Many examples exist from prokaryotes to complex multicellular organisms, in which cells from seemingly homogeneous or even recently clonal populations have very different and diverse responses to a single stimulus. This heterogeneity in response has been shown to play critical roles in processes as different as the generation of population fitness in cyanobacteria, to developmental patterning in Drosophila, and even differentiation of the vertebrate hematopoietic system1-2. Much of the observed heterogeneity involves cell fate decisions. Cell fate decisions are considered digital, in that the response is "all-or-none," as opposed to analog, or "graded" responses, in which intermediate phenotypes are stable. We wanted to explore heterogeneity in more transient cellular responses, and as a model chose to study murine bone marrow derived macrophage (BMDM) response to bacterial lipopolysaccharide (LPS). Despite BMDMs appearing to be a phenotypically homogenous population, we have observed heterogeneous digital cytokine production by them in response to LPS. Given that the observed cytokine production is digital, population level response is primarily controlled through the fraction of cells that make cytokine, rather than the level of cytokine made by a particular cell. Proper control of the cytokine-producing fraction would therefore be crucial for generating an adequate immune response while minimizing the risk of immunopathology. We investigated the idea that the phase of a macrophage with respect to its molecular clock may participate in the digital control of cytokine production in response to LPS. The molecular clock is a cell intrinsic oscillator present in nearly all mammalian cells, and participates in the circadian control of cellular physiology. Additionally, the phase of a cell within its clock can be entrained, or "reset," by numerous environmental stimuli, allowing for modulation of cellular function depending on environmental demands. We identified that two circadian clock proteins, NFIL3 and DBP, expressed in opposed phases of the molecular clock, oppositely modulate the likelihood that a macrophage will produce the cytokine interleukin (IL)-12p40 in response to LPS. NFIL3, a transcriptional repressor, and DBP, a transcriptional activator, have highly similar DNA binding sequences, and therefore are able to competitively bind DNA3. Our data suggests that NFIL3 and DBP are respectively able to repress and promote IL-12p40 production through competitive binding at the Il12b enhancer. Additionally, we and others have shown that Nfi13 expression can be induced by numerous immunomodulatory signals, including the anti-inflammatory cytokine IL-104. It is likely that changing environmental cues throughout the generation and resolution of an inflammatory response in part function by changing cell phase of clock, and therefore cell responsiveness to various signals. Finally, we show that there is circadian variation in the fraction of resident peritoneal macrophages that produce IL-12p40 in response to LPS. Together, this work demonstrates that cell phase within the molecular clock participates in the control of digital cellular responses. Additionally, this work suggests that modulation of cellular phase, and the degree to which populations are synchronized with respect to phase of clock, can be an important mechanism by which population level responses are controlled. In addition to the inflammatory response, proper regulation of digital cellular responses is likely important for a myriad of scenarios in which an "all-or-none" homogeneous population response would have devastating effects on the maintenance of organismal homoeostasis.
|School Location:||United States -- Connecticut|
|Source:||DAI-B 76/11(E), Dissertation Abstracts International|
|Keywords:||Cell Fate Decicions, Ciroation Clock, Immunology, Inmate Immunity|
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