Erythroblastic islands foster granulopoiesis in parallel to terminal erythropoiesis.

The erythroblastic island (EBI), composed of a central macrophage surrounded by maturing erythroblasts, is the erythroid precursor niche. Despite numerous studies, its precise composition is still unclear. Using multispectral imaging flow cytometry, in vitro island reconstitution, and single-cell RNA sequencing of adult mouse bone marrow (BM) EBI-component cells enriched by gradient sedimentation, we present evidence that the CD11b+ cells present in the EBIs are neutrophil precursors specifically associated with BM EBI macrophages, indicating that erythro-(myelo)-blastic islands are a site for terminal granulopoiesis and erythropoiesis. We further demonstrate that the balance between these dominant and terminal differentiation programs is dynamically regulated within this BM niche by pathophysiological states that favor granulopoiesis during anemia of inflammation and favor erythropoiesis after erythropoietin stimulation. Finally, by molecular profiling, we reveal the heterogeneity of EBI macrophages by cellular indexing of transcriptome and epitope sequencing of mouse BM EBIs at baseline and after erythropoietin stimulation in vivo and provide a searchable online viewer of these data characterizing the macrophage subsets serving as hematopoietic niches. Taken together, our findings demonstrate that EBIs serve a dual role as niches for terminal erythropoiesis and granulopoiesis and the central macrophages adapt to optimize production of red blood cells or neutrophils.

HMGB1-mediated restriction of EPO signaling contributes to anemia of inflammation.

Anemia of inflammation, also known as anemia of chronic disease, is refractory to erythropoietin (EPO) treatment, but the mechanisms underlying the EPO refractory state are unclear. Here, we demonstrate that high mobility group box-1 protein (HMGB1), a damage-associated molecular pattern molecule recently implicated in anemia development during sepsis, leads to reduced expansion and increased death of EPO-sensitive erythroid precursors in human models of erythropoiesis. HMGB1 significantly attenuates EPO-mediated phosphorylation of the Janus kinase 2/STAT5 and mTOR signaling pathways. Genetic ablation of receptor for advanced glycation end products, the only known HMGB1 receptor expressed by erythroid precursors, does not rescue the deleterious effects of HMGB1 on EPO signaling, either in human or murine precursors. Furthermore, surface plasmon resonance studies highlight the ability of HMGB1 to interfere with the binding between EPO and the EPOR. Administration of a monoclonal anti-HMGB1 antibody after sepsis onset in mice partially restores EPO signaling in vivo. Thus, HMGB1-mediated restriction of EPO signaling contributes to the chronic phase of anemia of inflammation.

Navigating the marrow sea towards erythromyeloblastic islands under normal and inflammatory conditions.

PURPOSE OF REVIEW: Terminal erythroid differentiation occurs in specialized niches called erythroblastic islands. Since their discovery in 1958, these niches have been described as a central macrophage surrounded by differentiating erythroblasts. Here, we review the recent advances made in the characterization of these islands and the role they could play in anaemia of inflammation. RECENT FINDINGS: The utilization of multispectral imaging flow cytometry (flow cytometry with microscopy) has enabled for a more precise characterization of the niche that revealed the presence of maturing granulocytes in close contact with the central macrophage. These erythromyeloblastic islands (EMBIs) can adapt depending on the peripheral needs. Indeed, during inflammation wherein inflammatory cytokines limit erythropoiesis and promote granulopoiesis, EMBIs present altered structures with increased maturing granulocytes and decreased erythroid precursors. SUMMARY: Regulation of the structure and function of the EMBI in the bone marrow emerges as a potential player in the pathophysiology of acute and chronic inflammation and its associated anaemia.

HMGB1-C1q complexes regulate macrophage function by switching between leukotriene and specialized proresolving mediator biosynthesis.

Macrophage polarization is critical to inflammation and resolution of inflammation. We previously showed that high-mobility group box 1 (HMGB1) can engage receptor for advanced glycation end product (RAGE) to direct monocytes to a proinflammatory phenotype characterized by production of type 1 IFN and proinflammatory cytokines. In contrast, HMGB1 plus C1q form a tetramolecular complex cross-linking RAGE and LAIR-1 and directing monocytes to an antiinflammatory phenotype. Lipid mediators, as well as cytokines, help establish a milieu favoring either inflammation or resolution of inflammation. This study focuses on the induction of lipid mediators by HMGB1 and HMGB1 plus C1q and their regulation of IRF5, a transcription factor critical for the induction and maintenance of proinflammatory macrophages. Here, we show that HMGB1 induces leukotriene production through a RAGE-dependent pathway, while HMGB1 plus C1q induces specialized proresolving lipid mediators lipoxin A4, resolvin D1, and resolvin D2 through a RAGE- and LAIR-1-dependent pathway. Leukotriene exposure contributes to induction of IRF5 in a positive-feedback loop. In contrast, resolvins (at 20 nM) block IRF5 induction and prevent the differentiation of inflammatory macrophages. Finally, we have generated a molecular mimic of HMGB1 plus C1q, which cross-links RAGE and LAIR-1 and polarizes monocytes to an antiinflammatory phenotype. These findings may provide a mechanism to control nonresolving inflammation in many pathologic conditions.

Nebulized in-line endotracheal dornase alfa and albuterol administered to mechanically ventilated COVID-19 patients: a case series.

BACKGROUND: Mechanically ventilated patients with COVID-19 have a mortality of 24-53%, in part due to distal mucopurulent secretions interfering with ventilation. DNA from neutrophil extracellular traps (NETs) contribute to the viscosity of mucopurulent secretions and NETs are found in the serum of COVID-19 patients. Dornase alfa is recombinant human DNase 1 and is used to digest DNA in mucoid sputum. Here, we report a single-center case series where dornase alfa was co-administered with albuterol through an in-line nebulizer system. METHODS: Demographic and clinical data were collected from the electronic medical records of five mechanically ventilated patients with COVID-19-including three requiring veno-venous extracorporeal membrane oxygenation-treated with nebulized in-line endotracheal dornase alfa and albuterol, between March 31 and April 24, 2020. Data on tolerability and response were analyzed. RESULTS: The fraction of inspired oxygen requirements was reduced for all five patients after initiating dornase alfa administration. All patients were successfully extubated, discharged from hospital and remain alive. No drug-associated toxicities were identified. CONCLUSIONS: Results suggest that dornase alfa will be well-tolerated by patients with severe COVID-19. Clinical trials are required to formally test the dosing, safety, and efficacy of dornase alfa in COVID-19, and several have been recently registered.

Coordination between innate immune cells, type I IFNs and IRF5 drives SLE pathogenesis.

Systemic lupus erythematosus (SLE) is a complex autoimmune disease which affects multiple organs. The type I interferon (IFN) gene signature and circulating autoantibodies are hallmarks of SLE. Plasmacytoid dendritic cells (pDCs) are considered the main producers of type I IFN and production is modulated by multiple other immune cell types. In SLE, essentially every immune cell type is dysregulated and aberrant deregulation is thought to be due, in part, to direct or indirect exposure to IFN. Genetic variants within or around the transcription factor interferon regulatory factor 5 (IRF5) associate with SLE risk. Elevated IFNα activity was detected in the sera of SLE patients carrying IRF5 risk polymorphisms who were positive for either anti-RNA binding protein (anti-RBP) or anti-double-stranded DNA (anti-dsDNA) autoantibodies. Neutrophils are also an important source of type I IFNs and are found in abundance in human blood. Neutrophil extracellular traps (NETs) are considered a potential source of antigenic trigger in SLE that can lead to type I IFN gene induction, as well as increased autoantibody production. In this review, we will focus on immune cell types that produce type I IFNs and/or are affected by type I IFN in SLE. In addition, we will discuss potential inducers of endogenous type I IFN production in SLE. Last, we will postulate how the different immune cell populations may be affected by an IRF5-SLE risk haplotype.

Genetic Versus Non-genetic Drivers of SLE: Implications of IRF5 Dysregulation in Both Roads Leading to SLE.

PURPOSE OF REVIEW: Systemic lupus erythematosus (SLE) is characterized by a breakdown of immune tolerance, resulting in inflammation and tissue destruction. While the primary causes of SLE are still obscure, the disorder is highly heritable. Genetic risk variants, on their own, are rarely causal or fully explain disease pathogenesis. We discuss the possibility that IRF5, a SLE susceptibility gene, has both genetic and non-genetic contributions to disease pathogenesis. RECENT FINDINGS: Genetic variants within and around IRF5 robustly associate with SLE risk. In SLE blood cells, IRF5 risk variants associate with elevated IRF5 expression and IFN production. Whether the observed increase in expression is due to risk variants or other disease-associated factors is not clear. Data from Irf5-/- mice backcrossed to multiple models of murine lupus support that IRF5's role in disease pathogenesis is non-genetic. Studies of IRF5 expression and function in genotyped healthy donors will address the question of whether IRF5 dysregulation in SLE is driven by genetic or non-genetic factors.

Interferon regulatory factor 5 (IRF5) suppresses hepatitis C virus (HCV) replication and HCV-associated hepatocellular carcinoma.

Hepatitis C virus (HCV) infection is a major risk factor for the development of chronic liver disease. The disease typically progresses from chronic HCV to fibrosis, cirrhosis, hepatocellular carcinoma (HCC), and death. Chronic inflammation associated with HCV infection is implicated in cirrhosis and HCC, but the molecular players and signaling pathways contributing to these processes remain largely unknown. Interferon regulatory factor 5 (IRF5) is a molecule of interest in HCV-associated HCC because it has critical roles in virus-, Toll-like receptor (TLR)-, and IFN-induced signaling pathways. IRF5 is also a tumor suppressor, and its expression is dysregulated in several human cancers. Here, we present first evidence that IRF5 expression and signaling are modulated during HCV infection. Using HCV infection of human hepatocytes and cells with autonomously replicating HCV RNA, we found that levels of IRF5 mRNA and protein expression were down-regulated. Of note, reporter assays indicated that IRF5 re-expression inhibited HCV protein translation and RNA replication. Gene expression analysis revealed significant differences in the expression of cancer pathway mediators and autophagy proteins rather than in cytokines between IRF5- and empty vector-transfected HCV replicon cells. IRF5 re-expression induced apoptosis via loss in mitochondrial membrane potential, down-regulated autophagy, and inhibited hepatocyte cell migration/invasion. Analysis of clinical HCC specimens supports a pathologic role for IRF5 in HCV-induced HCC, as IRF5 expression was down-regulated in livers from HCV-positive versus HCV-negative HCC patients or healthy donor livers. These results identify IRF5 as an important suppressor of HCV replication and HCC pathogenesis.

Insulin-like growth factor receptor signaling in breast tumor epithelium protects cells from endoplasmic reticulum stress and regulates the tumor microenvironment.

BACKGROUND: Early analyses of human breast cancer identified high expression of the insulin-like growth factor type 1 receptor (IGF-1R) correlated with hormone receptor positive breast cancer and associated with a favorable prognosis, whereas low expression of IGF-1R correlated with triple negative breast cancer (TNBC). We previously demonstrated that the IGF-1R acts as a tumor and metastasis suppressor in the Wnt1 mouse model of TNBC. The mechanisms for how reduced IGF-1R contributes to TNBC phenotypes is unknown. METHODS: We analyzed the METABRIC dataset to further stratify IGF-1R expression with patient survival and specific parameters of TNBC. To investigate molecular events associated with the loss of IGF-1R function in breast tumor cells, we inhibited IGF-1R in human cell lines using an IGF-1R blocking antibody and analyzed MMTV-Wnt1-mediated mouse tumors with reduced IGF-1R function through expression of a dominant-negative transgene. RESULTS: Our analysis of the Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) dataset revealed association between low IGF-1R and reduced overall patient survival. IGF-1R expression was inversely correlated with patient survival even within hormone receptor-positive breast cancers, indicating reduced overall patient survival with low IGF-1R was not due simply to low IGF-1R expression within TNBCs. Inhibiting IGF-1R in either mouse or human tumor epithelial cells increased reactive oxygen species (ROS) production and activation of the endoplasmic reticulum stress response. IGF-1R inhibition in tumor epithelial cells elevated interleukin (IL)-6 and C-C motif chemokine ligand 2 (CCL2) expression, which was reversed by ROS scavenging. Moreover, the Wnt1/dnIGF-1R primary tumors displayed a tumor-promoting immune phenotype. The increased CCL2 promoted an influx of CD11b+ monocytes into the primary tumor that also had increased matrix metalloproteinase (MMP)-2, MMP-3, and MMP-9 expression. Increased MMP activity in the tumor stroma was associated with enhanced matrix remodeling and collagen deposition. Further analysis of the METABRIC dataset revealed an increase in IL-6, CCL2, and MMP-9 expression in patients with low IGF-1R, consistent with our mouse tumor model and data in human breast cancer cell lines. CONCLUSIONS: Our data support the hypothesis that reduction of IGF-1R function increases cellular stress and cytokine production to promote an aggressive tumor microenvironment through infiltration of immune cells and matrix remodeling.

Interferon regulatory factor signaling in autoimmune disease.

Interferon regulatory factors (IRFs) play critical roles in pathogen-induced innate immune responses and the subsequent induction of adaptive immune response. Dysregulation of IRF signaling is therefore thought to contribute to autoimmune disease pathogenesis. Indeed, numerous murine in vivo studies have documented protection from or enhanced susceptibility to particular autoimmune diseases in Irf-deficient mice. What has been lacking, however, is replication of these in vivo observations in primary immune cells from patients with autoimmune disease. These types of studies are essential as the majority of in vivo data support a protective role for IRFs in Irf-deficient mice, yet IRFs are often found to be overexpressed in patient immune cells. A significant body of work is beginning to emerge from both of these areas of study - mouse and human.

A conserved region within interferon regulatory factor 5 controls breast cancer cell migration through a cytoplasmic and transcription-independent mechanism.

BACKGROUND: Migration of breast cancer cells out of a duct or lobule is a prerequisite for invasion and metastasis. However, the factors controlling breast cancer cell migration are not fully elucidated. We previously found that expression of the transcription factor interferon regulatory factor 5 (IRF5) is significantly decreased as a breast lesion progresses from a non-malignant stage to ductal carcinoma in situ and is eventually lost in ~80% of invasive ductal carcinomas examined. Human in vitro and murine in vivo models of invasive breast cancer confirmed an important role for IRF5 in regulating cell motility, invasion and/or metastasis; yet, the mechanism(s) by which this occurs is not known. Since IRF5 is primarily expressed in the cytoplasm of human mammary epithelial cells, we hypothesized that IRF5 may function in a transcription-independent manner to control intrinsic cell migration. RESULTS: A series of IRF5 deletion mutants were tested in cell motility, invasion and migration assays. A novel, conserved 10 amino acid domain was identified that regulates mammary epithelial cell migration. This region (∆115-125) is downstream of IRF5's DNA binding domain and therefore when absent, retains IRF5 transcription activity but loses cell migration control. An IRF5 construct with a mutated nuclear localization signal further confirmed that IRF5 controls migration in a cytoplasmic and transcription-independent manner. Candidate cytoskeletal molecules were identified in MDA-MB-231 cells to interact with IRF5 by immunoprecipitation and mass spectrometry analysis. α6-tubulin was independently confirmed to interact with endogenous IRF5 in MCF-10A cells. Alterations in F-actin bundling after staining EV- and IRF5-231 cells with phalloidin suggests that IRF5 may control cell migration/motility through its interaction with cytoskeletal molecules that contribute to the formation of F-actin networks. Last and most notably, we found that IRF5's control of cell migration is not restricted to mammary epithelial cells but functions in other epithelial cell types suggesting a more global role for this newly identified cell migratory function of IRF5. CONCLUSIONS: These findings are significant as they identify a new regulator of epithelial cell migration and provide specific insight into the mechanism(s) by which loss of IRF5 expression in mammary epithelial cells contributes to breast cancer metastasis.

IRF5 is a novel regulator of CXCL13 expression in breast cancer that regulates CXCR5(+) B- and T-cell trafficking to tumor-conditioned media.

Clinical studies using prognostic and predictive signatures have shown that an immune signal emanating from whole tumors reflects the level of immune cell infiltration--a high immune signal linked to improved outcome. Factors regulating immune cell trafficking to the tumor, however, are not known. Previous work has shown that expression of interferon regulatory factor 5 (IRF5), a critical immune regulator, is lost in ~80% of invasive ductal carcinomas examined. We postulated that IRF5-positive and -negative breast tumors would differentially regulate immune cell trafficking to the tumor. Using a focused tumor inflammatory array, differences in cytokine and chemokine expression were examined between IRF5-positive and -negative MDA-MB-231 cells grown in three-dimensional culture. A number of cytokines/chemokines were found to be dysregulated between cultures. CXCL13 was identified as a direct target of IRF5 resulting in the enhanced recruitment of B and T cells to IRF5-positive tumor-conditioned media. The ability of IRF5 to regulate mediators of cell migration was confirmed by enzyme-linked immunosorbent assay, chromatin immunoprecipitation assay, small interfering RNA knockdown and immunofluorescence staining of human breast tumor tissues. Analysis of primary immune cell subsets revealed that IRF5 specifically recruits CXCR5(+) B and T cells to the tumor; CXCR5 is the receptor for CXCL13. Analysis of primary breast tumor tissues revealed a significant correlation between IRF5 and CXCL13 expression providing clinical relevance to the study. Together, these data support that IRF5 directly regulates a network of genes that shapes a tumor immune response and may, in combination with CXCL13, serve as a novel prognostic marker for antitumor immunity.

B cell transcription factors: Potential new therapeutic targets for SLE.

B cells represent a critical arm of the adaptive immune system, and under normal circumstances, provide long lasting immunity to a wide range of pathogens. To achieve this, B cells differentiate into specialized subpopulations, which perform unique functions in response to antigen. A complex network of transcription factors regulates this transition as well as subsequent effector functions. Dysregulation of these transcription factors can lead to altered B cell biology and potentially autoimmunity. Systemic lupus erythematosus (SLE) is a complex autoimmune disease characterized by increased autoantibody production and B cell hyper-reactivity. It still remains unclear, however, what factors contribute to disease pathogenesis. Recent work has suggested dysregulation of B cell transcription factors may play a role in disease onset. Given the prominent role B cells play in the pathogenesis of SLE, it is important to have a full understanding of what transcription factors regulate B cell development, and how dysregulation of some of these transcription factors may contribute to the occurrence and/or pathogenesis of SLE. A comprehensive review of where this field currently stands, what new potential targets have recently been identified, and what functional information is still missing will be discussed.

Monocytes from Irf5-/- mice have an intrinsic defect in their response to pristane-induced lupus.

The transcription factor IFN regulatory factor (IRF)5 has been identified as a human systemic lupus erythematosus (SLE) susceptibility gene by numerous joint linkage and genome-wide association studies. Although IRF5 expression is significantly elevated in primary blood cells of SLE patients, it is not yet known how IRF5 contributes to SLE pathogenesis. Recent data from mouse models of lupus indicate a critical role for IRF5 in the production of pathogenic autoantibodies and the expression of Th2 cytokines and type I IFN. In the present study, we examined the mechanisms by which loss of Irf5 protects mice from pristane-induced lupus at early time points of disease development. We demonstrate that Irf5 is required for Ly6C(hi) monocyte trafficking to the peritoneal cavity, which is thought to be one of the initial key events leading to lupus pathogenesis in this model. Chemotaxis assays using peritoneal lavage from pristane-injected Irf5(+/+) and Irf5(-/-) littermates support an intrinsic defect in Irf5(-/-) monocytes. We found the expression of chemokine receptors CXCR4 and CCR2 to be dysregulated on Irf5(-/-) monocytes and less responsive to their respective ligands, CXCL12 and CCL2. Bone marrow reconstitution experiments further supported an intrinsic defect in Irf5(-/-) monocytes because Irf5(+/+) monocytes were preferentially recruited to the peritoneal cavity in response to pristane. Taken together, these findings demonstrate an intrinsic role for IRF5 in the response of monocytes to pristane and their recruitment to the primary site of inflammation that is thought to trigger lupus onset in this experimental model of SLE.

Role of interferon regulatory factor 5 (IRF5) in tumor progression: Prognostic and therapeutic potential.

Canonically, the transcription factor interferon regulatory factor 5 (IRF5) is a key mediator of innate and adaptive immunity downstream of pathogen recognition receptors such as Toll-like receptors (TLRs). Hence, dysregulation of IRF5 function has been widely implicated in inflammatory and autoimmune diseases. Over the last few decades, dysregulation of IRF5 expression has been also reported in hematologic malignancies and solid cancers that support a role for IRF5 in malignant transformation, tumor immune regulation, clinical prognosis, and treatment response. This review will provide an in-depth overview of the current literature regarding the mechanisms by which IRF5 functions as either a tumor suppressor or oncogene, its role in metastasis, regulation of the tumor-immune microenvironment, utility as a prognostic indicator of disease, and new developments in IRF5 therapeutics that may be used to remodel tumor immunity.

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

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.

Irf5-deficient mice are protected from pristane-induced lupus via increased Th2 cytokines and altered IgG class switching.

Polymorphisms in the transcription factor interferon (IFN) regulatory factor 5 (IRF5) have been identified that show a strong association with an increased risk of developing the autoimmune disease systemic lupus erythematosus (SLE). A potential pathological role for IRF5 in SLE development is supported by the fact that increased IRF5 mRNA and protein are observed in primary blood cells of SLE patients and this correlates with an increased risk of developing the disease. Here, we demonstrate that IRF5 is required for pristane-induced SLE via its ability to control multiple facets of autoimmunity. We show that IRF5 is required for pathological hypergammaglobulinemia and, in the absence of IRF5, IgG class switching is reduced. Examination of in vivo cytokine expression (and autoantibody production) identified an increase in Irf5(-/-) mice of Th2 cytokines. In addition, we provide clear evidence that loss of Irf5 significantly weakens the in vivo type I IFN signature critical for disease pathogenesis in this model of murine lupus. Together, these findings demonstrate the importance of IRF5 for autoimmunity and provide a significant new insight into how overexpression of IRF5 in blood cells of SLE patients may contribute to disease pathogenesis.

Interferon regulatory factor 5 activation in monocytes of systemic lupus erythematosus patients is triggered by circulating autoantigens independent of type I interferons.

OBJECTIVE: Genetic variants of interferon regulatory factor 5 (IRF-5) are associated with susceptibility to systemic lupus erythematosus (SLE). IRF-5 regulates the expression of proinflammatory cytokines and type I interferons (IFNs) believed to be involved in the pathogenesis of SLE. The aim of this study was to determine the activation status of IRF-5 by assessing its nuclear localization in the immune cells of SLE patients and healthy donors, and to identify SLE-associated triggers of IRF-5 activation. METHODS: IRF-5 nuclear localization in subpopulations of peripheral blood mononuclear cells from 14 genotyped SLE patients and 11 healthy controls was assessed using imaging flow cytometry. The activation and function of IRF-5 were examined after ex vivo stimulation of healthy donor monocytes with SLE serum or components of SLE serum. Cellular localization was determined by ImageStream flow cytometry, and cytokine expression was analyzed by quantitative polymerase chain reaction and enzyme-linked immunosorbent assay. RESULTS: IRF-5 was activated in a cell type-specific manner; monocytes from SLE patients had constitutively elevated levels of nuclear IRF-5, as compared to natural killer cells and T cells. SLE serum was identified as a trigger for IRF-5 nuclear accumulation; however, neither IFNα nor SLE immune complexes could induce nuclear localization. Instead, autoantigens composed of apoptotic/necrotic material triggered IRF-5 nuclear accumulation in monocytes. Production of the cytokines IFNα, tumor necrosis factor α, and interleukin-6 in monocytes stimulated with SLE serum or autoantigens was distinct, yet showed a correlation with the kinetics of IRF-5 nuclear localization. CONCLUSION: This study provides the first formal proof that IRF-5 activation is altered in the monocytes of SLE patients, which can be attributed, in part, to the SLE blood environment.

A New Methodology for the Quantification of Neutrophil Extracellular Traps in Patient Plasma.

Neutrophil extracellular traps (NETs) are web-like structures made up of decondensed chromatin fibers along with neutrophil granular proteins that are extruded by neutrophils after activation or in response to foreign microorganisms. NETs have been associated with autoimmune and inflammatory diseases such as systemic lupus erythematosus (SLE), rheumatoid arthritis, coronavirus disease 2019 (COVID-19), and others. There are reliable methods available to quantitate NETs from neutrophils, but their accurate quantification in patient plasma or serum remains a challenge. We developed a highly sensitive ELISA to detect NETs in serum/plasma and designed a novel smear immunofluorescence assay to detect NETs in as little as 1 µL of serum/plasma. We further validated our technology on plasma samples from SLE patients and healthy donors that carry interferon regulatory factor 5 genetic risk. The multiplex ELISA combines the use of three antibodies against myeloperoxidase (MPO), citrullinated histone H3 (CitH3), and DNA to detect the NET complexes with higher specificities. The immunofluorescence smear assay can visually detect intact structures of NETs in 1 µL of serum/plasma and provide similar results that correlate with findings from the multiplex ELISA. Furthermore, the smear assay is a relatively simple, inexpensive, and quantifiable method of NET detection for small volumes.

Identification of pan-cancer/testis genes and validation of therapeutic targeting in triple-negative breast cancer: Lin28a- and Siglece-based vaccination induces anti-tumor 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 with high immunogenicity are also lacking. Methods: Sequencing data from the Genotype-Tissue Expression (GTEx) and The Genomic Data Commons (GDC) databases was utilized 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 with 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 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 CT antigens as appealing targets in low mutation burden tumors. Importantly, tumor germness also correlated with markers of anti-tumor immunity. Vaccination of 4T1 tumor bearing mice with Siglece and Lin28a antigens resulted in increased T cell anti-tumor immunity and reduced primary tumor growth and lung metastases. Conclusion: Our results present a novel strategy for the identification of highly immunogenic CT antigens for the development of targeted vaccines that induce anti-tumor immunity and inhibit metastasis.