Adoptive transfer of bone marrow-derived dendritic cells decreases inhibitory and regulatory T-cell differentiation and improves survival in murine polymicrobial sepsis

Abstract

A decrease in the number of dendritic cells (DCs) is a major cause of post-sepsis immunosuppression and opportunistic infection and is closely associated with poor prognosis. Increasing the number of DCs to replenish their numbers post sepsis can improve the condition. This therapeutic approach could improve recovery after sepsis. Eighty C57BL/6 mice were subjected to sham or caecal ligation and puncture (CLP) surgery. Mice were divided into four groups: (i) Sham + vehicle, (ii) Sham + DC, (iii) CLP + vehicle, and (iv) CLP + DC. Bone-marrow-derived DCs (BMDCs) were administered at 6, 12 and 24 hr after surgery. After 3 days, we assessed serum indices of organ function (alanine aminotransferase, aspartate aminotransferase, creatinine, amylase and lipase), organ tissue histopathology (haematoxylin and eosin staining), cytokine [interferon-γ (IFN-γ), tumour necrosis factor-α, interleukin-12p70 (IL-12p70), IL-6 and IL-10] levels in the serum, programmed death-1 (PD-1) expression on T cells, regulatory T-cell differentiation in the spleen, and the survival rate (monitored for 7 days). BMDC transfer resulted in the following changes: a significant reduction in damage to the liver, kidney and pancreas in the CLP-septic mice as well as in the pathological changes seen in the liver, lung, small intestine and pancreas; significantly elevated levels of the T helper type 1 (Th1) cytokines IFN-γ and IL-12p70 in the serum; decreased levels of the Th2 cytokines IL-6 and IL-10 in the serum; reduced expression of PD-1 molecules on CD4(+) T cells; reduced the proliferation and differentiation of splenic suppressor T cells and CD4(+) CD25(+) Foxp3(+) regulatory T cells, and a significant increase in the survival rate of the septic animals. These results show that administration of BMDCs may have modulated the differentiation and immune function of T cells and contributed to alleviate immunosuppression, hence reducing organ damage and mortality post sepsis. Hence, the immunoregulatory effect of BMDC treatment has potential for the treatment of sepsis.

Keywords: dendritic cells; programmed death-1; regulatory T cells; sepsis.

Figure 1

Effect of bone-marrow-derived dendritic cell (BMDC) transfer on organ function and injury. Blood samples were obtained from mice 3 days after sham or caecal ligation and puncture (CLP) surgery and vehicle or BMDC transfer. Using standard laboratory techniques, the samples were tested for: (a) alanine aminotransferase (ALT), (b) aspartate aminotransferase (AST), (c) creatinine, (d) amylase and (e) lipase. The results are presented as the mean ± SD (n = 5 for each group). Error bars indicate the SD. *P < 0·05; **P < 0·01.

Figure 2

Pathological damage in organs. Tissue samples taken 3 days after surgery were prepared by routine histological techniques and stained with haematoxylin & eosin (H&E) for light microscopy analysis. Lung (a i), small intestine (b i), liver (c i), and pancreatic (d i) sections from mice in the caecal ligation and puncture (CLP) + vehicle group. Lung (a ii), small intestine (b ii), liver (c ii), and pancreatic (d ii) sections from mice in the CLP + dendritic cell (DC) group. The extent of pulmonary interstitial oedema; decrease in pulmonary ventilation; and necrosis of liver cells, small intestine villi and pancreatic acinar cells were all markedly reduced in the CLP + DC group, relative to damage in the CLP + vehicle group. Original magnification: × 400. Figures are representative of results from each experimental group.

Figure 3

Effect of bone-marrow-derived dendritic cell (BMDC) transfer on regulatory T-cell differentiation. Spleen mononuclear cells were labelled with monoclonal antibodies against CD4 and CD25, permeabilized, and intracellularly labelled with monoclonal antibody against Foxp3. The percentage of positive cells was determined by flow cytometry. Representative dot plots showing spleen mononuclear cells (a, R1), CD4⁺ T-cell subsets (a, R2), CD4⁺ CD25⁺ Foxp3⁺ regulatory T (Treg) cells (b, R3), and the percentage of CD4⁺ CD25⁺ Foxp3⁺ Treg cells (c). Error bars indicate the SD. **P < 0·01.

Figure 4

Effect of bone-marrow-derived dendritic cell (BMDC) transfer on inhibitory T-cell differentiation. Spleen mononuclear cells were labelled with a mixture of monoclonal antibodies against CD3ε, CD4, CD8a and programmed death 1 (PD-1). The percentage of positive cells was determined by flow cytometry. Representative dot plots showing CD4⁺ PD-1⁺ (a, R1) and CD8⁺ PD-1⁺ (b, R2) T-cell subsets and the percentage of CD4⁺ PD-1⁺ (c) and CD8⁺ PD-1⁺ (d) T cells. Error bars indicate the SD. **P < 0·01.

Figure 5

Effect of bone-marrow-derived dendritic cell (BMDC) transfer on caecal ligation and puncture (CLP) -induced mortality. Mortality in the different experimental groups (20 mice/group) was monitored for up to 7 days. The survival rate for the CLP + DC group was significantly higher than that for the CLP + vehicle group. *P < 0·05, CLP + vehicle versus Sham + vehicle/DC; ^†P < 0·05, CLP + DC versus CLP + vehicle.