Recombinant thrombomodulin does not promote bacterial propagation but alleviates oxidative stress in rats infected with methicillin-resistant Staphylococcus aureus


 Background

Recombinant thrombomodulin (rTM) has been used for treatment of sepsis-associated disseminated intravascular coagulation. Recent studies have suggested that anticoagulant therapy might dampen the protective role of immunothrombosis. We examined if rTM might worsen infectious diseases.
Methods

Male Sprague–Dawley rats with jugular-vein catheterization were divided into three groups: no anticoagulation; rTM pretreatment; rTM treatment at 6 h. Live methicillin-resistant Staphylococcus aureus (MRSA) was inoculated into the tail vein of rats. rTM was administered into the jugular-vein catheter before or 6 h after MRSA inoculation, while an equal volume of saline was administered in the no-anticoagulation group. Blood samples were collected from the jugular-vein catheter before, 6 h and 12 h after MRSA inoculation. Tissue samples were collected from anesthetized rats when moribund or 18 h after MRSA inoculation.
Results

The survival rate of rats in the no-anticoagulation group, rTM pretreatment group, and rTM treatment at 6-h group was 50%, 25%, and 75%, respectively. Bacterial burden in blood, lung, liver, and spleen was neither increased nor decreased in rats treated with rTM. The ratio of bacteria found in the extravascular space to those in the intravascular space was increased in rats treated with rTM. Metabolomics analysis revealed that rTM treatment alleviated oxidative stress, as evidenced by the decrease in levels of oxidized glutathione.
Conclusions

rTM does not promote bacterial propagation but alleviates oxidative stress in a rat model of bloodstream infection with MRSA.


Results
The survival rate of rats in the no-anticoagulation group, rTM pretreatment group, and rTM treatment at 6h group was 50%, 25%, and 75%, respectively. Bacterial burden in blood, lung, liver, and spleen was neither increased nor decreased in rats treated with rTM. The ratio of bacteria found in the extravascular space to those in the intravascular space was increased in rats treated with rTM. Metabolomics analysis revealed that rTM treatment alleviated oxidative stress, as evidenced by the decrease in levels of oxidized glutathione.
Conclusions rTM does not promote bacterial propagation but alleviates oxidative stress in a rat model of bloodstream infection with MRSA. Background Sepsis can be de ned as life-threatening organ dysfunction caused by dysregulated host responses to infection (1). Dysregulated host responses include feedforward neutrophil accumulation, overproduction of reactive oxygen species, release of neutrophil extracellular traps (NETs), and a metabolic shift towards decreased oxidation of fatty acids (2-4). Intravascular activation of coagulation is another important feature of the host response to infection (2, 5). Expression of tissue factor on monocytes and release of NETs from neutrophils are thought to be triggers for intravascular coagulation (5,6). Coagulation under certain circumstances has a major physiologic role in innate immune defense against infection, a process known as "immunothrombosis". However, uncontrolled immunothrombosis may lead to ischemic organ failure.
Several mechanisms have been proposed by which immunothrombosis promotes defense against infection (6). First, immunothrombosis limits microbial dissemination by arresting microbes within the clot (7). Second, thrombi form "barricades" which can limit microbial spread from one compartment to another (8,9). Third, brin supports leukocytes in eliminating microbes (10). Consequently, mice pretreated with coumadin have been shown to display increased hepatic bacterial burden and mortality after inoculation with Yersinia enterocolitica (11).
Anticoagulant therapy has been considered as a treatment option for sepsis. It is not e cacious in sepsis overall, but may provide bene cial effects in a speci c subpopulation with sepsis-associated disseminated intravascular coagulation (DIC) (12,13), possibly by alleviating ischemic organ failure.
Among anticoagulants, recombinant thrombomodulin (rTM) is a potential therapeutic agent for sepsisassociated DIC (14). Meta-analyses have suggested a potential survival bene t using this drug (15,16), and rTM is now used widely in Japan. However, one might assume that treatment with rTM "dampens" the protective role of immunothrombosis and, thus, may worsen infectious diseases. To validate the plausibility of this hypothesis, we examined bacterial burden in organs and blood in the presence or absence of rTM in methicillin-resistant Staphylococcus aureus (MRSA)-infected rats. We also examined if the timing of rTM administration, before or after MRSA inoculation, affected bacterial burden.

Model of MRSA infection in rats
Experiments involving animals were approved by the Animal Care and Use Committee of Kagoshima University and Shin Nippon Biomedical Laboratories.
Male Sprague-Dawley rats with jugular-vein catheterization were used for all experiments. MRSA (ATCC-43300) was incubated with agitation in Mueller-Hinton broth (Eiken Chemicals, Tokyo, Japan) for 2.5 h at 37 °C to achieve mid-log-phase growth. Then, MRSA was resuspended in saline, and 3.5 × 10 8 colonyforming units (CFUs) of MRSA was inoculated into the tail vein of rats at 0 h and 5 h. Anticoagulation therapy was conducted in some rats, for which rTM (1 mg/kg; Asahi Kasei Pharma, Tokyo, Japan) was administered into the jugular-vein catheter immediately before (rTM pretreatment group) or 6 h after the rst MRSA inoculation (rTM treatment at 6-h group). The general condition of rats was evaluated every 1 h. Rats were sacri ced when moribund or 18 h after the rst MRSA inoculation (n = 8 in each group).

Blood tests
Blood samples were collected from the jugular-vein catheter before, 6 h and 12 h after MRSA inoculation (n = 5 in each group). One portion of blood samples was used for bacteriology. Blood samples anticoagulated with ethylenediaminetetraacetic acid were used for automated counting of blood cells by an ADVIA120 system (Bayer Diagnostics, Leverkusen, Germany). Serum samples were used for measurement of levels of total bilirubin and creatinine by a clinical biochemistry analyzer (BioMajesty™ JCA-BM6070; Jeol, Tokyo, Japan). Citrated plasma samples were used for metabolomics analysis.

Bacteriology and pathology
Anesthetized rats were sacri ced when moribund or 18 h after MRSA inoculation. Small samples of liver, spleen, kidney, and lung were homogenized in sterile phosphate-buffered saline (PBS, 1 mL/g tissue), and used for bacteriology (n = 5 in each group). The remainder of the organs were xed in 10% neutralbuffered formalin, and used for pathology studies (n = 3 in each group). Samples of blood and tissue homogenates were serially diluted with sterile PBS, plated onto sheep blood agar plates (Nissui Pharmaceuticals, Tokyo, Japan), and incubated overnight at 37 °C before bacterial enumeration. Bacterial distribution in each organ was analyzed using Gram-stained tissue sections.

Metabolomics analysis
Metabolomics analysis was undertaken at a service facility of LSI Medience (Tokyo, Japan). Brie y, plasma samples (200 µL) collected before and 12 h after MRSA inoculation were mixed with methanol (800 µL) and shaken for 15 min at room temperature. After centrifugation, the precipitates were dissolved with 10% acetonitrile aqueous solution (200 µL). After addition of internal standards, they were analyzed by liquid chromatography-mass spectrometry (LC-MS) and capillary electrophoresis-mass spectrometry (CE-MS). Tuning and calibration were carried out with standard solutions provided by Agilent Technologies (Palo Alto, CA, USA).
LC-MS was done using a LC system (HP1200; Agilent Technologies) equipped with a C18 column (2 µm, 50 mm × 2.0 mm ID, CAPCELL PAK C18 IF; Shiseido, Tokyo, Japan) coupled with an electrospray ionization quadrupole time-of-ight mass spectrometer (6520; Agilent Technologies). Solvent A was composed of 5 mM ammonium acetate aqueous solution, whereas solvent B was acetonitrile.
Metabolites were eluted at a ow rate of 0.2 mL/min at 40 °C with a linear gradient of 10-90% solvent B over 10 min, followed by a further 5-min hold at 100% solvent B. The mass spectrometer was operated in positive and negative scan modes (m/z 60 to 1,200) with a capillary voltage of 3,500 V. The nebulizing gas pressure was 40 psi and the dry gas ow was 8 L/min at 350 °C.
Ionic metabolites were measured in the positive mode of a CE-time-of-ight mass spectrometer (6520; Agilent Technologies). Metabolites were separated in a fused-silica capillary (50 µm i.d.×100 cm total length; GL Science, Tokyo, Japan) lled with 1 mol/L of formic acid aqueous solution (cation mode), or 20 mM ammonium formate and 20 mM ammonium acetate aqueous solution (pH 10, anion mode) as the electrolyte. The sample solution was injected at 5 kPa for 15 s (~ 15 nL), and a voltage of 30 kV was applied. The capillary tray and sample tray were maintained at room temperature and 5 °C, respectively.
The sheath liquid was methanol/water (50% v/v) containing 5 mM ammonium acetate. The CE-time of ight mass spectrometer was operated in positive and negative scan modes (m/z 60 to 1,200). The capillary voltage was set at 3,500 V and the nitrogen gas (heater temperature = 250 °C) ow rate was set at 10 L/min.
A data le of MS was converted to csv format with csv convertor (Agilent Technologies). All peak positions (retention time and m/z) and areas were calculated by Markeranalysis (LSI Medience). All peak areas were aligned into one datasheet and the errors of peak intensities were corrected by internal standards. Noise peaks were deleted compared with the peaks detected in blank samples. Metabolites were identi ed by comparing the retention time and m/z with the standard data set established by LSI Medience. Mean fold-change and the Student's t-test were carried out for all detected peaks.

Statistical analyses
Statistical analyses were carried out using SPSS v26 (IBM. Armonk NY, USA). For comparison of independent groups, the Student's t-test was used. P < 0.05 was considered signi cant.

Results
To test whether anticoagulation with rTM could worsen infectious diseases, we rst analyzed the survival rates of MRSA-infected rats in the presence or absence of rTM administration. The survival rate of rats without rTM administration was 50% at the study endpoint. The survival rate of rats treated with rTM immediately before or 6 h after MRSA inoculation was 25% and 75%, respectively. Hence, the timing of rTM administration might be an important determinant of treatment e cacy.
Next, we conducted hematology and bacteriology studies to ascertain the effects of rTM administration on MRSA-induced disease. Leukopenia became evident 6 h after MRSA inoculation regardless of treatment (Fig. 2B). Thrombocytopenia was less prominent in rats treated with rTM when compared with those without anticoagulation, though the difference was not signi cant (Fig. 2C). Liver dysfunction and kidney dysfunction, as evidenced by an increase in the level of total bilirubin and creatinine, respectively, became prominent 12 h after MRSA inoculation regardless of treatment (Fig. 2D, E). The bacterial burden in samples of blood, lung, liver, and spleen was neither increased nor decreased in rats treated with rTM ( Fig. 3A, B). However, the ratio of bacteria found in the extravascular space to those found in the intravascular space was increased in rats treated with rTM (Fig. 3C, D). These ndings suggested that rTM treatment could allow bacterial passage from the intravascular space to the extravascular space, but bacterial burden in the whole organ may be only slightly affected by rTM treatment.
Next, we analyzed metabolites in the plasma of MRSA-infected rats comprehensively to ascertain the potential effects of rTM administration. A total of 209 metabolites of known structures were identi ed and pro led. The relative abundance and P-value of each metabolite in the rTM treatment at 6-h group with reference to the no-anticoagulation group or rTM pretreatment group are shown in Fig. 4A, and the identity of each metabolite is shown in Supplementary Table. Among the metabolites, levels of oxidized glutathione, glycodeoxycholate, and uridine 5′-diphosphate were decreased signi cantly in the rTM treatment at 6-h group compared with those in the no-anticoagulation group, whereas octanoylcarnitine levels were increased signi cantly (Fig. 4B). These ndings suggested that rTM treatment may alleviate oxidative stress, bile-acid stasis, nucleotide accumulation, and recover fatty-acid metabolism.

Discussion
Recent studies have raised fears that anticoagulant therapy might dampen the protective role of immunothrombosis and, thus, might worsen infectious diseases. Using a rat model of bloodstream infection with MRSA, we found that treatment with rTM neither increased bacterial burden nor worsened overall survival if rTM was used after the infection. Instead, rTM treatment alleviated oxidative stress, as evidenced by the decrease in the level of oxidized glutathione.
Bacterial passage from one compartment to another (e.g., from the intravascular space to the extravascular space) was facilitated by rTM treatment in our study. This nding might not be speci c to rTM but common to various anticoagulants, including hirudin (8) and heparin (data not shown). Thus, we cannot completely deny the potential risk of bacterial dissemination by anticoagulant therapy. However, the increase of bacteria in the extravascular space seemed to be accompanied by a decrease of bacteria in the intravascular space, and so the bacterial burden in the whole organ was little-affected by rTM treatment. Considering that rTM did not worsen the survival rate of MRSA-infected rats if used after the infection, rTM treatment might not dampen the protective role of immunothrombosis.
Metabolomics analysis revealed that the increase in the level of oxidized glutathione in response to MRSA infection was alleviated by rTM administration 6 h after MRSA infection. Glutathione plays a major part in cellular defenses against oxidative stress (17). In exchange for the reduction of target proteins, glutathione (also known as reduced glutathione or GSH) is converted to glutathione disul de (also known as oxidized glutathione or GSSG). The GSSG:GSH ratio is thought to be a marker of cellular toxicity and is associated with mortality in septic-shock patients (18). In the present study, the GSSG:GSH ratio was also increased in response to MRSA infection, and was decreased by rTM administration 6 h after MRSA infection (data not shown). These ndings suggest that rTM alleviates oxidative stress, possibly through the maintenance of vascular patency or its intrinsic antioxidant properties (19).
The timing of anticoagulation might also be controversial. Anticoagulant drugs have been administered before infection in studies for immunothrombosis (8,11) whereas rTM has been administered after infection onset in clinical settings (20). We administered rTM immediately before or 6 h after MRSA infection, and found that rTM administration 6 h after MRSA infection might be better in terms of improved survival. Although the best timing for clinical anticoagulation was not speci ed in our study, signs of organ dysfunction in addition to evident DIC might be important. Further studies are needed to gain insight into the appropriate timing for anticoagulation during sepsis treatment.
Our study had several limitations. First, it is not clear whether our ndings in rats can be transferred to humans. Although studies have shown that rTM is effective in mitigating DIC in rats and humans (20,21), activated protein C-dependent actions might not be fully expected in rats, unlike humans (22

Supplementary Files
This is a list of supplementary les associated with this preprint. Click to download. SupplementaryTableMetabolite.docx