Inhibition of necroptosis during inflamm-aging and pneumonia

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Necroptosis is a newly discovered cell death pathway. Regulated by receptor-interacting protein kinase (RIP)1 and RIP3, necroptosis involves the purposeful disruption of the cell membrane by the effecter molecule MLKL. Necroptosis is inflammatory due to the release of cytoplasmic components that act as alarmins. Necroptosis is thought to amplify and promote inflammatory circles that contribute to chronic disease. What role necroptosis plays during aging is unknown. For example, does it contribute to inflamm-aging? Pore-forming toxins produced by Streptococcus pneumoniae and other airway pathogens have been shown to trigger lung cell necroptosis and this is responsible for much of the injury that is observed. The elderly are in particular vulnerable to pneumonia, with lower respiratory tract infections being the 4th leading cause of death in those ≥65 years of age. Thus, inhibition of necroptosis is potential way to protect vulnerable individuals, such as the elderly, from the lung damage that occurs during bacterial pneumonia. We hypothesize that cell death by necroptosis increases with advanced age and this contributes to inflamm- aging. Also, that blocking necroptosis can protect the elderly from lung injury during pneumonia. Herein, we will leverage our expertise on aging, necroptosis, and bacterial pneumonia to test these hypotheses and improve our understanding of necroptosis and its impact during aging. We will: AIM 1. Determine the role of necroptosis on inflamm-aging and age-related decline in function. We have mice deficient in RIP3 and MLKL that cannot undergo necroptosis. We will compare pro-inflammatory cytokine and alarmin profiles in serum from WT, heterozygote, and KO mice at 6, 14, and 24 months of age. We will also examine aged WT mice treated for 7 days with two different necroptosis inhibitors. For all mice, we will examine the activation status of NFkB and MAPK in the thymus, lungs, heart, spleen, liver, kidney, brain, and visceral adipose by western and in immune cells (monocytes, B cells, T cells) by flow cytometry. We will also longitudinally measure activity, gait, and muscle strength to learn if blocking necroptosis impacts health status. AIM 2. Determine if blocking necroptosis protects aged animals against pneumonia. Alveolar macrophages and bone marrow derived macrophages from 6, 14 and 24 month old mice will be tested for their propensity to undergo necroptosis following exposure to the pore-forming toxin pneumolysin. Different aged WT, heterozygote, and MLKL KO mice, along with WT mice treated with necroptosis inhibitors will be intratracheally challenged with pneumolysin or infected with S. pneumoniae. Lung damage and disease severity will be assessed by measuring inflammatory cytokines, pathology, and bacterial burden, respectively. This proposal is in response to PAR-14-191:T1 Translational Research: Novel Interventions for Prevention and Treatment of Age-Related Conditions. We will determine the contribution of necroptosis to inflamm-aging and learn if blocking necroptosis protects against inflamm-aging related decline and pneumonia.