Identification and characterization of dendritic cell subtypes in preserved and cultured cadaveric human corneolimbal tissue on amniotic membrane

Lužnik, Zala; Kopitar, Andreja Nataša; Lapajne, Luka; Pižem, Jože; Ferrari, Stefano; Ihan, Alojz; Hawlina, Marko; Schollmayer, Petra

doi:10.1111/aos.13854

Cited by 6 publications

(4 citation statements)

References 41 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…We identified only a small population of CD86+ cells in a few, but not in all, samples. This is consistent with a study by Lužnik, reporting on only a very small percentage of CD86+ cells in the central area of the human cornea [36]. However, in our study the discrimination of CD86positive and negative populations was vague.…”

Section: Discussionsupporting

confidence: 92%

“…The first protocols for a respective FC analysis were established in mice which have a much thinner corneal stroma layer than humans, allowing for easier cell harvesting [10]. Two more recent studies, which examined human tissue, focused on herpetic infections of the cornea [34] and on subtypes of dendritic cells [36], whereby only the first study analyses the dynamics of APCs over time and both studies contain only few samples. The small number of previous studies on human cornea bespeaks the complexity of preparing corneal tissue for FC analysis although FC analysis is actually ideally suited to investigate the dynamics of APC – especially DCs and macrophages.…”

Section: Discussionmentioning

confidence: 99%

“…Due to its dense arrangement of fibrils and its greater corneal thickness compared to rabbits and mice [45, 46], the human cornea was inaccessible for a long time for obtaining single-cell suspensions for FC analyses. FC analyses of native DCs in human corneal single-cell suspensions have been reported before [34, 36]. To gain more insights into the myeloid cell composition of the cornea we addressed macrophage subtypes in human corneas and the possibility of a therapeutic modulation of their phenotype.…”

Section: Discussionmentioning

confidence: 99%

See 2 more Smart Citations

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

et al. 2021

View full text Add to dashboard Cite

Purpose: 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). Methods: 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. Results: 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 PTPRC (CD45), ITGAM (CD11b), CD14, and CD74. 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+, CD282+, CD86+, and CD284+) and M2 (CD163+ and CD206+) 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 (p < 0.05) while expression levels of various M1 macrophage markers were not significantly reduced by interleukin treatment. Conclusions: 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.

show abstract

Section: Discussionsupporting

confidence: 92%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

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

et al. 2021

View full text Add to dashboard Cite

show abstract

“…They are also nearby the limbal blood vessels, enabling travel in the circulation to further facilitate the immune responses. They also have a role in intravascular surveillance within the limbal vessels [197]. The impact of contact lenses and specifically materials and edge design on limbal dendritic cells has not yet been studied, but this area is worthy of research given the essential role these cells play in ocular surface defence.…”

Section: Sub-clinical Inflammatory Responsementioning

confidence: 99%