Microbiota Promotes Chronic Pulmonary Inflammation by Enhancing IL-17A and Autoantibodies.

RATIONALE: Changes in the pulmonary microbiota are associated with progressive respiratory diseases including chronic obstructive pulmonary disease (COPD). Whether there is a causal relationship between these changes and disease progression remains unknown. OBJECTIVES: To investigate the link between an altered microbiota and disease, we used a murine model of chronic lung inflammation that is characterized by key pathological features found in COPD and compared responses in specific pathogen-free (SPF) mice and mice depleted of microbiota by antibiotic treatment or devoid of a microbiota (axenic). METHODS: Mice were challenged with LPS/elastase intranasally over 4 weeks, resulting in a chronically inflamed and damaged lung. The ensuing cellular infiltration, histological damage, and decline in lung function were quantified. MEASUREMENTS AND MAIN RESULTS: Similar to human disease, the composition of the pulmonary microbiota was altered in diseased animals. We found that the microbiota richness and diversity were decreased in LPS/elastase-treated mice, with an increased representation of the genera Pseudomonas and Lactobacillus and a reduction in Prevotella. Moreover, the microbiota was implicated in disease development as mice depleted, or devoid, of microbiota exhibited an improvement in lung function, reduced inflammation, and lymphoid neogenesis. The absence of microbial cues markedly decreased the production of IL-17A, whereas intranasal transfer of fluid enriched with the pulmonary microbiota isolated from diseased mice enhanced IL-17A production in the lungs of antibiotic-treated or axenic recipients. Finally, in mice harboring a microbiota, neutralizing IL-17A dampened inflammation and restored lung function. CONCLUSIONS: Collectively, our data indicate that host-microbial cross-talk promotes inflammation and could underlie the chronicity of inflammatory lung diseases.

Targeting IL-1ß and IL-17A driven inflammation during influenza-induced exacerbations of chronic lung inflammation.

For patients with chronic lung diseases, such as chronic obstructive pulmonary disease (COPD), exacerbations are life-threatening events causing acute respiratory distress that can even lead to hospitalization and death. Although a great deal of effort has been put into research of exacerbations and potential treatment options, the exact underlying mechanisms are yet to be deciphered and no therapy that effectively targets the excessive inflammation is available. In this study, we report that interleukin-1ß (IL-1ß) and interleukin-17A (IL-17A) are key mediators of neutrophilic inflammation in influenza-induced exacerbations of chronic lung inflammation. Using a mouse model of disease, our data shows a role for IL-1ß in mediating lung dysfunction, and in driving neutrophilic inflammation during the whole phase of viral infection. We further report a role for IL-17A as a mediator of IL-1ß induced neutrophilia at early time points during influenza-induced exacerbations. Blocking of IL-17A or IL-1 resulted in a significant abrogation of neutrophil recruitment to the airways in the initial phase of infection or at the peak of viral replication, respectively. Therefore, IL-17A and IL-1ß are potential targets for therapeutic treatment of viral exacerbations of chronic lung inflammation.

Gut microbiota metabolism of dietary fiber influences allergic airway disease and hematopoiesis.

Metabolites from intestinal microbiota are key determinants of host-microbe mutualism and, consequently, the health or disease of the intestinal tract. However, whether such host-microbe crosstalk influences inflammation in peripheral tissues, such as the lung, is poorly understood. We found that dietary fermentable fiber content changed the composition of the gut and lung microbiota, in particular by altering the ratio of Firmicutes to Bacteroidetes. The gut microbiota metabolized the fiber, consequently increasing the concentration of circulating short-chain fatty acids (SCFAs). Mice fed a high-fiber diet had increased circulating levels of SCFAs and were protected against allergic inflammation in the lung, whereas a low-fiber diet decreased levels of SCFAs and increased allergic airway disease. Treatment of mice with the SCFA propionate led to alterations in bone marrow hematopoiesis that were characterized by enhanced generation of macrophage and dendritic cell (DC) precursors and subsequent seeding of the lungs by DCs with high phagocytic capacity but an impaired ability to promote T helper type 2 (TH2) cell effector function. The effects of propionate on allergic inflammation were dependent on G protein-coupled receptor 41 (GPR41, also called free fatty acid receptor 3 or FFAR3), but not GPR43 (also called free fatty acid receptor 2 or FFAR2). Our results show that dietary fermentable fiber and SCFAs can shape the immunological environment in the lung and influence the severity of allergic inflammation.

Dysregulation of allergic airway inflammation in the absence of microbial colonization.

RATIONALE: The incidence of allergic disorders is increasing in developed countries and has been associated with reduced exposure to microbes and alterations in the commensal bacterial flora. OBJECTIVES: To ascertain the relevance of commensal bacteria on the development of an allergic response, we used a model of allergic airway inflammation in germ-free (GF) mice that lack any exposure to pathogenic or nonpathogenic microorganisms. METHODS: Allergic airway inflammation was induced in GF, specific pathogen-free (SPF), or recolonized mice by sensitization and challenge with ovalbumin. The resulting cellular infiltrate and cytokine production were measured. MEASUREMENTS AND MAIN RESULTS: Our results show that the total number of infiltrating lymphocytes and eosinophils were elevated in the airways of allergic GF mice compared with control SPF mice, and that this increase could be reversed by recolonization of GF mice with the complex commensal flora of SPF mice. Exaggerated airway eosinophilia correlated with increased local production of Th2-associated cytokines, elevated IgE production, and an altered number and phenotype of conventional dendritic cells. Regulatory T-cell populations and regulatory cytokine levels were unaltered, but GF mice exhibited an increased number of basophils and decreased numbers of alveolar macrophages and plasmacytoid dendritic cells. CONCLUSIONS: These data demonstrate that the presence of commensal bacteria is critical for ensuring normal cellular maturation, recruitment, and control of allergic airway inflammation.

Bacterial-induced protection against allergic inflammation through a multicomponent immunoregulatory mechanism.

BACKGROUND: Airborne microbial products have been reported to promote immune responses that suppress asthma, yet how these beneficial effects take place remains controversial and poorly understood. METHODS: We exposed mice to the bacterium Escherichia coli and subsequently induced allergic airway inflammation through sensitization and intranasal challenge with ovalbumin. RESULTS: Pulmonary exposure to the bacterium Escherichia coli leads to a suppression of allergic airway inflammation. This immune modulation was neither mediated by the induction of a T helper 1 (Th1) response nor regulatory T cells; however, it was dependent on Toll-like receptor 4 (TLR4) but did not involve TLR desensitisation. Dendritic cell migration to the draining lymph nodes and activation of T cells was unaffected by prior exposure to E. coli, while dendritic cells in the lung displayed a less activated phenotype and had impaired antigen presentation capacity. Consequently, in situ Th2 cytokine production was abrogated. The suppression of airway hyper-responsiveness was mediated through the recruitment of gd T cells; however, the suppression of dendritic cells and T cells was mediated through a distinct mechanism that could not be overcome by the local administration of activated dendritic cells, or by the in vivo administration of tumour necrosis factor a. CONCLUSION: Our data reveal a localized immunoregulatory pathway that acts to protect the airways from allergic inflammation.

Murine endoglin-specific single-chain Fv fragments for the analysis of vascular targeting strategies in mice.

Endoglin has been identified as a promising cell surface antigen for vascular targeting approaches in cancer therapy, e.g. employing antibody molecules as targeting moieties. However, in vivo analysis of such strategies in mouse models requires antibodies recognizing endoglin on mouse endothelial cells. Here we describe the isolation of single-chain Fv fragments (scFvs) from phage display libraries, which bind to the extracellular region of mouse endoglin. One of these clones, scFv mE12, showed strong (K(d)=11 nM) and selective binding to purified endoglin and also to the endoglin-expressing mouse endothelioma cell line eEnd.2. This antibody recognized a linear epitope located in the N-terminal region (aa 27-361) of endoglin. Cell binding was further increased by generating a bivalent scFv-Fc fusion protein composed of scFv mE12 and the human gamma1 Fc part. Moreover, scFv mE12 was endowed with an additional cysteine residue in the linker region and applied for the generation of anti-endoglin scFv immunoliposomes capable of selectively binding to endoglin-expressing cells. Thus, anti-mouse endoglin scFv mE12 should be useful to analyze vascular targeting strategies in mice.

Surface coating of PLGA microparticles with protamine enhances their immunological performance through facilitated phagocytosis.

Surface modifications of poly(lactide-co-glycolide) microparticles with different polycationic electrolytes have mainly been studied for conjugation to antigens and/or adjuvants. However, the in vivo immunological effects of using surface charged particles have not been address yet. In this study, microparticles were coated or not with protamine, a cationic and arginine-rich electrolyte that confers microparticles with a positively surface charge. We then evaluated the potential of protamine-coatings to assist the induction of immune responses in mice. Interestingly, enhanced antibodies and T-cell responses were observed in mice treated with the coated particles. In vitro studies suggested that the improved immunological performance was mediated by an increased uptake. Indeed, protamine-coated particles that carried a plasmid were even internalised into non-phagocytic cells and to cause their transfection. These results open the way for further research into a novel technology that combines the use protamine for facilitated cell penetration of that and biodegradable microparticles for prolonged antigen or drug release.