Positive Selection in CD8+ T-Cell Epitopes of Influenza Virus Nucleoprotein Revealed by a Comparative Analysis of Human and Swine Viral Lineages.

UNLABELLED: Numerous experimental studies have demonstrated that CD8(+) T cells contribute to immunity against influenza by limiting viral replication. It is therefore surprising that rigorous statistical tests have failed to find evidence of positive selection in the epitopes targeted by CD8(+) T cells. Here we use a novel computational approach to test for selection in CD8(+) T-cell epitopes. We define all epitopes in the nucleoprotein (NP) and matrix protein (M1) with experimentally identified human CD8(+) T-cell responses and then compare the evolution of these epitopes in parallel lineages of human and swine influenza viruses that have been diverging since roughly 1918. We find a significant enrichment of substitutions that alter human CD8(+) T-cell epitopes in NP of human versus swine influenza virus, consistent with the idea that these epitopes are under positive selection. Furthermore, we show that epitope-altering substitutions in human influenza virus NP are enriched on the trunk versus the branches of the phylogenetic tree, indicating that viruses that acquire these mutations have a selective advantage. However, even in human influenza virus NP, sites in T-cell epitopes evolve more slowly than do nonepitope sites, presumably because these epitopes are under stronger inherent functional constraint. Overall, our work demonstrates that there is clear selection from CD8(+) T cells in human influenza virus NP and illustrates how comparative analyses of viral lineages from different hosts can identify positive selection that is otherwise obscured by strong functional constraint. IMPORTANCE: There is a strong interest in correlates of anti-influenza immunity that are protective against diverse virus strains. CD8(+) T cells provide such broad immunity, since they target conserved viral proteins. An important question is whether T-cell immunity is sufficiently strong to drive influenza virus evolution. Although many studies have shown that T cells limit viral replication in animal models and are associated with decreased symptoms in humans, no studies have proven with statistical significance that influenza virus evolves under positive selection to escape T cells. Here we use comparisons of human and swine influenza viruses to rigorously demonstrate that human influenza virus evolves under pressure to fix mutations in the nucleoprotein that promote escape from T cells. We further show that viruses with these mutations have a selective advantage since they are preferentially located on the "trunk" of the phylogenetic tree. Overall, our results show that CD8(+) T cells targeting nucleoprotein play an important role in shaping influenza virus evolution.

Palmitate and lipopolysaccharide trigger synergistic ceramide production in primary macrophages.

Macrophages play a key role in host defense and in tissue repair after injury. Emerging evidence suggests that macrophage dysfunction in states of lipid excess can contribute to the development of insulin resistance and may underlie inflammatory complications of diabetes. Ceramides are sphingolipids that modulate a variety of cellular responses including cell death, autophagy, insulin signaling, and inflammation. In this study we investigated the intersection between TLR4-mediated inflammatory signaling and saturated fatty acids with regard to ceramide generation. Primary macrophages treated with lipopolysaccharide (LPS) did not produce C16 ceramide, whereas palmitate exposure led to a modest increase in this sphingolipid. Strikingly, the combination of LPS and palmitate led to a synergistic increase in C16 ceramide. This response occurred via cross-talk at the level of de novo ceramide synthesis in the ER. The synergistic response required TLR4 signaling via MyD88 and TIR-domain-containing adaptor-inducing interferon beta (TRIF), whereas palmitate-induced ceramide production occurred independent of these inflammatory molecules. This ceramide response augmented IL-1ß and TNFα release, a process that may contribute to the enhanced inflammatory response in metabolic diseases characterized by dyslipidemia.

Macrophages modulate cardiac function in lipotoxic cardiomyopathy.

Diabetes is associated with myocardial lipid accumulation and an increased risk of heart failure. Although cardiac myocyte lipid overload is thought to contribute to the pathogenesis of cardiomyopathy in the setting of diabetes, the mechanism(s) through which this occurs is not well understood. Increasingly, inflammation has been recognized as a key pathogenic feature of lipid excess and diabetes. In this study, we sought to investigate the role of inflammatory activation in the pathogenesis of lipotoxic cardiomyopathy using the α-myosin heavy chain promoter-driven long-chain acylCoA synthetase 1 (MHC-ACS) transgenic mouse model. We found that several inflammatory cytokines were upregulated in the myocardium of MHC-ACS mice before the onset of cardiac dysfunction, and this was accompanied by macrophage infiltration. Depletion of macrophages with liposomal clodrolip reduced the cardiac inflammatory response and improved cardiac function. Thus, in this model of lipotoxic cardiac injury, early induction of inflammation and macrophage recruitment contribute to adverse cardiac remodeling. These findings have implications for our understanding of heart failure in the setting of obesity and diabetes.