Post-operative infection and sepsis in humans is associated with deficient gene expression of γc cytokines and their apoptosis mediators

Abstract

Introduction: Lymphocyte homeostasis is dependent on the γc cytokines. We hypothesised that sepsis in humans is associated with differential gene expression of the γc cytokines and their associated apoptosis mediators.

Methods: The study population consisted of a total of 60 patients with severe sepsis, 15 with gram negative bacteraemia, 10 healthy controls and 60 patients undergoing elective lung resection surgery. Pneumonia was diagnosed by CDC NNIC criteria. Gene expression in peripheral blood leukocytes (PBLs) of interleukin (IL)-2, 7, 15 and interferon (IFN)-γ, Bax, Bim, Bcl-2 was determined by qRT-PCR and IL-2 and IL-7 serum protein levels by ELISA. Gene expression of IL-2, 7 and IFN-γ was measured in peripheral blood leukocytes (PBL), cultured in the presence of lipopolysaccharide (LPS) and CD3 binding antibody (CD3ab)

Results: IL-2 gene expression was lower in the bacteraemia group compared with controls, and lower still in the sepsis group (P < 0.0001). IL-7 gene expression was similar in controls and bacteraemia, but lower in sepsis (P < 0.0001). IL-15 gene expression was similar in the three groups. Bcl-2 gene expression was less (P < 0.0001) and Bim gene expression was greater (P = 0.0003) in severe sepsis compared to bacteraemic and healthy controls. Bax gene expression was similar in the three groups.In lung resection surgery patients, post-operative pneumonia was associated with a perioperative decrease in IL-2 mRNA (P < 0.0001) and IL-7 mRNA (P = 0.003). IL-2 protein levels were reduced in sepsis and bacteraemia compared to controls (P = 0.02) but similar in pneumonia and non-pneumonia groups. IL-7 protein levels were similar in all groups.In cultured PBLs, IFN-γ gene expression was decreased in response to LPS and increased in response to CD3ab with sepsis: IL-7 gene expression increased in response to LPS in controls and to CD3ab with sepsis; Bcl-2 gene expression decreased in response to combined CD3ab and IL-2 with sepsis.

Conclusions: Patients with infection and sepsis have deficient IL-2 and IL-7 gene expression in PBLs. Aberrant cytokine gene expression may precede the onset of infection.

Figure 1

Cytokine gene expression levels in sepsis (n = 60), bacteraemia (n = 15) and controls (n = 10) as measured with qRT-PCR. IL-2, IL-17, IL-15, IFN-γ, Bcl-2, Bim and Bax are expressed as copy numbers of cytokine mRNA normalised to per 10 million β-Actin mRNA copy numbers. Values represented as medians and centiles 10 to 90% in parenthesis. Between groups analysis is by Wilcoxon Rank Sum test. Pairwise comparisons between control and bacteraemic (*), control and sepsis (#), p values are indicated where positive.

Figure 2

Cytokine protein levels in patients with sepsis (n = 56), bacteraemic (n = 15) and control (n = 10) study groups. Interleukin-2 (IL-2) and Interleukin-7 (IL-7) are expressed as picograms per milliliter (pg/mL). * denotes P-value < 0.05. N denotes number of subjects in each group. All values are median and interquartile range in parenthesis. Between group comparisons is by Wilcoxon Rank Sum test.

Figure 3

Cytokine mRNA gene expression in thoracic surgery patients. Cytokine mRNA gene expression was analysed at baseline Day 0 (24 hours pre surgery), Day 1 (24 hours post surgery) and Day 5 after surgery. POP, post-operative pneumonia (N = 19). Non POP, non post-operative pneumonia (N = 41). IL-2, IL-7, IL-15, Bcl-2 and Bim are expressed as copy numbers of cytokine mRNA per 10 million β-Actin mRNA copy numbers. Values represented as medians and centiles 10 to 90% in parenthesis. Between groups analysis is by Wilcoxon Rank Sum test. N denotes number of patients in each group.

Figure 4

Cytokine mRNA gene expression after in vitro stimulation of PBLs from septic (n = 5) and control (n = 5) patients. Interleukin (IL) -2, 7, Bcl-2 and Interferon gamma (IFN-_γ) gene expression are expressed as normalised copy numbers of cytokine mRNA per 10 million β-Actin mRNA copy numbers is plotted on y axis. PBL's were incubated with medium alone, or stimulated with 1 μg/ml lipopolysaccharide (LPS), or stimulated with 3 μg/ml anti-CD3 monoclonal antibody (CD3) or alternatively stimulated with 1 μg/M PMA and ionomycin. Type of stimulation is plotted on × axis. Figure 1 a, b, c, d in vitro stimulation of PBLs from control and septic patients in absence of recombinant IL-2 (rIL-2) and Figure 1e, f, g, h in vitro stimulation of PBLs after pre-treatment of PBLs with rIL-2.