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

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

LPS-TLR4 Signaling to IRF-3/7 and NF-κB Involves the Toll Adapters TRAM and TRIF

Toll–IL-1–resistance (TIR) domain–containing adaptor-inducing IFN-β (TRIF)–related adaptor molecule (TRAM) is the fourth TIR domain–containing adaptor protein to be described that participates in Toll receptor signaling. Like TRIF, TRAM activates interferon regulatory factor (IRF)-3, IRF-7, and NF-κB-dependent signaling pathways. Toll-like receptor (TLR)3 and 4 activate these pathways to induce IFN-α/β, regulated on activation, normal T cell expressed and secreted (RANTES), and γ interferon–inducible protein 10 (IP-10) expression independently of the adaptor protein myeloid differentiation factor 88 (MyD88). Dominant negative and siRNA studies performed here demonstrate that TRIF functions downstream of both the TLR3 (dsRNA) and TLR4 (LPS) signaling pathways, whereas the function of TRAM is restricted to the TLR4 pathway. TRAM interacts with TRIF, MyD88 adaptor–like protein (Mal)/TIRAP, and TLR4 but not with TLR3. These studies suggest that TRIF and TRAM both function in LPS-TLR4 signaling to regulate the MyD88-independent pathway during the innate immune response to LPS

Convalescent plasma in patients admitted to hospital with COVID-19 (RECOVERY): a randomised controlled, open-label, platform trial

SummaryBackground Azithromycin has been proposed as a treatment for COVID-19 on the basis of its immunomodulatoryactions. We aimed to evaluate the safety and efficacy of azithromycin in patients admitted to hospital with COVID-19.Methods In this randomised, controlled, open-label, adaptive platform trial (Randomised Evaluation of COVID-19Therapy [RECOVERY]), several possible treatments were compared with usual care in patients admitted to hospitalwith COVID-19 in the UK. The trial is underway at 176 hospitals in the UK. Eligible and consenting patients wererandomly allocated to either usual standard of care alone or usual standard of care plus azithromycin 500 mg once perday by mouth or intravenously for 10 days or until discharge (or allocation to one of the other RECOVERY treatmentgroups). Patients were assigned via web-based simple (unstratified) randomisation with allocation concealment andwere twice as likely to be randomly assigned to usual care than to any of the active treatment groups. Participants andlocal study staff were not masked to the allocated treatment, but all others involved in the trial were masked to theoutcome data during the trial. The primary outcome was 28-day all-cause mortality, assessed in the intention-to-treatpopulation. The trial is registered with ISRCTN, 50189673, and ClinicalTrials.gov, NCT04381936.Findings Between April 7 and Nov 27, 2020, of 16 442 patients enrolled in the RECOVERY trial, 9433 (57%) wereeligible and 7763 were included in the assessment of azithromycin. The mean age of these study participants was65·3 years (SD 15·7) and approximately a third were women (2944 [38%] of 7763). 2582 patients were randomlyallocated to receive azithromycin and 5181 patients were randomly allocated to usual care alone. Overall,561 (22%) patients allocated to azithromycin and 1162 (22%) patients allocated to usual care died within 28 days(rate ratio 0·97, 95% CI 0·87–1·07; p=0·50). No significant difference was seen in duration of hospital stay (median10 days [IQR 5 to >28] vs 11 days [5 to >28]) or the proportion of patients discharged from hospital alive within 28 days(rate ratio 1·04, 95% CI 0·98–1·10; p=0·19). Among those not on invasive mechanical ventilation at baseline, nosignificant difference was seen in the proportion meeting the composite endpoint of invasive mechanical ventilationor death (risk ratio 0·95, 95% CI 0·87–1·03; p=0·24).Interpretation In patients admitted to hospital with COVID-19, azithromycin did not improve survival or otherprespecified clinical outcomes. Azithromycin use in patients admitted to hospital with COVID-19 should be restrictedto patients in whom there is a clear antimicrobial indication

Multiple Regulatory Domains of IRF-5 Control Activation, Cellular Localization, and Induction of Chemokines That Mediate Recruitment of T Lymphocytes

Transcription factors of the interferon regulatory factor (IRF) family have been identified as critical mediators of early inflammatory gene transcription in infected cells. We recently determined that, besides IRF-3 and IRF-7, IRF-5 serves as a direct transducer of virus-mediated signaling. In contrast to that mediated by the other two IRFs, IRF-5-mediated activation is virus specific. We show that, in addition to Newcastle disease virus (NDV) infection, vesicular stomatitis virus (VSV) and herpes simplex virus type 1 (HSV-1) infection activates IRF-5, leading to the induction of IFNA gene subtypes that are distinct from subtypes induced by NDV. The IRF-5-mediated stimulation of inflammatory genes is not limited to IFNA since in BJAB/IRF-5-expressing cells IRF-5 stimulates transcription of RANTES, macrophage inflammatory protein 1β, monocyte chemotactic protein 1, interleukin-8, and I-309 genes in a virus-specific manner. By transient- transfection assay, we identified constitutive-activation (amino acids [aa] 410 to 489) and autoinhibitory (aa 490 to 539) domains in the IRF-5 polypeptide. We identified functional nuclear localization signals (NLS) in the amino and carboxyl termini of IRF-5 and showed that both of these NLS are sufficient for nuclear translocation and retention in infected cells. Furthermore, we demonstrated that serine residues 477 and 480 play critical roles in the response to NDV infection. Mutation of these residues from serine to alanine dramatically decreased phosphorylation and resulted in a substantial loss of IRF-5 transactivation in infected cells. Thus, this study defines the regulatory phosphorylation sites that control the activity of IRF-5 in NDV-infected cells and provides further insight into the structure and function of IRF-5. It also shows that the range of IRF-5 immunoregulatory target genes includes members of the cytokine and chemokine superfamilies

Induction of Tmolt 4 Leukemia Cell Death by 3,3-Disubstituted- 6,6-pentamethylene-1,5-diazabicyclo[3.1.0]hexane-2,4-diones: Specificity for Type II Inosine 5Ј-Monophosphate Dehydrogenase

ABSTRACT Inosine 5Ј-monophosphate dehydrogenase (IMPDH), the rate-limiting enzyme in the de novo pathway for synthesis of guanine nucleotides, is essential for normal cell proliferation and function. New derivatives of the 1,5-diazabicyclo[3.1.0]hexane-2,4-diones were synthesized and examined for antiproliferative effects and selective inhibition of human IMPDH type II activity. The 3,3-disubstituted-6,6-pentamethylene-1,5-diazabicyclo[3.1.0]hexane-2,4-diones proved to be effective antiproliferative agents in tumor cell lines derived from murine and human leukemias, lymphomas, uterine carcinoma, glioma, and breast effusion with ED 50 values (concentration of compound that inhibits 50% of cell growth) ranging from 3.3 to 16 M. The agents acted as antimetabolites suppressing de novo purine biosynthesis at the key regulatory enzyme IMPDH, resulting in the specific suppression of dGTP pool levels by 19 to 64% and DNA synthesis by 39 to 68%. The derivatives were specific inhibitors of IMPDH type II activity as opposed to type I, acting in a competitive manner with respect to inosine 5Ј-monophosphate, K i values of 44.2 to 62 M. In addition, effects of agents on Tmolt 4 cell growth and DNA synthesis could be reversed by coincubation with guanosine. Unlike mycophenolic acid and tiazofurin, the 6,6-pentamethylene-1,5-diazabicyclo[3.1.0]hexane-2,4-diones specifically targeted type II IMPDH, where activity is increased in replicating or neoplastic cells, and did not suppress type I activity, where expression is relatively unaffected by cell proliferation or transformation. Agents were not inhibitors of normal human lung fibroblast cell growth, WI-38, most likely due to the observed isoform selectivity