Mice lacking COX-2 in neutrophil has an exacerbated pain profile.
Figure 1A shows the intensity of mechanical hyperalgesia (variation of the threshold) 1h, 2h, 3h, 4h, 5h, 6h, and 24h after CG injection Cox-2fl/fl and Cox-2fl/fl: Mrp8cre+/-. Two-way ANOVA showed significant differences between groups (F7, 91=12.93; p ≤ 0.0001) and time (F7, 91=166.1; p ≤ 0.0001). The post hoc Bonferroni’s multiple comparison test revealed statistical differences between groups 1h (p ≤ 0.05), 2h, 3h, 4h, 5h, and 6h (p ≤ 0.001) except for 24h following the CG injection, showing that neutrophil Cox-2fl/fl: Mrp8cre+/- mice display more intense CG-induced hyperalgesia. This effect can also be observed by analyzing the area under the curve (p ≤ 0.001), showed in figure 1B. Note that 3h after CG injection we observe the peak of hyperalgesia in both groups and, because of this, we use this time point for all following experiments. Figure 1C shows the intensity of hyperalgesia 3h after CG injection for male and female, showing that both Cox-2fl/fl:Mrp8cre+/- gender display more intense CG-induced hyperalgesia (unpaired T-test; male: t=8.800 df=13, p ≤ 0.0001; female: t=3.974 df=10, p = 0.0026). It is important to mention that there is no statistical difference between males and females from the same group.
<Figure 1 near here>
The lack of COX-2 in neutrophils triggers an over-release of pro-inflammatory cytokines during inflammation
After observing higher inflammatory pain intensity in Cox-2fl/fl:Mrp8cre+/- group, we decided to evaluate the profile of pro-inflammatory cytokines released in the inflamed tissue of these mice. Figure 2A-D shows the level of cytokines released after Veh or CG injection in the hind paw for Cox-2fl/fl and Cox-2fl/fl:Mrp8cre+/- groups. We can observe that Cox-2fl/fl:Mrp8cre+/- mice over-release CXCL-1 and IL-1β when compared to Cox-2fl/fl. Figure 2A shows the CXCL-1 released level for both groups. One-way ANOVA showed significant differences between groups (F3,20 =27.83; p ≤ 0.0001). The post hoc Tukey`s multiple comparison test revealed statistical differences between Cox-2fl/fl Veh and Cox-2fl/fl CG (p ≤ 0.01); also, there is statistical differences between Cox-2fl/fl:Mrp8cre+/- Veh and Cox-2fl/fl:Mrp8cre+/- CG (p ≤ 0.001). Additionally, it is important to highlight the significant differences in CXCL1 release between the Cox-2fl/fl and Cox-2fl/fl:Mrp8cre+/- CG groups (p ≤ 0.001), (Cox-2fl/fl: Veh = 14.18 ± 2.268mg/mL; CG = 79.91 ± 11.98mg/mL; Cox-2fl/fl:Mrp8cre+/-: Veh = 46.48 ± 8.081mg/mL; CG = 162.3 ± 19.19mg/ mL). Figure 2B shows the IL-1β released level for both groups. One-way ANOVA showed significant differences between groups (F3,15 =71.30; p ≤ 0.0001). The post hoc Tukey`s multiple comparison test revealed statistical differences between Cox-2fl/fl Veh and Cox-2fl/fl CG (p ≤ 0.05); also, there is statistical differences between Cox-2fl/fl:Mrp8cre+/- veh and Cox-2fl/fl:Mrp8cre+/- CG (p ≤ 0.001), and once again, we highlight the differences in IL-1β release between the Cox-2fl/fl and Cox-2fl/fl:Mrp8cre+/- CG groups (p ≤ 0.001) (Cox-2fl/fl: Veh = 517.2 ± 105.3mg/mL; CG = 1773.00 ± 332.6mg/mL; Cox-2fl/fl:Mrp8cre+/-: Veh = 355.0 ± 92.33mg/mL; CG = 5532.0 ± 469.4mg/mL). Figure 2C shows the TNFα level for both groups. One-way ANOVA showed no significant differences between groups (F3,20 =1.744; p ≤ 0.1904) (Cox-2fl/fl: Veh 25.61 ± 4.405mg/mL; CG = 16.82 ± 3.328mg/ mL; Cox-2fl/fl:Mrp8cre+/-: Veh = 32.34 ± 6.803mg/mL; CG = 23.72 ± 4.088mg/mL). Figure 2D shows the IL-10 released level for both groups. One-way ANOVA showed no significant differences between groups (F3,16 = 3.053; p ≤ 0.05). (Cox-2fl/fl: Veh=0.121 ± 0.011 mg/mL; CG = 0.083 ± 0.016 mg/mL; Cox-2fl/fl:Mrp8cre+/- Veh = 0.097 ± 0.015 mg/mL; CG= 0.0595 ± 0.014 mg/mL).
<Figure 2 near here>
Mice lacking COX-2 in neutrophils showed long-lasting hyperalgesia following IL-1β injection.
Considering that Cox-2fl/fl: Mrp8cre+/- mice over-release IL-1β – a key cytokine for inflammatory hyperalgesia – during an inflammatory process, we tested if injecting IL-1β in the hind paw would trigger an exacerbated hyperalgesia in these mice. Our data showed that Cox-2fl/fl: Mrp8cre+/- mice display long-lasting hyperalgesia when compared to Cox-2fl/fl mice. Figure 3A shows the intensity of hyperalgesia at 20 min, 40 min, 60 min, 90 min, 120 min, 150 min, and 180 min after IL-1β injection for Cox-2fl/fl and Cox-2fl/fl: Mrp8cre+/- mice. Two-way ANOVA showed significant differences between groups (F6,54=21.23; p ≤ 0.0001) and time (F6,54=177.5; p ≤ 0.0001). The post hoc Bonferroni’s multiple comparison test revealed statistical differences between groups at two time points 150 min and 180 min after IL-1β injection (p ≤ 0.001). Figure 3B shows the area under the curve (AUC), showing an expressive increase in the hyperalgesia for Cox-2fl/fl: Mrp8cre+/- mice (p ≤ 0.001).
<Figure 3 near here>
COX-2 contributes to neutrophil migration during inflammatory pain.
Considering that leukocyte migration is necessary for the inflammatory hyperalgesia, we investigated the levels of neutrophil migration in CG-induced inflammation by MPO quantification in the hind paw and leukocyte migration by cytometry in the peritoneal washed. Figure 4A shows the quantity of MPO in the hind-paw tissue after Veh or CG injection. Our data showed a MPO increase in both groups after CG injection when compared to Veh (p ≤ 0.001; t=4.586 df=8 and t=7.341 df=7). We also demonstrated that Cox-2fl/fl:Mrp8cre+/- mice showed higher CG-induced MPO increase (p ≤ 0.001; t=4.604 df=8) when compared to Cox-2fl/fl mice (Cox-2fl/fl: Veh = 2.294 ± 0.279 U; CG = 4.432 ± 0.373U; Cox-2fl/fl:Mrp8cre+/-: Veh = 2.050 ± 0.194U; CG = 8.158 ± 0.718U). We further investigate the cell migratory profile for both groups during inflammation. Figure 4C-E shows the peritoneal percentage of Ly6G+CD11b+ neutrophils, CD11b+F4/80+ macrophages, and CD11b+CD11c+ dendritic cells on CD45+ compartment after CG or Veh injection, respectively. Figure 4B shows the representative flow cytometry dot plots of immune cells in peritoneal washing after Veh or CG i.p. injections. Figure 4C shows the percentage of neutrophils in peritoneal washing after Veh or CG i.p. injections for both groups. Our data showed that Cox-2fl/fl (p ≤ 0.01; t=3.630 df=15) and Cox-2fl/fl:Mrp8cre+/- (p ≤ 0.01; t=3.229 df=15) mice showed higher neutrophil infiltrate after CG injection when compared to Veh (Cox-2fl/fl: Veh = 13.16 ± 3.571; CG = 18.74 ± 1.689; Cox-2fl/fl:Mrp8cre+/-: Veh = 23.80 ± 1.554; CG = 34.28 ± 4.305). We also demonstrated that Cox-2fl/fl:Mrp8cre+/- mice exhibit higher neutrophil infiltrate after Veh (p ≤ 0.01; t=3.722 df=14) and CG (p ≤ 0.001; t=4.545 df=16) stimulus when compared to Cox-2fl/fl mice. Figure 5D shows the percentage of macrophages in peritoneal washing after Veh or CG i.p. injections for both groups. Our data showed no difference between groups (Cox-2fl/fl: Veh = 10.32 ± 1.318; CG = 12.76 ± 2.276; Cox-2fl/fl:Mrp8cre+/-: Veh = 8.852 ± 1.778; CG = 10.33 ± 2.407). Figure 5E shows the percentage of dendritic cells in peritoneal washing after Veh or CG i.p. injections for both groups. Our data showed no difference between groups (Cox-2fl/fl: Veh = 3.356 ± 0.991; CG = 2.934 ± 0.271; Cox-2fl/fl:Mrp8cre+/-: Veh = 2.512 ± 0.236; CG = 2.414 ± 0.216).
<Figure 4 near here>
CG-induced hyperalgesia in mice lacking COX-2 in neutrophils is dependent on neutrophil migration.
Considering the higher leukocyte migration in Cox-2fl/fl:Mrp8cre+/- mice, we tested if FC – a leukocyte migration inhibitor – would blocks the inflammatory hyperalgesia even in the Cox-2 absence in this cell-type. Figure 5 shows the intensity of hyperalgesia 3h after CG + Veh or CG + FC injection. Our data showed reduction in hyperalgesia intensity in both, Cox-2fl/fl and Cox-2fl/fl:Mrp8cre+/- FC groups when compared to CG groups, which demonstrates that leukocyte migration is necessary for inflammatory hyperalgesia regardless Cox-2 expression (Cox-2fl/fl: CG = 3.520 ± 0.276; FC = 0.620 ± 279; p< 0,0001; t=7.384 df=8; Cox-2fl/fl:Mrp8cre+/-: CG = 6.226 ± 0.586; FC = 1.478 ± 0.303; p< 0.0001; t=7.192 df=8).
<Figure 5 near here>
Mice lacking COX-2 in neutrophil shows cox-1 overexpression in the hind pawn after CG injection.
Once we observed an increase in inflammatory hyperalgesia intensity in mice lacking Cox-2 in neutrophils, we investigated if Cox isoenzymes would be differently expressed in the inflamed tissue of these mice. Figure 6A shows cox-1 and cox-2 mRNA expression in the hind pawn tissue for Cox-2fl/fl and Cox-2fl/fl:Mrp8cre+/- mice after Veh or CG injection. Our data shows no difference (p = 0.258) in cox-1 expression but statistical difference (p< 0.0001) in cox-2 expression between Veh and CG for Cox-2fl/fl mice (cox-1: Veh = 1.024 ± 0.1159; CG = 1.334 ± 0.2272; t=1.215 df=8; cox-2: Veh = 1.042 ± 0.1578; CG = 6.600 ± 0.6714; t=8.059 df=8). For Cox-2fl/fl:Mrp8cre+/- mice our data shows overexpression (p = 0.0067) of cox-1 after CG injection when compared to Veh, and almost undetected cox-2 expression (cox-1: Veh= 1.011 ± 0.068; CG = 1.467 ± 0.117; t=3.505 df=9; cox-2: Veh = 0.0179 ± 0.0137; CG = 0.0494 ± 0.029; t=0.980 df=8; p = 0.355).
Following this result, we hypothesized that another COX isoenzyme could be signaling replacing COX-2. We confirmed this hypothesis using VS, a specific COX-1 inhibitor, which reduced the inflammatory hyperalgesia in Cox-2fl/fl:Mrp8cre+/- mice, but not in Cox-2fl/fl. Figure 5B shows the intensity of hyperalgesia 3h after CG + Veh or CG + VS injection. Our data showed reduction in hyperalgesia intensity in Cox-2fl/fl:Mrp8cre+/- VS group but not in Cox-2fl/fl when compared to CG group (Cox-2fl/fl : CG = 3.682 ± 0.175; VS = 4.620 ± 0.324; p < 0,05; t=2.545; df=8; Cox-2fl/fl:Mrp8cre+/-: CG = 4,474 ± 0,409; VS = 2,594 ± 0,284; p < 0,01; t=3,771; df=8). Thus, these findings revealed the importance of neutrophils in inflammatory hyperalgesia even in the absence of neutrophil-derived Cox-2, in addition, at least in part, Cox-1 may be mediating hyperalgesia in Cox-2fl/fl:Mrp8cre+/- mice.
<Figure 6 near here>
Mice lacking COX-2 in neutrophil shows cox-1 overexpression and undetected expression of cox-2 after LPS stimulus in vitro.
Aiming to confirm our cell-specific knock out model, we isolated neutrophils from bone marrow and stimulated these cells using LPS in vitro, aiming to mimic an inflammatory environment. Figure 7 shows cox-1 and cox-2 mRNA expression in neutrophil culture from Cox-2fl/fl and Cox-2fl/fl:Mrp8cre+/- mice after LPS stimulus. Our data showed that Cox-2fl/fl:Mrp8cre+/- exhibit cox-1 overexpression when compared to Cox-2fl/fl (p ≤ 0.001; t=13.45 df=8), and, as expected, undetected expression of cox-2 (cox-2+/+: Cox 1 = 1.001 ± 0.021; Cox 2 = 1.017 ± 0.089; cox-2-/-: Cox 1 = 2.092 ± 0.078; Cox 2 = 0.011 ± 0.0005).
<Figure 7 near here>