Spillover. A single, seemingly simple word that conjures up images of bite wounds, devastating disease, and societal breakdown. Spillover events can occur when zoonotic diseases–diseases carried by animals–“cross over” to humans. As we continue expanding into regions previously occupied by animals, what was once a driving plot of apocalyptic zombie movies has become a present-day reality. From HIV/AIDS, Ebola, and avian influenza, to today’s current COVID-19 pandemic, zoonotic diseases pose an ever-looming threat to mankind. But how are animals able to harbor bacteria and viruses that are deadly to humans, without appearing affected by these diseases themselves? And what implications could this ability have for potential spillover events as the human population continues to expand and push into animal territories? One study from the Bryant group at the University of Cambridge set out to answer these questions, and assessed exactly how animals from the phylogenetic order Carnivora–a group including animals often in close contact with humans like dogs, cats, and mink–evolved the ability to carry potentially deadly diseases without displaying any detectable illnesses themselves.

Before delving into the group’s findings on how and why zoonotic diseases persist undetected in animal hosts, let’s break down a critical arm of immune protection: pathogen sensing and innate immune responses. Innate immune responses are broadly reactive to invading viruses and bacteria, and recognize similar “signatures,” or features, expressed across a variety of different pathogens. But how are these features recognized? To understand that, we need to explore one of the key components of innate immunity, the protein structure known as an inflammasome. 

Contained within a type of immune cell known as macrophages, inflammasomes are composed of multiple parts, the first of which is a receptor that recognizes features expressed by pathogens, the way a fingerprint scanner recognizes specific whorls and swirls on an individual’s fingers. There are several different proteins that can make up the receptor portions of inflammasome complexes, including: nucleotide-oligomerization domain leucine-rich-repeat receptors (NLRs, like NLRP1, NLRP3, and NLRC4), pyrin, or absent-in-melanoma 2 (AIM2) receptors. Inflammasome receptors are then attached to an adaptor protein known as apoptosis-associated speck-like protein (ASC), which is attached to a protein known as caspase (Fig 1). Upon encountering a pathogen, inflammasomes activate and process two chemicals that play an important role in inflammation, IL-1β and IL-18. Activated inflammasomes also cut another protein known as gasdermin D into its active form, allowing it to form holes in the cell’s membrane and cause cell death, or pyroptosis. There are multiple types of caspase proteins shared across animals in the order Carnivora, allowing for a variety of inflammasome responses following pathogen infection. The differences in these proteins and their activity could explain why some pathogens are able to persist, and subsequently spill over from, animals in the order Carnivora.

The group noted that while the caspases expressed in Carnivora differ from those expressed in humans (order: Primates) and mice (order: Rodentia), all Carnivora share a unique form of the protein, known as caspase-1/-4. These differences in caspase proteins could explain why inflammasome responses in Carnivora differ from those of other orders. To better understand the rate and magnitude at which Carnivora inflammasomes react to pathogens, Digby et al. used macrophages from both mice and dogs and infected them with the bacteria Salmonella enterica serovar Typhimurium (S. Typhimurium). While the mouse cells responded rapidly to the infection and burst within a couple of hours, the dog cells survived about six times as long. They also compared caspase-1/-4’s enzymatic abilities to those of its closest relatives in mice,  caspase-11 and caspase-1. The group found that caspase-1/-4 was highly active, so it was not the reason why the dog cells did not respond to S. Typhimurium infection. 

Using CRISPR-Cas9, the group deleted several different caspases from the dog cells and determined that the loss of caspase-8, but not caspase-1/-4, made the cells even more resistant to cell death and IL-1ꞵ production than before. This suggested that any inflammasome responses noted previously–however small they may were–were driven by caspase-8 rather than caspase-1/-4.

Finally, because inflammasome activation is so limited in dog cells, the group set out to examine whether the process of pyroptosis can occur at all in Carnivora. By imaging dog cells after S. Typhimurium infection, they determined that although the dog cells used gasdermin D to form pores in their cellular membranes, the cells did not rupture until late after infection. The group hypothesized that because inflammasome activation is so inefficient in Carnivora cells, the pores that do form as a result of gasdermin D activation are so insufficient that they do not lead to pyroptotic cell death.

Despite the inability to recognize pathogenic invasions and induce inflammasome-mediated cell death pathways, Carnivora do not appear to be more at risk for developing infections. The group hypothesized that the high-protein diets animals in this order evolved to adopt could potentially provide antimicrobial protection against any foodborne pathogens they may consume. Outside of their gastrointestinal systems, the weakened inflammasome responses experienced by Carnivora could play a role in other parts of their bodies, allowing them to harbor pathogens that could then be passed on to unsuspecting humans. While there still is a lot to learn, these findings could form the basis for understanding why other animals’ immune systems respond the way they do, and how that may impact humans as we continue to lay claim over their lands.