key: cord-0833640-6qknw382 authors: Hu, Xin; Zhang, Ye-An; Chen, Ben; Jin, Zi; Lin, Mei-Lin; Li, Ming; Mei, Hong-Xia; Lu, Jia-Chao; Gong, Yu-Qiang; Jin, Sheng-Wei; Zheng, Sheng-Xing title: Protectin DX promotes the inflammatory resolution via activating COX-2/L-PGDS-PGD(2) and DP(1) receptor in acute respiratory distress syndrome date: 2021-11-10 journal: Int Immunopharmacol DOI: 10.1016/j.intimp.2021.108348 sha: faf6031c8d1d346330607347877bd5fd5bd5585a doc_id: 833640 cord_uid: 6qknw382 PURPOSE: Acute respiratory distress syndrome (ARDS) is characterized by uncontrollable inflammation. Cyclooxygenase-2(COX-2) and its metabolite prostaglandins are known to promote the inflammatory resolution of ARDS. Recently, a newly discovered endogenous lipid mediator, Protectin DX (PDX), was also shown to mediate the resolution of inflammation. However, the regulatory of PDX on the pro-resolving COX-2 in ARDS remains unknown. MATERIAL AND METHODS: PDX (5 μg/kg) was injected into rats intravenously 12 hours after the lipopolysaccharide (LPS, 3mg/kg) challenge. Primary rat lung fibroblasts were incubated with LPS (1μg/ml) and/or PDX (100nM). Lung pathological changes examined using H&E staining. Protein levels of COX-2, PGDS and PGES were evaluated using western blot. Inflammatory cytokines were tested by qPCR, and the concentration of prostaglandins measured by using ELISA. RESULTS: Our study revealed that, COX-2 and L-PGDS has biphasic activation characteristics that LPS could induce induced by LPS both in vivo and in vitro.. The secondary peak of COX-2, L-PGDS-PGD(2) promoted the inflammatory resolution in ARDS model with the DP(1) receptor being activated and PDX up-regulated the inflammatory resolutionvia enhancing the secondary peak of COX-2/L-PGDS-PGD(2) and activating the DP(1) receptor. CONCLUSION: PDX promoted the resolution of inflammation of ARDS model via enhancing the expression of secondary peak of COX-2/L-PGDS-PGD(2) and activating the DP(1) receptor. PDX shows promising therapeutic potential in the clinical management of ARDS. lung injury in ALI/ARDS model via enhancing the endothelial barrier repairment. 12, 13 Both PGE 2 and PGD 2 are converted from PGH 2 by various synthases. PGE 2 synthases consist of cPGES, microsomal prostaglandin E synthase-1(mPGES-1) and microsomal prostaglandin E synthase-2 (mPGES-2). Various pro-inflammatory stimulations can up-regulate mPGES-1, m. Meanwhile, cPGES and mPGES-2 are constitutively expressed. 14 PGD 2 is induced by lipocalin-type prostaglandin D synthase (L-PGDS) and hematopoietic PGDS (H-PGDS). PGD 2 acts its important role via G protein-coupled receptor DP (DP 1 ) and the chemoattractant receptor-homologous molecule CRTH2 (DP 2 ). PGD 2 showing multiple pathophysiological characters via different receptors. 15 Primary lung fibroblasts, which are far from being bystander cells, are important to host defense in ARDS. After the inflammatory stimulation, fibroblasts are activated following the immune response, and secreting a large number of cytokines like interleukin 6 (IL-6) and interleukin 8 (IL-8). 16 Growing evidence manifested that fibroblasts-secreted growth factors promote the alveolar barrier functions and alleviate the lung injury induced by LPS. 17, 18 Our pervious study suggested that fibroblasts regulate the inflammatory resolution by producing proresolving mediators PGD 2 . 19 Formal studies have reported that endogenous lipid mediators and mechanisms can drive the resolution of inflammation the resolution of inflammation was driven by novel lipid mediators and endogenously triggered mechanisms. 20 Specialized pro-resolving lipid mediators (SPM) were identified as new genus, including Resolvins, Protectins and their aspirin-triggered forms.. 21, 22 Protectin DX (10S,17S-dihydroxydocosa-4Z,7Z,11E,13Z,15E,19 Zhexaenoic acid) is a newly discovered member of this genus, which derived from natural ω-3-fatty acid docosahexaenoic acid (DHA), 23 PDX possesses anti-inflammatory and inflammation pro-resolving bioactions. 24 A study reported that PDX maintains the integrity of lung epithelium, increases the alveolar fluid clearance of ARDS in rat. 25 PDX regulates inflammatory cell infiltration via resident macrophage in LPS-induced lung injury. 26 Moreover, PDX was shown to alleviate lung injury induced by LPS via inducing primary rat type II alveolar epithelial cells proliferation and inhibiting their apoptosis in vivo and in vitro. 27 Our studies confirmed that COX-2 has a biphasic activation pattern in LPS stimulated lung fibroblasts, showing that COX-2 and PGD 2 expression levels peaked at 6 hours and subsequently after 48 hours. 28 Moreover, NF-κB p50/50 was responsible for regulating the secondary expression peak of COX-2 in the resolution stage of the ARDS rats model. 29 However, the downstream mechanism of secondary peak COX-2 and PGD 2 in the resolution of inflammation remains unclear Furthermore, whether PDX promotes the resolution of inflammation by regulating secondary peak of the COX-2 and PGD 2 has not been proved yet. In this study, we hypothesize that the secondary peak of COX-2/L-PGDS-PGD 2 a promote the resolution of inflammation in the ARDS model. Moreover, we surmise that PDX plays a pro-resolving role in ARDS by enhancing the pro-resolving COX-2/L-PGDS-PGD 2 expressions and activating the DP 1 receptor. Protectin DX, NS-398( selective COX-2 inhibitor), AT-56 (L-PGDS inhibitor), BW245C (DP 1 receptor agonist), 15(R)-15-methyl-PGD 2 (CRTH2/DP 2 receptor agonist), MK-0524(DP 1 receptor antagonist) and CAY-10471(CRTH2/DP 2 receptor antagonist) were obtained from Cayman Chemical Rat primary lung fibroblasts and lung tissues were washed in iced PBS and harvested by using RIPA Primary lung fibroblasts were isolated from SD rats; isolation process was same as we described before. 30 The same part of the left lung of each rat was fixed in 10% paraformaldehyde for 24 hours. Lung tissues were embedded in paraffin wax, sectioned, and stained with H&E for light microscopy analysis. Acute lung injury scores were quantified by a single observer who was blinded to the treatment groups via the established histopathological scoring system. 31 PGE 2 and PGD 2 concentrations in fibroblasts cellular supernatants and rat homogenized lung tissues were measured as previously described. 32 ELISA kits were purchased from R&D Systems (Minneapolis, MN, USA), all procedures were performed according to the manufacturers' instructions. All analyzes were run in triplicate and repeated twice. Total RNA samples in lungs were isolated using TRIzol reagent (Takara Bio, Kusatsu, Japan) according to the manufacturer's protocol. The cDNA of mRNA was synthesized by the reverse transcription kit purchased from Thermo Scientific (Rockford, IL, USA). The expression of mRNA was detected by qPCR (Bio-Rad, Hercules, CA, USA) with TB Green® Premix Ex Taq™ PCR kit (Takara Bio, Kusatsu, Japan). The gene-specific primers used are listed in Table S1 and mRNA levels normalized to GAPDH. Data were calculated with using the 2-ΔΔCtmethod. All data were presented as mean ± SEM. All data were analyzed using one-way ANOVA, followed by a Tukey test for post hoc comparisons. P < 0.05 was considered as significant. Statistical analyses were performed using GraphPad Prism 8.0 software (GraphPad Software, San Diego, CA, USA). Pulmonary lung fibroblasts play an important role in inflammatory diseases and participate actively in immune response. 33 Herein, to find out the dynamic change of COX-2 expression in fibroblasts, cells were exposed to LPS for 0,6,12,24,48,72 hours ( Figure 1A ). We found that the COX-2 protein presents a biphasic expression character, COX-2 firstly peaked at 6 hours and secondary one at 48 hours ( Figure 1B) . Interestingly, we found that the L-PGDS also showed a biphasic expression, similar to that of COX-2 expression, peaking at 6 and 48 hours. ( Figure 1C ). Moreover, mPGES-1 only presented a single peak expression at 6 hours ( Figure 1D ). mPGES-2, as a constitutively expressed synthetase, 34 showed no significant changes after the LPS challenge ( Figure 1E ). PGE 2, as a pro-inflammatory prostaglandin, 35 highly expressed at 6 hours only ( Figure 1F ). In contrast, PGD 2 highly peaked both at 6 hours and 48 hours, and the secondary peak of PGD 2 was significantly higher than the first one ( Figure 1G ). We revealed that there was a biphasic activation character of COX-2/L-PGDS-PGD 2 in LPS-stimulated lung fibroblasts. To We have previously demonstrated that PDX ameliorates the wound repair of the lung epithelial barrier via ALX receptor. 27 Here we would like to know whether PDX enhances the secondary peak of COX-2/L-PGDS via ALX receptor. The ALX receptor antagonist, BOC-2(10μM) was added 30 minutes before the PDX treatment. As Figure 2D and Figure 2E presented, pre-treatment with BOC-2 reversed the promoting effect of PDX on the secondary peak of COX-2 and L-PGDS. suggesting the promoting effect of PDX on the expression of secondary peak COX-2, L-PGDS are via the ALX receptor. Next, to explore the activation character of COX-2 and L-PGDS in vivo, we established the self-limited ARDS model in SD rats by administrating low dosage of LPS intravenously ( Figure 3A ). Pathomorphological changes were detected by H&E staining, compared with the control group, the lung architecture in the LPS group showed most remarkable damage at 12 hours, as evidenced by the changes in lung injury score. The mRNA expression of IL-1β, IL-6 and TNF-α in the lungs ,peaked at 12 hours and decreased subsequently after the LPS challenge (data were shown in Figure S1 ). Moreover, COX-2 showed a biphasic expression pattern as presented in Figure 3B .COX-2 first peaked at 6 hours after LPS stimulation, and the secondary peak appeared at 24 hours. Our results also showed L-PGDS had a biphasic expression characteristic in vivo as same as in fibroblasts.The expression of L-PGDS firstly increased at 6 hours, and the secondary peak appeared at 24 hours ( Figure 3C ), mPGES-1 was highly expressed at 6 hours only ( Figure 3D ). mPGES-2 showed no significant change after the LPS stimulation ( Figure 3E ). The production of Next, to determine if secondary peak of COX-2 could play a pro-resolving role in the resolution of ARDS model. NS-398, a clinical-used selective COX-2 inhibitor, was administrated intravenously in rats 1 hour before(the NS-398+LPS group)or at 12 hours(the LPS+NS-398 group)after the LPS challenge ( Figure 4A ). Pathological features were detected at 24 hours as shown in Figure 4B . Compared with the NS-398+LPS group and LPS group, the LPS+NS-398 group revealed more distinct interstitial edema, hemorrhaging, thickening of alveolar walls, and inflammatory cells infiltration in the lung tissues. In contrast to the LPS group, the NS-398+LPS group showed less pathological damage. As shown in Figure 4C , acute lung injury score was quantified and found to be in consistent with the pathophysiological changes. In addition, compared with the NS-398+LPS group and the LPS group, relative mRNA levels of the proinflammatory cytokines: IL-1β, IL-6 and TNF-α were much higher in LPS+NS-398 group (Figure 4D-F) . These findings suggested that inhibition the latter peak was observed between LPS group and the NS-398+LPS group ( Figure 4I) . Inhibition of the COX-2 reduced the production of PGD 2 at 24 hours ( Figure 4J ). In summary, inhibition of the secondary peak COX-2 in the resolution stage of inflammation caused more significant lung damages compared to the inhibition of the first peak of COX-2 in the early stage of inflammation. Based on these findings, we surmise that the secondary peak of COX-2 plays a key role in promoting the resolution of inflammation in ARDS. We then attempted to figure out the function of the secondary peak of L-PGDS. AT-56 was administrated to rats at 12 hours after the LPS challenge ( Figure 5A ). H&E staining ( Figure 5B) revealed that, compared with the LPS group, LPS+AT-56 group displayed more interstitial edema, hemorrhage, and inflammatory cells infiltration in lung tissues. In accordance with this, the lung injury scores were also elevated ( Figure 5C) , along with the release of IL-1β, IL-6 and TNF-α ( Figure 5D -F). These results indicated that suppression of L-PGDS blocked ARDS resolution. ELISA results also proved that inhibiting the L-PGDS decreased the PGD 2 production at 24 hours ( Figure 5H ), while no significant difference was found in PGE 2 between the LPS group and the LPS+AT-56 group ( Figure 5G ). All results above indicated that the secondary peak COX-2/L-PGDS -PGD 2 was responsible for ARDS resolution in murine models. PDX have already been proved to enhance the repair of lung epithelial barrier in ALI/ARDS murine model . 27 To evaluate whether PDX promotes inflammatory resolution via activating ALX receptor and the COX-2/L-PGDS-PGD 2 expressions in rat ARDS model, PDX was given at 12 hours after the LPS exposure ( Figure 6A ). H&E staining result showed that PDX markedly alleviate the morphological and histological damages induced by LPS, consistent with a decrease in acute lung injury score (Figure 6B and 6C) . Administration of BOC-2 (the ALX receptor inhibitor) reversed the effect of PDX on both histological damages and the release of pro-inflammatory cytokines induced by LPS ( Figure 6B and 6D-F) , consistently with the acute lung injury score ( Figure 6C) . These findings suggested that PDX promotes the inflammation resolution via activating the ALX receptor. In addition, PDX significantly up-regulated the protein expression of COX-2 and L-PGDS during the resolution stage. PDX significantly promoted the expression of the pro-resolving mediator PGD 2 ( Figure 6J ). No significant difference was found in the PGE 2 level between the LPS group and the LPS+PDX group ( Figure 6I ). Pre-stimulation with BOC-2 reversed the improved effect of PDX on COX-2 and L-PGDS protein expression as well as PGD 2 secretion ( Figure 6G -H) . These results indicated that PDX facilitated the inflammatory resolution via activating ALX receptor and enhancing the expression of COX-2/L-PGDS as well as the production of PGD 2 . Previous studies demonstrated that the PGD 2 -DP 1 signaling pathway was responsible for the antiinflammatory function of ALI/ARDS. 12, 13 Herein, we investigated what kind of PGD 2 receptor plays the major role during the resolution phase in the murine ARDS model, BW-245C (DP 1 receptor agonist), 15(R)-15-methyl-PGD 2 (CRTH 2 /DP 2 receptor agonist), MK-0524(DP 1 receptor antagonist) or CAY-10471(CRTH 2 /DP 2 receptor antagonist) was intraperitoneally injected into rats at 12 hours after the LPS stimulation ( Figure 7A ). Morphological staining ( Figure 7B ) revealed that the DP 1 receptor agonist BW-245C significantly alleviated the pathological damage, while the DP 1 receptor antagonist (MK-0524) aggravated the lung injury. In comparison, the agonist or antagonist of the DP 2 receptor did not significantly influence the lung injury condition. As expected, the acute lung injury scores were in line with the findings of morphological staining ( Figure 7C) . Furthermore, mRNA levels of proinflammatory cytokines: IL-1β ( Figure 7D ), IL-6 ( Figure 7E) , TNF-α ( Figure 7F ) also validated the above results. Then, we investigated if PDX promoted the inflammatory resolution through the activation of the were given to rats with or without PDX ( Figure 8A ). Pathological staining ( Figure 8B ) showed that the DP 1 receptor agonist enhanced the pro-resolving function of PDX, while the DP 1 receptor antagonist suppressed the improved inflammatory resolution by the PDX treatment. Meanwhile, the DP 2 receptor did not significantly affect the resolution of inflammation promoted by PDX ( Figure 8B ). The acute lung injury scores were in accordance with the findings of morphological staining ( Figure 8C) . Furthermore, inflammatory cytokines levels (IL-1β, IL-6 and TNF-α) were downregulated after the DP 1 receptor agonist treatment, while up-regulated by DP 1 receptor antagonist ( Figure 8D-F) . In contrast, the DP 2 receptor did not affect the release of IL-1β, IL-6 and TNF-α ( Figure 8D-F) . Altogether, these data suggested that PDX promotes resolution of inflammation by activating the DP 1 receptor in vivo. Our study uncovered that COX-2/L-PGDS-PGD2 expressions have a dual activation induced by LPS. Importantly we found that the secondary peak of COX-2/L-PGDS-induced PGD2 was responsible for the pro-resolving process in ARDS. Moreover, we showed that the DP1 receptor was activated in inflammatory resolution. This study provides evidence for a new mechanism by which PDX may promote inflammation resolution of the ARDS model through improving the expression of the secondary peak of COX-2/L-PGDS-induced PGD 2. Interestingly, the ALX receptor antagonist, BOC-2, abrogated the effect of PDX on the COX-2/L-PGDS-PGD 2 . Altogether, these findings were summarized in Figure 9 , showed that PDX also promotes the inflammatory resolution via activating the ALX receptor and enhances the inflammatory resolution partly via activating the DP 1 receptor. COX-2 is catalyzed after the inflammatory stimuli immediately. Pro-inflammatory PGs were induced by COX-2, . 37 Fukunaga K. et al indicated that COX-2 plays a protective role in ALI/ARDS through COX-2-derived mediators, partly via enhancing the lipoxin signaling. 7 However, this study did not discuss the dynamic change of COX-2 expression in the ARDS model. In our LPS-stimulated ARDS model, we revealed that COX-2 was quickly and peaked at 6 hours, then peaked twice at 24 hours in vivo. The secondary peak of COX-2 displayed pro-resolving character which was distinguished from traditional concepts. Consistent with our study, Gilroy D.W. et al reported the biphasic expression of COX-2 in the carrageenin-induced pleurisy mice model . 8 However, the specific role of each COX-2 peak remains unclear. Herein, our findings suggested that COX-2 could be proinflammatory at early stage and be pro-resolving during the later stage of inflammation. Therefore, blindly using COXs inhibitors such as NSAIDs may postpone the resolution process of inflammatory diseases and cause unexpected damage to the ARDS patients.. This might be one of the reasons that the application of NSAIDS in patients with ARDS or sepsis in the clinical trials could not be an effective therapy . 6 Therefore, anti-inflammatory drugs should be used carefully depending on the stage of inflammation in the patients with ARDS. Our previous findings revealed that there is a biphasic expression of COX-2 induced-PGD 2 in LPSstimulated lung fibroblasts, 19 however we did not uncover the downstream PGs synthetases . In this study, we demonstrated that mPGES-1 peaked only at 6 hours assosiated with maximal PGE 2 synthesis. While L-PGDS had biphasic peaks at 6 and 48 hours assosiated with a biphasic PGD 2 synthesis. Previous studies indicated that L-PGDS alleviated the endothelial barrier injury in ALI/ARDS by promoting the production of PGD 2 in vivo. 13 We confirmed that the L-PGDS existed biphasic expression in rat ARDS model. Furthermore, we found that the secondary peak of COX-2/L-PGDS derived PGD 2 promoted the inflammatory resolution of ARDS model. PGs, such as PGE 2 , plays pro-inflammatory role in inflammation. 28 Previous studies indicated that PGD 2 promoted the resolution of inflammation. 38 In addition, PGD 2 performs its physiological function via the DP 1 and CRTH 2 (DP 2 ) receptors. We showed that the DP 1 receptor was activated in the resolution of ARDS. Administration of the agonist of the DP 1 receptor improved the resolution of inflammation, while the inhibition of the DP 1 receptor aggravated lung injuries. In our self-limited ARDS model, activation or inhibition of DP 2 receptors did not affect the resolution of inflammation. As previously described, the resolution of acute inflammatory diseases is an active process. 39 There is an internal, self-protective feature among the patient with ARDS. Thus, we established a self-limited ARDS model for further study. PDX, as an endogenous "braking signal", displays anti-inflammatory and pro-resolving characteristics. We already have determined that PDX could ameliorate the wound repair of the lung epithelial barrier. 27 In this study, we demonstrated that PDX significantly alleviated inflammatory injuries in ARDS via enhancing the expression of the secondary peak of COX-2/L-PGDS-and its metabolite PGD 2 . Additionally, we revealed that the pro-resolving effect of PDX was dependent on the activation of the DP 1 receptor. PDX exerts its effects via ALX receptor pathways. 25, 26, 27 Thus, our study used the ALX antagonist BOC-2. As expected, the ALX antagonist reversed the PDX induced improvement on COX-2/L-PGDS-PGD 2 expression both in vivo and in vitro, suggesting that PDX up-regulates COX-2/L-PGDS-PGD 2 signaling via the ALX/FPR2 receptor. Therefore, we found that the activation of the COX-2/L-PGDS-PGD 2 secondary peak and the DP 1 receptor may be the novel mechanism by which PDX exerts its pro-resolving effect on inflammation. The authors declare that they have no conflicts of interest for this work. and PGD 2 were detected by ELISA. All data are presented as mean ± SEM, n = 6. *p<0.05, **p<0.01,***p＜c0.001,****p < 0.0001. 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Prostaglandins and inflammation Incidence and outcomes of acute lung injury Signalling networks regulating cyclooxygenase-2 Hematopoietic prostaglandin D2 synthase controls the onset and resolution of acute inflammation through PGD2 and 15-deoxyDelta12 14 PGJ2 Inflammatory resolution: new opportunities for drug discovery Author Statement Xin Hu, designed and performed the experiments. Ye-An Zhang Sheng-Wei Jin, Sheng-Xing Zheng Department of Anesthesia and Critical Care, The Second Affiliated Hospital and Yuying Children's Hospital of Wenzhou Medical University, 109 Xueyuan Road The Second Affiliated Hospital and Yuying Children's Hospital of Wenzhou Medical University 2 and DP 1 receptor in acute respiratory distress syndrome Highlights 1.Cyclooxygenase-2 (COX-2) and Lipocalin-type prostaglandin D synthase (L-PGDS) have a biphasic activation character in ARDS model induced by lipopolysaccharide. 5.The pro-resolving effect of PDX was through the activation of DP 1 receptor.