Fight Aging!

Chronic inflammation is a serious issue in later life, contributing to the onset and progression of all of the common fatal age-related conditions. This unresolved inflammation arises for many reasons, such as the pro-inflammatory signaling produced by senescent cells, the reaction of immune cells to persistent pathogens, rising levels of molecular damage and stressed cells as a result of the aging tissue environment, and so forth. The challenge lies in identifying which of these mechanisms are the most influential. The only practical way to determine the relative importance of any specific contribution to chronic inflammation is to block just that mechanism, in isolation, and then see what happens.

This approach to discovery is illustrated in today's research materials. ASC specks are involved inside cells in the formation of inflammasome protein complexes that drive some of the mechanisms of the immune response. These ASC specks can also escape cells in significant numbers, however, and thereafter act as an inflammatory signal themselves, independently of the inflammasome. How important is this contribution to the inflamed tissue environment? In this paper, researchers report on the effects of clearing ASC specks from tissue, via a novel approach, and thereby quantifying the degree to which they cause inflammation.

New approach against chronic inflammation

The cells in our body have a sophisticated alarm system, the inflammasome. Its central component is the so-called ASC protein. In the event of danger, such as an attack by a pathogen, many of these molecules join together to form a large complex, the ASC speck. This ensures two things: First, its activity causes the cell to accumulate large quantities of messenger substances, which can be used to summon the help of the immune system. And secondly, numerous pores are formed in the cell membrane through which these alarm molecules can reach the outside and fulfill their task.

These holes ultimately lead to the demise of the cell. At some point, the cell basically explodes and empties its entire contents into the tissue. The messenger substances that are now abruptly released then act like a last great cry for help. This triggers the immune system to mount a strong inflammatory response that contains the infection. That is why this mechanism of innate immune defense is hugely important. However, in this process, ASC specks also accumulate in the tissue and may persist there for a long time. "We have now been able to show in mice that their activity activates the immune system even after the threat has been averted. This can result in chronic inflammation, which severely damages the tissue."

Researchers have now succeeded in preventing this undesirable effect. They used so-called nanobodies for this purpose. These agents are antibody fragments with a very simple structure. Researchers generated nanobodies that specifically target ASC and can dissolve the specks. "The mice in our experiments have rheumatoid and gout-like symptoms. After administration of the nanobody, the inflammation and also the general health of the rodents improved significantly."

Nanobodies dismantle post-pyroptotic ASC specks and counteract inflammation in vivo

Our previous attempt to target ASC specks using conventional antibodies (Abs) resulted in increased inflammation in a silica-induced model of peritonitis. Anti-ASC Abs promoted the uptake of extracellular ASC specks by phagocytes leading to increased IL-1β release from macrophages and immune cell infiltration into the peritoneal cavity. This common feature of conventional Abs used for therapy encouraged the development of alternative approaches, including single-domain antibody fragments, such as nanobodies (VHHs), which are derived from larger heavy chain-only Abs found in camelids. We recently generated a VHH against human ASC (VHHASC), which we over-expressed in the cytosol of cells to study the molecular mechanisms involved in ASC oligomerization. We showed that VHHASC binds the caspase-recruitment domain (ASCCARD) of ASC, preventing formation of CARD/CARD interactions necessary to form full ASC specks.

In this study, we tested the therapeutic potential of VHHASC and a newly generated VHH against murine ASC (VHHmASC) to target ASC specks in vitro and in vivo. We show that pre-incubation of extracellular ASC specks with VHHASC abrogated their inflammatory functions in vitro. Recombinant VHHASC rapidly disassembled pre-formed ASC specks and thus inhibited their ability to seed the nucleation of soluble ASC. Notably, VHHASC required prior cytosolic access to prevent inflammasome activation within cells, but it was effective against extracellular ASC specks released following caspase-1-dependent loss of membrane integrity, and pyroptosis. Finally, systemic treatment with VHHmASC efficiently dampened the inflammation in mouse models of acute gout or chronic rheumatoid arthritis (RA).