DB HS 1 Protección contra la humedad

Figure 5-7. Egg injection does not induce the expression of CD40, CD80, CD86 or OX40L on lamina propria DCs

The expression of CD40 (A), CD80 (B), CD86 (C) and OX40L (D) was assessed on small intestinal LP DC subsets from naive and egg injected animals. Median fluorescent intensity of isotype controls is represented as a dotted line. Data represent at least two independent experiments (mean ± SEM) with each point representing one animal. Mann-Whitney U tests were applied between naive and egg injected groups for statistical analysis.

We have shown that egg antigen is taken up by specialized DC subsets in the small intestine and colon and transported to individual draining lymph nodes.

There they interact with naive T cells and prime antigen specific immune responses. It has been reported that the uptake of antigen stimulates DCs and leads to the expression of costimulatory molecules that are required to efficiently communicate with naive T cells (Constant and Bottomly, 1997). We therefore addressed whether exposure to egg antigen also led to the upregulation of costimulatory molecules on DCs. We focused on CD40, CD80 and CD86, which represent costimulatory molecules that are upregulated during antigen-driven TLR activation, and OX40L which had been identified as a costimulatory molecule involved in Th2 priming (Jenkins et al., 2007). S. mansoni eggs were injected into the small intestinal lamina propria of C57BL/6 mice and the injected tissue was harvested after 24 hours. Tissues were digested with Collagenase VIII and cells stained for flow cytometry. The expression of costimulatory markers by DCs was assessed by measuring the median fluorescent intensity (MFI) for each antibody

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compared to its fluorescent isotype control. Hereby a shift in fluorescent intensity signified increased expression. We observed that small intestinal DCs expressed low levels of CD40. Hereby, the CD11b⁺ and DP DC subsets expressed higher amounts of CD40 compared to CD103⁺ DCs but no increase in CD40 expression was observed in egg injected tissues (Fig. 5-7 A). The expression of CD80 and CD86 by small intestinal DCs was also low. Their MFI was around 3,000 for CD11b⁺, DP and CD103⁺ DCs and lower for DN DCs. As observed for CD40, egg injection did not have an effect on the expression of CD80 or CD86 by any of the DC subsets (Fig. 5-7 B&C). We hypothesized that the expression of these classical costimulatory markers, which are associated with proinflammatory conditions, were not induced by egg antigens. However, we assumed that egg antigens might induce the expression of OX40L, which had been associated with Th2 cell priming (Jenkins et al., 2007). We observed that OX40L was weakly expressed by small intestinal DCs and that CD103⁺ DCs expressed lower levels than CD11b⁺ and DP DCs and DN DCs did not express any OX40L. Furthermore, levels of OX40L did not increase in any of the small intestinal DC subsets upon egg injection (Fig. 5-7 D). Thus, we concluded that egg injection into the small intestine did not affect the overall expression of conventional or Th2 inducing costimulatory molecules on lamina propria DCs 24 hours after injection. However, we were unsure if the injection of eggs into the small intestine represented a suitable model to study costimulatory marker upregulation, as the distribution of antigen and cell migration away from the lamina propria could not be controlled.

Figure 5-8. In vitro incubation with SEA only has limited effects on the expression of costimulatory markers of FACS sorted LP and lymph DCs

Survival of FACS sorted SI LP DC subsets after in vitro incubation with or without SEA (+/- SEA) for 18 hours (A). Representative expression of the costimulatory markers CD40, CD80, CD86 and OX40L is shown on SI LP DCs cultured with or without SEA for 18 hours (B). Viability of FACS sorted lymph DC subsets after in vitro incubation with or without SEA (+/- SEA) for 18 hours (C).

Representative MFI histograms of lymph DCs incubated with or without SEA for 18 hours are shown for CD40, CD80, CD86 and OX40L (D). Data represent at least two independent experiments (mean ± SEM) with each point representing cells from an individual animal.

We had observed that the expression of the costimulatory markers CD40, CD80, CD86 and OX40L by lamina propria DCs was not affected by intestinal egg injection. However, egg injections might not represent a suitable model to detect changes in costimulatory marker expression, as not all DCs might be exposed to the injected antigen. Indeed, we have shown that only 9% of total DCs take up fluorescent SEA in the small intestine. Thus, potential responses could be masked. Furthermore, DCs constantly migrate from the lamina propria to the draining lymph nodes and might not be present in the tissue at the time of harvest.

We therefore incubated FACS-sorted DCs with SEA in vitro to establish a controlled environment and ensure antigen availability to all cells. Small intestines of C57BL/6 mice were harvested and digested with Collagenase VIII. Cells were stained with fluorescent monoclonal antibodies against B220, MHCII, CD11c, CD64, Ly6C, CD103 and CD11b and total DCs purified by FACS sorting. DCs

7AAD staining solution was added to assess viability. We observed that after in vitro incubation the viability of small intestinal DC subsets was greatly affected.

When gating on the individual DC subsets and assessing their viability we observed that 35% of CD11b⁺ DCs were live, whereas 20% of DP and DN DCs and only 10% of CD103⁺ DCs were viable after the 18-hour incubation. No difference in survival was observed in SEA-containing cultures, suggesting that cell-intrinsic characteristics of the different subsets rather than the presence of antigen affected their survival in vitro (Fig. 5-8 A). The expression of costimulatory markers was assessed by comparing the MFIs between cells that were incubated with or without SEA. We observed that the expression of CD40 on total small intestinal DCs slightly decreased with SEA incubation whereas CD80 expression increased. However, these changes in MFI were not statistically significant and the expression of CD86 and OX40L by small intestinal DCs was unchanged after SEA incubation (Fig. 5-8 B). Supporting our previous observations, these data indicated that in vitro incubation with S. mansoni egg antigen only had minor effects on the expression of costimulatory markers by small intestinal DCs. However, in vitro incubation also severely affected cell viability, which could negatively influence cell stimulation and we suspected that the enzymatic digestion during DC isolation from the small intestine had a negative effect on cell survival.

As DCs collected from the lymph do not require enzymatic digestion we tested whether incubation with SEA would increase their expression of costimulatory markers. Contrary to small intestinal DCs, lymph DCs do not represent immature peripheral DCs, but are of a semi-mature state and express higher levels of MHCII, costimulatory markers and CCR7. However, contrary to fully mature MLN DCs, they still respond to TLR ligands and can acquire the ability to drive T cell differentiation, supporting our approach to incubate them with SEA in vitro (Cerovic et al., 2013). Steady state lymph was collected from MLNx C57BL/6 mice and stained for flow cytometry. Total lymph DCs were sort purified and incubated with or without 15 µg/ml SEA for 18 hours. After incubation cells were stained for the costimulatory markers CD40, CD80, CD86 and OX40L and a viability dye was added. We observed that lymph DCs had a higher viability after the 18-hour incubation than their small intestinal LP DC counterparts, indicating that the enzymatic digestion of LP DCs negatively affected their survival. 35% of CD11b⁺ lymph DCs, 40% of DP DCs, 20% of CD103⁺ DC and 30% of DN DCs were viable

DCs incubated with or without SEA (Fig. 5-8 C). Compared to small intestinal LP DCs the expression of CD86 was higher in lymph DCs, indicating their semi-mature state. Incubation with SEA decreased the expression of CD40 and CD86, whereas the MFIs of CD80 and OX40L were increased (Fig. 5-8 D). However, due to insufficient experimental replicates statistical analysis could not be performed, but MFI differences between lymph DCs incubated with or without SEA were greater compared to their small intestinal counterparts. Thus, lymph DCs expressed lower levels of CD40 and CD86, while expressing increased amounts of CD80 and OX40L after in vitro incubation with S. mansoni egg antigen.

5.1.5 CD301b⁺ CD11b-expressing dendritic cells preferentially