Thermoneutrality alters gastrointestinal antigen passage patterning and predisposes to oral antigen sensitization in mice

Food allergy is an emerging epidemic, and the underlying mechanisms are not well defined partly due to the lack of robust adjuvant free experimental models of dietary antigen sensitization. As housing mice at thermoneutrality (Tn) - the temperature of metabolic homeostasis (26-30°C) - has been shown to improve modeling various human diseases involved in inflammation, we tested the impact of Tn housing on an experimental model of food sensitization. Here we demonstrate that WT BALB/c mice housed under standard temperature (18-20°C, Ts) conditions translocated the luminal antigens in the small intestine (SI) across the epitheliu

Evaluation of Protective Efficacy of Respiratory Syncytial Virus Vaccine against A and B Subgroup Human Isolates in Korea

Human respiratory syncytial virus (HRSV) is a significant cause of upper and lower respiratory tract illness mainly in infants and young children worldwide. HRSV is divided into two subgroups, HRSV-A and HRSV-B, based on sequence variation within the G gene. Despite its importance as a respiratory pathogen, there is currently no safe and effective vaccine for HRSV. In this study, we have detected and identified the HRSV by RT-PCR from nasopharyngeal aspirates of Korean pediatric patients. Interestingly, all HRSV-B isolates exhibited unique deletion of 6 nucleotides and duplication of 60 nucleotides in the G gene. We successfully amplified two isolates (‘KR/A/09-8’ belonging to HRSV-A and ‘KR/B/10-12’ to HRSV-B) on large-scale, and evaluated the cross-protective efficacy of our recombinant adenovirus-based HRSV vaccine candidate, rAd/3xG, by challenging the immunized mice with these isolates. The single intranasal immunization with rAd/3xG protected the mice completely from KR/A/09-8 infection and partially from KR/B/10-12 infection. Our study contributes to the understanding of the genetic characteristics and distribution of subgroups in the seasonal HRSV epidemics in Korea and, for the first time, to the evaluation of the cross-protective efficacy of RSV vaccine against HRSV-A and -B field-isolates

Intranasal Delivery of Cholera Toxin Induces Th17-Dominated T-Cell Response to Bystander Antigens

Cholera toxin (CT) is a potent vaccine adjuvant, which promotes mucosal immunity to protein antigen given by nasal route. It has been suggested that CT promotes T helper type 2 (Th2) response and suppresses Th1 response. We here report the induction of Th17-dominated responses in mice by intranasal delivery of CT. This dramatic Th17-driving effect of CT, which was dependent on the B subunit, was observed even in Th1 or Th2-favored conditions of respiratory virus infection. These dominating Th17 responses resulted in the significant neutrophil accumulation in the lungs of mice given CT. Both in vitro and in vivo treatment of CT induced strongly augmented IL-6 production, and Th17-driving ability of CT was completely abolished in IL-6 knockout mice, indicating a role of this cytokine in the Th17-dominated T-cell responses by CT. These data demonstrate a novel Th17-driving activity of CT, and help understand the mechanisms of CT adjuvanticity to demarcate T helper responses

Cholera Toxin Promotes Th17 Cell Differentiation by Modulating Expression of Polarizing Cytokines and the Antigen-Presenting Potential of Dendritic Cells.

Cholera toxin (CT), an exotoxin produced by Vibrio cholera, acts as a mucosal adjuvant. In a previous study, we showed that CT skews differentiation of CD4 T cells to IL-17-producing Th17 cells. Here, we found that intranasal administration of CT induced migration of migratory dendritic cell (DC) populations, CD103+ DCs and CD11bhi DCs, to the lung draining mediastinal lymph nodes (medLN). Among those DC subsets, CD11bhi DCs that were relatively immature had a major role in Th17 cell differentiation after administration of CT. CT-treated BMDCs showed reduced expression of MHC class II and CD86, similar to CD11bhi DCs in medLN, and these BMDCs promoted Th17 cell differentiation more potently than other BMDCs expressing higher levels of MHC class II and CD86. By analyzing the expression of activation markers such as CD25 and CD69, proliferation and IL-2 production, we determined that CT-treated BMDCs showed diminished antigen-presenting potential to CD4+ T cells compared with normal BMDCs. We also found that CT-stimulated BMDCs promote activin A expression as well as IL-6 and IL-1β, and activin A had a synergic role with TGF-β1 in CT-mediated Th17 cell differentiation. Taken together, our results suggest that CT-stimulated DCs promote Th17 cell differentiation by not only modulating antigen-presenting potential but also inducing Th polarizing cytokines

Depletion of IL-2 recovers Th17 cell differentiation.

<p>Assessment of OT-II CD4⁺ T cell differentiation by untreated or CT-treated BMDCs pulsed with the indicated concentration of OVA_323-339 peptide in the absence (A and B) or presence of neutralizing IL-2 antibody (10 μg/ml) (A and C). *<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.001 (Student’s <i>t</i>-test). Dot plots and data are from one experiment representative of at least two independent experiments with similar results and average ± SEM of triplicate wells.</p

CD11b^hi DCs and CD103⁺ DCs migrated to the lung draining lymph node after CT administration and CD11b^hi DCs with the MHCII^loCD86^lo phenotype mediated IL-17-favored differentiation of CD4 T cell.

<p>Mice were intranasally administered 2 μg CT, and mediastinal lymph node (medLN) cells were prepared from the mice at day 1, day 2, or day 3 after or before CT administration. (A) Flow cytometry analysis of migratory DC subsets after CT administration. Five mice per group; migratory DC subsets in medLN (CD103⁺ DC and CD11b^hi DC); (B) Cell number of each DC subset after CT administration; (C-F) CD103⁺ DCs and CD11b^hi DCs were isolated from medLN cells of mice 2 day after intranasal CT administration, pulsed with 0.1 μM of OVA_323-339 peptide and washed and cultured with OT-II CD4⁺ T cells for 5 days. Stimulated OT-II CD4⁺ T cells were analyzed for expression of IL-17A and IFN-γ by flow cytometry after restimulation with PMA and ionomycin. (C) Flow cytometry analysis and (D and E) frequency of IL-17A- and IFN-γ-expressing CD4⁺ T cells. (F) Ratio of IL-17A⁺ to IFN-γ⁺ CD4⁺ T cells. (G) MHC class II and costimulatory molecules on DC subsets isolated from medLN of mice 2 day after administration of CT or PBS. *<i>p</i><0.05, **<i>p</i><0.01 (Student’s <i>t</i>-test). Data are from one experiment representative of two independent experiments with similar results. Dot plots and histograms are representative of four mice in A and G and quadruplicate wells in C and data are average ± SEM.</p

CT-treated BMDCs downregulate T cell activation and IL-2 production.

<p>OT-II CD4⁺ T cells were co-cultured with BMDCs or CT-BMDCs pulsed with the indicated concentration of OVA_323-339 peptide. At 16 h, OT-II CD4⁺ T cells were analyzed for expression of CD25 and CD69 (A-C). Histogram of CD25 and CD69 (A); Frequency of CD25-expressing or CD69-expressing CD4⁺ T cells (B); Intensity of CD25 or CD69 expression (C). CFSE-labeled OT-II CD4⁺ T cells were cultured alone or with BMDCs or CT-BMDCs pulsed with the indicated concentration of OVA_323-339 peptide. At day 4, CFSE dilution was assessed in OT-II CD4⁺ T cells (D). At day 5, IL-2 was detected in the co-culture of OT-II CD4⁺ T cells with BMDCs or CT-BMDCs by cytokine bead assay (eBioscience) (E). ***<i>p</i><0.001 (Student’s <i>t</i>-test). Histograms and data are from one experiment representative of two independent experiments. Data are average ± SEM of triplicate wells.</p

CT–treated BMDCs mediate Th17 cell differentiation by producing Th17 polarizing cytokines.

<p>(A) RT-PCR analysis of IL-6, IL-1β, TGF-β1 and activin A mRNA in BMDCs. BMDCs were cultured with CT (2 μg/ml) and harvested at 0, 6, or 20 h after CT treatment. mRNA levels of IL-6, IL-1β, TGF-β1 and activin βA were normalized by mRNA expression of GAPDH and β-actin. (B) Determination of cytokines in BMDC-conditioned media. BMDCs were cultured with CT (2 μg/ml) for 2 days, and culture media was removed to measure cytokines. IL-6 and IL-1β were assayed by multiplex bead cytokine assay kit following the manufacturer’s recommended protocol (eBioscience). TGF-β1 in BMDC-conditioned media was assayed for total (left) and active form (right) by ELISA (R&D Systems). (C and D) Intracellular staining of IFN-γ and IL-17A in OT-II CD4⁺ T cells stimulated with anti-CD3 and anti-CD28 antibody in the presence of BMDC-CM or CT-BMDC-CM for 5 days and restimulated with PMA and ionomycin after 5 days. Neutralizing antibodies and kinase inhibitors were also added in the culture as indicated. (E-G) BMDCs from <i>IL-6</i>^<i>-/-</i> mice were used for clarifying a role of IL-6 in Th17 differentiation promoted by CT-treated BMDCs. Frequency of IFN-γ⁺ or IL-17A⁺ CD4⁺ T cells (F) and ratio of IL-17⁺ cells to IFN-γ⁺ cells (G). *<i>p</i><0.5, **<i>p</i><0.01, ***<i>p</i><0.001 (Student’s <i>t</i>-test). Data are the representative of at least two independent experiments with similar results and average ± SEM of triplicate wells in A and D or duplicate wells in B, F and G.</p