The neuropeptide neuromedin U promotes autoantibody-mediated arthritis

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Abstract

Introduction: Neuromedin U (NMU) is a neuropeptide with pro-inflammatory activity. The primary goal of this study was to determine if NMU promotes autoantibody-induced arthritis. Additional studies addressed the cellular source of NMU and sought to define the NMU receptor responsible for its pro-inflammatory effects.Methods: Serum containing arthritogenic autoantibodies from K/BxN mice was used to induce arthritis in mice genetically lacking NMU. Parallel experiments examined whether NMU deficiency impacted the early mast-cell-dependent vascular leak response induced by these autoantibodies. Bone-marrow chimeric mice were generated to determine whether pro-inflammatory NMU is derived from hematopoietic cells or stromal cells. Mice lacking the known NMU receptors singly and in combination were used to determine susceptibility to serum-transferred arthritis and in vitro cellular responses to NMU.Results: NMU-deficient mice developed less severe arthritis than control mice. Vascular leak was not affected by NMU deficiency. NMU expression by bone-marrow-derived cells mediated the pro-arthritogenic effect. Deficiency of all of the known NMU receptors, however, had no impact on arthritis severity and did not affect the ability of NMU to stimulate intracellular calcium flux.Conclusions: NMU-deficient mice are protected from developing autoantibody-induced inflammatory arthritis. NMU derived from hematopoietic cells, not neurons, promotes the development of autoantibody-induced inflammatory arthritis. This effect is mediated by a receptor other than the currently known NMU receptors. © 2012 Rao et al.; licensee BioMed Central Ltd.

Figures

  • Figure 2 NMU-deficient mice have mast cells, leukocytes, platelets and an intact early vascular response to arthritogenic antibodies. (A) Mast cells were enumerated in sections of skin obtained from control B6 (n = 3) and NMU-KO (n = 4) mice and stained with toluidine blue. Cells were counted in five ×40 HPF per mouse and averaged. Plotted values represent the number of mast cells per HPF in the indicated groups (mean ± SEM). (B) Complete blood counts from control B6 and NMU-KO mice were performed. Numbers shown are × 109/L ± SD, with four mice per group. (C) Serum from K/BxN mice was injected intravenously into NMU-KO mice (red) or littermate control mice (black). Vascular leak in the hindpaw was monitored by intravital confocal microscopic evaluation of extravasation of the intravascular tracer, Angiosense 680. Each line represents one mouse. B6: C57BL/6; HPF: high power field; KO: knockout; MFI; mean fluorescence intensity; NMU: neuromedin U; SD: standard deviation; SEM: standard error of the mean.
  • Figure 1 NMU promotes autoantibody-mediated arthritis . Serum from arthritic K/BxN mice was injected into NMU-KO mice or littermate control mice on days 0 and 2. The development of arthritis was assessed using (A) arthritis scores and (B) ankle thickening measurements. NMU-KO mice had significantly lower arthritis scores (P < 0.001, repeated-measures ANOVA) and ankle thickening (P = 0.001, repeated measures ANOVA), leading to the conclusion that arthritis severity increases more quickly in wildtype mice relative to the NMU-KO mice. Data plotted are means ± SEM from 10 mice per group compiled from three independent experiments. P-values for individual timepoints calculated using twotailed Student’s T-test are also depicted: *P < 0.05; **P < 0.01; ***P < 0.001. (C) Representative ankle sections from the indicated mice at the peak of arthritis severity were stained with H&E. Arrows indicate inflammatory infiltrates. Original magnification ×5. (D) Serum was collected from the mice at the end of the experiment, diluted 1:900, and assayed by ELISA for the presence of anti-GPI IgG. Data plotted are arbitrary ELISA absorbance values (means ± SEM) from seven mice per group compiled from two independent experiments; P = 0.78. ANOVA: analysis of variance; ELISA: enzyme-linked immunosorbent assay; H&E: hematoxylin and eosin stain; GPI: glucose-6-phosphate isomerase; IgG, immunoglobulin G; KO: knockout; NMU: neuromedin U; SEM: standard error of the mean.
  • Figure 3 NMU production by bone marrow-derived cells promotes autoantibody-mediated arthritis. Bone marrow chimeric mice were generated in the four indicated combinations. Following bone marrow engraftment, serum from K/BxN mice was injected on days 0 and 2 and the development of arthritis was assessed at the indicated timepoints. Data plotted are means ± SEM and represent one of two independent experiments with a total of eight mice per genotype. B6®B6 versus NMU-KO®B6, P = 0.006; B6®B6 versus NMU-KO®NMU-KO, P = 0.022; B6®NMU-KO versus NMU-KO®B6, P = 0.007; B6®NMU-KO versus NMU-KO®NMU-KO, P = 0.027. There was no statistical difference between the two groups in which B6 mice were donors (P = 0.999) or between the two groups in which NMU-KO mice were donors (P = 0.880) (repeated-measures analysis of variance followed by post-hoc Tukey’s multiple comparison test). B6: C57BL/6; NMU: neuromedin U; SEM: standard error of the mean.
  • Figure 4 NMUR1 and NMUR2 are dispensable for the induction of autoantibody-mediated arthritis. Serum from K/BxN mice was injected into mice lacking NMUR1, NMUR2, both NMUR1 and NMUR2, or littermate controls animals on days 0 and 2. The development of arthritis was assessed by determination of (A) arthritis score and (B) ankle thickening. Data plotted are means ± SEM and are compiled from four experiments with eight to fourteen animals per genotype. For arthritis score (A), there were no statistical differences between the four groups. For ankle thickening (B), ankle thickening scores increased slightly more rapidly in the NMUR1-/- mice relative to the control mice (P = 0.042) and relative to the NMUR2-/- mice (P = 0.015). There were no statistically significant differences between the other groups of mice (repeatedmeasures analysis of variance followed by post-hoc Tukey’s multiple comparison test). SEM: standard error of the mean.
  • Figure 5 Mice lacking all known NMU receptors remain susceptible to autoantibody-mediated arthritis. Serum from K/BxN mice was injected into mice lacking NTSR1 or mice lacking NMUR1, NMUR2, and NTSR1 on days 0 and 2 (100 μL/injection). The development of arthritis was assessed by determination of (A) arthritis score and (B) ankle thickening. Data plotted are means ± SEM and represent one of three independent experiments with a total of eight to fifteen mice per group. There were no statistically significant differences between the groups (repeated-measures analysis of variance followed by post-hoc Tukey’s multiple comparison test). NMU: neuromedin U; SEM: standard error of the mean.
  • Figure 6 NMU triggered calcium flux remains intact in splenocytes lacking all known NMU receptors. Splenocytes from C57BL/6 mice (black) or mice lacking NMUR1, NMUR2 and NTSR1 (red) were loaded in vitro with the calcium sensitive dye Indo-1 and then stimulated with NMU at the 60 second timepoint at the indicated concentrations. Intracellular calcium flux was measured with flow cytometry and is plotted as the ratio of fluorescence emission at 405 nm (bound Indo-1) to fluorescence emission at 495 nm (free Indo-1) over time. Data are representative of three independent experiments. NMU: neuromedin U.

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Rao, S. M., Auger, J. L., Gaillard, P., Weissleder, R., Wada, E., Torres, R., … Binstadt, B. A. (2012). The neuropeptide neuromedin U promotes autoantibody-mediated arthritis. Arthritis Research and Therapy, 14(1). https://doi.org/10.1186/ar3732

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