A RUNX-CBFß-driven enhancer directs the Irf8 dose-dependent lineage choice between DCs and monocytes.

The transcription factor IRF8 is essential for the development of monocytes and dendritic cells (DCs), whereas it inhibits neutrophilic differentiation. It is unclear how Irf8 expression is regulated and how this single transcription factor supports the generation of both monocytes and DCs. Here, we identified a RUNX-CBFß-driven enhancer 56 kb downstream of the Irf8 transcription start site. Deletion of this enhancer in vivo significantly decreased Irf8 expression throughout the myeloid lineage from the progenitor stages, thus resulting in loss of common DC progenitors and overproduction of Ly6C+ monocytes. We demonstrated that high, low or null expression of IRF8 in hematopoietic progenitor cells promotes differentiation toward type 1 conventional DCs, Ly6C+ monocytes or neutrophils, respectively, via epigenetic regulation of distinct sets of enhancers in cooperation with other transcription factors. Our results illustrate the mechanism through which IRF8 controls the lineage choice in a dose-dependent manner within the myeloid cell system.

Lyn Kinase Suppresses the Transcriptional Activity of IRF5 in the TLR-MyD88 Pathway to Restrain the Development of Autoimmunity.

Interferon regulatory factor-5 (IRF5), a transcription factor critical for the induction of innate immune responses, contributes to the pathogenesis of the autoimmune disease systemic lupus erythematosus (SLE) in humans and mice. Lyn, a Src family kinase, is also implicated in human SLE, and Lyn-deficient mice develop an SLE-like disease. Here, we found that Lyn physically interacted with IRF5 to inhibit ubiquitination and phosphorylation of IRF5 in the TLR-MyD88 pathway, thereby suppressing the transcriptional activity of IRF5 in a manner independent of Lyn's kinase activity. Conversely, Lyn did not inhibit NF-κB signaling, another major branch downstream of MyD88. Monoallelic deletion of Irf5 alleviated the hyperproduction of cytokines in TLR-stimulated Lyn(-/-) dendritic cells and the development of SLE-like symptoms in Lyn(-/-) mice. Our results reveal a role for Lyn as a specific suppressor of the TLR-MyD88-IRF5 pathway and illustrate the importance of fine-tuning IRF5 activity for the maintenance of immune homeostasis.

Regulation and role of the transcription factor IRF5 in innate immune responses and systemic lupus erythematosus.

The transcription factor interferon regulatory factor-5 (IRF5) plays an important role in innate immune responses via the TLR-MyD88 (Toll-like receptor - myeloid differentiation primary response 88) pathway. IRF5 is also involved in the pathogenesis of the autoimmune disease systemic lupus erythematosus (SLE). Recent studies have identified new regulators, both positive and negative, which act on IRF5 activation events in the TLR-MyD88 pathway such as post-translational modifications, dimerization and nuclear translocation. A model of the causal relationship between IRF5 activation and SLE pathogenesis proposes that a loss of the negative regulation of IRF5 causes its hyperactivation, resulting in hyperproduction of type I interferons and other cytokines, and ultimately in the development of SLE. Importantly, to our knowledge, all murine models of SLE studied thus far have shown that IRF5 is required for the pathogenesis of SLE-like diseases. During the development of SLE-like diseases, IRF5 plays key roles in various cell types, including dendritic cells and B cells. It is noteworthy that the onset of SLE-like diseases can be inhibited by reducing the activity or amount of IRF5 by half. Therefore, IRF5 is an important therapeutic target of SLE, and selective suppression of its activity and expression may potentially lead to the development of new therapies.

Essential role of the IRF8-KLF4 transcription factor cascade in murine monocyte differentiation.

Monocytes regulate host defenses, inflammation, and tissue homeostasis. The transcription factor interferon regulatory factor-8 (IRF8) stimulates monocyte/macrophage differentiation, yet genome-wide understanding of the differentiation program initiated by IRF8 is lacking. By combining chromatin immunoprecipitation sequencing with gene expression profiling, we show that during IRF8-dependent monocyte differentiation, IRF8 binding occurs at both promoter-proximal and promotor-distal regions together with the transcription factor PU.1 and is associated with gene induction. Many of the promoter-distal IRF8 binding sites show an increase in histone H3 lysine 4 monomethylation, a signature for enhancers. However, about half the IRF8-induced genes were not bound by IRF8, suggesting the involvement of downstream transcription factors. Analysis of DNA motifs in cis-regulatory elements of these indirect IRF8 target genes predicted that Krüppel-like factor-4 (KLF4)-essential for Ly6C(+) monocyte development-is one such factor. Indeed, monocyte development in Irf8(-/-) mice is as defective as that in Klf4(-/-) chimeric mice. Moreover, Irf8(-/-) monocyte-dendritic cell progenitors do not express Klf4 messenger RNA. Introduction of KLF4 into an Irf8(-/-) myeloid progenitor cell line induced a subset of IRF8 target genes and caused partial monocyte differentiation. Taken together, our present results uncover genome-wide behavior of IRF8 and identify an IRF8-KLF4 axis that operates during monocyte differentiation.

High-mobility group box family of proteins: ligand and sensor for innate immunity.

Recent discoveries in signal-transducing innate receptors have illustrated the close link between innate and adaptive immunity. These advances revisit a fundamental issue of immunology, the recognition of self and non-self molecules by the immune system. Indeed, mounting evidence has been provided that the sensing of self-derived molecules by the immune system is important for health and disease. The high-mobility group box (HMGB) proteins, particularly HMGB1, are self-derived immune activators that have multiple functions in the regulation of immunity and inflammation. In this review, we summarize current knowledge of the function of HMGB proteins, as a ligand that can evoke inflammatory responses, and as a sensor for nucleic-acid-mediated immune responses.

HMGB proteins function as universal sentinels for nucleic-acid-mediated innate immune responses.

The activation of innate immune responses by nucleic acids is crucial to protective and pathological immunities and is mediated by the transmembrane Toll-like receptors (TLRs) and cytosolic receptors. However, it remains unknown whether a mechanism exists that integrates these nucleic-acid-sensing systems. Here we show that high-mobility group box (HMGB) proteins 1, 2 and 3 function as universal sentinels for nucleic acids. HMGBs bind to all immunogenic nucleic acids examined with a correlation between affinity and immunogenic potential. Hmgb1(-/-) and Hmgb2(-/-) mouse cells are defective in type-I interferon and inflammatory cytokine induction by DNA or RNA targeted to activate the cytosolic nucleic-acid-sensing receptors; cells in which the expression of all three HMGBs is suppressed show a more profound defect, accompanied by impaired activation of the transcription factors interferon regulatory factor 3 (IRF3) and nuclear factor (NF)-kappaB. The absence of HMGBs also severely impairs the activation of TLR3, TLR7 and TLR9 by their cognate nucleic acids. Our results therefore indicate a hierarchy in the nucleic-acid-mediated activation of immune responses, wherein the selective activation of nucleic-acid-sensing receptors is contingent on the more promiscuous sensing of nucleic acids by HMGBs. These findings may have implications for understanding the evolution of the innate immune system and for the treatment of immunological disorders.

Suppression of immune responses by nonimmunogenic oligodeoxynucleotides with high affinity for high-mobility group box proteins (HMGBs).

The activation of innate immune responses by nucleic acids is central to the generation of host responses against pathogens; however, nucleic acids can also trigger the development and/or exacerbation of pathogenic responses such as autoimmunity. We previously demonstrated that the selective activation of nucleic acid-sensing cytosolic and Toll-like receptors is contingent on the promiscuous sensing of nucleic acids by high-mobility group box proteins (HMGBs). From this, we reasoned that nonimmunogenic nucleotides with high-affinity HMGB binding may function as suppressing agents for HMGB-mediated diseases, particularly those initiated and/or exacerbated by nucleic acids. Here we characterize an array of HMGB-binding, nonimmunogenic oligodeoxynucleotides (ni-ODNs). Interestingly, we find that binding affinity is rather independent of nucleotide sequence, but is instead dependent on length and structure of the deoxyribose backbone. We further show that these ni-ODNs can strongly suppress the activation of innate immune responses induced by both classes of nucleic acid-sensing receptors. We also provide evidence for the suppressive effect of an ni-ODN, termed ISM ODN, on the induction of adaptive immune responses and in mouse models of sepsis and autoimmunity. We discuss our findings in relation to the critical role of HMGBs in initiating immune responses and the possible use of these ni-ODNs in therapeutic interventions.

Physical and functional interaction among Irf8 enhancers during dendritic cell differentiation.

The production of type 1 conventional dendritic cells (cDC1s) requires high expression of the transcription factor IRF8. Three enhancers at the Irf8 3' region function in a differentiation stage-specific manner. However, whether and how these enhancers interact physically and functionally remains unclear. Here, we show that the Irf8 3' enhancers directly interact with each other and contact the Irf8 gene body during cDC1 differentiation. The +56 kb enhancer, which functions from multipotent progenitor stages, activates the other 3' enhancers through an IRF8-dependent transcription factor program, that is, in trans. Then, the +32 kb enhancer, which operates in cDC1-committed cells, reversely acts in cis on the other 3' enhancers to maintain the high expression of Irf8. Indeed, mice with compound heterozygous deletion of the +56 and +32 kb enhancers are unable to generate cDC1s. These results illustrate how multiple enhancers cooperate to induce a lineage-determining transcription factor gene during cell differentiation.

DAI (DLM-1/ZBP1) is a cytosolic DNA sensor and an activator of innate immune response.

Central to innate immunity is the sensing of pathogen-associated molecular patterns by cytosolic and membrane-associated receptors. In particular, DNA is a potent activator of immune responses during infection or tissue damage, and evidence indicates that, in addition to the membrane-associated Toll-like receptor 9, an unidentified cytosolic DNA sensor(s) can activate type I interferon (IFN) and other immune responses. Here we report on a candidate DNA sensor, previously named DLM-1 (also called Z-DNA binding protein 1 (ZBP1)), for which biological function had remained unknown; we now propose the alternative name DAI (DNA-dependent activator of IFN-regulatory factors). The artificial expression of otherwise IFN-inducible DAI (DLM-1/ZBP1) in mouse fibroblasts selectively enhances the DNA-mediated induction of type I IFN and other genes involved in innate immunity. On the other hand, RNA interference of messenger RNA for DAI (DLM-1/ZBP1) in cells inhibits this gene induction programme upon stimulation by DNA from various sources. Moreover, DAI (DLM-1/ZBP1) binds to double-stranded DNA and, by doing so, enhances its association with the IRF3 transcription factor and the TBK1 serine/threonine kinase. These observations underscore an integral role of DAI (DLM-1/ZBP1) in the DNA-mediated activation of innate immune responses, and may offer new insight into the signalling mechanisms underlying DNA-associated antimicrobial immunity and autoimmune disorders.

Machine learning for prediction of immunotherapeutic outcome in non-small-cell lung cancer based on circulating cytokine signatures.

BACKGROUND: Immune checkpoint inhibitor (ICI) therapy has substantially improved the overall survival (OS) in patients with non-small-cell lung cancer (NSCLC); however, its response rate is still modest. In this study, we developed a machine learning-based platform, namely the Cytokine-based ICI Response Index (CIRI), to predict the ICI response of patients with NSCLC based on the peripheral blood cytokine profiles. METHODS: We enrolled 123 and 99 patients with NSCLC who received anti-PD-1/PD-L1 monotherapy or combined chemotherapy in the training and validation cohorts, respectively. The plasma concentrations of 93 cytokines were examined in the peripheral blood obtained from patients at baseline (pre) and 6 weeks after treatment (early during treatment: edt). Ensemble learning random survival forest classifiers were developed to select feature cytokines and predict the OS of patients undergoing ICI therapy. RESULTS: Fourteen and 19 cytokines at baseline and on treatment, respectively, were selected to generate CIRI models (namely preCIRI14 and edtCIRI19), both of which successfully identified patients with worse OS in two completely independent cohorts. At the population level, the prediction accuracies of preCIRI14 and edtCIRI19, as indicated by the concordance indices (C-indices), were 0.700 and 0.751 in the validation cohort, respectively. At the individual level, patients with higher CIRI scores demonstrated worse OS [hazard ratio (HR): 0.274 and 0.163, and p<0.0001 and p=0.0044 in preCIRI14 and edtCIRI19, respectively]. By including other circulating and clinical features, improved prediction efficacy was observed in advanced models (preCIRI21 and edtCIRI27). The C-indices in the validation cohort were 0.764 and 0.757, respectively, whereas the HRs of preCIRI21 and edtCIRI27 were 0.141 (p<0.0001) and 0.158 (p=0.038), respectively. CONCLUSIONS: The CIRI model is highly accurate and reproducible in determining the patients with NSCLC who would benefit from anti-PD-1/PD-L1 therapy with prolonged OS and may aid in clinical decision-making before and/or at the early stage of treatment.

A selective contribution of the RIG-I-like receptor pathway to type I interferon responses activated by cytosolic DNA.

The activation of the innate immune responses by DNA exposed within the cytosol has gained much attention and, in this context, several cytosolic DNA sensors have been identified. However, previous studies revealed the operation of redundant and complex mechanisms and it still remains to be clarified how the DNA-mediated evocation of diverse innate immune responses can be achieved. Here we show that two RIG-I-like receptors (RLRs), RIG-I and MDA5, known as cytosolic RNA receptors, nonredundantly function as cytosolic DNA receptors that lead to the selective activation of type I IFN genes. We demonstrate that overexpression of otherwise IFN-inducible RIG-I or MDA5 in IFN signal-deficient cells results in a marked enhancement of type I IFN gene induction upon cytosolic DNA stimulation, while in their absence the induction is impaired. Interestingly, the DNA-mediated induction of other cytokine genes was barely affected by the absence of RLRs. Indeed, unlike the RNA-RLR pathway that activates the transcription factors IRF3 and NF-kappaB, the DNA-RLR pathway is primarily responsible for the IRF3 activation critical for type I IFN gene transcription, illustrating a deliberate divergence of the DNA signaling pathways. Expectedly, the RLR pathway also contributes to intricate innate immune responses against infection by a DNA virus. Our study may provide insights into the complexity of host defense mechanisms that thwart immune evasion by DNA-containing pathogens.

Regulation of innate immune responses by DAI (DLM-1/ZBP1) and other DNA-sensing molecules.

DNA, whether it is microbe-derived or host-derived, evokes immune responses when exposed to the cytosol of a cell. We previously reported that DNA-dependent activator of IFN regulatory factors (DAI), also referred to as DLM-1/ZBP1, functions as a DNA sensor that activates the innate immune system. In the present study, we examined the regulation of the complex DNA-sensing system by DAI and other molecules. We first show that DAI directly interacts with DNA in vitro and that it requires three DNA-binding domains for full activation in vivo. We also show that the artificially induced dimerization of DAI results in the DNA-independent activation of type I IFN genes, thereby providing a better understanding for the molecular basis of DAI activation. Furthermore, we provide evidence for the presence of additional DNA sensors, either positively or negatively regulating cytosolic DNA-mediated innate immune responses. These results in toto provide insights into the mechanism of DAI activation and reveal the complex regulatory mechanisms underlying DNA-mediated protective and pathologic immune responses.

Irf5 siRNA-loaded biodegradable lipid nanoparticles ameliorate concanavalin A-induced liver injury.

RNA interference-based gene silencing drugs are attracting attention for treating various diseases. Lipid nanoparticles (LNPs) are carriers that efficiently deliver small interfering RNA (siRNA) to the cytoplasm of target cells. Recently, we developed potent and well-tolerated biodegradable LNPs with asymmetric ionizable lipids. Here, we evaluated the effect of LNPs on immune cells in mice. After intravenous administration, LNPs were efficiently incorporated into several tissue-resident macrophages, including liver macrophages, through an apolipoprotein E (ApoE)-independent mechanism. Administration of LNP-encapsulated siRNA against Irf5, encoding the transcription factor critical for inflammatory responses, sharply reduced its expression in macrophages in vivo, and persisted for as long as 7 days. The therapeutic potential of Irf5 siRNA-loaded LNPs in inflammatory diseases was tested in a concanavalin A (Con A)-induced hepatitis model, whose pathogenic mechanisms are dependent on cytokine secretion from macrophages. We found that Con A-induced liver injury was significantly attenuated after LNP injection. Serum aspartate transaminase, alanine aminotransferase, and inflammatory cytokine levels were significantly reduced in mice injected with Irf5 siRNA-loaded LNPs compared to those injected with control siRNA-loaded LNPs. Our results suggest that administering biodegradable LNPs to deliver siRNA is a promising strategy for treating inflammatory disorders.

Genetic and chemical inhibition of IRF5 suppresses pre-existing mouse lupus-like disease.

The transcription factor IRF5 has been implicated as a therapeutic target for the autoimmune disease systemic lupus erythematosus (SLE). However, IRF5 activation status during the disease course and the effects of IRF5 inhibition after disease onset are unclear. Here, we show that SLE patients in both the active and remission phase have aberrant activation of IRF5 and interferon-stimulated genes. Partial inhibition of IRF5 is superior to full inhibition of type I interferon signaling in suppressing disease in a mouse model of SLE, possibly due to the function of IRF5 in oxidative phosphorylation. We further demonstrate that inhibition of IRF5 via conditional Irf5 deletion and a newly developed small-molecule inhibitor of IRF5 after disease onset suppresses disease progression and is effective for maintenance of remission in mice. These results suggest that IRF5 inhibition might overcome the limitations of current SLE therapies, thus promoting drug discovery research on IRF5 inhibitors.

Combining IL-6 and SARS-CoV-2 RNAaemia-based risk stratification for fatal outcomes of COVID-19.

BACKGROUND: The coronavirus disease 2019 (COVID-19) pandemic rapidly increases the use of mechanical ventilation (MV). Such cases further require extracorporeal membrane oxygenation (ECMO) and have a high mortality. OBJECTIVE: We aimed to identify prognostic biomarkers pathophysiologically reflecting future deterioration of COVID-19. METHODS: Clinical, laboratory, and outcome data were collected from 102 patients with moderate to severe COVID-19. Interleukin (IL)-6 level and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA copy number in plasma were assessed with ELISA kit and quantitative PCR. RESULTS: Twelve patients died or required ECMO owing to acute respiratory distress syndrome despite the use of MV. Among various variables, a ratio of oxygen saturation to fraction of inspired oxygen (SpO2/FiO2), IL-6, and SARS-CoV-2 RNA on admission before intubation were strongly predictive of fatal outcomes after the MV use. Moreover, among these variables, combining SpO2/FiO2, IL-6, and SARS-CoV-2 RNA showed the highest accuracy (area under the curve: 0.934). In patients with low SpO2/FiO2 (< 261), fatal event-rate after the MV use at the 30-day was significantly higher in patients with high IL-6 (> 49 pg/mL) and SARS-CoV-2 RNAaemia (> 1.5 copies/µL) compared to those with high IL-6 or RNAaemia or without high IL-6 and RNAaemia (88% vs. 22% or 8%, log-rank test P = 0.0097 or P < 0.0001, respectively). CONCLUSIONS: Combining SpO2/FiO2 with high IL-6 and SARS-CoV-2 RNAaemia which reflect hyperinflammation and viral overload allows accurately and before intubation identifying COVID-19 patients at high risk for ECMO use or in-hospital death despite the use of MV.

Phos-tag Immunoblot Analysis for Detecting IRF5 Phosphorylation.

While the activation of the transcription factor interferon regulatory factor 5 (IRF5) is critical for the induction of innate immune responses, it also contributes to the pathogenesis of the autoimmune disease systemic lupus erythematosus (SLE). IRF5 phosphorylation is a hallmark of its activation in the Toll-like receptor (TLR) pathway, where active IRF5 induces type I interferon and proinflammatory cytokine genes. By using the phosphate-binding molecule Phos-tag, without either radioisotopes or phospho-specific antibodies, the protocol described here enables detection of the phosphorylation of both human and murine IRF5, as well as that of other proteins.

IRF8 inhibits C/EBPα activity to restrain mononuclear phagocyte progenitors from differentiating into neutrophils.

Myeloid progenitors lose their potential to generate neutrophils when they adopt the mononuclear phagocyte lineage. The mechanism underlying this lineage restriction remains unknown. We here report that the protein expression of IRF8, an essential transcription factor for the development of dendritic cells (DCs) and monocytes, sharply increases at the monocyte-DC progenitor (MDP) stage and remains high in common monocyte progenitors (cMoPs). Irf8(-/-) MDPs and cMoPs accumulate but fail to efficiently generate their downstream populations, instead giving rise to neutrophils in vivo. IRF8 physically interacts with the transcription factor C/EBPα and prevents its binding to chromatin in MDPs and cMoPs, blocking the ability of C/EBPα to stimulate transcription and neutrophil differentiation. A partial inhibition of C/EBP activity in Irf8(-/-) haematopoietic progenitors alleviates the neutrophil overproduction in vivo. Thus, IRF8 not only bestows monocyte and DC differentiation potential upon mononuclear phagocyte progenitors but also restrains these progenitors from differentiating into neutrophils.