Anaphylatoxin Receptor C3AR1 Promotesoptic Nerve Degeneration in DBA/2J Mice


 BackgroundThe risk of glaucoma increases significantly with age and exposure to elevated intraocular pressure, two factors linked with neuroinflammation. The complement cascade is a complex immune process with many bioactive end-products, including mediators of inflammation called anaphylatoxins. Complement cascade activation has been shown in glaucoma patients and models of glaucoma. However, the function of anaphylatoxin signaling in glaucoma is largely untested. Here,theC3AR1 anaphylatoxin receptor was genetically disrupted in DBA/2J mice, an ocular hypertensive model of glaucoma, to test its contribution to neurodegeneration. Methods A null allele of C3ar1 was backcrossed into DBA/2J mice. Development of iris disease, ocular hypertension, optic nerve degeneration, retinal ganglion cell activity, loss of RGCs, and myeloid cell infiltrationin C3ar1 deficient andsufficient DBA/2J mice were compared across multiple ages. RNA sequencing was performed on microglia from primary culture to determine global effects of C3ar1 on microglia gene expression. ResultsDeficiency in C3ar1 lowered the risk ofdegeneration in ocular hypertensive mice without affectingintraocular pressure elevation at 10.5 months of age. Differences were found in the percentage of mice affected, but not in individual characteristics of disease progression.The protective effect of C3ar1 deficiency was then overcome by additional aging and ocular hypertensive injury. Microglia and other myeloid derived cells were the primary cells identified that express C3ar1.In the absence of C3ar1, microglial expression of genes associated with neuroinflammation and other immune functions were differentially expressed compared to WT. A network analysis of these data suggested that the IL10 signaling pathway is a major interaction partner of C3AR1 signaling in microglia.ConclusionsC3AR1 was identified as a damaging neuroinflammatory factor. These datahelp suggest complement activation causesglaucomatous neurodegeneration through multiple mechanisms, including inflammation. Microglia and infiltrating myeloid cells expressed high levels ofC3ar1 and are the primary candidates to mediate its effects. C3AR1 appeared to be a major regulator ofmicrogliareactivity and neuroinflammatory function due to its interaction with IL10 signaling and other immune related pathways.Targeting myeloid-derived cells and anaphylatoxin signaling with therapiesis expected to improveneuroprotective therapeutic strategies.


Introduction
Glaucoma is a common disease that damages the optic nerve and impairs vision (1). Risk for glaucoma is greatly increasedafter middle age and by exposure to elevated intraocular pressure (IOP). Elevated IOP and aging areassociated withneuroin ammation, yet it remainsunclear when and how neuroin ammationbecomes damaging in glaucoma and how to intervene (2,3). These questions underlie a need to develop a comprehensive understanding of in ammatory processes in glaucoma.
A major type of in ammatory response observed in glaucoma patients is activation of the complement cascade (4)(5)(6). The complement cascade is activated by three distinct pathways, the classical, alternative, and mannose-binding lectin pathways, which play a key role in responding to tissue damage and infection. The nal product of the complement cascade, the membrane attack complex (MAC), has been identi ed in optic nerve tissue from ocular hypertensive patients. This suggests full activation of the complement cascade has occurred, including multiple steps that promote neuroin ammation. The major products of the complement cascade that promote neuroin ammationare theanaphylatoxins and the MAC (7,8). The anaphylatoxins arepolypeptides produced by the cleavage of complement components 3 and 5, and named C3a and C5a, respectively. C3a and C5a bind to cell surface receptors expressed on immune and non-immune cells to induce cytokine production and immune cell recruitment. The MAC is a complex formed on plasma membranes by complement components 5b, 6, 7, 8 and 9 as a result of opsonized antigens. Low levels of the MAC on target cells activate intracellular signaling pathways and high levels induce lysis. Sublytic levels of the MAC amplify in ammatory intracellular signaling pathways by activating the NFkB signaling and in ammasome pathways (9,10). Due to the potentially damaging role of in ammation in glaucoma and other neurodegenerative disorders, the anaphylatoxins and the MAC are predicted to be useful targets for developing anti-in ammatory therapies (11,12).
Research in animal models suggests that the complement cascade contributes to pathology in ocular hypertensive eyes (4,(13)(14)(15)(16)(17)(18)(19)(20). This includesmodels of glaucoma like DBA/2J mice, who develop an ocular hypertensive disease in which the complement component 1q complex(C1q)or C5exacerbates neuroin ammation, retinal ganglion cell loss and optic nerve degeneration (20)(21)(22)(23). These data further support the need to determine the function of pro-in ammatory products of the complement cascade afteran ocular hypertensive insult.To test the function of an anaphylatoxin in a chronic, age-related model of glaucoma, we backcrossed a null allele of the C3a receptor (C3ar1 -) into DBA/2J mice. C3AR1 is a Gprotein coupled receptorexpressed in cells in the nervous and immune systems (see review: (24)) and is implicated in neuropathology in several diseases (25)(26)(27)(28)(29). In DBA/2J mice, C3ar1-de ciency decreasedthe incidence of optic nerve damage and RGC loss at a time point consistent with the C3a anaphylatoxin promotingneurodegeneration.

Methods
Animals and husbandry. C.129S4-C3ar1 tm1Cge /J(C3ar1 -) mice were obtained from The Jackson Laboratory (Bar Harbor, ME, USA; stock number 005712) (30). The C3ar1 null allele was backcrossed onto DBA/2J (D2) for 10 generations to generate the congenic strain D2.129S4(C)-C3ar1 tm1Cge /Sj. Experimental cohorts of mice were produced by intercrossing heterozygous (C3ar1 +/-) mice. Mice of both sexes were used, with approximately equal numbers for each age group and genotype. Mice were housed with a 14-hour-light/10-hour-dark cycle as previously described (31). All animals were treated according to the guidelines of the Association for Research in Vision and Ophthalmology for use of animals in research. The Animal Care and Use Committee of The Jackson Laboratory approved all experimental procedures.
Clinical assessment. Assessment of iris disease was performed using a slit-lamp biomicroscope as previously reported (32) and mice were assessed every two months beginning at 6 months of age. IOP was measured by the microneedle method while mice were under anesthesia (ketamine/xylazine) (33,34). Mice were assessed every two months beginning at 8 months of age. Iris disease and IOP data were collected for at least 40 eyes of each age and genotype.
Optic nerve damage. Damaged axons stain darkly when treated with the sensitive chemical marker paraphenylenediamine (PPD) (35). We assessed optic nerve damage by staining cross-sections of the retro-orbital optic nerve with PPD. Two masked investigators assigned each optic nerve one of three damage levels: no or early (NOE; no readily detectible axon loss), moderate (MOD; less than 50% of axons damaged/lost), and severe (SEV; more than 50% of axons damaged/lost). This method of evaluating optic nerve damage has been carefully validated by counting axons (20,(36)(37)(38)(39). Glaucomatous axon damage was assessed in 10.5 and 12-month-old C3ar1 +/+ and C3ar1 -/mice (55 nerves for each age and genotype).
RGC soma loss. Eyes were xed overnight in 4% paraformaldehyde. Retinas were dissected, at-mounted, and Nissl-stained with cresyl violet as previously described (38). Images of 40x elds of the RGC layer were obtained using a Zeiss AxioImager. To account for regional variation in RGC density, two 40x elds were counted in each retinal quadrant equidistant to the periphery. The counts in the eight elds were averaged to obtain a single count per eye. Eight eyes were counted per optic nerve damage level and genotype. It is important to note that the RGC layer consists of roughly 50% RGCs. This limits the extent of total neuron loss measured because only RGCs die in standard DBA/2J mice. Loss of RGCs by Nisslstaining correlates well with loss of RGCs by axon count in optic nerves with severe damage (20,(36)(37)(38)(39).
Pattern electroretinography. PERG was performed as previously described (40). Brie y, mice were anaesthetized using ketamine / xylazine (34) and their body temperature was maintained at 37 °C. Eyes were stimulated asynchronously by contrast-reversal of gratings (0.05 cycles/degree, 100% contrast) generated on LED tablets. PERG signals were acquired using subcutaneous needles placed in the snout. Waveforms were determined using the average of three consecutive repetitions.
RNA isolation from cultured astrocytes and microglia. Primary mixed cortical cultures of microglia and astrocytes from three-day-old pups were generated and microglia were uorescently labeled and sorted as previously described (17). In brief, seventeen days after plating, cultures were dissociated (HyClone Trypsin .25%; Thermoscienti c) and resuspended in FACS buffer: HBSS (Gibco; Invitrogen 14025) supplemented with 2% BSA (Sigma-Aldrich, A7906) and containing 1U/ulSUPERaseIn™ RNase Inhibitor (Ambion; Life Technologies, AM2694). Cells were centrifuged at 1305g for 5 minutes and suspended in 50uL of fresh FACS buffer to wash. The cells were stained for 1 hour at 4°Cwith chicken anti-GFAP (Abcam, ab4674) to label astrocytes and rabbit anti-IBA1 (Wako, 016-20001) to label microglial cells.
RNA-sequencing and analysis of differentially expressed (DE) genes. The steps taken to produce sequencing libraries have been previously reported (17). In brief, starting with 5ng of high quality RNA, sequencing libraries were constructed using Ovation RNA-Seq V2 and TruSeq DNA sample prep kit v2 kits. Libraries were sequenced on a HiSeq 2000 sequencer from Illumina. Reads with 70% of their bases having a base quality score ≥ 30 were retained for further analysis. Read alignment and expression estimation were performed using TopHat v 2.0.7 (41) and HTSeq (42)with default parameters against mouse genome (build-mm10). DE genes between groups were identi ed using edgeR (v 3.8.5) (43)following the removal of lowly expressed genes (counts per million < 1 in more than two samples).
The DE gene set was analyzed using Ingenuity Pathway Analysis (IPA) software. Results for enrichment of IPA canonical pathways and upstream regulator terms are shown.

Myeloid-derived cell counting by ow cytometry
Mice were euthanized and eyes were immediately enucleated. Retinas, optic nerves, and spleens were dissected in ice-cold, lter sterilized HBSS (Gibco; 14175-095) and placed in HBSS with dispase (5 U/ml) (Stemcell Technologies), DNase I (2000 U/ml) (Worthington Biochemical) and SUPERase (1 U/μl) (ThermoFisher Scienti c). The tissues were shaken at 350 rpm for 60 minutes at 37°C in an Eppendorf Thermomixer R and then titrated with a 200 ul pipette to dissociate cells. Cells were centrifuged at ~3000 g for 5 minutes and suspended in a new solution by titration. Ovomucoid trypsin inhibitors (10 mg/ml) were added to the 2% BSA in HBSS block solution to inhibit proteases. Samples were kept on ice and protected from light for blocking and antibody incubations. Primary antibody solution contained anti-Cd11b, anti-CD45, anti-Cd11c, and DAPI. Samples were blocked for 1 hour, incubated with primary antibodies in block solution for 2 hours, washed 3x, incubated in secondary antibodies for 1 hour, washed 3x and then suspended in block solution for ow cytometry on BD Biosciences LSR II SORP. Tissue collected from the spleen and processed the same was used to guide analysis of the myeloid cell populations.
Statistics.Comparisons of mean IOP levels, RGC layer neuron counts, PERG amplitudes, and myeloid cell population numbers were comparisons between C3ar1 -/and C3ar +/+ mice at each age shown and performed using Student's t-tests. Each assay involved multiple comparisons and P < 0.01 was considered signi cant. Fisher's exact test of independence was used to compare the number of nerves at each grade level at a speci c age between C3ar1 -/and C3ar +/+ mice. P < 0.01 was considered signi cant.DE genes from RNA sequencing experiments were adjusted for multiple testing using FDR.
Genes were considered to be differentially expressed between C3ar1 -/and C3ar1 +/+ at FDR < 0.01. Ingenuity pathway analysis software was used to assess enrichment of terms (canonical pathways and upstream regulators) by DE genes. Benjamin-Hochberg adjusted P-values < 0.05 were considered signi cant. The complete list of genes detected by RNA sequencing was used as the background gene list. Expression data and analyses are provided in Tables 1-3.

Results
C3ar1de cient 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 de ciency 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 de cient mice, high IOP su cient 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)(37)(38)(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, signi cantly fewer eyes fromC3ar1 -/mice had degeneration( Figure 2B) suggesting thatC3ar1 de ciency decreased the risk of ocular hypertensive injury.By 12 months of age, C3ar1 de cient 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 de ciency 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 de ciency 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 de ciency 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)(50)(51)). In addition, higher levels of expression have beenobserved in subsets of microglia thought to mediate neuroin ammation, 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 speci c data from DBA/2J mice. ONH microglia and in ltrating monocytes express C3ar1at high levels, whileRGCs expressC3ar1 at a lower level ( Figure 4D, (54, 55)). Thus, C3ar1de ciency in microglia and monocytes may affect their function or number in the ONH of ocular hypertensive eyes based on these expression data.
C3ar1 de ciency altered the in ammatory phenotype of cultured microglia.
To determine how C3ar1 de ciencymay 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 neuroin ammatory genes expressed in the optic nerve head of DBA/2J mice, including C3 (17). Microglia were identi ed by uorescence-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 toC3ar1de ciency ( Figure 4C; N=6, FDR<0.005).
The biological pathways most signi cantly 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 ve genes incommon,suggested thatmost pathways were closely interrelated and relevant to neuroin ammation and immune cellrecruitment ( Figure 5B). Thus, thepathways altered by C3ar1 de ciencyregulate homeostatic and pathological responses in microglia and other immune cells. Upstream regulators of DE genes were analyzedto determine how C3ar1 de ciency may have this effect( Figure 5C). The most signi cantly enriched upstream regulators were 'TCL1A','IL10', and 'LDLR'. The endogenous regulator thathad the highest interconnectivity was the anti-in ammatory cytokine IL10 ( Figure 5D). In addition, the predicted regulator associated with the most DE genes was dexamethasone, a corticosteroid that prevents in ammation. These data show that C3ar1 de ciencysigni cantlyaltered the expression of in ammatory genes and signaling pathways in microglia.
C3ar1 de ciency altered myeloid cell populations in the optic nerve head.
In C3ar1 de cient DBA/2J mice, decreased anaphylatoxin signaling and other changes in in ammatory 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 ow 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 CD45 hi and Cd11c + monocytes ( Figure 6B). Thus, C3ar1 de ciency 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 bene cial 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.

Discussion
Interventions that targetcomplement activation are being evaluated in many types of neurological injury and disease (reviewed in (57).DBA/2J mice are a useful model for testingwhetherneurodegeneration caused by chronic ocular hypertension is prevented by targeting speci c components of the complement cascade. DBA/2J mice have an inborn de ciency in C5 that prevents the formation of both C5a and the MAC (which requires C5b). Therefore, optic nerve damage in these mice is independent of C5, which has been shown to be detrimental if present (21). However, optic nerve damage is still dependent on C1q based on the protection against ocular hypertension observed in C1qa-/mice (37). To determine how else C1q causes permanent damage and vision loss, there are a limited number of targetsremaining to investigate, such as C3, C4, and receptors for C1q.Disrupting C3ar1 tested a function of C3 and the importance of anaphylatoxin signaling. In fact, in the absence of C5a, C3AR1 is the only anaphylatoxin receptor with a potential ligand in DBA/2J mice.C3ar1 de ciencydecreased the percentage of mice with optic nerve damage at 10.5 months of age, providing evidence that anaphylatoxin signaling is a damaging form of neuroin ammation in a setting with elevated IOP.
Understanding why C3ar1 de ciency did not provide long-lasting protectionrequires understandingotherdamaging consequences of complement activation.Greater protection in DBA/2J mice has been achieved by disruptingC1qa(37)compared to C3ar1, suggesting that C1qa triggers multiple damaging responses. A therapy targeting sites opsonized by C3b and C4b, achieved by expression of CR2-Crry in retinal ganglion cells, has produced results more similar to C1qa de ciency (14).Crrywould be predicted to inhibit C3 convertase activity of the classical pathway (through C4b) and alternative pathway (through C3b) (58),severely limiting accumulation of both C3a and C3b in the treated DBA/2J mice. Theresults of treatment with CR2-Crry suggest that inhibition of C3a and C3b protects in an additive manner. DBA/2J mice that lack the C3b receptor CR3, by disruption ofItgam, are less vulnerable (54),butnot protected as well as C1qa -/and CR2-Crry treated mice.Thus, targeting C3ar1 and Itgamtogether mayprotect to a greater degree than targeting C3ar1 or Itgam alone and explain the effect of treatment with CR2-Crry or disrupting C1q.
Complement activation is primarily expected to guide a targeted immune cell response in DBA/2J mice, given that they lack C5. The microglial response to C1q and C3aenhances phagocytosis, promoting removal of neuronal blebs or dying neurons and limitingpro-in ammatory cytokine production (59)(60)(61).For C1q, this includes regulating dendritic and synaptic pruning during developmentand ocular hypertension in the retina (22,62).C3ar1 has also been implicated inmediatingsynaptic plasticity (25) and phagocytosis by microglia (63) in cell culture and a mouse model relevant to Alzheimer's disease. However, C3ar1 de ciency did not in uence PERG readings that occur in conjunction with synapse loss and dendritic remodeling. It is possible that C3AR1 signaling does not stronglyaffect microglial phagocytosis or microglia-mediated synapse loss in an ocular hypertensive setting and that C3AR1 signaling likely has detrimental effects in glaucoma through a different mechanism.
The biological pathways affected by C3ar1 de ciency in cultured microglia were neuroin ammatory pathways as opposed to more general cellular processes. This is consistent with microglia being the primary resident immune cells responsive to C3a in the central nervous system. The IL10 pathway altered in C3ar1 de cient microglia isan anti-in ammatory signaling pathway. In addition, many genes associated with general microglial reactivity were upregulated. Disrupting C3ar1 may have increased antiin ammatory signaling, prevented damaging in ammation, or both. C3a may also recruit monocytes that express C3ar1. A subclass of monocytes (CD11b-positive, CD45-hi, and Cd11c-positive) that express C3ar1increase in number in tissue affected by ocular hypertension (20), but how they are recruited is not known. C3a may in uence their recruitment based on ow cytometry data presented here, although this is unresolved.In some eyes from C3ar1 -/mice, the number of myeloid cells in the optic nerve head appeared to be increased as observed by ow cytometry. It is possible that myeloid cells have a protective role early in disease and that this increase helped prevent optic nerve damage. In this study, it was not feasible to thoroughly address these possibilities in more depth due to the spontaneous nature of IOP elevation, variability between eyes, and the unexpected increase in myeloid cell population variability in the ONH of C3ar1 -/mice. A larger study using DBA/2J mice or another model with chronic ocular hypertension could address how C3ar1 alters microglia and monocyte localization and function in this type of glaucoma. All of the hypotheses are consistent with the idea that targeting myeloid cells with therapy may improve disease outcomes in glaucoma.
In DBA/2J mice, ONH astrocytes express C3 (17), a marker associated with a neurotoxic phenotype in some conditions(64). However,C3de ciency was shown to increase vulnerability of the optic nerve to ocular hypertensive damage (17). This is counterintuitive to harmful effects of C3a and C3b was suggested to implicate early protective responses by astrocytes in glaucoma. The role of C3 in neuroprotective and neurotoxic functions of astrocytes needs to bedetermined.Astrocytes in DBA/2J mice may be capable of both neuroprotective and neurotoxic function that depends on the activation of speci c extracellular receptors. In this case, C3ar1 de ciency may protect bydecreasing the extracellular signals produced bymicroglia and in ltrating monocytes, including C1Q, IL1A, and TNF (64), that trigger the neurotoxic response.Testing the function of C1q receptors and C3 in astrocytes in DBA/2J micecould betterde ne the effects of complement activation and show whetherastrocytesdirectly contribute to optic nerve degeneration.

Conclusion
Anaphylatoxin signaling through C3AR1 promoted neurodegenerative processes in a model of glaucoma with chronic ocular hypertension and neuroin ammation. C3ar1de ciency caused changes toIL10 related signaling pathways in microglia, pathways predicted to have an important effect on microglia reactivity. In this regard, genetic and other factors that in uence expression of C3ar1, C3, or other members of the complement cascade may predispose people to bene cial or damaging neuroin ammatory responses by affecting microglial or astrocytic reactivity. Targeting myeloid cells and anaphylatoxin signaling with therapies will likely be a bene cial addition to neuroprotective therapeutic strategies.

Consent for publication
Not applicable.

Availability of data and materials
The datasets during and/or analysed during the current study available from the corresponding author on reasonable request.

Competing Interests
The authors declare that they have no competing interests.  with edges representing that more than 5 genes were shared between two pathways. This network identi ed that the pathways shown in A had common biological function related to neuroin ammation (salmon) and immune cell activation (yellow). (C) Top 20 upstream regulatorsin IPA rankedby P value for enrichment in regulating genes differentially expressed between C3ar1-/-and C3ar1+/+ microglia. (D) A network of upstream regulators was generated with edges representing that more than 5 genes were shared between two upstream regulators. Only endogenous upstream regulators were included in the network. IL10had the most connections to other upstream regulators (thick gray edges) and is a potential driver of changes associated with C3ar1 de ciency.

Supplementary Files
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