Single-cell RNAseq analysis for retinal CD45+CD11b+ cells 0, 1, 4 and 7 days after ocular injury.

(A) Principal component analysis shows the existence of 4 clusters with distinct transcriptional profiles assigned to the day of cell retrieval. CD45+CD11b+ cells undergo extensive changes in their transcriptome during retinal engraftment and by day 7 acquire an identical signature to naive microglia. (B) Singlec1+ gene is expressed in clusters 3 and 4, both assigned to day 1, suggestive of the presence of monocytes within the microglia sample. (C) tSNE analysis of cx3cr1, ccr2, p2ry12, tmem119, aif1, and fcrls expression in cluster 4 and (D) graphical representation of the expression of microglia markers p2ry12 tmem119, AIF1, and fcrls in the 4 clusters, including those representatives of microglia and monocytes signatures. (E) Flow cytometric analysis of the expression of CD45 marker in retinal CX3CR1+ cells before and 1, 4, and 7 days after injury. A transgenic CX3CR1+/EGFP mouse was used to show the expected CD45 profile of pure microglia from a non-chimera and without injury, while the BMT panels show the evolution in the experimental model. As reference we used a CX3CR1+/EFGP mouse, stained with CD45 markers. Double positive CD45+ CX3CR1+ cells represent only microglia, since naïve eyes do not have infiltration of monocytes[2]. To map infiltrating/engrafting monocytes, we used a CX3CR1+/EGFP bone marrow chimera. CX3CR1+ CD45+ infiltrating monocytes gradually transitioned their CD45 expression towards the expression of naïve microglia at 7 days.

Protein expression of P2RY12, TMEM119 and FCRLS by engrafted monocytes.

(A) CX3CR1+/EGFP::CCR2+/RFP bone marrow chimeras were used to differentiate engrafted monocytes from embryonic microglia, followed by immunostaining and flow cytometry to assess expression of P2RY12, TMEM119, and FCRLS proteins. Engrafted monocytes were recognized as EGFP+ RFP+ cells while embryonic microglia were EGFP− RFP− cells. (B) P2RY12 is not expressed at day 1 and day 7 after monocyte infiltration into the retina, however 55% of EGFP+ engrafted monocytes showed positive expression of P2RY12 at day 45. ***, P < 0.001. (C) Twenty-five percent of EGFP+ peripheral monocytes (white arrow) expressed TMEM119 at day 1. By day 7, all GFP+ engrafted monocytes are TMEM119+. TMEM119 expression in engrafted monocytes is retained at day 45. (B, C) Data displayed as Mean ± SEM, independent t test **P < 0.01; ***P < 0.001. Each dot represents 1 animal. Minimum of 3 animals per group. Yellow arrows indicate EGFP− microglia, white arrows GFP+ engrafted monocytes. Scare bar = 50 µm. (D-E) A CX3CR1+/EGFP::CCR2+/RFP bone marrow chimera was employed to assess FCRLS expression in monocytes and microglia by flow cytometry. BMT CX3CR1+ cells were labeled with a conjugated antibody against CX3CR1 (APC) which allowed differentiation between embryonic microglia (APC+ EGFP−) and engrafted monocytes (APC+ EGFP+). Blood monocytes had no FCRLS expression (grey). One day after infiltration, APC+ EGFP+ peripheral monocytes (purple) acquired strong FCRLS expression, which was comparable to naïve embryonic microglia. FCRLS expression was retained by engrafted monocytes sustained at day 7. At day 45, FCRLS expression was similar to embryonic microglia in the same injured tissue or to naïve microglia. Mean ± SEM, independent t-test,*P < 0.05. Each dot represents 1 anima. Minimum of 3 animals per group. MFI: Median Fluorescence Intensity, Mφ: macrophages, μG: microglia.

ATAC-seq analysis to assess chromatin accessibility for p2ry12, fcrls, aif1 (iba1), and tmem119 genes in engrafted monocytes and microglia

In ATAC seq, peak width refers to the horizontal extent of a peak in the ATAC-seq signal track. It represents the range over which chromatin accessibility is elevated. A wider peak indicates a broader region of accessible chromatin. Peak amplitude refers to the height or intensity of the peak in the ATAC-seq signal track. It represents the level of chromatin accessibility at the peak’s center. Higher amplitude suggests more frequent chromatin accessibility in that region. In the current study, circulating monocytes and naïve microglia were used as controls. Groups and color coding are listed in engrafted monocytes showed similar open chromatin peaks to circulating monocytes (blue arrows) but also displayed new open chromatin peaks not previously present in circulating monocytes (red arrows) but present in native (embryonic) microglia. One of the peaks under the p2ry12 gene in naïve microglia (red arrow) was absent in circulating monocytes but acquired upon engraftment into the retina (Fig. 3 B). The fcrls gene had similar open chromatin peak across all groups (Fig. 3 C), though naïve microglia had wider and higher peak as compared to circulating monocytes (blue peak compared to grey). After engraftment, the amplitude of this peak increased in monocytes and became similar to microglia at day 45 (blue peak compared to purple), (Fig. 3 C). Similarly, tmem119 chromatin was not accessible in circulating monocytes, however, it became accessible after monocyte engraftment into the retina (red arrow), (Fig. 3 D). Lastly, AIF1 gene (IBA1) had similar open chromatin peaks among the groups, corroborating the above transcriptional and protein findings showing gain of IBA1 expression by monocytes after engraftment into the retina (Fig. 3 E). (A) Color coding of analyzed groups. (B) p2ry12 gene contains 3 open chromatin peaks, 2 of the peaks (blue arrowhead) are similar between the groups, but the 3rd peak is present in microglia (red arrowhead) but not in circulating monocytes. Upon engraftment into the retina, monocytes acquire the 3rd peak (red arrowhead) which is retained throughout the study period (45 days). (C) Open chromatin peaks for fcrls gene appear similar between the groups, with differences only in the amplitude of the peaks at 45 days in microglia and engrafted monocytes which have higher peaks compared to circulating monocytes or monocytes during early engraftment into the retina (7 days). (D) Open chromatin peaks for aif1 gene (IBA1) appear similar between the groups, although microglia appeared to abolish one peak (red arrowhead) at 7 and 45 days after the injury. (E) tmem119 has only one open chromatin peak, which is present in microglia but not in circulating monocytes, but upon engraftment, peripheral monocytes acquire this distinct peak (red arrowhead). (F) Heat map analysis of consensus peaks. Chromatin accessibility is scaled by row (Z-score) to emphasize relative differences across samples. Each row represents a genomic peak, and each column represents a biological replicate. Blood monocyte, n=6; Engrafted Μφ, n=5, including 3 samples of 7 days post injury and 2 samples of 45 days post injury; Naive μG, n=4; Injured μG, n=5, including 2 samples of 7 days post injury and 3 samples of 45 days post injury. The analysis shows engrafted monocytes at day 7 and 45 cluster more closely with microglia than with circulating blood monocytes, visually confirming a shift in the epigenetic landscape. Monocytes undergo significant open chromatin alterations upon engraftment into the retina which enables differentiation from circulating monocytes to tissue-phagocytes. Monocytes increase chromatin accessible for genes p2ry12, tmem119, fcrls, and aif1 upon engraftment into the retina eventually acquiring a similar open chromatin signature to microglia.

ATAC-seq motif analysis for discovery of putative transcription factors regulating monocyte engraftment.

Motif analysis of differential open chromatin peaks identified by comparing naive microglia, retinal engrafted monocyte (7 and 45 days) compared to circulating monocytes. Blue section contains motifs assigned to transcription factors previously identified in human and mouse microglia, such as PU.1 (most common), CTCF, IRF, RUNX, MEF2, C/EBP, AP-1, MAF, and MEF. Green section contains enriched motifs assigned to novel transcription factors, such as STAT1, FOXN1, KLFs, ATF3, and Npas4. Red section contains previously reported disease associated motifs, such as MITF and NFKB1. Analysis of naive microglia identifies multiple reported factors but not MAF and MEF, which are identified only in engrafted monocytes. Engrafted monocyte at 7 and 45 days contain highly enriched motifs assigned to the above-mentioned transcription factors CEBP, IRF2, and ATF3. Disease-associated motifs assigned to MITF and NFKB1 are identified in engrafted monocytes but not in microglia. E-value <0.05 for statistically significant motifs.

Expression of conventional markers by microglia and engrafted monocytes.

(A) Development of a CX3CR1+/EGFP::CCR2+/RFP bone marrow chimera model to differentiate microglia from peripheral monocytes following flow cytometry. Microglia: EGFP− CX3CR1-APC+, engrafted monocytes: EGFP+CX3CR1-APC+. (B) Peripheral monocyte/macrophages repress CCR2 expression and enhance CX3CR1 expression during engraftment into the retina. Five distinct maturation phases of monocytes after engraftment are identified (Groups 1-5). A separate group of CCR−CX3CR1− cells, representing embryonic microglia (Group 6; µG), is retained throughout the study period (45 days). (C-D) Engrafted monocytes have increase expression of Ly6C at day 1 of infiltration, which is gradually suppressed during engraftment. At 45 days, the majority of engrafted monocytes (Group 5) have similar Ly6G expression as microglia (Group 6). ns, not significant, independent t test, ***P < 0.001. Each dot represents 1 animal. Minimum of 8 animals per group. (E-F) Engrafted monocytes exhibit sustained expression of CD45 at days 1 and 7 followed by repression in subpopulations of these cells (Group 5), and at day 45 reaching equal levels compared to retinal microglia (Group 6). Independent t test, **P < 0.01, ***P < 0.001. Each dot represents 1 animal. Minimum of 8 animals per group. (G) A clear population of CD45high and CD45int is present at day 1 and 7 after injury, but not at day 45 due to CD45 repression in engrafted monocytes.

Summary of the microglia and monocyte signature

Monocyte transition from highly amoeboid to highly ramified cells during engraftment into the retina, becoming morphometrically identical and indistinguishable from retinal microglia. These changes are accompanied by suppression of monocyte markers CCR2, Ly6C, and CD45, and upregulation of the tissue-resident macrophage marker CX3CR1+/GFP and microglia markers IBA1, TMEM119, P2RY12, and FCRLS. P2RY12 appears to be conditionally specific to microglia during early infiltration of monocytes) and to a subpopulation of engrafted monocytes.

Antibody information for flow Cytometry and immunostaining.

Both P2RY12 antibodies worked successfully in our experimental investigations.