It is now well established that dietary lipids are incorporated into macrophage and T-cell membrane microdomains, altering their structure and function. Within cell membranes, there are specific detergent-resistant domains in which key signal transduction proteins are localized. These regions are classified as “lipid rafts”. Rafts are composed mostly of cholesterol and sphingolipids and therefore do not integrate well into the fluid phospholipid bilayers causing them to form microdomains. Upon cell activation, rafts compartmentalize signal-transducing molecules, thus providing an environment conducive to signal transduction. In this review, we discuss recent novel data describing the effects of n-3 PUFA on alterations in the activation and functions of macrophages and T-cells. We believe that the modifications in these two disparate immune cell types are linked by fundamentally similar changes in membrane lipid composition and transmembrane signaling functions. We conclude that the outcomes of n-3 PUFA-mediated immune cell alterations may be beneficial (e.g., anti-inflammatory) or detrimental (e.g., loss of microbial immunity) depending upon the cell type interrogated.
Macrophage; tuberculosis; phagosome maturation; lipid rafts; polyunsaturated fatty acids; fish oil; fat-1 mice
We examined the ability of recombinant guinea pig IL-8 (CXCL8) to activate neutrophils upon infection with virulent M. tuberculosis. Using a Transwell insert culture system, contact-independent cell cultures were studied in which rgpIL-8-treated neutrophils were infected with virulent M. tuberculosis in the upper well, and AM were cultured in the lower well. IL-1β and TNF-α mRNA expression was significantly up-regulated by AM. Neutralizing anti-rgpTNF-α polyclonal antibody abrogated the response of AM to supernatants from the rgpIL-8 treated, infected neutrophils, while an anti-rgpIL-8 polyclonal antibody had no effect. This suggests that TNF-α produced by rgpIL-8 treated, infected neutrophils may play an important role in the activation of AM in the early response of the host against M. tuberculosis infection. Significant induction of apoptosis in M. tuberculosis-infected neutrophils was observed as compared to the uninfected neutrophils. Feeding of infected, apoptotic neutrophils to AM induced a significant up-regulation of TNF-α and IL-1β mRNA compared to AM exposed to staurosporine-treated apoptotic neutrophils. Suppressed intracellular mycobacterial growth was also seen in AM fed with infected, apoptotic neutrophils as compared to the AM infected with M. tuberculosis H37Rv alone. Taken together, these data suggest that neutrophil-macrophage interactions may contribute to host defense against M. tuberculosis infection.
Neutrophils; alveolar macrophages; IL-8; tuberculosis; guinea pig
n-3 polyunsaturated fatty acids (PUFA) are considered to be authentic immunosuppressors and appear to exert beneficial effects with respect to certain immune-mediated diseases. In addition to promoting T-helper 1 (Th1) cell to T-helper 2 (Th2) cell effector T-cell differentiation, n-3 PUFA may also exert anti-inflammatory actions by inducing apoptosis in Th1 cells. With respect to mechanisms of action, effects range from the modulation of membrane receptors to gene transcription via perturbation of a number of second messenger cascades. In this review, the putative targets of anti-inflammatory n-3 PUFA, activated during early and late events of T-cell activation will be discussed. Studies have demonstrated that these fatty acids alter plasma membrane micro-organization (lipid rafts) at the immunological synapse, the site where T-cells and antigen presenting cells (APC) form a physical contact for antigen initiated T-cell signaling. In addition, the production of diacylglycerol and the activation of different isoforms of protein kinase C (PKC), mitogen activated protein kinase (MAPK), calcium signaling, and nuclear translocation/activation of transcriptional factors, can be modulated by n-3 PUFA. Advantages and limitations of diverse methodologies to study the membrane lipid raft hypothesis, as well as apparent contradictions regarding the effect of n-3 PUFA on lipid rafts will be critically presented.
This study aims to evaluate immunization with polymeric microparticles containing recombinant antigen 85B (rAg85B) delivered directly to the lungs to protect against tuberculosis. rAg85B was expressed in Escherichia coli and encapsulated in poly(lactic-co-glycolic acid) microparticles (P-rAg85B). These were delivered as dry powders to the lungs of guinea pigs in single or multiple doses of homologous and heterologous antigens. Bacille Calmette–Guérin (BCG) delivered subcutaneously was employed as the positive control and as part of immunization strategies. Immunized animals were challenged with a low-dose aerosol of Mycobacterium tuberculosis (MTB) H37Rv to assess the extent of protection measured as reduction in bacterial burden (CFU) in the lungs and spleens of guinea pigs. Histopathological examination and morphometric analysis of these tissues were also performed. The heterologous strategy of BCG prime–P-rAg85B aerosol boosts appeared to enhance protection from bacterial infection, as indicated by a reduction in CFU in both the lungs and spleens compared with untreated controls. Although the CFU data were not statistically different from the BCG and BCG–BCG groups, the histopathological and morphometric analyses indicated the positive effect of BCG–P-rAg85B in terms of differences in area of tissue affected and number and size of granulomas observed in tissues. P-rAg85B microparticles appeared to be effective in boosting a primary BCG immunization against MTB infection, as indicated by histopathology and morphometric analysis. These encouraging observations are relevant to boosting adults previously immunized with BCG or exposed to MTB, commonly the case in the developing world, and should be followed by further assessment of an appropriate immunization protocol for maximum protection.
antigen 85B; BCG; boost immunization; microparticles; pulmonary delivery
n-3 polyunsaturated fatty acids (PUFA) are widely used for chemotheraphy/chemoprevention of chronic diseases. However, the molecular mechanism(s) by which the bioactive n-3 PUFA (eicosapentaenoic acid and docosahexaenoic acid) modulate effector pathways are not fully elucidated. Multiple experimental approaches, including use of animal models, cell lines, and human clinical trials, have been utilized to dissect the complex effectors. It is imperative to link these different experimental approaches together in order to interpret outcomes in the context of human physiology and pathophysiology. Unfortunately, the adoption of a broad array of model systems and a wide range of fatty acid exposures (i.e. doses) has made it difficult to interpret biological outcomes. Therefore, in this mini-review we discuss the impact of (a) molecular structure of bioactive fatty acids, (b) dose relevance relative to human consumption, (c) enrichment of fatty acids in sera and tissues following dietary intake, and (d) limitations of cell/tissue culture studies.
Objective. Mycobacterium tuberculosis infection is a major global threat to human health. The only tuberculosis (TB) vaccine currently available is bacillus Calmette-Guérin (BCG), although it has no efficacy in adults. Therefore, the development of a novel vaccine against TB for adults is desired.
Method. A novel TB vaccine expressing mycobacterial heat shock protein 65 (HSP65) and interleukin-12 (IL-12) delivered by the hemagglutinating virus of Japan- (HVJ)- envelope was evaluated against TB infection in mice. Bacterial load reductions and histopathological assessments were used to determine efficacy.
Results. Vaccination by BCG prime with IgHSP65+murine IL-12/HVJ-envelope boost resulted in significant protective efficacy (>10, 000-fold versus BCG alone) against TB infection in the lungs of mice. In addition to bacterial loads, significant protective efficacy was demonstrated by histopathological analysis of the lungs. Furthermore, the vaccine increased the number of T cells secreting IFN-γ.
Conclusion. This vaccine showed extremely significant protection against TB in a mouse model, consistent with results from a similar paper on cynomolgus monkeys. The results suggest that further development of the vaccine for eventual testing in clinical trials may be warranted.
The inflammatory response is designed to help fight and clear infection, remove harmful chemicals, and repair damaged tissue and organ systems. Although this process, in general, is protective, the failure to resolve the inflammation and return the target tissue to homeostasis can result in disease, including the promotion of cancer. A plethora of published literature supports the contention that dietary n-3 polyunsaturated fatty acids (PUFA), and eicosapentaenoic (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3) in particular, are important modulators of a host's inflammatory/immune responses. The following review describes a mechanistic model that may explain, in part, the pleiotropic anti-inflammatory and immunosuppressive properties of EPA and DHA. In this review, we focus on salient studies that address three overarching mechanisms of n-3 PUFA action: (i) modulation of nuclear receptor activation, i.e., nuclear factor-κB (NF-κB) suppression; (ii) suppression of arachidonic acid–cyclooxygenase-derived eicosanoids, primarily prostaglandin E2-dependent signaling; and (iii) alteration of the plasma membrane micro-organization (lipid rafts), particularly as it relates to the function of Toll-like receptors (TLRs), and T-lymphocyte signaling molecule recruitment to the immunological synapse (IS). We propose that lipid rafts may be targets for the development of n-3 PUFA-containing dietary bioactive agents to down-modulate inflammatory and immune responses and for the treatment of autoimmune and chronic inflammatory diseases.
Beside their health benefits, dietary omega 3 polyunsaturated fatty acids (n-3 PUFA) might impair host resistance to Mycobacterium tuberculosis (Mtb) by creating an immunosuppressive environment. We hypothesized that incorporation of n-3 PUFA suppresses activation of macrophage antimycobacterial responses and favors bacterial growth, in part, by modulating the IFNγ-mediated signaling pathway.
Murine macrophage-like J774A.1 cells were incubated with bovine serum albumin (BSA)-conjugated docosahexaenoic acid (DHA; 22:6n-3) or BSA alone, activated with recombinant IFNγ, and infected with a virulent strain (H37Rv) of M. tuberculosis. The fatty acid composition of macrophage membranes was modified significantly by DHA treatment. DHA-treated macrophages were less effective in controlling intracellular mycobacteria and showed impaired oxidative metabolism and reduced phagolysosome maturation. Incorporation of DHA resulted in defective macrophage activation, as characterized by reduced production of pro-inflammatory cytokines (TNFα, IL-6 and MCP-1), and lower expression of co-stimulatory molecules (CD40 and CD86). DHA treatment impaired STAT1 phosphorylation and colocalization of the IFNγ receptor with lipid rafts, without affecting surface expression of IFNγ receptor.
We conclude that DHA reduces the ability of J774A.1 cells to control M. tuberculosis in response to activation by IFNγ, by modulation of IFNγ receptor signaling and function, suggesting that n-3 PUFA-enriched diets may have a detrimental effect on host immunity to tuberculosis.
Previously, treatment with anti-gpTNFα antibody enhanced TNFα mRNA expression in pulmonary granulomas microdissected from non-vaccinated guinea pigs, and modified splenic granuloma architecture. In this study, pleural fluid, cells, and granulomatous tissues were collected 3, 5, and 8 days post-pleurisy induction in guinea pigs treated with anti-gpTNFα or normal serum control. Neutralizing TNFα reduced the percentage of macrophages in the pleural exudate while increasing the proportions of neutrophils and lymphocytes. Cell associated mycobacterial loads were increased in guinea pigs treated with anti-gpTNFα antibody. Cells from the pleural exudate in both treatment groups at day 3 expressed predominantly TNFα and IFNγ mRNA. By day 5, treatment with anti-gpTNFα antibody significantly reduced TNFα mRNA and increased TGFβ and iNOS mRNA expression, a transition which did not occur in the control group until day 8. TNFα mRNA overwhelmed the cytokine milieu of microdissected pleural granulomas in the control group at day 3 whereas TNFα, IFNγ, and TGFβ mRNA dominated the anti-gpTNFα-treated group. At day 8, granulomas from the control group began shifting towards an anti-inflammatory profile with increased levels of TGFβ mRNA. Neutralization of TNFα hastened the transition to an anti-inflammatory cytokine response in guinea pig pleural granulomas and exudate cells.
TUBERCULOUS PLEURISY; GUINEA PIG; TNFα
The molecular properties of immunosuppressive n-3 polyunsaturated fatty acids (PUFA) have not been fully elucidated. Using CD4+ T cells from wild type control and fat-1 transgenic mice (enriched in n-3 PUFA), we show that membrane raft accumulation assessed by Laurdan (6-dodecanoyl-2-dimethyl aminonaphthalene) labeling was enhanced in fat-1 cells following immunological synapse (IS) formation by CD3-specific Ab expressing hybridoma cells. However, the localization of PKCθ, PLCγ-1 and F-actin into the IS was suppressed. In addition, both the phosphorylation status of PLCγ-1 at the IS and cell proliferation as assessed by CFSE labeling and [3H]-thymidine incorporation were suppressed in fat-1 cells. These data imply that lipid rafts may be targets for the development of dietary agents for the treatment of autoimmune and chronic inflammatory diseases.
T cells; Signal Transduction; Cell Activation; fat-1; immunological synapse; lipid rafts; nutrition
Over the past several decades, data from both experimental animal studies and human clinical trials have shown that dietary n-3 polyunsaturated fatty acids (PUFA) exhibit anti-inflammatory bioactive properties, compared to n-6 PUFA. Collectively, these studies have identified multiple mechanisms by which n-3 PUFA affect immune cell responses. In this review, we discuss the putative targets of anti-inflammatory n-3 PUFA, specifically, cytokine production, antagonism of n-6 PUFA metabolism, binding to nuclear receptors as ligands, and the alteration of signaling protein acylation. In addition, we investigate the effect of n-3 PUFA on the coalescence of lipid rafts, specialized signaling platforms in the plasma membrane.
Diet; n-3 PUFA; chronic inflammation; CD4+ T-cells
The Mycobacterium tuberculosis dosR gene (Rv3133c) is part of an operon, Rv3134c-Rv3132c, and encodes a response regulator that has been shown to be upregulated by hypoxia and other in vitro stress conditions and may be important for bacterial survival within granulomatous lesions found in tuberculosis. DosR is activated in response to hypoxia and nitric oxide by DosS (Rv3132c) or DosT (Rv2027c). We compared the virulence levels of an M. tuberculosis dosR-dosS deletion mutant (ΔdosR-dosS [ΔdosR-S]), a dosR-complemented strain, and wild-type H37Rv in rabbits, guinea pigs, and mice infected by the aerosol route and in a mouse hollow-fiber model that may mimic in vivo granulomatous conditions. In the mouse and the guinea pig models, the ΔdosR-S mutant exhibited a growth defect. In the rabbit, the ΔdosR-S mutant did not replicate more than the wild type. In the hollow-fiber model, the mutant phenotype was not different from that of the wild-type strain. Our analyses reveal that the dosR and dosS genes are required for full virulence and that there may be differences in the patterns of attenuation of this mutant between the animal models studied.
In order to evaluate the effects of fatty acids on immune cell membrane structure and function, it is often necessary to maintain cells in culture. However, cell culture conditions typically reverse alterations in polyunsaturated fatty acid (PUFA) composition achieved by dietary lipid manipulation. Therefore, we hypothesized that T-cells from transgenic mice expressing the Caenorhabditis elegans n-3 desaturase (fat-1) gene would be resistant to the culture-induced loss of n-3 PUFA and, therefore, obviate the need to incorporate fatty acids or homologous serum into the medium. CD4+ T-cells were isolated from (i) control wild type (WT) mice fed a safflower oil-n-6 PUFA enriched diet (SAF) devoid of n-3 PUFA, (ii) fat-1 transgenic mice (enriched with endogenous n-3 PUFA) fed a SAF diet, or (iii) WT mice fed a fish oil (FO) based diet enriched in n-3 PUFA. T-cell phospholipids isolated from WT mice fed FO diet (enriched in n-3 PUFA) and fat-1 transgenic mice fed a SAF diet (enriched in n-6 PUFA) were both enriched in n-3 PUFA. As expected, the mol% levels of both n-3 and n-6 PUFA were decreased in cultures of CD4+ T-cells from FO-fed WT mice after 3 d in culture. In contrast, the expression of n-3 desaturase prevented the culture-induced decrease of n-3 PUFA in CD4+ T-cells from the transgenic mice. Carboxyfluorescein succinidyl ester (CFSE) -labeled CD4+ T-cells from fat-1/SAF vs. WT/SAF mice stimulated with anti-CD3 and anti-CD28 for 3 d, exhibited a reduced (P<0.05) number of cell divisions. We conclude that fat-1-containing CD4+ T-cells express a physiologically relevant, n-3 PUFA enriched, membrane fatty acid composition which is resistant to conventional cell culture-induced depletion.
n-3 fatty acid desaturase; Fish oil; Phospholipids; Lymphocyte
Recombinant adenovirus-vectored (Ad) tuberculosis (TB) vaccine platform has demonstrated great potential to be used either as a stand-alone or a boost vaccine in murine models. However, Ad TB vaccine remains to be evaluated in a more relevant and sensitive guinea pig model of pulmonary TB. Many vaccine candidates shown to be effective in murine models have subsequently failed to pass the test in guinea pig models.
Methods and Findings
Specific pathogen-free guinea pigs were immunized with BCG, AdAg85A intranasally (i.n), AdAg85A intramuscularly (i.m), BCG boosted with AdAg85A i.n, BCG boosted with AdAg85A i.m, or treated only with saline. The animals were then infected by a low-dose aerosol of M. tuberculosis (M.tb). At the specified times, the animals were sacrificed and the levels of infection in the lung and spleen were assessed. In separate studies, the long-term disease outcome of infected animals was monitored until the termination of this study. Immunization with Ad vaccine alone had minimal beneficial effects. Immunization with BCG alone and BCG prime-Ad vaccine boost regimens significantly reduced the level of M.tb infection in the tissues to a similar extent. However, while BCG alone prolonged the survival of infected guinea pigs, the majority of BCG-immunized animals succumbed by 53 weeks post-M.tb challenge. In contrast, intranasal or intramuscular Ad vaccine boosting of BCG-primed animals markedly improved the survival rate with 60% of BCG/Ad i.n- and 40% of BCG/Ad i.m-immunized guinea pigs still surviving by 74 weeks post-aerosol challenge.
Boosting, particularly via the intranasal mucosal route, with AdAg85A vaccine is able to significantly enhance the long-term survival of BCG-primed guinea pigs following pulmonary M.tb challenge. Our results thus support further evaluation of this viral-vectored TB vaccine in clinical trials.
Bioactive food components containing n-3 polyunsaturated fatty acids (PUFA) modulate multiple determinants which link inflammation to cancer initiation and progression. Therefore, in this study, fat-1 transgenic mice which convert endogenous n-6 PUFA to n-3 PUFA in multiple tissues, were injected with azoxymethane followed by 3 cycles of dextran sodium sulphate (DSS) to induce colitis-associated cancer. fat-1 mice exhibited a reduced number of colonic adenocarcinomas per mouse (1.05±0.29 vs 2.12±0.51, p=0.033), elevated apoptosis (p=0.03) and a decrease in n-6 PUFA-derived eicosanoids, compared to wild type (wt) mice. To determine whether the chemoprotective effects of n-3 PUFA could be attributed to its pleiotropic anti-inflammatory properties, colonic inflammation and injury scores were evaluated 5 d after DSS exposure followed by either a 3 d or 2 wk recovery period. There was no effect of n-3 PUFA at 3 d. However, following a 2 wk recovery period, colonic inflammation and ulceration scores returned to pretreatment levels compared to 3 d recovery only in fat-1 mice. For the purpose of examining the specific reactivity of lymphoid elements in the intestine, CD3+ T cells, CD4+ T helper cells and macrophages from colonic lamina propria were quantified. Comparison of 3 d vs 2 wk recovery time points revealed that fat-1 mice exhibited decreased (p<0.05) CD3+, CD4+ T helper, and macrophage cell numbers per colon as compared to wt mice. These results suggest that the anti-tumorigenic effect of n-3 PUFA may be mediated in part via its anti-inflammatory properties.
inflammation; chemoprevention; dextran sodium sulphate; tumor promotion
A growing body of epidemiological, clinical, and experimental evidence has underscored both the pharmacological potential and the nutritional value of dietary fish oil enriched in very long chain n-3 PUFAs such as docosahexaenoic acid (DHA, 22:6, n-3) and eicosapentaenoic acid (EPA, 20:5, n-3). The broad health benefits of very long chain n-3 PUFAs and the pleiotropic effects of dietary fish oil and DHA have been proposed to involve alterations in membrane structure and function, eicosanoid metabolism, gene expression and the formation of lipid peroxidation products, although a comprehensive understanding of the mechanisms of action has yet to be elucidated. In this review, we present data demonstrating that DHA selectively modulates the subcellular localization of lipidated signaling proteins depending on their transport pathway, which may be universally applied to other lipidated protein trafficking. An interesting possibility raised by the current observations is that lipidated proteins may exhibit different subcellular distribution profiles in various tissues, which contain a distinct membrane lipid composition. In addition, the current findings clearly indicate that subcellular localization of proteins with a certain trafficking pathway can be subjected to selective regulation by dietary manipulation. This form of regulated plasma membrane targeting of a select subset of upstream signaling proteins may provide cells with the flexibility to coordinate the arrangement of signaling translators on the cell surface. Ultimately, this may allow organ systems such as the colon to optimally decode, respond, and adapt to the vagaries of an ever-changing extracellular environment.
protein targeting; trafficking; fish oil; chemoprevention; colon cancer
The cytokine mRNA profiles of primary (arising from inhaled bacilli) and secondary (arising from hematogenous reseeding of the lung) granulomas from the lung lobes of bacillus Calmette-Guérin (BCG)-vaccinated and unimmunized guinea pigs challenged with virulent Mycobacterium tuberculosis by the pulmonary route were assessed in situ using laser capture microdissection (LCM) at 6 weeks after infection. The challenge dose chosen was so low that some lung lobes did not receive an implant from the airway. In unimmunized guinea pigs, some lobes contained either large, necrotic primary lesions or small, non-necrotic secondary lesions, or both. The lobes of BCG-vaccinated animals contained only non-necrotic primary tubercles, and no secondary lesions were visible. Real-time PCR analysis of the acquired RNA clearly demonstrated that primary tubercles from BCG-vaccinated guinea pigs were overwhelmed with mRNA from the anti-inflammatory cytokine, transforming growth factor (TGF)-β, with some IFN-γ and IL-12p40 mRNA. In contrast, primary lesions from unimmunized animals were dominated by proinflammatory TNF-α mRNA. The cytokine mRNA profile of secondary lesions from unimmunized animals was strikingly similar to the profile of primary lesions from BCG-vaccinated guinea pigs (i.e., a predominance of TGF-β mRNA with some IL-12p40 and IFN-γ mRNA), indicating that the lung lobes from which these lesions were retrieved had been naturally “vaccinated” by the time the bloodborne bacilli returned to the lung at 3 to 4 weeks after infection. Furthermore, cytokine mRNA analysis of splenic granulomas from nonvaccinated and vaccinated animals showed close resemblance to primary granulomas recovered from the lungs of the same animal, that is, high levels of TNF-α mRNA in unimmunized animals, and mostly TGF-β mRNA in BCG-vaccinated guinea pigs. Taken together, these data indicate that mycobacteria returning to the lungs of unimmunized guinea pigs 3 to 4 weeks after infection induce a local cytokine response that is fundamentally different from the response to inhaled bacilli and is reminiscent of the primary response in a vaccinated animal.
guinea pig; vaccine; tuberculosis; granuloma; cytokine
The plasma membrane of all eukaryotic cells contain heterogeneous self organizing intrinsically unstable liquid ordered domains or lipid assemblies in which key signal transduction proteins are localized. These assemblies are classified as “lipid rafts” (10–200 nm), which are composed mostly of cholesterol and sphingolipid microdomains and therefore do not integrate well into the fluid phospholipid bilayers. In addition, caveolae represent a subtype of lipid raft macrodomain that form flask-shaped membrane invaginations containing structural proteins, i.e., caveolins. With respect to the diverse biological effects of long chain polyunsaturated fatty acids (PUFA), increasing evidence suggests that n-3 PUFA and perhaps conjugated fatty acids uniquely alter the basic properties of cell membranes. Because of its polyunsaturation, docosahexaenoic acid (DHA) and possibly conjugated linoleic acid (CLA) are sterically incompatible with sphingolipid and cholesterol and, therefore, appear to alter lipid raft behavior and protein function. This review examines the evidence indicating that dietary sources of n-3 PUFA can profoundly alter the biochemical make up of lipid rafts/caveolae microdomains, thereby influencing cell signaling, protein trafficking, and cell cytokinetics.
membrane rafts; omega-3 fatty acids; conjugated fatty acids; microdomains
Understanding the physical characteristics of the local microenvironment in which Mycobacterium tuberculosis resides is an important goal that may allow the targeting of metabolic processes to shorten drug regimens. Pimonidazole hydrochloride (Hypoxyprobe) is an imaging agent that is bioreductively activated only under hypoxic conditions in mammalian tissue. We employed this probe to evaluate the oxygen tension in tuberculous granulomas in four animal models of disease: mouse, guinea pig, rabbit, and nonhuman primate. Following infusion of pimonidazole into animals with established infections, lung tissues from the guinea pig, rabbit, and nonhuman primate showed discrete areas of pimonidazole adduct formation surrounding necrotic and caseous regions of pulmonary granulomas by immunohistochemical staining. This labeling could be substantially reduced by housing the animal under an atmosphere of 95% O2. Direct measurement of tissue oxygen partial pressure by surgical insertion of a fiber optic oxygen probe into granulomas in the lungs of living infected rabbits demonstrated that even small (3-mm) pulmonary lesions were severely hypoxic (1.6 ± 0.7 mm Hg). Finally, metronidazole, which has potent bactericidal activity in vitro only under low-oxygen culture conditions, was highly effective at reducing total-lung bacterial burdens in infected rabbits. Thus, three independent lines of evidence support the hypothesis that hypoxic microenvironments are an important feature of some lesions in these animal models of tuberculosis.
Mouse virulence assessments of certain Mycobacterium tuberculosis mutants have revealed an immunopathology defect in which high tissue CFU counts are observed but the tissue pathology and lethality are reduced. M. tuberculosis mutants which grow and persist in the mouse lungs, but have attenuated disease progression, have the immunopathology (imp) phenotype. The antigenic properties of these strains may alter the progression of disease due to a reduction in host immune cell recruitment to the lungs resulting in disease attenuation and prolonged host survival.
In this study we focused on the mouse immune response to one such mutant; the M. tuberculosis ΔsigC mutant. Aerosol infection of DBA/2 and SCID mice with the M. tuberculosis ΔsigC mutant, complemented mutant and wild type strain showed proliferation of mutant bacilli in mouse lungs, but with decreased inflammation and mortality in DBA/2 mice. SCID mice shared the same phenotype as the DBA/2 mice in response to the ΔsigC mutant, however, they succumbed to the infection faster. Bronchoalveolar lavage (BAL) fluid analysis revealed elevated numbers of infiltrating neutrophils in the lungs of mice infected with wild type and complemented ΔsigC mutant strains but not in mice infected with the ΔsigC mutant. In addition, DBA/2 mice infected with the ΔsigC mutant had reduced levels of TNF-α, IL-1β, IL-6 and IFN-γ in the lungs. Similarly, there was a reduction in proinflammatory cytokines in the lungs of SCID mice. In contrast to the mouse model, the ΔsigC mutant had reduced initial growth in guinea pig lungs. A possible mechanism of attenuation in the ΔsigC mutant may be a reduction in neutrophilic-influx in the alveolar spaces of the lungs, and decreased proinflammatory cytokine secretion. In contrast to mouse data, the M. tuberculosis ΔsigC mutant proliferates slowly in guinea pig lungs, a setting characterized by caseating necrosis.
Our observations suggest that the immunopathology phenotype is associated with the inability to trigger a strong early immune response, resulting in disease attenuation. While macrophages and T cells have been shown to be important in containing M. tuberculosis disease our study has shown that neutrophils may also play an important role in the containment of this organism.
The early influx of neutrophils to the site of infection may be an important step in host resistance against Mycobacterium tuberculosis. In this study, we investigated the effect of M. tuberculosis infection on the ability of guinea pig neutrophils to produce interleukin-8 (IL-8; CXCL8) and tumor necrosis factor alpha (TNF-α) and to activate alveolar macrophages. Neutrophils and alveolar macrophages were isolated from naïve guinea pigs, cultured together or alone, and infected with virulent M. tuberculosis for 3, 12, and 24 h. IL-8 protein production in cocultures, as measured by using an enzyme-linked immunosorbent assay, was found to be additive at 24 h and significantly greater in M. tuberculosis-infected cocultures than in uninfected cocultures and in cultures of the infected neutrophils or macrophages alone. The IL-8 mRNA levels, determined by real-time reverse transcription-PCR, were elevated at 24 h in infected cocultures and infected cells cultured alone. In order to elucidate the contributions of neutrophils and their soluble mediators to the activation of alveolar macrophages, neutrophils and alveolar macrophages were cultured in a contact-independent manner by using a Transwell insert system. Neutrophils were infected with virulent M. tuberculosis in the upper wells, and alveolar macrophages were cultured in the lower wells. The release of hydrogen peroxide from alveolar macrophages exposed to soluble products from infected neutrophils was significantly increased compared to that from unexposed alveolar macrophages. Significant up-regulation of IL-1β and TNF-α mRNA levels in alveolar macrophages was observed at 24 and 30 h, respectively, compared to those in cells not exposed to soluble neutrophil products. Treatment with anti-guinea pig TNF-α polyclonal antibody completely abolished the response of alveolar macrophages to neutrophil products. This finding suggests that TNF-α produced by infected neutrophils may be involved in the activation of alveolar macrophages and hence may contribute to the containment of M. tuberculosis infection during the early period of infection.
Clinical and experimental evidence suggests that obesity-associated inflammation increases disease activity during colitis, attributed in part to the effects of Th17 cells. Using a model of concurrent obesity and colitis, we monitored changes in critical immune cell subsets and inflammatory biomarker expression in three key tissues: visceral adipose tissue, colon (local inflammatory site) and spleen (systemic inflammatory site), and we hypothesized that n-3 PUFA would reduce the percentage of inflammatory immune cell subsets and suppress inflammatory gene expression, thereby improving the disease phenotype. Obesity was induced in C57BL/6 mice by feeding a high fat (HF) diet (59.2% kcal) alone or an isocaloric HF diet supplemented with fish oil (HF-FO) for 12 weeks. Colitis was induced via a 2.5% trinitrobenzene sulfonic acid (TNBS) enema. The HF-FO diet improved the obese phenotype by reducing i) serum hormone concentrations (leptin and resistin), ii) adipose tissue mRNA expression of inflammatory cytokines (MCP-1, IFNγ, IL-6, IL17F and IL-21) and iii) total (F4/80+ CD11b+) and inflammatory adipose tissue M1 (F4/80+ CD11c+) macrophage content compared to HF (P<0.05). In addition, the HF-FO diet reduced both colitis-associated disease severity and colonic mRNA expression of the Th17 cell master transcription factor (RORγτ) and critical cytokines (IL-6, IL-17A, IL-17F, IL-21, IL-23 and IFNγ) versus HF (P<0.05). Compared to HF, the percentage of both splenic Th17 and Th1 cells were reduced by the HF-FO group (P<0.05). Under ex vivo polarizing conditions, the percentage of HF-FO derived CD4+ T cells that reached Th17 cell effector status was suppressed (P = 0.05). Collectively, these results indicate that n-3 PUFA suppress Th1/Th17 cells and inflammatory macrophage subsets and reconfigure the inflammatory gene expression profile in diverse tissue sites in obese mice following the induction of colitis.
Neutralization of tumor necrosis factor alpha (TNF-α) significantly down-regulated antigen-induced lymphoproliferation and the expression of interleukin-12 p40 and gamma interferon mRNA and enhanced the viability of intracellular attenuated and virulent mycobacteria in cocultures of immune T cells and macrophages obtained from Mycobacterium bovis BCG-vaccinated guinea pigs. This suggests the crucial role of TNF-α in the activation of a type 1 T-cell response against Mycobacterium tuberculosis infection.
Clinical tuberculosis (TB), whether noncavitary or cavitary, is the late stage of a chronic disease process, since Mycobacterium tuberculosis is a slowly growing organism. Our studies have shown that the profiles of antigenic proteins expressed by the in vivo bacteria that elicit antibodies differ in cavitary and noncavitary TB (K. Samanich, J. T. Belisle, and S. Laal, Infect. Immun. 69:4600-4609, 2001). To gain insight into antigenic proteins expressed during incipient, subclinical TB, an expression library of M. tuberculosis genomic DNA was screened with sera obtained during subclinical TB from guinea pigs infected with aerosols of M. tuberculosis H37Rv. One of the proteins recognized by antibodies elicited during subclinical TB infection of guinea pigs is the 309-kDa PPE55 (Rv3347c) protein. Genomic hybridization studies suggest that the PPE55 gene is specific to the M. tuberculosis complex and is present in a majority of clinical isolates tested. Antibodies to the C-terminal, ∼100-kDa fragment of PPE55 (PPE-C) were detectable in sera from 29/30 (97%) human immunodeficiency virus-negative/TB-positive (HIV− TB+) patients and 17/24 (71%) HIV+ TB+ patients tested but not in sera from purified-protein derivative-positive healthy controls, suggesting that the in vivo expression of PPE55 protein correlates with active M. tuberculosis infection. Anti-PPE-C antibodies were also detected in retrospective sera obtained months prior to manifestation of clinical TB from 17/21 (81%) HIV+ TB+ individuals tested, providing evidence that the protein is expressed during incipient, subclinical TB in HIV-infected humans. Thus, PPE55 is a highly immunogenic protein that may be useful for differentiating between latent TB and incipient, subclinical TB.
Tumor necrosis factor alpha (TNF-α) plays an important role in the host immune response to infection with the intracellular pathogen Mycobacterium tuberculosis. It is essential for the formation of protective tuberculous granulomas and regulates the expression of other cytokines which contribute to a protective immune response. Interleukin-12 (IL-12) is known to promote a Th1 response, which is essential for antimycobacterial resistance. Recombinant guinea pig TNF-α (rgpTNF-α) protein (17 kDa) was purified, and its bioactivity was confirmed by its cytotoxicity for L929 fibroblasts. High titers of polyclonal anti-gpTNF-α antibody were obtained by immunization of rabbits. Resident alveolar and peritoneal macrophages were isolated from guinea pigs and infected with either the H37Ra or H37Rv strain of M. tuberculosis. The mRNA levels for TNF-α and IL-12 p40 were measured using real-time PCR. IL-12 p40 mRNA was up-regulated in a dose-dependent manner by rgpTNF-α alone. In infected macrophages, a lower dose of rgpTNF-α intensified the mRNA levels of TNF-α and IL-12 p40. However, higher doses of rgpTNF-α suppressed TNF-α and IL-12 p40 mRNA. The antimycobacterial activity of macrophages was assessed by metabolic labeling of M. tuberculosis with [3H]uracil. Resident alveolar and peritoneal macrophages treated with anti-gpTNF-α antibody to block endogenous TNF-α exhibited increased intracellular mycobacterial growth. These data suggest that the dose of TNF-α is crucial to the stimulation of optimal expression of protective cytokines and that TNF-α contributes to the control of mycobacterial replication to promote host resistance against M. tuberculosis.