Sustained Down Regulation of Plasma Tetranectin Contributes to Liver Injury in Pneumonia-Associated Sepsis

Background: It was recently shown that tetranectin (TN) concentration in the plasma of sepsis patients was signicantly lower than healthy control and that exogenous TN reduced the mortality rate in septic mice. The aim of this study is to determine whether the reduction of plasma TN is a sepsis-specic host response and its impact on organ dysfunction in sepsis. Methods: The study was conducted in the Sepsis Laboratory at the Huaihe Hospital of Henan University in China. Thirty-seven healthy, 30 community-acquired pneumonia (CAP) and 363 sepsis with comorbid pneumonia (SWP) subjects were recruited. A murine model of polymicrobial sepsis was used to characterize the role of plasma TN in sepsis pathogenesis. Results: TN concentrations in plasma from both CAP and SWP subjects were lower than healthy controls, but not signicantly different between male and female sepsis patients, before and after the occurrence or the resolution of sepsis. In addition, plasma TN was not associated with the occurrence of septic shock or sepsis mortality in both genders. On the other hand, plasma TN negatively correlated with liver injury indicators in moribund SWP subjects. In mice of polymicrobial sepsis, a signicant decrease in plasma TN occurred within hours after the ligation and puncture of the cecum. Recombinant human TN induced signicant reductions of tissue injury markers of liver, but not other organs. In addition, exogenous TN selectively reduced the level of receptor-interacting protein kinase 3 among a panel of cell death markers in septic mouse liver. Conclusions: The dramatic and persistent down-regulation of plasma TN is not a sepsis-specic host response, but contributes to liver injury in pneumonia-associated sepsis.

but not signi cantly different between male and female sepsis patients, before and after the occurrence or the resolution of sepsis. In addition, plasma TN was not associated with the occurrence of septic shock or sepsis mortality in both genders. On the other hand, plasma TN negatively correlated with liver injury indicators in moribund SWP subjects. In mice of polymicrobial sepsis, a signi cant decrease in plasma TN occurred within hours after the ligation and puncture of the cecum. Recombinant human TN induced signi cant reductions of tissue injury markers of liver, but not other organs. In addition, exogenous TN selectively reduced the level of receptor-interacting protein kinase 3 among a panel of cell death markers in septic mouse liver.
Conclusions: The dramatic and persistent down-regulation of plasma TN is not a sepsis-speci c host response, but contributes to liver injury in pneumonia-associated sepsis.

Background
Sepsis is a life-threatening organ dysfunction caused by dysregulated host responses to infection and is a major cause of human death (1)(2)(3). The nature of organ dysfunction-induced host responses has not been adequately de ned. A growing body of evidence, especially the failure of virtually all in ammationtargeting clinical trials, suggests that in ammation may not be the direct cause of sepsis-associated organ dysfunctions, but may trigger sustained organ-damaging responses that are not sensitive to antiin ammation interventions (3)(4)(5)(6)(7)(8)(9)(10). Characterization of organ-speci c injurious host responses may aid in the development of sepsis therapies.
Sepsis patients often develop dysfunction in various organs, regardless of the location of initial infection (2,3). The heterogeneity of impacted organs contributes to the prevalent occurrence of multiple organ dysfunctions especially in moribund sepsis patients, and suggests inter-organ propagation of injurious factors likely through the circulation (1,3,7,10). The cause for the heterogeneity in impacted organs is poorly understood, but likely attributable to an imbalance of organ-speci c protective and injurious protective factors. Tetranectin (TN) is a 22 kDa protein encoded by the CLEC3B gene and was initially identi ed as a plasminogen-binding protein in human plasma (11). It was recently reported that the concentration of TN is signi cantly decreased in the serum of sepsis patients and septic mice, as compared with healthy controls (12). In addition, supplementation of recombinant human or mouse TN reduced the mortality rate of mice of polymicrobial sepsis (12). It is, however, not clear whether the reduction of TN concentration in septic plasma is a sepsis-or organ-speci c host response.
In this study, we determined plasma concentrations of TN in healthy controls, and subjects with community-acquired pneumonia (CAP), sepsis developed from pneumonia (PtoS), pneumonia after sepsis resolution (StoP) or sepsis with comorbid pneumonia (SWP), and correlated plasma concentrations of TN and values of a panel of clinical tests, re ecting the status of in ammation, and the function of most critical organ systems. In addition, we assessed the role of TN in organ injury using a rodent model of polymicrobial sepsis.

Methods
Detailed information of materials is summarized in S- Table 1.

Inclusion and exclusion criteria of human subjects
Subjects of both genders between 50 and 85 years of age were randomly quali ed initially, and subsequently screened with the following exclusion criteria: pregnancy, recent chemotherapy, use of immunosuppressants or steroid, and HIV or HBV positive.
Thirty-seven subjects with no existing medical conditions were enrolled as healthy controls. Thirty CAP subjects were diagnosed according to a recently updated guideline (13). Three hundred and sixty-three SWP subjects ful lled the Sepsis 3.0 (1) diagnosis criteria of sepsis as well as pneumonia. PtoS refers to CAP subjects that were later quali ed as SWP due to development of organ dysfunction, as determined by sequential [sepsis-related] organ failure assessment (SOFA), whereas StoP subjects were SWP patients that subsequently recovered from sepsis (SOFA < 1), but retained symptoms of pneumonia. Sepsis-associated pneumonia (SAP) is a combination of PtoS and StoP groups.
Comorbidities of SWP patients was based on diagnosis on discharge, which re ected assessments of the course of disease progression during hospitalization. Values of a panel of clinical tests (S- Table 2) were obtained from hospital record. The mortality was determined by hospital record or follow-up after discharge.
Human blood collection and plasma preparation Plasma preparation was conducted according to a protocol as previously described (14). Brie y, blood samples were obtained on the day of diagnosis of pneumonia or sepsis, centrifuged at 2,000g at 4 o C for 10 min within 2 hr after collection, and the resultant plasma was harvested, aliquoted and then kept at -80 o C until use.

Mouse model of polymicrobial sepsis
A total of 61 Balb/c male and 20 female mice (9-10 wks old, 23-27g) were obtained from the Model Animal Research Center of Nanjing University (Nanjing, China). A mouse model of polymicrobial sepsis was generated by the ligation and puncture of cecum (CLP), as previously described (15,16). For the characterization of changes in plasma TN in sepsis, CLP mice of both genders were allowed to survive 0, 6, 12, 24 and 48 hr before euthanization and subsequent intracardial blood collection. For the assessment of tissue injury, mice were allowed to survival to 6 and 18 hr post CLP. For the 6-hr survival group, mice received a single intravenous injection (via tail vein) either saline (+CLP+Saline) or recombinant TN (2 mg/kg, +CLP+TN) at 2 hr before euthanization. For the 18-hr survival group, mice were injected with either saline or TN once at 4 hr and once at 16 hr post CLP (2 hr before euthanization).
Blood and tissue samples were collected at the end of survival time.

TN ELISA
The concentration of TN in human plasma was determined using a human TN ELISA kit from Sino Biological (Beijing, China), according to a protocol provided by the manufacturer. One sample from each subject in healthy, CAP and SAP groups, and 1,132 samples from SWP groups (including multiple samples from 222 SWP subjects and single sample from the other 141 SWP subjects) were analyzed.

Direct bilirubin (DBIL), creatinine and blood urea nitrogen (BUN) assays
The levels of DBIL, creatinine and BUN in septic mouse plasma were determined using commercial test kits according to manufacturer's instructions.

Statistical analysis
Shapiro-Wilk normality test was performed to determine the distribution normality of variables. Variables with a normal distribution were analyzed using Student's t-test for comparison of two groups, or one-way ANOVA followed by Tukey test for multiple groups, and presented as mean + standard deviation; Variables with a nonparametric distribution were analyzed using the Mann-Whitney U test for two groups, or the Kruskal-Wallis ANOVA test followed by the Dunn's test for multiple groups, and presented as median and interquartile ranges. Spearman's rank-order correlation (r s ) analysis was performed to determine the correlation between plasma TN concentrations and values of clinical tests (S- Table 2). The statistical signi cance level was set at p < 0.05.

Patients characteristics
The demographics and basic clinical characteristics of human subjects are shown in Table 1 (1), dysfunction score of the respiratory system (SOFA_lung) ranked the highest. Accordant to the inclusion criteria, all SWP subjects had comorbidity of the respiratory system, followed by hypoproteinemia (55%) and gastrointestinal disorders (47%).
Plasma TN concentrations in healthy, pneumonia and sepsis subjects By de nition, infection is the necessary prelude for sepsis. In order to determine whether plasma TN is associated with sepsis pathogenesis, we compared the concentrations of TN in one plasma sample from each of plasma from 37 healthy, 30 CAP, 60 SAP and 148 SWP subjects, and multiple samples from 222 SWP subjects (1090 SWP samples in totality), using a human TN ELISA kit. The value of the rst samples (collected on the day of diagnosis) of all SWP subject was used as representative of the SWP group. Consistent with the reported range (12,(17)(18)(19), the average TN concentration in healthy individuals was 11.27 + 4.39 mg/L, the median concentration, however, was 2.12 mg/L (0.81-4.41, Fig. 1A). Compared with the healthy subjects, TN levels in CAP, SAP and sepsis plasma were 1.12 (0.76-1.40), 0.65 (0.32-1.61) and 0.92 mg/L (0.47-1.73), respectively (Fig. 1A), suggesting that plasma TN levels were reduced by 47.48%, 69.22% and 57% in CAP, SAP and SWP subjects, respectively. Table 2, the plasma TN concentration was generally higher in female than male patients with various comorbidities, but the difference was not statistically signi cant in all comorbidity groups except hypoproteinemia (p = 0.03). No signi cant differences were found in TN concentrations before  Fig. 1C). Moreover, no signi cant correlations were found between TN concentrations and the probability of occurrence of septic shock or 30 mortality rates of both genders, regardless of comorbidities.

As shown in
The association of plasma TN with organ dysfunction in sepsis patients Correlation analysis showed that TN concentrations in SWP plasma had the highest, but nevertheless moderate, correlation with brinogen among values of 42 clinical tests (r s = -0.29, Table 3). However, analysis of paired plasma samples (moderate and severe) from 31 moribund SWP subjects revealed a stronger and negative correlation between TN and liver dysfunction markers (Table 4), such as direct bilirubin (DBIL, r s = -0.46) and total bilirubin (r s = -0.42), indicating an inverse relation between plasma TN and liver injury in the course of sepsis-induced mortality.

The impact of recombinant TN on tissue injury in septic mice
To investigate the role of plasma TN in sepsis, we rst determined whether the induction of sepsis is accompanied by a down regulation of plasma TN in septic mice (Fig. 2). As expected, a signi cant reduction of plasma TN occurred at 6 hr post CLP, which persisted in the following 42 hr, in both genders ( Fig. 2A, B), demonstrating that the CLP-induced peritoneal infection caused a rapid and massive down regulation of plasma TN in a gender-independent manner.
As shown in Fig. 3 decreases in liver-speci c injury markers, ALT (p = 0.009) and DBIL (p = 0.03), respectively. In contrast to these liver injury markers, TN did not have any signi cant effects on non-selective tissue injury markers, such as AST and LDH, or markers for cardiac (CKM) and kidney (creatinine) injuries in septic mouse plasma. These results suggest that recombinant TN is protective of the liver in septic mice, corresponding to the correlation of plasma TN and the functional state of liver in sepsis patients.
Septic liver injury is associated with an activation of multiple mechanisms of cell death (20)(21)(22). As shown in Figure 4, we examined the levels of a panel of cell death markers in normal (-LPS) and sepsis mice. As shown in Figure 4, sepsis mouse liver (+CLP+Saline) contained signi cantly higher levels of RIP3, caspase-11 and GPX4, and lower levels of caspase-8, but no signi cant difference in caspase-1 at 18 after CLP, as compared with normal mice (-LPS). Intravenous administration of rHuTN (+LPS+TN) signi cantly reduced the level of RIP3 (p = 0.04), but not the others.

Discussion
By determining the plasma TN concentration of healthy, pneumonia and sepsis subjects and characterizing the correlation of plasma TN level and values of a variety of clinical parameters in sepsis patients, we found that both pneumonia in human and peritonitis in mouse can cause a dramatic down regulation of plasma TN. The reduction of TN concentration is likely not a sepsis-speci c event, and is not related to the occurrence or resolution of sepsis, but may contributes to the liver injury and dysfunction in moribund sepsis patients.
TN was initially identi ed as a plasminogen-binding protein in human plasma (11). A decrease in plasma TN has been observed in in ammatory diseases, such as ischemia, trauma and rheumatoid arthritis (17)(18)(19). Recently, Chen et al. found that administration of endotoxin or induction of peritonitis by CLP can cause a rapid decrease in plasma TN in mice (12). Our animal studies con rmed the nding by Chen et al. and, together, suggest that TN is a negative acute phase protein in a variety of infectious and noninfectious in ammatory diseases.
Compared with healthy subjects, plasma TN levels remained depressed in CAP, SAP and SWP subjects. Importantly, there was no signi cant change in the concentration of plasma TN before and after the development of sepsis in PtoS subjects, or the resolution of sepsis in StoP subjects. Moreover, the plasma TN level was not signi cantly associated with the occurrence of septic shock or 30-day mortality in both genders, regardless of comorbidities. These results suggest, apparently, that plasma TN is not a critical factor in the development, progression or prognosis of sepsis, at least in patients with comorbid pneumonia.
In contrast to the insigni cant role of plasma TN in sepsis patients, Chen et al. (12) showed in a mouse model of sepsis that a de ciency in TN expression was associated with increased mortality, whereas administrations of recombinant TN or TN domain-speci c monoclonal antibodies resulted in higher survival rates, suggesting an important role of TN in the (prevention of) pathogenesis of sepsis, at least in rodents.
The cause for this discrepancy is unknown. In SWP patients, the average dysfunction score of liver (0.53) was the lowest among all six organs (0.53-2.71, Table 1), indicating an overall very mild level of liver dysfunction. As a result, the association between plasma TN with liver dysfunction may have only very limited, and consequently insigni cant, impact on sepsis mortality in SWP patients. In septic mice, however, liver injury appears to be a prominent pathological component, as indicated by over 4-and 13fold increases in the plasma level of ALT and DBIL over control mice (Fig. 3). In fact, hepatocyte-speci c de ciency in the expression of a damage-associated molecular pattern, high mobility groups box-1, resulted in a marked reduction of the mortality rate of CLP mice (23). Thus, the extent of liver injury may have a substantial impact on the outcome in septic mice. Consequently, the apparently preferential liver protection by recombinant TN in septic mice may manifest in improved survival, as previously reported (12).
Sepsis-associated liver injury can be caused by activation of various mechanisms of cell death (20)(21)(22). Among a panel of cell injury markers, CLP induced most dramatic increases of RIP3 (26-fold) and caspase-11 (5-fold), a signi cant decrease of caspase-8, but did not have any signi cant impacts on caspase-1 and GPX4, in septic mouse liver. Administration of TN only caused signi cant reduction of RIP3, but not caspase-1, caspase-11, GPX4 and caspase-8, suggesting that the liver protective effect of TN is mediated, at least in part, through an inhibition of RIP3-mediated cell death.
The selective liver protection suggests a unique targeting mechanism of TN, which remains to be investigated due to a lack of understanding of TN receptors. Alternatively, a number of TN-binding factors have been identi ed in plasma, including plasminogen, brin, heparin hepatic growth factor and lipoprotein(a) (11,(24)(25)(26)(27). Hepatic receptors for TN, or its binding partners, deserve particular attention in the clari cation of the role of TN in sepsis.

Conclusion
In this study, we demonstrated that plasma TN is a negative APP. The dramatic and sustained down regulation of plasma TN is a respiratory infection-induced and sepsis-independent host response that may contribute to sepsis-associated liver injury, possibly by compromising the control of RIP3-mediated cell death. Supplementation of exogenous TN may hold therapeutic potential for the prevention or treatment of liver dysfunction in sepsis.