Liver International ISSN 1478-3223
CIRRHOSIS AND LIVER FAILURE
Enhanced LL-37 expression following vitamin D supplementation in patients with cirrhosis and spontaneous bacterial peritonitis Chong Zhang, Lianrong Zhao, Yang Ding, Qiuju Sheng, Han Bai, Ziying An, Tingting Xia, Jingyan Wang and Xiaoguang Dou Department of Infectious Diseases, Shengjing Hospital of China Medical University, Shenyang, China
Liver Int. 2016; 36: 68–75. DOI: 10.1111/liv.12888
Abstract Background & Aims: The morbidity and mortality of spontaneous bacterial peritonitis (SBP) are high among patients with cirrhosis; however, the mechanisms of SBP pathogenesis are poorly understood. This study aimed to determine the role of the vitamin D-LL-37 pathway in the pathogenesis and treatment in patients with cirrhosis and SBP. Methods: Serum 25-hydroxyvitamin D concentrations of 119 patients with chronic liver diseases were tested. Vitamin D receptor (VDR) and LL-37 in peritoneal leucocytes of cirrhotic and ascitic patients with SBP were detected and compared with those without SBP. Then the peritoneal macrophages of non-infected patients were cultured and activated by lipopolysaccharide (LPS) to analyse the changes of VDR and LL-37 expressions after incubation with vitamin D. Results: Vitamin D deficiency or insufficiency was found in all of patients with cirrhosis. LPS inhibited VDR and LL-37 expression in peritoneal macrophages [1.3-fold decrease (P = 0.003) and 20-fold decrease (P = 0.010) respectively]. However, vitamin D could reverse the inhibition of both VDR and LL-37 [1.5-fold increase (P = 0.001) and 2000-fold increase (P < 0.001) respectively]. The effect of the incubation time following vitamin D supplementation was significant for LL-37 expression, with a peak expression found at 36 h (P < 0.001). Conclusions: When vitamin D levels were low, bacteria inhibited VDR and LL-37 responses in peritoneal macrophages as a mechanism to evade antibacterial defence. Vitamin D supplementation could up-regulate peritoneal macrophage VDR and LL-37 expressions, which resulted in an enhanced immunological defence against SBP in patients with cirrhosis and ascites. Keywords LL-37 – peritoneal macrophages – spontaneous bacterial peritonitis – vitamin D
Spontaneous bacterial peritonitis (SBP) is the most common bacterial infection in decompensated cirrhosis, accounting for 25–31.1% of all cases (1, 2). And mortality rates of SBP are high, ranging from 10 to 46% in hospitalized patients (3), and the 5-year survival rate is only 15% (4). Despite the great deal of research focused on the prevention of and therapy for SBP, antibiotics are still the only effective treatment (5, 6). Because the spectrum of pathogens causing SBP
changes (the proportion of Gram-negative bacteria has declined) (2) and multidrug-resistant organisms have increased (accounting for 22–41.7% of the pathogens) (2, 7), the efficiency of traditional third-generation cephalosporins has decreased to less than 40% (7, 8). We are committed to finding a new treatment from the perspective of the antibacterial immunocompromise in patients with SBP to reduce the morbidity and mortality of SBP.
Abbreviations 1,25(OH)2D3, 1,25-dihydroxyvitamin D3; 25(OH)D, 25-hydroxyvitamin D; HBV, hepatitis B virus; HCV, hepatitis C virus; LPS, lipopolysaccharide; MELD, model for end-stage liver diseases; PMN, polymorphonuclear leukocyte; SBP, spontaneous bacterial peritonitis; TBIL, total bilirubin; TLRs, Tolllike receptors; VDR, Vitamin D receptor. Correspondence Professor Xiaoguang Dou, Department of Infectious Diseases, Shengjing Hospital of China Medical University, Shenyang 110022, China Tel: +86 189 4025 1121; Fax: +86 24 2452 0868 e-mail: [email protected] Handling Editor: Christophe Bureau Received 13 April 2015; Accepted 4 June 2015 Additional Supporting Information may be found at onlinelibrary.wiley.com/doi/10.1111/liv.12888/suppinfo
68
Liver International (2016) © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Zhang et al.
Enhanced LL-37 expression following vitamin D
Key points
Methods
• All of patients with cirrhosis were in a state of vitamin D deficiency or insufficiency. • Under low vitamin D circumstances, bacteria inhibited VDR and LL-37 responses in peritoneal macrophages as a mechanism to evade antibacterial defence. • Vitamin D supplementation could reverse the inhibition effect of LPS to up-regulate peritoneal macrophage VDR and LL-37 expressions, which resulted in an enhanced immunological defence against SBP in patients with cirrhosis and ascites. • The results of this study suggest that vitamin D could be an adjunct to antibiotic therapy in the prevention and treatment of SBP.
Patients
A decreased number of hepatocytes, reduced exposure to sunlight, decreased adipose tissue, malabsorption of vitamin D and altered hydroxylation of vitamin D in the liver are all causes of low levels of vitamin D in patients with cirrhosis (9). It was found that a low level of vitamin D was independently associated with an increased risk of bacterial infection in patients with cirrhosis (10); however, the exact mechanism is unclear. LL-37 is the only antibacterial peptide in humans that is regulated by vitamin D (11). Pathogens induce CYP27B1 and vitamin D receptor (VDR) up-regulation in macrophages by triggering Toll-like receptors (TLRs) 2/1. Then, 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] is synthesized intracellularly to trans-activate the endogenous antimicrobial peptide LL-37, which is mediated by VDR (12). Nielsen et al. (13) found that the concentration of LL-37 in the urinary tract and susceptibility to LL-37 could influence the likelihood of urinary tract infections. In addition, Barbeiro et al. (14) found that LL-37 was regulated through a complex pathway in septic shock. Thus, we speculated that innate immunodeficiency (an inadequate response of LL-37) in the peritoneal cavity caused by low vitamin D in cirrhosis patients might lead to susceptibility to bacterial infections with poor prognosis. Our previous study confirmed that cirrhosis patients with ascites had low levels of vitamin D and that LL-37 and its regulator VDR might be involved in SBP pathogenesis (15). This study aimed to further address this hypothesis and expand the study population to discuss the vitamin D status of chronic liver diseases with a focus on patients with cirrhosis, especially those with SBP. We investigated whether vitamin D supplementation could up-regulate LL-37 expression in peritoneal macrophages mediated by the VDR pathway and thus enhance local innate immune function. This study is valuable in understanding SBP pathogenesis and discovering new treatment strategies for SBP. Liver International (2016) © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
This study was approved by the Medical Ethic Committee of Shengjing Hospital of China Medical University. Clinical samples were used with informed consent. The subjects were 119 hospitalized patients with chronic hepatitis or cirrhosis in the Shengjing Hospital of China Medical University. All patients underwent routine laboratory testing, including liver and renal function tests, total and differential leucocytes counts and coagulation tests. The model for end-stage liver diseases (MELD) score and Child-Pugh classification of each cirrhotic patient were calculated. Furthermore, patients also underwent abdominal and pulmonary imaging studies, and their demographic and clinical characteristics were recorded. All patients with cirrhosis and ascites underwent diagnostic abdominal paracentesis to obtain a sample of ascitic fluid for a polymorphonuclear leukocyte (PMN) count. SBP was diagnosed if the patient’s PMN count from the ascitic fluid was greater than 250 cells/mm3 and/or if the patient had a positive ascitic fluid bacterial culture in the absence of clinical, radiological or laboratory data suggesting secondary peritonitis or other abdominal disorders (e.g. haemorrhagic ascites, pancreatitis, mycobacterial or fungal peritonitis, or carcinomatosis), as recommended by the American Association for the Study of Liver Diseases (16). Culture-negative neutrocytic ascites and bacterascites were included. Exclusion criteria for all the patients included vitamin D supplementation within the past 6 months. For patients with cirrhosis and ascites, exclusion criteria included the use of selective intestinal decontamination with norfloxacin or other antibiotics within 1 month prior to SBP or treatment of any infectious diseases with antibiotics within 2 weeks before diagnostic abdominal paracentesis. Measurement of serum and ascites 25(OH)D concentrations
Serum and ascites 25-hydroxyvitamin D [25(OH)D] concentrations were measured using a kit (IDS, Fountain Hills, AZ, USA) according to the manufacturer’s instruction. Samples were tested in triplicate. The reference ranges for vitamin D levels considered sufficient, insufficient and deficient were serum 25(OH)D concentrations of >75 nmol/L, 50–75 nmol/L and
CIRRHOSIS AND LIVER FAILURE
Enhanced LL-37 expression following vitamin D supplementation in patients with cirrhosis and spontaneous bacterial peritonitis Chong Zhang, Lianrong Zhao, Yang Ding, Qiuju Sheng, Han Bai, Ziying An, Tingting Xia, Jingyan Wang and Xiaoguang Dou Department of Infectious Diseases, Shengjing Hospital of China Medical University, Shenyang, China
Liver Int. 2016; 36: 68–75. DOI: 10.1111/liv.12888
Abstract Background & Aims: The morbidity and mortality of spontaneous bacterial peritonitis (SBP) are high among patients with cirrhosis; however, the mechanisms of SBP pathogenesis are poorly understood. This study aimed to determine the role of the vitamin D-LL-37 pathway in the pathogenesis and treatment in patients with cirrhosis and SBP. Methods: Serum 25-hydroxyvitamin D concentrations of 119 patients with chronic liver diseases were tested. Vitamin D receptor (VDR) and LL-37 in peritoneal leucocytes of cirrhotic and ascitic patients with SBP were detected and compared with those without SBP. Then the peritoneal macrophages of non-infected patients were cultured and activated by lipopolysaccharide (LPS) to analyse the changes of VDR and LL-37 expressions after incubation with vitamin D. Results: Vitamin D deficiency or insufficiency was found in all of patients with cirrhosis. LPS inhibited VDR and LL-37 expression in peritoneal macrophages [1.3-fold decrease (P = 0.003) and 20-fold decrease (P = 0.010) respectively]. However, vitamin D could reverse the inhibition of both VDR and LL-37 [1.5-fold increase (P = 0.001) and 2000-fold increase (P < 0.001) respectively]. The effect of the incubation time following vitamin D supplementation was significant for LL-37 expression, with a peak expression found at 36 h (P < 0.001). Conclusions: When vitamin D levels were low, bacteria inhibited VDR and LL-37 responses in peritoneal macrophages as a mechanism to evade antibacterial defence. Vitamin D supplementation could up-regulate peritoneal macrophage VDR and LL-37 expressions, which resulted in an enhanced immunological defence against SBP in patients with cirrhosis and ascites. Keywords LL-37 – peritoneal macrophages – spontaneous bacterial peritonitis – vitamin D
Spontaneous bacterial peritonitis (SBP) is the most common bacterial infection in decompensated cirrhosis, accounting for 25–31.1% of all cases (1, 2). And mortality rates of SBP are high, ranging from 10 to 46% in hospitalized patients (3), and the 5-year survival rate is only 15% (4). Despite the great deal of research focused on the prevention of and therapy for SBP, antibiotics are still the only effective treatment (5, 6). Because the spectrum of pathogens causing SBP
changes (the proportion of Gram-negative bacteria has declined) (2) and multidrug-resistant organisms have increased (accounting for 22–41.7% of the pathogens) (2, 7), the efficiency of traditional third-generation cephalosporins has decreased to less than 40% (7, 8). We are committed to finding a new treatment from the perspective of the antibacterial immunocompromise in patients with SBP to reduce the morbidity and mortality of SBP.
Abbreviations 1,25(OH)2D3, 1,25-dihydroxyvitamin D3; 25(OH)D, 25-hydroxyvitamin D; HBV, hepatitis B virus; HCV, hepatitis C virus; LPS, lipopolysaccharide; MELD, model for end-stage liver diseases; PMN, polymorphonuclear leukocyte; SBP, spontaneous bacterial peritonitis; TBIL, total bilirubin; TLRs, Tolllike receptors; VDR, Vitamin D receptor. Correspondence Professor Xiaoguang Dou, Department of Infectious Diseases, Shengjing Hospital of China Medical University, Shenyang 110022, China Tel: +86 189 4025 1121; Fax: +86 24 2452 0868 e-mail: [email protected] Handling Editor: Christophe Bureau Received 13 April 2015; Accepted 4 June 2015 Additional Supporting Information may be found at onlinelibrary.wiley.com/doi/10.1111/liv.12888/suppinfo
68
Liver International (2016) © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Zhang et al.
Enhanced LL-37 expression following vitamin D
Key points
Methods
• All of patients with cirrhosis were in a state of vitamin D deficiency or insufficiency. • Under low vitamin D circumstances, bacteria inhibited VDR and LL-37 responses in peritoneal macrophages as a mechanism to evade antibacterial defence. • Vitamin D supplementation could reverse the inhibition effect of LPS to up-regulate peritoneal macrophage VDR and LL-37 expressions, which resulted in an enhanced immunological defence against SBP in patients with cirrhosis and ascites. • The results of this study suggest that vitamin D could be an adjunct to antibiotic therapy in the prevention and treatment of SBP.
Patients
A decreased number of hepatocytes, reduced exposure to sunlight, decreased adipose tissue, malabsorption of vitamin D and altered hydroxylation of vitamin D in the liver are all causes of low levels of vitamin D in patients with cirrhosis (9). It was found that a low level of vitamin D was independently associated with an increased risk of bacterial infection in patients with cirrhosis (10); however, the exact mechanism is unclear. LL-37 is the only antibacterial peptide in humans that is regulated by vitamin D (11). Pathogens induce CYP27B1 and vitamin D receptor (VDR) up-regulation in macrophages by triggering Toll-like receptors (TLRs) 2/1. Then, 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] is synthesized intracellularly to trans-activate the endogenous antimicrobial peptide LL-37, which is mediated by VDR (12). Nielsen et al. (13) found that the concentration of LL-37 in the urinary tract and susceptibility to LL-37 could influence the likelihood of urinary tract infections. In addition, Barbeiro et al. (14) found that LL-37 was regulated through a complex pathway in septic shock. Thus, we speculated that innate immunodeficiency (an inadequate response of LL-37) in the peritoneal cavity caused by low vitamin D in cirrhosis patients might lead to susceptibility to bacterial infections with poor prognosis. Our previous study confirmed that cirrhosis patients with ascites had low levels of vitamin D and that LL-37 and its regulator VDR might be involved in SBP pathogenesis (15). This study aimed to further address this hypothesis and expand the study population to discuss the vitamin D status of chronic liver diseases with a focus on patients with cirrhosis, especially those with SBP. We investigated whether vitamin D supplementation could up-regulate LL-37 expression in peritoneal macrophages mediated by the VDR pathway and thus enhance local innate immune function. This study is valuable in understanding SBP pathogenesis and discovering new treatment strategies for SBP. Liver International (2016) © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
This study was approved by the Medical Ethic Committee of Shengjing Hospital of China Medical University. Clinical samples were used with informed consent. The subjects were 119 hospitalized patients with chronic hepatitis or cirrhosis in the Shengjing Hospital of China Medical University. All patients underwent routine laboratory testing, including liver and renal function tests, total and differential leucocytes counts and coagulation tests. The model for end-stage liver diseases (MELD) score and Child-Pugh classification of each cirrhotic patient were calculated. Furthermore, patients also underwent abdominal and pulmonary imaging studies, and their demographic and clinical characteristics were recorded. All patients with cirrhosis and ascites underwent diagnostic abdominal paracentesis to obtain a sample of ascitic fluid for a polymorphonuclear leukocyte (PMN) count. SBP was diagnosed if the patient’s PMN count from the ascitic fluid was greater than 250 cells/mm3 and/or if the patient had a positive ascitic fluid bacterial culture in the absence of clinical, radiological or laboratory data suggesting secondary peritonitis or other abdominal disorders (e.g. haemorrhagic ascites, pancreatitis, mycobacterial or fungal peritonitis, or carcinomatosis), as recommended by the American Association for the Study of Liver Diseases (16). Culture-negative neutrocytic ascites and bacterascites were included. Exclusion criteria for all the patients included vitamin D supplementation within the past 6 months. For patients with cirrhosis and ascites, exclusion criteria included the use of selective intestinal decontamination with norfloxacin or other antibiotics within 1 month prior to SBP or treatment of any infectious diseases with antibiotics within 2 weeks before diagnostic abdominal paracentesis. Measurement of serum and ascites 25(OH)D concentrations
Serum and ascites 25-hydroxyvitamin D [25(OH)D] concentrations were measured using a kit (IDS, Fountain Hills, AZ, USA) according to the manufacturer’s instruction. Samples were tested in triplicate. The reference ranges for vitamin D levels considered sufficient, insufficient and deficient were serum 25(OH)D concentrations of >75 nmol/L, 50–75 nmol/L and
