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Integrin and PD-1 Ligand Expression on Circulating Extracellular Vesicles in Systemic Inflammatory Response Syndrome and Sepsis

Kawamoto, Eiji*,†; Masui-Ito, Asami*,†; Eguchi, Akiko; Soe, Zay Yar*; Prajuabjinda, Onmanee*; Darkwah, Samuel*; Park, Eun Jeong*; Imai, Hiroshi; Shimaoka, Motomu*

doi: 10.1097/SHK.0000000000001228
Clinical Science Aspects
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ABSTRACT Extracellular vesicles (EVs) in the plasma mediate important intercellular communications in the pathogenesis of cancer and inflammatory diseases. EVs express integrins that regulate target specificities and programmed cell death ligand 1 and 2 (PD-L1 and 2) that suppress lymphocyte activation. However, the roles of these molecules on EVs in systemic inflammatory response syndrome (SIRS) and sepsis remain little understood. This study aimed to investigate how the EV expression of integrins and PD-1 ligands might differ in SIRS and sepsis, compared with healthy controls, and to correlate their expression with the clinical parameters reflecting pathogenesis. Twenty-seven SIRS patients without sepsis, 27 sepsis patients, and 18 healthy volunteers were included. EVs were isolated from plasma samples. The expression of three major integrins (β1, β2, β3 integrins) and PD-L1 and 2 were measured. The EV expression of β2 integrin and PD-L2 was significantly increased in sepsis patients compared with healthy controls. EV expression of PD-L1 was not elevated in sepsis and SIRS; however, circulating soluble PD-L1 levels were significantly higher in sepsis. Furthermore, EV expression of β2 integrin in sepsis patients correlated with hypotension and reduced kidney function. In addition, soluble PD-L1 levels correlated with sepsis severity, impaired kidney function, and impaired central nervous system function. These results suggest the potential involvements of the EV β2 integrin, as well as EV PD-L2 and soluble PD-L1, in the septic pathogenesis that occurs with the systemic immune activation leading to multiple organ dysfunctions.

*Department of Molecular Pathobiology and Cell Adhesion Biology, Mie University Graduate School of Medicine, Tsu-City, Mie, Japan

Department of Emergency and Disaster Medicine, Mie University Graduate School of Medicine, Tsu-City, Mie, Japan

Department of Gastroenterology and Hepatology, Mie University Graduate School of Medicine, Tsu-City, Mie, Japan

Address reprint requests to Motomu Shimaoka, MD, PhD, Department of Molecular Pathobiology and Cell Adhesion Biology, Mie University, Graduate School of Medicine, 2-174 Edobashi, Tsu-City, Mie 514-8507, Japan. E-mail: shimaoka@doc.medic.mie-u.ac.jp

Received 29 May, 2018

Revised 12 June, 2018

Accepted 8 July, 2018

Funding: This work was supported by JSPS KAKENHI (Grants-in-Aid for Scientific Research) (E.K., A.M.-I., A.E., E.J.P., M.S.) and a research grant from Asahi Kasei Pharma (E.K., M.S.).

The authors report no conflicts of interest.

Supplemental digital content is available for this article. Direct URL citation appears in the printed text and is provided in the HTML and PDF versions of this article on the journal's Web site (www.shockjournal.com).

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INTRODUCTION

Sepsis is a type of generalized and deregulated inflammation often triggered by infections. In sepsis, although aberrant activation of neutrophils and monocytes induces inflammatory tissue damage, the induction of lymphocyte unresponsiveness can also occur simultaneously, thereby causing immune-paralysis that can sustain infection and inflammation (1). Several soluble mediators (e.g., cytokines, lipid mediators, and high mobility group box-1) have been found in the circulation of septic shock patients, typically exercising important pathogenic roles in organ failure and tissue injury (1). Extracellular vesicles (EVs), which are small lipid bilayer particles that range from 50 to 1000 nm in size and which include exosomes and microvesicles, have recently emerged as a novel player potentially involved in sepsis pathogenesis (2). EVs are released from cells into the extracellular space, where they can play important roles in the intercellular communication not only between neighboring cells, but also between distant cells. EVs encapsulate bioactive molecules such as small RNAs, DNAs, and proteins, which upon delivery, modulate target cell functionality. Previous studies in sepsis focused on microRNAs and enzymes such as caspase-1 and nicotinamide adenine dinucleotide phosphate hydrogen, both found in EVs, can play important roles in disease pathogenesis (2).

In this study, we sought to investigate the expression profiles of receptors on the surface of EVs such as integrins and programmed cell death-1 (PD-1) ligands in sepsis and systemic inflammatory response syndrome (SIRS). Integrins comprise the family of α/β heterodimeric cell adhesion molecules that have been shown to regulate the biodistribution of cancer exosomes (3). Integrins on EVs are also involved in the binding and internalization to target cells (4). Thus, integrins on EVs might be important in regulating the fate determination of circulating EVs in sepsis. PD-1 ligands, including PD-L1 and PD-L2 on the cell surface, transmit signals through PD-1 on T lymphocytes that suppress immune activation, as shown in chronic inflammation and cancer (5). Although PD-L2 binds to PD-1 with a higher affinity than does PD-L1, the tissue and cellular distributions of PD-L1 are broader than those of PD-L2 (6). Expression of PD-L1 on leukocytes has been linked to immune defects in sepsis (7), suggesting the possibility that PD-1 ligands on EVs might also participate in immune deregulation such as the immune-paralysis observed in sepsis.

Thus far, the expression of some surface molecules on EV such as α-2-microgroblin (8), endoglin (CD105), platelet endothelial cell adhesion molecule-1 (CD31) (9), tissue factor, endothelial protein C receptor (10), CD41, and AnnexinV (11) have been studied in patients with septic shock. However, it has yet to be determined how the expression of integrins and PD-1 ligands on EVs might become altered in SIRS and sepsis. The major goals of this investigation were to characterize the EVs circulating in the plasma and to investigate whether the EV expressions of integrins and PD-1 ligands might be upregulated in SIRS and sepsis and, if so, to determine how they might correlate with the clinical parameters relevant to organ failure. Here, we have demonstrated that EV expression of β2 integrin and PD-L2 as well as soluble PD-L1 (sPD-L1) was significantly increased in sepsis. Furthermore, we have found that EV expression of β2 integrin correlated with hypotension and reduced kidney function, and that sPD-L1levels correlated with sepsis severity, impaired kidney function, and impaired central nervous system function.

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PATIENTS AND METHODS

The study protocol was reviewed and approved by the Institutional Review Board of the Mie University Graduate School of Medicine (#3027). Informed consent to participate in this study was obtained in all cases, either from patients admitted to the intensive care unit (ICU) at Mie University Hospital, Japan or from their close family members.

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Study design and characteristics of participants

This study enrolled 27 consecutive consented adult SIRS patients without sepsis, 27 consecutive consented adult sepsis patients, and 18 healthy volunteers as controls. SIRS was diagnosed according to the standard criteria (12). SIRS accompanied by bacterial infection, confirmed by cultures or clinical evaluation, was diagnosed as sepsis. Blood sample collection was performed within 1 week of admission. For a comparison, blood samples were also collected from 18 consented healthy adult volunteers. Blood samples were drawn into collection tubes containing 3.2% citrated sodium.

Platelet-free plasma fractions were prepared from blood samples as previously described (13). Briefly, red blood cells and leukocytes were excluded from blood sample by centrifugation at 1000 g for 10 min. Platelet and apoptotic body and debris were removed by centrifugation at 2000 g for 20 min. Platelet-free plasma samples were kept at −80°C until use.

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Isolation of EVs

EVs were isolated from platelet-free plasma as previously described (14) by centrifugation at 16,500 g for 1 h. Resulting EV samples were stored in aliquots at −80°C until analysis. The EV parameter measurements in the following sections were performed with the thawed samples. We confirmed in the preliminary experiments that single freeze–thaw procedures did not significantly affect the results of the EV parameter measurements as described in the following sections.

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Particle size measurements

The size distribution of EVs was measured by dynamic light scattering (DLS) as previously described (15) using Nano Partica equipment (Horiba Ltd., Kyoto, Japan).

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Plasma EV concentrations

The number of EVs in the plasma was measured as previously described (16). Briefly, plasma samples were treated with a fluorescent dye, Calcein-AM, which labeled intact EVs but not cellular debris and other nonvesicular particles (e.g., lipoprotein particles). The concentrations of fluorescently illuminated intact EVs in the plasma were measured by flow cytometry.

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Flow cytometric measurements of surface molecule expression of EVs immobilized on beads

Expression levels of molecules on the surface of EVs immobilized on beads were studied as previously described (17) with a minor modification. Briefly, 5 μg of EVs was mixed with 72 μg of 0.01% poly-L-lysine-treated 4 μm diameter latex beads (Thermo Fisher Scientific, Tokyo, Japan, catalog No. A37304) and incubated for 15 min at room temperature, and after the addition of 1 mL PBS, overnight at 4°C with rotation. To the samples, 110 μL of 1 M glycine were added, which were then mixed gently and left to stand on the bench at room temperature for 30 min. The samples were microcentrifuged for 3 min at 10,000 rpm and 4°C, and the supernatant was removed carefully. Pelleted beads were resuspended in 1 mL phosphate-buffered saline (PBS) containing 0.5% bovine serum albumin and micro-centrifuged for 3 min at 10,000 rpm and 4°C. Supernatant was removed and the resulting EV-coated beads were resuspended in 50 μL of PBS containing 0.5% bovine serum albumin.

Expressions of integrins and PD-1 ligands on EV-coated beads were measured by indirect immunofluorescent flow cytometry using Accuri flow cytometer equipment (BD Bioscience, Tokyo, Japan). Data were analyzed using BD Accuri C6 Plus software. The following primary monoclonal antibodies (mAbs) were used: anti-human integrin β1 mAb clone P5D2 (R&D systems, Minneapolis, MN), anti-integrin β2 mAbs anti-integrin clone TS1/18, β3 mAb clone CRC54 (Abcam INC, Japan), anti-PD-L1 clone M1H1 (eBioscience), anti-PD-L2 clone 176611 (Thermo Fisher), isotype control antibodies including mouse immunoglobulin G (IgG)1 clone MOPC-21 (Sigma-Aldrich), and mouse IgG2b clone PLPV219 (Abcam INC.). A fluorescein (FITC)-conjugated goat anti-mouse IgG (H+L) antibody (AffiniPure, Jackson ImmunoResearch, West Grove, Pa) was used as a secondary antibody. An Fc receptor-blocking reagent (Biolegend, San Diego, CA) was used to prevent unwanted antibody binding through Fc receptors.

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sPD-L1and sPD-L2 measurements

The concentrations of sPD-L1 and sPD-L2 in the plasma samples were measured using the enzyme-linked immunosorbent assay (ELISA) kits (Abcam, Japan), according to the manufacturer's instructions. Each sample was analyzed in duplicate.

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Statistical analyses

Statistical analyses were performed using SPSS software v.25.0 (IBM Corp, Armonk, NY). Results are presented as median ± inter quartile range, unless otherwise noted. Kruskal–Wallis tests were used for within 3-group comparisons. Mann–Whitney tests were used for 2-group comparisons. To compare the correlation between the EV parameters and the patient clinical and laboratory parameters obtained at the same date as the blood sampling, Spearman's rank correlation was calculated between each data set. A P value < 0.05 was considered statistically significant.

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A pilot investigation on a mouse sepsis model to study the EV expression of integrins and PD-1 ligands

The animal protocol used in this experiment was reviewed and approved by the Institutional Animal Care & Use Committee of Mie University Graduate School of Medicine (#27–6-rev2). Balb/c mice (12-w old, male) (CLEA Japan, Tokyo, Japan) were subjected to the cecal ligation and puncture (CLP) procedure, which involved an 18-G needle puncture to the cecum ligated at the three-fourth point from the end with No. 6 suture thread (Natsume Seisakusho, Tokyo, Japan). This procedure produced ∼50% survival rate at 48 h after the CLP. The blood samples were collected by a cardiac puncture 24 h after the CLP.

EVs were isolated from the blood plasma and the expression of integrins and PD-1 ligands on EVs were measured by flow cytometry as previously described (17) using the following antibodies: FITC-conjugated anti-β1 mAb (HMβ1–1) (Biolegend, San Diego, CA), FITC-conjugated anti-β2 mAbs (M18/2) (Biolegend), FITC-conjugated anti-β3 mAb (HMβ1–3) (Biolegend), PE-conjugated anti-PD-L1 (M1H5) (BD Biosciences), and PE-conjugated anti-PD-L2 (TY25) (Biolegend). FITC-conjugated American Hamster IgG (BD Biosciences), FITC-conjugated rat IgG2a (Biolegend), and PE-conjugated rat IgG2a (Biolegend) were used as isotype controls. Samples from healthy mice were used as controls.

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RESULTS

The number and the size of EVs in SIRS and sepsis

In this study, 27 patients who manifested SIRS but did not meet the diagnostic criteria for sepsis were assigned to the SIRS group, whereas the 27 patients who did meet the diagnostic criteria were placed in the sepsis group (Table 1). Comparisons of the demographic data between the SIRS and the sepsis groups revealed several major differences such as higher sequential organ failure assessment (SOFA) scores, higher 28-day mortality rates, lower blood pressure, higher CRP, and lower lymphocyte counts in the sepsis group than in the SIRS group (Table 2).

Table 1

Table 1

Table 2

Table 2

Flow cytometric analysis of Calcein-AM-labeled intact EVs in plasma showed that the sepsis group exhibited higher plasma EV concentrations than the SIRS group (Fig. 1A). However, there were no significant differences in the plasma EV concentrations between the SIRS group and the control group of 18 healthy volunteers (Fig. 1A).

Fig. 1

Fig. 1

EVs were then isolated from plasma samples using centrifugation and their size was measured by DLS analysis. The diameters of EVs, expressed as median (interquartile range), were 360 nm (124 nm–1444 nm) for the sepsis group, 265 nm (165 nm–776 nm) for the SIRS group, and 845 nm (514 nm–1317 nm) for the control group (Fig. 1B). Thus, the sizes of EVs in the SIRS group were significantly smaller and were more broadly distributed than those of the control group.

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Sepsis EVs express higher levels of β2 integrin

We studied the expression of integrins that are potentially involved in the regulation of organ-distributed EVs. We focused on 3 major integrin subfamilies: β1, β2, and β3. EVs from the sepsis group expressed higher surface β2 integrin levels compared with the control group (Fig. 2B, Fig S1, http://links.lww.com/SHK/A792). By contrast, there were no significant differences among the 3 groups in the expression of β1 and β3 integrins (Fig. 2A and C, Fig S1, http://links.lww.com/SHK/A792).

Fig. 2

Fig. 2

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SIRS and sepsis EVs express higher levels of PD-L2

We then investigated the expression of PD-1 ligands, PD-L1 and PD-L2, which are potentially involved in the negative regulation of lymphocyte activation and proliferation. EV expressions of PD-L1 in the sepsis and control groups were similar. Interestingly, EV expression of PD-L1 in the SIRS group was significantly decreased compared with those in the sepsis and control groups (Fig. 3A). By contrast, EV expressions of PD-L2 in the sepsis and SIRS groups were significantly higher than that of the control group (Fig. 3B, Fig S1, http://links.lww.com/SHK/A792).

Fig. 3

Fig. 3

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sPD-L1 levels increased in SIRS and sepsis

Thus far, we have found significant increases in the EV expression of PD-L2, but not PD-L1, in the sepsis and SIRS groups. Therefore, we sought to identify the sPD-L1 and sPD-L2 forms in circulation. ELISA-based measurements of the plasma sPD-L1 concentrations showed that the sepsis group expressed significantly higher sPD-L1 levels than the control group (Fig. 4A). sPD-L1 is thought to be released via proteolytic cleavage of the membrane-bound intact PD-L1 on the cell surface; however, whether sPD-L1 could be released from the surface of EVs remains to be elucidated. Our results showed that the EV expression of PD-L1 did not correlate with sPD-L1 levels in the sepsis and SIRS groups (Fig S2, http://links.lww.com/SHK/A792). In contrast to sPD-L1, no significant differences were observed in sPD-L2 levels in sepsis, SIRS, and control groups (Fig. 4B).

Fig. 4

Fig. 4

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Correlation of EV integrin and PD-1 ligand expression with clinical parameters

We next sought to investigate any possible correlations of the EV parameters with clinical parameters related to the sepsis pathogenesis (Fig. 5). Although we found many statistically significant correlations between the EV and clinical parameters in the SIRS and sepsis groups, we focused only on the EV parameters that were significantly increased in the sepsis and/or SIRS group. These included EV β2 integrin expression, EV PD-L2 expression, and plasma sPD-L1 levels (Fig. 5).

Fig. 5

Fig. 5

In the sepsis group, we found that EV β2 integrin expression correlated with hypotension (R = −0.56, P < 0.05) and impaired renal function, as indicated by reduced glomerular filtration rate (eGFR) (R = −0.38, P < 0.05). Although we did not find significant correlations of EV PD-L2 expression with clinical parameters, we did in terms of sPD-L1 levels and impaired renal function. This was evident based on reduced eGFR (R = −0.58, P < 0.05) and increased creatinine (R = 0.64, P < 0.05). We also noted the correlations of sPD-L1 with the following: dysregulated coagulation, as indicated by increased fibrin degradation products (R = 0.57, P < 0.05) and increased D-dimer (R = 0.46, P < 0.05), and worsened organ failures as shown by Glasgow Coma Scale (GCS) (R = −0.41, P < 0.05) and SOFA score (R = 0.40, P < 0.05) (Fig. 5B). In the SIRS group, EV PD-L2 expression levels correlated with reduced lymphocyte counts, reduced hematocrit, and reduced total protein levels (Fig. 5A).

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Subgroup analysis

There appears to be heterogeneity with respect to EV sizes and surface marker expression in the sepsis and SIRS samples. One might think that analyzing less heterogenous subgroups could produce additional valuable information. First, based on the mean diameters of EVs, samples in the SIRS and sepsis groups were subdivided into either a large EV subgroup (>1000 nm, n = 4, SIRS: n = 9, sepsis) or a small EV subgroup (<1000 nm, n = 23, SIRS; n = 18, sepsis). We then compared the EV expression of integrins and PD-L1 and PD-L2 between the large and small EV subgroups in the SIRS and sepsis parent groups (Fig S3, http://links.lww.com/SHK/A792), but did not observe any significant intersubgroup differences.

Second, based on the β2 integrin expression levels, samples in the sepsis parent group were subdivided into either a β2 integrin-positive (n = 19) or a β2 integrin-negative (n = 8) subgroups. Studying the correlations of the EV parameters (including sPD-L1) with the clinical parameters, we identified several significant intersubgroup differences (Fig S4, http://links.lww.com/SHK/A792). For example, a significant correlation of sPD-L1 levels with increased neutrophil counts was observed in the β2 integrin-positive subgroup. However, this did not hold true in either the β2 integrin-negative subgroup or the parent sepsis group.

Similarly, based on the EV PD-L1 or PD-L2 expression levels, samples in the SIRS and sepsis parent groups were subgrouped. We studied the correlations between the EV and clinical parameters and found several significant intersubgroup differences (Fig S5-S7, http://links.lww.com/SHK/A792). For example, a significant correlation of β2 integrin levels with liver injury, as indicated by increased liver enzymes (aspartate aminotransferase [AST], alanine aminotransferase [ALT]), was observed in the EV PD-L1-positive subgroup (Fig S5A, http://links.lww.com/SHK/A792). However, this was not the case in either the PD-L1-negative subgroup or the parent sepsis group. In addition, a significant correlation of sPD-L1 levels with reduced lymphocyte counts was observed in the PD-L1-positive sub-group, but not in the PD-L1-negative sub-group or the parent sepsis group.

Sepsis results from heterogenous etiologies, as is also the case with the present study. We examined subgroups of sepsis cases stemming from either pneumonia or nonpneumonia etiologies (Table S1, http://links.lww.com/SHK/A792). Comparisons between the pneumonia subgroup (n = 12) and the nonpneumonia subgroup (n = 15) revealed that, compared with the control group, sPD-L1 levels were significantly increased in the pneumonia subgroup, the nonpneumonia subgroup, and the parent sepsis group (Fig S8, http://links.lww.com/SHK/A792). However, EV expression levels of integrins, PD-L1, and PD-L2 did not differ significantly between the pneumonia and nonpneumonia subgroups (Fig S9-S12, http://links.lww.com/SHK/A792).

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A pilot investigation on a mouse sepsis model to study the EV expression of integrins and PD-1 ligands

To further substantiate our findings that EV expression of β2 integrins and PD-L2 were upregulated in sepsis patients, we performed a pilot animal investigation, using a mouse model of sepsis, that aimed to recapitulate the similar changes in the characteristic of EVs. Specifically, we utilized a CLP model, studying the expressions of integrins and PD-1 ligands on EVs isolated from sepsis and control mouse plasma samples. Unexpectedly, we were unable to observe any significant changes in the EV expression levels of integrins and PD-1 ligands (Fig S13, http://links.lww.com/SHK/A792).

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DISCUSSION

In this study, we have investigated the expression of integrins and PD-1 ligands on EVs as well as soluble forms of PD-1 ligands in SIRS and sepsis patients. We have shown that the sepsis patients exhibited increased EV expression of β2 integrin and PD-L2 as well as increased plasma levels of sPD-L1, compared with control group. EV expression of β2 integrin correlated with hypotension and reduced renal function, whereas sPD-L1levels correlated with severity of organ failures, reduced renal function, and impaired central nervous system function. In addition, the SIRS patients exhibited increased EV expression of PD-L2 as well as increased plasma levels of sPD-L1, compared with control group. EV expression of PD-L2 correlated with reduced lymphocyte counts, reduced hematocrit, and reduced total protein levels, whereas sPD-L1levels correlated with severity of organ failures and impaired central nervous system function. As discussed in the following paragraphs, the results in this study support the septic pathogenesis that the EV β2 integrin, as well as EV PD-L2 and sPD-L1increase in response to the systemic immune activation that leads to multiple organ dysfunctions.

EVs isolated in this study using a centrifugation at 16,500 g for 1 h predominantly included microvesicles, alternatively known as microparticles. Microvesicles are released from cells via the shedding of the plasma membrane, as opposed to exosomes which are much smaller in size and secreted from cells via endosomal pathways (2). Several previous studies have investigated microvesicles and microparticles in critically ill ICU patients. One previous study found that plasma microvesicle counts were higher in sepsis and burn patients (18), consistent with the current study showing that plasma microvesicle levels are higher in sepsis patients. Interestingly, some studies have shown that increased levels of plasma microparticles in critically ill patients are associated with a reduced risk of acute respiratory distress syndrome and sepsis (19, 20) and that microparticles expressing α-2-microgroblin may play a protective role in sepsis resulting from community-acquired pneumonia (8). By contrast, other studies have shown that increased levels of microvesicles correlated with poor outcomes in cases of septic shock (9–11).

One novel finding we have made is that EV β2 integrin expression levels were significantly increased in the sepsis group. Furthermore, we found significant correlations with clinical parameters, such as reduced blood pressure and reduced renal function, as shown by diminished eGFR. As β2 integrin is expressed only on leukocytes, β2 integrin-expressing EVs are thought to originate from leukocytes, including neutrophils, lymphocytes, and monocytes/macrophages. As expression of β2 integrin is upregulated upon leukocyte cellular activation (21), and activated leukocytes secrete β2 integrin-expressing EVs (22, 23), increased EV expression of β2 integrin suggests that enhanced leukocyte activation might be occurring systemically. This is consistent with the type of systemic inflammation that leads to aberrantly induced vascular dilatation and decreased blood pressure. Reduced eGFR could be caused either directly by an inflammatory injury to the kidney parenchymal and stromal cells and/or indirectly by renal hypoperfusion secondary to reduced blood pressure levels. Along with EVs from platelets and endothelial cells, EVs from leukocytes appear to represent a major fraction of the EVs found in the circulation of SIRS and sepsis patients (24). β2 integrin on EVs might interact with its ligand intercellular adhesion molecule-1, which is upregulated on inflamed endothelial cells, thereby, potentially regulating EV distribution in the body.

Another novel finding of our study is that EV PD-L2, but not PD-L1, expression levels were significantly increased in the sepsis and SIRS groups compared with the control group. PD-L2 was thought to be primarily expressed in macrophages and dendritic cells, but has recently been shown to be induced by microenvironmental stimulations in other various cell types (6). PD-L1 and PD-L2 are similar in that they both bind to and signal through PD-1. However, an important distinction is that PD-L1 is upregulated preferentially in Th1-type immune activation, whereas PD-L2 favors Th2-type stimulation (25), which might be relevant to immune suppression induced by sepsis and SIRS. PD-L1 and PD-L2 on EVs are assumed to be as functionally active to transmit the inhibitory signals through PD-1 as those on cells. However, the activities of PD-L1 and PD-L2 on EVs appear to change depending on the types of immune reactions involved. In delayed-type hypersensitivity responses, PD-L1 and PD-L2 on EVs were shown to be dispensable for damping T-cell activation (26). By contrast, in the tumor microenvironment, PD-L1 on EVs was found to be functionally active in signaling through PD-1 to suppress T-cell activation (27). The potential roles of PD-L1 and PD-L2 on EVs in the pathogenesis of sepsis currently warrant elucidation. They might, however, serve as alternative sources to bind to, and deliver, negative signals through PD-1 to suppress immune cell activation, as both PD-L1 and PD-L2 do on the cell surface. PD-L1 and PD-L2 interactions with PD-1 are thought to be involved in immune paralysis in SIRS and sepsis (7). It has been previously shown that sPD-L1 levels are associated with immunosuppression and infectious complications in acute pancreatitis patients (28). Our results have also shown a significant increase of EV PD-L2 expression and its correlation with reduced lymphocyte counts in the SIRS group. As compensatory anti-inflammatory response syndrome is thought to occur simultaneously with SIRS (29), this correlation might be related to a possible immune suppression in SIRS.

In our pilot animal investigation using a mouse CLP model, we were unable to observe the upregulation of EV β2 integrins and PD-L2, as opposed to our findings in sepsis patients. The reasons for these discrepancies are unclear. The major biogenesis pathways of EVs appear to be well conserved between mouse and human (2). One plausible explanation might be that the CLP model has a limited capacity to reflect the pathophysiological responses that occur in human sepsis. How well the global gene expression patterns elicited by the CLP model can mimic those by human sepsis remains a controversial issue (30).

Increased plasma sPD-L1 levels have been associated with survival in lymphoma (31) and atherosclerotic vascular pathologies in type 2 diabetes mellitus (32). sPD-L1 is biologically active in binding to PD-1, enabling it to either deliver negative signals to suppress T-cell functions (25) or to block the cellular PD-1/PD-L1 interactions that augment T-cell functions (32) depending on the pathological context. In sepsis, a study by Liu et al. (33) involving 91 sepsis patients demonstrated that sPD-L1 was increased in sepsis compared with healthy controls, was increased in nonsepsis survivors compared with sepsis survivors, and correlated with lymphocyte counts, platelet counts, and SOFA scores. In contrast, the study by Wilson et al. (34) involving 22 sepsis patients found no significant increase of sPD-L1. Consistent with Liu et al. (33), the present study showed that sPD-L1 levels in the plasma were significantly increased in the sepsis group compared with those of the control group. Furthermore, as reported by Liu et al(33), we have also shown a significant correlation of sPD-L1 levels with SOFA score in sepsis. In addition, our results revealed that sPD-L1 levels significantly correlated with impaired central nervous system function, as assessed by the GCS, as well as impaired renal function based on reduced eGFR and increased serum creatinine. These results suggest a possible link between sPD-L1 and organ failure. Increased sPD-L1 levels might be merely a consequence of aberrant activation of immune cells and nonimmune cells, resulting in the release of more sPD-L1 (6). Alternatively, increased sPD-L1 might lead to enhanced activity that blocks inhibitory signals via the PD-1/PD-L1 interaction, thereby exacerbating inflammation (32). Unlike sPD-L1, sPD-L2 did not altered in SIRS and sepsis, probably because of multiple factors such as different cellular origins and different biogenesis of sPD-L2 compared with sPD-L1 (6).

Sepsis patients are highly heterogenous with respect to their etiologies, disease progressions, and prognoses (35). A possible heterogeneity in the sizes and surface marker expression levels of EVs from sepsis and SIRS patients could make it difficult to identify the relevant underlying pathological changes underlying the disease. In an effort to examine less heterogenous samples, we subgrouped the sepsis samples depending on the EV sizes, the expression levels of the EV integrins PD-L1, and PD-L2, and on the sepsis etiologies. Subsequent subgroup analyses have produced several significant findings. For example, in the EV β2 integrin-positive subgroup, sPD-L1 levels positively correlated with neutrophil counts. A previous study found a similar correlation in lung cancer patients (33). Additionally, in the EV PD-L1-positive subgroup, EV β2 integrin expression levels correlated not only with hypotension but also with liver injury, as shown by the increased AST and ALT. These findings might point to the complexities of the deregulated immune system, intensifying inflammation both in the EV β2 integrin-positive subgroup and in the EV PD-L1-positive subgroup.

In addition, although all samples were obtained within 7 days of ICU admission and the majority of the samples were obtained within 4 days of ICU admission, differences in sampling time could lead to heterogeneity in the EV characteristics. The EV expression levels of PD-L1 and PD-L2 and sPD-L2 levels did not significantly differ at any sampling points in the SIRS and sepsis groups (Fig S14A&B, and Fig S15, http://links.lww.com/SHK/A792). By contrast, sPD-L1 levels in SIRS were higher in samples obtained at ICU day 7, compared with samples at ICU day 2 and 3 (Fig S14C&D, http://links.lww.com/SHK/A792). A future investigation would be needed to study the collecting of samples at different time points for the same patients, as such sample collection data were not available with the present patient cohorts.

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Acknowledgments

The authors would like to thank Dr. Hiroshi Yonekura at the Department of Pharmaco-epidemiology, Kyoto University Graduate School of Medicine, and Dr. Takumi Imai at the Department of Clinical Biostatistics, Kyoto University School of Public Health, for advice with statistical analysis.

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Keywords:

Extracellular vesicles; integrin; programmed death ligand-1 and ligand-2; sepsis

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