Cot/tpl2 (MAP3K8) mediates myeloperoxidase activity and hypernociception following peripheral inflammation

Abstract

Cot/tpl2 (also known as MAP3K8) has emerged as a new and potentially interesting therapeutic anti-inflammatory target. Here, we report the first study of Cot/tpl2 involvement in acute peripheral inflammation in vivo. Six hours after an intraplantar injection of zymosan, Cot/tpl2(-/-) mice showed a 47% reduction in myeloperoxidase activity, concomitant with a 46% lower neutrophil recruitment and a 40% decreased luminol-mediated bioluminescence imaging in vivo. Accordingly, Cot/tpl2 deficiency provoked a 25-30% reduction in luminol-mediated bioluminescence and neutrophil recruitment together with a 65% lower macrophage recruitment 4 h following zymosan-induced peritonitis. Significantly impaired levels of G-CSF and GM-CSF and of other cytokines such as TNFα, IL-1β, and IL-6, as well as some chemokines such as MCP-1, MIP-1β, and keratinocyte-derived chemokine, were detected during the acute zymosan-induced intraplantar inflammatory response in Cot/tpl2(-/-) mice. Moreover, Cot/tpl2 deficiency dramatically decreased the production of the hypernociceptive ligand NGF at the inflammatory site during the course of inflammation. Most importantly, Cot/tpl2 deficiency significantly reduced zymosan-induced inflammatory hypernociception in mice, with a most pronounced effect of a 50% decrease compared with wild type (WT) at 24 h following intraplantar injection of zymosan. At this time, Cot/tpl2(-/-) mice showed significantly reduced NGF, TNFα, and prostaglandin E(2) levels compared with WT littermates. In conclusion, our study demonstrates an important role of Cot/tpl2 in the NGF, G-CSF, and GM-CSF production and myeloperoxidase activity in the acute inflammatory response process and its implication in inflammatory hypernociception.

FIGURE 1.

MPO activity in the hindpaws of zymosan-injected WT and Cot/tpl2^−/− mice. A, sections of naive hindpaws from WT and Cot/tpl2^−/− mice stained with Masson's trichrome. Scale bar, 100 μm. B, Western blots of naive intraplantar lysates from WT and Cot/tpl2^−/− mice. C, means ± S.E. (error bars) of the Σ of the photon flux generated by the intraperitoneal injection of luminol at different times after zymosan injection (0.5, 6, and 24 h) in the hindpaws of WT and Cot/tpl2^−/− mice (n = 8 for each type of mice). D, luminol-mediated bioluminescence images of the hindpaws from WT and Cot/tpl2^−/− mice 6 h after the intraplantar zymosan injection. The images have been taken before and between 5 and 45 min after intraperitoneal injection of luminol at 5-min intervals (one representative experiment with one Cot/tpl2^−/− and two from WT mice is shown). E, representation of the photon influx recorded in D. F, measurement of MPO activity in the intraplantar tissue extracts of WT and Cot/tpl2^−/− mice hindpaws 6 h following zymosan (Zym) or just PBS (Cnt) injection. The value 1 is given to O.D./mg protein of WT intraplantar extracts injected only with PBS. The means ± S.E. of three independent experiments of four different pooled tissue extracts from each condition are shown. G, total number of Ly-6G⁺F4/80⁻ isolated from WT and Cot/tpl2^−/− intraplantar tissue 6 h following zymosan injection. The means ± S.E. of four independent experiments of six different pooled digested intraplantar tissues are shown.

FIGURE 2.

Recruitment of inflammatory cells during zymosan-induced peritonitis in WT and Cot/tpl2^−/− mice. Animals were injected intraperitoneally with zymosan (1 mg, 500 μl of PBS), killed 2 or 4 h later, and the peritoneal cells were obtained. A, total number of cells recruited at 2 and 4 h after zymosan-induced peritonitis in WT and Cot/tpl2^−/− mice (2 h, n = 5 each group; 4 h, n = 9 each group). B, representative FACS profile of F4/80⁺Ly-6G⁻ versus Ly-6G⁺F4/80⁻ staining of total isolated intraplantar cells obtained 6 h after the zymosan injection (left panel, WT; right panel, Cot/tpl2^−/− mice). Data are representative of six independent WT and Cot/tpl2^−/− animals. C, means ± S.E. (error bars) of the Ly-6G⁺F4/80⁻ (neutrophils) and F4/80⁺Ly-6G⁻ (macrophages) cells recruited at 4 h from the peritoneum of WT and Cot/tpl2^−/− mice (n = 6 for each group). D, total number of Ly-6G⁺F4/80⁻ and F4/80⁺Ly-6G⁻ cells calculated using the formula: % positive cells × 0.01 × total cell number obtained in A. The graph shows the means ± S.E. of six animals/group.

FIGURE 3.

Zymosan induction of cytokines and chemokines in intraplantar tissues of WT and Cot/tpl2^−/− mice. The concentration of MIP-1β, MIP-1α, KC, MCP-1, GM-CSF, G-CSF, TNFα, IL-1β, and IL-6 in the intraplantar tissue extracts of WT and Cot/tpl2^−/− mice was determined 2, 5, and 24 h after the injection of zymosan by FACS analysis with the CBA system. The means ± S.E. (error bars) of three independent experiments of three different pooled tissues extracts from each condition are shown.

FIGURE 4.

Involvement of Cot/tpl2 in the production of NGF, PGE₂, and LTB₄ synthesis by zymosan. A, concentration of NGF in the intraplantar tissue extracts of WT and Cot/tpl2^−/− mice hindpaws determined by ELISA 0, 2, 5, and 24 h after the injection of zymosan. The means ± S.E. (error bars) of three independent experiments of three different pooled tissues extracts from each condition are shown. B, Western blot showing COX2 expression in WT and Cot/tpl2^−/− BMDM stimulated with zymosan (10 μg/ml). As control of total protein, loaded total ERK2 levels were tested. One representative experiment of the four performed is shown. The PGE₂ concentration was measured in the supernatant of BMDM cells stimulated for 8 and 24 h. The means ± S.E. of two independent determinations of three different pooled supernatants from each condition are shown. C, PGE₂ concentration in intraplantar tissues of WT and Cot/tpl2^−/− mice 0, 2, 5, and 24 h after injection with zymosan (300 μg, 30 μl). D, expression of COX2 mRNA detected by quantitative RT-PCR. Total RNA was extracted from zymosan-injected intraplantar tissues. The means ± S.E. of two independent determinations of four different pooled tissue extracts from each condition are shown. E, LTB₄ concentration in the samples determined in C. C and E, means ± S.E. of two independent measurements of four different pooled tissue extracts from each condition.

FIGURE 5.

Paw edema following the induction of inflammation by zymosan in hindpaws of WT and Cot/tpl2^−/− mice. A, width and thickness of zymosan (left) and PBS (right) injected hindpaws from WT and Cot/tpl2^−/− mice before and at different times after injection. The values of hindpaw width and thickness are expressed as the value between the diameter measured after induction of inflammation divided by the basal (before) diameter in millimeters. Data are shown as the means ± S.E. (n = 7), the value obtained before the injection being considered as 100%. B, quantification of Evans Blue extravasation over 4 h in zymosan-injected hindpaws from WT and Cot/tpl2^−/− mice. Values of Evans Blue extravasation are expressed as the values between the inflamed plantar tissue and its corresponding control. The graph represents means ± S.E. (error bars) (n = 9 for each condition).

FIGURE 6.

Cot/tpl2^−/− mice develop less hyperalgesia than the WT. Mechanical hypernociception was assessed following zymosan injection (300 μg, 30 μl) over a period of 48 h in WT (n = 8) and Cot/tpl2^−/− mice (n = 7). A, time-response curve of zymosan-induced hypernociception in WT and Cot/tpl2^{−/−−/−} mice. The results are presented as the means ± S.E. (error bars). **, p = 0.004, two-way ANOVA, Tukey post hoc. B, area under the curve graph. The changes in nociceptive threshold and withdrawal latency were calculated for each mouse as the area under the curve versus time (over a 48-h period), and the results are presented as the means ± S.E. *, p = 0.019, Student's t test.