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A macrophage-based cell therapy approach for the treatment of non-healing diabetic wounds
Boodhoo, Kiara. 2023. A macrophage-based cell therapy approach for the treatment of non-healing diabetic wounds. Unpublished doctor's dissertation. Stellenbosch : Stellenbosch University [online]. Available at: https://scholar.sun.ac.za/items/b6c690c3-b303-4a9e-8833-0c650c058271
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| Format: | Thesis |
| Language: | English |
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Stellenbosch : Stellenbosch University
2023
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| _version_ | 1867614021446795264 |
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| access_status_str | Open Access |
| author | Boodhoo, Kiara |
| author2 | Van de Vyver, Mari |
| author_browse | Boodhoo, Kiara Van de Vyver, Mari |
| author_facet | Van de Vyver, Mari Boodhoo, Kiara |
| author_sort | Boodhoo, Kiara |
| collection | Thesis |
| dc_rights_str_mv | Stellenbosch University |
| description | Boodhoo, Kiara. 2023. A macrophage-based cell therapy approach for the treatment of non-healing diabetic wounds. Unpublished doctor's dissertation. Stellenbosch : Stellenbosch University [online]. Available at: https://scholar.sun.ac.za/items/b6c690c3-b303-4a9e-8833-0c650c058271 |
| format | Thesis |
| id | oai:scholar.sun.ac.za:10019.1/129391 |
| institution | Stellenbosch University (South Africa) |
| language | English |
| last_indexed | 2026-06-10T12:45:24.995Z |
| license_str | Other — see source repository |
| provenance_str_mv | Harvested via OAI-PMH from SUNScholar — Stellenbosch University Repository |
| publishDate | 2023 |
| publishDateRange | 2023 |
| publishDateSort | 2023 |
| publisher | Stellenbosch : Stellenbosch University |
| publisherStr | Stellenbosch : Stellenbosch University |
| record_format | dspace |
| source_str | SUNScholar — Stellenbosch University Repository |
| spelling | oai:scholar.sun.ac.za:10019.1/129391 A macrophage-based cell therapy approach for the treatment of non-healing diabetic wounds Boodhoo, Kiara Van de Vyver, Mari Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Medicine. Division of General Internal Medicine. Diabetes -- Complications Wound healing Macrophages -- Therapeutic use Cellular therapy Boodhoo, Kiara. 2023. A macrophage-based cell therapy approach for the treatment of non-healing diabetic wounds. Unpublished doctor's dissertation. Stellenbosch : Stellenbosch University [online]. Available at: https://scholar.sun.ac.za/items/b6c690c3-b303-4a9e-8833-0c650c058271 Thesis (PhD)--Stellenbosch University, 2023. ENGLISH ABSTRACT: Non-healing diabetic wounds are the leading cause of lower limb amputations globally. Persistent inflammation in diabetic wounds is associated with the inability of monocytic cells to switch from a phagocytic (M1) to a pro-regenerative (M2) phenotype and as consequence, the proliferative stage of healing does not commence. There are currently no effective treatments available. A targeted cell therapy approach could however restore the pathological wound microenvironment through paracrine signaling to enable healing. It is hypothesized that introducing M2-pro-regenerative macrophages into the wound environment could improve healing outcomes by dampening the inflammatory response and triggering the proliferative stage of healing to commence. In this research project, the efficacy of two different approaches to optimize macrophage phenotype in vitro were assessed prior to testing the efficacy of the optimized cell therapy in an in vivo wound model. The first approach investigated whether in vitro pre-treatment of monocytic (J774.1 A) cells - using a combination of endotoxin-induced immune tolerance (Tol) (Pam3CSK4) and M2 polarization (Pol) (IL-4) - could make these cells impervious to the pathological wound microenvironment and enhance the release of anti-inflammatory cytokines/growth factors. The effect of tolerance (Tol) and polarization (Pol) was assessed independently and in combination on the expression of intracellular (flow cytometry) and secreted (ELISA) cytokines with and without re-stimulation with wound fluid to define the optimal pretreatment conditions. Successive pre-treatment approach (endotoxin Tol followed by IL-4 Pol), dampened TNF-ɑ release and induced intracellular TGF-β production. To mimic a chronic wound microenvironment, the monocytes were differentiated into macrophages using GM-CSF prior to pre-treatment (optimal condition) and subsequently exposed to diabetic wound fluid. The data demonstrated that in the presence of wound fluid, the successive pre-treatment, promoted M2 polarization (CD206) of monocytic cells and significantly dampened the intracellular production of both pro-inflammatory (TNF-ɑ, IL-6) and anti-inflammatory (IL-10, TGF-β) cytokines. The second approach aimed to generate a macrophage cell line that does not produce pro-inflammatory TNF-α by knocking out the TNF-α gene using CRISPR Cas9 technology. This was attempted using induced pluripotent stem cells (iPSCs) and THP-1 monocytes but due to several challenges this approach was unsuccessful, and a blocking peptide (antibody) was used as alternative to inhibit the function of TNF-α within the secretome (conditioned media) of macrophages. The efficacy of these cell therapy approaches was subsequently tested in a murine full thickness excisional diabetic wound model. Full thickness excisional wounds induced on diabetic mice (B6.CgLepob, ob/ob) were divided into the following treatment groups (n=6 animals; n=12 wounds per group): 1) no treatment (Control); 2) M2 macrophages (M2Ф); 3) M2Ф conditioned media (M2Ф CM); 4) Conditioned media derived from Tolerized (PamC3SK) and Polarized (IL4) M2Ф (Tol/Pol M2Ф CM); 5) Tol/Pol M2Ф CM with TNF-α antibody (Tol/Pol M2Ф CMTNFα-). Wound healing dynamics were assessed histologically (SPOT wound score) on tissue sections from day 14 post wounding. Although all treatments improved healing rates compared to control, the Tol/Pol M2Ф CM treatment was the most effective with all wounds having progressed to the final stage of healing i.e. remodelling and displayed complete re-epithelialization on day 14 with signs of revascularization. This is a marked improvement when compared to the control group with wounds remaining in the inflammatory and proliferative stages of healing. The optimised macrophage-based cell therapeutic strategy has significant potential as a novel therapy for treating diabetic wounds. Further investigation is however required to determine the composition of the Tol/Pol M2Ф’s secretome to elucidate the mechanism of action responsible for promoting healing. AFRIKAANSE OPSOMMING: Nie-genesende diabetiese wonde is die hoof oorsaak van onderste ledemaat amputasies wêreld wyd. Langdurige inflammasie in ‘n diabetiese wond word geassosieer met die onvermoë van monosiete om van ‘n fagosiet (M1) na ‘n pro-herstellende (M2) fenotipe oor te skakel, wat tot die gevolg het dat die proliferasie fase van genesing nie kan plaasvind nie. Geen effektiewe behandeling is tans beskikbaar nie, alhoewel ‘n geteikende sel terapie benadering moontlik die patologiese wond mikro-omgewing kan herstel deur middel van parakriene sein om genesing te aktiveer. Daar word gehipotiseer dat die teenwoordigheid van M2-pro-herstellende makrofage in die wond omgewing moontlik genesing kan bevorder deur die inflammatoriese response te onderdruk en die proliferatiewe fase van genesing te bewerkstellig. In hierdie navorsingsprojek was twee verskillende benaderings getoets om makrofaag fenotipe in vitro te optimaliseer voordat die effektiwiteit van die geoptimaliseerde sel terapie in ‘n in vivo wond model getoets kon word. In die eerste benadering is monosiete (J774.1A) voor af in vitro behandel met ‘n kombinasie van endotoksiese-geïnduseerde immuun toleransie (Pam3CKSK4, Tol) en M2 polarisasie (IL-4, Pol) om vas te stel of dit die selle sal beskerm teen die patologiese wond mikroomgewing en die vrylating van anti-inflammatoriese sitokiene/groei faktore sal bewerkstellig. Die effek van toleransie en polarisasie was onafhanklik en in kombinasie geassesseer op die uitdrukking van intrasellulêre (vloei sitometrie) en vrygestelde sitokiene met of sonder herstimulasie met wondvloeistof om die optimale voorbehandeling kondisies te definieer. Die voorbehandeling benadering (endotoksientoleransie gevolg deur IL-4-voorbereiding) het TNF-α vrystelling gedemp en intrasellulêre TGF-β produksie bevorder. Om die kroniese wond mikro-omgewing na te boots was monosiet selle gedifferensieer met GM-CSF om sodoende makrofage te vorm voordat voorbehandeling (optimale kondisie) tesame met die blootsteling aan diabetiese wondvloeistof plaasgevind het. Die data demonstreer dat, die teenwoordigheid van wondvloeistof tesame met voorbehandeling, die M2 polarisasie (CD206) vanaf monosiet selle bevorder het en die intrasellulêre produksie van beide pro- (TNF-α, IL-6) en anti-inflammatoriese (IL-10, TGF-β) sitokiene merkwaardig verlaag het. Die tweede benadering is gefokus om ‘n makrofaag sel-lyn te skep wat nie TNF- α produseer nie deur die spesifieke geen te onderdruk deur middel van CRISPR Cas9 tegnologie. Hierdie benadering is getoets in beide geïnduseerde pluripotente stam selle (iPSCs) en THP-1 monosiet sel lyne, maar was onsuksesvol as gevolg van verskeie uitdagings. https://scholar.sun.ac.za 6 Alternatiewelik was ‘n teenliggaam (peptied blokker) gebruik om die funksie van TNF-α binne die selafskeiding (gekondisioneerde media) van makrofae te onderdruk. Die effektiwiteit van hierdie sel terapie was daarna getoets in ‘n muis volle dikte eksisie wond model. Volle dikte eksisie wonde wat op diabetiese muise (B6.CgLepob, ob/ob) geïnduseer was is verdeel in die volgende behandelingsgroepe (n=6 diere per groep): 1) geen behandeling (kontrole); 2) M2 Makrofage (M2Ф); 3) M2Ф gekondisioneerde media (M2Ф CM); 4) gekondisioneerde medium vanaf getolariseerde (PamC3SK) en gepolariseerde (IL4) M2Ф (Tol/Pol M2Ф CM); 5) Tol/Pol M2Ф CM met TNF-α teenliggaam (Tol/Pol M2Ф CMTNFα-). Wondgenesingsdinamika is geasseseer op wond seksies deur middel van histologie (SPOT wond telling) op dag 14. Alhoewel al die behandelings verbeterde genesing getoon het in vergelyking met die kontrole, was die Tol/Pol M2Ф CM behandeling die mees effektiefste en wonde het hermodulering en voltooide re-epitelisasie op dag 14 getoon met tekens van hervaskularisasie. Hierdie is ‘n merkbare verbetering in vergelyking met die kontrole groep waar wonde in die inflammatoriese en proliferatiewe fases van genesing gebly het. Alhowel die geoptimaliseerde makrofaag-gebaseerde sel terapie strategie merkwaardige potensiaal toon as nuwe terapie vir die behandeling van diabetiese wonde, word verdere ondersoek benodig om die spesifieke samestelling van die Tol/Pol M2 Ф selafskeiding verantwoordelik vir bevorderde genesing na te spoor. Doctoral 2023-09-29T12:05:12Z 2024-02-20T09:55:20Z 2023-09-29T12:05:12Z 2024-02-20T09:55:20Z 2023-10 Thesis https://scholar.sun.ac.za/handle/10019.1/129391 en Stellenbosch University 136 pages : illustration application/pdf Stellenbosch : Stellenbosch University |
| spellingShingle | Diabetes -- Complications Wound healing Macrophages -- Therapeutic use Cellular therapy Boodhoo, Kiara A macrophage-based cell therapy approach for the treatment of non-healing diabetic wounds |
| title | A macrophage-based cell therapy approach for the treatment of non-healing diabetic wounds |
| title_full | A macrophage-based cell therapy approach for the treatment of non-healing diabetic wounds |
| title_fullStr | A macrophage-based cell therapy approach for the treatment of non-healing diabetic wounds |
| title_full_unstemmed | A macrophage-based cell therapy approach for the treatment of non-healing diabetic wounds |
| title_short | A macrophage-based cell therapy approach for the treatment of non-healing diabetic wounds |
| title_sort | macrophage based cell therapy approach for the treatment of non healing diabetic wounds |
| topic | Diabetes -- Complications Wound healing Macrophages -- Therapeutic use Cellular therapy |
| url | https://scholar.sun.ac.za/handle/10019.1/129391 |
| work_keys_str_mv | AT boodhookiara amacrophagebasedcelltherapyapproachforthetreatmentofnonhealingdiabeticwounds AT boodhookiara macrophagebasedcelltherapyapproachforthetreatmentofnonhealingdiabeticwounds |