Specific detection of interferon regulatory factor 5 (IRF5): A case of antibody inequality

Interferon regulatory factor 5 (IRF5) is a member of the IRF family of transcription factors. IRF5 was first identified and characterized as a transcriptional regulator of type I interferon expression after virus infection. In addition to its critical role(s) in the regulation and development of host immunity, subsequent studies revealed important roles for IRF5 in autoimmunity, cancer, obesity, pain, cardiovascular disease, and metabolism. Based on these important disease-related findings, a large number of commercial antibodies have become available to study the expression and function of IRF5. Here we validate a number of these antibodies for the detection of IRF5 by immunoblot, flow cytometry, and immunofluorescence or immunohistochemistry using well-established positive and negative controls. Somewhat surprising, the majority of commercial antibodies tested were unable to specifically recognize human or mouse IRF5. We present data on antibodies that do specifically recognize human or mouse IRF5 in a particular application. These findings reiterate the importance of proper controls and molecular weight standards for the analysis of protein expression. Given that dysregulated IRF5 expression has been implicated in the pathogenesis of numerous diseases, including autoimmune and cancer, results indicate that caution should be used in the evaluation and interpretation of IRF5 expression analysis

Therapeutic Targeting of IRFs: Pathway-Dependence or Structure-Based?

The interferon regulatory factors (IRFs) are a family of master transcription factors that regulate pathogen-induced innate and acquired immune responses. Aberration(s) in IRF signaling pathways due to infection, genetic predisposition and/or mutation, which can lead to increased expression of type I interferon (IFN) genes, IFN-stimulated genes (ISGs), and other pro-inflammatory cytokines/chemokines, has been linked to the development of numerous diseases, including (but not limited to) autoimmune and cancer. What is currently lacking in the field is an understanding of how best to therapeutically target these transcription factors. Many IRFs are regulated by post-translational modifications downstream of pattern recognition receptors (PRRs) and some of these modifications lead to activation or inhibition. We and others have been able to utilize structural features of the IRFs in order to generate dominant negative mutants that inhibit function. Here, we will review potential therapeutic strategies for targeting all IRFs by using IRF5 as a candidate targeting molecule

Detection of neutrophil extracellular traps in patient plasma: method development and validation in systemic lupus erythematosus and healthy donors that carry IRF5 genetic risk

Neutrophil extracellular traps (NETs) are web-like structures extruded by neutrophils after activation or in response to microorganisms. These extracellular structures are decondensed chromatin fibers loaded with antimicrobial granular proteins, peptides, and enzymes. NETs clear microorganisms, thus keeping a check on infections at an early stage, but if dysregulated, may be self-destructive to the body. Indeed, NETs have been associated with autoimmune diseases such as systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), antiphospholipid syndrome (APS), psoriasis, and gout. More recently, increased NETs associate with COVID-19 disease severity. While there are rigorous and reliable methods to quantify NETs from neutrophils via flow cytometry and immunofluorescence, the accurate quantification of NETs in patient plasma or serum remains a challenge. Here, we developed new methodologies for the quantification of NETs in patient plasma using multiplex ELISA and immunofluorescence methodology. Plasma from patients with SLE, non-genotyped healthy controls, and genotyped healthy controls that carry either the homozygous risk or non-risk IRF5-SLE haplotype were used in this study. The multiplex ELISA using antibodies detecting myeloperoxidase (MPO), citrullinated histone H3 (CitH3) and DNA provided reliable detection of NETs in plasma samples from SLE patients and healthy donors that carry IRF5 genetic risk. An immunofluorescence smear assay that utilizes only 1 µl of patient plasma provided similar results and data correlate to multiplex ELISA findings. The immunofluorescence smear assay is a relatively simple, inexpensive, and quantifiable method of NET detection for small volumes of patient plasma

CD40-Mediated Activation of Vascular Smooth Muscle Cell Chemokine Production Through a Src-Initiated, MAKP-Dependent Pathway

The interaction between CD40 ligand (CD154) expressed on activated T cells and its receptor, CD40, has been shown to play a role in the onset and maintenance of autoimmune inflammation. Recent studies suggest that CD154+T cells also contribute to the regulation of atherogenesis due to their capacity to activate CD40+cells of the vasculature, including vascular smooth muscle cells (VSMC). The present study evaluated the signalling events initiated through CD40 ligation which culminate in VSMC chemokine production. CD40 ligation resulted in the phosphorylation/activation of mitogen-activated protein kinases (MAPKs), extracellular signal-regulated kinases 1 and 2 (ERK1/2), and p38, but not c-jun N-terminal kinase. Inhibition of both ERK1/2 and p38 activity abrogated CD40 stimulation of IL-8 and MCP-1 production. CD40-mediated induction of chemokines also showed dependence on the Src family kinase activity. The Src kinase inhibitor, PP2, was found to inhibit CD40-induced phosphorylation of ERK1/2 as well as activation of IκB kinase. An evaluation of Src kinases that may be important in CD40 signalling identified Lyn as a potential candidate. These data indicate that CD40 signalling in VSMC activates a Src family kinase-initiated pathway that results in the induction of MAPK activities required for successful induction of chemokine synthesis

A DNA helicase from Pisum sativum is homologous to translation initiation factor and stimulates topoisomerase I activity

DNA helicases play an essential role in all aspects of nucleic acid metabolism, by providing a duplex-unwinding function. This is the first report of the isolation of a cDNA (1.6 kb) clone encoding functional DNA helicase from a plant (pea, Pisum sativum). The deduced amino-acid sequence has eight conserved helicase motifs of the DEAD-box protein family. It is a unique member of this family, containing DESD and SRT motifs instead of DEAD/H and SAT. The encoded 45.5 kDa protein has been overexpressed in bacteria and purified to homogeneity. The purified protein contains ATP-dependent DNA and RNA helicase, DNA-dependent ATPase, and ATP-binding activities. The protein sequence contains striking homology with eIF-4A, which has not so far been reported as DNA helicase. The antibodies against pea helicase inhibit in vitro translation. The gene is expressed as 1.6 kb mRNA in different organs of pea. The enzyme is localized in the nucleus and cytosol, and unwinds DNA in the 3' to 5' direction. The pea helicase interacts with pea topoisomerase I protein and stimulates its activity. These results suggest that pea DNA helicase could be an important multifunctional protein involved in protein synthesis, maintaining the basic activities of the cell, and in upregulation of topoisomerase I activity. The discovery of such a protein with intrinsic multiple activity should make an important contribution to our better understanding of DNA and RNA transactions in plants

Identification of pan-cancer/testis genes and validation of therapeutic targeting in triple-negative breast cancer: Lin28a-based and Siglece-based vaccination induces antitumor immunity and inhibits metastasis

Background Cancer–testis (CT) genes are targets for tumor antigen-specific immunotherapy given that their expression is normally restricted to the immune-privileged testis in healthy individuals with aberrant expression in tumor tissues. While they represent targetable germ tissue antigens and play important functional roles in tumorigenesis, there is currently no standardized approach for identifying clinically relevant CT genes. Optimized algorithms and validated methods for accurate prediction of reliable CT antigens (CTAs) with high immunogenicity are also lacking.Methods Sequencing data from the Genotype-Tissue Expression (GTEx) and The Genomic Data Commons (GDC) databases was used for the development of a bioinformatic pipeline to identify CT exclusive genes. A CT germness score was calculated based on the number of CT genes expressed within a tumor type and their degree of expression. The impact of tumor germness on clinical outcome was evaluated using healthy GTEx and GDC tumor samples. We then used a triple-negative breast cancer mouse model to develop and test an algorithm that predicts epitope immunogenicity based on the identification of germline sequences with strong major histocompatibility complex class I (MHCI) and MHCII binding affinities. Germline sequences for CT genes were synthesized as long synthetic peptide vaccines and tested in the 4T1 triple-negative model of invasive breast cancer with Poly(I:C) adjuvant. Vaccine immunogenicity was determined by flow cytometric analysis of in vitro and in vivo T-cell responses. Primary tumor growth and lung metastasis was evaluated by histopathology, flow cytometry and colony formation assay.Results We developed a new bioinformatic pipeline to reliably identify CT exclusive genes as immunogenic targets for immunotherapy. We identified CT genes that are exclusively expressed within the testis, lack detectable thymic expression, and are significantly expressed in multiple tumor types. High tumor germness correlated with tumor progression but not with tumor mutation burden, supporting CTAs as appealing targets in low mutation burden tumors. Importantly, tumor germness also correlated with markers of antitumor immunity. Vaccination of 4T1 tumor-bearing mice with Siglece and Lin28a antigens resulted in increased T-cell antitumor immunity and reduced primary tumor growth and lung metastases.Conclusion Our results present a novel strategy for the identification of highly immunogenic CTAs for the development of targeted vaccines that induce antitumor immunity and inhibit metastasis

IRF5 suppresses metastasis through the regulation of tumor-derived extracellular vesicles and pre-metastatic niche formation

Abstract Metastasis is driven by extensive cooperation between a tumor and its microenvironment, resulting in the adaptation of molecular mechanisms that evade the immune system and enable pre-metastatic niche (PMN) formation. Little is known of the tumor-intrinsic factors that regulate these mechanisms. Here we show that expression of the transcription factor interferon regulatory factor 5 (IRF5) in osteosarcoma (OS) and breast carcinoma (BC) clinically correlates with prolonged survival and decreased secretion of tumor-derived extracellular vesicles (t-dEVs). Conversely, loss of intra-tumoral IRF5 establishes a PMN that supports metastasis. Mechanistically, IRF5-positive tumor cells retain IRF5 transcripts within t-dEVs that contribute to altered composition, secretion, and trafficking of t-dEVs to sites of metastasis. Upon whole-body pre-conditioning with t-dEVs from IRF5-high or -low OS and BC cells, we found increased lung metastatic colonization that replicated findings from orthotopically implanted cancer cells. Collectively, our findings uncover a new role for IRF5 in cancer metastasis through its regulation of t-dEV programming of the PMN