Immune suppression increases the incidence of invasive fungal infections, particularly those caused by the opportunistic mold Aspergillus fumigatus. Previous investigations revealed that members of the Toll-like receptor (TLR) family are not absolutely required for host defense against A. fumigatus in non-immunosuppressed hosts, suggesting that other pattern recognition receptors (PRRs) are involved. We show here that naive mice (i.e. not pharmacologically immunosuppressed) lacking the beta-glucan receptor Dectin-1 (Dectin-1−/−) are more sensitive to intratracheal challenge with A. fumigatus than control mice, exhibiting >80% mortality within 5 days, ultimately attributed to a compromise in respiratory mechanics. In response to A. fumigatus challenge, Dectin-1−/− mice demonstrated impaired interleukin (IL)-α, IL-1β, tumor necrosis factor (TNF)-α, CCL3/macrophage inflammatory protein (MIP)-α, CCL4/MIP-1β and CXCL1/KC production, which resulted in insufficient lung neutrophil recruitment and uncontrolled A. fumigatus lung growth. Alveolar macrophages from Dectin-1−/− mice failed to produce proinflammatory mediators in response to A. fumigatus, whereas neutrophils from Dectin-1−/− mice had impaired reactive oxygen species production and impaired killing of A. fumigatus. We further show that IL-17 production in the lung after A. fumigatus challenge was Dectin-1 dependent and that neutralization of IL-17 significantly impaired A. fumigatus clearance. Collectively, these results support a requisite role for Dectin-1 in in vivo defense against A. fumigatus.

Recent reports indicate that aerobic exercise improves the overall physical fitness and health of asthmatic patients. The specific exercise-induced improvements in the pathology of asthma and the mechanisms by which these improvements occur, however, are ill-defined; thus, the therapeutic potential of exercise in the treatment of asthma remains unappreciated. Using an OVA-driven mouse model, we examined the role of aerobic exercise in modulating inflammatory responses associated with atopic asthma. Data demonstrate that moderate intensity aerobic exercise training decreased leukocyte infiltration, cytokine production, adhesion molecule expression, and structural remodeling within the lungs of OVA-sensitized mice (n = 6–10; p < 0.05). Because the transcription factor NF-κB regulates the expression of a variety of genes that encode inflammatory mediators, we monitored changes in NF-κB activation in the lungs of exercised/sensitized mice. Results show that exercise decreased NF-κB nuclear translocation and IκBα phosphorylation, indicating that exercise decreased NF-κB activation in the lungs of sensitized mice (n = 6). Taken together, these results suggest that aerobic exercise attenuates airway inflammation in a mouse model of atopic asthma via modulation of NF-κB activation. Potential exists, therefore, for the amelioration of asthma-associated chronic airway inflammation through the use of aerobic exercise training as a non-drug therapeutic modality.

These studies provide evidence that cystic fibrosis transmembrane conductance regulator (CFTR) potentiates and accelerates regulatory volume decrease (RVD) following hypotonic challenge by an autocrine mechanism involving ATP release and signaling. In wild-type CFTR-expressing cells, CFTR augments constitutive ATP release and enhances ATP release stimulated by hypotonic challenge. CFTR itself does not appear to conduct ATP. Instead, ATP is released by a separate channel, whose activity is potentiated by CFTR. Blockade of ATP release by ion channel blocking drugs, gadolinium chloride (Gd 3؉ ) and 4,4-diisothiocyanatostilbene-2,2di-sulfonic acid (DIDS), attenuated the effects of CFTR on acceleration and potentiation of RVD. These results support a key role for extracellular ATP and autocrine and paracrine purinergic signaling in the regulation of membrane ion permeability and suggest that CFTR potentiates ATP release by stimulating a separate ATP channel to strengthen autocrine control of cell volume regulation.ATP and its metabolites function as potent autocrine and paracrine agonists that act within tissues to control cell function through activation of P2 purinergic receptors (1-3) expressed by all cells and tissues. Purinergic agonists are essential for many specialized physiological functions (1-10). In cystic fibrosis (CF), 1 ATP and a related triphosphate nucleotide, UTP, stimulate epithelial chloride (Cl Ϫ ) channels alternative to CFTR via purinergic receptors (11-16). Supraphysiological concentrations of ATP also stimulate CFTR (17). Metabolites of ATP can also act as Cl Ϫ secretagogues (15,16,18). Despite the diverse roles of purinergic signaling, the cellular mechanisms that govern ATP release are not fully defined. CFTR and related ATP-binding cassette (ABC) transporters such as mdr-1 or P-glycoprotein have been implicated as facilitators of ATP release in some cell models (14, 19 -24), while other laboratories have failed to show evidence of CFTRfacilitated ATP conduction or release (25-30).Release of ATP via a conductive pathway has been implicated as an essential autocrine regulator of cell volume in rat hepatoma cells (5). Moreover, ABC transporters have been shown to modulate volume-sensitive Cl Ϫ channels and cell volume (31-34). As such, we tested the hypotheses that CFTR facilitates ATP release under constitutive and hypotonic conditions for autocrine control of cell volume regulation. These hypotheses were also based on the fact that airway surface liquid on CF epithelia is hypertonic with respect to NaCl (35) and/or reduced in volume (36) or both (37, 38) when compared with non-CF epithelia. These airway surface liquid composition abnormalities may reflect an inability of CF epithelial cells to sense changes in external mucosal environment and/or an inability of CF cells to regulate their own cell volume.To this end, complimentary observations using a variety of techniques suggest that expression of CFTR enhances ATP release and modulates the dynamic relationship between cell volume, puriner...

Sensitization to fungi, such as the mold Aspergillus fumigatus, is increasingly becoming linked with asthma severity. We have previously shown that lung responses generated via the beta-glucan receptor Dectin-1 are required for lung defense during acute, invasive A. fumigatus infection. Unexpectedly, in an allergic model of chronic lung exposure to live A. fumigatus conidia, beta-glucan recognition via Dectin-1 led to the induction of multiple proallergic (Muc5ac, Clca3, CCL17, CCL22 and IL-33) and proinflammatory (IL-1β and CXCL1) mediators that compromised lung function. Attenuated proallergic and proinflammatory responses in the absence of Dectin-1 was not associated with changes in Ido (indoleamine 2,3-dioxygenase), Il12p35/Ebi3 (IL-35), IL-10 or TGF-β levels. Assessment of T helper responses demonstrated that purified lung CD4+ T cells produced IL-4, IL-13, IFN-γ and IL-17A, but not IL-22, in a Dectin-1 dependent manner. In contrast, we observed robust, Dectin-1 dependent IL-22 production by unfractionated lung digest cells. Intriguingly, the absence of IL-22 alone mimicked the attenuated proallergic and proinflammatory responses observed in the absence of Dectin-1, suggesting that Dectin-1 mediated IL-22 production potentiated responses that led to decrements in lung function. To this end, neutralization of IL-22 improved lung function in normal mice. Collectively, these results indicate that the beta-glucan receptor Dectin-1 contributes to lung inflammation and immunopathology associated with persistent fungal exposure via the production of IL-22.

Chemokines constitute a large family of secreted proteins that function as chemoattractants and activators of leukocytes. Astrocytes, the major glial cell type in the central nervous system (CNS), are a source of chemokine production within diseased brain. As such, we have examined the production of chemokines by human astroglioma cell lines and primary human astrocytes treated with a variety of stimuli, including LPS, TNF-a, IFN-g and IL-1b. In addition, IL-6 in conjunction with the soluble IL-6 receptor (sIL-6R), and hybrid IL-6 (H-IL-6), a highly active fusion protein of sIL-6R and IL-6, were tested for their ability to induce chemokine expression. The ®ndings presented herein demonstrate that both human astroglioma cell lines and primary human astrocytes express the CXC chemokines IP-10 and IL-8 and the CC chemokines MCP-1 and RANTES in response to TNF-a and IL-1b. IFN-g induced the expression of IP-10, but not of IL-8, MCP-1 or RANTES. Surprisingly, IL-6/sIL-6R and H-IL-6 had little or no effect on chemokine expression in these cells. The effect of TGF-b on chemokine expression in human astroglioma cell lines and astrocytes was also examined. TGF-b alone had little or no effect on RANTES, MCP-1 and IL-8 expression; however, TGF-b synergized with TNF-a to enhance MCP-1 expression in both astroglioma cells and primary astrocytes. An inhibitory effect of TGF-b on TNF-a and IL-1b induced RANTES and IL-8 expression was observed in human astroglioma cells. In contrast, TGF-b enhanced TNF-a and IL-1b induction of IL-8 production by human astrocytes. These ®ndings document a complex pattern of chemokine regulation by the pleiotropic cytokine TGF-b with both enhancing and inhibitory effects.

ATP and its metabolites regulate vascular tone; however, the sources of the ATP released in vascular beds are ill defined. As such, we tested the hypothesis that all limbs of an extracellular purinergic signaling system are present in vascular endothelial cells: ATP release, ATP receptors, and ATP receptor-triggered signal transduction. Primary cultures of human endothelial cells derived from multiple blood vessels were grown as monolayers and studied using a bioluminescence detection assay for ATP released into the medium. ATP is released constitutively and exclusively across the apical membrane under basal conditions. Hypotonic challenge or the calcium agonists ionomycin and thapsigargin stimulate ATP release in a reversible and regulated manner. To assess expression of P2X purinergic receptor channel subtypes (P2XRs), we performed degenerate RT-PCR, sequencing of the degenerate P2XR product, and immunoblotting with P2XR subtype-specific antibodies. Results revealed that P2X(4) and P2X(5) are expressed abundantly by endothelial cell primary cultures derived from multiple blood vessels. Together, these results suggest that components of an autocrine purinergic signaling loop exist in the endothelial cell microvasculature that may allow for "self-regulation" of endothelial cell function and modulation of vascular tone.

Introductory ParagraphThe coupling of hemoglobin sensing of physiological oxygen gradients to stimulation of nitric oxide (NO) bioactivity is an established principle of hypoxic blood flow. One mechanism proposed to explain this O 2 sensing/NO bioactivity linkage postulates an essential role for the conserved hemoglobin β93Cys residue and, specifically, for S-nitrosation of β93Cys to form S-nitrosohemoglobin (SNO-Hb) 1 . The SNO-Hb hypothesis, which conceptually linked hemoglobin and NO biology, has been debated intensely in recent years 2,3 . This debate has precluded a consensus on physiological mechanisms and on assessment of the potential role of SNO-Hb in pathology. Here we describe novel mouse models that express exclusively either human wild type hemoglobin or human hemoglobin in which the β93cys residue is replaced with alanine to assess the role of SNO-Hb in red cell mediated hypoxic vasodilation. Substitution of this residue, precluding hemoglobin S-nitrosation, did not change total red cell S-nitrosothiol levels but shifted S-nitrosothiol distribution to lower MWt species, consistent with the loss of SNO-Hb. Loss of β93cys resulted in no deficits in systemic nor pulmonary hemodynamics under basal conditions and, importantly, did not affect isolated red cell dependent hypoxic vasodilation. These results demonstrate that SNO-Hb is not essential for the physiologic coupling of erythrocyte deoxygenation with increased NO-bioactivity in vivo. *Co corresponding Authors: Rakesh P Patel, PhD, Department of Pathology, University of Alabama at Birmingham, 901 19 th street south, BMR 2, room 302, Birmingham, AL 35294, E mail: E-mail: rakeshp@uab.edu. Tim M Townes, PhD, Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, Kaul Human Genetics Bldg, room 502, 720 20 th street south, Birmingham, AL 35294, E mail: E-mail: ttownes@uab.edu. # these authors contributed equally to this work Author Contributions TSI, CWS, LCW, XT, DAV and KMP were responsible for performing experiments. TSI, CWS, DAV, RPP and TMT were responsible for planning all experiments, analyzing data and writing manuscript. MBR contributed to mass spectrometry assays, LS was responsible for exercise related studies, CGK and BGB for capillary density measurements, NP and JW contributed to blood pressure measurements and NA for assessment of pulmonary hemodynamics. JR did the ES cell injections to generate the chimeras. In addition to hemoglobin oxygen affinity, blood flow is a key component of the processes that match oxygen delivery to demand. Increased blood flow in response to hypoxia is a critical physiological response which does not correlate with dissolved oxygen tensions but does correlate with hemoglobin oxygen fractional saturation 4 . These observations have led to the concept that the red blood cell (RBC) itself is a regulator of flow and to the general paradigm that RBC/hemoglobin deoxygenation is coupled to the stimulation of vasodilation 1,5,6 . Three mechanisms for this coupling have been proposed (...

Extracellular nucleotides regulate NaCl transport in some epithelia. However, the effects of nucleotide agonists on NaCl transport in the renal inner medullary collecting duct (IMCD) are not known. The objective of this study was to determine whether ATP and related nucleotides regulate NaCl transport across mouse IMCD cell line (mIMCD-K2) epithelial monolayers and, if so, via what purinergic receptor subtypes. ATP and UTP inhibited Na(+) absorption [measured via Na(+) short-circuit current (I(Na)(sc))] and stimulated Cl(-) secretion [measured via Cl(-) short-circuit current (I(Cl)(sc))]. Using selective P2 agonists, we report that P2X and P2Y purinoceptors regulate I(Na)(sc) and I(Cl)(sc). By RT-PCR, two P2X receptor channels (P2X(3), P2X(4)) and two P2Y G protein-coupled receptors (P2Y(1), P2Y(2)) were identified. Functional localization of P2 purinoceptors suggest that I(Cl)(sc) is stimulated by apical membrane-resident P2Y purinoceptors and P2X receptor channels, whereas I(Na)(sc) is inhibited by apical membrane-resident P2Y purinoceptors and P2X receptor channels. Together, we conclude that nucleotide agonists inhibit I(Na)(sc) across mIMCD-K2 monolayers through interactions with P2X and P2Y purinoceptors expressed on the apical plasma membrane, whereas extracellular nucleotides stimulate I(Cl)(sc) through interactions with P2X and P2Y purinoceptors expressed on the apical plasma membrane.