Mapping intracellular cytokine pools in granulocytes: Dynamic storage and trafficking sites of interleukin-9 and interleukin-13 in human eosinophils

• Author / Creator

  Fayad, Nawell

• Eosinophils are immune cells with constantly evolving roles in both innate and adaptive immunity and have long been associated with the predominant pathology of the human airways, asthma. Eosinophils are specialized in their granulocytic nature, possessing several membrane-bound secretory organelles that store potent immunoregulatory mediators that can be rapidly and differentially released in the presence of an inflammatory stimulus. Among this arsenal of immunomodulatory factors are T helper 2 (Th2) cytokines that include interleukins (IL-) 4, 5, 9, and 13 which are pivotal proteins involved in immune cell crosstalk and pro-inflammation associated with asthma pathogenesis. While the pathobiological actions of eosinophils and their derived Th2 cytokines in asthma are increasingly appreciated and have translated to becoming approved therapeutic targets of asthma, the modes in which these cytokines, particularly IL-9 and IL-13, are delivered to the extracellular environment is not well known. In this study, I aimed to identify whether the secretory organelles crystalloid granules or recycling endosomes (REs) found in human blood eosinophils were associated with trafficking IL-9 and IL-13 in the presence of the inflammatory stimulus platelet activating factor (PAF). I hypothesize that REs act as a novel trafficking site for IL-9 and IL-13 upon stimulation in human eosinophils as REs have been implicated in exocytic transport of newly synthesized cytokines in different immune cells. Furthermore, crystalloid granules would mainly act as a storage site for these cytokines that could later be subsequently released. Human eosinophils were double-stained for IL-9 or IL-13 and transferrin receptor (TfnRc) as a marker of endosomal compartments or CD63 as a marker of crystalloid granules. Cells were imaged using super-resolution microscopy and the degree of colocalization between cytokines and secretory organelles of interest was measured with Pearson’s correlation coefficient and an object-based colocalization protocol. Intracellular cytokine levels were measured as mean immunofluorescence intensity values. Increases or decreases in the degree of colocalization between cytokines and secretory organelles of interest is suggestive of cytokine trafficking into or out of these membrane-bound compartments, respectively. Meanwhile elevated or reduced levels of intracellular cytokine following 60 min of PAF stimulation would be suggestive of de novo protein synthesis or cytokine secretion, respectively. In this study, IL-9 immunofluorescence intensity significantly increased upon PAF stimulation. Similarly, IL-9 colocalization with TfnRc significantly increased upon PAF stimulation while colocalization between IL-9 and CD63 transiently increased at 5 and 15 min of PAF stimulation prior to returning to baseline levels. Furthermore, 3.5 times the amount of immunolabelled IL-9 was found in TfnRc+ compartments compared to CD63+ granules in resting eosinophils. This data suggests for the first time that TfnRc+ endosomes are important trafficking sites of IL-9 in human eosinophils. It is also suggested that PAF induces de novo IL-9 synthesis and this newly made IL-9 is shuttled into TfnRc+ endosomes for release at the cell surface. Some newly made IL-9 may also be shuttled into CD63+ granules for storage and release. In contrast, both IL-13 colocalization with CD63 and IL-13 immunofluorescence intensity decreased upon PAF stimulation while IL-13 colocalization with TfnRc transiently increased at 5 min of PAF stimulation. This data suggests IL-13 is being shuttled out of CD63+ crystalloid granules for cytokine secretion and is predominantly preformed in these granules. Small amounts of IL-13 may also be shuttled into TfnRc+ compartments upon PAF stimulation as a static pool of intracellular IL-13 poised for release. Overall, we identified TfnRc+ endosomes in human eosinophils as a novel site for IL-9 and IL-13 trafficking. These novel trafficking sites provide potential therapeutic targets for asthma that aim to dampen the pro-inflammatory effects of eosinophils and Th2 cytokines in this disease.

• Subjects / Keywords

  • Recycling endosome
  • Asthma
  • Cytokine trafficking
  • Crystalloid granule
  • Interleukin-9
  • Interleukin-13
  • Eosinophil

• Graduation date

  Fall 2020

• Type of Item

  Thesis

• Degree

  Master of Science

• DOI

  https://doi.org/10.7939/r3-qn7a-cy90

• License

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