1. Patients with Deep Surgical Site Infection Have Suppressed Monocyte Function and Increased IRAK-M Expression
 Stephen Manek, Norman Galbraith, Samuel Walker, Sarah Gardner, Jane Carter, Susan Galandiuk, Hiram C. Polk, Jr.
Price Institute of Surgical Research, Hiram C. Polk Jr. MD Department of Surgery, University of Louisville School of Medicine, Louisville, KY1
Introduction
Methods
Results
Following trauma, major surgery, or infection approximately 1 in 6 individuals will have impaired monocyte function and display a decreased ability to clear a subsequent infection, increasing the likelihood of death.
Low levels of HLA-DR expression, a surface marker representing antigen presenting function, is predictive of nosocomial infection.
Decreased TNF-α production (a pro-inflammatory cytokine) in response to LPS demonstrates a suppressed monocyte inflammatory response.
IL-10, an anti-inflammatory cytokine, is typical of “M2” polarization which contributes to monocyte suppression.
Interleukin-1 receptor associated kinase M (IRAK-M) is an intracellular negative regulator of toll-like receptor pathways and could be a potential marker of monocyte dysfunction.
Ideally, physicians should be able to identify which patients have impaired monocyte function and adjust their treatment accordingly.
Monocyte HLA-DR expression (FACS) and cytokine levels (ELISA) were examined at 0h unstimulated and following ex-vivo lipopolysaccharide (LPS) stimulation (100 ng/mL) for 4h (Fig. 1).
Monocytes were isolated by magnetic bead positive selection and mRNA was extracted and analyzed via qRT-PCR, to compare the ratio of normalized mRNA expression (using RNU6) between patient and healthy control samples.
Mann-Whitney-U tests and Unpaired T-tests were used. *p<0.05, # p=0.057.
Patients had lower HLA-DR expression (0h, unstimulated) and after 4 h LPS stimulation compared against healthy controls (p<0.05) (Fig 2).
Decreased monocyte TNF-α production was observed in patients in response to LPS stimulation.
Patients also demonstrated increased circulating levels of plasma IL-10 as compared to the healthy controls (p<0.05).
IRAK-M mRNA
IRAK-M mRNA
Results
Fig 2. Monocyte function with and without LPS stimulation.
0 h unstim. TNF-α
0 h unstim. IL-10
0 h unstim. HLA-DR
4 h LPS stim. HLA-DR
4 h LPS stim. TNF-α
4 h LPS stim. IL-10
Healthy
controls
Initial patient samples
Follow-up patient samples
Initial patient sample
N = 10
N = 15
N = 9
N = 14
N = 8
N = 13
N = 11
Objective
N = 10
N = 10
N = 7
N = 10
N = 10
N = 7
Healthy
Controls
Follow-up patient samples
Healthy
Controls
Initial patient samples
Initial patient samples
Follow-up patient samples
To study impaired monocyte function (as assessed by monocyte
TNF-α production and HLA-DR expression) in patients with deep surgical site infection, as well as levels of IRAK-M gene expression that can inhibit inflammatory pathways.
Fig 3. Monocyte IRAK-M mRNA expression
Methods
Gene expression of IRAK-M, a known negative regulator of inflammation, was consistently upregulated in sick patients as compared to healthy controls (p<0.05) (Fig. 3).
Interestingly, IRAK-M mRNA expression appeared to decrease after clinical recovery (dark gray) although this did not reach statistical significance.
Patients with
Surgical Site Infection
(n=16)
Healthy Volunteers
(n=10)
Parameters of Immune Response:
0h unstim. and after 4 h ex-vivo LPS stimulation
Monocyte Isolation
(magnetic cell sorting)
Cytokines
Surface Markers
mRNA
Fig 4. Proposed mechanism
ELISA
Flow Cytometry
qRT-PCR
Conclusions
Fig 1. Experimental design
Monocytes from patients with deep surgical site infection showed lower levels of HLA-DR expression and TNF-α production following LPS stimulation, as expected.
IRAK-M was up-regulated in patients during infection with a return towards levels of healthy controls following recovery.
This suggests IRAK-M may be a useful biomarker for identifying patients with impaired monocyte function who might benefit from therapeutic intervention to restore immune function and hasten patient recovery.
Following IRB approval and informed consent, peripheral blood was taken from patients with deep surgical site infection and from healthy volunteers. For the patient group, blood samples were taken both during episodes of infection and after clinical recovery (median 60 d).
The majority of patients were hemodynamically stable and were diagnosed with deep surgical site infection, as defined by an abscess in an organ or cavity space. Sources of infection included intra-abdominal abscess (n=12), pneumonia (n=2), perineal abscess (n=1) and line sepsis (n=1).
Patients were aged 25-81, 6:10 (M:F). Healthy volunteers were aged 19-32, 7:3 (M:F).
Acknowledgements
John W. Price and Barbara Thruston Atwood Price Trust