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Purpose Immune reactions following corneal transplantation are the most common cause of transplant failure. However, the underlying mechanisms of corneal graft rejection are not yet fully understood but increasing evidence points to a crucial role of the innate immune system in this context. Using a human in vitro model, we aimed to assess the response of human macrophages to stimulation with human corneal tissue and whether corneal endothelial cells (CEC) have immune-modulating properties. Methods Human monocytes were isolated from peripheral blood mononuclear cells and differentiated into monocyte-derived macrophages (MDM). A standardized protocol was used for disaggregation of human corneas into fragments of defined sizes. MDMs were stimulated using processed corneal material with or without CEC. Lipopolysaccharide (LPS) or interferon-gamma (IFNγ) served as controls. RNA sequencing was applied to analyze the impact of differential stimulation of MDMs on their transcriptional profile. RNA sequencing results were validated using digital PCR. Results The transcriptional profile of MDMs was significantly modulated by the type of stimulus used for MDM activation as well as by the individual MDM donor. LPS- or IFNγ-stimulation resulted in distinct transcriptional alterations compared to unstimulated MDMs including an upregulation of various cytokines such as CCL3, 4, 5, 19 or CXCL9. Corneal tissue induced the differential expression of 45 genes when compared to unstimulated MDMs, with several metallothioneins (MTs) among the upregulated factors (MT1A, MT1E, MT1F, MT1G, MT1H, MT1L, MT1M, MT1X, MT2A). This effect was independent of the presence or absence of CEC. PCR validation confirmed induction of 3 different metallothioneins (MT1G, MT1H and MT2A) in MDMs stimulated by corneal tissue. Conclusions The MDM in vitro model proved to be a robust tool to study the effects of LPS, IFNγ and corneal tissue homogenates on the transcriptional activity of MDM. Human macrophages showed a distinct upregulation of various MTs when challenged with human corneal allogen with or without corneal endothelium, which might have an immune-modulatory effect. As a general observation, it appears that in MDM-based studies a significant donor-dependent effect on the transcriptional profile of MDMs needs to be considered and adjusted before downstream analysis.

This study aims to compare the potential of standard RNA-sequencing (RNA-Seq) and 3′ massive analysis of c-DNA ends (MACE) RNA-sequencing for the analysis of fresh tissue and describes transcriptome profiling of formalin-fixed paraffin-embedded (FFPE) archival human samples by MACE. To compare MACE to standard RNA-Seq on fresh tissue, four healthy conjunctiva from four subjects were collected during vitreoretinal surgery, halved and immediately transferred to RNA lysis buffer without prior fixation and then processed for either standard RNA-Seq or MACE RNA-Seq analysis. To assess the impact of FFPE preparation on MACE, a third part was fixed in formalin and processed for paraffin embedding, and its transcriptional profile was compared with the unfixed specimens analyzed by MACE. To investigate the impact of FFPE storage time on MACE results, 24 FFPE-treated conjunctival samples from 24 patients were analyzed as well. Nineteen thousand six hundred fifty-nine transcribed genes were detected by both MACE and standard RNA-Seq on fresh tissue, while 3251 and 2213 transcripts were identified explicitly by MACE or RNA-Seq, respectively. Standard RNA-Seq tended to yield longer detected transcripts more often than MACE technology despite normalization, indicating that the MACE technology is less susceptible to a length bias. FFPE processing revealed negligible effects on MACE sequencing results. Several quality-control measurements showed that long-term storage in paraffin did not decrease the diversity of MACE libraries. We noted a nonlinear relation between storage time and the number of raw reads with an accelerated decrease within the first 1000 days in paraffin, while the numbers remained relatively stable in older samples. Interestingly, the number of transcribed genes detected was independent on FFPE storage time. RNA of sufficient quality and quantity can be extracted from FFPE samples to obtain comprehensive transcriptome profiling using MACE technology. We thus present MACE as a novel opportunity for utilizing FFPE samples stored in histological archives., RNA of sufficient quality and quantity can be extracted from formalin-fixed paraffin-embedded (FFPE) samples to obtain comprehensive transcriptome profiling using the 3′ massive analysis of c-DNA ends (MACE) RNA-sequencing technology. Thus, MACE provides an opportunity for utilizing FFPE samples stored in histological archives.

Despite advances in surgical technique and postoperative care, fibrosis remains the major impediment to a marked reduction of intraocular pressure without the need of additional medication (complete success) following filtering glaucoma surgery. Several aspects specific to filtering surgery may contribute to enhanced fibrosis. Changes in conjunctival tissue structure and composition due to preceding treatments as well as alterations in interstitial fluid flow and content due to aqueous humor efflux may act as important drivers of fibrosis. In light of these pathophysiological considerations, current and possible future strategies to control fibrosis following filtering glaucoma surgery are discussed.

Glaucoma is a chronic neurodegenerative eye disease characterized by a loss of retinal ganglion cells associated with structural changes in the ocular outflow tract, disturbed intraocular pressure regulation and changes in tissue biomechanics. Earlier studies revealed a strong influence of ECM stiffness on protein expression patterns and growth factor signaling in cells of the ocular outflow tract (trabecular meshwork). To gain further insight into possible regulatory mechanisms, we asked whether ECM stiffness would alter expression of short non-coding microRNAs.Human trabecular meshwork cells were plated on tissue culture plastic or polyacrylamide gel substrata (E-moduli approx. 40 or 120kPa) of different stiffness and allowed to adjust for 7 days. Subsequently, miRNA expression levels were studied by Affymetrix arrays and qPCR. Genes targeted by at least five mechanoresponsive miRNAs (stiffness-related change >2 fold) were determined using R software. Protein expression of selected targets was assessed by western blot. At least 26 miRNAs were differentially expressed and 24 genes were predicted targets of 5 or more mechanoresponsive miRNAs. ECM stiffness-dependent protein expression changes were confirmed for the predicted targets NF-kB, AKT, E2F1, p53, p21 and cyclin-D1. These data suggest a mechanoresponsive regulation of miRNA levels with a possible role in cell differentiation and disease.