IL-17A is a therapeutic target in many autoimmune diseases. Most nonhematopoietic cells express IL-17A receptors and respond to extracellular IL-17A by inducing proinflammatory cytokines. The IL-17A signal transduction triggers two broad, TRAF6- and TRAF5-dependent, intracellular signaling pathways to produce representative cytokines (IL-6) and chemokines (CXCL-1), respectively. Our limited understanding of the cross-talk between these two branches has generated a crucial gap of knowledge, leading to therapeutics indiscriminately blocking IL-17A and global inhibition of its target genes. In previous work, we discovered an elevated expression of 14-3-3 proteins in inflammatory aortic disease, a rare human autoimmune disorder with increased levels of IL-17A. Here we report that 14-3-3ζ is essential for IL-17 signaling by differentially regulating the signal-induced IL-6 and CXCL-1. Using genetically manipulated human and mouse cells, and ex vivo and in vivo rat models, we uncovered a function of 14-3-3ζ. As a part of the molecular mechanism, we show that 14-3-3ζ interacts with several TRAF proteins; in particular, its interaction with TRAF5 and TRAF6 is increased in the presence of IL-17A. In contrast to TRAF6, we found TRAF5 to be an endogenous suppressor of IL-17A–induced IL-6 production, an effect countered by 14-3-3ζ. Furthermore, we observed that 14-3-3ζ interaction with TRAF proteins is required for the IL-17A–induced IL-6 levels. Together, our results show that 14-3-3ζ is an essential component of IL-17A signaling and IL-6 production, an effect that is suppressed by TRAF5. To the best of our knowledge, this report of the 14-3-3ζ-TRAF5 axis, which differentially regulates IL-17A–induced IL-6 and CXCL-1 production, is unique.

The ubiquitously expressed 14-3-3 family of proteins is evolutionarily conserved from yeast to mammals. Their involvement in humoral and cellular immune responses is emerging through studies in drosophila and humans. In humans, a select group of 14-3-3 isoforms are antigenic; however the determinants of their antigenicity are not known. Here, we show that although mammalian 14-3-3 proteins are mostly conserved, subtle differences between their isoforms may give rise to their antigenicity. We observed syntenic relations among all the isoforms of 14-3-3 for mammals, but not with that of birds or amphibians. However, the parasitic 14-3-3 isoforms, which have known antigenic properties, show unique sequence, structure and evolution compared to the human 14-3-3. Moreover we report, for the first time the existence of a bacterial 14-3-3 protein. Contrary to the parasitic isoforms, both bacterial and yeast 14-3-3 exhibited significant homology with mammalian 14-3-3 in protein sequence as well as structure. Furthermore, a human 14-3-3 inhibitor caused significant killing of Candida albicans, which could be due to the inhibition of the structurally similar yeast homologue of 14-3-3, BMH, which is essential for its life cycle. Overall, our bioinformatic analysis combined with the demonstration of a novel antifungal role of a peptide inhibitor of human 14-3-3 indicates that the sequences and structural similarities between the mammalian, bacterial and fungal proteins are likely determinants of the antigenic nature of these proteins. Further, we propose that molecular mimicry triggered by microbial infections with either yeast or bacteria may contribute to the antigenic role of human 14-3-3.

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-1β. Furthermore, the immunization with recombinant 14-3-3ζ protein suppressed IL-1β 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.

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.

Wastewater-based epidemiology (WBE) is a popular tool for the early indication of community spread of infectious diseases. WBE emerged as an effective tool during the COVID-19 pandemic and has provided meaningful information to minimize the spread of infection. Here, we present a combination of analyses using the correlation of viral gene copies with clinical cases, sequencing of wastewater-derived RNA for the viral mutants, and correlative analyses of the viral gene copies with the bacterial biomarkers. Our study provides a unique platform for potentially using the WBE-derived results to predict the spread of COVID-19 and the emergence of new variants of concern. Further, we observed a strong correlation between the presence of SARS-CoV-2 and changes in the microbial community of wastewater, particularly the significant changes in bacterial genera belonging to the families of Lachnospiraceae and Actinomycetaceae. Our study shows that microbial biomarkers could be utilized as prediction tools for future infectious disease surveillance and outbreak responses. Overall, our comprehensive analyses of viral spread, variants, and novel bacterial biomarkers will add significantly to the growing body of literature on WBE and COVID-19.