Th2-mediated Immune Responses Research Articles

Vascular and immunopathological role of Asymmetric Dimethylarginine (ADMA) in Experimental Autoimmune Encephalomyelitis.

Asymmetric dimethylarginine (ADMA) is an endogenous nitric oxide synthase (NOS) inhibitor/uncoupler inducing vascular pathology. Vascular pathology is an important factor for the development and progression of CNS pathology of MS, yet the role of ADMA in MS remains elusive. Patients with multiple sclerosis (MS) are reported to have elevated blood levels of ADMA, and mice with experimental autoimmune encephalomyelitis (EAE, an animal model of MS) generated by auto-immunization of myelin oligodendrocyte glycoprotein (MOG) and blood-brain barrier (BBB) disruption by pertussis toxin also had increased blood ADMA levels in parallel with induction of clinical disease. To explore the role of ADMA in EAE pathogenesis, EAE mice were treated with a daily dose of ADMA. It is of special interest that ADMA treatment enhanced the BBB disruption in EAE mice and exacerbated the clinical and CNS disease of EAE. ADMA treatment also induced the BBB disruption and EAE disease in MOG-immunized mice even without pertussis toxin treatment, suggesting the role of ADMA in BBB dysfunction in EAE. T-cell polarization studies also documented that ADMA treatment promotes TH 1- and TH 17-mediated immune responses but without affecting Treg-mediated immune response in EAE mice as well as in in vitro T-cell culture. Taken together, these data, for the first time, document the vascular and immunopathogenic roles of ADMA in EAE, thus pointing to the potential of ADMA-mediated mechanism as a new target of potential therapy for MS.

Immune gene expression in cyprinid herpesvirus-2 (CyHV-2)-sensitized peripheral blood leukocytes (PBLs) co-cultured with CyHV-2-infected goldfish fin cell line.

Goldfish is one of the preferred ornamental fish which is highly susceptible to cyprinid herpesvirus-2 (CyHV-2) infection. The present study aimed to analyse immune gene expression in a co-culture of CyHV-2-sensitized goldfish peripheral blood leukocytes (PBLs) with CyHV-2-infected fantail goldfish fin cell lines (FtGF). Goldfish were sensitized with intraperitoneal TCID50 dose (107.8±0.26/mL) of CyHV-2. After 2 weeks, PBLs were collected and co-cultured with CyHV-2-infected FtGF cells keeping both uninfected FtGF cells and PBL control groups. After 2 days of co-culture, WST-1 assay for cell proliferation was performed at 450 nm during the 2nd, 4th and 6th days of co-culture. The results showed a significant increase (p < 0.05) in cell density in CyHV-2-infected PBL and virus-infected FtGF cells during the 4th day post co-culture which confirmed effector cell generation. Expressions of few immune genes were checked taking RNA samples of CyHV-2-induced PBLs post co-culture with infected FtGF cells along with uninfected FtGF cells as control group at different time periods (2nd, 4th and 6th days) in triplicate. The results indicated increased expression of CD8α, IFNγ, b2m, MHC I, LMP 7, IL-10, IL-12 and GATA3 except Tapasin. From the above study, we concluded that goldfish showed both Th1- and Th2-mediated immune responses to CyHV-2. The current findings support the scope for further vaccine development against CyHV-2 for goldfish.

Macrophage-like iPS-derived Suppressor Cells Reduce Th1-mediated Immune Response to a Retinal Antigen

ABSTRACT Purpose To investigate the immunotherapeutic effects of macrophage-like induced pluripotent stem (iPS) cell-derived suppressor cells (SCs) in ocular immune response and experimental autoimmune uveoretinitis (EAU). Methods The genes of Oct3/4, Sox2, Klf4, and c-Myc were transferred to B cells enriched from the spleen cells of C57BL/6 mice by using retrovirus vectors. Transferred B cells were cultured for 17 days to obtain colonies of iPS cells. Through additional steps, iPS-SCs were induced. An antigen-specific T cell proliferation assay was performed with CD4+ T cells collected from draining lymph nodes of the mice immunized with human interphotoreceptor retinoid-binding protein (hIRBP) peptide and co-cultured with iPS-SCs. Cytokine concentrations in the culture supernatant were examined. Mice were immunized with hIRBP peptide to induce EAU. The iPS-SCs were administered into the mice one day before the induction of EAU. Results The iPS-SCs decreased hIRBP-specific T cell proliferation depending on the number of cells. Productions of tumor necrosis factor-α and interferon-γ were significantly decreased; however, transforming growth factor-β1, nitric oxide, interleukin (IL)-13, IL-17A, and IL-17 F levels were elevated in the supernatant when the collected T cells were co-cultured with iPS-SCs. The iPS-SCs had immunosuppressant effects even without cell-to-cell contact, and their effects were non-specific to the antigen preloaded on iPS-SCs. EAU was significantly milder in the mice administered iPS-SCs prior to immunization. Conclusions Macrophage-like iPS-SCs reduced Th1 immune response to a retinal antigen and Th1-mediated EAU in mice. These results showed the possibility of the application of iPS technology to the treatment of noninfectious ocular inflammation, endogenous uveitis, in the future.

The Effect of Immunosuppressive Adjuvant Kynurenine on Type 1 Diabetes Vaccine

Inducing antigen-specific tolerance is a promising treatment for preventing or reversing Type 1 diabetes (T1D). In contrast to a vaccine that induces immune responses against pathogens, a tolerogenic vaccine can suppress immunity against antigens causing diseases by administrating a mixture of self-antigens with an adjuvant that decreases the strength of antigen-specific response. Kynurenine (Kyn) is an endogenous substance that can inhibit the natural killer cell and T cell proliferation and promote the differentiation of naïve T cells into regulatory T cells (Tregs). In this study, we evaluated the efficacy of Kyn as a novel suppressive adjuvant. Kyn was co-immunized with GAD65 phage vaccine to induce Treg cells and tolerogenic responses for the prevention of T1D in NOD mouse model. Mice were subcutaneously immunized two times with 1011 Pfu (100μL,1012 Pfu/ml) GAD65 phage vaccine doses mixed with 200 μg of Kyn. Serum antibodies and cytokines were detected by ELISA and electrochemiluminescence, respectively. Flow cytometry assay was used to analyze DC and Treg. MTS was used for the analysis of spleen lymphocyte proliferation. RNA sequencing was used to investigate mRNA and miRNA expression profiles in spleen lymphocytes. Compared to GAD65 phage vaccine alone, co-immunization of Kyn and GAD65 phage vaccine resulted in the prevention of hyperglycemia in 60% of mice for at least one month. Further, Kyn enhances GAD65-specific Th2-mediated immune responses; regulates the Th1/Th2 imbalance and increases the secretion of Th2 cytokines and the number of CD4+CD25+Foxp3+T cells; suppresses DC maturation and GAD65-specific T lymphocyte proliferation. Moreover, we integrated Kyn related miRNA and mRNA expression profiles obtained from the spleen lymphocyte RNA-sequencing which was stimulated by Kyn in vitro. These data provide an important basis for understanding the mechanisms underlying Kyn as an immunosuppressive adjuvant which regulated the immune response. These findings suggest that Kyn can serve as an effective suppressive adjuvant candidate for Type 1 diabetes vaccines.

Lippia javanica (Zumbani) herbal tea infusion attenuates allergic airway inflammation via inhibition of Th2 cell activation and suppression of oxidative stress

BackgroundLippia javanica (lemon bush) is commonly used in the treatment of respiratory ailments, including asthma in southern African countries but there is no scientific evidence to support this claim. This study investigated the anti-inflammatory, antioxidant and anti-asthmatic effects of L. javanica using a rat model of asthma.MethodsA 5% w/v L. javanica tea infusion was prepared and characterised by liquid chromatography–mass spectrometer (LC-MS). Animals were intraperitoneally sensitized with ovalbumin (OVA) and subsequently challenged intranasal with OVA on day 15 except the control group. Animals were grouped (n = 5/group) for treatment: unsensitised control, sensitised control, sensitised + prednisolone and sensitised + L. javanica at 50 mg/kg/day and 100 mg/kg/day – equivalent to 1 and 2 cups of tea per day, respectively. After 2 weeks of treatment, bronchoalveolar lavage fluid (BALF) was collected for total and differential white blood cell (WBC) count. Nitric oxide (NO), lipid peroxidation and antioxidants were also assessed in BALF. Ovalbumin specific IgE antibody and inflammatory cytokines: IL-4, IL-5, IL-13 and TNF-alpha were measured in serum. Lung and muscle tissues were histological examined.ResultsL. javanica was rich in phenolic compounds. OVA sensitisation resulted in development of allergic asthma in rats. L. javanica treatment resulted in a reduction in total WBC count as well as eosinophils, lymphocytes and neutrophils in BALF. L. javanica inhibited Th2-mediated immune response, which was evident by a decrease in serum IgE and inflammatory cytokines: IL-4, IL-5, IL-13 and TNF-α. L. javanica treatment also reduced malondialdehyde (MDA) and NO, and increased superoxide dismutase, glutathione and total antioxidant capacity. Histology showed significant attenuation of lung infiltration of inflammatory cells, alveolar thickening, and bronchiole smooth muscle thickening.ConclusionL. javanica suppressed allergic airway inflammation by reducing Th2-mediated immune response and oxidative stress in OVA-sensitized rats which may be attributed to the presence of phenolic compound in the plant. This finding validates the traditional use of L. javanica in the treatment of respiratory disorders.

Characterization of the turbot (Scophthalmus maximus) interleukin-18: Identification of splicing variants, phylogeny, synteny and expression analysis

Interleukin-18 (IL-18) is a pro-inflammatory cytokine that belongs to the interleukin-1 (IL-1) family of cytokines. As occurs with IL-1β, it is synthetized as an inactive precursor peptide that is mainly processed by the cysteine protease caspase-1 in the inflammasome complex. In mammals, and in collaboration with IL-12, it has been described as an important cytokine controlling the Th1-mediated immune responses through the induction of IFN-γ. Although its function in mammals is well stablished, the activity of this cytokine in teleost remains to be elucidated. This could be due, among other things, to the absence of this gene in the fish model species zebrafish, but also to its complex regulation. As it was observed for rainbow trout and human, il18 splicing variants were also found in turbot, which could represent a regulatory mechanism of its bioactivity. In the case of turbot, three splicing variants were observed (SV1-3), and one of them showed an insertion of 10 amino acids in the middle of the potential caspase-1 cleavage position, reflecting that this is probably a form resistant to the processing by the inflammasome. Phylogenetic and three-dimensional analyses of turbot Il18 revealed that it is relatively well-conserved in vertebrates, although only a partial conservation of the gene synteny was observed between fish and mammals. As it was expected, turbot il18 splicing variants were mainly expressed in immune tissues under healthy conditions, and their expression was induced by a bacterial challenge, although certain inhibitions were observed after viral and parasitic infections. In the case of the viral challenge, il18 downregulations did not seem to be due to the effect of type I IFNs.

Abstract 1760: Role of Batf-dependant Th17 immune response in PTEN-null mouse model

Abstract Basic leucine zipper transcription factor ATF-like (BATF) belongs to the activator protein 1 (AP-1) family of transcription factors and has been shown to be predominantly expressed in cells of hematopoietic origin, especially in T cells. Mice deficient for BATF were first described to lack T helper cell type 17 (Th17). Th17 cells are a distinct lineage of CD4+ T helper cells that provide protective immunity to infections but are also involved in chronic inflammation and autoimmunity. However, the role of Th17 response in prostate cancer remains controversial. In this study, we took a genetic approach to explore the role of BATF-dependent Th17 cells in prostate cancers by interbreeding BATF-deficient (Batf-/-) mice with mice that are conditionally mutant for PTEN, an established preclinical model for prostate cancer. Batf+/+;Pten-/- and Batf+/-;Pten-/- mice presented similar prostate phenotypes thus were put into one group (named Batf+), which were compared to the Batf-/-;Pten-/- group (named Batf-). We found that Batf- prostates were clearly smaller than Batf+ prostates at 30 weeks. The genitourinary bloc weight was significantly heavier in Batf+ mice than in Batf- mice at 12 and 30 weeks. Batf+ mice developed significantly higher percent of invasive adenocarcinomas in different prostate lobes at 9, 12, and 30 weeks, respectively compared to Batf- mice. Tumors in Batf- mice exhibited lower rates of cellular proliferation and higher apoptosis, as well as reduced inflammatory cell infiltration and angiogenesis in the prostatic stroma. Moreover, Batf- mice exhibited significantly reduced mRNA and protein levels of Th17 cytokines, AP-1 family members and IL-23R, and reduced NF-kB pathway activity in both spleen and prostate tissues. CD4+ T cells from Batf- mice cultured under Th17 conditions for 4 days, expressed reduced AP-1 family-regulated genes compared to Batf+ mice. Prostate cancer cells (LNCaP) with overexpression of BATF in vitro exhibited increased IL-23R expression. Prostate tissues from Batf- mice cultured under IL-6 or Th17 conditions for 4 days had reduced IL-23R expression compared to that from Batf+ mice. Taken together, our results suggest that Batf promotes the initiation and progression of prostate adenocarcinoma via Th17-mediated inflammatory immune responses and that the Batf-IL-23-IL-17 axis is a critical pathway that is needed for prostate adenocarcinoma development and progression. This work was supported by the NIH-National Institute of General Medical Sciences COBRE on Aging and Regenerative Medicine (P20GM103629, PD: S. Michal Jazwinski; PI: Q.Z.) and the Carol Lavin Bernick Faculty Grant from Tulane University (PI: Q.Z.). Citation Format: Qiuyang Zhang, Sen Liu, Bing Zhang. Role of Batf-dependant Th17 immune response in PTEN-null mouse model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1760.

Zearalenone and deoxynivalenol reduced Th1-mediated cellular immune response after Listeria monocytogenes infection by inhibiting CD4+ T cell activation and differentiation

Based on the fact that mycotoxins and the food-borne bacteria coexist in the natural environment and pose a significant health hazard to humans and animals, it is important to investigate the immunosuppressive mechanism of ZEA (zearalenone), DON (deoxynivalenol), and their combination in bacterial infections. In this study, we established a mouse model of mycotoxin low-dose exposure combined with Listeria monocytogenes infection and investigated the effects of ZEA, DON and their combination on Th1-mediated anti-intracellular bacterial infection based on CD4+ T cell activation and differentiation using both in vitro and in vivo analyses. The present study showed that both ZEA and DON aggravated Listeria monocytogenes infection in mice and affected the activation of CD4+ T cells and Th1 differentiation, including the effects on costimulatory molecules CD28 and CD152 and on cross-linking of IL-12 and IL-12R, by inhibiting T cell receptor (TCR) signaling. When compared with ZEA, DON was found to have a greater impact on many related indicators. Surprisingly, the combined effects of ZEA and DON did not appear to enhance toxicity compared to treatment with the individual mycotoxins. Our findings more clearly revealed that exposure to low-dose ZEA and DON caused immunosuppression in the body by mechanisms including inhibition of CD4+ T cells activation and reduction of Th1 cell differentiation, thus exacerbating infection of animals by Listeria monocytogenes.

IDO and CD40 May Be Key Molecules for Immunomodulatory Capacity of the Primed Tonsil-Derived Mesenchymal Stem Cells.

Background: Tonsil-derived mesenchymal stem cells (T-MSCs) were reported to have suppressive effect on T cells, yet much remains unknown about the underlying mechanisms supporting this effect. We investigated the underlying mechanism of the immunomodulatory effect of T-MSCs on immune cell proliferation and cytokine production. Methods: We isolated T-MSCs from human palatine tonsil and evaluated the immunomodulatory capacity using RT-PCR, ELISA, and flow cytometry. Additionally, we assessed the expression of various soluble factors and several costimulatory molecules to detect the priming effect on T-MSCs. Results: T-MSCs significantly inhibited the immune cell proliferation and cytokine expression (TNF-α and IFN-γ) in the direct co-culture, but there was no suppressive effect in indirect co-culture. Additionally, we detected a remarkably higher expression of indoleamine 2,3-dioxygenase (IDO) in the primed T-MSCs having co-expression CD40. Moreover, immune cells or CD4+ T cells showed lower TNF-α, IFN-γ, and IL-4 expression when the primed T-MSC were added; whereas those findings were reversed when the inhibitor for IDO (not IL-4) or CD40 were added. Furthermore, T-bet and GATA3 levels were significantly decreased in the co-cultures of the primed T-MSCs and CD4+ T cells; whereas those findings were reversed when we added the neutralizing anti-CD40 antibody. Conclusions: Primed T-MSCs expressing IDO and CD40 may have immunomodulatory capacity via Th1-mediated and Th2-mediated immune response.

Co-delivery of antigens with TLR7/8 agonists leads to an improved Th1-mediated immune response to subunit vaccines.

Abstract When designing an effective and safe vaccine, the induction of a protective immune response to a pathogen without unacceptable levels of immunopathology is critical. The use of purified or recombinant antigens in combination with adjuvants that elicit a defined immune response is a promising strategy for eliciting a productive immune response while minimizing toxicity. Our group has developed libraries of novel toll-like receptor (TLR)7 and 8 ligands with a range of activity. Previous clinical trials have shown that the high potency of TLR7/8 adjuvants can lead to unacceptable toxicity. We hypothesized that direct conjugation of the adjuvant to the antigen will limit systemic distribution of the adjuvant, while boosting the immune response by ensuring co-delivery of antigen and adjuvant to antigen presenting cells. We demonstrate that covalent conjugation of TLR7/8 agonists to antigens results in greatly improved humoral and cell-mediated responses, without apparent systemic or local inflammation. This likely improves safety by preventing off-target immune responses from the engagement of PAMPs in the absence of appropriate antigens. When mice were immunized with an antigen/adjuvant conjugate we observed a switch towards IgG2a and away from IgG1 antigen-specific antibodies, plus greater Th1 cytokine production after restimulation of immune cells ex vivo. We hypothesize that co-delivery of a TLR7/8 agonist with antigen leads to activation of CD4+ T cells in the presence of Th1-skewing cytokines. The TLR7/8 agonist also appears to improve cross-presentation, inducing CD8+ T cells. We find this approach has great potential to enhance vaccine efficacy, while reducing potential immunopathology by TLR7/8 adjuvants.

Chitosan Nanoparticles Loaded with Truncated ORF2 Protein as an Oral Vaccine Candidate against Hepatitis E.

In a continuous effort to develop effective vaccines against hepatitis E (HE), oral vaccine nanoparticles using the truncated capsid protein p146 (aa460-605) are formulated and characterized. To improve the immunogenicity of p146, chitosan nanoparticles (CSNPs) are used as a mucosal delivery system. Next, the physical-chemical properties, cytotoxic effects in vitro, and immunogenicity in mice of the produced NPs are analyzed. The results show that the produced CS/p146 NPs are stable and well dispersive and display a near-spherical shape with a mean size of 200-300nm. The findings also demonstrate high encapsulation efficiency (65-73.9%) and loading capacity (27.7-67.5%) of the formulated nanoparticles. Further, the CS/p146 NPs exhibit low cytotoxicity and an obvious sustained-release effect in vitro. Immunogenicity experiments in mice indicate that CS/p146 NPs can induce antigen-specific systemic and mucosal immune responses higher than the purified p146 do. Besides, the expression levels and mRNA transcription of Interleukin (IL)-4 in spleen cells of CS/p146 NPs-immunized mice are higher than those of p146, indicating that a Th2-mediated cellular immune response is activated by the CS/p146 NPs. Overall, the synthesized CS/p146 NPs display promising properties as a potential HE oral vaccine candidate.

Anti-Asthmatic Effects of Saffron Extract and Salbutamol in an Ovalbumin-Induced Airway Model of Allergic Asthma

Introduction: Asthma is a chronic inflammatory disorder of the airways often characterized by airway remodeling and influx of inflammatory cells into the airways. Saffron (C. sativus) has been reported to possess anti-inflammatory, anti-allergic and immunomodulatory properties. Salbutamol is known to relax airway smooth muscles. Objective: To investigate the combined anti-asthmatic effect of C. sativus extract (CSE) and salbutamol in an ovalbumin (OVA)-induced asthma in rats. Materials and methods: Airway hyperresponsiveness (AHR) was induced in male Sprague-Dawley rats by OVA challenge and treated with CSE (30 mg/kg and 60 mg/kg i.p.) and salbutamol (0.5 mg/kg p.o) for 28 days. After the induction period, various hematological, biochemical, molecular (ELISA) and histological analyses were performed. Results: OVA-induced alterations observed in hematological parameters (total and differential cell counts observed in Bronchoalveolar Lavage Fluid (BALF) were significantly attenuated (p &lt; 0.01) by CSE (30 mg/kg and 60 mg/kg) and salbutamol (0.5 mg/kg). The treatment combination also significantly decreased (p &lt; 0.01) the levels of total protein and albumin in serum, BALF and lung tissues. Treatment with CSE and salbutamol significantly attenuated (p &lt; 0.01) increase in OVA induced Th2 cytokine levels (TNF-α, IL-1β, IL-4, IL-13). Histopathological analysis of lung tissue showed that combined effect of CSE and salbutamol treatment ameliorated OVA-induced inflammatory influx and ultrastructural aberrations. Conclusion: The results obtained from this study show that the combined effect of CSE and salbutamol exhibited anti-asthmatic properties via its anti-inflammatory effect and by alleviating Th2 mediated immune response. Thus, this treatment combination could be considered as a new therapeutic strategy for management of asthma.

Eosinophilic esophagitis: novel concepts regarding pathogenesis and clinical manifestations.

Eosinophilic esophagitis is a chronic disease whose incidence and prevalence are increasing, based on a genetic-driven interaction between environment and immune system. Several gene loci involved in the development of the disease have been identified. A two-step mechanism has been hypothesized: a thymic stromal lymphopoietin-induced allergic sensitization followed by upregulation of CAPN14-related esophageal-specific pathways. Environment seems to have a larger effect than genetic variants. Factors that could play a role are allergens, drugs, colonizing bacteria and possibly Helicobacter Pylori infection. Acting on these modifiable risk factors may be a tool to prevent the disease. EoE is characterized by a typical eosinophilic infiltrate limited to the esophageal epithelium, supported by a Th2-mediated immune response, found in other atopic conditions. The key of the pathogenesis is the disfunction of the epithelial barrier which allow the interaction between allergens and inflammatory cells. Eosinophilic-predominant inflammation leads to the typical wall remodeling, histologically characterized by epithelial and smooth muscle hyperplasia, lamina propria fibrosis and neo-angiogenesis. These alterations find their clinical expression in the pattern of symptoms: dysphagia, food impaction, chest pain, heartburn.

Eosinophilic esophagitis: novel concepts regarding pathogenesis and clinical manifestations

Eosinophilic esophagitis is a chronic disease whose incidence and prevalence are increasing, based on a genetic-driven interaction between environment and immune system. Several gene loci involved in the development of the disease have been identified. A two-step mechanism has been hypothesized: a TSLP-induced allergic sensitization followed by upregulation of CAPNA14-related esophageal-specific pathways. Environment seems to have a larger effect than genetic variants. Factors that could play a role are allergens, drugs, colonizing bacteria and possibly Helicobacter Pylori infection. Acting on these modifiable risk factors may be a tool to prevent the disease. EoE is characterized by a typical eosinophilic infiltrate limited to the esophageal epithelium, supported by a Th2-mediated immune response, found in other atopic conditions. The key of the pathogenesis is the disfunction of the epithelial barrier which allow the interaction between allergens and inflammatory cells. Eosinophilic-predominant inflammation leads to the typical wall remodeling, histologically characterized by epithelial and smooth muscle hyperplasia, lamina propria fibrosis and neo-angiogenesis. These alterations find their clinical expression in the pattern of symptoms: dysphagia, food impaction, chest pain, heartburn.

Clinical evaluation of rush immunotherapy using house dust mite allergen in Japanese asthmatics.

BackgroundAllergen immunotherapy (AIT) is a specific treatment of administering clinically important allergens to patients who have allergic diseases. In Japan, the standardized house dust mite (HDM) allergen for subcutaneous immunotherapy (SCIT) was approved in 2015, and we then introduced rush-immunotherapy (rush-IT) using the standardized HDM allergen for HDM-sensitive asthmatics. However, little data are available on the safety and effectiveness of rush-HDM-IT, especially for Japanese asthmatics.ObjectiveThe objective of this study was to examine the safety and clinical effectiveness of rush-IT using the standardized HDM for HDM-sensitive Japanese asthmatics.MethodsThirteen HDM-sensitive asthmatics who received rush-HDM-IT and 12 HDM-sensitive asthmatic controls were enrolled. To evaluate the safety, the number of systemic reaction (SR) events, including anaphylaxis, was assessed. To evaluate the effectiveness, changes in the treatment step, dose of inhaled corticosteroid, and asthma control after rush-HDM-IT and the subsequent maintenance SCIT were assessed. Changes in the HDM-induced production of type 2 cytokines from peripheral blood mononuclear cells were also evaluated.ResultsAmong the 12 patients who received rush-IT, 4 (30.7%) experienced a SR and 3 (23.1%) experienced anaphylaxis. However, the anaphylaxis was not severe (grade 3) in all cases, and they recovered in a short time. The treatment step of asthma was better and the dose of inhaled corticosteroid was lower in the rush-HDM-IT group than in the control group. The HDM-induced production of both interleukin (IL)-5 and IL-13 from peripheral blood mononuclear cells was significantly lower in the rush-HDM-IT group than in the control group.ConclusionRush-HDM-IT can be performed relatively safely in Japanese asthmatics. Furthermore, rush-HDM-IT and the subsequent maintenance SCIT provided clinical improvement in asthma patients, and was accompanied by the suppression of HDM-specific Th2-mediated systemic immune responses.

Antigen- and Epitope-Delivering Nanoparticles Targeting Liver Induce Comparable Immunotolerance in Allergic Airway Disease and Anaphylaxis as Nanoparticle-Delivering Pharmaceuticals

The targeting of natural tolerogenic liver sinusoidal endothelial cells (LSEC) by nanoparticles (NPs), decorated with a stabilin receptor ligand, is capable of generating regulatory T-cells (Tregs), which can suppress antigen-specific immune responses, including to ovalbumin (OVA), a possible food allergen. In this regard, we have previously demonstrated that OVA-encapsulating poly(lactic-co-glycolic acid) (PLGA) nanoparticles eliminate allergic airway inflammation in OVA-sensitized mice, prophylactically and therapeutically. A competing approach is a nanocarrier platform that incorporates pharmaceutical agents interfering in mTOR (rapamycin) or NF-κB (curcumin) pathways, with the ability to induce a tolerogenic state in nontargeted antigen-presenting cells system-wide. First, we compared OVA-encapsulating, LSEC-targeting tolerogenic nanoparticles (TNPs) with nontargeted NPs incorporating curcumin and rapamycin (Rapa) in a murine eosinophilic airway inflammation model, which is Treg-sensitive. This demonstrated roughly similar tolerogenic effects on allergic airway inflammation by stabilin-targeting NPOVA versus nontargeted NPs delivering OVA plus Rapa. Reduction in eosinophilic inflammation and TH2-mediated immune responses in the lung was accompanied by increased Foxp3+ Treg recruitment and TGF-β production in both platforms. As OVA incorporates IgE-binding as well as non-IgE-binding epitopes, the next experiment explored the possibility of obtaining immune tolerance by non-anaphylactic T-cell epitopes. This was accomplished by incorporating OVA323-339 and OVA257-264 epitopes in liver-targeting NPs to assess the prophylactic and therapeutic impact on allergic inflammation in transgenic OT-II mice. Importantly, we demonstrated that the major histocompatibility complex (MHC)-II binding (former) but not the MHC-I binding (latter) epitope interfered in allergic airway inflammation, improving TNPOVA efficacy. The epitope-specific effect was transduced by TGF-β-producing Tregs. In the final phase of experimentation, we used an OVA-induced anaphylaxis model to demonstrate that targeted delivery of OVA and its MHC-II epitope could significantly suppress the anaphylaxis symptom score, mast cell release, and the late-phase inflammatory response. In summary, these results demonstrate comparable efficacy of LSEC-targeting versus pharmaceutical PLGA nanoparticles, as well as the ability of T-cell epitopes to achieve response outcomes similar to those of the intact allergens.

Rosae multiflorae Fructus Extract Improves Trimellitic Anhydride-Induced Atopic Dermatitis-Like Symptoms.

Allergic disorders, including atopic dermatitis (AD), are closely linked to the activation of type 2 helper T (Th2) cells. The aim of this study was to investigate the possibility of using Rosae multiflorae fructus extract (RMFE) for AD treatment in the AD-like mouse model induced by treatment with trimellitic anhydride (TMA). Oral treatment of RMFE reduced the increase in ear thickness and suppressed inflammatory cytokine expression (interleukin [IL]-1β and tumor necrosis factor [TNF]-α) and Th2-associated immune responses (immunoglobulin [Ig] E and IL-4) in mouse ears. Furthermore, messenger RNA (mRNA) expression levels such as IL-4, IL-5, and IL-13, in draining lymph nodes were decreased by RMFE. Furthermore, we found that RMFE increased the level of heme oxygenase-1 (HO-1) through ERK and p38 pathways, reducing IL-2 production and CD4+ T cell proliferation, and inhibited STAT6 phosphorylation. Therefore, this study suggested that RMFE could be an effective treatment of AD induced by Th2-mediated immune responses by suppressing proliferation of CD4+ T cells via increased HO-1.

NLRX1 is a key regulator of immune signaling during invasive pulmonary aspergillosis.

Aspergillus fumigatus is an opportunistic fungal pathogen of immunocompromised patient populations. Mortality is thought to be context-specific and occurs via both enhanced fungal growth and immunopathogenesis. NLRX1 is a negative regulator of immune signaling and metabolic pathways implicated in host responses to microbes, cancers, and autoimmune diseases. Our study indicates loss of Nlrx1 results in enhanced fungal burden, pulmonary inflammation, immune cell recruitment, and mortality across immuno-suppressed and immuno-competent models of IPA using two clinically derived isolates (AF293, CEA10). We observed that the heightened mortality is due to enhanced recruitment of CD103+ dendritic cells (DCs) that produce elevated amounts of IL-4 resulting in a detrimental Th2-mediated immune response. Adoptive transfer of Nlrx1-/- CD103+ DCs in neutropenic NRG mice results in enhanced mortality that can be ablated using IL-4 neutralizing antibodies. In vitro analysis of CD103+ DCs indicates loss of Nlrx1 results in enhanced IL-4 production via elevated activation of the JNK/JunB pathways. Interestingly, loss of Nlrx1 also results in enhanced recruitment of monocytes and neutrophils. Chimeras of irradiated Nlrx1-/- mice reconstituted with wild type bone marrow have enhanced neutrophil recruitment and survival during models of IPA. This enhanced immune cell recruitment in the absence of Nlrx1 is mediated by excessive production of CXCL8/IL-8 family of chemokines and IL-6 via early and enhanced activation of P38 in response to A. fumigatus conidia as shown in BEAS-2B airway epithelial cells. In summary, our results point strongly towards the cell-specific and contextual function of Nlrx1 during invasive pulmonary aspergillosis and may lead to novel therapeutics to reduce Th2 responses by CD103+ DCs or heightened recruitment of neutrophils.

Optimally Fabricated Chitosan Particles Containing Ovalbumin Induced Cellular and Humoral Immunity in Immunized Mice

Subunit vaccines have been developed as promising vaccines with safety. However, subunit vaccines have difficulties in commercialization due to low efficiencies of delivery and immune response. As a result, studies of adjuvants that improve the immune-inducing ability of vaccines have been conducted globally. Aluminum salts (alum) have been extensively used as vaccine adjuvants, but they cannot induce potent cellular immunity. In this study, chitosan particles were fabricated by the precipitation-coacervation method on four different conditions. Ovalbumin (OVA), as a model antigen, was encapsulated in chitosan particles. The optimized fabrication conditions of chitosan particles were 8 mL/min drop rate of sodium sulfate and 0.5 mg/mL chitosan concentration in a sonication bath. Properties of the optimized chitosan particle were about 300 nm diameter, 0.1 polydispersity index, 16.2 mV zeta potential, and 90% loading efficiency. Chitosan particle-containing OVA showed 76% uptake efficiency by mouse macrophage cells and suitable cell viability. Immunization of mice by chitosan particles exhibited IgG1 titer and Interleukin-4 production of a similar level with the alum. Moreover, chitosan particles showed significantly enhanced IgG2 titer and Interferon-gamma production related to Th1-mediated cellular immune response. These results indicated that chitosan particles could be expected to be a promising adjuvant inducing cellular and humoral immunity for subunit vaccine delivery.

In vitro effects of 5 recombinant antigens of Eimeria maxima on maturation, differentiation, and immunogenic functions of dendritic cells derived from chicken spleen

Eimeria maxima possesses integral families of immunogenic constituents that promote differentiation of immune cells during host-parasite interactions. Dendritic cells (DCs) have an irreplaceable role in the modulation of the host immunity. However, the selection of superlative antigen with immune stimulatory efficacies on host DCs is lacking. In this study, 5 recombinant proteins of E. maxima (Em), including Em14-3-3, rhomboid family domain containing proteins (ROM) EmROM1 and EmROM2, microneme protein 2 (EmMIC2), and Em8 were identified to stimulate chicken splenic derived DCs in vitro. The cultured populations were incubated with recombinant proteins, and typical morphologies of stimulated DCs were obtained. DC-associated markers major histocompatibility complex class II, CD86, CD11c, and CD1.1, showed upregulatory expressions by flow cytometry assay. Immunofluorescence assay revealed that recombinant proteins could bind with the surface of chicken splenic derived DCs. Moreover, quantitative real-time PCR results showed that distinct gene expressions of Toll-like receptors and Wnt signaling pathway were upregulated after the coincubation of recombinant proteins with DCs. The ELISA results indicated that the DCs produced a significant higher level of interleukin (IL)-12 and interferon-γ secretions after incubation with recombinant proteins. While transforming growth factor-β was significantly increased with rEmROM1, rEmROM2, and rEmMIC2 as compared to control groups, and IL-10 did not show significant alteration. Taken together, these results concluded that among 5 potential recombinant antigens, rEm14-3-3 could promote immunogenic functions of chicken splenic derived DCs more efficiently, which might represent an effective molecule for inducing the host Th1-mediated immune response against Eimeria infection.