ImmunoTox Letter

December 2020

Report on the 27th Annual Meeting of the Japanese Society of Immunotoxicology(JSIT 2020)

Masashi Tsunoda
President of the 27th Annual Meeting of the JSIT
Department of Preventive Medicine and Public Health,National Defense Medical College, JapanThe 27th Annual Meeting of the Japanese Society of Immunotoxicology (JSIT 2020) was
held as the virtual meeting from September 26th to 27th, 2020. It was originally planned to beheld with the main theme of the annual meeting as “Past, Present and Future of Immunotoxicology”
at the Prism Tower of Kitasato University, Tokyo. However, because of the pandemic ofcoronavirus disease 2019 (COVID-19), under the decision of Board Meeting of the Society, it
was held as the virtual meeting.
After the decision of the Board Meeting, the organized committee of the JSIT 2020 faced on
the problem of high cost in case that the meeting was held on the virtual meeting. At that time,
Dr. Shigeru Hisada, one of Board members, offered to use the zoom webinar system of ASKA
Pharmaceutical Co. and the virtual meeting became possible. Dr. Hisada contributed greatly inmany ways such as the notice how to participate the virtual meeting and the management of thevirtual meeting on the days of the meeting.
Finally, the symposium “Immunotoxicology, past, present and future”, “The 10th JSIT Award”
and “The 10th JSIT prize for encouragement” lectures, the workshop “Development of the latest
technology for immunotoxicity evaluation” and Student and Young Scientist Session were held
as live via the zoom webinar. In addition, the poster presentation was held on the website of theannual meeting with the abstract book.
The number of posters was 19, and the number of the presentation of Student and Young
Scientist Session was 8. The 130 people were registered to participate in the meeting, and theactual number of the participant who attended to the meeting at least a day was 115.
On the first day, at the symposium “Immunotoxicology, past, present and future” which issame as the main theme of the annual meeting, Professor Ikuo Tsunoda, Department ofMicrobiology, Kindai University Faculty of Medicine, with the coauthor Dr. Shigematsu
Toriyama gave a lecture titled with “Role of gut microbiota in Theiler’s virus model for multiple
sclerosis: Max Theiler and Hideyo Noguchi”.

Professor Yasuo Yoshioka, Institute for Open andTransdisciplinary Research Initiatives, Osaka University, gave a lecture “Vaccine Safety: Current
No.2（19）38

ImmunoTox Letter

status and challenges in the development of safe potent vaccines”.

Professor Masashi Takano

gave a lecture “Carcinogenesis of ovarian clear cell carcinoma through toxic mechanism”.
On the second day, “The 10th JSIT Award” lecture was given by Dr. Tomoaki Inoue with thetitle “Investigation of novel in vivo evaluation methods for prediction of in vitro immunotoxicity”.
“The 10th JSIT prize for encouragement” lectures were given by Dr. Shigeki Aoki with the title“Investigation of idiosyncratic drug toxicity using HLA transgenic mice” and Dr. Takamasa Kido
with the title “Immune dysfunction derived from zinc deficiency exacerbates the Th2 cell-M2
macrophage pathway.
At the workshop “Development of the latest technology for immunotoxicity evaluation”, Dr.
Yoshitaka Shirasaki, Dr. Etsuhi Kuroda, Dr. Yoh-ichi Tagawa and Dr. Chiyomi Kubo gave lectures.
Among the poster presenters, Dr. Izumi Sasaki with his presentation “The roles of unfold
protein responses in cholera toxin B-induced interleukin-1 production” as the best poster of theannual meeting. Among the Student and Young Scientist Session, Dr. Yuuto Murata with hispresentation “Immunotoxicity of G-CSF through myeloid-derived suppressor cells in tumorbearing mice” was awarded as the best presenter among the session.
The annual meeting in this year was exceptionally held as the virtual meeting, however,
without serious trouble, and with many good discussions. I think it was held successfully. I
would like to express my gratitude for many people who contributed this meeting on this report.

Photo, Dr. Shigeru Hisada, Masashi Tsunoda and Professor Masashi Takano
at the meeting room of ASUKA Pharmaceutical Co. on September 26, 2020

No.2（20）39

ImmunoTox Letter

The Best Presentation Award

The roles of unfold protein responses
in cholera toxin B-induced interleukin-1 production

Izumi Sasaki
Department of Immunology,
Institute of advanced Medicine, Wakayama Medical University
Cholera toxin B (CTB) functions as an immune adjuvant. The response of CTB is dependent
on binding with membrane glycolipids, ganglioside GM1 (hereafter GM1). This binding
facilitates internalization of CTB. However, the underling mechanisms of CTB-induced immune
adjuvant effects are poorly understood. We have found that CTB, incorporated through GM1, caninduce IL-1 production from resident peritoneal macrophages (RPMs) prestimulated withlipopolysaccharides (LPS) through the pyrin inflammasome as well as the NLRP3 inflammasome(Int Immunol, 2019). However, it remains unclear how CTB activates the inflammasomes orinduces IL-1 production. In this study, we investigated the molecular mechanisms of CTBinduced IL-1 production.
First, we performed transcriptome analysis in CTB- and/or LPS-stimulated RPMs. In thestimulation of CTB+LPS, the expression of 510 genes was more than 2-fold upregulatedcompared with LPS alone stimulation. Pathway analysis of these genes showed enrichment for
the unfold protein response (UPR) genes. Endoplasmic reticulum (ER) stress is triggered byaccumulation of unfold proteins in the ER lumen. These unfold proteins are recognized by UPR
sensors which contains inositol-requiring enzyme 1-alpha (IRE1), Protein kinase R (PKR)-likeendoplasmic reticulum kinase (PERK) and Activating transcription factor 6 (ATF6). Then, thesesensors are activated and produce several transcription factors. IRE1 activation induces X-box
binding protein 1 (XBP1) mRNA splicing and generate transcription factor XBP1. Activation ofPERK induces the expression of transcription factor C/EBP homologous protein (CHOP).
Activation of ATF6 induces autoprocessing to its mature form. Subsequently, mature ATF6 canfunction as transcription factor. These transcription factors induce the expression of ER stressNo.2（21）40

ImmunoTox Letter

related genes such as chaperone proteins or apoptosis signaling molecules. These responses arecollectively termed as ER stress response or UPR. In RPMs, XBP1 splicing and the expression ofCHOP were induced in response to CTB+LPS. These inductions were abolished in GM1-deficient
RPMs. These results indicate that CTB could induce UPR in a GM1-dependent manner.
CTB is translocated from the cell surface through Golgi to the ER. This series of event isgenerally called as retrograde transport. However, the details of retrograde transport of CTB inRPMs are not fully characterized. Then, we investigated whether CTB is translocated to the ER.
In WT RPMs, FITC-labeled CTB was co-localized with IRE1. The co-localization did not occur
in GM1-deficient RPMs. The result suggests that CT is incorporated into the ER in a GM1dependent manner.
Finally, we analyzed the effects of inhibitors for UPR sensors. Inhibition of IRE1, but not
PERK and ATF6, decreased not only XBP1 mRNA splicing but also IL-1 production in RPMs
stimulated with CTB+LPS. In conclusion, our findings indicate that CTB is a novel ER stressinducer and IRE1 is involved in CTB-induced IL-1 production from RPMs.

The Student and Young Scientists Award

Immunotoxicity of G-CSF through myeloid-derived
suppressor cells in tumor-bearing mice

Yuuto Murata
Project for Vaccine and Immune Regulation,
Graduate School of Pharmaceutical Sciences,
Osaka University
It is honor to win The Student and Young Scientists Award at the 27th Annual Meeting ofJapanese Society of Immunotoxicology. I would like to sincerely appreciate the selection
committee and the president Dr. Tsunoda.
No.2（22）41

ImmunoTox Letter

Myeloid-derived suppressor cells (MDSCs) are immunosuppressive cells that promotetumor progression by inhibiting anti-tumor immunity. Granulocyte-colony stimulating factor(G-CSF) is a preventive agent for neutropenia, which is the major adverse reaction followingchemotherapy. On the other hand, some reports have mentioned G-CSF immunotoxicity through
MDSCs. Thus, we decided to elucidate this mechanism.
In order to investigate whether G-CSF enhanced the immunosuppressive activity ofMDSCs, we induced MDSCs in the presence of G-CSF and then co-cultured with CD4+ T cells.
It was revealed that these MDSCs demonstrated stronger suppression on the proliferation of T
cells than those induced without G-CSF. Next, we identified gamma-glutamyltranspeptidase(GGT) as a candidate for the enhanced immunosuppressive function of MDSCs by RNA-seq
analysis. GGT is the only hydrolase against extracellular glutathione. We confirmed that G-CSF
enhanced the GGT activity of MDSCs. In addition, the expression levels of Arginase-1 andiNOS, which are the immunosuppressive factors of MDSCs, were also increased by G-CSF.
Furthermore, these increases were thoroughly canceled by a GGT inhibitor. Consistent with this,the administration of this inhibitor into 3LL-tumor bearing mice resulted in the inhibition oftumor progression.
These results suggest that the enhancement of immunosuppressive function by G-CSF is
mediated by GGT. We previously reported that glutamate enhanced the immnosuppressivefunction of MDSCs (the 24th Annual Meeting, Biol Pharm Bull, 2018). Therefore, it isconsidered that the production of glutamate by degrading glutathione via GGT might be natureof G-CSF immunotoxicity.
In the end, I would like to express my greatest appreciation for Dr. Tachibana and all thepeople who supported and advised me for my research.

No.2（23）42

ImmunoTox Letter

The 9th Japanese Society of
Immunotoxicology Prize for Encouragement
Construction of adjuvant safety evaluation method using
human peripheral blood mononuclear cells (PBMC) andnovel human peripheral blood mouse and application tovaccine development
Eita Sasaki
Department of Safety Research on Blood and Biological Products,
National Institute of Infectious Diseases
It is my great pleasure and honor to be awarded the JSIT prize for encouragement. I would
like to express my sincere thanks to all of the members of the awarding committee.
High safety is required in vaccines because the vaccine is given to humans in a wide rangeof age groups, from children to the elderly. I have been conducting research with the idea that it
will be possible to obtain information from immunogenicity to toxicity by applying genomics
analysis. Such methods have already been applied to synthetic drugs, etc., but in biologicalproducts such as vaccines, they have been limited to assesse antibody production, body weightchanges and pathological analysis.
Since many adjuvants act on innate immunity, I suspect that early genomics data within 24hours after vaccination may contain information on the efficacy and toxicity of vaccine adjuvants.
I focused on inactivated influenza vaccines and developed safety evaluation system for
adjuvanted influenza vaccines using biomarker genes in lung.
On the other hand, these data were experiments in mice. So I wondered if this test method
reflects effects in humans? I first performed analysis the changes of biomarker genes expression
patterns by vaccine addition in human peripheral blood mononuclear cells (PBMC) culture. I
found that in PBMC, biomarker genes expression fluctuations related to type 1 interferon (IFN)
were similar to those obtained in mouse in vivo. Although some cytokines and chemokines do not
cross in humans and mice, by using NOD/Shi-scid-IL2rγnull (NOG) mice receiving humanperipheral blood mononuclear cells (hu-PBL-NOG mice), it has been revealed that somebiomarker genes expression changes in human PBMC by vaccinations are occurred due tointeraction between human PBMC and lung parenchyma cell. Although the humanized mousemodels are not easy to apply to safety tests due to instability of the model and cost issue, it has
been found to be an excellent tool for predicting extrapolation to humans.
In the end, I would like to express my deepest appreciation to all of the people who involved,
supported and advised me for my research.
No.2（24）43

ImmunoTox Letter

The Student and Young Scientists Award

Association between non-alcholic steatohepatitis andimmunologic factor

Kinuko Uno
Department of Food and Nutrition Science, Faculty of Agriculture,
Graduate School of Tokyo University of Agriculture
Laboratory of Food Safety Assessment Science
Non-alcoholic steatohepatitis (NASH) is a lifestyle related disease that has developed fatty liver,inflammation and fibrosis despite absence of the alcohol drinking career. NASH has a risk factor ofthe development cirrhosis and liver cancer. Therefore, it needs to be managed before the conditionworsens.
We examined the liver lesions over time from early stage to serious hepatic lesion in Fischer 344
rats fed choline- deficient, methionine- lowered, amino acid-defined (CDAA) diet, the feeding periods
were set for 3 days, 2, 4, 13, 26 and 52 weeks. CDAA diet caused oxidative stress, inflammation andfibrosis from the early period, and these lesions have progressed over time.
“Liver-Gut Axis” contribute to the initiation and promotion of hepatic inflammation and fibrosis.
The second bile acid, LPS and inflammatory cytokines flow into the liver from intestinal tract, andthese factors affect hepatic stellate cell (HSC) and Kupffer cell though the toll like receptors (TLR),
which induced senescence of HSC and production of inflammatory cytokines (Seki E, et al., Nat Med,2007). Failure of intestinal barrier system and lipid metabolism causes influx of the related factors
including such as bile acid and LPS, etc. from intestinal tract. These changes were also observed inrats fed the CDAA diet, which also revealed that the enterohepatic axis is involved in NASH in theexperimental model.
CD68- positive macrophage and Th1 type cytokines were increased in the same model, and M1
macrophage is classified as pro-inflammatory macrophage and activated by Th1 type cytokines andLPS. CD44 is a hyaluronic acid receptor, deletion of CD44 enhanced the polarization of the M2
macrophage, CD44 has also been reported that the major player in the role of NASH. (StephaniePatouraux, et al., Hepatology, 2017). From these information, I would like to perform the detailed
analysis about relationship polarization of macrophage and CD44 based on the study of immunology
and toxicology.
No.2（25）44