Sensitization of colonic nociceptors by IL-13 is dependent on JAK and p38 MAPK activity

Abstract

The effective management of visceral pain is a significant unmet clinical need for those affected by gastrointestinal diseases, such as inflammatory bowel disease (IBD). The rational design of novel analgesics requires a greater understanding of the mediators and mechanisms underpinning visceral pain. Interleukin-13 (IL-13) production by immune cells residing in the gut is elevated in IBD, and IL-13 appears to be important in the development of experimental colitis. Furthermore, receptors for IL-13 are expressed by neurons innervating the colon, though it is not known whether IL-13 plays any role in visceral nociception per se. To resolve this, we used Ca²⁺ imaging of cultured sensory neurons and ex vivo electrophysiological recording from the lumbar splanchnic nerve innervating the distal colon. Ca²⁺ imaging revealed the stimulation of small-diameter, capsaicin-sensitive sensory neurons by IL-13, indicating that IL-13 likely stimulates nociceptors. IL-13-evoked Ca²⁺ signals were attenuated by inhibition of Janus (JAK) and p38 kinases. In the lumbar splanchnic nerve, IL-13 did not elevate baseline firing, nor sensitize the response to capsaicin application, but did enhance the response to distention of the colon. In line with Ca²⁺ imaging experiments, IL-13-mediated sensitization of the afferent response to colon distention was blocked by inhibition of either JAK or p38 kinase signaling. Together, these data highlight a potential role for IL-13 in visceral nociception and implicate JAK and p38 kinases in pronociceptive signaling downstream of IL-13.

Keywords: IL-13; JAK; inflammatory bowel disease; nociception; p38 MAPK.

Graphical abstract

Figure 1.

IL-13 increases [Ca²⁺]_i in capsaicin-sensitive DRG neurons. A: representative traces from individual (i) and averaged (ii) neuronal responses to 30 nM IL-13 (n = 63 neurons). B: the percentage of DRG neurons stimulated by IL-13 was significantly lower than nonresponsive neurons. C: IL-13-sensitive neurons were smaller in diameter than nonresponders (n = 644 neurons). D: example traces from (i) individual neurons responsive to both IL-13 and 1 µM capsaicin and (ii) response profiles of responder populations (black, IL-13⁺/cap⁺; dark gray, IL-13⁺/cap⁻, light gray: IL-13⁻/cap⁺). E: representative Venn diagram of overlapping responses in neurons stimulated with IL-13 and capsaicin (n = 5, N = 5). F: size distribution of IL-13- and/or capsaicin-sensitive neurons. IL/13/capsaicin cosensitive and capsaicin-sensitive neurons were smaller in diameter than nonresponders (n = 488 neurons). DRG, dorsal root ganglion.

Figure 2.

Activation of DRG sensory neurons by IL-13 is JAK1/2- and p38 MAPK-dependent. The proportion of IL-13-sensitive neurons was significantly reduced by pan-JAK inhibition with pyridone 6 (P6) (n = 5), JAK1/2 inhibition with ruxolitinib (Rux) (n = 5), and p38 MAPK inhibition with SB203580 (SB) (n = 5). DRG, dorsal root ganglion. **P < 0.01.

Figure 3.

IL-13 evokes colonic afferent hypersensitivity to ramp distension via JAK and p37 MAPK. A: example rate histograms and neurograms showing LSN activity with accompanying pressure trace showing sequential (x3) ramp distensions (0–80 mmHg) from vehicle- and IL-13 (100 nM)-treated preparations, demonstrating the sensitization of colonic afferent responses to noxious distension after IL-13 perfusion. B: IL-13 perfusion shifted the afferent pressure-response curve (Ramp 5) at distension pressures greater than 60 mmHg compared with vehicle (*P < 0.05, **P < 0.01, vehicle vs. IL-13), whereas coapplication of either P6 (##P < 0.01, IL-13 vs. IL-13 + P6) or SB (&&P < 0.01, &&&P < 0.001 IL-13 vs. IL-13 + SB) abrogated the effect of IL-13. Application of P6 or SB alone had no effect on the pressure-response curve compared with vehicle (curves not shown for clarity, but these groups were included in two-way analysis). C: IL-13 potentiated the area under the pressure-response curve (AUC) compared with vehicle. This effect of IL-13 on distension-evoked afferent firing was abolished by incubation of the tissue with P6 or SB. Comparisons to the IL-13-treated group: *P < 0.05, **P < 0.01. D: peak afferent activity was significantly increased following IL-13 pretreatment; an effect abolished by coapplication of P6 or SB. Comparisons to the IL-13-treated group: *P < 0.05. E: spontaneous baseline afferent activity was indistinguishable between all treatment groups. F: colonic compliance was unchanged between sequential ramp distensions within treatment groups and no differences between treatment groups were observed. LSN, lumbar splanchnic nerve.

Figure 4.

IL-13 does not sensitize the colonic afferent response to capsaicin. A: example rate histograms and accompanying neurograms of LSN activity, illustrating similar afferent responses to 1 µM capsaicin after incubation with IL-13 (100 nM) compared with vehicle pretreatment. B: afferent firing was unchanged in the 10-min postcapsaicin application (P = 0.979, N = 8). C: IL-13 did not alter response AUC 0- to 10-min postcapsaicin application (P = 0.818, unpaired t test; N = 8). D: peak afferent firing remained constant between vehicle- and IL-13-treated tissues (P = 0.534, N = 8). E: the time taken for nerve activity to return to baseline firing following capsaicin application was unchanged by IL-13 incubation (P = 0.649, N = 8). LSN, lumbar splanchnic nerve.