July 13, 2022

Commented [Expert1]: Please revise the date before
submission.

Joanne Kwak-Kim
Chief Editor
Journal of Reproductive Immunology

Dear Editor:
Please find enclosed our manuscript entitled “Interleukin-18 levels and mouse Leydig cell apoptosis
during lipopolysaccharide-induced acute inflammatory conditions,” which we request that you consider
for publication as an Original Article in Journal of Reproductive Immunology.

This study investigates the effect of interleukin-18 on Leydig cell apoptosis. We have previously
demonstrated that endogenous IL-18 induces testicular germ cell apoptosis during acute inflammation
when plasma IL-18 levels are very high. Moreover, we have demonstrated an IL-18-mediated decrease ingerm cell apoptosis during the recovery phase of inflammation when plasma IL-18 levels are low.
However, the impact of acute inflammation and IL-18 on the Leydig cells remains unclear. In this study,
we aimed to understand the role of IL-18 in the testes during acute inflammation by examining thepathways mediating Leydig cell apoptosis in response to lipopolysaccharide (LPS) or high levels of IL-18.
We stimulated a mouse Leydig cell line TM3 and a mouse macrophage cell line RAW264.7 with LPS orrecombinant IL-18 (rIL-18). We assessed the expression of inflammatory cytokines, caspase cleavage,and markers of apoptotic pathways (TNF-α, TNFR1, Fas, FasL, and FADD). LPS stimulation increased
caspase 3 cleavage in Leydig cells. IL-18 protein expression in RAW264.7 cells was examined todetermine if high IL-18 levels in the testes after inflammation were derived from immune cells. IL-18
increased significantly in RAW264.7 macrophage cells on LPS stimulation. In the TM3 cells, high-dose
rIL-18 (10 or 100 ng/mL) upregulated TNF-α, Fas, and FADD, promoted cleavage of caspase-8 and -3,and lead to Leydig cell apoptosis via a death-receptor-mediated pathway. These results indicate that highdose IL-18 derived from macrophages are harmful to Leydig cells during acute inflammation. Therefore,reducing IL-18 overexpression could be a new therapeutic approach for preventing Leydig cell apoptosis
as a result of acute inflammation.

We believe that the findings of this study are relevant to the scope of your journal and will be of interest
to its readership. All authors have significantly contributed this study. The authors report no conflicts ofinterest.

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Commented [Expert2]: Authors are encouraged to suggest thenames and email address of prospective reviewers. The names ofundesired reviewers can also be stated.

This manuscript has not been published elsewhere and is not under consideration by another journal. Themanuscript has been carefully reviewed by an experienced editor who specializes in editing papers writtenby scientists whose native language is other than English.
We look forward to hearing from you at your earliest convenience.

Sincerely,
Name of author
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Enago | Disclaimer: Any unauthorized reprint or use of this material is prohibited. No part of this report may be
reproduced or transmitted in any form or by any means (electronic or mechanical), including photocopying,recording, or by any information storage and retrieval system, without written permission from the author/publisher.

Commented [Expert3]: Please mention these details before
submission to the journal.