Search

Background: Mitochondria regulate a myriad of cellular needs and functions. Dysregulation of mitochondrial control within airway epithelial cells has been implicated in the pro-inflammatory response to allergens in asthmatics. Because of their multifaceted nature, mitochondrial structure needs to be tightly regulated through fission and fusion. Dynamin Related Protein 1 (DRP1), a cytosolic GTPase, is a key driver of mitochondrial fission. During allergic asthma, airway epithelial mitochondria appear smaller and structurally altered. The role of DRP1-mediated mitochondrial fission, however, has not been fully elucidated in allergic airway disease. Methods: We used a Human Bronchial Epithelial Cell line (HBECs), primary Mouse Tracheal Epithelial Cells (MTECs), and conditional ablation of DRP1 in lung epithelial cells to investigate mitochondrial fission and its impact on the pro-inflammatory response to House Dust Mite (HDM) in vitro and in vivo. Results: Our data suggest that, following HDM challenge, mitochondrial fission is rapidly upregulated in airway epithelial cells and precedes production of pro-inflammatory cytokines and chemokines. Further, deletion of DRP1 in lung epithelial cells lead to decreased mitochondrial fission and enhanced pro-inflammatory signaling in response to HDM. Analysis of lung epithelial specific DRP1 deletion in mice demonstrated enhanced Airway Hyper Responsiveness (AHR), inflammation, differential mucin transcription, and epithelial cell death. Conclusions: Mitochondrial fission is rapidly upregulated in airway epithelial cells following HDM exposure, prior to epithelial release of pro-inflammatory cytokines and chemokines. Deletion of DRP1, a necessary pro- fission protein, reduces fission and enhances the pro-inflammatory epithelial response to HDM, exacerbating the allergic response.

Introduction: Emphysema is an important component of chronic obstructive pulmonary disease (COPD) for which effective therapies are lacking. Emphysema is proposed to be caused by chronic inflammation induced by noxious particles but is also associated with lung development. Cysteinyl-leukotrienes (LTC4, LTD4, and LTE4, cysLTs) are inflammatory lipid mediators derived from arachidonic acid and the cysLT1 receptor expression is reportedly increased in COPD patients with severe exacerbations. However, whether cysLT synthesis influence the development emphysema is unknown. Aim: To investigate the influence of the constitutive synthesis of cysLTs on lung structure. Methods: Lung structure and function were assessed in leukotriene C4 synthase knockout (LTC4S-KO) mice, ranging in age from 1-40 weeks, by the measurement of pulmonary mechanics using FlexiVent and the mean linear intercept (MLI), respectively. To examine the involvement of immune cells, differential cell counts in bronchoalveolar lavage (BAL) and immunohistochemistry of lung were performed. Emphysema-related molecules including protease/antiprotease expression were assessed by qPCR and ELISA. Results: Histological examination revealed premature emphysematous changes in the lungs, in evidence as early as 5 weeks of age. Static compliance and the K value (Salazar-Knowles equation) were higher and tissue elastance was lower in LTC4S-KO mice than in age matched wild type (WT) mice. Additionally, MLI was higher in LTC4S-KO mice. In BAL fluid from LTC4S-KO mice, hemosiderin-laden macrophages were increased. Conclusions: LTC4S deficiency in vivo leads to emphysematous changes spontaneously, and it may be caused by abnormal maturation of lung.

Background Inhaled oxidative toxicants present in ambient air cause airway epithelial injury, inflammation, and airway hyperresponsiveness. Effective adaptation to such environmental insults is essential for the preservation of pulmonary function, whereas failure or incomplete adaptation to oxidative injury can render the host susceptible to the development of airway disease. Objective We sought to explore the mechanisms of airway adaptation to oxidative injury. Methods For a model to study pulmonary adaptation to oxidative stress–induced lung injury, we exposed mice to repeated nose-only chlorine gas exposures. Outcome measures were evaluated 24 hours after the last chlorine exposure. Lung mechanics and airway responsiveness to methacholine were assessed by using the flexiVent. Inflammation and antioxidant responses were assessed in both bronchoalveolar lavage fluid and lung tissue. Using both loss or gain of function and genomic approaches, we further dissected the cellular and molecular mechanisms involved in pulmonary adaptation. Results Repeated exposures to oxidative stress resulted in pulmonary adaptation evidenced by abrogation of neutrophilic inflammation and airway hyperresponsiveness. This adaptation was independent of antioxidant mechanisms and regulatory T cells but dependent on residential alveolar macrophages (AMs). Interestingly, 5% of AMs expressed forkhead box P3, and depletion of these cells abolished adaptation. Results from transcriptomic profiling and loss and gain of function suggest that adaptation might be dependent on TGF-β and prostaglandin E2. Conclusion Pulmonary adaptation during oxidative stress–induced lung injury is mediated by a novel subset of forkhead box P3–positive AMs that limits inflammation, favoring airway adaptation and host fitness through TGF-β and prostaglandin E2.

Oxidative stress in allergic asthma may result from oxidase activity or proinflammatory molecules in pollens. Signaling via TLR4 and its adaptor Toll–IL-1R domain–containing adapter inducing IFN-β (TRIF) has been implicated in reactive oxygen species–mediated acute lung injury and in Th2 immune responses. We investigated the contributions of oxidative stress and TLR4/TRIF signaling to experimental asthma induced by birch pollen exposure exclusively via the airways. Mice were exposed to native or heat-inactivated white birch pollen extract (BPEx) intratracheally and injected with the antioxidants, N-acetyl-l-cysteine or dimethylthiourea, prior to sensitization, challenge, or all allergen exposures, to assess the role of oxidative stress and pollen-intrinsic NADPH oxidase activity in allergic sensitization, inflammation, and airway hyperresponsiveness (AHR). Additionally, TLR4 signaling was antagonized concomitantly with allergen exposure, or the development of allergic airway disease was evaluated in TLR4 or TRIF knockout mice. N-acetyl-l-cysteine inhibited BPEx-induced eosinophilic airway inflammation and AHR except when given exclusively during sensitization, whereas dimethylthiourea was inhibitory even when administered with the sensitization alone. Heat inactivation of BPEx had no effect on the development of allergic airway disease. Oxidative stress–mediated AHR was also TLR4 and TRIF independent; however, TLR4 deficiency decreased, whereas TRIF deficiency increased BPEx-induced airway inflammation. In conclusion, oxidative stress plays a significant role in allergic sensitization to pollen via the airway mucosa, but the pollen-intrinsic NADPH oxidase activity and TLR4 or TRIF signaling are unnecessary for the induction of allergic airway disease and AHR. Pollen extract does, however, activate TLR4, thereby enhancing airway inflammation, which is restrained by the TRIF-dependent pathway.

Modulation of adaptive immune responses via the innate immune pattern recognition receptors, such as the TLRs, is an emerging strategy for vaccine development. We investigated whether nasal rather than intrapulmonary application of Protollin, a mucosal adjuvant composed of TLR2 and TLR4 ligands, is sufficient to elicit protection against murine allergic lower airway disease. Wild-type, Tlr2−/−, or Tlr4−/− BALB/c mice were sensitized to a birch pollen allergen extract (BPEx), then received either intranasal or intrapulmonary administrations of Protollin or Protollin admixed with BPEx, followed by consecutive daily BPEx challenges. Nasal application of Protollin or Protollin admixed with BPEx was sufficient to inhibit allergic lower airway disease with minimal collateral lung inflammation. Inhibition was dependent on TLR4 and was associated with the induction of ICOS in cells of the nasal mucosa and on both CD4+Foxp3+ and CD4+Foxp3− T cells of the draining lymph nodes (LNs), as well as their recruitment to the lungs. Adoptive transfer of cervical LN CD4+ICOS+, but not CD4+ICOS−, cells inhibited BPEx-induced airway hyperresponsiveness and bronchoalveolar lavage eosinophilia. Thus, our data indicate that expansion of resident ICOS-expressing CD4+ T cells of the cervical LNs by nasal mucosal TLR4 stimulation may inhibit the development of allergic lower airway disease in mice.

Background: Clinical data have associated Th17 cells, IL-17 and neutrophilia with severe asthma. The objective of these experiments is to assess how T cell specific overexpression of IL-17 modifies OVA-induced airway inflammatory responses using an adoptive transfer murine model. Methods: Recombinant retroviruses encoding either EGFP alone (pAP2) or both EGFP and IL-17 were used to transduce CD4+ T cells from OVA-sensitized donor BALB/c mice, re-stimulated with OVA ex vivo for targeted retroviral transduction. CD4+ T cells were isolated using magnetic selection and adoptively transferred intraperitoneally into naïve mice on day 0. Recipients were then anaesthetized and challenged intranasally with OVA daily for 5 days. Mice were sacrificed 24h after the final challenge. Results: Recombinant retroviruses efficiently transduced CD4+ T cells after re-stimulation with OVA such that 0.17% of T cells transduced with control virus expressed IL-17 vs 4.65% of T cells transduced with IL-17pAP2. Supernatants from IL-17pAP2 transduced cells produced abundant IL-17, which was undetectable in pAP2 transduced cells. Following adoptive transfer and challenge, IL-17 levels in the bronchoalveolar lavage fluid (BALF) of recipients from the IL-17pAP2 group were greater than those from the pAP2 control group. BALF levels of inflammatory cells were increased in each group. This model will allow us to define how allergic airway inflammation is modified by T cell specific IL-17 overproduction.