IFN Regulatory Factor Research Articles

Extracellular CIRP activates STING to exacerbate hemorrhagic shock.

Stimulator of IFN genes (STING) activates TANK-binding kinase 1 (TBK1) and IFN regulatory factor 3 (IRF3) to produce type I IFNs. Extracellular cold-inducible RNA-binding protein (eCIRP) is released from cells during hemorrhagic shock (HS). We hypothesized that eCIRP activates STING to induce inflammation and acute lung injury (ALI) after HS. WT and STING–/– mice underwent controlled hemorrhage by bleeding, followed by fluid resuscitation. Blood and lungs were collected at 4 hours after resuscitation. Serum ALT, AST, LDH, IL-6, and IFN-β were significantly decreased in STING–/– mice compared with WT mice after HS. In STING–/– mice, the levels of pTBK1 and pIRF3, and expression of TNF-α, IL-6, and IL-1β mRNAs and proteins in the lungs, were significantly decreased compared with WT HS mice. The 10-day mortality rate in STING–/– mice was significantly reduced. I.v. injection of recombinant mouse CIRP (rmCIRP) in STING–/– mice showed a significant decrease in pTBK1 and pIRF3 and in IFN-α and IFN-β mRNAs and proteins in the lungs compared with rmCIRP-treated WT mice. Treatment of TLR4–/–, MyD88–/–, and TRIF–/– macrophages with rmCIRP significantly decreased pTBK1 and pIRF3 levels and IFN-α and IFN-β mRNAs and proteins compared with WT macrophages. HS increases eCIRP levels, which activate STING through TLR4/MyD88/TRIF pathways to exacerbate inflammation.

Zebrafish Uba1 Degrades IRF3 through K48-Linked Ubiquitination to Inhibit IFN Production.

Fish IFN regulatory factor 3 (IRF3) is a crucial transcription factor in the IFN activation signaling pathway, which leads to IFN production and a positive cycle. Unrestricted IFN expression results in hyperimmune responses and therefore, IFN must be tightly regulated. In the current study, we found that zebrafish Ub-activating enzyme (Uba1) negatively regulated IRF3 via the K-48 ubiquitin proteasome degradation of IRF3. First, ifn expression stimulated by spring viraemia of carp virus infection was blunted by the overexpression of Uba1 and enhanced by Uba1 knockdown. Afterward, we found that Uba1 was localized in the cytoplasm, where it interacted with and degraded IRF3. Functional domains analysis revealed that the C-terminal ubiquitin-fold domain was necessary for IRF3 degradation by Uba1 and the N-terminal DNA-binding domain of IRF3 was indispensable for the degradation by Uba1.The degradation of IRF3 was subsequently impaired by treatment with MG132, a ubiquitin proteasome inhibitor. Further mechanism analysis revealed that Uba1 induced the K48-linked Ub-proteasomal degradation of IRF3. Finally, the antiviral capacity of IRF3 was significantly attenuated by Uba1. Taken together, our study reveals that zebrafish Uba1 interacts with and activates the ubiquitinated degradation of IRF3, providing evidence of the IFN immune balance mechanism in fish.

Nonstructural Protein NSs Hampers Cellular Antiviral Response through LSm14A during Severe Fever with Thrombocytopenia Syndrome Virus Infection.

The nonstructural protein (NSs) of severe fever with thrombocytopenia syndrome virus (SFTSV) plays multiple functions in the virus life cycle. Proteomic screening for host proteins interacting with NSs identified the cellular protein LSm14A. LSm14A, a member of the LSm family involved in RNA processing in the processing bodies, binds to viral RNA or synthetic homolog and mediates IFN regulatory factor 3 activation and IFN-β induction. NSs interacted with and colocalized with LSm14A, and this interaction effectively inhibited downstream phosphorylation and dimerization of IFN regulatory factor 3, resulting in the suppression of antiviral signaling and IFN induction in several cell types of human origin. Knockdown of NSs resulted in the suppression of SFTSV replication in host cells. Viral RNA bound to LSm14A-NSs protein complex during the interaction. A newly discovered LRRD motif of NSs functioned to interact with LSm14A. Altogether, our data demonstrated a mechanism used by SFTSV to inhibit host innate immune response.

Human Cytomegalovirus UL23 Attenuates Signal Transducer and Activator of Transcription 1 Phosphorylation and Type I Interferon Response.

Human cytomegalovirus (HCMV), the human beta-herpesvirus, can cause severe syndromes among both immunocompromised adult patients and newborns. Type I interferon (IFN-I) exerts an important effect to resist infections caused by viruses such as HCMV, while IFN evasion may serve as a key determining factor for viral dissemination and disease occurrence within hosts. In this study, UL23, a tegument protein of HCMV, was confirmed to be a key factor for negatively regulating the type I IFN immune response. A detailed analysis indicated that the viral UL23 protein increases the IFN-I antiviral resistance during HCMV infections. Furthermore, UL23 was shown to significantly reduce the levels of IFN-stimulated genes (ISGs) and promoter activity of IFN-I-stimulated response element. Mechanically, UL23 was discovered to impair the signal transducer and activator of transcription 1 (STAT1) phosphorylation, although it was not found to affect phosphorylation and expression of STAT2, Janus activated kinase 1, or tyrosine kinase 2, which are associated with IFN-I signal transduction pathway. Additionally, a significantly reduced nuclear expression of STAT1 but not of IFN regulatory factor 9 or STAT2 was observed. Findings of this study indicate that HCMV UL23 is a viral antagonist that acts against the cellular innate immunity and reveal a possible novel effect of UL23 on IFN-I signaling.

Monoallelic IRF5 deficiency in B cells prevents murine lupus.

Gain-of-function polymorphisms in the transcription factor IFN regulatory factor 5 (IRF5) are associated with an increased risk of developing systemic lupus erythematosus. However, the IRF5-expressing cell type(s) responsible for lupus pathogenesis in vivo is not known. We now show that monoallelic IRF5 deficiency in B cells markedly reduced disease in a murine lupus model. In contrast, similar reduction of IRF5 expression in macrophages, monocytes, and neutrophils did not reduce disease severity. B cell receptor and TLR7 signaling synergized to promote IRF5 phosphorylation and increase IRF5 protein expression, with these processes being independently regulated. This synergy increased B cell–intrinsic IL-6 and TNF-α production, both key requirements for germinal center (GC) responses, with IL-6 and TNF-α production in vitro and in vivo being substantially lower with loss of 1 allele of IRF5. Mechanistically, TLR7-dependent IRF5 nuclear translocation was reduced in B cells from IRF5-heterozygous mice. In addition, we show in multiple lupus models that IRF5 expression was dynamically regulated in vivo with increased expression in GC B cells compared with non-GC B cells and with further sequential increases during progression to plasmablasts and long-lived plasma cells. Overall, a critical threshold level of IRF5 in B cells was required to promote disease in murine lupus.

Teleost-Specific MxG, a Traitor in the Mx Family, Negatively Regulates Antiviral Responses by Targeting IPS-1 for Proteasomal Degradation and STING for Lysosomal Degradation

Abstract IFN-β promoter stimulator-1 (IPS-1)– and stimulator of IFN genes (STING)-mediated type I IFNs play a critical role in antiviral responses. Myxovirus resistance (Mx) proteins are pivotal components of the antiviral effectors induced by IFNs in many species. An unprecedented expansion of Mx genes has occurred in fish. However, the functions and mechanisms of Mx family members remain largely unknown in fish. In this study, we found that grass carp (Ctenopharyngodon idella) MxG, a teleost-specific Mx protein, is induced by IFNs and viruses, and it negatively regulates both IPS-1- and STING-mediated antiviral responses to facilitate grass carp reovirus, spring viremia of carp virus, and cyprinid herpesvirus-2 replication. MxG binds and degrades IPS-1 via the proteasomal pathway and STING through the lysosomal pathway, thereby negatively regulating IFN1 antiviral responses and NF-κB proinflammatory cytokines. MxG also suppresses the phosphorylation of STING IFN regulatory factor 3/7, and it subsequently downregulates IFN1 and NF-κB1 at the promoter, transcription, and protein levels. GTPase and GTPase effector domains of MxG contribute to the negative regulatory function. On the contrary, MxG knockdown weakens virus replication and cytopathic effect. Therefore, MxG can be an ISG molecule induced by IFNs and viruses, and degrade IPS-1 and STING proteins in a negative feedback manner to maintain homeostasis and avoid excessive immune responses after virus infection. To our knowledge, this is the first identification of a negative regulator in the Mx family, and our findings clarify a novel mechanism by which the IFN response is regulated.

Clinically used JAK inhibitor blunts dsRNA-induced inflammation and calcification in aortic valve interstitial cells.

Calcific aortic valve disease (CAVD) is the most prevalent valvulopathy worldwide. Growing evidence supports a role for viral and cell-derived double-stranded (ds)-RNA in cardiovascular pathophysiology. Poly(I:C), a dsRNA surrogate, has been shown to induce inflammation, type I interferon (IFN) responses, and osteogenesis through Toll-like receptor 3 in aortic valve interstitial cells (VIC). Here, we aimed to determine whether IFN signaling via Janus kinase (JAK)/Signal transducers and activators of transcription (STAT) mediates dsRNA-induced responses in primary human VIC. Western blot, ELISA, qPCR, calcification, flow cytometry, and enzymatic assays were performed to evaluate the mechanisms of dsRNA-induced inflammation and calcification. Poly(I:C) triggered a type I IFN response characterized by IFN-regulatory factors gene upregulation, IFN-β secretion, and STAT1 activation. Additionally, Poly(I:C) promoted VIC inflammation via NF-κB and subsequent adhesion molecule expression, and cytokine secretion. Pretreatment with ruxolitinib, a clinically used JAK inhibitor, abrogated these responses. Moreover, Poly(I:C) promoted a pro-osteogenic phenotype and increased VIC calcification to a higher extent in cells from males. Inhibition of JAK with ruxolitinib or a type I IFN receptor blocking antibody blunted Poly(I:C)-induced calcification. Mechanistically, Poly(I:C) promoted VIC apoptosis in calcification medium, which was inhibited by ruxolitinib. Moreover, Poly(I:C) co-operated with IFN-γ to increase VIC calcification by synergistically activating extracellular signal-regulated kinases and hypoxia-inducible factor-1α pathways. In conclusion, JAK/STAT signaling mediates dsRNA-triggered inflammation, apoptosis, and calcification and may contribute to a positive autocrine loop in human VIC in the presence of IFN-γ. Blockade of dsRNA responses with JAK inhibitors may be a promising therapeutic avenue for CAVD.

Bovine viral diarrhea virus NS4B protein interacts with 2CARD of MDA5 domain and negatively regulates the RLR-mediated IFN-β production

Bovine viral diarrhea virus (BVDV) is an important member of the family Flaviviridae and often causes immunosuppression. Previous studies have suggested that BVDV envelope protein Erns and the nonstructural autoprotease Npro can inhibit host innate immune responses. Herein, we found that BVDV NS4B, as a nonstructural protein necessary for replication, is involved in antagonizing the main RNA virus sensing pathway. Overexpression of BVDV NS4B protein significantly inhibited Sendai virus (SeV)-induced interferon-β promoter activity, IFN-β mRNA and IFN regulatory factor 3 (IRF3) phosphorylation levels. We also discovered that BVDV NS4B protein significantly inhibited RIG-I like receptor (RLRs)-mediated interferon-β (IFN-β) promoter activity and endogenous MDA5 mRNA levels. In addition, the BVDV NS4B protein directly interacts with N-terminal CARDs of MDA5, and co-localized with MDA5 or MDA5–2CARD in the cytoplasm. In summary, the results of this study indicate that the BVDV NS4B protein acts as an interferon-β antagonist through inhibiting the MDA5-mediated signal transduction pathway. Our study provides an in-depth understanding of the molecular mechanisms of BVDV evading the host's natural immune response.

Targeted Transcriptomics of Frog Virus 3 in Infected Frog Tissues Reveal Non-Coding Regulatory Elements and microRNAs in the Ranaviral Genome and Their Potential Interaction with Host Immune Response.

BackgroundFrog Virus 3 (FV3) is a large dsDNA virus belonging to Ranaviruses of family Iridoviridae. Ranaviruses infect cold-blood vertebrates including amphibians, fish and reptiles, and contribute to catastrophic amphibian declines. FV3 has a genome at ~105 kb that contains nearly 100 coding genes and 50 intergenic regions as annotated in its reference genome. Previous studies have mainly focused on coding genes and rarely addressed potential non-coding regulatory role of intergenic regions.ResultsUsing a whole transcriptomic analysis of total RNA samples containing both the viral and cellular transcripts from FV3-infected frog tissues, we detected virus-specific reads mapping in non-coding intergenic regions, in addition to reads from coding genes. Further analyses identified multiple cis-regulatory elements (CREs) in intergenic regions neighboring highly transcribed coding genes. These CREs include not only a virus TATA-Box present in FV3 core promoters as in eukaryotic genes, but also viral mimics of CREs interacting with several transcription factors including CEBPs, CREBs, IRFs, NF-κB, and STATs, which are critical for regulation of cellular immunity and cytokine responses. Our study suggests that intergenic regions immediately upstream of highly expressed FV3 genes have evolved to bind IRFs, NF-κB, and STATs more efficiently. Moreover, we found an enrichment of putative microRNA (miRNA) sequences in more than five intergenic regions of the FV3 genome. Our sequence analysis indicates that a fraction of these viral miRNAs is targeting the 3’-UTR regions of Xenopus genes involved in interferon (IFN)-dependent responses, including particularly those encoding IFN receptor subunits and IFN-regulatory factors (IRFs).ConclusionsUsing the FV3 model, this study provides a first genome-wide analysis of non-coding regulatory mechanisms adopted by ranaviruses to epigenetically regulate both viral and host gene expressions, which have co-evolved to interact especially with the host IFN response.

Porcine Epidemic Diarrhea Virus Membrane Protein Interacted with IRF7 to Inhibit Type I IFN Production during Viral Infection.

Porcine epidemic diarrhea virus (PEDV) is a highly pathogenic porcine enteropathogenic coronavirus causing severe enteritis and lethal watery diarrhea in piglets. PEDV infection suppresses the synthesis of type I IFN, and multiple viral proteins of PEDV have been shown to target the adaptors of innate immune pathways to inhibit type I IFN production. In this study, we identified PEDV membrane (M) protein as a new antagonist of type I IFN production in both human embryonic kidney HEK293T cells and porcine kidney PK-15 cells and determined the antagonistic mechanism used by M protein to target IFN regulatory factor 7 (IRF7), an important regulator of type I IFN production. IRF7 is phosphorylated and activated by TBK1 and IKKε in response to viral infection. We found that PEDV M protein interacted with the inhibitory domain of IRF7 and significantly suppressed TBK1/IKKε-induced IRF7 phosphorylation and dimerization of IRF7, leading to the decreased expression of type I IFN, although it did not affect the interaction between TBK1/IKKε and IRF7. As expected, overexpression of M protein significantly increased PEDV replication in porcine cells. The M proteins of both epidemic PEDV strains and vaccine strain showed similar antagonistic effect on type I IFN production, and the 1-55 region of M protein was essential for disruption of IRF7 function by interacting with IRF7. Taken together, our data identified a new, to our knowledge, IFN antagonist of PEDV, as well as a novel, to our knowledge, antagonistic mechanism evolved by PEDV to inhibit type I IFN production.

Herpes Simplex Virus Type 2 Glycoprotein D Inhibits NF-κB Activation by Interacting with p65.

NF-κB plays a crucial role in regulating cell proliferation, inflammation, apoptosis, and immune responses. HSV type 2 (HSV-2) is one of the most predominant sexually transmitted pathogens worldwide, and its infection increases the risk of HIV type 1 (HIV-1) acquisition and transmission. HSV-2 glycoprotein D (gD), highly homologous to HSV-1 gD, is essential for viral adhesion, fusion, entry, and spread. It is known that HSV-1 gD can bind herpesvirus entry mediator (HVEM) to trigger NF-κB activation and thereby facilitate viral replication at the early stage of infection. In this study, we found that purified HSV-2 gD triggered NF-κB activation at the early stage of infection, whereas ectopic expression of HSV-2 gD significantly downregulated TNF-α-induced NF-κB activity as well as TNF-α-induced IL-6 and IL-8 expression. Mechanistically, HSV-2 gD inhibited NF-κB, but not IFN-regulatory factor 3 (IRF3), activation and suppressed NF-κB activation mediated by overexpression of TNFR-associated factor 2 (TRAF2), IκB kinase α (IKKα), IKKβ, or p65. Coimmunoprecipitation and binding kinetic analyses demonstrated that HSV-2 gD directly bound to the NF-κB subunit p65 and abolished the nuclear translocation of p65 upon TNF-α stimulation. Mutational analyses further revealed that HSV-2 gD interacted with the region spanning aa 19-187 of p65. Findings in this study together demonstrate that HSV-2 gD interacts with p65 to regulate p65 subcellular localization and thereby prevents NF-κB-dependent gene expression, which may contribute to HSV-2 immune evasion and pathogenesis.

Oxidative stress-induced senescence mediates inflammatory and fibrotic phenotypes in fibroblasts from systemic sclerosis patients.

SSc is an autoimmune connective tissue disorder characterized by inflammation and fibrosis. Although constitutive activation of fibroblasts is proposed to be responsible for the fibrotic and inflammatory features of the disease, the underlying mechanism remains elusive, and effective therapeutic targets are still lacking. The aim of this study was to evaluate the role of oxidative stress-induced senescence and its contribution to the pro-fibrotic and pro-inflammatory phenotypes of fibroblasts from SSc patients. Dermal fibroblasts were isolated from SSc (n = 13) and healthy (n = 10) donors. Fibroblasts' intracellular and mitochondrial reactive oxygen species (ROS) were determined by flow cytometry. Mitochondrial function was measured by Seahorse XF24 analyser. Fibrotic and inflammatory gene expressions were assessed by qPCR and key pro-inflammatory components of the fibroblasts' secretome (IL-6 and IL-8) were quantified by ELISA. Compared with healthy fibroblasts, SSc fibroblasts displayed higher levels of both intracellular and mitochondrial ROS. Oxidative stress in SSc fibroblasts induced the expression of fibrotic genes and activated the TGF-β-activated kinase 1 (TAK1)-IκB kinase β (IKKβ)-IFN regulatory factor 5 (IRF5) inflammatory signalling cascade. These cellular responses paralleled the presence of a DNA damage response, a senescence-associated secretory phenotype and a fibrotic response. Treatment of SSc fibroblasts with ROS scavengers reduced their pro-inflammatory secretome production and fibrotic gene expression. Oxidative stress-induced cellular senescence in SSc fibroblasts underlies their pro-inflammatory and pro-fibrotic phenotypes. Targeting redox imbalance of SSc fibroblasts enhances their in vitro functions and could be of relevance for SSc therapy.

Prevention of Acute Lung Injury by a Novel CD14-Inhibitory Receptor Activator of the NF-κB Ligand Peptide in Mice.

Although CD14 has been implicated in the initiation of multiple TLR-mediated inflammatory responses to sepsis and sepsis-related acute lung injury (ALI), an inhibitor of CD14, except for neutralizing Abs, has not been developed. A partial peptide, microglial healing peptide 1 with N-terminal acetylation and C-terminal amidation (MHP1-AcN), derived from the receptor activator of the NF-кB ligand, was recently found to inhibit multiple TLR signaling in the macrophages. Therefore, we hypothesized that the inhibitory effect of MHP1-AcN might be through the inhibition of CD14, a common coreceptor for multiple TLRs. In cultured mouse macrophages, MHP1-AcN was shown to bind to CD14 and compete with LPS for competitive inhibition of CD14, resulting in inhibition of TLR4 signaling, including NF-кB and IFN regulatory factor 3 activation and nuclear translocation. In addition to TLR2, TLR4, and TLR7, MHP1-AcN also inhibited TLR3 signaling and Escherichia coli DNA-induced, CD14-dependent TLR9 signals; however, CpG oligodeoxynucleotide-induced, CD14-independent TLR9 signals were not inhibited in the mouse macrophages. In sepsis-induced ALI mouse model, MHP1-AcN treatment showed the reduction in the expression of IL-6 and CCL2 in both the serum and lung tissues. IL-6 levels in the bronchoalveolar lavage fluid and pathological score were also decreased by MHP1-AcN. Thus, MHP1-AcN, a novel CD14 inhibitor, could be a promising agent for treating sepsis-induced ALI.

Sustained IL-4 priming of macrophages enhances the inflammatory response to TLR7/8 ligand R848.

Macrophages (Mϕ) are highly plastic, and can acquire a variety of functional phenotypes depending on the presence of different stimuli in their local environment. Mφ stimulated by interleukin (IL)-4 induce an alternative activation state and function as anti-inflammatory cells and promote tissue repair. However, there is overwhelming evidence that IL-4 can play a role in promoting inflammation. In asthma and allergic inflammation, IL-4 mediates proinflammatory responses that lead to tissue damage. Thus the effect of IL-4 on the outcome of the immune responses is greatly influenced by other cofactors and cytokines present in the microenvironment. R848 (resiquimod), a TLR7/8 agonist is a novel vaccine adjuvant, triggering a strong Th1-skewed response but its efficacy as a vaccine adjuvant shows variable results. It is not currently known whether the presence of IL-4 can dampen or enhance immunity in response to TLR7 agonists. In the present study, we sought to investigate the impact of IL-4-induced Mφ polarization on the outcome of R848 stimulation. The activation marker expression and production of cytokines were measured in murine spleen-derived Mφ. Protein expression levels of innate recognition molecules and transcription factors involved, including retinoic-acid inducible gene I, mitochondrial antiviral signaling protein, stimulator of interferon genes (STING), and IFN regulatory factors were evaluated in activated Mφ. These play a crucial role in the control of viral replication and optimal CD8+ T cell priming. We report that sustained priming with IL-4 alone promotes an antiviral response in Mφ, and enhances proinflammatory responses to R848 treatment. This highlights the need for better understanding of IL-4 proinflammatory functions and its potential use as a broad-acting antiviral in combination with R848 may be used in combination with other therapies to target the innate arm of immunity against emerging infections.

Nur77 Attenuates Inflammasome Activation by Inhibiting Caspase-1 Expression in Pulmonary Vascular Endothelial Cells

Inflammasomes are intracellular multiprotein complexes that help trigger and maintain the inflammatory response as part of the innate immune system. Recently, it has been increasingly recognized that aberrant inflammasome activation is critically involved in endothelial dysfunction in a variety of human diseases, such as atherosclerosis, acute lung injury (ALI), and type 2 diabetes. The molecular mechanisms underlying endothelial inflammasome activation, however, have not been completely elucidated. In the present study, we identified orphan nuclear receptor Nur77 as a novel regulator in controlling inflammasome activation in vascular endothelial cells (ECs). We demonstrated that LPS-induced inflammasome activation was significantly inhibited by ectopic overexpression of Nur77, predominantly through transcriptional suppression of caspase-1 expression in vascular ECs. Consistent with this observation, we found that LPS-induced inflammasome activation was significantly augmented in lung ECs isolated from Nur77-knockout mice. Mechanistically, we showed that Nur77-induced inhibition of caspase-1 expression was due to an inhibition of IRF1 (IFN regulatory factor 1) expression and its subsequent binding to the caspase-1 promoter. Importantly, in a mouse model of LPS-induced ALI, Nur77 knockout led to a marked activation of caspase-1 in the lung, increased alveolar and circulating IL-1β levels, and exacerbated ALI, all of which were substantially inhibited by administration of caspase-1 inhibitor. Together, our results support the presence of an important role for Nur77 in controlling inflammasome activation in vascular ECs and suggest that Nur77 could be a novel therapeutic target for the treatment of human diseases associated with aberrant inflammasome activation, such as ALI and atherosclerosis.

Deletion of Myeloid Interferon Regulatory Factor 4 (Irf4) in Mouse Model Protects against Kidney Fibrosis after Ischemic Injury by Decreased Macrophage Recruitment and Activation.

AKI is characterized by abrupt and reversible kidney dysfunction, and incomplete recovery leads to chronic kidney injury. Previous studies by us and others have indicated that macrophage infiltration and polarization play key roles in recovery from AKI. The role in AKI recovery played by IFN regulatory factor 4 (IRF4), a mediator of polarization of macrophages to the M2 phenotype, is unclear. We used mice with myeloid or macrophage cell-specific deletion of Irf4 (MΦ Irf4-/- ) to evaluate Irf4's role in renal macrophage polarization and development of fibrosis after severe AKI. Surprisingly, although macrophage Irf4 deletion had a minimal effect on early renal functional recovery from AKI, it resulted in decreased renal fibrosis 4 weeks after severe AKI, in association with less-activated macrophages. Macrophage Irf4 deletion also protected against renal fibrosis in unilateral ureteral obstruction. Bone marrow-derived monocytes (BMDMs) from MΦ Irf4-/- mice had diminished chemotactic responses to macrophage chemoattractants, with decreased activation of AKT and PI3 kinase and increased PTEN expression. PI3K and AKT inhibitors markedly decreased chemotaxis in wild-type BMDMs, and in a cultured macrophage cell line. There was significant inhibition of homing of labeled Irf4-/- BMDMs to postischemic kidneys. Renal macrophage infiltration in response to AKI was markedly decreased in MΦ Irf4-/- mice or in wild-type mice with inhibition of AKT activity. Deletion of Irf4 from myeloid cells protected against development of tubulointerstitial fibrosis after severe ischemic renal injury in mice, due primarily to inhibition of AKT-mediated monocyte recruitment to the injured kidney and reduced activation and subsequent polarization into a profibrotic M2 phenotype.

Cleavage of TANK-Binding Kinase 1 by HIV-1 Protease Triggers Viral Innate Immune Evasion.

Type-I interferons (IFN-I) are the innate immune system’s principal defense against viral infections. Human immunodeficiency virus-1 (HIV-1) has evolved several ways to suppress or evade the host’s innate immunity in order to survive and replicate to sustain infection. Suppression of IFN-I is one among the multiple escape strategies used by HIV-1 to prevent its clearance. HIV-1 protease which helps in viral maturation has also been observed to cleave host cellular protein kinases. In this study we performed a comprehensive screening of a human kinase library using AlphaScreen assay and identified that TANK binding kinase-1 (TBK1) was cleaved by HIV-1 protease (PR). We demonstrate that PR cleaved TBK1 fails to phosphorylate IFN regulatory factor 3 (IRF3), thereby reducing the IFN-I promoter activity and further reveal that the PR mediated suppression of IFN-I could be counteracted by protease inhibitors (PI) in vitro. We have also revealed that mutations of HIV-1 PR that confer drug resistance to PIs reduce the enzyme’s ability to cleave TBK1. The findings of this study unearth a direct link between HIV-1 PR activity and evasion of innate immunity by the virus, the possible physiological relevance of which warrants to be determined.

TSLP-Driven Chromatin Remodeling and Trained Systemic Immunity after Neonatal Respiratory Viral Infection.

Our studies have previously shown a role for persistent TSLP production in the lungs of mice after early-life respiratory syncytial virus (RSV) infection that leads to an altered immune phenotype, including accumulation of "inflammatory" dendritic cells (DC). This study investigates the role of TSLP driving systemic trained immunity in DC in early-life RSV-infected mice. Bone marrow-derived DCs (BMDC) from early-life RSV-infected mice at 4 wk postinfection showed enhanced expression of costimulatory molecules and cytokines, including Tslp, that regulate immune cell function. The adoptive transfer of BMDC grown from early-life RSV-infected mice was sufficient to exacerbate allergic disease development. The addition of recombinant TSLP during differentiation of BMDC from naive mice induced a similar altered phenotype as BMDC grown from early-life RSV-infected mice, suggesting a role for TSLP in the phenotypic changes. To assess the role of TSLP in these changes, global transcriptomic characterization of TSLPR-/- BMDC infected with RSV was performed and showed a higher upregulation of type 1 IFN genes and concomitant downregulation of inflammatory genes. Assay for transposase-accessible chromatin using sequencing analysis demonstrated that TSLPR-/- BMDC had a parallel gain in physical chromatin accessibility near type 1 genes and loss in accessibility near genes related to RSV pathology, with IFN regulatory factor 4 (IRF4) and STAT3 predicted as top transcription factors binding within differentially accessible regions in wild-type. Importantly, these studies show that in the absence of TSLP signaling, BMDC are able to mount an appropriate type 1 IFN-associated antiviral response to RSV. In summary, RSV-induced TSLP alters chromatin structure in DC to drive trained innate immunity and activates pathogenic gene programs in mice.

The IKZF1–IRF4/IRF5 Axis Controls Polarization of Myeloma-Associated Macrophages

The bone marrow niche has a pivotal role in progression, survival, and drug resistance of multiple myeloma cells. Therefore, it is important to develop means for targeting the multiple myeloma bone marrow microenvironment. Myeloma-associated macrophages (MAM) in the bone marrow niche are M2 like. They provide nurturing signals to multiple myeloma cells and promote immune escape. Reprogramming M2-like macrophages toward a tumoricidal M1 phenotype represents an intriguing therapeutic strategy. This is especially interesting in view of the successful use of mAbs against multiple myeloma cells, as these therapies hold the potential to trigger macrophage-mediated phagocytosis and cytotoxicity. In this study, we observed that MAMs derived from patients treated with the immunomodulatory drug (IMiD) lenalidomide skewed phenotypically and functionally toward an M1 phenotype. Lenalidomide is known to exert its beneficial effects by modulating the CRBN-CRL4 E3 ligase to ubiquitinate and degrade the transcription factor IKAROS family zinc finger 1 (IKZF1). In M2-like MAMs, we observed enhanced IKZF1 levels that vanished through treatment with lenalidomide, yielding MAMs with a bioenergetic profile, T-cell stimulatory properties, and loss of tumor-promoting capabilities that resemble M1 cells. We also provide evidence that IMiDs interfere epigenetically, via degradation of IKZF1, with IFN regulatory factors 4 and 5, which in turn alters the balance of M1/M2 polarization. We validated our observations in vivo using the CrbnI391V mouse model that recapitulates the IMiD-triggered IKZF1 degradation. These data show a role for IKZF1 in macrophage polarization and can provide explanations for the clinical benefits observed when combining IMiDs with therapeutic antibodies.

Composition, Spatial Characteristics, and Prognostic Significance of Myeloid Cell Infiltration in Pancreatic Cancer.

Although abundant myeloid cell populations in the pancreatic ductal adenocarcinoma (PDAC) microenvironment have been postulated to suppress antitumor immunity, the composition of these populations, their spatial locations, and how they relate to patient outcomes are poorly understood. To generate spatially resolved tumor and immune cell data at single-cell resolution, we developed two quantitative multiplex immunofluorescence assays to interrogate myeloid cells (CD15, CD14, ARG1, CD33, HLA-DR) and macrophages [CD68, CD163, CD86, IFN regulatory factor 5, MRC1 (CD206)] in the PDAC tumor microenvironment. Spatial point pattern analyses were conducted to assess the degree of colocalization between tumor cells and immune cells. Multivariable-adjusted Cox proportional hazards regression was used to assess associations with patient outcomes. In a multi-institutional cohort of 305 primary PDAC resection specimens, myeloid cells were abundant, enriched within stromal regions, highly heterogeneous across tumors, and differed by somatic genotype. High densities of CD15+ARG1+ immunosuppressive granulocytic cells and M2-polarized macrophages were associated with worse patient survival. Moreover, beyond cell density, closer proximity of M2-polarized macrophages to tumor cells was strongly associated with disease-free survival, revealing the clinical significance and biologic importance of immune cell localization within tumor areas. A diverse set of myeloid cells are present within the PDAC tumor microenvironment and are distributed heterogeneously across patient tumors. Not only the densities but also the spatial locations of myeloid immune cells are associated with patient outcomes, highlighting the potential role of spatially resolved myeloid cell subtypes as quantitative biomarkers for PDAC prognosis and therapy.