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.