Relationship between lymphocytes and idiopathic macular hole

A macular hole (MH) is a full-thickness anatomic defect that extends from the internal limiting membrane (ILM) to the photoreceptor layer of the macula [1]. Gass described MH in terms of four clinical stages in 1995: stage I is a central yellow spot or yellow ring with loss of foveolar depression, but no vitreofoveal separation; stage II is a full-thickness hole < 400 μm in diameter; stage III is a full-thickness hole ≥ 400 μm in diameter with persistent hyaloid attachment; and stage IV is a full-thickness hole ≥ 400 μm with complete posterior vitreous detachment [2]. Many causes of macular holes have been identified, including trauma, uveitis, and high myopia [3]. However, systemic or ocular causes of idiopathic macular holes (IMHs) remain elusive [4]. IMHs cause severe impairment of central vision predominantly in women older than 60 years [5].

Although the exact pathogenesis of IMHs is unclear, the literature provides several explanations for the etiology of the disease: (1) anterior–posterior traction by the posterior vitreous cortex on the macular fovea has been extensively accepted as a possible cause [6, 7]; (2) ILM contains collagen fibrils, proteoglycans, basement membranes and plasma membrane of Müller cells, and the proliferation and contraction of the ILM may be one of the reasons for MH enlargement [8]; and (3) Tornambe proposed the hydration theory, in which a break in the inner retinal layer leads to the accumulation of vitreous fluid in the middle and outer retinal tissues [9]. Based on these theories, vitrectomy with peeling of the ILM combined with gas or silicone oil tamponade has become a common surgical method for the treatment of IMHs.

However, there are different views on treatment options for IMHs. Tornambe [9] suggested that not all patients with MH should undergo surgery with ILM removal. If optical coherence tomography (OCT) shows only direct traction of the posterior hyaloid on the inner retinal defect, simply removing the posterior hyaloid should suffice for the permanent closure of the MH. Eckardt et al. [10] found evidence of necrosis or degeneration in Müller cells of the inner boundary membrane that was removed surgically. Hayashi [11] found mild inflammation on fluorescein angiography in a recurrent MH. After initiation of topical steroid treatment, inflammation was reduced and the MH was subsequently closed. Previous studies on eye inflammations have used IMH patients as controls because they were considered free of inflammation; however, Li and Wang [4, 12] found that after IMH onset, there were cytokines, such as IL-17, IL-14, and IL-13, in the aqueous humor. Therefore, several researchers have reported medical therapies for MH, including topical steroids [13, 14], nonsteroidal anti-inflammatory drugs [15, 16], and topical anhydrase inhibitors [17‐19]. In addition, these drugs have been combined with intravitreal injections of steroids or bevacizumab [20].

Clinical laboratory markers of systemic immunoinflammatory status derived from complete blood counts include lymphocyte count, leukocyte count, neutrophil count, monocyte count, eosinophil count, basophil count, platelet count, neutrophil-to-lymphocyte ratio (NLR), monocyte-to-lymphocyte ratio (MLR), platelet-to-lymphocyte ratio (PLR), and systemic immune-inflammation index (SII, platelet × neutrophil/lymphocyte). Furthermore, some of these markers have been evaluated in ocular diseases, such as central retinal artery occlusion [21], anterior ischemic optic neuropathy [22], retinopathy of prematurity [23], and diabetic macular edema [24].

However, no studies have investigated the relationship between these immunoinflammatory markers and IMH. Therefore, this study aimed to explore whether they are involved in the development and progression of IMH.

This retrospective study was performed at the Ophthalmology Department of the Affiliated Jinling Hospital, Medical School of Nanjing University, from January 2013 to January 2022.

OCT is currently used for detailed assessment of IMHs and regarded as the diagnostic gold standard [25]. This study included 36 patients with IMH (IMH group) and 36 sex-and-age-matched patients with cataracts (control group). According to Gass staging, the IMH group was classified using OCT into three categories: stage II (11 patients), stage III (13 patients), and stage IV (12 patients). The study was conducted in accordance with the Declaration of Helsinki and informed consent was obtained from each participant. Ethics committee approval was obtained from the ethics committee of the Affiliated Jinling Hospital, Medical School of Nanjing University, approval number 2023DZKY-008–01.

A total of 72 participants were included in this study: 36 in the IMH group and 36 in the control group. The median (IQR) ages of the IMH and control groups were 63.00 (IQR 61.00–65.00) and 65.00 (IQR 62.00–69.00) years, respectively (p = 0.079). The study included 15 men and 57 women. The sex distribution was similar between the two groups (p = 0.772). The clinical characteristics and complete blood counts were shown in Table 1. The lymphocyte levels were significantly higher in the IMH group than in the control group (p = 0.029). There were no significant differences in other data between the two groups.

To the best of our knowledge, this is the first study to demonstrate a close relationship between serum inflammatory markers and IMH. In our study, the lymphocyte counts in the IMH group were significantly higher than those in the control group. There were no significant differences in other data between the two groups.

Our clinical results are consistent with those of preliminary studies. Studies have shown that owing to the anatomical structure of the retina, T lymphocytes can contact Müller cells after passing through the capillary wall. In vitro studies have shown that Müller cells inhibit the proliferation of T helper lymphocytes, and experiments have shown that specific destruction of Müller cells in animals increases the incidence of experimental autoimmune uveitis [28]. Müller cells are damaged during the formation of a MH; afterwards, T helper lymphocytes may proliferate. Once IMH forms, the retina undergoes a process called reactive gliosis [29]. Lymphocytes disrupt the blood-retinal barrier and migrate to the vitreous cavity. There, lymphocytes come into contact with transitional pigment cells and glial cells, secreting cytokines that promote inflammatory responses and enhance macrophage-mediated phagocytosis [30]. Significant differences in cytokine levels in the aqueous humor were found between patients with IMHs and those with cataracts. Significant changes in cytokine levels in patients with IMHs are due to inflammatory responses [4]. In addition, lymphocytes in the vitreous cavity can promote the transformation of glial cells into fibroblasts and accelerate tissue hyperplasia repair [4]. The recruitment of innate immune cells, which leads to the recruitment of T cells and other immune cells, can also induce damage repair [31]. We hypothesized that lymphocytes are not pathogenic factors in IMH, but play an important role in the tissue repair of IMH.

Lymphocytes and neutrophils are important components of leukocytes that mediate adaptive and innate immunity. Neutrophils play an important role in initiating and regulating immune processes [32]. Meanwhile, lymphocytes are specific inflammatory mediators with regulatory and protective effects. Consistent evidence suggests that the infiltration of lymphocytes into solid tumors is a beneficial prognostic marker [33]. Platelets can bind to leukocytes and endothelia and influence the function of inflammatory factors. Blood cell parameters, including lymphocyte counts, neutrophil counts, monocyte counts, platelet count, NLR, MLR, PLR, and SII, serve as simple inflammatory markers in diseases. Once a high inflammatory response occurs, the counts of neutrophils, platelets, and monocyte increase overall, whereas lymphocyte counts decrease overall. However, in our study, there were no significant changes in the counts of neutrophils, monocytes, and platelets, whereas lymphocyte counts increased in the IMH group. We assumed that during the MH formation phase, lymphocyte activation increased the release of related inflammatory factors and promoted MH repair. Inflammation and cytokines may be involved in the formation of MH; however, IMH is not a hyper-inflammatory disease.

With the advent of OCT, which is a useful diagnostic tool for fundus disorders, observation of the progression and prediction of visual outcomes after IMH surgery has become convenient. In our study, we investigated various measurements and indices, including BD, MIN, H, IRC, MHI, THI, DHI, HFF, and MHCI. These parameters were used to assess macular morphology and predict postoperative anatomical outcomes.

We investigated the correlation between lymphocyte counts and these parameters. In the IMH group, we found that lymphocyte count had significantly negative correlations with BD and MIN, but had significantly positive correlations with MHI, THI, and MHCI. Some researchers found that BD and MIN might reflect tangential traction during the formation of IMH and have negative correlations with IMH closure rates [34, 35]. MHI is highly correlated with postoperative anatomical outcomes and is a good predictor of anatomically successful closure of IMH. THI was found to have a significant positive correlation with optimal postoperative correction [36]. When the MHCI is between 0.7 and 1.0, successful closure with normal foveal morphology may be achieved [37]. Our ROC curve analysis showed similar results. Regarding the cut-off points of the ROC curves, lymphocyte count was significantly higher in the group with MIN ≤ 499.61 μm, and it was significantly higher in the MHI ≥ 0.47, THI ≥ 1.2, and MHCI ≥ 0.81 groups. According to previous studies, lymphocytes can accelerate tissue hyperplasia repair [4]. Therefore, we hypothesized that the MH healed better when the MIN was ≤ 499.61 μm, MHI was ≥ 0.47, THI was ≥ 1.2, or MHCI was ≥ 0.81.

This study had some limitations. First, the sample size was relatively small, and the data were obtained from a single center. Second, this was a retrospective study. The complete blood count of the patients used in this study was extracted from only one blood test, which may not accurately predict the persistence of blood parameters over time. Further investigation is needed, especially regarding the blood samples of the postoperative patients. Meanwhile, third, the study lacked additional examinations of blood or eye tissues to test inflammatory markers such as cytokines and laser flare cell values.

Inflammation might not be an initiating factor, but it plays a relatively important role in IMH formation. Furthermore, lymphocytes might be involved in the developmental and postoperative recovery phases of IMH. The result of the study could provide new insights into the development and progression of IMH. This was a retrospective study, and we used complete blood counts as test specimens; however, the number of clinical cases was insufficient. In addition, the lack of detailed analysis of inflammation was a limitation in our study. In the future, intraocular tissue samples from patients with MHs should be used to further clarify the role of lymphocytes in the pathogenesis of IMH.