High levels of circulating GM-CSF+CD4+ T cells are predictive of poor outcomes in sepsis patients: a prospective cohort study

Abstract

Granulocyte colony-stimulating factor (GM-CSF), produced by CD4⁺ T cells, has recently been implicated in the pathogenesis of inflammatory diseases, such as multiple sclerosis and juvenile arthritis. However, the role of GM-CSF-producing CD4⁺ T cells in sepsis remains unknown. This study reports peripheral changes in GM-CSF-producing CD4⁺ T cells in septic patients and the possible underlying mechanism by which GM-CSF influences the outcome of sepsis. Forty-three septic patients, 20 SIRS patients, and 20 healthy controls were enrolled in this study and followed for 28 days to assess mortality. We measured the peripheral frequency of GM-CSF⁺CD4⁺ T cells and recorded their associated relationship with disease progression. Our data demonstrated that peripheral GM-CSF-producing CD4⁺ T cells were significantly higher in septic patients than in both SIRS patients and healthy controls. These cells exhibit a memory phenotype and impaired IFN-γ-secreting capacity in sepsis patients. Using a receiver operating curve analysis with 8.01% as a cut-off point, the percentage of GM-CSF⁺CD4⁺ T cells could predict the outcome of septic patients^. Combined with the increase in GM-CSF-producing CD4⁺ T cells, inflammatory cytokines IL-1β and IL-6 were also upregulated. Using an in vitro neutrophil model, we found that GM-CSF inhibited C3aR expression, while inducing IL-8 production. Furthermore, this effect was transferrable in plasma from sepsis patients and was attenuated by inhibition of GM-CSF using an anti-GM-CSF antibody. These results indicate that GM-CSF-producing CD4⁺ T cells may serve as a marker of sepsis severity. Thus, targeting GM-CSF overproduction may benefit sepsis patients.

Keywords: CD4+ T cells; GM-CSF; SIRS; sepsis.

Fig. 1

GM-CSF is primarily produced by CD4⁺ T cells. a Representative dot plots illustrating the source of GM-CSF in lymphocyte population. b Pooled data showing the proportion of CD3⁺ and CD3⁻ T cells from GM-CSF-positive cells. c Pooled data showing the proportion of CD4⁺ T cells, CD8⁺ T cells, NK cells, and B cells from GM-CSF-positive cells. d Representative dot plots showing expression of GM-CSF in different lymphocyte populations. e Pooled data illustrating expression of GM-CSF among CD3⁺ T cells and CD3⁻ T cells in different groups. f Pooled data showing expression of GM-CSF in CD4⁺ T cells, CD8⁺ T cells, NK cells, and B cells in different groups. Each group included eight subjects for pooled data collection. *P < 0.05; **P < 0.01

Fig. 2

Phenotype and cytokine production of GM-CSF⁺CD4⁺ T cells. a Representative dot plots showing expression of CD45RO and CD27 on GM-CSF⁺CD4⁺ T cells. b Pooled data showing expression of CD45RO and CD27 on GM-CSF⁺CD4⁺ T cells. c Representative dot plots showing expression of IFN-γ and IL-17A in the population of GM-CSF⁺CD4⁺ T cells. d Pooled data showing expression of IFN-γ in GM-CSF⁺ CD4⁺ T cells in different groups. e Representative dot plots showing expression of IFN-γ in CD4⁺ T cells and CD8⁺ T cells. f Pooled data showing expression of IFN-γ in CD4⁺ T cells in different groups.  g Pooled data showing expression of IFN-γ in CD8⁺ T cells in different groups. Each group included eight subjects for pooled data collection. *P < 0.05; **P < 0.01

Fig. 3

Dynamics of GM-CSF⁺ CD4⁺ T-cell expression in sepsis patients. a Pooled data showing expression of GM-CSF⁺ CD4⁺ T cells in healthy controls and patients with SIRS and sepsis. b, c Increasing GM-CSF⁺ CD4⁺ T-cell expression is associated with sepsis severity. d Differential expression of GM-CSF⁺ CD4⁺ T cells in blood and ascites from patients with sepsis caused by intra-abdominal infection. *P < 0.05; **P < 0.01

Fig. 4

Increase of GM-CSF⁺CD4⁺ T-cell expression is positively associated with sepsis severity. a, b GM-CSF⁺CD4⁺ T-cell levels are positively correlated with neutrophil counts and SOFA scores in sepsis patients. c Receiving operating curve of GM-CSF⁺ CD4⁺ T cells on Day 0 for predicting mortality. d Kaplan–Meier survival curves of septic patients showing that patients with high levels of GM-CSF⁺CD4⁺ T cells ( > 8.01%) have increased mortality compared with patients with low levels of GM-CSF⁺ CD4⁺ T cells ( ≤ 8.01%). Follow-up period: 28 days. P < 0.01

Fig. 5

Pro-inflammatory cytokine production in sepsis patients is associated with increased GM-CSF⁺ CD4⁺ T cells. a Pooled data showing that production of pro-inflammatory cytokines, including IL-1β, IL-6, and TNF-α, is associated with sepsis outcome. b Sepsis patients with high proportions of GM-CSF⁺CD4⁺ T cells ( > 8.01%) have increased expression of IL-1β and IL-6 compared with patients with low proportions of GM-CSF⁺ CD4⁺ T cells ( ≤ 8.01%). *P < 0.05; NS no significance

Fig. 6

Increased GM-CSF production is associated with decreased C3aR expression and increased IL-8 production in neutrophils. a–c Representative dot plots and pooled data showing expression of C3aR on neutrophils following incubation with different doses of GM-CSF. d C3aR on healthy neutrophils incubated with sepsis patient plasma before and after blocking with an anti-GM-CSF antibody. e–g Representative dot plots and pooled data showing production of IL-8 in neutrophils following incubation with different doses of GM-CSF. h IL-8 in healthy neutrophils incubated with sepsis patient plasma before and after blocking with anti-GM-CSF antibody. *P < 0.05; **P < 0.01