Untangling the Immune and Injury Response in the Liver as a Result of Simulated Spaceflight Conditions

Introduction:: Activation of the innate immune system is of peak concern in spaceflight biology; the development of an inflammatory physiological state due to microgravity and radiation exposure directly impacts every physiological system in the body. This work aims to enable the goals of expanding human exploration in space set forth by the National Aeronautics and Space Administration (NASA). Stresses of spaceflight include microgravity and radiation which we replicate with a rodent hindlimb disuse model combined with ionizing radiation exposure. The interest in spaceflight and liver health has been prompted by multiple observations of rapid onset fatty liver pathology in mice flown on the International Space Station. Lipid accumulation in the liver is a sign of Non-Alcoholic Fatty Liver Disease (NAFLD), a disease which impacts 1 in 4 adults in the world. Liver damage, in response to injury or chronic stressors, is defined by inflammation and fibrotic scarring. The process of the liver injury response is mediated by the innate immune system including pro-inflammatory and anti-inflammatory macrophages. It is hypothesized that early lipid accumulation in the liver initiates an immune response amplified by systemic inflammation due to radiation and microgravity exposure in spaceflight.

Materials and Methods:: This investigation looks at the recruited macrophage and resident Kupffer cell populations in the liver. We ask how both population size and inflammatory action changes under hindlimb unloading (HU) with or without acute 1 Gy gamma exposure (IR). To isolate the non-immune related effects of HU and IR we employed a C-C motif chemokine 2 (CCR2) knock out (KO) animal model, where immune cell recruitment is impaired. Further, we tested the effectiveness of the powerful antioxidant Pyrroloquinoline Quinone (PQQ), in mediating liver injury. Macrophages were isolated from dissociated liver tissue and purified via MerTK+ Magnetic Assisted Cell Sorting (MACS). RNA was extracted and purified using Qiagen RNeasy isolation kits. A portion of unpurified cells was fixed in 2% paraformaldehyde and stained for quantification via flow cytometry; specifically looking for monocyte derived macrophages, infiltrating monocytes, and resident Kupffer cells. Additionally, to investigate liver health we carried out metabolomics and liver enzyme function assays.

Results, Conclusions, and Discussions:: Results: We found circulating blood monocytes dramatically increased in response to 30-day HU as compared to normally loaded animals. PQQ only minorly reduced the volume of circulating monocytes in HU or IR conditions. Interestingly, KO and WT animals shared similar circulating monocyte populations but differed in ability for those immune cells to migrate into the tissue. Metabolomic profiling of the liver tissue showed several key changes in hepatocyte function. We found that acyl-carnitines, necessary intermediates for fatty acid transport into mitochondria, were elevated in our HU and IR experimental groups. Additionally, we saw significantly reduced intermediates in the taurine metabolism pathway, a key component in bile acid synthesis in hepatocytes. RNA sequencing and analysis is currently underway to identify alterations in macrophage immunometabolic and inflammatory state in response to HU and gamma radiation.


Discussion & Conclusions: Here we show that changes in liver health due to simulated spaceflight conditions on earth do not align with the pathology seen in spaceflight. However, we have shown that these conditions are capable of driving recruitment of monocyte derived macrophages to the tissue, leading to an increased inflammatory presence. These results suggest monocyte release from bone marrow is stimulated in a CCR2 independent manor, while it remains essential in trafficking from the blood vessel into the liver tissue. Further we know that these simulated spaceflight conditions were effective in causing a level of metabolic disruption in essential hepatocyte functions. The results presented here are indicative of reduced mitochondrial efficiency, possibly contributing to the increased liver lipid content. Future analysis is aimed at better understanding how these functional changes differ in the absence of recruited immune cells.


Acknowledgements (Optional): : Special thanks to all members of the Blaber Lab, Brookhaven National Laboratory NASA Space Radiation Lab Support Staff, and the RPI BioResearch Core Staff. This work is funded by NASA Grant #80NSSC20K0289 and by the Rensselaer Polytechnic Institute Biomedical Engineering Department.

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