Glaucoma has a huge impact on public health worldwide and primary open-angle glaucoma (POAG) is responsible for over 70% of glaucoma [8]. Current consensus on glaucoma is not solely based on intraocular pressure (IOP), especially in cases of normal-tension glaucoma (NTG). Although high IOP is a well-established risk factor for glaucoma, there is increasing evidence that other factors, such as vascular and neurodegenerative mechanisms [20, 21], also play a role in the development and progression of the disease. Lowering IOP is currently the only intervention available [22]. However, clinical evidence indicates that lowering IOP does not prevent progression in all POAG patients [23]. Thus, non-IOP factors are involved in POAG. Considering these findings, the treatment of glaucoma has shifted from a sole focus on lowering IOP to a more individualized approach that considers other factors that may be contributing to the disease. Previous studies have revealed inflammation and immune response mediate the process of POAG from several different aspects [9], yet much remains to be understood with regards to cellular processes regulating the maintenance of the TM and its relevance to IOP.
Recent advances have identified the significant associations between gut microbiota and several disorders in multiple organs besides the digest tract[37, 38]. Emerging evidence has suggested gut microbiota could be the regulator of several central nervous system disorder, including the ocular system disorders[39]. Although data from clinical filed showing the possible link between gut microbiota and the occurrence of POAG[29,40−42], the mechanism underlying its connections and the key molecules remained unknown.
In this study, we aimed at discovering the key genes and pathways in POAG that are gut microbiota related. After analysis, NFKB1, IL18, TLR9, FKBP2, KITLG, and HDAC4 were recognized as hub genes in POAG patients and in the database of gut microbiota regulation. They showed the most remarkable correlation with regulation and stimulation of leukocytes such as macrophages. Thus, we conclude these 6 hub genes might play important roles via affecting the activity of macrophages during the progression of POAG and in regulating gut microbiota. We further verified the expression level of 4 hub genes (NFKB1, TLR9, HDAC4 and IL18) through RT-qPCR. The above 4 hub genes were shown to be regulating immune response from different aspects. Based on our RT-qPCR results, the mRNA level of all 4 genes were increased in H2O2 treated group compared to control.
NFKB1 has been reported to be playing a critical role in suppressing inflammation, aging and cancer[43]. NFKB1−/− mice have reduced numbers of conventional and plasmacytoid dendritic cells and in response to TLR9 stimulation[44]. Moreover, NFKB1−/− bone marrow-derived macrophages show an increased expression of interferon-inducible genes, including IFN-β, in response to TLR-4 and TLR-9 stimulation[45]. Toll like receptor 9 (TLR9) is an intracellular TLR, expressed in different immunological and non-immunological cells[46]. Gut microbiota are a source of TLR ligands, including TLR9. Though its role in glaucoma via regulating gut microbiota remained mystical, previous research have indicated that TLR9 deficiency induces osteoclastic bone loss via gut microbiota-associated systemic chronic inflammation[47], thus, it is possible that TLR9 mediate the occurrence of glaucoma through chronic inflammation process. Targeting immune responses mediated by TLR9 is a potential therapeutic strategy for preventing disease-associated inflammation, and autoimmune diseases[48]. Several previous studies have also described a wide range of oxidative stress-related makers which are found in glaucomatous patients, including the activation of the NFKB pathway and the up-regulation of pro-inflammatory cytokines[49].
Little is known of what the function of HDAC4 in glaucoma is, but emerging evidence have shown HDAC4 has its importance in regulating neurogenesis[50]. In vivo selective inhibition of HDAC4 via MS-275 (entinostat) or LMK-235 could prevent ongoing RGC degeneration[51]. Reduced HDAC4 expression is associated with blood-brain barrier (BBB) breakdown contributing to ischemia/reperfusion injury-induced infarct in ischemic stroke model rats, while increased HDAC4 expression ameliorates BBB injury, contributing to the reduced infarct volume[52].
IL18 is a pro-inflammatory cytokine which has varies function in different cell types[53]. As an important regulator for both innate and acquired immune response, IL18 plays an important role in inflammatory/autoimmunity diseases. Research have shown the intestinal epithelial cell secretion of IL18 is necessary for gut microbiota homeostasis(dysbiosis)[54]. Meanwhile, increased IL18 expression was observed in glaucomatous trabecular meshwork[55]. Our bioinformatic prediction showed downregulation of IL18 while the RT-qPCR result showed significantly increased mRNA level of IL18, and RT-qPCR result is consistent with most previous research. In that case, we hypothesized the reason for the downregulation of IL18 showed in bulk RNA-seq data is due to the small sample number.
Pathways enriched in both GO and KEGG database are mostly related to the activity of leukocytes especially macrophages. Substantial amount of research concerning the role of the immune system in glaucoma has been performed in the recent years. Researchers have provided evidence of macrophages participate in the process of glaucomatous pathogenesis [56], studies analyzing the trabecular meshwork of patients with POAG found macrophages in this tissue by scanning electron microscopy[57], and the presence of macrophage inhibitory factor were observed in healthy human doners’ TM. Meanwhile, it is shown using macrophage activator drug Zymosan could induce neurodegenerative effect in glaucoma model[58]. Moreover, TM has been shown to display characters of several cell types including express behavior pattern of macrophages when operate to maintain homeostasis of IOP [59], which further support the accuracy of our bioinformatic analysis. It seems like macrophages played the role of the ‘double sided sword’ in glaucoma. Still, deeper discovery of the protective factors of the macrophages is needed to bring forward a therapeutic approach.
In this study, we utilized a hydrogen peroxide (H2O2) model to mimic glaucomatous ocular hypertension in vitro. H2O2 induces acute oxidative stress to TM which leads to the pathogenesis of POAG[60], this model was proved to be a useful platform which resulted in TM cellular dysfunction without destruction of TM structure[61], which made this model suitable for the hypothesis that gut microbiota dysbiosis regulates TM cellularity through innate immune response.
Gut microbiota and its metabolites contributes to a variety of diseases with inflammation components via regulation of macrophages, for example, intracranial aneurysm [62], IBD(inflammatory bowel disease)[63], Alzheimer’s disease[64], and more. Gut microbiota derived metabolite SCFAs (short-chain fatty acids) promotes anti-inflammatory functions of macrophages[65], as another gut microbiota metabolite TMAO(trimethylamine-N-oxide) induces M1 macrophage polarization[66]. Currently, it is still unclear how gut microbiota effects glaucoma via regulation of macrophages, it will be crucial to testify this mechanism in the real world. The gut-eye-axis is a relatively new concept that has gained attention in the scientific community in recent years.
Utilizing non-ocular laboratory examinations to help with early identification of ocular diseases has been proved to have a great potential[67, 68], for example: key molecules and pathways affected by glaucoma pathology can be developed into predictive diagnosis strategy[69]. In our case, we identified hub genes and pathways linked POAG with gut microbiota, which could be further used as biomarkers indicating POAG at an early stage from common non-invasive examination including fecal examinations.
While in this study, we discovered hub genes linked POAG with gut microbiota, those hub genes are also common inflammation related genes that could be altered during either POAG, or microbiota alone. Thus, the gut-eye-axis hypothesis is still an emerging field of research, and further studies are needed to fully understand the mechanisms behind this relationship and to establish a causal link towards future predictive and preventive medicine.
In the end, our study has its limitations. We showed gut microbiota could be influencing the occurrence of glaucoma via affecting macrophages, but lack of evidence from patients with glaucoma and gut microbiota dysbiosis at the same time. Moreover, sample number of POAG patients is relatively small(n = 4). As a matter of fact, human trabecular meshwork tissue is very rare, and incomparable with other animal or in vitro cell models. Considering our results are statistically significant, we believe that the four groups of samples can still reflect the situation of POAG. In a word, as a pilot study, our observations provide expanding insight of the gut-eye-axis and possible therapeutic target for future predictive, preventive and personalized treatment of glaucoma.