Sepsis is a life-threatening condition caused by a dysregulated host response to infection (1). The hunt for a cure has been ongoing for years, but, except for growing alertness, early identification, and rapid onset with antibiotics and organ supportive care, drugs specifically targeting causal molecules or pathways responsible for the dysregulated immune response inducing sepsis, are still missing (2). As such, clinical trials in sepsis generally lack effectiveness (3, 4), and a need for precision medicine is gaining increasingly more attention since the beginning of the century (5).
Throughout the last few years, an ongoing debate on whether intravenous vitamin C administration to septic patients is beneficial has dominated the field (6). Also known as ascorbic acid, vitamin C is a low molecular weight anti-oxidant, with both anti-inflammatory and immune-enhancing properties due to its capacity to accept and donate electrons in human cells (7). Its role in sepsis is commonly explained through its attenuating effect in oxidative stress and inflammation, among others (6, 8). It has also been shown to suppress tumor necrosis factor (TNF)-induced NF-κB-activation in human cells and mice (9, 10), overturn myeloperoxidase (MPO) expression in lung cells (11), lower histamine levels in patients (12), and decrease high mobility group box 1 secretion in mice (13). Vitamin C acts both as a nuclear transcription factor and a cytokine and is highly concentrated in several immune cells, such as leukocytes, lymphocytes, and macrophages, enhancing their maturation (14), chemotaxis, proliferation, phagocytosis, and interferon production (15). Furthermore, several studies have also demonstrated the antimicrobial effect of vitamin C, both in vitro and in vivo(16–18), even opening the path to its use in the prevention and treatment of COVID-19 (19, 20).
On the other hand, hydrocortisone is commonly used in clinical sepsis, although the evidence is debated, short- and long-term mortality appears to be unaffected, still, its use is recommended for patients with fluid- and vasopressor refractory shock (2, 21). The primary anti-inflammatory action of this and other glucocorticoids is to repress the transcriptional activity of multiple proinflammatory genes such as NF-κB and AP-1. As such, since some of its effects overlap with those of vitamin C, these two drugs are speculated to act synergistically in the setting of bacterial sepsis (22).
The rationale to use vitamin C in sepsis has been highlighted after the retrospective study by Marik and colleagues (23) which demonstrated that a cocktail of hydrocortisone, ascorbic acid, and thiamine reduced sepsis mortality from 40.4% to 8.5% in the treatment group. An absolute and relative reduction in mortality of 32% and 79%, respectively, is biologically implausible, leading to wide critics due to insufficient methodological setup. Furthermore, this finding led to the conduction of randomized controlled trials which demonstrated a lack of effect by this combined treatment regimen (24–29), even undermining its findings by showing higher 90-day mortality in the treatment group (26). However, vitamin C and hydrocortisone are still used in the treatment of sepsis patients as the in vitro anti-inflammatory effects are still thought to be of importance for septic patients.
Innate immunity plays a key role in sepsis pathophysiology (30), by activation of upstream pattern recognition receptors resulting in downstream signaling through a multitude of different pathways. The complement system and the Toll-like receptors (TLRs) have proven to be important, independently acting but also interconnected through extensive cross-talk and responsible for a robust and redundant host response (31, 32). Within this complex network featured by a myriad of mediators, evidence suggests an increased activation of radical oxygen species (33). In this situation, anti-oxidants like vitamin C appear as intuitively relevant drugs. However, preclinical data examining the effect of vitamin C and hydrocortisone on bacteria-induced inflammation is limited (16–18).
Thus, the present study aimed to explore the effect of vitamin C, hydrocortisone, and the combination thereof, in a human whole-blood model of inflammation and study key aspects of the innate immune response to Gram-negative and Gram-positive bacteria in the presence and the absence of these drugs.
MATERIALS AND METHODS
Equipment and reagents
Endotoxin-free tubes from Thermo Fischer Scientific NUNC (Roskilde, Denmark), sterile phosphate-buffered saline (PBS) with Ca2+ and Mg2+, and ethylenediaminetetraacetic acid (EDTA) from Sigma–Aldrich (Steinheim, Germany) were used. Lepirudin (Refludan®) from Pharmion (Windsor, UK) was diluted in PBS to a concentration of 2.5 mg/mL and used as an anticoagulant. Ascorbic acid 150 mg/mL (Pascorbin®) was obtained from Pascoe Natural Healthcare (Gießen, Germany), whereas hydrocortisone 50 mg/mL (Solu-Cortef®) was obtained from Pfizer (New York, NY).
Heat-inactivated Escherichia coli strain LE392 (ATCC 33572) and live Staphylococcus aureus Cowan strain 1 (ATCC 12598) were purchased from the American Type Culture Collection (Manassas, VA). Both bacteria are clinically relevant, representing the most common pathogens of Gram-negative and Gram-positive origin. Previously experiments using the same whole-blood model have demonstrated that live and dead or heat-inactivated strains of E. coli and S. aureus induced a similar pronounced and broad inflammation (34). Concentrations of both bacteria were adjusted to provoke noticeable inflammation, and differences in concentrations chosen (107 bacteria/mL of E. coli and 108 bacteria/mL of S. aureus) to obtain reasonable similar responses.
The soluble terminal C5b-9 complement complex (TCC) was quantified by an established in-house enzyme-linked immunosorbent assay (ELISA) previously described (35) and later modified (36). Briefly, the capture antibody is the monoclonal anti-C9 neoepitope (clone aE11) and the detection antibody is the monoclonal biotinylated anti-C6 (clone 9C4). The results are expressed in “complement arbitrary units” (CAU) per milliliter with Stock Complement Standard number 2 as standard (set to 1,000 CAU/mL) (37) and zymosan-activated human serum as the positive control.
Human proinflammatory cytokine interleukin 6 (IL-6) was quantified through an ELISA-based commercial kit from R&D Systems (Minneapolis, MN). The samples were diluted at 1:10 and analyzed according to the manufacturer's provided protocol (catalog number DY206, lot P223708). Proinflammatory cytokines, namely tumor necrosis factor and interleukins 1β (IL-1β), 6 (IL-6), and 8 (IL-8), as well as the anti-inflammatory IL-10, were also measured using a multiplex cytokine panel assay from Bio-Rad Laboratories (Hercules, CA) named Bio-Plex Pro™ Human Cytokine 27-plex Panel (catalog #M500KCAF0Y). The samples were analyzed according to the manufacturer's provided protocol (standard lot # 64301206, control lot # 64347577).
Human granulocyte activation marker MPO was quantified through an ELISA-based commercial kit from R&D Systems (Minneapolis, MN). The samples were diluted 1:80 to 1:4000 and analyzed according to the manufacturer's provided protocol (catalog number DY3174, lot P132450).
Human platelet activation marker beta-thromboglobulin (β-TG) was quantified through an ELISA-based commercial kit from Diagnostica Stago (Asnières-sur-Seine, France). The samples were diluted 1:500 to 1:2,500 and analyzed according to the manufacturer's provided protocol (Asserachrom, REF 00950, lot 256065).
Ex vivo human whole-blood model of inflammation
The human whole-blood model of inflammation used has previously been described in detail (38). Briefly, fresh human whole blood was collected into 4.5-mL NUNC tubes and anticoagulated with a specific thrombin-inhibitor, lepirudin at a final concentration of 50 μg/mL. Unlike other anticoagulants, lepirudin is inert for complement activation thus allowing cross-talk with complement and the remaining inflammatory network in the whole blood (38). Baseline samples were preincubated with PBS for 5 min at 4°C (samples on slushed ice) after the blood was drawn, then processed immediately by adding EDTA. The rest of the samples was preincubated with PBS or drug for 5 min at 4°C, before adding PBS, E. coli to a final concentration of 1 × 107 bacteria/mL or S. aureus to a final concentration of 1 × 108 bacteria/mL, then incubated for 120 min at 37 °C. At the end of the incubation, EDTA was added to a final concentration of 20 mM, stopping complement activation. The tubes were centrifuged for 20 min at 3,000g at 4 °C. Plasma was collected and stored at −70°C until analyzed.
Vitamin C and hydrocortisone titration and combination experiments
Different concentrations of vitamin C and hydrocortisone were assessed initially by measuring the effect on two different key inflammatory mediators, interleukin 6 (IL-6), and the soluble form of the terminal complement complex (sC5b-9 or TCC). The final concentrations used were equivalent to what is used in the clinical setting, 1.5 g of vitamin C and 50 mg of hydrocortisone (23, 26). Given a blood volume of 5 L, these concentrations of vitamin C and hydrocortisone are equivalent to an instant concentration of 300 μL and 10 μL, respectively.
Incubation of whole blood for CD11b analysis
Whole blood was drawn from six healthy human donors (three males and three females) and treated as described before. Bacterial activation with E. coli (adjusted to 1 × 108 bacteria/mL) or S. aureus, and PBS (control) was performed for 15 min at 37 °C, after which the blood was fixed with 0.5% (v/v) paraformaldehyde at 37°C for 4 min. Fixed blood was stained with anti-CD11b phycoerythrin, anti-CD14 PerCP, and anti-CD45 V450, all antibodies from Becton Dickinson (San Jose, CA), for 15 min at room temperature in the dark. The red cells were lysed with EasyLyse™ (Dako, Agilent, Santa Clara, CA) for 15 min and within 1 h run on a NovoFlow cytometer (ACEA, Agilent), with a threshold on CD45 to exclude debris. Monocytes and granulocytes were gated in a side-scatter/CD14 dot-plot. Expression of the activation marker CD11b (also known as CR3) on granulocytes and monocytes was measured as the mean fluorescence intensity. The data were analyzed by NovoExpress (ACEA, Agilent).
Incubation of whole blood for oxidative burst analysis
Samples were treated as described for CD11b quantification and the oxidative burst was quantified through Phagoburst™, a commercial reagent kit for the quantification of the oxidative burst activity of monocytes and granulocytes in heparinized human whole blood from Celonic (Heidelberg, Germany), according to the manufacturer's provided protocol, except for adding DNA-stain. Gating was performed in a forward-scatter/side-scatter dot plot. The flow cytometer and the data analysis software were the same as used for CD11b quantification.
The data were analyzed using GraphPad Prism version 8.3.0 (San Diego, CA) for Windows by one- or two-way ANOVA with Geisser-Greenhouse correction and Dunnett's multiple comparisons test for the comparison of multiple columns. A P-value < 0.05 was considered statistically significant.
Each blood donor provided informed written consent, in addition to the approval of the study by the Regional Ethical Committee (REK Sør, Ref. number S-04114).
Vitamin C and hydrocortisone titration
To assess the effect of vitamin C and hydrocortisone on complement activation and the formation of IL-6, incremental doses of vitamin C and hydrocortisone, covering the clinical concentrations used, were added to human whole blood (n = 6) and incubated with E. coli 107 bacteria/mL (EC) or without (Sham), for 120 min at 37°C. Non-incubated baseline samples (T0) were used for comparison (Fig. 1). Neither vitamin C nor hydrocortisone provoked a statistically significant dose–response effect in the formation of TCC or expression of IL-6. The symbols with dark fill in Figure 1 indicate the concentration that was used in the experiments below.
Complement, granulocyte, and platelet activation measured in plasma
The potential effect of vitamin C, hydrocortisone, and the combination thereof on either E. coli or S. aureus bacterial-induced activation of complement, granulocytes, and platelets were examined in human whole blood (Fig. 2). Neither vitamin C, hydrocortisone, nor the combination thereof had any significant attenuating effect on E. coli (Fig. 2, left panels) or S. aureus (Fig. 2, right panels) induced complement activation measured by TCC, granulocyte activation measured by MPO or platelet activation measured by β-TG.
Inflammatory cytokines release measured in plasma
The potential effect of vitamin C, hydrocortisone, and the combination thereof on bacterial-induced cytokine release (IL-1β, IL6, IL-8, and TNF) with E. coli (Fig. 3, left panels) or S. aureus (Fig. 3, right panels) was examined in human whole blood. E. coli-induced TNF formation was attenuated by hydrocortisone alone (19%, P = 0.01) and by the combination of vitamin C and hydrocortisone (31%, P < 0.01). The release of all other cytokines, induced by either E. coli or S. aureus, was not affected by vitamin C, hydrocortisone, or the combination thereof (Fig. 3).
Monocyte and granulocyte activation measured by surface markers in flow cytometry
The potential effect of vitamin C, hydrocortisone, and the combination thereof on E. coli- (left panels) or S. aureus- (right panels) induced upregulation of CD11b on monocytes and granulocytes was examined in human whole blood (Fig. 4). Compared to the uninhibited bacteria-induced activation, there were no significant differences in the expression of CD11b on either monocytes or granulocytes (Fig. 4).
Oxidative burst measured by reactive oxygen species in flow cytometry
The release of reactive oxygen species was measured by oxidative burst (Fig. 5). Compared to the E. coli-induced activation, vitamin C alone significantly attenuated oxidative burst in monocytes by 14% (P = 0.04) and in granulocytes by 19% (P = 0.02) (Fig. 5, left panels). Hydrocortisone alone attenuated burst in granulocytes by 16% (P = 0.02). The combined inhibition with vitamin C and hydrocortisone reduced the release of reactive oxygen species in monocytes by 8% (P = 0.04) and in granulocytes by 15% (P = 0.03). S. aureus-induced oxidative burst in monocytes and granulocytes were not affected by any inhibition (Fig. 5, right panel).
In the present study, we explored the effect of vitamin C and hydrocortisone on E. coli- and S. aureus-induced inflammation in human whole blood, measuring complement activation, leukocyte activation, platelet activation, and cytokine release. Instead of testing a biological hypothesis by starting at the bench before going to the bedside, we went from bedside to bench, trying to elucidate any rationale for the use of vitamin C in septic patients. In general, hydrocortisone demonstrated anti-inflammatory properties on TNF release, whereas both hydrocortisone and vitamin C showed effects on oxidative burst when E. coli was used. No effects were seen by neither of the drugs using S. aureus.
Different concentrations of vitamin C and hydrocortisone were assessed initially by measuring the effect on two different key inflammatory mediators, interleukin 6 (IL-6) and the soluble form of TCC (sC5b-9). Both mediators appear early in the sepsis pathophysiology, reflect different inflammatory pathways, and are robust measurable markers (39). However, we found no differences in the measured level of these two markers over a broad concentration range including the doses equivalent to what is used in the clinical practice (Fig. 1) (26). Thus, we decided to continue the experiments with these doses.
As expected, both E. coli and S. aureus induced a pronounced complement activation and release of both MPO and β-thromboglobulin. Complement can be regarded as a master alarm system of innate immunity, where in particular the potent anaphylatoxin C5a, appears to be one of the main key players in sepsis (40). Controlled activation of these immunological responses is of prime importance in maintaining homeostasis and immune surveillance. MPO facilitates the neutrophil-mediated killing of bacteria by enhancing the production of oxygen and nitrogen. Uncontrolled MPO release exaggerates inflammation and can also lead to tissue damage in the absence of inflammation (41). Moreover, one previous animal study has described a decrease of MPO release with vitamin C in septic damaged mice lungs (11). Similarly, β-thromboglobulin secreted from platelet granules participates in the inflammation but its excess release will increase sepsis-related impairments (42). However, in our experiments, we could not see attenuating effects of either vitamin C or hydrocortisone on these central markers.
E. coli and S. aureus also induced a clear-cut cytokine response assessed by increased formation of the classical proinflammatory cytokines IL-1β, IL6, IL-8, and TNF, reflecting the “cytokine storm.” Again, vitamin C alone did not have any attenuating effects on either E. coli- or S. aureus-induced inflammation. TNF-induced phosphorylation inhibition of NF-κB has been described (9, 10); however, it occurs downstream of the TNF receptor, so a change in the free concentration would not be expected. On the other hand, hydrocortisone significantly decreased E. coli-induced TNF formation. This observation is in line with previous studies, describing suppression of TNF formation in human monocytes by hydrocortisone (43). The role of low-dose hydrocortisone in septic patients has been investigated in numerous randomized clinical trials, but its use is still controversial. The Surviving Sepsis Campaign suggests in general against its use, except to those patients with shock not responding to fluid and vasoactive treatment (2). Notably, hydrocortisone did not have any effect on S. aureus-induced inflammation.
An essential part of host defense against pathogens is oxidative burst. The NADPH oxidase (NOX2) is the enzyme responsible for the creation of superoxide anions, enabling oxidative damage and destruction of neutrophil- and macrophage-engulfed pathogens (44). Furthermore, it is well known that sepsis and septic shock leads to an augmentation of free radical oxygen species, potentially causing increased damage to the host (33). It is proposed that arachidonic acid and its derivatives may have antagonistic roles on NOX2 activity (45), advocating the use of vitamin C. In our study, we observed that vitamin C attenuated oxidative burst in monocytes and granulocytes by 14% and 19%, respectively, whereas hydrocortisone attenuated burst in granulocytes by 16%. Among all different inflammatory readouts explored, reduction of oxidative burst by vitamin C was the only significantly affected read-out.
In this ex vivo model, we have previously used a completely different strategy to attenuate the inflammatory response to the two bacteria. By targeting upstream pattern recognition molecules of innate immunity, the complement system and the TLR accessory molecule CD14, we have demonstrated a global downstream attenuating effect on all inflammatory mediators investigated (31). This dual inhibition blocked ex vivo E. coli-induced inflammation with 80% to 99% (46, 47), whereas S. aureus-induced inflammation was attenuated to a similar extent (48). Furthermore, in vivo studies showed a reduction of the cytokine storm to the same extent as ex vivo, and most important, significantly increased the survival in mice and pigs (49, 50). As compared to these previous observations demonstrating an efficient attenuation of the inflammatory response, it is uncertain whether the effect by vitamin C observed in the present ex vivo study would be of biological significance, and if it would be of therapeutical efficacy if translated into the clinic.
Although distant from what happens in clinical sepsis, we staged this bacterial-induced inflammation through a well-used and reliable ex vivo whole-blood model allowing cross-talk to occur between all inflammatory branches within human blood (38). Even though our “clean” ex vivo model is not comparable to the in vivo situation, involving the circulatory system, endothelial cells, and organ interaction with companion systemic processes, it is still surprising that vitamin C and its combination with hydrocortisone had only a reduced effect on two of the nine biomarkers induced by E. coli and no effect on the inflammatory response induced by S. aureus, given the large effect referred in the most optimistic studies (23). When implementing vitamin C to clinical use, one would expect thorough and broad documentation including preclinical studies advocating beneficial effects. Instead, the treatment regimen appears to be based on case reports and sparse references not always sepsis-specific (6–9, 11–18, 23), i.e., not on data justifying the clinical use. Worryingly, this drug keeps being administered to patients and funds being spent in large clinical trials without a solid scientific rationale for its use. To our knowledge, no properly conducted randomized controlled trial has demonstrated that vitamin C increases survival in sepsis and septic shock. On the contrary, several studies recently published document no effect (26, 29).
In conclusion, bacteria-induced inflammation in a physiologically relevant ex vivo whole-blood model showed significant attenuation by vitamin C, hydrocortisone, or the combination thereof for two of the nine biomarkers, namely, TNF and oxidative burst. This effect was seen for E. coli although no effect was observed with S. aureus. The findings of this preclinical study might partly explain the pathophysiological reason for the randomized clinical trials with negative results, linking bedside findings to what we found on the bench.
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