Fig 2: CCL1 is involved in the neuronal excitability and mechanical allodynia by activating astrocytes.(A) Immunofluorescence imaging showed BTZ-activated astrocytes (turquoise) in close proximity to macrophages (green) and vascular endothelial cells (red). Scale bars, 20 μm. (B) Schematic of two-photon imaging configuration. (C) Representative image showed the increased GCaMP6s (green) in spinal cord astrocytes (SR101, red) following CCL1 incubation. Scale bar, 150 μm. (D) Example signal trace (left) and histogram of area under the curve (AUC) (right) of calcium signaling value from the spinal dorsal horn astrocyte in mice treated with CCL1. n = 3. (E to G) R243 incubation prevented CCL1-induced calcium signaling increase in GCaMP6s+ astrocytes. Representative image (E) and signal trace (F) show R243 blocked CCL1-induced calcium up-regulation. Scale bar, 20 μm. AUC quantification (G). n = 3. (H to J) Response of astrocytes to recombinant CCL1 during bath application of TTX (1 μM). Representative image (H) and signal trace (I) showed that TTX did not inhibit the increase in intracellular calcium signaling induced by CCL1. Scale bar, 20 μm. AUC quantification (J). n = 3. (K to M) Application of CNO increased the astrocyte intracellular calcium signaling in the female with injection of AAV2/5-GfaABC1D-CRE and AAV-DIO-hM3Dq-mCherry. Scale bar, 20 μm. (N) The action potential number in female mouse spinal cord slice was increased by CNO application. Representative trace (left) and statistical data (right) were shown. n = 3. (O) Application with CNO decreased the paw withdrawal threshold in female mice with intraspinal injection of AAV2/5-GfaABC1D-CRE and AAV-DIO-hM3Dq-mCherry. n = 5. Significance: not significant (n.s.) and *P < 0.05.

Fig 3: ICAM-1 and IL-1α released by endothelial cells synergistically promote macrophage infiltration in female mice following BTZ.(A) Il1a, Ccl5, and Ccl1 mRNA levels in the dorsal horn of female mice. n = 5 to 8. (B) Immunofluorescence image of IL-1α (green) in vascular endothelial cells (red). Scale bars, 50 μm (low) or 20 μm (high). (C) Double immunofluorescence staining of CCL1 (green) and vascular endothelial cells (red). Scale bars, 50 μm (low) or 20 μm (high). (D and E) Coimmunostaining image of F4/80 (green) and CD31 (red) in the spinal dorsal horn and the statistical graph. Scale bar, 200 μm. n = 5. (F) Intrathecal injection of IL-1α siRNA attenuated the mechanical allodynia induced by BTZ. n = 5. (G) Immunofluorescence image shows IL-1R1 (red) expression in macrophages (green). Scale bar, 10 μm. (H and I) Intrathecal IL-1R1 injection inhibited the up-regulation of F4/80 (green) and CD31 (red) in the near vascular of the spinal dorsal horn. Scale bar, 200 μm (H). The number of F4/80high cells in the dorsal horn of the spinal cord [(I), left]. The percentage of F4/80high area among CD31+ area [(I), right]. n = 4 to 5. (J and K) Immunostaining shows F4/80 (green) and CD31 (red) expression in the spinal dorsal horn. Scale bar, 200 μm (J). The number of F4/80high cells in the dorsal horn of the spinal cord [(K), left]. The percentage of F4/80high area among CD31+ area [(K), right]. n = 5. (L) Intrathecal injection of IL-1α siRNA attenuated the mechanical allodynia induced by BTZ. n = 5. Significance: *P < 0.05, #P < 0.05, **P < 0.01, and ##P < 0.01.

Fig 4: The mechanism underlying the earlier pain induced by BTZ in females.(A) Increased ICAM-1 promotes monocyte adhesion to vascular endothelial cells following BTZ treatment. (B) IL-1α/ICAM-1 collaboratively promote monocyte transmigration across the vascular wall. (C) Infiltrated macrophage-released CCL1 enhances neuron excitability directly or indirectly via the activated astrocytes.

Fig 5: Succinate potentiated the production of CCL1 in CD4+ T cells to promote myofibroblast activation. (A) Heatmap showing the expression of Pde1a, Ccl1, Ccl3, and Klb in control and succinate-treated CD4+ T cells. (B) qPCR analysis for Ccl1 and Ccl3 in CD4+ T cells treated with DMSO or dimethyl succinate (DS) (n = 6). (C) Immunoblot and (D) quantification analysis of CCL1 in the UUO kidneys from mice treated with succinate or DMM (n = 3). (E) Immunoblot and (F) quantification analysis of Col I and αSMA in NIH-3T3 cells treated with PBS or rmCCL1 (n =3). (G) Immunoblot and (H) quantification analysis of pERK and ERK in NIH-3T3 cells treated with PBS or rmCCL1 (n =3). (I) qPCR analysis for Col1a2 and Acta2 in NIH-3T3 fibroblasts co-cultured with succinate-preconditioned CD4+ T cells or untreated CD4+ T cells (n = 5). The results represent mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001.