Abstract
Aim
The ability of neutrophil CD16 (nCD16) expression to predict outcome in complicated intra-abdominal infections (cIAIs) has not yet been studied; therefore we aimed to evaluate its potential prognostic value in such patients.
Methods
Between November 2018 and August 2021 a single-center prospective study was performed in the Department of Surgical Diseases at a University Hospital Stara Zagora. A flow cytometry was used to measure the levels of nCD16 before surgery and on the 3rd postoperative day (POD) in 62 patients with cIAIs.
Results
We observed a mortality rate of 14.5% during hospitalization. Survivors had significantly higher perioperative expression of nCD16 than non-survivors (P = 0.02 preoperatively and P = 0.006 postoperatively). As predictor of favorable outcome we found a good predictive performance of preoperative nCD16 (AUROC = 0.745) and a very good predictive performance of postoperative levels (AUROC = 0.846). An optimal preoperative threshold nCD16 = 34.75 MFI permitted prediction of survival with sensitivity and specificity of 66.7% and 77.8%, respectively. A better sensitivity of 72.5% and specificity of 85.7% were observed for threshold = 54.8 MFI on the 3rd POD.
Conclusion
Perioperative neutrophil CD16 expression shows a great potential as a predictor of favorable outcome in patients with cIAIs.
Introduction
On a global scale, the complicated intra-abdominal infections (cIAIs) are associated with high levels of morbidity, prolonged hospital stay in Intensive Care Units and poor prognosis [1]. They constitute a serious potential threat for human health, regardless of their age group, race or socio-economic status. Although over the years, various factors impacting the mortality have been studied, at present, satisfactory methods for prognosis of cIAIs adopted in the clinical practice are not existent. Prediction of disease severity using biomarkers is a simple and rapid way to provide information about the outcome and possible options for modifying the treatment plan.
Cluster of differentiation (CD) 16, is a Fragment crystallizable receptor (FcγRIII) which is found on the surface of neutrophils, natural killer (NK) cells, monocytes, macrophages and certain T cells [2, 3]. CD16 has been identified as Fc receptors FcγRIIIa (CD16a) and FcγRIIIb (CD16b), which are involved in signal transduction [4, 5]. FcγRIIIa is expressed on mast cells, macrophages and NK cells as a transmembrane receptor, while FcγRIIIb (nCD16) is only expressed on neutrophils [6, 7]. In the absence of infection, resting neutrophils constitutively express CD16 in contrast to CD64, whose expression on neutrophils is maintained at low levels and rapidly increases in the presence of pathogen-associated molecular patterns (PAMPs), components of the complement system, and proinflammatory cytokines [8]. It has been proven that nCD16 plays a significant role in neutrophil degranulation. FcγRIIIa and FcγRIIIb together are able to activate degranulation, phagocytosis, and oxidative burst, allowing neutrophils to eliminate already opsonized pathogens [4–6]. Decreased expression of CD16 is associated with an increased risk of death and may reflect an increased release of immature neutrophils from the bone marrow following inflammatory stimuli in a systemic immune response [9]. The immature neutrophil progenitors, metamyelocytes are associated with intermediate expression of CD16, whereas myelocytes do not express CD16 [10, 11]. Patients with a systemic inflammatory response or sepsis who die within a week of blood sampling have higher levels of myelocytes and metamyelocytes [12]. In sepsis, the concentration of serum nCD16 increases with the severity of the condition [13], and in patients with severe trauma, nCD16 expression is reduced [14]. Decreased expression of CD16 on neutrophils has been reported in severe systemic inflammatory response [15] and is associated with an increased risk of mortality in the critically ill [9].
The data in the available literature regarding the prognostic qualities of nCD16 is rather scarce, and is lacking in cIAIs. Therefore, we aimed to study the prognostic potential of this biomarker in such patients.
Material and methods
Study population and data collection
This prospective study was performed in the Department of Surgical Diseases (DSD) at a University Hospital “Prof. Dr. Stoyan Kirkovich” Stara Zagora on sixty-two patients with complicated intra-abdominal infections over a 34-month period (November 2018 to August 2021). All patients with cIAIs over 18 years who underwent emergency abdominal surgery were included. The registration number of ethical approval from the hospital ethical committee is № РД-10-275/05.04.2018. The preoperative diagnosis was determined stepwise by clinical evaluation, imaging methods, and laboratory tests, and the final diagnosis was made based on the intraoperative finding.
Expression of cell surface CD16 on neutrophils (nCD16) was measured preoperatively (day 0) and on the 3rd postoperative day (POD). For comparison, we have studied 31 healthy controls with a similar distribution of sex and age to the patients and lack of comorbidity. Before surgery, neutrophil CD16 expression was assessed in 60 (96.8%) patients, as in 2 (3.2%) the peripheral blood sample was clotted, and on the 3rd POD in 58 (93.5%) patients – in 2 (3.2%) the blood sample was clotted and 2 (3.2%) didn't survive before the day of sampling.
Flow cytometry
All peripheral whole blood samples were taken in EDTA anticoagulated tube and stored at room temperature until flow cytometric testing. Flow cytometric analyses were performed 1–3 h after blood sampling. After mixing well, 100-μL aliquots of blood were incubated for 15 min in the dark at room temperature with ready-to-use monoclonal antibodies (anti-CD16PC7, anti-CD14PE, corresponding mouse isotype control) all purchased from Beckman Coulter (USA). Lyse/no wash procedure was performed using the automated TQ-Prep Workstation and Immunoprep Reagent system (Beckman Coulter). A minimum of 100,000 events for each sample were collected on a Cytomics FC500 flow cytometer and analyzed using CXP software (Beckman Coulter). Relative measurement of CD16 expression was obtained by determining the mean fluorescence intensity (MFI) of neutrophils.
Initially neutrophils were labeled as a distinct population based on side scatter/CD14 dot-plot analysis (Fig. 1A and C), after which CD16 expression (mono-parametric histogram Fig. 1B and D) was also measured as MFI relative to the entire neutrophil population [16].
Gating strategy of measurement of CD16 expression on neutrophils in peripheral blood in patients (A, B) and in healthy controls (C, D), determined by flow cytometry. (A) CD14 vs side scatter (SS) dot-plot histogram allowing discrimination between neutrophils (58.5% in BD gate) and monocytes (3.7% in Y gate). (B) Monoparametric histogram showing the expression of CD16 on gated neutrophils as mean fluorescence intensity (MFI) relative to the entire neutrophil population – MFI = 8.46. (C) CD14 vs side scatter (SS) dot-plot histogram allowing discrimination between neutrophils (36.6% in BD gate) and monocytes (6.0% in Y gate). (D) Monoparametric histogram showing the expression of CD16 on gated neutrophils as mean fluorescence intensity MFI) relative to the entire neutrophil population – MFI = 133.0
Citation: European Journal of Microbiology and Immunology 14, 1; 10.1556/1886.2023.00046
Statistical analysis
For statistical analysis, we used statistical software SPSS version 19 for Windows (IBM, Chicago, Illinois, USA). Pre- and postoperative cutoff values of nCD16 were obtained from receiver operating characteristic (ROC) curves drawn for each group according to 28‐day survival. Continuous variables were compared using Mann-Whitney U test (IQR) or Student's t-test (SD), and categorical variables were compared Fisher's exact test or χ2 test. P-values <0.05 were considered statistically significant.
Ethics statement
The study was approved by the Ethics Committee of the University Hospital “Prof. Dr Stoyan Kirkovich” Stara Zagora (№ РД-10-275/05.04.2018). All procedures performed in the study involving human participants were in accordance with the ethical standards of the 1964 WMA Helsinki Declaration and its later amendments or comparable ethical standards.
Results
Patients characteristics
The observed in-hospital mortality was 14.5%. The patients who died had higher median age than survivors: 79 (61–86) years vs. 65 (47.5–75) years, P = 0.032. Fatal outcome was associated with diffuse peritonitis (P = 0.024), unlike gender (P = 1.000), comorbidity (P = 0.423) and source of infection (P = 0.466) (Table 1).
Patients characteristics
| Variable | Total population | Survivors (n = 53) | Non-Survivors (n = 9) | P value |
| Sex, n (%) | 1.000 | |||
| male/female | 35 (56.5)/27 (43.5) | 30 (85.7)/23 (85.2) | 5 (14.3)/4 (14.8) | |
| Age, years (IQR) | 65 (49.5–76.25) | 65 (47.5–75) | 79 (61–86) | 0.032 |
| Peritonitis, n (%) | 0.024 | |||
| Local | 37 (59.7) | 35 (66) | 2 (22.2) | |
| Diffuse | 25 (40.3) | 18 (34) | 7 (77.8) | |
| Source, n (%) | 0.466 | |||
| Appendicular | 15 (24.2) | 14 (26.4) | 1 (11.1) | |
| Hepatobiliary | 22 (35.4) | 19 (35.8) | 3 (33.3) | |
| Gastroduodenal | 12 (19.3) | 9 (17) | 3 (33.3) | |
| Colorectal | 7 (11.3) | 6 (11.3) | 1 (11.1) | |
| Jejunoileal | 2 (3.2) | 1 (1.9) | 1 (11.1) | |
| Gynecological | 4 (6.4) | 4 (7.5) | 0 (0) | |
| Comorbidity, n (%) | 45 (72.6) | 37 (69.8%) | 8 (88.9%) | 0.423 |
| Cardiovascular | 38 (61.3) | 31 (58.5) | 7 (77.8) | 0.462 |
| Endocrine | 8 (12.9) | 8 (15.1) | 0 (0) | 0.59 |
| Neurologic | 7 (11.3) | 5 (9.4) | 2 (22.2) | 0.266 |
| Excretory | 5 (8.1) | 3 (5.7) | 2 (22.2) | 0.149 |
| Oncologic | 3 (4.8) | 3 (5.7) | 0 (0) | 1.000 |
Inflammatory biomarkers
Perioperative concentrations of leucocytes (P = 0.141 before surgery and P = 0.194 after surgery) and C-reactive protein (P = 0.085 before surgery and P = 0.083 after surgery), as well as preoperative percentages of neutrophils (P = 0.99) showed no association with final outcome. In contrast, higher neutrophil levels on postoperative day 3 discriminated successfully deceased patients from those who survived (73.3 ± 9.7 vs 84.3 ± 9.7%, P = 0.007) (Table 2).
Perioperative levels of inflammatory biomarkers
| Variable | Total population | Survivors | Non-survivors | P value |
| WBC0, ×109/L ±SD | 12.3 ± 6.1 | 12.8 ± 6.1 | 9.5 ± 5.5 | 0.141 |
| WBC3, ×109/L (IQR) | 8.7 (6.8–12.8) | 8.3 (6.7–12.4) | 13.6 (7.9–14.9) | 0.194 |
| Neu0, % ±SD | 79.7 ± 8.7 | 79.7 ± 8.4 | 79.8 ± 11.1 | 0.99 |
| Neu3, % ±SD | 74.9 ± 10.2 | 73.3 ± 9.7 | 84.3 ± 9.7 | 0.007 |
| CRP0, mg L−1 ±SD | 177.7 ± 103.9 | 168.3 ± 106.4 | 232.8 ± 68.5 | 0.085 |
| CRP3, mg L−1 ±SD | 143.9 ± 70.6 | 138.1 ± 69.9 | 187.3 ± 64.4 | 0.083 |
| nCD160, MFI (IQR) | 44.25 (29.58–73.33) | 48.8 (31.5–75.6) | 25.5 (10.7–49.2) | 0.02 |
| nCD163, MFI ±SD | 70.72 ± 30.82 | 74.78 ± 30.2 | 41.16 ± 16.23 | 0.006 |
Neutrophil CD16
On the 3rd POD (nCD163) we established significantly higher levels of neutrophil CD16 expression compared to the preoperatively measured (nCD160): 71.05 (48.48–89.95) MFI vs. 44.25 (29.58–73.33) MFI, P = 0.001 (Fig. 2A). The healthy controls had more than 2 times higher levels of nCD16 than patients with cIAIs before surgery: 98.8 (87.5–116) MFI vs. 44.25 (29.58–73.33) MFI, P < 0.0001 (Fig. 2B).
Box-plot and dot-plot of nCD16: (A) Comparing CD16 expression on neutrophils in patients with cIAIs before surgery (n = 60) and postoperatively (n = 58) using box-plot and dot-plot (B) Comparing CD16 expression on neutrophils in patients with cIAIs (n = 60) and healthy controls (n = 31) using box-plot and dot-plot nCD16 – neutrophil CD16 expression; 3rd POD – third postoperative day; cIAIs – complicated intra-abdominal infections
Citation: European Journal of Microbiology and Immunology 14, 1; 10.1556/1886.2023.00046
Preoperative nCD16 expression successfully discriminated patients with a favorable outcome, whereas higher values were associated with lower mortality. The median nCD160 in survivors was almost twice as high compared to non-survivors - 48.8 (31.5–75.6) MFI vs. 25.5 (10.7–49.2), P = 0.02, and this regularity was preserved on the 3rd POD - 74.78 ± 30.2 MFI vs. 41.16 ± 16.23 MFI, P = 0.006 (Table 2).
Preoperatively, we found a good prognostic ability of nCD16 (AUROC = 0.745) and a very good on the 3rd POD (AUROC = 0.846) (Fig. 3). The favorable outcome was successfully predicted at a preoperative threshold >34.75 MFI with a sensitivity of 66.7% and specificity of 77.8%, as well as postoperatively at a threshold nCD163 value >54.8 MFI with a sensitivity of 72.5% and a specificity of 85.7% (Table 3).
ROC Curves of nCD16: (A) ROC Curve of nCD16 before surgery (n = 60) for prediction of survival (AUROC = 0.745) (B) ROC Curve of nCD16 on postoperative day 3 (n = 58) for prediction of survival (AUROC = 0.846) ROC Curve – receiver operating characteristic curve nCD16 – neutrophil CD16 expression
Citation: European Journal of Microbiology and Immunology 14, 1; 10.1556/1886.2023.00046
Sensitivity, Specificity and AUROCs A. preoperatively B. on the 3rd POD
| Cut-off | Sensitivity, % | Specificity, % | AUROC | Std. Error | 95% CI | P-value | ||
| Lower bound | Upper bound | |||||||
| nCD160 | 34.75 | 66.7 | 77.8 | 0.745 | 0.096 | 0.558 | 0.932 | 0.02 |
| nCD163 | 54.8 | 72.5 | 85.7 | 0.846 | 0.056 | 0.736 | 0.955 | 0.003 |
Discussion
Despite advances in diagnostics, surgical management, and antimicrobial therapy in recent years, complicated intra-abdominal infections are still associated with high morbidity and mortality [1, 17]. They affect large and heterogeneous groups of patients, which makes it difficult to create a general treatment algorithm and emphasizes the need for an individual approach for each patient. The main pre-condition for the effective treatment of the cIAIs includes early diagnosis and prognosis, a suitable antimicrobial therapy and timely and appropriate source control. The early prognostic assessment could significantly reduce the disease severity and improve the outcome by introducing changes with regard to the aggressiveness of the therapeutic approach.
In case of intra-abdominal infection, the local inflammatory response in the peritoneal cavity attracts neutrophils, which arrive at the site within 2–4 h [18]. They are first-line defense cells with function to recognize and respond to infection depending on normal surface receptor expression [9]. On neutrophils, surface expression of the high- and low-affinity receptors for the IgG heavy chain, CD64 and CD16, mediate effector functions [9, 19]. Neutrophils have a short lifespan in circulation (<24 h), but in the presence of infection, their life is prolonged by action of the inflammatory mediators [20]. A delay in neutrophil apoptosis is observed, and that allows their continued function, whereat specific regulatory mechanisms are required to limit the extent of neutrophil activity and restore homeostasis [21]. This is achieved by inducing neutrophil apoptosis. Apoptotic cells undergo several characteristic changes, including changes in the cell membrane that allow their engulfment by macrophages [21, 22]. The level of surface expression of CD16 is proven to be an indicator of the apoptotic status of neutrophils, which was assessed by a very strong positive correlation between lower surface levels of CD16, chromatin condensation, DNA fragmentation, and loss of cellular functions [23].
CD16 expression on neutrophils was found to be a biomarker associated with sepsis [15], which has never been investigated in patients with cIAIs. According to Hsu et al. [15], nCD16 decreases in parallel with an increase in the severity of sepsis. In vivo et in vitro nCD16 expression is significantly decreased by bacterial membrane components, IgG opsonized particles and cytokines [9, 24]. Resting neutrophils constitutively express CD16, which we confirmed by the significantly higher expression in healthy controls compared to cIAIs (98.8 MFI vs. 44.25 MFI, P < 0.0001). In contrast, neutrophil expression of CD64 in the absence of infection is maintained at low levels, but the appearance of PAMPs, components of the complement system and pro-inflammatory cytokines leads to higher expression [8]. In the same cohort, but in another paper published earlier this year [25] we confirmed these findings by the observation of higher nCD64 expression before surgery in patients with cIAIs than in healthy controls (74.4% vs. 45.5%, respectively, P < 0.0001).
Neutrophil CD16 plays a significant role in activating degranulation, phagocytosis, and the oxidative burst, allowing neutrophils to eliminate already opsonized pathogens [4]. Decreased CD16 expression is associated with an increased risk of mortality and reflects increased release following inflammatory stimuli in systemic immune response to immature bone marrow neutrophils that have low or absent CD16 expression [9]. In our study postoperative levels of nCD16 were significantly higher than preoperative (71.05 MFI vs. 44.25 MFI, respectively, P = 0.001), possibly due to decreased release of immature neutrophils from the bone marrow and successful control of the systemic inflammatory response. We found almost two-fold lower expression of nCD16 in non-survivors both preoperatively (25.5MFI vs. 48.8 MFI. P = 0.02) and on the 3rd POD (41.16 MFI vs. 74.78 MFI, P = 0.006). Our previously published paper investigating prognostic value of nCD64 in the same cohort [25] described significantly higher preoperative expression in non-survivors compared to survivors (96.8% vs. 71.85%, P = 0.02). The same observation was also found after surgery (P = 0.024), as in the patients with a favorable outcome the levels were reduced to 56.3%, while non-survivors had evidence of a persistent pro-inflammatory reaction, as they kept the expression of nCD64 high: 90.7%. Both these finding suggested that the systemic pro-inflammatory response is a leading factor in determining the risk of fatal outcome. We observed good prognostic ability of nCD16 preoperatively (AUROC = 0.745) and very good of 3rd POD (AUROC = 0.846). Favorable outcome was successfully predicted at a preoperative cut-off >34.75 MFI with a sensitivity of 66.7% and a specificity of 77.8%, as well as postoperatively at a cut-off value > 54.8 MFI with a sensitivity of 72.5% and a specificity of 85.7%. So far, nCD16 has not been investigated as a prognostic biomarker in patients with cIAIs. In sepsis, Hanna et al. [9], like us, reported that decreased nCD16 expression was associated with an increased risk of death.
This is the first study (to the best of our knowledge) that analyzes the prognostic performance of nCD16 in patients with cIAIs. As limitation we can highlight the single-center experience and the small sample size.
Conclusion
The perioperative expression of CD16 on neutrophils shows prognostic qualities as a significant predictor of survival in patients with complicated intra-abdominal infections.
Funding
The authors declare that no funds, grants, or other support were received during the preparation of this manuscript.
Conflict of interest/Competing interests
The authors declare no conflicts of interest or competing interests.
Consent for publication
All authors approved the final draft of the manuscript and gave their consent for publication.
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