Figure S1. Regulatory T cells do not infiltrate the spinal cord or DRG after PSNL.
(A) Foxp3-GFP mice were subjected to PSNL, and the frequencies of viable CD45 cells and CD4 cells after the nerve digestion protocol are shown (n= 3-5).
(B) The frequency of CD4+Foxp3+ viable cells was evaluated by flow cytometry 14 days after injury (n= 3-5).
(C) Gating strategy used for Treg cell analysis of injured nerves.
(E) Gating strategy used for the analysis of spinal cord (top) and dorsal root ganglia (L3, L4, and L5; bottom) from Foxp3-GFP mice subjected to PSNL 14 days after nerve injury.
(D-G) Representative dot plots (D) and frequency of CD4+Foxp3+ cells (F) were evaluated by flow cytometry, and Foxp3 mRNA expression (G) was determined by qPCR (n=3-4).
(H-K) Representative dot plots (I) and frequency of CD4+Foxp3+ cells (J) were evaluated by flow cytometry, and Foxp3 mRNA expression (K) was determined by qPCR (n=3-4).
Data were analyzed through t-tests (D) or one-way ANOVA with Tukey’s posttest (C, G, H), and the values in the graphs are expressed as the mean ± SEM.
Figure S2. Depletion of Foxp3+ cells in the spleen of DEREG mice.
(A) Injured DEREG mice were treated with diphtheria toxin (500 ng/mouse/day, i.p.) for 2 consecutive days, and the frequency of CD4+Foxp3+ cells in the spleen was evaluated 14 days after PSNL induction (n=3-4 mice/group).
Data were analyzed through t-tests, and the values in the graphs are expressed as the mean ± SEM with ***P< 0.001 compared to vehicle-treated mice.
Figure S3. Treg cells counteract neuropathic pain. (A-B) Injured DEREG mice were treated with diphtheria toxin (500 ng/mouse/day, i.p.) for 2 consecutive days, and the paw withdrawal threshold (A) and frequency of response (B, 0.008 g filament) were evaluated in these mice before
(C) pTregs isolated from spleen and lymph nodes from Foxp3-GFP mice were transferred (1 x 106 CD4+Foxp3+ cells/ mouse, i.v. ) to injured C57BL/6 mice 5-8 h after PSNL induction (D), and the paw withdrawal threshold was evaluated in both the cell transfer and saline-injected groups up to 10 days after peripheral injury (n= 5-6).
Figure S4. Expression of Th1-, Th2- and Th17-related genes and pSTAT-1 after PSNL in the absence of Treg cells
(A- D) Bar graphs of the heatmap represented in Figure 5A. Injured DEREG mice were treated
(A, B), Th17 (C) and Th2 (D) were evaluated 14 days post PSNL by real-time PCR (n= 5- 6). (E) Western blot showing the expression of pSTAT-1 in injured sciatic nerves from DTx- and vehicle- treated DEREG mice (n=2) 7 days after PSNL. (F) Full-size membrane showing the expression of the IFNγRα subunit (top) and B-actin (bottom) in injured nerves from DTx- and vehicle-treated DEREG mice (n=3; relative to Figure 5E). (G) Full-size membrane showing the expression of pSTAT1 (top) and GAPDH (bottom) in injured nerves from DTx- and vehicle-treated DEREG mice (relative to Figure 5F). Data were analyzed through one-way ANOVA (B) and Tukey’s posttest, and the values in the graphs are expressed as the mean ± SEM with *P<0.05; **P< 0.01;
***P< 0.001 and ##P< 0.01; ###P< 0.001 compared to injured mice 7 days after PSNL.
Figure S5. The absence of Treg cells induces neuronal damage and macrophage activation in DRGs after PSNL.
(A- B) Injured DEREG mice were treated with diphtheria toxin (500 ng/mouse/day, i.p.) or vehicle for 2 consecutive days. (A) Full-size membranes relative to figure 6A showing the expression of ATF3 (A, top) and B-actin (A, bottom) in ipsilateral DRGs (L3, L4, L5, n=3). (B) Full-size membranes relative to figure 6C showing the expression of IBA-1 (B, top) and B-actin (B, bottom) in ipsilateral DRGs (L3, L4, L5) by Western blotting (n=3).
Figure S6. Treg cell depletion does not change the frequency of myeloid cells in DRGs after PSNL.
(A-B) Injured DEREG mice were treated with diphtheria toxin (500 ng/mouse/day, i.p.) or vehicle for 2 consecutive days. The frequency of CD45+CD11b+ cells in the ipsilateral DRGs (L3, L4 and L5) is shown.
Sequences
Gene Sense (5´-3´) Antisense (5´-3´)
Atf3 Taqman: Mm00476032_m1 Gapdh Taqman: Mm03302249_g1 Foxp3 Taqman: Mm00475162_m1
Gapdh GGGTGTGAACCACGAGAAAT CCTTCCACAATGCCAAAGTT
Ifng TGCATCTTGGCTTTGCAGCTCTCCTCATGGC TGGACCTGTGGGTTGTTGACCTCAAACTTGGC
Tbx21 CCACCTGTTGTGGTCCAAGTT CATTCGCCGTCCTTGCTTAG
Stat1 GACTTCAGACACAGAAATCAACTC TTGACAAAGACCACGCCTT
Rorc GAGTTTGCCAAGCGGCTTT TCCATTGCTCCTGCTTTCAGT
Entpd1 AACTCTCTGCAATTCGTCTCT ATGTCCTTGGCCAGTTTCTG
Tgfb ACCGCAACAACGCCATCTAT TCAAAAGACAGCCACTCAGGC
Il10 AACAAAGGACCAGCTGGACAAC GCAACCCAAGTAACCCTTAAAGTC
Aif1 GCTTCAAGTTTGGACGGCAG TGAGGAGCCATGAGCCAAAG
Cx3cr1 GCCTCTGGTGGAGTCTGCGTG CGCCCAAATAACAGGCCTCAGCA
Itgam GAGTCTGCCTCCGTGTCCGC TACGTGAGCGGCCAGGGTCT
Table 1. Sequence of the primers used in the present study