CCL2 and ACKR2 transcripts are upregulated following human brain injury.

<p>(A-B) Levels of CCL2 (A) and ACKR2 (B) transcripts were assessed by qPCR in brain tissue from TBI patients that had died < 17 minutes (acute; n = 9), < 3 h (early; n = 8), or > 6 h (late; n = 6) after injury, and in patients whose cause of death was not brain injury (control; n = 8). ANOVA reported a significant increase in CCL2 expression in the late group versus the acute group (* = p < 0.05) and in the late contralateral (late CL; dotted boxes) group versus the control group (# = p < 0.05), and no significant effect of time on ACKR2 transcript expression. (C) Correlation of ACKR2 transcript levels with time.</p

Neurological severity score (NSS).

<p>Neurological severity score (NSS).</p

Transient increase in brain lesion volumes in ACKR2^-/- after CHI.

<p>(A) Brain lesion volume was assessed by H&E staining at day 1 (n = 8), 3 (n = 9), and 7 (n = 5) after CHI. ACKR2^-/- mice (white columns) were found to have significantly larger lesions as compared to WT mice (black columns) at day 1 post-CHI (* = p < 0.05). No significant differences were observed at later time points. ANOVA did not detect any significant effect of genotype. (B) Representative low and high magnification images of an ACKR2^-/- mouse brain at 24 h post-CHI.</p

Skull morphometric analysis in WT and ACKR2^-/- mice.

<p>Skull morphometric analysis in WT and ACKR2^-/- mice.</p

Functional recovery is similar in WT and ACKR2^-/- mice over 7 days post-CHI.

<p>Neurological testing was conducted for four groups of mice at indicated time points following CHI or sham surgery. WT mice (full lines) received CHI (black triangles; n = 5) or sham surgery (black dots; n = 3), ACKR2^-/- mice (dotted lines) were also assigned to either CHI (white triangles; n = 7) or sham surgery (white dots; n = 4). Sham animals did not show any significant sign of neurological deficit. ANOVA confirmed a significant effect of time-point on NSS in CHI animals, but did not detect difference between genotypes in functional recovery.</p

Macrophage recruitment does not differ in ACKR2^-/- and WT mice.

<p>(A-B) CCL2 transcript (A) and protein (B) levels were assessed in WT (black columns) and ACKR2^-/- mice (white columns) at indicated time points after CHI (n = 4–10), or 2 h after sham surgery (n = 4). CCL2 transcript and protein levels were significantly upregulated at all time-points assessed compared to sham levels (p < 0.01 at all time points). AVOVA detected no difference between genotypes, but multiple comparisons revealed a significant reduction in CCL2 levels between 12 and 24 h in WT (p < 0.05) but not ACKR2^-/- mice. (C) The volume of F4/80-positive cells was measured in the injured cortex at indicated time points. ANOVA detected no difference between genotypes at any time point analysed. (D) Accumulation of activated, F4/80-positive macrophages and microglia at 3 days post-CHI shown at low power and higher magnification (inlet). There are macrophages with a round, amoeboid morphology and activated microglia, with typical processes extending from the cell body (scale bar = 500 μm).</p

Analysis of skeletal bone and skull does not reveal overt differences in bone morphology between WT and ACKR2^-/- mice.

<p>(A) Representative total body (left) and cranium (right) X-ray images of WT and ACKR2^-/- mice. (B) Schematic representation of the skull region analysed by histology in the CHI model (red rectangle). The black arrow indicates the bregma, the red arrow the direction of cut. Representative H&E images of the skull bone histology in WT and ACKR2^-/<i>-</i> mice are shown on the right. Scale bar: upper panels, 500 μm; lower panels, 200 μm. (C) Representative H&E images of long bones histology in WT and ACKR2^-/ mice. Scale bar: 500 μm.</p

Regulation of ACKR2 and CCL2 following CHI and in <i>ex vivo</i> inflamed astrocytes.

<p>(A) ACKR2 transcript level was detected by qPCR in brain tissue of WT animals at indicated time points after CHI. Animal numbers per time point: WT sham n = 10; WT CHI 2h n = 7; WT CHI 4h n = 13; WT CHI 24h n = 6. (B) Following LPS challenge there was no change in ACKR2 expression in cultured astrocytes from WT (black columns) or CCL2^-/- (white columns) animals across the time points, although post-hoc analysis detected a significant difference in ACKR2 expression at 2 h (* = p < 0.05), with higher levels in WT compared to CCL2^-/- astrocytes. (C) CCL2 transcript level increased significantly with time in both WT (black columns) and ACKR2^-/- (white columns) astrocytes, with peak expression seen 4 h after LPS challenge. At all time points the expression of CCL2 was lower in ACKR2^-/- astrocytes as compared to WT cells. * = p < 0.05.</p