Candida albicans elicits protective allergic responses via platelet mediated T helper 2 and T helper 17 cell polarization.

Fungal airway infection (airway mycosis) is an important cause of allergic airway diseases such as asthma, but the mechanisms by which fungi trigger asthmatic reactions are poorly understood. Here, we leverage wild-type and mutant Candida albicans to determine how this common fungus elicits characteristic Th2 and Th17 cell-dependent allergic airway disease in mice. We demonstrate that rather than proteinases that are essential virulence factors for molds, C. albicans instead promoted allergic airway disease through the peptide toxin candidalysin. Candidalysin activated platelets through the Von Willebrand factor (VWF) receptor GP1bα to release the Wnt antagonist Dickkopf-1 (Dkk-1) to drive Th2 and Th17 cell responses that correlated with reduced lung fungal burdens. Platelets simultaneously precluded lethal pulmonary hemorrhage resulting from fungal lung invasion. Thus, in addition to hemostasis, platelets promoted protection against C. albicans airway mycosis through an antifungal pathway involving candidalysin, GP1bα, and Dkk-1 that promotes Th2 and Th17 responses.

Divergent functions for airway epithelial matrix metalloproteinase 7 and retinoic acid in experimental asthma.

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.

NCP2/RHD4/SAC7, SAC6 and SAC8 phosphoinositide phosphatases are required for PtdIns4P and PtdIns(4,5)P2 homeostasis and Arabidopsis development.

Phosphoinositides play important roles in plant growth and development. Several SAC domain phosphoinositide phosphatases have been reported to be important for plant development. Here, we show functional analysis of SUPPRESSOR OF ACTIN 6 (SAC6) to SAC8 in Arabidopsis, a subfamily of phosphoinositide phosphatases containing SAC-domain and two transmembrane motifs. We isolated an Arabidopsis mutant ncp2 that lacked cotyledons in seedling and embryo in pid, a background defective in auxin signaling and transport. NCP2 encodes RHD4/SAC7 phosphoinositide phosphatase. SAC6, SAC7 and SAC8 exhibit overlapping and specific expression patterns in seedling and embryo. The sac6 sac7 embryos either fail to develop into seeds, or have three or four cotyledons. The embryo development of sac7 sac8 and sac6 sac7 sac8 mutants is significantly delayed or lethal, and the seedlings are arrested at early stages. Auxin maxima are decreased in double and triple sac mutants. The contents of PtdIns4P and PtdIns(4,5)P2 in sac6 sac7 and sac7 sac8 mutants are dramatically increased. Protein trafficking of the plasma membrane (PM)-localized protein PIN1 and PIN2 from trans-Golgi network/early endosome back to PM is delayed in sac7 sac8 mutants. These results indicate that SAC6-SAC8 are essential for maintaining homeostasis of PtdIns4P and PtdIns(4,5)P2, and auxin-mediated development in Arabidopsis.

AtMOB1 Genes Regulate Jasmonate Accumulation and Plant Development.

The MOB1 proteins are highly conserved in yeasts, animals, and plants. Previously, we showed that the Arabidopsis (Arabidopsis thaliana) MOB1A gene (AtMOB1A/NCP1) plays critical roles in auxin-mediated plant development. Here, we report that AtMOB1A and AtMOB1B redundantly and negatively regulate jasmonate (JA) accumulation and function in Arabidopsis development. The two MOB1 genes exhibited similar expression patterns, and the MOB1 proteins displayed similar subcellular localizations and physically interacted in vivo. Furthermore, the atmob1a atmob1b (mob1a/1b) double mutant displayed severe developmental defects, which were much stronger than those of either single mutant. Interestingly, many JA-related genes were up-regulated in mob1a/1b, suggesting that AtMOB1A and AtMOB1B negatively regulate the JA pathways. mob1a/1b plants accumulated more JA and were hypersensitive to exogenous JA treatments. Disruption of MYC2, a key gene in JA signaling, in the mob1a/1b background partially alleviated the root defects and JA hypersensitivity observed in mob1a/1b. Moreover, the expression levels of the MYC2-repressed genes PLT1 and PLT2 were significantly decreased in the mob1a/1b double mutant. Our results showed that MOB1A/1B genetically interact with SIK1 and antagonistically modulate JA-related gene expression. Taken together, our findings indicate that AtMOB1A and AtMOB1B play important roles in regulating JA accumulation and Arabidopsis development.

Novel acute hypersensitivity pneumonitis model induced by airway mycosis and high dose lipopolysaccharide.

BACKGROUND: Inhalation of fungal spores is a strong risk factor for severe asthma and experimentally leads to development of airway mycosis and asthma-like disease in mice. However, in addition to fungal spores, humans are simultaneously exposed to other inflammatory agents such as lipopolysaccharide (LPS), with uncertain relevance to disease expression. To determine how high dose inhalation of LPS influences the expression of allergic airway disease induced by the allergenic mold Aspergillus niger (A. niger). METHODS: C57BL/6J mice were intranasally challenged with the viable spores of A. niger with and without 1 µg of LPS over two weeks. Changes in airway hyperreactivity, airway and lung inflammatory cell recruitment, antigen-specific immunoglobulins, and histopathology were determined. RESULTS: In comparison to mice challenged only with A. niger, addition of LPS (1 µg) to A. niger abrogated airway hyperresponsiveness and strongly attenuated airway eosinophilia, PAS+ goblet cells and TH2 responses while enhancing TH1 and TH17 cell recruitment to lung. Addition of LPS resulted in more severe, diffuse lung inflammation with scattered, loosely-formed parenchymal granulomas, but failed to alter fungus-induced IgE and IgG antibodies. CONCLUSIONS: In contrast to the strongly allergic lung phenotype induced by fungal spores alone, addition of a relatively high dose of LPS abrogates asthma-like features, replacing them with a phenotype more consistent with acute hypersensitivity pneumonitis (HP). These findings extend the already established link between airway mycosis and asthma to HP and describe a robust model for further dissecting the pathophysiology of HP.

A Novel Animal Model of Emphysema Induced by Anti-Elastin Autoimmunity.

Loss of immune tolerance to self-antigens can promote chronic inflammation and disrupt the normal function of multiple organs, including the lungs. Degradation of elastin, a highly insoluble protein and a significant component of the lung structural matrix, generates proinflammatory molecules. Elastin fragments (EFs) have been detected in the serum of smokers with emphysema, and elastin-specific T cells have also been detected in the peripheral blood of smokers with emphysema. However, an animal model that could recapitulate T cell-specific autoimmune responses by initiating and sustaining inflammation in the lungs is lacking. In this study, we report an animal model of autoimmune emphysema mediated by the loss of tolerance to elastin. Mice immunized with a combination of human EFs plus rat EFs but not mouse EFs showed increased infiltration of innate and adaptive immune cells to the lungs and developed emphysema. We cloned and expanded mouse elastin-specific CD4+ T cells from the lung and spleen of immunized mice. Finally, we identified TCR sequences from the autoreactive T cell clones, suggesting possible pathogenic TCRs that can cause loss of immune tolerance against elastin. This new autoimmune model of emphysema provides a useful tool to examine the immunological factors that promote loss of immune tolerance to self.

NCP1/AtMOB1A Plays Key Roles in Auxin-Mediated Arabidopsis Development.

MOB1 protein is a core component of the Hippo signaling pathway in animals where it is involved in controlling tissue growth and tumor suppression. Plant MOB1 proteins display high sequence homology to animal MOB1 proteins, but little is known regarding their role in plant growth and development. Herein we report the critical roles of Arabidopsis MOB1 (AtMOB1A) in auxin-mediated development in Arabidopsis. We found that loss-of-function mutations in AtMOB1A completely eliminated the formation of cotyledons when combined with mutations in PINOID (PID), which encodes a Ser/Thr protein kinase that participates in auxin signaling and transport. We showed that atmob1a was fully rescued by its Drosophila counterpart, suggesting functional conservation. The atmob1a pid double mutants phenocopied several well-characterized mutant combinations that are defective in auxin biosynthesis or transport. Moreover, we demonstrated that atmob1a greatly enhanced several other known auxin mutants, suggesting that AtMOB1A plays a key role in auxin-mediated plant development. The atmob1a single mutant displayed defects in early embryogenesis and had shorter root and smaller flowers than wild type plants. AtMOB1A is uniformly expressed in embryos and suspensor cells during embryogenesis, consistent with its role in embryo development. AtMOB1A protein is localized to nucleus, cytoplasm, and associated to plasma membrane, suggesting that it plays roles in these subcellular localizations. Furthermore, we showed that disruption of AtMOB1A led to a reduced sensitivity to exogenous auxin. Our results demonstrated that AtMOB1A plays an important role in Arabidopsis development by promoting auxin signaling.

Antielastin autoimmunity in tobacco smoking-induced emphysema.

Chronic obstructive pulmonary disease and emphysema are common destructive inflammatory diseases that are leading causes of death worldwide. Here we show that emphysema is an autoimmune disease characterized by the presence of antielastin antibody and T-helper type 1 (T(H)1) responses, which correlate with emphysema severity. These findings link emphysema to adaptive immunity against a specific lung antigen and suggest the potential for autoimmune pathology of other elastin-rich tissues such as the arteries and skin of smokers.

Host gastric corpus microenvironment facilitates Ascaris suum larval hatching and infection in a murine model.

Ascariasis (roundworm) is the most common parasitic helminth infection globally and can lead to significant morbidity in children including chronic lung disease. Children become infected with Ascaris spp. via oral ingestion of eggs. It has long been assumed that Ascaris egg hatching and larval translocation across the gastrointestinal mucosa to initiate infection occurs in the small intestine. Here, we show that A. suum larvae hatched in the host stomach in a murine model. Larvae utilize acidic mammalian chitinase (AMCase; acid chitinase; Chia) from chief cells and acid pumped by parietal cells to emerge from eggs on the surface of gastric epithelium. Furthermore, antagonizing AMCase and gastric acid in the stomach decreases parasitic burden in the liver and lungs and attenuates lung disease. Given Ascaris eggs are chitin-coated, the gastric corpus would logically be the most likely organ for egg hatching, though this is the first study directly evincing the essential role of the host gastric corpus microenvironment. These findings point towards potential novel mechanisms for therapeutic targets to prevent ascariasis and identify a new biomedical significance of AMCase in mammals.

Dual protective mechanisms of matrix metalloproteinases 2 and 9 in immune defense against Streptococcus pneumoniae.

A localized and effective innate immune response to pathogenic bacterial invasion is central to host survival. Identification of the critical local innate mediators of lung defense against such pathogens is essential for a complete understanding of the mechanism(s) underlying effective host defense. In an acute model of Streptococcus pneumoniae lung infection, deficiency in matrix metalloproteinase (MMP)2 and MMP9 (Mmp2/9(-/-)) conferred a survival disadvantage relative to wild-type mice treated under the same conditions. S. pneumoniae-infected Mmp2/9(-/-) mice recruited more polymorphonuclear leukocytes to the lung but had higher bacterial burdens. Mmp2/9(-/-) mice showed significantly higher levels of IL-17A, IP-10, and RANTES in the lung. Although MMP2-dependent cleavage partially inactivated IL-17A, MMP9 was critical for effective bacterial phagocytosis and reactive oxygen species generation in polymorphonuclear neutrophils. These data demonstrate critical nonredundant and protective roles for MMP2 and MMP9 in the early host immune response against S. pneumoniae infection.

Toll-like receptor 4 and CD11b expressed on microglia coordinate eradication of Candida albicans cerebral mycosis.

The fungal pathogen Candida albicans is linked to chronic brain diseases such as Alzheimer's disease (AD), but the molecular basis of brain anti-Candida immunity remains unknown. We show that C. albicans enters the mouse brain from the blood and induces two neuroimmune sensing mechanisms involving secreted aspartic proteinases (Saps) and candidalysin. Saps disrupt tight junction proteins of the blood-brain barrier (BBB) to permit fungal brain invasion. Saps also hydrolyze amyloid precursor protein (APP) into amyloid ß (Aß)-like peptides that bind to Toll-like receptor 4 (TLR4) and promote fungal killing in vitro while candidalysin engages the integrin CD11b (Mac-1) on microglia. Recognition of Aß-like peptides and candidalysin promotes fungal clearance from the brain, and disruption of candidalysin recognition through CD11b markedly prolongs C. albicans cerebral mycosis. Thus, C. albicans is cleared from the brain through innate immune mechanisms involving Saps, Aß, candidalysin, and CD11b.

Chronic exposure to carbon black ultrafine particles reprograms macrophage metabolism and accelerates lung cancer.

Chronic exposure to airborne carbon black ultrafine (nCB) particles generated from incomplete combustion of organic matter drives IL-17A-dependent emphysema. However, whether and how they alter the immune responses to lung cancer remains unknown. Here, we show that exposure to nCB particles increased PD-L1+ PD-L2+ CD206+ antigen-presenting cells (APCs), exhausted T cells, and Treg cells. Lung macrophages that harbored nCB particles showed selective mitochondrial structure damage and decreased oxidative respiration. Lung macrophages sustained the HIF1α axis that increased glycolysis and lactate production, culminating in an immunosuppressive microenvironment in multiple mouse models of non-small cell lung cancers. Adoptive transfer of lung APCs from nCB-exposed wild type to susceptible mice increased tumor incidence and caused early metastasis. Our findings show that nCB exposure metabolically rewires lung macrophages to promote immunosuppression and accelerates the development of lung cancer.

Transient Ascaris suum larval migration induces intractable chronic pulmonary disease and anemia in mice.

Ascariasis is one of the most common infections in the world and associated with significant global morbidity. Ascaris larval migration through the host's lungs is essential for larval development but leads to an exaggerated type-2 host immune response manifesting clinically as acute allergic airway disease. However, whether Ascaris larval migration can subsequently lead to chronic lung diseases remains unknown. Here, we demonstrate that a single episode of Ascaris larval migration through the host lungs induces a chronic pulmonary syndrome of type-2 inflammatory pathology and emphysema accompanied by pulmonary hemorrhage and chronic anemia in a mouse model. Our results reveal that a single episode of Ascaris larval migration through the host lungs leads to permanent lung damage with systemic effects. Remote episodes of ascariasis may drive non-communicable lung diseases such as asthma, chronic obstructive pulmonary disease (COPD), and chronic anemia in parasite endemic regions.

Arabidopsis AGC protein kinases IREH1 and IRE3 control root skewing.

AGC protein kinases play important roles in plant growth and development. Several AGC kinases in Arabidopsis have been functionally characterized. However, the "AGC Other" subfamily, including IRE, IREH1, IRE3 and IRE4, has not been well understood. Here, we reported that ireh1 mutants displayed a root skewing phenotype, which can be enhanced by ire3 mutation. IREH1 and IRE3 were expressed in roots, consistent with their function in controlling root skewing. The fluorescence intensities of the microtubule marker KNpro:EGFP-MBD were decreased in ireh1, ire3 and ireh1 ire3 mutants compared to wild type. The microtubule arrangements in ireh1 and ireh1 ire3 mutants were also altered. IREH1 physically interacted with IRE3 in vitro and in planta. Thus, our findings demonstrate that IREH1 and IRE3 protein kinases play important roles in controlling root skewing, and maintaining microtubule network in Arabidopsis.

Elastin-Specific Autoimmunity in Smokers With Thoracic Aortic Aneurysm and Dissection is Independent of Chronic Obstructive Pulmonary Disease.

Background Thoracic aortic aneurysm ( TAA ) and dissection ( TAD ) are characterized by progressive disorganization of the aortic wall matrix, including elastin, a highly immunogenic molecule. Whether acquired autoimmune responses can be detected in TAA / TAD patients who are smokers is unknown. The objectives of this study were to determine whether TAA / TAD smokers have increased T-cell responses to human elastin fragments, and to determine whether autoimmune responses in TAA / TAD smokers are dependent on chronic obstructive pulmonary disease. Methods and Results In a cross-sectional study (N=86), we examined peripheral blood CD 4+ T cell responses to elastin fragments in never-, former-, or current-smokers with or without TAA / TAD . CD 4+ T cells were co-cultured with irradiated autologous peripheral blood CD 1a+/ CD 14+ antigen presenting cells pulsed with or without elastin fragments to measure cytokine production. Baseline plasma concentration of anti-elastin antibodies and elastin-degrading enzymes (eg, matrix metalloproteinase-9, and -12, and neutrophil elastase) were measured in the same cohort. elastin fragment-specific CD 4+ T cell expression of interferon-γ, and anti-elastin antibodies were dependent on history of smoking in TAA / TAD patients but were independent of chronic obstructive pulmonary disease. Matrix metalloproteinase-9, and -12, and neutrophil elastase plasma concentrations were also significantly elevated in ever-smokers with TAA / TAD . Conclusions Cigarette smoke is associated with loss of self-tolerance and induction of elastin-specific autoreactive T- and B-cell responses in patients with TAA / TAD . Development of peripheral blood biomarkers to track immunity to self-antigens could be used to identify and potentially prognosticate susceptibility to TAA / TAD in smokers.

Cigarette smoke-induced reduction of C1q promotes emphysema.

Alteration of innate immune cells in the lungs can promote loss of peripheral tolerance that leads to autoimmune responses in cigarette smokers. Development of autoimmunity in smokers with emphysema is also strongly linked to the expansion of autoreactive T helper (Th) cells expressing interferon gamma (Th1), and interleukin 17A (Th17). However, the mechanisms responsible for enhanced self-recognition and reduced immune tolerance in smoker with emphysema remain less clear. Here we show that C1q, a component of the complement protein 1 complex (C1), is downregulated in lung CD1a+ antigen presenting cells (APCs) isolated from emphysematous human, and mouse lung APCs after chronic cigarette smoke exposure. C1q potentiated the function of APCs to differentiate CD4+ T cells to Tregs, while it inhibited Th17 cell development and proliferation. Mice deficient in C1q that were exposed to chronic smoke exhibited exaggerated lung inflammation marked by increased Th17 cells, while reconstitution of C1q in the lungs enhanced Tregs abundance, dampened smoke-induced lung inflammation, and reversed established emphysema. Our findings demonstrate that cigarette smoke-mediated loss of C1q could play a key role in reduced peripheral tolerance, which could be explored to treat emphysema.

Electronic cigarettes disrupt lung lipid homeostasis and innate immunity independent of nicotine.

Electronic nicotine delivery systems (ENDS) or e-cigarettes have emerged as a popular recreational tool among adolescents and adults. Although the use of ENDS is often promoted as a safer alternative to conventional cigarettes, few comprehensive studies have assessed the long-term effects of vaporized nicotine and its associated solvents, propylene glycol (PG) and vegetable glycerin (VG). Here, we show that compared with smoke exposure, mice receiving ENDS vapor for 4 months failed to develop pulmonary inflammation or emphysema. However, ENDS exposure, independent of nicotine, altered lung lipid homeostasis in alveolar macrophages and epithelial cells. Comprehensive lipidomic and structural analyses of the lungs revealed aberrant phospholipids in alveolar macrophages and increased surfactant-associated phospholipids in the airway. In addition to ENDS-induced lipid deposition, chronic ENDS vapor exposure downregulated innate immunity against viral pathogens in resident macrophages. Moreover, independent of nicotine, ENDS-exposed mice infected with influenza demonstrated enhanced lung inflammation and tissue damage. Together, our findings reveal that chronic e-cigarette vapor aberrantly alters the physiology of lung epithelial cells and resident immune cells and promotes poor response to infectious challenge. Notably, alterations in lipid homeostasis and immune impairment are independent of nicotine, thereby warranting more extensive investigations of the vehicle solvents used in e-cigarettes.

IL17A Regulates Tumor Latency and Metastasis in Lung Adeno and Squamous SQ.2b and AD.1 Cancer.

Somatic mutations can promote malignant transformation of airway epithelial cells and induce inflammatory responses directed against resultant tumors. Tumor-infiltrating T lymphocytes (TIL) in early-stage non-small cell lung cancer (NSCLC) secrete distinct proinflammatory cytokines, but the contribution of these TILs to tumor development and metastasis remains unknown. We show here that TILs in early-stage NSCLC are biased toward IL17A expression (Th17) when compared with adjacent tumor-free tissue, whereas Th17 cells are decreased in tumor infiltrating locoregional lymph nodes in advanced NSCLC. Mice in which Pten and Smad4 (Pts4d/d ) are deleted from airway epithelial cells develop spontaneous tumors, that share genetic signatures with squamous- (SQ.2b), and adeno- (AD.1) subtypes of human NSCLC. Pts4d/d mice globally lacking in IL17a (Pts4d/dIl17a-/- ) showed decreased tumor latency and increased metastasis. Th17 cells were required for recruitment of CD103+ dendritic cells, and adoptive transfer of IL17a-sufficient CD4+ T cells reversed early tumor development and metastasis in Pts4d/dIl17a-/- mice. Together, these findings support a key role for Th17 cells in TILs associated with the Pts4d/d model of NSCLC and suggest therapeutic and biomarker strategies for human SQ2b and AD1 lung cancer. Cancer Immunol Res; 6(6); 645-57. ©2018 AACR.

Overlapping and independent contributions of MMP2 and MMP9 to lung allergic inflammatory cell egression through decreased CC chemokines.

The mechanisms that initiate allergic lung inflammation are relevant to expression of diseases such as asthma, but the factors underlying resolution of inflammation are equally important. Previously, we demonstrated the importance of matrix metalloproteinase 2 (MMP2) for airway egression of lung eosinophils, a critical anti-inflammatory mechanism without which mice are rendered highly susceptible to lethal asphyxiation. Here we show that leukocyte MMP9 is the dominant airway MMP controlling inflammatory cell egression. The allergic lung phenotype of MMP9-/- mice was similar to WT and was not altered by concomitant deletion of the MMP2 gene (double knockout; dko). However, inflammatory cells accumulated aberrantly in the lungs of allergen-challenged MMP9-/- and dko mice and fewer eosinophils and neutrophils were present in bronchoalveolar lavage. These aberrant cellular trafficking patterns were explained by disruption of transepithelial chemokine gradients, in MMP2-/- mice affecting only eotaxin (CCL11), but in MMP9-/- and dko mice involving eotaxin, MARC (CCL7), and TARC (CCL17). Thus, by establishing multiple transepithelial chemokine gradients, MMP9 is broadly implicated in the resolution of allergic inflammation, an essential protective mechanism that overlaps with a more limited role played by MMP2.

Nanoparticulate carbon black in cigarette smoke induces DNA cleavage and Th17-mediated emphysema.

Chronic inhalation of cigarette smoke is the major cause of sterile inflammation and pulmonary emphysema. The effect of carbon black (CB), a universal constituent of smoke derived from the incomplete combustion of organic material, in smokers and non-smokers is less known. In this study, we show that insoluble nanoparticulate carbon black (nCB) accumulates in human myeloid dendritic cells (mDCs) from emphysematous lung and in CD11c(+) lung antigen presenting cells (APC) of mice exposed to smoke. Likewise, nCB intranasal administration induced emphysema in mouse lungs. Delivered by smoking or intranasally, nCB persisted indefinitely in mouse lung, activated lung APCs, and promoted T helper 17 cell differentiation through double-stranded DNA break (DSB) and ASC-mediated inflammasome assembly in phagocytes. Increasing the polarity or size of CB mitigated many adverse effects. Thus, nCB causes sterile inflammation, DSB, and emphysema and explains adverse health outcomes seen in smokers while implicating the dangers of nCB exposure in non-smokers.