IL-4 mRNA Expression Research Articles

A potent and selective CXCR2 antagonist improves neuroimmune dysregulation through the inhibition of NF-κB and notch inflammatory signaling in the BTBR mouse model of autism.

Autism comprises a broad range of neurodevelopmental disorders characterized by social communication deficits and repetitive and stereotyped behaviors. Chemokine receptor CXCR2 is expressed on neurons and is upregulated in neurological disorders. BTBR T+ Itpr3tf/J (BTBR) mice, a model for autism that shows the core features of ASD. Here, we studied the anti-inflammatory effect of a potent and selective CXCR2 antagonist SB332235 in the BTBR mice. The CXCR2 antagonist represents a promising therapeutic agent for several neuroinflammatory disorders. In this study, we investigated the effects of SB332235 administration on NF-κB-, Notch-1-, Notch-3-, GM-CSF-, MCP-1-, IL-6-, and IL-2- and TGF-β1-expressing CD40+ cells in BTBR and C57BL/6 (C57) mice in the spleen cells by flow cytometry. We further assessed the effect of SB332235 treatment on NF-κB, Notch-1, GM-CSF, MCP-1, IL-6, and IL-2 mRNA expression levels in the brain tissue by RT-PCR. We also explored the effect of SB332235 administration on NF-κB, GM-CSF, IL-6, and TGF-β1 protein expression levels in the brain tissue by western blotting. The SB332235-treated BTBR mice significantly decreases in CD40+NF-κB+, CD40+Notch-1+, CD40+Notch-3+, CD40+GM-CSF+, CD40+MCP-1+, CD40+IL-6+, and CD40+IL-2+, and increases in CD40+TGF-β1+ in the spleen cells. Our results further demonstrated that BTBR mice treated with SB332235 effectively decreased NF-κB, Notch-1, GM-CSF, MCP-1, IL-6, and IL-2, increasing TGF-β1 mRNA and protein expression levels in the brain tissue. In conclusion, these results indicate that SB332235 elicits an anti-inflammatory response by downregulating the inflammatory mediators and NF-κB/Notch inflammatory signaling in BTBR mice. This could represent a promising novel therapeutic target for autism treatment.

Study of the proviral load levels and mRNA expression of cytokines in peripheral blood mononuclear cells and somatic milk cells in cattle with different BLV infection profiles.

The retrovirus bovine leukemia virus (BLV) might produce abnormal immune function, associated with susceptibility to developing other infectious diseases, including mastitis. This study aimed to determine the proviral load and cytokines gene expression in peripheral blood mononuclear cells (PMBC) and milk somatic cells (SC) in BLV-infected and non-infected cattle. Of 27 BLV-infected cows in PBMC, 17 (62.96%) had a high proviral load (HPL), and 10 (37.04%) had a low proviral load (LPL). All SC samples had low proviral load (LPL-SC). Higher IFN-γ and IL-10 expression, and lower IL-12 and IL-6 expression, were found in PBMC from BLV-infected compared to BLV non-infected cattle. Moreover, higher IFN-γ, IL-12, and IL-6 expression, and lower IL-10 expression were observed in cattle with LPL-PBMC compared to HPL-PBMC. In milk samples, lower IFN-γ and higher IL-12 mRNA expression were observed in LPL-SC compared to BLV non-infected cattle in SC. IL-10 and IL-6 expression mRNA was significantly lower in LPL-SC than in SC from BLV non-infected cattle. This study shows that milk SC maintains lower proviral load levels than PBMC. This first report on Th1 and Th2 cytokines expression levels in SC may be relevant to future control strategies for BLV infection, mastitis, and udder health management.

Evaluation of Effects of Spirulina Extracts on Immunologic Dysfunction and Inflammation Associated with Aflatoxin B1 Induced Toxicity in Mice

The contamination of foods by various mycotoxins has been reported as a major public health concern across the world. The most predominant type of fungal toxins are aflatoxins which are synthesized by certain fungi that contaminate agricultural crops or produce. The main aflatoxin producing fungi are Aspergillus flavus and Aspergillus parasiticus which contaminate food crops in the farm and after harvesting. Out of all the types of aflatoxins, the most potent type is aflatoxin B1. The mechanism of toxicity and health effects of aflatoxins have been studied widely and it has been shown that aflatoxin B1 leads to liver necrosis, inflammation and liver cancer. The use of natural products as a remedy to health consequences of aflatoxins in humans and animals is gaining popularity. Owing to its anti-inflammatory effects, Spirulina plantesis has been studied for its immunomodulatory effects. This study evaluated the effects of spirulina extract on aflatoxin B1 (AFB1) induced immune dysfunction and inflammation. Male BALB/c mice weighing 28-34g were randomly placed into 6 groups and orally treated as follows: Group 1 was not treated but received food and water for entire experimental period. Group 2 received 200 µg/kg b.w of aflatoxin B1 orally. Group 3 received 1g/kg b.w of activated charcoal and an hour later 200 µg/kg b.w of aflatoxin B1 orally. Group 4, 5 and 6 received 50mg/kg, 100mg/kg and 150mg/kg b.w of Spirulina plantesis respectively then an hour later each group received 200 µg/kg b.w of aflatoxin B1 orally. Treatments were done on a daily basis for 14 days. At the last day of the experiment, all the mice were denied food and water for 12 hours, thereafter sacrificed and samples processed for immunological studies. The results indicated that body weight significantly increased when treated with 100mg/kg spirulina+AFB1 and 150mg/kg spirulina+AFB1 groups in compared to AFB1 treated group (p < 0.05). AFB1 was shown to increase serum level of IFN- γ and IL 2 and decrease levels of IL 4. Treatment with spirulina extract had no significant effect on the serum concentrations of IL-4 and IL-2 (p > 0.05) in comparison with aflatoxin B1 treated group, the serum levels of IFN- γ and IL-2 reduced significantly (p<0.05). Treatment with spirulina extract at different doses had no significant effect on serum levels of immunoglobulins A, G and M (p < 0.05). The mRNA expression of IL-4 was downregulated while that of TNFα, and IFNγ were upregulated. The results showed that increasing mRNA expressions of TNFα, and IFNγ as a result of AFB1was prevented (p≤0.01) by administration of spirulina extract. These findings suggests that spirulina extract could be used as a remedy to AFB1 induced immune dysfunction and inflammation.

Immunogenicity and protective efficacy of an inactivated Newcastle disease virus vaccine encapsulated in poly-(lactic-co-glycolic acid) nanoparticles

An immunization experiment was conducted in specific pathogen-free chickens with the inactivated Newcastle disease virus (NDV) vaccine encapsulated in the poly-(lactic-co-glycolic) acid (PLGA) nanoparticles (NP) to evaluate its immunogenicity and protective efficacy. The NDV vaccine was prepared by inactivating one virulent Indian strain of NDV belonging to Genotype VII by using beta-propiolactone. PLGA nanoparticles encapsulating inactivated NDV were prepared by the solvent evaporation method. Scanning electron microscopy and zeta sizer analysis revealed that the (PLGA+NDV) NP were spherical, with an average size of 300 nm, having a zeta potential of -6 mV. The encapsulation efficiency and loading efficiency were 72% and 2.4%, respectively. On immunization trial in chicken, the (PLGA+NDV) NP induced significantly (P < 0.0001) higher levels of HI and IgY antibodies with the peak HI titer of 28 and higher expression of IL-4 mRNA. The consistency of higher antibody levels suggests slow and pulsatile release of the antigens from the (PLGA+NDV) NP. The nano-NDV vaccine also induced cell mediated immunity with higher expression of IFN-γ indicating strong Th1 mediated immune responses in contrast to the commercial oil adjuvanted inactivated NDV vaccine. Further, the (PLGA+NDV) NP afforded 100% protection against the virulent NDV challenge. Our results suggested that PLGA NP have adjuvant potential on induction of humoral as well as Th1 biased cell mediated immune responses and also enhanced protective efficacy of the inactivated NDV vaccine. This study provides an insight for development of PLGA NP based inactivated NDV vaccine using the same genotype circulating in the field as well as for other avian diseases at exigencies.

Th1 and Th2 epitopes of Cowdria polymorphic gene 1 of Ehrlichia ruminantium.

Cowdriapolymorphic gene 1 (cpg1, Erum2510, ERUM_RS01380) has been shown to induce 30% and 100% protection in sheep immunised by deoxyribonucleic acid (DNA) prime combined with DNA boost and DNA prime combined with protein boost, respectively, against heartwater infection via needle challenge. To localise its antigenic regions for inclusion in a multi-epitope DNA vaccine against heartwater, Erum2510 was cleaved into five overlapping subfragments. These subfragments were expressed individually in anEscherichia colihost expression system and evaluated for their ability to induce proliferative responses, Th1 and Th2 cytokines (interferon gamma [IFN-γ] and interleukin 4 [IL-4]) via enzyme-linked immunospot (ELISpot), quantitative real time polymerase chain reaction (qRT-PCR) and flow cytometry. Recombinant (r)proteins 3 and 4 were shown to induce immunodominant Th1 and Th2 immune responses characterised by the secretion of effector cytokines IFN-γ and IL-4 in addition to differential messenger ribonucleic acid (mRNA) expression of tumour necrosis factor (TNF), IL-2, IL-1, IL-18, IL-10, transforming growth factor (TGF), granulocyte-macrophage colony-stimulating factor (GM-CSF) and inducible nitric oxide synthase (iNOS). Thirty-seven overlapping synthetic peptides (16 mer) spanning the lengths of these immunodominantrproteins were synthesised and assayed. A peptide pool comprising p9 and p10 derived fromrprotein 3 induced a Th1-biased immune response. A peptide pool comprising p28 and p29 derived from rprotein 4 induced a mixed Th1 and Th2 immune response characterised by secretion of IFN-γ and differential mRNA expression of IL-1, IL-2, IL-10, IL-12, iNOS, TGF, TNF and GM-CSF. Only one of the peptides (p29) induced secretion of IL-4. Phenotypic analysis showed significant activation of cluster of differentiation 8+ (CD8+), cluster of differentiation 4+ (CD4+) and B+ lymphocyte populations. Findings suggest that Erum2510rproteins and synthetic peptides can induce both cellular and humoral immune responses, thereby implicating their importance in protection against heartwater.Contribution:This study will facilitate the design of an effective multi-epitope DNA vaccine against heartwater that will contribute to control this economically important disease in sub-Saharan Africa and beyond.

Sputum Interleukin-32 in childhood asthma: correlation with IL-1β

_Background:_ Asthma is an airway disorder where inflammatory cytokines are partly responsible for exacerbating the disease. Interleukin (IL)-32, generally referred to as natural killer cell (NK4) transcript 4, is described as an immunoregulator involved in the stimulation of anti-/pro-inflammatory cytokines. The abnormal presence of IL-32 has been observed during inflammatory diseases, particularly in asthma. This study aims to characterize IL-32 in the inflammatory process in patients with severe asthma. _Methods:_ IL-32 and IL-1β levels in the supernatant of induced sputum obtained from 59 asthma patients (mild: 20 patients; moderate: 20 patients; severe: 19 patients) were measured using ELISA. Sputum IL-32 mRNA expression was measured by RT- PCR. _Results:_ Sputum IL-32 was significantly elevated at the protein (_p= 0.00019_) and mRNA expression (_p &lt; 0.0001_) in asthmatics compared to non-asthmatic controls. IL-32 was elevated in severe asthmatic patients (_p &lt; 0.0001_) compared to mild-moderate asthma. Severe asthmatics allergic to house dust mites expressed higher sputum IL-32 levels than severe asthmatics without allergy (_p = 0.0001_). A significant association was found between sputum IL-32 and IL-32 mRNA (r =0.531_; p = 0.0159_) in severe asthma. In the same way, sputum IL-1β and IL-32 were significantly correlated (r = 0.476; _p = 0.0337_). _Conclusion:_ The level of IL-32 in induced sputum may be associated with asthma severity.

Photobiomodulation activates undifferentiated macrophages and promotes M1/M2 macrophage polarization via PI3K/AKT/mTOR signaling pathway

Macrophages are the main mediators of the inflammatory response and play a major role in the onset and maintenance of periodontitis. Studies revealed that photobiomodulation (PBM) can change the polarization state of macrophages and inflammation reduction, although the cellular mechanisms are not fully elucidated. Here, the present study explored the effect of PBM (980 nm) on undifferentiated and M1-type macrophages and the underlying mechanism. RAW264.7 cells were exposed to laser irradiation under different laser parameters (0.5, 5.0, and 10.0 J/cm2) with or without LY294002 (an inhibitor of PI3K pathway). Then, confocal laser microscopy was used to observe cell differentiation; qPCR was performed to examine the gene expression and western blotting was used to detect the protein in the PI3K/AKT/mTOR pathway and activated macrophage markers. The obtained results revealed that 980 nm PBM increased the mRNA expression of iNOS, Il-10, Arg1, and Il-12 along with the inflammatory cytokines Tnfα, IL-1β, and Il-6 in M0-type macrophages in dose-dependent manner. More interestingly, PBM at 5 J/cm2 decreased the mRNA expression of iNOS, Il-12, Tnfα, IL-1β, and Il-6 and increased the expression of Arg1 and Il-10 by M1-type macrophages, along with the elevated expression of phosphorylation of AKT and mTOR. Moreover, PBM-induced M1-type macrophage polarization was significantly attenuated via LY294002 treatment. These suggest that 980 nm PBM could activate M0-type macrophages and increase M2/M1 ratio via the PI3K/AKT/mTOR pathway.

Sputum Interleukin-32 in childhood asthma: correlation with IL-1β

_Background:_ Asthma is an airway disorder where inflammatory cytokines are partly responsible for exacerbating the disease. Interleukin (IL)-32, generally referred to as natural killer cell (NK4) transcript 4, is described as an immunoregulator involved in the stimulation of anti-/pro-inflammatory cytokines. The abnormal presence of IL-32 has been observed during inflammatory diseases, particularly in asthma. This study aims to characterize IL-32 in the inflammatory process in patients with severe asthma. _Methods:_ IL-32 and IL-1β levels in the supernatant of induced sputum obtained from 59 asthma patients (mild: 20 patients; moderate: 20 patients; severe: 19 patients) were measured using ELISA. Sputum IL-32 mRNA expression was measured by RT- PCR. _Results:_ Sputum IL-32 was significantly elevated at the protein (_p= 0.00019_) and mRNA expression (_p &lt; 0.0001_) in asthmatics compared to non-asthmatic controls. IL-32 was elevated in severe asthmatic patients (_p &lt; 0.0001_) compared to mild-moderate asthma. Severe asthmatics allergic to house dust mites expressed higher sputum IL-32 level than severe asthmatics without allergy (_p = 0.0001_). A significant association was found between sputum IL-32 and IL-32 mRNA (r =0.531_; p = 0.0159_) in severe asthma. In the same way, sputum IL-1β and IL-32 were significantly correlated (r = 0.476; _p = 0.0337_). _Conclusion:_ The level of IL-32 in induced sputum may be associated with asthma severity.

Effect of customised supplement on haemato-biochemical profile, serum minerals, metabolic hormones, antioxidant capacity and gene expression in crossbred calves

Present experiment examined the supplementary effect of a tailor-made supplement to farmers’-based diet in crossbred calves. Male crossbred calves (15) were randomly allocated in 3 dietary treatments consisting of 5 calves in each. The dietary treatments were: Control- cereal straw-based diet with concentrate mixture as per the farmers’ practices; CS (customised supplement)- control diet with additional customised supplement @ 0.25% of BW; SD-standard diet. Serum glucose was higher in SD than control, however, CS had an intermediate response. The serum macro (Ca and i-P) and trace (Zn, Cu, Fe and Mn) minerals were higher in SD and CS than control. The serum T3 and T4 hormones were significantly higher in SD and CS than control group. The serum growth hormone (GH) and insulin-like growth factor-1 (IGF-1) were significantly higher in SD than control groups, however, SD had an intermediate position. The total antioxidant capacity (TAOC) was significantly higher in SD and CS than control group. The relative mRNA expression of cytokines, viz. IL-2 and IL-4 was significantly higher in SD and CS than control group. The relative mRNA expression of leptin (LEP) was significantly higher and ghrelin (GHRL) was significantly lower in SD than control group, however, CS had a transitional position. Thus, it can be concluded that supplementation of the customised supplement (@ 0.25% BW) to farmers’-based diet significantly improved the serum glucose concentration, metabolic hormone profile, antioxidant capacity and relative mRNA expression of cytokines and genes involved in energy metabolism in crossbred calves.

Cytokine expression profile of B. melitensis-infected goat monocyte-derived macrophages

Brucella parasitize the macrophage where is able to replicate and modulate the immune response in order to establish a chronic infection. The most adequate response to control and eliminate Brucella infection is a type 1 (Th1) cell-mediated effector immunity. Research in immune response of B. melitensis-infected goats is relatively scarce. In this study, we first evaluated changes in the gene expression of cytokines, a chemokine (CCL2) and the inducible nitric oxide synthase (iNOS) of goat macrophage cultures derived from monocytes (MDMs) infected for 4 and 24 h with Brucella melitensis strain 16 M. TNFα, IL-1β and iNOS, and IL-12p40, IFNγ and also iNOS were significantly expressed (p < 0.05) at 4 and 24 h respectively, in infected compared to non-infected MDMs. Therefore, the in vitro challenge of goat MDMs with B. melitensis promoted a transcriptional profile consistent with a type 1 response. However, when the immune response to B. melitensis infection was contrasted between MDM cultures phenotypically restrictive or permissive to intracellular multiplication of B. melitensis 16 M, it was observed that the relative IL-4 mRNA expression was significantly higher in permissive macrophage cultures with respect to restrictive cultures (p < 0.05), independently of the time p.i. A similar trend, although non-statistical, was recorded for IL-10, but not for pro-inflammatory cytokines. Thus, the up-expression profile of inhibitory instead of pro-inflammatory cytokines could explain, in part, the difference observed in the ability to restrict intracellular replication of Brucella. In this sense, the present results make a significant contribution to the knowledge of the immune response induced by B. melitensis in macrophages of its preferential host species.

MCP-1/IL-12 ratio expressions correlated with adventitial collagen depositions in renal vessels and IL-4/IFN-γ expression correlated with interstitial collagen depositions in the kidneys of dogs with canine leishmaniasis

Collagen deposition is a common event in chronic inflammation, and canine Leishmaniosis (CanL) is generally associated with a long and chronic evolution. Considering that the kidney shows fibrinogenic changes during CanL, and the balance of cytokines/chemokines regulates the profibrinogenic and antifibrinogenic immune responses differently, it can be hypothesized that the balance of cytokines/chemokines can be differentially expressed in the renal tissue in order to determine the expression of collagen depositions in the kidneys. This study aimed to measure collagen deposition and to evaluate cytokine/chemokine expressions in the kidney by means of qRT-PCR in sixteen Leishmania-infected dogs and six uninfected controls. Kidney fragments were stained with hematoxylin & eosin (H&E), Masson’s Trichrome, Picrosirius Red, and Gomori’s reticulin. Intertubular and adventitial collagen depositions were evaluated by the morphometric approach. Cytokine RNA expressions were measured by means of qRT-PCR to identify molecules involved in chronic collagen depositions in kidneys with CanL. Collagen depositions were related to the presence of clinical signs, and more intense intertubular collagen depositions occurred in infected dogs. Adventitial collagen deposition, as morphometrically measured by the average area of the collagen, was more intense in clinically affected dogs than in subclinically infected dogs. TNF-α/TGF-β, MCP1/IL-12, CCL5/IL-12, IL-4/IFN-γ, and IL-12/TGF-β expressions were associated with clinical manifestations in dogs with CanL. The IL-4/IFN-α ratio was more commonly expressed and upregulated in clinically affected dogs, and downregulated in subclinically infected dogs. Furthermore, MCP-1/IL-12 and CCL5/IL-12 were more commonly expressed in subclinically infected dogs. Strong positive correlations were detected between morphometric values of interstitial collagen depositions and MCP-1/IL-12, IL-12, and IL-4 mRNA expression levels in the renal tissues. Adventitial collagen deposition was correlated with TGF-β, IL-4/IFN-γ, and TNF-α/TGF-β. In conclusion, our results showed the association of MCP-1/IL-12 and CCL5/IL-12 ratios with an absence of clinical signs, as well as an IL-4/IFN-α ratio with adventitial and intertubular collagen depositions in dogs with visceral leishmaniosis.

Sputum Interleukin-32 in childhood asthma: correlation with IL-1β

_Background:_ Asthma is an airway disorder where inflammatory cytokines are partly responsible for exacerbating the disease. Interleukin (IL)-32, generally referred to as natural killer cell (NK4) transcript 4, is described as an immunoregulator involved in the stimulation of anti-/pro-inflammatory cytokines. The abnormal presence of IL-32 has been observed during inflammatory diseases, particularly in asthma. This study aims to characterize IL-32 in the inflammatory process in patients with severe asthma. _Methods:_ IL-32 and IL-1β levels in the supernatant of induced sputum obtained from 59 asthma patients (mild: 20 patients; moderate: 20 patients; severe: 19 patients) were measured using ELISA. Sputum IL-32 mRNA expression was measured by RT- PCR. _Results:_ Sputum IL-32 was significantly elevated at the protein (_p= 0.00019_) and mRNA expression (_p &lt; 0.0001_) in asthmatics compared to non-asthmatic controls. IL-32 was elevated in severe asthmatic patients (_p &lt; 0.0001_) compared to mild-moderate asthma. Severe asthmatics allergic to house dust mites expressed higher sputum IL-32 level than severe asthmatics without allergy (_p = 0.0001_). A significant association was found between sputum IL-32 and IL-32 mRNA (r =0.531_; p = 0.0159_) in severe asthma. In the same way, sputum IL-1β and IL-32 were significantly correlated (r = 0.476; _p = 0.0337_). _Conclusion:_ The level of IL-32 in induced sputum may be associated with asthma severity.

IL17A mRNA Staining Distinguishes Palmoplantar Psoriasis from Hyperkeratotic Palmoplantar Eczema in Diagnostic Skin Biopsies

Acral dermatoses, including hyperkeratotic palmoplantar eczema (HPE), palmoplantar psoriasis (PP), and mycosis fungoides palmaris et plantaris (MFPP), can be challenging to diagnose clinically and histopathologically. In this setting, cytokine biomarkers may be able to help provide diagnostic clarity. Therefore, we evaluated IL-17A, IFN-γ, and IL-13 expression in PP, HPE, and MFPP and compared their expression profiles with nonacral sites. We used biopsy specimens from the Yale Dermatopathology database, selecting cases of HPE (n= 12), PP (n= 8), MFPP (n= 8), normal acral skin (n= 9), nonacral eczema (n= 10), and nonacral psoriasis (n= 10) with classic clinical and histopathologic features. IL17A mRNA expression by RNA in situ hybridization differentiated PP (median score 63.1 [interquartile range 9.4-104.1]) from HPE (0.8 [0-6.0]; P= 0.003), MFPP (0.6 [0-2.6]; P= 0.003), and normal acral skin (0 [0-0]; P < 0.001). Unexpectedly, both PP and HPE showed co-expression of IFNG and IL13 mRNA. In contrast, nonacral psoriasis and eczema showed divergent patterns of IFNG and IL13 mRNA expression. Taken together, we show that IL17A mRNA expression may be a useful biomarker of PP, and we further show that acral dermatoses exhibit distinct immunology compared to nonacral sites, with implications for clinical management.

PLGA nanoparticle with Amomum longiligulare polysaccharide 1 increased the immunogenicity of infectious bursal disease virus VP2 protein

ABSTRACT 1. The purpose of this study was to create ALP1-VP2-PLGA nanoparticle (AVPN) and to study the immunogenicity of AVPN. AVPN was prepared and observed by scanning and transmission electron microscopies. 2. Chickens were divided into five groups and vaccinated with normal saline, VP2 protein, ALP1 and VP2 protein, AVPN or PLGA, respectively. After 28 days, the immune organ indexes were calculated; specific antibody levels in blood were detected by enzyme-linked immunosorbent assay (ELISA). Additionally, the spleen and bursa of Fabricius were determined by HE staining, immunological cytokine mRNA levels in bursa of Fabricius were detected by qPCR andchicken body weight was determined. 3. The results indicated that AVPN was a spherical nanoparticle with a diameter of about 85 nm. It increased bursal indexes and IBDV-specific antibody levels and promoted the expression of IL-2 mRNA in blood and TNF-α and IgG mRNA in bursa of Fabricius. This promoted growth. 4. This study suggested that AVPN can increase immunogenicity of VP2 protein, and it could possibly be used as an IBDV subunit vaccine.

Analyses of Transcriptomics upon IL-1β-Stimulated Mouse Chondrocytes and the Protective Effect of Catalpol through the NOD2/NF-κB/MAPK Signaling Pathway

The overall objective of this study was to investigate the mechanism of inflammation on chondrocyte injury and the protective effect of catalpol on chondrocytes in an inflammatory environment. Chondrocytes were isolated and cultured from the knee joints of three-day-old newborn mice. Alcian Blue staining and the immunocytochemistry staining of type II collagen were used to identify the purity of chondrocytes. Primary chondrocytes were stimulated by IL-1β (10 ng/mL) and subjected to transcriptome analysis. Differentially expressed genes (DEGs) were further analyzed based on Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses. In this experimental study, we performed the viability assay to determine the effects of different concentrations of catalpol on the cell viability of chondrocytes. Chondrocytes were seeded in six-well plates and exposed to 10 μM catalpol 2 h prior to treatment with IL-1β (10 ng/mL). Quantitative real-time (qPCR) and Western blotting were performed to evaluate the RNA and protein expression, respectively. Based on the results of transcriptomics analysis, we found the NOD2 signaling pathway, the NF-kappa B signaling pathway, and the MAPK signaling pathway showed significant changes in chondrocyte damage caused by inflammation. Catalpol (10 μM and 100 μM) could significantly reduce NO, IL-6, IL-1β, and TNF-α in supernatant of chondrocytes. Catalpol significantly inhibited the mRNA expression of IL-1, IL-6, and IL-12 in chondrocytes induced by IL-1β. Catalpol markedly inhibited MMP3, MMP13 mRNA, and protein levels. Catalpol could significantly reduce TNF-α mRNA levels in inflammatory chondrocytes. Inflammation causes significant increases in mRNA levels and protein levels of NOD2, mRNA levels, and protein levels were markedly suppressed by catalpol. In addition, catalpol could significantly increase IKBα protein levels and significantly lower intranuclear P65 levels. Catalpol significantly lowered the phosphorylation protein levels of ERK, p38, and JNK. Our transcriptomic analysis demonstrated that the activation of NOD2 and its downstream pathways, NF-κB and MAPK, is an important cause of the inflammatory injury to chondrocytes induced by IL-1β. Catalpol inhibited the activation of the NOD2 signaling pathway, which reduced the phosphorylation of ERK, p38, and JNK, inhibited the degradation of IκBα, inhibited p65 translocation into the nucleus, reduced the release of inflammatory cytokines, and attenuated the inflammatory damage to chondrocytes.

Restoration of IL-11 and IL-15 cytokine production post calcium modulators and ROS treatment can assist viral clearance both in vitro and in vivo.

Hepatitis B virus (HBV) infection alters the cytokines production to establish persistent infection. A reversion of cytokines back to their normal state can be a promising therapeutic approach to establish an optimal host immune response. We investigated the alteration in expression of IL-15 and IL-11 after HBV infection in vitro and in vivo in PBMCs of 63 individuals divided into various HBV-infected patient groups. The mRNA expression was evaluated post-anti-oxidant and calcium modulators treatment by Real-time qPCR. In vitro mRNA expression of both cytokines, post-infection was down-regulated considerably. Interestingly, in line with in vitro results, both cytokines' in vivo expression was intensively down-regulated in chronic HBV-infected individuals rather than healthy controls. Both cytokines' expression was up-regulated in cases of recovery compared with the inactive carriers and chronic HBV-infected individuals. IL-15 mRNA expression was significantly up-regulated in both cell lines post EGTA and Ru360 treatment while a significant increase was observed in the HepAD38 cell line with NAC and BAPTA treatment. IL-11 mRNA expression was significantly up-regulated in the HepG2 cell line after all modulator treatments, whereas in the HepAd38 cell line it was observed after BAPTA treatment. Our results thus indicate that viral infection tends to down-regulate the expression of cytokines and an in vivo up-regulation is an indication of recovery. Treatment of anti-oxidants and calcium modulators has resulted in the successful restoration of these cytokines thus pointing towards the use of calcium modulators to boost natural antiviral cytokine production.

P009 IL-3 receptor signalling suppresses chronic intestinal inflammation by controlling mechanobiology and tissue egress of regulatory T cells

Abstract Background The pathogenesis of inflammatory bowel diseases (IBD) such as Crohn’s disease (CD) and ulcerative colitis (UC) is still incompletely understood. However, despite having been described back in the 1980s and reported to be involved in chronic inflammatory arthritis, the role of interleukin 3 (IL-3) signaling via the IL-3 receptor (IL-3R) in the development and perpetuation of chronic intestinal inflammation remains largely unexplored. In this study, we therefore aimed to explore the role of IL-3 signaling via IL-3R in experimental colitis as well as human IBD. Methods We analysed IL-3 expression in lamina propria mononuclear cells (LPMCs) from patients with IBD and from mice with experimental colitis. We compared the development of T cell transfer colitis in Rag1-/- mice after transfer of Il3r-/- and Il3r+/+ naïve T cells and investigated the underlying mechanisms by immunofluorescence and functional cell trafficking assays. The mechanical properties of Il3r-/- and Il3r+/+ T cells were determined by real-time deformability cytometry (RT-DC) and atomic force microscopy (AFM) and cytoskeleton structure was analysed by scanning electron microscopy (SEM) and fluorescence recovery after photobleaching (FRAP). Results The expression of IL-3 and IL3+ T cells were increased in the gut of patients with IBD and IL-3 expression was associated to shorter flare-free survival. In vivo, experimental chronic colitis upon T cell transfer was exacerbated in the absence of IL-3 or IL-3R signalling. This was attributable to IL-3-induced changes in kinase signalling and actin cytoskeleton structure, resulting in increased mechanical deformability and enhanced egress of regulatory T (Treg) cells from the inflamed colon mucosa. Similarly, IL-3 controlled mechanobiology in human Treg cells. (A) IL3 mRNA expression in colon tissue from patients.(B) Flare-free survival of IBD patients.(C) Colitis in Rag1–/– mice after transfer of naïve CD4+ T cells from Il3r+/+ and Il3r-/- mice. Mini-endoscopy (left) and IVIS (right).(D) RT-DC plot (left) and quantification (right) of thymic lymphocytes from Il3r-/- and Il3r+/+ mice.(E) Lightsheet microscopy (left) and quantification of TReg index (right, TReg fraction in Ozanimod/DATK32 per fraction in placebo) in mLNs from Rag1-/- mice with transfer colitis induced by Il3r-/- or Il3r+/+ T cells. Conclusion We uncover a crucial role of IL-3R signalling in regulating TReg mechanobiology and tissue egress. Together, our data demonstrate a central – probably counter-regulatory – role for IL-3 receptor signalling in Treg cells in the pathogenesis of murine and human chronic intestinal inflammation and therefore suggest that IL-3 might be a novel target for future therapeutic approaches in IBD.

α7 nicotinic acetylcholine receptor agonist improved brain injury and impaired glucose metabolism in a rat model of ischemic stroke.

Vagus nerve stimulation through the action of acetylcholine can modulate inflammatory responses and metabolism. α7 Nicotinic Acetylcholine Receptor (α7nAChR) is a key component in the biological functions of acetylcholine. To further explore the health benefits of vagus nerve stimulation, this study aimed to investigate whether α7nAChR agonists offer beneficial effects against poststroke inflammatory and metabolic changes and to identify the underlying mechanisms in a rat model of stroke established by permanent cerebral ischemia. We found evidence showing that pretreatment with α7nAChR agonist, GTS-21, improved poststroke brain infarction size, impaired motor coordination, brain apoptotic caspase 3 activation, dysregulated glucose metabolism, and glutathione reduction. In ischemic cortical tissues and gastrocnemius muscles with GTS-21 pretreatment, macrophages/microglia M1 polarization-associated Tumor Necrosis Factor-α (TNF-α) mRNA, Cluster of Differentiation 68 (CD68) protein, and Inducible Nitric Oxide Synthase (iNOS) protein expression were reduced, while expression of anti-inflammatory cytokine IL-4 mRNA, and levels of M2 polarization-associated CD163 mRNA and protein were increased. In the gastrocnemius muscles, stroke rats showed a reduction in both glutathione content and Akt Serine 473 phosphorylation, as well as an elevation in Insulin Receptor Substrate-1 Serine 307 phosphorylation and Dynamin-Related Protein 1 Serine 616 phosphorylation. GTS-21 reversed poststroke changes in the gastrocnemius muscles. Overall, our findings, provide further evidence supporting the neuroprotective benefits of α7nAChR agonists, and indicate that they may potentially exert anti-inflammatory and metabolic effects peripherally in the skeletal muscle in an acute ischemic stroke animal model.

Evaluation of the goat cellular immune response to rBtuB-Hia-FlgK peptides from Brucella melitensis

Brucellosis is a zoonosis caused by Brucella; B. melitensis is the most prevalent species in goats and humans. Previously, three B. melitensis peptides, rBtuB-Hia-FlgK showed antigen-specific immune responses in rodent models. The goal of this study was to evaluate the goat Th1/Th2 immune response to B. melitensis peptides. Twenty-eight animals were separated into four groups and were immunized with the rBtuB-Hia-FlgK peptides cocktail, adjuvant, PBS and Rev-1 vaccine, respectively. Peripheral blood samples were collected on days 0, 15, and 80 post-inoculation. The CD4+ and CD8+ T cells proliferation, and cytokine production of the Th-1 (IL-2, IL-12, TNF-α, and IFN-γ) and Th-2 profiles (IL-4, IL-5, and IL-10) were evaluated. An increase of CD4+/CD8+ at 15 days post-vaccination was observed and continued until the 80th. In addition, the IFN-γ, TNF-α, and IL-2 mRNA expression were typically induced by the 15th day, but only IFN-γ levels were observed at day 80 post-immunization. Brucella pathogenesis is distinguished by the presence of a large amount of Th-1 cytokines. Although a reduced amount of IFN-γ in the culture supernatant was accurately detected compared with Rev-1 after 15 days, it could be influenced by the sampling schedule, as a higher cytokine production might be induced as early as the first-week post-vaccination. The results indicate that rBtuB-Hia-FlgK induced an immune response similar to the Rev-1 vaccine. The possible use of inert molecules with the unique ability to typically induce cellular response similar to attenuated vaccine represents an attractive option that should not be ruled out.

Interleukin-33 Ameliorates Murine Systemic Lupus Erythematosus and Is Associated with Induction of M2 Macrophage Polarisation and Regulatory T Cells

The innate cytokine IL-33 is increasingly recognised to possess biological effects on various immune cells. We have previously demonstrated elevated serum level of soluble ST2 in patients with active systemic lupus erythematosus suggesting involvement of IL-33 and its receptor in the lupus pathogenesis. This study sought to examine the effect of exogenous IL-33 on disease activity of pre-disease lupus-prone mice and the underlying cellular mechanisms. Recombinant IL-33 was administered to MRL/lpr mice for 6 weeks, whereas control group received phosphate-buffered saline. IL-33-treated mice displayed less proteinuria, renal histological inflammatory changes, and had lower serum levels of pro-inflammatory cytokines including IL-6 and TNF-α. Renal tissue and splenic CD11b+ extracts showed features of M2 polarisation with elevated mRNA expression of Arg1, FIZZI, and reduced iNOS. These mice also had increased IL-13, ST2, Gata3, and Foxp3 mRNA expression in renal and splenic tissues. Kidneys of these mice displayed less CD11b+ infiltration, had downregulated MCP-1, and increased infiltration of Foxp3-expressing cells. Splenic CD4+ T cells showed increased ST2-expressing CD4+Foxp3+ population and reduced IFN-γ+ population. There were no differences in serum anti-dsDNA antibodies and renal C3 and IgG2a deposit in these mice. Exogenous IL-33 was found to ameliorate disease activity in lupus-prone mice with induction of M2 polarisation, Th2 response, and expansion of regulatory T cells. IL-33 likely orchestrated autoregulation of these cells through upregulation of ST2 expression.