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Castillo-Velázquez U Aranday-Cortés E Gutiérrez-Pabello JA 《Veterinary microbiology》2011,151(1-2):51-59
The aim of this study was to evaluate the influence of macrophage alternative activation in the intracellular pathogen natural disease resistance phenotype of the host. Macrophage monolayers from resistant (R) (3) or susceptible (S) (3) cattle donors were treated with 10 ng/ml of bovine recombinant IL-4 (rbIL-4), and infected with virulent and avirulent Mycobacterium bovis (MOI 10:1). Bactericidal assays were performed to assess the bacterial phagocytic index and intracellular survival. Total RNA was reverse transcribed and used to analyze the relative changes in gene expression of IL-10, IL-12, IL-18 IL-1β, TNF-α, MCP-1, MCP-2, IL-6, MIP-1, MIP-3, iNOS, ARGII and SLAM by real time PCR. Cell supernatants were collected and nitric oxide and arginase production was assessed. Apoptosis induction was measured by TUNEL. IL-4 treatment increased the phagocytic index in both R and S macrophages; however intracellular survival was augmented mainly in S macrophages. Alternative activation decreased gene expression of pro-inflammatory cytokines, nitric oxide production and DNA fragmentation mainly in R macrophages. On the other hand, arginase production was not different between R and S macrophages. Alternative activation modifies the macrophage response against M. bovis. IL-4 treatment minimized the functional differences that exist between R and S macrophages. 相似文献
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Cytokine gene expression in ileal tissues of cattle infected with Mycobacterium paratuberculosis was evaluated. The effects of infection with M. paratuberculosis on cytokine production may influence immune regulation at the site of colonization, resulting in the chronic inflammatory state associated with the latter stages of this disease. Ileal samples were obtained at necropsy from noninfected control cows (n=8) and clinically infected cows (n=7) and processed for immunohistochemistry and in situ hybridization. Cows infected with M. paratuberculosis were in the latter stages of disease with clinical signs such as weight loss, watery diarrhea, and inappetence. Among cytokines we studied, interleukin-1alpha (IL-1alpha), IL-1beta, IL-6, and interferon-gamma (IFN-gamma) were expressed significantly more in infected animals than in noninfected control animals. The expression of tumor necrosis factor-alpha (TNF-alpha), however, was not different between the two groups of cattle. In addition, immunohistochemical staining demonstrated that the number of resident macrophages in the ileum of infected animals was three times greater than that of noninfected cows. In contrast to this, ileal tissues from noninfected control animals contained 1.5 times more neutrophils than the ileal tissues from cows infected with M. paratuberculosis. These data demonstrate that localized ileal cytokine production is different between cows chronically infected with M. paratuberculosis and noninfected control cows. 相似文献
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de Almeida DE Colvin CJ Coussens PM 《Veterinary immunology and immunopathology》2008,125(3-4):234-245
Mycobacterium avium subspecies paratuberculosis (MAP) is an intracellular pathogen that survives in the host's intestinal macrophages and causes chronic enteritis in ruminants. The subclinical stage of MAP infection is accompanied by loss of pro-inflammatory T(H)1 response, and a predominant, but ineffective, antibody-mediated T(H)2 response. How MAP interacts with the bovine immune system and suppresses T(H)1 responses is unclear. Studies carried out in our lab and others indicate that when peripheral blood mononuclear cells (PBMCs) from subclinical MAP-infected cattle are stimulated with MAP-antigen, IL-10 is up-regulated and leads to suppression of IFN-gamma expression in MAP-antigen-reactive effector T cells. IL-10 up-regulation and reduction in IFN-gamma would favor MAP survival and proliferation in macrophages. Depletion studies in PBMCs from MAP-infected cattle also revealed that the MAP responsive T-cell population that produces IL-10 is CD4(+) and CD25(+). Therefore, we hypothesize that cattle infected with MAP develop regulatory T (Treg) cells capable of producing IL-10 that in turn limits peripheral and tissue-specific T(H)1 immune responses. The aim of this review is to summarize current thinking regarding Treg cells and provide preliminary evidence that infection of cattle with MAP may lead to development of Treg cells. 相似文献
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Role of the MAPK(ERK) pathway in regulation of cytokine expression by Mycobacterium avium subsp paratuberculosis-exposed bovine monocytes 总被引：1，自引：0，他引：1
OBJECTIVE: To evaluate the role of the mitogen-activated protein kinase extracellular signal-regulated kinase (MAPK(ERK)) pathway in the interaction between Mycobacterium avium subsp paratuberculosis (MAP) organisms and bovine monocytes. SAMPLE POPULATION: Monocytes obtained from healthy adult Holstein dairy cows that were not infected with MAP organisms. PROCEDURES: Monocytes and MAP organisms were incubated together with or without a specific inhibitor of the MAPK(ERK) pathway (PD98059), and the capacity of monocytes to express tumor necrosis factor alpha (TNF)-alpha and interleukin (IL)-10 and -12, produce nitric oxide, acidify phagosomes, kill MAP organisms, and undergo apoptosis was evaluated. RESULTS: The MAPK(ERK) pathway was activated within 10 minutes after addition of MAP organisms to monocytes. Addition of PD98059 to monocyte-MAP mixtures decreased monocyte TNF-alpha and IL-12 mRNA expression but had no effect on IL-10 mRNA expression. Treatment with PD98059 failed to induce significant alterations in phagosome acidification, organism killing, nitric oxide production, or apoptosis of MAP-exposed monocytes. CONCLUSIONS AND CLINICAL RELEVANCE: Results indicated that the MAPK(ERK) pathway was activated during the interaction of MAP organisms with monocytes, which initiated TNF-alpha and IL-12 mRNA expression but failed to initiate antimicrobial activity. The MAPK(ERK) pathway may be involved in initiating proinflammatory and proimmune responses in MAP infection in cattle. 相似文献
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旨在探讨STAT6介导的巨噬细胞极化对布鲁氏菌胞内生存的影响。本研究采用布鲁氏菌光滑株S2308（S2308）和粗糙型疫苗株RB51（RB51）侵染巨噬细胞。利用qRT-PCR检测M1型巨噬细胞标志因子p65、NOS2和IL-1β,M2型巨噬细胞标志因子STAT6、ARG1、IL-10的mRNA表达水平;流式细胞术检测M1型标记分子CD86和M2型标记分子CD206的表达;Western blot检测p-STAT6蛋白及抑制剂AS对蛋白的抑制作用;ELISA检测M1型细胞因子TNF-α、IL-12和M2型细胞因子IL-4、IL-10的表达量;最后对胞内菌落进行CFU计数。qRT-PCR结果显示,在感染8、12 h时可显著诱导M1型因子mRNA转录表达,72 h时低表达,而M2型因子在72 h时高表达;流式细胞术结果显示,S2308感染12 h可显著诱导CD86的表达,感染72 h可显著诱导CD206的表达,但RB51对二者无影响;Western blot结果显示,S2308菌株在感染72 h时激活STAT6信号通路,而RB51几乎不激活该通路,抑制剂AS在2 μmol·L-1浓度时抑制效果最佳;ELISA结果显示,AS抑制剂可显著抑制IL-4、IL-10的释放,并促进TNF-α、IL-12的释放;CFU计数结果显示,S2308组的胞内菌呈先降低后显著上升趋势,加入AS抑制剂后可显著抑制布鲁氏菌胞内复制。布鲁氏菌S2308在感染后期能够通过STAT6诱导M1型巨噬细胞向M2型转化,并促进Th2型细胞因子的释放,从而有利于布鲁氏菌的胞内生存。而RB51几乎不激活该通路,不影响胞内生存。 相似文献
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McGuire K Manuja A Russell GC Springbett A Craigmile SC Nichani AK Malhotra DV Glass EJ 《Veterinary immunology and immunopathology》2004,99(1-2):87-98
The pathogenic mechanisms involved in tropical theileriosis, caused by the tick-borne protozoan parasite Theileria annulata, are unclear. Pathology is associated with the schizont stage of the parasite, which resides within bovine macrophages. Breed-specific differences in pathology have been observed in cattle, several Bos indicus breeds are relatively resistant to tropical theileriosis whilst Bos taurus cattle are highly susceptible. Infected cells express pro-inflammatory cytokines and it has been hypothesized that these cytokines play a major role in the pathology of the disease. Therefore, using quantitative RT-PCR we investigated the expression of the key candidates, interleukin 1 beta (IL-1beta), IL-6 and tumour necrosis factor alpha (TNF-alpha), in T. annulata low passage infected cell lines derived ex vivo from experimental infection of resistant and susceptible cattle. mRNA for each cytokine was detected in all cell lines investigated at levels higher than those observed in resting monocytes. However, the analyses did not identify any breed-specific differences. Therefore, these results are not consistent with the hypothesis that differential regulation of infected cell derived pro-inflammatory cytokines (IL-1beta, IL-6 and TNF-alpha) accounts for the breed-related differences in resistance and susceptibility to T. annulata infection. Other, currently unknown mechanisms may be of greater importance. 相似文献
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本试验用布鲁氏菌强、弱毒株侵染小鼠巨噬细胞RAW264.7,旨在探讨NF-κB信号通路与布鲁氏菌强、弱毒株在胞内生存的关系。采用光滑型牛布鲁氏菌2308、粗糙型牛布鲁氏菌RB51在不同感染复数下侵染小鼠巨噬细胞RAW264.7,侵染0、4、8、24 h后,裂解细胞收集蛋白,Western blotting检测布鲁氏菌对激活NF-κB信号通路的影响。利用不同浓度的NF-κB信号通路抑制剂处理小鼠巨噬细胞RAW264.7,然后用布鲁氏菌在不同感染复数下侵染小鼠巨噬细胞RAW264.7,ELISA试剂盒检测细胞因子TNF-α、IL-1β、IL-6的表达量;同时对胞内菌CFU进行计数。结果显示粗糙型牛布鲁氏菌RB51可以强烈激活NF-κB信号通路,光滑型牛布鲁氏菌2308对其激活作用较弱;同时对NF-κB信号通路的激活具有浓度依赖性,在感染复数为80:1、侵染时间为8 h时光滑型牛布鲁氏菌2308和粗糙型牛布鲁氏菌RB51对NF-κB激活程度最强,且该通路参与产生TNF-α、IL-1β和IL-6;NF-κB信号通路抑制剂BAY11-7082影响布鲁氏菌在胞内的生存。因此,粗糙型牛布鲁氏菌RB51胞内存活与NF-κB信号通路密切相关,为进一步研究布鲁氏菌的胞内致病机制奠定基础,也为布鲁氏菌新型药物的研发、家畜布鲁氏菌病预防和治疗提供科学依据。 相似文献
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By the infection of Brucella virulent strain and attenuated strain in mice macrophage RAW264.7,the assay was aimed to explore the relationship between NF-κB signaling pathways and Brucella virulent strain and attenuated strain in intracellular survival.Use different MOI Brucella (2308,RB51,16M and M5) to infect mice macrophage RAW264.7,after 0,4,8 and 24 h infected,cracking cell and collecting supernatant,we detected the effect of Brucella on activation of NF-κB signaling pathway by Western blotting.Different concentrations of NF-κB signaling pathway inhibitor were incubated with mice macrophage RAW264.7,with different multiplicities of infection (MOI) of Brucella infecting cells,ELISA kits to detect the expressions of TNF-α,IL-1β and IL-6 cytokine;At the same time,count the number of intracellular bacteria of CFU.The results showed that rough cattle Brucella strains RB51 could strongly activate NF-κB signaling pathway,smooth cattle Brucella strains 2308 was weak in the activation;At the same time,the activation of NF-κB signaling pathway was concentration dependent.When the MOI was 80,infection time was 8 h,NF-κB activation degrees of rough cattle Brucella strains RB51 and smooth cattle Brucella strains 2308 were the strongest,and this pathway was involved in producing TNF-α and IL-6;NF-κB signaling pathway inhibitor BAY11-7082 affected Brucella intracellular survival.So rough cattle Brucella strains RB51 intracellular survival and NF-κB signaling pathway activity were closely related.The results laid the foundation for the further study of Brucella intracellular pathogenesis,also provided scientific basis for the research of new drugs to Brucella,and prevention and treatment of brucellosis. 相似文献
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Pereira-Suárez AL Estrada-Chávez C Arriaga-Díaz C Espinosa-Cueto P Mancilla R 《Veterinary pathology》2006,43(5):709-717
In murine models the inducible nitric oxide synthase (iNOS) and the natural resistance associated macrophage protein (NRAMP1) play major roles in host defense against mycobacteria. iNOS regulates nitric oxide (NO) production, which is noxious for ingested mycobacteria, and NRAMP1 displays pleiotropic antimicrobial effects, including upregulation of iNOS expression. Little is known about the role of these molecules in bovine tuberculosis (TB). In this work we demonstrate by Western blot a high expression of NRAMP1 in peripheral blood mononuclear cells (PBMCs), alveolar macrophages (obtained by bronchioalveolar lavage), and lymph node granulomas from 8 Holstein-Freisian cattle with autopsy-proven bovine TB. Immunohistochemistry revealed the abundant expression of NRAMP1 and iNOS in lymph node and lung granulomas. Immunoreactivity was abundant in the cytoplasm of many epithelioid macrophages and multinucleated giant cells of the Langhans type. A striking accumulation of nitrotyrosine (NT), an indicator of iNOS activity and local NO production, was observed in granuloma cells, particularly in multinucleated Langhans cells. This study shows that the expression of NRAMP1 and iNOS is costimulated in granulomas, which are protective T-cell reactions against mycobacteria. 相似文献
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《Veterinary microbiology》2015,175(1):50-57
The zinc-finger protein A20 has crucial physiological functions as a dual inhibitor of macrophage activation and apoptosis in tumor necrosis factor receptor1 (TNFR1) signaling pathway. Brucella infection can induce A20 expression in macrophages. Here, we hypothesize that A20 promotes Brucella intracellular growth via inhibition of activation and apoptosis of macrophages. To test this hypothesis, we stably incorporated mouse A20-shRNA into the RAW264.7 cells by lentiviral gene transfer to successfully knockdown A20. A20-deficient RAW264.7 cells were subsequently challenged with Brucella abortus and colony formation units (CFUs) of bacteria, TNFα production, NF-kB activation, macrophages apoptosis and cell death were evaluated. The A20 knockdown was shown to effectively promote B. abortus-stimulated TNFα release, NF-kB activation and macrophage cell death, which suppressed B. abortus intracellular replication. Unexpectedly, deficiency of A20 failed to lead to B. abortus-induced macrophage apoptosis. A20 deficiency coupled NF-kB inhibition promoted caspase-8 dependent B. abortus-induced macrophage apoptosis. These findings provide a novel mechanism by which Brucella intracellular growth within macrophages occurs through up-regulation of A20 thereby limiting activation and macrophages cell death. 相似文献
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A Chelmonska-Soyta R B Miller L Ruhnke S Rosendal 《Canadian journal of veterinary research》1994,58(4):275-280
Ureaplasma diversum is a pathogen in the bovine reproductive tract. The objective of the research was to study interactions with macrophages and lymphocytes which might elucidate aspects of pathogenetic mechanisms of this organism. We studied the activation of murine macrophages of C3H/HeN (LPS-responder) and C3H/HeJ (LPS-low-responder) genotype for TNF-alpha, IL-6, IL-1 and nitric oxide production and blastogenic response of C3H/HeJ splenocytes after Ureaplasma diversum stimulation. Live and heat-killed U. diversum induced TNF-alpha, IL-6 and IL-1 in peritoneal macrophage cultures of both C3H/HeN and C3H/HeJ mice in a dose dependent manner. Interferon-gamma modulated the cytokine production, by increasing the production of TNF-alpha, IL-6 and nitric oxide, but IL-1 secretion was only enhanced in C3H/HeJ macrophages stimulated by live ureaplasmas. Supernatant of U. diversum sonicate was mitogenic for murine spleen lymphocytes. The blastogenic response was dose dependent, and stimulation with both U. diversum and Concanavalin A seemed to have an additive effect. These results suggest that U. diversum, similar to other mycoplasmas, activates murine macrophages and lymphoid cells. The studies should be repeated with bovine cells in order to elucidate pathogenetic aspects of inflammation in cattle caused by U. diversum. 相似文献
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Based on epidemiological and clinical observations, different strains of Mycobacterium avium subsp. paratuberculosis (MAP) are suspected to significantly differ in their virulence for ruminants. In the pathogenesis of paratuberculosis, macrophages represent the principal target cell for MAP. In order to judge the ability of different MAP-genotypes to modulate macrophage responses, the cytokine responses of the monocyte cell line THP-1 were studied after challenge with three different MAP strains under standardized conditions. The bovine field isolate J1961 (major Type II) and the ovine field isolate JIII-86 (Type III) were compared with the laboratory adapted reference strain ATCC 19698 (Type II). Strains were shown by three different typing methods (IS900-RFLP-, MIRU-VNTR-, and SSR-analysis) to substantially differ in several genotypic features. Macrophage function was assessed by quantifying mRNA of the cytokines TNF-α, IL-1?, and IL-10 by quantitative RT-PCR. Secreted TNF-α protein was measured by a cytotoxicity test, IL-1? and IL-10 using ELISA tests. The three MAP strains of various genotypes differ in their effect on human macrophages depending on challenge dose and infection time. These differences concerned both the mRNA level and secreted protein amounts of proinflammatory cytokines TNF-α, IL-1β and anti-inflammatory cytokine IL-10. Type III strain produced less IL-10 and IL-1β mRNA and protein but more TNF-α protein at 2h than the Type II strains. In summary, our results support the hypothesis that strain characteristics might have relevance for the host response towards MAP and, consequently, for the pathogenesis of paratuberculosis. 相似文献
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本研究对具核梭杆菌(F.nucleatum)感染鼠巨噬细胞进行共同培养之后,分别在10个时段采取上清液和巨噬细胞,用ELISA、qRT-PCR法和扫描电镜检测TNF-α、 IL-1α、 IL-6和IL-8细胞因子,并观察巨噬细胞的超微形态变化。结果显示,4种细胞因子均有高水平表达;IL-8在共培养6～72 h呈高水平表达;IL-6和TNF-α表达水平也较高;IL-1从3 h表达,12 h达最高值,48 h后无表达。qRT-PCR检测结果显示,TNF-α的RNA表达呈波浪式,分别于6,60 h各出现1个高峰值;IL-1α和IL-6呈现正态表达,从3 h开始明显表达至60 h达峰值,此后缓慢下降;IL-8从6 h逐渐上升,至72 h仍呈高水平表达。超微形态观察显示,在F.nucleatum感染的前24 h,巨噬细胞呈收缩状,其表面和周围有大量发育良好F.nucleatum,并呈团块状黏附,巨噬细胞逐渐伸出伪足。30 h后F.nucleatum逐渐发育不良,小而细,而巨噬细胞表面粗糙,有许多大小不一的结节,伪足变粗大。48 h巨噬细胞表面出现胞吐孔,少量细小的F.nucleatum仍然黏附于... 相似文献
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Inflammatory cytokines are suspected to contribute to the pathogenesis of bovine pneumonic pasteurellosis (BPP) through neutrophil recruitment, leukocyte activation, and the induction of a broad array of soluble inflammatory mediators. An in vivo experimental model of BPP was used to characterize the pulmonary expression kinetics of tumor necrosis factor alpha (TNFalpha), interleukin-1 beta (IL-1beta), and interleukin-8 (IL-8) genes and proteins during the acute phase of disease development. Cytokine expression in bronchoalveolar lavage (BAL) fluid, BAL cells, and pneumonic lung parenchyma was quantitated by northern blot analysis, enzyme-linked immunosorbent assay (ELISA), and in situ hybridization at 2, 4, 8, 16, and 24 hours after endobronchial inoculation of Pasteurella (Mannheimia) haemolytica. Expression of TNFalpha, IL-1beta, and IL-8 was significantly increased in the airways and lung lesions of infected calves as compared with mock-infected controls. Although kinetic patterns varied, peak levels of cytokine mRNA occured within 8 hours postinfection (PI), and peak cytokine concentrations occurred within 16 hours PI. In all samples, IL-8 was expressed to the greatest extent and TNFalpha was least expressed. Expression of TNFalpha was restricted to alveolar macrophages. Alveolar and interstitial macrophages produced IL-1beta and IL-8 in the first 4 hours; bronchial and bronchiolar epithelial cells were also significant sources of IL-8 during this period. By 8 hours PI, neutrophils were the dominant source of both IL-1beta and IL-8. These findings demonstrate a spatial and temporal association between pulmonary expression of inflammatory cytokines and acute lung pathology, supporting the hypothesis that cytokines contribute to inflammatory lung injury in BPP. 相似文献
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Effect of infection by bovine viral diarrhea virus (BVDV) in vitro on interleukin-1 activity of bovine monocytes 总被引：2，自引：0，他引：2
The effect of bovine viral diarrhea virus (BVDV) infection in vitro on the interleukin-1 (IL-1) activity of bovine monocytes was studied. Supernatants from BVDV-infected monocytes suppressed IL-1-stimulated proliferation of mouse thymocytes and masked lipopolysaccharide-stimulated IL-1 activity of bovine monocytes in the mouse comitogen thymocyte assay. Suppression of mouse thymocyte proliferation was restored by the addition of IL-1. IL-1 inhibitory activity was induced both by the prototype variants BVDV/NADL cytopathic and BVDV/NY-1 noncytopathic and by BVDV variants isolated from persistently infected cattle. Suppressed IL-1 activity was also found in supernatants from monocytes from persistently infected cattle following infection with BVDV in vitro. No differences in levels of IL-1 mRNA synthesis were detected between BVDV-infected and uninfected monocytes by RNA-cDNA hybridization. These results suggest that infection of bovine monocytes with BVDV results in the production and/or activation of a soluble inhibitor of IL-1 activity. 相似文献