FC 099DECLINING PERITONEAL HOST DEFENCES REVEALED BY EX-VIVO CYTOKINE RELEASE ASSAY OF PERITONEAL DIALYSIS EFFLUENT CELLS

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
Rebecca Herzog ◽  
Lisa Daniel-Fischer ◽  
Isabel Sobieszek ◽  
Christoph Aufricht ◽  
Klaus Kratochwill
Keyword(s):  
Ex Vivo ◽  
Significant Proportion ◽  
Host Defence ◽  
Clinical Staging ◽  
Continuous Exposure ◽  
Immune Competence ◽  
Cytokine Release ◽  
Acute Peritonitis ◽  
Tnf Α ◽  
Release Assay

Abstract Background and Aims Infectious complications occur in a significant proportion of PD patients, limiting long-term applicability. Reduced peritoneal immune-competence, caused by the continuous exposure to PD-fluids, has been described as a therapy-related pathomechanisms, prompting the need for a tool to assess the functional peritoneal immune status. We established an ex-vivo stimulation assay to test host defence mechanisms in only 9ml of PD-effluent. The aim of this study was to analyse basal inflammation and immune-competence in the general PD population at routine conditions to evaluate the assay as surrogate parameter of immune competence and linking it to PD vintage and clinical outcome parameters. Method 147 of 284 (51.8%) adult and paediatric PD patients treated between April 2013 and September 2020 at the local Department of Nephrology were included in the analysis. The study was approved by the local ethics committee and was conducted in accordance with the Declaration of Helsinki. Patients were exclusively treated with neutral pH/multi-chamber PD fluids during the glucose dwells. The majority of the 558 included PD-effluent samples were obtained during standard 4-hours peritoneal equilibration tests (PET) with 3.86% glucose containing PDF. Samples from the pre-PET dwell and at PET time points 1-hour and 4-hours were collected and immediately processed. Additional effluent samples were obtained during unscheduled hospitalization and in the event of an acute peritonitis. Effluent samples were collected directly from the drainage bags into standard 9 ml additive-free sample tubes. For ex-vivo stimulation, 100 ng/ml toll-like receptor (TLR) 4 agonist LPS and TLR2 agonist Pam3Cys were added to the effluent in the 9 ml collection tubes in duplicates and incubated at 37°C for 24h. Unstimulated samples kept in parallel were used as controls. IL-6 and TNF-α concentrations were measured with ELISA in the supernatants. Results Ex-vivo stimulation of peritoneal cells significantly increased the IL-6 and TNF-α release compared to unstimulated controls and resulted in a dwell-time dependent increase, with a significant lower cytokine released at the 1h PET time point. To assess local inflammation IL-6 levels of crude effluent were determined. IL-6 concentrations remained stable over time on PD. Interestingly, we were able to show higher IL-6 levels in CAPD patients in comparison to APD. As chronic exposure to PD-fluids has been shown to dampen the peritoneal immune competence, consecutive peritoneal effluent bags, obtained from patients were analysed. In this subcohort of 183 4h-PET effluents we found a decline in cytokine secretion with time on PD (IL-6 r=-0.27, p=0.00015, TNFa r=-0.25, p=0.00071). In a subgroup the ex-vivo cytokine release of effluent samples from patients with an acute peritonitis was assessed. IL-6 levels of acute peritonitis effluent samples did not differ from the stimulated IL-6 levels of effluent samples without acute peritonitis (2.45 pg/mL vs 2.31 pg/mL, p=0.85, t-test) suggesting that the assay seemingly represents the in-vivo host-defence cytokine release accurately. Conclusion The study provides evidence of a correlation of declining local host defence and duration of PD-therapy. It supports the hypothesis of PD duration-dependent progressive impairment of the ability of the peritoneal immune cells to secrete cytokines in response to a pathogenic stimulus and thereby dampening the global peritoneal immuno-competence. This suggests the utility of this clinically feasible ex-vivo induced cytokine-release assay in peritoneal effluent as a surrogate of the functional peritoneal immune competence. Future analyses need to evaluate the assay as a tool to predict common clinical outcomes and define reference values to facilitate stratification of patient populations, clinical staging and to guide novel therapeutic interventions.

scholarly journals P1176DECLINING PERITONEAL HOST DEFENCES REVEALED BY EX-VIVO CYTOKINE RELEASE ASSAY OF PERITONEAL DIALYSIS EFFLUENT CELLS

2020 ◽  
Vol 35 (Supplement_3) ◽  
Author(s):  
Rebecca Herzog ◽  
Andreas Vychytil ◽  
Christoph Aufricht ◽  
Klaus Kratochwill
Keyword(s):  
Peritoneal Dialysis ◽  
Ex Vivo ◽  
Infectious Complications ◽  
Host Defence ◽  
Clinical Staging ◽  
Continuous Exposure ◽  
Immune Competence ◽  
Cytokine Release ◽  
Outcome Parameters ◽  
Release Assay

Abstract Background and Aims Infectious complications occur in a significant proportion of patients treated with peritoneal dialysis (PD), limiting its long-term applicability. Reduced peritoneal immune-competence, caused by the continuous exposure to PD-fluids, has been described as a therapy-related pathomechanisms, prompting the need for a tool to assess the functional peritoneal immune status. We established an ex-vivo stimulation assay to test host defence mechanisms in only 9ml of crude PD-effluent. The assay was used in two randomized clinical trials investigating immune-modulatory effects of a novel additive (alanyl-glutamine) to PD-fluid. Here we aimed to evaluate the assay as a method to assess peritoneal immune-competence in the general PD-population and linking it to PD vintage and outcome parameters. Method 462 PD-effluent samples, originating from 148 adult and paediatric PD patients, were stimulated ex-vivo (24h, 37°C) with toll-like receptor (TLR)-agonists lipopolysaccharide (LPS) and Pam3Cys. Unstimulated samples of each effluent were used as controls. Interleukin 6 (IL-6) and tumor necrosis factor α (TNF-α) concentrations and the predictive value of the assay for clinical outcome parameters were analyzed using Wilcoxon matched-pairs signed rank test and a mixed model logistic regression analysis. Results Ex-vivo stimulation of peritoneal cells resulted in a dwell-time dependent increase of cytokine release, with a significant lower cytokine released at the 1h PET time point (normalized to unstimulated controls). Ratios between stimulated PDE-samples and unstimulated controls, accounting for a potential increase in basal inflammation, significantly declined with PD therapy duration. Correlation of the cytokine levels with the predefined clinical parameters revealed predictive potential of the assay for the occurrence of infectious complications, as well as of ultrafiltration and technique failure. Conclusion In summary, the current study provides evidence of a correlation of declining local host defence and duration of PD-therapy. These data support the hypothesis of PD-duration dependent progressive impairment of the ability of the peritoneal immune cells to secrete cytokines in response to a pathogenic stimulus and thereby dampening the global peritoneal immuno-competence. The results suggest the utility of this clinically feasible ex-vivo induced cytokine-release assay in crude peritoneal effluent as a surrogate of the functional peritoneal immune competence. Future analyses will have to evaluate the assay as a tool to predict common clinical outcomes and define reference values to facilitate stratification of patient populations, clinical staging and to guide novel therapeutic interventions.

scholarly journals Organ Injury and Cytokine Release Caused by Peptidoglycan Are Dependent on the Structural Integrity of the Glycan Chain

2004 ◽  
Vol 72 (3) ◽  
pp. 1311-1317 ◽  
Author(s):  
Anders E. Myhre ◽  
Jon Fredrik Stuestøl ◽  
Maria K. Dahle ◽  
Gunhild Øverland ◽  
Christoph Thiemermann ◽  
...  
Keyword(s):  
Structural Integrity ◽  
Inflammatory Responses ◽  
Ex Vivo ◽  
Organ Injury ◽  
Enzyme Linked Immunosorbent Assay ◽  
Cytokine Release ◽  
Ex Vivo Model ◽  
Glycan Chain ◽  
Tnf Α ◽  
Glutamyl Transferase

ABSTRACT Several studies have implicated a role of peptidoglycan (PepG) as a pathogenicity factor in sepsis and organ injury, in part by initiating the release of inflammatory mediators. We wanted to elucidate the structural requirements of PepG to trigger inflammatory responses and organ injury. Injection of native PepG into anesthetized rats caused moderate but significant increases in the levels of alanine aminotransferase, aspartate aminotransferase, γ-glutamyl transferase, and bilirubin (markers of hepatic injury and/or dysfunction) and creatinine and urea (markers of renal dysfunction) in serum, whereas PepG pretreated with muramidase to digest the glycan backbone failed to do this. In an ex vivo model of human blood, PepG containing different amino acids induced similar levels of the cytokines tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6), IL-8, and IL-10, as determined by plasma analyses (enzyme-linked immunosorbent assay). Hydrolysis of the Staphylococcus aureus cross-bridge with lysostaphin resulted in moderately reduced release of TNF-α, IL-6, IL-8, and IL-10, whereas muramidase digestion nearly abolished the ability to induce cytokine release and IL-6 mRNA accumulation in CD14+ monocytes compared to intact PepG. However, additional experiments showed that muramidase-treated PepG synergized with lipopolysaccharide to induce TNF-α and IL-10 release in whole blood, despite its lack of inflammatory activity when administered alone. Based on these studies, we hypothesize that the structural integrity of the glycan chain of the PepG molecule is very important for the pathogenic effects of PepG. The amino acid composition of PepG, however, does not seem to be essential for the inflammatory properties of the molecule.

scholarly journals Evaluation of a Whole-Blood Cytokine Release Assay for Use in Measuring Endotoxin Activity of Group B Neisseria meningitidis Vaccines Made from Lipid A Acylation Mutants

2009 ◽  
Vol 17 (1) ◽  
pp. 98-107 ◽  
Author(s):  
Mark B. Stoddard ◽  
Valerian Pinto ◽  
Paul B. Keiser ◽  
Wendell Zollinger
Keyword(s):  
Neisseria Meningitidis ◽  
Whole Blood ◽  
Lipid A ◽  
Mononuclear Cells ◽  
Membrane Vesicle ◽  
Cytokine Release ◽  
Tnf Α ◽  
Release Assay ◽  
Group B ◽  
Lal Assay

ABSTRACT Bacterial endotoxin interacts with the human immune system via complex immunological pathways. The evaluation of endotoxicity is important in the development of safe vaccines and immunomodulatory therapeutics. The Limulus amebocyte lysate (LAL) assay is generally accepted by the FDA for use for the quantification of lipopolysaccharide (LPS), while the rabbit pyrogen test (RPT) is used to estimate pyrogenicity during early development and production. Other in vitro assays, such as cytokine release assays with human whole blood (WB) or peripheral blood mononuclear cells (PBMCs), have also been used and may better estimate the human immunological response to products containing novel LPS molecules. In this study, WB and PBMC interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-α) release assays were used to estimate the endotoxic activities of purified LPS and native outer membrane vesicle (NOMV) vaccines derived from wild-type (hexa-acylated lipid A) and genetically detoxified (penta- and tetra-acylated lipid A) group B Neisseria meningitidis. A method for quantification of the differences in endotoxicity observed in the WB and PBMC assays is elucidated. The LAL assay was shown to be relatively insensitive to lipid A variations, and the RPT was less sensitive than the cytokine release assay with WB. The IL-6 and TNF-α assays with WB but not the assays with PBMCs distinguished between vaccines containing LPS from penta- and tetra-acylated strains. The high degree of sensitivity of the WB system to LPS variations and the presumed relevance of the use of human tissues to predict toxicity in humans suggest that this assay may be particularly well suited for the safety evaluation of vaccines and therapeutics containing acylation variants of LPS.

Generation of TNFα and Interleukin-6 by Peritoneal Macrophages after Overnight Dwells with Bicarbonate- or Lactate-Buffered Dialysis Fluid

2002 ◽  
Vol 22 (6) ◽  
pp. 663-669 ◽  
Author(s):  
Tomasz Liberek ◽  
Monika Lichodziejewska–Niemierko ◽  
Wanda Knopinska–Posluszny ◽  
Thomas P. Schaub ◽  
Judith Kirchgessner ◽  
...  
Keyword(s):  
Ex Vivo ◽  
Peritoneal Macrophages ◽  
Cytokine Release ◽  
Dialysis Fluid ◽  
Tnf Α ◽  
Peritoneal Dialysis Fluid ◽  
Initial Ph ◽  
Elisa Technique

Objective In order to evaluate the biocompatibility profile of a newly designed peritoneal dialysis fluid (PDF), we evaluated peritoneal leukocyte (PMΦ) cytokine release following overnight in vivo dwells using standard, lactate-buffered, single-chamber bag PDF (Lac-PDF) and purely bicarbonate-buffered, double-chamber bag PDF containing 34 (Bic-PDF) or 39 (Bic Hi-PDF) mmol/L bicarbonate. Design A randomized, open, crossover clinical trial with single weekly test dwells was performed in stable, long-term continuous ambulatory PD patients ( n = 8). During 8-hour overnight dwells, PMΦ were exposed to different PDF containing 1.5% glucose. After drainage, peritoneal cells were isolated and incubated with RPMI 1640 medium for 2 or 3 hours, with and without stimulation by lipopolysaccharide (LPS). Ex vivo release of tumor necrosis factor (TNF)-α and interleukin (IL)-6 was measured by specific ELISA technique. Results After pre-exposure to Lac-PDF, PMΦ generated 242 ± 279 pg TNFα/106 cells and 157 ± 105 pg IL-6/106 cells. When pre-exposed to Bic-PDF and Bic Hi-PDF, TNFα and IL-6 production of PMΦ was not significantly different from Lac-PDF. After LPS stimulation (100 ng/mL), PMΦ secretion of TNFα and IL-6 pre-exposed to three PDF revealed no significant differences between groups: TNFα was 2864 ± 1216, 2910 ± 1202, and 3291 ± 558 pg/106 cells after overnight dwells with Lac-PDF, Bic-PDF, and Bic Hi-PDF, respectively. Comparably, LPS-stimulated (100 pg/mL) PMΦ showed IL-6 secretion of 891 ± 335, 1380 ± 1149, and 1442 ± 966 pg/106 cells for Lac-PDF, Bic-PDF, and Bic Hi-PDF. Conclusion After long-term overnight dwells, initial pH, the different buffers, and varying glucose degradation product levels of PDF do not strongly affect PMΦ function with respect to cytokine release. The lack of significant differences between fluids may result from the complete dialysate equilibration achieved during the overnight intraperitoneal dwell.

scholarly journals No Inhibition of Anti-Viral and Anti-Leukemia Effects By Extracorporeal Photopheresis Therapy

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 3399-3399
Author(s):  
Ming NI ◽  
Lei Wang ◽  
Angela Hückelhoven ◽  
Leopold Sellner ◽  
Brigitte Neuber ◽  
...  
Keyword(s):  
T Cells ◽  
Nk Cells ◽  
Cd8 T Cells ◽  
Research Funding ◽  
Clinical Staging ◽  
Cytokine Release ◽  
Release Assay ◽  
Ifn Γ ◽  
Support Research

Abstract Introduction: Graft-versus-host disease (GvHD), a severe complication of allogeneic hematopoietic stem cell transplantation (allo-HSCT), results in post-transplant morbidity and mortality. Steroids with strong immunosuppressive and anti-inflammatory effects remain the gold standard for initial management of GvHD. However, high doses of steroids are usually accompanied by an increased risk of infections and secondary malignancies. ECP, due to its good clinical response, is recommended as a second-line treatment for GvHD. Notably, no clinical data showed that ECP therapy results in relapse and infection. However, the mechanism of action behind that is barely known. Therefore, we conducted this study to figure out how ECP preserves the anti-viral and anti-leukemia effects. Materials and Methods: 34 patients with steroid-refractory/resistant aGvHD ≥ °II and moderate to severe cGvHD received ECP therapy at the University Hospitals Heidelberg, Greifswald and Tel Hashomer. ECP therapy was performed according to the current European guidelines. For clinical staging of aGvHD under ECP treatment patients were evaluated according to Glucksberg criteria, for quality of life according to Karnofsky and for clinical staging of cGvHD according to NIH criteria. Phenotypical analysis of different cellular subsets was evaluated by multicolor flow cytometry. The quantity and quality of virus-specific CD8+ T cells were evaluated by Tetramer staining and IFN-γ-ELISPOT. NK activity in terms of killing function and cytokine release function was monitored by chromium-51 release assay and intracellular cytokine staining. Results: ECP seems to be favorable in the treatment of GvHD. Clinically, an overall response of 75% for aGvHD and 78% for cGvHD patients was achieved. A steroid-sparing effect in addition to the resolution of GvHD manifestations was observed in responders. Our patients showed no increased susceptibility to infections. On the cellular level, the frequency of cytotoxic CD8+ T cells, the most important mediators of GvL activity, as well as CD4+CD8+ T cells, γδ T cells, NKT cells and CD56dimCD57+NKG2C+ NK cells as other well-established protective cell subsets remained constant under ECP therapy. Moreover, neither frequency nor IFN-γ release of CMV specific CD8+ T cells was hampered by ECP. 51Cr-release assay revealed stable functional cytotoxicity of NK cells. Additionally, ECP therapy did not affect the capacity of cytokine release by NK cells, including quality, quantity and multifunctional release. Furthermore, no significant influence of ECP therapy on antigen recall function of NK cells, CD4+ and CD8+ T cells could be observed. Conclusion: ECP therapy represents an attractive strategy to treat GvHD. Moreover, our results reflect that ECP therapy preserves immunity against infections as well as the GvL effect via maintaining the quality and quantity of effector cells. Disclosures Hilgendorf: Novartis: Other: Travel support, Research Funding; Medac: Other: Travel support, Research Funding.

scholarly journals Modulation of Cytokine Release in Ex Vivo-Stimulated Blood from Borreliosis Patients

2001 ◽  
Vol 69 (2) ◽  
pp. 687-694 ◽  
Author(s):  
Isabel Diterich ◽  
Luc Härter ◽  
Dieter Hassler ◽  
Albrecht Wendel ◽  
Thomas Hartung
Keyword(s):  
Ex Vivo ◽  
Interleukin 10 ◽  
Cytokine Response ◽  
Healthy Controls ◽  
Cytokine Release ◽  
Necrosis Factor Alpha ◽  
Cell Counts ◽  
Tnf Α ◽  
Ifn Γ

ABSTRACT In lipopolysaccharide-stimulated blood from 71 late-stage borreliosis patients, the ex vivo cytokine release capacity of tumor necrosis factor alpha (TNF-α) and gamma interferon (IFN-γ) was reduced to 28% ± 5% and to 31% ± 5% (P ≤ 0.001), respectively, compared to that of 24 healthy controls. White blood cell counts were normal in both groups. To investigate direct interactions between the pathogen and the immune cells, blood from healthy controls was exposed in vitro to live or heat-killedBorrelia or to Borrelia lysate. Compared to the pattern induced by bacterial endotoxins, a reduced release of TNF-α and IFN-γ and an enhanced secretion of interleukin-10 and granulocyte colony-stimulating factor was found. In blood from 10 borreliosis patients stimulated with Borrelia lysate, TNF-α formation was decreased to 31% ± 14% and IFN-γ formation was decreased to 8% ± 3% (P ≤ 0.001) compared to the cytokine response of blood from healthy controls (n = 24). We propose to consider anti-inflammatory changes in the blood cytokine response capacity elicited by Borrelia as a condition that might favor the persistence of the spirochete.

Elevated level of circulatory sTLT1 induces inflammation through SYK/MEK/ERK signalling in coronary artery disease

2019 ◽  
Vol 133 (22) ◽  
pp. 2283-2299
Author(s):  
Apabrita Ayan Das ◽  
Devasmita Chakravarty ◽  
Debmalya Bhunia ◽  
Surajit Ghosh ◽  
Prakash C. Mandal ◽  
...  
Keyword(s):  
Risk Factors ◽  
Coronary Artery Disease ◽  
Coronary Artery ◽  
Chronic Inflammation ◽  
Ex Vivo ◽  
Human Subjects ◽  
Critical Role ◽  
Atherosclerotic Cardiovascular Disease ◽  
Tnf Α ◽  
Artery Disease

Abstract The role of inflammation in all phases of atherosclerotic process is well established and soluble TREM-like transcript 1 (sTLT1) is reported to be associated with chronic inflammation. Yet, no information is available about the involvement of sTLT1 in atherosclerotic cardiovascular disease. Present study was undertaken to determine the pathophysiological significance of sTLT1 in atherosclerosis by employing an observational study on human subjects (n=117) followed by experiments in human macrophages and atherosclerotic apolipoprotein E (apoE)−/− mice. Plasma level of sTLT1 was found to be significantly (P<0.05) higher in clinical (2342 ± 184 pg/ml) and subclinical cases (1773 ± 118 pg/ml) than healthy controls (461 ± 57 pg/ml). Moreover, statistical analyses further indicated that sTLT1 was not only associated with common risk factors for Coronary Artery Disease (CAD) in both clinical and subclinical groups but also strongly correlated with disease severity. Ex vivo studies on macrophages showed that sTLT1 interacts with Fcɣ receptor I (FcɣRI) to activate spleen tyrosine kinase (SYK)-mediated downstream MAP kinase signalling cascade to activate nuclear factor-κ B (NF-kB). Activation of NF-kB induces secretion of tumour necrosis factor-α (TNF-α) from macrophage cells that plays pivotal role in governing the persistence of chronic inflammation. Atherosclerotic apoE−/− mice also showed high levels of sTLT1 and TNF-α in nearly occluded aortic stage indicating the contribution of sTLT1 in inflammation. Our results clearly demonstrate that sTLT1 is clinically related to the risk factors of CAD. We also showed that binding of sTLT1 with macrophage membrane receptor, FcɣR1 initiates inflammatory signals in macrophages suggesting its critical role in thrombus development and atherosclerosis.

Pentoxifylline and Oxypurinol: Potential Drugs to Prevent the “Cytokine Release (Storm) Syndrome” Caused by SARS-CoV-2?

2020 ◽  
Vol 26 (35) ◽  
pp. 4515-4521
Author(s):  
Francisco J. López-Iranzo ◽  
Ana M. López-Rodas ◽  
Luis Franco ◽  
Gerardo López-Rodas
Keyword(s):  
Lung Parenchyma ◽  
Interstitial Tissue ◽  
Cytokine Release ◽  
Phase I Clinical Trials ◽  
Infected People ◽  
Tnf Α ◽  
Transient Loss ◽  
Anti Inflammatory ◽  
And Control ◽  
Inflammatory Action

Background: COVID-19, caused by SARS-CoV-2, is a potentially lethal, rapidly-expanding pandemic and many efforts are being carried out worldwide to understand and control the disease. COVID-19 patients may display a cytokine release syndrome, which causes severe lung inflammation, leading, in many instances, to death. Objective: This paper is intended to explore the possibilities of controlling the COVID-19-associated hyperinflammation by using licensed drugs with anti-inflammatory effects. Hypothesis: We have previously described that pentoxifylline alone, or in combination with oxypurinol, reduces the systemic inflammation caused by experimentally-induced pancreatitis in rats. Pentoxifylline is an inhibitor of TNF-α production and oxypurinol inhibits xanthine oxidase. TNF-α, in turn, activates other inflammatory genes such as Nos2, Icam or IL-6, which regulate migration and infiltration of neutrophils into the pulmonary interstitial tissue, causing injury to the lung parenchyma. In acute pancreatitis, the anti-inflammatory action of pentoxifylline seems to be mediated by the prevention of the rapid and presumably transient loss of PP2A activity. This may also occur in the hyperinflammatory -cytokine releasing phase- of SARS-CoV-2 infection. Therefore, it may be hypothesized that early treatment of COVID-19 patients with pentoxifylline, alone or in combination with oxypurinol, would prevent the potentially lethal acute respiratory distress syndrome. Conclusion: Pentoxifylline and oxypurinol are licensed drugs used for diseases other than COVID-19 and, therefore, phase I clinical trials would not be necessary for the administration to SARS-CoV-2- infected people. It would be worth investigating their potential effects against the hyperinflammatory response to SARS-CoV-2 infection.

TNF-α Suppresses Autophagic Flux in Acinar Cells in IgG4-Related Sialadenitis

2019 ◽  
Vol 98 (12) ◽  
pp. 1386-1396 ◽  
Author(s):  
X. Hong ◽  
S.N. Min ◽  
Y.Y. Zhang ◽  
Y.T. Lin ◽  
F. Wang ◽  
...  
Keyword(s):  
Acinar Cell ◽  
Cell Injury ◽  
Ex Vivo ◽  
Secretory Granules ◽  
Acinar Cells ◽  
Autophagic Flux ◽  
C6 Cells ◽  
Tnf Α ◽  
Α Level

IgG4-related sialadenitis (IgG4-RS) is a newly recognized immune-mediated systemic fibroinflammatory disease that affects salivary glands and leads to hyposalivation. Tumor necrosis factor–α (TNF-α) is a critical proinflammatory cytokine involved in several salivary gland disorders, but its role and mechanism regarding acinar cell injury in IgG4-RS are unknown. Here, we found that TNF-α level was significantly increased in serum and submandibular gland (SMG) of patients and that serum TNF-α level was negatively correlated with saliva flow rate. Ultrastructural observations of IgG4-RS SMGs revealed accumulation of large autophagic vacuoles, as well as dense fibrous bundles, decreased secretory granules, widened intercellular spaces, swollen mitochondria, and expanded endoplasmic reticulum. Expression levels of LC3 and p62 were both increased in patients’ SMGs. TNF-α treatment led to elevated levels of LC3II and p62 in both SMG-C6 cells and cultured human SMG tissues but did not further increase their levels when combined with bafilomycin A1 treatment. Moreover, transfection of Ad-mCherry-GFP-LC3B in SMG-C6 cells confirmed the suppression of autophagic flux after TNF-α treatment. Immunofluorescence imaging revealed that costaining of LC3 and the lysosomal marker LAMP2 was significantly decreased in patients, TNF-α–treated SMG-C6 cells, and cultured human SMGs, indicating a reduction in autophagosome-lysosome fusion. Furthermore, the ratio of pro/mature cathepsin D was elevated in vivo, ex vivo, and in vitro. TNF-α also appeared to induce abnormal acidification of lysosomes in acinar cells, as assessed by lysosomal pH and LysoTracker DND-26 fluorescence intensity. In addition, TNF-α treatment induced transcription factor EB (TFEB) redistribution in SMG-C6 cells, which was consistent with the changes observed in IgG4-RS patients. TNF-α increased the phosphorylation of extracellular signal–regulated kinase (ERK) 1/2, and inhibition of ERK1/2 by U0126 reversed TNF-α–induced TFEB redistribution, lysosomal dysfunction, and autophagic flux suppression. These findings suggest that TNF-α is a key cytokine related to acinar cell injury in IgG4-RS through ERK1/2-mediated autophagic flux suppression.

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