Type-1 and type-2 lung granulomas, respectively, elicited by bead immobilized Mycobacteria bovis and Schistosoma mansoni egg antigens (Ags) display different patterns of chemokine expression. This study tested the hypothesis that chemokine expression patterns were related to upstream cytokine signaling. Using quantitative transcript analysis, we defined expression profiles for 16 chemokines and then examined the in vivo effects of neutralizing antibodies against interferon-γ (IFN-γ), interleukin (IL)-4, IL-10, IL-12, and IL-13. Transcripts for CXCL2, −5, −9, −10, and −11 and the CCL chemokine, CCL3, and lymphotactin (XCL1), were largely enhanced by Th1-related cytokines, IFN-γ or IL-12. Transcripts for CCL11, CCL22, CCL17, and CCL1 were enhanced largely by Th2-related cytokines, IL-4, IL-10, or IL-13. Transcripts for CCL4, CCL2, CCL8, CCL7, and CCL12 were potentially induced by either Th1- or Th2-related cytokines, although some of these showed biased expression. IFN-γ and IL-4 enhanced the greatest complement of transcripts, and their neutralization had the greatest anti-inflammatory effect on type-1 and type-2 granulomas, respectively. Th1/Th2 cross-regulation was evident because endogenous Th2 cytokines inhibited type-1, whereas Th1 cytokines inhibited type-2 biased chemokines. These findings reveal a complex cytokine–chemokine regulatory network that dictates profiles of local chemokine expression during T cell–mediated granuloma formation.
Chemokines are key mediators of leukocyte recruitment during pathogenic insult and also play a prominent role in homeostasis. While most chemokine receptors bind to multiple chemokines, CCR6 is unique in that this receptor is one of only a few that can bind only a single chemokine ligand, CCL20. CCR6 is an important receptor that is involved in regulating several aspects of mucosal immunity, including the ability to mediate the recruitment of immature dendritic cells (DCs) and mature DCs, and professional antigen presenting cells (APCs) to the sites of epithelial inflammation. Further, CCR6 mediates the homing of both CD4+ T (T-helper; Th) cells and DCs to the gut mucosal lymphoid tissue. DCs, which are known to be essential immune cells in innate immunity and in the initiation of adaptive immunity, play a central role in initiating a primary immune response Herein, we summarize the role of CCR6 in immune responses at epithelial and mucosal sites in both the lung and gut based on a review of the current literature.
CCR6; innate immunity; mucosal immunity; dendritic cell
C57BL/6 mice were maintained for up to 18-months on high-fat and low-fat diets with or without a multi-mineral-supplement derived from the skeletal remains of the red marine algae Lithothamnion calcareum. Numerous grossly observable liver masses were visible in animals on the “western-style” high-fat diet sacrificed at 12 and 18 months. The majority of the masses were in male mice (20 out of 100 males versus 3 out of 100 females; p=0.0002). There were more liver masses in animals on the high-fat diet than on the low-fat diet (15 out of 50 on high-fat versus 5 out of 50 on low-fat; p=0.0254). The multi-mineral supplement reduced the number of liver masses in mice on both diets (3 out of 25 male mice in the low-fat diet group without the supplement versus 1 out of 25 mice with supplement; 12 of 25 male mice in the high-fat diet group without the supplement versus 3 of 25 mice with supplement [p=0.0129]). Histological evaluation revealed a total of 17 neoplastic lesions (9 adenomas and 8 hepatocellular carcinomas), and 18 pre-neoplastic lesions. Out of 8 hepatocellular carcinomas, 7 were found in unsupplemented diet groups. Steatosis was widely observed in livers with and without grossly observable masses, but the multi-mineral supplement had no effect on the incidence of steatosis or its severity. Taken together, these findings suggest that a multi-mineral-rich natural product can protect mice against neoplastic and pre-neoplastic proliferative liver lesions that may develop in the face of steatosis.
Calcium; hepatocellular carcinoma; liver disease; minerals; trace elements
Although arachidonic acid cascade has been shown to be involved in sepsis, little is known about the role of prostaglandin D2 and its newly found receptor, chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2), on the septic response. Severe sepsis is associated with the failure of neutrophil migration. To investigate whether CRTH2 influences neutrophil recruitment and the lethality during sepsis, sepsis was induced by cecal ligation and puncture (CLP) surgery in mice. CRTH2 knockout (−/−) mice were highly resistant to CLP-induced sepsis, which was associated with lower bacterial load and lower production of TNF-α, IL-6, and CCL3. IL-10, an anti-inflammatory cytokine, was higher in CRTH2−/− mice, blunting CLP-induced lethality in CRTH2−/− mice. Neutrophil accumulation in the peritoneum was more pronounced after CLP in CRTH2−/− mice, which was associated with higher CXCR2 level in circulating neutrophils. Furthermore, sepsis caused a decrease in the level of acetylation of histone H3, an activation mark, at the CXCR2 promoter in WT neutrophils, suggesting that CXCR2 expression levels are epigenetically regulated. Finally, both pharmacological depletion of neutrophils and inhibition of CXCR2 abrogated the survival benefit in CRTH2−/− mice. These results demonstrate that genetic ablation of CRTH2 improved impaired neutrophil migration and survival during severe sepsis, which was mechanistically associated with epigenetic mediated CXCR2 expression. Thus, CRTH2 is a potential therapeutic target for polymicrobial sepsis.
The present study addressed the modulatory role of CCR4 in TLR9-mediated innate immunity and explored the underlying molecular mechanisms. Our results demonstrated that CCR4-deficient mice were resistant to both septic peritonitis induced by cecal ligation and puncture (CLP) and CpG DNA/D-galactosamine-induced shock. In bone marrow-derived macrophages (BMMΦs) from CLP-treated CCR4-deficient mice, TLR9-mediated pathways of MAPK/AP-1, PI3K/Akt, and IκB kinase (IKK) /NF-κB were impaired compared to WT cells. While TLR9 expression was not altered, the intensity of internalized CpG DNA was increased in CCR4-deficient macrophages when compared to WT macrophages. Pharmacological inhibitor studies revealed that impaired activation of JNK, PI3K/Akt, and/or IKK/NF-κB could be responsible for decreased proinflammatory cytokine expression in CCR4-deficient macrophages. Interestingly, the CCR4-deficient BMMΦs exhibited an alternatively activated (M2) phenotype and the impaired TLR9-mediated signal transduction responses in CCR4-deficient cells were similar to the signaling responses observed in WT BMMΦs skewed to an alternatively activated phenotype. These results indicated that macrophages deficient in CCR4 impart a regulatory influence on TLR9-mediated innate immunity.
Chemokines; Toll-like receptors; Macrophages
Granulomas represent a spectrum of inflammatory sequestration responses that may be initiated by a variety of agents, including non-infectious environmental factors and infectious microbial pathogens. Although this reaction is designed to be protective, the associated tissue injury is often responsible for a profound degree of pathology. While many of the mechanisms that sustain the development of the granuloma are enigmatic, it is accepted that the maintenance of this inflammatory process is dependent upon dynamic interactions between an inciting agent, inflammatory mediators, various immune and inflammatory cells, and structural cells of the involved tissue. The best studied of the host-dependent processes during granuloma development is the innate and adaptive immune response. The innate immune response by antigen-presenting cells [APCs; dendritic cells (DCs) and macrophages] is initiated quickly to protect from overwhelming pathogens, but with time, can also activate the adaptive immune response. APCs, essential regulators of the innate immune response, can respond to microbial ligands through Toll-like receptors (TLRs), which function in the recognition of microbial components and play an important role to link the innate and adaptive immune responses. CD4+ T helper (Th) cells are essential regulators of adaptive immune responses and inflammatory diseases. Recently, the Notch system has been shown to be an important bridge between APCs and T cell communication circuits. In the present review, we discuss recent findings that explore the mechanisms in the linkage of innate and adaptive immunity, including granulomatous formation though TLRs and Notch activation.
Notch signaling; Toll-like receptor; dendritic cell; T helper cell; innate immunity; acquired immunity
Ligands from dying cells are a source of Toll-like receptor (TLR) activating agents. Although TLR3 is known to respond to RNA from necrotic cells, the relative importance of this response in vivo during acute inflammatory processes has not been fully explored. We observed the involvement of TLR3 activation during experimental polymicrobial septic peritonitis and ischemic gut injury in the absence of an exogenous viral stimulus. In TLR3-deficient mice, increased chemokine/cytokine levels and neutrophil recruitment characterized the initial inflammatory responses in both injury models. However, the levels of inflammatory chemokines and tumor necrosis factor α quickly returned to baseline in tlr3−/− mice, and these mice were protected from the lethal effects of sustained inflammation. Macrophages from tlr3−/− mice responded normally to other TLR ligands but did not respond to RNA from necrotic neutrophils. Importantly, an immunoneutralizing antibody directed against TLR3 attenuated the generation of inflammatory chemokines evoked by byproducts from necrotic neutrophils cultured with wild-type macrophages. In vivo, anti-TLR3 antibody attenuated the tissue injury associated with gut ischemia and significantly decreased sepsis-induced mortality. Collectively, these data show that TLR3 is a regulator of the amplification of immune response and serves an endogenous sensor of necrosis, independent of viral activation.
Current dogma supports the concept that the expression of a disease-inducing signature cytokine phenotype is important to the maintenance stage of chronic lung disorders. This cytokine phenotype has been characterized as a polarization toward type 2 cytokines, which are profibrotic and immunoregulatory. The biology of this latter activity could mechanistically explain pathogen-induced exacerbation of chronic lung inflammation, as a skewed cytokine profile in the lung alters dendritic cell function, activates fibroblasts, and facilitates a subsequent “second hit” by an infectious pathogen. In this setting, cytokine biology is also linked to Toll-like receptors (TLRs) in the maintenance of lung immunity, as the activity of this receptor–ligand system by both leukocytes and stromal cells is likely an important component of disease chronicity. The participation of dendritic cells via TLRs in chronic lung disease could facilitate communication circuits established between antigen-presenting cells and lymphocytes. Data suggest that TLR activation via myeloid differentiation factor 88 adaptor protein leads to the induction of a Notch ligand known as Delta-like-4 on dendritic cells that activate the Notch receptor on T cells, promoting a helper T-cell type 1 cytokine response. It is likely that the evolution of host defense signals designed to recognize patterns emitted from a hostile microbial environment may now be superimposed on adaptive immunity and provide the underpinning to support the maintenance of chronic lung disease.
virus; dendritic cell; innate immunity
Toll like receptors (TLR) bridge innate immunity and host responses, including inflammation. Sterile inflammation such as a venous thrombus (VT) may involve TLR signaling, including TLR9.
Methods and Results
TLR9 signaling on thrombus resolution was investigated using a mouse model of stasis VT. VT were significantly larger in TLR9 −/− mice as compared with WT at 2 and 8 days, despite a 2 fold increase in thrombus PMN at 2d, and monocytes at 8d, while thrombus collagen and neovascularization was 55% and 37% less at 8d. Coincidently, decreased fibrinogen and increased thrombin-antithrombin complex were observed in TLR9 −/− mice thrombi. Vein wall IFNα, IL1α, and IL2 was significantly reduced in TLR9 −/− mice as compared to WT. Thrombus cell death pathway markers were not significantly altered at 2d, but caspase 1 was reduced in TLR9 −/− thrombi at 8d. MyD88 confers TLR9 intracellular signaling, but MyD88−/− mice had similar VT resolution as WT. However, inhibition of the NOTCH ligand delta-like 4 was associated with larger VT. Finally, stimulation with a TLR9 agonist was associated with smaller VT.
TLR9 signaling is integral for early and mid VT resolution through modulation of sterile inflammation, maintaining a TH1 milieu, and effects on the thrombosis pathway.
Previous studies have suggested that neonates rely heavily on innate immunity for their antimicrobial response to bacterial infections. However, the innate immune response by neonates to bacterial infection remains poorly characterized. Here, we show that in a murine model of neonatal polymicrobial sepsis, CXC ligand 10 (CXCL10) concentrations increase in the blood and peritoneum concordant with the peritoneal recruitment of granulocytes and macrophages. Additionally, CXC receptor 3 (CXCR3) expression on elicited peritoneal macrophages and granulocytes increases following sepsis. Blockade of CXCL10 worsens not only recruitment and phagocytic function of peritoneal granulocytes and macrophages but also survival. Deletion of CXCR3 also significantly increases mortality to a septic challenge. Finally, we demonstrate that the protective adjuvant effect of pretreatment with a Toll-like receptor 4 agonist to neonatal sepsis is dependent on an endogenous CXCL10 response and that pretreatment of neonates with CXCL10 can also significantly improve macrophage and granulocyte function and modestly improve outcome to polymicrobial sepsis. Together, these data suggest a critical role for CXCL10 signaling during neonatal sepsis.
TLRs are a family of receptors that mediate immune system pathogen recognition. In the respiratory system, TLR activation has both beneficial and deleterious effects in asthma. For example, clinical data indicate that TLR6 activation exerts protective effects in asthma. Here, we explored the mechanism or mechanisms through which TLR6 mediates this effect using mouse models of Aspergillus fumigatus–induced and house dust mite antigen–induced (HDM antigen–induced) chronic asthma. Tlr6–/– mice with fungal- or HDM antigen–induced asthma exhibited substantially increased airway hyperresponsiveness, inflammation, and remodeling compared with WT asthmatic groups. Surprisingly, whole-lung levels of IL-23 and IL-17 were markedly lower in Tlr6–/– versus WT asthmatic mice. Tlr6–/– DCs generated less IL-23 upon activation with lipopolysaccharide, zymosan, or curdlan. Impaired IL-23 generation in Tlr6–/– mice also corresponded with lower levels of expression of the pathogen-recognition receptor dectin-1 and expansion of Th17 cells both in vivo and in vitro. Exogenous IL-23 treatment of asthmatic Tlr6–/– mice restored IL-17A production and substantially reduced airway hyperresponsiveness, inflammation, and lung fungal burden compared with that in untreated asthmatic Tlr6–/– mice. Together, our data demonstrate that TLR6 activation is critical for IL-23 production and Th17 responses, which both regulate the allergic inflammatory response in chronic fungal-induced asthma. Thus, therapeutics targeting TLR6 activity might prove efficacious in the treatment of clinical asthma.
Previous epidemiological studies in humans and experimental studies in animals indicate that survivors of severe sepsis exhibit deficiencies in the activation and effector function of immune cells. In particular, CD4+ T lymphocytes can exhibit reduced proliferative capacity and improper cytokine responses following sepsis. To further investigate the cell-intrinsic defects of CD4+ T cells following sepsis, splenic CD4+ T cells from sham surgery and post-septic mice were transferred into lymphopenic mice. These recipient mice were then subjected to both TH1-(purified protein derivative) and TH2-(Schistosoma mansoni egg antigen) driven models of granulomatous lung inflammation. Post-septic CD4+ T cells mediated smaller TH1 and larger TH2 lung granulomas as compared to mice receiving CD4+ T cells from sham surgery donors. However, cytokine production by lymph node cells in antigen restimulation assays indicated increased pan-specific cytokine expression by post-septic CD4+ T cell recipient mice in both TH1 and TH2 granuloma models. These include increased production of TH2 cytokines in TH1 inflammation, and increased production of TH1 cytokines in TH2 inflammation. These results suggest that cell-intrinsic defects in CD4+ T cell effector function can have deleterious effects on inflammatory processes post-sepsis, due to a defect in the proper regulation of TH-specific cytokine expression.
Immunosuppression following severe sepsis remains a significant human health concern, as long-term morbidity and mortality rates of patients who have recovered from life-threatening septic shock remain poor. Mouse models of severe sepsis indicate this immunosuppression may be partly due to alterations in myeloid cell function; however, the effect of severe sepsis on subsequent CD4+ T cell responses remains unclear. In the present study, CD4+ T cells from mice subjected to an experimental model of severe sepsis (cecal ligation and puncture, CLP) were analyzed in vitro. CD4+ CD62L+ T cells from CLP mice exhibited reduced proliferative capacity and altered gene expression. Additionally, CD4+ CD62L+ T cells from CLP mice exhibit dysregulated cytokine production after in vitro skewing with exogenous cytokines, indicating a decreased capability of these cells to commit to either the TH1 or TH2 lineage. Repressive histone methylation marks were also evident at promoter regions for the TH1 cytokine interferon-γ (IFN-γ) and the TH2 transcription factor GATA-3 in naïve CD4+ T cells from CLP mice. These results provide evidence that CD4+ T cell subsets from postseptic mice exhibit defects in activation and effector function, possibly due to chromatin remodeling proximal to genes involved in cytokine production or gene transcription.
Sepsis; CD4+ T cell; Inflammation; Epigenetics; Mouse
Studies in humans and animal models indicate that profound immunosuppression is one of the chronic consequences of severe sepsis. This immune dysfunction encompasses deficiencies in activation of cells in both the myeloid and lymphoid cell lineages. As a result, survivors of severe sepsis are at risk of succumbing to infections perpetrated by opportunistic pathogens that are normally controlled by a fully functioning immune system. Recent studies have indicated that epigenetic mechanisms may be one driving force behind this immunosuppression, through suppression of proinflammatory gene production and subsequent immune cell activation, proliferation and effector function. A better understanding of epigenetics and post-septic immunosuppression can improve our diagnostic tools and may be an important potential source of novel molecular targets for new therapies. This review will discuss important pathways of immune cell activation affected by severe sepsis, and highlight pathways of epigenetic regulation that may be involved in post-septic immunosuppression.
sepsis; immunosuppression; histone modification; gene regulation; inflammation; macrophages; dendritic cells; T lymphocytes
Rationale: Accumulating evidence supports the hypothesis that the continuous host response to a persistent challenge can polarize the cytokine environment toward a Th2 cytokine phenotype, but the mechanisms responsible for this skewing are not clear.
Objectives: We investigated the role of Toll-like receptor 9 (TLR9) in a Th2-driven pulmonary granulomatous response initiated via the embolization of Schistosoma mansoni eggs to the lungs of mice.
Methods: Mice were intravenously injected with S. mansoni eggs. Histological and flow cytometric analysis, cytokine measurement, adoptive transfer of bone marrow (BM)-derived dendritic cells (DCs), and in vitro T-cell treatments with antigen-presenting cells were examined.
Measurements and Main Results: In comparison to wild-type mice, TLR9−/− mice showed increased pulmonary granuloma size, augmented collagen deposition, increased Th2 cytokine phenotype, and impaired accumulation of DCs. BM-derived DCs, but not macrophages, recovered from animals with developed Th2-type lung granulomas promoted the production of type 2 cytokines from CD4+ T cells. BM-derived DCs from TLR9−/− mice induced impaired Th1 cytokine and enhanced Th2 cytokine production by T cells, compared with DCs from WT mice. Macrophages from TLR9−/− mice expressed a significantly higher alternatively activated (M2) phenotype characterized by increased “found in inflammatory zone-1” (FIZZ1) and arginase-1 expression. The adoptive transfer of BM-derived DCs from syngeneic WT mice into TLR9−/− mice restored the granuloma phenotype seen in WT mice.
Conclusions: These studies suggest that TLR9 plays an important mechanistic role in the maintenance of the pulmonary granulomatous response.
granuloma; pulmonary fibrosis; innate immunity; dendritic cell; macrophage
Influenza virus is a common cause of respiratory infection and morbidity, which is often due to deleterious host immune responses directed against the pathogen. We investigated the role of IL-1 receptor-associated kinase-M (IRAK-M), an inhibitor of MyD88-dependent TLR signaling, in modulating the innate inflammatory response during influenza pneumonia using a murine model. The intranasal administration of influenza resulted in the upregulation of IRAK-M mRNA and protein levels in the lungs within 2 d after infectious challenge. Pulmonary influenza infection in mice deficient in IRAK-M (IRAK-M−/−) resulted in substantially increased mortality compared with similarly treated wild-type animals. Increased mortality in IRAK-M−/− mice was associated with enhanced early influx of neutrophils, high permeability edema, apoptosis of lung epithelial cells, markedly increased expression of inflammatory cytokines/chemokines, and release of neutrophil-derived enzymes, including myeloperoxidase and neutrophil elastase. Early viral clearance was not different in mutant mice, whereas viral titers in lungs and blood were significantly higher in IRAK-M−/− mice compared with wild-type animals. Increased lethality observed in IRAK-M−/− mice after influenza challenge was abrogated by Ab-mediated blockade of CXCR2. Collectively, our findings indicate that IRAK-M is critical to preventing deleterious neutrophil-dependent lung injury during influenza infection of the respiratory tract.
The activation and differentiation of T cells are dependent upon numerous initiating events that are influenced by the immune environment, nature of the antigen, as well as the activation state of APCs. In the present studies we have investigated the role of a specific notch ligand, delta-like 4 (Dll4). In particular, our data have indicated that Dll4 is inducible by pathogen-associated signals through TLR activation on DC but not early response inflammatory cytokines, IL-1 and IL-18 that also activate cells via MyD88 adapter pathway. Our observations from in vitro cultures with ovalbumin specific TCR transgenic cells (DO11.10) confirmed earlier reports demonstrating that Dll4 inhibits Th2 cytokine production. Furthermore, Dll4 enhances the generation of IL-17 producing T cells in the presence of additional skewing cytokines, IL-6 and TGFβ. In the absence of notch signals IL17 production was significantly reduced even under specific skewing conditions. These studies further demonstrate that Dll4 upregulates RORγt expression in T cells and that both RORγt and IL17 gene promoters are direct transcriptional notch targets that augment the differentiation of Th17 cell populations. Thus, facilitation of efficient T cell differentiation may depend upon the activation of T cells via specific notch ligand stimulation.
Interleukin-33 is a member of the IL-1 cytokine family whose functions are mediated and modulated by the ST2 receptor. IL-33-ST2 expression and interactions have been explored in mouse macrophages but little is known about the effect of IL-33 on human macrophages. The expression of ST2 transcript and protein levels, and IL-33-mediated effects on M1 (i.e. classical activation) and M2 (i.e. alternative activation) chemokine marker expression in human bone marrow-derived macrophages were examined.
Human macrophages constitutively expressed the membrane-associated (i.e. ST2L) and the soluble (i.e. sST2) ST2 receptors. M2 (IL-4 + IL-13) skewing stimuli markedly increased the expression of ST2L, but neither polarizing cytokine treatment promoted the release of sST2 from these cells. When added to naïve macrophages alone, IL-33 directly enhanced the expression of CCL3. In combination with LPS, IL-33 blocked the expression of the M2 chemokine marker CCL18, but did not alter CCL3 expression in these naive cells. The addition of IL-33 to M1 macrophages markedly increased the expression of CCL18 above that detected in untreated M1 macrophages. Similarly, alternatively activated human macrophages treated with IL-33 exhibited enhanced expression of CCL18 and the M2 marker mannose receptor above that detected in M2 macrophages alone.
Together, these data suggest that primary responses to IL-33 in bone marrow derived human macrophages favors M1 chemokine generation while its addition to polarized human macrophages promotes or amplifies M2 chemokine expression.
Sepsis results in a profound state of immunosuppression, which is temporally associated with impaired leukocyte function. The mechanism of leukocyte reprogramming in sepsis is incompletely understood. In this study, we explored mechanisms contributing to dysregulated inflammatory cytokine expression by pulmonary macrophages during experimental sepsis. Pulmonary macrophages (PM) recovered from the lungs of mice undergoing cecal ligation and puncture (CLP) display transiently reduced expression of some, but not all innate genes in response to LPS. Impaired expression of TNF-α and iNOS was associated with reduced acetylation and methylation of specific histones (AcH4 and H3K4me3) and reduced binding of RNA polymerase II to the promoters of these genes. Transient impairment in LPS-induced cytokine responses in septic PM temporally correlated with induction of IRAK-M mRNA and protein, which occurred in a MyD88-dependent fashion. PM isolated from IRAK-M−/− mice were largely refractory to CLP-induced impairment in cytokine expression, chromatin remodeling, recruitment of RNA polymerase II, and induction of histone deacetylase-2 observed during sepsis. Our findings indicate that systemic sepsis induces epigenetic silencing of cytokine gene expression in lung macrophages, and IRAK-M appears to be a critical mediator of this response.
Helminthiasis and tuberculosis (TB) coincide geographically and there is much interest in exploring how concurrent worm infections might alter immune responses against bacilli and might necessitate altered therapeutic approaches. A DNA vaccine that codifies heat shock protein Hsp65 from M. leprae (DNAhsp65) has been used in therapy during experimental tuberculosis. This study focused on the impact of the co-existence of worms and TB on the therapeutic effects of DNAhsp65.
Mice were infected with Toxocara canis or with Schistosoma mansoni, followed by coinfection with M. tuberculosis and treatment with DNAhsp65. While T. canis infection did not increase vulnerability to pulmonary TB, S. mansoni enhanced susceptibility to TB as shown by higher numbers of bacteria in the lungs and spleen, which was associated with an increase in Th2 and regulatory cytokines. However, in coinfected mice, the therapeutic effect of DNAhsp65 was not abrogated, as indicated by colony forming units and analysis of histopathological changes. In vitro studies indicated that Hsp65-specific IFN-γ production was correlated with vaccine-induced protection in coinfected mice. Moreover, in S. mansoni-coinfected mice, DNA treatment inhibited in vivo TGF-β and IL-10 production, which could be associated with long-term protection.
We have demonstrated that the therapeutic effects of DNAhsp65 in experimental TB infection are persistent in the presence of an unrelated Th2 immune response induced by helminth infections.
From 14 diseases considered by WHO as Neglected Tropical Diseases, four involve helminth infections, such as schistosomiasis and soil-transmitted helminthiasis. Toxocariasis is a soil-transmitted worm highly prevalent in many developing countries, while schistosomiasis causes an annual mortality of 14,000 deaths per year, with 200–300 million infected people and 10% at risk of infection worldwide. Additionally, tuberculosis (TB) remains one of the leading causes of morbidity and mortality in many settings, particularly in the world's poorest countries. Mycobacteria and helminths are co-endemic and induce opposing patterns of immune responses in the host, recognized as Th1 and Th2 respectively. These co-existing patterns could be associated with the failure of TB vaccines. In this sense, we investigated the inflammatory and immune response in a coinfection model with T. canis or S. mansoni and M. tuberculosis analyzing the effects of an immunotherapy that has previously shown efficacy in experimental TB. This immunotherapy is based on a DNA vaccine that codifies a mycobacterial heat shock protein (hsp65), which can prevent TB in a prophylactic and also therapeutic setting. In this work, we show that helminth coinfection does not abrogate the therapeutic effects of DNAhsp65 vaccine against TB.
Tuberculosis (TB) granulomas are organized collections of immune cells comprised of macrophages, lymphocytes and other cells that form in the lung as a result of immune response to Mycobacterium tuberculosis (Mtb) infection. Formation and maintenance of granulomas are essential for control of Mtb infection and are regulated in part by a pro-inflammatory cytokine, tumor necrosis factor-α (TNF). To characterize mechanisms that control TNF availability within a TB granuloma, we developed a multi-scale two compartment partial differential equation model that describes a granuloma as a collection of immune cells forming concentric layers and includes TNF/TNF receptor binding and trafficking processes. We used the results of sensitivity analysis as a tool to identify experiments to measure critical model parameters in an artificial experimental model of a TB granuloma induced in the lungs of mice following injection of mycobacterial antigen-coated beads. Using our model, we then demonstrated that the organization of immune cells within a TB granuloma as well as TNF/TNF receptor binding and intracellular trafficking are two important factors that control TNF availability and may spatially coordinate TNF-induced immunological functions within a granuloma. Further, we showed that the neutralization power of TNF-neutralizing drugs depends on their TNF binding characteristics, including TNF binding kinetics, ability to bind to membrane-bound TNF and TNF binding stoichiometry. To further elucidate the role of TNF in the process of granuloma development, our modeling and experimental findings on TNF-associated molecular scale aspects of the granuloma can be incorporated into larger scale models describing the immune response to TB infection. Ultimately, these modeling and experimental results can help identify new strategies for TB disease control/therapy.
Tuberculosis is a common and deadly infectious disease caused by a highly successful bacterium, Mycobacterium tuberculosis (Mtb). Multiple host immune factors control the formation of a self-organizing aggregate of immune cells termed a granuloma in the lungs after inhalation of Mtb. One such factor, tumor necrosis factor-α (TNF), is a protein that regulates inflammatory immune responses. Availability of TNF within a TB granuloma has been proposed to have a critical role in the protective immunity against TB. However, direct measurement of the level of TNF in a granuloma is not experimentally feasible. Therefore, we develop a mathematical model based on an experimental model of granuloma developed in mice to predict TNF availability in a granuloma. We measure values of critical model parameters and explore mechanisms that influence TNF availability in the granuloma. We find that cellular organization in a granuloma and intracellular trafficking of TNF control TNF availability in a granuloma. Further, our model analysis also highlights anti-TNF drug properties that determine their TNF neutralization power. Our findings complement and extend those of recent studies on the role of TNF in the immune response against TB.
This article reviews current animal models and laboratory studies investigating the pathophysiology of lung contusion (LC), a common and severe condition in patients with blunt thoracic trauma. Emphasis is on studies elucidating cells, mediators, receptors and processes important in the innate pulmonary inflammatory response that contribute to LC injury. Surfactant dysfunction in the pathogenesis of LC is also discussed, as is the potential role of epithelial cell or neutrophil apoptosis. Studies examining combination injuries where LC is exacerbated by secondary insults like gastric aspiration in trauma patients are also noted. The need for continuing mechanism-based research to further clarify the pathophysiology of LC injury, and to define and test potential therapeutic interventions targeting specific aspects of inflammation or surfactant dysfunction to improve clinical outcomes in patients with LC, is also emphasized.
The role of Toll-like receptor 9 (TLR9) in antifungal responses in the immunodeficient and allergic host is unclear. We investigated the role of TLR9 in murine models of invasive aspergillosis and fungal asthma. Neutrophil-depleted TLR9 wild-type (TLR9+/+) and TLR9-deficient (TLR9−/−) mice were challenged with resting or swollen Aspergillus fumigatus conidia and monitored for survival and lung inflammatory responses. The absence of TLR9 delayed, but did not prevent, mortality in immunodeficient mice challenged with resting or swollen conidia compared to TLR9+/+ mice. In a fungal asthma model, TLR9+/+ and TLR9−/− mice were sensitized to soluble A. fumigatus antigens and challenged with resting or swollen A. fumigatus conidia, and both groups of mice were analyzed prior to and at days 7, 14, and 28 after the conidium challenge. When challenged with resting conidia, TLR9−/− mice exhibited significantly lower airway hyper-responsiveness compared to the TLR9+/+ groups. In contrast, A. fumigatus-sensitized TLR9−/− mice exhibited pulmonary fungal growth at days 14 and 28 after challenge with swollen conidia, a finding never observed in their allergic wild-type counterparts. Increased fungal growth in allergic TLR9−/− mice correlated with markedly decreased dectin-1 expression in whole lung samples and isolated dendritic cell populations. Further, whole lung levels of interleukin-17 were lower in allergic TLR9−/− mice compared to similar TLR9+/+ mice. Together, these data suggest that TLR9 modulates pulmonary antifungal immune responses to swollen conidia, possibly through the regulation of dectin-1 expression.
Chronic obstructive pulmonary disease (COPD) is characterized by chronic airway inflammation. Cigarette smoke has been considered a major player in the pathogenesis of COPD. The inflamed airways of COPD patients contain several inflammatory cells including neutrophils, macrophages,T lymphocytes, and dendritic cells (DCs). The relative contributions of these various inflammatory cells to airway injury and remodeling are not well documented. In particular, the potential role of DCs as mediators of inflammation in the smoker's airways and COPD patients is poorly understood. In the current study we analyzed the effects of cigarette smoke extract on mouse bone marrow derived DC and the production of chemokines and cytokines were studied. In addition, we assessed CSE-induced changes in cDC function in the mixed lymphocyte reaction (MLR) examining CD4+ and CD8+ T cell proliferation. Cigarette smoke extract induces the release of the chemokines CCL3 and CXCL2 (but not cytokines), via the generation of reactive oxygen species (ROS). In a mixed-leukocyte reaction assay, cigarette smoke-primed DCs potentiate CD8+T cell proliferation via CCL3. In contrast, proliferation of CD4+T cells is suppressed via an unknown mechanism. The cigarette smoke-induced release of CCL3 and CXCL2 by DCs may contribute to the influx of CD8+T cells and neutrophils into the airways, respectively.