Granulocyte-colony-stimulating factor (G-CSF) signaling in spinal microglia drives visceral sensitization following colitis

Abstract

Pain is a main symptom of inflammatory diseases and often persists beyond clinical remission. Although we have a good understanding of the mechanisms of sensitization at the periphery during inflammation, little is known about the mediators that drive central sensitization. Recent reports have identified hematopoietic colony-stimulating factors as important regulators of tumor- and nerve injury-associated pain. Using a mouse model of colitis, we identify the proinflammatory cytokine granulocyte-colony-stimulating factor (G-CSF or Csf-3) as a key mediator of visceral sensitization. We report that G-CSF is specifically up-regulated in the thoracolumbar spinal cord of colitis-affected mice. Our results show that resident spinal microglia express the G-CSF receptor and that G-CSF signaling mediates microglial activation following colitis. Furthermore, healthy mice subjected to intrathecal injection of G-CSF exhibit pronounced visceral hypersensitivity, an effect that is abolished by microglial depletion. Mechanistically, we demonstrate that G-CSF injection increases Cathepsin S activity in spinal cord tissues. When cocultured with microglia BV-2 cells exposed to G-CSF, dorsal root ganglion (DRG) nociceptors become hyperexcitable. Blocking CX3CR1 or nitric oxide production during G-CSF treatment reduces excitability and G-CSF-induced visceral pain in vivo. Finally, administration of G-CSF-neutralizing antibody can prevent the establishment of persistent visceral pain postcolitis. Overall, our work uncovers a DRG neuron-microglia interaction that responds to G-CSF by engaging Cathepsin S-CX3CR1-inducible NOS signaling. This interaction represents a central step in visceral sensitization following colonic inflammation, thereby identifying spinal G-CSF as a target for treating chronic abdominal pain.

Keywords: G-CSF; colitis; microglia; nociceptors; visceral pain.

Fig. 1.

G-CSF and its receptor, expressed on microglia, are increased in the spinal cord during acute DSS-induced colitis. Acute colitis was induced in mice with 2.5% DSS for 7 d. After blood removal, G-CSF levels were determined in the thoracolumbar (T12–L1) spinal cord by luminex technology in control (white bar, n = 10) or colitis (black bar, n = 7) mice (A). Mononuclear cells of the spinal cord were isolated, and CD45^low CD11b⁺ cells (microglia) were analyzed using flow cytometry for their expression of G-CSFR represented by increased binding of specific G-CSFR antibody (gray histogram, representative of three independent experiments) compared with control IgG (dotted histogram) (B). (C, Left) Iba-1 expression was quantified by immunostaining in spinal cord sections of control and DSS colitis mice. (C, Right) Intensity of the immunostaining was measured using Image J on a total of 35 sections from three independent experiments. (D) G-CSFR expression level was determined by Western blot in spinal cord of control (white bar, n = 6) or colitis (black bar, n = 6) mice. Statistical analyses were performed using Mann–Whitney U test; *P < 0.05; ***P < 0.001. (Scale bar: 100 μm.)

Fig. S1.

Inflammatory state of spinal cord during acute colitis. (A) Measure of G-CSF and IL6 in the serum of control (white bar, n = 10) or DSS colitis (black bar, n = 7) mice. (B) Measure of IL1β, IL6, and TNFα levels from the thoracolumbar (T12–L1) spinal cord of control (white bar, n = 10) or DSS colitis (black bar, n = 7) mice. (C) Spinal cord mononuclear cells were isolated, and CD45low CD11b+ cells (microglia) were analyzed using flow cytometry. Expression of G-CSF was determined after in vitro stimulation with phorbol myristate acetate (PMA) and ionomycin for 3 h and is represented by increased binding of specific G-CSF antibody (gray histogram) compared with control IgG (dotted histogram). This is a representative result from three independent experiments. (D) Expression of G-CSFR mRNA was assessed by qPCR in the thoracolumbar spinal cord of control (white bar, n = 4) or colitis (black bar, n = 5) mice.

Fig. 2.

G-CSF signaling in microglia induces visceral hypersensitivity. (A) Representative electromyogram recording elicited by 45 mmHg pressure at 12 h of intrathecal PBS or G-CSF (20 ng) injection. (B) VMR to colorectal distension after treatment with PBS (n = 4), G-CSF (20 ng; n = 8), anti–G-CSFR antibody (G-CSF-Rab 1 µg; n = 5; ***P < 0.001), or a combination of both (n = 5). (C) Representative electromyogram recording elicited by 45 mmHg pressure at 12 h of intrathecal PBS or G-CSF (20 ng) injection in mice that received control or the PLX 5622 diet for 2 wk. (D) VMR to colorectal distension after treatment with PBS (control diet: n = 15, PLX diet: n = 10) or G-CSF (control diet: n = 16, PLX diet: n = 11). Results are expressed as fold increase in VMR (±SEM) for control diet (white bar) or PLX diet (black bar) animals. Statistical analysis was performed using either repeated-measures two-way ANOVA and Bonferroni post-hoc test (B; *P < 0.05 G-CSF; ***P < 0.001 vs. PBS; $P < 0.05 G-CSF vs. G-CSF+G-CSF Receptor antibody) or the Mann–Whitney U test (D; *P < 0.05; ***P < 0.001).

Fig. S2.

Ablation of microglia by PLX 5622 has no effect on basal visceral sensitivity. Iba-1 expression was assessed by immunostaining (A), and G-CSFR expression was assessed by Western blot (B) in spinal cord of control-diet or PLX-fed mice. (C) Visceral sensitivity was measured in control (blue circle), G-CSF–treated (green square) or PLX mice (red square), and PLX mice treated with G-CSF (green square).

Fig. 3.

G-CSF–treated microglia increases excitability of DRG neurons through Cathepsin S-CX3CR1-iNOS signaling. (A) Mice were treated intrathecally with either PBS or G-CSF (20 ng) 12 h before evaluating Cathepsin S activity in thoracolumbar spinal cord lysate (n = 3 experiments). (B) Cartoon illustrating the coculture system experiment. BV2 microglia cells were plated into the upper chamber of a transwell and treated with G-CSF for 16 h. G-CSF was removed, and DRG neurons were plated in the lower chamber of the transwell for 16 h of coculture and then used for electrophysiological recordings. (Lower) A representative trace of spontaneous activity (SA) in a small-size TRPV1-sensitive DRG neuron cocultured with vehicle- or G-CSF–primed BV2 cells. (C) Exposure of BV2 microglia to G-CSF depolarizes the resting membrane potential (RMP) of DRG neurons and increases the percentage of neurons with SA (5/28 in control, 17/30 for G-CSF treated, 10/31 for G-CSF combined with CX3CR1 Ab-treated, 3/11 for G-CSF combined with Cat S inhibitor-treated, 5/15 for G-CSF combined with L-NAME, and 4/10 for G-CSF combined with Apyrase). (Right) G-CSF induces a decrease in the AP threshold and an increase in spontaneous AP discharge. These parameters are reversed by cotreatment with CX3CR1 Ab, Cathepsin S inhibitor, or L-NAME, but not apyrase. (D) Representative AP discharge evoked by 100-, 200-, and 300-pA current injections (1 s) in control (blue circle) and G-CSF (red circle) and for different conditions of treatment (E). (F) Measure of VMR to colorectal distension in mice injected with PBS (n = 9), G-CSF (20 ng; n = 12), or G-CSF + CX3CR1 Ab (5 µg; n = 9) 12 h before recording. Results indicate mean ± SEM (*P < 0.05; **P < 0.01). Statistical analysis was performed using one-way (C and E) or two-way ANOVA (A, D, and F) followed by Bonferroni post hoc test (*P < 0.05; **P < 0.01; $P < 0.05; $$P < 0.01).

Fig. S3.

Role of iNOS in G-CSF–induced hyperexcitability. Exposure to L-NAME (100 µM) alone has no effect on the resting membrane potential (A), the AP threshold (B), or spontaneous (C) and evoked (D) AP discharge. Results indicate mean ± SEM. Statistical analysis was performed using one-way ANOVA followed by Bonferroni post hoc test. (E) Western blot analysis of iNOS level from spinal cord samples (T12–L1) collected from G-CSF–treated mice. (Lower) Quantification of relative integrated density values from Western blots corrected from GAPDH and normalized to control PBS.

Fig. 4.

Blocking G-CSF receptor signaling in the spinal cord alleviates DSS-induced postinflammatory visceral hypersensitivity. (A) Colitis was induced with 2.5% DSS for 5 d, after which mice received only water for the next 5 wk. During the recovery period, mice were treated twice a week with either G-CSFR–blocking antibodies or control IgG. A healthy control group that did not receive DSS was intrathecally injected with PBS. (B) Visceromotor responses to colorectal distension post-DSS–induced colitis in G-CSF-Rab (green squares; n = 11) and control IgG (red circles; n = 6) treated mice compared with healthy mice injected with PBS (blue triangle; n = 4). Data are expressed as mean of VMR ± SEM. Statistical analysis was performed using repeated-measures two-way ANOVA and the Bonferroni post hoc test (*P < 0.05; **P < 0.01; ***P < 0.001). (C) Schematic representation of G-CSF–mediated visceral sensitization in the spinal cord. (1) Acute DSS colitis increases G-CSF in the spinal cord. (2) G-CSF binds to its receptor on microglia and (3) induces the secretion of Cathepsin S. (4) Cathepsin S triggers the release of soluble fractalkine that activates its receptor CX3CR1 at the microglial cell surface. (5) This drives a hyperexcitability of DRG neurons through a NO-dependent process and the establishment of visceral sensitization. (6) Inhibition of either G-CSFR or CX3CR1 prevents both DRG hyperexcitability and visceral hypersensitivity.

Fig. S4.

G-CSFR antibody does not alter disease recovery. Mice were treated for 7 d with 2.5% DSS and injected with either control IgG (n = 3) or G-CSF-RAb (n = 3) on days 2, 5, and 7 of DSS regimen. Macroscopic damages (A) and myeloperoxidase activity (B) were evaluated at day 7 of DSS. (C) Schematic representation of the experimental design to assess acute DSS-induced visceral hypersensitivity; colitis was induced by 2.5% DSS regimen and mice received intrathecal injection of either IgG (red circle n = 13) or G-CSF-RAb (green square n = 10). A control group receiving only water was injected with PBS IT (blue triangle n = 11) at similar time points. On day 5, visceral hypersensitivity was assessed by measuring visceromotor response to colorectal distension.

Fig. S5.

Chronic intrathecal treatment with G-CSF-RAb does not alter disease recovery post colitis. Colitis was induced by adding 2.5% DSS in the drinking water of mice. On day 5, DSS mice were on water regimen for 5 wk. During the recovery period, mice were treated twice a week with either G-CSFR–blocking antibody (blue square; n = 11) or control IgG (red circle; n = 6). A control group that was not exposed to DSS received intrathecal PBS (n = 4). Recovery of colitis was monitored by weighing mice weekly (A) and macroscopic scoring of colonic tissue damages (B) as previously described (46).