Aqueous humour cytokine profiles after Descemet’s membrane endothelial keratoplasty

The aim of this study was to compare aqueous humour (AqH) cytokine profiles before and after Descemet’s membrane endothelial keratoplasty (DMEK) in eyes with bullous keratopathy (BK) and age-matched normal eyes. A total of 49 participants (mean age 75.0 ± 13.5 years) were divided into three groups: (1) BK patients scheduled for DMEK (BK group); (2) patients after DMEK (DMEK group; average postoperative time 1,018 ± 460 days); and (3) control participants without corneal endothelial disease scheduled for cataract surgery (control group). AqH (0.2 mL) was collected from all participants, and the levels of various AqH cytokines were simultaneously measured using a multiplex bead immunoassay. The DMEK group exhibited significantly lower concentrations of several pro-inflammatory cytokines, such as IL-1β, IL-5, IL-6, IL-10, and IL-8, and granulocyte colony stimulating factor than the BK group. In addition, the levels of IL-1β and IL-5 were significantly lower in the DMEK group than in the control group. The AqH levels of several pro-inflammatory cytokines were significantly reduced after DMEK, indicating that regeneration of the endothelial pump owing to the transplantation of a healthy endothelium might have a positive effect on anterior chamber inflammation.


Results
Characteristics of the participants. The characteristics of the study patients are summarised in Table 1.
The mean age of the patients was 75.0 years (range, 54-89 years), and 14 of the 49 patients (28.6%) were males. Mean axial length (AXL) was 23.6 ± 1.4 mm, and mean intraocular pressure (IOP) was 13.6 ± 3.2 mmHg. The reasons for endothelial dysfunction were pseudophakic bullous keratopathy (12 eyes in the BK group, 12 eyes in the DMEK group), Fuchs endothelial corneal dystrophy (two eyes in the BK group; one eye in the DMEK group), pseudoexfoliation syndrome (one eye in the BK group, one eye in the DMEK group), and chemical burn (one eye in the BK group).
In the DMEK group, the time from DMEK surgery until AqH collection was 1,018 ± 460 days. In all patients, the indication for DMEK was pseudophakic BK. All patients in the DMEK group had been using low-dose steroid eye drops (fluorometholone 0.1%; Santen, twice a day). The best-corrected visual acuity (BCVA) was 0.06 ± 0.04 logarithm of the minimum angle of resolution (logMAR). In the DMEK group, corneal ECD on the day of AqH collection was 979 ± 357 cells/mm 2 . One eye showed allograft rejection on the day of AqH collection and was excluded from further statistical analyses. In the BK group, all patients were on the waiting list for DMEK surgery. Some patients had been using topical steroids (n = 10) or topical non-steroidal anti-inflammatory drugs (NSAIDs) (n = 1). All eyes in the control group were scheduled for cataract surgery, and none of the patients had been using anti-inflammatory drugs, such as steroids or NSAIDs. In the DMEK group, no clinical feature of anterior chamber inflammation (keratic precipitates [KPs], synechiae, or flare) was detected except for one eye with graft rejection. On the contrary, most of BK patients showed some KPs although the clear picture was not preserved owing to oedematous and hazy cornea. There was no inflamed eye in the control group.
All data in our cohort are shown as a dataset (Supplementary Data set).
In addition, AqH cytokine levels in one eye that was newly diagnosed with graft rejection after DMEK were measured. This eye showed high cytokine levels of IP-10 (6247.25 pg/mL; compared to a mean of 18.77 pg/mL in the control group), G-CSF (1568.14 pg/mL, compared to a mean of 10.51 pg/mL in the control group), MCP-1 (3158.59 pg/mL, compared to a mean of 866.9 pg/mL in the control group), IFN-γ (13.37 pg/mL, compared to a mean of 1.94 pg/mL in the control group), and IL-6 (29837.1 pg/mL, compared to a mean of 54.8 pg/mL in the control group).
A major strength of this study is the post-operative evaluation of AqH cytokine profiles in eyes that underwent DMEK. Although the existence of a correlation between immune responses and graft failure following DMEK has been reported 24 , no comprehensive evaluation of AqH cytokine profiles following DMEK has been performed, especially during the stable and quiescent post-operative period. The average duration of the post-operative period in the DMEK group was 1,018 ± 460 days, which is a relatively long-term follow-up after DMEK. Our results showed that the concentrations of some AqH cytokines (IFN-γ, IL-8, and IL-10) in the DMEK group were significantly higher than in the control group (Fig. 1), whereas the concentrations of other cytokines (IL-1β and Importantly, the concentrations of all the analysed cytokines were remarkably lower in the DMEK group than in the BK group, most of which were statistically significant (Fig. 1). This finding clearly demonstrates that the transplanted healthy endothelium has a positive effect on anterior chamber inflammation in eyes with BK. Previous studies have indicated that CEnCs play an important role in maintaining the immune-privileged status of the cornea (Fig. 2) 26,27 . Thus, the higher concentrations of inflammatory cytokines observed in eyes with BK may be attributable to the absence of healthy and protective CEnCs. After transplantation, transplanted healthy CEnCs might inhibit these inflammatory cytokines and restore the silent anterior chamber environment. Therefore, any surgical method providing healthy corneal endothelium, such as PKP and Descemet stripping automated endothelial keratoplasty (DSAEK), might show the similar effects on the anterior chamber inflammation. CEnCs may play an important role in anterior chamber inflammation and immunity, for example, via the programmed death-1 (PD-1)/B7-H1 co-stimulatory pathways 26 , or vasoactive intestinal peptides 28 .
Interestingly, we obtained cytokine profiles from a patient presenting with allograft rejection following DMEK. We detected relatively high levels of several inflammatory cytokines, including IP-10, G-CSF, MCP-1, IFN-γ, and IL-6. IP-10 is induced by IFN-γ, which is related to the Th1 pathway 29 , whereas G-CSF, MCP-1, and IL-6 are associated with macrophages 30 . Notably, the up-regulation of IL-6 levels has already been reported in cases presenting with allograft rejection following penetrating keratoplasty (PKP) 31 . Despite the differences between PKP and DMEK, our results are consistent with those of this study.
Some of our study subjects had used topical steroids prior to the collection of AqH. However, we did not detect any significant difference between the concentration of cytokines in the AqH of eyes treated with topical steroids and those that were not treated 19,20 . In fact, concentrations of some cytokines were higher in eyes treated with topical steroids than in those that were not. Although topical steroids play an essential role in preventing allograft rejection 32 , our results also show that high AqH concentrations of at least some inflammatory cytokines remain in the eyes after uneventful DMEK, regardless of the use of low-dose steroid eye drops. Several studies have described the positive effects of using topical steroids and NSAIDs in endothelial keratoplasty 33,34 ; thus, proper treatments that reduce inflammatory responses following DMEK, such as steroids, NSAIDs, or 'anticytokine' eye drops, should certainly be considered.
Our study was beset with some limitations, such as the relatively small number of patients and the lack of evaluation of flare by laser flare-cell photometry, or of a cytokine profile time course following DMEK. Moreover, future prospective studies are necessary to compare changes in cytokine levels at earlier phases (1, 3, 6, and 12 months) after different types of keratoplasty, such as PKP, DSAEK, and DMEK.
Taken together, our current study clearly demonstrates that after DMEK, significant changes in AqH cytokine profiles occur. These changes may be attributable to the immunomodulatory effect of transplanted CEnCs. As DMEK may be useful to reduce anterior chamber inflammation in patients with BK, early intervention should be considered in treating inflammation in these eyes. However, a certain level of inflammation persists even after uneventful DMEK (compared to healthy eyes without corneal transplants), which might warrant the development of specific anti-inflammatory drugs that endow patients with long-term graft survival.

Methods
Study design and approval. The current cross-sectional study was approved by the Institutional Review  www.nature.com/scientificreports/ The experimental procedures followed all institutional guidelines, and written informed consent was obtained from all patients before each procedure.

Collection of AqH.
Between September 1 and September 30, 2020, we collected AqH (0.2 mL per participant) at Yokohama Minami Kyosai Hospital from all study participants. Briefly, after the sterilisation of eyes with an antiseptic agent (povidone-iodine), approximately 0.2 mL of AqH was collected under a surgical microscope using a 30-gauge needle. The samples were immediately stored at − 80 °C until the proteins were analysed. The participants were divided into three groups: (1) eyes with BK scheduled for DMEK (BK group), (2) eyes that had previously received DMEK (DMEK group), and (3) control eyes without any signs of corneal disease scheduled for cataract surgery (control group). Patients with additional ocular diseases, including glaucoma, retinal diseases, or uveitis, were excluded from the study 35 . Post-operatively, 1.5% levofloxacin (Cravit; Santen) was administered four times daily for 1 week.
Ophthalmic examinations. In all participants, BCVA, logMAR, central corneal thickness (CCT), ECD, AXL, and IOP were measured prior to the collection of AqH. BCVA was measured as decimal visual acuity and converted to logMAR units for statistical analysis. CCT was measured using corneal tomography (SS1000; Tomey Corporation, Aichi, Japan) and evaluated by a corneal specialist (HT). ECD was evaluated using a specular microscope (FA3509; Konan Medical Hyogo, Japan). AXL was measured via optical biometry (IOL Master 500, Carl Zeiss Meditec, Oberkochen, Germany).

Statistical analysis.
Statistical analyses were performed using the JMP Pro software (version 13.2.0; SAS Institute, Cary, NC, USA). The Kruskal-Wallis test with Dunn's multiple comparisons test was used to compare age, BCVA, AXL, IOP, and CCT among the three groups. Nominal variables, such as sex, were compared using Pearson's chi-square test. Cytokine concentrations were analysed using ANOVA followed by Tukey's test, after logarithmic transformation because these data showed a lognormal distribution 27 . Statistical significance was set at p < 0.05.

Data availability
All data generated or analysed during this study are included in this published article and in its Supplementary Information files.