Bioactivation of latent transforming growth factor beta1 by Mycobacterium tuberculosis in human mononuclear phagocytes

Biologically active transforming growth factor beta 1 (TGFbeta1) has been identified at sites of Mycobacterium tuberculosis (MTB) infection in the lung; however, the underlying mechanism(s) for its activation is not clear. Here using an enzyme-linked immunospot assay for TGFbeta1, we show that human blood monocytes (MN) and alveolar macrophages (AM) produce bioactive TGFbeta1 upon stimulation by MTB. However, only MTB-stimulated MN increased TGFbeta1 production on a per cell basis. The frequency of TGFbeta1-producing MN was reduced by an inhibitor of plasmin, bdellin, indicating a role for plasmin pathways in the bioactivation of cytokine. The expression of urokinase plasminogen activator receptor (uPAR) mRNA and both surface and soluble uPAR (CD87) was increased in MTB-activated MN. However, antibody neutralization of uPAR suppressed bioactive TGFbeta1 in MN alone. Thus, the more immature MN, which are continuously recruited to the lung during tuberculosis (TB), have a higher capacity to bioactivate TGFbeta1 by expression of components of the plasmin pathway. Excess production and bioactivation of TGFbeta1 at sites of MTB infection may undermine host immune responses during TB.     

Modulation of the immune response by transforming growth factor beta.

For the past several years immunologists have been fascinated by a series of experiments showing that transforming growth factor beta (TGF beta) suppresses T- and B-lymphocyte growth as well as IgM and IgG production by B cells. Moreover, while exerting chemotactic activity on monocytes and inducing expression of interleukin-1 and interleukin-6 by these cells, TGF beta interferes with bacterially induced tumor necrosis factor alpha production, oxygen radical formation and the adhesiveness of granulocytes to endothelial cells. These mechanisms may provide the basis for the effect of TGF beta to prevent the microvascular changes associated with brain edema formation in bacterial meningitis. Given the potential of lymphocytes as well as macrophages to produce TGF beta 1, this cytokine may exert negative feedback signals on the immune response, provided the cytokine is processed from its latent form to the bioactive homodimer. Potent effects of TGF beta have been observed in experimental animals including the inhibition of the generation of virus-specific cytotoxic T cells and antiviral antibodies as well as the diminution of cellular infiltrates with decreased major histocompatibility complex class-II expression and CD8+ T cells in the tissue of virally infected animals. TGF beta may also be of importance in tumor immunology. By the production of bioactive TGF beta as detected in glioblastoma and acute T-cell leukemia, tumor cells may induce an immunodeficiency state and escape immune surveillance. In inflammation, monitoring of TGF beta in the tissue will bring light on the immune regulation in acute and chronic inflammatory diseases.     

Influence of Mycobacterium bovis BCG vaccination on cellular immune response of guinea pigs challenged with Mycobacterium tuberculosis

Mycobacterium bovis bacillus Calmette-Guérin (BCG) currently remains the only licensed vaccine for the prevention of tuberculosis. In this study, we used a newly described flow cytometric technique to monitor changes in cell populations accumulating in the lungs and lymph nodes of na?ve and vaccinated guinea pigs challenged by low-dose aerosol infection with virulent Mycobacterium tuberculosis. As anticipated, vaccinated guinea pigs controlled the growth of the challenge infection more efficiently than controls did. This early phase of bacterial control in immune animals was associated with increased accumulation of CD4 and CD8 T cells, including cells expressing the activation marker CD45, as well as macrophages expressing class II major histocompatibility complex molecules. As the infection continued, the numbers of T cells in the lungs of vaccinated animals waned, whereas the numbers of these cells expressing CD45 increased. Whereas BCG vaccination reduced the influx of heterophils (neutrophils) into the lungs, an early B-cell influx was observed in these vaccinated animals. Overall, vaccine protection was associated with reduced pathology and lung damage in the vaccinated animals. These data provide the first direct evidence that BCG vaccination accelerates the influx of protective T-cell and macrophage populations into the infected lungs, diminishes the accumulation of nonprotective cell populations, and reduces the severity of lung pathology.     

Effect of transforming growth factor beta 1 on neonatal rat latent transforming growth factor beta binding protein 2 in cardiomyocytes

目的 探讨转化生长因子β1 (TGF-β1)在诱导心肌细胞表达转化生长因子结合蛋白2(LTBP2)中的作用及信号传导通路.方法 培养乳鼠心肌细胞;实时定量聚合酶链式反应、蛋白质印迹和免疫细胞化学方法检测不同时间和不同浓度的TGF-β1对大鼠乳鼠心肌细胞LTBP2基因及蛋白表达的影响;用TGF-β1相关信号通路阻断剂探讨TGF-β1调节LTBP2表达改变的信号传导机制.结果 LTBP2基因表达随着TGF-β1浓度增加(0、2、5及10 μg/L)而明显升高,在5μg/L时刺激最强(P＜0.05);5μg/L的TGF-β1刺激下心肌细胞内LTBP2基因和蛋白表达的升高呈时间依赖性,均在12 h最高,24 h开始呈下降趋势(P＜0.05或P＜0.01);免疫细胞化学结果显示TGF-β1明显升高LTBP2的表达.信号传导通路研究显示TGF-β1在心肌细胞内主要通过ERK信号通路和PI3K信号通路诱导LTBP2的表达.结论TGF-β1在乳鼠心肌细胞内通过ERK信号通路和PI3K信号通路上调LTBP2的表达.     

转化生长因子β1对乳鼠心肌细胞转化生长因子结合蛋白2表达的影响

 目的：探讨转化生长因子β1（TGF-β1）在诱导心肌细胞表达转化生长因子结合蛋白2（LTBP2）中的作用及信号传导通路。 方法：培养乳鼠心肌细胞;实时定量聚合酶链式反应（Real-time PCR）、蛋白质印迹和免疫细胞化学方法检测不同时间和不同浓度的TGF-β1对大鼠乳鼠心肌细胞LTBP2基因及蛋白表达的影响;用TGF-β1相关信号通路阻断剂探讨TGF-β1调节LTBP2表达改变的信号传导机制。 结果：LTBP2基因表达随着TGF-β1浓度增加（0、2、5、10 ng/mL）而明显升高,在5 ng/mL时刺激最强（P < 0.05）;5 ng/mL的TGF-β1刺激下心肌细胞内LTBP2基因和蛋白表达的升高呈时间依赖性,均在12 h最高,24 h开始呈下降趋势（P < 0.05或P<0.01）;免疫细胞化学结果显示TGF-β1明显升高LTBP2的表达。信号传导通路研究显示TGF-β1在心肌细胞内主要通过ERK信号通路和PI3K信号通路诱导LTBP2的表达。 结论：TGF-β1在乳鼠心肌细胞内通过ERK信号通路和PI3K信号通路上调LTBP2的表达。     

Selective induction of transforming growth factor beta in human monocytes by lipoarabinomannan of Mycobacterium tuberculosis.

The induction of macrophage-deactivating (interleukin-10 [IL-10] and transforming growth factor beta [TGF-beta] and macrophage-activating (IL-1, IL-6, and tumor necrosis factor alpha [TNF-alpha] cytokines by lipoarabinomannan (LAM) from pathogenic Mycobacterium tuberculosis Erdman and H37Rv strains (ManLAM) and nonpathogenic mycobacteria (AraLAM) in human blood monocytes was examined. ManLAM was significantly less potent in induction of TNF-alpha, IL-1, IL-6, and IL-10 protein and mRNA, whereas its ability to induce TGF-beta was similar to that of AraLAM. Differences in induction of TNF-alpha mRNA by the two LAM preparations only became apparent at late time points of culture (24 h). The induction of TNF-alpha and IL-1 by purified protein derivative of M. tuberculosis was significantly stronger than that by ManLAM. Pretreatment of monocytes with ManLAM did not, however, interfere with cytokine induction by lipopolysaccharide or AraLAM. The extensive mannosyl capping of arabinose termini of ManLAM may underlie the lack of ability to induce some cytokines (IL-1, TNF-alpha, and IL-10) and the retained ability to induce TGF-beta. The latter may have a role in shifting the cytokine milieu in favor of survival of M. tuberculosis.     

How B cells shape the immune response against Mycobacterium tuberculosis

Extensive work illustrating the importance of cellular immune mechanisms for protection against Mycobacterium tuberculosis has largely relegated B‐cell biology to an afterthought within the tuberculosis (TB) field. However, recent studies have illustrated that B lymphocytes, through a variety of interactions with the cellular immune response, play previously underappreciated roles in shaping host defense against non‐viral intracellular pathogens, including M. tuberculosis. Work in our laboratory has recently shown that, by considering these lymphocytes more broadly within their variety of interactions with cellular immunity, B cells have a significant impact on the outcome of airborne challenge with M. tuberculosis as well as the resultant inflammatory response. In this review, we advocate for a revised view of TB immunology in which roles of cellular and humoral immunity are not mutually exclusive. In the context of our current understanding of host defense against non‐viral intracellular infections, we review recent data supporting a more significant role of B cells during M. tuberculosis infection than previously thought.     

Boosting with poxviruses enhances Mycobacterium bovis BCG efficacy against tuberculosis in guinea pigs

Tuberculosis is rising in the developing world due to poor health care, human immunodeficiency virus type 1 infection, and the low protective efficacy of the Mycobacterium bovis BCG vaccine. A new vaccination strategy that could protect adults in the developing world from tuberculosis could have a huge impact on public health. We show that BCG boosted by poxviruses expressing antigen 85A induced unprecedented 100% protection of guinea pigs from high-dose aerosol challenge with Mycobacterium tuberculosis, suggesting a strategy for enhancing and prolonging the efficacy of BCG.     

Tissue factor expression by myeloid cells contributes to protective immune response against Mycobacterium tuberculosis infection

Tissue factor (TF) is a transmembrane glycoprotein that plays an essential role in hemostasis by activating coagulation. TF is also expressed by monocytes/macrophages as part of the innate immune response to infections. In the current study, we determined the role of TF expressed by myeloid cells during Mycobacterium tuberculosis (M. tb) infection by using mice lacking the TF gene in myeloid cells (TF^Δ) and human monocyte derived macrophages (MDMs). We found that during M. tb infection, a deficiency of TF in myeloid cells was associated with reduced inducible nitric oxide synthase (iNOS) expression, enhanced arginase 1 (Arg1) expression, enhanced IL‐10 production and reduced apoptosis in infected macrophages, which augmented M. tb growth. Our results demonstrate that a deficiency of TF in myeloid cells promotes M2‐like phenotype in M .tb infected macrophages. A deficiency in TF expression by myeloid cells was also associated with reduced fibrin deposition and increased matrix metalloproteases (MMP)‐2 and MMP‐9 mediated inflammation in M. tb infected lungs. Our studies demonstrate that TF expressed by myeloid cells has newly recognized abilities to polarize macrophages and to regulate M. tb growth.     

Specificity of a protective memory immune response against Mycobacterium tuberculosis.

We have investigated the memory T-cell immune response to Mycobacterium tuberculosis infection. C57BL/6J mice infected with M. tuberculosis were found to generate long-lived memory immunity which provided a heightened state of acquired resistance to a secondary infection. The T-cell response of memory immune mice was directed to all parts of the bacilli, i.e., both secreted and somatic proteins. Major parts of the memory T-cell repertoire were maintained in a highly responsive state by cross-reactive restimulation with antigens present in the normal microbiological environment of the animals. A resting non-cross-reactive part of the memory repertoire was restimulated early during a secondary infection to expand and produce large amounts of gamma interferon. The molecular target of these T cells was identified as a secreted mycobacterial protein with a molecular mass of 3 to 9 kDa.     

Mononuclear cell function in Mycobacterium tuberculosis infected guinea pigs

In this study mononuclear cell function was studied in the lymph glands, spleen, and peripheral blood of Mycobacterium tuberculosis infected guinea pigs. Adherent cells from draining lymph nodes and spleens of infected animals spontaneously produced a factor which inhibited normal lymphocyte proliferative responses. As it has previously been shown that this factor activates a population of suppressor T cells, resident lymphocytes in the lymph nodes and spleen were examined and were shown to inhibit normal lymphocyte functions. It is suggested that adherent cells ingesting M. tuberculosis spontaneously release a suppressor cell activating factor (SCAF) which locally activates lymphocytes to become suppressor cells. Even at a time of overwhelming infection, peripheral blood adherent cells could not be shown to release SCAF and peripheral blood suppressor cells could not be identified. Although peripheral blood lymphocyte proliferative responses to PHA were normal in infected animals, their ability to produce the lymphokine macrophage inhibition factor was considerably reduced after the second week of infection. This dissociation between lymphocyte proliferation and lymphokine production is similar to that previously described in humans overwhelming tuberculosis.     

Glutamine synthetase GlnA1 is essential for growth of Mycobacterium tuberculosis in human THP-1 macrophages and guinea pigs

To assess the role of glutamine synthetase (GS), an enzyme of central importance in nitrogen metabolism, in the pathogenicity of Mycobacterium tuberculosis, we constructed a glnA1 mutant via allelic exchange. The mutant had no detectable GS protein or GS activity and was auxotrophic for L-glutamine. In addition, the mutant was attenuated for intracellular growth in human THP-1 macrophages and avirulent in the highly susceptible guinea pig model of pulmonary tuberculosis. Based on growth rates of the mutant in the presence of various concentrations of L-glutamine, the effective concentration of L-glutamine in the M. tuberculosis phagosome of THP-1 cells was approximately 10% of the level assayed in the cytoplasm of these cells (4.5 mM), indicating that the M. tuberculosis phagosome is impermeable to even very small molecules in the macrophage cytoplasm. When complemented by the M. tuberculosis glnA1 gene, the mutant exhibited a wild-type phenotype in broth culture and in human macrophages, and it was virulent in guinea pigs. When complemented by the Salmonella enterica serovar Typhimurium glnA gene, the mutant had only 1% of the GS activity of the M. tuberculosis wild-type strain because of poor expression of the S. enterica serovar Typhimurium GS in the heterologous M. tuberculosis host. Nevertheless, the strain complemented with S. enterica serovar Typhimurium GS grew as well as the wild-type strain in broth culture and in human macrophages. This strain was virulent in guinea pigs, although somewhat less so than the wild-type. These studies demonstrate that glnA1 is essential for M. tuberculosis virulence.     

Reactivity of neuroglia in guinea pigs sensitized by Mycobacterium tuberculosis

In guinea pigs with experimental allergic encephalomyelitis (EAE) induced by sensitization withMycobacterium tuberculosis cells mixed with petrolatum and Arlacel the reactivity of the glia was shown to change regularly depending on the stage and severity of the pathological process. Changes in reactivity determined by inhibition of glial migration in tissue culture were specific with respect to antigens from nerve tissue. The study of the mechanism of this phenomenon showed that similar changes in reactivity can be induced in a culture of glial cells from intact animals if blood serum or regional lymph gland cells taken from actively sensitized guinea pigs are added. It cannot yet be concluded from the available results that changes in reactivity of the glia to products of brain tissue destruction in guinea pigs with EAE are completely attributable to the action of humoral or cellular elements which penetrate into the CNS from the bloodstream or lymphatic tissue.     

The SecA2 secretion factor of Mycobacterium tuberculosis promotes growth in macrophages and inhibits the host immune response

The SecA protein is present in all bacteria, and it is a central component of the general Sec-dependent protein export pathway. An unusual property of Mycobacterium tuberculosis is the presence of two SecA proteins: SecA1, the essential "housekeeping" SecA, and SecA2, the accessory secretion factor. Here, we report that a DeltasecA2 mutant of M. tuberculosis was defective for growth in the early stages of low-dose aerosol infection of C57BL/6 mice, a time during which the bacillus is primarily replicating in macrophages. Consistent with this in vivo phenotype, we found that the DeltasecA2 mutant was defective for growth in macrophages from C57BL/6 mice. The DeltasecA2 mutant was also attenuated for growth in macrophages from phox(-/-) mice and from NOS2(-/-) mice. These mice are defective in the reactive oxygen intermediate (ROI)-generating phagocyte oxidase and the reactive nitrogen intermediate (RNI)-generating inducible nitric oxide synthase, respectively. This indicated a role for SecA2 in the intracellular growth of M. tuberculosis that is independent of protecting against these ROIs or RNIs. Macrophages infected with the DeltasecA2 mutant produced higher levels of tumor necrosis factor alpha, interleukin-6, RNI, and gamma interferon-induced major histocompatibility complex class II. This demonstrated a function for M. tuberculosis SecA2 in suppressing macrophage immune responses, which could explain the role of SecA2 in intracellular growth. Our results provide another example of a relationship between M. tuberculosis virulence and inhibition of the host immune response.     

Neutralizing antibodies against transforming growth factor beta potentiate the proliferation of Ki-1 positive lymphoma cells. Further evidence for negative autocrine regulation by transforming growth factor beta.

Activated lymphocytes and malignant lymphoma cells derived from them (Ki-1 positive lymphoma cells) share similar mechanisms of proliferation. To further examine the inhibitory role of endogenous transforming growth factor beta (TGF beta) in Ki-1 positive lymphoma cells, the authors studied anti-TGF beta antibodies and measured their effect on proliferation. A monoclonal antibody (T1A5) prepared against a unique antigenic epitope of high molecular weight Hodgkin's TGF beta and a polyclonal rabbit antibody prepared against highly purified 25,000 D porcine platelet TGF beta 1 were used. Both antibodies are shown here to inhibit the biological activity of Hodgkin's TGF beta and to crossreact with their respective antigens in immunoblotting. DNA synthesis by Ki-1 lymphoma cells was increased 138-fold by anti-TGF beta 1 antibody and 262-fold by anti-Hodgkin's TGF beta. Exogenous TGF beta 1 suppression was completely reversed by anti-TGF beta 1 antibody and IL-2-induced proliferation was markedly potentiated (41 fold). L-428 Reed-Sternberg cells secrete physiologically active TGF beta but have fewer than 500 TGF beta receptor sites per cell; no significant proliferative response was measured for either anti-TGF beta 1 or anti-Hodgkin's TGF beta. These results show the suppressive effect of exogenous TGF beta 1 on indolent Ki-1 lymphoma cells and suggest that the endogenous secretion of high molecular weight physiologically active TGF beta is important in maintaining the indolent nature of this low-grade Ki-1 positive lymphoma.     

Cross-clade neutralizing antibodies against HIV-1 induced in rabbits by focusing the immune response on a neutralizing epitope

Studies were performed to induce cross-clade neutralizing antibodies (Abs) by testing various combinations of prime and boost constructs that focus the immune response on structurally-conserved epitopes in the V3 loop of HIV-1 gp120. Rabbits were immunized with gp120 DNA containing a V3 loop characterized by the GPGR motif at its tip, and/or with gp120 DNA with a V3 loop carrying the GPGQ motif. Priming was followed by boosts with V3-fusion proteins (V3-FPs) carrying the V3 sequence from a subtype B virus (GPGR motif), and/or with V3 sequences from subtypes A and C (GPGQ motif). The broadest and most consistent neutralizing responses were generated when using a clade C gp120 DNA prime and with the V3_B-FP boost. Immune sera displayed neutralizing activity in three assays against pseudoviruses and primary isolates from subtypes A, AG, B, C, and D. Polyclonal Abs in the immune rabbit sera neutralized viruses that were not neutralized by pools of human anti-V3 monoclonal Abs. Greater than 80% of the neutralizing Abs were specific for V3, showing that the immune response could be focused on a neutralizing epitope and that vaccine-induced anti-V3 Abs have cross-clade neutralizing activity.     

Vaccination of guinea pigs with nutritionally impaired avirulent mutants of Mycobacterium bovis protects against tuberculosis

Four nutritionally impaired strains of Mycobacterium bovis produced by illegitimate recombination were tested for their ability to protect guinea pigs against intratracheal challenge with virulent M. bovis. All four strains and M. bovis BCG induced significant levels of protection as measured by the reduced spread of infection to the spleen and liver. In animals vaccinated with BCG or two of the other strains, the bacterial counts from the lungs were significantly lower than those of the nonvaccinated animals.     

转化生长因子β1在兔房水中的浓度变化

背景：转化生长因子β1可参与角膜损伤后的修复。 目的：观察转化生长因子β1滴眼液滴眼后房水中的浓度变化规律。 方法：将新西兰大白兔随机分为5组,分别给予PBS和质量浓度为0.5,1.0,2.0,4.0 mg/L的转化生长因子β1滴眼液滴右眼。 结果与结论：通过裂隙灯观察兔角膜和结膜结构,各组兔眼均无结膜分泌物、球结膜充血、角膜水肿增厚、角膜后沉着物、前房炎性反应及晶状体混浊改变。ELISA检测结果显示,与PBS组比较,质量浓度2.0和4.0 mg/L转化生长因子β1滴眼液能有效提高兔眼房水中转化生长因子β1的质量浓度(P < 0.01),角膜穿透性良好,在房水中可以达到有效的治疗浓度。     

Exploring the immune response against Mycobacterium tuberculosis for a better diagnosis of the infection

Tuberculosis (TB) still represents a monumental problem, with more than two million deaths every year worldwide. The current diagnostics for TB offer sub-optimal accuracy both for the active and the latent form of infection and are often based on technologies unaffordable in low-income settings. The tuberculin skin test was the first diagnostic based on an acquired immune response towards Mycobacterium tuberculosis (MTB). Advances in molecular and cellular biology and the elucidation of the mechanisms governing the relation between MTB and the human immune system form the basis for new and more accurate assays, potentially able to fill the gaps and limits of classical diagnostics. However, the process of validating new tests is still complex and hampered by specific questions regarding TB immunology and natural history. We present here a summary of the current approaches to validate new diagnostics based on the detection of immunological biomarkers of TB infection.