Th17 immune responses in the chicken
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Welch2015.docx (10.73Mb)
Date
04/07/2015Author
Welch, Louise Michelle
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Abstract
In recent years, the subsets of mammalian CD4+ T cells and their repertoire of
effector cytokines has expanded beyond the original Th1/Th2 paradigm, to include
natural (n) and inducible (i) regulatory T cells (Treg), Th17, Th9, Th22 and follicular
T helper (Tfh) cells. Whilst Th1, Th2 and nTreg immune responses have been
described in the chicken, the existence of other Th cell subsets is yet to be
determined. To investigate Th17 immune responses in the chicken, the mammalian
components of these responses currently unannotated in the chicken genome, IL-23
p19 and the IL-23R, were identified and cDNAs cloned. A chicken IL-23 flexiconstruct,
containing IL-23 p19 and p40 joined by a linker, was designed.
Recombinant chicken IL-23 protein (rchIL-23) was expressed and purified.
Bioactivity of rchIL-23 was demonstrated by increased mRNA expression of chIL-
17F and chIL-22 in rchIL-23-stimulated splenocytes. Monoclonal antibodies which
identify chIL-12/chIL-23 p40 also recognised purified rchIL-23. Further, chIL-23
p19 mRNA levels were measured and detected in a wide range of tissues but was not
up-regulated in stimulated splenocytes, thymocytes or bursal cells.
Messenger RNA (mRNA) expression levels of Th17 cytokines (chIL-17A, chIL-17F,
chIL-21, chIL-22 and chIL-23) were measured in a chicken tissue panel, in
stimulated splenocytes, thymocytes and bursal cells, as well as during infections
previously described as initiating typical Th1 or Th2 adaptive immune responses in
the chicken. Chicken IL-17A mRNA expression levels were up-regulated in
susceptible chickens during infection with Marek’s disease virus (a disease which
typically drives a Th1 immune response), but were down-regulated in resistant birds.
Chicken CD4+ T cells were sorted by fluorescence-activated cell sorting (FACS) and
recombinant Th17-associated cytokines used to attempt to drive the cells towards a
Th17 phenotype, as measured by expression of mRNA for chIL-17A and chIL-23R.
The sorted chicken CD4+ cells failed to proliferate or respond to Th17 cytokine
stimulation.
ChIL-23R was also correctly identified and cloned as cDNA, and its mRNA
expression measured in a panel of unstimulated and stimulated tissues and cells. The
chIL-23R mRNA levels were detected in a wide range of tissues as well as
stimulated splenocytes, thymocytes and bursal cells. Future work would seek to
positively identify Th17 cells in the chicken and determine the role of Th17 immune
responses against avian diseases.