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Association of protein disulfide isomerase family A, member 4, and inflammation in people living with HIV

  • Author Footnotes
    # These authors contributed equally to this work.
    Ning-Chi Wang
    Footnotes
    # These authors contributed equally to this work.
    Affiliations
    Division of Infectious Diseases and Tropical Medicine, Department of Internal Medicine, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan
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  • Author Footnotes
    # These authors contributed equally to this work.
    Hsuan-Wei Chen
    Footnotes
    # These authors contributed equally to this work.
    Affiliations
    Division of Gastroenterology, Department of Internal Medicine, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan
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  • Te-Yu Lin
    Correspondence
    Correspondence author: Te-Yu Lin, Division of Infectious Diseases and Tropical Medicine, Department of Internal Medicine, Tri-Service General Hospital, National Defense Medical Center, Number. 325, Section 2, Cheng-Kung Road, Neihu 114, Taipei, Taiwan, Tel: +886-2-87927257, Fax: +886-2-87927258
    Affiliations
    Division of Infectious Diseases and Tropical Medicine, Department of Internal Medicine, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan
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  • Author Footnotes
    # These authors contributed equally to this work.
Open AccessPublished:November 11, 2022DOI:https://doi.org/10.1016/j.ijid.2022.11.010

      Highlights

      • Plasma protein disulfide isomerase family A member 4 (PDIA4) levels in people living with HIV were higher than those in the healthy controls.
      • Plasma PDIA4 levels reduced significantly after combination antiretroviral therapy.
      • Plasma PDIA4 levels correlated with a decreasing trend in the HIV viral load.
      • Plasma PDIA4 levels are closely associated with inflammation in people living with HIV.

      Abstract

      Objectives

      : Protein disulfide isomerase (PDI) family members are specific endoplasmic reticulum proteins associated with inflammation, obesity, and cancer. In HIV infection, the role of PDI family A, member 4 (PDIA4), is unclear. This study aimed to clarify the association between plasma PDIA4 levels and inflammation in people living with HIV (PLWH).

      Methods

      : In this study, 287 PLWH and 74 healthy participants were enrolled. The plasma PDIA4 values, demographic data, laboratory data, and other inflammatory markers were recorded. The association between PDIA4 level and inflammatory extent was analyzed using logistic regression and Spearman rank-order correlations. Other results were analyzed using Student's t-test or chi-square test.

      Results

      : In PLWH, the PDIA4 levels were positively associated with the inflammatory markers, interleukin 6 (r = 0.209, p = 0.001), and tumor necrosis factor-α (r = 0.162, p = 0.01) levels, but not with high-sensitivity C-reactive protein levels. Moreover, the plasma PDIA4 level of PLWH decreased after anti-viral treatment (p = 0.0001).

      Conclusion

      : Plasma PDIA4 levels are closely associated with inflammation in PLWH and have a positive correlation with the viral load during anti-viral therapy.

      GRaphical Abstract

      Keywords

      Introduction

      HIV infection can cause CD4 T-cell depletion and an immunodeficiency state. HIV causes immune activation that is associated with massive CD4 T-cell depletion in the gut-associated lymphoid tissue (
      • Guadalupe M
      • Reay E
      • Sankaran S
      • Prindiville T
      • Flamm J
      • McNeil A
      • et al.
      Severe CD4+ T-cell depletion in gut lymphoid tissue during primary human immunodeficiency virus type 1 infection and substantial delay in restoration following highly active antiretroviral therapy.
      ). Both innate and adaptive immune responses are involved in HIV infection. Furthermore, cytokines secreted by the T cells changed during infection, and dysregulation of the cytokine profile contributes to the pathogenesis of the disease by impairing cell-mediated immunity (
      • Kedzierska K
      • Crowe SM.
      Cytokines and HIV-1: interactions and clinical implications.
      ,
      • Ramana KV.
      Effect of highly active antiretroviral therapy (HAART) on human immunodeficiency virus disease pathogenesis and progression.
      ).
      Combination antiretroviral therapy (cART) includes the combination of different types of highly effective antiretroviral agents, including nucleoside/nucleotide reverse transcriptase inhibitors (NRTIs), non-NRTIs (nNRTIs), protease inhibitors (PIs), integrase strand transfer inhibitors (INSTIs), and entry inhibitors. This effective treatment suppresses viral replication and increases the CD4 T-cell counts in most cases, but it only partly reduces T-cell activation, cell death, and soluble inflammatory markers (
      • Lederman MM
      • Calabrese L
      • Funderburg NT
      • Clagett B
      • Medvik K
      • Bonilla H
      • et al.
      Immunologic failure despite suppressive antiretroviral therapy is related to activation and turnover of memory CD4 cells.
      ;
      • Massanella M
      • Negredo E
      • Pérez-Alvarez N
      • Puig J
      • Ruiz-Hernández R
      • Bofill M
      • et al.
      CD4 T-cell hyperactivation and susceptibility to cell death determine poor CD4 T-cell recovery during suppressive HAART.
      ). The residual chronic inflammation and persistent immune activation may increase morbidity and mortality in people living with HIV (PLWH) receiving cART (
      • Wada NI
      • Jacobson LP
      • Margolick JB
      • Breen EC
      • Macatangay B
      • Penugonda S
      • et al.
      The effect of HAART-induced HIV suppression on circulating markers of inflammation and immune activation.
      ). A previous study demonstrated that interleukin 6 (IL-6) and high-sensitivity C-reactive protein (hs-CRP) are linked to a higher risk of subsequent mortality (
      • Neaton JD
      • Neuhaus J
      • Emery S.
      Soluble biomarkers and morbidity and mortality among people infected with HIV: summary of published reports from 1997 to 2010.
      ). Furthermore, a sub-study of AIDS Clinical Trials Group shows that tumor necrosis factor-α (TNF-α) levels are significantly associated with increased risk of AIDS and non–AIDS-related events (
      • McComsey GA
      • Kitch D
      • Sax PE
      • Tierney C
      • Jahed NC
      • Melbourne K
      • et al.
      Associations of inflammatory markers with AIDS and non-AIDS clinical events after initiation of antiretroviral therapy: AIDS clinical trials group A5224s, a substudy of ACTG A5202.
      ).
      The endoplasmic reticulum (ER) plays a crucial role in protein folding, assembly, and secretion. Disruption of ER homeostasis may lead to the accumulation of misfolded or unfolded proteins in the ER lumen. ER stress markers such as glucose-regulated protein GRP 78, calreticulin, calnexin, and protein disulfide isomerases (PDIs) are associated with lipid metabolism, insulin resistance, cardiovascular disease, type 2 diabetes mellitus, and obesity (
      • Hotamisligil GS.
      Endoplasmic reticulum stress and the inflammatory basis of metabolic disease.
      ,
      • Lee J
      • Ozcan U.
      Unfolded protein response signaling and metabolic diseases.
      ). PDIs, oxidoreductases of the thioredoxin superfamily, are expressed mainly in the ER of eukaryotic cells (
      • Appenzeller-Herzog C
      • Ellgaard L.
      The human PDI family: versatility packed into a single fold.
      ). They participate in the redox-dependent chaperones process, catalyzing the formation and rearrangement of disulfide bonds, which is an important physiological function of the ER (
      • Grek C
      • Townsend DM.
      Protein disulfide isomerase superfamily in disease and the regulation of apoptosis.
      ;
      • Okumura M
      • Kadokura H
      • Inaba K.
      Structures and functions of protein disulfide isomerase family members involved in proteostasis in the endoplasmic reticulum.
      ). PDIs are also expressed on the cell surface of hepatocytes, lymphocytes, and platelets. Protein disulfide isomerase family A, member 4 (PDIA4), a member of the PDI family, has been found to be located in the cytoplasm and nucleus (
      • Su SC
      • Chien CY
      • Chen YC
      • Chiang CF
      • Lin FH
      • Kuo FC
      • et al.
      PDIA4, a novel ER stress chaperone, modulates adiponectin expression and inflammation in adipose tissue.
      ,
      • Turano C
      • Coppari S
      • Altieri F
      • Ferraro A.
      Proteins of the PDI family: unpredicted non-ER locations and functions.
      ).
      PDIs have been implicated as potential therapeutic targets for influenza A and B viruses (
      • Kim Y
      • KO Chang
      Protein disulfide isomerases as potential therapeutic targets for influenza A and B viruses.
      ). Additionally, PDIA4 is involved in genome uncoating during human astrovirus cell entry (
      • Aguilar-Hernández N
      • Meyer L
      • López S
      • DuBois RM
      • Arias CF.
      Protein disulfide isomerase A4 is involved in genome uncoating during human astrovirus cell entry.
      ). The reductase activity of surface-associated PDI is important in the HIV fusogenic process (
      • Markovic I
      • Stantchev TS
      • Fields KH
      • Tiffany LJ
      • Tomiç M
      • Weiss CD
      • et al.
      Thiol/disulfide exchange is a prerequisite for CXCR4-tropic HIV-1 envelope mediated T-cell fusion during viral entry.
      ). Several studies have indicated that ER stress markers are elevated in the inflammatory state (
      • Galligan JJ
      • Petersen DR.
      The human protein disulfide isomerase gene family.
      ;
      • Spee P
      • Subjeck J
      • Neefjes J.
      Identification of novel peptide binding proteins in the endoplasmic reticulum: ERp72, calnexin, and grp170.
      ). HIV induces a chronic inflammatory condition; however, there is limited knowledge about the clinical significance of ER stress-induced PDIA4 in PLWH. Hence, this study aimed to assess whether plasma PDIA4 levels are associated with inflammation in PLWH receiving treatment.

      Patients and methods

      Study population and setting

      PLWH are provided with free-of-charge inpatient and outpatient medical services at designated hospitals in Taiwan. These services include management of opportunistic infections, cART prescription, and laboratory measurement of HIV RNA viral load, CD4 T lymphocyte count, viral hepatitis, blood cell count, renal and liver function, glucose, total cholesterol, triglyceride, low-density lipoprotein cholesterol (LDL), high-density lipoprotein cholesterol, and uric acid levels. Plasma HIV RNA load, CD4 lymphocyte count, and liver and renal function tests are conducted every 3 months in the 1st year of cART initiation. These laboratory monitoring schedules can be adjusted to every 6 months thereafter if the HIV RNA load remains <50 copies/ml. This study enrolled 361 adults who followed up at the Tri-Service General Hospital from July 2018 to June 2019. This study was approved by the Institutional Review Board of Tri-Service General Hospital.

      Study design

      In this cross-sectional study, we reviewed the medical records, physical examinations, clinical symptoms, and laboratory results of PLWH. HIV treatment-naïve patients aged ≥20 years and eligible for cART were assessed for inclusion in this study. We excluded PLWH who had active opportunistic infections or could not attend regular follow-up visits post cART treatment. Blood samples of PLWH (n = 260) who had received at least three effective cART regimens, such as two NRTIs plus nNRTI, PI, or INSTI for ≥6 months, were obtained once in the analysis. In addition, we enrolled additional PLWH (n = 27,) and their blood samples were collected at three different points: before treatment (baseline), 6-month, and 12-month post cART treatment. Participants in the control group (n = 74) were randomly selected from the health examination department, and their clinical and laboratory data were collected. Traditional inflammatory markers, such as TNF-α, IL-6, and hs-CRP, were also measured using the sandwich enzyme-linked immunosorbent assay method.

      Measurement of PDIA4 level

      The PDIA4 protein levels were measured using a sandwich enzyme-linked immunosorbent assay kit (USCN Life Science Inc., USA). Briefly, each 100μl plasma sample was directly added to the micro-test plate coated with an antibody specific to PDIA4, followed by incubation at 37°C for 2 hours. Avidin conjugated with horseradish peroxidase was then added to each microplate well and allowed to incubate, followed by aspiration, the addition of 100μl of Detection Reagent A, and incubation for 1 hour at 37°C. After washing thrice, 100μl of Detection Reagent B was added, followed by incubation for 30 minutes at 37°C. After washing five times, 90μl of 3,3′,5,5′-tetramethylbenzidine substrate solution was added, followed by incubation for 15-25 minutes at 37°C. Subsequently, only the wells containing PDIA4, biotin-conjugated antibodies, and enzyme-conjugated avidin changed color. The enzyme-substrate reaction was terminated by the addition of 50μl of sulfuric acid solution, and the color change was measured spectrophotometrically at a wavelength of 450 ±10 nm. The concentration of PDIA4 in the samples was determined by comparing the optical density of the samples to the standard curve.

      Statistical analysis

      Descriptive continuous variables are presented as means and SDs, and categorical variables as numbers and percentages. Statistical comparisons between the two groups were performed using Student's t-test (unpaired t-test) or chi-square test, according to the type of data. Biochemical variables associated with PDIA4 levels were analyzed using Spearman rank-order correlations and partial correlation analysis after adjusting for age. Multivariate logistic regression analysis was used to study the independent factors of plasma PDIA4 levels and other covariates. All statistical analyses were performed using GraphPad Prism 7.0 software (version 18.0; SPSS Inc., Chicago, Illinois).

      Results

      Demographics and biochemical values of healthy participants and PLWH receiving cART

      From July 2018 to June 2019, 484 PLWH met the inclusion criteria, and the recruitment strategy is shown in Figure 1. The demographics and biochemical values of the healthy participants and PLWH are presented in Table 1. The PLWH were younger than the healthy participants (38.3 ± 12.2 vs 46 ± 12.8 years; p < 0.001) and had significantly lower total cholesterol levels (168 ± 36.9 vs 193 ± 34.1 mg/dl; p < 0.001). However, the PLWH showed higher triglyceride (147 ± 20.1 vs 102 ± 47.6 mg/dl; p < 0.001), creatinine (0.92 ± 0.18 vs 0.78 ± 0.18 mg/dl; p < 0.001), and PDIA4 (24.7 ± 18.8 vs 15 ± 28.1 ng/ml; p = 0.001) levels than those of the healthy participants.
      Figure 1
      Figure 1Study flow.
      Abbreviations: cART, combination antiretroviral therapy; PLWH, people living with HIV.
      Table 1Demographic and biochemical variables among healthy participants and PLWH receiving combination antiretroviral therapy.
      Healthy participants(n = 74)PLWH receiving cART (n = 260)P-value
      Age, years46 ± 12.838.3 ± 12.2<0.001*
      Body mass index, kg/m223.6 ± 3.423.4 ± 3.50.679
      Fasting glucose, mg/dl85.97 ± 0.997.1 ± 21.2<0.001*
      Total cholesterol, mg/dl193 ± 34.1168 ± 36.9<0.001*
      Triglyceride, mg/dl102 ± 47.6147 ± 20.1<0.001*
      Creatinine, mg/dl0.78 ± 0.180.92 ± 0.18<0.001*
      Alanine aminotransferase, U/l21.5 ± 15.641.3 ± 21.50.428
      Protein disulfide isomerase family A, member 4, ng/ml15 ± 28.124.7 ± 18.80.001*
      Note: The demographics and biochemical values of healthy participants and PLWH are presented. *p < 0.05.
      Abbreviations: cART, combination antiretroviral therapy; PLWH, people living with HIV.

      Demographics and laboratory values stratified by different treatment regimens

      The demographic and biochemical variables of the PLWH stratified according to different treatment regimens are shown in Table 2. Age, body mass index, hepatitis B surface antigen positivity, anti-hepatitis C virus positivity, highly active antiretroviral duration, log10 HIV viral load, CD4 count, CD8 count, CD4/CD8 ratio, white blood cells, and the platelet, fasting glucose, total cholesterol, LDL, high-density lipoprotein cholesterol, triglyceride, creatinine, uric acid, aspartate aminotransferase, alanine aminotransferase, total bilirubin, albumin, TNF-α, IL-6, and hs-CRP levels were similar among the three treatment groups of PLWH. PLWH receiving nNRTI had higher hemoglobin levels than those of the other two treatment groups (15.1 ± 1.2 vs 14.7 ± 1.5 vs 14.7 ± 1.5 g/dl; p = 0.026). PLWH of the INSTI group had higher blood urea nitrogen (15 ± 5 vs 14.5 ± 4.45 vs 13.4 ± 3.36 mg/dl; p = 0.027), and PDIA4 (28.1 ± 24.9 vs 24.9 ± 16.5 vs 19.3 ± 10.7 ng/ml; p = 0.024) levels than those of the other two treatment groups.
      Table 2Demographic and biochemical variables of PLWH stratified by treatment regimen.
      nNRTI (n = 123)
      Among PLWH received nNRTI group, 68 patients received efavirenz; 44 patients received rilpivirine; and 11 patients received nevirapine.
      PI (n = 57)
      Among PLWH received PI group, 20 patients received atazanavir; 29 patients received lopinavir/ritonavir; and eight patients received darunavir/ritonavir.
      INSTI (n = 80)
      Among PLWH received INSTI group, 25 patients received raltegravir, and 55 patients received dolutegravir.
      p-value
      Age, years38.5 ± 1236.9 ± 11.239 ± 12.30.591
      Body mass index, kg/m223.5 ± 3.8423.5 ± 3.423.8 ± 3.10.78
      Hepatitis B virus surface antigen positivity, n (%)15(12.2)9(15.8)6(7.5)0.308
      Anti-hepatitis C virus positivity, n (%)6(4.9)4(7)7(8.8)0.564
      Highly active antiretroviral therapy duration, months30.6 ± 29.227.0 ± 28.139.4 ± 36.10.14
      HIV viral load, log10 copies/ml0.42 ± 0.920.24 ± 0.910.51 ± 1.050.283
      CD4, cells/mm3546 ± 242536 ± 259571 ± 2740.689
      CD8, cells/mm3886 ± 398913 ± 392902 ± 4260.905
      CD4/CD8 ratio0.71 ± 0.390.68 ± 0.440.74 ± 0.440.688
      White blodd cell, x 103 cells/mm35.77 ± 1.616.43 ± 1.915.95 ± 1.720.056
      Hemoglobin, g/dl15.1 ± 1.214.7 ± 1.414.7 ± 1.50.026
      Platelet, cells/mm3220 ± 53.4239.7 ± 62.2228 ± 57.80.103
      Fasting glucose, mg/dl94.3 ± 11.396.8 ± 15.2101.5 ± 32.60.061
      Total cholesterol, mg/dl179 ± 38.4168 ± 36.1164 ± 35.10.485
      Low-density lipoprotein cholesterol, mg/dl101 ± 27.5103 ± 25.998.8 ± 27.70.66
      High-density lipoprotein cholesterol, mg/dl44.4 ± 10.943.8 ± 9.6240.9 ± 7.930.681
      Triglyceride, mg/dl155 ± 128131 ± 100148 ± 1210.471
      Blood urea ntrogne, mg/dl13.4 ± 3.3614.5 ± 4.4515 ± 50.027
      Creatinine, mg/dl0.92 ± 0.170.91 ± 0.170.94 ± 0.210.632
      Uric acid, mg/dl5.87 ± 1.256.13 ± 1.66.03 ± 1.370.467
      Aspirate aminotransferase, U/l38.2 ± 17124.8 ± 14.722.2 ± 8.810.595
      Alanine aminotransferase, U/l26.5 ± 2131.2 ± 60.871 ± 3820.33
      Total bilirubin, mg/dl0.81 ± 0.690.82 ± 0.710.83 ± 0.80.988
      Albumin, mg/dl4.52 ± 0.364.49 ± 0.474.53 ± 0.370.828
      Tumor necrosis factor-α, ng/ml0.9 ± 1.181.06 ± 1.321.1 ± 1.330.512
      Interleukin -6, pg/ml8.48 ± 25.27.29 ± 14.88.81 ± 19.70.915
      High-sensitivity C-reactive protein, mg/l4.67 ± 8.843.66 ± 6.444.99 ± 8.770.637
      Protein disulfide isomerase family A, member 4, ng/ml24.9 ± 16.519.3 ± 10.728.1 ± 24.90.024
      Note: We stratified PLWH into three groups according to different treatment regimens. Continuous data are presented as means±SD, and categorical data as numbers (%).
      Abbreviations: CD, clusters of differentiation; INSTI, integrase inhibitor; nNRTI, non-nucleoside reverse transcriptase inhibitor; PLWH, people living with HIV; PI, protease inhibitor.
      a Among PLWH received nNRTI group, 68 patients received efavirenz; 44 patients received rilpivirine; and 11 patients received nevirapine.
      b Among PLWH received PI group, 20 patients received atazanavir; 29 patients received lopinavir/ritonavir; and eight patients received darunavir/ritonavir.
      c Among PLWH received INSTI group, 25 patients received raltegravir, and 55 patients received dolutegravir.

      Demographic and biochemical variables of cART-naïve, 6-month, and 12-month post cART patients

      The demographic and biochemical variables of cART-naïve, 6-month, and 12-month post cART patients are shown in Table 3. PLWH receiving cART 6-month and 12-month dramatically reduce HIV viral load (0.67 ± 1.40 vs 4.83 ± 1.29 log10 copies/ml; p < 0.0001; 0.34 ± 0.66 vs 4.83 ± 1.29 log10 copies/ml; p < 0.0001) and elevation CD4 count (511.6 ± 226.5 vs 287.5 ± 174.3 cells/mm3; p = 0.0002; 569.0 ± 228.0 vs 287.5 ± 174.3 cells/mm3; p < 0.0001) compared to naïve status. However, HIV viral load and CD4 count were similar between PLWH receiving cART 6-month and 12-month group. PLWH cART-naïve patients have higher PDIA4 levels than those receiving cART 6-month and 12-month group (77.4 ± 60.1 vs 57.6 ± 39.5 ng/ml; p = 0.02; 77.4 ± 60.1 vs 42.0 ± 30.0 ng/ml; p = 0.0001).
      Table 3Demographic and biochemical variables of cART-naïve, 6-month and 12-month post cART patients.
      cART-naïve (n = 27)Six-month post cART (n = 27)Twelve-month post cART (n = 27)p-value
      cART naïve vs 6-month post cART.
      p-value
      cART naïve vs 12-month post cART.
      p-value
      Six-month post cART vs 12-month post cART.
      Body weight, kg62.8±11.863.7±12.265.7±13.50.770.390.57
      Body mass index, kg/m221.2±3.2321.5±3.2822.2±3.710.720.290.48
      HIV viral load, log10 copies/ml4.83±1.290.67±1.400.34±0.66<0.0001<0.00010.28
      CD4 count, cells/mm3287.5±174.3511.6±226.5569.0±228.00.0002<0.00010.36
      CD8 count, cells/mm31041.5±703.1975.6±451.4892.1±428.10.690.360.49
      CD4/CD8 ratio0.35±0.230.60±0.380.73±0.340.005<0.00010.22
      White blood cell, cells/mm35.69±1.946.03±1.776.54±2.140.500.130.34
      Hemoglobin, mg/dl13.7±2.1114.6±2.0814.8±1.360.110.020.65
      Platelet, x 103 cells/mm3222.6±80.3246.8±77.4275.4±68.70.270.010.16
      Fasting glucose, mg/dl90.9±18.892.2±18.696.0±15.70.790.280.42
      Total cholesterol, mg/dl153.2±32.6161.8±31.4155.9±27.60.330.740.47
      Low-density lipoprotein cholesterol, mg/dl97.1±23.6103.6±24.2102.6±22.70.330.390.88
      Triglyceride, mg/dl127.0±80.4124.4±53.7116.1±59.40.890.580.59
      Blood urea nitrogen, mg/dl11.4±2.8912.2±2.6813.0±4.130.290.100.39
      Creatinine, mg/dl0.74±0.140.87±0.130.90±0.140.0010.00020.49
      Uric acid, mg/dl5.98±1.426.62±2.336.25±1.420.230.490.48
      Aspirate aminotransferase, U/l36.7±33.827.2±25.635.0±55.40.250.890.51
      Alanine aminotransferase, U/l34.3±32.430.3±42.444.6±101.30.700.620.50
      Total bilirubin, mg/dl0.83±0.560.76±0.270.65±0.240.580.130.10
      Protein disulfide isomerase family A, member 4, ng/ml77.4±60.157.6±39.542.0±30.00.020.00010.11
      Note: We compare biochemical variables between cART-naïve, 6-month and 12-month post cART. Continuous data are presented mean ± SD.
      Abbreviations: cART, combination antiretroviral therapy; DC, clusters of differentiation.
      a cART naïve vs 6-month post cART.
      b cART naïve vs 12-month post cART.
      c Six-month post cART vs 12-month post cART.

      Positive correlation between inflammatory markers and PDIA4 levels in PLWH

      To emphasize the association between inflammatory markers and PDIA4 plasma levels, an age-adjusted Spearman partial correlation was used (Table 4). The TNF-α, IL-6, and hs-CRP levels and white blood cell count correlated positively with the PDIA4 levels. Additionally, TNF-α and IL-6 showed significant differences (p < 0.05). In contrast, the platelet count and CD4 and CD8 T lymphocyte counts were inversely correlated with PDIA4 levels.
      Table 4Age-adjusted Spearman partial correlation coefficients between Protein disulfide isomerase A4 and inflammatory markers.
      Correlation coefficients (n = 260)
      rp-value
      Tumor necrosis factor-α, ng/ml0.1620.01
      Interleukin-6, pg/ml0.2090.001
      High-sensitivity C-reactive protein, mg/l0.0250.694
      White blood cell count, cells/mm30.010.873
      Platelet count, x 103 cells/mm3-0.1060.089
      Clusters of differentiation 4 T lymphocyte count, cells/mm3-0.0560.374
      Clusters of differentiation 8 T lymphocyte count, cells/mm3-0.0290.64
      Note: Potential inflammatory markers and protein disulfide isomerase family A, member 4 levels in people living with HIV were analyzed using Spearman partial correlation analysis

      Factors associated with high PDIA4 levels in PLWH

      Table 5 shows an analysis of the potential factors that may influence the PDIA4 levels in PLWH using univariate and multivariate logistic regression analyses. The HIV viral load and TNF-α and IL-6 levels showed a significant positive correlation with elevated PDIA4 levels (p < 0.05), but age, lipid profile, CD4 T lymphocyte count, and other biochemical markers did not show a significant correlation. Moreover, TNF-α showed a significant association with high PDIA4 levels in the multivariate analysis.
      Table 5Logistic analysis to identify factors associated with high protein disulfide isomerase family A, member 4 values in people living with HIV.
      VariablesUnivariate analysisMultivariate analysis
      Odds ratio (95% confidence interval)p-valueOdds ratio (95% confidence interval)p-value
      Age (ref: <30)
      31-400.73 (0.37-1.43)0.356
      41-500.77 (0.37-1.64)0.503
      >500.50 (0.24-1.07)0.073
      Hepatitis B virus surface antigen positivity0.46 (0.21-0.99)0.069
      Anti-hepatitis C virus positivity2.35 (0.87-6.33)0.091
      DM1.15 (0.35-3.86)0.817
      Biochemical data
      Glucose ante cibum (ref: <100 mg/dl)0.95 (0.52-1.72)0.858
      Total cholesterol (ref: <200 mg/dl)1.05 (0.54-2.03)0.449
      Triglyceride (ref: <150 mg/dl)1.06 (0.61-1.85)0.827
      Low-density lipoprotein (ref: <100 mg/dl)1.24 (0.56-2.73)0.591
      Aspirate aminotransferase (ref: <40 U/l)0.37 (0.13-1.11)0.076
      Alanine aminotransferase (ref: <40 U/l)0.54 (0.27-1.07)0.078
      Blood urea nitrogen (ref: <25 mg/dl)2.06 (0.22-18.7)0.521
      Creatinine (ref: <1 mg/dl)1.34 (0.68-2.65)0.398
      Total bilirubin (ref: <1.2 mg/dl)1.62 (0.79-3.30)0.188
      White blood cell count (ref: <4 × 103 cells/mm3)3.29 (0.94-11.5)0.062
      Platelet (ref: <150 × 103 cells/mm3)1.74 (0.56-5.33)0.336
      HIV viral load, log 10 copies/ml2.31(1.06-5.05)0.036
      CD4 count (ref: >500)
      <2002.15 (0.69-6.72)0.190
      201-5000.85 (0.49-1.46)0.551
      CD4/CD8 ratio (ref: <0.8)1.35 (0.79-2.30)0.275
      Tumor necrosis factor-α4.22 (2.27-7.86)<0.0014.32 (2.29-8.17)<0.001
      Interleukin-62.22 (1.13-4.37)0.021
      High-sensitivity C-reactive protein0.96 (0.50-1.92)0.959
      Note: Multivariable analysis used forward logistic regression and adjusted for other variables. Univariate and multivariate analysis of factors associated with high protein disulfide isomerase family A, member 4 levels were conducted.
      Abbreviation: DC, clusters of differentiation; DM, diabetes mellitus.

      Patients with high PDIA4 levels presenting high IL-6 and TNF-α levels

      In Figure 2, the PLWH were stratified by the PDIA4 levels (<20, 20-50, and >50 ng/ml) (Figure 2a). The IL-6 levels were significantly higher in the group with PDIA4 levels >50 ng/ml than in the other groups (Figure 2b). Furthermore, the TNF-α levels were higher in the group with PDIA4 levels >20 ng/ml than in the other groups (Figure 2c). There was no difference in the inflammatory marker, hs-CRP, among these groups (Figure 2d).
      Figure 2
      Figure 2People living with HIV with high protein disulfide isomerase family A, member 4 levels presenting high interleukin-6 and tumor necrosis factor-α levels.
      People living with HIV were stratified by protein disulfide isomerase family A, member 4 levels (<20, 20-50, and >50 ng/ml) (a), and their interleukin-6 (b), tumor necrosis factor-α (c) and high-sensitivity C-reactive protein (d) levels were analyzed; *p < 0.05.

      Positive correlation between PDIA4 levels, and HIV viral load

      To study the trend of PDIA4 level change in correlation with viral activity, 27 patients were followed up for their PDIA4 levels and viral load during the highly active antiretroviral therapy (HAART) course. We recorded the PDIA4 levels and HIV viral load before and during the treatment course (at 6 months and 12 months). PLWH responded well to HAART and showed a significantly reduced viral load after 6 months of treatment (Figure 3a). Notably, the PDIA4 levels also showed a decreasing trend during the HAART course (Figure 3b). The change in the PDIA4 levels during the treatment course showed a significantly positive correlation with the viral logic response (r = 0.242, p = 0.029) (Figure 3c).
      Figure 3
      Figure 3Association between the PDIA4 levels and viral load HIV.
      In total, 27 people living with HIV were followed up for PDIA4 levels and viral load during the highly active antiretroviral therapy course. We recorded the PDIA4 levels and HIV viral load before treatment (a) and during the treatment course at 24 weeks and 48 weeks (b). The correlation between the change in PDIA4 expression and the viral logic response during the treatment course was analyzed (c); *p < 0.05.
      Abbreviation: PDIA4, protein disulfide isomerase family A, member 4.

      Discussion

      HIV infection is characterized by dysregulated production of pro-inflammatory cytokines and leads to persistent chronic inflammation. Our results showed that the plasma PDIA4 levels in PLWH were significantly higher than those in the healthy controls. The PDIA4 levels reduced significantly after cART and correlated with a decreasing trend in the HIV viral load. Moreover, PDIA4 levels positively correlated with the TNF-α levels.
      PDIA4, comprising 645 amino acids, is upregulated in tumor cell lines and human cancer tissue (
      • Li H
      • Liu Q
      • Xiao K
      • He Z
      • Wu C
      • Sun J
      • et al.
      PDIA4 correlates with poor prognosis and is a potential biomarker in glioma.
      ,
      • Wang Z
      • Zhang H.
      • Cheng Q.
      PDIA4: the basic characteristics, functions and its potential connection with cancer.
      ). Furthermore, the PDIA4 level is elevated in metabolic syndrome, and it is involved in the pathogenesis of diabetes. (
      • Chien CY
      • Hung YJ
      • Shieh YS
      • Hsieh CH
      • Lu CH
      • Lin FH
      • et al.
      A novel potential biomarker for metabolic syndrome in Chinese adults: circulating protein disulfide isomerase family A, member 4.
      ;
      • Kau TF
      • Hsu SW
      • Huang SH
      • Chang CLT
      • Feng CS
      • Huang MG
      • et al.
      Pdia4 regulates β-cell pathogenesis in diabetes: molecular mechanism and targeted therapy.
      ). Pickup et al. reported the role of inflammation and activated innate immunity in the pathogenesis of type 2 diabetes (
      • Pickup JC.
      Inflammation and activated innate immunity in the pathogenesis of type 2 diabetes.
      ). Persistent systemic inflammation remains a characteristic feature despite continuous cART in PLWH (
      • Hunt PW
      • Lee SA
      • Siedner MJ.
      Immunologic biomarkers, morbidity, and mortality in treated HIV infection.
      ;
      • Legarth RA
      • Ahlström MG
      • Kronborg G
      • Larsen CS
      • Pedersen C
      • Pedersen G
      • et al.
      Long-term mortality in HIV-infected individuals 50 years or older: a nationwide, population-based cohort study.
      ). We have demonstrated that the plasma PDIA4 levels in PLWH were significantly higher than those in the healthy controls, implying a higher inflammatory state exists in the former than in the latter, and a similar trend is observed in diabetes patients.
      The current HARRT regimen comprises different regimens and may have various effects on the patient's inflammatory status (
      • Hattab S
      • Guihot A
      • Guiguet M
      • Fourati S
      • Carcelain G
      • Caby F
      • et al.
      Comparative impact of antiretroviral drugs on markers of inflammation and immune activation during the first two years of effective therapy for HIV-1 infection: an observational study.
      ;
      • Massanella M
      • Ouchi D
      • Marfil S
      • Llibre JM
      • Puertas MC
      • Buzón MJ
      • et al.
      Different plasma markers of inflammation are influenced by immune recovery and cART composition or intensification in treated HIV infected individuals.
      ). Our results revealed that the PDIA4 level was the highest in PLWH receiving INSTIs, followed by those receiving nNRTIs and those receiving PIs. The possible explanation is that INSTI-treated individuals have a higher chance of weight gain (
      • Sax PE
      • Erlandson KM
      • Lake JE
      • Mccomsey GA
      • Orkin C
      • Esser S
      • et al.
      Weight gain following initiation of antiretroviral therapy: risk factors in randomized comparative clinical trials.
      ). A recent study showed that the expression of PDIA4 in the adipose tissue was upregulated in obese participants when compared with lean participants (
      • Su SC
      • Hung YJ
      • Lin FH
      • Hsieh CH
      • Lu CH
      • Chien CY
      • et al.
      Circulating protein disulfide isomerase family member 4 is associated with type 2 diabetes mellitus, insulin sensitivity, and obesity.
      ). Therefore, PLWH receiving INSTI regimen present higher PDIA4 levels than those treated with the other regimens.
      HIV infection is accompanied by a robust inflammatory cytokine response, and several studies have linked surrogate markers to the clinical outcomes (
      • Hunt PW.
      HIV and inflammation: mechanisms and consequences.
      ;
      • Neuhaus J
      • Jacobs DR
      • Baker JV
      • Calmy A
      • Duprez D
      • La Rosa AL
      • et al.
      Markers of inflammation, coagulation, and renal function are elevated in adults with HIV infection.
      ). Traditional soluble inflammatory markers, such as TNF-α and IL-6, can be biomarkers predicting the state of inflammation and treatment outcomes in PLWH. Ledwaba et al. found that elevated pre-ART levels of IL-6 are strongly associated with early mortality after commencing ART (
      • Ledwaba L
      • Tavel JA
      • Khabo P
      • Maja P
      • Qin J
      • Sangweni P
      • et al.
      Pre-ART levels of inflammation and coagulation markers are strong predictors of death in a South African cohort with advanced HIV disease.
      ). Baker et al. also found that higher IL-6 levels during entry in The Strategic Timing of AntiRetroviral Treatment trial were associated with an increased risk of AIDS events (
      • Baker JV
      • Sharma S
      • Grund B
      • Rupert A
      • Metcalf JA
      • Schechter M
      • et al.
      Systemic inflammation, coagulation, and clinical risk in the START trial.
      ). McComsey et al. showed that higher TNF-α levels are associated with increased risk of AIDS-related and non-AIDS-related clinical events after cART initiation (
      • McComsey GA
      • Kitch D
      • Sax PE
      • Tierney C
      • Jahed NC
      • Melbourne K
      • et al.
      Associations of inflammatory markers with AIDS and non-AIDS clinical events after initiation of antiretroviral therapy: AIDS clinical trials group A5224s, a substudy of ACTG A5202.
      ). In our study, age-adjusted Spearman partial correlation coefficients revealed that PDIA4 positively correlated with TNF-α and IL-6 (Table 4). Additionally, logistic regression analysis of the association between biochemical variables and PDIA4 levels showed that PDIA4 levels positively correlated with TNF-α (p < 0.001) and IL-6 (p = 0.021) (univariate analysis). Moreover, TNF-α showed a significant correlation with PDIA4 in the multivariable logistic analysis (Table 5). Our findings that PDIA4 levels positively correlated with the TNF-α and IL-6 levels imply that PDIA4 could be a potential surrogate marker of inflammation in PLWH.
      The cART effectively suppresses HIV viral replication after initiation. Several pro-inflammatory cytokines are elevated in PLWH and markedly decrease after treatment (
      • Hamlyn E
      • Stöhr W
      • Cooper DA
      • Fisher M
      • Tambussi G
      • Schechter M
      • et al.
      The effect of short-course antiretroviral therapy initiated in primary HIV-1 infection on interleukin-6 and D-dimer levels.
      ;
      • Roff SR
      • Noon-Song EN
      • Yamamoto JK.
      The significance of interferon-γ in HIV-1 pathogenesis, therapy, and prophylaxis.
      ). Our study showed a significant viral load decline at 6 months and 12 months during the treatment course (Table 3 and Figure 3a). Baker et al. found a reduction in the IL-6 levels after cART initiation (
      • Baker JV
      • Neuhaus J
      • Duprez D
      • Kuller LH
      • Tracy R
      • Belloso WH
      • et al.
      Changes in inflammatory and coagulation biomarkers: a randomized comparison of immediate versus deferred antiretroviral therapy in patients with HIV infection.
      ). We also demonstrated that PDIA4 levels markedly decreased at 6 months and 12 months during the treatment course (Figure 3b). Osuji et al. found that pro-inflammatory cytokines, TNF-α, and IL-6 significantly decreased in PLWH at 24 weeks and 48 weeks of cART treatment (
      • Osuji FN
      • Onyenekwe CC
      • Ahaneku JE
      • Ukibe NR.
      The effects of highly active antiretroviral therapy on the serum levels of pro-inflammatory and anti-inflammatory cytokines in HIV infected subjects.
      ). Furthermore, they found a significant positive correlation between viral load and the IL-6 and TNF-α levels. Our study demonstrated a significant positive correlation between viral load and PDIA4 levels, implying that PDIA4 could be a good surrogate inflammatory marker in PLWH.
      Our study has several limitations. First, as this was a cross-sectional study, it was difficult to assess the causality relationship of PDIA4 in PLWH. Second, only 27 PLWH cART-naïve patients were recruited in the observation cohort, which is a relatively small sample size; therefore, future studies with a larger population size are warranted. Third, biological PDIA4 pathways are complex and interconnected; hence, we were unable to interpret the observational data in a comprehensive manner.
      Our study demonstrates a significant association between PDIA4 levels and inflammation in PLWH. Moreover, PDIA4 levels are associated with HIV viral load and TNF-α and IL-6 levels. The plasma PDIA4 level may be considered a potential surrogate marker of inflammation in PLWH.

      Funding

      This study was supported by grants from the Ministry of Science and Technology, Taiwan (MOST106-2314-B-016-026-MY3, MOST111-2314-B-016-033), National Defense Medical Center (MND-MAB-110-013), and Tri-Service General Hospital (TSGH-C108-141, TSGH-E-109238, TSGH-E-110206).

      Ethical approval

      This study was approved by the Institutional Review Board of Tri-Service General Hospital (No. 2-105-05-027).

      Author contributions

      All authors made a significant contribution to the work reported in terms of conception, study design, execution, acquisition of data, analysis and interpretation, or areas all of these; took part in drafting, revising, or critically reviewing the article; gave final approval of the version to be published; have agreed on the journal to which the article has been submitted; and agree to be accountable for all aspects of the work.

      Declaration of competing interests

      The authors have no competing interests to declare.

      Acknowledgment

      We thank all the patients who participated in this study.

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