Impaired CD4+ T-cell proliferation and effector function correlates with repressive histone methylation events in a mouse model of severe sepsis

Abstract

Immunosuppression following severe sepsis remains a significant human health concern, as long-term morbidity and mortality rates of patients who have recovered from life-threatening septic shock remain poor. Mouse models of severe sepsis indicate this immunosuppression may be partly due to alterations in myeloid cell function; however, the effect of severe sepsis on subsequent CD4(+) T-cell responses remains unclear. In the present study, CD4(+) T cells from mice subjected to an experimental model of severe sepsis (cecal ligation and puncture (CLP)) were analyzed in vitro. CD4(+)CD62L(+) T cells from CLP mice exhibited reduced proliferative capacity and altered gene expression. Additionally, CD4(+)CD62L(+) T cells from CLP mice exhibit dysregulated cytokine production after in vitro skewing with exogenous cytokines, indicating a decreased capability of these cells to commit to either the T(H)1 or T(H)2 lineage. Repressive histone methylation marks were also evident at promoter regions for the T(H)1 cytokine IFN-gamma and the T(H)2 transcription factor GATA-3 in naïve CD4(+) T cells from CLP mice. These results provide evidence that CD4(+) T-cell subsets from post-septic mice exhibit defects in activation and effector function, possibly due to chromatin remodeling proximal to genes involved in cytokine production or gene transcription.

FIGURE 1

Percentages and total numbers of CD4+ T cells and T cell subsets in the spleen of postseptic mice. Spleens from sham surgery (“sham”) or cecal ligation and puncture (“CLP”) mice were analyzed via flow cytometry for the presence of CD4+ T cells and various CD4+ T cell subsets. Total numbers of cells were obtained using a hemacytometer and trypan blue staining for the enumeration of viable cells. A) Percentage and B) total numbers of viable lymphocytes were calculated using forward and size scatter profiles for gating on viable lymphocytes. C) Percentage and D) total numbers of CD4+ T cells were obtained from the viable cell gate using antibodies to CD3e and CD4. E) Percentages and F) total numbers of CD44^hi CD62L+ T cells, and G) percentages and H) total numbers of CD44^lo CD62L+ T cells were obtained from the CD4+ T cell gate. Data presented represents the mean ± SEM, representative of two separate experiments, n=5-6 per group. (*) = p<0.05 vs. sham.

FIGURE 2

Surface marker profiles of splenic CD4+ CD62L+ T cells from sham and CLP mice at 14 days following surgery. Spleens from (A) sham and (B) CLP mice 14 days following surgery were analyzed via flow cytometry for the presence of CD4+ CD62L+ T cells, and these cells were analyzed for the surface expression of CD44 and CCR7. Flow diagrams are representative of spleens from individual animals, n=5-6 mice per group. Data is representative of two separate experiments.

FIGURE 3

CD4+ CD62L+ T cells from CLP mice exhibit decreased proliferative capacity in vitro in response to polyclonal stimulus. (A) CD4+ CD62L- and (B) CD4+ CD62L+ T cells from sham and CLP mice were isolated from spleens 14 days following surgery utilizing bead antibodies and magnetic columns (MACS), and were stimulated for 72 hours in vitro with 1μg/ml plate-bound αCD3 and 3 μg/ml soluble αCD28 in 96-well flat-bottom plates. Where indicated, cell culture media was supplemented with 10 U/ml recombinant IL-2. Data presented represents the mean ± SEM, representative of three separate experiments utilizing pooled spleens from 5-6 mice per group. C) Representative flow diagrams of in vitro restimulated CD4+ CD62L+ T cells from sham and CLP mice. Following stimulation with αCD3/αCD28, cells were analyzed for viability by flow cytometry using antibodies to CD3e. and CD4 (for gating), and LIVE/DEAD dye exclusion (Invitrogen). Non-viable cells are identified by bright staining with the LIVE/DEAD dye. D) Percentage of viable CD4+ cells (LIVE/DEAD^lo) in cultures of restimulated CD4+ CD62L+ T cells from sham and CLP mice. Data represents the mean ± SEM of triplicate cultures using pooled spleens from 5-6 mice per group. (*) = p<0.05 vs. sham.

FIGURE 4

Phosphorylation of signal transduction proteins in sham and CLP CD4+ CD62L+ T cells following in vitro polyclonal stimulus. CD4+ CD62L+ T cells from sham and CLP mice were isolated from spleens 14 days following surgery utilizing bead antibodies and magnetic columns (MACS) and were cultured for the indicated timepoints (0-20 hours) in the presence of αCD3/αCD28. At the indicated timepoints, total cellular protein was isolated using cell lysis reagents (Bio-Rad), the protein lysate was clarified via centrifugation, and analyzed for the relative abundance of both total and phosphorylated (A) JNK and (B) ERK1/2 using a multiplex bead assay technique (Luminex). Y-axis values represent the relative ratio of phosphoprotein to total protein in each sample. Values represent the mean ± SEM of triplicate cell cultures for each timepoint, with cells isolated from the spleens of 3-6 mice per group. (*) = p<0.05 vs sham at each individual timepoint indicated.

FIGURE 5

mRNA expression in splenic sham and CLP CD4+ CD62L+ T cells during ex vivo rest and restimulation. CD4+ CD62L+ T cells from sham and CLP mice were isolated from spleens 14 days following surgery utilizing bead antibodies and magnetic columns (MACS), and were either (A) rested in minimal media or (B) restimulated with 1μg/ml plate-bound αCD3 and 3μg/ml soluble αCD28 for 6 hours directly ex vivo. Following either rest or restimulation, total RNA from cell cultures was isolated utilizing a spin column method (Qiagen) and converted to cDNA following the manufacturer's protocol (SA Biosciences). Gene expression was then analyzed using a 96-well superarray containing primers for genes involved with immune cell activation and effector function (T_H1-T_H2-T_H3 superarray, SA Biosciences). Plates were analyzed using a ABI standard 7500 real-time PCR system, and data was analyzed using the manufacturer's web-based software suite. Data reported represents genes that were up- or down-regulated above the average amount for all genes, and p-values below 0.05 were considered statistically significant. mRNAs that were not significantly up- or down-regulated are not shown. Values represent the mean of two separate experiments, n=3 replicate plates per experimental condition.

FIGURE 6

Cytokine expression of splenic CD4+ CD62L+ T cells following in vitro skewing in the presence of exogenous cytokines. CD4+ CD62L+ T cells from sham and CLP mice were isolated from spleens 14 days following surgery utilizing bead antibodies and magnetic columns (MACS) and were cultured for 4 days in the presence of polyclonal stimulus (αCD3/αCD28) and exogenous cytokines. (A) For T_H1 skewing, cells were cultured in the presence of rIL-12 (10ng/ml) and αIL-4 (10μg/ml). (B) For T_H2 skewing, cells were cultured in the presence of rIL-4 (10ng/ml) and αIL-12 and α IFN-γ (both at 10μg/ml). Following skewing, cells were enumerated using a hemacytometer and vital dye, re-plated in equivalent numbers and rested for 3 days in minimal media, and were then restimulated with αCD3/αCD28 for an additional 48 hours. Cell culture supernatants were harvested and analyzed utilizing a multiplex cytometric bead assay (Luminex). Data presented represents the mean ± SEM, representative of two separate experiments, n=3 replicate wells for each cell type. (*) = p<0.05 vs. sham.

FIGURE 7

Methylation of histones associated with promoter regions of genes involved in T_H lineage commitment. CD4+ CD62L+ T cells from sham and CLP mice were isolated from spleens 14 days following surgery utilizing bead antibodies and magnetic columns (MACS), and were analyzed via chromatin immunoprecipitation (ChIP) assay using antibodies directed to (A) methylation of lysine 4 on histone 3 (H3K4) or (B) methylation of lysine 27 on histone 3 (H3K27) ChIP-enriched genomic DNA was analyzed via quantitative real-time PCR for the promoter regions of indicated genes, and were compared to a pre-enriched sample (“input”) to generate relative values. Data presented represents the mean ± SEM, representative of three separate experiments, n=3 replicates. (*) = p<0.05 vs. sham.