Increases in inflammatory and CD14^dim/CD16^pos/CD45^pos patrolling monocytes in sepsis: correlation with final outcome

Immune status in sepsis varies during the course of the disease. The early stages are characterized by a phase of excessive inflammation also known as the “cytokine storm”. In this stage, blood monocytes are overfunctioning for the production of cytokines. As the syndrome progresses, the proinflammation stage is followed by a phase of immune-suppression that is characterized by the inability for cytokine secretion [1]. In this stage monocytes are functionally deactivated, and this is shown by the decreased expression of the human leukocyte antigen (HLA) class II on their cell surface [2]. However, it is also possible that these two phases of proinflammation and anti-inflammation may occur simultaneously. Monocytes can be classified into three phenotypical and functionally distinct subpopulations based on the expression of the lipopolysaccharide receptor CD14 and of the FcγIII receptor CD16 on their cell membranes. According to this classification, monocytes are divided into the “classical or inflammatory monocytes” characterized by a bright expression of CD14 and lack of expression of CD16, the “intermediate monocytes” that express both receptors; and the “nonclassical” monocytes with patrolling function that express predominantly CD16 [3, 4]. Data on the contribution of patrolling monocytes to the pathogenesis of sepsis are still missing. Regarding the role of classical monocytes, available data are mainly focused on the change in their percentage whereas data on their absolute counts are missing [5, 6].

The aim of the present study was to investigate the changes in the absolute counts of monocyte subpopulations in sepsis. For reasons of study homogeneity, the study was limited to patients with infections either proven or highly suspected to be caused by Gram-negative bacteria.

The study flowchart is shown in Fig. 1. The study was divided into two phases. In the first phase, absolute counts of CD14^pos/HLA-DR^pos/CD45^pos monocytes were determined in 70 patients and 10 healthy volunteers. Demographic characteristics of patients participating in both study phases are shown in Additional file 1 (Table S1).

Absolute cell counts of CD14^pos/HLA-DR^pos/CD45^pos monocytes on days 1 and 3 did not differ between patients allocated to the two groups of treatment (Additional file 2: Figure S1A). Since all-cause mortality was similar in the two groups [7], data from both groups were pooled and analyzed together.

Absolute cell counts of CD14^pos/HLA-DR^pos/CD45^pos monocytes on day 1 were significantly higher in survivors compared to nonsurvivors (Fig. 2a). Based on this observation, a ROC curve was designed and a cutoff value of 337 cells/mm³ could discriminate between survivors and nonsurvivors with PPV of 91.1% (Fig. 2b, c). These results were further confirmed after survival analysis showing prolonged survival among patients with more than 337 cells/mm³ on day 1 (Fig. 2d). The OR for unfavorable outcome among patients with more than 337 cells/mm³ was 0.21 (95% CI 0.06–0.78; p = 0.020). Median (IQR) count of CD14^pos/HLA-DR^pos/CD45^pos monocytes on day 1 was 533.7 (846.1)/mm³ among patients without acute kidney injury (AKI) and 852.7 (1181.9)/mm³ among patients with AKI (p = 0.275). Among patients remaining in phase 1 of the study after day 3, median (IQR) count of CD14^pos/HLA-DR^pos/CD45^pos monocytes on day 7 was 192 (494.8)/mm³ among survivors and 321.9 (365.3)/mm³ among nonsurvivors (p = 0.620); the respective values on day 10 were 900.5 (704.8)/mm³ and 254.4 (820.3)/mm³ (p = 0.312).

These results generated the question whether the defined cutoff for CD14^pos/HLA-DR^pos/CD45^pos monocytes on day 1 is an independent predictor of final outcome. As shown in Additional file 3 (Table S2), the only variables that differed significantly between nonsurvivors and survivors at baseline were Acute Physiology and Chronic Health Evaluation (APACHE) II and Sequential Organ Failure Assessment (SOFA) scores. In the logistic regression analysis, APACHE II score, SOFA score, and an absolute CD14^pos/HLA-DR^pos/CD45^pos monocyte count on day 1 greater than 337 cells/mm³ were entered as independent variables; 28-day outcome was the dependent variable. Analysis showed that an absolute CD14^pos/HLA-DR^pos/CD45^pos monocyte count on day 1 greater than 337 cells/mm³ was independently associated with favorable 28-day outcome (Table 1).

Since the impact of the absolute count of CD14^pos/HLA-DR^pos/CD45^pos monocytes on survival was confirmed after the first phase, we proceeded with the second phase where absolute counts of subpopulations of monocytes were measured in 55 patients and 10 healthy volunteers (Fig. 1). Three monocyte subpopulations were defined: CD14^pos/CD16^neg/CD45^pos and CD14^pos/CD16^pos/CD45^pos monocytes analyzed and reported together as inflammatory monocytes, and CD14^dim/CD16^pos/CD45^pos reported as patrolling monocytes. The absolute counts of both inflammatory and patrolling monocytes were significantly greater among patients than healthy controls (Fig. 3a, b); no differences were found between patients without and with AKI (Fig. 3c, d). Among patients remaining in phase 2 of the study after day 3, median (IQR) counts of inflammatory monocytes was 641 (592.3)/mm³ on day 7 (p = 0.204 vs controls) and 669 (519)/mm³ on day 10 (p = 0.049 vs controls). Those of CD14^dim/CD16^pos/CD45^pos patrolling monocytes were 14 (20)/mm³ on day 7 (p = 0.043 vs controls) and 25.5 (28.5)/mm³ on day 10 (p = 0.011 vs controls).

As a next step, we explored if the described changes in the subpopulations of monocytes are associated with final outcome. The absolute cell counts on days 1 and 3 did not differ between patients allocated to placebo or clarithromycin treatment (Additional file 2: Figure S1B and C). Since all-cause mortality was similar in the two groups [7], data from both groups were pooled and analyzed together.

No differences in cell counts of inflammatory monocytes were observed between survivors and nonsurvivors for any day of sampling (Fig. 4a). Regarding CD14^dim/CD16^pos/CD45^pos patrolling monocytes, significant differences between survivors and nonsurvivors were found on day 3 (Fig. 4b). ROC analysis revealed that a cutoff value of 27 cells/mm³ could discriminate between survivors and nonsurvivors with PPV 94.1% (Fig. 4c, d). This finding was further confirmed after survival analysis (Fig. 4e). The OR for unfavorable outcome among patients with more than 27 cells/mm³ was 0.09 (95% CI 0.01–0.80; p = 0.031).

These results generated the question whether the defined cutoff for CD14^dim/CD16^pos/CD45^pos patrolling monocytes on day 3 is an independent predictor of final outcome. As shown in Additional file 4 (Table S3), the only variables that differed significantly between nonsurvivors and survivors at baseline were age, APACHE II score, and SOFA score. In the logistic regression analysis, age, APACHE II score, SOFA score, and an absolute count of CD14^dim/CD16^pos/CD45^pos patrolling monocytes on day 3 greater than 27 cells/mm³ were entered as independent variables; 28-day outcome was the dependent variable. Analysis showed that an absolute count of CD14^dim/CD16^pos/CD45^pos patrolling monocytes on day 3 greater than 27 cells/mm³ was independently associated with favorable 28-day outcome (Table 1).

Next, we asked the question whether clinical signs at admission could predict low counts of patrolling monocytes on day 3. The frequencies of acute pyelonephritis and intra-abdominal infection were significantly different between patients with ≤ 27 cells/mm³ or greater than 27 cells/mm³ CD14^dim/CD16^pos/CD45^pos patrolling monocytes (Table 2). These two clinical characteristics were entered into the logistic regression analysis as independent variables. Analysis revealed that the presence of an intra-abdominal infection was the only variable associated with low CD14^dim/CD16^pos/CD45^pos patrolling monocyte counts on day 3 (Table 3).

Circulating counts of inflammatory and patrolling monocytes are significantly increased up to at least 10 days in the course of Gram-negative sepsis. Exploratory analysis also identified an association between circulating CD14^dim/CD16^pos/CD45^pos patrolling monocytes on the third day of follow-up and 28-day outcome; the greater number of this subpopulation, the lower is the risk for unfavorable outcome. The presence of acute intra-abdominal infection on admission is a predictive sign for low circulating patrolling monocytes counts on day 3. It was also found that more than 337 CD14^pos/HLA-DR^pos/CD45^pos monocytes/mm³ on admission is associated with favorable outcome.

HLA-DR is expressed on activated CD14 monocytes that are capable of antigen presentation and perpetuation of the inflammatory reaction. In sepsis, the expression of HLA-DR on circulating CD14 monocytes and the number of HLA-DR molecules (mHLA-DR) on the monocyte surface are decreased and this is interpreted as an index of sepsis-induced immunosuppression [9, 10]. To date, decreased mHLA-DR is considered the gold standard for the identification of immunosuppression [11]. However, several problems seem to apply for the universal application of mHLA-DR as a diagnostic tool, which are mainly associated with the inter-laboratory variability of the assay.

Recent studies enrolling between 79 and 93 patients [12‐14] (i.e., close to the enrolment of the present study) showed that the decrease in HLA-DR expression is greater in septic shock and among nonsurvivors as early as the first day of sampling. The expression of HLA-DR on CD14 monocytes was further deepening upon worsening on days 3 and 7 of follow-up. However, in these studies the percentage of CD14 cells expressing HLA-DR and mHLA-DR were studied and the absolute count of circulating CD14^pos/HLA-DR^pos/CD45^pos cells was not reported as in our study. This may explain why the absolute count of circulating CD14^pos/HLA-DR^pos/CD45^pos cells was different between survivors and nonsurvivors only on day 1 and not on the next days of follow-up. It may also be the case that the provided association of the absolute count of circulating CD14^pos/HLA-DR^pos/CD45^pos cells on day 1 is a reflection of the pathobiology of the host but that it cannot serve as a biomarker for the follow-up of the patient.

An expansion of CD16-expressing monocytes usually occurs in sepsis with a simultaneous reduction in inflammatory monocytes [5]. In two previous studies, one in sepsis and another in acute pancreatitis, the absolute counts of cells highly expressing both CD14 and CD16 were measured [15, 16]; however, the counts of patrolling monocytes were not reported.

At a functional level, patrolling monocytes are poorly phagocytic, have high antigen-presenting capacity, and express higher levels of Toll-like receptors (TLRs) compared with the other CD14 monocyte subpopulations. In addition, they bear a proinflammatory function by producing high levels of tumor necrosis factor (TNF)α and interleukin (IL)-1β but not IL-10 upon stimulation with lipopolysaccharide (LPS) [5, 17]. In a recent publication from our group we found that failure of adequate production of proinflammatory cytokines by circulating mononuclear cells 48 h after admission for sepsis was interpreted as sustaining the immunosuppression and it was associated with poor outcome. This defective production continued for at least 10 days [18]. Since CD14^dim/CD16^pos/CD45^pos cells are the monocyte subpopulation providing most of the TNFα production, the low cell count of patrolling monocytes on the third day in nonsurvivors could be associated with the immunosuppression of sepsis.

The current study is powered to define the absolute count of CD14^pos/HLA-DR^pos/CD45^pos on day 1 that impact on final outcome. Although the number of patients enrolled during the second phase of the study is rather small to provide definitive associations with final outcome, it clearly reveals a main pathobiological feature of Gram-negative sepsis—i.e., the increase in the studied monocyte subpopulations. However, the independent associations of the proposed cutoffs of circulating CD14^pos/HLA-DR^pos/CD45^pos cells on day 1 and of the circulating CD14^dim/CD16^pos/CD45^pos cells on day 3 with the final outcome proved through logistic regression analysis are remarkable.

This study shows that circulating counts of inflammatory and patrolling monocytes are greatly increased in Gram-negative sepsis. Low absolute counts of CD14^dim/CD16^pos/CD45^pos patrolling monocytes on day 3 are associated with unfavorable outcome. The presence of acute intra-abdominal infection at baseline can predict the low absolute count of patrolling monocytes on day 3. In addition, an association with unfavorable outcome is also provided by low absolute counts of CD14^pos/HLA-DR^pos/CD45^pos on day 1. The present findings cannot support the use of the defined cutoffs of CD14^pos/HLA-DR^pos/CD45^pos and of CD14^dim/CD16^pos/CD45^pos cells as biomarkers for sepsis since validation by another independent cohort is warranted. However, they strongly provide evidence for the independent role of CD14^pos/HLA-DR^pos/CD45^pos cells at baseline and of the CD14^dim/CD16^pos/CD45^pos cells during progression of sepsis as determinants of the outcome of the septic host.