Hospital-Acquired Pneumonia After Lung Resection Surgery Is Associated With Characteristic Cytokine Gene Expression

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Original Research CHEST INFECTIONS

Hospital-Acquired Pneumonia After Lung Resection Surgery Is Associated With Characteristic Cytokine Gene Expression Mary White, MB; Ignacio Martin-Loeches, PhD; Matthew W. Lawless, PhD; Michael J. O’Dwyer, PhD; Derek G. Doherty, PhD; Vincent Young, MB; Dermot Kelleher, MD; Ross McManus, PhD; and Thomas Ryan, MB

Background: Infection in humans has been linked with altered cytokine gene transcription. It is unclear whether this phenomenon is a consequence of an established disease process or precedes the infective process. The primary end point of this study was to determine whether hospitalacquired pneumonia (HAP) was associated with differential gene expression of interferon (IFN)-g, tumor necrosis factor (TNF)-a, and IL-23p19. The secondary end point was to identify whether alteration in gene expression preceded the clinical onset of infection. Methods: Sixty consecutive patients undergoing elective thoracic surgery were recruited. HAP was diagnosed as per National Nosocomial Infection Surveillance guidelines. Messenger RNA (mRNA) and protein levels were analyzed preoperatively and 24 h and 5 days postoperatively. Results: Forty-one patients had an uncomplicated recovery. Nineteen patients developed HAP. IL-6, IL-10, IL-12p35, IL-23p19, IL-27p28, TNF-a, and IFN-g mRNA and protein levels of IL-6, IL-23, and IFN-g in peripheral blood leukocytes were analyzed before surgery and 24 h and 5 days postsurgery. IL-23p19 mRNA levels were reduced in the pneumonia group (median, 4.19; 10th-90th centile range, 3.90-4.71) compared with the nonpneumonia group (4.50; 3.85-5.32) day 1 postsurgery (P 5.02). IFN-g mRNA levels were reduced in the pneumonia group (2.48; 1.20-3.20) compared with nonpneumonia group (2.81; 2.10-3.26) (P 5.03) day 5 postsurgery. Results are expressed as log to base 10 copy numbers of cytokine mRNA per 10 million b-actin mRNA copy numbers. All values are given as median and 10th to 90th centile range. Conclusions: Cytokine gene expression is altered immediately following surgery in patients with postoperative HAP. CHEST 2011; 139(3):626–632 Abbreviations: cDNA 5 complementary DNA; ELISA 5 enzyme-linked immunosorbent assay; HAP 5 hospital-acquired pneumonia; IFN-g 5 interferon-g; mRNA 5 messenger RNA; PBL 5 peripheral blood leukocyte; PCR 5 polymerase chain reaction; TNF 5 tumor necrosis factor

surgery is frequently complicated by postThoracic operative hospital-acquired pneumonia (HAP),

with an associated mortality rate of 30%.1 Although risk factors for HAP after thoracic surgery are reported,2 these consist of demographic factors and indices of pulmonary dysfunction, which are largely immutable. Few studies have investigated the inflammatory or immune response after thoracic surgery and none have identified any link between the occurrence of infection in this setting and an underlying immune suppression. The cytokine-mediated immune response may be important in generating an initial predisposition for the occurrence and subsequent outcome of infection. Infection after surgery has been linked with

down-regulation of interferon-g (IFN-g) production by monocytes and inhibition of inducible IL-12 production.3 Trauma patients who develop sepsis exhibit greater IL-10 levels in peripheral blood.4 Furthermore, sepsis after esophageal surgery has been linked with allelic variation of the tumor necrosis factor (TNF) gene.5,6 Similarly, the occurrence of sepsis in patients with infection has been related to increased IL-10 and reduced IFN-g and TNF-a messenger RNA (mRNA) expression compared with patients who tolerate infection without consequent end-organ dysfunction.7 The IL-12 family of cytokines, including IL-23 and IL-27, are important regulators of innate and adaptive

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immune response to infection.8 We recently demonstrated an association between IL-23 and IL-27 gene expression and the occurrence of sepsis in patients with infection.9 Collectively, these and other studies indicate a link between the human host response to infection and diminished cytokine-mediated bactericidal proinflammatory response.3,6 However, these studies focused on patients with established infection or severe sepsis, and at a single time point, and did not report on the initial cytokine response at the onset of infection. Thoracic surgical patients have a high incidence of postoperative pneumonia and provide an ideal model to study the onset of infection. We hypothesize the host response to HAP is associated with diminished bactericidal inflammatory response, identifiable prior to the clinical onset of pneumonia rather than consequential to the inflammatory process. A prospective study was performed to observe changes in cytokine gene expression after thoracic surgery and to determine whether the occurrence of postoperative HAP is related to characteristic expression of cytokines, which are critical mediators of the immune response.

Materials and Methods Patients Sixty patients scheduled for lung resection surgery for noninfective pathology were consented for inclusion. Our institutional ethical review committee approved this study. Patients with HIV infection, taking cytotoxic drugs, or receiving long-term oral steroid therapy were excluded. Patients were examined daily for clinical evidence of HAP. Patients were diagnosed as having an HAP based on the Centers for Disease Control and Prevention definition.10 Treatment decisions for all study participants were made by the attending physician. Antimicrobial prophylaxis was provided for 24 h after surgery as per institutional protocol. Manuscript received February 10, 2010; revision accepted July 9, 2010. Affiliations: From the Department of Anaesthesia and Intensive Care Medicine (Drs White, Martin-Loeches, O’Dwyer, and Ryan), and the Department of Cardiothoracic Surgery (Dr Young), St James Hospital, Dublin, Ireland; Department of Clinical Medicine (Drs White, Lawless, Kelleher, and McManus), and the Department of Immunology (Dr Doherty), Institute of Molecular Medicine, Trinity College Dublin, Dublin, Ireland; and the Critical Care Department (Dr Martin-Loeches), Joan XXIII University Hospital, University Rovira i Virgili, IISPV, CIBER Enfermedades Respiratorias (CIBERes) Tarragona, Spain. Funding/Support: This study was funded by the Royal City of Dublin Hospital Trust Fund and the Association of Anaesthetists of Great Britain and Ireland. Correspondence to: Mary White, MB, Department of Anaesthesia and Critical Care Medicine, St. James Hospital, Dublin 8, Ireland; e-mail: [email protected] © 2011 American College of Chest Physicians. Reproduction of this article is prohibited without written permission from the American College of Chest Physicians (http://www.chestpubs.org/ site/misc/reprints.xhtml). DOI: 10.1378/chest.10-0016 www.chestpubs.org

Blood Sampling Blood sampling was carried out 24 h before and 24 h and 5 days after surgery. The peripheral blood leukocytes (PBLs) were isolated by density gradient centrifugation of supernatant using Lymphoprep (Nycomed Pharma; Oslo, Norway). Cells were lysed and stored at 280°C until RNA extraction. Serum was obtained from whole blood clotted for 30 min. Total RNA Extraction and Reverse Transcription RNA was isolated from lysed PBLs using a commercially available kit (Qiagen; West Sussex, England) following manufacturer’s instructions. To avoid amplification of contaminating genomic DNA, all samples were treated with RNase-free DNase (Qiagen) for 15 min. The quantity and purity of extracted RNA was measured with a spectrophotometer (Eppendorf BioPhotometer; Eppendorf AG; Hamburg, Germany). Total RNA was reverse transcribed as follows: 11.15 mL water containing 500 ng of total RNA was first incubated at 65°C for 10 min. Then 18.85 mL of the reverse transcription mix containing the following components was added: (1) 3 mL 0.1 M dithiothreitol; (2) 4.5 mL dimethyl sulfoxide; (3) 2 mL 100 mM random primers (Invitrogen Corp; Carlsbad, California); (4) 1.25 mL moloney murine leukemia virus reverse transcriptase (Invitrogen Corp); (5) 6 mL 5X first strand buffer (Invitrogen Corp); (6) 1.5 mL 4 mM deoxynucleotide triphosphate mix (Promega Corp; Madison, Wisconsin); and (7) 0.6 mL RNasin (Promega Corp) 10 U/mL. The samples were then incubated at 37°C for 1 h and stored at 220°C. Primers and Probes Primer and probes used in this study were synthesized at Applied Biosystems (Foster City, California). IL-27 (Hs00377366_m1), TNF-a (Hs00174128_m1), and IL-10 (Hs00174086_m1) primers and probes were obtained as a precustomized mix. b-actin, IL-6, IL-23, IFN-g, and IL-12p35 primers and probes were customized as per Stordeur et al.11 Real-Time Polymerase Chain Reaction Polymerase chain reaction (PCR) was carried out in an ABI Prism 7000 (Applied Biosystems). Reactions were performed in triplicate. Thermocycling was carried out in a 20 mL final volume containing: (1) water up to 20 mL; (2) 10 mL Mastermix (Applied Biosystems); (3) 300, 600, or 900 nM forward and reverse primers; (4) 200 nM Taqman Probe (Applied Biosystems; Warrington, England) or 1 mL of precustomized primer/probe mix with default primer and probe concentrations; (5) 0.8 mL standard dilution or 2.4 mL complementary DNA (cDNA). After an initial denaturation step at 95°C for 10 min, temperature cycling was initiated. Each cycle consisted of 95°C for 15 s and 60°C for 60 s, the fluorescence being read at the end of this second step. In total, 40 cycles were performed. Standard Curves and Expression of the Results DNA standards consisted of a cloned PCR product that included the quantified amplicon prepared by PCR from a cDNA population containing the target mRNA. In order to quantify transcript levels a standard curve was constructed for each selected mRNA target from 10-fold serial dilutions of the relevant standard. All standard curves showed correlation coefficients . 0.99. The efficiency of the standard curves for all target cDNA was . 96%. The mRNA copy numbers were then calculated for each patient sample using the standard curve to convert the obtained crossing threshold value into mRNA copy numbers. Results were then expressed in absolute copy numbers after normalization CHEST / 139 / 3 / MARCH, 2011

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against b-actin mRNA (mRNA copy numbers of cytokine mRNA per 10 million b-actin). The efficiency of the standard curves for all target cDNA was . 96%. The mRNA copy numbers were calculated for each patient sample using the standard curve to convert the obtained crossing threshold value into mRNA copy numbers. Results were expressed in absolute copy numbers after normalization against b-actin mRNA (mRNA copy numbers per 10 million b-actin mRNA copy numbers). Cytokine Enzyme-Linked Immunosorbent Assay Serum IL-6, IL-23, and IFN-g concentrations were measured by enzyme-linked immunosorbent assay (ELISA) (R&D Systems; Oxfordshire, England) following manufacturer’s instructions. The lower limit of detection for IL-6 was 1.68 pg/mL, IL-23 was 3.59 pg/mL, and IFN-g was 6.59 pg/mL. All samples were tested in triplicate. Statistical Analysis Discrete variables were expressed as counts (%) and continuous variables as mean and SD, unless stated otherwise; all statistical tests were two-sided with P values , .05 considered significant. Differences in categorical variables were calculated using twosided likelihood ratio x2 test or Fisher exact test, and the MannWhitney U test or Kruskal-Wallis test were used for continuous variables, when appropriate. The significance level of univariate analyses are reported without correction for multiple comparisons. The relation between nonparametric continuous variables was analyzed using Spearman rank correlation. Data analysis was performed using the JMP statistical package (SAS Insitute; Cary, North Carolina).

Results In this study, 19 of 60 patients with a thoracotomy developed HAP day 2 to day 5 postsurgery. Patient demographics, indication for surgery, and preoperative pulmonary dysfunction were similar in patients with HAP and non-HAP groups (Table 1). All patients were extubated at the end of surgery in the operating room and transferred to the postanesthesia recovery area. The duration of surgery and duration of onelung ventilation are listed in Table 1. Preoperative cytokine mRNA levels in PBLs were similar in patients in HAP and non-HAP groups (Table 2). On the first postoperative day, IL-23 mRNA levels were significantly lower in PBLs in HAP compared with non-HAP groups (Table 2). On the fifth postoperative day, the HAP group had lower levels of IFN-g mRNA (Table 2). Cytokine mRNA levels on the first postoperative day were compared with preoperative values by calculating a ratio of preoperative to postoperative levels (ie, preoperative levels divided by postoperative levels), such that a ratio . 1 represents a perioperative decrease in respective cytokine mRNA. When cytokine mRNA levels on the first postoperative day were compared with preoperative baseline cytokine mRNA levels, a greater perioperative decrease in IL-23 mRNA (P 5 .0006) and TNF-a mRNA (P 5 .0044) was noted in patients with HAP. There was no relation between the occurrence of

Table 1—Patient Demographic Data Characteristic Gender, M (F), No. Age, y BMI, kg/m2 Karnofsky index score, mean 6 SD NSCLC Currently smoking Recent cessation Nonsmoker Cardiovascular comorbidity Preoperative FEV1, % Type of operation, No. Lobectomy Pneumonectomy Neoadjuvant chemotherapy Perioperative antimicrobial cover Anesthesia time, min One-lung ventilation time, min Postoperative PPV, No.

Postoperative Non-HAP

Postoperative HAP

P Value

29 (12) 64.5 6 1.34 25.9 6 0.92 90.24 6 1.17

13 (6) 63.7 6 1.9 27.65 6 1.35 88.95 6 1.72

.86 .74 .29 .54

24 (58) 16 (39) 15 (36) 10 (24) 14 (34)

8 (47) 8 (42) 8 (42) 3 (16) 7 (37)

.82 … … .18 .57

82.12 6 2.28

82.34 6 3.36

.95

39 2 5 (12.2)

14 5 2 (10.5)

… .03 .96

41 (100)

19 (100)

ns

233.0 6 40 85.5 6 14.9

238.6 6 52 88.0 6 9.6

.51 .19

1

ns

2

Values given as mean 6 SD or No. (%) unless otherwise indicated. F 5 female; HAP 5 hospital-acquired pneumonia; M 5 male; nonHAP 5 absence of hospital-acquired pneumonia; ns 5 not significant; NSCLC 5 non-small cell lung cancer; PPV 5 positive pressure ventilation.

HAP and perioperative change in cytokine mRNA on day 5 (Table 3). When analyzing these changes in cytokine mRNA, allowing for multiple comparisons of seven cytokine changes at two time points, with 14 comparisons, the perioperative changes in IL-23 on day 1 after surgery would still retain significance, whereas the perioperative change in TNF-a on day 1 after surgery would be of borderline significance. On the first postoperative day, IL-23 mRNA decreased compared with preoperative values in 34 patients; 18 of these 34 patients developed HAP, whereas none of the other patients developed HAP (P 5 .00002). A patient with a decrease in IL-23 mRNA on the first day after surgery had a 2.4-fold (95% CI, 1.7-3.6) relative risk of developing HAP. Likewise, on the first postoperative day, TNF-a mRNA decreased compared with preoperative values in 26 patients; 13 of these 26 patients developed HAP, whereas five of the remaining 33 patients developed HAP (P 5 .005). Patients with a perioperative decrease in TNF-a mRNA had a 2.7-fold (95% CI, 1.3-3.9) relative risk of developing HAP. Nineteen patients had combined decrease in IL-23 and TNF mRNA, and 13 of these developed HAP, whereas only five of the other 38 patients developed HAP (P , .0001). Patients with combined decrease in cytokine mRNA had a 4.7-fold (95% CI, 2.1-10.3) relative risk of developing HAP. Again, allowing for multiple comparisons

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Table 2—Cytokine mRNA Levels 24 h Before Surgery

Day 1

Day 5

Cytokine

Non-HAP

HAP

Non-HAP

HAP

Non-HAP

HAP

No. IL-6 No. IL-10 No. IL-12p35 No. IL-23p19 No. IL-27p28 No. TNF-a No. IFN-g No.

41 3.27 (2.44-4.58) 41 2.75 (1.90-3.53) 38 4.08 (3.74-4.67) 40 4.55 (3.30-5.19) 40 2.56 (1.19-3.0) 37 4.77 (3.70-5.87) 41 2.80 (2.17-3.37) 38

19 3.16 (1.58-4.97) 19 2.86 (2.68-3.43) 19 4.03 (3.68-4.77) 19 4.70 (4.16-5.12) 19 2.60 (2.16-2.81) 18 4.69 (3.70-5.88) 19 2.95 (2.03-3.47) 17

41 2.84 (2.12-3.98) 41 3.31 (2.38-4.03) 39 4.0 (3.60-4.69) 39 4.50 (3.85-5.32) 39 2.49 (1.48-3.09) 40 4.62 (3.86-6.10) 41 2.53 (2.10-3.58) 40

19 2.73 (1.38-4.33) 19 3.51 (2.67-4.25) 19 3.88 (3.28-4.52) 18 4.19 (3.90-4.71)a 18 2.47 (2.00-2.79) 17 4.61 (3.47-5.42) 18 2.52 (1.45-3.20) 18

41 3.10 (1.97-4.80) 33 3.09 (1.84-3.47) 37 3.94 (3.52-4.61) 35 4.57 (3.65-5.25) 39 2.40 (1.51-2.82) 37 4.76 (3.95-5.89) 38 2.81 (2.10-3.26) 35

19 3.03 (2.27-4.77) 15 3.27 (2.26-3.74) 15 3.95 (3.64-4.62) 17 4.5 (3.55-5.60) 15 2.45 (1.88-3.02) 14 4.9 (4.10-5.48) 17 2.48 (1.20-3.2)b 15

Cytokine mRNA levels before surgery and on days 1 and 5 after surgery. IL-6, IL-10, IL-12p35, IL-23, IL-27, TNF-a, IFN-g are expressed as log to base 10 copy numbers of cytokine mRNA per 10 million b-actin mRNA copy numbers. No. denotes number of patients in each group. Values represented as medians, with centiles 10%-90% in parentheses. Between-groups analysis is by Wilcoxon rank sum test. IFN-g 5 interferon-g; mRNA 5 messenger RNA; TNF-a 5 tumor necrosis factor-a. See Table 1 legend for expansion of other abbreviations. aP 5 .02 for HAP vs non-HAP. bP 5 .03 for HAP vs non HAP.

of changes for seven cytokines, the level of significance remains valid. When cytokine mRNA levels on the fifth postoperative day were compared with preoperative values, the reduction in IFN-g expression was greater in patients who developed HAP (n 5 13; median, 2.4; 10th-90th centile, 0.4-66.5) than the non-HAP group (n 5 32; median, 1.04; 10th-90th centile, 0.3-0.8; P 5 .05). However, this change would not retain statistical significance after allowing for multiple comparisons. HAP was observed with greater frequency after pneumonectomy. There was no association between a decrease in IL-23 mRNA, either as an absolute value or as a category, and pneumonectomy. In a multivariate analysis of the relation between HAP and type of surgery and perioperative change in IL-23, both pneumonectomy (P 5 .02) and decrease in IL-23 (P 5 .005) remained independent risk factors for HAP.

Cytokine Protein Measurement by ELISA Cytokine protein measurement by ELISA on IL-6, IL-23, and IFN-g in both groups was carried out preoperatively and on days 1 and 5 postoperatively (Table 4). There was no difference in protein expression between HAP and non-HAP groups and no correlation between cytokine gene and protein expression. When cytokine protein levels on the first postoperative day were compared with preoperative values, there was no relation between the occurrence of HAP and perioperative change in cytokine protein expression on day 1 (Table 5). Discussion In this study, cytokine gene expression was altered immediately after surgery in patients who subsequently developed HAP. Thoracic surgery is

Table 3—Perioperative Changes in Cytokine mRNA on the First Day After Surgery Cytokine mRNA No. IL-23 Decrease in IL-23 No. TNF-a Decrease in TNF-a Decrease IL-23, TNF-a

HAP

Non-HAP

18 2.84 (1.39-7.95) 18/18 (100%) 18 1.63 (0.24-18.6) 13/18 (72%) 13/18 (72%)

39 0.83 (0.1-6.92) 16/39 (41%) 41 0.79 (0.15-3.67) 13/41 (32%) 6/39 (15%)

Relative Risk

P Value

2.4 (1.7-3.6)

.0006 , .0001

2.3 (1.3-39) 4.7 (2.1-10.3)

.004 .005 , .0001

Data represent the ratio of preoperative mRNA to postoperative mRNA (the preoperative value divided by the postoperative value). Values are given as median and 10th to 90th centile range. Comparison is by Wilcoxon rank sum test. Categoric data are presented as percentage in parentheses, with comparison by Fisher exact test. Relative risk refers to the relative risk for HAP in patients with a perioperative decrease in cytokine mRNA. No. denotes number of patients in each group. See Table 1 and 2 legends for expansion of abbreviations. www.chestpubs.org

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Table 4—Cytokine Protein Levels by ELISA Days 0, 1, and 5 Cytokine Protein Level, pg/mL IL-6 Day 0 Day 1 Day 5 IL-23 Day 0 Day 1 Day 5 IFN-g Day 0 Day 1 Day 5

Table 5—Relative Change in Cytokine Protein by ELISA in the First 24 h After Surgery Cytokine

Non-HAP (n 5 36)

HAP (n 5 17)

P Value

54.73 (38.76-286.49) 69.42 (26.43-286.55) 59.59 (14.35-221.37) 56.02 (13.17-238.56) 49.84 (7.34-302.12) 94.09 (2.71-158.44)

.61 .52 .52

83.67 (23.56-486.30) 92.95 (18.67-364.88) 78.41 (11.67-366.66) 87.09 (11.45-831.17) 79.96 (39.67-359.34) 91.63 (9.03-599.11)

.78 .54 .34

27.55 (10.09-67.44) 21.85 (12.06-51.58) 21.41 (17.41-29.02)

.26 .63 .95

23.11 (15.62-41.48) 22.56 (12.91-35.88) 22.03 (14.20-39.02)

Cytokine protein levels in thoracic study groups on days 0, 1, and 5 postsurgery. Values are median and interquartile range. Betweengroup comparisons is by Wilcoxon rank sum test. See Table 1 and 2 legends for expansion of abbreviations.

generally elective surgery, on a sterile body cavity, in patients with a defined disease process, with no infection at the time of surgery, and with a significant incidence of HAP within the first week after surgery. Therefore, it provides an interesting model of the onset of infection after surgery. IFN-g is produced by natural killer cells and the CD4 Th1cells and with TNF-a is pivotal in the generation of a robust bactericidal phagocytic response to infection.12 There is ample evidence that these two cytokines are essential for immune competence. In this regard, humans deficient in IFN-g develop recurring gram-negative infections,13 and carriage of polymorphic IFN-g receptor alleles has been linked to greater risk for TB infection.14 Animal models of sepsis demonstrate the central role of IFN-g in the immune response to sepsis.15 Pharmacologic TNF-a antagonism is associated with increased susceptibility to opportunistic infection16 and prior attempts to treat septic shock with a TNF-a antagonist have proven harmful.17 Lymphocyte TNF-a production is attenuated in families of patients who succumb to meningococcal infection,18 and persistent septic shock is associated with decreased IFN-g and TNF-a gene expression in PBLs.7 Thus, there is a physiologic basis for the link between the occurrence of infection and deficient TNF-a and IFN-g. Members of the IL-12 family of cytokines, which includes IL-23 and IL-27, are master regulators of the immune response to infection.7 IL-12, the prototypic cytokine in this family, is produced by antigenpresenting cells and promotes IFN-g production by both natural killer cells and CD4 cells.19 In this study, there was no relation between IL-12 gene expression in PBLs and the occurrence of infection, whereas

IL-6 IL-23 IFN-g

Non-HAP (n 5 36)

HAP (n 5 17)

0.99 (0.73-1.29) 0.87 (0.11-1.47) 1.09 (0.57-1.60)

1.03 (0.65-2.17) 1.03 (0.44-9.57) 1.10 (0.43-1.32)

Data represent the ratio of preoperative protein to postoperative protein (the preoperative value divided by the postoperative value). All values are given as median and 10th to 90th centile range. P was not significant. Comparison is by Wilcoxon rank sum test. See Table 1 and 2 legends for expansion of abbreviations.

there was a clear relation between the occurrence of infection and a reduction in IL-23 gene expression. These results are in accordance with studies by this group, which investigated gene expression of the IL-12 family of cytokines in patients with severe sepsis.7 Although in vitro studies have reported that IL-12 is an important regulator of IFN-g gene expression, there was no correlation between IL-12 and IFN-g gene expression in this study. In contrast to IL-12, deficient expression of IL-23 in PBLs is associated with the occurrence of infection in this study and with the occurrence of sepsis and adverse outcome in sepsis.9 IL-23 is a pleiotropic cytokine, which acts on many cell lines, including macrophages and CD4 cells.20 The importance of IL-23 in generating an appropriate bactericidal response to infection was analyzed in IL-23-deficient mice; it was apparent that IL-23 deficiency increases mortality in an animal model of pneumonia, and this effect may in turn be related to deficient IL-17 production.21 However, an alternate animal model indicated that IL-17 might be of primary importance in generating an adequate host response to infection in the absence of adequate IFN-g.22 In this study there was a significant correlation between TNF-a and IL-23 gene expression. Thus, it is apparent from these collective human studies of PBL gene expression that the IL-23/TNF-a axis is of considerable importance in the initiation of the human protective immune response to infection. In this study, the prototypic antiinflammatory cytokine IL-10 was not linked with the occurrence of infection, whereas the novel antiinflammatory cytokine IL-27 decreased in patients who developed HAP. The association between IL-27 and IL-10 has been previously described in a laboratory model that defined IL-27 as a regulator of IL-10.23 Thus, in these postoperative patients the onset of infection was linked to a deficiency in the expression of genes encoding proinflammatory proteins that are involved in generating a bactericidal response to infection, rather than a predominant antiinflammatory response. These data differ from those of other studies that reported an excess antiinflammatory cytokine

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response in patients with established infection and sepsis.17,24-26 The discordance between these findings may be explained by methodologic difference; in contrast to prior studies, the present study tested human cytokine response at the time of the onset of clinical infection rather than in patients with established infection. By inference, these results suggest that the well-described antiinflammatory response in infection and sepsis, although important, may not be a primary or causal event.27 In this and other studies of cytokine gene expression in patients with sepsis there was little or no correlation between mRNA and protein levels.7 This discordance in assay results may be related to post-transcriptional regulation of gene expression or alternatively may reflect differential gene expression in disparate cell populations. Thus, the results of assays of cytokine proteins produced predominantly by polymorphs, endothelial cells, and hepatocytes may be discordant with assays of cytokine gene expression in PBL.28 Although assays of cytokine gene expression in specific cell subgroups, such as PBL, may not reflect the global inflammatory response to infection, PBLs are important immune regulatory cells, and in sepsis, PBLs exhibit distinct patterns of gene expression in toll receptorassociated signaling pathways that are different from those of polymorphs.29 However, the predominance of polymorphs in the whole blood of patients with sepsis may obscure the significance of gene expression in the immune regulatory cells included in the buffy coat layer. Further work is required to confirm the cellular source of IL-23, TNF-a, and IFN-g within the buffy coat layer and to identify the cellular site of action of IL-23 in these cells. The extent of tissue trauma obviously has a considerable effect on the inflammatory and immune response to surgery, and this effect may be mediated via cell surface receptors such as the toll family of receptors.30 However, in this study the majority of patients had seemingly uneventful surgery, with relatively brief duration of one-lung ventilation, and were extubated in the operating room. Yet, there was considerable variation in the cytokine gene transcription in response to surgery, and this variation was linked with a propensity to develop postoperative respiratory tract infection. Thus it is plausible that there is an underlying factor that modified cytokine gene transcription after surgery. The mechanism for the individual variability in cytokine gene transcription is not addressed in this study and will require further investigation. The present study has several potential limitations that should be addressed. There is no gold standard definition of HAP and the diagnosis remains controversial. Clinical diagnosis of pneumonia is sensitive but not specific. Invasive diagnostic strategies for HAP generally use bronchoscopy to obtain quantitawww.chestpubs.org

tive cultures. Although more specific, bronchoscopic diagnosis is invasive and is not routine practice in our institution for postoperative thoracotomy patients. We used the Centers for Disease Control National Nosocomial Infection Surveillance pneumonia flow diagram,10 as this algorithm incorporates both clinical and microbiologic data. The simple algorithms based on cytokine gene expression, characterizing patients at high risk for postoperative HAP, may potentially be of use in identifying patients at high risk for developing HAP and initiating therapy with antimicrobial agents in a preemptive manner. This individualized approach to preventing postoperative HAP would represent a significant change from current practice, which consists of treating patients by protocol with an array of interventions, which presently almost exclusively consist of physical interventions, such as chest physiotherapy or incentive spirometry, and which are of dubious merit.31 If this concept proved to be valid then it might be possible to significantly reduce the nosocomial infection rate after thoracotomy, and indeed after other major surgeries. Further study is required to validate this concept. In conclusion, this study suggests a link between postoperative HAP and changes in cytokine gene expression that may occur before the clinical onset of pneumonias. Simple algorithms based on cytokine gene expression may potentially be of use in identifying patients at high risk for developing nosocomial respiratory infection and initiating therapy with antimicrobial agents promptly. Acknowledgments Author contributions: Dr White: contributed to the conception and design of this study, acquisition of data, analysis and interpretation of data, and manuscript preparation. Dr Martin-Loeches: contributed to acquisition and interpretation of data and manuscript preparation. Dr Lawless: contributed to acquisition and interpretation of data and manuscript preparation. Dr O’Dwyer: contributed to acquisition and interpretation of data and manuscript preparation. Dr Doherty: contributed to the conception and design of this study and manuscript preparation. Dr Young: contributed to the conception and design of this study and manuscript preparation. Dr Kelleher: contributed to the conception and design of this study and manuscript preparation. Dr McManus: contributed to the conception and design of this study, acquisition of data, analysis and interpretation of data, and manuscript preparation. Dr Ryan: contributed to the conception and design of this study, acquisition of data, analysis and interpretation of data, and manuscript preparation. Financial/nonfinancial disclosures: The authors have reported to CHEST the following conflicts of interest: Drs White, Young, Kelleher, McManus, and Ryan have a patent pending on the relation of cytokine mRNA levels to the risk of developing nosocomial pneumonia after thoracic surgery. Drs McManus and Ryan are cosupervisors of a project funded by the Irish Health Research Board into gene expression patterns in infection and sepsis. CHEST / 139 / 3 / MARCH, 2011

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Drs Martin-Loeches, Lawless, O’Dwyer, and Doherty have reported to CHEST that no potential conflicts of interest exist with any companies/organizations whose products or services may be discussed in this article. Other contributions: This research was conducted in St James Hospital, Dublin, and the Institute of Molecular Medicine, Trinity College, Dublin, Ireland.

References 1. Dales RE, Dionne G, Leech JA, Lunau M, Schweitzer I. Preoperative prediction of pulmonary complications following thoracic surgery. Chest. 1993;104(1):155-159. 2. Arozullah AM, Khuri SF, Henderson WG, Daley J; Participants in the National Veterans Affairs Surgical Quality Improvement Program. Development and validation of a multifactorial risk index for predicting postoperative pneumonia after major noncardiac surgery. Ann Intern Med. 2001;135(10): 847-857. 3. Döcke WD, Randow F, Syrbe U, et al. Monocyte deactivation in septic patients: restoration by IFN-gamma treatment. Nat Med. 1997;3(6):678-681. 4. Sherry RM, Cue JI, Goddard JK, Parramore JB, DiPiro JT. Interleukin-10 is associated with the development of sepsis in trauma patients. J Trauma. 1996;40(4):613-616. 5. Mira JP, Cariou A, Grall F, et al. Association of TNF2, a TNF-alpha promotor polymorphism, with septic shock susceptibility and mortality: a multicenter study. JAMA. 1996; 282(6):561-568. 6. Azim K, McManus R, Brophy K, Ryan A, Kelleher D, Reynolds JV. Genetic polymorphisms and the risk of infection following esophagectomy. positive association with TNFalpha gene -308 genotype. Ann Surg. 2007;246(1):122-128. 7. O’Dwyer MJ, Mankan AK, Stordeur P, et al. The occurrence of severe sepsis and septic shock are related to distinct patterns of cytokine gene expression. Shock. 2006;26(6):544-550. 8. Hunter CA. New IL-12-family members: IL-23 and IL-27, cytokines with divergent functions. Nat Rev Immunol. 2005; 5(7):521-531. 9. O’Dwyer MJ, Mankan AK, White M, et al. The human response to infection is associated with distinct patterns of interleukin 23 and interleukin 27 expression. Intensive Care Med. 2008;34(4):683-691. 10. Centers for Disease Control and Prevention. CDC definitions for nosocomial infections, 1988. Am Rev Respir Dis. 1989;139(4):1058-1059. 11. Stordeur P, Poulin LF, Craciun L, et al. Cytokine mRNA quantification by real-time PCR. J Immunol Methods. 2002; 259(1-2):55-64. 12. Boehm U, Klamp T, Groot M, Howard JC. Cellular responses to interferon-gamma. Annu Rev Immunol. 1997;15(15):749-795. 13. Zhang SY, Boisson-Dupuis S, Chapgier A et al. Inborn errors of interferon (IFN)-mediated immunity in humans: insights into the respective roles of IFN-alpha/beta, IFN-gamma, and IFN-lambda in host defense. Immunol Rev. 2008;226:29-40. 14. Condos R, Rom WN, Schluger NW. Treatment of multidrugresistant pulmonary tuberculosis with interferon-gamma via aerosol. Lancet. 1997;349(9064):1513-1515.

15. Muenzer JT, Davis CG, Dunne BS, Unsinger J, Dunne WM, Hotchkiss RS. Pneumonia after cecal ligation and puncture: a clinically relevant “two-hit” model of sepsis. Shock. 2006; 26(6):565-570. 16. Dhainaut JF, Vincent JL, Richard C, et al. CDP571, a humanized antibody to human tumor necrosis factor-alpha: safety, pharmacokinetics, immune response, and influence of the antibody on cytokine concentrations in patients with septic shock. CPD571 Sepsis Study Group. Crit Care Med. 1995; 23(9):1461-1469. 17. Fisher CJ Jr, Agosti JM, Opal SM, et al; The Soluble TNF Receptor Sepsis Study Group. Treatment of septic shock with the tumor necrosis factor receptor:Fc fusion protein. N Engl J Med. 1996;334(26):1697-1702. 18. Westendorp RG, Langermans JA, Huizinga TW, et al. Genetic influence on cytokine production and fatal meningococcal disease. Lancet. 1997;349(9046):170-173. 19. Del Vecchio M, Bajetta E, Canova S, et al. Interleukin-12: biological properties and clinical application. Clin Cancer Res. 2007;13(16):4677-4685. 20. Uhlig HH, McKenzie BS, Hue S, et al. Differential activity of IL-12 and IL-23 in mucosal and systemic innate immune pathology. Immunity. 2006;25(2):309-318. 21. Happel KI, Dubin PJ, Zheng M, et al. Divergent roles for IL-23 and IL-12 in host defense against Klebsiella pneumoniae. JEM. 2005;202(6):761-769. 22. Rudner XL, Happel KI, Young EA, et al. Interleukin-23 (IL-23)-IL-17 cytokine axis in murine Pneumocystis carinii infection. Infect Immun. 2007;75(6):3055-3061. 23. Miyazaki Y, Yoshida H. Regulation of immune responses by interleukin-27. Immunol Rev. 2008;226:234-247. 24. van Dissel JT, van Langevelde P, Westendorp RG, Kwappenberg K, Frölich M. Anti-inflammatory cytokine profile and mortality in febrile patients. Lancet. 1998;351(9107):950-953. 25. Oberholzer A, Oberholzer C, Moldawer LL. Sepsis syndromes: understanding the role of innate and acquired immunity. Shock. 2001;16(2):83-96. 26. Bone RC. Sir Isaac Newton, sepsis, SIRS, and CARS. Crit Care Med. 1996;24(7):1125-1128. 27. Osuchowski MF, Welch K, Siddiqui J, Remick DG. Circulating cytokine/inhibitor profiles reshape the understanding of the SIRS/CARS continuum in sepsis and predict mortality. J Immunol. 2006;177(3):1967-1974. 28. White M, Lawless MW, O’Dwyer MJ, et al. Transforming growth factor beta-1 and interleukin-17 gene transcription in peripheral blood mononuclear cells and the human response to infection. Cytokine. 2010;50(3):322-327. 29. Salomao R, Brunialti MK, Gomes NE, et al. Toll-like receptor pathway signaling is differently regulated in neutrophils and peripheral mononuclear cells of patients with sepsis, severe sepsis, and septic shock. Crit Care Med. 2009;37(1):132-139. 30. Zhang Q, Raoof M, Chen Y, et al. Circulating mitochondrial DAMPs cause inflammatory responses to injury. Nature. 2010;464(7285):104-107. 31. Paul WL, Downs JB. Postoperative atelectasis: Intermittent positive pressure breathing, incentive spirometry, and facemask positive end-expiratory pressure. Arch Surg. 1981;116(7): 861-863.

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