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Immune cell dysfunction and eicosanoid imbalance in chronic diabetic wounds
Thesis (PhD)--Stellenbosch University, 2025.
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Stellenbosch : Stellenbosch University
2025
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| access_status_str | Open Access |
| author | Dhanraj, Priyanka |
| author2 | Van de Vyver, Mari |
| author_browse | Dhanraj, Priyanka Van de Vyver, Mari |
| author_facet | Van de Vyver, Mari Dhanraj, Priyanka |
| author_sort | Dhanraj, Priyanka |
| collection | Thesis |
| dc_rights_str_mv | Stellenbosch University |
| description | Thesis (PhD)--Stellenbosch University, 2025. |
| format | Thesis |
| id | oai:scholar.sun.ac.za:10019.1/134600 |
| institution | Stellenbosch University (South Africa) |
| language | English |
| last_indexed | 2026-06-10T12:46:09.042Z |
| license_str | Other — see source repository |
| provenance_str_mv | Harvested via OAI-PMH from SUNScholar — Stellenbosch University Repository |
| publishDate | 2025 |
| publishDateRange | 2025 |
| publishDateSort | 2025 |
| publisher | Stellenbosch : Stellenbosch University |
| publisherStr | Stellenbosch : Stellenbosch University |
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| spelling | oai:scholar.sun.ac.za:10019.1/134600 Immune cell dysfunction and eicosanoid imbalance in chronic diabetic wounds Dhanraj, Priyanka Van de Vyver, Mari Vlok, Mare Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Medicine. Division of General Internal Medicine. Diabetes -- Complications Foot -- Ulcers Wounds and injuries -- Infections Inflammation -- Immunological aspects Thesis (PhD)--Stellenbosch University, 2025. Dhanraj, P. 2025. Immune cell dysfunction and eicosanoid imbalance in chronic diabetic wounds. Unpublished doctoral dissertation. Stellenbosch: Stellenbosch University [online]. Available: https://scholar.sun.ac.za/items/cdfb4c67-dd7e-4700-8f9d-2e8a901b25ad ENGLISH ABSTRACT: Diabetic foot ulcers (DFUs) are a severe and challenging complication of diabetes mellitus (DM) and due to the complex nature, treatment strategies have proven ineffective These wounds often exhibit signs of infection and persistent inflammation, suggesting that dysregulated immune responses underlie poor healing outcomes. However, limited information exists regarding the acute inflammatory response following injury, which involves the recruitment and activation of innate immune cells. Given the complex interplay between the pathogenesis of DM and immune function, investigating immune cell responsiveness in DFUs is crucial. This research project hypothesised that pre-existing immune deficits related to disease pathogenesis impair the recruitment and activation of immune cells in the early stages post-injury. This impairment likely disrupts the crucial inflammatory phase of wound healing, making these wounds more vulnerable to infection. The dissertation consisted of three independent studies; each aimed at addressing a specific aspect of this hypothesis. Study 1 utilised a murine full-thickness excisional wound model comparing wild-type control (C57BL/6J) and obese diabetic B6.Cg-Lepob/J (ob/ob) mice. The expression of pro-inflammatory (LTB4, PGE2, TxA2) and pro-resolving (MaR1, RvE1, RvD1, PD) lipid mediators (eicosanoids) (ELISAs) was assessed during early stages post-injury (6h) and subsequent transition through the phases of healing, compared on days 2 and 7. Wound healing dynamics (SPOT wound score) and immune cell infiltration (MPO, TNF-α) were assessed using immunohistochemistry. Diabetic wounds exhibited a five-fold reduction in cellular infiltration (p<0.05) and delayed expression of TNF-α and MPO compared to controls.No difference was, however, detected in eicosanoid expression, suggesting that the immune cells were unresponsive to the initial eicosanoid signals. This supports the notion that the onset of the acute inflammatory response is delayed in diabetic wounds. Study 2 employed a retrospective sample selection from a biobank to perform secondary analysis on wound tissue samples that either responded to therapeutic intervention (healers) or that became infected and failed to heal (non-healers). These samples were all derived from a study utilising the same obese diabetic full thickness wound model as in study 1; whereby the therapeutic interventions were anti-inflammatory cellular tissue-based products, namely, amniotic membrane (AmR) and umbilical cord blood serum (UCBS). Eicosanoid expression (ELISAs), immune cell infiltration (immunohistochemistry), and the proteome (LC-MS/MS) were compared between untreated controls, healers, and non-healers on day 14 post-wounding. Non-healing wounds exhibited excessive bacterial aggregation with increased neutrophil (Ly6G) infiltration. Proteomic analysis (LC-M/MS) indicated that highly expressed proteins in the non-healers were mostly involved in innate immune cell recruitment, whilst the activity of metabolic processes was downregulated. The spontaneous infection that caused wounds to deteriorate (non-healing) was only evident in 50% of the treatment groups and not in any of the untreated controls. This coincided with reduced LTB4 expression, indicating potentially impaired neutrophil antimicrobial activity. The non-healing wounds furthermore failed to transition into the proliferative phase of healing. Study 3, therefore, compared the functional responsiveness of patient-derived peripheral blood mononuclear cells (PBMCs) from self-reported healthy controls and patients with Type 2 diabetes mellitus (T2DM) at different stages of disease progression. Ex vivo testing evaluated the activation status of PBMCs (ROS production, Ca2+ flux), and functional responsiveness (chemotaxis, phagocytosis). Despite large individual variability, differences were detected between well- and poorly controlled T2DM patients. Altered Ca2+ flux and migration capacity (velocity, distance) were observed, with associations noted between these parameters and age, blood glucose, and HbA1c levels, confirming a link between disease progression and immune cell functionality. Taken together, this research project indicated a delayed and diminished acute immune response in diabetic wounds. It confirms that anti-inflammatory and growth-promoting therapies pose a risk of weakening already compromised acute immune responses, further making the host vulnerable to opportunistic infections. Infection subsequently creates a perpetuating cycle of inflammation and tissue damage, a hallmark of DFUs. Furthermore, disease progression influences immune cell functionality and signalling. This work highlights the importance of shifting research and therapeutic strategies toward understanding and modulating acute immune responses in diabetic patients, with the potential to improve DFU prevention and treatment outcomes. AFRIKAANSE OPSOMMING: Diabetiese voetswere (DVS) is 'n ernstige komplikasie van diabetes mellitus (DM) met geen effektiewe behandelingsstrategie nie. Hierdie wonde toon dikwels tekens van infeksie en aanhoudende inflammasie, wat daarop dui dat wanfunksionerende immuunreaksies onderliggend is aan swak genesingsuitkomste. Daar is egter beperkte inligting beskikbaar oor die akute inflammatoriese reaksie ná besering, wat die werwing en aktivering van aangebore immuunselle behels. Gegewe die komplekse wisselwerking tussen die patogenese van DM en immuunfunksie, is dit van kardinale belang om immuunselreaktiwiteit in DVS te ondersoek. Hierdie navorsingsprojek het gehipotetiseer dat voorafbestaande immuungebreke wat met die siektepatogenese verband hou, die werwing en aktivering van immuunselle in die vroeë stadiums ná besering benadeel. Hierdie benadeling ontwrig waarskynlik die kritieke inflammatoriese fase van wondgenesing en maak hierdie wonde meer kwesbaar vir infeksie. Die proefskrif het uit drie onafhanklike studies bestaan, elk gerig op 'n spesifieke aspek van hierdie hipotese. Studie 1 het 'n muriene model van volle-dikte eksisionele wonde gebruik waarin wilde-tipe kontrole muise (C57BL/6J) vergelyk is met vetsugtige diabetiese B6.Cg-Lepob/J (ob/ob) muise. Die uitdrukking van pro-inflammatoriese (LTB4, PGE2, TxA2) en pro-oplossende (MaR1, RvE1, RvD1, PD) lipiedmediators (ELISA’s) is geëvalueer tydens vroeë stadiums ná besering (6 uur) en gedurende die daaropvolgende genesingsfases op dag 2 en 7. Wondgenesingsdinamika (SPOT-wondtelling) en immuunselinfiltrasie (MPO, TNF-α) is geassesseer met behulp van immunohistochemie. Verminderde sellulêre infiltrasie (x5-voudig) (p<0.05) en beduidende vertragings in die uitdrukking van TNF-α (proinflammatoriese sitokien) en MPO (neutrofielmerker) was duidelik in diabetiese wonde in vergelyking met die kontrole. Geen verskil is egter in eikosanoïeduitdrukking waargeneem nie, wat daarop dui dat die immuunselle nie op die aanvanklike eikosanoïedseine gereageer het nie. Dit ondersteun die idee dat die aanvang van die akute inflammatoriese reaksie in diabetiese wonde vertraag is. Studie 2 het 'n retrospektiewe steekproefkeuse uit 'n biobank gebruik om sekondêre analise op wondweefselmonsters uit te voer wat óf op terapeutiese intervensie gereageer het (genesers), óf wat besmet geraak het en nie genees het nie (nie-genesers). Hierdie monsters is almal afgelei van 'n studie wat dieselfde vetsugtige diabetiese vol-dikte wondmodel as in studie 1 gebruik het, met terapeutiese intervensies wat bestaan uit antiinflammatoriese sellulêre weefselgebaseerde produkte was, naamlik amniotiese membraan (AmR) en naelstringbloedserum (UCBS). Eikosanoïeduitdrukking (ELISA’s), immuunselinfiltrasie (immunohistochemie), en die proteoom (LC-MS/MS) is op dag 14 na wondvorming tussen onbehandelde kontroles, genesers en nie-genesers vergelyk. Niegenesende wonde het oormatige bakteriële ophoping en verhoogde neutrofiel (Ly6G) infiltrasie getoon. Proteomiese analise (LC-M/MS) het aangedui dat hoogs uitgedrukte proteïene in die nie-genesers meestal met aangebore immuunselwerwing geassosieer is, terwyl metaboliese prosesse onderdruk is. Die spontane infeksie wat daar toe gelei het dat die wonde agteruitgaan (nie-genesing), is slegs in 50% van die behandelingsgroepe waargeneem, en in geen van die onbehandelde kontroles nie. Dit het saamgeval met verminderde LTB4-uitdrukking, wat dui op moontlik benadeelde neutrofiel anti-mikrobiese aktiwiteit. Die nie-genesende wonde het verder versuim om na die proliferatiewe fase van genesing oor te gaan. Studie 3 het dus die funksionele reaktiwiteit van pasiënt-afgeleide perifere bloed mononukleêre selle (PBMC’s) van self-gerapporteerde gesonde kontroles en Tipe 2- diabetespasiënte (T2DM) in verskillende stadiums van siekteprogressie vergelyk. Eks vivotoetsing het die aktiveringsstatus van PBMC’s (ROS-produksie, Ca2+-vloei) en funksionele reaktiwiteit (chemotaksis, fagositose) geëvalueer. Ten spyte van groot individuele variasie, is verskille tussen goed en swak beheerde T2DM-pasiënte waargeneem. Veranderde Ca2+-vloei en migrasiekapasiteit (snelheid, afstand) is waargeneem, met assosiasies tussen hierdie parameters en ouderdom, bloedglukose en HbA1c-vlakke, wat 'n verband tussen siekteprogressie en immuunselfunksionaliteit bevestig. Gesamentlik het hierdie projek 'n vertraagde en verminderde akute immuunreaksie in diabetiese wonde aangedui. Dit bevestig dat anti-inflammatoriese en groei-stimulerende terapieë die risiko inhou om reeds verswakte akute immuunreaksies verder te benadeel, wat die gasheer kwesbaar maak vir opportunistiese infeksies. Infeksie skep vervolgens 'n voortdurende siklus van inflammasie en weefselskade, 'n kenmerk van DVS. Verder beïnvloed siekteprogressie die funksionaliteit en seine van immuunselle. Hierdie werk beklemtoon die belangrikheid daarvan om navorsings- en terapeutiese strategieë te verskuif na die begrip en modulering van akute immuunreaksies in diabetiese pasiënte om moontlik DVS-voorkoming en -behandelingsuitkomste te verbeter. Doctoral 2025-12-18T07:31:10Z 2025-12-18T07:31:10Z 2025-12 Thesis https://scholar.sun.ac.za/handle/10019.1/134600 en Stellenbosch University xiii, 96 pages : illustrations application/pdf Stellenbosch : Stellenbosch University |
| spellingShingle | Diabetes -- Complications Foot -- Ulcers Wounds and injuries -- Infections Inflammation -- Immunological aspects Dhanraj, Priyanka Immune cell dysfunction and eicosanoid imbalance in chronic diabetic wounds |
| title | Immune cell dysfunction and eicosanoid imbalance in chronic diabetic wounds |
| title_full | Immune cell dysfunction and eicosanoid imbalance in chronic diabetic wounds |
| title_fullStr | Immune cell dysfunction and eicosanoid imbalance in chronic diabetic wounds |
| title_full_unstemmed | Immune cell dysfunction and eicosanoid imbalance in chronic diabetic wounds |
| title_short | Immune cell dysfunction and eicosanoid imbalance in chronic diabetic wounds |
| title_sort | immune cell dysfunction and eicosanoid imbalance in chronic diabetic wounds |
| topic | Diabetes -- Complications Foot -- Ulcers Wounds and injuries -- Infections Inflammation -- Immunological aspects |
| url | https://scholar.sun.ac.za/handle/10019.1/134600 |
| work_keys_str_mv | AT dhanrajpriyanka immunecelldysfunctionandeicosanoidimbalanceinchronicdiabeticwounds |