IL-2 mRNA Research Articles

Efficacy of fungal immunomodulatory protein to promote swine immune responses against porcine reproductive and respiratory syndrome virus infection

Fungal immunomodulatory protein (FIP) is one of the bioactive compounds of edible mushrooms, which has been shown to trigger type 1 T helper (Th1) pathway activation in research with mice. This study was designated to assess immunomodulatory effects of recombinant FIP-Flammulina velutipes (rFIP-fve) on swine and the protective efficacy against PRRSV infection. In the in vitro evaluations, rFIP-fve significantly triggered up-regulation of IL-2 and IFN-γ mRNA in porcine PBMCs and stimulated natural killer cytotoxicity. Porcine pulmonary alveolar macrophages (PAMs) treated with rFIP-fve showed prolonged life times, up-regulation of both MHC I and II molecules and enhanced abilities to present antigen. In the in vivo trial, two doses of 2 mg rFIP-fve significantly reduced drops in the CD4/CD8 ratio after PRRSV challenge, and the cytokine mRNA profile of PBMC revealed a tendency of IFN-γ up-regulation and a decrease in IL-10 in the rFIP-treated group. Moreover, administration of rFIP-fve also decreased the PRRSV viremia with 1 log10 in titer (p = 0.07) and alleviated the severity of clinical signs after PRRSV challenge. Conclusively, these results illustrate the in vitro and in vivo immunological changes of rFIP-fve administered to pigs and reveal its potential to be used as an immunomodulatory therapeutic against PRRSV infection.

Early-life heterologous rhinovirus infections induce an exaggerated asthma-like phenotype

Early-life wheezing-associated respiratory tract infection by rhinovirus (RV) is a risk factor for asthma development. Infants are infected with many different RV strains per year. We previously showed that RV infection of 6-day-old BALB/c mice induces a mucous metaplasia phenotype that is dependent on type 2 innate lymphoid cells (ILC2s). We hypothesized that early-life RV infection alters the response to subsequent heterologous infection, inducing an exaggerated asthma-like phenotype. Wild-type BALB/c mice and Rorafl/flIl7rcre mice lacking ILC2s were treated as follows: (1) sham on day 6 of life plus sham on day 13 of life, (2) RV-A1B on day 6 plus sham on day 13, (3) sham on day 6 plus RV-A2 on day 13, and (4) RV-A1B on day 6 plus RV-A2 on day 13. Mice infected with RV-A1B at day 6 and sham at day 13 showed an increased number of bronchoalveolar lavage eosinophils and increased expression of IL-13 mRNA but not expression of IFN-γ mRNA (which is indicative of a type 2 immune response), whereas mice infected with sham on day 6 and RV-A2 on day 13 of life demonstrated increased IFN-γ expression (which is a mature antiviral response). In contrast, mice infected with RV-A1B on day 6 before RV-A2 infection on day 13 showed increased expression of IL-13, IL-5, Gob5, Muc5b, and Muc5ac mRNA; increased numbers of eosinophils and IL-13-producing ILC2s; and exaggerated mucus metaplasia and airway hyperresponsiveness. Compared with Rorafl/fl mice, Rorafl/flIl7rcre mice showed complete suppression of bronchoalveolar lavage eosinophils and mucous metaplasia. Early-life RV infection alters the response to subsequent heterologous infection, inducing an intensified asthma-like phenotype that is dependent on ILC2s.

Disodium cromoglycate treatment reduces TH2 immune response and immunohistopathological features in a murine model of Eosinophilic Esophagitis

Eosinophilic esophagitis (EoE) is an emergent chronic disease of the esophagus. The immunopathological process in EoE is characterized by Th2 immune response and prominent eosinophilic influx, in response to common food allergens. The classical treatment consists of allergen elimination diet and systemic/topical corticosteroid therapy. Nevertheless, patients do not always comply to treatment, and the prolonged corticosteroid therapy can cause side effects, therefore, there is an immediate need for new therapeutic approach for EoE. Disodium cromoglicate (DSCG) is a substance broadly used in allergic asthma treatment, and a well-known mast cell activation stabilizer. However, its effect in EoE have not been evaluated yet. This study aimed to assess the effects of DSCG treatment in an EoE experimental model. Male Balb/C mice were subcutaneously sensitized for five days with OVA, and subsequently orally OVA-challenged, DSCG administration was performed between the OVA-challenges. DSCG treatment not only reduced eosinophilic and mast cell influx, as well as reduced fibrosis. In addition, tslp, GATA3, IL-5, FoxP3 and IL-10 mRNA expression were reduced in esophageal mucosa, associated with lower Th2 (CD3+CD4+GATA3+IL4+) and B cells (CD19+CD40+) number in peripheral lymphoid organs. In conclusion, the data demonstrate DSCG treatment was effective in reducing mast cell activation and Th2 immune response, important immunopathological EoE features. Therefore, the use of DSCG as an EoE treatment can be considered a promising therapeutic approach to treat this disease.

Expression and Protective Functions of the sst4 Somatostatin Receptor in the Indomethacin‐Induced Gastrointestinal Mucosal Injury Model of the Mouse

IntroductionGastrointestinal ulceration is associated with high mortality despite advanced management, therefore, understanding of the mucosal protective mechanisms is important. Somatostatin is present in the endocrine cells and sensory nerves, and its analogues were shown to reduce mucosal blood flow, pepsin and gastric acid secretion. Our group discovered that its anti‐inflammatory and analgesic actions are exerted via the somatostatin receptor subtype 4 (sst4). Therefore, we investigated its expression and role in gastrointestinal injury.MethodsSst4 gene‐deleted (sst4−/−) and wildtype mice were treated s.c. with indomethacine (IDM; 35 mg/kg) or vehicle. RNA scope in situ hybridization was performed to localize, qPCR to quantify sst4 and somatostatin mRNA. Macroscopic and microscopic lesions in the stomach and small intestine were evaluated after 4 and 48 h. Plasma protein extravasation was measured by fluorescent, neutrophil myeloperoxidase (MPO) activity bioluminescent ex vivo imaging, TNFα, IL‐1 and COX2 mRNA by qPCR.ResultsSomatostatin mRNA is present in the neuroendocrine D‐cells and significantly upregulated 48 h after IDM. Sst4 mRNA in located in the myenteric plexus neurons, it’s expression did not change in response to mucosal injury.IDM‐induced gastric and small intestinal macroscopic lesions (4, 24 h), gastric MPO activity (24, 48 h) and plasma extravasation (30 min) were significantly greater in sst4−/− mice. TNFα, IL‐1, COX2 expressions significantly increased after 4 h in the stomach of sst4−/− mice, but in wild‐types the same pattern was observed only later, at the 48 h time‐point. IDM‐evoked histopathological alterations and small intestinal length reduction were not altered by the sst4 deletion.ConclusionThis is the first evidence for sst4 expression in myenteric plexus neurons and its protective roles against chemically‐induced gastrointestinal mucosal damage and inflammation. This suggests the safety of sst4 agonist drug candidates in the gastrointestinal tract, and even their potential use for gastroprotective indication.Support or Funding InformationSupported by: GINOP‐2.3.2‐15‐2016‐00050 – PEPSYS, GINOP‐2.3.2 STAY ALIVE, EFOP‐3.6.2‐16‐2017‐00008, ÚNKP‐19‐3‐III‐PTE‐211

Higher immune response induced by vaccination in Houhai acupoint relates to the lymphatic drainage of the injection site

Houhai acupoint (HA) is a site for acupuncture stimulation, located in the fossa between the anus and tail base in animals. To evaluate HA as a potential immunization site, the immune responses were compared when HA and the conventional site nape were vaccinated in rats. The results showed that injection of a porcine epidemic diarrhea virus (PEDV) vaccine in HA induced significantly higher IgG, IgG1, IgG2, splenocyte proliferation and mRNA expression of IL-2, IL-4 and IFN-γ than in the nape. To search for the underlying mechanisms, the draining lymph nodes for HA and the nape were investigated. When rats were injected in HA with Indian ink, 11 lymph nodes including caudal mesenteric lymph node and bilateral gluteal lymph nodes, posterior inguinal lymph nodes, lumbar lymph nodes, internal iliac lymph nodes and popliteal lymph nodes were visibly stained with the ink and injection of a model antigen ovalbumin (OVA) in HA resulted in detection of OVA by western blotting while in the same lymph nodes only a pair of lymph nodes (central brachial lymph nodes) were observed when Indian ink or OVA was injected in the nape. IL-2 mRNA expression was detected in all the lymph nodes when PEDV vaccine was injected. Therefore, the enhanced immune response elicited by vaccination in HA may be attributed to more lymphocytes activated.

IL-1β prevents ILC2 expansion, type 2 cytokine secretion, and mucus metaplasia in response to early-life rhinovirus infection in mice.

Early-life wheezing-associated respiratory infection with human rhinovirus (RV) is associated with asthma development. RV infection of 6-day-old immature mice causes mucous metaplasia and airway hyperresponsiveness which is associated with the expansion of IL-13-producing type 2 innate lymphoid cells (ILC2s) and dependent on IL-25 and IL-33. We examined regulation of this asthma-like phenotype by IL-1β. Six-day-old wild-type or NRLP3-/- mice were inoculated with sham or RV-A1B. Selected mice were treated with IL-1 receptor antagonist (IL-1RA), anti-IL-1β, or recombinant IL-1β. Rhinovirus infection induced Il25, Il33, Il4, Il5, Il13, muc5ac, and gob5 mRNA expression, ILC2 expansion, mucus metaplasia, and airway hyperresponsiveness. RV also induced lung mRNA and protein expression of pro-IL-1β and NLRP3 as well as cleavage of caspase-1 and pro-IL-1β, indicating inflammasome priming and activation. Lung macrophages were a major source of IL-1β. Inhibition of IL-1β signaling with IL-1RA, anti-IL-1β, or NLRP3 KO increased RV-induced type 2 cytokine immune responses, ILC2 number, and mucus metaplasia, while decreasing IL-17 mRNA expression. Treatment with IL-1β had the opposite effect, decreasing IL-25, IL-33, and mucous metaplasia while increasing IL-17 expression. IL-1β and IL-17 each suppressed Il25, Il33, and muc5ac mRNA expression in cultured airway epithelial cells. Finally, RV-infected 6-day-old mice showed reduced IL-1β mRNA and protein expression compared to mature mice. Macrophage IL-1β limits type 2 inflammation and mucous metaplasia following RV infection by suppressing epithelial cell innate cytokine expression. Reduced IL-1β production in immature animals provides a mechanism permitting asthma development after early-life viral infection.

Identification of a novel splice variant for mouse and human interleukin-5.

Expression of interleukins and their receptors is often regulated by alternative splicing. Alternative isoform of IL-5 receptor α-chain is well studied; however, no data on functional alternative splice variants of IL-5 has been reported up today. In the present study, we describe a novel splice variant for the mouse and human IL-5. The new form was found during analysis of PCR-products amplified from different mouse lymphoid tissues with a pair of primers designed to clone full-length mIL-5 ORF. A single short isoform of mIL-5 was detected along with the canonical full-length mRNA in ConA-stimulated lymphoid cells isolated from spleen, thymus, lymph nodes and blood. It was 30–40 nt shorter, and less abundant than classical form. The sequence analysis of an additional form of mIL-5 revealed that it lacks exon-2 (δ2). Using RT-PCR with the splice-specific primers we obtained an additional evidence for δ2 form expression. To verify whether mIL-5δ2 transcript is translated into protein, the coding sequences corresponding to full and δ2 forms of mIL-5 were cloned into an expression plasmid. After transfection into the human 293T cell line, we found that the short form of mIL-5 protein is expressed in cells and secreted into the supernatant, but at the reduced level than that detected for full isoform of mIL-5. Fluorescence microscopy examination revealed a partial translocation of mIL-5δ2 into cytoplasm, whereas mIL-5 resided mostly within endoplasmic reticulum. This can explain why the level of δ2 protein expression was reduced. Using a similar set of experimental approaches, we received the evidence that the human IL-5 mRNA has the δ2 splice form (hIL-5δ2) as well. It can be firmly detected by RT-PCR in PHA-activated mononuclear cells isolated from peripheral blood of healthy persons or patients with asthma. Altogether, our results showed that the human and mouse IL-5 have an alternative mRNA splice isoform, which loses exon-2, but nevertheless is expressed at protein level. However, more comprehensive studies will be required for evaluation of IL-5δ2 expression, regulation, biological function and clinical significance.

Single-Cell Analysis of Cytokine mRNA and Protein Expression by Flow Cytometry.

Understanding how immune cells respond to external stimuli such as pathogens or drugs is a key component of biomedical research. Critical to the immune response are the expression of cell-surface receptors and the secretion of cytokines, which are tightly regulated by gene expression and protein synthesis. Previously, cytokine mRNA expression levels have been measured from bulk analysis of heterogeneous or sorted cell populations, and the correlation between cytokine mRNA expression and protein levels using these techniques can be highly variable. Flow cytometry is used to monitor changes in cell-surface and intracellular proteins, but some proteins such as cytokines may be transient and difficult to measure. Thus, a flow cytometry method that can simultaneously measure cytokine mRNA and protein levels in single cells is a very powerful tool. We defined a flow cytometry method that combines the conventional measurement of T cell surface proteins (CD45, CD3, CD4, CD8) and intracellular cytokines (IL-2, INF-γ) with fluorescent in situ hybridization and branched DNA technology for amplification and detection of IL-2 and INF-γ mRNA transcripts in activated T cells. This method has been applied to frozen peripheral mononuclear blood cells (PBMCs) and frozen blood samples, making it applicable to clinical trial specimens that require shipment to the test site. In CD4+ cells from activated PBMCs, the concordance between mRNA and protein levels was 41% for IL-2 and 21% for and INF-γ. In CD8+ cells from activated PBMCs, the concordance was 15% for IL-2 and 32% for INF-γ. © 2020 by John Wiley & Sons, Inc. Basic Protocol: Detection of IL-2 and IFN-γ mRNA and protein expression in frozen PBMCs Alternate Protocol: Detection of IL-2 and IFN-γ mRNA and protein expression in frozen blood.

Selected cytokine expression in dogs with alergic conjunctivitis: Correlation with disease activity

IntroductionCanine allergic conjunctivitis (cAC) is described as the most frequent ocular manifestation associated with canine atopic dermatitis (cAD). ObjectivesClinical and immunological characterization of cAD through IL-6, TNF-α and IL-12 mRNA expression quantification in canine conjunctivae. ProceduresTwenty client-owned dogs with both cAC and cAD and twenty-one healthy controls were enrolled and clinician assessed CADESI-04 and grade of ocular signs were calculated. Conjunctival biopsies were performed on all animals and relative quantification of the interleukins mRNA expression performed by qRT-PCR. The correlation between cytokine gene expression and cAC score was evaluated, as well as CADESI-04 values. ResultsThe qRT-PCR showed a significant gene upregulation of respectively 291.48 (p = 1.306e-09) and 4.85 (p = .00033) folds on IL-6 and IL-12 in dogs with allergic conjunctivitis compared to the control group. Regarding the average expression of TNF-α there were no statistical significant differences between both groups (p = .18). Higher cAC scores were associated with enhanced gene expression of TNF-α and IL-12. No correlation was found between the cytokine gene expression levels and the CADESI-04 values. ConclusionAn increase of IL6 and IL12 in cAC was found in the studied population. These two cytokines may be potential immunotherapy targets cAC classification.

Measuring CMI responses using the PrimeFlow RNA assay: A new method of evaluating BVDV vaccination response in cattle

Current methods for evaluating bovine viral diarrhea virus (BVDV) vaccination response typically rely on measurement of humoral responses as determined by virus neutralizing antibody titers (VNT) against BVDV. While VNT are correlated with increased protection, research has also shown that cell mediated immunity (CMI) is an important component of a protective response against BVDV. For example, improved protection against BVDV by modified-live viral (MLV) vaccines as compared to killed vaccines is thought to be due to better CMI induced by the MLV. The goal of this work was to evaluate the cell mediated response in vaccinated calves using a novel PrimeFlow RNA assay that incorporates cell surface marker staining with intracellular RNA expression of cytokines and viral RNA detection. Results from this study evaluating mRNA for IFN-γ and IL-2 at 24 h post-BVDV stimulation are similar to previous studies in which IFN-γ was detected in the CD4+ and CD8+ T cell population. However, a novel observation was the detection of IFN-γ mRNA in the NK cell population in vaccinated animals. The NK cell population contributed a significant portion of the IFN-γ produced. This study also demonstrated a decrease in the frequency and amount of BVDV in PBMCs, harvested from vaccinated calves and exposed to BVDV in vitro. Collectively data from this study highlights the association between an increase in IFN-γ and a decreased infection rate of isolated PBMC’s, based on the frequency and amount of BVDV positive cells following in vitro exposure. This new method combines not only the ability to evaluate cellular responses, but also the ability to understand potential antiviral properties associated with cellular responses. This is the first assay to describe and simultaneously measure CMI responses and intracellular viral RNA quantity as a method to evaluate protective responses associated with vaccination.

Optimal identification of human conventional and nonconventional (CRTH2–IL7Rα–) ILC2s using additional surface markers

Human type 2 innate lymphoid cells (ILC2s) are identified by coupled detection of CRTH2 and IL7Rα on lineage negative (Lin-) cells. Type 2 cytokine production by CRTH2-IL7Rα- innate lymphoid cells (ILCs) is unknown. We sought to identify CRTH2-IL7Rα- type 2 cytokine-producing ILCs and their disease relevance. We studied human blood and lung ILCs from asthmatic and control subjects by flow cytometry, ELISA, RNA sequencing, quantitative PCR, adoptive transfer to mice, and measurement of airway hyperreactivity by Flexivent. We found that IL-5 and IL-13 were expressed not only by CRTH2+ but also by CRTH2-IL7Rα+ and CRTH2-IL7Rα- (double-negative [DN]) human blood and lung cells. All 3 ILC populations expressed type 2 genes and induced airway hyperreactivity when adoptively transferred to mice. The frequency of type 2 cytokine-positive IL7Rα and DN ILCs were similar to that of CRTH2 ILCs in the blood and lung. Their frequency was higher in asthmatic patients than in disease controls. Transcriptomic analysis of CRTH2, IL7Rα, and DN ILCs confirmed the expression of mRNA for type 2 transcription factors in all 3 populations. Unexpectedly, the mRNA for GATA3 and IL-5 correlated better with mRNA for CD30, TNFR2, ICOS, CCR4, and CD200R1 than for CRTH2. By using a combination of these surface markers, especially CD30/TNFR2, we identified a previously unrecognized ILC2 population. The commonly used surface markers for human ILC2s leave a majority of type 2 cytokine-producing ILC2s unaccounted for. We identified top GATA3-correlated cell surface-expressed genes in human ILCs by RNA sequencing. These new surface markers, such as CD30 and TNFR2, identified a previously unrecognized human ILC2 population. This ILC2 population is likely to contribute to asthma.

Lessons Learned from Experimental Human Model of Zinc Deficiency.

Zinc is an essential element for humans, and its deficiency was documented in 1963. Nutritional zinc deficiency is now known to affect over two billion subjects in the developing world. Conditioned deficiency of zinc in many diseases has also been observed. In zinc-deficient dwarfs from the Middle East, we reported growth retardation, delayed sexual development, susceptibility to infections, poor appetite, and mental lethargy. We never found a zinc-deficient dwarf who survived beyond the age of 25 y. In an experimental model of human mild zinc deficiency, we reported decreased thymulin (a thymopoietic hormone) activity in Th1 cells, decreased mRNAs of IL-2 and IFN-gamma genes, and decreased activity of natural killer cells (NK) and T cytotoxic T cells. The effect of zinc deficiency on thymulin activity and IL-2 mRNA was seen within eight to twelve weeks of the institution of zinc-deficient diet in human volunteers, whereas lymphocyte zinc decreased in 20 weeks and plasma zinc decreased in 24 weeks after instituting zinc-deficient diet. We hypothesized that decreased thymulin activity, which is known to proliferate Th1 cells, decreased the proliferation differentiation of Th1 cells. This resulted in decreased generation of IL-2 and IFN-gamma. We observed no effect in Th2 cell function; thus, zinc deficiency resulted in an imbalance of Th1 to Th2 function resulting in decreased cell-mediated immunity. Zinc therapy may be very useful in many chronic diseases. Zinc supplementation improves cell-mediated immunity, decreases oxidative stress, and decreases generation of chronic inflammatory cytokines in humans. Development of sensitive immunological biomarkers may be more sensitive than an assay of zinc in plasma and peripheral blood cells for diagnosis of marginal zinc deficiency in human.

Human Monocytes/Macrophage Inflammatory Cytokine Changes Following in vivo and in vitro Schistomam manoni Infection.

IntroductionEpidemiological and animal studies indicate that helminth infections have positive effects due to their potential to protect against autoimmune diseases. Here, we aim to assess the effect of S. mansoni infection on immune modulation of human monocytes and their potential protection against autoimmune disease development both in vivo and in vitro.Materials and MethodsMonocytes were isolated from helminth-infected Ethiopians (MHIE), and from Dutch healthy volunteers (MHV). The MHV were stimulated in vitro with S. mansoni soluble egg antigens (SEA) or soluble worm antigens (SWA). In addition, phenotypical changes were studied directly, as well as after culturing for 6 days in the presence of human serum to obtain macrophages. Q-PCR, flow cytometry, multiplex bead immunoassay, and live-cell imaging were employed during analysis.ResultsMHIE showed elevated transcripts of SOCS-1 and TNF-α compared to MHV. Similarly, MHV that were stimulated with SEA demonstrated enhanced levels of SOCS-1, IL-10, and IL-12 mRNA, compared to control MHV. Remarkably, the SEA-treated monocytes showed a much higher motility than control monocytes, a hallmark of a patrolling phenotype. Furthermore, in vitro cultured macrophages that were stimulated by SEA exhibited enhanced mRNA levels of SOCS-1, IL-10, TNF-α, IL-12 and TGF-β, compared to control macrophages.ConclusionMacrophages from MHIE as well as SEA-treated MHV show an intermediate activation phenotype with both pro-inflammatory and anti-inflammatory characteristics in vitro. The observed pro-inflammatory properties might reflect a recent response of the cells due to contact with a pathogen, whereas the anti-inflammatory properties might contribute to helminth-induced protection against inflammatory diseases. Large-scale study is recommended to consolidate the findings of the present study.

Macrophage migration inhibitory factor modulates cytokine expression in the trophoblast cell line HTR-8/SVneo.

Extravillous trophoblasts are specific placental cells that invade the uterine stroma and spiral arteries modifying and adjusting them to pregnancy. Many pregnancy pathologies are associated with impairment of this process, including preeclampsia and intrauterine growth restriction, among others. Macrophage migration inhibitory factor (MIF) is a pleiotropic cytokine that is abundant at the fetomaternal interface. Previous results from our group showed that MIF participates in trophoblast invasion and modulates the expression of molecules known to mediate stromal and endovascular trophoblast invasion. In this study we investigated the possibility that MIF could act as a regulator of cytokines known to modulate trophoblast invasion using the normal extravillous trophoblast-derived cell line HTR-8/SVneo. Expression of trophoblast MIF was attenuated by MIF mRNA-specific small interfering RNAs. Cytokine expression was assessed at the mRNA and protein levels using real-time quantitative polymerase chain reaction and flow cytometry respectively. Knockdown of MIF led to a significant decrease in mRNA for IL-1β (IL1B) and IL-8 (CXCL8) and interleukin (IL)-8 protein. The addition of recombinant human MIF to cell culture medium increased IL-6 after 24h treatment and IL-6 and IL-8 after 72h treatment. Cell viability was not affected by MIF silencing or rhMIF treatment. The results of this study imply that at least some of the effects of MIF on trophoblast invasion could be mediated through autocrine or paracrine modulation of trophoblast cytokine production.

Sika deer (Cervus nippon) velvet antler ameliorates 2,4-dinitrochlorobenzene-induced atopic dermatitis-like clinical signs in a rodent model

Context Deer velvet is a rarely used component in traditional Chinese medicine and has beneficial effects against several diseases. As a substance that covers the bone and cartilage of immature antlers, deer velvet is a natural cytokine ‘storeroom’ that is rich in protein and proteoglycans. Recently, proteoglycans have been shown to have beneficial effects against inflammation. Aims To determine whether antler extract possesses therapeutic effects in a mouse model of atopic dermatitis (AD) and to explore the underlying mechanisms of action. Methods BALB/c mice were randomly divided into the following groups: control, AD, and AD + antler groups. We established an in vivo AD model by repeatedly exposing the ears of mice to Dermatophagoides farinae extract (house dust-mite extract) and 2,4-dinitrochlorobenzene once per week for 4 weeks. On the day after induction, ear thickness was measured. Antler extract (100 mg/kg) was administered orally once a day for 26 days. After 4 weeks of treatment with antler extract, the epidermal and dermal ear thickness, mast-cell infiltration, spleen weight, and lymph-node weight were measured. In addition, the mRNA levels of several pathogenic cytokines in the ears were measured. The concentrations of IL-4, IL-5, IL-10, IL-31 and IL-17 mRNA in the skin lesions of each group were measured by quantitative polymerase chain reaction. Key results Epidermal and dermal ear thickness, mast-cell infiltration, lymph-node weight, and gene expression levels of pathogenic cytokines in ear tissue were diminished following oral administration of antler extract, unlike in the control group. Conclusions The results of the present study strongly suggest that antler extract exhibits therapeutic activity against atopic dermatitis via regulation of inflammatory response. Implications Further exploration of the mechanisms of action of antler extract will be important for clinical application.

Utilization of ict-based feeding system on egg production, egg quality, blood parameters and caecal microflora in laying hens

Smart poultry farm application technology that enables adequate maintenance and management of a livestock production environment was developed as a remote control method through the convergence of information and communications technology. The objective of this study was to investigate the improvement of egg production and egg-laying quality of laying hens in a smart poultry feeding system (SP) and its working mechanism. A total of 400 laying hens (50-week-old; Hy-line brown) were divided into two groups, in a randomized block design, namely, a conventional feeding system (CON) group and SP group using an enriched cage system of animal welfare type. The birds were reared for 10 weeks. Egg production and egg quality parameters were higher (P<0.05) in the SP group than those in the CON group. Blood RBC, HCT, Hb, platelet count, plateletcrit and electrolytes (Na+, K+, Cl”) were higher in the SP group than those in the CON group while helerphils: lymphocyte ratio, ALT, and AST were lower in the SP group (P<0.05). Concentrations of HCO3 and blood partial presure levels were higher in the SP group than those in the CON group whereas partal pressure and total CO2 content in the blood was lower in the SP group (P<0.05). Blood IL-2 and IL-6 mRNA expression levels were higher (P<0.05) in the SP group than those in the CON group. Count of caecal Lactobacillus was higher in the SP group than those in the CON group while that of E. coli, total aerobic bacteria, and coliform bacteria were lower (P<0.05) in the SP group. These results of the blood parameters, immune cell growth, and caecal microflora balance indicate that the smart poultry feeding system can increase the productivity of laying hens in comparison to the conventional feeding system.

Effects of Pelargonium sidoides and Coptis Rhizoma 2 : 1 Mixed Formula (PS + CR) on Ovalbumin-Induced Asthma in Mice.

Pelargonium sidoides (PS) is traditionally used to treat respiratory and gastrointestinal infections, dysmenorrhea, and hepatic disorders in South Africa. Coptis Rhizoma (CR) is used to treat gastroenteric disorders, cardiovascular diseases, and cancer in East Asia. In the present study, we intended to observe the possible beneficial antiasthma effects of PS and CR on the ovalbumin- (OVA-) induced asthma C57BL/6J mice. Asthma in mice was induced by OVA sensitization and subsequent boosting. PS + CR (300 and 1,000 mg/kg; PO) or dexamethasone (IP) was administered once a day for 16 days. The changes in the body weight and gains, lung weights and gross inspections, total and differential cell counts of leukocytes in bronchoalveolar lavage fluid (BALF), serum OVA-specific immunoglobulin E (OVA-sIgE) levels, interleukin-4 (IL-4) and IL-5 levels in BALF and lung tissue homogenate, and IL-4 and IL-5 mRNA levels in lung tissue homogenates were analyzed with lung histopathology: mean alveolar surface area (ASA), alveolar septal thickness, numbers of inflammatory cells, mast cells, and eosinophils infiltrated in the alveolar regions, respectively. Significant increases in lung weights, total and differential cell counts of leukocytes in BALF, serum OVA-sIgE levels, and IL-4 and IL-5 levels in BALF and lung tissue homogenate were observed in OVA control as compared to those of intact control. In addition, OVA control showed a significant decrease in mean ASA and increases in alveolar septal thickness, numbers of inflammatory cells, mast cells, and eosinophils infiltrated in alveolar regions. However, these allergic and inflammatory asthmatic changes were significantly inhibited by PS + CR in a dose-dependent manner. In this study, PS + CR showed dose-dependent beneficial effects on OVA-induced asthma in mice through anti-inflammatory and antiallergic activities. Therefore, it is expected that PS + CR have enough potential as a new therapeutic agent or as an ingredient of a medicinal agent for various allergic and inflammatory respiratory diseases including asthma.

High-fat diet-induced obesity worsens TH2 immune response and immunopathologic characteristics in murine model of eosinophilic oesophagitis.

Eosinophilic oesophagitis (EoE) is an emergent chronic immune-mediated disease of the oesophagus, which affects both children and adults. It is clinically characterized by dysphagia, food impaction and oesophageal eosinophilia. Epidemiological studies indicate that obesity can worsen allergic symptoms; however, its effect on EoE immunopathological response has not been evaluated yet. This study aimed to assess the effect of obesity on allergic inflammation and T helper-2 profile in an EoE experimental model. Obesity was induced by high-fat feeding. After 7weeks of diet, male BALB/c mice were subcutaneously sensitized and orally challenged with OVA. Obesity itself induced a significant mast cell and eosinophil accumulation in the oesophagus, trachea, gut and lung. After allergy induction, this number was higher, when compared to lean-allergic mice. Moreover, obese-allergic mice showed higher remodelling area, in the oesophagus, associated with higher IL-5 and TSLP mRNA expression. In contrast, FoxP3 and IL-10 were less expressed in comparison with lean-allergic mice. In addition, the amount of CD11c+ MHCII+ PDL1+ dendritic cells was reduced, while the number of CD11c+ MHCII+ CD80+ DCs and CD3+ CD4+ GATA3+ IL-4+ cells was increased in obese-allergic mice in the spleen and lymph nodes when compared to lean-allergic mice. Obesity aggravated the immune histopathological characteristics in the EoE experimental model, which was associated with the reduction in the regulatory profile, and the increased inflammatory cells influx, related to the TH 2 profile. Altogether, the data provide new knowledge about obesity as a risk factor, worsening EoE symptoms, and contribute for future treatment strategies for this specific profile.

Endogenous Toll-like Receptor 2 Modulates Th1/Treg-Promoting Dendritic Cells in Mice Corneal Transplantation Model

ABSTRACT Purpose Endogenous toll-like receptor (TLR) 2 is linked to allograft rejection in corneal transplantation. TLR2 also could modulate dendritic cell (DC) phenotype, resulting in T cell polarization. Thus, we investigated the role of endogenous TLR2 on DC development and T cell polarization during corneal rejection. Materials and Methods Corneas of BALB/c mice were transplanted into the eyes of C57BL/6 wild-type (WT) recipients and TLR2−/- (KO) recipients. Graft survival and TLR2 mRNA expression were assessed. At day 14 after transplantation, to study endogenous TLR2 effects on DC development and function, surface expression of MHC classⅡ (MHCⅡ), CD86, CD80 and CD40 in ipsilateral cervical draining lymph nodes (DLNs) is measured by flow cytometry, and DC phenotype in corneas is detected by immunofluorescence. The levels of IL-12, IL-10 and IL-4 in corneas were measured by real time-qPCR (RT-qPCR). The ability of DCs to stimulate T cell polarization was assessed by IFN-γ expressions via RT-qPCR and immunohistochemistry. Results TLR2 mRNA expression in corneas was peaked at day 14 post-transplantation in WT group. KO group improved corneal allograft survival compared to the WT group. In addition, the KO group decreased expression of CD86, CD80 and CD40 on DCs compared to the WT group. There was no difference in MHCⅡ expression in two groups. The CD11c+MHCⅡ+CD40high DCs could not be detected in corneas of the KO group. Moreover, the KO group decreased IL-12 (Th1-promoting cytokines) mRNA expression and increasing IL-10 (Treg-promoting cytokines) mRNA expression compared to the WT group. IL-4 (Th2-promoting cytokines) mRNA expression gained no difference between the two groups. The IFN-γ (Th1 cytokines) expression was significantly decreased in the KO group compared to the WT group. Conclusions Endogenous TLR2 may contribute to allogeneic corneal rejection via Th1 immunity by activating Th1-promoting DCs and suppressing Treg-promoting DCs.

Electroacupuncture improves inflammatory reaction and insulin sensitivity in insulin-resistant obese rats

To observe the effect of electroacupuncture (EA) on inflammatory reaction and insulin sensitivity in insulin-resistant obese (OIR) rats. Thirteen male Wistar rats were randomly selected as the control group and fed with common diet. The other 39 rats were fed with high-fat diet for 8 weeks to establish OIR model and then randomized into model, EA and sham EA groups. EA (2 Hz, 1 mA) was applied to unilateral "Zusanli" (ST36), "Fenglong" (ST40), "Zhongwan" (CV12) and "Guanyuan" (CV4) for 15 min, once every other day for 8 weeks, and sham EA was applied to unilateral 4 control spots about 5 mm lateral to the aforementioned 4 acupoints after shallowly inserting acupuncture needles, but without electric current output. After 8 weeks' intervention, the body weight was recorded and the glucose infusion rate (GIR) measured using hyperinsulinemic-euglycemic clamp technique. At the 6th week of intervention, glucose contents of intraperitoneal glucose tolerance test (IPGTT) and intraperitoneal insulin tole-rance test (IPITT) were measured. The levels of serum insulin (INS) and inflammatory factors as C-reactive protein (CRP), IL-6 and TNF-α were measured by using ELISA at the end of the treatment. The expression levels of IL-6, TNF-α, monocyte chemoattractant protein-1(MCP-1), IL-10 and IL-1β proteins and mRNAs in the abdominal adipose tissues were detected by Western blot and quantitative real time-PCR, separately. The CD68 expression (displaying infiltration of macrophages) of adipose tissue was detected using immunohistochemistry. After modeling, the contents of glucose of IPGTT at 30, 60 and 120 min and those of IPITT at 15, 30, 60 and 120 min, serum INS, CRP, IL-6 and TNF-α, as well as the expression levels of IL-6, TNF-α, IL-1β and MCP-1 proteins and mRNAs and CD68 protein were significantly increased (P<0.01, P<0.05), while the levels of GIR and IL-10 protein and mRNA were obviously decreased in the model group in comparison with those of the control group (P<0.01), suggesting an increase of inflammation and a decline of INS sensitivity. Following the interventions, the increased contents of glucose of IPGTT and IPITT, serum INS, CRP, IL-6 and TNF-α, expression levels of IL-6, TNF-α, IL-1β and MCP-1 proteins and mRNAs and CD68 protein, and the decreased levels of GIR and IL-10 protein and mRNA were evidently reversed in the EA group compared with the model group (P<0.01, P<0.05) rather than those in the sham EA group (P>0.05). EA can reduce the level of inflammation and improve insulin sensitivity in OIR rats.