Osteocalcin Alleviates Lipopolysaccharide-Induced Acute Inflammation via Activation of GPR37 in Macrophages
, Chunhua Liu 1, Hongchao Li 1, Haiyang Yang 1,2, Jianhao Wu 3, Jing Liu 3, Yanjiao Li 4, Xuhui Chen 5, Jianyang Xu 3 and Xiang Li 1,*Round 1
Reviewer 1 Report
Type of manuscript: Article
Title: Osteocalcin alleviates lipopolysaccharide-induced acute inflammation via activation of GPR37 in macrophages
Zhengjiang Qian , Chunhua Liu, Hongchao Li , Haiyang Yang ,Jianhao Wu , Jing Liu, Yanjiao Li , Xuhui Chen , Jianyang Xu , Xiang Li
Journal: Biomedicines
In this article, the authors have investigated the role of OCN in the inflammatory response in WT mice treated with LPS and OCN-/- mice. They have demonstrated both in vivo and in vitro that OCN treatment significantly attenuates LPS-induced acute inflammation in WT and OCN-/- mice, while the protective action of OCN was absent in GPR37-/- mice. Indeed, the in vitro studies further showed that OCN triggers intracellular responses via interaction with GPR37 in peritoneal macrophages, modulating inflammatory factors release and phagocytic function.
In this way, the authors have demonstrated that OCN plays an anti-inflammatory role in LPS-induced acute inflammation through activation of GPR37.
The paper is well structured and well written and the experimental procedure are well done, precise and multiple. I personally recommend the publication of this work.
I have only a suggestion: the authors could be to add some information on the GPR37 signalling pathway in the introduction.
Author Response
Reviewer: 1
In this article, the authors have investigated the role of OCN in the inflammatory response in WT mice treated with LPS and OCN-/- mice. They have demonstrated both in vivo and in vitro that OCN treatment significantly attenuates LPS-induced acute inflammation in WT and OCN-/- mice, while the protective action of OCN was absent in GPR37-/- mice. Indeed, the in vitro studies further showed that OCN triggers intracellular responses via interaction with GPR37 in peritoneal macrophages, modulating inflammatory factors release and phagocytic function.
In this way, the authors have demonstrated that OCN plays an anti-inflammatory role in LPS-induced acute inflammation through activation of GPR37.
The paper is well structured and well written and the experimental procedure are well done, precise and multiple. I personally recommend the publication of this work.
I have only a suggestion: the authors could be to add some information on the GPR37 signalling pathway in the introduction.
Response: According to the suggestion, we have added some information on the GPR37 signaling pathway at the end of the third paragraph in introduction section.
Thanks for the helpful comment.
Reviewer 2 Report
The authors presented involvement of OCN as key player in an anti-inflammatory function in LPS-induced acute inflammation. Quality of data is good, images are clear and representative, results are consistent with the conclusions. The limitations of the study were discussed. However, some aspects need to be elucidated:
- How do you support the statement that OCN may also take actions in macrophages via GPR37? How do you propose this could be investigated?
- How can you explain that OCN-treated macrophages could attenuate the inflammatory
responses in LPS exposed mice?
- How can you explain that OCN treatment could suppress LPS-induced upregulation of p-p65 level in macrophage?
Author Response
Reviewer: 2
The authors presented involvement of OCN as key player in an anti-inflammatory function in LPS-induced acute inflammation. Quality of data is good, images are clear and representative, results are consistent with the conclusions. The limitations of the study were discussed. However, some aspects need to be elucidated:
Point 1: How do you support the statement that OCN may also take actions in macrophages via GPR37? How do you propose this could be investigated?
Response 1: Thanks for the great comments.
In this study, the statement that OCN takes action in macrophages via GPR37 is supported by the following observation. First, OCN can trigger intracellular responses in macrophage by interaction with GPR37 as displayed in Figure 3, in which we showed that OCN treatment induced the changes of intracellular calcium (iCa2+), cAMP and pERK levels in macrophage from WT mice, but these action of OCN was absent in macrophage from GPR37-/- mice. Moreover, we further observed that OCN treatment can inhibit LPS-induced release of pro-inflammatory factors (TNFa and IL6) and phagocytic function in macrophage from WT mice, but these action of OCN was resisted in macrophage from GPR37-/- mice (Figure 4 and Figure 5). Similarly, our in vivo study also illustrated that OCN treatment could alleviate LPS-caused detrimental effects in WT mice, but these protective actions of OCN were abolished in GPR37-/- mice (Figure 6). Therefore, by combination of the results from both in vitro and in vivo studies, we concluded that OCN plays a functional role in macrophage through interaction with GPR37.
Point 2: How can you explain that OCN-treated macrophages could attenuate the inflammatory responses in LPS exposed mice?
Response 2: We are grateful to the important comment.
As showed in Figure 2, OCN treatment inhibits the expression of pro-inflammatory genes (IL-6, TNFa), but promotes the expression of anti-inflammatory genes (IL10, TGFb and Arg1), indicating that OCN is prone to promote macrophage as an anti-inflammatory phenotype. Moreover, OCN stimulation also enhanced the phagocytic function of macrophage (Figure 5). Therefore, the phenotypes and functions of macrophages could be modulated by the treatment of OCN, and the transfer of OCN-treated macrophage may play a protective role in LPS exposed mice. However, the exact mechanism should be further studies in future work as described in the discussion section.
Point 3: How can you explain that OCN treatment could suppress LPS-induced upregulation of p-p65 level in macrophage?
Response 3: Thanks for the careful reviewing and great comment.
The suppression of LPS-induced upregulation of p-p65 level by OCN treatment is coincident with the anti-inflammatory function of OCN in macrophage expose to LPS (Figure 1 and Figure2). Moreover, the inhibition of p-p65 by OCN was absent in macrophage derived from GPR37-/- (Figure 4), thus p65 NFkB might be a crucial mediator in the downstream of OCN/GPR37 signaling. However, more insight work should be performed to explore the underlying regulatory network. The related discussion are presented in the 4th paragraph of discussion section.
