Discussion

Dendritic cells play a crucial role in regulating the immune response and are involved in the pathogenesis of IBD.²⁷ We and others^5,7 have reported that mucosal DCs in inflamed tissue are exposed to hypoxia, and this finding caused us to focus on the role of dendritic HIF-1 in a murine model of colitis. Recent studies have demonstrated that epithelial HIF-1 exerts a protective effect in intestinal inflammation.^7,8 In the current report we show for the first time that dendritic HIF-1α also exerts a pivotal influence on the control of colitis. The absence of dendritic HIF-1α leads to severe intestinal inflammation with increased levels of inflammatory cytokines and impaired induction of regulatory T cells.

Inflammatory cytokines such as IL-6 and IL-23 are strongly involved in the pathogenesis of IBD.^28,29 IL-6 can inhibit Treg-mediated suppression in a murine model of colitis.³⁰ It has been shown that IL-23 promotes inflammation by IL-6 and IL-17 and drives a Th17 immune response.²⁹ A recent study demonstrated that lamina propria DCs constitutively express IL-23³¹; this finding reveals an additional influence of DCs on IBD. In the present study we found higher levels of IL-6 and IL-23 in DSS-treated CD11cCre/HIF-1α^+f/+f mice than in HIF-1α^+f/+f mice. In addition, we looked for lipocalin2, a known regulator of the gut´s immune response.¹⁵ Lipocalin2 is a member of the lipocalin protein family and is apically secreted by epithelial cells. Chassaing et al. measured fecal lipocalin2 levels with ELISA and reported that these levels are up-regulated in murine models of colitis.³² After DSS treatment, our knock-out mice exhibited elevated levels of lipocalin2, a finding indicating severer inflammation (Figure 2D). Therefore, in future experiments fecal measurements of lipocalin2 may prove useful in monitoring the progression of intestinal inflammation.

We further aimed to analyze the influence of dendritic HIF-1α on the production of type I IFN in a murine colitis model. Wobben et al. found that dendritic

IFN-α production is HIF-1α dependent.¹¹ We confirmed these findings in vivo, because our knock-out mice exhibited less expression of IFN-α4 and IFN-α12 mRNA after DSS treatment. Type I IFNs are known to exert protective effects in intestinal inflammation by preventing epithelial barrier dysfunction and inhibiting the inflammatory response of activated macrophages.^33,34 This finding is in line with our finding that HIF-1α^+f/+f mice exhibit fewer infiltrated macrophages than do CD11cCre/HIF-1α^+f/+f mice in DSS colitis (Figure 2E).

Tregs can limit inappropriate inflammatory responses and are protective in intestinal inflammation.³⁵ A subset of intestinal DCs can induce Tregs to control inflammation. This is why we looked for the impact of dendritic HIF-1α on Treg induction. Tregs are defined by the expression of Foxp3.²³ We found fewer Foxp3-positive cells in our knock-out mice, and this reduced number of cells may lead to severer inflammation. The induction of Tregs is further dependent on the release of retinoic acid (RA) and TGF-β by DCs.²² RA is converted from retinal by aldehyde dehydrogenases (Aldhs). Aldh1a2 is found primarily in DCs in the mesenteric lymph nodes.²⁰ Moreover, RA can inhibit the IL-6–mediated induction of proinflammatory Th17 cells.³⁶ DSS treatment did not induce higher levels of TGF-β, Aldh1a2, and retinoic acid receptor α (RARα) in our DSS-treated knock-out mice (Figure 5B-D). Consistent with the findings of Fernández-Martínez et al., who reported crosstalk between HIF-1α and RARβ,³⁷ we have now shown a connection between dendritic HIF-1α and RARα expression. Transcription of RARα is directly induced by RA.²¹ Together with the decrease in Aldh1a2 expression, this finding leads to the conclusion that HIF-1α–deficient dendritic cells secrete less RA and consequently induce fewer Tregs.

Treg induction also relies on DC-secreted IL-10.³⁸ Spontaneous colitis develops in IL-10 knock-out mice after a few weeks.³⁹ Several studies have demonstrated that HIF-1α influences IL-10 production. Cai et al. showed that HIF-1α activates IL-10 production in myocytes and is required for remote preconditioning of the heart.⁴⁰ HIF-1α–dependent IL-10 secretion is also required for lung interstitial macrophages to prevent airway allergy.⁴¹ In the current study we found for the first time that dendritic loss of HIF-1α directly leads to diminished levels of IL-10 in vitro

(Figure 4A-B) and in the lymph nodes in vivo (Figure 5A), with a severe impact on intestinal inflammation. The fact that we could not detect significant changes in IL-10 mRNA expression in the colon (Figure 4D) may be due to the small numbers of DCs in colon tissue.

Intestinal DCs can induce the specific gut-homing markers α4β7 integrin and CCR9 on Tregs.⁴² RA enhances the expression of α4β7 integrin and CCR9.²⁰ We found that the expression of β7 integrin and CCR9 was significantly higher in DSS-treated HIF-1α^+f/+f mice than in knock-out mice (Figure 4E-F), a finding indicating that dendritic HIF-1α is needed for adequate Treg homing.

Rimoldi et al recently reported that intestinal epithelial cells (IECs) and DCs interact.¹⁸ IECs release TSLP, which conditions mucosal DCs to noninflammatory tolerogenic DCs. Jang et al. demonstrated that the expression of TSLP in keratinocytes is HIF-1α dependent.⁴³ We were unable to detect differences in the expression of TSLP in the colon because the IECs in our model express active HIF-1α. However, expression of the TSLP receptor (TSLPR) was induced by LPS only in BmDCs from HIF-1α^+f/+f mice (Figure 4C). Correspondingly, in vivo only HIF-1α^+f/+f mice exhibited increased levels of TSLPR in DSS colitis (Figure 4E).

Furthermore, we detected an additional interaction between IECs and DCs.

We found that knock-out of dendritic HIF-1α leads to increased production of MUC1 - 3. Mucins are produced primarily by goblet cells and play a crucial role in mucosal protection and repair.²⁶ The expression of mucins in intestinal inflammation depends on the severity and degree of colitis. Hoebler et al. found that the expression of MUC1 and MUC3 was higher in DSS-induced colitis, whereas the expression of MUC2 did not change.⁴⁴ In contrast, Dharmani et al. found that the expression of MUC2 and MUC3 did not decrease in a DSS model.⁴⁵ Clinical studies also found diverging findings in patients with IBD.^26,46,47 Van der Sluis et al. reported that MUC2 knock-out mice develop spontaneous colitis and are more prone to DSS colitis.⁴⁷ Furthermore, recent studies demonstrated an interaction between IL-6 and the production of mucin. Yokoigawa et al. found that mucins secreted from colon cancer cells induce the production of IL-6.⁴⁸ In contrast, Li et al. showed that IL-6 modulates mucin expression of colorectal cancer cells by up-regulating MUC1 and

down-regulating MUC2.⁴⁹ Other studies have demonstrated IL-6–mediated induction of MUC2 and MUC3 in colon carcinoma cells.^50,51 In our DSS-treated knock-out mice, higher levels of IL-6 were associated with higher levels of mucins. This association may result from a compensatory protective mechanism in the intestinal epithelium, because mucins are also involved in mucosal repair.^52,53

Although new therapeutic strategies for IBD focus primarily on T cells, it is noteworthy that the phenotype of DCs can also affect intestinal inflammation. In the present study we demonstrated that loss of dendritic HIF-1α leads to severer intestinal inflammation with impaired induction of Tregs and enhanced production of mucins. Targeting intestinal dendritic cells may therefore be an additional therapeutic option in the treatment of IBD.