RESUMEN

Exposure to storage mite (SM) and house dust mite (HDM) allergens is a risk factor for sensitization and asthma development; however, the related immune responses and their pathology have not been fully investigated. The HDMs Dermatophagoides farinae and Dermatophagoides pteronyssinus and SM Tyrophagus putrescentiae are potent allergens that induce asthma. Most SM-related studies have focused on the allergic reactions of individuals by measuring their immunoglobulin (Ig)E expression. Considering the limited research on this topic, the present study aims to investigate the differences in the immune responses induced by HDMs and SMs and histologically analyze lung tissues in a mouse asthma model to understand the differential effects of HDM and SM. The results revealed that all mite species induced airway inflammation. Mice challenged with T. putrescentiae had the highest airway resistance and total cell, eosinophil, and neutrophil counts in the bronchoalveolar lavage fluid (BALF). The SM-sensitized groups showed more severe lesions and mucus hypersecretions than the HDM-sensitized groups. Although the degree of HDM and SM exposure was the same, the damage to the respiratory lung tissue was more severe in SM-exposed mice, which resulted in excessive mucin secretion and increased fibrosis. Furthermore, these findings suggest that SM sensitization induces a more significant hypersensitivity response in mucosal immunity than HDM sensitization in asthma models.

Asunto(s)

Asma , Pulmón , Pyroglyphidae , Animales , Ratones , Pyroglyphidae/inmunología , Pulmón/inmunología , Pulmón/patología , Asma/inmunología , Asma/patología , Femenino , Neumonía/inmunología , Neumonía/patología , Líquido del Lavado Bronquioalveolar/inmunología , Líquido del Lavado Bronquioalveolar/citología , Modelos Animales de Enfermedad , Ratones Endogámicos BALB C , Acaridae/inmunología , Alérgenos/inmunología , Eosinófilos/inmunología , Eosinófilos/patología

RESUMEN

Background: The clinical efficacy of allergen-specific immunotherapy (AIT) for Alternaria alternata (A. alt) and Dermatophagoides farinae (Der f) extracts remains largely unknown in China. We sought to retrospectively evaluate the efficacy caused by AIT agents manufactured in China of patients who are sensitized to A. alt and Der f. Methods: Patients aged 5-27 years with asthma and perennial allergic rhinitis (AR), and AIT with A. alt and Der f were recruited, and then classified into two groups: A. alt-AIT (n = 31) and A. alt + Der f-AIT group (n = 39). All data were gathered retrospectively, including biological parameters, pulmonary function, and symptom and medication scores. Results: 70 patients who underwent A. alt and Der f AIT were enrolled. A significant improvement was observed in the values of FEV1% (P < 0.0001) and MEF 25 (P = 0.023) of lung function. Both the rhinitis symptoms and combined symptoms and medication scores for asthma decreased after AIT (by 45.3% and 80.3%, respectively, P < 0.0001 for each). Nearly 67% improvement rate (P < 0.0001) occurred in rhinoconjunctivitis quality of life, and a great increase existed in Asthma Control Test (ACT) score (P < 0.0001) after at least 1 year AIT, although there were no significant changes between these two groups. Besides, no significance was displayed in specific IgE to different allergens. Conclusion: AIT with A. alt and Der f extracts had clinical efficacy for many patients in China, with a reduction of symptom and medication scores, and great improvement in spirometry function.

RESUMEN

BACKGROUND: Precision dosing in sublingual immunotherapy (SLIT) has become a hotspot gradually, yet no standardized dose adjustment pattern for house dust mite (HDM)-SLIT. This study aims to investigate the clinical feasibility of the dynamic maintenance dose ascending regimen for individualized SLIT. METHODS: A total of 258 allergic rhinitis (AR) patients treated with HDM-SLIT were included in this retrospective study. Patients were divided into the regular dose (RD) group (n = 101) and the high dose (HD) group (n = 157) according to different maintenance dosages of SLIT. In the RD group, patients received the fixed dose recommended by the manufacturer. In the HD group, patients received a maximum tolerance dose determined by dynamic dose ascending. The clinical efficacy was evaluated by combined symptom and medication score (CSMS) and visual analogue scale score (VAS) at the baseline, 0.5-year, 1-year, and 2-year. The safety was evaluated by adverse events (AEs). RESULTS: Significant reductions of CSMS and VAS at 0.5-year, 1-year, and 2-year were observed in both the RD group and the HD group compared to the baseline (P < 0.05). In addition, greater improvements in these clinical parameters from 0.5- to 2-year were found in the HD group compared to the RD group (P < 0.05). For subgroup analysis in the HD group, no significant differences in CSMS and VAS were observed among subgroups of patients <14 years old and patients ≥14 years old (P > 0.05). No serious AEs in the two groups and no significant differences were observed between the AE incidence rate of the RD group and HD group during the incremental and maintenance phases. CONCLUSIONS: The 2-year HDM-SLIT with dynamic maintenance dose ascending regimen offers an "optimal" treatment for AR patients while maintaining safety. This study introduced a pattern for individualized dose adjustment in clinical practice, offering potential benefits for AR patients.

RESUMEN

BACKGROUND: Dermatophagoides farinae proteins (DFPs) are abundantly expressed in D. farinae; however, their functions remain unknown. Our previous transcriptome sequencing analyses revealed that the basal expression of DFP1 and DFP2 in D. farinae was high and, more importantly, upregulated under temperature stress. Therefore, DFPs were speculated to exert a temperature stress response function. RESULTS: Real-time quantitative polymerase chain reaction detection revealed that both DFP1 and DFP2 were significantly upregulated under temperature stress. Particularly, DFP1 was upregulated under cold stress. Electrophoresis of D. farinae total proteins revealed an increased abundance of DFP1 and DFP2 (40-55 kDa bands) under temperature stress, which was corroborated by the mass spectrometry results. After silencing DFP1 and DFP2 further, temperature stress led to decreases in gene expression and survival rates. Moreover, DFP1 was identified as the upstream regulator of DFP2. CONCLUSION: This study highlights the temperature stress response functions of DFP1 and DFP2 at the mRNA and protein levels. These results provide important insights for applying DFP1 and DFP2 as potential target genes for the molecular prevention and control of D. farinae to prevent allergic diseases. The newly established methods provide methodological guidance for the study of genes with unknown functions in mites.

Asunto(s)

Dermatophagoides farinae , Estrés Fisiológico , Animales , Temperatura , Proteínas de Artrópodos/genética , Proteínas de Artrópodos/metabolismo

RESUMEN

Papain (PN) is a proteolytic enzyme derived from Carica Papaya L. While the pharmacological effects of PN have not been extensively studied compared to its enzymatic activity, PN also holds potential benefits beyond protein digestion. This study aimed to investigate the potential effects of PN against skin inflammation in house dust mite Dermatophagoides farinae body (Dfb)-exposed NC/Nga atopic dermatitis (AD) mice and human HaCaT keratinocytes and their underlying mechanisms. The effects of PN on the skin were assessed via histological examination, measurements of transepidermal water loss (TEWL), quantitative reverse transcription-polymerase chain reaction, Western blotting, and enzyme-linked immunosorbent assay. Our findings indicated that the oral intake of PN decreased the severity scores of lesions resembling AD, TEWL, and the levels of inflammatory cytokines and serum immunoglobulin E in Dfb-induced AD mice, along with a reduction in epidermal thickness and mast cell infiltration. Additionally, PN inhibited the activation of the mitogen-activated protein kinases (MAPKs) and the signal transducer and activator of transcription (STAT) pathways in Dfb-induced AD mice and HaCaT keratinocytes. Moreover, PN improved survival and reduced ROS production in H2O2-damaged HaCaT keratinocytes and enhanced the expression of antioxidant enzymes in Dfb-induced AD mice. Concludingly, the oral administration of PN suppressed inflammatory mediators and downregulated the MAPKs/STAT pathway, suggesting its potential role in AD pathogenesis.

RESUMEN

Dermatophagoides farina (D. farinae) and Dermatophagoides pteronyssinus (D. pteronyssinus) are the prevalent kinds of house dust mites (HDMs). HDMs are common inhalant allergens that cause a range of allergic diseases, such as rhinitis, atopic dermatitis, and asthma. The epidemiology of these diseases is associated with exposure to mites. Therefore, in the present study, a method named multiplex loop-mediated isothermal amplification (LAMP) was developed to detect environmental dust mites. The multiplex LAMP assay allows amplification within a single tube and has an ITS plasmid detection limit as low as 40 fg/µL for both single dust mites and mixed dust mites (D. pteronyssinus and D. farinae), which is up to ten times more sensitive than classical PCR techniques. Furthermore, the multiplex LAMP method was applied to samples of single dust mites and clinical dust to confirm its validity. The multiplex LAMP assay exhibited higher sensitivity, simpler instrumentation, and visualization of test results, indicating that this method could be used as an alternative to traditional techniques for the detection of HDMs.

Asunto(s)

Dermatophagoides farinae , Dermatophagoides pteronyssinus , Técnicas de Amplificación de Ácido Nucleico , Animales , Dermatophagoides pteronyssinus/genética , Dermatophagoides farinae/genética , Técnicas de Amplificación de Ácido Nucleico/métodos , Técnicas de Diagnóstico Molecular/métodos , Sensibilidad y Especificidad

RESUMEN

BACKGROUND: Dermatophagoides pteronyssinus and Dermatophagoides farinae belong to the family Pyroglyphidae (subfamily: "Dermatophagoidinae") and have the respective allergenic proteins of Der p1, Der p2, and Der p23 and Der f1 and Der f2. Euroglyphus maynei, belongs to the family Pyroglyphidae (subfamily: "Pyroglyphinae") and its main allergenic protein is Eur m1, a source of sensitization. Sensitization to D. pteronyssinus and D. farinae is assessed through skin tests, while sensitization to E. maynei is assessed less frequently. OBJECTIVE: This experimental work aims to analyze the prevalence of sensitization to E. maynei in patients with respiratory allergies treated at M. Albanesi Allergy and Immunology Unit in Bari, Italy, and the sequence homology of major allergenic proteins of E. maynei with D. farinae and D. pteronyssinus was analyzed. METHODS: In this real-life study, 65 patients were enrolled. In particular, patients with respiratory allergy were subjected to skin prick tests for common respiratory allergens, including Euroglyphus maynei. The sequence homology analysis was performed between the major allergenic proteins of E. maynei and those of D. pteronyssinus and D. farinae. RESULTS: Sensitization to E. maynei accounts for 41.5% of patients. All patients with E. maynei sensitization had concomitant sensitization to D. farinae and D. pteronyssinus. The analysis of sequence homology of Der p1 and Der f1 proteins with the sequence of Eur m1 protein demonstrated an identity of 84.4% and 86%, respectively. CONCLUSIONS: Nearly 50% of house dust mites-sensitized patients have a concomitant sensitization to E. maynei. The cross-sensitization could be due to Der f1, Der p1, and Eur m1 similarity.

Asunto(s)

Alérgenos , Antígenos Dermatofagoides , Biología Computacional , Hipersensibilidad Respiratoria , Pruebas Cutáneas , Humanos , Animales , Masculino , Antígenos Dermatofagoides/inmunología , Femenino , Adulto , Persona de Mediana Edad , Prevalencia , Hipersensibilidad Respiratoria/epidemiología , Hipersensibilidad Respiratoria/inmunología , Hipersensibilidad Respiratoria/diagnóstico , Italia/epidemiología , Alérgenos/inmunología , Pyroglyphidae/inmunología , Proteínas de Artrópodos/inmunología , Adulto Joven , Adolescente , Anciano

RESUMEN

The functions of highly expressed genes DFP1 and DFP2 in Dermatophagoides farinae remain unknown. DFP1 and DFP2 have been abundantly annotated and were up-regulated under temperature stress at 43 °C and -10 °C in our previous RNA-seq study, indicating that DFP1 and DFP2 may have temperature stress response function. Here, we amplified, cloned, and sequenced to obtain the complete coding sequences of DFP1 and DFP2 and predicted their protein characteristics using bioinformatics analysis. Then, prokaryotic expression systems were constructed and found that DFP1 was expressed in Escherichia coli Rosetta-gami 2 (DE3) but not BL21 (DE3); DFP2 was expressed in both BL21 (DE3) and Rosetta-gami 2 (DE3), with higher expression in BL21 (DE3). Finally, the growth curves of bacteria were drawn and indicated that the DFP1- and DFP2-pET32a carrying recombinant bacteria grew better than the respectiveonly pET32a carrying control bacteria after heat and cold stress. This study confirms for the first time that DFP1 and DFP2 respond to temperature stress at the protein level. The constructed prokaryotic expression systems will provide an experimental foundation for future antibody preparation for western blotting detection to confirm the temperature-stress response functions of DFP1 and DFP2.

Asunto(s)

Dermatophagoides farinae , Escherichia coli , Animales , Dermatophagoides farinae/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Estrés Fisiológico/genética , Clonación Molecular , Temperatura , Respuesta al Choque por Frío/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/genética , Respuesta al Choque Térmico/genética

RESUMEN

Atopic dermatitis (AD) is a chronic inflammatory skin disease characterized by skin barrier dysfunction and chronic inflammatory responses. Reynoutria japonica, known as Huzhang in traditional Chinese Medicine, can enhance blood circulation to eliminate wind pathogens and terminate coughing. Despite pharmacological evidence supporting the efficacy of R. japonica in suppressing edema-induced skin inflammation or connective tissue diseases, its pharmaceutical potential for treating AD-like skin inflammation remains unexplored. This study investigated the possible effects of R. japonica ethanol extract (RJE) on Dermatophagoides farinae extract (DfE)-induced AD-like skin inflammation in NC/Nga mice. To elucidate the underlying mechanisms by which RJE inhibits skin inflammation, we examined the effect of RJE on IFN-γ/TNF-α-induced signal transducer and activator of transcription (STAT) signaling in human epidermal keratinocytes (HEKs) and human dermal fibroblasts (HDFs). Our findings revealed that RJE mitigates DfE-induced AD-like symptoms and skin barrier disruptions in mouse skin lesions. Moreover, RJE attenuated DfE-induced mast cell infiltration and serum levels of inflammatory cytokines (IL-1α, IL-1ß, IL-6, IL-23, IFN-γ, TNF-α, and GM-CSF). RJE also inhibited IFN-γ/TNF-α-induced chemokine levels and STAT3 phosphorylation in HEKs and HDFs. Virtual binding analysis of the RJE components suggested that emodin-8-ß-D-glucoside binds to Janus kinase (JAK) 1/2, thereby suppressing STAT signaling, which was confirmed by Western blot analysis. In conclusion, our results suggest that RJE may alleviate DfE-induced skin barrier dysfunction by inhibiting JAK/STAT signaling and the proinflammatory immune response through the suppression of inflammatory mediators in AD-like skin disease. These findings suggest that RJE has potential as an effective therapy for AD management.

Asunto(s)

Dermatitis Atópica , Dermatophagoides farinae , Quinasas Janus , Factores de Transcripción STAT , Transducción de Señal , Animales , Dermatitis Atópica/tratamiento farmacológico , Dermatitis Atópica/patología , Dermatitis Atópica/inducido químicamente , Transducción de Señal/efectos de los fármacos , Ratones , Factores de Transcripción STAT/metabolismo , Quinasas Janus/metabolismo , Humanos , Glucósidos/farmacología , Citocinas/metabolismo , Masculino , Piel/efectos de los fármacos , Piel/patología , Piel/metabolismo , Emodina/farmacología , Emodina/análogos & derivados , Queratinocitos/efectos de los fármacos , Queratinocitos/metabolismo , Extractos Vegetales/farmacología , Inflamación/tratamiento farmacológico , Inflamación/metabolismo , Inflamación/patología

RESUMEN

Dermatophagoides farinae (Acari: Pyroglyphidae) has been reported as one of the major sources of indoor allergens that trigger allergic disease in humans. In this study, the genetic diversity and differentiation of nine geographic populations of D. farinae were investigated by analyzing mitochondrial and nuclear genes (COI, Cytb, COI+Cytb, and ITS). The results showed high genetic diversity across the D. farinae populations. The BX (Benxi) population showed the lowest genetic diversity, possibly due to climatic causes. Significant genetic differentiation was observed among D. farinae populations based on mitochondrial genes. The analysis of molecular variance (AMOVA) results elucidated that the contribution to the rate of variation was primarily from among populations. Phylogenetic analysis and haplotype network based on mitochondrial genes both indicated significant geographic structure among D. farinae populations. The nine geographic populations of D. farinae were divided into two groups with the Qinling Mountains-Huai River Line serving as the boundary for spatial analysis of molecular variance analysis (SAMOVA). However, the Mantel test analysis showed no association between genetic differentiation and geographic distance because of the high level of gene flow among some populations through the transportation of stored food. Overall, these results indicate both significant genetic differentiation among D. farinae populations, but also significant gene exchange between them. Results from the analysis of the nuclear gene ITS differed from the mitochondrial genes due to differences in molecular markers between mitochondrial genes and nuclear genes. These observations improve our understanding of the genetic diversity and structure of D. farinae populations.

Asunto(s)

Dermatophagoides farinae , Variación Genética , Animales , Dermatophagoides farinae/genética , Filogenia , China , Haplotipos , Proteínas de Artrópodos/genética , Filogeografía

RESUMEN

Background: Dermatophagoides farinae (DFA) is an important species of house dust mites (HDMs) that causes allergic diseases. Previous studies have focused on allergens with protein components to explain the allergic effect of HDMs; however, there is little knowledge on the role of microRNAs (miRNAs) in the allergic effect of HDMs. This study aimed to unravel the new mechanism of dust mite sensitization from the perspective of cross-species transport of extracellular vesicles-encapsulated miRNAs from HDMs. Methods: Small RNA (sRNA) sequencing was performed to detect miRNAs expression profiles from DFA, DFA-derived exosomes and DFA culture supernatants. A quantitative fluorescent real-time PCR (qPCR) assay was used to detect miRNAs expression in dust specimens. BEAS-2B cells endocytosed exosomes were modeled in vitro to detect miRNAs from DFA and the expression of related inflammatory factors. Representative dfa-miR-276-3p and dfa-novel-miR2 were transfected into BEAS-2B cells, and then differentially expressed genes (DEGs) were analyzed by RNA sequencing. Protein-protein interaction (PPI) network analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway and Gene Ontology (GO) terms enrichment analyses were performed on the first 300 nodes of DEGs. Results: sRNA sequencing identified 42 conserved miRNAs and 66 novel miRNAs in DFA, DFA-derived exosomes, and DFA culture supernatants. A homology analysis was performed on the top 18 conserved miRNAs with high expression levels. The presence of dust mites and miRNAs from HDMs in living environment were also validated. Following uptake of DFA-derived exosomes by BEAS-2B cells, exosomes transported miRNAs from DFA to target cells and produced pro-inflammatory effects in corresponding cells. RNA sequencing identified DEGs in dfa-miR-276-3p and dfa-novel-miR2 transfected BEAS-2B cells. GO and KEGG enrichment analyses revealed the role of exosomes with cross-species transporting of DFA miRNAs in inflammatory signaling pathways, such as JAK-STAT signaling pathway, PI3K/AKT signaling pathway and IL-6-mediated signaling pathway. Conclusion: Our findings demonstrate the miRNAs expression profiles in DFA for the first time. The DFA miRNAs are delivered into living environments via exosomes, and engulfed by human bronchial epithelial cells, and cross-species regulation may contribute to inflammation-related processes.

Asunto(s)

Exosomas , Hipersensibilidad , MicroARNs , Animales , Humanos , MicroARNs/genética , MicroARNs/metabolismo , Dermatophagoides farinae/genética , Dermatophagoides farinae/metabolismo , Exosomas/genética , Exosomas/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Células Epiteliales/metabolismo , Pyroglyphidae , Inflamación/genética , Inflamación/metabolismo , Hipersensibilidad/metabolismo , Alérgenos/metabolismo , Polvo , Expresión Génica

RESUMEN

Understanding the house dust mites (HDMs) microbiome is crucial due to its potential effects on the development of allergic diseases. In 1998, our laboratory collected Dermatophagoides farinae and D. pteronyssinus from beds in a Korean household and began cultivating these HDMs. Our laboratory has been actively investigating several topics about HDMs in recent years, including the bacterial and fungal microbiome and their interactions, as well as the impact of the HDM microbiome on airway inflammation. To study the D. farinae microbiome, we employed high-throughput sequencing of the 16S rDNA amplicons. The results revealed that the two most abundant bacteria were Enterococcus faecalis and Bartonella spp. In contrast, we found almost no bacteria in D. pteronyssinus. By inoculating bacteria to HDMs, we found that D. farinae is more susceptible to bacteria than D. pteronyssinus. This susceptibility was associated with the presence of certain fungal species in D. pteronyssinus. Additionally, we have recently made efforts to produce HDMs with reduced levels of symbiotic bacteria. We believe that standardizing and controlling the microbiome in HDMs are crucial steps for the future development and improvement of allergic immunotherapies.

RESUMEN

OBJECTIVE: To investigate the mechanisms underlying allergic conjunctivitis caused by conjunctival epithelial cell damage, neutrophil migration and neutrophil extracellular traps (NETs) formation induced by crude extracts of Dermatophagoides farinae mite (CDM). METHODS: Human conjunctival epithelial cells were stimulated with 500, 1 000, 2 000, 4 000 ng/mL, and the expression levels of interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ) and IL-8 were detected using quantitative real-time PCR (qPCR) assay and enzyme-linked immunosorbent assay (ELISA). The culture supernatant of human conjunctival epithelial cells was collected and co-cultured with neutrophils. Neutrophil migration was measured using Transwell migration assay, and the expression of NETs markers myeloperoxidase (MPO) and citrullinated histone H3 (CitH3) was quantified using immunofluorescence staining. Neutrophils were stimulated with phorbol 12-myristate 13-acetate (PMA), and then NETs were collected for treatment of human conjunctival epithelial cells. Cell apoptosis was detected using flow cytometry, and the levels of IL-6, TNF-α, IFN-γ and IL-8 were measured in the cell culture supernatant using ELISA. RESULTS: Treatment with CDM at concentrations of 2 000 ng/mL and 4 000 ng/mL up-regulated IL-6, TNF-α, IFN-γ and IL-8 expression in human conjunctival epithelial cells. Following treatment with CDM at concentrations of 2 000 ng/mL and 4 000 ng/mL, the culture supernatant of human conjunctival epithelial cells promoted neutrophil migration and induced increases in the staining intensity of MPO and CitH3. In addition, increased NETs triggered the apoptosis of human conjunctival epithelial cells and IL-6, TNF-α, IFN-γ and IL-8 secretion in the culture supernatant of human conjunctival epithelial cells. CONCLUSIONS: CDM induces human conjunctival epithelial cell damages, thereby promoting neutrophil migration and NETs formation, while the release of NETs further aggravates human conjunctival epithelial cell damages.

Asunto(s)

Trampas Extracelulares , Animales , Humanos , Neutrófilos , Interleucina-8/metabolismo , Dermatophagoides farinae , Interleucina-6/metabolismo , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/metabolismo , Células Epiteliales , Interferón gamma/metabolismo , Acetato de Tetradecanoilforbol/metabolismo , Acetato de Tetradecanoilforbol/farmacología

RESUMEN

House dust mites (HDMs) are a major source of indoor allergens that cause airway allergic disease. Dermatophagoides farinae, a predominant species of HDMs in China, has demonstrated pathogenic role in allergic disorders. Exosomes derived from human bronchoalveolar lavage fluid have been strongly associated with allergic respiratory diseases progression. However, the pathogenic role of D. farinae-derived exosomes in allergic airway inflammation has remained unclear until now. Here, D. farinae was stirred overnight in phosphate-buffered saline, and the supernatant was used to extract exosomes by ultracentrifugation. Then, shotgun liquid chromatography-tandem mass spectrometry and small RNA sequencing were performed to identify proteins and microRNAs contained in D. farinae exosomes. Immunoblotting, Western blotting, and enzyme-linked immunosorbent assay demonstrated the specific immunoreactivity of D. farinae-specific serum IgE antibody against D. farinae exosomes, and D. farinae exosomes were found to induce allergic airway inflammation in a mouse model. In addition, D. farinae exosomes invaded 16-HBE bronchial epithelial cells and NR8383 alveolar macrophages to release the inflammation-related cytokines interleukin-33 (IL-33), thymic stromal lymphopoietin, tumor necrosis factor alpha, and IL-6, and comparative transcriptomic analysis of 16-HBE and NR8383 cells revealed that immune pathways and immune cytokines/chemokines were involved in the sensitization of D. farinae exosomes. Taken together, our data demonstrate that D. farinae exosomes are immunogenic and may induce allergic airway inflammation via bronchial epithelial cells and alveolar macrophages. IMPORTANCE Dermatophagoides farinae, a predominant species of house dust mites in China, has displayed pathogenic role in allergic disorders, and exosomes derived from human bronchoalveolar lavage fluid have been strongly associated with allergic respiratory diseases progression. However, the pathogenic role of D. farinae-derived exosomes in allergic airway inflammation has remained unclear until now. This study, for the first time, extracted exosomes from D. farinae, and sequenced their protein cargo and microRNAs using shotgun liquid chromatography-tandem mass spectrometry and small RNA sequencing. D. farinae-derived exosomes trigger allergen-specific immune responses and present satisfactory immunogenicity, as revealed by immunoblotting, Western blotting, and enzyme-linked immunosorbent assay and may induce allergic airway inflammation via bronchial epithelial cells and alveolar macrophages. Our data provide insights into the mechanisms of allergic airway inflammation caused with D. farinae-derived exosomes and the treatment of house dust mite-induced allergic airway inflammation.

Asunto(s)

Exosomas , MicroARNs , Enfermedades Respiratorias , Animales , Ratones , Humanos , Dermatophagoides farinae/genética , Inflamación , Alérgenos/genética , Citocinas

RESUMEN

Atopic dermatitis and psoriasis are prevalent chronic inflammatory skin diseases that are characterized by dysfunctional skin barriers and substantially impact patients' quality of life. Vitamin D3 regulates immune responses and keratinocyte differentiation and improves psoriasis symptoms; however, its effects on atopic dermatitis remain unclear. Here, we investigated the effects of calcitriol, an active form of vitamin D3, on an NC/Nga mouse model of atopic dermatitis. We observed that the topical application of calcitriol decreased the dermatitis scores and epidermal thickness of NC/Nga mice with atopic dermatitis compared to untreated mice. In addition, both stratum corneum barrier function as assessed by the measurement of transepidermal water loss and tight junction barrier function as evaluated by biotin tracer permeability assay were improved following calcitriol treatment. Moreover, calcitriol treatment reversed the decrease in the expression of skin barrier-related proteins and decreased the expression of inflammatory cytokines such as interleukin (IL)-13 and IL-33 in mice with atopic dermatitis. These findings suggest that the topical application of calcitriol might improve the symptoms of atopic dermatitis by repairing the dysfunctional epidermal and tight junction barriers. Our results suggest that calcitriol might be a viable therapeutic agent for the treatment of atopic dermatitis in addition to psoriasis.

Asunto(s)

Dermatitis Atópica , Psoriasis , Ratones , Animales , Dermatitis Atópica/metabolismo , Calcitriol/uso terapéutico , Colecalciferol/farmacología , Calidad de Vida , Piel/metabolismo , Citocinas/metabolismo , Interleucina-13/metabolismo , Psoriasis/tratamiento farmacológico , Psoriasis/metabolismo , Modelos Animales de Enfermedad

RESUMEN

Dermatophagoides farinae is considered to be an important factor causing some allergic diseases, such as urticaria, allergic rhinitis, asthma, and other interrelated diseases. Avoiding exposure to allergens is the most effective way to reduce allergic reactions. In this study, we successfully established a loop-mediated isothermal amplification (LAMP) method for the detection of D. farinae DNA target internal transcribed spacer (ITS) and D. farinae 1 allergen (Der f 1) genes. The turbidity-monitoring system and visual fluorescent reagents were used to verify the test results of LAMP assay. Following optimization of the primers and reaction temperatures, the amplification sensitivity, specificity, and efficiency of the method for detecting D. farinae were assessed. There was no cross-reaction with other arthropod species that are commonly found in indoor environmental dust, such as Dermatophagoides pteronyssinus, Alophagoides ovatus, Periplaneta americana, Anopheles sinensis, and Musca domestica. Furthermore, the sensitivity of LAMP assay for detecting D. farinae DNA was 10 times greater than that of conventional PCR. The positive detection rate by the LAMP method was greater than the conventional PCR for both single D. farinae mites and D. farinae mites in indoor dust. A new type of LAMP method for D. farinae based on the Der f 1 and ITS genes was, therefore, successfully established. This study is the first time to detect the D. farinae allergen using LAMP assay. This assay could be useful as a model for the rapid detection of allergens produced by other house dust mites in the future.

Asunto(s)

Alérgenos , Rinitis Alérgica , Animales , Alérgenos/análisis , Polvo , Dermatophagoides farinae , ADN , Antígenos Dermatofagoides/análisis

RESUMEN

Receptor-interacting protein kinase (RIP) family 1 signaling has complex effects on inflammatory processes and cell death, but little is known concerning allergic skin diseases. We examined the role of RIP1 in Dermatophagoides farinae extract (DFE)-induced atopic dermatitis (AD)-like skin inflammation. RIP1 phosphorylation was increased in HKCs treated with DFE. Nectostatin-1, a selective and potent allosteric inhibitor of RIP1, inhibited AD-like skin inflammation and the expression of histamine, total IgE, DFE-specific IgE, IL-4, IL-5, and IL-13 in an AD-like mouse model. The expression of RIP1 was increased in ear skin tissue from a DFE-induced mouse model with AD-like skin lesions and in the lesional skin of AD patients with high house dust mite sensitization. The expression of IL-33 was down-regulated after RIP1 inhibition, and the levels of IL-33 were increased by over-expression of RIP1 in keratinocytes stimulated with DFE. Nectostatin-1 reduced IL-33 expression in vitro and in the DFE-induced mouse model. These results suggest that RIP1 can be one of the mediators that regulate IL-33-mediated atopic skin inflammation by house dust mites.

Asunto(s)

Dermatitis Atópica , Animales , Ratones , Antígenos Dermatofagoides , Citocinas/farmacología , Dermatitis Atópica/patología , Dermatophagoides farinae , Modelos Animales de Enfermedad , Inmunoglobulina E , Inflamación/patología , Interleucina-33/farmacología , Extractos Vegetales/farmacología , Pyroglyphidae , Piel/patología

RESUMEN

Dermatophagoides farinae is an important house dust mite species that causes allergies in humans worldwide. In houses, these mites are commonly found in actively used mattresses and pillows, which provide food (i.e. sloughed skin and microorganisms), moisture, and increased temperature for faster mite development. In mattresses, feeding mites prefer the upper sector, as close as possible to the resting human (temperature 32-36 °C, humidity between 55 and 59%). However, mites that are not actively feeding prefer staying at deeper zones of the mattress. Here, we analyzed mite responses to different temperatures (15-35 °C) and relative humidity (62-94% RH) in terms of their population size growth and respiration (CO2 production) using lab mite cultures. The intrinsic rate of population increase had a single maximum at approximately 28 °C and 85% RH. At 30 °C, there were two respiration peaks at RH 90% (smaller peak) and 65% (larger peak). Therefore, there is a mismatch between the optimal temperature/humidity for the population size increase vs. respiration. We propose preliminary hypotheses explaining the two respiration peaks and suggest that future research should be done to elucidate the nature of these peaks.

Asunto(s)

Dermatophagoides farinae , Crecimiento Demográfico , Humanos , Animales , Humedad , Temperatura , Dermatophagoides farinae/fisiología , Alérgenos , Polvo , Antígenos Dermatofagoides

RESUMEN

Objective To investigate the mechanisms underlying allergic conjunctivitis caused by conjunctival epithelial cell damage, neutrophil migration and neutrophil extracellular traps (NETs) formation induced by crude extracts of Dermatophagoides farinae mite (CDM). Methods Human conjunctival epithelial cells were stimulated with 500, 1 000, 2 000, 4 000 ng/mL, and the expression levels of interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ) and IL-8 were detected using quantitative real-time PCR (qPCR) assay and enzyme-linked immunosorbent assay (ELISA). The culture supernatant of human conjunctival epithelial cells was collected and co-cultured with neutrophils. Neutrophil migration was measured using Transwell migration assay, and the expression of NETs markers myeloperoxidase (MPO) and citrullinated histone H3 (CitH3) was quantified using immunofluorescence staining. Neutrophils were stimulated with phorbol 12-myristate 13-acetate (PMA), and then NETs were collected for treatment of human conjunctival epithelial cells. Cell apoptosis was detected using flow cytometry, and the levels of IL-6, TNF-α, IFN-γ and IL-8 were measured in the cell culture supernatant using ELISA. Results Treatment with CDM at concentrations of 2 000 ng/mL and 4 000 ng/mL up-regulated IL-6, TNF-α, IFN-γ and IL-8 expression in human conjunctival epithelial cells. Following treatment with CDM at concentrations of 2 000 ng/mL and 4 000 ng/mL, the culture supernatant of human conjunctival epithelial cells promoted neutrophil migration and induced increases in the staining intensity of MPO and CitH3. In addition, increased NETs triggered the apoptosis of human conjunctival epithelial cells and IL-6, TNF-α, IFN-γ and IL-8 secretion in the culture supernatant of human conjunctival epithelial cells. Conclusions CDM induces human conjunctival epithelial cell damages, thereby promoting neutrophil migration and NETs formation, while the release of NETs further aggravates human conjunctival epithelial cell damages.

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Objective@#To investigate the role of miR-142-3p in alleviation of house dust mite induced allergic airway inflammation among children, so as to provide insights into unraveling the pathogenesis of allergic airway inflammation.@*Methods@#Serum samples were collected from 15 patients with house dust mite induced allergic asthma and 15 healthy children in Jiangnan University Medical Center from September to November 2022, and serum miR-142-3p expression was quantified using a fluorescent quantitative real time PCR (qPCR) assay. The levels of interleukin 6 (IL 6) and tumor necrosis factor α (TNF α) were measured in the cell culture supernatant using enzyme linked immunosorbent assay (ELISA), and the expression of high mobility group box 1 (HMGB1) was detected at transcriptional and translational lvels using qPCR and Western blotting assays. The negative regulation of the HMGB1 gene by miR 142 3p was identified using a dual luciferase gene reporter assay, and the expression of downstream regulatory proteins was determined in human normal lung epithelial cells (BEAS 2B) cells transfected with miR 142 3p using Western blotting. In addition, female C57BL/6 mice at ages of 6-8 weeks were randomly assigned to the phosphate buffer saline (PBS) group, house dust mite sensitized airway inflammation group and house dust mite sensitized airway inflammation + miR 142 3p intervention group. Mouse airway inflammation was evaluated using hematoxylin eosin staining, and the expression of inflammatory cells and inflammatory cytokines were detected in mouse bronchoalveolar lavage fluid (BALF) using Giemsa staining and ELISA.@*Results@#Lower serum miR-142-3p expression was quantified among children with house dust mite induced allergic asthma than among healthy controls (1.33±0.21 vs. 4.74±0.62, t=5.22, P <0.05). Stimulation with dermatophagoides farinae extract (DFE) resulted in a reduction in miR-142-3p expression in BEAS-2B cells (0.82±0.25), while transfection with miR-142-3p mimics resulted in a rise in miR-142-3p expression in BEAS-2B cells (0.55±0.14)( t=3.31, 3.94, P <0.05). Pre treatment with miR-142-3p reduced the expression of IL 6(2.25±0.46)and TNF α(6.58±1.95) ( t=4.86, 3.38, P <0.05) in BEAS 2B cells stimulated with DFE, and treatment with miR-142-3p mimics resulted in a reduction in TLR4 and NF-κB expression in BEAS-2B cells via negative regulation of the HMGB1 expression. In addition, treatment with miR-142-3p was found to alleviate inflammatory cell infiltration in lung tissues of house dust mite sensitized mice, and results in a reduction in interleukin 4 (IL-4)[(107.60±10.43)pg/mL], interleukin 5 (IL 5)[(95.78±13.14)pg/mL] and HMGB1[(2.52±0.87)pg/mL] expression in BALF ( t=10.32, 7.29, 2.90, P <0.05).@*Conclusion@#miR-142-3p alleviates house dust mite induced allergic airway inflammation among children via negative regulation of the HMGB1/TLR4/NF-κB pathway.