A role for transient receptor potential vanilloid 4 in tonicity-induced neurogenic inflammation

Abstract

Background and purpose: Changes in extracellular fluid osmolarity, which occur after tissue damage and disease, cause inflammation and maintain chronic inflammatory states by unknown mechanisms. Here, we investigated whether the osmosensitive channel, transient receptor potential vanilloid 4 (TRPV4), mediates inflammation to hypotonic stimuli by a neurogenic mechanism.

Experimental approach: TRPV4 was localized in dorsal root ganglia (DRG) by immunofluorescence. The effects of TRPV4 agonists on release of pro-inflammatory neuropeptides from peripheral tissues and on inflammation were examined.

Key results: Immunoreactive TRPV4 was detected in DRG neurones innervating the mouse hindpaw, where it was co-expressed in some neurones with CGRP and substance P, mediators of neurogenic inflammation. Hypotonic solutions and 4alpha-phorbol 12,13-didecanoate, which activate TRPV4, stimulated neuropeptide release in urinary bladder and airways, sites of neurogenic inflammation. Intraplantar injection of hypotonic solutions and 4alpha-phorbol 12,13-didecanoate caused oedema and granulocyte recruitment. These effects were inhibited by a desensitizing dose of the neurotoxin capsaicin, antagonists of CGRP and substance P receptors, and TRPV4 gene knockdown or deletion. In contrast, antagonism of neuropeptide receptors and disruption of TRPV4 did not prevent this oedema. TRPV4 gene knockdown or deletion also markedly reduced oedema and granulocyte infiltration induced by intraplantar injection of formalin.

Conclusions and implications: Activation of TRPV4 stimulates neuropeptide release from afferent nerves and induces neurogenic inflammation. This mechanism may mediate the generation and maintenance of inflammation after injury and during diseases, in which there are changes in extracellular osmolarity. Antagonism of TRPV4 may offer a therapeutic approach for inflammatory hyperalgesia and chronic inflammation.

Figure 1

Effects of intraplantar injections of 4α PDD, distilled water (dH₂O), 10% NaCl and 0.9% NaCl on paw diameter (A), MPO (B) and histology of paw tissues (C). Scale bar = 200 µm. 4αPDD, dH₂O and 10% NaCl increased paw diameter and paw MPO activity, and disrupted the structure of the tissue compared with 0.9% NaCl. *P < 0.05 compared with 0.9% NaCl, n = 16 per group for all except for histology, n = 5 per group. 4αPDD, 4α-phorbol 12,13-didecanoate; MPO, myeloperoxidase.

Figure 2

Effects of capsaicin, CGRP_8-37, SR140333 and vehicle (control) on paw diameter (A–C) and MPO (D) of mice after intraplantar injections of 4αPDD (A), distilled water (dH₂O, B), 10% NaCl (C) and 0.9% NaCl (D). Capsaicin, CGRP_8-37 and SR140333 inhibited the effects of 4αPDD and dH₂O on diameter and MPO activity, but had little or no effect on responses to 10% NaCl. *P < 0.05 compared with vehicle, n = 8 per group. 4αPDD, 4α-phorbol 12,13-didecanoate; CGRP, calcitonin gene-related peptide; MPO, myeloperoxidase.

Figure 3

Localization of TRPV4, CGRP and SP in DiI-labelled DRG (L4-L5) innervating the mouse paw. DiI was injected into the plantar surface of the hind paw to retrogradely label neurones innervating this tissue. TRPV4-LI was detected in some DiI-labelled neurones containing CGRP-LI and SP-LI (arrows). However, some neurones expressing TRPV4-LI did not contain detectable DiI, CGRP-LI or SP-LI (arrow heads). Pre-absorption of the antibody abolished staining (control). Scale bar = 40 µM in top and middle rows and 50 µM in bottom row. CGRP, calcitonin gene-related peptide; DRG, dorsal root ganglia; LI, like-immunoreactivity; SP, substance P; TRPV, transient receptor potential vanilloid 4 (TRPV).

Figure 4

Effects of 4αPDD and hypotonic solution on the release of SP (A, B) and CGRP (C, D) from mouse urinary bladder (left) and airways (right). 4αPDD and hypotonic solution stimulated release of CGRP and SP, and the effects were abolished by removal of extracellular Ca²⁺ ions (-Ca²⁺) and by capsaicin (Caps) treatment. Neuropeptide release was measured over 20 min. *P < 0.05 compared with basal; #P < 0.05 compared with 100 µM 4αPDD and hypotonic solution. 4αPDD, 4α-phorbol 12,13-didecanoate; CGRP, calcitonin gene-related peptide; DRG, dorsal root ganglia; SP, substance P.

Figure 7

Effects of TRPV4 deletion on paw diameter (A–C) and MPO (D) of mice after intraplantar injections of 0.9% NaCl (A), 4αPDD (B), or distilled water (dH₂O, C). TRPV4-deficient mice (TRPV4^−/−) showed significantly less oedema and MPO activity than did wild-type littermates (TRPV4^+/+). *P < 0.05 compared with TRPV4^+/+ (A–C) or compared with 0.9% NaCl (D); #P < 0.05 to TRPV4^+/+; n = 5 for each group of mice that received 4αPDD or distilled water, n = 7 for TRPV4^+/+ and n = 8 for TRPV4^−/− that received 0.9% NaCl. MPO, myeloperoxidase; TRPV, transient receptor potential vanilloid 4 (TRPV).

Figure 6

Effects of TRPV4 down-regulation with siRNA on paw diameter (A–D) and MPO (E) of mice after intraplantar injections of 0.9% NaCl (A), 4αPDD (B), distilled water (dH₂O, C) or 10% NaCl (D). TRPV4 siRNA inhibited the effects of 4αPDD and distilled water on diameter and MPO activity, but did not affect the responses to 10% NaCl. *P < 0.05 compared with control siRNA (A–D) or 0.9% NaCl (E); #P < 0.05 compared with control siRNA (E), n = 6 in each group. MPO, myeloperoxidase; TRPV, transient receptor potential vanilloid 4 (TRPV).

Figure 5

Down-regulation of TRPV4 with siRNA. Vehicle (DEPC water), control siRNA or TRPV4 siRNA was injected intrathecally and DRG were collected after 4 days to verify uptake of fluorescent 6FAM-siRNA and to localize TRPV4-LI (A). Both control and TRPV4 6FAM-siRNA were detected in many cells of the DRG. TRPV4-LI was detected in DRG after control siRNA, and this signal was prevented by pre-adsorpion of antibody (control). Arrows show neurones that have taken up siRNA (6FAM positive) and that did not express TRPV4-LI. Scale bar = 50 µM. Injection of TRPV4 siRNA down-regulated TRPV4-LI. TRPV4 siRNA also reduced the number of neurones with detectable TRPV4-LI (B). *P < 0.05 compared with DEPC water. DRG, dorsal root ganglia; LI, like-immunoreactivity; TRPV, transient receptor potential vanilloid 4 (TRPV).

Figure 8

Effects of TRPV4 down-regulation with siRNA (A, C) or TRPV4 deletion (B, D) on control non-inflamed and formalin-induced changes in paw volume (A, B) and MPO (C, D). In (A) and (C), controls received an intraplantar injection of 0.9% NaCl and an intrathecal injection of TRPV4 siRNA. In (D), control TRPV4^+/+ and TRPV4^–/– mice received an intraplantar injection of 0.9% NaCl. TRPV4 siRNA-treated and TRPV4^–/– mice showed a reduced inflammatory response to formalin with a diminished oedema and MPO activity. *P < 0.05 compared with control non-inflamed (A, C); #P < 0.05 compared with control siRNA + formalin (A, C) or TRPV4^+/+ (B, D), n = 6 for all mice treated with siRNA, n = 7 for TRPV4^+/+ and n = 8 for TRPV4^−/− mice that received formalin. MPO, myeloperoxidase; TRPV, transient receptor potential vanilloid 4 (TRPV).