Effect of CCR2 drug inhibition on sm-EAN-associated inflammatory demyelination.

<p>Representative digital photomicrographs of toluidine-blue stained, 1 μm semi-thin plastic embedded axial sections of mouse sciatic nerves show multiple foci of demyelinated axons associated with infiltrated mononuclear cells (double white asterisk) in vehicle controls (A), compared to the preserved myelin architecture and lack of inflammation seen in CCR2INB treated mice (B). Clusters of thinly myelinated axons (double black asterisk) surrounded by large myelinated axons, suggestive of recent multifocal axonal injury and repair are commonly seen in human IVIg-treated mice, associated with scattered foci of mononuclear cells (black arrows; C). Higher magnification digital photomicrographs further demonstrate the mononuclear cell-mediated demyelination in Vehicle-treated controls as expected in untreated sm-EAN (white arrows). Active leukocyte extravasation at an endoneurial microvessel (EMV) is also seen (D). Foci of mononuclear cells (white arrow) are rarely seen in CCR2INB treated (white arrow; E); these are more common in human IVIg treated mice (black arrows; F). Scale bar  =  100 μm (A–C), 50 μm (D–F).</p

Effect of CCR2 gene deletion on BPNM-induced splenocyte proliferation <i>in vitro</i>.

<p>Bar histographs depicting the mean absorbance of cultured splenocytes (optical density at 485 nm minus optical density at 595 nm; directly proportional to the number of viable cells) following treatment with bovine peripheral nerve myelin (BPNM) or phytohemagglutinin (PHA) show no significant increases in mean absolute proliferation in response to BPNM irrespective of CCR2 genotype compared to basal culture conditions (A–D). Significantly increased splenocyte proliferation to PHA verifies splenocyte viability and proliferative capacity (A–C). Deletion of one or both CCR2 gene alleles is associated with a small but significant relative increase in mean BPNM-induced splenocyte proliferation relative to basal proliferation on a mouse-per-mouse basis <i>in vitro</i>, with no significant difference observed between CCR2HT and CCR2KO mice (E). N = 9 mice per genotype, with each assay performed in triplicate. * indicates p<0.05, N.S. not significant.</p

Effect of CCR2 gene deletion on Toll-like receptor (TLR) expression at expected sm-EAN onset.

<p>Representative merged digital indirect fluorescent immunohistochemistry photomicrographs of 10 μm frozen acetone fixed, axial sciatic nerve sections stained to detect galactocerebroside (GCB; Schwann cell and myelin marker; red) and TLR2 (A–D; green) or TLR4 (E–H; green) demonstrate increased TLR expression co-localizing with Schwann cells (white arrows: yellow/orange immunoreactivity) in all CCR2 genotypes at sm-EAN onset, prior to significant mononuclear cell infiltration, compared to control mice not affected with sm-EAN. TLR2 or TLR4 expression may be multifocal (B and G), focally diffuse (D and F) or honeycomb (C and H) in early sm-EAN. Representative digital autoradiographs following western blot of sciatic nerve protein homogenates demonstrate similar TLR2 (I) and TLR4 (K) expression for all CCR2 genotypes, using β-actin as an internal protein loading control. Molecular weights in KDa are in brackets. No statistically significant differences are seen in TLR2 or TLR4 expression between the CCR2 genotypes based on semi-quantitative spot densitometry analyses relative to β-actin (J and L). N = 3 mice per genotype, with assays performed in duplicate experiments. WT =  wild type, HT = heterozygote, KO = knockout; N.S. not significant</p

PCR verification of CCR2 genotype.

<p>Representative digital images of ethidium bromide-stained agarose gels of resolved tail snip genomic DNA (A and B) and splenocyte cDNA (C and D) demonstrate mouse CCR2 genotype. Mice that lack CCR2 genomic DNA (A) and express the neomycin cassette (B) are knockout (KO), while wild type (WT) mice express CCR2 without the neomycin cassette. Heterozygote (HT) mice express both. KO mouse splenocytes do not express CCR2 cDNA, in contrast to HT and WT mice (C). GAPDH serves as an internal loading control and housekeeping gene for splenocyte cDNA loading (D).</p

Effect of CCR2 drug inhibition on the behavioral features of sm-EAN.

<p>Treatment with RS 102895, a specific CCR2 inhibitor (CCR2INB; N = 9) from days 13–17 post-induction (early effector phase) results in rapid and persistent improvement in muscle strength supported by significantly lower mean NMSS compared to Vehicle (N = 10) and human IVIg (N = 8) treated mice. Statistically significant differences are observed between Human IVIg and Vehicle-treated mice from day 23 post-induction (6 days after completing drug therapy) with no further worsening in human IVIg treated mice (A). The yellow bar indicates the drug treatment phase, * indicates p<0.05 relative to both Vehicle and IVIg treated mice, # indicates p<0.05 comparing human IVIg and Vehicle treated mice. Bar histographs summarizing mean motor electrophysiology from the bilateral dorsal caudal tail (DCTN; B–D) and sciatic (ScN; E–G) nerves obtained from each mouse at expected maximal severity demonstrate significantly higher conduction velocities and shorter total waveform durations following CCR2INB treatment compared to Vehicle and human IVIg (hIVIg) treated mice. CCR2INB and hIVIg treatment result in higher compound motor action potential (CMAP) amplitudes than Vehicle treated controls (implying protection from axonal degeneration or distal conduction block), with no significant differences seen between the former two treatments (B and E). Despite small but significantly increased mean conduction velocities following hIVIg treatment compared to Vehicle controls (C and F), no significant differences are observed in total waveform duration (D and G) implying residual axonal dyssynchrony (demyelination or early remyelination) following IVIg treatment. * indicates p<0.05, N.S. not significant</p

Effect of CCR2 gene deletion on CCL2 expression at expected sm-EAN onset and maximal severity.

<p>Representative digital autoradiographs following western blot of sciatic nerve protein homogenates demonstrate CCL2 expression as presumed dimers (d), tetramers (t) and higher-order oligomers (o) at expected disease onset in all CCR2 genotypes, with β-actin serving as an internal protein loading control (A). CCL2 oligomerization is expected during <i>in vivo</i> secretion, and this has been demonstrated by western blot <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090463#pone.0090463-Yao1" target="_blank">[31]</a>. No statistically significant differences are seen in mean total CCL2 expression at disease onset between the CCR2 genotypes based on semi-quantitative spot densitometry analyses relative to β-actin (B). At expected maximal severity, CCL2 expression predominantly occurs as presumed tetramers (t) and higher-order oligomers (o) with loss of dimer expression in all CCR2 genotypes (C). A small, but statistically significant increase in mean total CCL2 expression is observed in CCR2HT compared to both CCR2WT and CCR2KO, with no difference observed between CCR2WT and CCR2KO mice (D). Numbers in A and C represent molecular weights in kDa. Data are obtained from three mice per genotype, in quadruplicate for expected disease onset experiments and triplicate for expected maximal severity experiments. WT =  wild type, HT = heterozygote, KO = knockout; N.S. not significant.</p

Effect of CCR2 gene deletion on sm-EAN associated inflammatory demyelination.

<p>Representative digital photomicrographs of toluidine-blue stained, 1 μm semi-thin axial plastic embedded mouse sciatic nerve sections show foci of demyelinated axons associated with infiltrated mononuclear cells (white asterisk) and intra-endoneurial edema (paler background; white arrow) in CCR2WT mice (A), with an example of more confluent demyelinated segments seen in a CCR2HT mouse (B). These are in contrast to the normal appearing honeycomb appearance of myelinated axons seen within the endoneurium of CCR2KO mice (C). Scale bar  =  100 μm. Quantitative analyses demonstrate statistically significant increase in mean total endoneurial area [associated with leukocyte infiltration and intra-endoneurial edema] (D), total demyelinated area (E) and percentage demyelinated area (F) per section in CCR2WT and CCR2HT mice compared to CCR2KO mice. No significant differences are seen between CCR2WT and CCR2HT mice. At least 30 sections separated by > 50 μm from 6 mice per genotype were analyzed. * indicates p<0.05, N.S. not significant.</p

Antibodies used for immunohistochemistry and western blot.

<p>Abbreviations: FITC: fluorescein, H+L: heavy and light chain, HRP: horseradish peroxidase, N/A: not applicable, TXRD: Texas Red.</p

Comparative mean complete blood counts and leukocyte subset differentials in treated sm-EAN mice.

<p>Numbers in brackets represent standard errors of the means. N = 10 for vehicle, 8 for CCR2 inhibitor RS 108295 and 7 for human IVIg treated mice. ∧ Indicates p-value <0.05 relative to vehicle controls.</p

Effect of CCR2 gene deletion on the histopathological features of sm-EAN.

<p>Representative digital indirect fluorescent immunohistochemistry photomicrographs of 10 μm frozen acetone fixed, axial sciatic nerve sections show foci of demyelination (white asterisk) associated with mononuclear cell infiltrates in CCR2WT (A) and CCR2HT (F) mice, compared to the normal uniform honeycomb appearance seen with S100β staining (Schwann cell and myelin marker) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090463#pone.0090463-Xia1" target="_blank">[9]</a> observed with CCR2KO mice (K). No obvious qualitative difference in axonal density (heavy chain neurofilament [NF-H] staining) is seen between the genotypes in these sections (B, G and L). Diffuse infiltration of F4/80+ monocytes/macrophages is seen in CCR2WT (C) and CCR2HT (H) mice, compared to a few foci observed in CCR2KO mice (M). Multiple foci of CD3+ T-cells and CD19+ B-cells are seen in CCR2WT (D and E) and CCR2HT (I and J) mice [white arrows], while these cells are rarely seen in CCR2KO mice (N and O). Cells are detected by the 4', 6-diamidino-2-phenylindole (DAPI) nuclear stain in all images. Scale bar  =  100 μm. Quantitative analyses show significant differences in the mean total number of mononuclear cells (DAPI+) between CCR2WT, CCR2HT and CCR2KO mice (P, S and V), with a significant step-wise reduction in mean numbers (Q) and percentages (R) of F4/80+ monocytes/macrophages with CCR2 gene allele deletion per section. Significant reduction in CD3+ T cell numbers (T) and percentages (U), as well as CD19+ B cell numbers (W) and percentages (X) per section is seen in CCR2KO relative to both CCR2WT and CCR2HT mice, with no significant differences between the latter two genotypes. At least 4 sections per mouse separated by > 30 μm were analyzed from 5 mice per genotype for each immune cell marker. * indicates p<0.05, N.S. not significant.</p