Macrophage-derived netrin-1 contributes to endometriosis-associated pain, 2021, Ding et al.

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Macrophage-derived netrin-1 contributes to endometriosis-associated pain

Spoiler: Authors

Shaojie Ding; Xinyue Guo; Libo Zhu; Jianzhang Wang; Tiantian Li; Qin Yu; Xinmei Zhang

BACKGROUND
Endometriosis-associated pain can be considered a type of neuropathic pain. Netrin-1 is an axon guidance cue that regulates axonal attraction or rejection in neural injury and regeneration. However, whether netrin-1 plays a role in endometriosis-associated pain remains unclear. This study aimed to determine the role of netrin-1 in endometriosis-related pain.

METHODS
Peripheral blood, peritoneal fluid, and endometrial tissues were sampled from women with (n=37) and without endometriosis (n=23). Lipopolysaccharide (LPS) and interferon gamma (IFN-γ) were used to stimulate human monocytic cell lines (THP-1) and rat alveolar macrophage-derived cell lines (NR8383) to induce M1 phenotype macrophages. Serum netrin-1 concentrations, endometrial expression levels of netrin-1, and its receptors including deleted in colorectal cancer (DCC), A2B adenosine receptor (A2BAR), uncoordinated B receptor (UNC5B), uncoordinated C receptor (UNC5C) and Down’s syndrome cell adhesion molecule (DSCAM) were assessed. The polarization phenotypes of the peritoneal macrophages were identified by detecting the marker expression of M1/M2 macrophages via flow cytometry. The expression levels of M1 markers and netrin-1 in THP-1/NR8383 cells were determined.

RESULTS
The expression levels of netrin-1 in serum and endometriotic lesions were significantly higher in women with endometriosis, and were positively correlated with the severity of endometriosis-associated pain. Netrin-1 was co-expressed with CD68 (a macrophage marker) in endometriotic lesions and was synthesized and secreted by THP-1 and NR8383 cells in the process of M1 polarization. In women with endometriosis, peritoneal macrophages were polarized towards the M1 phenotype. In addition, increased expression of DCC and A2BAR, and decreased expression of UNC5B, UNC5C and DSCAM were found in endometriotic lesions.

CONCLUSIONS
These results suggest that netrin-1 production by macrophages in endometriotic lesions may play an important role in endometriosis-associated pain.

Web | DOI | PDF | Annals of Translational Medicine | Open Access

 

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Some summary quotes —

In 2000, Anaf et al. were first to report S100-labeled nerves infiltrating in endometriotic lesions, with the percentage of nerves being significantly higher in the lesions of women who suffered severe endometriosis-associated pain. In subsequent studies, elevated specific makers for sensory, sympathetic, and parasympathetic nerves and growth factors such as nerve growth factor (NGF), brain derived neurotrophic factor (BDNF) and neurite growth factor 2 (NEGF2) were identified in different types of endometriotic lesions, all of which correlated with endometriosis-associated pain. Therefore, endometriosis associated pain was considered to be a type of neuropathic pain.

Netrins are members of the laminin superfamily which share a similar amino acid terminal sequence. The name netrin is derived from the Sanskrit word “netr”, meaning “to guide”, because of their role in axon guidance. Until now, three secreted netrins (netrins 1, 3 and 4), and two glycosylphosphatidylinositol-anchored membrane proteins, netrins G1 and G2, have been identified in mammals. Netrin Gs regulate axon guidance by forming synaptic interactions between neurons with the transmembrane netrin G ligands NGL1 and 2. The secreted netrins can bind to the receptors of deleted in colorectal cancer (DCC), neogenin or uncoordinated (UNC5) A–D, causing axonal attraction or rejection. Recent studies have shown that netrins also participate in angiogenesis, cell proliferation, migration and tumorigenesis by binding to the receptors of DCC, neogenin, UNC5, Down’s syndrome cell adhesion molecule (DSCAM), CD146 and A2B adenosine receptor (A2BAR).

It has been demonstrated that netrin-1, the most studied guidance cue, triggers an attraction effect through DCC and neogenin, or a repulsion effect via UNC5 A-D. […] In the process of peripheral neural injury and regeneration, increased levels of netrin-1 are mainly produced by Schwann cells. Netrin-1 expression has also been found to be increased in hypoxic conditions

The interaction between macrophages and nerve fibers contributes to neuroinflammation and pain generation in endometriosis. A large number of up-regulated molecules released by nerve fibers in endometriotic lesions such as monocyte chemotactic protein-1 (MCP-1), colony-stimulating factor 1 (CSF-1) and leukemia inhibitory factor (LIF), are responsible for the recruitment of macrophages from vessels within the lesions. Conversely, infiltrating macrophages in the lesions can in turn mediate neurogenesis by secreting neurotrophins, semaphorins, and vascular epithelial growth factor (VEGF).

The serum concentrations of netrin-1 in women with endometriosis were significantly higher than those from women without endometriosis (P<0.05)

The mRNA expression levels of netrin-1 in endometriotic lesions were significantly higher than those in the eutopic endometrium from women with (P<0.01) or without endometriosis (P<0.01), and were positively correlated with the severity of endometriosis-associated pain (r=0.83, P<0.0001).

IHC results showed that DCC was mainly expressed in the epithelial and stromal cells and the interstitium in endometriotic lesions

Since netrin-1 is an axon guide molecule, we speculated that netrin-1 mediates endometriosis-associated pain by promoting nerve fiber infiltration in endometriotic lesions. In fact, netrin-1 has bi-functionality on axonal guidance. It has been reported that netrin-1 leads to axon attraction by binding to the DCC receptor or repulsion by binding to the UNC5A–D receptors in the same cells. […] The bi-functionality on axon guidance is based on the crystal structure of netrin-1 and its receptors.

Our study demonstrated that endometriotic lesions showed significantly higher expression levels of DCC and A2BAR and lower expression levels of UNC5B and UNC5C compared with the eutopic endometrium, which further supports the proposal that netrin-1 is responsible for endometriosis-associated pain by promoting nerve infiltration in endometriotic lesions. Dorsal root ganglion (DRG) neurons express DCC, neogenin and UNC5A–D receptors.

In endometriosis, macrophage retention in endometriotic lesions increases the local concentration of netrin-1, which may play a role in the infiltration of nerve fibers. In tumorigenesis, netrin-1 promotes cell survival, proliferation, invasion and migration in different types of cancer […] Although endometriosis is a benign gynecological disease, it demonstrates malignant behaviors in its adhesion, proliferation, invasion, metastasis and recurrence. Thus, elevated netrin-1 may also be involved in the growth of endometriotic lesions.

In the in vitro studies, we found that netrin-1 was synthesized and secreted by THP-1 and NR8383 cells in the process of M1 polarization. However, it has been reported that endothelial cells, as well as epithelial cells, can also synthesize and secrete netrin-1.