokine expression balance in human primary macrophages stimulated with multiple non-pathogenic Gram-positive bacteria used in the food industry and as probiotic substances. Bacteria representing Lactobacillus, Bifidobacterium, Lactococcus, Leuconostoc, Propionibacterium and Streptococcus species induced anti-inflammatory IL-10 production, although quantitative differences between the bacteria were observed. S. thermophilus was able to induce IL-12 production, while the production of IL-12 induced by other bacteria remained at a low level. The highest anti-inflammatory potential was seen with bifidobacteria, as evidenced by high IL-10/IL-12 induction ratios. All studied non-pathogenic bacteria were able to stimulate the expression of suppressor of cytokine signalling (SOCS) 3 that controls the expression of proinflammatory cytokine genes. Lactobacillus and Streptococcus species induced SOCS3 mRNA expression directly in the absence of protein synthesis and indirectly via bacteria-induced IL-10 production, as demonstrated by experiments with cycloheximide (CHX) and anti-IL-10 antibodies, respectively. The mitogen-activated protein kinase (MAPK) p38 signalling pathway played a key role in bacteria-induced SOCS3 gene expression. Enhanced IL-10 production and SOCS3 gene expression induced by live non-pathogenic Lactobacillus and Streptococcus is also likely to contribute to their immunoregulatory effects in vivo.     

Identification and purification of a receptor on macrophages for the dengue virus-induced suppressor cytokine.

Dengue type 2 virus (DV)-induced suppressor cytokine (SF) binds to macrophages to transmit the suppressor signal to recruit the second subpopulation of suppressor T cells. The present study was undertaken to identify and purify the receptor for SF (SF-R) on macrophages. The binding of 125I-SF to macrophages was saturable and reversible. Scatchard analysis showed the presence of both high (54,000/cell) and low (1.78 x 10(6)/cell) affinity receptor sites. The binding of 125I-SF to macrophages was inhibited by pretreatment of macrophages with anti-SF antiserum but not by a heterologous antiserum. Normal mouse peritoneal macrophage membrane was solubilized with Triton-X-100 and the components separated by low pressure liquid chromatography (LPLC) to purify SF-R. The presence of SF binding moiety (SF-R) was screened at each step of purification. The purified SF-R resolved into two bands of 45-50 kD mol. wt on SDS-PAGE. 125I-SF+SF-R complex run on SDS-PAGE showed a single band at about 55-60 kD mol. wt by autoradiography. Anti-SF-R antiserum reacted with SF-R in a Western blot test; the reaction was abolished by pretreatment of the blots with proteinase K, but not by pretreatment with periodic acid. SF-R was composed of two polypeptide chains (alpha and beta) which were obtained in pure form by high performance liquid chromatography (HPLC) of dithiothreitol- and iodoacetamide-treated SF-R. Only the beta chain bound SF.     

大鼠细胞因子信号转导抑制因子-1基因的原核克隆表达和免疫学分析

目的 克隆表达大鼠细胞因子信号转导抑制因子-1基因(SOCS-1).方法 将大鼠SOCS-1全长编码基因的PCR产物,克隆到原核表达质粒pET-28a(+)中,构建重组质粒pET-28a(+)-ratSOCS-1.转化大肠埃希菌BL-21/DE3,用异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达,应用镍离子金属螯合剂亲和层析柱从表达产物中纯化重组蛋白,通过十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)对表达产物和重组蛋白进行鉴定.应用纯化的重组蛋白免疫新西兰大白兔,抗体滴度达到1:10000以上后进行末次免疫,2周后心脏取血、测定滴度,分离制备免疫血清.用红细胞裂解法制备大鼠外周血白细胞裂解全蛋白.将阴性血清(未用重组蛋白免疫的新西兰大白兔的血清)、免疫血清及兔抗组氨酸标签抗体转入各蛋白条带,用Western免疫印迹检测重组蛋白的免疫学活性.结果 PCR、双酶切及DNA测序分析均表明重组质粒pET-28a(+)-ratSOCS-1构建成功.SDS-PAGE结果可见转化了重组质粒的大肠埃希菌全菌和经超声裂解后的菌体沉淀的样品均在相对分子质量约26 000处有一明显的蛋白条带,经亲和层析获得的纯化重组蛋白有特异的单一条带与上述条带一致,而在超声裂解后的菌体上清中相同位置却未见有蛋白表达条带.Western免疫印迹表明重组蛋白、大鼠外周血白细胞裂解蛋白中相对分子质量约24000的蛋白条带、兔抗组氨酸标签抗体均可被免疫血清识别,分别出现清晰的相对分子质量26000、24 000、26000的反应条带,表明其具有免疫活性.结论 SOCS-1基因可以在大肠埃希菌BL-21/DE3中获得表达,其纯化重组蛋白具有良好的抗原性和免疫活性.