Nosocomial infections (NIs), also often called care-related infections, still pose a major problem as recently confirmed in a large investigation (1). Their prevalence is now around 5% in hospitalized patients, and up to 19% in critically ill patients. In the absence of either proper hygiene or immune suppressive drug administration, clinicians readily accept immune paralysis as a cause of their occurrence (2–4). NIs are also thought to be associated with transient immune cellular defects (5) whose improvement is expected to ameliorate the outcome of the initial disease. The high prevalence of NI in previously healthy persons undergoing a major medical stress suggests the existence of multifactorial mechanisms able to trigger common innate immune defects that favor the occurrence of care-related infections. Multiple trauma provides an attractive model to study the time-dependent immune consequences of an acute and severe disease (early after its onset) for two reasons: the exact beginning of the stress is usually defined; trauma frequently occurs in healthy young persons, which excludes confounding factors such as associated diseases or treatments. As in other severe injuries, the immune response to multiple trauma includes not only a severe hyperinflammatory response called systemic immune inflammatory response syndrome (SIRS) but also immune paralysis often leading to NI (1–5).
Taking into account that some transient innate defense dysfunction may be related to the innate immune system (6, 7) and severity of the shock could be a factor involved in the occurrence of NI, we focused our attention on chromogranin A in the plasma (pCGA) (8). CGA is a protein shown to connect the stress-activated adrenal medulla and immunity (9), and pCGA is a predictor of severity in critically ill patients (9). More recently, pCGA was evaluated after burn trauma showing that high concentration predicts organ dysfunction (10). CGA is enclosed in storage vesicles of chromaffin cells from the adrenal medulla and released with catecholamines upon splanchnic stimulation in vitro and in vivo(11). This acidic protein is also present in neutrophils (12), and under the action of proteases, it is converted into numerous peptides with physiologically relevant functions (13, 14). VS-I (CGA1–76), the major natural CGA-derived peptide (13), has already been recognized as a biomarker of severity in acutely ill patients (15).
In this study we postulated that CGA processing would play a role in driving some depression in monocytes’ responses after trauma-related injury. We used both the clinical setting of trauma in humans and an in vitro model of NF-kappa B and AP-1 responses in genes upregulation in monocytes. Our data demonstrate a previously unidentified role of CGA in the occurrence of NI through one of its derived peptide on immune cells. These findings support the notion that preventing the proteolysis of CGA may attenuate the immune depression after trauma.
MATERIALS AND METHODS
CGA assessment in plasma from trauma patients
This study was approved by our institutional review board for human experimentation. Over a period of 1 year, trauma victims requiring critical care were prospectively screened and included in this study if formal consent for participation could be obtained from the patient or next of kin. Exclusion criteria were as follows: age under 18 and known reason for increased CGA release (i.e., steroid treatment, neuroendocrine tumor, etc.), independently of acute stress. Mortality was defined as death occurring before day 28 after admission. Diagnosis of NI was based on classical criteria: the early-onset NIs used in our study were infections detected after 48 h of hospital admission in patients without previous contact with healthcare services and not related with disease incubation that could have started before admission (16).
Organ failure scores were assessed during the first days after admission (SAPS II, ISS, SOFA, delta Sofa max). Healthy controls were staff.
Preparation of blood samples
Blood samples were collected as early as possible within 6 h of admission by vascular puncture into plasma-separator tubes (Becton Dickinson, France), immersed in ice or stored at 4°C until transported to the laboratory. Plasma was separated by centrifugation at 1,500 g for 10 min at room temperature and stored in 200 μL aliquots at −20°C until analysis. In nine randomly chosen patients, one tube was further collected every 12 h after the first sampling for 3 days to study the kinetic of the plasma CGA concentration.
The CGA assay is a sandwich ELISA (Cisbio Bioassays, France) with two monoclonal antibodies against human CGA amino acid sequences 145 to 197 and 198 to 245 (17). Procalcitonin concentration was measured on the Kryptor system (Brahms Diagnostic) according to the assay manufacturer's recommendations; C-reactive protein was measured by immune turbidimetry, creatinine using an enzymatic method (Siemens ADVIA, Paris, France), and lactate in whole blood using a lactate oxydase amperometric method (Roche, Cobas b221, Germany).
In vitro studies on monocytes
The human monocytic THP-1 cell line (ATCC) was maintained at 37°C, under 5% CO2, in RPMI supplemented with 10% fetal calf serum and 2 mM l-glutamine.
Preparation of synthetic rhodaminated peptides
Rhodaminated synthetic peptides Rho-CGA47–70 and Rho-CGA7–40 were prepared on an Applied Biosystem 433A peptide synthesizer (Foster City), using the stepwise solid-phase approach with 9-fluorenylmethoxycarbonyl (Fmoc) chemistry. Thereafter, the synthetic peptides were purified by RP-HPLC on a Macherey Nagel Nucleosil RP 300-7C18 column (10 × 250 mm; particle size 7 μm and pore size 100 nm). Rhodamine fluorophore 5(−)-carboxytetramethyl rhodamine was conjugated with peptides at the N-terminal end as previously described (18). Synthetic peptides were analyzed by mass spectrometry and automated Edman sequencing on an Applied Sequencing System (Applied Biosystems, Foster City). Maldi-Tof mass measurements were carried out on an Ultraflex TOF/TOF (Bruker Daltonics).
THP-1 cells were seeded at 1 × 106 cells/mL and treated with 20 μM of Rho-CGA47–70 or Rho-CGA7–40 for 5 min or 15 min. Cells were then washed twice with cold PBS. Using a cytospincytocentrifuge (CytoSpin 4 Cytocentrifuge, Thermo Fisher), 2 × 105 cells were deposited onto a glass slide and fixed for 20 min at room temperature with 4% paraformaldehyde. Finally, coverslips were mounted onto the glass slides using Vectashield Mounting Medium with DAPI (Vector Laboratories). Images were acquired using a fluorescence microscope (Nikon Eclipse TE200) at 60× magnification.
THP-1 were transiently transfected with a pNF-kappa B-Luc (a generous gift from Prof. Carine Van Lint, Université Libre de Bruxelles, Belgium) or pAP1-Luc reporter plasmids (Stratagene) using Lipofectamine LTX (Invitrogen). Twelve hours posttransfection cells were treated with 20 μM of CGA47–66 for 1, 6, or 24 h. Cells were then washed twice, lysed, and luciferase activities were measured (Dual Luciferase Reporter Assay System, Promega). The assay was normalized to total protein content (Bradford assay). The first control, with a relative luciferase activity of 1, corresponds to transfection by pNF-kappa B-Luc or pAP1-Luc reporter plasmids before treatment with peptide. Negative control corresponds to the treatment with CGA7–40.
Variables were expressed as medians (interquartile range [25–75]) or means ± SD as indicated. Differences between continuous variables were tested using the Mann–Whitney U test; differences between categorical variables were tested using Fisher exact test. Univariate analyses were performed using a nonparametric analysis of variance by the Kruskal–Wallis test, followed by Scheffé's method analysis. When necessary, ANOVA for repeated nonparametric measures was used. Receiver-operating curves (ROCs) were constructed for biological biomarkers at the best cutoff level to predict NI occurrence, and the areas under the curve (AUC) were assessed and then compared using the Z-statistic with correction for the correlation introduced by studying the same sample. The threshold of statistical significance was P < 0.05.
In vivo assessments
Samples were obtained from 31 patients among 40 screened within 6 h from admission (Fig. 1). Nine patients were excluded because they chronically took proton pump inhibitors (n = 4), no consent was obtained (n = 2), or they died within 2 days (n = 3). The baseline characteristics of the study population are presented in Table 1. None of the patients had previous significant medical history. The patients suffered predominantly from blunt trauma (n = 28) due to motor vehicle accidents, a fall from a great height or gunshot (n = 3). The length of stay in ICU ranged from 2 to 40 days (mean 13 ± 11 days). The entire cohort required mechanical ventilation and circulatory support with norepinephrine. None needed renal support with hemodialysis over the first 3 days. Surgical intervention under general anesthesia was required immediately after admission in 22/31 patients, and 100% received blood transfusion in the first 24 h (mean transfusion requirement of 8 ± 15 units). Ten patients died within 28 days (mortality among study population 32%) either from intracranial hypertension (n = 7) or from multiple organ failure subsequent to hemorrhagic shock (n = 3). After day 2, 11/31 patients (35%) developed NI. Among these patients, nine were randomly chosen for a longitudinal survey of pCGA concentration over 66 h after admission (Fig. 2) and demographic and clinical characteristics (Table 2).
Plasma release of CGA after trauma
After the initial injury, the first concentration of CGA was significantly increased compared with healthy controls (112.3 [88.73–144.3] ng/mL vs. 19.5 [2.3–36.6] ng/mL, P < 0.0001) (Table 1). As indicated in Figure 2, in a subset of nine patients undergoing serial sampling, from its initial peak, the concentration of CGA showed a progressive decrease but remained significantly higher than the control values throughout the survey (P < 0.001).
Nosocomial infection occurrence according to plasma CGA concentration at admission
Admission values of CGA were found to be significantly higher in those patients who developed NI (NI+) (111.1 [87.28–149.0] ng/mL vs. 63.08 [33.67–204.7] ng/mL in patients without infections (NI−), P = 0.003 (Fig. 3). Using ROC curves, CGA concentration at admission predicted the occurrence of NI with a sensitivity of 100% and a specificity of 70% at the level of 67.25 ng/mL in the whole study population (n = 31) (data not shown). AUC for this prediction was 0.837 (95% CI, 0.67–0.94), which was significantly greater than AUC for C-reactive protein (0.557, 95% CI, 0.377–0.726, P = 0.04), but no different from the AUC for procalcitonin (0.705, 95% CI, 0.524–0.848, P = 0.243). At the same CGA concentration, the positive likelihood ratio of the prediction was 3.33, whereas the negative likelihood ratio was zero.
In vitro tests
Vasostatin-I (VS-I; CGA1–76) (Fig. 4) corresponds to the predominant natural CGA-derived fragment (13) and is highly conserved during evolution (12). In a previous study, we reported that significant amounts of VS-I are detected on admission in critically ill patients and that a plasma concentration above 3.97 ng/mL is associated with poor outcome (15). As VS-I has never been reported as interacting with immune cells, we decided to test whether VS-I-derived fragments have the ability to impact on inflammation. The first peptide we tested corresponds to CGA7–40, (Fig. 4) which, as has previously been reported, binds biological membranes with the disulfide bridge (19). The second peptide we tested is CGA47–70, including chromofungin (CGA47–66), which has already been described as a cell-penetrating peptide (18, 20) (Fig. 4).
This in vitro study includes two parts: the confocal microscopy of the interaction of the two rhodaminated peptides with THP1 cells; the effects of CGA47–70 against NF-kappa B and AP-1 activities.
We investigated the ability of Rho-CGA47–70 and Rho-CGA7–40 to penetrate the outer membrane of THP-1 cells, using a human monocytic cell line as a model. The cellular internalization was visualized via confocal microscopy after 5 and 15 min of treatment. Figure 5A indicates that CGA47–70 was significantly internalized by cells, in line with its positive charges. The fluorescence was predominantly detected at 5 and 15 min at the nuclear level. We note an increase of the nuclear fluorescence after 15 min as compared with after 5 min (Fig. 5A). Fluorescence was also detected at the perinuclear region with a lower level. In contrast, fluorescence of Rho-CGA7–40 could not be observed within cells (Fig. 5B), showing a specific behavior forRho-CGA47–70.
CGA47–66 inhibits the proinflammatory transcription factors NF-kappa B and AP-1
We carried out luciferase assays, and THP-1 cells were then transfected with the NF-kappa B-Luc or AP-1-Luc reporter construct and incubated with CGA47–70 for 1, 6, and 24 h. Surprisingly, CGA47–66 was able to inhibit both NF-kappa B and AP-1-mediated transcription in a time-dependent manner (Fig. 6, columns 2–4 and columns 6–8, respectively). To be more precise, it was capable of inhibiting 95% of NF-kappa B activity and 70% of AP-1 activity at 24 h posttreatment (Fig. 6, columns 4 and 8, respectively). As NF-kappa B and AP-1 play a critical role in amplifying and perpetuating the inflammatory process by controlling the expression of numerous inflammatory genes (21), these results collectively reveal an anti-inflammatory potential for CGA47–66.
In our study population of multiple trauma patients, CGA levels were higher at admission and throughout the first 66 h after injury. Also, the plasma concentrations of CGA were significantly higher when care-related infection occurred. The stress induced by a life-threatening disease is thought to be responsible for the release of CGA within the plasma (9, for review); herein, the early damage control by surgery may also have partially contributed to such an increase (22). These data raise the question of a possible mechanism through which CGA and its endogenous fragments may contribute to a possible immunosuppression and the development of infection in trauma patients that are at risk of NI (4). Among the endogenous fragments, VS-I (CGA1–76) is predominantly produced and CGA47–66 is able to penetrate into immune cells (20). One recent report demonstrates that orally given VS-I protects mice against inflammatory colitis by reducing the cytokine-induced increase of permeability of intestinal epithelial cells and by promoting healing of injured cells (23). Production of CGA47–66 containing fragments may occur within the plasma or at cell surfaces by enzymes that are upregulated after a life-challenging trigger. As a consequence of acute illness, several proteolytic enzymes are activated and upregulated in a time-dependent manner in injured tissue and in circulating mononuclear cells when full-blown SIRS is occurring (24): peptidases, ADAMTS proteins and matrix metallo proteases (25–27). In addition, some strains of Staphylococcus aureus release a glutamyl endopeptidase, able to break a bond after a glutamic residue, and may therefore contribute to the production of several fragments such as CGA47–60 and CGA47–70(18). Interestingly, numerous patients of ours developed care-related lung infection due to S. aureus, which may induce the production of CGA-derived fragments able to trigger or to increase an immune defect.
To further characterize such an immune defect we report that CGA47–70 enters readily the monocytes, whereas its control peptide (CGA7–40) does not. CGA47–66 is a cell-penetrating peptide, as shown previously by our group (18, 20) and in vitro we showed that these cells exposed only to the CGA47–70 peptide display a rapid onset but long-lasting decrease of both NF-kappa B and AP-1 activities (Fig. 6). In contrast, another VS-I-derived peptide, (CGA7–40), was not able to modify the level of activity of these two transcriptional factors. These results suggest a subsequent downregulation of their target genes (28), which are critical for the fine regulation of the balance between pro- and anti-inflammatory factors. In a recent paper, it has also been reported that a cell-penetrating domain of human beta-defensin 3 (hBD3–3) displays anti-inflammatory activity (29). Comparison of the sequences of CGA47–70 and hBD3–3 is reported in Figure Supp. 1, Supplemental Digital Content 1, http://links.lww.com/SHK/A655. The authors demonstrated that hBD3–3 downregulated nuclear factor kappa B-dependent inflammation by suppressing the degradation of phosphorylated-I kappa B alpha and by downregulating active nuclear factor kappa B p65. As far as our peptide is concerned, CGA47–66 has been shown to develop anticalcineurin activities (20) resembling those of cyclosporin and FK 506 (30), two immunosuppressive drugs.
Our study has limitations: we have used a small and specific population (i.e., multiple trauma patients), and different stresses may induce CGA processing in relation with the previous medical history of patients.
In conclusion, we speculate that in vivo the occurrence of NIs s might be related with the VS-I processing into smaller fragments but the relevance of action of CGA47–66 needs further investigation of its role in vivo. These data support the need to rethink the concept of some forms of endogenous healthcare-associated infections: some of them may not be iatrogenic, but a consequence of immune deficiency due to severity of initial injury.
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