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The effects of c-reactive protein (CRP) isoforms on inflammation and wound healing processes

Hall, Nicola, Rachel (2017) The effects of c-reactive protein (CRP) isoforms on inflammation and wound healing processes. Doctoral thesis (PhD), Manchester Metropolitan University.

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Abstract

C-reactive protein (CRP) is a homopentameric acute-phase inflammatory protein that exhibits elevated expression during inflammation. CRP can be found in its pentameric form (nCRP) or monomeric form (mCRP). To date there has been little research investigating the effects of these CRP isoforms in wound healing processes and age-related impaired healing, which is known to have an altered inflammatory response. The study investigated CRP isoform localisation in murine wounds (detected by immunohistochemistry) and circulating blood (measured by immunoblotting) using models of acute healing in young adults and age-related impaired healing (n=3). Using in vitro inflammatory cell assays developed with the monocytic U937 cell line this study investigated the effects of CRP isoforms in nitric oxide release (detected by an immunoassay), phagocytosis (measured by a bacterial recovery assay), apoptosis (detected via DNA fragmentation) and cytokine secretion (measured by a multiplex immunoassay) in both monocyte and macrophage-like cells (n=6). Pharmacological inhibitors were used to determine the potential CRP-induced pathways or mechanisms. Both isoforms were localised to sites of inflammation, implying that they may play an active inflammatory role during wound repair. Moreover, the ratio of CRP isoforms (mCRP:nCRP) reflected the degree of inflammation, with higher values indicating elevated mCRP localisation and pronounced inflammation in the model of age-related impaired healing. In in vitro inflammatory cell assays, nCRP exhibited a more anti-inflammatory effect leading to significantly (P=0.01) increased phagocytosis, increased apoptosis, decreased nitric oxide (NO) production, and reduced overall pro-inflammatory cytokine production in monocytes and macrophages. The mCRP isoform significantly (P<0.001) increased NO production and overall levels of pro-inflammatory cytokines, thus confirming its pro-inflammatory nature indicated in the literature. This research highlighted that estrogen mediated CRP-induced responses in a cell type- and status-specific manner, being pro-inflammatory in circulating cells (monocytes) but anti-inflammatory in wound tissue cells (mature activated macrophages). The profile of CRP isoforms shifted in age-related (estrogen-deprived) impaired healing to relatively higher levels of mCRP compared to nCRP, suggesting impaired healing in the elderly may result from the dissociation of nCRP to mCRP following the decline in estrogen with increasing age. In conclusion, this study has provided novel data showing that both isoforms of CRP may differentially mediate inflammatory responses during wound healing and may play key roles in age-related impaired healing and may ultimately provide potential therapeutic strategies for the treatment of chronic wounds in the elderly.

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