Rat Dendritic Cells Research Articles

Vitamin D3 alleviates intestinal injury in necrotizing enterocolitis and lipopolysaccharide-induced inflammatory response in dendritic cells in rats.

Necrotizing enterocolitis (NEC) is a serious condition that predominantly affects premature infants and involves an aberrant immune response and inflammatory cytokine release resulting in intestinal epithelial damage. The current study investigated the immunoregulatory effects of vitamin D3 on the maturation and activation of dendritic cells (DCs) and the antiinflammatory impact on the intestines in a neonatal rat model of NEC.Materials and methods: Inflammatory damage to intestinal tissue was assessed via morphological changes and apoptosis and DC expression of costimulatory molecules, inflammatory factors, and immunoregulatory factors by immunohistochemical staining, quantitative real-time PCR, and immunofluorescence. The fluorescein isothiocyanate-ovalbumin (FITC-OVA) uptake assay was used to analyze DC endocytosis. Vitamin D3 administration attenuated intestinal damage and apoptosis, inhibiting CD86 and increasing CD80 expression. Lipopolysaccharide (LPS)-challenged DC2.4 cells in vitro showed upregulated CD86, tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), inducible nitric oxide synthase (iNOS), and indoleamine 2,3-dioxygenase 1 (IDO-1) expression, which were all reduced by vitamin D3, except for IDO-1. LPS inhibited CD80 expression, which was restored by vitamin D3 treatment, and endocytic capacity was improved. Vitamin D3 ameliorated intestinal damage in neonatal rats with NEC and exerted antiinflammatory and immunomodulatory effects on DCs. Vitamin D3 has potential as a supplementary treatment for NEC patients.

Glomerulonephritis and autoimmune vasculitis are independent of P2RX7 but may depend on alternative inflammasome pathways.

P2RX7, an ionotropic receptor for extracellular adenosine triphosphate (ATP), is expressed on immune cells, including macrophages, monocytes, and dendritic cells and is upregulated on nonimmune cells following injury. P2RX7 plays a role in many biological processes, including production of proinflammatory cytokines such as interleukin (IL)‐1β via the canonical inflammasome pathway. P2RX7 has been shown to be important in inflammation and fibrosis and may also play a role in autoimmunity. We have developed and phenotyped a novel P2RX7 knockout (KO) inbred rat strain and, taking advantage of the human‐resembling unique histopathological features of rat models of glomerulonephritis, we induced three models of disease: nephrotoxic nephritis, experimental autoimmune glomerulonephritis, and experimental autoimmune vasculitis. We found that deletion of P2RX7 does not protect rats from models of experimental glomerulonephritis or the development of autoimmunity. Notably, treatment with A‐438079, a P2RX7 antagonist, was equally protective in WKY WT and P2RX7 KO rats, revealing its ‘off‐target’ properties. We identified a novel ATP/P2RX7/K+ efflux‐independent and caspase‐1/8‐dependent pathway for the production of IL‐1β in rat dendritic cells, which was absent in macrophages. Taken together, these results comprehensively establish that inflammation and autoimmunity in glomerulonephritis is independent of P2RX7 and reveals the off‐target properties of drugs previously known as selective P2RX7 antagonists. Rat mononuclear phagocytes may be able to utilise an ‘alternative inflammasome’ pathway to produce IL‐1β independently of P2RX7, which may account for the susceptibility of P2RX7 KO rats to inflammation and autoimmunity in glomerulonephritis. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

IFNγ-stimulated dendritic cell extracellular vesicles can be nasally administered to the brain and enter oligodendrocytes.

Extracellular vesicles secreted from IFNγ-stimulated rat dendritic cells (referred to here as IFNγ-DC-EVs) contain miRNAs which promote myelination (including but not limited to miR-219), and preferentially enter oligodendrocytes in brain slice cultures. IFNγ-DC-EVs also increase myelination when nasally administered to naïve rats. While we can infer that these extracellular vesicles enter the CNS from functional studies, here we demonstrate biodistribution throughout the brain after nasal delivery by way of imaging studies. After nasal administration, Xenolight DiR-labelled IFNγ-DC-EVs were detected 30 minutes later throughout the brain and the cervical spinal cord. We next examined cellular uptake of IFNγ-DC-EVs by transfecting IFNγ-DC-EVs with mCherry mRNA prior to nasal administration. mCherry-positive cells were found along the rostrocaudal axis of the brain to the brainstem. These cells morphologically resembled oligodendrocytes, and indeed cell-specific co-staining for neurons, astrocytes, microglia and oligodendrocytes showed that mcherry positive cells were predominantly oligodendrocytes. This is in keeping with our prior in vitro results showing that IFNγ-DC-EVs are preferentially taken up by oligodendrocytes, and to a lesser extent, microglia. To confirm that IFNγ-DC-EVs delivered cargo to oligodendrocytes, we quantified protein levels of miR-219 mRNA targets expressed in oligodendrocyte lineage cells, and found significantly reduced expression. Finally, we compared intranasal versus intravenous delivery of Xenolight DiR-labelled IFNγ-DC-EVs. Though labelled IFNγ-DC-EVs entered the CNS via both routes, we found that nasal delivery more specifically targeted the CNS with less accumulation in the liver. Taken together, these data show that intranasal administration is an effective route for delivery of IFNγ-DC-EVs to the CNS, and provides additional support for their development as an EV-based neurotherapeutic that, for the first time, targets oligodendrocytes.

A Hybrid Glioma Tumor Cell Lysate Immunotherapy Vaccine Demonstrates Good Clinical Efficacy in the Rat Model.

BackgroundConventional immunotherapy for glioma is not only expensive but also demonstrates less-than-desired clinical efficacy. In this study, we evaluated the immunotherapeutic efficacy of a tumor cell lysate-based hybrid glioma vaccine developed using a molecular-based approach.MethodsFirst, the ability of the autologous (9L-cell lysate) and allogeneic (C6-cell lysate) vaccines against glioma, individually and in combination, to activate Fischer344 rat dendritic cells (DCs) was determined. Next, the activated DCs were co-cultured with T lymphocytes and screened for the optimal DC-to-T-cell ratio. The in vitro efficacy of the DC/T-cell vaccine formulations subjected to different immunogen treatments and co-cultured with glioma cells was evaluated based on glioma cell viability and monocyte chemoattractant protein (MCP)-2 and interferon (IFN)-γ secretion. Subsequently, the efficacy of the 9L + C6 hybrid vaccine was evaluated in 32 glioma rat models, randomly allocated to the following five treatment groups: blank control, tumor, vaccine treatment, thymosin treatment, and vaccine + thymosin treatment (combined treatment). Changes in survival duration, intracranial tumor volume, peripheral blood immune-cell (CD4+ T, CD8+ T, and natural killer [NK] cell) count, and serum cytokine (interleukin [IL]-2, IL-10) levels were assessed in these groups.ResultsThe hybrid vaccine demonstrated the highest glioma cell apoptosis and the lowest cell viability and promoted MCP-2 and IFN-γ secretion in vitro. The vaccine treatment and combined treatment groups demonstrated longer survival duration, lower intracranial tumor volume, and higher immune cell glioma tissue infiltration and IL-2 secretion than the untreated tumor group, indicating the vaccine’s good in vivo efficacy. Thymosin treatment had minimal effect in enhancing anti-glioma immunity.ConclusionWe demonstrated the feasibility of combining autologous and allogeneic tumor cell lysates to stimulate specific host cell immune response against glioma cells. The good clinical efficacy of our developed glioma hybrid vaccine in rat models suggests its potential clinical application.

Determination of the quantity and topography dynamics of the LCA+-dendritic cells in rat thymus in early postnatal period in norm and after the prenatal administration of the dexamethasone

Purpose was to study the dynamics of LCA + -dendritic thymus cells in newborn rats in the postnatal period after administration of dexamethasone in the intrauterine period. Materials and methods. The study was performed on 144 white nonlinear rats on the 1 st –3 rd , 5 th , 9 th , 14 th , 21 st , 30 th days after birth. There are 3 groups with 48 rats in each group: 1 – intact group; 2 – experimental group of rats, who received peritoneal injection of 0.05 ml of dexamethasone (in dilution 1:40) on the 18 th day of the prenatal period, and a control group of rats receiving 0.05 ml of 0.9 % NaCl according to M. A. Voloshyn. To study the dynamics of dendritic cells in the thymus carrying the α-D mannose receptors, LCA-lectin was used. Results . It has been established that the maximum content of LCA + -dendritic thymus cells in the intact (3.92 ± 0.65), experimental (3.41 ± 0.49) and control (4.07 ± 0.53) groups occurs on the 1 st day after birth. The minimum number of LCA + -dendritic cells was detected on the 5 th day in the intact (1.88 ± 0.37) and control (1.73 ± 0.34) groups, and their minimum in the experimental group (2.35 ± 0.48) was determined on the 30 th day after birth. In the experimental group from the 5 th day to the 9 th day a significantly higher number of LCA + -dendritic cells than in the comparison groups was detected. Conclusions. The introduction of dexamethasone in the prenatal period leads to the significant increase in the LCA + -dendritic cell content in the experimental group from the 5 th to the 9 th day of life, which indirectly coincides with the increase in the number of peripheral and mitotic dividing lymphocytes in the thymus of rats in the postnatal period.

Immunomodulatory potential of particular Trichinella spiralis muscle larvae excretory–secretory components

Excretory–secretory antigens of Trichinella spiralis muscle larvae can induce the semi-matured status of rat dendritic cells. This may at least partly be the consequence of transient activation of extracellular signal-regulated kinases 1/2 (ERK1/2). Here we investigated the potential of several components of excretory–secretory antigens (native fraction containing 45, 49 and 53kDa proteins and recombinant Tsp53, representing one of the constituents of this fraction) to demonstrate previously observed effects of excretory–secretory antigens on dendritic cells in vitro, characterised by establishment of a particular phenotype (very low MHC II expression, moderate CD86 expression and significant ICAM-1 expression) and functional properties (low production of pro-inflammatory cytokine IL-12p70, and high production of IL-10 and TGF-β). Dendritic cells activated by these components were able to provoke proliferation of naïve T cells and their polarisation towards Th2 and anti-inflammatory responses. The investigated antigens had almost the same capacity to induce IL-4 and IL-10 production from T cells as excretory–secretory antigens, but failed to induce significant TGF-β synthesis. It could be concluded that the investigated excretory–secretory antigens components can largely reproduce the immunomodulatory effects of the complete excretory–secretory antigens and therefore may be considered as molecules important for creation of the anti-inflammatory milieu achieved by the parasite.

Effects of ganoderma lucidum polysaccharide peptide on phenotypic and functional maturation of dendritic cells from rat peripheral blood

Objective To investigate the effects of ganoderma (GL-PP) lucidum polysaccharide peptide on the phenotype and function of rat dendritic cells (DC). Methods Monocytes isolated from rat peripheral blood were induced into DC by being cultured with granulocyte macrophage-colony stimulating factor (GM-CSF) and interleukin (IL)-4. Flow cytometry, enzyme linked immunosorbent assay (ELISA) and mixed lymphocyte response were used to determine the effect of GL-PP on DC. Results The ratio of cell uptake fluoresceine isothiocyanate (FITC)- dextran (FITC-dextran) in GL-PP group [(35.33±4.62)%, (28.18±3.84)%, (26.52±2.01)%] was significantly lower than the control group [(58.42±3.84)%, P<0.01]; The expression of surface molecules major histocompatibility complex (MHC)-Ⅱ, CD40, CD80 and CD86[(92.6±1.42)%, (33.90±1.25)%, (89.97±1.19)%, (88.93±0.66)%] in GL-PP group (12.5 μg/ml) was significantly higher than the control group [(79.43±0.80)%, (5.47±0.21)%, (76.63±0.51)%, (68.63±0.78)%, P<0.01]. The allogeneic T cell-stimulatory capacity of DC in GL-PP group [(110.77±3.53)%, (112.14±4.33)%, (110.77±3.56)%] was enhanced compare with the control group [(100.00±1.78)%, P<0.01]. In addition, GL-PP could promote secretion of interleukin (IL)-12p70 and IL-10 from DC. Conclusion These data suggest that GL-PP promotes the phenotypic and functional maturation of DCs. Key words: Ganoderma lucidum polysaccharide; Dendritic cells; Immunomodulatory

Estradiol enhances capacity of TLR-matured splenic dendritic cells to polarize CD4+ lymphocytes into IL-17/GM-CSF-producing cells in vitro

There are little data on modulatory effects of estrogens on rat dendritic cell (DC) responses to inflammatory stimuli, and consequently their ability to activate and polarize CD4+ T lymphocyte-mediated immune responses. Splenic conventional DCs from young female Albino Oxford rats were activated in vitro with LPS (TLR4 agonist) or R848 (TLR7/8 agonist) in the presence and absence of 17β-estradiol (E2), and their allostimulatory and CD4+ lymphocyte polarizing ability in mixed leukocyte culture (MLC) were studied. Irrespective of the E2 presence, LPS and R848 up-regulated the expression of MHC II on DCs, so they exhibited enhanced allostimulatory capacity in co-culture with CD4+ lymphocytes. On the other hand, E2 promoted stimulatory action of both TLRs on OX62+ DC IL-23 production, augmented their stimulatory effects on IL-6 and IL-1β production, but diminished their enhancing effects on the expression IL-10 and IL-27 by DCs. Consequently, in MLC, OX62+ DCs activated/matured in the co-presence of E2 and either LPS or R848 increased the levels of IL-17, the signature Th17 cell cytokine, when compared with those activated/matured in the absence of E2. GM-CSF levels were also increased in these MLC. Given that the expression of IL-7 mRNA was diminished in DCs activated/matured in the co-presence of E2 and TLR, this increase most likely did not reflect enhanced differentiation of Th cells producing GM-CSF only (Th-GM). ConclusionsE2 augments capacity of LPS- and R848-activated/matured DCs from young rat spleen to induce differentiation of IL-17- and GM-CSF-producing cells.

Effects of gene silence on immune function of colonic dendritic cells in rats with irritable bowel syndrome visceral hypersensitivity

Objective To explore the role and mechanism of protein disulfide isomerase A3 (PDIA3) in abnormal colonic mucosa immune response of irritable bowel syndrome (IBS) rats with visceral hypersensitivity. Methods A total of 48 SD rats were divided into blank control group (n=12), empty virus group (IBS-1) (n=12), PDIA3 silencing group (IBS-2) (n=12) and model control group(IBS-3) (n=12). The expression of CD103 in ileocecus colonic tissues was detected by immunohistochemistry. Dendritic cells (DC) of mesenteric lymph node were isolated by flow cytometry. CD4+ /CD8+ T cells of spleen were separated by immune-magnetic beads sorting technique. DC and CD4+ /CD8+ T cells were co-cultured. The proliferation ability of lymphocytes promoted by DC was measured by methyl thiazolyl tetrazolium (MTT). The secretion levels of interlcukin (IL)-4 and IL-9 of CD4+ /CD8+ T cells stimulated by DC were determined by enzyme linked immunosorbent assay (ELISA) method. Independent sample t-test was performed for statistical analysis. Results The cell number of CD103 positive DC of rats colon of blank control group, IBS-3 group was 6.25±1.14 and 10.83±1.03(t=10.07, P<0.05); that of IBS-2 group was 7.42±0.90, and compared with that of IBS-3 group, the difference was statistically significant (t=-9.25, P<0.05). The MTT value of CD4+ T cells proliferation stimulated by DC of blank control group and IBS-3 group was 0.54±0.01 and 0.60±0.01(t=3.373, P<0.05); that of IBS-2 group was 0.53±0.01, and compared with that of IBS-3 group, the difference was statistically significant (t=-3.139, P<0.05). The MTT value of CD8+ T cells proliferation stimulated by DC was 0.52±0.01 and 0.59±0.00(t=3.539, P<0.01); that of IBS-2 group was 0.54±0.01, and compared with that of IBS-3 group, the difference was statistically significant (t=-3.183, P<0.05). The level of IL-4 secreted by CD4+ T cells promoted by DC of blank control group and IBS-3 group was 10.24±0.09 and 16.61±1.00 (t=3.222, P<0.05); that of IBS-2 group was 11.75±0.54, and compared with that of IBS-3 group, the difference was statistically significant (t=-3.539, P<0.01). The level of IL-9 secreted by CD4+ T cells stimulated by DC of blank control group and IBS-3 group was 15.86±10.19 and 43.51±11.32 (t=4.529, P<0.05); that of IBS-2 group was 29.05±2.09, and compared with that of IBS-3 group, the difference was statistically significant (t=-6.841, P<0.01). The level of IL-4 secreted by CD8+ T cells promoted by DC of blank control group and IBS-3 group was 7.35±0.12 and 13.91±0.57 (t=19.557, P<0.01); that of IBS-2 group was 8.63±0.24, and compared with that of IBS-3 group, the difference was statistically significant (t=-14.782, P<0.01). The level of IL-9 secreted by CD8+ T cells stimulated by DC of blank control group and IBS-3 group was 29.12±5.14 and 60.70±11.02 (t=4.122, P<0.05); that of IBS-2 group was 37.17±2.65, and compared with that of IBS-3 group, the difference was statistically significant (t=-3.255, P<0.05). Conclusions Protein disulfide isomerase A3 may mediate the process of DC activating T lymphocyte levels of related cytokines, cause abnormal immune response of colonic mucosa and promote the genesis of IBS visceral hypersensitivity. Key words: Protein disulfide isomrase A3; Dendritic cells; Interleukin-4; Interleukin-9; CD4-positive T-lymphocytes; CD8-positive T-lymphocytes; Visceral hypersensitivity

Three distinct subsets of thymic epithelial cells in rats and mice defined by novel antibodies.

AimThymic epithelial cells (TECs) are thought to play an essential role in T cell development and have been detected mainly in mice using lectin binding and antibodies to keratins. Our aim in the present study was to create a precise map of rat TECs using antibodies to putative markers and novel monoclonal antibodies (i.e., ED 18/19/21 and anti-CD205 antibodies) and compare it with a map from mouse counterparts and that of rat thymic dendritic cells.ResultsRat TECs were subdivided on the basis of phenotype into three subsets; ED18+ED19+/−keratin 5 (K5)+K8+CD205+ class II MHC (MHCII)+ cortical TECs (cTECs), ED18+ED21−K5−K8+ Ulex europaeus lectin 1 (UEA-1)+CD205− medullary TECs (mTEC1s), and ED18+ED21+K5+K8dullUEA-1−CD205− medullary TECs (mTEC2s). Thymic nurse cells were defined in cytosmears as an ED18+ED19+/−K5+K8+ subset of cTECs. mTEC1s preferentially expressed MHCII, claudin-3, claudin-4, and autoimmune regulator (AIRE). Use of ED18 and ED21 antibodies revealed three subsets of TECs in mice as well. We also detected two distinct TEC-free areas in the subcapsular cortex and in the medulla. Rat dendritic cells in the cortex were MHCII+CD103+ but negative for TEC markers, including CD205. Those in the medulla were MHCII+CD103+ and CD205+ cells were found only in the TEC-free area.ConclusionBoth rats and mice have three TEC subsets with similar phenotypes that can be identified using known markers and new monoclonal antibodies. These findings will facilitate further analysis of TEC subsets and DCs and help to define their roles in thymic selection and in pathological states such as autoimmune disorders.

Exogenous IL-10 induces corneal transplantation immune tolerance by a mechanism associated with the altered Th1/Th2 cytokine ratio and the increased expression of TGF-β

The aim of the present study was to examine the effect of exogenous IL-10 transfected rat dendritic cells (DCs) in corneal allografts. Rat lymphocyte separation medium and a cytokine induction method was used to extract and culture precursor cells of rat bone marrow-derived dendritic cells. A corneal transplant model was established, with Sprague-Dawley (SD) rats as the recipients and Wistar rats as the donors. Flow cytometry (FCM) was used to detect the expression of CD83, which indicates a mature dendritic cell, and a specific cell surface stimulatory molecule CD86. Western blot analysis was used to detect interleukin (IL)-10 protein expression and RT-PCR was used to detect cytokine IL-10, IL-2 and TGF-β mRNA expression in each group. Compared with the other groups, the survival time of corneal grafts in the IL-10-green fluorescent protein (GFP)-DC group was significantly prolonged and H&E staining demonstrated mild graft edema and inflammatory cell infiltration. There was a high expression of IL-10 and a low expression of the surface antigens, CD83 and CD86, with a lower proliferation of T lymphocytes in the IL-10-GFP-DC group. The expression of IL-10 and TGF-β in the IL-10-GFP-DC group was higher than that in the other groups, while the expression of IL-2 was lower. The transfection of the IL-10 gene inhibited dendritic cell maturation. IL-10 gene-modified immature dendritic cells (imDC) were able to inhibit corneal allograft rejection and induce immune tolerance to prolong the survival time of the corneal graft. The Th1/Th2 deviation and the high expression of TGF-β may lead to immune tolerance.

Cytomegalovirus Expresses the Chemokine Homologue vXCL1 Capable of Attracting XCR1 + CD4 − Dendritic Cells

Cytomegaloviruses (CMV) have developed various strategies to escape the immune system of the host. One strategy involves the expression of virus-encoded chemokines to modulate the host chemokine network. We have identified in the English isolate of rat CMV (murid herpesvirus 8 [MuHV8]) an open reading frame encoding a protein homologous to the chemokine XCL1, the only known C chemokine. Viral XCL1 (vXCL1), a glycosylated protein of 96 amino acids, can be detected 13 h postinfection in the supernatant of MuHV8-infected rat embryo fibroblasts. vXCL1 exclusively binds to CD4(-) rat dendritic cells (DC), a subset of DC that express the corresponding chemokine receptor XCR1. Like endogenous rat XCL1, vXCL1 selectively chemoattracts XCR1(+) CD4(-) DC. Since XCR1(+) DC in mice and humans have been shown to excel in antigen cross-presentation and thus in the induction of cytotoxic CD8(+) T lymphocytes, the virus has apparently hijacked this gene to subvert cytotoxic immune responses. The biology of vXCL1 offers an interesting opportunity to study the role of XCL1 and XCR1(+) DC in the cross-presentation of viral antigens.

Immunomodulatory effects of polysaccharides isolated from Hericium erinaceus on dendritic cells

Hericium erinaceus (H. erinaceus; HE) polysaccharides (HE-PS) have been shown to have immunomodulatory activity. We found that the bioactive components of β-glucan derivatives consisted of 20% in HE-PS. We used an analytic platform for investigating the effects of HE-PS on the maturation of rat dendritic cells (DCs), which are derived from rat bone marrow hematopoietic cells (BMHCs). The results showed that treatment with 50μg/mL HE-PS changed the morphology of the DCs to an active form in parallel with a significant two fold increase in MHC class II and CD80/86 surface antigens compared to the control. Furthermore, endocytosis by the DCs was significantly reduced at the same dosage. IL-12, IFN-γ and IL-10 cytokine secretion was significantly increased by 2.7, 1.5 and 1.6-fold, respectively, compared to the control after treatment with 50μg/mL of HE-PS. This study used a powered analysis platform to show that HE-PS induces DCs activation and modulates the TH1 immune response. Thus, HE-PS has potential as an immunopotentiating agent that could be further developed in the health food industry.

OP0238 Patients with Ankylosing Spondylitis Have an Increased Proportion of CD16+ Dendritic Cells, Able to Induce CCR6-Expression on CD4+ T Cells

BackgroundAnkylosing Spondylitis (AS) is a chronic systemic inflammatory disease primarily affecting the axial skeleton. The genetic association between AS and HLA-B27 was discovered 40 years ago, however the pathogenic role...

OX62+OX6+OX35+ rat dendritic cells are unable to prime CD4+ T cells for an effective immune response following acute burn injury

Co-stimulatory molecules expressed on Dendritic Cells (DCs) function to coordinate an efficient immune response by T cells in the peripheral lymph nodes. We hypothesized that CD4+ T cell-mediated immune suppression following burn injury may be related to dysfunctional DCs residing in gut associated lymphoid tissues (GALT), such as Mesenteric Lymph Nodes (MLN). Therefore, we studied co-stimulatory molecules expressed on burn rat MLN DCs as an index of functional DCs that would mount an effective normal CD4+ T cell immune response. In a rat model of 30% Total Body Surface Area (TBSA) scald burn, OX62+OX6+OX35+ DCs and CD4+ T cells were isolated from MLN of day 3 post-burn and sham control rats. DCs were tested for their expression of co-stimulatory molecules, and prime CD4+ T cell (DC:CD4+T cell co-culture assays) to determine an effector immune response such as CD4+ T cell proliferation. The surface receptor expressions of MLN DCs co-stimulatory molecules, i.e., MHC-II, CD40, CD80 (B7-1), and CD86 (B7-2) were determined by Flow cytometry (quantitatively) and confocal microscopy (qualitatively). Tritiated thymidine and CFDA-SE determined CD4+ T cell proliferation following co-incubation with DCs. Cytokine milieu of MLN (IL-12 and IL-10) was assessed by mRNA determination by RT-PCR. The results showed down-regulated expressions of co-stimulatory markers (CD80, CD86, CD40 and MHC-II) of MLN DCs obtained from burn-injured rats, as well as lack of ability of these burn-induced DCs to stimulate CD4+ T cell proliferation in co-culture assays, as compared to the sham rats. Moreover, anti-CD40 stimulation of affected burn MLN DCs did not reverse this alteration. Furthermore, a marked up-regulation of mRNA IL-10 and down-regulation of mRNA IL-12 in burn MLN as compared to sham animals was also observed. To surmise, the data indicated that dysfunctional OX62+OX6+OX35+ rat MLN DCs may contribute to CD4+ T-cell-mediated immune suppression observed following acute burn injury.

Age-associated shift in rat dendritic cell T-helper polarizing capacity

Event Abstract Back to Event Age-associated shift in rat dendritic cell T-helper polarizing capacity Biljana Bufan1, Zorica Stojić-Vukanić1, Nevena Arsenović-Ranin1, Duško Kosec2, Ivan Pilipovic2, Milica Perišić2, Jasmina Đikić3 and Gordana Leposavić3* 1 University of Belgrade-Faculty of Pharmacy, Department of Microbiology and Immunology, Serbia 2 Institute of Virology, Vaccines and Sera "Torlak" , Immunology Research Centre "Branislav Jankovic" , Serbia 3 University of Belgrade-Faculty of Pharmacy, Department of Physiology, Serbia Almost all cellular components of innate and adaptive immunity undergo age-related remodeling. The findings on age-related changes in human and mouse dendritic cells (DCs) are conflicting, whereas there is no data on the influence of aging on rat DCs. In attempt to fill this gap, freshly isolated splenic conventional OX62+ DCs from 3- (young) and 26-month-old (aged) Albino Oxford rats were examined for subset composition, cell surface expression of activation markers (CD80, CD86 and CD40 and MHC II molecules) and endocytic capacity using flow cytometric analysis (FCA). In addition, splenic OX62+ DCs isolated from rats of both ages were cultured in the presence or in the absence of LPS. These cells were examined for the activation marker and TNF-α, IL-6, IL-12, IL-23, TGF-β1, IL-10 expression using FCA, and RT-PCR and ELISA, respectively. Moreover, the allostimulatory capacity of OX62+ DCs and allogeneic CD4+ T cell cytokine (IFN-γ, IL-4 and IL-17) production in MLR was quantified using FCA and ELISA, respectively. It was found that aging: i) in OX62+ DCs population leads to a shift in CD4+:CD4- cell ratio towards CD4- cells and ii) influences OX62+ DCs maturation capacity (judging by activation marker expression and efficiency of endocytosis) by affecting action of intrinsic (TNF-α and IL-10) and extrinsic regulatory factor expression. Furthermore, in LPS-matured OX62+ DCs from aged rats TNF-α, IL-12, IL-23 and IL-6 expression was increased, while IL-10 expression was diminished. Moreover, in MLR, OX62+ DCs from aged rats exhibited enhanced Th1/Th17 driving force and diminished allostimulatory capacity. Acknowledgements This work was supported by the Ministry of Education, Science and Technological Development of the Republic of Serbia [grant number 175050]. Keywords: rat, splenic conventional dendritic cells, Aging, Cytokine expression, Th polarization Conference: 15th International Congress of Immunology (ICI), Milan, Italy, 22 Aug - 27 Aug, 2013. Presentation Type: Abstract Topic: Innate immunity Citation: Bufan B, Stojić-Vukanić Z, Arsenović-Ranin N, Kosec D, Pilipovic I, Perišić M, Đikić J and Leposavić G (2013). Age-associated shift in rat dendritic cell T-helper polarizing capacity. Front. Immunol. Conference Abstract: 15th International Congress of Immunology (ICI). doi: 10.3389/conf.fimmu.2013.02.00138 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 11 Mar 2013; Published Online: 22 Aug 2013. * Correspondence: Prof. Gordana Leposavić, University of Belgrade-Faculty of Pharmacy, Department of Physiology, Belgrade, Serbia, gordana.leposavic@pharmacy.bg.ac.rs Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Biljana Bufan Zorica Stojić-Vukanić Nevena Arsenović-Ranin Duško Kosec Ivan Pilipovic Milica Perišić Jasmina Đikić Gordana Leposavić Google Biljana Bufan Zorica Stojić-Vukanić Nevena Arsenović-Ranin Duško Kosec Ivan Pilipovic Milica Perišić Jasmina Đikić Gordana Leposavić Google Scholar Biljana Bufan Zorica Stojić-Vukanić Nevena Arsenović-Ranin Duško Kosec Ivan Pilipovic Milica Perišić Jasmina Đikić Gordana Leposavić PubMed Biljana Bufan Zorica Stojić-Vukanić Nevena Arsenović-Ranin Duško Kosec Ivan Pilipovic Milica Perišić Jasmina Đikić Gordana Leposavić Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Immunomodulating Activity of Nymphaea rubra Roxb. Extracts: Activation of Rat Dendritic Cells and Improvement of the TH1 Immune Response

Polysaccharides play a key role in enhancing immune function and facilitating cellular communication. Here, we purified Nymphaea rubra Roxb. polysaccharides (NR-PS) by treating them with pullulanase. They were then cultured with immature dendritic cells (DCs) derived from rat bone marrow hematopoietic cells (BMHCs). After treatment with bioactive NR-PS with a degree of polymerization (DP) value of 359.8, we found that the DCs underwent morphological changes indicative of activation. CD80/86 (87.16% ± 8.49%) and MHC class II (52.01% ± 10.11%) expression levels were significantly up-regulated by this treatment compared to the controls (65.45% ± 0.97% and 34.87% ± 1.96%). In parallel, endocytosis was also reduced (167.94% ± 60.59%) after treatment with 25 μg/mL of NR-PS as measured by the medium fluorescence intensity compared to the control (261.67% ± 47.26%). Furthermore, the DCs after treatment with 25 μg/mL NR-PS showed increased IL-12 (102.09 ± 10.16 to 258.78 ± 25.26 pg/mL) and IFN-γ (11.76 ± 0.11 to 15.51 ± 1.66 pg/mL) secretion together with reduced IL-10 secretion (30.75 ± 3.35 to 15.37 ± 2.35 pg/mL), which indicates a TH1 immune response. In conclusion, NR-PS exhibits stimulatory effects on rat DCs and promotes the secretion of TH1 cytokines. Taken together, our studies are the first to show that NR-PS is an immunomodulator affecting the maturation and functioning of DCs.

Composition analysis and immuno-modulatory effect of okra (Abelmoschus esculentus L.) extract

The aim of this study was to analyse the composition of okra (Abelmoschus esculentus L.) extract and investigate the effect of A. esculentus L. polysaccharides (AE-PS) on the maturation and function of dendritic cells (DCs) derived from rat bone marrow hematopoietic cells (BMHCs) in vitro. BMHC-derived immature DCs (BMHC-imDCs) were extracted from rats and treated with AE-PS. The hydrolysed okra extract contained 0.6% β-1, 3-D-glucan. AE-PS induced the presence of polymorphic nuclei and elongated protrusion in the BHMC-imDCs, indicating DC activation. Treatment with 100 μg/mL of AE-PS increased the MHC class II and CD80/86 expression levels by 41% and 42%, respectively. Treated cells had reduced endocytosis activity. The secretion of IL-12 and IFN-γ increased significantly by 120% and 75%, respectively, when treated with 100 μg/mL of AE-PS. Moreover, IL-10 production was reduced by 66%. In conclusion, AE-PS exhibits stimulatory effects on rat dendritic cells and promotes the secretion of T(H)1 cytokines.

Inhibition of T-cell proliferation by induction of IDO in spleen-derived dendritic cells of rats

Objective To investigate the expression chenges of indoleamine 2,3-dioxygenase (IDO) mRNA and protein in dendritic cells (DCs) derived from rat spleen in vitro as well as the effect of IDO on proliferation of allogeneic T lymphocytes in terms of different concentrations of interferon (IFN)-γ.Methods We first extracted DCs from the spleen of rats,then measured the expression of DC-specific molecules and surface molecules CD80 and CD86 in rats by flow cytometry.DCs were induced by IFN-γ at differenct concentrations (0,100,300,500 U/ml).Real-time polymerase chain reactio (PCR) and Western blotting were used to detect the relative expression levels of IDO mRNA and protein in DCs.Allogeneic mixed lymphocyte reaction (MLR) was applied to test the effect of DCs induced with different concentrations of IFN-γ on allogeneic T lymphocyte proliferation.Results The results showed that the spleenderived DCs from rats cultured for 10 days had a typical dendritic morphology,whose expression rate of OX62 was above 80％ and positive expression rate of CD80 and CD86 was more than 75％ and 90％ respectively.The expression levels of IDO mRNA ( 1.010 ±0.094,1.340 ±0.114,1.700 ±0.087,2.080±0.150) and protein (0.861 ±0.612,1.155 ±0.059,1.308 ±0.068,1.755 ±0.118) were increased gradually with the increase of concentrations of IFN-γ,which showed significant difference between any two groups.As compared with the control group,the groups induced by the IFN-γ had a decrease in T lymphocyte proliferation of DCs (P ＜ 0.05 ).Besides,with the increased concentration of IFN-γ,the rate of T lymphocyte proliferation was decreased gradually and there was significant difference between ant two groups (P ＜0.05).Conclusion The IFN-γ can induce the increased expression of IDO in spleen-derived DCs from rats,and the decreased stimulation capacity of spleen DCs to the antigen-specific T cell response. Key words: Dendritic cell; Indoleamine 2,3-dioxygenase; γ-interferon; T lymphocyte

The association of non-specific chronic inflammation with dendritic cells in a rat model of chronic obstructive pulmonary disease

To evaluate the changes in the number and maturation of lung tissue dendritic cells (DCs) and to assess the chronic inflammation in a cigarette smoke-induced COPD model in rats. Thirty male F344 rats were randomly divided into 3 groups (n = 10): a control group, a smoke-exposure group and a smoking cessation group. Rat lung pathomorphological changes were observed by hematoxylin-eosin (HE) stain. Lung tissue CD(11c)(+) DCs, CD(86)(+) DCs and CD(8)(+) T cell numbers were observed by immunohistochemistry method. Flow cytometry was used for detection of CD(11c)(+)/CD(86)(+) DCs and CD(11c)(+)/MHCII(+) DCs proportions. The airway inflammatory pathological score and the mean linear intercept (MLI) obtained from he smoke-exposure group and the smoking cessation group (390 ± 33, 324 ± 28) and [(68 ± 11) µm, (58 ± 9) µm] were higher than those in the control group (56 ± 13) and (36 ± 6) µm (F = 459.85 and 34.03, all P < 0.05). In the smoke-exposure group and the smoking cessation group, the positive rate of CD(11c)(+) DCs [(1.47 ± 0.12)%, (1.30 ± 0.17)%], and the positive rate of CD(86)(+) DCs [(1.26 ± 0.18)%, (1.02 ± 0.08)%] were higher than those in the control group [(0.96 ± 0.08)%, (0.65 ± 0.03)%] (F = 6.55 and 30.26, all P < 0.05), but there was no significant difference between the smoke-exposure group and the smoking cessation group (t = 1.10 and 1.47, all P>0.05). In the smoke-exposure group and the smoking cessation group, CD(8)(+) T positive rate [(2.72 ± 0.15)%, (2.35 ± 0.23)%] was higher than that in the control group [(1.39 ± 0.11)%] (F = 16.07, P < 0.05). CD(11c)(+)/CD(86)(+) DCs and CD(11c)(+)/MHCII(+)DCs percentages [(5.5 ± 0.4)%, (4.8 ± 0.4)%], [(4.2 ± 0.4)%, (3.3 ± 0.3)%] decreased in the smoke-exposure group and the smoking cessation group as compared to the control group [(8.0 ± 0.5)%, (6.1 ± 0.5)%] (F = 14.34 and 12.82, all P < 0.05). There was no significant difference between all the above index from the smoke-exposure group and the smoking cessation group (t = 1.10 and 2.11, all P > 0.05). Cigarette smoke exposure induced increased DCs transmigrated and influenced the maturation of DCs in COPD rats. Even after smoking cessation, non-specific chronic inflammation was still present, suggesting that DCs number and maturation abnormality may be involved in the chronic inflammation of COPD.