Epigenetic regulation of dendritic cell-derived interleukin-12 facilitates immunosuppression after a severe innate immune response

Abstract

Patients who survive sepsis have significant deficiencies in their immune responses caused by poorly understood mechanisms. We have explored this phenomenon by studying dendritic cells (DCs) recovered from animals surviving severe peritonitis-induced sepsis, using the well-established cecal ligation and puncture (CLP) model. Immediately after the initiation of sepsis there is a depletion in DCs from the lung and spleen, which is followed by repopulation of these cells back to the respective organs. DCs recovered from surviving animals exhibited a significant and chronic suppression of interleukin-12 (IL-12), a key host defense cytokine. The suppression of DC-derived IL-12 persisted for at least 6 weeks after CLP and was not due to immunoregulatory cytokines, such as IL-10. Using chromatin immunoprecipitation (ChIP) techniques, we have shown that the deficiency in DC-derived IL-12 was due to epigenetic alterations. Specifically, IL-12 expression was regulated by stable reciprocal changes in histone H3 lysine-4 trimethylation (H3K4me3) and histone H3 lysine-27 dimethylation (H3K27me2), as well as changes in cognate histone methyltransferase (HMT) complexes on the Il12p35 and Il12p40 promoters. These data implicate histone modification enzymes in suppressing DC-derived IL-12, which may provide one of the mechanisms of long-term immunosuppression subsequent to the septic response.

Figure 1

Depletion of tissue mDCs followed by reconstitution after experimental peritonitis. (A) At different time points after CLP procedure, spleen and lung were collected and dispersed. The cells were stained with FITC–anti-CD11c, PerCP-Cy5.5–anti-CD11b and PE–anti-I-A^b. Myeloid dendritic cells (mDCs) were characterized as CD11c⁺CD11b⁺MHCII^hi. *P ≤ .05 compared with mDC number observed in spleen or lung from sham mice. Error bars represent SEM. (B) At day 11 after CLP procedure, spleens were collected and dispersed. Expression of CD40, CD80, CD86, and MHCII were investigated on the cell surface of CD11c⁺CD11b⁺ mDCs. Black line indicates isotype control; dashed line, sham group; gray line, CLP group.

Figure 2

Impaired IL-12 production by splenic DCs and lung DCs day 11 after experimental peritonitis. (A) Purified splenic DCs from septic or control mice on day 11 after surgery were stimulated by a series of TLR agonists. Splenic DC mRNA levels of the 2 IL-12 subunits and IL-10 were determined by Taqman. (B) Splenic DC protein levels of IL-12p70 and IL-10 were measured by Bio-Plex. (C) Purified lung DCs from septic or sham mice on day 11 after surgery were stimulated by a series of TLR agonists, and protein levels of IL-12p70 and IL-10 were measured by Bio-Plex. Results are representative of 3 to 5 independent experiments and are expressed as mean plus or minus SEM. *P ≤ .05 compared with cytokine levels measured in splenic DCs from sham mice.

Figure 3

Deficient IL-12 production in postseptic DCs was not due to excessive IL-10 or other extrinsic effector molecules. Purified splenic DCs from septic or control mice on day 11 after surgery were stimulated by LPS, in the absence or presence of 1 or 10 μg/mL anti-IL-10 antibody. Protein levels of IL-12p70 and IL-10 were determined by Bio-Plex. Results are representative of 3 independent experiments. *P ≤ .05 compared with cytokine levels measured in splenic DCs from sham mice.

Figure 4

DC IL-12 suppression is a long-term consequence of severe experimental sepsis. Purified splenic DCs from septic or control mice 6 weeks after surgery were stimulated with a number of TLR agonists. IL-12 and IL-10 expression was determined at the mRNA level (A) and protein level (B). Results represent 3 independent experiments. *P ≤ .05 compared with cytokine levels measured in splenic DCs from sham mice. Error bars represent SEM.

Figure 5

Th2-polarized capacity of postseptic splenic DCs. Purified splenic DCs from septic or control mice on day 11 after surgery were pulsed with 5 μg/mL OVA_323–339 peptide and cocultured with splenic CD4⁺ T cells isolated from naive TCR transgenic OT II mice. Twenty-four hours later, the supernatants were collected to measure cytokine protein levels by Bio-Plex. Results represent 3 independent experiments and are expressed as means plus or minus SEM. *P ≤ .05 compared with cytokine protein levels in the supernatants of coculture between CD4⁺ T cells and sham splenic DCs.

Figure 6

Alterations in histone H3 methylation on Il12 promoters in postseptic splenic DCs. Splenic DCs were isolated from sham and CLP mice at either day 11 (A) or 6 weeks (B) after surgery. ChIP assay was performed to determine the histone H3 methylation status at the promoter regions of Il12p35 and Il12p40, respectively. The results shown are representative of 2 to 3 independent experiments and are expressed as means plus or minus SEM. *P ≤ .05 compared with splenic DCs from sham mice.

Figure 7

Altered recruitment of HMT complexes at Il12 promoters in postseptic splenic DCs. Splenic DCs were isolated from sham and CLP mice at either day 11 (A) or 6 weeks (B) after surgery. Using appropriate antibodies directed against the core components of the MLL and PRC2 complexes, recruitment of the core components to the promoter regions of Il12p35 and Il12p40 was determined by ChIP assay. The results shown are representative of 2 to 3 independent experiments and are expressed as means plus or minus SEM. *, α P ≤ .05 compared with splenic DCs from sham mice.