Ovalbumin Stimulation Research Articles

Immunomodulation in allergic rhinitis: Insights from Th2 cells and NLRP3/IL-18pathway.

Allergic rhinitis (AR) is characterized by nasal symptoms such as rubbing and sneezing, often triggered by allergen exposure. The purpose of this study is to dissect the roles of NLRP3-mediated immune modulation and macrophage pyroptosis in modulating T cell differentiation within the context of ovalbumin (OVA)-induced AR in mice. OVA-induced AR was established in mice, evaluating nasal symptoms, macrophage infiltration, cytokine levels, and T cell differentiation. Manipulations using NLRP3-/-, ASC-/- mice, clodronate liposome treatment, and NLRP3 inhibitor MCC950 were performed to assess their impact on AR symptoms and immune responses. Following OVA stimulation, increased nasal symptoms were observed in the OVA group along with augmented GATA3 expression and elevated IL-4 and IL-1b levels, indicative of Th2 polarization and cellular pyroptosis involvement. NLRP3-/- and ASC-/- mice exhibited reduced CD3+ T cells post OVA induction, implicating cellular pyroptosis in AR. Macrophage depletion led to decreased IgE levels, highlighting their involvement in allergic responses. Further investigations revealed enhanced macrophage pyroptosis, influencing Th1/Th2 differentiation in AR models. IL-18 released through NLRP3-mediated pyroptosis induced Th2 differentiation, distinct from IL-1b. Additionally, MCC950 effectively mitigated AR symptoms by modulating Th2 responses and reducing macrophage infiltration. This comprehensive study unravels the pivotal role of NLRP3-mediated immune modulation and macrophage pyroptosis in Th1/Th2 balance regulation in OVA-induced AR. Targeting NLRP3 pathways with MCC950 emerged as a promising strategy to alleviate AR symptoms, providing insights for potential therapeutic interventions in AR management.

Effect and mechanism of Maxing Shigan Decoction on reducing inflammatory response in rats with cough variant asthma via TLR4/MyD88/NF-κB and p38 MAPK signaling pathways

This study aims to investigate the effect and mechanism of Maxingshigan Decoction on inflammation in the rat model of cough variant asthma(CVA). The SPF-grade SD rats of 6-8 weeks were randomized into normal, model, Montelukast sodium, and low-, medium-, and high-dose Maxing Shigan Decoction groups, with 8 rats in each group. The CVA rat model was induced by ovalbumin(OVA) and aluminum hydroxide sensitization and ovalbumin stimulation. The normal group and model group were administrated with equal volume of normal saline by gavage, and other groups with corresponding drugs by gavage. After the experiment, the number of white blood cells in blood and the levels of interleukin-6(IL-6), interleukin-10(IL-10), and tumor necrosis factor-α(TNF-α) in the serum were measured. The lung tissue was stained with hematoxylin-eosin(HE). Western blot was employed to determine the protein levels of nuclear factor-κB(NF-κB), Toll-like receptor 4(TLR4), myeloid differentiation protein(MyD88), and mitogen-activated protein kinase(MAPK) in the lung tissue. Real-time PCR was carried out to measure the mRNA levels of TLR4 and MyD88 in the lung tissue. Compared with the normal group, the model group showed increased white blood cells, elevated IL-6 and TNF-α levels(P<0.01), lowered IL-10 level(P<0.01), up-regulated protein levels of TLR4, MyD88, p-p65/NF-κB p65, and p-p38 MAPK/p38 MAPK(P<0.01) and mRNA levels of TLR4 and MyD88(P<0.01) in the lung tissue. HE staining showed obvious infiltration of inflammatory cells around the airway and cell disarrangement in the model group. Compared with the model group, Montelukast sodium and high-dose Maxing Shigan Decoction reduced the white blood cells, lowered the IL-6 and TNF-α levels(P<0.01), and elevated the IL-10 level(P<0.01). Moreover, they down-regulated the protein levels of TLR4, MyD88, p-p65/NF-κB p65, p-p38 MAPK/p38 MAPK in the lung tissue(P<0.01) and the mRNA levels of TLR4 and MyD88 in the lung tissue(P<0.01). HE staining showed that Montelukast sodium and high-dose Maxing Shigan Decoction reduced inflammatory cell infiltration and cell disarrangement. The number of white blood cells, the levels of IL-10 and TNF-α in the serum, the protein levels of TLR4, MyD88, p-p65/NF-κB p65, and p-p38 MAPK/p38 MAPK, and the mRNA levels of TLR4 and MyD88 in the lung tissue showed no significant differences between the Montelukast sodium group and high-dose Maxing Shigan Decoction group. Maxing Shigan Decoction can inhibit airway inflammation in CVA rats by inhibiting the activation of TLR4/MyD88/NF-κB and p38 MAPK signaling pathways.

NLRP3 Inflammasome Deficiency Alleviates Inflammation and Oxidative Stress by Promoting PINK1/Parkin-Mediated Mitophagy in Allergic Rhinitis Mice and Nasal Epithelial Cells.

Accumulating evidence indicates that oxidative stress and inflammation are the pathological basis of allergic diseases. Inhibition of NOD-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome could ameliorate allergic rhinitis (AR). Here, we explored the effects and mechanisms that underlie NLRP3 inhibition on oxidative stress and inflammation in AR. Ovalbumin (OVA)-induced AR murine model was established using wild-type (WT) and NLRP3-deficient mice. HNEpCs were stimulated with interleukin (IL)-13 with MCC950 pretreatment or PTEN-induced putative kinase 1 (PINK1) siRNA. The indicators of oxidative stress, inflammation, apoptosis, and mitophagy were determined both in vivo and in vitro. NLRP3 knockout (KO) reduced the frequency of nasal rubbing and sneezing, the infiltration of eosinophils, the number of mast cells, and the accumulation of goblet cells in AR mice after OVA stimulation. The NLRP3 KO AR mice exhibited the increased concentrations of OVA-specific immunoglobulin E (OVA-sIgE), IL-1β, IL-4, IL-13, IL-6, TNF-α, and the upregulated level of IFN-γ. NLRP3 KO significantly inhibited oxidative stress, and also markedly decreased apoptosis in the nasal mucosa of AR mice. Moreover, evaluated protein expressions of PINK1, enzyme 3 (E3) ubiquitin ligase PRKN (Parkin), and LC3 II, decreased expression of TOM20, as well as the increased colocalization of LC3 with mitochondria were observed in NLRP3 KO AR mice. In vitro, IL-13 exposure increased the levels of NLRP3 and IL-1β. Inhibition of NLRP3 using MCC950 enhanced PINK1/Parkin-mediated mitophagy but attenuated inflammation, oxidative stress, and apoptosis. However, PINK1 knockdown abrogated mitophagy and also reversed the protective effects of MCC950 on inflammation, oxidative stress, and apoptosis in HNEpCs stimulated with IL-13. Inhibition of NLRP3 inflammasome exerts the protective effects on AR by facilitating mitophagy regulated by PINK1/Parkin signaling pathway.

Mitochondrial dynamics modulate the allergic inflammation in a murine model of allergic rhinitis.

Allergic rhinitis (AR) is a common allergic disorder, afflicting thousands of human beings. Aberrant mitochondrial dynamics are important pathological elements for various immune cell dysfunctions and allergic diseases. However, the connection between mitochondrial dynamics and AR remains poorly understood. This study aimed to determine whether mitochondrial dynamics influence the inflammatory response in AR. In the present study, we established a murine model of AR by sensitization with ovalbumin (OVA). Then, we investigated the mitochondrial morphology in mice with AR by transmission electron microscopy and confocal fluorescence microscopy, and evaluated the role of Mdivi-1 (an inhibitor of mitochondrial fission) on allergic symptoms, inflammatory responses, allergic-related signals, and reactive oxygen species formation. There was a notable enhancement in mitochondrial fragmentation in the nasal mucosa of mice following OVA stimulation, whereas Mdivi-1 prevented aberrant mitochondrial morphology. Indeed, Mdivi-1 alleviated the rubbing and sneezing responses in OVA-sensitized mice. Compared with vehicle-treated ones, mice treated with Mdivi-1 exhibited a reduction in interleukin (IL)-4, IL-5, and specific IgE levels in both serum and nasal lavage fluid, and shown an amelioration in inflammatory response of nasal mucosa. Meanwhile, Mdivi-1 treatment was associated with a suppression in JAK2 and STAT6 activation and reactive oxygen species generation, which act as important signaling for allergic response. Our findings reveal mitochondrial dynamics modulate the allergic responses in AR. Mitochondrial dynamics may represent a promising target for the treatment of AR.

CTRP3 regulates NF-κB and TGFβ1/Smad3 pathways to alleviate airway inflammation and remodeling in asthmatic mice induced by OVA.

Asthma is a common illness with chronic airway inflammation. C1q/tumor necrosis factor (TNF)-related protein 3 (CTRP3) plays a vital role ininflammatory response, but its effect on asthma is imprecise. Herein, we analyzed the functions of CTRP3 in asthma. The BALB/c mice were randomized into four groups: control, ovalbumin (OVA), OVA+vector, and OVA+CTRP3. The asthmatic mice model was established by OVA stimulation. Overexpression of CTRP3 was implemented by the transfection of corresponding adeno-associated virus 6 (AAV6). The contents of CTRP3, E-cadherin, N-cadherin, smooth muscle alpha-actin (α-SMA), phosphorylated (p)-p65/p65, transforming growth factor-beta 1 (TGFβ1), and p-Smad3/Smad3 were determined by Western blot analysis. The quantity of total cells, eosinophils, neutrophils, and lymphocytes in bronchoalveolar lavage fluid (BALF) was assessed by using a hemocytometer. The contents of tumor necrosis factor-α and interleukin-1β in BALF were examined by enzyme-linked immunesorbent serologic assay. The lung function indicators and airway resistance (AWR) were measured. The bronchial and alveolar structures were evaluated by hematoxylin and eosin staining and sirius red staining. The CTRP3 was downregulated in mice of OVA groups; however, AAV6-CTRP3 treatment markedly upregulated the expression of CTRP3. Upregulation of CTRP3 diminished asthmatic airway inflammation by decreasing the number of inflammatory cells and the contents of proinflammatory factors. CTRP3 markedly lessened AWR and improved lung function in OVA-stimulated mice. Histological analysis found that CTRP3 alleviated OVA-induced airway remodeling in mice. Moreover, CTRP3 modulated NF-κB and TGFβ1/Smad3 pathways in OVA-stimulated mice. CTRP3 alleviated airway inflammation and remodeling in OVA-induced asthmatic mice via regulating NF-κB and TGFβ1/Smad3 pathways.

ADP Ribosylation Factor 6 Relieves Airway Inflammation and Remodeling by Inhibiting Ovalbumin Induced-Epithelial Mesenchymal Transition in Experimental Asthma, Possibly by Regulating of E2F Transcription Factor 8

ABSTRACT Background Childhood asthma is a major global health concern. ADP-ribosylation factor 6 (ARF6) is a low-molecular-weight GTPase; however, its role in childhood asthma remains unclear. Methods Ovalbumin (OVA)-challenged neonatal mice and transforming growth factor-β1 (TGF-β1)-induced BEAS-2B cells were used as in vivo and in vitro models of childhood asthma, respectively. Results Upon OVA stimulation, ARF6 expression was upregulated in the lung tissue. Neonatal mice administered SehinH3 (an ARF6 inhibitor) exhibited improved pulmonary pathological injury, along with reduced inflammatory cell infiltration in the lungs and cytokine release in bronchial alveolar lavage fluid and serum (interleukin [IL]−3, IL−5, IL−13, IgE, and OVA-specific IgE). SehinH3 treatment restrained epithelial – mesenchymal transition (EMT) in the lungs of asthmatic mice, as evidenced by increased E-cadherin and decreased N-cadherin and α-smooth muscle actin expression. Different TGF-β1 exposures to BEAS-2B cells induced a time- and dose-dependent increase in ARF6 expression in vitro. Upon TGF-β1 stimulation, ARF6 knockdown repressed EMT and SehinH3 treatment caused similar results in BEAS-2B cells. The transcription factor E2F8 is involved in diverse biological functions and its increased expression was confirmed in vivo and in vitro. Dual-luciferase assays confirmed that E2F8 binds to the ARF6 promoter and promotes its transcriptional activity. In vitro results revealed that E2F8 silencing suppressed EMT, whereas rescue experiments showed that ARF6 overexpression partly reversed these phenomena. Conclusion Our study showed that ARF6 is associated with childhood asthma progression and may be positively regulated by E2F8. These results provide insight into the pathogenesis and treatment of childhood asthma.

Inotodiol suppresses allergic inflammation in allergic rhinitis mice.

Inotodiol has been proven to have antitumor, antiviral, anti-inflammatory, and antiallergic properties. This study investigated the immunomodulatory capability of inotodiol in allergic rhinitis (AR) mice. Forty BALB/c mice were divided into four groups, 10 mice each: control (CON), AR with phosphate-buffered saline (PBS) treatment (AR), inotodiol treatment (AR+Ino), and dexamethasone treatment (AR+Dex). Episodes of sneezing and nose rubbing were counted. Cytokines in nasal lavage fluid (NLF) and immunoglobulin in blood serum were measured. Nasal mucosae from each group were used for protein, reverse transcriptase-polymerase chain reaction (RT-PCR), and histological analyses. Splenocytes were cultured for evaluation of cytokine production in each group. Symptoms of rubbing and sneezing improved in the group of AR+Ino and AR+Dex than in the AR. NLF in the AR+Ino and AR+Dex also showed a significant decrease in interleukin (IL)-5, IL-10, and IL-13 compared to the AR. In addition, the number of eosinophils, goblet cells, and mast cells were notably lower in the nasal mucosae of the AR+Ino and AR+Dex. IL-4 and IL-17A in the AR+Ino and AR+Dex groups were decreased compared to the AR. Chemokines related to mast cell degradation were also decreased in the AR+Ino and AR+Dex groups. Total immunoglobulin (Ig)E, specific IgE and ovalbumin (OVA)-specific IgG1, and histamine levels were also significantly lower in the AR+Ino and AR+Dex groups. IL-10 and IL-13 were notably increased in the splenocytes of the AR after OVA stimulation, whereas the other groups showed no change. These results indicate inotodiol can help suppress allergic responses by immunomodulation activities.

Chryseriol attenuates the progression of OVA-induced asthma in mice through NF-κκB/HIF-1αα and MAPK/STAT1 pathways

Asthma is a hackneyed chronic inflammatory disease of the airway. Chryseriol (CSR) is a kind of flavonoid, and has the effect of bronchiectasis, indicating its potential application for treating respiratory diseases. However, the functions of CSR in asthma have not been reported till now. The histopathologic changes of the lung tissues were assessed by hematoxylin and eosin staining. The cell apoptosis was identified through terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling assay. Total numbers of eosinophils, neutrophils, and macrophages were assessed under microscope. The levels of interleukin (IL)-1β, IL-4, IL-5, and IL-13 were detected by enzyme-linked-immunosorbent serologic assay. The airway hyper-responsiveness (AHR) was evaluated by the whole body plethysmography. The levels of methane dicarboxylic aldehyde, superoxide dismutase, glutathione S-transferase, and glutathione in lung homogenates were confirmed by using corresponding commercial kits. The protein expressions were examined by Western blot analysis. The ovalbumin (OVA) was utilized to establish asthma mouse model. At first, it was revealed that CSR treatment reduced lung injury in OVA-stimulated mice. Moreover, cell apoptosis was enhanced after OVA stimulation but was attenuated by CSR treatment. In addition, CSR treatment decreased the infiltration of inflammatory cells and the production of inflammatory factors in OVA-treated mice. Further investigations demonstrated that CSR treatment relieved AHR in OVA-stimulated mice. The oxidative stress was strengthened in OVA-treated mice, but these effects were relieved by CSR treatment. Lastly, it was discovered that CSR treatment retarded nuclear factor kappa B (NF-κB)/hypoxia-inducible factor 1 alpha (HIF-1α) and p38 mitogen-activated protein kinase (MAPK)/signal transducer and activator of transcription 1 (STAT1) pathways in OVA-triggered asthma mice. Our findings proved that CSR attenuated the progression of OVA-induced asthma in mice through inhibiting NF-κB/HIF-1α and MAPK/STAT1 pathways. This work might highlight the functions of CSR in the treatment of asthma.

The Lipid-Soluble Forms of Choline Enhance Ex Vivo Responses from the Gut-Associated Immune System in Young Female Rat Offspring

In humans, the development of gut-associated lymphoid tissue (GALT) occurs in the first years of life and can be influenced by diet. The objective of this study was to determine the effect of dietary choline on the development of gut-associated lymphoid tissue (GALT). Three feeding trials were conducted in female Sprague-Dawley rats. Beginning 3 d before parturition (studies 1 and 3) or at day 10 of gestation (study 2), control dams consumed a 100% free choline (FC) diet until the end of the lactation period. In studies 1 and 3, test dams consumed a high-glycerophosphocholine (HGPC) diet [75% glycerophosphocholine (GPC), 12.5% phosphatidylcholine (PC), 12.5% FC] and a 100% PC diet, respectively (both 1 g of choline/kg diet). In study 2, test dams consumed a high-sphingomyelin (SM) and PC (SMPC) diet (34% SM, 37% PC, 17% GPC, 7% FC, 5% phosphocholine) or a 50% PC diet (50% PC, 25% FC, 25% GPC), both 1.7 g of choline/kg diet. Immune cell phenotypes and ex vivo cytokine production by mitogen-stimulated immune cells were measured. Feeding of the HGPC diet lowered T-cell IL-2 (44%), IFN-γ (34%), and TNF-α (55%) production in mesenteric lymph nodes (MLNs) compared with control. Feeding both SMPC and 50% PC diets during the lactation and weaning periods increased IL-2 (54%) and TNF-α (46%) production after T-cell stimulation compared with control. There was a lower production of IL-2 (46%), IL-6 (66%), and TNF-α (45%), and a higher production of IL-10 (44%) in both SMPC and 50% PC groups following ovalbumin stimulation compared with control in MLNs. Feeding a diet containing 100% PC increased the production of IFN-γ by 52% after T-cell stimulation compared with control. Feeding a diet containing a mixture of choline forms with a high content of lipid-soluble forms during both the lactation and weaning periods enhances ex vivo immune responses from the GALT in female Sprague-Dawley offspring.

Nonylphenol exacerbates ovalbumin-induced allergic rhinitis via the TSLP-TSLPR/IL-7R pathway and JAK1/2-STAT3 signaling in a mouse model

Nonylphenol (NP) can be widely used as a plasticizer, surfactant, antioxidant, textile printing, dyeing additive, and pesticide emulsifier. Animal studies have shown that NP aggravates ovalbumin (OVA)-induced allergic rhinitis (AR); however, the exact mechanism underlying its action has not yet been detailed. This study aimed to explore the aggravation of the AR inflammatory response following NP exposure and its possible mechanism. The AR mouse model was constructed using OVA. Under NP exposure, allergic nasal symptoms were observed, eosinophil infiltration was assessed by Sirius red staining, and the levels of IL-4, IL-5, and IL-13 in nasal mucosa samples were detected using cytometric bead array. The mRNA levels of OX40/OX40L and GATA3 in nasal mucosa were detected by qPCR, and the expression levels of the TSLP and JAK1/2-STAT3 signaling pathway components were also identified. Our results suggest that NP exposure exacerbated allergic nasal symptoms and that eosinophils accumulated in nasal mucosa after OVA challenge. The levels of the typical T helper 2 cytokines, as well as the mRNA levels of OX40/OX40L and GATA3, were elevated in the nasal mucosa of OVA-challenged mice exposed to NP. In addition, NP exposure elevated the TSLP, TSLPR, IL-7R, p-JAK1, p-JAK2, and p-STAT3 levels in the nasal mucosa after OVA stimulation. Overall, the present study suggests NP can exacerbate OVA-induced AR inflammatory responses; furthermore, this aggravating effect of NP may be related to the TSLP-TSLPR/IL-7R and JAK1/2-STAT3 signaling pathways.

CD226 Deficiency Alleviates Murine Allergic Rhinitis by Suppressing Group 2 Innate Lymphoid Cell Responses.

Allergic rhinitis (AR) is an immunoglobulin E-mediated type 2 inflammation of the nasal mucosa that is mainly driven by type 2 helper T cells (Th2) and type 2 innate lymphoid cells (ILC2s). CD226 is a costimulatory molecule associated with inflammatory response and is mainly expressed on T cells, natural killer cells, and monocytes. This study is aimed at elucidating the role of CD226 in allergic inflammatory responses in murine AR using global and CD4+ T cell-specific Cd226 knockout (KO) mice. AR nasal symptoms were assessed based on the frequency of nose rubbing and sneezing. Hematoxylin and eosin and periodic acid–Schiff staining and quantitative real-time PCR methods were used to determine eosinophils, goblet cells, and ILC2-associated mRNA levels in the nasal tissues of mice. CD226 levels on ILC2s were detected using flow cytometry, and an immunofluorescence double staining assay was employed to determine the number of ILC2s in the nasal mucosa. The results showed that global Cd226 KO mice, but not CD4+ T cell-specific Cd226 KO mice, exhibited attenuated AR nasal symptoms. Eosinophil recruitment, goblet cell proliferation, and Th2-inflammatory cytokines were significantly reduced, which resulted in the alleviation of allergic and inflammatory responses. ILC2s in the murine nasal mucosa expressed higher levels of CD226 after ovalbumin stimulation, and CD226 deficiency led to a reduction in the proportion of nasal ILC2s and ILC2-related inflammatory gene expression. Hence, the effect of CD226 on the AR mouse model may involve the regulation of ILC2 function rather than CD4+ T cells.

MiR-146a-5p Attenuates Allergic Airway Inflammation by Inhibiting the NLRP3 Inflammasome Activation in Macrophages

Objective: Asthma is a common inflammatory respiratory disease with increasing incidence worldwide. This study aimed to investigate the mechanism of miR-146a-5p in reducing allergic airway inflammation by inhibiting NLRP3 inflammasome activation in macrophages. Methods: Allergic mouse models were established by ovalbumin stimulation, and mice were treated with miR-146a-5p agomir and oe-TIRAP 3 h before OVA stimulation. The pathological changes of lung tissues were observed by hematoxylin-eosin staining. The airway hyperresponsiveness of mice were examined. The miR-146a-5p level was detected by RT-qPCR. The inflammatory cytokines (IL-18/TNF-α) and anti-inflammatory cytokine IL-10 levels in bronchoalveolar lavage fluid and serum IgE levels were examined by ELISA. Airway inflammation in mice was detected after miR-146a-5p overexpression. The levels of NLRP3/ASC/caspase1 proteins and macrophage M1/M2 surface markers in mouse lung tissues were examined using immunohistochemistry, Western blot, and flow cytometry. The targeting relationship between miR-146a-5p and TIRAP was verified by dual-luciferase assay. The p65 levels in the cytoplasm/nucleus of mouse lung tissue were measured. Results: miR-146a-5p was downregulated in the lung tissues of allergic mice, and miR-146a-5p overexpression alleviated airway inflammation in asthmatic mice. miR-146a-5p suppressed NLRP3 inflammasome activation in macrophages of allergic mice, reduced NLRP3/ASC/caspase1 protein levels in lung tissues, blocked M1 polarization, and promoted M2 polarization. miR-146a-5p targeted TIRAP. TIRAP overexpression partially reversed the promoting effect of miR-146a-5p on M2 polarization. miR-146a-5p can inhibit the activation of the TIRAP/NF-κB pathway. Conclusion: miR-146a-5p inhibited NLRP3 inflammasome activation in macrophages in the lung tissue of allergic mice, prevented pro-inflammatory phenotype M1 polarization, and promoted anti-inflammatory phenotype M2 polarization by targeting the TIRAP/NF-κB pathway, thus alleviating airway inflammation in allergic asthma.

MicroRNA-214-3p facilitates M2 macrophage polarization by targeting GSK3B.

Allergic rhinitis (AR) is a chronic inflammatory disease of the nasal mucosa. M2 macrophage polarization can reduce inflammation and repair tissue injury during AR development. Studies have substantiated the involvement of miRNAs in AR pathogenesis. Herein, the molecular mechanism of miR-214-3p in AR development was explored. To mimic the AR environment, ovalbumin (OVA) was used to treat macrophages. MiR-214-3p and glycogen synthase kinase 3 beta (GSK3B) expression in nasal mucus tissues and macrophages was assessed by RT-qPCR. The M2 phenotypic signature of CD206 in macrophages was assessed by flow cytometry. The protein expression of GSK3B and M2 macrophage markers (ARG-1 and IL-10) was evaluated by western blotting. The correlation between miR-214-3p and GSK3B was validated by a luciferase reporter assay. We found that miR-214-3p was overexpressed in macrophages and nasal mucus tissues from AR patients. MiR-214-3p facilitated M2 polarization of macrophages upon OVA stimulation. Mechanistically, miR-214-3p targeted the GSK3B 3' untranslated region in macrophages. In addition, GSK3B was downregulated in macrophages and nasal mucus tissues from AR patients. In rescue assays, GSK3B downregulation reversed the inhibitory effects of miR-214-3p silencing on M2 polarization of macrophages treated with OVA. Overall, miR-214-3p facilitates M2 macrophage polarization by targeting GSK3B.

Bruceine D ameliorates the balance of Th1/Th2 in a mouse model of ovalbumin-induced allergic asthma via inhibiting the NOTCH pathway.

Allergic asthma is a heterogeneous inflammatory disorder triggered by inhaled allergens, leading to airflow obstruction, bronchial inflammation, and airway hyperresponsiveness (AHR). T helper (Th) 2 cell-mediated immune response and airway inflammation are the key features of allergic asthma. Bruceine D (BD) is a bioactive compound extracted from the seeds of Brucea javanica. The present study aimed to investigate the effects of increased doses of BD on AHR, secretion of Th1-/Th2-associated cytokines, and inflammatory cell infiltration in ovalbumin (OVA)-induced allergic asthma mice. The results showed that BD reduced OVA-induced inflammatory cell infiltration and bronchial hyperresponsiveness into the peribronchial tissues and perivascular areas. Mice treated with BD also showed significantly decreased expressions of Th2-associated cytokines (i.e., interleukin (IL)-4, IL-5, and IL-13) and elevated production of Th1-associated cytokines (i.e., interferon gamma and IL-2) following OVA stimulation. BD treatment dose-dependently inhibited OVA-induced accumulation of inflammatory cells in asthmatic mice. Further analysis revealed that OVA exposure upregulated pulmonary expressions of NOTCH signaling receptors, a group of transmembrane proteins that communicate signals upon binding to transmembrane ligands expressed on adjacent cells, while BD treatment significantly abolished OVA-induced activation of the NOTCH pathway. In conclusion, BD protected mice against OVA-induced allergic asthma by reducing AHR and restoring the Th1/Th2 balance through the NOTCH signaling pathway. Our findings highlighted the potential of BD as a therapeutic agent for allergic asthma.

A Diet High in Lipid Soluble Forms of Choline Modulates Gut-Associated Immune Function in Sprague-Dawley Dams

ObjectivesOral tolerance is the state of unresponsiveness to dietary antigens that are not harmful to the host, a process that begins in the gut and influences both local and peripheral immune responses. Lipid soluble forms of choline have been shown to beneficial affect peripheral immune responses during pregnancy and lactation, yet little is known about the impact of the different choline forms on the gut-associate lymphoid tissue (GALT). Therefore, the objective of this study was to determine the effect of feeding buttermilk-derived choline forms on the GALT in lactating dams. MethodsSprague-Dawley dams were randomized to consume one of three diets, all containing 1.9 g/kg of total choline: 1-Control (100% free choline (FC)), 2-Buttermilk (37% phosphatidylcholine (PC), 34% sphingomyelin, 17% glycerophosphocholine (GPC), 7% FC, 5% phosphocholine) and 3-Placebo (50% PC, 25% FC, 25% GPC). Immune cell phenotypes of mesenteric lymph nodes (MLN) and Peyer’s patches (PP) were measured by flow cytometry. Ex vivo cytokine production by immune cells isolated from MLN stimulated with Concanavalin A (ConA) and Ovalbumin (OVA) was measured by ELISA. ResultsIn MLN, dams from the buttermilk and placebo groups had a higher production of IL-10 after both ConA and OVA stimulation and a higher proportion of helper T cells expressing co-inhibitory marker (CD4+CD152+) when compared to the control diet (both P < 0.05). No changes were found in IL-2, TNF-α, or IL-6 production and the proportion of regulatory T cells (CD3+CD4+CD25+FoxP3+) among groups. In PP, dams from the buttermilk group had a higher proportion of CD45RA + cells (B cells) compared to both the placebo and control diets and dams from the placebo diet had a higher proportion of dendritic cells (DC, OX62+OX6+) compared to both the buttermilk and control diets (both P < 0.05). ConclusionsCompared to the control diet (only FC), the buttermilk and placebo diets appear to beneficially modulate oral tolerance by increasing the production of IL-10 (an important mediator) and could be partially explain by the higher proportion of CD4+CD152+cells in MLN. In PP, the higher proportion of B cells in the buttermilk and DC in the placebo groups, suggests that dams from these groups may have better antigen presentation function, which may further assist the induction of oral tolerance. Funding SourcesDairy Farmers of Canada, NSERC, CONACYT.

Immunomodulative Effects of Chamaecyparis obtusa Essential Oil in Mouse Model of Allergic Rhinitis.

The present study aims to investigate the immunomodulatory effects of essential oil from Chamaecyparis obtusa (EOCO) in an ovalbumin (OVA)-induced allergic rhinitis (AR) mouse model. BALB/c mice were intraperitoneally sensitized and stimulated with OVA. From day 22 to 35, 0.01% and 0.1% ECOC was intranasally administered 1 h before OVA stimulation. Nasal symptoms, as well as serum total and OVA-specific immunoglobulin (Ig) E levels, were measured. Interleukin (IL)-4, IL-10, interferon (IFN)-γ, and tumor necrosis factor (TNF)-α levels in nasal lavage fluid (NLF) and their production by activated splenocytes were measured. Histological changes in the sinonasal mucosa were evaluated through hematoxylin and eosin and periodic acid-Schiff staining procedure. Th cytokines and their transcription factor mRNA expressions were determined using reverse-transcription polymerase chain reaction. Intranasal EOCO administration significantly suppressed allergic symptoms, OVA-specific IgE level, sinonasal mucosal inflammatory cell infiltration, and mucus-producing periodic acid-Schiff (PAS) positive cell count. EOCO also significantly inhibited IL-4, IL-10, and TNF-α levels in NLF and activated splenocytes. Th2 and Treg related cytokines and their transcription factors in sinonasal mucosa were significantly suppressed through intransal EOCO instillation. In conclusion, repetitive EOCO intranasal instillation showed anti-inflammatory and anti-allergic effects by suppressing nasal symptoms and inhibiting the production and expression of inflammatory mediators in the OVA-induced AR mouse model.

Low-Dose LPS Induces Tolerogenic Treg Skewing in Asthma

The mechanism(s) underlying endotoxin tolerance in asthma remain elusive. As the endotoxin lipopolysaccharide (LPS) affects the expression of the regulatory T-cell (Treg)-suppressive glucocorticoid-induced tumor necrosis factor receptor ligand (GITRL) on antigen-presenting dendritic cells (DCs), we hypothesized that LPS-induced changes in DC GITRL expression may impact Treg-mediated T-helper (Th) cell suppression and the induction of endotoxin tolerance. Here, we propose a novel mechanism by which low-dose LPS inhalation in neonatal mice induces endotoxin tolerance, thereby offering protection from later asthma development. Three-day old wild-type and Toll-like receptor 4 (TLR4)-deficient neonatal mice were exposed to low-dose LPS (1 μg) intranasally for 10 consecutive days prior to ovalbumin (OVA)-induced asthma to better understand the tolerogenic mechanism(s) of low-dose LPS pre-exposure. In vivo findings were validated using in vitro co-culturing studies of primary CD11c+ DCs and CD4+ T-cells with or without low-dose LPS pre-exposure before OVA stimulation. Low-dose LPS pre-exposure upregulated the Treg response and downregulated pathogenic Th2 and Th17 responses through promoting apoptosis of Th2 and Th17 cells. Low-dose LPS pre-exposure downregulated DC GITRL expression and T-cell GITR expression. Artificial DC GITRL expression abrogated the tolerogenic Treg-skewing effect of low-dose LPS pre-exposure. Low-dose LPS pre-exposure inhibited TRIF/IRF3/IFNβ signaling and upregulated expression of tolerogenic TRIF/IRF3/IFNβ negative regulators in a TLR4-dependent manner. This tolerogenic DC GITRL downregulation was attributable to TRIF/IRF3/IFNβ signaling inhibition. Low-dose LPS pre-exposure produces tolerogenic Treg skewing in neonatal asthmatic mice, a phenomenon attributable to TLR4-dependent TRIF/IRF3/IFNβ-mediated DC GITRL downregulation.

CCR5 attenuates neutrophilic airway inflammation exacerbated by infection with rhinovirus

Human rhinovirus (hRV) is the most common cause of asthma exacerbation characterized by clinical and pathophysiological heterogeneity. Steroid-sensitive, Th2 type-eosinophilic asthma has been somewhat studied, but hRV-induced neutrophilic asthma exacerbation is poorly understood. Here, CCR5 was found to play a role in attenuating neutrophilic airway inflammation in hRV-induced asthma exacerbation using chicken ovalbumin (OVA)-based model. CCR5 deficiency resulted in exacerbated neutrophilic asthmatic responses in airways following hRV infection. CCR5-deficient mice showed enhanced mucus expression and altered expression of tight junction proteins in lung tissues. CCR5-deficient mice were also manifested with influx of CD45+CD11b+Siglec-F+Gr-1+ neutrophils, along with enhanced production of IL-17A, IFN-γ, IL-6, IL-1β cytokines in inflamed tissues. In contrast, CCR5-deficient mice elicited down-regulation of Th2-related cytokine proteins following hRV infection. More interestingly, the lack of CCR5 altered the equilibrium of CD4+FoxP3+ Tregs and IL-17+CD4+ Th17 in inflamed tissues. CCR5-deficient mice showed increased frequency and absolute number of IL-17-producing CD4+ Th17 cells in lung tissues compared to wild-type mice, whereas the reduced infiltration of CD4+FoxP3+ Treg cells was observed. CCR5 deficiency resulted in the skewed production of Th17 and Th1 cytokines in lymph nodes and lungs upon OVA stimulation. Likewise, CCR5-deficient mice showed enhanced expression of Th17-inducing cytokines (IL-1β, IL-6, and TNF-α) in lung tissues. These results imply that CCR5 deficiency facilitates Th17 airway inflammation during hRV-induced asthma exacerbation, along with suppressing Th2 responses. Furthermore, our results suggest that CCR5 attenuates hRV-induced neutrophilic airway inflammation through conserving the equilibrium of CD4+Foxp3+ Treg cells and IL-17+CD4+ Th17 cells in hRV-induced asthma exacerbation.

PF356 CHRONIC LYMPHOCYTIC LEUKEMIA INDUCES SKEWING OF (ANTIGEN‐SPECIFIC) T CELL RESPONSES TOWARDS A DYSFUNCTIONAL SHORT‐LIVED EFFECTOR PHENOTYPE

Background:In recent years, CAR T cells showed to be highly effective in B cell malignancies such as acute lymphocytic leukemia. However, for chronic lymphocytic leukemia (CLL) efficacy of autologous T cell therapies is rather limited. T cells of CLL patients have a dysfunctional, terminally differentiated phenotype, which might be linked with the lack of success of T cell therapies. Recently it was shown that presence of memory T cell subsets in the CAR T cell infusion product associated with complete responses in CLL (Fraietta et al., 2018).Aims:It remains to be elucidated how CLL cells influence T cell skewing and whether this affects T cell responses in vivo. In this context, we functionally investigated acute T cell responses in the TCL1 mouse model, which features similar T cell dysfunction as observed in CLL patients (Gorgun et al., 2009). We applied a set‐up that allows to study the response against acute infection of (1) endogenous CD8+ T cells, which have been exposed to leukemic cells during CLL development, and (2) the antigen‐specific response of naïve, transferred OT‐I T cells.Methods:C57BL/6J (CD45.2+) mice were injected i.p. with 20x106 TCL1 splenocytes or PBS. When CD5+ leukemia reached 70% cells in blood, 50.000 naïve ovalbumin‐specific CD8+ T cells (OT‐I, CD45.1+) were injected i.v. Next, all mice were infected with mCMV‐OVA and sacrificed 7 days later. Splenocytes were analyzed directly or after in vitro ovalbumin stimulation by FACS.Results:(1) Already prior to infection, we found a skewing towards antigen‐experienced cells in the endogenous CD8+ compartment of leukemic mice. After mCMV‐OVA infection, a population of naïve CD8+ T cells persisted in control mice, but in leukemic mice the complete CD8+ compartment differentiated into effector cells. Within the effector compartment, we found a skewing to the short‐lived effector cell (SLEC) phenotype in the leukemic mice, while memory precursor effector cells (MPEC) were reduced. This skewing towards SLEC associated with increased effector‐associated T‐bet expression.(2) Analysis of the OT‐I cells allowed to perform detailed study of the impact of leukemia on an antigen‐specific response. Seven days after infection, spleens of leukemic and control mice contained similar frequencies of differentiated OT‐I cells, that had an effector phenotype. Also here, TCL1‐derived OT‐I cells showed a decreased percentage of MPEC, while SLECs were increased (Figure 1), indicating impaired memory formation. In correlation with skewing towards SLECs in leukemic mice, these OT‐I cells showed reduced expression of memory‐associated transcription factor Bcl6, while T‐bet was also increased. Earlier it was shown that Bcl6 is able to repress glycolysis, essential for T cell effector function (Oestreich et al., 2014). In line with reduced Bcl6 expression, we found increased glucose uptake in TCL1‐derived OT‐I cells. Despite the skewing towards SLEC phenotype, the TCL1‐derived OT‐I cells showed decreased production of IFNγ and TNFα and decreased degranulation (CD107a expression) upon in vitro restimulation.Summary/Conclusion:In this murine model of CLL, both bystander T cells as well as acute antigen‐specific T cell responses are driven towards an SLEC phenotype with signs of functional impairment, and metabolic alterations. This approach may provide crucial insight into the interplay between CLL and T cells, and the underlying failure of cancer immune surveillance.image

Correction: Notch Ligand Delta-Like 4-Pretreated Dendritic Cells Alleviate Allergic Airway Responses by Enhancing IL-10 Production.

[This corrects the article DOI: 10.1371/journal.pone.0063613.].