Akirin2 enhances antibacterial ability via interacting with 14-3-3ζ in V. splendidus-challenged Apostichopus japonicus.

Akirin2 is pivotal for regulating host immunological responses in vertebrates, including antibacterial immunity and inflammation. However, the functional significance of Akirin2 in invertebrates remains largely unexplored. In this study, we cloned the complete cDNA sequence of Akirin2 from A. japonicus (AjAkirin2) and elucidated its immunological mechanism upon pathogen infection. The whole AjAkirin2 cDNA sequence spanned 1014 bp, which comprised a 630 bp open reading frame encoding 209 amino acids, a 230 bp 5'-untranslated region (UTR), and a 154 bp 3'-UTR. Spatial expression analysis displayed constitutive expression of AjAkirin2 in all examined tissues. Both mRNA and protein expression abundance of the AjAkirin2 showed considerably high in coelomocytes of sea cucumbers challenged with Vibrio splendidus or stimulated with lipopolysaccharide. In addition, we found that sea cucumbers with 107 CFU/mL V. splendidus infection had a lower survival rate upon AjAkirin2 knockdown. Mechanistically, the result of GST-pull down and co-IP assays indicated that AjAkirin2 directly interacted with Aj14-3-3ζ. Moreover, we also detected that AjAkirin2 positively regulated Aj14-3-3ζ expression in sea cucumber coelomocytes. Furthermore, the knockdown of AjAkirin2 or Aj14-3-3ζ resulted in increasing intracellular bacteria load and suppressed the expression of key genes of the NF-κB signaling pathway (p65 and p105) and inflammatory cytokines including IL-17, VEGF, and MMP-1. In summary, these results confirmed the critical role of AjAkirin2 in mediating innate immune responses against V. splendidus infection via interaction with Aj14-3-3ζ and thereby exerting antibacterial function.

Coat proteins of necroviruses target 14-3-3a to subvert MAPKKKα-mediated antiviral immunity in plants.

Mitogen-activated protein kinase (MAPK) cascades play an important role in innate immunity against various pathogens in plants and animals. However, we know very little about the importance of MAPK cascades in plant defense against viral pathogens. Here, we used a positive-strand RNA necrovirus, beet black scorch virus (BBSV), as a model to investigate the relationship between MAPK signaling and virus infection. Our findings showed that BBSV infection activates MAPK signaling, whereas viral coat protein (CP) counteracts MAPKKKα-mediated antiviral defense. CP does not directly target MAPKKKα, instead it competitively interferes with the binding of 14-3-3a to MAPKKKα in a dose-dependent manner. This results in the instability of MAPKKKα and subversion of MAPKKKα-mediated antiviral defense. Considering the conservation of 14-3-3-binding sites in the CPs of diverse plant viruses, we provide evidence that 14-3-3-MAPKKKα defense signaling module is a target of viral effectors in the ongoing arms race of defense and viral counter-defense.

A translational study: Involvement of miR-21-5p in development and maintenance of neuropathic pain via immune-related targets CCL5 and YWHAE.

Neuropathic pain occurs in more than half of the patients suffering from peripheral neuropathies. We investigated the role of microRNA (miR)-21 in neuropathic pain using a murine-human translational approach. We applied the spared nerve injury (SNI) model at the sciatic nerve of mice and assessed the potential analgesic effect of perineurial miR-21-5p inhibitor application. Immune-related targets of miR-21-5p were determined by a qRT-PCR based cytokine and chemokine array. Bioinformatical analysis identified potential miR-21-5p targets interacting with CC-chemokine ligand (CCL)5. We validated CCL5 and tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein (YWHAE), an interaction partner of miR-21-5p and CCL5, by qRT-PCR in murine common peroneal and tibial nerves. Validated candidates were then investigated in white blood cell and sural nerve biopsy samples of patients with focal to generalized pain syndromes, i.e. small fiber neuropathy (SFN), polyneuropathy (PNP), and nerve lesion (NL). We showed that perineurial miR-21-5p inhibition reverses SNI-induced mechanical and heat hypersensitivity in mice and found a reduction of the SNI-induced increase of the pro-inflammatory mediators CCL5 (p < 0.01), CCL17 (p < 0.05), and IL-12ß (p < 0.05) in miR-21-5p inhibitor-treated mice. In silico analysis revealed several predicted and validated targets for miR-21-5p with CCL5 interaction. Among these, we found lower YWHAE gene expression in mice after SNI and perineurial injections of a scrambled oligonucleotide compared to naïve mice (p < 0.05), but this was not changed by miR-21-5p inhibition. Furthermore, miR-21-5p inhibition led to a further increase of the SNI-induced increase in TGFß (p < 0.01). Patient biomaterial revealed different systemic expression patterns of miR-21-5p, with higher expression in SFN and lower expression in NL. Further, we showed higher systemic expression of pro-inflammatory mediators in white blood cells of SFN patients compared to healthy controls. We have conducted a translational study comparing results from animal models to human patients with three different neuropathic pain syndromes. We identified CCL5 as a miR-21 dependent common player in the mouse SNI model and the human painful disease SFN.

14-3-3ζ: A suppressor of inflammatory arthritis.

Inflammatory arthritis (IA) is a common disease that affects millions of individuals worldwide. Proinflammatory events during IA pathogenesis are well studied; however, loss of protective immunity remains underexplored. Earlier, we reported that 14-3-3zeta (ζ) has a role in T-cell polarization and interleukin (IL)-17A signal transduction. Here, we demonstrate that 14-3-3ζ knockout (KO) rats develop early-onset severe arthritis in two independent models of IA, pristane-induced arthritis and collagen-induced arthritis. Arthritic 14-3-3ζ KO animals showed an increase in bone loss and immune cell infiltration in synovial joints. Induction of arthritis coincided with the loss of anti-14-3-3ζ antibodies; however, rescue experiments to supplement the 14-3-3ζ antibody by passive immunization did not suppress arthritis. Instead, 14-3-3ζ immunization during the presymptomatic phase resulted in significant suppression of arthritis in both wild-type and 14-3-3ζ KO animals. Mechanistically, 14-3-3ζ KO rats exhibited elevated inflammatory gene signatures at the messenger RNA and protein levels, particularly for IL-1ß. Furthermore, the immunization with recombinant 14-3-3ζ protein suppressed IL-1ß levels, significantly increased anti-14-3-3ζ antibody levels and collagen production, and preserved bone quality. The 14-3-3ζ protein increased collagen expression in primary rat mesenchymal cells. Together, our findings indicate that 14-3-3ζ causes immune suppression and extracellular remodeling, which lead to a previously unrecognized IA-suppressive function.

Role of the Chaperone Protein 14-3-3ε in the Regulation of Influenza A Virus-Activated Beta Interferon.

The NS1 protein of the influenza A virus plays a critical role in regulating several biological processes in cells, including the type I interferon (IFN) response. We previously profiled the cellular factors that interact with the NS1 protein of influenza A virus and found that the NS1 protein interacts with proteins involved in RNA splicing/processing, cell cycle regulation, and protein targeting processes, including 14-3-3ε. Since 14-3-3ε plays an important role in retinoic acid-inducible gene I (RIG-I) translocation to mitochondrial antiviral-signaling protein (MAVS) to activate type I IFN expression, the interaction of the NS1 and 14-3-3ε proteins may prevent the RIG-I-mediated IFN response. In this study, we confirmed that the 14-3-3ε protein interacts with the N-terminal domain of the NS1 protein and that the NS1 protein inhibits RIG-I-mediated IFN-ß promoter activation in 14-3-3ε-overexpressing cells. In addition, our results showed that knocking down 14-3-3ε can reduce IFN-ß expression elicited by influenza A virus and enhance viral replication. Furthermore, we found that threonine in the 49th amino acid position of the NS1 protein plays a role in the interaction with 14-3-3ε. Influenza A virus expressing C terminus-truncated NS1 with a T49A mutation dramatically increases IFN-ß mRNA in infected cells and causes slower replication than that of virus without the T-to-A mutation. Collectively, this study demonstrates that 14-3-3ε is involved in influenza A virus-initiated IFN-ß expression and that the interaction of the NS1 protein and 14-3-3ε may be one of the mechanisms for inhibiting type I IFN activation during influenza A virus infection. IMPORTANCE Influenza A virus is an important human pathogen causing severe respiratory disease. The virus has evolved several strategies to dysregulate the innate immune response and facilitate its replication. We demonstrate that the NS1 protein of influenza A virus interacts with the cellular chaperone protein 14-3-3ε, which plays a critical role in retinoic acid-inducible gene I (RIG-I) translocation that induces type I interferon (IFN) expression, and that NS1 protein prevents RIG-I translocation to the mitochondrial membrane. The interaction site for 14-3-3ε is the RNA-binding domain (RBD) of the NS1 protein. Therefore, this research elucidates a novel mechanism by which the NS1 RBD mediates IFN-ß suppression to facilitate influenza A viral replication. Additionally, the findings reveal the antiviral role of 14-3-3ε during influenza A virus infection.

TNFR2/14-3-3ε signaling complex instructs macrophage plasticity in inflammation and autoimmunity.

TNFR1 and TNFR2 have received prominent attention because of their dominance in the pathogenesis of inflammation and autoimmunity. TNFR1 has been extensively studied and primarily mediates inflammation. TNFR2 remains far less studied, although emerging evidence demonstrates that TNFR2 plays an antiinflammatory and immunoregulatory role in various conditions and diseases. Herein, we report that TNFR2 regulates macrophage polarization, a highly dynamic process controlled by largely unidentified intracellular regulators. Using biochemical copurification and mass spectrometry approaches, we isolated the signaling molecule 14-3-3ε as a component of TNFR2 complexes in response to progranulin stimulation in macrophages. In addition, 14-3-3ε was essential for TNFR2 signaling-mediated regulation of macrophage polarization and switch. Both global and myeloid-specific deletion of 14-3-3ε resulted in exacerbated inflammatory arthritis and counteracted the protective effects of progranulin-mediated TNFR2 activation against inflammation and autoimmunity. TNFR2/14-3-3ε signaled through PI3K/Akt/mTOR to restrict NF-κB activation while simultaneously stimulating C/EBPß activation, thereby instructing macrophage plasticity. Collectively, this study identifies 14-3-3ε as a previously unrecognized vital component of the TNFR2 receptor complex and provides new insights into the TNFR2 signaling, particularly its role in macrophage polarization with therapeutic implications for various inflammatory and autoimmune diseases with activation of the TNFR2/14-3-3ε antiinflammatory pathway.

14-3-3ζ and 14-3-3ε are involved in innate immune responses in Pacific abalone (Haliotis discus hannai).

The 14-3-3 proteins play important roles in various cellular processes by binding to different ligands, but little is known about these proteins in mollusks. In this study, two 14-3-3 cDNAs were identified from the Pacific abalone Haliotis discus hannai (designated 14-3-3ζ and 14-3-3ε), possessing 59.40% identity with each other. Both genes were predominantly expressed in the gills of unchallenged abalones, and their mRNA signals could also be detected in several other tissues, including the mantle, hepatopancreas and ovary. However, after Vibrio harveyi challenge, hemocytes were induced significantly (p < 0.01). Meanwhile, phagocytosis was inhibited, but apoptosis, reactive oxygen species formation, and caspase 3 expression were significantly induced (p < 0.01), and they were all suppressed with 14-3-3ζ knockdown (p < 0.01). The differences were that silencing 14-3-3ε reverted the decline in the phagocytic rate derived from bacterial infection, while ROS formation was not influenced significantly. In addition, the expression levels of several antimicrobial peptide and proinflammatory cytokine genes were also decreased with the silencing of 14-3-3 genes. However, with the knockdown of 14-3-3ζ, the expression of 14-3-3ε was further significantly increased (p < 0.01), and vice versa. Overall, our results suggested that 14-3-3ζ and 14-3-3ε should play important roles in innate immunity against V. harveyi infection.

Bioinformatic prediction and identification of immunogenic epitopes of the antigenic 14-3-3 protein of Echinococcus multilocularis.

INTRODUCTION: Alveolar echinococcosis is a high-risk parasitic disease caused by the larval stage of Echinococcus multilocularis. The study aimed to predict and identify the dominant Th1/Th2 and B cell epitopes of the antigen protein 14-3-3 beta:alpha from Echinococcus multilocularis. METHODS: A comparison of the four amino acid sequences of 14-3-3 beta:alpha was respectively derived from Echinococcus multilocularis, Rattus norvegicus, Canis lupus familiaris, and Homo sapiens was carried out by CLUSTALW to provide a basis for excluding similar epitopes. The amino acid sequence information was analyzed by SOPMA and the homology model was established by Swiss-Model. IEDB and SYFPEITHI were used to predict T cell epitopes. According to the Bcepred and ABCpred, the B cell epitopes were comprehensively predicted and analyzed. The dominant epitopes were validated by Lymphocyte Proliferation, ELISA, ELISpot, and Flow cytometry. RESULTS: Eight potential epitopes of 14-3-3 from Echinococcus multilocularis were screened according to the results of prediction and analysis: 14-3-31-15, 14-3-36-21, 14-3-371-86, 14-3-3144-157, 14-3-3145-166, 14-3-3146-160, 14-3-3153-161, and 14-3-3164-177. The 3D structure model of the protein was constructed and the location distribution of potential epitope was ascertained. Respectively, the epitopes of the dominant antigen of B cells were validated as 14-3-3145-166 and 14-3-3164-177; the Th1 dominant antigen epitopes were 14-3-36-21, 14-3-3145-166; and the Th2 dominant epitopes was 14-3-3145-166. CONCLUSION: In this study, two dominant antigen epitopes of B cells, two Th1 dominant antigen epitopes, and one Th2 dominant antigen epitope were validated. Our work provides a basis for the subsequent development of efficient and safe vaccines targeting epitopes of Echinococcus multilocularis.

The role of 14-3-3 proteins in cell signalling pathways and virus infection.

14-3-3 proteins are highly conserved in species ranging from yeast to mammals and regulate numerous signalling pathways via direct interactions with proteins carrying phosphorylated 14-3-3-binding motifs. Recent studies have shown that 14-3-3 proteins can also play a role in viral infections. This review summarizes the biological functions of 14-3-3 proteins in protein trafficking, cell-cycle control, apoptosis, autophagy and other cell signal transduction pathways, as well as the associated mechanisms. Recent findings regarding the role of 14-3-3 proteins in viral infection and innate immunity are also reviewed.

Protective Effect Against Toxoplasmosis in BALB/c Mice Vaccinated With Recombinant Toxoplasma gondii MIF, CDPK3, and 14-3-3 Protein Cocktail Vaccine.

Toxoplasma gondii can infect almost all endotherm organisms including humans and cause life-threatening toxoplasmosis in immunocompromised individuals, which leads to serious public health problems. Developing an excellent vaccine against this disease is impending. In present study, we formulated a cocktail protein vaccine including the TgMIF, TgCDPK3, and Tg14-3-3 proteins, which play critical roles in T. gondii infection. The recombinant protein vaccines were constructed and assessed by vaccination in BALB/c mice. We organized the mice in various protein combination groups of vaccines, and all mice were immunized with corresponding proteins at 0, 2, and 4 weeks. The specific protective effects of the vaccines on mice against T. gondii were analyzed by the mensuration of cytokines, serum antibodies, splenocyte proliferation assay, survival time, and parasite cyst burden of mice after the challenge. The study indicated that mice immunized with all three multicomponent proteins vaccine triggered a strong immune response with highest levels of IFN-γ production and IgG antibody compared with the other two protein combinations and controls. Moreover, there was an increase in IL-4 production and antigen-specific lymphocyte proliferation. The parasite cysts were significantly reduced (resulting in an 82.7% reduction), and survival time was longer in immunized mice with three multicomponent proteins compared with the other groups of mice. The enhanced humoral and cell-mediated immunity indicated that the protein cocktail vaccine containing three antigens provided effective protection for mice. These results indicated that recombinant TgMIF, TgCDPK3, and Tg14-3-3 multicomponent proteins were potential candidates for vaccine against toxoplasmosis.

STAT3 couples with 14-3-3σ to regulate BCR signaling, B-cell differentiation, and IgE production.

BACKGROUND: STAT3 or dedicator of cytokinesis protein 8 (Dock8) loss-of-function (LOF) mutations cause hyper-IgE syndrome. The role of abnormal T-cell function has been extensively investigated; however, the contribution of B-cell-intrinsic dysfunction to elevated IgE levels is unclear. OBJECTIVE: We sought to determine the underlying molecular mechanism of how STAT3 regulates B-cell receptor (BCR) signaling, B-cell differentiation, and IgE production. METHODS: We used samples from patients with STAT3 LOF mutation and samples from the STAT3 B-cell-specific knockout (KO) mice Mb1CreStat3flox/flox mice (B-STAT3 KO) to investigate the mechanism of hyper-IgE syndrome. RESULTS: We found that the peripheral B-cell homeostasis in B-STAT3 KO mice mimicked the phenotype of patients with STAT3 LOF mutation, having decreased levels of follicular and germinal center B cells but increased levels of marginal zone and IgE+ B cells. Furthermore, B-STAT3 KO B cells had reduced BCR signaling following antigenic stimulation owing to reduced BCR clustering and decreased accumulation of Wiskott-Aldrich syndrome protein and F-actin. Excitingly, a central hub protein, 14-3-3σ, which is essential for the increase in IgE production, was enhanced in the B cells of B-STAT3 KO mice and patients with STAT3 LOF mutation. The increase of 14-3-3σ was associated with increased expression of the upstream mediator, microRNA146A. Inhibition of 14-3-3σ with R18 peptide in B-STAT3 KO mice rescued the BCR signaling, follicular, germinal center, and IgE+ B-cell differentiation to the degree seen in wild-type mice. CONCLUSIONS: Altogether, our study has established a novel regulatory pathway of STAT3-miRNA146A-14-3-3σ to regulate BCR signaling, peripheral B-cell differentiation, and IgE production.

The 14-3-3/SLP76 protein-protein interaction in T-cell receptor signalling: a structural and biophysical characterization.

The SH2 domain-containing protein of 76 kDa, SLP76, is an important adaptor protein that coordinates a complex protein network downstream of T-cell receptors (TCR), ultimately regulating the immune response. Upon phosphorylation on Ser376, SLP76 interacts with 14-3-3 adaptor proteins, which leads to its proteolytic degradation. This provides a negative feedback mechanism by which TCR signalling can be controlled. To gain insight into the 14-3-3/SLP76 protein-protein interaction (PPI), we have determined a high-resolution crystal structure of a SLP76 synthetic peptide containing Ser376 with 14-3-3σ. We then characterized its binding to 14-3-3 proteins biophysically by means of fluorescence polarization and isothermal titration calorimetry. Furthermore, we generated two recombinant SLP76 protein constructs and characterized their binding to 14-3-3. Our work lays the foundation for drug design efforts aimed at targeting the 14-3-3/SLP76 interaction and, thereby, TCR signalling.

Activation of innate immunity by 14-3-3 ε, a new potential alarmin in osteoarthritis.

OBJECTIVE: The innate immune system plays a central role in osteoarthritis (OA). We identified 14-3-3ε as a novel mediator that guides chondrocytes toward an inflammatory phenotype. 14-3-3ε shares common characteristics with alarmins. These endogenous molecules, released into extracellular media, are increasingly incriminated in sustaining OA inflammation. Alarmins bind mainly to toll-like receptor 2 (TLR2) and TLR4 receptors and polarize macrophages in the synovium. We investigated the effects of 14-3-3ε in joint cells and tissues and its interactions with TLRs to define it as a new alarmin involved in OA. DESIGN: Chondrocyte, synoviocyte and macrophage cultures from murine or OA human samples were treated with 14-3-3ε. To inhibit TLR2/4 in chondrocytes, blocking antibodies were used. Moreover, chondrocytes and bone marrow macrophage (BMM) cultures from knockout (KO) TLRs mice were stimulated with 14-3-3ε. Gene expression and release of inflammatory mediators [interleukin 6 (IL-6), monocyte chemoattractant protein-1 (MCP-1), tumor necrosis factor alpha (TNFα)] were evaluated via reverse transcription quantitative polymerase chain reaction (RT-qPCR) and ELISA. RESULTS: In vitro, 14-3-3ε induced gene expression and release of IL6 and MCP1 in the treated cells. The inflammatory effects of 14-3-3ε were significantly reduced following TLRs inhibition or in TLRs KO chondrocytes and BMM. CONCLUSIONS: 14-3-3ε is able to induce an inflammatory phenotype in synoviocytes, macrophages and chondrocytes in addition to polarizing macrophages. These effects seem to involve TLR2 or TLR4 to trigger innate immunity. Our results designate 14-3-3ε as a novel alarmin in OA and as a new target either for therapeutic and/or prognostic purposes.

Tumour necrosis factor alpha promotes secretion of 14-3-3η by inducing necroptosis in macrophages.

BACKGROUND: 14-3-3η is an intracellular protein also detected in the serum and synovial fluid of patients with rheumatoid arthritis (RA). It is closely related to disease activity and anti-cyclic citrullinated peptide antibody levels. However, the main source of 14-3-3η and the mechanism of its release into the extracellular space remain unclear. Addressing these two points was the main goal of the current study. METHODS: The source of 14-3-3η was investigated by immunostaining RA synovial tissue. Fibroblast-like synoviocytes, CD4+ cells, and macrophages were selected as candidates among the various cell types in the synovial tissue. Phosphorylation of mixed-lineage kinase domain-like pseudokinase (MLKL) and cell death of macrophages were studied by phalloidin staining and electron microscopy after stimulation with an oxidative stress inducer (diamide) or tumour necrosis factor (TNF)-α. Extracellular 14-3-3η protein levels were examined by western blotting. RESULTS: Macrophages from the synovial tissue from RA, but not osteoarthritis, showed dense and widespread cytoplasmic staining for the 14-3-3η protein, co-localized with peptidylarginine deiminase 4. Swelling and membrane rupture of macrophages were induced by treatment with TNF-α, but not interleukin (IL) 6/soluble IL-6 receptor (sIL-6R). Increased MLKL phosphorylation followed by necroptosis was also induced in TNF-α-stimulated macrophages. Necrostatin-1, a necroptosis inhibitor, antagonized MLKL phosphorylation. High levels of 14-3-3η were detected in the culture supernatants of macrophages stimulated with diamide and TNF-α, but not IL-6/sIL-6R. CONCLUSIONS: Macrophages that highly express 14-3-3η undergo TNF-α-induced necroptosis with damage to the cellular structure, resulting in the secretion of 14-3-3η into the extracellular space. The current study provides a novel mechanism for 14-3-3η level increase in the RA synovial fluid.

Clinical diagnostic significance of 14-3-3η protein, high-mobility group box-1, anti-cyclic citrullinated peptide antibodies, anti-mutated citrullinated vimentin antibodies and rheumatoid factor in rheumatoid arthritis.

Introduction: Circulating markers of rheumatoid arthritis (RA) include the 14-3-3η protein, high-mobility group box-1 (HMGB1), anti-cyclic citrullinated peptide (anti-CCP) antibodies, anti-mutated citrullinated vimentin (anti-MCV) antibodies and rheumatoid factor (RF). We set out to determine which two markers in combination provided best discriminatory power for this disease.Methods: We recruited 108 RA patients, 102 non-RA patients (SLE, AS, Sjogren's syndrome, MCTD) and 90 healthy controls. Sensitivity, specificity, positive predictive value, negative predictive value, positive likelihood ratio, negative likelihood ratio and the Youden index of each analyte were calculated and binary logistic regression analysis and receiver operating characteristic (ROC) curve were performed to evaluate their diagnostic value for RA alone and in paired combination.Results: As expected, all markers were elevated in RA patients (P < 0.05). Binary logistic regression analysis showed that 14-3-3η had the highest odds ratio (95% CI) at 2.4 (1.9-2.8). Anti-CCP and anti-MCV had the highest areas under the curves [AUC (95% CI)] at 0.85 (0.78-0.90) and 0.85 (0.78-0.91) respectively (both P < 0.001). In serial detection (one marker followed by another), no combination had a Youden index >0.6. In parallel analysis (both considered together) several combinations had a Youden index >0.7, of which the highest (0.78) was anti-CCP with anti-MCV, with a sensitivity of 93.3% and specificity of 84.7%.Conclusions: Despite individual increases in serum 14-3-3η, HMGB1, anti-CCP, anti-MCV and RF, the combination of anti-CCP and anti-MCV might be of great help for diagnostic in RA, and so should be considered as routine tests for this disease.

Diagnostic performance of 14-3-3η and anti-carbamylated protein antibodies in Rheumatoid Arthritis in Han population of Northern China.

OBJECTIVES: As we already know, Rheumatoid arthritis (RA) cannot be excluded when the rheumatoid factor (RF) or anti-cyclic citrullinated peptide antibody (anti-CCP) is negative. Here, we determined the application value of 14-3-3η protein, anti-carbamylated proteins antibodies (anti-CarP), as well as their potential role to diagnose RA together with RF or anti-CCP. METHOD: Serum levels of anti-CCP, RF, 14-3-3η and anti-CarP antibodies were detected in 291 RA patients, 223 patients with autoimmune diseases except RA, and 156 healthy subjects recruited from Han population of Northern China. We examined the differences in the levels of these indicators among groups and compared the correlations between any two of the indicators. At the same time, a total of 12 testing strategies were established for comparison to maximize the diagnostic value. RESULT: The levels of RF, anti-CCP, anti-CarP and 14-3-3η were significantly higher in RA patients (12.5;[9.36-15.7], 30.7;[25.7-35.6], 1.90;[1.70-2.01], 15.8;[10.8-20.8], respectively) compared with either interference-control group (1.24;[1.07-1.41], 0.64;[0.42-0.86], 0.51;[0.46-0.57], 0.33;[0.23-0.44], respectively) (p < 0.0001) or healthy-control group (1.03;[0.99-1.08], 0.49;[0.38-0.59], 0.28;[0.21-0.35], 0.55;[0.27-0.85], respectively) (p < 0.0001). Among all 12 detection strategies, the YI and κ value of a novel strategy that either double-positive of any 2 markers or single-positive of anti-CCP can be diagnosed as RA had the highest diagnostic value. CONCLUSION: The results of our study demonstrated that in Han population of Northern China, anti-CarP antibodies and 14-3-3η protein can be treated as valuable indicators of RA, especially when combined with RF and anti-CCP, the detection value is maximized.

Role of tristetraprolin phosphorylation in paediatric patients with inflammatory bowel disease.

BACKGROUND: Intestinal inflammation and epithelial injury are the leading actors of inflammatory bowel disease (IBD), causing an excessive pro-inflammatory cytokines expression. Tristetraprolin (TTP), an mRNA binding protein, plays a role in regulating the inflammatory factors, recognizing specific sequences on the 3' untranslated region of cytokine mRNAs. TTP activity depends on its phosphorylation state: the unphosphorylated TTP degrades pro-inflammatory cytokine mRNAs; on the contrary, the phosphorylated TTP fails to destabilize mRNAs furthering their expression. The phospho-TTP forms a complex with the chaperone protein 14-3-3. This binding could be one of the factors that promote intestinal inflammation as a cause of disease progression. AIM: To assess if TTP phosphorylation has a role in paediatric IBD. METHODS: The study was carried out on a cohort of paediatric IBD patients. For each patient enrolled, a specimen of inflamed and non-inflamed colonic mucosa was collected. Furthermore, the experiments were conducted on macrophages differentiated from blood samples of the same patients. Macrophages from healthy donors' blood were used as controls. Co-immunoprecipitation assay and immunoblotting analyses were performed to observe the formation of the phospho-TTP/14-3-3 complex. In the same samples TNF-α expression was also evaluated as major factor of the pro-inflammatory activity. RESULTS: In this work we studied indirectly the phosphorylation of TTP through the binding with the chaperone protein 14-3-3. In inflamed and non-inflamed colon mucosa of IBD paediatric patients immunoblot assay demonstrated a higher expression of the TTP in inflamed samples respect to the non-inflamed; the co-immunoprecipitated 14-3-3 protein showed the same trend of expression. In the TNF-α gene expression analysis higher levels of the cytokine in inflamed tissues compared to controls were evident. The same experiments were conducted on macrophages from IBD paediatric patients and healthy controls. The immunoblot results demonstrated a high expression of both TTP and co-immunoprecipitated 14-4-3 protein in IBD-derived macrophages in comparison to healthy donors. TNF-α protein levels from macrophages lysates showed the same trend of expression in favour of IBD paediatric patients compared to healthy controls. CONCLUSION: In this work, for the first time, we describe a relation between phospho-TTP/14-3-3 complex and IBD. Indeed, a higher expression of TTP/14-3-3 was recorded in IBD samples in comparison to controls.

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

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 ex vivo effects on human peripheral blood mononuclear cells (PBMC) proliferation, polarization, and cytokine production. Exogenous 14-3-3ζ promoted PBMC proliferation and T cell polarization toward Th1 and Th17 populations. Significant increases in IFN-γ and IL-17 levels were observed in the presence of 14-3-3ζ. A specific increase in Th1 cells and IFN-γ production provided strong evidence for MHC class II presentation of 14-3-3ζ antigen. Particularly HLA-DRB1*0401 allele strongly promoted 14-3-3ζ-induced IFN-γ producing cells. In contrast, prednisolone treatment suppressed both 14-3-3ζ-induced T cell polarization and cytokine production. Overall, we show that MHC presentation and the adaptor functions of 14-3-3ζ participate in promoting IFN-γ 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 14-3-3ζ with human PBMC, thereby providing the basis of its immunological role in human diseases.

Production of recombinant 14-3-3 protein and determination of its immunogenicity for application in serodiagnosis of strongyloidiasis.

BACKGROUND: Strongyloides stercoralis is the fourth most important intestinal nematode worldwide. The parasite load and larvae count are often low, thus conventional methods are not sufficiently sensitive to detect the infection. In this study we developed an immunoglobulin G-based enzyme-linked immunosorbent assay (ELISA) method to detect antibodies against S. stercoralis 14-3-3 protein in patients' sera. METHODS: S. stercoralis RNA was extracted and following complementary DNA synthesis, the 708-bp fragment of 14-3-3 protein was amplified by polymerase chain reaction and cloned into the pET28a+ expression vector. The 30-kDa recombinant 14-3-3 protein was expressed in Escherichia coli BL21 (DE3) cells and purified by affinity chromatography. Finally, its immunoreactivity was assessed by indirect ELISA and western blotting. RESULTS: The S. stercoralis 14-3-3 gene was successfully amplified and cloned into an expression vector. The 30-kDa recombinant protein was purified by affinity chromatography. An ELISA developed in-house detected infected patients' sera with 96% sensitivity. CONCLUSIONS: We concluded that the recombinant 14-3-3 protein has enough sensitivity and specificity for detection of strongyloidiasis in human sera and could be applied for serodiagnosis.

Autoantibody against 14-3-3 zeta: a serological marker in detection of gastric cancer.

PURPOSE: Autoantibody to 14-3-3 zeta was identified in gastric cancer (GC) by serological proteome analysis (SERPA) in our previous study. We comprehensively evaluated its ability to detect GC, determined its association with clinical characteristics, and explored its temporal change in GC patients before and after gastrectomy resection in this study. METHODS: Anti-14-3-3 zeta antibody was examined by immunoassay in sera from 465 GC patients and 465 normal individuals, and also in 69 serial sera from 26 GC patients before and after resection. RESULTS: The frequency of anti-14-3-3 zeta were significantly higher in GC group than in control group, with AUC of 0.627. The appearance of anti-14-3-3 zeta showed no difference in different tumor stage, tumor size, tumor differentiation, and lymphatic metastasis, but was higher in GC patients with family tumor history than without family tumor history. When anti-14-3-3 zeta was combined with clinical markers (CEA, CA199 and CA724), the sensitivity increased to 52.7%. In the follow-up analysis, the titer of anti-14-3-3 zeta was higher in post-resection sera than pre-resection sera, and no difference was observed in CEA, CA199 and CA724. Anti-14-3-3 zeta showed an increase from negative status to positive status in six patients after resection, while other three clinical markers presented different change in GC patients after resection. CONCLUSIONS: Autoantibody against 14-3-3 zeta could be a potential diagnostic biomarker and improve the sensitivity of CEA, CA199 and CA724 in diagnosis of GC. Further largescale studies will be needed to validate its performance in GC patients after resection.