In the present study, we investigated the function of resolution in macrophages from diabetic patients with acute ischemic stroke, and found that SPMs secretion was downregulated upon stimulated with ox-LDL, thus might lead to uncontrolled inflammation. Moreover, therapeutic administration of RvD2 enhanced inflammation resolution in these macrophages ex vivo. We also identified that RvD2 could protect against cerebral ischemia injury in DM mice with pMCAO and promote inflammation resolution in vivo. These findings uncovered new knowledge about uncontrolled inflammation in DM-related AIS, and provided evidence of potential novel treatment methods targeted on SPMs.
Inflammatory responses are activated both in the brain and in peripheral circulation after AIS and contribute to stroke prognosis.[21–24] DM is an independent risk factor for AIS, and often worsens the clinical symptoms and prognosis of AIS, where unresolved inflammation plays a key role therein [25–27]. SPM-mediated resolution of inflammation is a programmed process to counter-regulate pro-inflammatory responses . The disturbed balance between SPMs and pro-inflammatory forces may contribute to inflammatory diseases. To evaluate the balance of these two forces, the ratios between SPMs and LTB4 have been applied as an index in many studies[19, 29–31]. In our study, we showed that this index was reduced in macrophages from AIS patients compared to those from nonAIS patients. Such a reduction was further ananlyzed by dividing the patients into DM and nonDM groups. We then found that DM condition contributed to such a reduction prominently, as SPMs/LTB4 ratios were significantly decreased in DM+AIS patients compared with DM patients without AIS, but there was no difference between control group and nonDM+AIS group. This result is consistent with our recent report that RvD2/LTB4 is reduced in the plasma of AIS patients with DM compared to those without DM. Such a finding revealed that disturbed resolution may be a critical cause for excessive inflammation in DM-related pathophysiological conditions, e.g. AIS.
We further explored possible causes for reduced SPMs secretion by analyzing the synthesis enzymes of SPMs. We found that macrophages from DM+AIS patients expressed less 15-LOX-1 comared to nonDM+AIS patients and DM patients. On the other hand, there was no difference with regard to 5-LOX among the patient groups. 15-LOX-1 is the key enzyeme involved in the biosynthesis of most SPMs. It has been recently reported that α-hemolysin activates 15-LOX-1 in M2 macrophages to enhance the production of SPMs, and depletion of 15-LOX-1 expression abolished the effects of α-hemolysin on SPMs synthesis. On the other hand, overexpression of 15-LOX-1 ameliorated diabetic peripheral neuropathy and improved nerve recovery via increasing the production of SPMs . Thus, the reduced expression of synthesis enzyme 15-LOX-1 may be one of the causes for insufficient SPM production[36, 37].
The above results of disturbed resolution of inflammation is related to the excessive inflammation in AIS patients with DM. We found that macrophages from AIS patients with DM were more M1-polarized, and the pro-inflammatory pathway markers p-p38/p38 and p-p65/p65 were also increased in these patients. These data are consistent with previous studies reporting overactivated pro-inflammatory signals in DM. It has been demonstrated that macrophages are pushed towards a pro-inflammatory M1 spectrum in DM , which is in line with our data about lower CD206/iNOS ratio in DM patients with AIS. Furthermore, MAPK and NFκB pathways have been considered as the main activated inflammatory signals in DM [39, 40]. Thus, the decreased relative levels of SPMs may be a key etiology of unresolved inflammation in DM-related AIS. Our data explain the abnormal inflammation from a novel perspective, resolution of inflammation.
Our findings in peripheral macrophages of DM patients with AIS may represent a state of unresolved inflammation in the brain. During AIS, peripheral macrophages invade the brain and participate in the inflammatory response in the infarct area [41, 42]. Meanwhile, microglia, which serve as the resident tissue macrophages in central nervous system, are also activated after AIS [24, 43]. The invading macrophages and resident microglia respond to the ischemia pathophysiology, including neuronal death, blood-brain barrier disrupt and brain edema. Similar to many other situations, the inflammatory response after AIS partially help to clean out the brain debris, and prepare the brain for further recovery of homeostasis in the resolution stage. However, unresolved inflammatory response can lead to secondary injury of the inflamed tissue, and thus exaggerate the primary injury, i.e. disruption of cerebral blood flow in AIS patients with DM [44, 45]. SPMs-based therapeutic strategy thus becomes promising in DM-related AIS.
Among the SPMs analyzed in our study, RvD2 has been previously implicated in DM-associated inflammatory disease. In a study of hind limb ischemia, RvD2 treatment rescued DM-impaired revascularization through activation of its receptor GPR18 . In the aorta of Apoe−/− mice fed a high-fat diet, the level of RvD2 were reduced and correlated to plaque stability. In mice from diet-induced obesity, treatment with RvD2 inhibited inflammation and adiposity, and improved glucose tolerance . Thus, we chose RvD2 as a representative SPM to test the treatment effects on DM-related AIS. Firstly, we found macrophages in AIS patients with DM were skewed to M1 polarization, as shown by decreased CD206/iNOS ratio. And the pro-inflammatory MAPK and NFκB pathways were overactivated, as shown by increased ratios of p-p38/p38 and p-p65/p65. Similar result were found in microglia cells in the brain of DM+pMCAO mice. Then, we treated the human macrophages with RvD2 ex vivo, and injected RvD2 i.c.v in DM+pMCAO mice in vivo. The results showed that intervention with RvD2 switched macrophages/microglia to M2 polarization and downregulated the MAPK and NFκB inflammatory pathway markers ex vivo and in vivo. In turn, such an effect of RvD2 on macrophages/microglia contributed to the protection against brain injury caused by DM-related AIS. In DM+pMCAO mice, RvD2 reduced infarction volume, improved neurological function and increased neuron survival by inhibiting inflammation and promote resolution. These results were consistent with recent studies about the treatment effects of RvD2 on resolution of inflammation. Giannakis N and his colleague reported that pro-resolving Ly6Clo macrophages could release more SPMs and less pro-inflammatory mediators like LTB4. RvD2 treatment increased the number of macrophages expressing Ly6Clo, and thus benefit tissue repair in muscle injury . In a cardiovascular study, treatment with RvD2 shifted macrophages to a pro-resolving phenotype, and prevented the progression of atherosclerosis .