Time- and Stimulus-Dependent Characteristics of Innate Immune Cells in Organ-Cultured Human Corneal Tissue

Abstract

<b><i>Purpose:</i></b> The pattern of immune cells infiltrating the corneal stroma has been extensively studied in mice, but data on human tissue have been far less elaborate. To further characterize the number and differentiation state of resident immune cells in organ-cultured human corneal tissue, we employed a comprehensive bioinformatic deconvolution (xCell) of bulk RNA-sequencing (RNA-seq) data, immunohistochemistry (IHC), and flow cytometry (FC). <b><i>Methods:</i></b> A transcriptome-based analysis of immune cell types in human corneal samples was performed. The results were validated by IHC, focusing on the identification of pro-inflammatory (M1) and regulatory (M2) macrophages. A protocol was established to identify these 2 different macrophage populations in human corneal tissue by means of FC. Subsequently, corneal samples in organ culture were differentially stimulated by IL-10, IL-4 & IL-13, or LPS and macrophage populations were evaluated regarding their response to these stimuli. Furthermore, cell survival was analyzed in correlation with time in organ culture. <b><i>Results:</i></b> xCell-based mathematical deconvolution of bulk RNA-seq data revealed the presence of CD8 T cells, Th17 cells, dendritic cells, and macrophages as the predominant immune cell types in organ-cultured human corneal tissue. Furthermore, RNA-seq allowed the detection of different macrophage marker genes in corneal samples, including <i>PTPRC</i> (CD45), <i>ITGAM</i> (CD11b), <i>CD14</i>, and <i>CD74</i>. Our RNA-seq data showed no evidence of a relevant presence of monocytes in human corneal tissue. The presence of different macrophage subtypes was confirmed by IHC. The disintegration and subsequent FC analysis of human corneal samples showed the presence of both M1 (HLA-DR<sup>+</sup>, CD282<sup>+</sup>, CD86<sup>+</sup>, and CD284<sup>+</sup>) and M2 (CD163<sup>+</sup> and CD206<sup>+</sup>) macrophage subtypes. Furthermore, we found that the total number of macrophages in corneal samples decreased more than the total cell count with increasing tissue culture time. Treatment with IL-10 led to higher total cell counts per cornea and to an increased expression of the M2 marker CD163 (<i>p</i> < 0.05) while expression levels of various M1 macrophage markers were not significantly reduced by interleukin treatment. <b><i>Conclusions:</i></b> Regarding different macrophage populations, untreated human corneas showed more M1 than M2 macrophages. With increasing organ culture time, these macrophages decreased. In terms of cell dynamics, adding interleukins to the organ culture medium influenced the phenotype of macrophages within the cornea as detected by FC. Modifying the immunomodulatory properties of human grafts appears a promising approach to further reduce the risk of graft rejection in patients. In this context, treatment with interleukins was more effective in upregulating M2 macrophages than in suppressing M1 macrophages in corneal tissue.

Transcriptional Profiling Provides New Insights into Organ Culture-Induced Changes in Human Donor Corneas

“…In accordance with previous findings, this study demonstrated that the corneal immune cell profile changes over culture time [ 8 , 9 ], with decreasing numbers over time for almost all immune cell populations. It is well known that corneal grafts which were in culture for more than 21 days showed less subsequent rejection than grafts stored for shorter times [ 8 ], a finding which is explained by decreasing numbers of competent immune cells in the cornea with increasing time in culture [ 9 ]. Adding modulatory factors to the culture medium that affect the differentiation status of immune cells in the graft have been demonstrated to reduce the risk of rejection [ 10 ].…”

“…This ensures a thorough quality control as well as detailed testing for potential infectious pathogens before transplantation. It is well known that organ culture influences donor corneas, with most of the evidence being focused on the effect of culture conditions on endothelial cell density and corneal graft survival after transplantation [ 8 , 9 ]. However, apart from that, it is still largely unknown which other molecular processes may be altered by corneal tissue culture.…”

“…For example, grafts that were kept in culture for more than 21 days showed less subsequent rejection than grafts stored for shorter times [ 8 ]. This phenomenon is associated with the fact that the number of competent immune cells in the cornea decreases with time in culture [ 9 ]. Furthermore, it has been proposed to prepare tissue for transplantation by adding factors to the culture medium that affect the differentiation status of immune cells in the graft, thereby reducing the subsequent risk of rejection [ 9 ].…”

“…This phenomenon is associated with the fact that the number of competent immune cells in the cornea decreases with time in culture [ 9 ]. Furthermore, it has been proposed to prepare tissue for transplantation by adding factors to the culture medium that affect the differentiation status of immune cells in the graft, thereby reducing the subsequent risk of rejection [ 9 ]. In animal experiments, successful approaches adding interleukin-10 (IL-10) to the corneal culture have been reported [ 10 ].…”

Clinical Course of Different Types of Immune Reactions following Keratoplasty

“…Risk factors for IR include properties of the graft (e.g., antigen mismatch, storage time in organ culture [9]) and features of the recipient [10,11]. Most importantly, vascularization of the graft bed leads to enhanced trafficking of antigen-presenting cells as well as adaptive immune cells and thus increases the risk of graft rejection.…”