The development of emphysema in humans and mice exposed to cigarette smoke is promoted by activation of an adaptive immune response. Lung myeloid dendritic cells (mDCs) derived from cigarette smokers activate autoreactive Th1 and Th17 cells. mDC-dependent activation of T cell subsets requires expression of the SPP1 gene, which encodes osteopontin (OPN), a pleiotropic cytokine implicated in autoimmune responses. The upstream molecular events that promote SPP1 expression and activate mDCs in response to smoke remain unknown. Here, we show that peroxisome proliferator-activated receptor γ (PPARG/Pparg) expression was downregulated in mDCs of smokers with emphysema and mice exposed to chronic smoke. Conditional knockout of PPARγ in APCs using Cd11c-Cre Pparg(flox/flox) mice led to spontaneous lung inflammation and emphysema that resembled the phenotype of smoke-exposed mice. The inflammatory phenotype of Cd11c-Cre Pparg(flox/flox) mice required OPN, suggesting an antiinflammatory mechanism in which PPARγ negatively regulates Spp1 expression in the lung. A 2-month treatment with a PPARγ agonist reversed emphysema in WT mice despite continual smoke exposure. Furthermore, endogenous PPARγ agonists were reduced in the plasma of smokers with emphysema. These findings reveal a proinflammatory pathway, in which reduced PPARγ activity promotes emphysema, and suggest that targeting this pathway in smokers could prevent and reverse emphysema.

Pathogenic lung antigen presenting cells (APCs) are abundant in the lungs of smokers with emphysema and are capable of inducing T helper type 1 (Th1) and Th17 cell differentiation. Mice exposed to chronic cigarette smoke develop emphysema and harbor CD11b+CD11c+ APCs that can transfer the disease to naïve mice. Proteolytic products from activated innate immune cells cleave complement proteins but the contribution of their downstream signaling in APC-driven lung parenchyma destruction is less clear. We show here that relative to control smokers, complement 3 (C3) is increased in the plasma and its cleaved products are deposited on the lung tissue of smokers with advanced lung disease. Matrix metalloproteinase 12 and neutrophils elastase directly cleave human C3, and generate multiple activated signaling products. Compared to wild type, C3 deficient mice exposed to chronic smoke showed reduced CD11b+CD11c+ cells in the lungs and were protected against emphysema development. C3a and C5a, two of the major anaphylatoxin and chemoattractant mediators downstream of C3 activation, are present in the lungs of mice exposed to smoke, but only C3a signaling was critical in emphysema development because deficiency in C3a receptor (C3aR) but not C5aR phenocopied C3-/- mice in this model. These findings suggest a critical role for C3aR in the pathogenesis of smoke induced lung disease, and could be explored to develop specific new therapeutic targets for emphysema treatment.

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 (T(H)1) and T(H)17 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 T(H)17 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 T(H)17 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.

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.

The innate immune response of airway epithelial cells to aeroallergen likely initiates the development of T cell responses that are central to allergic inflammation. Although proteolytically active allergens induce the expression of interleukin (IL)-25/IL-17E, we show that epithelial matrix metalloproteinase 7 (MMP7) is expressed in human asthma, and is required for maximal activity of this cytokine in promoting T helper type 2 cell differentiation. Allergen-challenged MMP7-/- mice showed reduced airway hyperreactivity, allergic inflammatory cytokine, and increased expression of retinal dehydrogenase (RALDH)-1. Inhibition of RALDH-1 restored the asthma phenotype in MMP7-/- mice and inhibited lung T regulatory cell responses while exogenous administration of retinoic acid attenuated the asthma phenotype. Thus, MMP7 coordinates allergic lung inflammation by activating IL-25 while simultaneously inhibiting retinoid-dependent T regulatory cell development.

A bivalent influenza virus (INFV) A vaccine composed of purified HA and NA surface proteins have long been available. Disadvantages of this vaccine include annual reformulation. We have developed a novel T cell-based vaccine comprised of short and highly conserved peptides derived from M2, HA, and NP peptides formulated with TLR ligands in a lipid matrix. We hypothesized that IN immunization with short and well conserved peptides will confer protection to lethally infected mice. The tri-peptide (TP) vaccine was given IN to mice on d-21, -14, and -7 prior to INFV A and B aerosol infection (d0). Control animals received only sham liposomes (DLPC). For memory studies, mice were infected on 60d or 90d post treatment. 100% survival was observed in TP treated vs. DLPC treated mice (<10%). PAS staining of lung sections showed reduced inflammation associated with TP treatment. Although TP treatments were not long lasting, it conferred partial protection to boosted mice (40% survival), and 17% NP+ CD8 T lungs cells were observed after booster. Lung viral burden was reduced by 2 log TP treated vs. naïve mice (d2). Divergent trends of IFN-γ and IL-6 production between TP treated and naïve animals on d6 and d8 post infection were observed. Our findings indicate that T cells play an important role in viral clearance and mediation of flu. Future studies will aim at the understanding of the recruitment and egress of these T cell populations.

The innate immune response of airway epithelial cells to airborne allergens initiates the development of T cell responses that are central to allergic inflammation. Although proteinase allergens induce the expression of interleukin 25, we show here that epithelial matrix metalloproteinase 7 (MMP7) was expressed during asthma and was required for the maximum activity of interleukin 25 in promoting the differentiation of T helper type 2 cells. Allergen-challenged Mmp7(-/-) mice had less airway hyper-reactivity and production of allergic inflammatory cytokines and higher expression of retinal dehydrogenase 1. Inhibition of retinal dehydrogenase 1 restored the asthma phenotype of Mmp7(-/-) mice and inhibited the responses of lung regulatory T cells, whereas exogenous administration of retinoic acid attenuated the asthma phenotype. Thus, MMP7 coordinates allergic lung inflammation by activating interleukin 25 while simultaneously inhibiting retinoid-dependent development of regulatory T cells.

Bronchial asthma is a respiratory tract disease characterized by airway hyper‐responsiveness, mucus hyper‐production and inflammation. Airway epithelial cells are the initial sites where allergens are recognized. However, the role of epithelial cells in modulation of airway inflammation is not fully understood. One critical group of enzymes that regulate inflammatory processes in the lung is Matrix metalloproteases (MMP). We hypothesize that epithelial specific MMP7 modulates asthmatic phenotype by modifying inflammatory mediators produced by airway epithelium. We found that MMP7 null mice had reduced airway hyper‐reactivity in response to allergen challenge as well as fewer eosinophils in broncho‐alveolar lavage (BAL) fluid compared to WT mice. IL‐25 and Eotaxin, the critical mediators for asthma phenotype, were also reduced in MMP7−/− mice. Proteomic analysis of BAL fluid identified that Retinaldehydrogenase‐1 (RALDH‐1), a rate limiting epithelial specific enzyme for retinoic acid production, was up regulated in MMP7−/− asthmatic mice. In vivo inhibition of RALDH‐1, increased eotaxin protein expression in the airway and rescued the asthma phenotype in MMP7 −/− mice. Together, our data supports the role of MMP7 in modifying airway inflammation in asthma. We propose that identifying in vivo substrates for MMP7 may provide novel therapeutics for treatment of asthma.This work is funded by NIH grant (U19AI070973) to FK.

A fullerene-paclitaxel conjugate has been synthesized as a slow-release drug for aerosol liposome delivery of paclitaxel for lung cancer therapy. The conjugate was designed to release paclitaxel via enzymatic hydrolysis and subsequently has shown a half-life of release of 80 min in bovine plasma. A liposome formulation of the conjugate has been prepared using dilauroylphosphatidylcholine (DLPC), and its IC50 is virtually identical to the IC50 for a paclitaxel-DLPC formulation in human epithelial lung carcinoma A549 cells. With both clinically relevant kinetics of hydrolysis and significant cytotoxicity in tissue culture, the conjugate holds promise for enhanced therapeutic efficacy of paclitaxel in vivo.

9-Nitrocamptothecin (9-NC) dilauroylphosphatidylcholine (DLPC) liposome aerosol was evaluated for potential toxicity in an 8-wk, subacute toxicity study in dogs. Fourteen adult dogs were divided into 2 groups with 10 animals in the 9-NC-DLPC treatment group and 4 animals in the DLPC-only vehicle control group. 9-NC-DLPC was administered to the animals using an Aerotech II nebulizer flowing at 10 L/min. Full-face exposures for 60 min were conducted for 5 consecutive days a week for 8 wk. The estimated deposited aerosol dose was 24.7 microg/kg/day. Animals in the vehicle control group received aerosolized DLPC only. Body weight, food consumption, urinalysis, in-life observations, hematology, plasma chemistry, and necropsy and histopathology were monitored before and during the treatment period and in a subset of animals for 2 wk following the end of treatment. Animals were observed for signs of pharmacologic and/or toxicologic effects three times on days of dosing and once daily on nondosing days. 9-NC-DLPC liposomes administered as a small-particle aerosol were determined to be nontoxic to dogs when given for 5 days/wk, for a duration of 8 wk. DLPC-only liposome also had no toxic effects.

Purpose: The plant alkaloid camptothecin (CPT) has shown significant antitumor activity against a wide variety of human tumors xenografted in nude mice. In previous studies we have found that administration of dilauroylphosphatidylcholine (DLPC) liposome aerosols containing 9-nitrocamptothecin (9-NC) inhibits the growth of human breast, colon and lung cancer xenografts. The purpose of this study was to analyze the pharmacokinetics and tissue distribution of inhaled CPT formulated in DLPC liposomes. Methods: C57BL/6 mice with subcutaneous Lewis lung carcinoma, Swiss nu/nu mice with human lung carcinoma xenografts and BALB/c mice without tumors were used for pharmacokinetic studies of CPT administered as a liposome aerosol and BALB/c mice were given CPT intramuscularly. Results: After 30 min inhalation of CPT liposome aerosol, drug was deposited in the lungs (310 ng/g) and was followed promptly by the appearance of high concentrations in the liver (192 ng/g) and with lesser amounts appearing in other organs. Drug concentration in the brain was 61 ng/g. After intramuscular injection of CPT dissolved in DMSO, drug was released from the site of injection very slowly and accumulated mainly in the liver (136 ng/g. Only trace amounts appeared in the lungs (2-4 ng/g). These results demonstrate a prompt pulmonary and later systemic distribution of CPT following liposome aerosol administration. Conclusions: The substantial concentrations of CPT in lungs and other organs following inhalation of liposome aerosol suggest the possible benefit of it and of its more active derivative, 9-NC, in the treatment of lung, liver, kidney and brain cancer in humans.