Expression of proBDNF/p75NTR in peripheral blood lymphocytes of patients with sepsis and its impact on lymphocyte differentiation

Abstract

Objectives: Sepsis is a life-threatening organ dysfunction caused by the host's imbalanced response to infection. Due to lack of effective treatments, it has always been the difficulty and focus of clinical treatment of sepsis. Studies have shown that pro-brain-derived neurotrophic factor (proBDNF) binds to the high-affinity total neurotrophic factor p75 neurotrophin receptor (p75^NTR), which activates downstream signaling cascades and disrupts immunological inflammation and plays an important role in the progression of sepsis. This study aims to explore the expression changes of lymphocyte-derived proBDNF/p75^NTR in patients with sepsis and its effect on lymphocyte differentiation.

Methods: From the healthy donors (control group, n=40) and sepsis patients (sepsis group, n=40) admitted to the hospital for the first time, peripheral blood samples and blood routine clinical detection indicators were obtained. By using flow cytometry, the proportion of lymphocyte subsets and their expression of proBDNF/p75^NTR were examined. The peripheral blood lymphocytes were isolated from the control group and incubated with lipopolysaccharide (LPS). Flow cytometry analysis technology was used to detect the expression of proBDNF/p75^NTR on LPS-treated lymphocyte subsets. On this basis, we investigated the effects on lymphocyte differentiation by inhibiting p75^NTR.

Results: White blood cell count, neutrophil count, and neutrophil percentage of the patients in the sepsis group at admission were significantly higher than those in the control group; on the contrary, lymphocyte count and lymphocyte percentage in the sepsis group were lower than those in the control group (all P<0.001). The patients in the sepsis group had considerably greater neutrophil/lymphocyte and monocyte/lymphocyte ratios than those in the control group (both P<0.05). In the peripheral blood of sepsis patients, proBDNF expression was upregulated on CD19⁺ B cells, whereas p75^NTR expression was elevated on B cells, CD4⁺ T cells, and CD8⁺ T cells (all P<0.05). ProBDNF/p75^NTR expression was upregulated by LPS stimulation in vitro in peripheral blood cells of the control group (P<0.05), and this tendency was similar to the expression alterations in peripheral lymphocytes of the sepsis group. Inhibition of p75^NTR increased CD4⁺ T cell and CD19⁺ B cell percentages, cytokine expression of IL-4 and IL-10, and reduced IL-1β and IL-6 production (all P<0.05).

Conclusions: The immunosuppressive state of sepsis patients is indicated by a reduction in lymphocyte count and an increase in the proportion of inactive neutrophils. ProBDNF/p75^NTR expression is upregulated in the peripheral blood lymphocytes of sepsis patients, and p75^NTR inhibition may control lymphocyte differentiation involved in sepsis progression.

Keywords: lymphocytes; p75 neurotrophin receptor; pro-brain-derived neurotrophic factor; sepsis.

图1

脓毒症组和对照组的淋巴细胞亚群百分比 Figure 1 Percentage of lymphocytes subsets of peripheral blood in the sepsis group and the control group A: Gateing strategy; B: Percentage of CD3⁺ T cells; C: Percentage of CD4⁺ T cells; D: Percentage of CD8⁺ T cells; E: Percentage of CD19⁺ B cells. *P<0.05, **P<0.01. ns: No differences; Con: Control; SSC: Side scatter; APC-Cy7-CD45: APC/Cyanine 7 anti-human CD45 antibody; PE-Cy7-CD3: PE/Cyanine 7 anti-human CD3 antibody; BV510-CD8: BV510 anti-human CD8 antibody.

图2

脓毒症组和对照组淋巴细胞亚群上proBDNF/p75^NTR 的表达 Figure 2 Expression of proBDNF/p75^NTR on lymphocytes subpopulations of the sepsis group and the control group A-D: ProBDNF MFI on CD3⁺ T cells (A), CD4⁺ T cells (B), CD8⁺ T cells (C), and CD19⁺ B cells (D). E-H: P75^NTR MFI on CD3⁺ T cells (E), CD4⁺ T cells (F), CD8⁺ T cells (G), and CD19⁺ B cells (H). *P<0.05, **P<0.01. ns: No differences; Con: Control; MFI: Mean fluorescence intensity; proBDNF: Pro-brain-derived neurotrophic factor; p75^NTR: P75 neurotrophin receptor.

图3

LPS刺激上调健康对照组外周血淋巴细胞proBDNF/p75^NTR 的表达 Figure 3 Expression of proBDNF/p75^NTR of lymphocytes subpopulations in healthy control after LPS stimulation A-D: ProBDNF MFI on CD3⁺ T cells (A), CD4⁺ T cells (B), CD8⁺ T cells (C), and CD19⁺ B cells (D). E-H: P75^NTR MFI on CD3⁺ T cells (E), CD4⁺ T cells (F), CD8⁺ T cells (G), and CD19⁺ B cells (H). *P<0.05. ns: No differences; MFI: Median fluorescence intensity; proBDNF: Pro-brain-derived neurotrophic factor; p75^NTR: P75 neurotrophin receptor; LPS: Lipopolysaccharide.

图4

P75^ECD-Fc干预对淋巴细胞分化的影响 Figure 4 Effects of p75^ECD-Fc therapy on lymphocyte differentiation A: Method of lymphocyte subsets’ gating; B-E: Proportion of CD3⁺ (B), CD4⁺ (C), CD8⁺ (D) T cells, and CD19⁺ B cells (E) in the control-Fc group and the p75^ECD-Fc group, respectively; F: Relative mRNA levels of lymphocyte-related cytokines. *P<0.05, **P<0.01, ***P<0.001. IFN-γ: Interferon-γ; p75^ECD-Fc: Extracellular domain of p75^NTR; TGF-β: Transforming growth factor-β; ns: No differences.