Modulation of leukocyte adhesion and cell migration by human neuraminidase enzymes

• Author / Creator

  Howlader, Md Amran

• The human neuraminidases (NEU1, NEU2, NEU3, and NEU4) are a family of enzymes implicated in pathologies including cancer and diabetes. Our group has developed selective inhibitors for these enzymes that have been employed as tools to understand their biological roles. Several reports have linked neuraminidase activity to the regulation of cell migration in cancerous and normal cells, as well as to the function of leukocytes. This thesis has investigated the role of human neuraminidase enzymes (hNEU) in cell migration and adhesion.
  In Chapter 2, we used an in vitro cell migration assay on fibronectin (FN) coated surfaces to investigate the role of these enzymes in integrin-mediated cell migration. We observed that neuraminidase inhibition caused significant retardation of cell migration in breast cancer (MDA-MB-231) and prostate cancer (PC-3) cell lines when treated with inhibitors of NEU3 and NEU4. In contrast, inhibition of NEU1 caused a significant increase in cell migration for these same cell lines. Both NEU1 and NEU3 inhibition caused significant reduction of β1 integrin endocytosis in both PC-3 and MDA-MB-231 cell lines. We concluded that blockade of human neuraminidase enzymes with isoenzyme-selective inhibitors has significant potential in the development of anti-cancer or wound healing therapeutics.
  In Chapter 3, we investigated the influence of hNEU on the adhesion of leukocytes in vitro. Within the plasma membrane, glycoconjugate-receptor interactions play an important role in the regulation of cell-cell interactions. We investigated the mechanism and activity of the hNEU isoenzyme, NEU3, on T cell adhesion receptors. The enzyme is known to prefer glycolipid substrates and we confirmed that exogenous enzyme altered the glycolipid composition of cells. Enzymatic activity of NEU3 resulted in re-organization of LFA-1 into large clusters on the membrane. This change was facilitated by an increase in the lateral mobility of LFA-1 upon NEU3 treatment. Changes to the lateral mobility of LFA-1 were specific for NEU3 activity, and we observed no significant change in diffusion when cells were treated with a bacterial NEU (NanI). Furthermore, we found that NEU3 treatment of cells increased surface expression levels of LFA-1. We observed that NEU3-treated cells had suppressed LFA-1 adhesion to an ICAM-1 coated surface using an in vitro static adhesion assay. These results suggest that modulation of glycoconjugate composition contributes to the regulation of integrin activity. Additionally, NEU3 may be implicated as a negative regulator of the inflammatory cascade through its regulation of LFA-1.
  In Chapter 4, we investigated the role of NEU isoenzymes in acute inflammation in mice with an in vivo air pouch model. We observed that mice showed significantly reduced leukocyte recruitment in animals with reduced NEU1 or NEU3 enzyme activity. In contrast, knocking out the NEU4 isoenzyme (NEU4 KO) or NEU4 in combination with NEU3 (NEU3/4 DKO), showed significantly increased leukocyte recruitment. These effects were quantified on leukocyte subpopulations including monocytes, neutrophils, macrophages, and NK cells in isoenzyme selective neuraminidase KO mice. Changes in NEU expression corresponded to changes in systemic cytokine levels, with reduced expression of G-CSF, IL-6, TNF-γ, IP-10, and RANTES in NEU1 KO mice treated with LPS. LPS treated NEU4 KO mice had reduced expression of G-CSF, TNF-Ύ, and IP-10, and increased expression of IL-21. Immunohistochemistry (IHC) of tissue collected from the air pouch model confirmed changes to leukocyte recruitment. Using isoenzyme-selective inhibitors designed in our laboratory, we tested the hNEU enzymes as anti-inflammatory targets. A selective NEU4 inhibitor increased leukocyte recruitment and could act as an immune stimulant. The results show human neuraminidase enzymes are a modulator of acute inflammation.

• Subjects / Keywords

  • Inflammation
  • LFA-1
  • Adhesion
  • Migration
  • Endocytosis
  • Neuraminidase

• Graduation date

  Spring 2020

• Type of Item

  Thesis

• Degree

  Doctor of Philosophy

• DOI

  https://doi.org/10.7939/r3-yx86-mv28

• License

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