Fig 1: The effect of CCR2 and CX3CR1 deletion on macrophage and CD8+ T cell number, activation status and cytokine release in the nerve of L31 mice. (A) Quantitative analysis from flow cytometry showed a reduced number of macrophages (CD45+CD11b+F4/80+) in the nerve of L31/CX3CR1KO mice compared to L31 and L31/CCR2KO mice. Of note, two distinct subsets of macrophages were detected in L31 mice, which were distinguished by varying expression level of F4/80 and CD11b. Only one subset (F4/80highCD11blow) was seen in L31/CX3CR1KO and L31/CCR2KO mice, although the latter had significantly higher number. (B) The number of activated macrophages (CD86+) were significantly reduced in L31/CX3CR1KO mice. (C) Frequency and absolute number of infiltrating CD8+ T cells were significantly diminished in nerve of L31/CX3CR1KO mice. Real-time quantitative PCR showed a significantly reduced/undetectable expression of pro-inflammatory molecules (D) IFN-γ, (E) TNF-α, (F) IL-6, and (G) IL-1β, in sciatic nerve of L31/CX3CR1KO mice. (H) The mRNA level of the anti-inflammatory molecule, IL-10 was significantly enhanced in L31/CX3CR1KO mice. Disease was induced by PSNL and experiments done 30 days post PSNL. Quantification in a-c is shown as number of cells per a segment of 2 cm long sciatic nerve. n = 5-8/group; student’s t test; t*p < 0.05; **p < 0.01; ***p < 0.001.
Fig 2: Clotrimazole induced dendritic cell activation by regulating Chop expression. (A and B) DC2.4 cells were treated with clotrimazole or DMSO for 24 hours, and the expression of Chop was detected by WB (A) or qPCR (B). (C and D) DC2.4 cells were treated with clotrimazole or DMSO, then transfected with OVA (100 µg/mL) for 20 hours, then co-culture with B3Z for an additional 24 hours, after which B3Z cell activation was measured by LacZ activity and IL-2 production (C), the expression of Chop was detected by WB and qPCR (D). (E) DC2.4 cells were treated with clotrimazole or DMSO for 24 hours, after which the expression of CD86 was detected by flow cytometry and qPCR. (F) DC2.4 cells were treated with DMSO, clotrimazole, bafilomycin or clotriamzole +bafilomycin for 24 hours, after which the protein level of CHOP was detected by WB. (G) DC2.4 cells were treated with DMSO or clotrimazole for 24 hours, after which the protein level of CHOP was detected by WB. (H) DC2.4 cells were treated with DMSO, clotrimazole, lactate or clotriamzole +lactate for 24 hours, after which the protein level of CHOP was detected by WB. Data in A, C, E–H are the representative result of three repeated experiments. Data in (B, D and E) are shown as mean±SD of three replicates from one representative experiment. *p<0.05, **p<0.01, ***p<0.001, by one-way analysis with Bonferroni’s post-test. OVA, antigen ovalbumin; CLT, clotrimazole; DMSO, dimethyl sulfoxide; WB, Western blot; SCR, scramble; CHOP, C/EBP homologous protein; NT, no lactate treatment.
Fig 3: Focal inflammatory reaction appeared in the DRG and the roots of pre-symptomatic L31/CD4-/- mice, preceding the disease onset. While there were only few resident macrophages and no CD8+ T cells in the DRG and the roots of wild type mice (A), both types of immune cells were found in these two organs in pre-symptomatic L31/CD4-/- mice (B). Some mice showed a slightest degree of infiltration, presumably the earliest pathologic changes (B-upper panels), some had significant amount of both Iba1+ macrophages and CD8+ T cells with increased B7.2 expression (B-bottom panels) suggesting the status close to the disease onset (B). In symptomatic L31/CD4-/- mice, the DRG and the roots were fulfilled with infiltrated Iba1+ macrophages and CD8+ T cells. Both expressed high levels of B7.2 (C). Scale bar: 100 μm.
Fig 4: Massive infiltration of immune cells in sciatic nerves of L31/CD4-/- mice. Cell infiltration in the diseased sciatic nerves was first evidenced by the significant increase of DAPI labelled cell number (A-B). Immunohistochemistry analysis demonstrated that while there was a slight increase of elongated (insert) Iba-1+ cells in the pre-symptomatic L31/CD4-/- mice, nerves from the symptomatic L31/CD4-/- mice were submerged with round-shaped (insert) infiltrated Iba-1+ macrophages (A). While there were abundant CD8+ T cells found in the sciatic nerves of symptomatic L31 mice, no CD8+ T cell infiltration was detected in pre-symptomatic L31/CD4-/- mice (B). Quantitative analysis with FACS (n = 3/group) confirmed the immunohistochemistry observation (**: p < 0.01, ***: p < 0.001) (C). In symptomatic L31 mouse sciatic nerves, infiltrates could either group in small foci (D-left) or be distributed diffusely (A/B-right). Fragmented myelin were found within Iba-1+macrophages (yellow signals within Iba-1+ cells). There were less infiltrates in the area where myelin remained in healthy shape (D-left). Majority of infiltrated Iba-1+ macrophages had high levels of B7.2 expression (D-middle) and CD8+ T cells were in close apposition with Iba-1+ macrophages (D-right). Scale bar: 200 μm.
Fig 5: An HSD activates peripheral nerve macrophages. a Representative flow cytometry dot plots showed an increase in the frequency of peripheral nerve macrophages (CD45+CD11b+F4/80+) at 1 and 3 months of HSD feeding. b Histograms depicted the absolute macrophage (CD45+CD11b+F4/80+) number, as well as those that were CD86+ macrophages. A quantitative analysis indicated a significant increase at 1 and 3 months of HSD feeding, and the number remained elevated after shifting HSD-fed mice to a ND, n = 6–9/group. c Histograms depicted the absolute macrophage (CD45+CD11b+F4/80+) number as well as those that were CD206+ macrophages. An HSD favored the differentiation of CD86+ macrophages in the nerve more than those of CD206+ macrophages, n = 6–9/group. d A representative flow cytometry histogram showed the percentage of Ki67+macrophages (CD45+CD11b+F4/80+) in the sciatic nerves of HSD and ND fed mice. A quantitative analysis showed that HSD did not trigger significant macrophage proliferation in nerves, n = 6–8/group. e Longitudinal sections of mouse sciatic nerves were stained with CD16/32 (FcγII/III receptor), F4/80, and DAPI, showing an increased F4/80+ macrophage density in HSD nerves primarily colocalized with CD16/32. f Real-time quantitative PCR showed an elevated expression of IL-1β and IL-6 in the peripheral nerves at three months of HSD feeding, n = 6–10/group. Data were combined with from male and female mice. All data was presented as mean ± SEM and analyzed with an unpaired t test, **p < 0.01, ***p < 0.001; red and green ## represents statistics for CD86+ or CD206+ macrophages, respectively
Supplier Page from Thermo Fisher Scientific for CD86 (B7-2) Antibody PE