Annexin 1 Reduces Dermatitis-Induced Itch and Cholestatic Itch through Inhibiting Neuroinflammation and Iron Overload in the Spinal Dorsal Horn of Mice

Abstract

The unclear pathogenesis of chronic itch originating from several systemic disorders poses challenges to clinical intervention. Recent studies recapitulate the spinal neurocircuits associated with neuroinflammation and synaptic plasticity responsible for pruriceptive sensations. The resolution of nociception and inflammation by Annexin 1 (ANXA1) has been identified. Given that pain and itch share many neural mechanisms, we employed two mice models of chronic itch to study the underlying targets and therapeutic potential of ANXA1, comprising allergic contact dermatitis-induced itch and cholestatic itch. Herein, we report that spinal expression of ANXA1 is down-regulated in mice with dermatitis-induced itch and cholestatic itch. Repetitive injections of ANXA1-derived peptide Ac2-26 (intrathecal, 10 μg) reduce itch-like scratching behaviors following dermatitis and cholestasis. Single exposure to Ac2-26 (intrathecal, 10 μg) alleviates the established itch phenotypes. Moreover, systemic delivery of Ac2-26 (intravenous, 100 μg) is effective against chronic dermatitis-induced itch and cholestatic itch. Strikingly, Ac2-26 therapy inhibits transferrin receptor 1 over-expression, iron accumulation, cytokine IL-17 release and the production of its receptor IL-17R, as well as astrocyte activation in the dorsal horn of spinal cord in mouse with dermatitis and cholestasis. Pharmacological intervention with iron chelator deferoxamine impairs chronic itch behaviors and spinal iron accumulation after dermatitis and cholestasis. Also, spinal IL-17/IL-17R neutralization attenuates chronic itch. Taken together, this current research indicates that ANXA1 protects against the beginning and maintenance of long-term dermatitis-induced itch and cholestatic itch, which may occur via the spinal suppression of IL-17-mediated neuroinflammation, astrocyte activation and iron overload.

Keywords: Annexin 1; IL-17; chronic itch; iron overload; transferrin receptor 1.

Figure 1

Spinal therapy of ANXA1-derived peptide Ac2-26 reduces dermatitis-induced chronic itch in mice. (A) Experimental design for intrathecal (i.t.) treatment with Ac2-26 in DNFB-induced chronic pruritus. (B) Scratching bouts after DNFB exposure. n = 6 mice/group. (C) The expression of ANXA1 in the spinal dorsal horn after DNFB exposure was measured. n = 4 mice/group. (D) Pre-administrations of Ac2-26 prevent DNFB-induced scratching behaviors. n = 6 mice/group. (E) Single injection of Ac2-26 (i.t., 10 μg) on day 7 after DNFB exposure attenuates the established persistent scratching behaviors. n = 6 mice/group. All behavioral and biochemical results are expressed as medians with interquartile ranges and individual data plots. **** p < 0.0001 vs. group Control.

Figure 2

ANXA1-derived peptide Ac2-26 down-regulates spinal IL-17 release, IL-17R expression, astrocyte activation and iron overload after dermatitis in mice. Repetitive injections of Ac2-26 (intrathecal, 10 μg) daily for three consecutive days were performed from day 0 to day 2 following DNFB exposure. The dorsal horns of spinal cord were collected on day 3 following DNFB exposure. (A) The expression of IL-17 in the spinal dorsal horn was measured by ELISA assay. (B) The expression of IL-17R in the spinal dorsal horn was measured by RT-qPCR. (C) The expression of GFAP in the spinal dorsal horn was measured by ELISA assay. (D) The iron concentration in the spinal dorsal horn was measured. (E–H) Western blot showed the changes of spinal iron metabolism-related proteins (TfR1, IRP1 and DMT1) after DNFB exposure and Ac2-26 treatment, respectively. n = 4 mice/group. All biochemical results are expressed as medians with interquartile ranges and individual data plots.

Figure 3

Spinal therapy with ANXA1-derived peptide Ac2-26 reduces cholestatic itch in mice. (A) Experimental design for intrathecal (i.t.) treatment with Ac2-26 in BDL-induced chronic pruritus. (B) Serum levels of total bilirubin were measured after sham and BDL surgery. n = 4 mice/group. (C) Scratching bouts after BDL surgery. n = 6 mice/group. (D) The expression of ANXA1 in the spinal dorsal horn after BDL surgery was measured. n = 4 mice/group. (E) Pre-administration of Ac2-26 prevents BDL-induced scratching behaviors. n = 6 mice/group. (F) Single injection of Ac2-26 (i.t., 10 μg) on day 21 after BDL surgery attenuates the established persistent scratching behaviors. n = 6 mice/group. All behavioral and biochemical results are expressed as medians with interquartile ranges and individual data plots. **** p < 0.0001 vs. sham group.

Figure 4

ANXA1-derived peptide Ac2-26 down-regulates spinal IL-17 release, IL-17R expression, astrocyte activation and iron overload after cholestasis in mice. Repetitive injections of Ac2-26 (intrathecal, 10 μg) daily for three consecutive days were performed from day 11 to day 13 following BDL surgery. The dorsal horns of spinal cords were collected on day 14 following BDL surgery. (A) The expression of IL-17 in the spinal dorsal horn was measured by ELISA assay. (B) The expression of IL-17R in the spinal dorsal horn was measured by RT-qPCR. (C) The expression of GFAP in the spinal dorsal horn was measured by ELISA assay. (D) The iron concentration in the spinal dorsal horn was measured. (E–H) Western blot showed the changes in spinal iron metabolism-related proteins (TfR1, IRP1 and DMT1) after BDL surgery and Ac2-26 treatment, respectively. n = 4 mice/group. All biochemical results are expressed as medians with interquartile ranges and individual data plots.

Figure 5

Spinal inhibition of IL-17 cascade reduces dermatitis-induced chronic itch and cholestatic itch in mice. (A) Intrathecal injection of anti-IL-17 (2 μg) and IL-17R Ab (2 μg) were performed on day 7 following DNFB intervention. The therapy with anti-IL-17 and IL-17R Ab attenuated the established persistent scratching behaviors. n = 6 mice/group. (B) Intrathecal injections of anti-IL-17 (2 μg) and IL-17R Ab (2 μg) were performed on day 21 following BDL operation. The therapy with anti-IL-17 and IL-17R Ab attenuated the established persistent scratching behaviors. n = 6 mice/group. (C–F) Spinal expression of TfR1 and iron concentration after anti-IL-17 and IL-17R Ab treatment were measured in mice with DNFB exposure and BDL surgery, respectively. n = 4 mice/group. (G,H) The expression of GFAP in the spinal dorsal horn was measured by ELISA assay. n = 4 mice/group. All behavioral and biochemical results are expressed as medians with interquartile ranges and individual data plots.

Figure 6

Iron chelation reduces dermatitis-induced chronic itch and cholestatic itch in mice. (A) Experimental design for intrathecal (i.t.) treatment with iron chelator DFO in DNFB-induced chronic pruritus. (B) Spinal iron concentration after DNFB and DFO treatment was measured. The dorsal horns of spinal cord were collected on day 3 following DNFB exposure. n = 4 mice/group. (C) Pre-administration of DFO prevented DNFB-induced scratching behaviors. n = 6 mice/group. (D) Single injection of DFO (i.t., 20 μg) on day 7 after DNFB exposure attenuates the established persistent scratching behaviors. n = 6 mice/group. (E) Experimental design for intrathecal (i.t.) treatment with iron chelator DFO in BDL-induced chronic pruritus. (F) Spinal iron concentration after BDL surgery and DFO treatment was measured. The dorsal horns of spinal cord were collected on day 14 following BDL surgery. n = 4 mice/group. (G) Pre-administration of DFO prevented BDL-induced scratching behaviors. n = 6 mice/group. (H) Single injection of DFO (i.t., 20 μg) on day 21 after BDL surgery attenuates the established persistent scratching behaviors. n = 6 mice/group. All behavioral and biochemical results are expressed as medians with interquartile ranges and individual data plots.

Figure 7

Systemic administration of ANXA1-derived peptide Ac2-26 reduces dermatitis-induced chronic itch and cholestatic itch in mice. (A) Experimental design for intravenous (i.v.) treatment with Ac2-26 in DNFB-induced chronic pruritus. (B) Pre-administration of Ac2-26 prevented DNFB-induced scratching behaviors. n = 6 mice/group. (C) Single injection of Ac2-26 (i.v., 100 μg) on day 7 after DNFB exposure attenuates the established persistent scratching behaviors. n = 6 mice/group. (D) Experimental design for intravenous (i.v.) treatment with Ac2-26 in BDL-induced chronic pruritus. (E) Pre-administration of Ac2-26 prevented BDL-induced scratching behaviors. n = 6 mice/group. (F) Single injection of Ac2-26 (i.v., 100 μg) on day 21 after BDL surgery attenuates the established persistent scratching behaviors. n = 6 mice/group. All behavioral results are expressed as medians with interquartile ranges and individual data plots.