With this research we demonstrate that children with septic shock have reduced PPARα expression in whole blood derived RNA. The magnitude of that reduction is commensurate with disease severity as measured by PRISM scores; the sicker the child the lower the PPARα expression. To our knowledge, this is the first reported association between PPARα and severity of disease in sepsis.
These findings are significant because they indicate that dysregulation or loss of PPARα activity may play a central role in the immunopathogenesis of sepsis beyond its known function as a metabolic regulator. Indeed, these two activities of potential immune and metabolic coordination may be intimately associated. Recently, the intersection between metabolic disease and inflammation has been an active field of research and a high degree of crosstalk is observed between specific cells and tissues of both systems (9
). PPARα activity may be necessary for the initiation, maintenance, or resolution of an appropriate immune response and the absence of PPARα from the regulatory milieu can contribute substantially to life threatening disease processes. Evidence in support of this hypothesis was provided in our animal studies.
Mice that lack a functional PPARα gene are more susceptible to death using a model of surgically induced polymicrobial sepsis treated with antibiotics. Antibiotics were used specifically to mimic the clinical arena where physicians encounter and treat sepsis syndromes. PPAR alpha null mice experienced earlier onset of mortality than wild type mice and had overall increased mortality, indicating that PPARα is involved early in the development of sepsis. This pattern of mortality was conserved across two other models of sepsis, which strengthens the conclusion that PPARα null mice have a more susceptible phenotype.
We have also shown that PPARα null mice, compared to wild type mice, have reduced levels of many inflammatory cytokines at 24 hours after the initiation of sepsis. There were no statistically significant differences in cytokine levels between the two groups at 3 or 6 hours after CLP. This was a surprising observation due to the significant body of research that ascribes an anti-inflammatory role to PPARα in other models of disease and inflammation (22
). Thus, its absence in sepsis should have permitted the evolution of a hyperinflammatory state after CLP. We initially anticipated that an early hypercytokinemia would have contributed substantially to the observed increase in mortality. The results obtained, however, indicated that a hypoinflammatory state is developing even at this early time point. This conclusion is corroborated by evidence of decreased splenocyte activation and increased tissue bacterial load.
Splenic dendritic cells and macrophages from PPARα null animals, when compared to those from wild type mice, exhibited decreased surface expression of MHCII, an antigen presentation protein upregulated in activated antigen presenting cells. CD69, a marker of T cell activation, was lower as well on PPARα null CD8 T cells. It is not known whether decreased activation results in decreased cytokine levels or vice versa.
The finding of increased tissue bacterial load is important. PPARα knock out animals had more bacteria in their lung and splenic tissues at 24 hours than wild type counterparts. No statistical difference in bacterial load was seen between study groups in the blood and peritoneal fluid. This is likely due to the administration of antibiotics which preferentially cleared those body fluids, but probably had less tissue penetration. Even if overwhelming bacteremia was not a factor in sepsis mortality in our antibiotic treated model, tissue microbial persistence points toward a functional deficit in host defense mechanisms in the PPARα knockout mice. This may be attributable to increased bacterial tissue penetration and colonization, permissive bacterial replication, or decreased immunologic bacterial clearance compared to wildtype mice. Reduced bacterial killing could be due to the decreased level of circulating chemokines which possibly impaired phagocyte tissue extravasation and chemotaxis. It does not appear to be directly related to macrophage phagocytosis or oxidative burst.
Despite the association observed between PPARα expression levels and disease severity in septic children, it is yet unclear whether the relationship is causal or coincidental. PPARα expression in the blood could possibly be reduced by any number of variables that are also known to be associated independently with severe sepsis and increased mortality including lymphocyte apoptosis, mitochondrial bioenergetic failure, and metabolic derangements induced by progressive organ failure. Furthermore, whole blood derived mRNA represents only the expression patterns of circulating leukocytes and may not mirror PPARα expression or activity in other tissues less accessible to sampling. Although PPARα in those tissues is likely relevant to organ function and survival in sepsis we feel that our initial approach of evaluating whole blood derived mRNA is viable as a hypothesis-generating tool because of its relative ease of sampling and because it allows a biologically relevant assessment of the immunologic response in sepsis.
However, our experimental data from mice indicate that individuals without PPARα are less able to survive in the face of a septic challenge. As noted above, we did not observe the anticipated hyperinflammatory storm, but rather a relative immunosuppressed state in the PPARα null animals. This finding, though unexpected, fits well with much current thinking that a significant portion of actual human mortality from sepsis results from a state of relative immunoparalysis (30
). A possible pro-inflammatory role for PPARα, however, does stand at variance with what is currently understood about its related family members PPAR. A substantial body of research has ascribed to PPAR an anti-inflammatory influence on multiple cellular processes (6
). It is not currently known how PPARα and PPARγ interact with each other or with the third member of the family PPARβ/δ. It may be that while PPAR has potent anti-inflammatory properties, those of PPARα are less significant, but that PPARα’s activation of important bioenergetic pathways in leukocytes is essential for their normal activation and function.
In conclusion, our data indicate that decreased or absent PPARα expression confers a survival disadvantage in sepsis and potentiates a functionally immunosuppressed state. Further research is needed to evaluate the effect of PPARα activation and to elucidate the specific mechanisms by which this nuclear receptor impinges upon the immunologic response in sepsis.