Figure 1.
α-NETA selectively inhibits chemerin-stimulated CMKLR1 association with β-arrestin2.
(A) Chemerin triggered the association of β-arrestin2 with CMKLR1 in a concentration-dependent manner, as measured by enzymatic complementation assay and quantified by chemiluminescence. For each point the mean ± SEM of n = 9 independent experiments is shown. (B) α-NETA was tested for potency in inhibiting chemerin-mediated β-arrestin2 association with CMKLR1. CMKLR1/β-ARR2 CHO cells were incubated with the indicated concentrations of α-NETA and 7 nM recombinant chemerin (background signal was evaluated in the absence of α-NETA and chemerin). α-NETA was tested for selectivity by evaluating inhibition of chemerin-mediated β-arrestin2 association with GPR1 or CXCL12-mediated β-arrestin2 association with CXCR7. For each point the mean ± SEM of n = 18-19 independent experiments is shown for CMKLR1 and CXCR7, and the mean ± SEM of triplicate wells for one experiment is shown for GPR1. (C) α-NETA was tested for potency and selectivity in inhibiting chemerin-mediated CMKLR1+ cell migration. CMKLR1+ L1.2 cells, CCR9+ MOLT4 cells, CXCR4+ U937 cells, and CXCR5+ NC-37 cells were incubated with the indicated concentrations of α-NETA and then tested for transwell migration to their cognate attractant ligands (7 nM chemerin, 250 nM CCL25, 100 nM CXCL12, 150 nM CXCL13, respectively). Chemotaxis for each cell type was normalized to its maximum migration signal, and cell migration at each concentration of α-NETA was compared with vehicle (0.1% DMSO) for inhibition. For each point the mean ± SEM of 3–5 wells from n = 1–3 independent experiments is shown. *p<0.05 by Student's t-test.
Table 1.
α-NETA target selectivity.
Table 2.
Structure-activity relationship: charged ethyltrimethylamine critical for target potency.
Figure 2.
α-NETA was spiked into 10% captisol (A) or mouse plasma (B) and incubated at the indicated temperatures. At the indicated times, the samples were quenched and analyzed by mass spectrometry. The stability of Benfluorex (positive control compound) was also determined (B). Mean ± SD of triplicate wells for each point is displayed.
Figure 3.
α-NETA is safe and suppresses clinical EAE.
(A) C57BL/6 mice were injected s.c. with α-NETA (10 mg/kg) or vehicle daily for 16 days. The mice were then euthanized and vital organs weighed. Each symbol represents an individual mouse, and the bars indicate mean ± SEM, n = 3 mice per group. (B) EAE was induced in C57BL/6 mice by active immunization with MOG35-55/CFA. Mice received α-NETA (10 mg/kg daily, s.c. injection) beginning at the time of disease induction and were monitored daily for clinical disease as described in Materials and Methods. Control mice received either captisol vehicle or no treatment. The pooled data from five independent experiments with 7–10 mice per group is displayed, mean clinical score ± SEM. For days 1–15, n = 52 mice per group; for days 16–27, n = 24–25 mice per group. * p<0.05 by Mann-Whitney U test. (C) EAE was induced in C57BL/6 mice by passive transfer of MOG35–55-reactive lymphocytes derived from actively immunized EAE mice. Recipient mice received either vehicle or α-NETA (10 mg/kg) by daily s.c. injection beginning at the time of transfer and were scored daily for clinical disease. The pooled data from three independent experiments with 3–5 mice per group is displayed, mean clinical score ± SEM. * p<0.05 by Mann-Whitney U test.
Table 3.
Clinical EAE in actively immunized mice treated with α-NETAa.
Table 4.
Clinical EAE in adoptive transfer model treated with α-NETAa.
Figure 4.
Reduced histological EAE in α-NETA-treated mice.
(A) Brain and spinal cord tissues were harvested 16 days after active EAE induction from mice treated with α-NETA or vehicle control. Histological changes in meninges (left panel) and parenchyma (right panel) were evaluated as described in Materials and Methods. Results are pooled from two independent experiments with similar results, each with 4–6 mice per group. mean ± SEM, *p<0.05 as by Student's t test. (B) Representative spinal cord sections are shown from actively immunized vehicle- (left panel) or α-NETA- (right panel) treated mice that were euthanized at 16 days p.i. Meningeal and parenchymal mononuclear cell infiltrates typical of acute EAE in the spinal cord of a vehicle-treated mouse (left panel). Less meningeal and parenchymal infiltration, as well as reduced myelin loss (indicated by Luxol fast blue staining intensity), in the spinal cord of α-NETA-treated mice with EAE (right panel). Bar, 50 µm. (C) At day 16 p.i., mononuclear cells isolated from the CNS and spleen of EAE mice treated with α-NETA or vehicle control were enumerated by flow cytometry. n = 7 mice/group; mean ± SEM; *p<0.05 by Student's t test. (D) CNS mononuclear cells were isolated at day 16 p.i. from EAE mice treated with α-NETA or vehicle control. Cells were then stained with fluorophore-labeled monoclonal antibodies to identify the indicated leukocyte subsets: T cells (CD3+), B cells (B220+), dendritic cells (DC; CD3-B220-NK1.1-CD11c+) and macrophages (Mac; F4/80+CD11b+). *p<0.05 by Student's t test. Two independent experiments yielded similar results.
Table 5.
α-NETA limits accumulation of NK cells in the spleen during EAEa.
Figure 5.
Recall proliferation and cytokine responses of lymphocytes from α-NETA-treated mice and induction of EAE by adoptive transfer of MOG-reactive lymphocytes.
MOG35–55/CFA-immunized mice received α-NETA or captisol vehicle once daily. Ten days after immunization, spleen cells and draining LN cells were harvested and re-stimulated in vitro with the indicated concentrations of MOG35–55. After 72 h of stimulation, (A) proliferation and (B) cytokine production by spleen cells (left panel) or LN cells (right panel) were evaluated. Proliferation data are presented as mean cpm ± SEM (triplicate wells); ELISA data are presented as mean cytokine concentration ± SEM (triplicate wells). (C) EAE was induced in C57BL/6 mice by passive transfer of MOG35–55-reactive lymphocytes derived from vehicle-treated (n = 4) or α-NETA-treated mice (n = 7) as described in the Materials and Methods. The pooled data from two independent experiments is displayed, mean clinical score + SEM.
Figure 6.
α-NETA suppresses clinical EAE in CCRL2 KO mice.
(A) EAE was induced in CCRL2 KO mice by active immunization with MOG35-55/CFA. Mice received α-NETA (10 mg/kg daily, s.c. injection) beginning at the time of disease induction and were monitored daily for clinical disease. Control mice received either captisol vehicle or no treatment. The pooled data from two independent experiments with 5–6 mice per group is displayed, mean clinical score ± SEM. * p<0.05 by Mann-Whitney U test.
Table 6.
Clinical EAE in actively immunized CCRL2 KO mice treated with α-NETAa.
Table 7.
Clinical EAE in actively immunized CMKLR1 KO mice treated with α-NETAa.