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14-3-3?-A Novel Immunogen Promotes Inflammatory Cytokine Production.

ABSTRACT: The presence of autoantibodies against 14-3-3? in human autoimmune diseases indicates its antigenic function. However, neither the cause nor the consequence of this newly-identified antigenic function of 14-3-3? protein is known. To address this, we investigated the immunological functions of 14-3-3? by studying ex vivo effects on human peripheral blood mononuclear cells (PBMC) proliferation, polarization, and cytokine production. Exogenous 14-3-3? promoted PBMC proliferation and T cell polarization toward Th1 and Th17 populations. Significant increases in IFN-? and IL-17 levels were observed in the presence of 14-3-3?. A specific increase in Th1 cells and IFN-? production provided strong evidence for MHC class II presentation of 14-3-3? antigen. Particularly HLA-DRB1*0401 allele strongly promoted 14-3-3?-induced IFN-? producing cells. In contrast, prednisolone treatment suppressed both 14-3-3?-induced T cell polarization and cytokine production. Overall, we show that MHC presentation and the adaptor functions of 14-3-3? participate in promoting IFN-? and IL-17 production, two of the cytokines commonly associated with autoimmune diseases. To the best of our knowledge, this is the first report describing the ex vivo antigenic function of 14-3-3? with human PBMC, thereby providing the basis of its immunological role in human diseases.

SUBMITTER: McGowan J

PROVIDER: S-EPMC6667649 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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14-3-3ζ-A Novel Immunogen Promotes Inflammatory Cytokine Production.

McGowan Jenna J Peter Cara C Chattopadhyay Saurabh S Chakravarti Ritu R

Frontiers in immunology 20190724

The presence of autoantibodies against 14-3-3ζ in human autoimmune diseases indicates its antigenic function. However, neither the cause nor the consequence of this newly-identified antigenic function of 14-3-3ζ protein is known. To address this, we investigated the immunological functions of 14-3-3ζ by studying <i>ex vivo</i> effects on human peripheral blood mononuclear cells (PBMC) proliferation, polarization, and cytokine production. Exogenous 14-3-3ζ promoted PBMC proliferation and T cell p ...[more]

PMID: 31396202

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Project description:BackgroundMitogen-activated protein kinase (MAPK)-activated protein kinase-2 (MK2) is activated downstream of p38 MAPK and regulates stability of mRNAs encoding inflammatory cytokines. CC-99677 is a novel, irreversible, covalent MK2 inhibitor under development for the treatment of ankylosing spondylitis (AS) and other inflammatory diseases. As part of a phase I clinical trial to assess safety and tolerability, we evaluated target engagement, pharmacokinetics, and pharmacodynamics of CC-99677.MethodsThe MK2 inhibitor CC-99677 was evaluated for its effect on cytokine expression in vitro in peripheral blood mononuclear cells (PBMCs) from healthy donors and patients with a definitive AS diagnosis. A novel in vitro model was developed to compare the potential for tachyphylaxis of CC-99677 and p38 inhibitors in THP-1 cells. The effect of CC-99677 on tristetraprolin (TTP) and cytokine mRNA was assessed in stimulated human monocyte-derived macrophages. In a first-in-human study, thirty-seven healthy volunteers were randomly assigned to daily oral doses of CC-99677 or placebo, and blood was collected at pre-specified time points before and after dosing. CC-99677 concentrations were assessed in the plasma, and CC-99677 binding to MK2 was evaluated in PBMCs. Ex vivo stimulation of the whole blood was conducted from participants in the first-in-human study to assess the pharmacodynamic effects.ResultsIn vitro, CC-99677 inhibited tumor necrosis factor (TNF), interleukin (IL)-6, and IL-17 protein production in samples of monocytes and macrophages from AS patients and healthy volunteers via an mRNA-destabilization mechanism. In the in vitro model of tachyphylaxis, CC-99677 showed a differentiated pattern of sustained TNF protein inhibition compared with p38 inhibitors. CC-99677 reduced TTP phosphorylation and accelerated the decay of inflammatory cytokine mRNA in lipopolysaccharide-stimulated macrophages. Administration of CC-99677 to healthy volunteers was safe and well-tolerated, with linear pharmacokinetics and sustained reduction of ex vivo whole blood TNF, IL-6, and chemokine synthesis.ConclusionsCC-99677 inhibition of MK2 is a promising approach for the treatment of inflammatory diseases and may overcome the limitations of p38 MAPK inhibition.Trial registrationClinicalTrials.gov NCT03554993 .

Project description:The major cause of death in SARS-CoV-2 infected patients is due to de-regulation of the innate immune system and development of cytokine storm. SARS-CoV-2 infects multiple cell types in the lung, including macrophages, by engagement of its spike (S) protein on angiotensin converting enzyme 2 (ACE2) receptor. ACE2 receptor initiates signals in macrophages that modulate their activation, including production of cytokines and chemokines. IL-1R-associated kinase (IRAK)-M is a central regulator of inflammatory responses regulating the magnitude of TLR responsiveness. Aim of the work was to investigate whether SARS-CoV-2 S protein-initiated signals modulate pro-inflammatory cytokine production in macrophages. For this purpose, we treated PMA-differentiated THP-1 human macrophages with SARS-CoV-2 S protein and measured the induction of inflammatory mediators including IL6, TNFα, IL8, CXCL5, and MIP1a. The results showed that SARS-CoV-2 S protein induced IL6, MIP1a and TNFα mRNA expression, while it had no effect on IL8 and CXCL5 mRNA levels. We further examined whether SARS-CoV-2 S protein altered the responsiveness of macrophages to TLR signals. Treatment of LPS-activated macrophages with SARS-CoV-2 S protein augmented IL6 and MIP1a mRNA, an effect that was evident at the protein level only for IL6. Similarly, treatment of PAM3csk4 stimulated macrophages with SARS-CoV-2 S protein resulted in increased mRNA of IL6, while TNFα and MIP1a were unaffected. The results were confirmed in primary human peripheral monocytic cells (PBMCs) and isolated CD14+ monocytes. Macrophage responsiveness to TLR ligands is regulated by IRAK-M, an inactive IRAK kinase isoform. Indeed, we found that SARS-CoV-2 S protein suppressed IRAK-M mRNA and protein expression both in THP1 macrophages and primary human PBMCs and CD14+ monocytes. Engagement of SARS-CoV-2 S protein with ACE2 results in internalization of ACE2 and suppression of its activity. Activation of ACE2 has been previously shown to induce anti-inflammatory responses in macrophages. Treatment of macrophages with the ACE2 activator DIZE suppressed the pro-inflammatory action of SARS-CoV-2. Our results demonstrated that SARS-CoV-2/ACE2 interaction rendered macrophages hyper-responsive to TLR signals, suppressed IRAK-M and promoted pro-inflammatory cytokine expression. Thus, activation of ACE2 may be a potential anti-inflammatory therapeutic strategy to eliminate the development of cytokine storm observed in COVID-19 patients.

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14-3-3?-A Novel Immunogen Promotes Inflammatory Cytokine Production.

Publications

14-3-3ζ-A Novel Immunogen Promotes Inflammatory Cytokine Production.

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