Outlines

A research group, including Specially Appointed Assistant Professor (full-time) Xuhao Huang (a doctoral student at the Graduate School of Science of the University of Osaka at the time of the research), Professor Kazuya Kabayama, and Specially appointed Professor (full time) Koichi Fukase of the Institute for Radiation Science, the University of Osaka , in collaboration with Project Assistant Professor Hirotaka Kano of Keio University, Professor Shinya Hanashima of Tottori University, and Professor Kei-ichiro Inamori of Tohoku Medical and Pharmaceutical University, was the first in the world to clarify that differences in the long-chain fatty acid of the cell membrane lipid sphingomyelin (SM) are key to turning on and off the innate immune system.

In particular, the short-chain fatty acid (SM C12) was found to induce strong pro-inflammatory cytokine (IL-1α, IL-6, etc.) in macrophages and cause pyroptosis (inflammatory cell death accompanied by cell swelling and rupture) that is dependent on gasdermin D, a protein that forms holes in the cell membrane that allow water to flow in. Furthermore, the researchers demonstrated that SM C12 directly binds to human caspase-4 (and mouse caspase-11) and is a novel ligand that activates intracellular inflammatory sensors.

These findings not only represent a major advance in our understanding of innate immunity, but may also lead to the development of treatments to control excessive inflammation.

Fig. 1 Mechanism of sphingomyelins (SM) with different fatty acid chain lengths. All SMs other than those with 12 (C12) or 14 (C14) carbon chains are abundant in mammalian cells.

Credit: Kazuya Kabayama

Research Background

Human bodies have a mechanism called innate immunity that quickly detects foreign invaders such as bacteria and viruses and initiates a defensive response. One of the receptors that plays a central role is Toll-like receptor 4 (TLR4). When TLR4 detects foreign substances such as bacteria-derived lipopolysaccharides (LPS), it triggers an inflammatory response and acts to protect the body. However, excessive activation of TLR4 causes inflammatory disorders, and leads to serious symptoms such as sepsis. It is also known to be related to the worsening of chronic inflammation and autoimmune disorders. Therefore, understanding the mechanisms that appropriately control TLR4 function is a crucial medical challenge.

On the other hand, human cell membranes are rich in lipids called sphingolipids. Until now, lipids have mainly been thought of as "materials" that determine the structure of cell membranes, but in recent years, it has become clear that slight differences in lipid structure are deeply involved in the control of immunity and inflammation.

Among these, sphingomyelin (SM) exists in a variety of molecular species depending on the chain length of its fatty acids (Fig. 1), and the function of each species in the body remained unknown for a long time.

Furthermore, much remains unknown about how caspase-4/11, the intracellular sensor that triggers inflammatory cell death (pyroptosis) is regulated by lipids in the body. Against this background, the question of how slight differences in the chemical structure of lipids, such as their chain length, can switch on and off the innate immune system has attracted great interest in both the fields of immunology and lipid biology.

This research group tackled this important question head-on, aiming to clarify how differences in lipid chain length control the function of innate immunity.

Research Contents

The research group first used several types of sphingomyelin (SM) with different fatty acid chain lengths (C12, C14, C16, C24, etc.) to closely examine how immune cells called macrophages react. Then, they found that only SM C12, which has short-chain fatty acid, triggered a strong inflammatory response and produced large amounts of pro-inflammatory cytokine such as IL-1α, IL-6, and TNFα.

Furthermore, cells treated with SM C12 exhibited morphological changes characteristic of inflammatory cell death (pyroptosis), such as swelling and rupture of the cells. This indicates that short-chain sphingomyelin induces highly inflammatory pyroptosis.

Next, the research group investigated through which receptor SM C12 acts on cells. As a result, they found that the TLR4/MD2 complex, which combines the molecules TLR4 and MD2, is essential for this reaction.

In fact, the use of a drug (TAK-242) that suppresses the function of TLR4 significantly reduced SM C12-induced cytokine release and pyroptosis. Furthermore, testing using TLR4/MD2-expressing cells also confirmed that SM C12 activates NF-κB via TLR4.

On the other hand, an interesting discovery was made. Conversely, it was found that long-chain fatty acid SM (C16-C24) has the function of suppressing the inflammatory response caused by LPS. In other words, differences in SM chain length determined whether inflammation was enhanced or suppressed. Furthermore, it was found that C14 SM only elicits an inflammatory response at high concentrations, showing that the difference of two carbons, C12 and C14, significantly influences inflammatory activity.
In addition, the research group clarified that SM C12 is a new type of ligand that enters cells and directly binds to molecules called caspase-4/11. Normally, when caspase-4/11 finds a foreign substance such as LPS that has entered a cell, it aggregates to form clumps (oligomers) and then acts on a protein called gasdermin D to open holes in the cell membrane to allow water to flow in, causing the cell to burst. It is confirmed in this study that SM C12, same as LPS, also induces this oligomerization.

Furthermore, computer-based molecular docking analysis (a method for predicting how molecules will bind to each other) indicated that SM C12 enters deeply into a region of TLR4/MD2 known as the active pocket, and binds in a manner like that of Lipid A, the active form of LPS. This is an important result that explains the structural reason why SM C12 can strongly activate TLR4. On the other hand, it was also revealed that the reason why long-chain fatty acid SM (C16-C24) do not easily bind to TLR4 is because the binding pattern is significantly different from that of SM C12.

These research results revealed that SM C12 is a special lipid that can activate two different molecules, "TLR4, which works on the outside of cells," and "caspase-4/11, which works on the inside of cells," at different times (Fig. 2). This demonstrates that the length of the fatty acid chains in sphingomyelin is crucial for precisely controlling the function of innate immunity. Furthermore, SM C12 is a rare molecule that has rarely been identified in the body until now, and its properties are known to be more similar to lipids found in bacteria.

Therefore, they are highly likely to act as "signals of dangers coming from outside" for innate immunity, that is pathogen-associated molecular patterns (PAMPs). On the other hand, SM C14, which has a slightly longer fatty acid chain, is known to exist in very small amounts in mammalian cells. It indicates that SM C14 may act as a "signal that notifies cells of damage," or damage-associated molecular patterns (DAMPs), which stimulate the immune system by leaking out when cells are damaged. In the future, the study group plans to conduct research to further understand the relationship between innate immunity and lipids, using lipids produced by bacteria and lipids that appear in the blood during illness.

Fig. 2 SM C12 activates the TLR4 signaling pathway and induces inflammatory cell death (pyroptosis)

Credit: Kazuya Kabayama

Social Impact of Research

This study demonstrated that slight structural differences in chain length of lipids are key to greatly affecting immune function. This discovery is expected to lead to the development of new methods to suppress excessive inflammation that occurs in infections and autoimmune diseases. Furthermore, this finding may be applicable to the design of vaccine adjuvants that appropriately enhance immune system, as well as new immunotherapy strategies to fight cancer. In other words, this study clarified the important mechanism of how lipids affect the immune system, and its findings could make a significant contribution to the development of future medicine.

Notes

The article, “Modulation function of sphingomyelin molecular species in TLR4 signaling and cell death,” was published in American Scientific Journal Cell Reports at DOI: https://doi.org/10.1016/j.celrep.2025.116568.

Links

• Professor Kabayama’s Profile
• Professor Kabayama’s Lab.