Luz Roberts

Cleavage of Fibrinogen by Proteinases Elicits Allergic Responses Through Toll-Like Receptor 4

Allergy Induction Proteinases found in fungi and other allergens elicit allergic inflammation, but how they do so is far from clear. It is also unclear how pattern recognition receptors, which detect invading microbes, drive allergic inflammation. Millien et al. (p. 792) shed light on this puzzle by showing that, in mice, induction of allergic inflammation requires proteinase-dependent cleavage of the clotting factor fibrinogen, leading to generation of a ligand that activates the pattern-recognition receptor, Toll-like receptor 4 (TLR4). Cleaved fibrinogen signals through TLR4 to activate the innate immune system and recruit cells to the airway, which drives both allergic responses and antifungal immunity. Allergic inflammation requires proteinase-dependent cleavage of fibrinogen that activates innate immunity through Toll-like receptor 4. Proteinases and the innate immune receptor Toll-like receptor 4 (TLR4) are essential for expression of allergic inflammation and diseases such as asthma. A mechanism that links these inflammatory mediators is essential for explaining the fundamental basis of allergic disease but has been elusive. Here, we demonstrate that TLR4 is activated by airway proteinase activity to initiate both allergic airway disease and antifungal immunity. These outcomes were induced by proteinase cleavage of the clotting protein fibrinogen, yielding fibrinogen cleavage products that acted as TLR4 ligands on airway epithelial cells and macrophages. Thus, allergic airway inflammation represents an antifungal defensive strategy that is driven by fibrinogen cleavage and TLR4 activation. These findings clarify the molecular basis of allergic disease and suggest new therapeutic strategies.

Lung Myeloid Dendritic Cells Coordinately Induce T H 1 and T H 17 Responses in Human Emphysema

Specialized immune cells in the lungs of patients with emphysema create an inflammatory environment that drives lung destruction in a characteristic autoimmune reaction. Dendritic Cells of Destruction Underlie Emphysema Tobacco smoke is never good for your lungs, and in some people it sets up a destructive process called emphysema. In this disease, air sacs that normally exchange carbon dioxide for oxygen become enlarged, ultimately losing their elastic recoil and physiological function. Breathing becomes labored. Even uninfected lungs with emphysema show signs of a complex immune response, with an accumulation of immune cells. To attack the difficult chicken-or-the-egg problem presented by this disease, Shan et al. sorted out which of these cells serve as the ringleaders in orchestrating this immune reaction and, in the process, found the telltale presence of T helper 17 (TH17) cells—a recently identified hallmark of autoimmune inflammation. Cigarette smoke causes irritation in the lung and activates a general defensive reaction via the innate immune system. When this system cannot restore tissue health, the more precise adaptive immune system comes into play. The authors of Shan et al. now show that specialized professional antigen-presenting cells—called dendritic cells—are recruited by a chemoattractant into the lung, where they induce naïve CD4 T cells to develop into TH1 cells. These immune agents then help cytotoxic T cells to target damaged host lung tissue for destruction. Also induced by the dendritic cells are TH17 cells. These specialized T lymphocytes normally protect the barriers between the body and the environment (the skin and the gut lining, for example), but they also congregate at sites in which the body is erroneously attacking itself, as in autoimmune diseases such as rheumatoid arthritis and colitis. The cytokine interleukin-17A secreted by the TH17 cells coordinates their contribution to destruction of the lung in emphysema by causing lung macrophages to secrete two critical molecules: CCL20, a chemoattractant for the dendritic cells, which then set up an inflammatory positive feedback loop, and matrix metalloproteinase 12 (MMP12), a potent enzyme that destroys a lung endogenous protective proteinase called α1-antitrypsin. In the industrialized world, the ultimate cause of emphysema is usually smoking, but in developing countries, smoke from cooking fires and pollution are important factors in the development of this disease, which is a leading cause of death worldwide. Even after removal of the respiratory irritant, the disease progression is only slowed and existing lung damage is irreversible. Medications can ease the shortness of breath but are not a cure. A lung transplant or partial lung removal is a last resort available only to a few fortunate individuals (see Cypel et al. in this issue). Identification of the cellular players—like the dendritic and TH17 cells described by Shan et al.—through which smoke causes lung destruction is a key to discovering drugs that effect damage control. Moreover, the injurious cellular cycles established in the emphysematous lung are likely not unique, and their elucidation will undoubtedly uncover clues to other immune-related diseases that are associated with smoking. Exposure to tobacco smoke activates innate and adaptive immune responses that in long-term smokers have been linked to diseases of the lungs, cardiovascular system, joints, and other organs. The destruction of lung tissue that underlies smoking-induced emphysema has been associated with T helper 1 cells that recognize the matrix protein elastin. Factors that result in the development of such autoreactive T cells in smokers remain unknown but are crucial for further understanding the pathogenesis of systemic inflammatory diseases in smokers. Here, we show that lung myeloid dendritic cells were sufficient to induce T helper 1 and T helper 17 responses in CD4 T cells. T helper 1 and 17 cells are invariably present in lungs from patients with emphysema but not in lungs from normal individuals. Interleukin-17A, a canonical T helper 17 cytokine, enhanced secretion of CCL20, a chemoattractant for dendritic cells, and matrix metalloproteinase 12, a potent elastolytic proteinase, from lung macrophages. Thus, although diverse lung factors potentially contribute to T helper effector differentiation in vivo, lung myeloid dendritic cells direct the generation of pathogenic T cells and support a feedback mechanism that sustains both inflammatory cell recruitment and lung destruction. This mechanism may underlie disease in other elastin-rich organs and tissues.

Cigarette Smoke Induction of Osteopontin (SPP1) Mediates T H 17 Inflammation in Human and Experimental Emphysema

The destruction of lung tissue in emphysema is orchestrated by antigen-presenting cells that have been activated by smoke. When Smoke Gets in Your Lungs Even without the warning label on the cigarette box, everyone knows that tobacco smoke is bad for you. But what really happens inside the lung to cause disease? Shan et al. have married studies in patients with emphysema and in mice exposed to tobacco smoke for several months to identify some of the crucial events that cause one serious lung disease—emphysema. They find that the antigen-presenting cells of the immune system are culpable; indeed, transfer of these cells from a mouse with emphysema into a healthy mouse induces disease. Further, they identify the signaling molecules in these cells that are activated by smoke (osteopontin and the transcription factor Irf7), which may prove to be useful targets for therapies. First, the authors verified that mice exposed to smoke carried similar immune cells in their lungs as do human smokers. These cells, including their secretory products IFN-γ and IL-17A, trigger the lung tissue destruction that robs emphysema patients of their ability to breathe. What activates these immune responses? The authors finger antigen-presenting cells by transferring these cells from diseased mice to healthy ones, showing that the disease transfers along with them. Then, tracing the process one step back, the authors look for smoke-induced genes in these antigen-presenting cells and identify osteopontin and Irf7, a transcription factor regulated by Spp1, as key mediators of smoke-induced cell activation. There are earlier steps in the process that have yet to be elucidated, but the cause of the havoc wreaked inside the lung by tobacco smoke is getting clearer. As a bonus, another cell type that participates in the mayhem unleashed in the emphysemic lung was uncovered. The γδ T cell, usually very rare, was unexpectedly found to be a good guy. Induced along with the destructive cells, these cells curtailed the damage but ultimately lost the battle. Perhaps bolstering the abilities of the helpful γδ T cells could ultimately help to treat serious immunogenic destructive processes in the lung. Smoking-related lung diseases are among the leading causes of death worldwide, underscoring the need to understand their pathogenesis and develop new effective therapies. We have shown that CD1a+ antigen-presenting cells (APCs) from lungs of patients with emphysema can induce autoreactive T helper 1 (TH1) and TH17 cells. Similarly, the canonical cytokines interferon-γ (IFN-γ) and interleukin-17A (IL-17A) are specifically linked to lung destruction in smokers, but how smoke activates APCs to mediate emphysema remains unknown. Here, we show that, in addition to increasing IFN-γ expression, cigarette smoke increased the expression of IL-17A in both CD4+ and γδ T cells from mouse lung. IL-17A deficiency resulted in attenuation of, whereas lack of γδ T cells exacerbated, smoke-induced emphysema in mice. Adoptive transfer of lung APCs isolated from mice with emphysema revealed that this cell population was capable of transferring disease even in the absence of active smoke exposure, a process that was dependent on IL-17A expression. Spp1 (the gene for osteopontin) was highly expressed in the pathogenic lung APCs of smoke-exposed mice and was required for the TH17 responses and emphysema in vivo, in part through its inhibition of the expression of the transcription factor Irf7. Thus, the Spp1-Irf7 axis is critical for induction of pathological TH17 responses, revealing a major mechanism by which smoke activates lung APCs to induce emphysema and identifying a pathway that could be targeted for therapeutic purposes.

Cyclosporin A aerosol improves the anticancer effect of paclitaxel aerosol in mice.

The objective of this study was to assess the effect of cyclosporin A liposome aerosol on the anticancer activity of paclitaxel (PTX) liposome aerosol against renal cell carcinoma (Renca) pulmonary metastases in mice. Cyclosporin A (CsA) was administered as a liposome aerosol for one-half hour before starting one-half hour treatment with PTX liposome aerosol (CsA/PTX), and in a second groups of animals cyclosporin A liposome aerosol was given before PTX for one-half hour and also later by mixing a second dose of cyclosporin A aerosol with PTX aerosol and extending the treatment period to one hour (CsA/PTX + CsA). In one experiment, PTX and CsA/PTX aerosols were significantly more effective compared to untreated controls against renal cell cancer as measured by lung weights and tumor surface areas. CsA/PTX was significantly better that PTX alone as measured by lung weights and tumor area. In a second experiment, tumor areas of PTX and CsA/PTX treated mice were significantly reduced compared to untreated controls and CsA/PTX treated mice had significantly smaller tumor areas than PTX treated mice. In contrast, tumor numbers were not significantly fewer than controls in either therapeutic group. In a third experiment, tumor numbers and tumor areas were significantly fewer in mice treated with CsA/PTX and CsA/PTX + CsA compared to untreated controls. Mice treated with CsA/PTX + CsA had significantly fewer tumors and less tumor area than mice receiving CsA/PTX. While PTX treated mice were not different than untreated controls with respect to tumor numbers or tumor volumes, PTX treated mice had significantly greater tumor numbers and tumor areas than CsA/PTX and CsA/PTX + CsA treated mice. Co-administration of CsA with PTX demonstrated significant dose dependent anticancer effects against renal cell pulmonary metastases in mice. Toxicity manifested by weight loss was associated with the highest dose of CsA.

Overexpression of Methyl-CpG Binding Protein 2 Impairs TH1 Responses

Duplication of the MECP2 gene in humans and mice results in immune dysregulation due in part to the suppression of IFN-γ production from T helper cells. The Immune Response in a Bind Children with MeCP2 (methyl-CpG binding protein 2) duplication syndrome have severe neurological defects caused by the overexpression of the MECP2 gene, which modulates expression of genes that influence neuronal and brain function. Many of these children also have recurrent respiratory infections; however, the role of MECP2 duplication in the immune system has been unclear. Now, Yang et al. have found that MeCP2 duplication may affect T helper type 1 (TH1) immunity. The authors examined patients with MECP2 duplication and found immunological abnormalities, including differences in memory T and B cells and NK cells. Indeed, mice that overexpress MeCP2 had an immunological defect as well. The immune system has evolved specialized functions to ward off particular types of infections. These MeCP2-overexpressing mice could not mount a sufficient TH1 response to control an infection by the parasite Leishmania major but could fight off an airway fungal infection—a TH2 response. Immune cells from children with MeCP2 duplication syndrome also had defects in the interferon-γ–mediated TH1 response. Yang et al. show that this defect may be caused by MeCP2-suppressing expression of IFN-γ in TH1 cells. This study paves the way for preventing infectious complications in children with MeCP2 duplication syndrome. The DNA binding protein methyl-CpG binding protein 2 (MeCP2) critically influences neuronal and brain function by modulating gene expression, and children with overexpression of the MECP2 gene exhibit postnatal neurological syndromes. We demonstrate that some children with MECP2 duplication also display variable immunological abnormalities that include reductions in memory T and B cells and natural killer cells and immunoglobulin assay responses. Moreover, whereas mice with MeCP2 overexpression were unable to control infection with the intra-macrophage parasite Leishmania major and secrete interferon-γ (IFN-γ) from involved lymph nodes, they were able to control airway fungal infection by Aspergillus niger and mount protective T helper cell type 2 (TH2)–dependent allergic responses. Relative to normal T cells, TH cells from children and mice with MECP2 duplication displayed similar impairments in IFN-γ secretion and TH1 responses that were due to both MeCP2-dependent suppression of IFN-γ transcription and sequestration of the IFN-γ locus as assessed by chromatin immunoprecipitation assay. Thus, overexpressed MeCP2 aberrantly suppresses IFN-γ secretion from TH cells, potentially leading to a partially immunodeficient state. Our findings establish a rational basis for identifying, treating, and preventing infectious complications potentially affecting children with MECP2 duplication.

Necessary and Sufficient Role for T Helper Cells To Prevent Fungal Dissemination in Allergic Lung Disease

ABSTRACT Mucosal immune responses to fungal infection range from T helper type 2 (Th2) cell-directed allergic inflammation to Th1-predominant neutrophilic inflammation, but the mechanisms directing these divergent mucosal immune outcomes and the role of T cells in host defense against mucosal fungal infections are not known. Here we examined the mouse mucosal immune responses to 12 filamentous environmental fungal species over a broad range of exposure doses and determined the requirement of T cells for host defense. For all tested fungi, low-grade conidium exposures induced Th2- and eosinophil-predominant allergic lung disease, whereas higher exposures led to rapid conversion to neutrophil- and Th1 cell-predominant inflammation, a phenomenon we term immune phenotype switching. All fungal exposure doses were further linked to the secretion of interleukin-17A (IL-17A). Fungal infections with Curvularia lunata and Aspergillus fumigatus were typically confined to the airway during allergic inflammation but became locally invasive and disseminated to the brain at higher conidium challenge doses, in association with predominant Th1 responses. Fungal dissemination occurred at relatively low challenge doses with the conidia of Aspergillus fumigatus administered to recombinase activating gene 1 (Rag-1)-deficient mice, which lack B and T cells, but B cell-deficient μMT mice and T helper cell-reconstituted Rag-1-deficient mice were comparable to wild-type mice in preventing fungal dissemination. Our findings demonstrate that Th2 cell-predominant allergic responses followed by immune phenotype switching and fungal dissemination are highly predictable outcomes with progressive fungal infectious burdens and that T helper cell responses are protective against lethal fungal dissemination.

Autoreactive T Cells in Human Smokers is Predictive of Clinical Outcome

Cross-sectional studies have suggested a role for activation of adaptive immunity in smokers with emphysema, but the clinical application of these findings has not been explored. Here we examined the utility of detecting autoreactive T cells as a screening tool for emphysema in an at-risk population of smokers. We followed 156 former and current (ever)-smokers for 2 years to assess whether peripheral blood CD4 T cell cytokine responses to lung elastin fragments (EFs) could discriminate between those with and without emphysema, and to evaluate the relevance of autoreactive T cells to predict changes during follow-up in lung physiological parameters. Volunteers underwent baseline complete phenotypic assessment with pulmonary function tests, quantitative chest CT, yearly 6-min walk distance (6MWD) testing, and annual measurement of CD4 T cell cytokine responses to EFs. The areas under the receiver operating characteristic curve to predict emphysema for interferon gamma (IFN-γ), and interleukin 6 (IL-6) responses to EFs were 0.81 (95% CI of 0.74–0.88) and 0.79 (95% CI of 0.72–0.86) respectively. We developed a dual cytokine enzyme-linked immunocell spot assay, the γ-6 Spot, using CD4 T cell IFN-γ and IL-6 responses and found that it discriminated emphysema with 90% sensitivity. After adjusting for potential confounders, the presence of autoreactive T cells was predictive of a decrease in 6MWD over 2 years (decline in 6MWD, −19 m per fold change in IFN-γ; P = 0.026, and −26 m per fold change in IL-6; P = 0.003). In support of the human association studies, we cloned CD4 T cells with characteristic T helper (Th)1 and Th17 responses to EFs in the peripheral blood of ever-smokers with emphysema, confirming antigenicity of lung elastin in this population. These findings collectively suggest that the EF-specific autoreactive CD4 T cell assay, γ-6 Spot, could provide a non-invasive diagnostic tool with potential application to large-scale screening to discriminate emphysema in ever-smokers, and predict early relevant physiological outcomes in those at risk.

Multistrain influenza protection induced by a nanoparticulate mucosal immunotherapeutic

All commercial influenza vaccines elicit antibody responses that protect against seasonal infection, but this approach is limited by the need for annual vaccine reformulation that precludes efficient responses against epidemic and pandemic disease. In this study we describe a novel vaccination approach in which a nanoparticulate, liposome-based agent containing short, highly conserved influenza-derived peptides is delivered to the respiratory tract to elicit potent innate and selective T cell-based adaptive immune responses. Prepared without virus-specific peptides, mucosal immunostimulatory therapeutic (MIT) provided robust, but short-lived, protection against multiple, highly lethal strains of influenza in mice of diverse genetic backgrounds. MIT prepared with three highly conserved epitopes that elicited virus-specific memory T-cell responses but not neutralizing antibodies, termed MITpep, provided equivalent, but more durable, protection relative to MIT. Alveolar macrophages were more important than dendritic cells in determining the protective efficacy of MIT, which induced both canonical and non-canonical antiviral immune pathways. Through activation of airway mucosal innate and highly specific T-cell responses, MIT and MITpep represent novel approaches to antiviral protection that offer the possibility of universal protection against epidemic and pandemic influenza.

Respiratory tract allergic disease and atopy: experimental evidence for a fungal infectious etiology

Allergic asthma is an obstructive lung disease linked to environmental exposures that elicit allergic airway inflammation and characteristic antigen-specific immunoglobulin reactions termed atopy. Analyses of asthma pathogenesis using experimental models have shown that T helper cells, especially T helper type 2 (Th2) cells and Th2 cytokines such as interleukin 4 (IL-4) and IL-13, are critical mediators of airway obstruction following allergen challenge, but the environmental initiators of lung Th2 responses are less defined. Our studies demonstrate that fungal-derived proteinases that are commonly found in home environments are requisite immune adjuvants capable of eliciting robust Th2 responses and allergic lung disease in mice. We have further shown that common household fungi readily infect the mouse respiratory tract and induce both asthma-like disease and atopy to otherwise innocuous bystander antigens through the secretion of proteinases. These findings support the possibility that asthma and atopy represent a reaction to respiratory tract fungal infection, suggesting novel means for diagnosis and therapy of diverse allergic disorders.

Mechanisms of allergy and clinical immunologyCD11a polymorphisms regulate TH2 cell homing and TH2-related disease

BACKGROUND TH2-dependent diseases vary in severity according to genotype, but relevant gene polymorphisms remain largely unknown. The integrin CD11a is a critical determinant of allergic responses, and allelic variants of this gene might influence allergic phenotypes. OBJECTIVE We sought to determine major CD11a allelic variants in mice and human subjects and their importance to allergic disease expression. METHODS We sequenced mouse CD11a alleles from C57BL/6 and BALB/c strains to identify major polymorphisms; human CD11a single nucleotide polymorphisms were compared with allergic disease phenotypes as part of the international HapMap project. Mice on a BALB/c or C57BL/6 background and congenic for the other strains CD11a allele were created to determine the importance of mouse CD11a polymorphisms in vivo and in vitro. RESULTS Compared with the C57BL/6 allele, the BALB/c CD11a allele contained a nonsynonymous change from asparagine to aspartic acid within the metal ion binding domain. In general, the BALB/c CD11a allele enhanced and the C57BL/6 CD11a allele suppressed TH2 cell-dependent disease caused by the parasite Leishmania major and allergic lung disease caused by the fungus Aspergillus niger. Relative to the C57BL/6 CD11a allele, the BALB/c CD11a allele conferred both greater T-cell adhesion to CD54 in vitro and enhanced TH2 cell homing to lungs in vivo. We further identified a human CD11a polymorphism that significantly associated with atopic disease and relevant allergic indices. CONCLUSIONS Polymorphisms in CD11a critically influence TH2 cell homing and diverse TH2-dependent immunopathologic states in mice and potentially influence the expression of human allergic disease.