Selective cleavage of fibrinogen by diverse proteinases initiates innate allergic and antifungal immunity through CD11b.

Proteinases are essential drivers of allergic airway disease and innate antifungal immunity in part through their ability cleave the clotting factor fibrinogen (FBG) into fibrinogen cleavage products (FCPs) that signal through Toll-like receptor 4 (TLR4). However, the mechanism by which FCPs engage TLR4 remains unknown. Here, we show that the proteinases from Aspergillus melleus (PAM) and other allergenic organisms rapidly hydrolyze FBG to yield relatively few FCPs that drive distinct antifungal mechanisms through TLR4. Functional FCPs, termed cryptokines, were characterized by rapid loss of the FBG α chain with substantial preservation of the ß and γ chains, including a γ chain sequence (Fibγ390-396) that binds the integrin Mac-1 (CD11b/CD18). PAM-derived cryptokines could be generated from multiple FBG domains, and the ability of cryptokines to induce fungistasis in vitro and innate allergic airway disease in vivo strongly depended on both Mac-1 and the Mac-1-binding domain of FBG (Fibγ390-396). Our findings illustrate the essential concept of proteinase-activated immune responses and for the first time link Mac-1, cryptokines, and TLR4 to innate antifungal immunity and allergic airway disease.

Blocking KV1.3 channels inhibits Th2 lymphocyte function and treats a rat model of asthma.

Allergic asthma is a chronic inflammatory disease of the airways. Of the different lower airway-infiltrating immune cells that participate in asthma, T lymphocytes that produce Th2 cytokines play important roles in pathogenesis. These T cells are mainly fully differentiated CCR7(-) effector memory T (TEM) cells. Targeting TEM cells without affecting CCR7(+) naïve and central memory (TCM) cells has the potential of treating TEM-mediated diseases, such as asthma, without inducing generalized immunosuppression. The voltage-gated KV1.3 potassium channel is a target for preferential inhibition of TEM cells. Here, we investigated the effects of ShK-186, a selective KV1.3 channel blocker, for the treatment of asthma. A significant proportion of T lymphocytes in the lower airways of subjects with asthma expressed high levels of KV1.3 channels. ShK-186 inhibited the allergen-induced activation of peripheral blood T cells from those subjects. Immunization of F344 rats against ovalbumin followed by intranasal challenges with ovalbumin induced airway hyper-reactivity, which was reduced by the administration of ShK-186. ShK-186 also reduced total immune infiltrates in the bronchoalveolar lavage and number of infiltrating lymphocytes, eosinophils, and neutrophils assessed by differential counts. Rats with the ovalbumin-induced model of asthma had elevated levels of the Th2 cytokines IL-4, IL-5, and IL-13 measured by ELISA in their bronchoalveolar lavage fluids. ShK-186 administration reduced levels of IL-4 and IL-5 and induced an increase in the production of IL-10. Finally, ShK-186 inhibited the proliferation of lung-infiltrating ovalbumin-specific T cells. Our results suggest that KV1.3 channels represent effective targets for the treatment of allergic asthma.

Airway surface mycosis in chronic TH2-associated airway disease.

BACKGROUND: Environmental fungi have been linked to TH2 cell-related airway inflammation and the TH2-associated chronic airway diseases asthma, chronic rhinosinusitis (CRS) with nasal polyps (CRSwNP), and allergic fungal rhinosinusitis (AFRS), but whether these organisms participate directly or indirectly in disease pathology remains unknown. OBJECTIVE: To determine the frequency of fungus isolation and fungus-specific immunity in patients with TH2-associated and non-TH2-associated airway disease. METHODS: Sinus lavage fluid and blood were collected from sinus surgery patients (n = 118) including patients with CRSwNP, patients with CRS without nasal polyps, patients with AFRS, and non-CRS/nonasthmatic control patients. Asthma status was determined from medical history. Sinus lavage fluids were cultured and directly examined for evidence of viable fungi. PBMCs were restimulated with fungal antigens in an enzyme-linked immunocell spot assay to determine total memory fungus-specific IL-4-secreting cells. These data were compared with fungus-specific IgE levels measured from plasma by ELISA. RESULTS: Filamentous fungi were significantly more commonly cultured in patients with TH2-associated airway disease (asthma, CRSwNP, or AFRS: n = 68) than in control patients with non-TH2-associated disease (n = 31): 74% vs 16%, respectively (P < .001). Both fungus-specific IL-4 enzyme-linked immunocell spot (n = 48) and specific IgE (n = 70) data correlated with TH2-associated diseases (sensitivity 73% and specificity 100% vs 50% and 77%, respectively). CONCLUSIONS: The frequent isolation of fungi growing directly within the airways accompanied by specific immunity to these organisms only in patients with TH2-associated chronic airway diseases suggests that fungi participate directly in the pathogenesis of these conditions. Efforts to eradicate airway fungi from the airways should be considered in selected patients.

Seeking common pathophysiology in asthma, atopy and sinusitis.

Asthma and chronic sinusitis are inexplicably common airway diseases that are linked to atopy and allergic inflammation. T helper type 2 (Th2) cells and the associated cytokines are believed to play crucial pathogenic roles in asthma, but the environmental factors that instigate allergic airway disease remain poorly understood. Environmental proteinases are highly allergenic and are candidate inducers of airway Th2 responses. Determining the proteinases and their sources that are relevant to airway disease, however, remains challenging. In this Opinion, we summarize the evidence that implicates fungi as both a relevant source of allergenic proteinases and a potential cause of asthma, atopy and chronic sinusitis through airway infection. Clarification of the extrinsic causes of these processes will markedly improve diagnosis, prognosis and therapy.

IL-33-responsive innate lymphoid cells are an important source of IL-13 in chronic rhinosinusitis with nasal polyps.

RATIONALE: Chronic rhinosinusitis (CRS) without nasal polyps (CRSsNP) and CRS with nasal polyps (CRSwNP) are associated with Th1 and Th2 cytokine polarization, respectively; however, the pathophysiology of CRS remains unclear. The importance of innate lymphoid cells in Th2-mediated inflammatory disease has not been clearly defined. OBJECTIVES: The objective of this study was to investigate the role of the epithelial cell-derived cytokine IL-33 and IL-33-responsive innate lymphoid cells in the pathophysiology of CRS. METHODS: Relative gene expression was evaluated using quantitative real-time polymerase chain reaction. Innate lymphoid cells in inflamed ethmoid sinus mucosa from patients with CRSsNP and CRSwNP were characterized using flow cytometry. Cytokine production from lymphoid cells isolated from inflamed mucosa of patients with CRS was examined using ELISA and intracellular cytokine staining. MEASUREMENTS AND MAIN RESULTS: Elevated expression of ST2, the ligand-binding chain of the IL-33 receptor, was observed in inflamed sinonasal mucosa from CRSwNP compared with CRSsNP and healthy control subjects. An increased percentage of innate lymphoid cells was observed in inflamed sinonasal mucosa from CRSwNP compared with CRSsNP. ST2(+) innate lymphoid cells are a consistent source of IL-13 in response to IL-33 stimulation. Significant induction of IL-33 was observed in epithelial cells derived from patients with CRSwNP compared with patients with CRSsNP in response to stimulation with Aspergillus fumigatus extract. CONCLUSIONS: These data suggest a role for sinonasal epithelial cell-derived IL-33 and an IL-33-responsive innate lymphoid cell population in the pathophysiology of CRSwNP demonstrating the functional importance of innate lymphoid cells in Th2-mediated inflammatory disease.

Tracheobronchial mycosis in a retrospective case-series study of five status asthmaticus patients.

The etiology of status asthmaticus (SA), a complication of severe asthma, is unknown. Fungal exposure, as measured by fungal atopy, is a major risk factor for developing asthma, but the relationship of fungi in SA per se has not previously been reported. In this five patient retrospective case series study, lower respiratory tract cultures were performed on bronchoalveolar lavage or tracheal aspirate fluid, comparing standard clinical laboratory cultures with a specialized technique in which respiratory mucus was removed prior to culture. We show that mucolytic treatment allows an increased detection of fungal growth, especially yeast, from the lower airways of all SA patients. We also demonstrate that inhalation of the yeast Candida albicans readily induces asthma-like disease in mice. Our observations suggest that SA may represent a fungal infectious process, and support additional prospective studies utilizing anti-fungal therapy to supplement conventional therapy, broad-spectrum antibiotics and high-dose glucocorticoids, which can promote fungal overgrowth.

Proteinases as molecular adjuvants in allergic airway disease.

BACKGROUND: Asthma and related respiratory tract allergic diseases are among the most common chronic diseases of adults and children. Despite their importance, disease course cannot be predicted and treatment remains non-specific and potentially hazardous, with no means for cure. Improved clinical management of asthma will require an improved understanding of the fundamental factors that initiate allergic inflammation, especially T helper type 2 (T(H)2) cell induction. SCOPE OF REVIEW: In this review, we explore the Proteinase Hypothesis of allergic airway disease, considering specifically how organismal proteinases contribute to the expression of allergic disease and potentially important proteinase signaling pathways. MAJOR CONCLUSIONS: Proteinases from diverse sources (bacteria, fungi, plants) may cause occupational asthma by acting as immune adjuvant factors that specifically elicit T(H)2 cell-dependent allergic inflammation. However, more conventional allergic airway diseases (asthma, allergic sinusitis) are more likely to arise from contained fungal or viral infections of the airway in which proteinases are produced and serve as major virulence factors. Proteinases may elicit allergic disease by disrupting numerous cellular proteins, potentially including Toll like receptor (TLR) 4, but critical proteinase-activated signaling pathways remain largely unknown. GENERAL SIGNIFICANCE: Clarification of how proteinases cause allergic disease, specifically confirming an infectious basis for airway proteinase exposure, will likely radically advance how asthma and related respiratory tract disorders are diagnosed and treated. This article is part of a Special Issue entitled Biochemistry of Asthma.

Necessary and sufficient role for T helper cells to prevent fungal dissemination in allergic lung disease.

Mucosal immune responses to fungal infection range from T helper type 2 (Th2) cell-directed allergic inflammation to Th1-predominant neutrophilic inflammation, but the mechanisms directing these divergent mucosal immune outcomes and the role of T cells in host defense against mucosal fungal infections are not known. Here we examined the mouse mucosal immune responses to 12 filamentous environmental fungal species over a broad range of exposure doses and determined the requirement of T cells for host defense. For all tested fungi, low-grade conidium exposures induced Th2- and eosinophil-predominant allergic lung disease, whereas higher exposures led to rapid conversion to neutrophil- and Th1 cell-predominant inflammation, a phenomenon we term immune phenotype switching. All fungal exposure doses were further linked to the secretion of interleukin-17A (IL-17A). Fungal infections with Curvularia lunata and Aspergillus fumigatus were typically confined to the airway during allergic inflammation but became locally invasive and disseminated to the brain at higher conidium challenge doses, in association with predominant Th1 responses. Fungal dissemination occurred at relatively low challenge doses with the conidia of Aspergillus fumigatus administered to recombinase activating gene 1 (Rag-1)-deficient mice, which lack B and T cells, but B cell-deficient µMT mice and T helper cell-reconstituted Rag-1-deficient mice were comparable to wild-type mice in preventing fungal dissemination. Our findings demonstrate that Th2 cell-predominant allergic responses followed by immune phenotype switching and fungal dissemination are highly predictable outcomes with progressive fungal infectious burdens and that T helper cell responses are protective against lethal fungal dissemination.

Telomerase-immortalized human fibroblasts retain UV-induced mutagenesis and p53-mediated DNA damage responses.

Immortalized cells frequently have disruptions of p53 activity and lack p53-dependent nucleotide excision repair (NER). We hypothesized that telomerase immortalization would not alter p53-mediated ultraviolet light (UV)-induced DNA damage responses. DNA repair proficient primary diploid human fibroblasts (GM00024) were immortalized by transduction with a telomerase expressing retrovirus. Empty retrovirus transduced cells senesced after a few doublings. Telomerase transduced GM00024 cells (tGM24) were cultured continuously for 6 months (>60 doublings). Colony forming ability after UV irradiation was dose-dependent between 0 and 20J/m2 UVC (LD50=5.6J/m2). p53 accumulation was UV dose- and time-dependent as was induction of p48(XPE/DDB2), p21(CIP1/WAF1), and phosphorylation on p53-S15. UV dose-dependent apoptosis was measured by nuclear condensation. UV exposure induced UV-damaged DNA binding as monitored by electrophoretic mobility shift assays using UV irradiated radiolabeled DNA probe was inhibited by p53-specific siRNA transfection. p53-Specific siRNA transfection also prevented UV induction of p48 and improved UV survival measured by colony forming ability. Strand-specific NER of cyclobutane pyrimidine dimers (CPD) within DHFR was identical in tGM24 and GM00024 cells. CPD removal from the transcribed strand was nearly complete in 6h and from the non-transcribed strand was 73% complete in 24h. UV-induced HPRT mutagenesis in tGM24 was indistinguishable from primary human fibroblasts. These wide-ranging findings indicate that the UV-induced DNA damage response remains intact in telomerase-immortalized cells. Furthermore, telomerase immortalization provides permanent cell lines for testing the immediate impact on NER and mutagenesis of selective genetic manipulation without propagation to establish mutant lines.

XP-A cells complemented with Arg228Gln and Val234Leu polymorphic XPA alleles repair BPDE-induced DNA damage better than cells complemented with the wild type allele.

Functional effects of Arg228Gln and Val2343Leu XPA polymorphisms on benzo[a]pyrene-r-7,t-8-dihydrodiol-t-9,10-epoxide-(+/-)-anti (BPDE) survival and repair were investigated in SV40 immortalized XP12RO cells complemented with wild type and polymorphic XPA cDNAs in an inducible cDNA expression system. In contrast to previous studies showing little impact of XPA polymorphisms on UV survival and repair, cells complemented with polymorphic XPAs displayed improved BPDE survival and repair as compared to wild type XPA-complemented cells. Survival after BPDE treatment was measured using AlamarBlue reduction and colony forming ability. Cells expressing low levels of either polymorphic XPA had equivalent or improved survival compared to wild type XPA-complemented cells (XPAwt cells). XPA induction improved BPDE survival in Arg228Gln (R228Q cells) and Val234Leu (V234L cells) complemented cells, but not XPAwt cells. BPDE-induced DNA damage repair was measured both by reactivation after transfection of a luciferase reporter plasmid reacted with BPDE in vitro, and by removal of adducts from genomic DNA of BPDE-treated cells. BPDE-induced DNA damage repair in R228Q and V234L cells expressing XPA at very low levels was similar to repair in XPAwt cells expressing XPA at normal levels. XPA induction improved repair in R228Q and V234L cells but not in XPAwt cells. Our findings suggest that both Arg228Gln and Val234Leu XPAs function better than wild type XPA for BPDE adduct removal. These observations differ from UV repair results suggesting that the differences are lesion specific. The location of the polymorphisms within the putative poly(ADP-ribose) binding domain suggests that poly(ADP-ribose) interaction is important in repair.

Preferential uptake of antioxidant carbon nanoparticles by T lymphocytes for immunomodulation.

Autoimmune diseases mediated by a type of white blood cell-T lymphocytes-are currently treated using mainly broad-spectrum immunosuppressants that can lead to adverse side effects. Antioxidants represent an alternative approach for therapy of autoimmune disorders; however, dietary antioxidants are insufficient to play this role. Antioxidant carbon nanoparticles scavenge reactive oxygen species (ROS) with higher efficacy than dietary and endogenous antioxidants. Furthermore, the affinity of carbon nanoparticles for specific cell types represents an emerging tactic for cell-targeted therapy. Here, we report that nontoxic poly(ethylene glycol)-functionalized hydrophilic carbon clusters (PEG-HCCs), known scavengers of the ROS superoxide (O2•-) and hydroxyl radical, are preferentially internalized by T lymphocytes over other splenic immune cells. We use this selectivity to inhibit T cell activation without affecting major functions of macrophages, antigen-presenting cells that are crucial for T cell activation. We also demonstrate the in vivo effectiveness of PEG-HCCs in reducing T lymphocyte-mediated inflammation in delayed-type hypersensitivity and in experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis. Our results suggest the preferential targeting of PEG-HCCs to T lymphocytes as a novel approach for T lymphocyte immunomodulation in autoimmune diseases without affecting other immune cells.

Polymorphisms in the human xeroderma pigmentosum group A gene and their impact on cell survival and nucleotide excision repair.

Polymorphisms in DNA repair genes may contribute to defects in DNA repair and increased susceptibility to cancer. The xeroderma pigmentosum group A (XPA) gene is required for nucleotide excision repair (NER) and mutations in XPA highly predispose humans to skin cancer. We examined DNA samples from 189 individuals for polymorphisms in the XPA gene. First, SSCP analysis was used to examine each of the six exons and their intron boundaries. One frequent single nucleotide polymorphism (SNP) in the untranslated region of exon 1 and two rare SNPs which produce the changes Arg228Gln and Val234Leu in the coding region of exon 6 were identified. Quite surprisingly, no sequence variants were found within the coding regions or the adjacent intron boundaries of exons 1-5. Ecdysone-inducible expression vectors containing wild type XPA cDNA or cDNAs representing the two polymorphisms that we identified in exon 6 were created and independently introduced into the XPA deficient cell line XP12RO-SV. Transcription-coupled repair (TCR), global genome repair (GGR) and cell survival following UV irradiation were studied in each cell line in the absence or presence of the ecdysone hormone analog, ponasterone A. No substantial difference in repair or cell survival was found in cells complemented with wild type or polymorphic alleles of XPA. A 10-fold increase in the expression of XPA by addition of ponasterone A resulted in faster removal of 6-4 photoproducts from the total genomes of cells complemented with wild type or polymorphic alleles of XPA but had no significant impact on TCR or global genome repair of cyclobutane pyrimidine dimers (CPDs). Since our SSCP analysis failed to detect significant numbers of polymorphisms we directly sequenced exons 4-6 in a subset of our samples. One additional rare SNP, which produces the change Leu252Val was found in exon 6 and four rare SNPs and one rare single nucleotide deletion were found in intron 4. Hence, the XPA gene appears to be a cold spot for genetic variation and rare polymorphisms in the coding region of the gene do not reduce NER or cell survival after UV irradiation.