Delayed-type Hypersensitivity Assay Research Articles

Pigment Dispersion Contributes to Ocular Immune Privilege in a DBA/2J Mouse Model of Pigmentary Glaucoma.

To investigate the effects of anterior chamber pigment dispersion on ocular immune privilege and the possible mechanisms involved in a DBA/2J mouse model of pigmentary glaucoma. DBA/2J mice were utilized as a pigment dispersion model, and age-matched C57BL/6J mice were used as the control group in this study. Proteins in the aqueous humor (AH) and serum were quantified using the bicinchoninic acid assay. Immune cells in the AH were detected using hematoxylin and eosin staining and immunocytochemistry. The expression of TGF-β2 in the AH and cytokine levels (IL-10, IFN-γ) in serum were measured using ELISA. Anterior chamber-associated immune deviation (ACAID) was induced in DBA/2J mice by injecting antigens into the anterior chamber. Delayed-type hypersensitivity (DTH) assays were used to assess the induction of ACAID. In DBA/2J mice, before and after pigment dispersion, following anterior chamber injection of pigment particles, and after ACAID modeling, the expression of regulatory T cells (Tregs) was detected using flow cytometry. Compared to C57BL/6J mice, the protein concentration, immune cell count, and TGF-β2 levels in the AH were elevated in DBA/2J mice. Protein concentration and IL-10 levels in serum were increased, while IFN-γ levels were decreased in DBA/2J. Additionally, the expression of Treg cells in the spleen of DBA/2J mice was significantly increased after pigment dispersion and anterior chamber injection of pigment particles. At 3 and 6 months, DTH responses in DBA/2J mice were not inhibited, thus preventing ACAID induction. However, the opposite was observed at 9 months in DBA/2J mice. Furthermore, the ACAID group exhibited an augmented expression of Treg cells. Dispersion of pigment particles in the anterior chamber of the eye enhances the state of ocular immune privilege by influencing the immunosuppressive microenvironment and inducing more Treg cells to reestablish ACAID.

Immunotoxicity of 2-Acetyl-4-tetrahydroxybutylimidazole in BALB/c mice with different vitamin B6 nutritional statuses

Caramel color is a widely used food pigment, and 2-Acetyl-4-tetrahydroxybutylimidazole (THI) is a by-products of Class III caramel color. Some studies have shown that THI can reduce the number of peripheral blood lymphocytes. However, the comprehensive mechanism of THI immunotoxicity requires further study. In this study, the effects of THI on lymphocyte count, humoral immunity, cellular immunity and nonspecific immunity were determined and the effect of the nutritional status of VB6 on THI immunotoxicity was evaluated. Female BALB/c mice were divided into 3 groups and fed chow containing different doses of VB6: VB6-normal (6 mg/kg VB6), VB6-deprived (0.5 mg/kg VB6) or VB6-enhanced (12 mg/kg VB6) feed. Each group was further divided into 4 subgroups and treated with THI (0.5, 2.5 or 12.5 mg/kg bw) or the solvent control by gavage for 30 days. The thymic cortical thickness was measured with ViewPoint; the proportions of major immune cells and T cells in peripheral blood and tissues were detected via flow cytometry; the transformation and proliferation abilities of T and B cells were detected via T and B lymphocyte proliferation assays; NK cell activity was assessed via lactate dehydrogenase assays; humoral immune function was assessed via plaque-forming cell assays; and the immune function of T lymphocytes was assessed via delayed type hypersensitivity assays. The results showed that compared with those in the corresponding control group, the white blood cell count and lymphocyte count decreased significantly in all the VB6-deprived groups, in the 2.5 and 12.5 mg/kg VB6 groups, and in the 12.5 mg/kg VB6-enhanced group. With increasing THI dose, the thymic cortical layer became thinner. In the thymus, THI increased the proportions of CD3+ T cells and mature CD8+ T cells and decreased the proportions of immature double-positive, double-negative T cells and CD69-expressing lymphocytes. The proportions of naïve T cells and Tcm (central memory T) cells related to homing decreased. The proportion of mature T cells in the spleen decreased significantly. The proliferation of T cells stimulated by ConA decreased after THI exposure. VB6-deficient mice were more sensitive to THI immunotoxicity, and supplementation with VB6 had a certain protective effect on these mice. The results of the PFC and NK cell activity assays indicated that THI exposure might not affect humoral immune or innate immune function.

Effects of 2-acetyl-4-tetrahydroxybutylimidazole exposure during gestation and lactation on maternity and offspring immune function in Balb/c mice

2-Acetyl-4-tetrahydroxybutylimidazole (THI), a by-product of Class Ⅲ caramel color, is generally recognized to cause lymphopenia in mammals. However, it remains unknown whether THI exposure during gestation and lactation causes damage to the immune system of offspring. In this study, pregnant Balb/c mice were gavaged with 0, 0.5, 2.5 and 12.5 mg/kg THI from gestation day (GD) 6 to postanal day (PND) 21, after which we treated another batch of dams from GD6 to PND21 and the offspring for 3 weeks after weaning with 0, 2, 10, 50 mg/L THI in drinking water respectively, and investigated the immunological anomalies of dams and offspring. The results showed that lymphopenia was observed in dams but not in weaning pups on PND21, which were exposed to THI during gestation and lactation. 2 mg/L THI and 2.5 mg/kg THI began to cause a remarkable reduction of the numbers of white blood cells and lymphocytes in dams. Besides both the cellular and the humoral immune response was not affected in weaning pups, which were measured by plaque-forming cell (PFC) assay and delayed-type hypersensitivity (DTH) assay respectively. Furthermore, THI could be detected in the plasma of dams with a dose-dependent manner, but not in that of both female and male weaning pups. In both male and female offspring being treated with 10 and 50 mg/L THI for another 3 weeks after weaning, lymphocytopenia was observed and T lymphocytes including CD4+ and CD8+ cells were significantly reduced in their spleens except lymph nodes. 10 and 50 mg/L THI treatment increased CD4+ and CD8+ single positive cells in thymus of female and male weaning mice. Mitogen-induced proliferation ability of T cells in the spleen and lymph nodes was impaired in female weaning mice exposed 50 mg/L THI, while male weaning mice treated with 10 and 50 mg/L THI showed impairment in the spleen but not lymph nodes. Based on the results in this study, no observed adverse effect level (NOAEL) for 3-week THI treatment in weaning mice was considered to be 2 mg/L (0.30 mg/kg bw for female mice and 0.34 mg/kg bw for male mice). And NOAEL for THI treatment in dams might be set to 0.5 mg/kg bw/day. Collectively from the perspective of NOAEL, offspring are not more sensitive than dams or adult mice.

Extracellular Vesicle-associated GARP/TGFβ:LAP Mediates "Infectious" Allo-tolerance.

C57BL/6 mice were tolerized by i.p. injection of CBA/J splenocytes followed by anti-CD40L/CD154 antibody treatment on days 0, 2, and 4. On day 35, spleen and lymph nodes were extracted and isolated lymphocytes were restimulated with sonicates of CBA splenocytes overnight. sEVs were extracted from culture supernatants by ultracentrifugation (100 000g) and assayed for (a) the presence of TGFβ:LAP associated with tetraspanins CD81,CD63, and CD9 by enzyme-linked immunosorbent assay; (b) GARP, critical to membrane association of TGFβ:LAP and to activation from its latent form, as well as various TGFβ receptors; and (c) TGFβ-dependent function in 1° and 2° immunosuppression of tetanus toxoid-immunized B6 splenocytes using trans-vivo delayed-type hypersensitivity assay. After tolerization, CBA-restimulated lymphocytes secreted GARP/TGFβ:LAP-coated extracellular vesicles. Like IL35 subunits, but unlike IL10, which was absent from ultracentrifuge pellets, GARP/TGFβ:LAP was mainly associated with CD81+ exosomes. sEV-bound GARP/TGFβ:LAP became active in both 1° and 2° immunosuppression, the latter requiring sEV uptake by "bystander" T cells and reexpression on the cell surface. Like other immune-suppressive components of the Treg exosome, which are produced in a latent form, exosomal GARP/TGFβ:LAP produced by allo-specific regulatory T cells undergoes either immediate activation (1° suppression) or internalization by naive T cells, followed by surface reexpression and subsequent activation (2°), to become suppressive. Our results imply a membrane-associated form of TGFβ:LAP that, like exosomal IL35, can target "bystander" lymphocytes. This new finding implicates exosomal TGFβ:LAP along with Treg-derived GARP as part of the infectious tolerance network.

<i>Camphora</i> Augments Humoral Mediated Immunity and Decreases Delayed type Hypersensitivity in BALB/c Mice

Background: Immunomodulation encompasses all therapeutic interventions aimed at modifying the immune response. The immune response augments are desirable to prevent infection in immunodeficiency states and fight established diseases. In this context, the present study investigated the effect of the homeopathic medicine, Camphora, in 6CH, 30CH, and 200CH potencies on immunomodulation in experimental animals. The acute oral toxicity study was also carried out in 6CH, 30CH, and 200CH potencies to determine the safe dose volume for the immunomodulatory study. Methodology: Acute oral toxicity studies of Camphora in 6CH, 30CH, and 200CH potencies were carried out as per OECD guideline 423 with slight modifications in Wistar albino rats. Humoral immunity, i.e., primary and secondary humoral responses, was assessed by measuring the hemagglutination titre of sheep red blood cells. Delayed-Type Hypersensitivity (DTH) was evaluated by measuring footpad thickness in BALB/c mice. Results: Camphora in 6CH, 30CH and 200CH potencies at a dose volume of 2000 µl/kg did not cause any mortality in the rats when administered as a single dose. Camphora in 6CH, 30CH and 200CH potencies showed augmented primary and secondary humoral responses against the SRBC antigen in BALB/c mice. However, the values were statistically non-significant except in the case of 6 CH potency (p<0.01), which showed statistically significant primary anti-SRBC antibodies. In the DTH assay, Camphora in 6CH, 30CH and 200CH potencies significantly decreased the paw volume ratio after 24 hrs of SRBC injection in the paw, thus insinuating its role in reducing cell-mediated immunity. Camphora in 6CH, 30CH and 200CH potencies also showed enhanced antibody titres and decreased paw volume compared to vehicle control, i.e. dispensing alcohol, suggesting that the effect was imminent because of Camphora. Conclusion: The study results indicate that Camphora in 6CH, 30CH, and 200 CH potencies is safe up to a dose volume of 2000 µl/kg when administered as a single dose, augments the primary and secondary humoral immunity, and decreases DTH in experimental animals. The current study’s findings suggest that Camphora might be useful as an immunomodulator in treating immune system disorders and infectious diseases and require further investigation to investigate its mechanism of action.

Immunosuppressive Effect of Methanol Extract and Ethyl Acetate Fraction of Breadfruit [Artocarpus altilis (Park.) Fosberg] Leaves

Background: Artocarpus sp. contained a variety of flavonoids, some of which have been shown to reduce inflammation. In vitro anti-inflammatory activity of breadfruit (Artocarpus altilis (Park.) Fosberg) has been demonstrated. Chronic inflammation can occur when immune defence mechanisms are activated by inflammatory conditions. Objectives: This study examined breadfruit leaf methanol extract and ethyl acetate fraction immunosuppressive effects. Material and Methods: The bioactive compounds in breadfruit leaves were extracted in methanol via maceration and fractionation in ethyl acetate via liquid-liquid extraction. The in vivo immunosuppressive effect was evaluated in male Swiss albino mice by humoral antibody titer, delayed-type hypersensitivity, and phagocytic index assays. Results: According to phytochemical screening, flavonoids and steroids were found in the extract and its ethyl acetate fraction. The breadfruit leaf extract and fraction showed an immunosuppressive effect through phagocytic index<1, and the thymus gland index was significantly decreased (p<0.05) compared to control. The breadfruit leaf extract and the ethyl acetate fraction were able to inhibit the production of antibodies and DTH reactions, two types of immune responses. Conclusions: The extract of breadfruit leaves and the ethyl acetate fraction showed immunosuppressive properties.

Contemporary uses of old folks: the immunomodulatory and toxic potential of fenbufen

Fenbfen is used for pain, pyrexia and in the management of osteoarthritis, rheumatoid arthritis and other musculoskeletal disorders. The present research was planned to examine the immunomodulatory activity of fenbufen in different models of cell-mediated immunity (CMI) and humoral immunity (HI). The CMI was evaluated by delayed-type hypersensitivity (DTH) and cyclophosphamide-induced neutropenia assays while HI was appraised by hemagglutination (HA) assay by administering fenbufen at 2, 6 and 10 mg.kg-1 and azathioprine 40 mg.kg-1 (as standard therapy) to albino mice by intraperitoneal route. The ex vivo immunomodulatory action was determined by red blood cell (RBC) membrane stabilization and protein denaturation assays. The results showed that fenbufen treatment had significantly (p<0.05-p<0.001) reduced white blood cells, hemoglobin content, and red blood cells in the healthy and neutropenic mice. A significant (p<0.001) reduction in activities of superoxide dismutase and catalase and glutathione contents, and enhancement of malondialdehyde level were observed in neutropenic mice that were restored by fenbufen treatment. It suppressed DTH reaction after 24, 48 and 72 h post topical application of 2, 4-dinitrofluorobenzene (DNFB). Fenbufen or azathioprine treated groups also showed a significant reduction in the antibody titer against human RBCs induced immune activation in mice as compared to the disease control mice. Fenbufen showed IC50 of 14.0, 50.5 and 66.2 μg.ml-1 whereas, diclofenac sodium showed IC50 of 61.0, 126 and 50.5 μg/ml in RBCs membrane stabilization, egg albumin and bovine serum albumin denaturation assays respectively. The current study shows that fenbufen might have potential immunomodulatory activity against CMI and HI. It can be utilized to treat immune system disorders.

IL10- and IL35-Secreting MutuDC Lines Act in Cooperation to Inhibit Memory T Cell Activation Through LAG-3 Expression.

Dendritic cells (DCs) are professional antigen-presenting cells involved in the initiation of immune responses. We generated a tolerogenic DC (tolDC) line that constitutively secretes interleukin-10 (IL10-DCs), expressed lower levels of co-stimulatory and MHCII molecules upon stimulation, and induced antigen-specific proliferation of T cells. Vaccination with IL10-DCs combined with another tolDC line that secretes IL-35, reduced antigen-specific local inflammation in a delayed-type hypersensitivity assay independently on regulatory T cell differentiation. In an autoimmune model of rheumatoid arthritis, vaccination with the combined tolDCs after the onset of the disease impaired disease development and promoted recovery of mice. After stable memory was established, the tolDCs promoted CD4 downregulation and induced lymphocyte activation gene 3 (LAG-3) expression in reactivated memory T cells, reducing T cell activation. Taken together, our findings indicate the benefits of combining anti-inflammatory cytokines in an antigen-specific context to treat excessive inflammation when memory is already established.

NFATc3 regulation of collagen V expression contributes to cellular immunity to collagen type V and hypoxic pulmonary hypertension.

Chronic hypoxia (CH)-induced pulmonary hypertension (PH) results, in part, from T helper-17 (TH17) cell-mediated perivascular inflammation. However, the antigen(s) involved is unknown. Cellular immunity to collagen type V (col V) develops after ischemia-reperfusion injury during lung transplant and is mediated by naturally occurring (n)TH17 cells. Col5a1 gene codifies for the α1-helix of col V, which is normally hidden from the immune system within type I collagen in the extracellular matrix. COL5A1 promoter analysis revealed nuclear factor of activated T cells, cytoplasmic 3 (NFATc3) binding sites. Therefore, we hypothesized that smooth muscle NFATc3 upregulates col V expression, leading to nTH17 cell-mediated autoimmunity to col V in response to CH, representing an upstream mechanism in PH development. To test our hypothesis, we measured indexes of PH in inducible smooth muscle cell (SMC)-specific NFATc3 knockout (KO) mice exposed to either CH (380 mmHg) or normoxia and compared them with wild-type (WT) mice. KO mice did not develop PH. In addition, COL5A1 was one of the 1,792 genes differentially affected by both CH and SMC NFATc3 in isolated intrapulmonary arteries, which was confirmed by RT-PCR and immunostaining. Cellular immunity to col V was determined using a trans vivo delayed-type hypersensitivity assay (Tv-DTH). Tv-DTH response was evident only when splenocytes were used from control mice exposed to CH but not from KO mice, and mediated by nTH17 cells. Our results suggest that SMC NFATc3 is important for CH-induced PH in adult mice, in part, by regulating the expression of the lung self-antigen COL5A1 protein contributing to col V-reactive nTH17-mediated inflammation and hypertension.

Identification of PD1-mediated regulation of antitumor antigen response in patients with NSCLC using the trans vivo DTH assay.

ObjectivesEmerging evidence has shown a role for tumor antigen-specific regulation in cancer. Identifying individuals with pre-existing regulatory responses may be key to understand those who are more likely to respond to Programmed Death-1 (PD-1) or PD-1 Ligand 1 (PD-L1) checkpoint blockade. We hypothesized that a functional assay could identify the role of PD-1/PD-L1 interactions on tumor-specific immune cells in the peripheral blood in patients with advanced non-small-cell lung cancer (NSCLC).MethodsWe performed the trans vivo delayed-type hypersensitivity assay to identify the role of PD-1/PD-L1-mediated tumor-specific immune regulation in ten patients with advanced NSCLC.ResultsThe majority of patients had PD-1-mediated anergic immune responses towards their tumor antigens. Eight out of nine of these patients did not respond to their own tumor antigens but responded in the presence of anti-PD-1 antibody (‘PD-1 anergy’ phenotype). A minority (3/9) also had ‘active’ PD-1-mediated immune suppressive regulatory responses. Our results suggest that PD-1-anergy is a common feature of NSCLC immune responses, whereas PD-1-mediated immune suppression is present only in a minority of patients. The latter was associated with poor clinical outcomes in our sample.ConclusionsOverall, our results indicate that bystander suppression or the ‘anergy-only’ phenomenon may be novel biomarkers in NSCLC and suggest prediction value based on these phenotypes.

EVALUATION OF IN VIVO IMMUNOMODULATORY ACTIVITY OF AQUEOUS AND ETHANOLIC EXTRACT OF CAPPARIS MOONII

Objective: The present study was intended to evaluate the in vivo immunomodulatory activity of aqueous and ethanolic extract of dried fruits of Capparis moonii Wight. 
 Methods: The effect of both the extracts was evaluated by carbon clearance assay in mice, and they showed a significant increase in the phagocytic index at 50, 100, and 200 mg/kg dose. The effect of the extracts on delayed-type hypersensitivity (DTH) and antibody titer assay was evaluated in ovalbumin immunized mice.
 Results: Both extracts showed a significant effect on DTH response and stimulation of antibody titer at 200 mg/kg dose. The effects of the extracts in cyclophosphamide-induced myelosuppression mice were also statistically significant.
 Conclusion: The studies demonstrated specific and non-specific immunostimulating properties of both the ethanol and aqueous extract of C. moonii fruits in various in vivo experimental methods suggesting its therapeutic usefulness in immunocompromised conditions.

Evaluation of immunomodulatory effects of lomefloxacin in mice

Lomefloxacin is a flouroquinolone antibiotic that is quite efficacious against many gram negative and gram positive pathogens. Lomefloxacin evince antibacterial effects by modifying DNA gyrase in gram negative pathogens and topoisomerase IV in gram positive pathogens. This study is designed to assess the immunomodulatory effects of lomefloxacin in male albino mice. Three doses of lomefloxacin 12.5 mg/kg, 25 mg/kg and 50 mg/kg were used and delayed type hypersensitivity assay, cyclophosphamide induced neutropenic assay, carbon clearance assay, heamagglutination assay and mice lethality test were performed to evaluate the effects of lomefloxacin on immune system of mice. DTH assay has depicted the significant immunosuppressant potential of lomefloxacin at 25 mg/kg and 50 mg/kg dose. Total leukocyte count have exhibited highly significant reduction (P<0.001) in leukocyte count after cyclophosphamide administration. Differential leukocyte count has shown significant (P<0.01) reduction in lymphocyte count, whereas, highly significant (P<0.001) increase in monocyte count and significant (P<0.05) increase neutrophil count has been observed. In carbon clearance assay, highly significant (P<0.01) increase in phagocytic index has been noted at 12.5 mg/kg and 25 mg/kg doses. Humoral immune system responses are suppressed in dose dependent manner by both heamagglutination assay (P<0.001) and mice lethality assay (P<0.001). Results clearly depict that lomefloxacin possess quite significant immunomodulatory potential.

Pulsation Activates Mechanosensitive Piezo1 to Form Long-Term Hematopoietic Stem Cells

The temporal and spatial origin and development of long-term, self-renewing hematopoietic stem cells (LT-HSC) remain a mystery. The first set of definitive HSCs is born from the hemogenic endothelial cells residing in the ventral wall of the dorsal aorta (DA) of the aorta-gonad-mesonephros region during embryonic development. Blood flow- and shear-stress-mediated nitric oxide-induced vasodilation are responsible for the endothelial-to-HSC transition (EHT). However, it remains unknown why the ventral wall, and not the dorsal wall, of the DA is the restricted site of the EHT when blood flows through the entire DA and exerts shear stress on both the ventral and dorsal sides of the DA. Using single-particle tracking and fast Fourier Transform analyses of pulsating blood vessels, we demonstrate that the circumferential strain in the ventral wall, and not dorsal wall, is concurrent with and responsible for the magnitude, the site, and timing of the HSC formation. We extended our findings by developing a bioreactor to establish the functional link between pulsation in the blood vessels and HSC formation. Using serial transplant, limiting dilution, and serial replating assays, we found that pulsation mediated circumferential stretching of hemogenic endothelial cells or Piezo1 activation (Yoda1) yields 3-times higher amounts of Long Term (LT)-HSC formation; which reconstitute to normal multi-lineage adult blood. Using delayed-type hypersensitivity assay, adult globin expression, MPO enzyme activity, immunoglobulins, and T-cell receptor rearrangement analyses, we found that circumferential stretching or Piezo1 activation-derived HSCs reconstitute to functional T and B cells, adult erythrocytes, and myeloid cells. Our Piezo1fl/flxScl-Cre conditional knockout, gene-silencing, &amp; confocal imaging further demonstrate that circumferential stretching of blood vessels activates Piezo1; which enhances epigenetic regulator Dnmt3b expression to stimulate the EHT. Our CUT&amp;RUN CHIP-Sequencing &amp; MASSArray methylation analyses demonstrate that Dnmt3b suppresses endothelial genes during EHT. To analyze the conserved role of PIEZO1-mediated mechanosensitive mechanisms in human hematopoiesis, we employed directed differentiation of constitutive RUNX1-mCherry human induced pluripotent stem cells (iPSCs) to hemogenic endothelial cells. We found that Yoda1-mediated PIEZO1 activation stimulated human endothelial-to-hematopoietic transition. In conclusion, pulsation-mediated circumferential strain activates Piezo1 to stimulate the endothelial-to-HSC transition via the induction of Dnmt3b expression. This leads to the formation of long-term self-renewing HSCs, which can engraft and reconstitute to multi-lineage, adult blood upon serial transplantations. Our identification of a novel biomechanical cue unravels the physiological mystery in HSC formation in the ventral wall of the DA. We also establish its cross-talk with mechanosensitive and epigenetic mechanisms to produce functional, long-term HSCs that reconstitute to form normal adult blood. This yields the therapeutic promise of developing transgene-free LT-HSC-based cellular therapies for the treatment of human blood disorders. Disclosures No relevant conflicts of interest to declare.

Evaluation of the Induction of Cell-Mediated Immunity Against Candida albicans in a Model of Cutaneous Infection in Newborn 0-Day-Old Mice.

Candida albicans is a commensal fungus of the skin and mucous membranes in humans, but it is also responsible for mucocutaneous and systemic infections in immunocompromised patients like low birth weight neonates and premature newborns. The epicutaneous application of C. albicans is widely used to study the immune response against this pathogen in adult mice models. However, the immune response of newborns against infections caused by the genus Candida is poorly understood. In order to mimic premature human infection, we developed a model of C. albicans epicutaneous infection in newborn mice. We found that yeasts were able to colonize while the pseudohyphae invaded the epidermis. Recruitment of polymorphonuclear and mononuclear cells at the infection zone was observed. Fungal invasion, fungal burden and cellular infiltration displayed a time- and dose-dependent response. Interestingly, newborn mice were able to control C. albicans primary infection. Finally, we showed that the epicutaneous infection of C. albicans in newborn mice at birth results in the induction of cell-mediated immunity as evinced by delayed-type hypersensitivity assays.

Abstract 127: The Lung-Associated Self-Antigen Type V Collagen is Regulated by Nuclear Factor of Activated T Cells Isoform c3 in Chronic Hypoxia

Chronic hypoxia (CH)-induced pulmonary hypertension (PH) is a progressive and often fatal consequence of chronic lung diseases, chronic exposure to high altitude and sleep apnea. PH results from arterial remodeling, enhanced vasoreactivity, and Th17 cell-mediated perivascular inflammation. Naturally occurring “nTH17” cells, can be detected in fetal life and are in homeostatic equilibrium with natural T regulatory cells. nTH17 cells are specific for a limited range of self-antigens, including type V collagen (colV). ColV is normally hidden from the immune system within type I col in the extracellular matrix of the lungs. We have shown that type V collagen is a self-antigen uncovered by CH. On the other hand, we have shown that mice lacking nuclear factor of activated T cells isoform c3 (NFATc3) globally and specifically in smooth muscle (SMC-NFATc3 KO) are protected from CH-induced PH. No differences in cardiac function were detected between SMC-NFATc3 KO and control mice using a Vevo LAZR-X ultrasound system before CH. In silico analysis of Col5A1 promoter shows NFATc3 binding sites. Therefore, we hypothesized that smooth muscle NFATc3 upregulates Col5A1 expression contributing to the development of acute autoreactivity to collagen V in response to CH, representing an upstream mechanism in PH development. Col5A1 was one of the 1792 genes differentially affected by both CH and smooth muscle NFATc3 in isolated pulmonary arteries (RNA Illumina Sequencing). Col5A1 mRNA increased significantly after 5 days of CH in control mice (fold change from normoxia±SD=2.2±0.8, n=5) vs. SMC-NFATc3 KO (0.8±0.3, n=3, p&lt;0.03). Cellular autoimmunity to colV was determined using a trans-vivo delayed-type hypersensitivity assay (TV-DTH). Splenocytes from 5 days CH-exposed control mice displayed a TV-DTH colV response significantly higher than that from CH-exposed SMC-NFATc3 KO mice (footpad swelling [10 -4 ”] ± SD of naïve recipient mice injected with cells from CH control mice 25.6±4.2, n=8 vs. CH SMC-NFATc3 KO mice 5.3±0.5, n=7; p&lt;0.0001). Our results suggest that smooth muscle NFATc3 is important for CH-induced PH in adult mice, in part, by regulating the expression of the lung self-antigen ColV contributing to pulmonary vascular inflammation.

Anti-inflammatory and immune-modulatory properties of anemoside B4 isolated from Pulsatilla chinensis in vivo

BackgroundPulsatilla chinensis is commonly used for the treatment of cancers and inflammatory disorders in China. Our recent studies showed that anemoside B4, its major ingredient, possessed notable antioxidant and protected cisplatin-induced acute kidney injury in vivo. Furthermore, we found the protective effect might be involved its anti-inflammation activities. However, its anti-inflammatory mechanisms are not clear. PurposeIn the present study, we extensively investigated the anti-inflammatory and immune-modulatory properties of anemoside B4 in vivo. MethodsTo carry out this work, the xylene-induced ear edema and LPS-induced systemic inflammation of mice model was also used to evaluate the anti-inflammatory activity. Then, anti-inflammatory mechanism of anemoside B4 was further determined by pro-inflammatory cytokines production using enzyme-linked immunosorbent assay (ELISA) and nuclear factor-κ-gene binding (NF-κB) pathway activation by Western blot. At last, immuno-modulatory effects were observed by splenocyte proliferation assay, delayed type hypersensitivity assay (DTH) and T cell subtype assay in mice. Results12.5–50 mg/kg anemoside B4 significantly suppressed xylene-induced mice ear edema. Furthermore, it ameliorated LPS-induced kidney and lung inflammation damage, which inhibited pro-inflammatory response by NF-κB pathway in mice. In addition, anemoside B4 decreased CD4+/CD8+ ratio, inhibited splenic lymphocyte proliferation and decreased DNFB-induced changes of ear thickness. ConclusionFrom these data, it can be concluded that anemoside B4 presented anti-inflammatory and immune-modulatory activities in vivo, and potentially be a novel natural anti-inflammatory drug candidate for treating inflammatory disorder.

Operational immune tolerance towards transplanted allogeneic pancreatic islets in mice and a non-human primate.

Patients with autoimmune type 1 diabetes transplanted with pancreatic islets to their liver experience significant improvement in quality of life through better control of blood sugar and enhanced awareness of hypoglycaemia. However, long-term survival and efficacy of the intrahepatic islet transplant are limited owing to liver-specific complications, such as immediate blood-mediated immune reaction, hypoxia, a highly enzymatic and inflammatory environment and locally elevated levels of drugs including immunosuppressive agents, all of which are injurious to islets. This has spurred a search for new islet transplant sites and for innovative ways to achieve long-term graft survival and efficacy without life-long systemic immunosuppression and its complications. We used our previously established approach of islet transplant in the anterior chamber of the eye in allogeneic recipient mouse models and a baboon model of diabetes, which were treated transiently with anti-CD154/CD40L blocking antibody in the peri-transplant period. Survival of the intraocular islet allografts was assessed by direct visualisation in the eye and metabolic variables (blood glucose and C-peptide measurements). We evaluated longitudinally the cytokine profile in the local microenvironment of the intraocular islet allografts, represented in aqueous humour, under conditions of immune rejection vs tolerance. We also evaluated the recall response in the periphery of the baboon recipient using delayed-type hypersensitivity (DTH) assay, and in mice after repeat transplant in the kidney following initial transplant with allogeneic islets in the eye or kidney. Results in mice showed >300days immunosuppression-free survival of allogeneic islets transplanted in the eye or kidney. Notably, >70% of tolerant mice, initially transplanted in the eye, exhibited >400days of graft survival after re-transplant in the kidney without immunosuppression compared with ~30% in mice that were initially transplanted in the kidney. Cytokine and DTH data provided evidence of T helper 2-driven local and peripheral immune regulatory mechanisms in support of operational immune tolerance towards the islet allografts in both models. We are currently evaluating the safety and efficacy of intraocular islet transplantation in a phase 1 clinical trial. In this study, we demonstrate immunosuppression-free long-term survival of intraocular islet allografts in mice and in a baboon using transient peri-transplant immune intervention. These results highlight the potential for inducing islet transplant immune tolerance through the intraocular route. Therefore, the current findings are conceptually significant and may impact markedly on clinical islet transplantation in the treatment of diabetes.

Comparison and Evaluation of Different Seed Extracts of Trachyspermum ammi for Immunomodulatory Effect on Cell-Mediated Immunity through Delayed-Type Hypersensitivity Assay Skin Thickness Method.

Objective:The aim and objective of this study was to find the immunostimulant and immunomodulatory effect of T. ammi seed extracts.Methods:Seeds of T. ammi were extracted using three different solvents n-hexane, chloroform, and methanol by using soxhlet apparatus. To assess the immunomodulatory effect, delayed-type hypersensitivity (DTH) assay method was used and by the DTH assay, the effect of T. ammi on the skin thickness of rats was estimated. To find the exact dose for administration, acute toxicity test was performed using crude methanolic extract at a dose of 400, 800, 1600, and 3200mg/kg. After acute toxicity test, 500mg/kg dose was determined as safe for therapeutic effect and immunomodulatory effect was evaluated at this dose. Dose of 500mg/kg was administered to Wistar rats daily for 14 days and skin thickness of rats was measured at 24, 48, and 72h.Results:Results were obtained from six groups of rats, which were positive control group, negative control group, and the groups receiving the test drugs. Standard drug was the combination of sodium selenite, vitamin E, and sodium chloride and it showed more positive results as compared to that of test drug. Furthermore, among the three extracts, methanol extract showed more effectiveness on skin thickness.Conclusion:There was a meaningful difference was observed between the skin thickness of rats which shows that T. ammi have good immunomodulatory as well as immunostimulant activity.

Abstract P280: Type V Collagen is a Lung-Associated Self-Antigen Uncovered by Chronic Hypoxia

Hypoxic pulmonary hypertension (PH) is a progressive and often fatal consequence of chronic hypoxia (CH) exposure (chronic lung diseases, high altitude and sleep apnea). We recently demonstrated that T helper 17 (T H 17) cells are localized in the perivascular region of pulmonary arteries and contribute to CH-induced PH, at least in part, by causing pulmonary arterial (PA) remodeling. Naturally occurring “nT H 17” cells, can be detected as early as week 17 of fetal life in humans, and are in homeostatic equilibrium with natural T regulatory (nTregs) cells. nT H 17 cells are specific for a limited range of self-antigens, including type V collagen (colV). ColV is normally sequestered within type I col in the extracellular matrix of the lungs hidden from the immune system. Activation of matrix metalloproteinase-2, which is induced by CH, can expose colV. Therefore, we tested the hypothesis that ColV is a lung-associated self-antigen uncovered by CH. We found colV immunoreactivity only in lungs from mice exposed to hypobaric CH for 5 days vs. controls and colV mRNA expression is significantly increased in isolated PA (log fold change mean±SEM, normoxia= 0.22±0.08 vs. CH=0.48±0.02, n=3, p=0.02) suggesting that CH uncovers hidden colV. We then, determined cellular autoimmunity to colV using a trans-vivo delayed-type hypersensitivity assay (TV-DTH). Splenocytes obtained from 5 days CH-exposed mice displayed a TV-DTH colV response significantly higher than that from normoxic mice (footpad swelling [10 -4 ”] normoxia=11.8±1.1 vs. CH= 35.6±3.0, n=14, p=0.0001), while there was minimal response to col I. To determine that reactivity to colV contributes to CH-induced PH, we induced peripheral tolerance to colV by administering 25 μl of 0.08 μg/μl of bovine colV (Sigma) in PBS in each nare or PBS every two days for two weeks prior and during CH (21 days). Right ventricular systolic pressure (RVSP) was higher in vehicle-treated compared to colV-treated mice (mmHg CH vehicle= 32.4±0.3 vs. CH colV= 27.8±0.9, n=4, p=0.0085) suggesting that oral tolerance to col V attenuated PH. These results are ground-breaking suggesting that acute autoreactivity to colV develops after exposure to hypoxia. Next, we will determine whether a disrupted balance of nTh17/nTreg mediates this response.

Glycolytic Bias in mTORC2 Deficient Dendritic Cells Potentiates Antigen-Specific Immunity and Accelerates Graft Rejection

Background/Hypothesis The mechanistic target of rapamycin (mTOR) is known to function in two discrete complexes: mTOR complex 1 (mTORC1) and mTORC2. The function of mTORC1 in dendritic cells (DCs) has been studied extensively using rapamycin (RAPA) as an inhibitor. RAPA inhibition of mTORC1 prevents DC maturation, leading to decreased T effector cell proliferation and increased regulatory T cell (Treg) differentiation. Our group has recently demonstrated that mTORC2 deletion in DCs leads to both an enhanced pro-inflammatory DC phenotype and Th1/Th17 allogeneic T cell polarization and proliferation. However, the underlying mechanism has not been resolved. In addition, the role of mTORC2 in DCs in the context of transplantation has not been defined. We hypothesized that ablation of mTORC2 in DCs would alter metabolic activity, resulting in augmented antigen-specific T cell responses and accelerated graft rejection. Methods To assess the role of mTORC2 in DCs in transplantation, we used models of non-MHC mismatched skin transplantation in which either the graft donor or recipient was deficient in mTORC2 specifically in conventional CD11c+ DC (TORC2DC-/-). Graft survival was monitored; Banff rejection scoring was performed by a blinded pathologist. T cell infiltration and collagen degradation in the graft were determined by immunohistochemistry. To ascertain whether skin-resident TORC2DC-/- could augment inflammatory responses, we performed a cell-mediated delayed-type hypersensitivity assay. As mTORC2 has been implicated in cytoskeletal dynamics, we measured DC migration into secondary lymphoid tissue. To elucidate the role of mTORC2 in regulating DC metabolism we analyzed glycolytic capacity and mitochondrial respiratory activity of wild-type (WT) DC and TORC2DC-/- using a Seahorse XF Bioanalyzer. Mitochondrial mass and activity were determined via flow cytometric analysis of MitoTracker Green and TMRE uptake, respectively. ATP production was assessed using a luciferase-based luminescence assay. Results/Conclusions We demonstrate for the first time, that TORC2DC-/- deficiency in either skin graft donors or recipients accelerates immune-mediated rejection. TORC2DC-/- mice also exhibit enhanced T cell and inflammatory monocytic infiltration in the course of delayed-type hypersensitivity responses. These effects are not due to differential migration of TORC2DC-/- to secondary lymphoid tissue. TORC2DC-/- utilize an altered metabolic program, wherein glycolytic function is enhanced as compared to WT DCs. This metabolic phenotype corresponds with increased viability of TORC2DC-/- after stimulation, which may allow these TORC2DC-/- to persist in secondary lymphoid tissue longer than WT DCs. These findings reveal a novel role for mTORC2 in regulating DC immunometabolism, and may provide a basis for therapeutic targeting of DC metabolism to regulate immune responses in transplantation. NIH T32 AI74490.