Preprint Club
A cross-institutional Journal Club Initiative

Keywords

● Resident Tissue Macrophage

● Short-Lived Macrophage

● Conventional Outflow Tract

● Intraocular Pressure

Main Findings

In this preprint, the authors investigate the roles of resident tissue macrophages (RTMs) and short-lived macrophages in modulating intraocular pressure (IOP) homeostasis in the eye, as elevated IOP is a major risk factor for developing glaucoma. Previous studies have implicated an involvement of eye-resident macrophages in glaucoma pathogenesis and progression. For example, limbal macrophages were shown to promote Schlemm’s canal morphogenesis and prevent intraocular hypertension via activation of αvβ3 signalling, while increased infiltrating CD163+ macrophages were observed in the trabecular meshwork and Schlemm’s canal of postmortem glaucoma patients, suggesting a multifaceted role for macrophages in regulating ocular homeostasis. However, the roles of ontogenetically distinct outflow tract RTMs and short-lived macrophages in modulating IOP remain unknown. To assess the ontogeny, spatial distribution, and function of macrophages across the ocular space in the steady state, the authors employ a variety of elegant transgenic systems. Using a tamoxifen-inducible Cx3cr1YFP-CreERT2: iDTR mouse line, they demonstrated that RTMs are found predominantly in the trabecular network, while the use of Ms4a3Cre:RosaTdTom and Ccr2RFP mice revealed that monocyte-derived, short-lived macrophages are abundant in the distal vessels of the conventional outflow tract. These differences in ontogeny and spatial distribution suggest these two macrophage subpopulations play different roles in maintaining ocular homeostasis. Specifically, depletion of RTMs in tamoxifen-pulsed Cx3cr1YFP-CreERT2: iDTR mice via subconjunctival injection of diphtheria toxin (DT) resulted in elevated IOP and increased outflow resistance. The authors provided evidence that alterations in IOP may result from aberrant extracellular matrix turnover in the trabecular meshwork and Schlemm’s canal. In line with their observations that RTMs modulate ocular outflow, depletion of short-lived macrophages in Ccr2RFP/RFPknockout mice or pharmacological inhibition using the anti-CCR2 antibody MC-21 failed to present evidence that these macrophages control IOP. Based on these findings, the authors conclude that RTMs in the conventional outflow tract support the maintenance of IOP in the steady state.

Limitations

● While these experiments provide a detailed overview of the roles of outflow tract macrophages on IOP homeostasis at steady state, one may wonder how the loss of RTMs and short-lived macrophages may affect IOP in the context of disease. How would IOP change during inflammation or prolonged RTM depletion? Would RTM-deficient animals eventually develop glaucoma? Along these lines, would depletion of RTMs during fully manifested glaucoma further exacerbate the disease? Would depleting short-lived macrophages in glaucomatous mice via MC-21 treatment be beneficial in terms of reducing IOP towards more tolerable levels? While the study is conducted in the steady state, adding a disease model would greatly advance the authors’ findings.

● Normal IOP in mice ranges from 10-20 mmHg. However, in this preprint, the authors demonstrated that depletion of RTMs resulted in IOP levels of 18.4±0.4 mmHg. While significantly increased compared to baseline readings (16.9±0.3 mmHg) and control mice (16.1±0.3 mmHg), these levels still fall within normal range. While these findings suggest that RTMs contribute to IOP, their alteration is likely not sufficient to lead to glaucoma. Previous studies demonstrated increased levels of infiltrating T cells and antibodies specific for heat shock proteins in the eyes of glaucomatous mice and patients, with their elevation corresponding to more severe optic neurodegeneration, suggesting that multiple immune defects may be required to develop glaucoma.

● While the authors claim that RTMs are the key players in regulating IOP homeostasis, the roles of short-lived macrophages remain unclear. In Figure 3, mice depleted of short-lived macrophages exhibit decreased IOP independent of changes to outflow resistance and ECM remodelling. It would be interesting to expand on this data and confirm whether the increased IOP observed in DT-treated Cx3cr1YFP-CreER: iDTR mice is directly related to the loss of RTM-intrinsic functions, or caused by an influx of monocyte-derived, short-lived macrophages infiltrating the now empty niche and pushing towards a glaucomatous state. These findings would be valuable, as short-lived macrophages represent potential therapeutic targets for minimizing glaucoma pathogenesis.

● The authors’ evidence supporting the role of outflow tract RTMs in modulating ECM deposition is limited. Glaucomatous eyes are known to exhibit histological features consistent with fibrosis in the trabecular meshwork (https://doi.org/10.1002/ca.23263). Would depleting RTMs and short-lived macrophages drive morphological changes in collagen or elastin deposition? Histochemical assessments via Sirius Red and Verhoeff's staining would be useful additions in the contexts of steady state and glaucoma.

● While the authors provide spatial and functional characterization of ontogenetically distinct macrophages across the conventional outflow tract, additional functional assessments of outflow tract macrophages on glaucoma pathogenesis would be greatly appreciated. It would be interesting to measure the levels of retinal ganglion cell death via TUNEL staining in RTM-depleted versus control eyes. Similarly, in Ccr2RFP/RFPmice or mice treated with MC-21, would depleting short-lived macrophages result in increased apoptosis of retinal ganglion cells, possibly connecting the authors’ observations to a failure in optic nerve homeostasis?

● Glaucoma is an age-related disease. Is IOP different in aged versus young animals? Along this line, do aged mice possess altered levels of RTMs and short-lived macrophages in the conventional outflow tract, and would putative differences track with changes to IOP? Could depletion of either RTMs or short-lived macrophages poise IOP towards pathologic or homeostatic states, respectively?

Significance/Novelty

While eye-resident immune cells have previously been implicated in glaucoma pathogenesis, their roles in the maintenance of IOP—a major risk factor for developing glaucoma—have not been well established. This preprint represents a first-of-its-kind study to reveal a direct role of outflow tract RTMs in modulating IOP. Using a reductionist approach, the authors determine that RTMs, rather than short-lived macrophages, contribute to IOP maintenance potentially via regulation of extracellular matrix homeostasis across the conventional outflow tract. These findings highlight the importance of macrophage heterogeneity—particularly their ontogeny, spatial localization, and function—in modulating tissue homeostasis, ultimately revealing a previously uncharacterized role of RTMs in maintaining IOP at steady state. These findings are fundamental in paving the way towards the development of novel and effective therapeutics against glaucoma.

Credit

Reviewed by Alex Lac as part of a cross-institutional journal club between the Icahn School of Medicine at Mount Sinai, the University of Oxford, the Karolinska Institute and the University of Toronto.

The author declares no conflict of interests in relation to their involvement in the review.