Viral infection and the subsequent immune responses such as the expression of interferon beta (ifnb1) show extreme levels of cell to cell variability. A fraction of cells get infected and a fraction of infected cells induce an ifnb1 response. These responding cells then signal to coordinate appropriate immune responses required to clear infection. The mechanism of propagation of this response at the single cell level is critical to generate an appropriate defense against the virus, yet is incompletely understood.
Interesting work on cell to cell variability has been done using transfected ifnb1 reporter constructs. However, this approach has several limitations. The reporter systems introduce multiple copies of the reporter construct in each cell, which does not reflect the conditions in the intact cells where only two of the interferon promoter and gene are present. This alters the ifnb1 enhanceosome stoichiometry from the one present in a normal physiological environment, and potentially distorts the patterns of single cell responses observed. In addition, reporter constructs integrate the response that occur over many hours, which makes it difficult to measure the expression dynamics that occur early after exposure to infection. Such measurements in the intact cells would be helpful for understanding the mechanisms underlying the propagation of this immune response.
In order to obtain sensitive and accurate measurements of changes in gene expression in infected single cells, we used single-cell single-molecule mRNA imaging to directly and simultaneously count the transcripts of ifnb1 and that of a virus (Newcastle disease virus) gene Hemagglutinin-neuraminidase hn . This experimental approach enabled us to measure the single-cell responses from the very early stages of infection, in primary immune cells. Simultaneous measurement of ifnb1 and the viral gene hn high lighted the variation in responses across cells, the temporal evolution of the expression of the two genes and their single cell correlation following infection.
We find that the single cell ifnb1 response to virus infection shows a temporally dispersed (asynchronous) pattern. A small fraction of infected cells respond very early and more ifnb1 expressing cells are recruited at later time after infection. In contrast, the single ifnb1 response to the toll like receptor stimulant LPS, follows a highly synchronous pattern, where in a large number of cells showed an ifnb1 response around the same time after treatment. These results suggest that the temporal evolution of single cell ifnb1 responses was likely dependent on the type of the inducing stimuli. Furthermore we observed that extracellular signaling plays an important role in introducing cell-to-cell variability in ifnb1gene induction in response to virus infection. Inhibition of extracellular signaling converted the response to virus infection into an early synchronous LPS-like response. Thus extracellular signaling shapes the temporally dispersed pattern of single cell ifnb1 response to viral infection.
The pattern of ifnb1 responses to virus infection involves an increase in the amplitude of the response per cell as well as an increase in the number of responding cells over time of infection. These properties may enable cells to fine tune the ifnb1 responses gradually. This strategy of mounting an antiviral cytokine response may be useful in calibrating the immune response such that an appropriate antiviral response is generated and cellular toxicity resulting from excessive cytokine expression is usually avoided.
|Advisor:||Sealfon, Stuart C.|
|Commitee:||Basler, Christopher, Kleinstein, Steven, Sobie, Eric, Ting, Adrian|
|School:||Icahn School of Medicine at Mount Sinai|
|Department:||Pharmacology and System Biology|
|School Location:||United States -- New York|
|Source:||DAI-B 75/08(E), Dissertation Abstracts International|
|Keywords:||Cell to cell variability, Fluorescent in situ hybridization assay, Interferon beta, Response propagation, Single cell, Single stranded RNA virus|
Copyright in each Dissertation and Thesis is retained by the author. All Rights Reserved
The supplemental file or files you are about to download were provided to ProQuest by the author as part of a
dissertation or thesis. The supplemental files are provided "AS IS" without warranty. ProQuest is not responsible for the
content, format or impact on the supplemental file(s) on our system. in some cases, the file type may be unknown or
may be a .exe file. We recommend caution as you open such files.
Copyright of the original materials contained in the supplemental file is retained by the author and your access to the
supplemental files is subject to the ProQuest Terms and Conditions of use.
Depending on the size of the file(s) you are downloading, the system may take some time to download them. Please be