Date of Award

Fall 12-2022

Document Type


Degree Name

Master of Science (MS)


Pharmaceutical Sciences

First Advisor

Ajay Sharma

Second Advisor

Jennifer Totonchy

Third Advisor

Rennolds Ostrom


The present study is designed to investigate whether irradiation as used for conditioning regimen to ablate recipient’s diseased bone marrow prior to transplantation could trigger macrophage activation and polarization and whether macrophages could cause the transdifferentiation of ocular surface fibroblasts into myofibroblasts, thus contributing to oGVHD-associated fibrosis.

Bone marrow cells were cultured in M-CSF to obtain M0 macrophages, which were subsequently polarized into M1 and M2 phenotypes using IFN-g + LPS and IL-4, respectively.

The macrophages were exposed to 7 Gy radiation. The effect of irradiation on macrophage activation markers, phagocytosis, and apoptosis was assessed using real-time PCR, confocal microscopy, and flow cytometry. Ocular surface fibroblasts were co-cultured using membrane inserts placed on top of cultured M0, M1, and M2 macrophages for 3 days. Myofibroblast formation was assessed using a-SMA immunostaining and gene expression. The effect of macrophages on profibrotic mediators in the fibroblasts was quantified using real-time PCR. Finally, chemokine release from macrophages was analyzed using real-time PCR and bead-based immunoassay.

Our data demonstrates successful generation of M0 macrophages and their polarization into M1 and M2 phenotypes as confirmed by gene quantification and flow cytometry for macrophage markers CD11b, F4/80, CD86, CD206, iNOS, and arginase-1. Our data demonstrates that irradiation caused an increase in macrophage pro-inflammatory cytokines IL-1b, TNF-a, and IL-6 and chemokine CCL2. While irradiation did not cause increase in M1 markers, there was a robust increase in M2 markers. Furthermore, irradiation significantly increased macrophage phagocytosis without compromising their viability. Our data also
demonstrates that M0 and M2 macrophages induced significant increase of a-SMA expression in ocular surface fibroblasts and a concomitant notable increase in RAS components and TGF-b1 receptor, but no change in PDGF and M-CSF expression was noted. Finally, our data demonstrates that both M1 and M2 macrophages showed increased gene expression and secretion of chemokines CCL17 and CCL22.

In conclusion, our data demonstrates that macrophages could play a key role in the pathology of oGVHD-associated fibrosis due to their likely activation in response to irradiation and cross-talk with ocular surface fibroblasts, resulting in their transdifferentiation to myofibroblasts.

Creative Commons License

Creative Commons License
This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 4.0 License.



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