Our current study demonstrated that the polarization of macrophages affected the expression of OT signaling system in the gut. In vitro, M1 macrophages inhibited the expression of the OT signaling system through secretion of pro-inflammatory factors, including IL-1β, IL-6 and TNF-α, and the intracellular STAT3, NF-κB pathways in enteric neurons. On the other hand, M2 macrophages promoted the expression of the OT signaling system in enteric neurons through secretion of TGF-β and the intracellular Smad2/3-Peg3 pathway. This is the first time to report that macrophages polarization differentially regulated the OT and OTR expression in enteric neurons.
OT is a stress hormone, which changes during infection and inflammation [36–38]. As we know, in response to LPS stimulation, the recruited M1 macrophages produced large amounts of pro-inflammatory cytokines, such as IL-1β, IL-6, TNF-α. IL-4 treatment induced the M2 macrophages polarization and released anti-inflammatory cytokines such as TGF-β and IL-10 . We found that the pro-inflammatory cytokines, including IL-1β, IL-6 and TNF-α inhibited OT and OTR expression in enteric neurons, while TGF-β exerted an excitatory effect. This data was consistent with the observation that M1 supernatant inhibited the OT and OTR expression and M2 supernatant increased it. So, we thought the cytokines in the M1 supernatant that inhibited the OT and OTR expression in enteric neurons might be IL-1β, IL-6 and TNF-α, and the cytokines in M2 supernatant that increased OT and OTR expression might be TGF-β. Moreover, pretreatment of the antagonists of these cytokines abolished the effect of M1 and M2 supernatant. So, we believed that following activation, the M1 macrophages decreased the OT and OTR expression in enteric neurons through pro-inflammatory cytokines IL-1β, IL-6 and TNF-α, and M2 macrophages increased them through anti-inflammatory cytokine TGF-β. After these inflammatory factors bound to their receptors, then they participated in biological processes through a series of signaling pathways such as JAK-STAT3 and NF-κB [28, 29, 40, 41]. In agreement with the above observations, our results showed that the pro-inflammatory factors IL-6 and TNF-α activated STAT3 signaling pathway, and IL-1β activated NF-κB signaling pathway to inhibit the expression of OT and OTR. The data of the experiments that STAT3/NF-κB signaling pathway were blocked further prove these results. One of the interesting finding of the present study was that M2 polarized macrophages increased the expression of OT and OTR in enteric neurons, which might be mediated by TGF-β. TGF-β mainly regulated downstream cell responses through Smad and non-Smad signaling pathways [31, 42]. Our experiments confirmed that TGF-β activated Smad2/3 and promoted the expression of OT and OTR. Both LY2109761(TGF-β receptor inhibitor) and SIS3HCL (Smad3 blocker) completely abolished the excitatory effect of TGF-β on the excitatory effect of OT and OTR. So, we believed that TGF-β upregulated the expression of OT and OTR in enteric neurons mainly through intracellular Smad2/3 pathway.
In order to confirm the correlation between macrophages polarization and OT/OTR expression in vivo, we used DSS induced colitis and investigated the temporal relationship between the inflammation and OT/OTR expression. We found that the disease activity index and the levels of pro-inflammatory cytokines in the colon increased and reached the highest level at the second week, so we defined this period as the inflammatory phase. In this period, correlated with the activation of M1 macrophages and the increased of the pro-inflammatory cytokines, the expression of OT and OTR in ENS were also decreased. Three weeks later after the DSS administration, the disease activity of the mice gradually returned to normal, and the expression of M2 macrophages markers and the anti-inflammatory cytokine began to increase and reached to the highest level at fourth week and returned to normal at the fifth to seventh week. We defined this period as the recovery period. In this period, correlated with the polarization of macrophages to M2 type and the release of TGF-β, the expression of OT/OTR in ENS was also increased. This observation further supported our hypothesis that following the acute inflammation, there were dynamic changes of the OT/OTR expression correlated to the different polarization of the macrophages in gut. The M1 macrophages polarization correlated to the decrease of OT/OTR expression in inflammatory period, and the M2 macrophages polarization correlated with the increase of OT/OTR expression in the recovery period. As far as we know, this is the first report about the dynamic changes of the OT/OTR expression in ENS following the DSS induced colitis. Although we found that OT inhibited the polarization of macrophages to M1 type and promoted it to M2 type, and OTR deficient mice are more susceptible to DSS induced colitis [20, 38], the anti-inflammatory effect of OT might not be very important at the acute inflammatory period, because during this period, with the release of pro-inflammatory cytokines from M1 macrophages, the expression of OT and OTR in ENS was decreased. But on the other hand, it was interesting to find that the OT/OTR expression was increased during the recovery period, which might be promoted by the polarization of M2 macrophages and the released TGF-β. During the recovery period of the acute inflammation, the function of the polarization of M2 macrophages and the released anti-inflammatory cytokines were mainly to confine the inflammation and promote the proliferation of the damaged tissue[12, 23, 43–45]. During this period, there was a positive feedback between the M2 macrophages activity and expression of OT and OTR in ENS, so it was possible that OT signaling system in ENS was involved in tissue repair following the local acute inflammation in gut.
D-mannose prevented acute lung injury by regulating PPAR-γ and TGF-β level , and inhibited LPS-induced macrophages activation by impairing IL-1β production. In vivo, D-mannose improved endotoxemia and relieve colitis . Therefore, we used D-mannose to induce polarization of M2 macrophages in the mice. We found that following D-mannose administration, the expression iNOS in gut were significantly down-regulated, but TGF-β, CD206, Arg1 were significantly increased. The expression of OT and OTR in colon was also increased. The above data indicated that D-mannose inhibited the polarization of M1 macrophages and promoted the polarization of M2 macrophages. The increase of OT and OTR expression following treatment of D-mannose might be caused by the polarization of M2 macrophages. In order to further testify this hypothesis, we used clodronate liposomes to deplete macrophages in vivo. The upregulation of OT and OTR expression was abolished by the depletion of the macrophages. So, we believed that, in vivo, D-mannose increased the OT and OTR expression in ENS through polarization of macrophages to M2 type. Another interesting finding in these experiments was that, following D-mannose administration, the size and weight of the gut were increased, although the histological structure of the intestine did not change. This change was macrophages dependent. Therefore, we thought that it might be caused by the cytokines such as TGF-β released from polarized M2 macrophages and OT released from the ENS.
As is known, Peg3 is widely expressed in various tissues of humans and mice, encodes a DNA-binding protein involved in the control of specific target genes of different cell functions  and plays a role as a transcriptional regulator for the expression of OTR. In Peg3-KO mice, the expression of OTR in hypothalamus and breast were up-regulated . In this study, we first proved that M2 supernatant and TGF-β promoted the expression of OT by suppressing the expression of peg3 in enteric neurons. But the M0/M1 macrophages and pro-inflammatory factors did not affect the expression of Peg3.
Notably, many reports indicated that the ENS cooperated with intestinal macrophages to maintain intestinal immune homeostasis [47, 48]. The unique location between mucosal macrophages and enteric neurons has attracted great attention from researchers . Emerging data suggested that MMs secreted cytokines to protect nerves from damage and promote nerve regeneration . These might be the results of the interaction between the ENS and the immune system. In this study, we found that, polarization of macrophages affected expression of enteric neurons OT signaling system. In the recent study, we reported that OT signaling system regulated the polarization of macrophages. This ‘‘feedback loop’’ between macrophages and enteric neurons could be a pivotal ‘‘controller’’ that decided whether the organism tends to be homeostasis or leads to disease.