Granulocyte macrophage colony-stimulating factor and interferon gamma act through different mechanisms in reversing post-operative immune suppression

Introduction: We have previously demonstrated that the incidence of infection following major abdominal surgery is 35 %1 and is associated with the extent of post-operative immune suppression2. It remains unclear which clinically available immune stimulant may best suited as an adjunct to prevent post-operative infection.

Objectives: To determine whether Granulocyte macrophage colony-stimulating factor (GM-CSF) and interferon gamma (IFNγ) act through different pathways in reversing defects in monocyte antigen presentation following major abdominal surgery.

Methods: Serum was collected from 12 patients undergoing elective abdominal surgery (Research ethics approved). The median patient age was 68 years (IQR 48-74), median operation time was 270mins (IQR 207-320) and 50 % (6) developed nosocomial infection.

Pooled healthy peripheral blood mononuclear cells (PMBCs) were incubated with media containing 30 % serum taken either pre-operatively or 24 hrs post operatively and then with the addition of GM-CSF (10 ng/ml) and INFg 250 IU/ml).

Monocyte human leucocyte antigen-DR (mHLA-DR) membrane density (geometric mean fluorescent intensity) was characterised using flow cytometry following a 20-hour incubation. Cells were then sorted on a BD FACS ARIA IIIu system using CD14 positive selection. Messenger RNA (mRNA) was extracted from the sorted population (>90 % purity post sorting). Genes of interest were quantified using polymerase chain reaction (PCR) using TaqMan® labelled primers and real time PCR (ABI HT7900).

Flow cytometry data was analysed using FlowJo. Continuous variables were analysed using a Wilcoxin signed-rank test (JMP (version 11) statistical software).

Results: PMBCs incubated with post-operative serum demonstrated a significant reduction in mHLA-DR membrane density (Fig. 19, p = 0.001). The reduction in mHLA-DR density was prevented when co-incubated with GM-CSF and IFNγ (Fig. 19). Incubation with IFNγ but not GM-CSF increased expression of HLA-DRα chain (p = 0.01), Cathepsin S (CTSS) (p = 0.001), suppressor of cytokine signalling 3 (SOCS3) (p = 0.01) and March 1 (p = 0.002) (Fig. 20).

Conclusions: These results suggest that these immune stimulants, GM-CSF and IFNγ, exert their effects on monocyte antigen presentation through different signalling pathways. The increased gene expression associated with IFNγ may be indicative of potential therapeutic benefit in reversing post-operative immune suppression.