Pper quartiles (grey boxes), 95 confidence intervals (T-bars) and possible outliers (u) for each aggressive group per cytokine. Significant group difference was determined using student 10781694 T-test with Bonferroni correction (a #0.005) and bootstrap (see methods). CPA indicates the chronic physical aggression SPDP Crosslinker site trajectory group and CG the control group. MANOVA combining all 10 cytokines: F(10) = 2.9, P = 0.019. *** P#0.0001, ** P#0.001, * P#0.005, # P#0.01 from Student T-test (two-tailed). doi:10.1371/journal.pone.0069481.gmany confounders into the analyses. We did adjust for one of the most likely confounder, family adversity. Childhood family adversity is a well known risk factor for chronic physical aggression [4] as well as immune response deficits [39]. Even with our small samples size, the significant group differences for cytokine levels were maintained when we adjusted for childhood family adversity in the regression analysis. As expected the two groups were also significantly different on other variables that are known to be strongly associated with chronic physical aggression trajectories from childhood to adolescence: childhood hyperactivity, adolescence physical violence and adulthood criminal behavior (Table 1) [2,5]. Although cytokine levels have been shown to associate with psychiatric diseases such as major depression [51] the two groups of males were not significantly different on levels of anxiety and presence of psychiatric diagnoses (Table 1). We also determined whether physical health problems could explain the cytokine leveldifferences between the two groups. Two members of the control group had cardiovascular disease and two others had respiratory disease. Excluding these subjects from our MedChemExpress SPDP Crosslinker analysis did not change the significant cytokine differences observed between the two groups. We quantified CRP levels, a well-known marker of infection, and found no differences between CPA and control groups (Table 1). Because our small sample size prevents the use of many confounders, we attempted to control for the three main confounders; family adversity, hyperactivity and CRP levels. Results showed that the CPA group was still significantly associated with lower level of two cytokines (IL-4 and IL-8). There were no differences in age between the groups and no significant correlations were found between age and cytokine levels. Taken together, these results suggest that chronic physical aggression during childhood is a predictor of cytokine levels during early adulthood.Aggression and Cytokine Levels in PlasmaDiurnal variation has been reported for IL-6 [52], TNF-a [53], IL-4 [54], IL-13 [55], IFNc, IL-10 and IL-1 [56]. In general, their levels peak at night and/or early morning. To account for theses variations, all the blood samples were taken during daytime between 13:00 and 20:00. Future studies are needed to determine whether similar results would be obtained for IL-1a, IL-4, IL-6, IL-8 and IL-10 when samples are taken at different time points during the day. However, the relatively high correlation between samples at 26 and 28 years (R = 0.554, P = 1.48E-17) suggests that one daytime sample is a relatively robust assessment.ConclusionsThis study has several implications. The results suggest that cytokines may be involved in chronic physical aggression, hence that a peripheral immune component may play a key role in regulating these behavioral states. We also showed that measuring the levels of a panel of 4 cytokines in plas.Pper quartiles (grey boxes), 95 confidence intervals (T-bars) and possible outliers (u) for each aggressive group per cytokine. Significant group difference was determined using student 10781694 T-test with Bonferroni correction (a #0.005) and bootstrap (see methods). CPA indicates the chronic physical aggression trajectory group and CG the control group. MANOVA combining all 10 cytokines: F(10) = 2.9, P = 0.019. *** P#0.0001, ** P#0.001, * P#0.005, # P#0.01 from Student T-test (two-tailed). doi:10.1371/journal.pone.0069481.gmany confounders into the analyses. We did adjust for one of the most likely confounder, family adversity. Childhood family adversity is a well known risk factor for chronic physical aggression [4] as well as immune response deficits [39]. Even with our small samples size, the significant group differences for cytokine levels were maintained when we adjusted for childhood family adversity in the regression analysis. As expected the two groups were also significantly different on other variables that are known to be strongly associated with chronic physical aggression trajectories from childhood to adolescence: childhood hyperactivity, adolescence physical violence and adulthood criminal behavior (Table 1) [2,5]. Although cytokine levels have been shown to associate with psychiatric diseases such as major depression [51] the two groups of males were not significantly different on levels of anxiety and presence of psychiatric diagnoses (Table 1). We also determined whether physical health problems could explain the cytokine leveldifferences between the two groups. Two members of the control group had cardiovascular disease and two others had respiratory disease. Excluding these subjects from our analysis did not change the significant cytokine differences observed between the two groups. We quantified CRP levels, a well-known marker of infection, and found no differences between CPA and control groups (Table 1). Because our small sample size prevents the use of many confounders, we attempted to control for the three main confounders; family adversity, hyperactivity and CRP levels. Results showed that the CPA group was still significantly associated with lower level of two cytokines (IL-4 and IL-8). There were no differences in age between the groups and no significant correlations were found between age and cytokine levels. Taken together, these results suggest that chronic physical aggression during childhood is a predictor of cytokine levels during early adulthood.Aggression and Cytokine Levels in PlasmaDiurnal variation has been reported for IL-6 [52], TNF-a [53], IL-4 [54], IL-13 [55], IFNc, IL-10 and IL-1 [56]. In general, their levels peak at night and/or early morning. To account for theses variations, all the blood samples were taken during daytime between 13:00 and 20:00. Future studies are needed to determine whether similar results would be obtained for IL-1a, IL-4, IL-6, IL-8 and IL-10 when samples are taken at different time points during the day. However, the relatively high correlation between samples at 26 and 28 years (R = 0.554, P = 1.48E-17) suggests that one daytime sample is a relatively robust assessment.ConclusionsThis study has several implications. The results suggest that cytokines may be involved in chronic physical aggression, hence that a peripheral immune component may play a key role in regulating these behavioral states. We also showed that measuring the levels of a panel of 4 cytokines in plas.
