[Practical Case Sharing] Elucidation of the mechanism of anti-inflammatory effect of erythromycin in pneumonia and periodontitis using knockout mice (Dr. Maekawa, Niigata University)
Summary: Associate Professor Tomoki Maekawa of Niigata University School of Dentistry commissioned Setsuro Tech Inc. (Setsuro Tech) to produce knockout mice of DEL-1, an anti-inflammatory molecule, in December 2017, and we delivered seven F0 mice in April of the following year. Then, on August 6, 2020, the international academic journal JCI Insight published a paper including the analysis results of the DEL-1 knockout mice produced by our company. Therefore, we asked him about the background of his research and future prospects.
Erythromycin, a component for periodontitis treatment, has other effects than antibacterial effects.
Dr. Maekawa said that I am a dentist, and I did academic research as I worked as a dentist in a hospital when I was in Japan. I did say “when I was in Japan” since I am now an associate professor of not only Niigata University in Japan, but also the University of Pennsylvania in the United States. Since 2019, I have been conducting research at the University of Pennsylvania as part of an international collaborative research project funded by a Grant-in-Aid for Scientific Research.
Originally, I was supposed to have already returned to Japan. However, due to the novel coronavirus infection (COVID-19), I am still staying in the US (as of September 2020). The paper published in JCI Insight is a presentation of the work I was engaged at Niigata University.
My research subject is periodontitis. Periodontitis is a disease in which inflammation of gums spreads to the supporting structures of teeth, leading to bone destruction and eventually tooth loss. As a drug treatment for periodontitis, erythromycin, a macrolide antibacterial drug, is used to eliminate the bacteria that cause periodontitis.
On the other hand, it has been reported that macrolides have not only antibacterial effects but also immunomodulatory and anti-inflammatory effects. One example of this is a clinical report that COVID-19 improved pneumonia symptoms. In addition, macrolides are used to treat non-infectious diseases such as cystic fibrosis and COPD (chronic obstructive pulmonary disease). However, the details of how macrolides exert their anti-inflammatory effects are still unclear.
Erythromycin induces upregulation of DEL-1 expression and inhibition of inflammation
Macrolide antimicrobials are considered to have anti-inflammatory effects because they inhibit neutrophil-induced inflammation. Developmental endothelial locus-1 (DEL-1) protein, which is expressed on vascular endothelial cells, is known as a molecule that blocks neutrophil migration. We have previously shown that DEL-1 inhibits inflammatory reaction by antagonizing the integrin of neutrophils and preventing neutrophils from migrating out of blood vessels. Previously, we reported that preventing neutrophils from reaching the brain suppressed inflammation in mice with experimental autoimmune encephalomyelitis. We also reported that DEL-1 suppressed inflammatory bone resorption in monkeys. Therefore, we hypothesized that the macrolide antimicrobial agent erythromycin may exert anti-inflammatory effects by inducing the expression of DEL-1.
Administration of erythromycin to mice with experimental pneumonia caused by bacterial endotoxin lipopolysaccharide (LPS) resulted in the production of DEL-1 in lung tissue as well as a reduction in neutrophils and alleviation of alveoli. Erythromycin treatment also improved the survival rate when lethal doses of LPS were administered. These effects were not observed with other macrolide antimicrobial agents such as penicillin or Josamycin, suggesting that they were specific to erythromycin.
All of the above experiments were performed in wild-type mice. However, in order to demonstrate the anti-inflammatory effect to erythromycin in a DEL-1-dependent manner, it was necessary to show that erythromycin cannot exert anti-inflammatory effects in DEL-1 knockout mice. This is the reason why I asked Setsuro Tech to produce DEL-1 knockout mice.
The knockout mice have improved the quality of the paper significantly.
Dr. Maekawa said, although we were concerned about the recessive lethality in knockout mice, fortunately, DEL-1 knockout mice survived. His team found that, with an LPS-induced pneumonia model, erythromycin did not cause a reduction in neutrophils, alleviation of pneumonia symptoms, or increase in survival rate in the DEL-1 knockout mice. Similarly, in mice with periodontitis, erythromycin treatment did not improve the symptoms in DEL-1 knockout mice. These results clearly demonstrated that erythromycin suppresses inflammation in pneumonia and periodontitis through the induction of DEL-1 expression.
Taken all, Dr. Maekawa stated that he and his team believed that demonstration of the lack of DEL-1 using knockout mice is a decisive factor for the quality of this paper. In order to show that the anti-inflammatory effect of erythromycin is mediated by DEL-1, it was essential to conduct experiments using knockout mice. The results became convincing with in vivo data provided by knockout mice. With a detailed pathway analysis, we found that erythromycin acts on the growth hormone secretagogue receptor (GHSR) to induce the expression of DEL-1.
In fact, when we submitted this paper, a reviewer suggested that we should also analyze the results of GHSR knockout mice. Although we were not able to verify the results this time, we deeply felt the importance of knock out mice.
As an application of this research, we would like to develop anti-inflammatory and bone regenerating drugs targeting DEL-1. It is known that the expression level of DEL-1 decreases with aging, which is considered to be one of the causes of inflammation and bone melting. We would like to conduct a clinical trial to investigate changes in the expression of DEL-1 and bone density by administering erythromycin. However, since erythromycin is an antibacterial drug, there is a concern about resistant bacteria. Therefore, we are also working on finding a DEL-1 inducer among variants of erythromycin that do not have antibacterial properties. Since DEL-1 is involved in the inhibition of bone resorption, we are also analyzing the phenotype of bone in DEL-1 knockout mice, which has yielded very interesting results. We are planning to publish a paper to show these results in the very near soon.
Feedback from Prof. Maekawa group for the technical supporting of Setsuro Tech
Prof. Maekawa stated, “when we ordered the production of DEL-1 knockout mice, one of the things that helped our team significantly is that Setsuro Tech was responsible for the design of guide RNA. Our team was not experts in mice or genome editing, and they were not very familiar with genome-editing by CRISPR/Cas9, including guide RNA design. In this paper, we cited two papers provided by Setsuro Tech in the Methods with regard to the production of knockout mice. However, we found that Setsuro Tech now has a template for paper publication, so that we will use that template next time. Also, the price was very low. When I come back to Japan, I want to ask Setsuro Tech to produce knockout mice with the deletion of each domain of DEL-1. Also, I will contact Setsuro Tech in the near future to ask about the production of Flox mice since Flox mice production service is currently available, which was not in 2017 when I made an order of knockout mice.”
Prof. Maekawa claimed that researchers can improve the quality of papers by using knockout mice. In addition, at the end of this interview, although he said that even there are many companies overseas that provide knockout mouse production service, I am supporting Japanese companies, and it was a great encouragement for Setsuro Tech.
Setsuro Tech produces and delivers not only knockout mice but also point mutation and humanized mice (replacing the gene sequence of mouse protein with human sequence). Please feel free to contact us.
Erythromycin inhibits neutrophilic inflammation and mucosal disease by upregulating DEL-1.
Maekawa T, Tamura H, Domon H, Hiyoshi T, Isono T, Yonezawa D, Hayashi N, Takahashi N, Tabeta K, Maeda T, Oda M, Ziogas A, Alexaki VI, Chavakis T, Terao Y, Hajishengallis G.
JCI Insight. 2020 Aug 6;5(15):e136706. doi: 10.1172/jci.insight.136706.
Dr. Maekawa is an Associate Professor at the Center for Advanced Oral Function Education and Research, Niigata University Graduate School of Medical and Dental Sciences, and an Associate Professor at the University of Pennsylvania, USA. D. in Dentistry from Niigata University Graduate School of Medical and Dental Sciences.
He completed his doctorate at Niigata University Graduate School of Medical and Dental Sciences and is a certified periodontist by the Japanese Society of Periodontology. He specializes in immunology and periodontal system dentistry.