C3ar1deficient DBA/2J mice developed elevated intraocular pressure
DBA/2J mice inherit a depigmenting iris disease that leads to high IOP and glaucoma(32, 38). Immune cells that are likely to express C3ar1contribute to iris damage and the development of ocular hypertension(44, 45). To determine whether C3ar1 deficiency affected iris disease or IOP elevation, eyes of C3ar1-/- mice and their C3ar1+/+ littermates were examined regularly beginning at 6 months of age. No differences between genotypes were observed in the onset and progression of the iris disease (Figure 1A) or IOP elevation (Figure 1B). In C3ar1 deficient mice, high IOP sufficient to cause ocular hypertensive damage was observed, similar to standard DBA/2J mice(38).
C3ar1 promoted glaucomatous degeneration in D2 mice
The presence of optic nerve degeneration in an eyecan be explicitly determined by identifyingdegenerating axons and scarred regions withaxonloss in the optic nerve(Figure 2A) (20, 36-39).The percentage of eyes with optic nerve degenerationin C3ar1-/- and C3ar1+/+mice was compared at 10.5 and 12 months of age.At 10.5 months of age, significantly fewer eyes fromC3ar1-/-mice had degeneration(Figure 2B) suggesting thatC3ar1 deficiency decreased the risk of ocular hypertensive injury.By 12 months of age, C3ar1 deficient mice were no longer protected from glaucomatous degeneration(Figure 2B). Thus,C3ar1was not the sole trigger for degeneration, but did promote optic nerve damage.
Eyes from C3ar1-/- micewith healthy optic nerveshad a normalnumber of RGC layer neurons, suggesting thatC3ar1 deficiency had not caused abnormal loss of RGCs or amacrine cells (Figure 2C,D). In eyes with optic nerve degeneration, the loss of RGC layer neurons was independent ofC3ar1 genotype (Figure 2C,D).The observedloss of approximately half of RGC layer neurons is consistent with cell loss due to optic nerve injury, where the majority of RGCs die and amacrine cells are not affected(36, 39). These data indicate that C3ar1-/- mice had the same type of injury as standard D2 mice.
To investigate changes in RGC function in C3ar1-/- mice, pattern electroretinography was used. PERG amplitude is a sensitive measure of RGC activity and detects RGC dysfunction in ocular hypertensive DBA/2J mice (46, 47). PERG amplitude wasrecorded at 4 months of age, prior to the elevation of IOP, and 10 months of age, when lower amplitudes areexpected due to ocular hypertension and not due to the degeneration that typically occurs at slightly older ages. C3ar1 deficiency had no effect on the average PERG amplitude in young mice. C3ar1-/- mice also had a similar decrease in PERG amplitude due to chronically elevated IOP as C3ar1+/+ mice. Thus, C3ar1 deficiency did not prevent changes in RGC activity associated with ocular hypertension (Figure 2C).
Ocular hypertension affects C3ar1expression in the optic nerve head
In DBA/2J mice, observable injury occurs at the optic nerve head (ONH) prior to other regions of the optic nerve (36). At this same time point, the expression of C3ar1 increased in the ONH (2.0 to 3.4-fold; q<0.05), but not in the retina (1.0-fold; q=0.85) based on publicly available data (48). In the healthy brain it is well established that microglia primarily express C3ar1, with low or no expression in other cells (Figure 4A,B, (49-51)). In addition, higher levels of expression have beenobserved in subsets of microglia thought to mediate neuroinflammation, such as disease-associated microglia in 5xFAD mice, a widely-used mouse model of Alzheimer’s disease and Ccl3/Ccl4-positive microglia in aged and white matter-injured brain, as well as at embryonic and postnatal ages of development (Figure 4C, (52, 53)). This expression pattern is consistent with cell-type specific data from DBA/2J mice. ONH microglia and infiltrating monocytes express C3ar1at high levels, whileRGCs expressC3ar1 at a lower level (Figure 4D, (54, 55)). Thus, C3ar1deficiency in microglia and monocytes may affect their function or number in the ONH of ocular hypertensive eyes based on these expression data.
C3ar1 deficiency altered the inflammatory phenotype of cultured microglia.
To determine how C3ar1 deficiencymay alter microglia function, RNA sequencing was performed on microglia sorted fromprimary co-cultures of postnatally-derivedastrocytes and microglia.In culture, where gene expression is more uniform compared to DBA/2J mice,glial cells express many neuroinflammatory genes expressed in the optic nerve head of DBA/2J mice, including C3(17). Microglia were identified by fluorescence-activated cell sortingas IBA1-positive and GFAP-negative cells(Figure 4A). The selected cells expressed high levels of genes associated withmicroglia and low levels of genes associated with astrocytes (Figure 4B). 408 genes were differentially expressed (DE) in microglia due toC3ar1deficiency (Figure 4C; N=6, FDR<0.005).
The biological pathways most significantly enriched in DE genes included ‘role of pattern recognition receptors in recognition of bacteria and viruses’, ‘phagosome formation’, and ‘TREM1 signaling’ (Figure 5A).A network of the top 20 enriched pathways,with connections based on having more than five genes incommon,suggested thatmost pathways were closely interrelated and relevant to neuroinflammation and immune cellrecruitment (Figure 5B). Thus, thepathways altered by C3ar1 deficiencyregulate homeostatic and pathological responses in microglia and other immune cells. Upstream regulators of DE genes were analyzedto determine how C3ar1 deficiency may have this effect(Figure 5C). The most significantly enriched upstream regulators were ‘TCL1A’,’IL10’, and ‘LDLR’. The endogenous regulator thathad the highest interconnectivity was the anti-inflammatory cytokine IL10 (Figure 5D). In addition, the predicted regulator associated with the most DE genes was dexamethasone, a corticosteroid that prevents inflammation. These data show that C3ar1 deficiencysignificantlyaltered the expression of inflammatory genes and signaling pathways in microglia.
C3ar1 deficiency altered myeloid cell populations in the optic nerve head.
In C3ar1 deficient DBA/2J mice, decreased anaphylatoxin signaling and other changes in inflammatory gene expressionare likely tochangethelocalization or reactivity of microglia and monocytes. To investigate this in DBA/2J mice, thepopulation of myeloid-derived cellsin the retina and the optic nerve headwas assessed by flow cytometry at 10 months of age (Figure 6A). In the retina, no difference was observed between C3ar1+/+and C3ar1-/- mice in the percentage of myeloid-derived cells, including CD45hi and Cd11c+ monocytes (Figure 6B). Thus, C3ar1 deficiency did not have a general effect on the number ofthese cells in neural tissue exposed to ocular hypertension. In contrast to the retina, the ONHis a verysmallregion of tissue more sensitive toocular hypertensive stress anda location where myeloid cellslikely have beneficial and harmful effects at different stages of disease(20, 56);(54).In the ONH of C3ar1-/- mice the number of myeloid cells was more variable compared to in C3ar1+/+ mice(Figure 6C). These data suggest a role for C3ar1 in regulating myeloid cells in ONH under chronic ocular hypertensive stress.