Date of Graduation

8-2022

Document Type

Dissertation

Degree Name

Doctor of Philosophy in Cell & Molecular Biology (PhD)

Degree Level

Graduate

Department

Cell & Molecular Biology

Advisor/Mentor

Durdik, Jeannine M.

Committee Member

Tipsmark, Christian K.

Second Committee Member

Ivey, D. Mack

Third Committee Member

Rath, Narayan C.

Keywords

Chicken; Fish; Macrophage; Mice; Temperature

Abstract

Fever is an essential component of the immune response. Baseline body temperatures vary in different species, and fever represents increased temperature over baseline. Fever initiates and enhances immune responses as well as creates an environment in which the body has advantages over pathogens. Macrophages are often the first line of cells that come in contact with pathogens, asthey reside in and traffic through tissues. They are important for their engulfment of pathogens that results in the digestion of the pathogen, and they also produce nitric oxide and cytokines that contribute to immune responses in a variety of ways, including initiating adaptive immunity and directing the production and activity of other immune cells. On this context, we have studied the role of temperature variation on macrophage physiology and function in vitro in mice, chickens and fish. During exposure to a variety of temperatures and stimuli and immunomodulators such as lipopolysaccharide (LPS), poly I:C, and peptidoglycan, the metabolism and activity was measured, in both macrophage cell lines and primary cells, at various times using protein synthesis, production of nitric oxide, and cytokine mRNA production.

In mice, with a macrophage cell line, RAW264.7, we find that the incubation at fever temperature of 39°C induced a much faster reprogramming from mitochondria-based oxidative phosphorylation to aerobic glycolysis in response to macrophage stimulation. High temperature also increased protein synthesis in general and specifically iNOS and its product NO, IL-6, IL-1α, IL-1β, IFNα, IFNβ and IL-12, but not TNFα or IL34, in LPS-stimulated RAW264.7 cells. Testing primary macrophages from both young and aged mice, we find that peritoneal resident macrophages show similar outcomes with fever temperature. Cells from young mice have larger enhancement in protein synthesis and IL-34 responses than cells from aged mice. For many responses, baseline levels are already very high in cells from aged mice. In mouse bone marrow-derived macrophage (BMDM) cultures, we find that increasing the temperature shows some increases in both iNOS mRNA and NO production. Again, the responses are greater in cells from aged mice, males and females, than from young mice. While protein synthesis is enhanced by fever temperature for stimulated BMDMs from both young and aged mice, there was no temperature effect on MHC class II expression levels.

Based on the tests of the effect of fever temperature ranges for 3 species -mice, chicken, and rainbow trout, - via NO production by stimulated macrophages, we found increases of 2 degrees for mice and 1 degree for chickens was optimal, while fish showed increasing responses over a 7-degree range. Above these temperatures, cells from all three species showed substantial drops in responses. In chickens, primary splenic cells as well as the chicken macrophage cell line HTC showed increases in NO production at 42°C than 41°C. Interestingly, substantial increases in protein synthesis were also observed at 42°C alone, with further increases with stimulation with LPS.

Both mice and chickens are homeotherms thus, and it was interesting to examine the fever responses of macrophages from a poikilothermic species. The RTS11 cell line derived from the spleen of rainbow trout, and primary cells from rainbow trout head kidney were utilized here for studies on the influence of varying temperature on fish macrophage functions. Even without stimulation, cells from both sources showed increases from 16°C to 19°C in baseline nitric oxide production and protein synthesis. Cytokine mRNA production responses peaked at later times in fish cells than in mouse or chicken cells. TNFα showed some increase with LPS and temperature, IL-1B1 showed highest production at 19°C with all stimuli, Il-1B2 only increased with LPS and high temperature, IL-12 showed a strong temperature effect alone and with polI:C at 2h stimulation. Collectively, our results indicate that initiating fever temperatures has major positive effects on macrophage function increasing both speed and magnitude of the responses. However, this thermomodulation of macrophage responses is wired differently in different species in terms of the actual temperature levels detected. Further, macrophages from a poikilothermic species show a relatively more variable responses to temperature-mediated functional modulation than cells from homeothermic species do. Our data make future work on the molecular evolution of the regulatory circuits involved of great interest.

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