12/15-Lipoxygenase Deficiency Impairs Neutrophil Granulopoiesis and Lung Proinflammatory Responses to Aspergillus fumigatus.

Development of invasive aspergillosis correlates with impairments in innate immunity. We and others have recently shown that arachidonic acid metabolism pathways, specifically the cyclooxygenase-2 (COX-2) and 5-lipoxygenase (5-LOX) pathways, participate in the induction of protective innate immune responses during invasive aspergillosis. Based on the high degree of cooperation and interconnection within the eicosanoid network, we hypothesized that 12/15-LOX is also active during invasive aspergillosis. We report in this study that mice deficient in the gene encoding 12/15-LOX (Alox15) are profoundly susceptible to invasive aspergillosis. Decreased survival correlated with increased fungal burden and evidence of increased lung damage. These defects were associated with very early (6 and 12 h) 12/15-LOX-dependent inflammatory cytokine (IL-1α, IL-1ß, and TNF-α) and chemokine (CCL3 and CCL4) production. Neutrophil levels in the lung were blunted in the absence of 12/15-LOX, although neutrophil antifungal activity was intact. However, lower neutrophil levels in the lungs of Alox15 -/- mice were not a result of impaired recruitment or survival; rather, Alox15 -/- mice demonstrated impaired neutrophil granulopoiesis in the bone marrow intrinsically and after fungal exposure. Employing a lower inoculum to allow for better survival allowed the identification of 12/15-LOX-dependent induction of IL-17A and IL-22. Impaired IL-17A and IL-22 production correlated with reduced invariant NKT cell numbers as well as lower IL-23 levels. Together, these data indicate that 12/15-LOX is a critical player in induction of the earliest aspects of the innate immune response to Aspergillus fumigatus.

Role of fibroblast growth factor 23 and klotho cross talk in idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive fibrosing interstitial pneumonia that mainly affects the elderly. Several reports have demonstrated that aging is involved in the underlying pathogenic mechanisms of IPF. α-Klotho (KL) has been well characterized as an "age-suppressing" hormone and can provide protection against cellular senescence and oxidative stress. In this study, KL levels were assessed in human plasma and primary lung fibroblasts from patients with idiopathic pulmonary fibrosis (IPF-FB) and in lung tissue from mice exposed to bleomycin, which showed significant downregulation when compared with controls. Conversely, transgenic mice overexpressing KL were protected against bleomycin-induced lung fibrosis. Treatment of human lung fibroblasts with recombinant KL alone was not sufficient to inhibit transforming growth factor-ß (TGF-ß)-induced collagen deposition and inflammatory marker expression. Interestingly, fibroblast growth factor 23 (FGF23), a proinflammatory circulating protein for which KL is a coreceptor, was upregulated in IPF and bleomycin lungs. To our surprise, FGF23 and KL coadministration led to a significant reduction in fibrosis and inflammation in IPF-FB; FGF23 administration alone or in combination with KL stimulated KL upregulation. We conclude that in IPF downregulation of KL may contribute to fibrosis and inflammation and FGF23 may act as a compensatory antifibrotic and anti-inflammatory mediator via inhibition of TGF-ß signaling. Upon restoration of KL levels, the combination of FGF23 and KL leads to resolution of inflammation and fibrosis. Altogether, these data provide novel insight into the FGF23/KL axis and its antifibrotic/anti-inflammatory properties, which opens new avenues for potential therapies in aging-related diseases like IPF.

IL-33 Signaling Regulates Innate IL-17A and IL-22 Production via Suppression of Prostaglandin E2 during Lung Fungal Infection.

Members of the IL-1 family play protective and regulatory roles in immune defense against the opportunistic mold Aspergillus fumigatus In this study, we investigated the IL-1 family member IL-33 in lung defense against A. fumigatus IL-33 was detected in the naive lung, which further increased after exposure to A. fumigatus in a dectin-1-independent manner. Mice deficient in the receptor for IL-33 (Il1rl1-/-) unexpectedly demonstrated enhanced lung clearance of A. fumigatus IL-33 functioned as a negative regulator of multiple inflammatory cytokines, as IL-1α, IL-1ß, IL-6, IL-17A, and IL-22 were significantly elevated in fungal-exposed Il1rl1-/- mice. Subsequently, IL-33 administration to normal mice attenuated fungal-induced IL-17A and IL-22, but not IL-1α, IL-1ß, or IL-6, production. IL-33-mediated regulation of IL-17A and IL-22 did not involve the modulation of IL-23 but rather PGE2; PGE2 was significantly increased in fungal-exposed Il1rl1-/- mice, and normal mice produced less PGE2 after fungal exposure when administered IL-33, suggesting that IL-33-mediated regulation of IL-17A and IL-22 occurred at the level of PGE2 This was confirmed by in vivo cyclooxygenase 2 inhibition, which attenuated fungal-induced IL-17A and IL-22, as well as IL-1α, IL-1ß, and IL-6, production in Il1rl1-/- mice, resulting in impaired fungal clearance. We also show that a PGE2 receptor agonist increased, whereas a PGE2 synthase inhibitor decreased, the levels of IL-17A and IL-22 but not IL-1α, IL-1ß, or IL-6. This study establishes novel mechanisms of innate IL-17A/IL-22 production via PGE2 and regulation of the PGE2/IL-17A/IL-22 axis via IL-33 signaling during lung fungal exposure.

Acidic Mammalian Chitinase Negatively Affects Immune Responses during Acute and Chronic Aspergillus fumigatus Exposure.

Chitin is a polysaccharide that provides structure and rigidity to the cell walls of fungi and insects. Mammals possess multiple chitinases, which function to degrade chitin, thereby supporting a role for chitinases in immune defense. However, chitin degradation has been implicated in the pathogenesis of asthma. Here, we determined the impact of acidic mammalian chitinase (AMCase) (Chia) deficiency on host defense during acute exposure to the fungal pathogen Aspergillus fumigatus as well as its contribution to A. fumigatus-associated allergic asthma. We demonstrate that chitin in the fungal cell wall was detected at low levels in A. fumigatus conidia, which emerged at the highest level during hyphal transition. In response to acute A. fumigatus challenge, Chia-/- mice unexpectedly demonstrated lower A. fumigatus lung burdens at 2 days postchallenge. The lower fungal burden correlated with decreased lung interleukin-33 (IL-33) levels yet increased IL-1ß and prostaglandin E2 (PGE2) production, a phenotype that we reported previously to promote the induction of IL-17A and IL-22. During chronic A. fumigatus exposure, AMCase deficiency resulted in lower dynamic and airway lung resistance than in wild-type mice. Improved lung physiology correlated with attenuated levels of the proallergic chemokines CCL17 and CCL22. Surprisingly, examination of inflammatory responses during chronic exposure revealed attenuated IL-17A and IL-22 responses, but not type 2 responses, in the absence of AMCase. Collectively, these data suggest that AMCase functions as a negative regulator of immune responses during acute fungal exposure and is a contributor to fungal asthma severity, putatively via the induction of proinflammatory responses.

Targeting Cytokines as Evolving Treatment Strategies in Chronic Inflammatory Airway Diseases.

Cytokines are key players in the initiation and propagation of inflammation in chronic inflammatory airway diseases such as chronic obstructive pulmonary disease (COPD), bronchiectasis and allergic asthma. This makes them attractive targets for specific novel anti-inflammatory treatment strategies. Recently, both interleukin-1 (IL-1) and IL-6 have been associated with negative health outcomes, mortality and a pro-inflammatory phenotype in COPD. IL-6 in COPD was shown to correlate negatively with lung function, and IL-1beta was induced by cigarette smoke in the bronchial epithelium, causing airway inflammation. Furthermore, IL-8 has been shown to be a pro-inflammatory marker in bronchiectasis, COPD and allergic asthma. Clinical trials using specific cytokine blockade therapies are currently emerging and have contributed to reduce exacerbations and steroid use in COPD. Here, we present a review of the current understanding of the roles of cytokines in the pathophysiology of chronic inflammatory airway diseases. Furthermore, outcomes of clinical trials in cytokine blockade as novel treatment strategies for selected patient populations with those diseases will be discussed.

Human intestinal stromal cells promote homeostasis in normal mucosa but inflammation in Crohn's disease in a retinoic acid-deficient manner.

Intestinal stromal cells (SCs), which synthesize the extracellular matrix that gives the mucosa its structure, are newly appreciated to play a role in mucosal inflammation. Here, we show that human intestinal vimentin+CD90+smooth muscle actin- SCs synthesize retinoic acid (RA) at levels equivalent to intestinal epithelial cells, a function in the human intestine previously attributed exclusively to epithelial cells. Crohn's disease SCs (Crohn's SCs), however, synthesized markedly less RA than SCs from healthy intestine (normal SCs). We also show that microbe-stimulated Crohn's SCs, which are more inflammatory than stimulated normal SCs, induced less RA-regulated differentiation of mucosal dendritic cells (DCs) (circulating pre-DCs and monocyte-derived DCs), leading to the generation of more potent inflammatory interferon-γhi/interleukin-17hi T cells than normal SCs. Explaining these results, Crohn's SCs expressed more DHRS3, a retinaldehyde reductase that inhibits retinol conversion to retinal and, thus, synthesized less RA than normal SCs. These findings uncover a microbe-SC-DC crosstalk in which luminal microbes induce Crohn's disease SCs to initiate and perpetuate inflammation through impaired synthesis of RA.

Fibroblast Growth Factor Receptor 4 Deficiency Mediates Airway Inflammation in the Adult Healthy Lung?

Fibroblast growth factor receptor (FGFR) 4 has been shown to mediate pro-inflammatory signaling in the liver and airway epithelium in chronic obstructive pulmonary disease. In past reports, FGFR4 knockout (Fgfr4 -/- ) mice did not show any lung phenotype developmentally or at birth, unless FGFR3 deficiency was present simultaneously. Therefore, we wanted to know whether the loss of FGFR4 had any effect on the adult murine lung. Our results indicate that adult Fgfr4 -/- mice demonstrate a lung phenotype consisting of widened airway spaces, increased airway inflammation, bronchial obstruction, and right ventricular hypertrophy consistent with emphysema. Despite downregulation of FGF23 serum levels, interleukin (IL) 1ß and IL-6 in the Fgfr4 -/- lung, and abrogation of p38 signaling, primary murine Fgfr4 -/- airway cells showed increased expression of IL-1ß and augmented secretion of IL-6, which correlated with decreased airway surface liquid depth as assessed by micro-optical coherence tomography. These findings were paralleled by increased ERK phosphorylation in Fgfr4 -/- airway cells when compared with their control wild-type cells. Analysis of a murine model with constitutive activation of FGFR4 showed attenuation of pro-inflammatory mediators in the lung and airway epithelium. In conclusion, we are the first to show an inflammatory and obstructive airway phenotype in the adult healthy murine Fgfr4 -/- lung, which might be due to the upregulation of ERK phosphorylation in the Fgfr4 -/- airway epithelium.

IL-1RA regulates immunopathogenesis during fungal-associated allergic airway inflammation.

Severe asthma with fungal sensitization (SAFS) defines a subset of human asthmatics with allergy to 1 or more fungal species and difficult-to-control asthma. We have previously reported that human asthmatics sensitized to fungi have worse lung function and a higher degree of atopy, which was associated with higher IL-1 receptor antagonist (IL-1RA) levels in bronchoalveolar lavage fluid. IL-1RA further demonstrated a significant negative association with bronchial hyperresponsiveness to methacholine. Here, we show that IL-1α and IL-1ß are elevated in both bronchoalveolar lavage fluid and sputum from human asthmatics sensitized to fungi, implicating an association with IL-1α, IL-1ß, or IL-1RA in fungal asthma severity. In an experimental model of fungal-associated allergic airway inflammation, we demonstrate that IL-1R1 signaling promotes type 1 (IFN-γ, CXCL9, CXCL10) and type 17 (IL-17A, IL-22) responses that were associated with neutrophilic inflammation and increased airway hyperreactivity. Each of these were exacerbated in the absence of IL-1RA. Administration of human recombinant IL-1RA (Kineret/anakinra) during fungal-associated allergic airway inflammation improved airway hyperreactivity and lowered type 1 and type 17 responses. Taken together, these data suggest that IL-1R1 signaling contributes to fungal asthma severity via immunopathogenic type 1 and type 17 responses and can be targeted for improving allergic asthma severity.

The Effects of the Anti-aging Protein Klotho on Mucociliary Clearance.

α-klotho (KL) is an anti-aging protein and has been shown to exert anti-inflammatory and anti-oxidative effects in the lung and pulmonary diseases such as chronic obstructive pulmonary disease (COPD) and cystic fibrosis. The current study investigated the direct effect of KL on the bronchial epithelium in regards to mucociliary clearance parameters. Primary human bronchial and murine tracheal epithelial cells, cultured, and differentiated at the air liquid interface (ALI), were treated with recombinant KL or infected with a lentiviral vector expressing KL. Airway surface liquid (ASL) volume, airway ion channel activities, and expression levels were analyzed. These experiments were paired with ex vivo analyses of mucociliary clearance in murine tracheas from klotho deficient mice and their wild type littermates. Our results showed that klotho deficiency led to impaired mucociliary clearance with a reduction in ASL volume in vitro and ex vivo. Overexpression or exogenous KL increased ASL volume, which was paralleled by increased activation of the large-conductance, Ca2+-activated, voltage-dependent potassium channel (BK) without effect on the cystic fibrosis transmembrane conductance regulator (CFTR). Furthermore, KL overexpression downregulated IL-8 levels and attenuated TGF-ß-mediated downregulation of LRRC26, the γ subunit of BK, necessary for its function in non-excitable cells. In summary, we show that KL regulates mucociliary function by increasing ASL volume in the airways possibly due to underlying BK activation. The KL mediated BK channel activation may be a potentially important target to design therapeutic strategies in inflammatory airway diseases when ASL volume is decreased.

FGF23 Induction of O-Linked N-Acetylglucosamine Regulates IL-6 Secretion in Human Bronchial Epithelial Cells.

The hexosamine biosynthetic pathway (HBP) generates the substrate for the O-linked ß-N-acetylglucosamine (O-GlcNAc) modification of proteins. The HBP also serves as a stress sensor and has been reported to be involved with nuclear factor of activated T-cells (NFAT) activation, which can contribute to multiple cellular processes including cell metabolism, proliferation, and inflammation. In our previously published report, Fibroblast Growth Factor (FGF) 23, an important endocrine pro-inflammatory mediator, was shown to activate the FGFR4/phospholipase Cγ (PLCγ)/nuclear factor of activated T-cells (NFAT) signaling in chronic inflammatory airway diseases such as cystic fibrosis (CF) and chronic obstructive pulmonary disease (COPD). Here, we demonstrate that FGF23 increased the O-GlcNAc modification of proteins in HBECs. Furthermore, the increase in O-GlcNAc levels by FGF23 stimulation resulted in the downstream activation of NFAT and secretion of interleukin-6 (IL-6). Conversely, inhibition of FGF23 signaling and/or O-GlcNAc transferase (OGT)/O-GlcNAc reversed these effects. Collectively, these data suggest that FGF23 induced IL-6 upregulation and secretion is, at least, partially mediated via the activation of the HBP and O-GlcNAc levels in HBECs. These findings identify a novel link whereby FGF23 and the augmentation of O-GlcNAc levels regulate airway inflammation through NFAT activation and IL-6 upregulation in HBECs. The crosstalk between these signaling pathways may contribute to the pathogenesis of chronic inflammatory airway diseases such as COPD and CF as well as metabolic syndromes, including diabetes.

Innate Lung Defense during Invasive Aspergillosis: New Mechanisms.

Invasive aspergillosis (IA) is one of the most difficult to treat and, consequently, one of the most lethal fungal infections known to man. Continued use of immunosuppressive agents during chemotherapy and organ transplantation often leads to the development of neutropenia, the primary risk factor for IA. However, IA is also becoming more appreciated in chronic diseases associated with corticosteroid therapy. The innate immune response to Aspergillus fumigatus, the primary agent in IA, plays a pivotal role in the recognition and elimination of organisms from the pulmonary system. This review highlights recent findings about innate host defense mechanisms, including novel aspects of innate cellular immunity and pathogen recognition, and the inflammatory mediators that control infection with A. fumigatus.