Two recent articles have proposed that animals, like plants, have evolved two different strategies to protect against infectious diseases: resistance and tolerance (called in this article “resilience” to distinguish it from previously described uses of the term “tolerance”) (1
). The mechanisms underlying resistance to infection (the ability to limit pathogen burden) are reasonably well studied and are related to induction of inflammation by the host (1
). In contrast, the mechanisms that underlie resilience (the ability to tolerate infections for an equivalent pathogen load by limiting harmful consequences of inflammation) are less well studied (1
). Resistance and resilience are often inversely correlated, and there is a tradeoff between the two frequently opposing strategies (2
). There is little known about the mechanisms that determine the balance of this trade-off between different vertebrate species.
Most animal models are utilized for the purpose of generating results that can be extrapolated to human diseases. For example, in drug development, rodent studies are part of routine pre-clinical studies that determine progression to clinical studies in humans. Mice are the most commonly utilized species in biomedical research. Advantages of mice as opposed to other species as the top choice of species for animal models include: cost, size, public acceptance, availability of reagents, rapid generation time, and ease of genetic manipulation. However, a problem with this approach for the study of inflammation is that rodents are highly resilient to most models of induced inflammation compared to humans. One of the most common assays used to assess novel pathways of inflammation by academic investigators and pharmaceutical companies alike consists of challenging mice with the Gram-negative bacterial cell wall molecule, lipopolysaccharide (LPS), which activates cells through Toll-like receptor 4 (TLR4). Most wild type mice are highly resilient to challenge with LPS. The dose of LPS utilized in most in vivo
studies is 1–25 mg/kg, which leads to death in about half of the mice (3
). This dose is about 1,000,000 times the 2–4 ng/kg dose of LPS utilized in human volunteer studies to induce fever and cytokines (7
), and about 1000–10,000 times the dose required to induce severe disease with shock in humans (8
). It is generally assumed that this marked difference in resilience to pro-inflammatory molecules such as LPS is related to cellular differences between species. The studies reported here were undertaken to better understand the mechanism(s) involved.
Direct comparison of the sensitivity of macrophage stimulating agonists between mice and humans to agents other than LPS is limited by the safety of challenging humans. However, the resilience of mice to challenge with inflammatory agonists is probably not limited to LPS. For example, mice are at least several orders of magnitude more resilient to the superantigen Staphylococcal enterotoxin B compared to rabbits (10
), which are similar to humans in sensitivity to LPS (12
Acute lethal toxicity from bacteria, LPS, or superantigens occurs through the release of cytokines that induce fever and shock prior to death. Tumor necrosis factor (TNF) is the primary cytokine involved because antibodies directed to TNF block lethality induced by LPS and bacteria (12
). This process is believed to be macrophage dependent, because replacement of macrophages in mice unable to respond to LPS due to a mutation in TLR4 with macrophages from sensitive animals restores sensitivity (15
). Many assays have been developed to mimic this concept in vitro
by utilizing isolated macrophages in cell culture to which different agonists are added, followed by quantification of TNF. This type of in vitro
assay often forms the basis of studies to assess inflammatory pathways or candidate anti-inflammatory drugs for further development.
The discrepancy of several orders of magnitude in the sensitivity of mice and humans to LPS in vivo is not paralleled by the response of macrophages from the two species when the macrophages are removed and tested in cell culture after 1 to 24 hours incubation. Low and roughly similar concentrations of LPS (pg/ml) or other TLR agonists readily stimulate TNF and other mediator production from primary mouse macrophages or human (blood) monocytes or cell culture lines from each species when grown in cell culture under identical conditions. This dichotomy in response suggested to us that cells of the macrophage lineage might behave differently in vitro than in vivo.
To explore potential mechanisms that would explain the difference in sensitivity between mice and humans to pro-inflammatory macrophage stimuli, we studied the production of cytokines from human and mouse macrophages in response to different agonists in whole blood and in the microenvironment of mouse and human serum. These studies indicated that proteins in mouse serum markedly suppress the induction of pro-inflammatory cytokines compared to human serum. We broadened these studies to include sera from additional species that vary markedly in sensitivity to LPS and found a striking inverse correlation between ex vivo macrophage suppression by sera from different species and the published lethal sensitivity to LPS in these same species. These data support the concept that one mechanism for the large difference in resilience between species to pro-inflammatory macrophage stimuli may be regulation by proteins in serum rather than intrinsic cellular differences.