Innate immunity recovers earlier than acquired immunity during severe postoperative immunosuppression.

Background: Postoperative immune suppression, particularly a loss of cell-mediated immunity, is commonly seen after surgery and is associated with worse outcome, i.e. delayed wound healing, infections, sepsis, multiple-organ failure and cancer recurrence. However, the recovery of immune cells focusing on differences between innate and acquired immunity during severe postoperative immunosuppression is not investigated.
Methods: In this retrospective randomized controlled trial (RCT) subgroup analysis, 10 postoperatively immune suppressed patients after esophageal or pancreatic resection were analyzed. Innate and acquired immune cells, the expression of human leukocyte antigen-D related on monocytes (mHLA-DR), lipopolysaccharide (LPS)-induced monocytic TNF-α and IL-10 secretion ex vivo, Concanavalin A (Con A)-induced IFN-γ, TNF-α, IL-2, IL-4, IL-5 and IL-10 release were measured preoperatively (od) until day 5 after surgery (pod5). Recovery of immune cells was defined by a significant decrease respectively increase after a significant postoperative alteration. Statistical analyses were performed using nonparametric statistical procedures.
Results: Postoperative alterations of innate immune cells recovered on pod2 (eosinophils), pod3 (neutrophils) and pod5 (mHLA-DR, monocytic TNF-α and IL-10 secretion), whereas alterations of acquired immune cells (lymphocytes, T cells, T helper cells, and cytotoxic T cells) did not recover until pod5. Peripheral blood T cells showed an impaired production of the T helper (Th) 1 cytokine IFN-γ upon Con A stimulation on pod1, while Th2 specific cytokine release did not change until pod5.Conclusions: Innate immunity recovered earlier than acquired immunity during severe postoperative immunosuppression. Furthermore, we found a more anti- than pro-inflammatory T cell function on the first day after surgery, while T cell counts decreased.

RCT Entities:   Population Interventions Outcomes

Introduction

Postoperative immune suppression particularly a loss of cell-mediated immunity is commonly seen after surgery due to an increased release of immune suppressing hormones such as catecholamines, prostaglandins and cortisol depending on the amount of surgical stress and tissue damage 1, 2. Blood transfusion, hypothermia, dehydration and anesthetics can further attenuate immunity 3-6. An impaired immunity after surgery is associated with worse outcome, i.e. delayed wound healing, infections, sepsis, multiple-organ failure and cancer recurrence 1, 7-12. In particular, postoperative immunosuppression comprises decreased numbers of natural killer (NK) cells, T lymphocytes, as well as an impaired function of T lymphocytes and monocytes including a suppressed expression of human leukocyte antigen-D related on monocytes (mHLA-DR) 1, 13-19. B lymphocytes seem to be less effected 1, 20. Furthermore, increasing numbers of T regulatory (Treg) cells and neutrophils often occur after surgery 13, 21. While major surgery may suppress cellular immunity for several days, humoral immunity remains relatively intact 8. However, the recovery of immune cells focusing on differences between innate and acquired immunity during severe postoperative immunosuppression has not been investigated, yet.

Patients and Methods

Study Participants and Treatment

This retrospective subgroup analysis of a previously published study of our research group 22, 23 investigated innate and acquired immune cells as well as monocytic and T cell immune function during severe postoperative immune suppression (mHLA-DR ≤ 10,000 antigens per cell on pod1) that were measured in 10 out of 20 patients of the placebo group of the bigger cohort until pod5 after elective esophageal or pancreatic resection (measurement had to be stopped after 10 patients for economic reasons, no selection of the patients; Figure 1). All patients received guideline-based anesthesiological and surgical treatment according to our standard operating procedures 24.

Figure 1

Consort diagram. 10 patients of the placebo group were analyzed in this subgroup analyses: immune cells and functional parameters were determined in these patients without any prior selection.

Measurement of parameters of immune function

Blood samples were drawn from od until pod5. mHLA-DR and further parameters of immune function were measured from od until pod5. Expression of mHLA-DR was determined by cytometric analysis using a highly standardized quantitative assay as described earlier 25. For determination of soluble mediators, ethylene diamine tetraacetic acid (EDTA) and heparin plasma samples were collected and stored at -80°C until assay. All immunological parameters were analyzed in collaboration with the Institute of Medical Immunology and Berlin-Brandenburg Center for Regenerative Therapies (BCRT), Charité - Universitätsmedizin Berlin, Berlin, Germany. White blood cell differential count was measured in a standard hematology analyzer (Sysmex). For flow cytometry analysis, lymphocyte subpopulations were identified using the following antibody combinations: CD45 for leukocytes, CD3+ for T lymphocytes, CD3+CD4+ for T helper cells (Th), CD3+CD8+ for cytotoxic T cells, CD2+CD3-CD16+ for natural killer (NK) cells and CD19+ for B lymphocytes. Cell phenotyping was performed by flow cytometry/fluorescence-activated cell sorting (FACS) on a FACSCalibur™ using CELLQuest™ Software (BD Biosciences). LPS-induced monocytic tumor necrosis factor alpha (TNF-α) and Interleukin (IL)-10 secretion ex vivo as well as Concanavalin A (Con A)-induced interferon gamma (IFN-γ), TNF-α, IL-2, IL-4, IL-5 and IL-10 release were determined as described earlier 23.

Statistical analysis

Data were expressed according to their scaling as arithmetic mean ± standard deviation (SD), median [25%, 75% quartiles], or frequencies [%]. After exploratory data analysis, all tests were accomplished by means of non-parametric exact statistical tests. Longitudinal data were analyzed using nonparametric univariate procedures. For each immune parameter, we first tested over all six time points (od until pod5). In a second step, we determined the first significant postoperative alteration by comparing postoperative and presurgical values. The specific time point was then compared with further time points to determine the recovery of immune parameters, i.e. a significant decrease respectively increase after a significant postoperative alteration. A two-tailed p-value < 0.05 was considered statistically significant. All tests should be understood as constituting exploratory data analysis, so that no adjustments for multiple testing have been made. Numerical calculations were performed using IBM© SPSS© Statistics, Version 23.

Results

Study population

Basic patient characteristics, intraoperative- and outcome parameters are shown in Table 1. Some of the results were already shown to analyze group differences after postoperative immune stimulation 23.

Table 1

Basic patient characteristics, intraoperative and outcome parameters.

	Placebo group (n = 10)
Age [years]	62 (55-69)
Gender male/female [n]	7/3
Body Mass Index [kg/m²]	25.5 (24.2-27.5)
Pancreatic/esophageal resection [n]	6/4
ASA score II/III [n]	7/3
Smokers/non-smokers [n]	4/6
AUDIT score	3 (0-6)
Non-diabetes/diabetes [n]	9/1
Metabolic equivalent (MET) <4/4-10/>10	0/8/2
Surgical time [min]	308 (280-378)
Intraop. blood loss [mL]	600 (313-950)
Intraop. mean blood glucose [mg/dL]	127 (122-142)
Intraop. max. blood lactate [mmol/L]	1.0 (0.8-1.3)
Intraop. mean systolic blood pressure [mmHg]	113 (109-117)
APACHE II score on admission to ICU	12 (9-16)
SAPS II score on admission to ICU	22 (12-27)
SOFA score on admission to ICU	2 (1-4)
TISS 28 score on admission to ICU	32 (27-36)
ICU stay [d]	3.2 (2.4-4.9)
Hospital stay [d]	14.4 (11.5-20.6)
Survived/deceased [n]	10/0

Continuous quantities in median (25%-75% percentiles), frequencies with n (%); ASA, American Society of Anesthesiologists; AUDIT score, Alcohol Use Disorders Identification Test; APACHE, Acute Physiology and Chronic Health Evaluation; SAPS, Simplified Acute Physiology Score; SOFA, Sequential Organ Failure Assessment; TISS, Therapeutic Intervention Scoring System; ICU, Intensive Care Unit.

Innate immune cells

Neutrophils showed significant differences from od until pod5 (p < 0.001; Figure 2A), increased on pod1 (p = 0.005) and recovered on pod3 (p = 0.037). Eosinophils differed from od until pod5 (p = 0.002; Figure 2B), decreased on pod1 (p = 0.018) and recovered on pod2 (p = 0.043). Basophils differed from od until pod5 (p = 0.035; Figure 2C) and decreased on pod3 (p = 0.047). NK cell counts showed significant differences from od until pod5 (p < 0.001; Figure 2D), decreased on pod2 (p = 0.024) and again on pod3 (p = 0.015). Monocytes differed from od until pod5 (p < 0.001; Figure 2E), increased on pod1 (p = 0.007) and again on pod5 (p = 0.050). mHLA-DR showed significant differences from od until pod5 (p < 0.001; Figure 2F), decreased on pod1 (p = 0.005) and recovered on pod5 (p = 0.008).

Figure 2

Neutrophils, eosinophils, basophils, natural killer (NK) cells, monocytes and mHLA-DR from day of surgery before surgery (od) until day 5 after surgery (pod5). Neutrophils increased on pod1 and recovered on pod3, eosinophils decreased on pod1 and recovered on pod2, basophils decreased on pod3, NK cells decreased on pod2 and again on pod3 compared to pod2. Monocytes increased on pod1 and again on pod5 compared to pod1. mHLA-DR decreased on pod1 and recovered on pod5. **P<0.01, *P<0.05 represent the first significant differences between pre- and postsurgical values. ^P<0.05 represents the second significant alteration compared to the prior significant alteration. #P<0.05 represents recovery, i.e. the first significant in- respectively decrease after significant postoperative alteration. Error bars with 95% confidence intervals.

Function of innate immune cells

TNF-α release of LPS-stimulated monocytes significantly decreased on pod2 (p = 0.038; Figure 3A) and recovered on pod5 (p = 0.050). IL-10 release of LPS-stimulated monocytes decreased on pod3 (p = 0.038; Figure 3B) and recovered on pod5 (p = 0.012).

Figure 3

TNF-α and IL-10 release of LPS-stimulated monocytes from day of surgery before surgery (od) until day 5 after surgery (pod5). TNF-α decreased on pod2 and recovered on pod5, IL-10 decreased on pod3 and recovered on pod5. *P<0.05 represents the first significant differences between pre- and postsurgical values. #P<0.05 represents recovery, i.e. the first significant in- respectively decrease after significant postoperative alteration. Error bars with 95% confidence intervals.

Acquired immune cells und subsets

Lymphocytes showed significant differences from od until pod5 (p = 0.007; Figure 4A) and decreased on pod1 (p = 0.015). B cells did not show any significant differences (Figure 4B). T cells showed significant differences from od until pod5 (p = 0.027; Figure 4C) and decreased on pod1 (p = 0.011). T helper cells decreased on pod1 (p = 0.007). Cytotoxic T cells showed significant differences from od until pod5 (p = 0.016; Figure 4E) and decreased on pod2 (p = 0.005). The ratio of T helper and cytotoxic T cells significantly increased on pod3 (p = 0.011; Figure 4F) compared to pod1.

Figure 4

Lymphocytes, B cells, T cells, T helper cells and cytotoxic T cells from day of surgery before surgery (od) until day 5 after surgery (pod5). Lymphocytes decreased on pod1, B cells did not show any significant differences. T cells and T helper cells decreased on pod1 and cytotoxic T cells decreased on pod2. The ratio of T helper and cytotoxic T cells increased on pod3 compared to pod1. **P<0.01, *P<0.05 represent the first significant differences between pre- and postsurgical values. Error bars with 95% confidence intervals.

Function of acquired immune cells

After stimulation of whole-blood cultures for 24 h with Con A, the cytokines for Th1 and Th2 responsiveness IFN-γ, TNF-α, IL-2, IL-4, IL-5 and IL-10 were measured. While the Th1 cytokine IFN-γ decreased on pod1 (p = 0.028; Figure 5A), TNF-α and IL-2 did not change significantly after surgery (Figure 5B, C). The Th2 cytokines IL-4, IL-5 and IL-10 did not show any differences after surgery (Figure 5D, E, F).

Figure 5

Con A-induced lymphocytic IFN-γ, TNF-α, IL-2, IL-4, IL-5 and IL-10 secretion from day of surgery before surgery (od) until day 5 after surgery (pod5). The Th1 cytokine IFN-γ decreased on pod1, TNF-α and IL-2 did not significantly change after surgery. The Th2 cytokines IL-4, IL-5 and IL-10 did not significantly change after surgery. *P<0.05 represents the first significant differences between pre- and postsurgical values. Error bars with 95% confidence intervals.

Discussion

The major finding of this subgroup analysis is that innate immunity recovered earlier than acquired immunity during severe postoperative immunesuppression. To the best of our knowledge, no other study has investigated differences in recovery between innate and acquired immune cells during severe postoperative immunosuppression after major cancer surgery, yet.

In general, innate and acquired immune defense play a key role in eliminating of infective pathogens and malignancies. When stimulated by pathogens, immune cells of the innate immune system produce cytokines and other co-stimulatory molecules, whereas the adaptive immune system is essential for immunologic memory and release of antibodies for more specific immune responses 26. Interactions between innate and acquired immunity, i.e. monocytes and T lymphocytes with antigen presentation and consequent T cell response are essential for adequate immune function 27. Particularly in postsurgical patients, profound immune alterations occur with a highly attenuated and restricted immunity 1, which can be measured by a decreased mHLA-DR 12.

We included immune suppressed patients with a mHLA-DR concentration not higher than 10,000 antibodies per monocyte on day one after surgery, which indicates a highly suppressed immune function. Therefore, all patients showed a postoperative immune suppressed state: counts of basophils, eosinophils, NK cells, lymphocytes except of B cells, as well as function of monocytes (mHLA-DR, TNF-α and IL-10 release of LPS-stimulated monocytes) and T cells (IFN-γ release after stimulation) decreased, whereas counts of neutrophils and monocytes increased. Immune alterations during the postoperative period are well described and in accordance with our findings 1, 13-19.

The exact pathological mechanism for an impaired postoperative immune function is still speculative. Perioperatively secreted catecholamines and prostaglandins are assumed to be a major cause of postoperative immune suppression following anesthesia 2, 6. Latest research suggests that so-called alarmins released depending on tissue damage might lead to a pronounced pro-inflammatory response 28. The initial pro-inflammatory response aims to activate immunity to the site of injury and induces a systemic anti-inflammatory state whose physiological effect should prevent the formation of inflammatory tissue and organ damage with the negative effect of leading to a pronounced postoperative immunosuppression 29.

We found an earlier recovery of innate immune cells compared to acquire immune cells. Concretely, innate immune cells recovered on pod2 (eosinophils), pod3 (neutrophils) and pod5 (monocytic function), whereas counts of acquired immune cells (lymphocytes, T cells, T helper and cytotoxic T cells) did not recover until pod5. B cells did not decrease postoperatively. In the present study, we additionally investigated changes in lymphocyte subsets in patients with postoperative immunosuppression. Our findings suggest that the homeostasis of T cells is perturbed in immune suppressive patients after surgery. The decreased numbers of T cells in peripheral blood of immunosuppressed patients may reflect an increased rate of apoptosis of these cells. Clinical research showed an influence of surgical procedures on circulating blood lymphocyte apoptosis 30. Considering functional parameters of T cells, Th1 specific cytokines were decreased and Th2 specific cytokines were unchanged on day one after surgery, which suggests an anti-inflammatory state immediately postoperatively. The imbalance of Th1 and Th2 cytokines is associated with an increased susceptibility to postoperative infections 31. Our results therefore suggest that particularly acquired immune cells are highly vulnerable to postoperative immunosuppression, and compared to innate immune cells remain suppressed for a longer period of time.

It is of major importance to minimize postoperative immunosuppression due to its high impact on outcome regarding sepsis and cancer recurrence 8, 9, 11. Adequate perioperative pain control particularly epidural analgesia was shown to reduce postoperative immune suppression after major abdominal surgery 32. Furthermore, perioperative hypothermia must be avoided 5. Another approach might be postoperative immune stimulation, which was shown to reduce infection days 22. The impact of this stimulation on cancer metastases and recurrence is unknown and should be further investigated.

This study reveals several limitations. First of all, it is a retrospective subgroup analysis. Secondly, we analyzed only a small sample size of 10 patients, i.e. some results might possibly be not significant. Thirdly, patients were analyzed only until pod5. The course of immune cells and function after this period is unknown. Finally, the optimal threshold level for mHLA-DR (≤ 10,000 mAb/cell in our study) used to stratify patients with severe surgery-induced immunosuppression is unclear. Studies suggest values between 5,000 and 10,000 mAb/cell as indicator of severely impaired immune function in critically ill patients 25, 33, 34.

Conclusions

Postoperative innate immunity recovered earlier than acquired immunity during severe postoperative immunosuppression. Furthermore, we found a more anti- than pro-inflammatory T cell function on the first day after surgery, while T cell counts decreased. Further research should focus on strategies to avoid postoperative immune suppression and improve outcome.