Myeloid PPARδ mutation delayed macrophage accumulation in muscle
after injury, regulated CCR2 expression and affected CCL2-mediated chemotaxis.
A-C: Cross-sections of TA muscles 3-, 7- and 15-dpi were immunolabeled with
anti-F4/80. Representative images of F4/80+ macrophages in PPARδ WT (A)
and PPARδ mutant (B) muscles at 3-dpi. Representative F4/80+ cells are
indicated by arrows. C: Numbers of F4/80+ cells per volume of sectioned tissue
at 3-, 7- and 15-dpi. D: Cross-section of TA muscles 3-dpi from
Ppard WT and mutant mice were immunolabeled with anti-F4/80
(green) and anti-Ki67 (red). Nuclei stained blue with DAPI. Arrow indicates an
example of an F4/80+ cell with nuclear Ki67. E: Proportion of F4/80+ cells
co-expressing nuclear Ki67 in PPARδ WT and PPARδ mutant muscles.
F, G: Muscle sections were co-labeled with anti-F4/80 (green) and anti-CCR2
(red). Arrows indicate examples of F4/80+ cells that are CCR2+ at 3-dpi in
PPARδ WT and PPARδ mutant muscles. Bars = 10 μm. H: Numbers
of F4/80+ cells that were CCR2+ per volume of sectioned tissue at 3-dpi in
PPARδ WT (F) and PPARδ mutant (G) muscles. H: Proportion of F4/80+
cells that were CCR2+ at 3-dpi in PPARδ WT and PPARδ mutant
muscles. I: QPCR analysis of BMDMs demonstrated that Ppard
mutation reduced Ccr2 expression. Values in each data set were
normalized to Ppard WT BMDMs and set at 1. # indicates
significant difference (P < 0.05) from Ppard WT within a
time point. P-values based on two-tailed t-test. N = 4-5 for each data set. J: A
chemotaxis assay was used to assess whether PPARδ affects macrophage
mobility or chemotaxis. CCL2 increased chemotaxis but the Ppard
mutation completely abrogated the chemotactic effect. Macrophage migration was
unaffected by PPARδ in the absence of CCL2. * indicates significant
difference (P < 0.05) from nontreated BMDMs within same genotype. #
indicates significant difference (P < 0.05) from Ppard
WT within a treatment group. P-values based ANOVA with Tukey’s multiple
comparison test. N = 6 for each data set. Data are presented as mean ±
SEM. K, L: QPCR analysis of non-injured or injured muscle at 7-dpi showed that
Ppard mutation did not affect Ccl2
expression (K) but decreased Ccr2 expression in non-injured
muscle and increased Ccr2 expression at 7-dpi (L). Values in
each data set were normalized to Ppard WT BMDMs and set at 1. #
indicates significant difference (P < 0.05) from Ppard
WT with same treatment conditions. P-values based on two-tailed t-test. N = 5
for each data set.