Discussion
In this study, we generated diverse DCs from the bone marrow of wild-type mice that were stimulated with LPS or IL-10 and delivered these cells to OVA-sensitized/challenged mice. Their impact on pulmonary allergic inflammation, memory CD4+ T cells and Tregs was determined. Apart from the downregulation of costimulatory molecules, the upregulation of IL-10 and TGF-β secreted from specific DCs can facilitate tolerance10. Our results show that DClps were phenotypically similar to previously reported tolDCs27, 28, which exhibited signally reduced levels of MHCII and CD80 and enhanced levels of inhibitory cytokines (IL-10 and TGF-β) relative to DCia. Our observations further confirm that DCs expressing low levels of costimulatory molecules and high levels of anti-inflammatory cytokines might take on a decided tolerance advantage.
In vivo studies showed intravenous transfer of DC10 reportedly markedly impeded airway allergic inflammation in murine models of OVA-induced asthma14, 29. Moreover, the intravenous delivery of LPS-induced tolDCs notably prohibited EAE development in a mouse model6, and Zheng et al. proved that LPS-activated plasmacytoid DCs effectively alleviated experimental chronic kidney disease30. Intriguingly, there is conflicting evidence the intranasal administration of DClps producing high levels of IL-10 enhanced airway inflammation31, and the intravenous infusion of DC10 failed to reverse the asthma phenotype in sensitized mice32. Whether DClps can be used to ameliorate allergic asthma has not been completely explored. The salient discoveries from our study show for the first time that the intraperitoneal transfer of DClps after OVA challenge considerably reduced inflammatory cell infiltration and the lung injury score in a model of asthma, suggesting that the delivery routes of DClps might influence their protective effect in treated animals. On the other hand, the injection of DCia had negligible regulatory effects on airway inflammation; therefore, our data clearly indicate the pivotal functional differences between LPS-stimulated and nonstimulated DCs in vivo.
When DClps migrate to the lung has not been clarified, nor had it been assessed whether DClps stably occupy the lung in vivo. Huang et al. previously reported that DC10 that were intraperitoneally delivered predominantly migrated to the lungs over 1 week and were visibly reduced within 2 weeks, which corresponded to the time span over which lung inflammation improved13. On the other side, DC10 treatment have been shown to induce progressive decreases in Th2 responses (cytokines, eosinophilia, and IgE) to levels near background over the next 2-5 weeks33. Others have confirmed that LPS activation is required for migratory activity and antigen presentation by tolDCs generated with dexamethasone34. Similarly, our DClps tracking data showed that injected cells well migrated to lungs but were active for only 1 week. It remains unexplored why DClps in this study only affected tolerance over 7 days while others have observed impacts over 2-5 weeks13, 33, but the findings were not contradictory since the modelling time of animals and some treatment conditions differed in these studies.
Allergen-specific Th2 memory cells have been viewed as fundamental for the development of allergic asthma in both human and animal models, and DCs regulate the generation of memory T cells at each stage via the interplay between these cells35. We thus rechallenged the animals with OVA and assessed pulmonary memory CD4+ T cells, as described above. Of note, we showed that DClps and DC10, but not DCia, markedly decreased the number and proliferation activity of CD4+CD44highmemory T cells. Similarly, our MLR assay data confirm that IL-4 secretion was reduced in both TCM and TEMcells from DClps-treated mice, but IFN-γ was reduced only in TEM cells. Nonetheless, there was no difference in the proportion of TCM and TEM cells in CD4+CD44high memory T cells among the groups. To some extent, this might suppress the Th2-skewing immune response, thereby reducing the airway inflammation in treated mice. In addition, ample experimental evidence supports that TCM and TEM cells can be converted to the other cell type in many conditions 36; hence, their exact mutual proportion need to be investigated in the future.
We showed that the number of Tregs in the lungs differed negligibly between the groups, as determined by intracellular staining. Interestingly, these data agree well with previous data showing that DC10 treatment ex vivo and in vivo did not augment the number of CD4+CD25+Foxp3+ T cells but rather contributed to their activation24, 33, 37. It is important to note that Zhou et al. demonstrated that LPS-stimulated DCs cannot modulate Treg-associated molecules on CD4+ T cells such as CD25 and Foxp3 ex vivo38. Others have also highlighted that the induction of tolerance does not necessarily lead to elevation in the number of CD4+CD25+ Tregs, but it does unequivocally enhance the expression of IL-10, CTLA4 and LAG-3 in OVA-sensitized mice39 and house dust mite-sensitized mice12, but we did not evaluate these specific markers in our experiments. Nevertheless, there is contradictory evidence showing that regulatory DCs prohibited both antigen-specific IgE production and allergic responses via the expansion of CD4+CD25+Foxp3+ Tregs16, and Tregs were shown to remarkably increase in kidney-draining lymph nodes and kidneys after the adoptive transfer of DClps30; thus, wherein we were unable to unequivocally conclude that DClps have an ignorable impact on the number of Tregs in different diseases.
DCs determine the differentiation of distinct T cell subsets and complex network of environmental signals and intrinsic cellular mechanisms affect DCs’ tolerogenicity. Signal transducer and activator of transcription (STAT) is crucial in cytokines-induced cell responses, for example, IFN-γ induced STAT1 phosphorylation and IL-12 induced STAT4 phosphorylation can promote Th1 differentiation, whereas IL-4 induced STAT6 phosphorylation promotes Th2 differentiation. Medoff et al. demonstrated that STAT6 in BMDCs was sufficient for the production of C-C motif chemokine ligand such as CCL17, CCL22, which are critical for Th2 lymphocyte recruitment to allergic airways40. In present study, there is a trend of diminished expression of py-STAT4 in DClps group compared to OVA group, but it did not reach statistical significance (p>0.05). Py-STAT6 in DClps and DC10 groups were obviously decreased compared to OVA group, which indicate DClps adoptive transfer reversed OVA-sensitized airway inflammation by inhibiting Th2-mediated inflammation.
In short, BMDCs stimulated with LPS exhibited a tolerogenic phenotype. The intraperitoneal transfer of DClps inhibited the development of Th2 allergic responses by suppressing memory T cells and decreasing STAT6 phosphorylation in a mouse model of OVA-induced asthma. Further insight into the molecular mechanism and optimal process underlying the DClps-mediated immune tolerance should be investigated in the future.