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INTRODUCTION: TNFα-inducible matrix metalloproteinases play a critical role in the process of airway remodeling in respiratory inflammatory disease including asthma. The cationic host defense peptide LL-37 is elevated in the lungs during airway inflammation. However, the impact of LL-37 on TNFα-driven processes is not well understood. Here, we examined the effect of LL-37 on TNFα-mediated responses in human bronchial epithelial cells (HBECs). METHODS: We used a slow off-rate modified aptamer-based proteomics approach to define the HBEC proteome altered in response to TNFα. Abundance of selected protein candidates and signaling intermediates was examined using immunoassays, ELISA and Western blots, and mRNA abundance was examined by qRT-PCR. RESULTS: Proteomics analysis revealed that 124 proteins were significantly altered, 12 proteins were enhanced by ≥2-fold compared to unstimulated cells, in response to TNFα. MMP9 was the topmost increased protein in response to TNFα, enhanced by â¼10-fold, and MMP13 was increased by â¼3-fold, compared to unstimulated cells. Furthermore, we demonstrated that LL-37 significantly suppressed TNFα-mediated MMP9 and MMP13 in HBEC. Mechanistic data revealed that TNFα-mediated MMP9 and MMP13 production is controlled by SRC kinase and that LL-37 enhances related upstream negative regulators, namely, phospho-AKT (T308) and TNFα-mediated TNFAIP3 or A20. CONCLUSIONS: The findings of this study suggest that LL-37 may play a role in intervening in the process of airway remodeling in chronic inflammatory respiratory disease such as asthma.
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Remodelación de las Vías Aéreas (Respiratorias) , Péptidos Catiónicos Antimicrobianos , Asma , Bronquios , Catelicidinas , Células Epiteliales , Metaloproteinasa 13 de la Matriz , Metaloproteinasa 9 de la Matriz , Factor de Necrosis Tumoral alfa , Humanos , Péptidos Catiónicos Antimicrobianos/metabolismo , Asma/inmunología , Asma/metabolismo , Células Cultivadas , Células Epiteliales/metabolismo , Metaloproteinasa 13 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Proteómica , Mucosa Respiratoria/inmunología , Transducción de Señal , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Biological sex impacts disease prevalence, severity and response to therapy in asthma, however preclinical studies often use only one sex in murine models. Here, we detail sex-related differences in immune responses using a house dust mite (HDM)-challenge model of acute airway inflammation, in adult mice of two different strains (BALB/c and C57BL/6NJ). Female and male mice were challenged (intranasally) with HDM extract (~ 25 µg) for 2 weeks (N = 10 per group). Increase in serum HDM-specific IgE showed a female bias, which was statistically significant in BALB/c mice. We compared naïve and HDM-challenged mice to define immune responses in the lungs by assessing leukocyte accumulation in the bronchoalveolar lavage fluid (BALF), and profiling the abundance of 29 different cytokines in BALF and lung tissue lysates. Our results demonstrate specific sex-related and strain-dependent differences in airway inflammation. For example, HDM-driven accumulation of neutrophils, eosinophils and macrophages were significantly higher in females compared to males, in BALB/c mice. In contrast, HDM-mediated eosinophil accumulation was higher in males compared to females, in C57BL/6NJ mice. Differences in lung cytokine profiles indicated that HDM drives a T-helper (Th)17-biased response with higher IL-17 levels in female BALB/c mice compared to males, whereas female C57BL/6NJ mice elicit a mixed Th1/Th2-skewed response. Male mice of both strains showed higher levels of specific Th2-skewed cytokines, such as IL-21, IL-25 and IL-9, in response to HDM. Overall, this study details sex dimorphism in HDM-mediated airway inflammation in mice, which will be a valuable resource for preclinical studies in allergic airway inflammation and asthma.
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Asma , Pyroglyphidae , Femenino , Masculino , Ratones , Animales , Alérgenos , Caracteres Sexuales , Ratones Endogámicos C57BL , Dermatophagoides pteronyssinus , Inflamación , Ratones Endogámicos BALB C , CitocinasRESUMEN
BACKGROUND: The heterodimer interleukin (IL)-17A/F is elevated in the lungs in chronic respiratory disease such as severe asthma, along with the pro-inflammatory cytokine tumor necrosis factor-α (TNF-α). Although IL-17A/F and TNF-α are known to functionally cooperate to exacerbate airway inflammation, proteins altered by their interaction in the lungs are not fully elucidated. RESULTS: We used Slow Off-rate Modified Aptamer-based proteomic array to identify proteins that are uniquely and/or synergistically enhanced by concurrent stimulation with IL-17A/F and TNF-α in human bronchial epithelial cells (HBEC). The abundance of 38 proteins was significantly enhanced by the combination of IL-17A/F and TNF-α, compared to either cytokine alone. Four out of seven proteins that were increased > 2-fold were those that promote neutrophil migration; host defence peptides (HDP; Lipocalin-2 (LCN-2) and Elafin) and chemokines (IL-8, GROα). We independently confirmed the synergistic increase of these four proteins by western blots and ELISA. We also functionally confirmed that factors secreted by HBEC stimulated with the combination of IL-17A/F and TNF-α uniquely enhances neutrophil migration. We further showed that PI3K and PKC pathways selectively control IL-17A/F + TNF-α-mediated synergistic production of HDPs LCN-2 and Elafin, but not chemokines IL-8 and GROα. Using a murine model of airway inflammation, we demonstrated enhancement of IL-17A/F, TNF-α, LCN-2 and neutrophil chemokine KC in the lungs, thus corroborating our findings in-vivo. CONCLUSION: This study identifies proteins and signaling mediated by concurrent IL-17A/F and TNF-α exposure in the lungs, relevant to respiratory diseases characterized by chronic inflammation, especially neutrophilic airway inflammation such as severe asthma.
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Innate defense regulator (IDR) peptides show promise as immunomodulatory therapeutics. However, there is limited understanding of the relationship of IDR peptide sequence and/or structure with its immunomodulatory activity. We previously reported that an IDR peptide, IDR-1002, reduces airway hyperresponsiveness (AHR) and inflammation in a house dust mite (HDM)-challenged murine model of airway inflammation. Here, we examined the sequence-to-function relationship of IDR-1002 in HDM-challenged mice and human bronchial epithelial cells (HBEC). We demonstrated that the tryptophan (W8) in the central hydrophobic region of IDR-1002 is required for the peptide to (i) suppress the pro-inflammatory cytokine IL-33, and induce anti-inflammatory mediators IL-1RA and stanniocalcin-1 in HBEC, and (ii) reduce IL-33 abundance, and eosinophil and neutrophil infiltration, in the lungs of HDM-challenged mice, without affecting the capacity to improve AHR, suggesting multimodal activity in vivo. Findings from this study can be used to design IDR peptides with targeted impact on immunomodulation and pathophysiology in respiratory diseases.
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Antiinflamatorios/farmacología , Péptidos Catiónicos Antimicrobianos/química , Inmunomodulación/efectos de los fármacos , Triptófano/química , Sustitución de Aminoácidos , Animales , Antiinflamatorios/química , Antiinflamatorios/uso terapéutico , Péptidos Catiónicos Antimicrobianos/farmacología , Péptidos Catiónicos Antimicrobianos/uso terapéutico , Asma/tratamiento farmacológico , Modelos Animales de Enfermedad , Células Epiteliales/citología , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Femenino , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Proteína Antagonista del Receptor de Interleucina 1/metabolismo , Interleucina-33/metabolismo , Ratones , Ratones Endogámicos BALB C , Infiltración Neutrófila/efectos de los fármacos , Análisis de Componente Principal , Estructura Secundaria de Proteína , Pyroglyphidae/patogenicidad , Triptófano/metabolismoRESUMEN
IL-33 induces airway inflammation and hyper-responsiveness in respiratory diseases. Although defined as a therapeutic target, there are limited studies that have comprehensively investigated IL-33-mediated responses in the lungs in vivo. In this study, we characterized immunological and physiological responses induced by intranasal IL-33 challenge, in a mouse model. We identified specific cytokines, IL-4, IL-5, IL-6, IL-10, IP-10 and MIP1-α, that are increased in bronchoalveolar lavage and lung tissues by IL-33. Using transcriptomics (RNA-Seq) we demonstrated that 2279 transcripts were up-regulated and 1378 downregulated (≥ 2-fold, p < 0.01) in lung tissues, in response to IL-33. Bioinformatic interrogation of the RNA-Seq data was used to predict biological pathways and upstream regulators involved in IL-33-mediated responses. We showed that the mRNA and protein of STAT4, a predicted upstream regulator of IL-33-induced transcripts, was significantly enhanced in the lungs following IL-33 challenge. Overall, this study provides specific IL-33-induced molecular targets and endpoints that can be used as a resource for in vivo studies, e.g. in preclinical murine models examining novel interventions to target downstream effects of IL-33.
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Interleucina-33/inmunología , Pulmón/inmunología , Pulmón/metabolismo , Modelos Animales , Transcriptoma , Administración Intranasal , Animales , Femenino , Interleucina-33/administración & dosificación , Pulmón/patología , Ratones , Ratones Endogámicos BALB C , RNA-SeqRESUMEN
Antimicrobial peptides, also known as host defence peptides, are immunomodulatory molecules required to resolve infections. Antimicrobial peptides and proteins (APPs) are important in the control of infections in the lungs. Despite evidence that APPs exhibit a wide range of immune functions and modulate inflammation, the effect of inflammatory cytokines on the expression of APPs is not completely defined. In this study, we profiled the expression of 39 different APPs in human bronchial epithelial cells (HBEC) using Slow Off-rate Modified Aptamer (SOMAmer)-based protein array, in the presence and absence of three different inflammatory cytokines (IL-17, TNF and IFN-γ). Expression of 13 different APPs was altered in response to IL-17, TNF or IFN-γ. Independent validations of selected proteins from the proteomics screen i.e., those that were significantly enhanced by >2-fold change (p < 0.01) using western blots conclusively demonstrated that inflammatory cytokines alter the expression of APPs differentially. For example, the abundance of cathepsin S was enhanced by only IFN-γ, whereas lipocalin-2 was increased by IL-17 alone. Abundance of elafin increased in presence of IL-17 or TNF, but decreased in response to IFN-γ. Whereas the abundance of cathepsin V decreased following stimulation with IL-17, TNF and IFN-γ. The results of this study demonstrate that inflammatory cytokines alter the expression of APPs disparately. This suggests that the composition of the inflammatory cytokine milieu may influence APPs abundance and thus alter the processes required for infection control and regulation of inflammation in the lungs.
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BACKGROUND: Exacerbation in asthma is associated with decreased expression of specific host defence peptides (HDPs) in the lungs. We examined the effects of a synthetic derivative of HDP, innate defence regulator (IDR) peptide IDR-1002, in house dust mite (HDM)-challenged murine model of asthma, in interleukin (IL)-33-challenged mice and in human primary bronchial epithelial cells (PBECs). METHODS: IDR-1002 (6 mg/kg per mouse) was administered (subcutaneously) in HDM-challenged and/or IL-33-challenged BALB/c mice. Lung function analysis was performed with increasing dose of methacholine by flexiVent small animal ventilator, cell differentials in bronchoalveolar lavage performed by modified Wright-Giemsa staining, and cytokines monitored by MesoScale Discovery assay and ELISA. PBECs stimulated with tumour necrosis factor alpha (TNF-α) and interferon gamma (IFN-γ), with or without IDR-1002, were analysed by western blots. RESULTS: IDR-1002 blunted HDM challenge-induced airway hyper-responsiveness (AHR), and lung leucocyte accumulation including that of eosinophils and neutrophils, in HDM-challenged mice. Concomitantly, IDR-1002 suppressed HDM-induced IL-33 in the lungs. IFN-γ/TNF-α-induced IL-33 production was abrogated by IDR-1002 in PBECs. Administration of IL-33 in HDM-challenged mice, or challenge with IL-33 alone, mitigated the ability of IDR-1002 to control leucocyte accumulation in the lungs, suggesting that the suppression of IL-33 is essential for the anti-inflammatory activity of IDR-1002. In contrast, the peptide significantly reduced either HDM, IL-33 or HDM+IL-33 co-challenge-induced AHR in vivo. CONCLUSION: This study demonstrates that an immunomodulatory IDR peptide controls the pathophysiology of asthma in a murine model. As IL-33 is implicated in steroid-refractory severe asthma, our findings on the effects of IDR-1002 may contribute to the development of novel therapies for steroid-refractory severe asthma.
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Péptidos Catiónicos Antimicrobianos/farmacología , Asma/tratamiento farmacológico , Citocinas/metabolismo , Inmunomodulación/efectos de los fármacos , Hipersensibilidad Respiratoria/tratamiento farmacológico , Animales , Asma/inmunología , Asma/metabolismo , Western Blotting , Líquido del Lavado Bronquioalveolar/citología , Técnicas de Cultivo de Célula , Modelos Animales de Enfermedad , Ensayo de Inmunoadsorción Enzimática , Células Epiteliales/efectos de los fármacos , Células Epiteliales/inmunología , Femenino , Humanos , Pulmón/metabolismo , Pulmón/patología , Cloruro de Metacolina/farmacología , Ratones , Ratones Endogámicos BALB C , Pyroglyphidae/inmunología , Hipersensibilidad Respiratoria/inmunología , Hipersensibilidad Respiratoria/metabolismoRESUMEN
We investigate the nonlinear evolution of the morphological deformation of a solid-liquid interface of a binary melt under rapid solidification conditions near two absolute stability limits. The first of these involves the complete stabilization of the system to cellular instabilities as a result of large enough surface energy. We derive nonlinear evolution equations in several limits in this scenario and investigate the effect of interfacial disequilibrium on the nonlinear deformations that arise. In contrast to the morphological stability problem in equilibrium, in which only cellular instabilities appear and only one absolute stability boundary exists, in disequilibrium the system is prone to oscillatory instabilities and a second absolute stability boundary involving attachment kinetics arises. Large enough attachment kinetics stabilize the oscillatory instabilities. We derive a nonlinear evolution equation to describe the nonlinear development of the solid-liquid interface near this oscillatory absolute stability limit. We find that strong asymmetries develop with time. For uniform oscillations, the evolution equation for the interface reduces to the simple form f^{''}+(ßf^{'})^{2}+f=0, where ß is the disequilibrium parameter. Lastly, we investigate a distinguished limit near both absolute stability limits in which the system is prone to both cellular and oscillatory instabilities and derive a nonlinear evolution equation that captures the nonlinear deformations in this limit. Common to all these scenarios is the emergence of larger asymmetries in the resulting shapes of the solid-liquid interface with greater departures from equilibrium and larger morphological numbers. The disturbances additionally sharpen near the oscillatory absolute stability boundary, where the interface becomes deep-rooted. The oscillations are time-periodic only for small-enough initial amplitudes and their frequency depends on a single combination of physical parameters, including the morphological number, as well as the amplitude. The critical amplitude, at which solutions loose periodicity, depends on a single combination of parameters independent of the morphological number that indicate that non-periodic growth is most commonly present for moderate disequilibrium parameters. The spatial distribution of the interface develops deepening roots at late times. Similar spatial distributions are also seen in the limit in which both the cellular and oscillatory modes are close to absolute stability, and the roots deepen with larger departures from the two absolute stability boundaries.
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House dust mite (HDM) challenge is commonly used in murine models of allergic asthma for preclinical pathophysiological studies. However, few studies define objective readouts or biomarkers in this model. In this study we characterized immune responses and defined molecular markers that are specifically altered after HDM challenge. In this murine model, we used repeated HDM challenge for two weeks which induced hallmarks of allergic asthma seen in humans, including airway hyper-responsiveness (AHR) and elevated levels of circulating total and HDM-specific IgE and IgG1. Kinetic studies showed that at least 24â h after last HDM challenge results in significant AHR along with eosinophil infiltration in the lungs. Histologic assessment of lung revealed increased epithelial thickness and goblet cell hyperplasia, in the absence of airway wall collagen deposition, suggesting ongoing tissue repair concomitant with acute allergic lung inflammation. Thus, this model may be suitable to delineate airway inflammation processes that precede airway remodeling and development of fixed airway obstruction. We observed that a panel of cytokines e.g. IFN-γ, IL-1ß, IL-4, IL-5, IL-6, KC, TNF-α, IL-13, IL-33, MDC and TARC were elevated in lung tissue and bronchoalveolar fluid, indicating local lung inflammation. However, levels of these cytokines remained unchanged in serum, reflecting lack of systemic inflammation in this model. Based on these findings, we further monitored the expression of 84 selected genes in lung tissues by quantitative real-time PCR array, and identified 31 mRNAs that were significantly up-regulated in lung tissue from HDM-challenged mice. These included genes associated with human asthma (e.g. clca3, ear11, il-13, il-13ra2, il-10, il-21, arg1 and chia1) and leukocyte recruitment in the lungs (e.g. ccl11, ccl12 and ccl24). This study describes a biosignature to enable broad and systematic interrogation of molecular mechanisms and intervention strategies for airway inflammation pertinent to allergic asthma that precedes and possibly potentiates airway remodeling and fibrosis.
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Amphiphilic aminoglycosides (AAGs) are an emerging source of antibacterials to combat infections caused by antibiotic-resistant bacteria. Mode-of-action studies indicate that AAGs predominately target bacterial membranes, thereby leading to depolarization and increased permeability. To assess whether AAGs also induce host-directed immunomodulatory responses, we determined the AAG-dependent induction of cytokines in macrophages in the absence or presence of lipopolysaccharide (LPS). Our results show for the first time that AAGs can boost the innate immune response, specifically the recruitment of immune cells such as neutrophils required for the resolution of infections. Moreover, AAGs can selectively control inflammatory responses induced in the presence of endotoxins to prevent septic shock. In conclusion, our study demonstrates that AAGs possess multifunctional properties that combine direct antibacterial activity with host-directed clearance effects reminiscent of those of host-defense peptides.