In the present study, we have successfully repeated visualization of hyperreflective VRICs, previously described in the literature as MLCs, of patients with active uveitis using averaged en face SS-OCTA images, and we have demonstrated how their number, size, and density decrease when the inflammation gets under control. Since van Furth and colleagues
15 introduced the “mononuclear phagocytic system” concept to classify highly phagocytic mononuclear cells and their precursors in 1972, knowledge regarding their role in the human body progressed exponentially. Specifically in the eye, the retina contains resident microglia, a type of myeloid-derived cell primarily consisting of macrophages. These microglia are strategically positioned within the glial limitans surrounding the inner retinal vessels and are distributed throughout the retinal tissue.
16 Microglia express several markers associated with macrophages, including cluster of differentiation 14 (CD14), CD11b, and epidermal growth factor (EGF)-like module-containing mucin-like hormone receptor-like 1 (EMR1), also known as F4/80 in the animal model.
16 Monocyte-derived macrophages infiltrating the retina can differentiate into microglia-like cells but are distinct from resident microglia.
17,18 These monocyte-derived macrophages can remain in the retina for extended periods, adopting a microglia-like morphology and gene expression profile, although they exhibit differences in gene expression, such as class II major histocompatibility complex (MHCII) levels.
17,18 The infiltration of monocyte-derived macrophages into the retina is part of the immune response to degeneration, and their presence can be crucial in disease states affecting the retina.
19
Over the last 2 years, there has been a growing interest in utilizing OCTA and SS-OCT for the visualization of retinal microglia, which appear as hyperreflective bodies that the existing literature identifies as macrophage-like cells.
20–24 However, the term “macrophage” implies cells of myeloid origin. Many tissues, including retina and brain, have immune cells of both yolk sac and myeloid origin. Given the marked context-specific development of both types, we decided not to refer to these hyperreflective bodies by their origin (MLC) because that cannot be determined. We thus adopted the term VRICs to refer to their location. Hyperreflective VRICs are increased in numbers in inflammatory retinal conditions, including both proliferative and non-proliferative diabetic retinopathy, as well as retinal vein and artery occlusions. Within our research group, we have adopted a methodology involving concurrent OCTA and en face OCT imaging of the same retinal region (as pioneered by Castanos et al.
7) and have incorporated sequential scans of the identical area using tracking mechanisms to enhance image clarity and to cross-sectionally visualize VRICs in uveitis and make comparisons with healthy controls.
9,10
Furthermore, it is important to note that the term “macrophage-like cells” remains a speculative designation. Thus far, only studies conducted ex vivo or in animal models have provided insights into the dynamic behavior over time of microglia, monocyte-derived macrophages, and hyalocytes under various physiological conditions, elucidating alterations in cell morphology and motility in response to injury.
1,2,25 All investigations applying readily available retinal imaging modalities for in vivo visualization of these hyperreflective bodies have presented static snapshots taken at a single time point. Thus, we cannot affirm with certainty that the hyperreflective bodies are cells (in particular, microglial cells) that are increasing and moving around the retina surface after an insult. As such, we believe that referring to these hyperreflective bodies by their location (VRICs), instead of assuming their nature (MLCs), is a more scientifically sound option.
To illustrate the mobility of hyperreflective bodies observed in en face SS-OCTA images at the retinal surface, we conducted seven 6 × 6 OCTA scans within 1 hour on a healthy eye belonging to one of the authors. Subsequently, we processed the seven en face averaged images using ImageJ to generate a video stack, enabling us to track the positions of these hyperreflective bodies over time. The accompanying
Supplementary Movie S1 demonstrates that these hyperreflective bodies (which we term VRICs and the literature suggests may be MLCs), exhibit movement over the course of 7 hours, characterized by pseudopodic extensions. This dynamic behavior of hyperreflective bodies on the surface of a healthy retina strongly supports their classification as microglial cells. These resident macrophage-like or dendritic-like cells continually survey their surrounding environment, poised to respond promptly to any retinal insults.
Indeed, during three-dimensional migration, macrophages can adopt either an ameboid or mesenchymal migratory movement. When they engage in ameboid migration, these cells assume a rounded or polarized shape as they move through the extracellular matrix. In our study, VRICs showed an ameboid movement pattern on the retinal surface and along blood vessels.
26 This is aligned with their critical immune patrolling and response functions, as well as chasing chemotactic signals and revealing an increase in shape plasticity, which confirmed the outcome of previous studies performed in vitro.
27
After establishing the provisional nature of hyperreflective corpuscles as VRICs, our prospective study demonstrated a significant increase in their number (552.5 cells) and density (15.3 cells/mm
2) in patients with active uveitis when compared to the corresponding measurements in healthy controls (as reported by Pichi et al.
10). These findings in inflamed eyes align with our group's prior results (average cell count of 546.1 cells and an average density of 15.17 cells/mm
2, in patients with active posterior uveitis). This phenomenon may be attributed to the infiltration of perivascular macrophages originating from circulating monocytes, which enter the retina following an insult.
28 As reported by London et al.,
29 it has been demonstrated that these cells differ from resident microglia, which appear to play no role as effector cells in human posterior uveitis.
8
The VRIC increase in size in active uveitis might be an indirect feature of the proinflammatory phenotype of these hyperreflective bodies.
30 Although our initial measurements of these VRICs align with previous findings regarding their detection in active uveitis,
9,10,22 our current study focused on monitoring their dynamics over two distinct time intervals, coinciding with the administration of appropriate anti-inflammatory therapy (prednisolone, 1 mg/kg). Our research demonstrated a significant reduction in their quantity, dimension, and concentration at T1 (1–2 months into treatment compared to baseline) (
P = 0.007,
P = 0.009, and
P = 0.007, respectively). Furthermore, no significant further changes were observed at T2 (3–4 months from baseline) (
P = 0.5,
P = 0.3, and
P = 0.5, respectively), indicating a return to their baseline state. These findings correspond with a positive correlation initially observed between VRIC size and density and inflammatory markers (particularly strong for AC cells and moderately so for vitreous haze) at the baseline, which weakens at T1 and disappears at T2. Collectively, these data suggest that VRICs promptly respond to ocular inflammation by presenting antigens and recruiting circulating monocytes, thereby contributing to the mounting of an immune response characterized by AC cells and vitreous haze. When an appropriate anti-inflammatory treatment is initiated, VRICs revert to a steady state, diminishing in number, size, and concentration, even in cases where inflammation has not been fully resolved and AC and vitreous cells and proteins continue to decrease without complete resolution.
This absence of any significant change in VRICs after T1 could be explained by their measurements returning to the levels of healthy controls.
10 These data give rise to various hypotheses on potential discrepancies of the immune response between the two immune compartments of the eye. The different levels of correlation might be interpreted first as an unmatched immune response between the anterior chamber driven by the anterior chamber–associated immune deviation and the immune compartment of the posterior pole.
31 On the other hand, this might be simplistically a slower clearance of the AC cells in the attempt to restore the immune homeostasis, albeit poor scientific literature is available regarding the aqueous humor dynamic in uveitis. Further analysis and exploration are undoubtedly necessary for a better understanding of cell interplay in the context of our research.
32
The present study has several limitations. First, the sample size of the patients studied was small, and there was heterogeneity in terms of inflammatory pathologies, which may impact the VRIC measurements. A second limitation is the heterogeneity in the timing between T1 and T2 visits; however, even if the timings were not standardized, we ensured that at T1 the uveitic subjective parameters had decreased and that at T2 the uveitis was completely quiet. Furthermore, we have not scaled the images per axial length to the correct lateral scale of the OCTA image; however, given that we were imaging the same subject at different time points, the axial length was a constant for the same subject and that limitation was partially overcome.
In addition, although our methodology allowed us to appreciate the VRIC interplay with the retinal surface immune compartment, this represents only a bidimensional representation of the potential migratory activity of VRICs. The
x- and
y-planes are the only ones explored in our manuscript, as we currently have no information on the
z-plane that might identify VRICs through the neuroretinal layers.
26 Although it might be perceived as marginal, this biodynamic aspect may significantly contribute to defining the macrophage migration dynamic, clarifying whether they adopt a prevalent ameboid or mesenchymal migratory pattern or behave differently depending on the plane that they interact with. In addition, the potential migration toward the retinal pigment epithelium might lead to further hypotheses on the interplay of VRICs with the choroidal immune compartment. Furthermore, it would be interesting to break down the different uveitis entities to segregate VRIC behavior in different uveitis subsets and identify whether the primary signal might come from the retina or the choroid, which still remains poorly explored.
Despite these limitations, the strength of our study lies in highlighting the dynamic nature of VRICs in uveitic patients. Traditionally, the retina was considered an immune-privileged site, presumed to have limited immune cell involvement. On the other hand, recent advances in research have unveiled the active and multifaceted roles of these immune cells in maintaining retinal homeostasis and responding to various challenges. In retinal diseases, although macrophages can play a role in phagocytosing damaged retinal cells and modulating the inflammatory response, dendritic cells excel in antigen presentation as essential bridge builders between the innate and adaptive immune systems. They capture and process antigens and present them to T cells, initiating the immune response. Their presence in the retina during uveitic conditions underscores their participation in the pathogenesis of these diseases.
Considering this evolving understanding of the intricate interactions between these immune cells and retinal tissues, researchers are exploring innovative therapeutic approaches. Among these approaches, dendritic cell–based vaccines have emerged. These vaccines aim to modulate the immune response by targeting specific antigens related to retinal diseases, allowing for precise regulation of the immune response. By harnessing dendritic cells and their associated CD markers, these vaccines can potentially ameliorate intraocular inflammation and represent a highly specific and technologically advanced avenue for managing and treating retinal diseases. These advances open new horizons for personalized and effective treatment strategies for retinal diseases, and an effective in vivo visualization of those cells could play an effective role in future uveitis treatments.