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Frequency of CD4+CXCR5+ TFH cells in patients with hepatitis b virus-associated membranous nephropathy
International Immunopharmacology 22 (2014) 98–106
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International Immunopharmacology journal homepage: www.elsevier.com/locate/intimp
Frequency of CD4+CXCR5+ TFH cells in patients with hepatitis b virus-associated membranous nephropathy☆ Yong Liu a,1, Pingwei Zhao a,1, Zhihui Qu b, Desalegn Admassu Ayana c, Yanfang Jiang a,⁎ a b c
Key Laboratory of Zoonosis Research, Ministry of Education, The First Hospital of Jilin University, Changchun, 130032, China Department of Nephrology, The First Hospital of Jilin University, Changchun 130021, China Department of Medical Laboratory Sciences, Haramaya University, Dire Dawa, Ethiopia
a r t i c l e
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Article history: Received 6 April 2014 Received in revised form 6 June 2014 Accepted 11 June 2014 Available online 24 June 2014 Keywords: TFH cell HBV-MN IL-21 IL-4
a b s t r a c t The frequency of different subsets of CD4+CXCR5+ TFH cells and serum cytokine levels were analyzed in a total of 14 patients with newly diagnosed hepatitis B virus-associated membranous nephropathy (HBV-MN), 12 individuals with immune-tolerant HBV infection (HBV-IT) and 12 healthy controls (HC). Serum cytokine levels were measured before and 10–12 weeks after treatment. Significantly higher frequency of CD4+CXCR5+, CD4+CXCR5+ICOS+ and CD4+CXCR5+PD-1+ TFH cells, and higher serum levels of IL-17A, IFN-γ, IL-2, IL-10, IL-4 and IL-21 were detected in HBV-MN patients compared to the HC. The percentage of CD4+CXCR5+ TFH cells and serum IL-21 level in HBV-MN patients were also higher than the HBV-IT. The percentage of CD4+CXCR5+ TFH cell was negatively correlated with the value of eGFR, and the percentage of CD4+CXCR5+ICOS+ TFH cells was positively correlated with the 24-h urinary protein concentration. Notably, the percentage of CD4+CXCR5+PD-1+ TFH cells was positively correlated with serum IL-21 level and 24-h urinary protein concentration. Treatment with prednisone or/and immunosuppressive drugs significantly reduced the frequency of CD4+CXCR5+, CD4+CXCR5+PD-1+ TFH cells and serum IL-21 level, but increased IL-4 and IL-10 levels in the patients. CD4+CXCR5+ TFH cells, especially CD4+CXCR5+PD-1+ TFH cells may participate in the pathogenesis of HBV-MN. © 2014 Elsevier B.V. All rights reserved.
1. Introduction HBV infection is a well-recognized cause of glomerulonephritis, and HBV-associated membranous nephropathy (HBV-MN) is one of the most common extrahepatic manifestations of HBV infection. Its incidence closely parallels the incidence of HBV infection, especially for chronic HBV carriers through horizontal transmission in the general population [1]. HBV-MN, a kidney disease caused by hepatitis B virus infection, is a common secondary membranous nephropathy. The most common clinical features of HBV-MN are proteinuria and edema. It is considered that deposition of hepatitis antigen–antibody immune complexes in the subepithelial space results in the pathogenesis of HBV-MN. This assumption is based on the presence of HBV in kidney tissue, mainly the glomerular area [2]. In children, HBV-MN is usually self-limited with only rare progression to renal failure. However, in adults, the ☆ Funding source: This study was supported by grants from the National Natural Science Foundation of China (Nos. 30972610 and 81273240), Jilin Province Science and Technology Agency (No. 20110716), the Health Department Research Projects of Jilin Province (2009Z054) and Norman Bethune Program of Jilin University (2012206). ⁎ Corresponding author at: The First Hospital of Jilin University, Changchun 130032, China. Tel.: +86 13756660113; fax: +86 0431 84808391. E-mail address: [email protected] (Y. Jiang). 1 These authors made equal contributions to this study.
http://dx.doi.org/10.1016/j.intimp.2014.06.024 1567-5769/© 2014 Elsevier B.V. All rights reserved.
natural disease course of HBV-MN may be more relentless, slowly progressing to renal failure. At present, one of the important mechanisms of HBV-MN is based on the deposition of situ composites of HBeAg in subepithelial area. HBeAg form subepithelial immune complex through non-immune mechanism in the basement membrane because of its small molecular weight [3]. Two studies show that HBV can promote the inflammatory reaction in renal tissue. A study by Xia et al. [4] found that activation of NF- B by HBx protein might play a critical role in the transcriptional induction of inflammatory genes and HBx-induced chemotaxis. Yang et al. indicate [5] that HBeAg can bind to the IL-1 receptor accessory protein, express IL-1 sensitive gene induced by NF- B pathway, and promote the occurrence of inflammatory reaction. T cells are necessary for IgG responses and regulate the development of HBV-MN. However, it is still unclear how different types of CD4+ helper T cells regulate the pathogenesis of HBV-MN. CD4+ T cells have a crucial role in helping B cells produce antibodies in response to challenge with foreign antigens. Two studies described CD4+ T cells residing in the B cell follicle expressed CXCR5 [6,7]. These cells are more commonly known as T follicular helper (TFH) cells. Characteristics of TFH cells are increasing the expression of numerous molecules including inducible T-cell co-stimulator (ICOS), program death 1 (PD-1) and CD40-ligand (CD40L), the transcription factor Bcl-
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6 and regulatory cytokines of IL-21 [8–10]. ICOS appears to be important for TFH cell development, and PD-1 is a critical regulator of the function of TFH cells [11,12]. IL-21 was a cytokine preferentially expressed by TFH cells and served as an important regulator of humoral responses by directly regulating B cells proliferation and class switching [13]. Increased numbers of circulating TFH cells and aberrant activation of TFH cells have been associated with the development of autoimmune diseases [9,10]. Although HBV-MN is a complicated disease, an immune complex in the subepithelial space is a common and consistent characteristic among the patients. There currently is no information on how these different subsets of TFH cells exist in HBV-MN patients and whether TFH cells participate in the pathogenesis of HBV-MN. Therefore, we sought to explore the role of TFH cells in patients with HBV-MN. Currently, patients with HBV-MN are usually treated with antiviral drug, and sometimes need antivirus combined with corticosteroids or/and immunosuppressive therapy. Lamivudine has been recommended as the most important antivirus drug for HBV-MN, but the incidence of lamivudine-resistant HBV strains is up to 20% per year [2]. Anti-hypertension and anti-coagulation drugs, such as angiotensin-converting enzyme (ACE) inhibitors, angiotensin II receptor antagonists, aspirin or dipyridamole are also used to treat patients. However, it is unclear how these therapeutic strategies affect different subsets of TFH cells and immune measures in patients with HBV-MN. In this study, we measured the frequency of circulating CD4+CXCR5+ TFH cells in patients with HBV-MN, HBV-IT and healthy controls to investigate the potential relationship between the percentages of different subsets of TFH cells and the values of laboratory measures before and after treatment.
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First Hospital of Jilin University from November 2011 to May 2014. Written informed consent was obtained from individual participants. The experimental protocol was established, according to the guidelines of the Declaration of Helsinki and was approved by the Human Ethics Committee of First Hospital of Jilin University (Jilin University, Changchun, China). Histological examination of biopsied kidney tissues was conducted and HBV was detected in renal tissue of the fourteen HBV-MN patients (Fig. 1). Exclusion criteria include (1) rapidly progressive HBV-MN (with rapid renal function deterioration or historically necrotic capillaries and cellular crescents), (2) secondary HBV-MN, such as Henoch–Schonlein purpura, lupus nephritis, and other primary glomerulonephritis, (3) pregnancy or planning for pregnancy, or (4) diabetes mellitus, neoplasia, active peptic ulcer disease and recent infection. In this study, another 12 gender-, age- and ethnicity-matched healthy controls (HC) were recruited during the same period and they had no history of any chronic inflammatory disease. Their demographic and clinical characteristics were recorded. 2.2. Treatment and follow up A total of fourteen patients were treated with prednisone (methylprednisolone, 32 mg/d) or/and immunosuppressive (tacrolimus capsules, 1 mg/d), and not with antiviral drug. Six patients were also treated with benazepril (10 mg/d) or valsartan (75 mg/d, Novartis Pharma, Beijing, China). In addition, individual patients were treated with aspirin (100 mg/d, Bayer, Germany) or dipyridamole (25 mg/d, Yunpeng Pharmaceutical, Shanxi, China) if the patient had a high risk of a hypercoagulable state. Patients visited the clinical office monthly and were followed for at least 10–12 weeks after beginning treatment.
2. Materials and methods 2.3. Blood sampling and analyses 2.1. Patients A total of fourteen patients with newly diagnosed HBV-MN and twelve patients with immune-tolerant HBV infection (HBV-IT) were recruited in the inpatient service of the Department of Nephrology, the
Fasting venous blood samples were collected from individual patients before and 10–15 weeks after treatment and blood samples were also collected from HBV-IT and healthy controls. Blood sample was used for preparing peripheral blood mononuclear cells (PBMCs)
Fig. 1. Histological examination of biopsied kidney tissues was conducted and HBV was detected in renal tissue. Needle biopsy specimens were obtained from all HBV-MN patients during routine treatment procedures. Subsequently, these paraffin-embedded samples were sliced (2–3 μm) and stained. Such as the kidney biopsy is shown from a representative HBV-MN patient. (A–B) Massive HBcAg and small amounts of HBsAg were detected in the glomerular capillary by immunofluorescent. As indicated by the arrows, along the glomerular capillary distribution of HBV antigen. (C) Histological examination of biopsied kidney tissues was conducted using periodic acid-silver methenamine (PASM) staining. As indicated by the arrows, glomerular basement membrane was stained dark and thickening. (D) Histological examination of biopsied kidney tissues was conducted using periodic acid silver methenamine–martius yellow (PASM-MAS) staining. As indicated by the arrows, Glomerular basement membrane sediments were stained red. All images are shown at ×400 magnification.
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by density-gradient centrifugation using Ficoll-Paque Plus (Amersham Biosciences, Little Chalfont, UK) and the remaining blood samples were centrifuged for preparing serum samples. The number of lymphocytes were examined and the concentrations of serum triglycerides, cholesterol, albumin, uric acid, ALT and AST were determined using ADVIA 1650 biochemical analyzer (Bayer, Pittsburg, PA, USA). In addition, 24 h urine sample was collected and urine protein concentration and microscopic hematuria were measured. Estimation of glomerular filtration rate (eGFR) was performed using the modified eGFR formula [14]. 2.4. Flow cytometry analysis Peripheral blood mononuclear cells (PBMCs) were isolated by density-gradient centrifugation using Ficoll-Paque Plus (Amersham Biosciences, Little Chalfont, UK). Human PBMCs at 106/tube were stained in duplicate with PE-anti-CXCR5 (Biolegend, San Diego, USA) and PerCPanti-CD4, Alexa-flour-anti-CD278, FITC-anti-CD279, or isotype-matched control IgG (Beckton Dickinson, San Jose, USA) at room temperature for 30 minutes, respectively. After being washed with PBS, the cells were subjected to flow cytometry analysis using a FACSCalibur (Beckton Dickinson) and FlowJo software (v7.6.2) [15]. The cells were gated on the forward scatter of living cells and then centered on CD4+ T cells. Subsequently, the CD4+CXCR5+, CD4+CXCR5+ICOS+, CD4+CXCR5+PD-1+, and CD4+CXCR5+ICOS+PD-1+ TFH cells were determined by flow cytometry analysis, and at least 50,000 events per sample were analyzed. 2.5. Measurement of serum IL-21 by enzyme-linked immunosorbent assay (ELISA) The concentrations of serum IL-21 in HBV-MN patients and healthy controls were measured by ELISA using human IL-21 ELISA kit, according to the manufacturer's instruction (Roche Diagnostics, Lewes, UK). The detection limit for human IL-21 was 10 pg/ml. 2.6. Cytometric bead array (CBA) analysis of serum cytokines The concentrations of serum IFN-γ, TNF-α, IL-2, IL-4, IL-6, IL-10 and IL-17A were detected by CBA [16], according to the manufacturer's protocol (BD Biosciences) with minor modifications. Briefly, the individual sera (25 μl/each) were tested in duplicate on a FACSAria II, as described before [17] using the CellQuest Pro and CBA software (Becton Dickinson) to quantify serum cytokine concentrations. 2.7. Circulating HBV-DNA quantification Peripheral blood (2 ml) was collected from HBV-MN patients. HBVDNA was detected by QF-PCR using hepatitis B virus nucleic acid
detection kit, according to the manufacturer's instruction (Haoyuan Biological Technology Co., Ltd. Shanghai, China), and PCR amplification was performed using ABI Prism 7500 (AB, USA). The detection limit was 50 IU/ml. All HBV-MN patients had undetectable HBV-DNA levels (b500 copies/ml). 2.8. Statistical analysis All data are expressed as median, and range. The difference between two groups was analyzed by the Kruskal–Wallis H nonparametric test using the SPSS 19.0 software. The relationship between variables was analyzed by the Spearman's rank correlation test. A two-sided P-value of b 0.05 was considered statistically significant. 3. Results 3.1. Frequency of circulating CXCR5+CD4+ TFH cells in the HBV-MN patients There was no significant age and gender difference between HBVMN patients, HBV-IT, and the HCs (Table 1). Furthermore, there was no significant difference in the concentrations of serum uric acid, triglycerides, cholesterol, ALT and AST, as well as the numbers of lymphocytes between these three groups of subjects. As expected, microscopic hematuria and 24-h urinary proteins were significantly higher in the patients than the HC, but the values of eGFR and serum albumin were significantly less in the patients compared to the HC, suggesting that those patients had kidney function impairment. TFH cells can promote B cell activation, expansion and differentiation. To investigate the potential role of TFH cells in the development of HBVMN, we characterized the percentages of peripheral blood CD4+CXCR5+ TFH cells in CD4+ T cells among the patients, HBV-IT and HCs by flow cytometry analysis. We found that the percentages of CD4+CXCR5+ TFH cells in CD4+ T cells in the patients were significantly higher than the HBV-IT and HCs (17.80 (6.46–29.90) vs.14.53 (7.50–21.00), P = 0.046; 17.80 (6.46–29.90) vs.12.85 (9.54–26.67), P = 0.023, Fig. 2B). The percentages of CD4+CXCR5+ICOS+ and CD4+CXCR5+PD-1+ TFH cells in the patients were also significantly higher than the HCs (6.42 (4.55–9.30) vs.5.25 (3.14-8.24), P = 0.023; 8.34 (5.45–13.40) vs.6.75 (4.22–9.84), P = 0.015, Fig. 2B). However, there was no significant difference in the frequency of circulating CD4+CXCR5+ICOS+ and CD4+CXCR5+PD-1+ TFH cells between HBV-MN patients and HBV-IT, and there was also no significant difference in the frequency of circulating CD4+CXCR5+ICOS+PD-1+ TFH cells among HBV-MN patients, HBVIT and HCs. TFH cells can secrete IL-21, which has been shown to regulate B cell differentiation and proliferation. In this study, we examined the
Table 1 The demographic and clinical characteristics of participants.
Age, years Female/Male Lymphocytes, 109/L Serum albumin, g/L ALT, U/L AST, U/L Serum uric acid, μmol/L Triglycerides, mmol/L Cholesterol, mmol/L Urinary proteins, g/24 h Urea nitrogen, mmol/L eGFR, ml/min/1.73 m2 Microscopic hematuria, rbc/hpf Data shown are median and range, except specified. ⁎ P b 0.05 vs. the HC.
HBV-MN (n = 14)
Healthy controls (n = 12)
HBV-IT (n = 12)
57 (26–77) 3/11 2.16 (1.38–3.50) 21.6 (15.6–27.9)⁎ 18 (5–35) 17 (11–33) 350 (269–635) 3.56 (0.74–7.89) 6.80 (4.54–11.95) 6.44 (2.19–13.90)⁎ 5.83 (3.36–11.17) 72.20 (11.74–118.34)⁎ 14.7 (0.6–76.7)⁎
53 (25–75) 3/9 1.12 (0.41–1.77) 40.3 (35.7–48.9) 16 (9–32) 18 (9–31) 315 (230–430) 1.13 (0.35–1.60) 4.05 (2.95–5.95) 0.05 (0–0.12) 5.01 (3.71–6.14) 99.35 (90.24–112.04) 1 (0–2)
50 (23–64) 4/8 1.24 (0.40–2.01) 37.8 (33.5–44.8) 20 (15–40) 21 (14–39) 340 (234–450) 1.26 (0.51–3.01) 4.23 (3.02–7.34) 0.06 (0–0.13) 5.21 (3.63–7.12) 96.24 (87.45–109.87) 1 (0–2)
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Fig. 2. Flow cytometry analysis of TFH cells. PBMCs from HBV-MN patients before treatment as well as HBV-IT, HC were stained with anti-CD4, anti-CXCR5, anti-ICOS, and anti-PD-1. The cells were gated initially on living lymphocytes, and then on CD4+ T cells, and subsequently, CD4+CXCR5+TFH cells. The last, the frequency of CD4+CXCR5+ICOS+, CD4+CXCR5+PD-1+, and CD4+CXCR5+ICOS+PD-1+ TFH cells was analyzed by flow cytometry. (A) Flow cytometry analysis. (B) The percentage of CD4+CXCR5+ T cells among CD4+ T cells and CD4 + CXCR5 + ICOS+, CD4 + CXCR5 + PD-1+, and CD4 + CXCR5 + ICOS+PD-1+T cells among CD4+CXCR5+ T cells. Data shown are representative dot plug or expressed as the mean % of different subsets of TFH cells in total CD4+T cells or CD4+CXCR5+ TFH cells in individual subjects from two separate experiments. The horizontal lines represent the median values.
concentration of serum IL-21 using ELISA. The concentration of serum IL21 was significantly higher in HBV-MN patients compared to the HBV-IT (P b 0.05) and the HCs (P b 0.05, Fig. 3A). We also found that the concentrations of serum IL-17A, IFN-γ, IL-2, IL-4 and IL-10 were significantly
higher in HBV-MN patients compared to the HCs (P b 0.05, Fig. 3B–F). In brief, these data suggest a higher frequency of different subsets of CD4+CXCR5+ TFH cells and significantly elevated levels of serum cytokines in the patients.
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Fig. 3. Analysis of serum cytokines in HBV-MN patients. The levels of serum IL-2, IL-4, IL-10, IL-17A, and IFN-γ were tested by CBA in HBV-MN patients and HCs, and the level of serum IL-21 was tested by ELISA in HBV-MN and HBV-IT patients, respectively. Data are expressed as the mean values of individual participants from two separate experiments. The horizontal lines indicate the median values of different groups.
3.2. The relation between the percentages of CD4+CXCR5+, CD4+CXCR5+ICOS+, and CD4+CXCR5+PD-1+ TFH cells and other clinical parameters in HBV-MN patients The percentage of circulating CD4+CXCR5+ TFH cells was negatively correlated with the values of eGFR (r = −0.8645, P = 0.0056, Fig. 4A). The percentage of circulating CD4+CXCR5+PD-1+ TFH cells was positively correlated with serum IL-21 level (r = 0.7203, P = 0.0037, Fig. 4B), and 24-h urinary protein concentration (r = 0.8260, P = 0.0115, Fig. 4C). The percentage of circulating CD4+CXCR5+ICOS+ TFH cells was positively correlated with the 24-h urinary protein concentration (r = 0.8249, P = 0.0117, Fig. 4D), but no significant association with serum IL-21 level (r = 0.4987, P = 0.0695, Fig. 4E). In addition, a positive correlation was observed between serum IL-21 level and 24-h urinary protein concentration (r = 0.8087, P = 0.0151, Fig. 4F). These data suggest that CD4 +CXCR5 + and CD4 +CXCR5 +ICOS+ , especially CD4 +CXCR5+ PD-1 + TFH cells response, may be associated with the pathogenesis of HBV-MN. 3.3. Clinical parameters, frequency of CD4+CXCR5+TFH cells and the serum cytokines levels in HBV-MN patients following treatment Only six patients have complete records and other eight patients were lost to follow-up. The impact of treatment on the values of clinical
parameters, the frequency of circulating CD4+CXCR5+ TFH cells and serum cytokines levels in six patients were assessed for 10–12 weeks. The 24-h urinary protein concentration decreased and serum albumin concentration increased significantly, but there was no significant difference in the values of other clinical parameters (Table 2). In addition, the post-treatment percentages of circulating CD4+CXCR5+ (P = 0.038), CD4+CXCR5+ICOS+ (P = 0.048), and CD4+CXCR5+PD-1+ TFH cells (P = 0.011) reduced significantly compared to the pretreatment level (Fig. 5A–C). Similarly, post treatment serum IL-21 level was significantly decreased as compared to the pretreatment level (P = 0.004 Fig. 5D), but the post-treatment levels of serum IL-4 and IL-10 were significantly increased compared to the pretreatment level (P = 0.025 and P = 0.011, respectively, Fig. 5E–F). Collectively, treatment dramatically improved proteinuria, which was associated with a reduction in the frequency of different subsets of TFH cells in the patients. 4. Discussion Previous research has shown that one of the most important mechanisms of HBV-MN is the deposition of hepatitis Ag and Ab immune complexes in the subepithelial space. Some scholars showed that, mainly IgG antibody, especially IgG4 was the dominant IgG subclass in primary membranous glomerulonephritis, however, in secondary
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Fig. 4. Correlation analysis of clinicopathological features of IgAN with the percentages of circulating TFH cells in HBV-MN patients. It is well known that renal function can be influenced by either benazepril or valsartan through non-immunologic factors. So, the patients treated with those drugs can be excluded when analyzing the relationship between TFH and renal function. (A) The values of eGFR are negatively associated with the percentages of CD4+CXCR5+ TFH cells. (B–C) The concentrations of serum IL-21, 24-h urinary proteins are positively correlated with the percentage of CD4+CXCR5+ PD-1+TFH cells. (D) The concentrations of 24-h urinary proteins are positively correlated with the percentage of CD4+CXCR5+ ICOS+TFH cells. (E) The level of serum IL-21 and the percentage of CD4+CXCR5+ICOS+TFH cells have no significant statistical significance. (F) The level of serum IL-21 is positively correlated with the percentage of CD4+CXCR5+ ICOS+TFH cells.
membranous glomerulonephritis IgG1 is dominant [18]. It is also known that the generation of a high-affinity long-lived antibody response requires the presence of CD4+ T cells. The newly discovered CD4+ CXCR5+ T cell subset, namely follicular helper T cells, key function is Table 2 The effect of treatment on the values of clinical measures in the follow-up HBV-MN patients.
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Lymphocytes, 10 /L Serum albumin, g/L ALT, U/L AST, U/L Serum uric acid, μmol/L Triglycerides, mmol/L Cholesterol, mmol/L Urinary proteins, g/24 h Urea nitrogen, mmol/L eGFR, ml/min/1.73 m2 Microscopic hematuria, rbc/hpf
Before treatment
After treatment
2.25 (1.38–2.93) 24.2 (15.6–27.9) 16 (5–24) 17 (11–33) 410 (302–603) 4.23 (3.18–7.89) 7.24 (4.54–11.95) 7.47 (3.67–13.90) 5.46 (3.36–6.93) 57.50 (11.74–118.34) 13.5 (0.6–76.7)
1.65 (1.27–2.76) 34.2 (25.4–40.3)⁎ 15 (9–27) 16 (10–30) 377 (297–403) 1.98 (1.34–4.01) 6.02 (4.56–8.35) 2.58 (1.01–5.69)⁎ 4.67 (3.27–5.98) 65.24 (50.35–110.28) 12.4 (0.6–45.7)
Data shown are median and range. ⁎ P b 0.05 vs. the values before treatment.
to help B cells to support their activation, expansion and differentiation, and the formation of the GC [19]. Recently, two studies have demonstrated that circulating CD4+CXCR5+ T cells secrete IL-21 and CXCL13, express ICOS, and induce antibody production from naive B cells, suggesting that blood CD4+CXCR5+ T cells share functional properties with TFH cells [20, 21]. In this study, we found a significantly higher frequency of CD4+CXCR5+, CD4+CXCR5+ICOS+ and CD4+CXCR5+PD-1+ TFH cells in HBV-MN patients compared to the HC and the percentage of CD4+CXCR5+ TFH cells in HBV-MN patients was also found to be higher than the HBV-IT. Furthermore, the concentrations of serum IL-17A, IFN-γ, IL-2, IL-4, IL-10 and IL-21 were significantly higher in HBV-MN patients than the HC and IL-21 level was higher than the HBV-IT. Consistently, an increase in circulating CD4+CXCR5+, CD4+CXCR5+ICOS+ and CD4+CXCR5+PD-1+ TFH cells has been detected in patients with autoimmune myasthenia gravis, Graves' disease, SLE and rheumatoid arthritis [22–25]. Our data provided first hand evidence that the circulating TFH cells are increased in HBV-MN patients, suggesting that CD4+CXCR5+, CD4+CXCR5+ICOS+ and CD4+CXCR5+PD-1+ TFH cells may contribute in the pathogenesis of HBV-MN. The inducible costimulatory molecule (ICOS), a member of the CD28 family, is highly
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Fig. 5. Altered frequency of TFH cells and concentrations of serum cytokines in HBV-MN patients following treatment. The change of the percentages of different subsets of TFH cells and the levels of serum cytokines in six HBV-MN patients when compared before and after the treatment. Data are expressed as the mean or concentrations of individual subjects from two separate experiments. (A) The percentages of CD4+CXCR5+ cells in the total CD4+T cells in individual patients pre- and post-treatment. (B–C) The percentage of CD4+CXCR5+ICOS+ and CD4+CXCR5+PD-1+TFH cells in total CD4+CXCR5+ TFH cells in individual patients pre- and post-treatment. (D–F) The levels of serum IL-21, IL-4 and IL-10 in individual patients pre- and post-treatment.
expressed in the activated TFH cells. ICOS combined with the B cell surface ICOSL, not only stimulate the differentiation of activated TFH cells but also promote B cells to produce antibodies [26]. Thus, ICOS may promote B cells to produce hepatitis Ab, resulting in immune complexes deposit in the subepithelial space and the happening of HBN-MN. Consistently, the percentage of peripheral blood ICOS+TFH cell was significantly higher in HBV-MN patients than the HC and the percentage of ICOS+ TFH cell was positively correlated with the 24-h urinary protein concentration. The PD-1 molecule is expressed on activated T cells, particularly on TFH cells in the GC. PD-1 promotes cognate T-B interactions and provides an inhibitory signal to TFH cells in the GC [27]. Interestingly, we found that the percentage of circulating PD-1+ TFH cells was positively correlated with serum IL-21 level, and 24-h urinary protein concentration. Serum IL-21 level was also positively correlated with 24-h urinary protein concentration in HBV-MN patients. Similarly, some studies reported that the percentages of CD4+CXCR5+PD-1+ TFH cells and serum IL-21 level were significantly and positively correlated with level of serum autoantibodies and disease activity in patients with SLE and rheumatoid arthritis [28,29]. Now, the researchers showed that circulating precursor CCR7loPD-1hiCXCR5+CD4+ T cells promote antibody response upon antigen re-exposure [30]. All of these show that
the PD-1 is associated with autoantibodies. A previous study has shown that PD-1 regulates selection and survival in the GC, impacting the quantity and quality of long-lived PCs, and promotes IL-21 and IL-4 production [31]. Our result also indicates that CD4+CXCR5+PD-1+ TFH cells promote antibody response upon antigen exposure and the production of IL-21. Consequently, it is possible that high expression of PD-1 may provide an inhibitory signal to T cells and freeze T cells in a TFH state, allowing sustained production of IL-21 and IL-4 in the B cell follicle [31]. IL-21 maintains expression of Bcl-6 in GC B cells and contributes to GC formation and affinity maturation [32,33], promoting the production of antibodies. As a result, the produced anti-HBV interacts with antigens to form immune complexes that deposit in the subepithelial space, leading to renal injury. Thereby, the percentages of circulating CD4+CXCR5+ICOS+ and CD4+CXCR5+PD-1+ TFH cells, especially CD4 +CXCR5+ PD-1 + TFH cells may serve as biomarkers for evaluating the disease progression in HBV-MN patients. Some studies indicate that TFH cells are involved in the immune response in HBV infection and their increase in number reflects the activation of the immune response [34,35]. Studies found that the percentages of CD4+CXCR5+ [34], CD4+CXCR5+ICOS+ and CD4+CXCR5+PD-1+ [35] TFH cells were significantly higher in immune-tolerant chronic
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hepatitis B (CHB) patients compared to the healthy controls, consistent with our study. Because our experimental group also belongs to immune-tolerant CHB, it is difficult to explain why the increased percentage of TFH cells is caused by HBV-MN. However, we found that the percentage of CD4+CXCR5+ TFH cells was significantly higher in HBV-MN patients compared to HBV-IT. Xing et al. [34] found that decreased level of IL-21 in HBV-IT suggests the decrease in TFH cells activity. Our data showed that IL-21 level was significantly higher in HBV-MN patients compared to the HBV-IT and the HCs. This suggests that IL-21 may participate in the pathogenesis of HBV-MN. IL-21 is the major cytokine of TFH cells and also a key factor affecting the formation of germinal centers. Moreover, we found that the percentages of PD1+TFH cells were positively correlated with serum IL-21 levels in HBV-MN patients. Thus, these results indicate that the role of TFH cells in pathogenesis of HBV-MN is different from the immune-tolerant CHB, this may be because HBx can cause immune cells and inflammatory mediator disorder, which play an important role in the progress of hepatitis B virus associated glomerulonephritis [36]. Therefore, our data can suggest that CD4+CXCR5+ and CD4+CXCR5+ICOS+, especially CD4+CXCR5+PD-1+ TFH cells, may participate in the pathogenesis of HBV-MN. Serum HBsAg and HBeAg are undetectable in the fourteen patients and the domestic HBV-DNA quantities are all within the normal range. In this study, while monitoring HBV-DNA quantity and liver enzyme situation, patients were treated with glucocorticoids or/and immunosuppressives, but not with antiviral drug. Glucocorticoids inhibit cellular and humoral immunity, inflammation, fibroblast and blood capillary hyperplasia, as well as reduce vascular permeability, which play a role in anti-inflammatory and immunosuppressive effect, and reduce urinary protein. In patients treated with prednisone or/and immunosuppressive drugs, the clinical symptoms improved obviously and we found that the frequency of circulating TFH cells and serum IL-21 level were significantly reduced after 10–12 weeks of treatment compared to the pretreatment level. Feng et al. [28] observed a significant reduction in the TFH cells after corticosteroid treatment and they also found that TFH proportions were significantly decreased when dexamethasone was added to the culture system in vitro studies. Our results are consistent with theirs. This indicates that the different subsets of TFH cells may be involved in the pathogenesis of HBV-MN and the proportion of circulating TFH cells was down-regulated by corticosteroids in a dose-dependent manner. After encountering a specific antigen, naive CD4+ T cells activate and differentiate into various effector T-cell subsets, such as Th1, Th2, and Th17 cells. It is known that Th1 cells mainly secret IFN-γ and Th2 cells mainly secret IL-4. Accumulating data suggest that Th17 cells are highly proinflammatory, and IL-17A, as an effector cytokine, is involved in the pathogenesis of host defense, autoimmune responses, and more recently in chronic inflammatory diseases [37]. Treg cells controls these effector T cells, thereby regulating immune responses and preventing autoimmunity and tissue inflammation [38]. Previous research has shown that Th1 and Th17 effector cells can induce glomerular nephritis [39,40]. In our study, we found that the levels of serum IL-17A, IFN-γ, IL-2, IL-4, IL-10 and IL-21 in HBV-MN patients were significantly higher than the HC. The concentration of serum IL-21 was significantly decreased and serum IL-4 and IL-10 were significantly increased after treatment, while the levels of serum IL-2, IL-6, IFN-γ and IL-17A have no significant difference, compared to the pretreatment level. These data suggest that Th1 and Th17 effector cells may participate in the pathogenesis of HBV-MN, and promote the inflammatory response. It is possible that Th1 and Th17 stimulate anti-inflammatory Th2 and Treg that increase the expression of IL-4 and IL-10 during the pathogenesis of HBV-MN though further research is needed to support this assumption. In conclusion, our result suggests that CD4+CXCR5+ and CD4+ CXCR5+ICOS+, especially CD4+CXCR5+PD-1+ TFH cells, may participate in the pathogenesis of HBV-MN and serve as a biomarker for
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evaluating disease severity. The limitations of the study include single time-point measurement for some patients, a very small sample size, and lack of long-term follow-up and functional studies of different subsets of TFH cells as well as information from the affected kidney. Thus, further research on the precise roles of TFH cells in the pathogenesis of HBV-MN in a bigger population is warranted. References [1] Liao MT, Chang MH, Lin FG, Tsai IJ, Chang YW, Tsau YK. Universal hepatitis B vaccination reduces childhood hepatitis B virus-associated membranous nephropathy. Pediatrics 2011;128(3):e600–4. [2] Moon JY, Lee SH. Treatment of hepatitis B virus-associated membranous nephropathy: lamivudine era versus post-lamivudine era. Korean J Intern Med 2012;27(4):394–6. [3] Takekoshi Y, Tanaka M, Miyakawa Y, Yoshizawa H, Takahashi K, Mayumi M. Free “small” and IgG-associated “large” hepatitis Be antigen in the serum and glomerular capillary walls of two patients with membranous glomerulonephritis. N Engl J Med 1979;300(15):814–9. [4] Xia LM, Huang WJ, Wu JG, Yang YB, Zhang Q, Zhou ZZ, et al. HBx protein induces expression of MIG and increases migration of leukocytes through activation of NFkappaB. Virology 2009;385(2):335–42. [5] Yang CY, Kuo TH, Ting LP. Human hepatitis B viral e antigen interacts with cellular interleukin-1 receptor accessory protein and triggers interleukin-1 response. J Biol Chem 2006;281(45):34525–36. [6] Breitfeld D, Ohl L, Kremmer E, Eilwart J, Sallusto F, Lipp M, et al. Follicular B helper T cells express CXC chemokine receptor 5 localize to B cell follicles, and support immunoglobulin production. J Exp Med 2000;192(11):1545–52. [7] Schaerli P, Willimann K, Lang AB, Lipp M, Loetscher P, Moser B. CXC chemokine receptor 5 expression defines follicular homing T cells with B cell helper function. 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Methods of estimating GFR-different equations including CKD-EPI. Clin Biochem Rev 2011;32(2):75–9. [15] Jiang Y, Ma Z, Xin G, Yan H, Li W, Xu H, et al. Th1 and Th2 immune response in chronic hepatitis B patients during a long-term treatment with adefovir dipivoxil. Mediators Inflamm 2010:143026. [16] Morgan E, Varro R, Sepulveda H, Ember JA, Apgar J, Wilson J, et al. Cytometric bead array: a multiplexed assay platform with applications in various areas of biology. Clin Immunol 2004;110(3):252–66. [17] Tárnok A, Hambsch J, Chen R, Varro R. Cytometric bead array to measure six cytokines in twenty-five microliters of serum. Clin Chem 2003;49(6):1000–2. [18] Huang CC, Lehman A, Albawardi A, Satoskar A, Brodsky S, Nadasdy G, et al. IgG subclass staining in renal biopsies with membranous glomerulonephritis indicates subclass switch during disease progression. Mod Pathol 2013;26(6):799–805. [19] Deenick EK, Ma CS. The regulation and role of T follicular helper cells in immunity. Immunology 2011;134(4):361–7. [20] Morita R, Schmitt N, Bentebibel SE, Ranganathan R, Bourdery L, Zurawski G, et al. Human blood CXCR5 + CD4 + T cells are counterparts of T follicular cells and contain specific subsets that differentially support antibody secretion. Immunity 2011;34(1):108–21. [21] Chevalier N, Jarrossay D, Ho E, Avery DT, Ma CS, Yu D, et al. CXCR5 expressing human central memory CD4 T cells and their relevance for humoral immune responses. J Immunol 2011;186(10):5556–68. [22] Luo C, Li Y, Liu W, Feng H, Wang H, Huang X, et al. Expansion of circulating counterparts of follicular helper T cells in patients with myasthenia gravis. J Neuroimmunol 2013;256(1-2):55–61. [23] Zhu C, Ma J, Liu Y, Tong J, Tian J, Chen J, et al. Increased frequency of follicular helper T cells in patients with autoimmune thyroid disease. J Clin Endocrinol Metab 2012;97(3):943–50. [24] Simpson N, Gatenby PA, Wilson A, Malik S, Fulcher DA, Tangye SG, et al. Expansion of circulating T cells resembling follicular helper T cells is a fixed phenotype that identifies a subset of severe systemic lupus erythematosus. Arthritis Rheum 2010;62(1):234–44. [25] Ma J, Zhu C, Ma B, Tian J, Baidoo SE, Mao C, et al. Increased frequency of circulating follicular helper T cells in patients with rheumatoid arthritis. Clin Dev Immunol 2012;201:827480. [26] McAdam AJ, Greenwald RJ, Levin MA, Chernova T, Malenkovich N, Ling V, et al. ICOS is critical for CD40-mediated antibody class switching. Nature 2001;409(6816):102–5. [27] Dorfman DM, Brown JA, Shahsafaei A, Freeman GJ. Programmed death-1 (PD-1) is a marker of germinal center-associated T cells and angioimmunoblastic T-cell lymphoma. Am J Surg Pathol 2006;30(7):802–10.
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[35] Feng J, Lu L, Hua C, Qin L, Zhao P, Wang J, et al. High frequency of CD4+ CXCR5+ TFH cells in patients with immune-active chronic hepatitis B. PLoS One 2011;6(7): e21698. [36] Wang X, Zhou Y, Zhu N, Wang L, Gu LJ, Yuan WJ. The deposition of Notch1 in hepatitis B virus-associated nephropathy and its role in hepatitis B virus X protein-induced epithelial–mesenchymal transdifferentiation and immunity disorder in renal tubular epithelial cells. J Viral Hepat 2014. http://dx.doi.org/10.1111/jvh. 12244. [37] Rodrigues-Díez R, Aroeira LS, Orejudo M, Bajo MA, Heffernan JJ, Rodrigues-Díez RR, et al. IL-17A is a novel player in dialysis-induced peritoneal damage. Kindey Int 2014. http://dx.doi.org/10.1038/ki. [38] Ifuku M, Miyake K, Watanebe M, Ito K, Abe Y, Sasatomi Y, et al. Various roles of Th cytokine mRNA expression in different forms of glomerulonephritis. Am J Nephrol 2013;38(2):115–23. [39] Summers SA, Steinmetz OM, Li M, Kausman JY, Semple T, Edgtton KL, et al. Th1 and Th17 cells induce proliferative glomerulonephritis. J Am Soc Nephrol 2009;20(12):2518–24. [40] Ooi JD, Kitching AR, Holdsworth SR. Review: T helper 17 cells: Their role in glomerulonephritis. Nephrology (Carlton) 2010;15(5):513–21.
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International Immunopharmacology journal homepage: www.elsevier.com/locate/intimp
Frequency of CD4+CXCR5+ TFH cells in patients with hepatitis b virus-associated membranous nephropathy☆ Yong Liu a,1, Pingwei Zhao a,1, Zhihui Qu b, Desalegn Admassu Ayana c, Yanfang Jiang a,⁎ a b c
Key Laboratory of Zoonosis Research, Ministry of Education, The First Hospital of Jilin University, Changchun, 130032, China Department of Nephrology, The First Hospital of Jilin University, Changchun 130021, China Department of Medical Laboratory Sciences, Haramaya University, Dire Dawa, Ethiopia
a r t i c l e
i n f o
Article history: Received 6 April 2014 Received in revised form 6 June 2014 Accepted 11 June 2014 Available online 24 June 2014 Keywords: TFH cell HBV-MN IL-21 IL-4
a b s t r a c t The frequency of different subsets of CD4+CXCR5+ TFH cells and serum cytokine levels were analyzed in a total of 14 patients with newly diagnosed hepatitis B virus-associated membranous nephropathy (HBV-MN), 12 individuals with immune-tolerant HBV infection (HBV-IT) and 12 healthy controls (HC). Serum cytokine levels were measured before and 10–12 weeks after treatment. Significantly higher frequency of CD4+CXCR5+, CD4+CXCR5+ICOS+ and CD4+CXCR5+PD-1+ TFH cells, and higher serum levels of IL-17A, IFN-γ, IL-2, IL-10, IL-4 and IL-21 were detected in HBV-MN patients compared to the HC. The percentage of CD4+CXCR5+ TFH cells and serum IL-21 level in HBV-MN patients were also higher than the HBV-IT. The percentage of CD4+CXCR5+ TFH cell was negatively correlated with the value of eGFR, and the percentage of CD4+CXCR5+ICOS+ TFH cells was positively correlated with the 24-h urinary protein concentration. Notably, the percentage of CD4+CXCR5+PD-1+ TFH cells was positively correlated with serum IL-21 level and 24-h urinary protein concentration. Treatment with prednisone or/and immunosuppressive drugs significantly reduced the frequency of CD4+CXCR5+, CD4+CXCR5+PD-1+ TFH cells and serum IL-21 level, but increased IL-4 and IL-10 levels in the patients. CD4+CXCR5+ TFH cells, especially CD4+CXCR5+PD-1+ TFH cells may participate in the pathogenesis of HBV-MN. © 2014 Elsevier B.V. All rights reserved.
1. Introduction HBV infection is a well-recognized cause of glomerulonephritis, and HBV-associated membranous nephropathy (HBV-MN) is one of the most common extrahepatic manifestations of HBV infection. Its incidence closely parallels the incidence of HBV infection, especially for chronic HBV carriers through horizontal transmission in the general population [1]. HBV-MN, a kidney disease caused by hepatitis B virus infection, is a common secondary membranous nephropathy. The most common clinical features of HBV-MN are proteinuria and edema. It is considered that deposition of hepatitis antigen–antibody immune complexes in the subepithelial space results in the pathogenesis of HBV-MN. This assumption is based on the presence of HBV in kidney tissue, mainly the glomerular area [2]. In children, HBV-MN is usually self-limited with only rare progression to renal failure. However, in adults, the ☆ Funding source: This study was supported by grants from the National Natural Science Foundation of China (Nos. 30972610 and 81273240), Jilin Province Science and Technology Agency (No. 20110716), the Health Department Research Projects of Jilin Province (2009Z054) and Norman Bethune Program of Jilin University (2012206). ⁎ Corresponding author at: The First Hospital of Jilin University, Changchun 130032, China. Tel.: +86 13756660113; fax: +86 0431 84808391. E-mail address: [email protected] (Y. Jiang). 1 These authors made equal contributions to this study.
http://dx.doi.org/10.1016/j.intimp.2014.06.024 1567-5769/© 2014 Elsevier B.V. All rights reserved.
natural disease course of HBV-MN may be more relentless, slowly progressing to renal failure. At present, one of the important mechanisms of HBV-MN is based on the deposition of situ composites of HBeAg in subepithelial area. HBeAg form subepithelial immune complex through non-immune mechanism in the basement membrane because of its small molecular weight [3]. Two studies show that HBV can promote the inflammatory reaction in renal tissue. A study by Xia et al. [4] found that activation of NF- B by HBx protein might play a critical role in the transcriptional induction of inflammatory genes and HBx-induced chemotaxis. Yang et al. indicate [5] that HBeAg can bind to the IL-1 receptor accessory protein, express IL-1 sensitive gene induced by NF- B pathway, and promote the occurrence of inflammatory reaction. T cells are necessary for IgG responses and regulate the development of HBV-MN. However, it is still unclear how different types of CD4+ helper T cells regulate the pathogenesis of HBV-MN. CD4+ T cells have a crucial role in helping B cells produce antibodies in response to challenge with foreign antigens. Two studies described CD4+ T cells residing in the B cell follicle expressed CXCR5 [6,7]. These cells are more commonly known as T follicular helper (TFH) cells. Characteristics of TFH cells are increasing the expression of numerous molecules including inducible T-cell co-stimulator (ICOS), program death 1 (PD-1) and CD40-ligand (CD40L), the transcription factor Bcl-
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6 and regulatory cytokines of IL-21 [8–10]. ICOS appears to be important for TFH cell development, and PD-1 is a critical regulator of the function of TFH cells [11,12]. IL-21 was a cytokine preferentially expressed by TFH cells and served as an important regulator of humoral responses by directly regulating B cells proliferation and class switching [13]. Increased numbers of circulating TFH cells and aberrant activation of TFH cells have been associated with the development of autoimmune diseases [9,10]. Although HBV-MN is a complicated disease, an immune complex in the subepithelial space is a common and consistent characteristic among the patients. There currently is no information on how these different subsets of TFH cells exist in HBV-MN patients and whether TFH cells participate in the pathogenesis of HBV-MN. Therefore, we sought to explore the role of TFH cells in patients with HBV-MN. Currently, patients with HBV-MN are usually treated with antiviral drug, and sometimes need antivirus combined with corticosteroids or/and immunosuppressive therapy. Lamivudine has been recommended as the most important antivirus drug for HBV-MN, but the incidence of lamivudine-resistant HBV strains is up to 20% per year [2]. Anti-hypertension and anti-coagulation drugs, such as angiotensin-converting enzyme (ACE) inhibitors, angiotensin II receptor antagonists, aspirin or dipyridamole are also used to treat patients. However, it is unclear how these therapeutic strategies affect different subsets of TFH cells and immune measures in patients with HBV-MN. In this study, we measured the frequency of circulating CD4+CXCR5+ TFH cells in patients with HBV-MN, HBV-IT and healthy controls to investigate the potential relationship between the percentages of different subsets of TFH cells and the values of laboratory measures before and after treatment.
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First Hospital of Jilin University from November 2011 to May 2014. Written informed consent was obtained from individual participants. The experimental protocol was established, according to the guidelines of the Declaration of Helsinki and was approved by the Human Ethics Committee of First Hospital of Jilin University (Jilin University, Changchun, China). Histological examination of biopsied kidney tissues was conducted and HBV was detected in renal tissue of the fourteen HBV-MN patients (Fig. 1). Exclusion criteria include (1) rapidly progressive HBV-MN (with rapid renal function deterioration or historically necrotic capillaries and cellular crescents), (2) secondary HBV-MN, such as Henoch–Schonlein purpura, lupus nephritis, and other primary glomerulonephritis, (3) pregnancy or planning for pregnancy, or (4) diabetes mellitus, neoplasia, active peptic ulcer disease and recent infection. In this study, another 12 gender-, age- and ethnicity-matched healthy controls (HC) were recruited during the same period and they had no history of any chronic inflammatory disease. Their demographic and clinical characteristics were recorded. 2.2. Treatment and follow up A total of fourteen patients were treated with prednisone (methylprednisolone, 32 mg/d) or/and immunosuppressive (tacrolimus capsules, 1 mg/d), and not with antiviral drug. Six patients were also treated with benazepril (10 mg/d) or valsartan (75 mg/d, Novartis Pharma, Beijing, China). In addition, individual patients were treated with aspirin (100 mg/d, Bayer, Germany) or dipyridamole (25 mg/d, Yunpeng Pharmaceutical, Shanxi, China) if the patient had a high risk of a hypercoagulable state. Patients visited the clinical office monthly and were followed for at least 10–12 weeks after beginning treatment.
2. Materials and methods 2.3. Blood sampling and analyses 2.1. Patients A total of fourteen patients with newly diagnosed HBV-MN and twelve patients with immune-tolerant HBV infection (HBV-IT) were recruited in the inpatient service of the Department of Nephrology, the
Fasting venous blood samples were collected from individual patients before and 10–15 weeks after treatment and blood samples were also collected from HBV-IT and healthy controls. Blood sample was used for preparing peripheral blood mononuclear cells (PBMCs)
Fig. 1. Histological examination of biopsied kidney tissues was conducted and HBV was detected in renal tissue. Needle biopsy specimens were obtained from all HBV-MN patients during routine treatment procedures. Subsequently, these paraffin-embedded samples were sliced (2–3 μm) and stained. Such as the kidney biopsy is shown from a representative HBV-MN patient. (A–B) Massive HBcAg and small amounts of HBsAg were detected in the glomerular capillary by immunofluorescent. As indicated by the arrows, along the glomerular capillary distribution of HBV antigen. (C) Histological examination of biopsied kidney tissues was conducted using periodic acid-silver methenamine (PASM) staining. As indicated by the arrows, glomerular basement membrane was stained dark and thickening. (D) Histological examination of biopsied kidney tissues was conducted using periodic acid silver methenamine–martius yellow (PASM-MAS) staining. As indicated by the arrows, Glomerular basement membrane sediments were stained red. All images are shown at ×400 magnification.
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by density-gradient centrifugation using Ficoll-Paque Plus (Amersham Biosciences, Little Chalfont, UK) and the remaining blood samples were centrifuged for preparing serum samples. The number of lymphocytes were examined and the concentrations of serum triglycerides, cholesterol, albumin, uric acid, ALT and AST were determined using ADVIA 1650 biochemical analyzer (Bayer, Pittsburg, PA, USA). In addition, 24 h urine sample was collected and urine protein concentration and microscopic hematuria were measured. Estimation of glomerular filtration rate (eGFR) was performed using the modified eGFR formula [14]. 2.4. Flow cytometry analysis Peripheral blood mononuclear cells (PBMCs) were isolated by density-gradient centrifugation using Ficoll-Paque Plus (Amersham Biosciences, Little Chalfont, UK). Human PBMCs at 106/tube were stained in duplicate with PE-anti-CXCR5 (Biolegend, San Diego, USA) and PerCPanti-CD4, Alexa-flour-anti-CD278, FITC-anti-CD279, or isotype-matched control IgG (Beckton Dickinson, San Jose, USA) at room temperature for 30 minutes, respectively. After being washed with PBS, the cells were subjected to flow cytometry analysis using a FACSCalibur (Beckton Dickinson) and FlowJo software (v7.6.2) [15]. The cells were gated on the forward scatter of living cells and then centered on CD4+ T cells. Subsequently, the CD4+CXCR5+, CD4+CXCR5+ICOS+, CD4+CXCR5+PD-1+, and CD4+CXCR5+ICOS+PD-1+ TFH cells were determined by flow cytometry analysis, and at least 50,000 events per sample were analyzed. 2.5. Measurement of serum IL-21 by enzyme-linked immunosorbent assay (ELISA) The concentrations of serum IL-21 in HBV-MN patients and healthy controls were measured by ELISA using human IL-21 ELISA kit, according to the manufacturer's instruction (Roche Diagnostics, Lewes, UK). The detection limit for human IL-21 was 10 pg/ml. 2.6. Cytometric bead array (CBA) analysis of serum cytokines The concentrations of serum IFN-γ, TNF-α, IL-2, IL-4, IL-6, IL-10 and IL-17A were detected by CBA [16], according to the manufacturer's protocol (BD Biosciences) with minor modifications. Briefly, the individual sera (25 μl/each) were tested in duplicate on a FACSAria II, as described before [17] using the CellQuest Pro and CBA software (Becton Dickinson) to quantify serum cytokine concentrations. 2.7. Circulating HBV-DNA quantification Peripheral blood (2 ml) was collected from HBV-MN patients. HBVDNA was detected by QF-PCR using hepatitis B virus nucleic acid
detection kit, according to the manufacturer's instruction (Haoyuan Biological Technology Co., Ltd. Shanghai, China), and PCR amplification was performed using ABI Prism 7500 (AB, USA). The detection limit was 50 IU/ml. All HBV-MN patients had undetectable HBV-DNA levels (b500 copies/ml). 2.8. Statistical analysis All data are expressed as median, and range. The difference between two groups was analyzed by the Kruskal–Wallis H nonparametric test using the SPSS 19.0 software. The relationship between variables was analyzed by the Spearman's rank correlation test. A two-sided P-value of b 0.05 was considered statistically significant. 3. Results 3.1. Frequency of circulating CXCR5+CD4+ TFH cells in the HBV-MN patients There was no significant age and gender difference between HBVMN patients, HBV-IT, and the HCs (Table 1). Furthermore, there was no significant difference in the concentrations of serum uric acid, triglycerides, cholesterol, ALT and AST, as well as the numbers of lymphocytes between these three groups of subjects. As expected, microscopic hematuria and 24-h urinary proteins were significantly higher in the patients than the HC, but the values of eGFR and serum albumin were significantly less in the patients compared to the HC, suggesting that those patients had kidney function impairment. TFH cells can promote B cell activation, expansion and differentiation. To investigate the potential role of TFH cells in the development of HBVMN, we characterized the percentages of peripheral blood CD4+CXCR5+ TFH cells in CD4+ T cells among the patients, HBV-IT and HCs by flow cytometry analysis. We found that the percentages of CD4+CXCR5+ TFH cells in CD4+ T cells in the patients were significantly higher than the HBV-IT and HCs (17.80 (6.46–29.90) vs.14.53 (7.50–21.00), P = 0.046; 17.80 (6.46–29.90) vs.12.85 (9.54–26.67), P = 0.023, Fig. 2B). The percentages of CD4+CXCR5+ICOS+ and CD4+CXCR5+PD-1+ TFH cells in the patients were also significantly higher than the HCs (6.42 (4.55–9.30) vs.5.25 (3.14-8.24), P = 0.023; 8.34 (5.45–13.40) vs.6.75 (4.22–9.84), P = 0.015, Fig. 2B). However, there was no significant difference in the frequency of circulating CD4+CXCR5+ICOS+ and CD4+CXCR5+PD-1+ TFH cells between HBV-MN patients and HBV-IT, and there was also no significant difference in the frequency of circulating CD4+CXCR5+ICOS+PD-1+ TFH cells among HBV-MN patients, HBVIT and HCs. TFH cells can secrete IL-21, which has been shown to regulate B cell differentiation and proliferation. In this study, we examined the
Table 1 The demographic and clinical characteristics of participants.
Age, years Female/Male Lymphocytes, 109/L Serum albumin, g/L ALT, U/L AST, U/L Serum uric acid, μmol/L Triglycerides, mmol/L Cholesterol, mmol/L Urinary proteins, g/24 h Urea nitrogen, mmol/L eGFR, ml/min/1.73 m2 Microscopic hematuria, rbc/hpf Data shown are median and range, except specified. ⁎ P b 0.05 vs. the HC.
HBV-MN (n = 14)
Healthy controls (n = 12)
HBV-IT (n = 12)
57 (26–77) 3/11 2.16 (1.38–3.50) 21.6 (15.6–27.9)⁎ 18 (5–35) 17 (11–33) 350 (269–635) 3.56 (0.74–7.89) 6.80 (4.54–11.95) 6.44 (2.19–13.90)⁎ 5.83 (3.36–11.17) 72.20 (11.74–118.34)⁎ 14.7 (0.6–76.7)⁎
53 (25–75) 3/9 1.12 (0.41–1.77) 40.3 (35.7–48.9) 16 (9–32) 18 (9–31) 315 (230–430) 1.13 (0.35–1.60) 4.05 (2.95–5.95) 0.05 (0–0.12) 5.01 (3.71–6.14) 99.35 (90.24–112.04) 1 (0–2)
50 (23–64) 4/8 1.24 (0.40–2.01) 37.8 (33.5–44.8) 20 (15–40) 21 (14–39) 340 (234–450) 1.26 (0.51–3.01) 4.23 (3.02–7.34) 0.06 (0–0.13) 5.21 (3.63–7.12) 96.24 (87.45–109.87) 1 (0–2)
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Fig. 2. Flow cytometry analysis of TFH cells. PBMCs from HBV-MN patients before treatment as well as HBV-IT, HC were stained with anti-CD4, anti-CXCR5, anti-ICOS, and anti-PD-1. The cells were gated initially on living lymphocytes, and then on CD4+ T cells, and subsequently, CD4+CXCR5+TFH cells. The last, the frequency of CD4+CXCR5+ICOS+, CD4+CXCR5+PD-1+, and CD4+CXCR5+ICOS+PD-1+ TFH cells was analyzed by flow cytometry. (A) Flow cytometry analysis. (B) The percentage of CD4+CXCR5+ T cells among CD4+ T cells and CD4 + CXCR5 + ICOS+, CD4 + CXCR5 + PD-1+, and CD4 + CXCR5 + ICOS+PD-1+T cells among CD4+CXCR5+ T cells. Data shown are representative dot plug or expressed as the mean % of different subsets of TFH cells in total CD4+T cells or CD4+CXCR5+ TFH cells in individual subjects from two separate experiments. The horizontal lines represent the median values.
concentration of serum IL-21 using ELISA. The concentration of serum IL21 was significantly higher in HBV-MN patients compared to the HBV-IT (P b 0.05) and the HCs (P b 0.05, Fig. 3A). We also found that the concentrations of serum IL-17A, IFN-γ, IL-2, IL-4 and IL-10 were significantly
higher in HBV-MN patients compared to the HCs (P b 0.05, Fig. 3B–F). In brief, these data suggest a higher frequency of different subsets of CD4+CXCR5+ TFH cells and significantly elevated levels of serum cytokines in the patients.
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Fig. 3. Analysis of serum cytokines in HBV-MN patients. The levels of serum IL-2, IL-4, IL-10, IL-17A, and IFN-γ were tested by CBA in HBV-MN patients and HCs, and the level of serum IL-21 was tested by ELISA in HBV-MN and HBV-IT patients, respectively. Data are expressed as the mean values of individual participants from two separate experiments. The horizontal lines indicate the median values of different groups.
3.2. The relation between the percentages of CD4+CXCR5+, CD4+CXCR5+ICOS+, and CD4+CXCR5+PD-1+ TFH cells and other clinical parameters in HBV-MN patients The percentage of circulating CD4+CXCR5+ TFH cells was negatively correlated with the values of eGFR (r = −0.8645, P = 0.0056, Fig. 4A). The percentage of circulating CD4+CXCR5+PD-1+ TFH cells was positively correlated with serum IL-21 level (r = 0.7203, P = 0.0037, Fig. 4B), and 24-h urinary protein concentration (r = 0.8260, P = 0.0115, Fig. 4C). The percentage of circulating CD4+CXCR5+ICOS+ TFH cells was positively correlated with the 24-h urinary protein concentration (r = 0.8249, P = 0.0117, Fig. 4D), but no significant association with serum IL-21 level (r = 0.4987, P = 0.0695, Fig. 4E). In addition, a positive correlation was observed between serum IL-21 level and 24-h urinary protein concentration (r = 0.8087, P = 0.0151, Fig. 4F). These data suggest that CD4 +CXCR5 + and CD4 +CXCR5 +ICOS+ , especially CD4 +CXCR5+ PD-1 + TFH cells response, may be associated with the pathogenesis of HBV-MN. 3.3. Clinical parameters, frequency of CD4+CXCR5+TFH cells and the serum cytokines levels in HBV-MN patients following treatment Only six patients have complete records and other eight patients were lost to follow-up. The impact of treatment on the values of clinical
parameters, the frequency of circulating CD4+CXCR5+ TFH cells and serum cytokines levels in six patients were assessed for 10–12 weeks. The 24-h urinary protein concentration decreased and serum albumin concentration increased significantly, but there was no significant difference in the values of other clinical parameters (Table 2). In addition, the post-treatment percentages of circulating CD4+CXCR5+ (P = 0.038), CD4+CXCR5+ICOS+ (P = 0.048), and CD4+CXCR5+PD-1+ TFH cells (P = 0.011) reduced significantly compared to the pretreatment level (Fig. 5A–C). Similarly, post treatment serum IL-21 level was significantly decreased as compared to the pretreatment level (P = 0.004 Fig. 5D), but the post-treatment levels of serum IL-4 and IL-10 were significantly increased compared to the pretreatment level (P = 0.025 and P = 0.011, respectively, Fig. 5E–F). Collectively, treatment dramatically improved proteinuria, which was associated with a reduction in the frequency of different subsets of TFH cells in the patients. 4. Discussion Previous research has shown that one of the most important mechanisms of HBV-MN is the deposition of hepatitis Ag and Ab immune complexes in the subepithelial space. Some scholars showed that, mainly IgG antibody, especially IgG4 was the dominant IgG subclass in primary membranous glomerulonephritis, however, in secondary
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Fig. 4. Correlation analysis of clinicopathological features of IgAN with the percentages of circulating TFH cells in HBV-MN patients. It is well known that renal function can be influenced by either benazepril or valsartan through non-immunologic factors. So, the patients treated with those drugs can be excluded when analyzing the relationship between TFH and renal function. (A) The values of eGFR are negatively associated with the percentages of CD4+CXCR5+ TFH cells. (B–C) The concentrations of serum IL-21, 24-h urinary proteins are positively correlated with the percentage of CD4+CXCR5+ PD-1+TFH cells. (D) The concentrations of 24-h urinary proteins are positively correlated with the percentage of CD4+CXCR5+ ICOS+TFH cells. (E) The level of serum IL-21 and the percentage of CD4+CXCR5+ICOS+TFH cells have no significant statistical significance. (F) The level of serum IL-21 is positively correlated with the percentage of CD4+CXCR5+ ICOS+TFH cells.
membranous glomerulonephritis IgG1 is dominant [18]. It is also known that the generation of a high-affinity long-lived antibody response requires the presence of CD4+ T cells. The newly discovered CD4+ CXCR5+ T cell subset, namely follicular helper T cells, key function is Table 2 The effect of treatment on the values of clinical measures in the follow-up HBV-MN patients.
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Lymphocytes, 10 /L Serum albumin, g/L ALT, U/L AST, U/L Serum uric acid, μmol/L Triglycerides, mmol/L Cholesterol, mmol/L Urinary proteins, g/24 h Urea nitrogen, mmol/L eGFR, ml/min/1.73 m2 Microscopic hematuria, rbc/hpf
Before treatment
After treatment
2.25 (1.38–2.93) 24.2 (15.6–27.9) 16 (5–24) 17 (11–33) 410 (302–603) 4.23 (3.18–7.89) 7.24 (4.54–11.95) 7.47 (3.67–13.90) 5.46 (3.36–6.93) 57.50 (11.74–118.34) 13.5 (0.6–76.7)
1.65 (1.27–2.76) 34.2 (25.4–40.3)⁎ 15 (9–27) 16 (10–30) 377 (297–403) 1.98 (1.34–4.01) 6.02 (4.56–8.35) 2.58 (1.01–5.69)⁎ 4.67 (3.27–5.98) 65.24 (50.35–110.28) 12.4 (0.6–45.7)
Data shown are median and range. ⁎ P b 0.05 vs. the values before treatment.
to help B cells to support their activation, expansion and differentiation, and the formation of the GC [19]. Recently, two studies have demonstrated that circulating CD4+CXCR5+ T cells secrete IL-21 and CXCL13, express ICOS, and induce antibody production from naive B cells, suggesting that blood CD4+CXCR5+ T cells share functional properties with TFH cells [20, 21]. In this study, we found a significantly higher frequency of CD4+CXCR5+, CD4+CXCR5+ICOS+ and CD4+CXCR5+PD-1+ TFH cells in HBV-MN patients compared to the HC and the percentage of CD4+CXCR5+ TFH cells in HBV-MN patients was also found to be higher than the HBV-IT. Furthermore, the concentrations of serum IL-17A, IFN-γ, IL-2, IL-4, IL-10 and IL-21 were significantly higher in HBV-MN patients than the HC and IL-21 level was higher than the HBV-IT. Consistently, an increase in circulating CD4+CXCR5+, CD4+CXCR5+ICOS+ and CD4+CXCR5+PD-1+ TFH cells has been detected in patients with autoimmune myasthenia gravis, Graves' disease, SLE and rheumatoid arthritis [22–25]. Our data provided first hand evidence that the circulating TFH cells are increased in HBV-MN patients, suggesting that CD4+CXCR5+, CD4+CXCR5+ICOS+ and CD4+CXCR5+PD-1+ TFH cells may contribute in the pathogenesis of HBV-MN. The inducible costimulatory molecule (ICOS), a member of the CD28 family, is highly
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Fig. 5. Altered frequency of TFH cells and concentrations of serum cytokines in HBV-MN patients following treatment. The change of the percentages of different subsets of TFH cells and the levels of serum cytokines in six HBV-MN patients when compared before and after the treatment. Data are expressed as the mean or concentrations of individual subjects from two separate experiments. (A) The percentages of CD4+CXCR5+ cells in the total CD4+T cells in individual patients pre- and post-treatment. (B–C) The percentage of CD4+CXCR5+ICOS+ and CD4+CXCR5+PD-1+TFH cells in total CD4+CXCR5+ TFH cells in individual patients pre- and post-treatment. (D–F) The levels of serum IL-21, IL-4 and IL-10 in individual patients pre- and post-treatment.
expressed in the activated TFH cells. ICOS combined with the B cell surface ICOSL, not only stimulate the differentiation of activated TFH cells but also promote B cells to produce antibodies [26]. Thus, ICOS may promote B cells to produce hepatitis Ab, resulting in immune complexes deposit in the subepithelial space and the happening of HBN-MN. Consistently, the percentage of peripheral blood ICOS+TFH cell was significantly higher in HBV-MN patients than the HC and the percentage of ICOS+ TFH cell was positively correlated with the 24-h urinary protein concentration. The PD-1 molecule is expressed on activated T cells, particularly on TFH cells in the GC. PD-1 promotes cognate T-B interactions and provides an inhibitory signal to TFH cells in the GC [27]. Interestingly, we found that the percentage of circulating PD-1+ TFH cells was positively correlated with serum IL-21 level, and 24-h urinary protein concentration. Serum IL-21 level was also positively correlated with 24-h urinary protein concentration in HBV-MN patients. Similarly, some studies reported that the percentages of CD4+CXCR5+PD-1+ TFH cells and serum IL-21 level were significantly and positively correlated with level of serum autoantibodies and disease activity in patients with SLE and rheumatoid arthritis [28,29]. Now, the researchers showed that circulating precursor CCR7loPD-1hiCXCR5+CD4+ T cells promote antibody response upon antigen re-exposure [30]. All of these show that
the PD-1 is associated with autoantibodies. A previous study has shown that PD-1 regulates selection and survival in the GC, impacting the quantity and quality of long-lived PCs, and promotes IL-21 and IL-4 production [31]. Our result also indicates that CD4+CXCR5+PD-1+ TFH cells promote antibody response upon antigen exposure and the production of IL-21. Consequently, it is possible that high expression of PD-1 may provide an inhibitory signal to T cells and freeze T cells in a TFH state, allowing sustained production of IL-21 and IL-4 in the B cell follicle [31]. IL-21 maintains expression of Bcl-6 in GC B cells and contributes to GC formation and affinity maturation [32,33], promoting the production of antibodies. As a result, the produced anti-HBV interacts with antigens to form immune complexes that deposit in the subepithelial space, leading to renal injury. Thereby, the percentages of circulating CD4+CXCR5+ICOS+ and CD4+CXCR5+PD-1+ TFH cells, especially CD4 +CXCR5+ PD-1 + TFH cells may serve as biomarkers for evaluating the disease progression in HBV-MN patients. Some studies indicate that TFH cells are involved in the immune response in HBV infection and their increase in number reflects the activation of the immune response [34,35]. Studies found that the percentages of CD4+CXCR5+ [34], CD4+CXCR5+ICOS+ and CD4+CXCR5+PD-1+ [35] TFH cells were significantly higher in immune-tolerant chronic
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hepatitis B (CHB) patients compared to the healthy controls, consistent with our study. Because our experimental group also belongs to immune-tolerant CHB, it is difficult to explain why the increased percentage of TFH cells is caused by HBV-MN. However, we found that the percentage of CD4+CXCR5+ TFH cells was significantly higher in HBV-MN patients compared to HBV-IT. Xing et al. [34] found that decreased level of IL-21 in HBV-IT suggests the decrease in TFH cells activity. Our data showed that IL-21 level was significantly higher in HBV-MN patients compared to the HBV-IT and the HCs. This suggests that IL-21 may participate in the pathogenesis of HBV-MN. IL-21 is the major cytokine of TFH cells and also a key factor affecting the formation of germinal centers. Moreover, we found that the percentages of PD1+TFH cells were positively correlated with serum IL-21 levels in HBV-MN patients. Thus, these results indicate that the role of TFH cells in pathogenesis of HBV-MN is different from the immune-tolerant CHB, this may be because HBx can cause immune cells and inflammatory mediator disorder, which play an important role in the progress of hepatitis B virus associated glomerulonephritis [36]. Therefore, our data can suggest that CD4+CXCR5+ and CD4+CXCR5+ICOS+, especially CD4+CXCR5+PD-1+ TFH cells, may participate in the pathogenesis of HBV-MN. Serum HBsAg and HBeAg are undetectable in the fourteen patients and the domestic HBV-DNA quantities are all within the normal range. In this study, while monitoring HBV-DNA quantity and liver enzyme situation, patients were treated with glucocorticoids or/and immunosuppressives, but not with antiviral drug. Glucocorticoids inhibit cellular and humoral immunity, inflammation, fibroblast and blood capillary hyperplasia, as well as reduce vascular permeability, which play a role in anti-inflammatory and immunosuppressive effect, and reduce urinary protein. In patients treated with prednisone or/and immunosuppressive drugs, the clinical symptoms improved obviously and we found that the frequency of circulating TFH cells and serum IL-21 level were significantly reduced after 10–12 weeks of treatment compared to the pretreatment level. Feng et al. [28] observed a significant reduction in the TFH cells after corticosteroid treatment and they also found that TFH proportions were significantly decreased when dexamethasone was added to the culture system in vitro studies. Our results are consistent with theirs. This indicates that the different subsets of TFH cells may be involved in the pathogenesis of HBV-MN and the proportion of circulating TFH cells was down-regulated by corticosteroids in a dose-dependent manner. After encountering a specific antigen, naive CD4+ T cells activate and differentiate into various effector T-cell subsets, such as Th1, Th2, and Th17 cells. It is known that Th1 cells mainly secret IFN-γ and Th2 cells mainly secret IL-4. Accumulating data suggest that Th17 cells are highly proinflammatory, and IL-17A, as an effector cytokine, is involved in the pathogenesis of host defense, autoimmune responses, and more recently in chronic inflammatory diseases [37]. Treg cells controls these effector T cells, thereby regulating immune responses and preventing autoimmunity and tissue inflammation [38]. Previous research has shown that Th1 and Th17 effector cells can induce glomerular nephritis [39,40]. In our study, we found that the levels of serum IL-17A, IFN-γ, IL-2, IL-4, IL-10 and IL-21 in HBV-MN patients were significantly higher than the HC. The concentration of serum IL-21 was significantly decreased and serum IL-4 and IL-10 were significantly increased after treatment, while the levels of serum IL-2, IL-6, IFN-γ and IL-17A have no significant difference, compared to the pretreatment level. These data suggest that Th1 and Th17 effector cells may participate in the pathogenesis of HBV-MN, and promote the inflammatory response. It is possible that Th1 and Th17 stimulate anti-inflammatory Th2 and Treg that increase the expression of IL-4 and IL-10 during the pathogenesis of HBV-MN though further research is needed to support this assumption. In conclusion, our result suggests that CD4+CXCR5+ and CD4+ CXCR5+ICOS+, especially CD4+CXCR5+PD-1+ TFH cells, may participate in the pathogenesis of HBV-MN and serve as a biomarker for
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evaluating disease severity. The limitations of the study include single time-point measurement for some patients, a very small sample size, and lack of long-term follow-up and functional studies of different subsets of TFH cells as well as information from the affected kidney. Thus, further research on the precise roles of TFH cells in the pathogenesis of HBV-MN in a bigger population is warranted. References [1] Liao MT, Chang MH, Lin FG, Tsai IJ, Chang YW, Tsau YK. Universal hepatitis B vaccination reduces childhood hepatitis B virus-associated membranous nephropathy. Pediatrics 2011;128(3):e600–4. [2] Moon JY, Lee SH. Treatment of hepatitis B virus-associated membranous nephropathy: lamivudine era versus post-lamivudine era. Korean J Intern Med 2012;27(4):394–6. [3] Takekoshi Y, Tanaka M, Miyakawa Y, Yoshizawa H, Takahashi K, Mayumi M. 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