Ex Vivo Evaluation of the Sepsis Triple Therapy High-Dose Vitamin C in Combination with Vitamin B1 and Hydrocortisone in a Human Peripheral Blood Mononuclear Cells (PBMCs) Model

Abstract

Sepsis is an extremely complex clinical syndrome, usually involving an excessive inflammatory response including an overshooting cytokine release that damages tissue and organs of the patient. Due to the severity of this condition, it is estimated that over 11 million people die from sepsis each year. Despite intensive research in the field, there is still no specific therapy for sepsis. Many sepsis patients show a marked deficiency of vitamin C. 9 out of 10 sepsis patients have a hypovitaminosis C, and every third patient even shows a clinical deficiency in the scurvy range. In addition, low vitamin C levels of intensive care sepsis patients correlate with a higher need for vasopressors, higher Sequential Organ Failure Assessment (SOFA) scores, and increased mortality. Based on this observation and the conducted clinical trials using vitamin C as sepsis therapy in intensive care patients, the aim of the present ex vivo study was to evaluate the effects of high-dose vitamin C alone and in a triple combination supplemented with vitamin B1 (thiamine) and hydrocortisone on the lipopolysaccharide (LPS)-induced cytokine response in peripheral blood mononuclear cells (PBMCs) from healthy human donors. We found that all corticosteroid combinations strongly reduced the cytokine response on RNA- and protein levels, while high-dose vitamin C alone significantly diminished the PBMC mediated secretion of the cytokines interleukin (IL)-10, IL-23, and monocyte chemo-attractant protein (MCP-1), which mediate the inflammatory response. However, vitamin C showed no enhancing effect on the secretion of further cytokines studied. This data provides important insights into the possible immunomodulatory function of vitamin C in an ex vivo setting of human PBMCs and the modulation of their cytokine profile in the context of sepsis. Since vitamin C is a vital micronutrient, the restoration of physiologically adequate concentrations should be integrated into routine sepsis therapy, and the therapeutic effects of supraphysiological concentrations of vitamin C in sepsis patients should be further investigated in clinical trials.

Keywords: cytokine release; hydrocortisone; sepsis; vitamin B1; vitamin C.

Figure 1

PBMC viability analysis and cytokine secretion analysis after vitamin C treatment. (A) The viability of the PBMCs was measured with the Countess^TM II FL Automated Cell Counter and a trypan blue staining. An untreated control and three different vitamin C concentrations were examined, as well as an LPS-treated control and the three vitamin C concentrations under LPS (10 ng/mL) stimulation. Shown are the mean values in percent viability and the standard deviations, with one separate data point for each donor of isolated PBMCs in all 12 donors after 6 h of treatment with different vitamin C concentrations (0.2–2 mM). (B) Heat map analysis of the cytokine secretion detected with the LEGENDplex^TM human inflammation panel 1 via flow cytometry, after stimulation of the cells with different vitamin C concentrations (0.2–2 mM) with (10 ng/mL) or without LPS; shown is the fold induction compared to the control w/o LPS. Additional data analysis stratified according to gender is available as Figure S2. IFN = interferon; IL = interleukin; LPS = bacterial lipopolysaccharides; MCP = monocyte chemo-attractant protein; PBMCs = peripheral blood mononuclear cells; TNF = tumor necrosis factor; Vit. C = vitamin C.

Figure 2

Detailed cytokine secretion analysis after vitamin C treatment. (A–H). The secreted cytokines after 6 h treatment were quantified using LEGENDplex^TM followed by flow cytometry. An untreated control and three different vitamin C concentrations (0.2–2 mM) were studied, as well as an LPS-treated (10 ng/mL) control and the three vitamin C concentrations under LPS stimulation. Shown are the mean values and standard deviations with one separate data point for each donor, with a measurement in duplicates for each donor. *: p ≤ 0.05; **: p ≤ 0.01; ***: p ≤ 0.001. Additional data analysis stratified according to gender is available as Figure S3. IL = interleukin; LPS = bacterial lipopolysaccharides; MCP = monocyte chemo-attractant protein; TNF = tumor necrosis factor; Vit. C = vitamin C.

Figure 3

PBMC viability analysis and cytokine secretion analysis after vitamin C, vitamin B1, and hydrocortisone treatment. (A) The viability of PBMCs measured by Countess™ II FL Automated Cell Counter and a trypan blue staining) was at a comparable level in all donors (6 females (F), 6 males (M)) after the start of the 6 h combination treatment of Vit. C, B1, or HC alone or in combination and with (10 ng/mL) or without LPS. (B) Heat map analysis of the cytokine secretion measured with the LEGENDplex^TM human inflammation panel 1 via flow cytometry, after treatment of the cells for 6 h with either Vit. C (1 mM), B1 (1 µM), and HC (2 µM) alone or in combination and with or without LPS; shown is the fold induction compared to the control w/o LPS. Additional data analysis stratified according to gender is available as Figure S4. B1 = vitamin B1; HC = hydrocortisone; IFN = interferon; IL = interleukin; LPS = bacterial lipopolysaccharides; MCP = monocyte chemo-attractant protein; PBMCs = peripheral blood mononuclear cells; TNF = tumor necrosis factor; Vit. C = vitamin C.

Figure 4

Detailed cytokine secretion analysis after vitamin C, vitamin B1, and hydrocortisone treatment. (A–H). The amount of the cytokines was determined using LEGENDplex^TM followed by flow cytometry after treatment of the cells for 6 h with either vitamin C, vitamin B1, or hydrocortisone alone or in combination and with or without LPS. LPS (10 ng/mL); Vit. C = Vitamin C (1 mM); B1 (1 µM); HC (2 µM); *: p ≤ 0.05; **: p ≤ 0.01; ***: p ≤ 0.001; ****: p ≤ 0.0001. Additional data analysis stratified according to gender is available as Figure S5. B1 = vitamin B1; HC = hydrocortisone; IL = interleukin; LPS = bacterial lipopolysaccharides; MCP = monocyte chemo-attractant protein; TNF = tumor necrosis factor; Vit. C = vitamin C.

Figure 5

Real-time PCR analysis of TNF-α, MCP-1, IL-6, and IL-23 after vitamin C, vitamin B1, and hydrocortisone treatment. (A–D). PBMCs were treated for 6 h with either vitamin C (1 mM), vitamin B1 (1 µM), or hydrocortisone (2 µM) alone or in combination and with (10 ng/mL) or without LPS. QRT-PCR results show levels of TNF-α (A), MCP-1 (B), IL-6 (C), and IL-23 (D) mRNA expression in comparison with untreated cells and normalized to the LPS stimulated control. TBP and β-actin mRNA expression were used as housekeeping genes. ***: p ≤ 0.001; ****: p ≤ 0.0001. Additional data analysis stratified according to gender is available as Figure S6. B1 = vitamin B1; HC = hydrocortisone; IFN = interferon; IL = interleukin; LPS = bacterial lipopolysaccharides; MCP = monocyte chemo-attractant protein; PBMCs = peripheral blood mononuclear cells; qRT-PCR = quantitative reverse transcription-polymerase chain reaction; TBP = TATA box binding protein; TNF = tumor necrosis factor; Vit. C = vitamin C.