Histones-mediated lymphocyte apoptosis during sepsis is dependent on p38 phosphorylation and mitochondrial permeability transition
Authors: Zhen-Hui Guo
Shu-Yuan Ni
Gui-Ming Chen
Ping Chang
Yu-Sheng Li
Jing Cai
Zhan-Guo Liu
Journal: PLoS ONE (2013)
Discipline: Multidisciplinary
Keywords: Lymphocyte apoptosis; extracellular histones; sepsis; p38 phosphorylation; mitochondrial injury
Affiliations: Department of ICU, Southern Medical University, Zhujiang Hospital, Guangzhou, China.

Abstract:  Lymphocyte apoptosis is one reason for immunoparalysis seen in sepsis, although the triggers are unknown. We hypothesized that molecules in plasma, which are up-regulated during sepsis, may be responsible for this. In this study, peripheral lymphocyte apoptosis caused by extracellular histones was confirmed both in mouse and human primary lymphocytes, in which histones induced lymphocyte apoptosis dose-dependently and time-dependently. To identify which intracellular signal pathways were activated, phosphorylation of various mitogen-activated protein kinases (MAPKs) were evaluated during this process, and p38 inhibitor (SB203580) was used to confirm the role of p38 in lymphocyte apoptosis induced by histones. To investigate the mitochondrial injury during these processes, we analyzed Bcl2 degradation and Rhodamine 123 to assess mitochondrial-membrane stability, via cyclosporin A as an inhibitor for mitochondrial permeability transition (MPT). Then, caspase 3 activation was also checked by western-blotting. We found that p38 phosphorylation, mitochondrial injury and caspase 3 activation occurred dose-dependently in histones-mediated lymphocyte apoptosis. We also observed that p38 inhibitor SB203580 decreased lymphocyte apoptotic ratio by 49% (P&lt;0.05), and inhibition of MPT protected lymphocytes from apoptosis. Furthermore, to investigate whether histones are responsible for lymphocyte apoptosis, various concentrations of histone H4 neutralization antibodies were co-cultured with human primary lymphocytes and plasma from cecal ligation and puncture (CLP) mice or sham mice. The results showed that H4 neutralization antibody dose-dependently blocked lymphocyte apoptosis caused by septic plasma in vitro. These data demonstrate for the first time that extracellular histones, especially H4, play a vital role in lymphocyte apoptosis during sepsis which is dependent on p38 phosphorylation and mitochondrial permeability transition. Neutralizing H4 can inhibit lymphocyte apoptosis, indicating that it could be a potential target in clinical interventions for sepsis associated immunoparalysis. 

Retracted: Yes

PubPeer comments:
 December 2020 retraction. The Retraction Notice is found here: https: journals.plos.org plosone article?id=10.1371 journal.pone.0244473 and reads:  Following the publication of this article [1], concerns were raised regarding the flow cytometry data presented in Fig 2 and Fig 3, the sample size employed in the study, and the statistical analyses reported: * The 0 u00b5g ml panel of Fig 2A and the 0 h panel of Fig 2B look similar. * The 100 u00b5g ml panel and the 200 u00b5g ml of Fig 2A, and the 2 h panel of Fig 2B look similar. * The GAPDH panel of Fig 3A looks similar to the GAPDH panel of Fig 4B. Raw image data were provided in support of these panels, including four independent replicates of the p-p38 experiment and two replicates of the Bcl2 experiment. These data suggest that the wrong image was reported for GAPDH in Fig 4B, and that the GAPDH panel shown in Fig 3A is not the matched internal control for the p38 and p-p38 data included in the figure. * The sample group represented in Fig 2 and Fig 3 include only three replicates per condition, with heterogeneous variances between the four groups, raising concerns about the power of the study. A sample size computation to determine if the study is powered to test the hypothesis was not reported in the article, and a statistical advisor commented that the study was not sufficiently powered to support the conclusions drawn in the study. * Concerns were raised about the use of parametric analyses (multiple Student t-tests) in Figs. 2 and 3 as the data did not conform to a normal distribution. The statistical advisor indicated that non-parametric tests ought to have been used instead, and that an analysis by ANOVA of the available results from the original experiments did not yield statistically significant results. In response to the first two points, above, the authors provided comments, dot plots and quantification data, and offered replacement panels. However, their comments, data, and replacement panels did not fully resolve the concerns. The authors indicated that the raw.fcs data are no longer available for the flow cytometry experiments reported in Fig 2. In post-publication discussions, the authors noted that they used the following formula to compute sample size: n = u03a82 (u03a3(Si2) K) [u03a3(Xiu2013X)2 (K-1)] [2u20134]. The authors noted that they determined that a sample size of three was sufficient to test the studyu2019s hypothesis. In response to the fifth bullet point, above, the authors noted that in their view non-parametric tests should not be used for sample sizes 10, and that when they reanalysed the available data from the original experiments using one-way ANOVA they obtained statistically significant results supporting dose-dependent and time-dependent effects. A second independent statistical advisor reviewed their reanalyses and indicated that the sample sizes within the groups are too small to allow for an estimation of between-group variation, and that the error bars show within subject variance as opposed to within group variance. While some image issues were addressed by the original data provided in post-publication discussions, concerns remain about the overall preparation of the figures, statistical analyses and whether the studyu2019s results adequately support the articleu2019s conclusions. As a result, the PLOS ONE Editors retract this article. ZGL, SYN, GMC, PC, and YSL agree with the retraction. JC and ZHG either did not respond directly or could not be reached. Here is an illustration of the duplications noted for Figure 2, as reported to the journal in May 2014. ![file] ,