Manifestation of lipopolysaccharide-induced tolerance in neuro-glial primary cultures of the rat afferent somatosensory system

Abstract Objective Bacterial lipopolysaccharide (LPS) may contribute to the manifestation of inflammatory pain within structures of the afferent somatosensory system. LPS can induce a state of refractoriness to its own effects termed LPS tolerance. We employed primary neuro-glial cultures from rat dorsal root ganglia (DRG) and the superficial dorsal horn (SDH) of the spinal cord, mainly including the substantia gelatinosa to establish and characterize a model of LPS tolerance within these structures. Methods Tolerance was induced by pre-treatment of both cultures with 1 µg/ml LPS for 18 h, followed by a short-term stimulation with a higher LPS dose (10 µg/ml for 2 h). Cultures treated with solvent were used as controls. Cells from DRG or SDH were investigated by means of RT-PCR (expression of inflammatory genes) and immunocytochemistry (translocation of inflammatory transcription factors into nuclei of cells from both cultures). Supernatants from both cultures were assayed for tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) by highly sensitive bioassays. Results At the mRNA-level, pre-treatment with 1 µg/ml LPS caused reduced expression of TNF-α and enhanced IL-10/TNF-α expression ratios in both cultures upon subsequent stimulation with 10 µg/ml LPS, i.e. LPS tolerance. SDH cultures further showed reduced release of TNF-α into the supernatants and attenuated TNF-α immunoreactivity in microglial cells. In the state of LPS tolerance macrophages from DRG and microglial cells from SDH showed reduced LPS-induced nuclear translocation of the inflammatory transcription factors NFκB and NF-IL6. Nuclear immunoreactivity of the IL-6-activated transcription factor STAT3 was further reduced in neurons from DRG and astrocytes from SDH in LPS tolerant cultures. Conclusion A state of LPS tolerance can be induced in primary cultures from the afferent somatosensory system, which is characterized by a down-regulation of pro-inflammatory mediators. Thus, this model can be applied to study the effects of LPS tolerance at the cellular level, for example possible modifications of neuronal reactivity patterns upon inflammatory stimulation.

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Sensitization of primary cultures from rat dorsal root ganglia with lipopolysaccharide (LPS) requires a robust inflammatory response

Inflammation Research ◽

10.1007/s00011-021-01534-2 ◽

2021 ◽

Author(s):

Franz Nürnberger ◽

Stephan Leisengang ◽

Daniela Ott ◽

Jolanta Murgott ◽

Rüdiger Gerstberger ◽

...

Keyword(s):

Inflammatory Response ◽

Dorsal Root Ganglia ◽

Dorsal Root ◽

Primary Cultures ◽

Somatosensory System ◽

Low Doses ◽

Tnf Α ◽

Wide Range ◽

Ratio Imaging ◽

Pre Treatment

Abstract Objective We investigated whether it is possible to induce a state of “LPS-sensitization” in neurons of primary cultures from rat dorsal root ganglia by pre-treatment with ultra-low doses of LPS. Methods DRG primary cultures were pre-treated with low to ultra-low doses of LPS (0.001–0.1 µg/ml) for 18 h, followed by a short-term stimulation with a higher LPS-dose (10 µg/ml for 2 h). TNF-α in the supernatants was measured as a sensitive read out. Using the fura-2 340/380 nm ratio imaging technique, we further investigated the capsaicin-evoked Ca2+-signals in neurons from DRG, which were pre-treated with a wide range of LPS-doses. Results Release of TNF-α evoked by stimulation with 10 µg/ml LPS into the supernatant was not significantly modified by pre-exposure to low to ultra-low LPS-doses. Capsaicin-evoked Ca2+-signals were significantly enhanced by pre-treatment with LPS doses being above a certain threshold. Conclusion Ultra-low doses of LPS, which per se do not evoke a detectable inflammatory response, are not sufficient to sensitize neurons (Ca2+-responses) and glial elements (TNF-α-responses) of the primary afferent somatosensory system.

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Primary culture of the rat spinal dorsal horn: a tool to investigate the effects of inflammatory stimulation on the afferent somatosensory system

Pflügers Archiv - European Journal of Physiology ◽

10.1007/s00424-020-02478-y ◽

2020 ◽

Vol 472 (12) ◽

pp. 1769-1782 ◽

Cited By ~ 1

Author(s):

Stephan Leisengang ◽

Franz Nürnberger ◽

Daniela Ott ◽

Jolanta Murgott ◽

Rüdiger Gerstberger ◽

...

Keyword(s):

Spinal Cord ◽

Transcription Factors ◽

Substance P ◽

Primary Culture ◽

Primary Cultures ◽

Somatosensory System ◽

Microglial Cells ◽

Prostaglandin E ◽

Short Term ◽

Stimulation Of

AbstractOne maladaptive consequence of inflammatory stimulation of the afferent somatosensory system is the manifestation of inflammatory pain. We established and characterized a neuroglial primary culture of the rat superficial dorsal horn (SDH) of the spinal cord to test responses of this structure to neurochemical, somatosensory, or inflammatory stimulation. Primary cultures of the rat SDH consist of neurons (43%), oligodendrocytes (35%), astrocytes (13%), and microglial cells (9%). Neurons of the SDH responded to cooling (7%), heating (18%), glutamate (80%), substance P (43%), prostaglandin E2 (8%), and KCl (100%) with transient increases in the intracellular calcium [Ca2+]i. Short-term stimulation of SDH primary cultures with LPS (10 μg/ml, 2 h) caused increased expression of pro-inflammatory cytokines, inflammatory transcription factors, and inducible enzymes responsible for inflammatory prostaglandin E2 synthesis. At the protein level, increased concentrations of tumor necrosis factor-α (TNFα) and interleukin-6 (IL-6) were measured in the supernatants of LPS-stimulated SDH cultures and enhanced TNFα and IL-6 immunoreactivity was observed specifically in microglial cells. LPS-exposed microglial cells further showed increased nuclear immunoreactivity for the inflammatory transcription factors NFκB, NF-IL6, and pCREB, indicative of their activation. The short-term exposure to LPS further caused a reduction in the strength of substance P as opposed to glutamate-evoked Ca2+-signals in SDH neurons. However, long-term stimulation with a low dose of LPS (0.01 μg/ml, 24 h) resulted in a significant enhancement of glutamate-induced Ca2+ transients in SDH neurons, while substance P-evoked Ca2+ signals were not influenced. Our data suggest a critical role for microglial cells in the initiation of inflammatory processes within the SDH of the spinal cord, which are accompanied by a modulation of neuronal responses.

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Geranylgeraniol Inhibits Lipopolysaccharide-Induced Inflammation in Mouse-Derived MG6 Microglial Cells via NF-κB Signaling Modulation

International Journal of Molecular Sciences ◽

10.3390/ijms221910543 ◽

2021 ◽

Vol 22 (19) ◽

pp. 10543

Author(s):

Wahyu Dwi Saputra ◽

Hiroki Shono ◽

Yusuke Ohsaki ◽

Halima Sultana ◽

Michio Komai ◽

...

Keyword(s):

Nuclear Translocation ◽

Microglial Cells ◽

Side Chain ◽

Mrna Levels ◽

Inflammatory Reactions ◽

Inflammatory Conditions ◽

Tnf Α ◽

Related Proteins ◽

Microglial Inflammation ◽

Pro Inflammatory Cytokines

Persistent inflammatory reactions in microglial cells are strongly associated with neurodegenerative pathogenesis. Additionally, geranylgeraniol (GGOH), a plant-derived isoprenoid, has been found to improve inflammatory conditions in several animal models. It has also been observed that its chemical structure is similar to that of the side chain of menaquinone-4, which is a vitamin K2 sub-type that suppresses inflammation in mouse-derived microglial cells. In this study, we investigated whether GGOH has a similar anti-inflammatory effect in activated microglial cells. Particularly, mouse-derived MG6 cells pre-treated with GGOH were exposed to lipopolysaccharide (LPS). Thereafter, the mRNA levels of pro-inflammatory cytokines were determined via qRT-PCR, while protein expression levels, especially the expression of NF-κB signaling cascade-related proteins, were determined via Western blot analysis. The distribution of NF-κB p65 protein was also analyzed via fluorescence microscopy. Thus, it was observed that GGOH dose-dependently suppressed the LPS-induced increase in the mRNA levels of Il-1β, Tnf-α, Il-6, and Cox-2. Furthermore, GGOH inhibited the phosphorylation of TAK1, IKKα/β, and NF-κB p65 proteins as well as NF-κB nuclear translocation induced by LPS while maintaining IκBα expression. We showed that GGOH, similar to menaquinone-4, could alleviate LPS-induced microglial inflammation by targeting the NF-kB signaling pathway.

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1441LRP5 and PCSK9 in inflammatory cells immunomodulation

European Heart Journal ◽

10.1093/eurheartj/ehz748.0076 ◽

2019 ◽

Vol 40 (Supplement_1) ◽

Author(s):

M Borrell ◽

J Crespo ◽

L Badimon

Keyword(s):

Protein Expression ◽

Nuclear Translocation ◽

Primary Cultures ◽

Inflammatory Cells ◽

Blood Cholesterol ◽

Foam Cell Formation ◽

Cholesterol Levels ◽

Tnf Α ◽

Lipid Loading

Abstract Background Atherosclerosis, the leading cause of cardiovascular diseases, is driven by high blood cholesterol levels and chronic inflammation. The disruption of the hepatic interaction between Proprotein Convertase Subtilisin/Kexin 9 (PCSK9) and Low-Density Lipoprotein Receptor (LDLR) downregulates blood cholesterol levels and reduces cardiovascular events. Recent data suggest that other members of the LDLR superfamily may be targets of PCSK9. Purpose The aim of this work is to determine if LDLR-related protein 5 (LRP5) is a PCSK9 target, and to study the role of PCSK9 and LRP5 in foam cell formation and hence, in the mechanism of lipid accumulation and atherosclerotic plaque formation. Methods Intracellular protein and lipid localization, cholesteryl esters (CE) accumulation; quantification of structural and inflammatory proteins expression and immunoprecipitation analyses were performed in primary cultures of human inflammatory cells (monocytes and macrophages) silenced for LRP5 or PCSK9 and challenged with modified LDLs. Results We first show that LRP5 is needed for macrophage lipid uptake since LRP5-silenced macrophages have less intracellular CE accumulation. In LDL treated macrophages internalization of LRP5-bound LDL starts after 30 minutes of incubation and lasts up to 24hours. The SREBP-2 promoter is not involved in LRP5 regulation but it does regulate macrophage PCSK9 expression. Immunoprecipitation experiments show that LRP5 forms a complex with PCSK9 in lipid-loaded macrophages. Finally we studied the role of PCSK9 and LRP5 in the inflammatory response by TLR4/NFkB signaling pathway. We show decreased TLR4 protein expression levels and decreased nuclear translocation of NFκB in PCSK9 silenced-inflammatory cells after lipid loading indicating a downregulation of the proinflammatory pathway TLR4/NFκB. Increased gene expression is observed in TNF-α and IL1β after lipid-loading that is abolished in PCSK9-silenced macrophages. Furthermore release of the proinflammatory cytokines TNF-α and IL1β is decreased in PCSK9-silenced macrophages. LRP5 protein expression is increased in lipid-loaded macrophages independent of the presence or absence of PCSK9. Conclusions These results demonstrate that, in human macrophages, LRP5 is internalized with lipids. Furthermore, PCSK9 and LRP5 can form a complex in the cytoplasm of lipid-loaded macrophages opening the possibility that PCSK9 induces lysosomal LRP5 degradation in a similar manner than it does with LDLR. Finally we also show that PCSK9 gene interference decreases inflammation and supports a role for PCSK9 as an inflammatory mediator in atherosclerosis. Acknowledgement/Funding CN16/11/00411-LB; TERCEL RD16/0011/018-LB; FIS2016-02014 -MBP; SEC2015 to MBP

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Divergent regulation of 92-kDa gelatinase and TIMP-1 by HBECs in response to IL-1β and TNF-α

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.1997.273.4.l866 ◽

1997 ◽

Vol 273 (4) ◽

pp. L866-L874 ◽

Cited By ~ 19

Author(s):

P. M. Yao ◽

B. Maitre ◽

C. Delacourt ◽

J. M. Buhler ◽

A. Harf ◽

...

Keyword(s):

Mrna Level ◽

Primary Cultures ◽

Gelatin Zymography ◽

Tissue Inhibitor Of Metalloproteinase ◽

Type I ◽

Control Mechanisms ◽

Mrna Levels ◽

Gelatinase Activity ◽

Twofold Increase ◽

Tnf Α

In this study, we addressed the question of whether human bronchial epithelial cells (HBECs) contribute to the regulation of 92-kDa gelatinase activity by secreting tissue inhibitor of metalloproteinase (TIMP)-1. We investigated expression of 92-kDa gelatinase and TIMP-1 in response to lipopolysaccharide (LPS) and to the proinflammatory cytokines interleukin (IL)-1β and tumor necrosis factor (TNF)-α. Confluent HBECs from explants were cultured in plastic dishes coated with type I and III collagen. We demonstrated that TIMP-1 was expressed at both the protein and mRNA levels by primary cultures of HBECs. Gelatin zymography of HBEC-conditioned media showed that exposure of HBECs to LPS, IL-1β, or TNF-α induced a twofold increase in the latent form of 92-kDa gelatinase production, as well as its activation. Also, quantitative reverse transcriptase (RT)-polymerase chain reaction (PCR) demonstrated a twofold increase in the 92-kDa mRNA level in response to both cytokines. In contrast, TIMP-1 production evaluated by immunoblotting was unchanged in the presence of LPS and IL-1β and was clearly decreased in the presence of TNF-α. Quantitative RT-PCR demonstrated that TIMP-1 mRNA levels remained unchanged in response to LPS or IL-1β but decreased by 70% in the presence of TNF-α. All of these results strongly suggest that the control mechanisms regulating the expression of 92-kDa gelatinase and TIMP-1 by HBECs in response to inflammatory stimuli are divergent and result in an imbalance between 92-kDa gelatinase and TIMP-1 in favor of the metalloproteinase. Such an imbalance may contribute significantly to acute airway inflammation.

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Tumor necrosis factor-α inhibits renin gene expression

AJP Regulatory Integrative and Comparative Physiology ◽

10.1152/ajpregu.00142.2002 ◽

2002 ◽

Vol 283 (5) ◽

pp. R1046-R1051 ◽

Cited By ~ 37

Author(s):

Vladimir Todorov ◽

Markus Müller ◽

Frank Schweda ◽

Armin Kurtz

Keyword(s):

Gene Expression ◽

Knockout Mice ◽

Primary Cultures ◽

Cellular Level ◽

Renin Synthesis ◽

Tnf Α ◽

Renin Gene ◽

Renin Mrna ◽

Vascular Proliferation ◽

Factor Α

Renin, produced in renal juxtaglomerular (JG) cells, is a fundamental regulator of blood pressure. Accumulating evidence suggests that cytokines may directly influence renin production in the JG cells. TNF-α, which is one of the key mediators in immunity and inflammation, is known to participate in the control of vascular proliferation and contraction and hence in the pathogenesis of cardiovascular diseases. Thus TNF-α may exert its effects on the cardiovascular system through modulation of renal renin synthesis. Therefore we have tested the effect of TNF-α on renin transcription in As4.1 cells, which represent transformed mouse JG cells, and in native mouse JG cells in culture. Renin gene expression was also determined in mice lacking the gene for TNF-α (TNF-α knockout mice). TNF-α inhibited renin gene expression via an inhibition of the transcriptional activity, targeting the proximal 4.1 kb of the renin promoter in As4.1 cells. TNF-α also attenuated forskolin-stimulated renin gene expression in primary cultures of mouse JG cells. Mice lacking the TNF-α gene had almost threefold higher basal renal renin mRNA abundance relative to the control strain. The general physiological regulation of renin expression by salt was not disturbed in TNF-α knockout mice. Our data suggest that TNF-α inhibits renin gene transcription at the cellular level and thus may act as a modulator of renin synthesis in (physio)pathological situations.

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Long Noncoding RNA CIR Promotes Chondrocyte Extracellular Matrix Degradation in Osteoarthritis by Acting as a Sponge For Mir-27b

Cellular Physiology and Biochemistry ◽

10.1159/000480532 ◽

2017 ◽

Vol 43 (2) ◽

pp. 602-610 ◽

Cited By ~ 39

Author(s):

Yu-Fei Li ◽

Shu-Hua Li ◽

Yong Liu ◽

Ya-Tong Luo

Keyword(s):

Extracellular Matrix ◽

Noncoding Rna ◽

Mrna Level ◽

Cartilage Degradation ◽

Degenerative Joint Disease ◽

Cellular Level ◽

Joint Disease ◽

Small Interfering Rnas ◽

Tnf Α ◽

Regulatory Functions

Background/Aims: Osteoarthritis (OA) is a degenerative joint disease characterized by articular cartilage degradation. The degradation of the extracellular matrix (ECM) of chondrocyte is closely associated with the destruction of joints in OA patients. lncRNAs are non-coding segments of RNA that possess important regulatory functions at the cellular level and in a variety of pathophysiological processes. The present study was conducted to investigate whether lncRNA-CIR regulated the expression of MMP13 as a sponge of miR-27 in OA. Methods: Primary cultured chondrocytes were challenged by IL-1β and TNF-α to simulate OA conditions. qRT-PCR was performed to detect the miR-27, lncRNA-CIR, MMP13 mRNA expression levels. Western blot was applied to detect MMP13 protein expression. Soluble sGAG secretion/ formation was analysed by the dimethylmethylene blue (DMMB) assay. lncRNA-CIR overexpression or inhibition was performed using overexpression plasmid and small interfering RNAs (siRNAs), respectively. Results: lncRNA-CIR significantly up-regulated in OA patients, concomitantly down-regulated miR-27 and up-regulated MMP13. Bioinformatics analysis predicted miR-27 was the target of both lncRNA-CIR and MMP13. Overexpression of lncRNA-CIR significantly increased the expression of MMP13, while miR-27 remarkably suppressed the expression of MMP13, Accompanying with the increases of mRNA level, protein level and relative luciferase activity. Conclusion: The present findings indicated that lncRNA-CIR/miR-27/MMP13 axis involved in the degradation of the ECM of chondrocyte in OA.

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Menaquinone-4 Suppresses Lipopolysaccharide-Induced Inflammation in MG6 Mouse Microglia-Derived Cells by Inhibiting the NF-κB Signaling Pathway

International Journal of Molecular Sciences ◽

10.3390/ijms20092317 ◽

2019 ◽

Vol 20 (9) ◽

pp. 2317 ◽

Cited By ~ 1

Author(s):

Wahyu Dwi Saputra ◽

Nao Aoyama ◽

Michio Komai ◽

Hitoshi Shirakawa

Keyword(s):

Inflammatory Cytokines ◽

Nuclear Translocation ◽

Mrna Level ◽

Underlying Mechanism ◽

Inflammatory Reactions ◽

Cell Responses ◽

Tnf Α ◽

The Central Nervous System ◽

The Brain ◽

Microglial Inflammation

The overactivation of microglia is known to trigger inflammatory reactions in the central nervous system, which ultimately induce neuroinflammatory disorders including Alzheimer’s disease. However, increasing evidence has shown that menaquinone-4 (MK-4), a subtype of vitamin K2, can attenuate inflammation in the peripheral system. Whereas it was also observed at high levels within the brain, its function in this organ has not been well characterized. Therefore, we investigated the effect of MK-4 on microglial activation and clarified the underlying mechanism. Mouse microglia-derived MG6 cells were exposed to lipopolysaccharide (LPS) either with or without MK-4 pretreatment. Cell responses with respect to inflammatory cytokines (Il-1β, Tnf-α, and Il-6) were measured by qRT-PCR. We further analyzed the phosphorylation of TAK1, IKKα/β, and p65 of the NF-κB subunit by Western blotting. We observed that in LPS-induced MG6 cells, MK-4 dose-dependently suppressed the upregulation of inflammatory cytokines at the mRNA level. It also significantly decreased the phosphorylation of p65, but did not affect that TAK1 and IKKα/β. Furthermore, the nuclear translocation of NF-κB in LPS-induced MG6 cells was inhibited by MK-4. These results indicate that MK-4 attenuates microglial inflammation by inhibiting NF-κB signaling.

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Piper sarmentosum Roxb. Root Extracts Confer Neuroprotection by Attenuating Beta Amyloid-Induced Pro-Inflammatory Cytokines Released from Microglial Cells

Current Alzheimer Research ◽

10.2174/1567205016666190228124630 ◽

2019 ◽

Vol 16 (3) ◽

pp. 251-260 ◽

Cited By ~ 4

Author(s):

Elaine Wan Ling Chan ◽

Emilia Tze Ying Yeo ◽

Kelly Wang Ling Wong ◽

Mun Ling See ◽

Ka Yan Wong ◽

...

Keyword(s):

Alzheimer’S Disease ◽

Mrna Expression ◽

Inflammatory Mediators ◽

Beta Amyloid ◽

Microglial Cells ◽

Root Extracts ◽

Tnf Α ◽

P38α Mapk ◽

Anti Inflammatory ◽

Bv2 Microglial Cells

<P>Background: Alzheimer’s disease (AD) is a multifactorial neurodegenerative disorder that eventually leads to severe cognitive impairment. Although the exact etiologies of AD still remain elusive, increasing evidence suggests that neuroinflammation cascades mediated by microglial cells are associated with AD. Piper sarmentosum Roxb. (PS) is a medicinal plant reported to possess various biological properties, including anti-inflammatory, anti-psychotic and anti-oxidant activity. However, little is known about the anti-inflammatory activity of PS roots despite their traditional use to treat inflammatory- mediated ailments. Objective: This study aimed to evaluate the anti-inflammatory and neuroprotective properties of extracts obtained from the roots of PS against beta-amyloid (Aβ)-induced microglial toxicity associated with the production of pro-inflammatory mediators. Method: BV2 microglial cells were treated with hexane (RHXN), dichloromethane (RDCM), ethyl acetate (REA) and methanol (RMEOH) extracts of the roots of PS prior to activation by Aβ. The production and mRNA expression of pro-inflammatory mediators were evaluated by Griess reagent, ELISA kits and RT-qPCR respectively. The phosphorylation status of p38α MAPK was determined via western blot assay. BV2 conditioned medium was used to treat SH-SY5Y neuroblastoma cells and the neuroprotective effect was assessed using MTT assay. Results: PS root extracts, in particular RMEOH significantly attenuated the production and mRNA expression of IL-1β, IL-6 and TNF-α in Aβ-induced BV2 microglial cells. In addition, RHXN, REA and RMEOH extracts significantly reduced nitric oxide (NO) level and the inhibition of NO production was correlated with the total phenolic content of the extracts. Further mechanistic studies suggested that PS root extracts attenuated the production of cytokines by regulating the phosphorylation of p38α MAPK in microglia. Importantly, PS root extracts have protective effects against Aβ-induced indirect neurotoxicity either by inhibiting the production of NO, IL-1β, IL-6, and TNF-α in BV2 cells or by protecting SHSY5Y cells against these inflammatory mediators. Conclusions: These findings provided evidence that PS root extracts confer neuroprotection against Aβ- induced microglial toxicity associated with the production of pro-inflammatory mediators and may be a potential therapeutic agent for inflammation-related neurological conditions including Alzheimer’s disease (AD).</P>

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Modulation of ACE-2 mRNA by inflammatory cytokines in human thyroid cells: a pilot study

Endocrine ◽

10.1007/s12020-021-02807-w ◽

2021 ◽

Author(s):

Francesca Coperchini ◽

Gianluca Ricci ◽

Laura Croce ◽

Marco Denegri ◽

Rubina Ruggiero ◽

...

Keyword(s):

Inflammatory Cytokines ◽

Combination Treatment ◽

Primary Cultures ◽

Human Thyroid ◽

Thyroid Cell ◽

Mrna Levels ◽

Thyroid Cells ◽

Tnf Α ◽

Ifn Γ ◽

Pro Inflammatory Cytokines

Abstract Introduction Angiotensin-converting-enzyme-2 (ACE-2) was demonstrated to be the receptor for cellular entry of SARS-CoV-2. ACE-2 mRNA was identified in several human tissues and recently also in thyroid cells in vitro. Purpose Aim of the present study was to investigate the effect of pro-inflammatory cytokines on the ACE-2 mRNA levels in human thyroid cells in primary cultures. Methods Primary thyroid cell cultures were treated with IFN-γ and TNF-α alone or in combination for 24 h. ACE-2 mRNA levels were measured by RT-PCR. As a control, the levels of IFN-γ inducible chemokine (CXCL10) were measured in the respective cell culture supernatants. Results The mean levels of ACE-2 mRNA increased after treatment with IFN-γ and TNF-α in all the thyroid cell preparations, while the combination treatment did not consistently synergically increase ACE-2-mRNA. At difference, CXCL10 was consistently increased by IFN-γ and synergically further increased by the combination treatment with IFN-γ + TNF-α, with respect to IFN-γ alone. Conclusions The results of the present study show that IFN-γ and, to a lesser extent TNF-α consistently increase ACE-2 mRNA levels in NHT primary cultures. More interestingly, the combined stimulation (proven to be effective according to the synergic effect registered for CXCL10) produces different responses in terms of ACE-2 mRNA modulation. These results would suggest that elevated levels of pro-inflammatory cytokines could facilitate the entering of the virus in cells by further increasing ACE-2 expression and/or account for the different degree of severity of SARS-COV-2 infection. This hypothesis deserves to be confirmed by further specific studies.

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