Beneficial effects of citrulline enteral administration on sepsis-induced T cell mitochondrial dysfunction

Abstract

Severe sepsis induces a sustained immune dysfunction associated with poor clinical behavior. In particular, lymphopenia along with increased lymphocyte apoptosis and decreased lymphocyte proliferation, enhanced circulating regulatory T cells (Treg), and the emergence of myeloid-derived suppressor cells (MDSCs) have all been associated with persistent organ dysfunction, secondary infections, and late mortality. The mechanisms involved in MDSC-mediated T cell dysfunction during sepsis share some features with those described in malignancies such as arginine deprivation. We hypothesized that increasing arginine availability would restore T cell function and decrease sepsis-induced immunosuppression. Using a mouse model of sepsis based on cecal ligation and puncture and secondary pneumonia triggered by methicillin-resistant Staphylococcus aureus inoculation, we demonstrated that citrulline administration was more efficient than arginine in increasing arginine plasma levels and restoring T cell mitochondrial function and proliferation while reducing sepsis-induced Treg and MDSC expansion. Because there is no specific therapeutic strategy to restore immune function after sepsis, we believe that our study provides evidence for developing citrulline-based clinical studies in sepsis.

Keywords: T cell; citrulline; mitochondria; sepsis.

Fig. 1.

The impact of sepsis on immune functions, metabolic disturbances, and mitochondrial properties. (A) Animals underwent CLP or sham laparotomy and received antibiotic therapy and fluid resuscitation to create a clinically relevant sepsis model. The immune dysfunction following sepsis was studied 5 d after surgery. (B) T cell apoptosis was assessed by flow cytometry as the percentage of AnnexinVpos/DAPIneg CD3pos cells in peripheral blood (Left; Sham n = 19; CLP n = 20) and spleen (Right, Sham n = 11; CLP n = 11). (C) Peripheral blood T cell growth was determined by the incorporation of [3H] thymidine after 48 h of in vitro stimulation by anti-CD3/anti-CD28 antibodies. The data are expressed as the ratios of the counts per minute (cpm) obtained with stimulated versus nonstimulated T cells (Sham n = 19; CLP n = 20). (D) Spleen T cell proliferation was determined by CFSE dilution under in vitro stimulation by anti-CD3/anti-CD28 antibodies. The data are expressed as the proportion (%) of proliferative T cells among living T cells (Sham n = 11; CLP n = 11). (E) Peripheral blood CD3pos T cell mitochondrial mass was analyzed by flow cytometry using MitoTracker probe (Left, Sham n = 20; CLP n = 11) while maximal mitochondrial OCR was determined using Seahorse XF-24 extracellular flux analyzer (Right, Sham n = 5; CLP n = 5). (F) Spleen CD3pos T cell ATP production (Left) and maximal mitochondrial OCR (Right) were defined by CellTiter-Glo Luminescent Cell viability assay and Seahorse XF-24 extracellular flux analyzer, respectively (Sham n = 7; CLP n = 7). (G) The quantification of Treg (Sham n = 20; CLP n = 13) and monocytic and granulocytic myeloid-derived suppressor cells (M-MDSC and G-MDSC, Sham n = 11; CLP n = 11) by flow cytometry within peripheral blood of CLP versus sham mice. (H) Plasmatic amino acid concentrations were assessed by high-performance liquid chromatography (HPLC). Arginase activity was defined by the ornithine-to-arginine ratio (Sham n = 10; CLP n = 14). *P < 0.05; **P < 0.01; ***P < 0.001; ns: nonsignificant.

Fig. 2.

The impact of CLP on ALI and secondary infection severity. (A) Animals underwent CLP or sham laparotomy and received antibiotic therapy and fluid resuscitation to create a clinically relevant sepsis model. Sepsis-induced ALI was evaluated 5 d after surgery. The second set of experiments studied the severity of a secondary infection following sepsis (MRSA-induced pneumonia). The severity of the secondary infection was evaluated 12 h after pneumonia initiation. (B) The ALI score was determined by pathological examination of lung tissues 5 d after surgery in CLP and sham mice, and representative images of lung tissues show the differences between sham and septic mice (Sham n = 6; CLP n = 6). (Scale bar, 100 µm.) (C) Bacterial counts were determined 12 h after the tracheal instillation of MRSA in sham and CLP mice by quantitative cultures of BALF (Sham n = 12; CLP n = 9), spleen, and kidney (Sham n = 14; CLP n = 10) homogenates for 24 h. (D) The ALI score was determined by pathological examination of lung tissues 12 h after MRSA instillation in mice that underwent CLP or sham procedure (Sham n = 7; CLP n = 7). (Scale bar, 100 µm.) (E and F) The Kaplan–Meier survival curves of CLP versus sham mice without (Left, Sham n = 107; CLP = 109) and with secondary pneumonia induction (Right, Sham n = 12; CLP n = 11). *P < 0.05; **P < 0.01; ***P < 0.001; ns: nonsignificant.

Fig. 3.

The effects of citrulline administration on post-septic immunoparalysis and mitochondrial dysfunctions. (A) The animals underwent CLP and received antibiotic therapy and fluid resuscitation. From day 0 to 5, the mice were enterally fed by citrulline (150 mg/kg/day), arginine (150 mg/kg/day), or an isonitrogenous placebo. The immune dysfunctions were studied 5 d after surgery. (B) Plasmatic amino acid concentrations were assessed by HPLC. Arginase activity was defined by the ornithine-to-arginine ratio (Sham n = 10; CLP n = 14) (Placebo n = 10, Arginine n = 13, and Citrulline n = 13). (C) The quantification of Treg and M-MDSC and G-MDSC by flow cytometry within peripheral blood of CLP mice (Placebo n = 14, Arginine n = 12, and Citrulline n = 14). (D) T cell apoptosis was assessed by flow cytometry as the percentage of AnnexinVpos/DAPIneg CD3pos cells in peripheral blood (Placebo n = 15, Arginine n = 15, and Citrulline n = 15) and spleen (Placebo n = 8, Arginine n = 8, and Citrulline n = 8) of CLP mice. (E) Peripheral blood T cell growth was determined by the incorporation of [3H] thymidine after 48 h of in vitro stimulation by anti-CD3/anti-CD28 antibodies. The data are expressed as the ratios of the counts per minute (cpm) obtained with stimulated versus nonstimulated T cells (Placebo n = 6, Arginine n = 7, and Citrulline n = 12). (F) Spleen T cell proliferation was determined by CFSE dilution under in vitro stimulation by anti-CD3/anti-CD28 antibodies. The data are expressed as the proportion (%) of proliferative T cells among living T cells. (Placebo n = 8, Arginine n = 8, and Citrulline n = 8). (G) Peripheral blood CD3pos T cell mitochondrial mass was analyzed by flow cytometry using MitoTracker probe (Left, Placebo n = 7, Arginine n = 4, and Citrulline n = 8), while maximal mitochondrial OCR was determined using Seahorse XF-24 extracellular flux analyzer (Right, Placebo n = 4, Arginine n = 4, and Citrulline n = 4). (H) Spleen CD3pos T cell ATP production (Left) and maximal mitochondrial OCR (Right) were defined by CellTiter-Glo Luminescent Cell viability assay and Seahorse XF-24 extracellular flux analyzer, respectively (Placebo n = 7, Arginine n = 7, and Citrulline n = 7). *P < 0.05; **P < 0.01; ***P < 0.001; ns: nonsignificant.

Fig. 4.

Citrulline mediated ALI‘s protection and improvement in secondary infection severity. (A) The animals underwent CLP or sham laparotomy and received antibiotic therapy and fluid resuscitation to create a clinically relevant sepsis model. From day 0 to 5, the mice were enterally fed by citrulline (150 mg/kg/day), arginine (150 mg/kg/day), or an isonitrogenous placebo. Sepsis-induced ALI was evaluated 5 d after surgery. The second set of experiments studied the severity of a secondary infection following sepsis (MRSA-induced pneumonia). The severity of the secondary infection was evaluated 12 h after pneumonia initiation. (B) The ALI score determined by pathological examination of lung tissues 5 d after CLP, and representative images of lung tissues show the differences between the three groups of mice (Placebo n = 9, Arginine n = 7, and Citrulline n = 9) (Sham n = 6; CLP n = 6). (Scale bar, 100 µm.) The ALI scores were higher in placebo and arginine mice than in citrulline mice. (C) Bacterial counts were determined 12 h after the instillation of MRSA in mice by quantitative cultures of BALF, spleen, and kidney homogenates at 24 h was performed. Bacteriological secondary pneumonia severity assessment showed lower MRSA concentrations in BALF (Placebo n = 8, Arginine n = 13, and Citrulline n = 13), spleen, and kidneys (Placebo n = 15, Arginine n = 21, and Citrulline n = 20) of citrulline-fed mice. (D) The ALI score was determined by the pathological examination of lung tissues 12 h after the secondary infection initiation, and representative images of lung tissues show the differences between the three groups of mice (Placebo n = 7, Arginine n = 8, and Citrulline n = 10). (Scale bar, 100 µm.) The ALI scores were higher in placebo and arginine mice than in citrulline mice. *P < 0.05, ***P < 0.001; ns, nonsignificant.