Transcription In Macrophages Research Articles

Abstract 2056: PI3Kgamma inhibition reduces inflammation and promotes survival in cancer and SARS-CoV-2

Abstract Solid tumors are characterized by extensive immune suppressive inflammation, vascular leak, fibrosis and organ damage. Similarly, SARS-CoV-2 infections induce aberrant pulmonary and systemic inflammation, vascular leak, coagulation, fibrosis and fatal organ damage. We previously demonstrated that macrophages in solid tumors strongly expressed phosphatidylinositol 3-kinase gamma (PI3Kγ), a signaling protein that coordinately controls granulocyte and monocyte trafficking to tumors as well as wound-healing-type macrophage transcription in cancer and fibrosis. We also observed that macrophages in COVID-19 lungs strongly expressed PI3Kγ. To identify therapeutic strategies to suppress COVID-19-associated inflammation, we characterized lung tissue of COVID-19 patients using multiplex immunohistochemistry and tissue transcriptomics. Lungs of deceased patients exhibited substantial infiltration by neutrophils and wound-healing macrophages, fibrosis and alveolar type II cell depletion. In animal models of lung inflammation, bacterial infections, viral infection and SARS-CoV-2 infection, PI3Kγ deletion or inhibition with the cancer therapeutic IPI-549 (eganelisib) suppressed pulmonary and systemic inflammation, reduced lung damage, and promoted survival. These studies demonstrate the essential role of PI3Kγ in inflammatory diseases as well as cancer and support the use of PI3Kγ inhibitors such as eganelisib to suppress inflammation and promote survival in pulmonary infections like SARS-CoV-2 and cancer. Citation Format: Judith A. Varner, Anghesom Ghebremedhin. PI3Kgamma inhibition reduces inflammation and promotes survival in cancer and SARS-CoV-2 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2056.

Abstract 3889: Identification of ADRN-specific, MES-specific, and pan-subtype therapeutic targets in neuroblastoma

Abstract Background: Neuroblastoma is an extracranial solid tumor of childhood that arises from the developing sympathetic nervous system. Half of patients with high-risk disease do not survive despite multimodal therapy, and survival after relapse is unlikely. Recent studies have characterized two distinct neuroblastoma cell subtypes: the adrenergic (ADRN) subtype of sympathetic noradrenergic-like identity and the mesenchymal (MES) subtype of neural crest-like identity. ADRN-like cells are more aggressive and proliferative, while the MES-like cells are chemotherapy resistant. Neuroblastoma tumors are heterogenous and comprised of both ADRN and MES cell subtypes, which likely leads to differential responses to therapy. Thus, to eliminate cells that may lead to tumor relapse, it is critical to develop therapies against both neuroblastoma subtypes. Methods: To identify candidate subtype-specific therapeutic targets, we used ssGSEA to classify human neuroblastoma tumors into ADRN- or MES-dominant and performed differential expression. We validated subtype-specific targets in additional datasets and models, including transdifferentiated neuroblastoma cell lines (ADRN-to-MES) after inducible overexpression of the MES transcription factor PRRX1. Results: We show ADRN-specific expression of known preclinical and clinical neuroblastoma targets with limited or no expression in MES-dominant tumors. We propose CD276 (B7-H3) and L1CAM as pan-subtype targets with similar expression in both subtypes and stable expression after ADRN-to-MES transdifferentiation. In MES-specific neuroblastoma patient samples and cell lines, we identify the receptor tyrosine kinase AXL as a candidate MES target. MES neuroblastoma cell lines are more sensitive to small molecule AXL inhibitors (Cabozantinib, NPS-1034, and ONO-7475) compared to ADRN cell lines. We also observe higher expression of PDL1 and immunosuppressive chemokines in MES-dominant tumors and cell lines. Finally, we detect AXL and Gas6 transcripts in tumor-resident macrophages and fibroblasts, which may further promote immune evasion. Conclusion: Here we have identified and prioritized ADRN-specific, MES-specific, and pan-subtype neuroblastoma therapeutic targets and suggest AXL-targeted therapy may eliminate both MES-dominant neuroblastoma cells and immune cell populations that contribute to an immunosuppressive microenvironment. Citation Format: Nathan M. Kendsersky, Nathaniel W. Mabe, Alvin Farrel, Liron D. Grossmann, Michal Odrobina, Kimberly Stegmaier, John M. Maris. Identification of ADRN-specific, MES-specific, and pan-subtype therapeutic targets in neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3889.

Abstract 933: Macrophage-derived C/EBPb promotes breast tumorigenesis

Abstract Breast cancer growth and progression relies on complex interactions between tumor cells and surrounding stromal cells, including immune cells. Infiltrating immune cells, including macrophages, have been shown to promote tumor progression and metastasis, and efforts have aimed at the tumor microenvironment as a therapeutic strategy. Although tumor-promoting roles of macrophages are well-established, the specific macrophage factors and transcription factor-mediated regulatory programs that drive breast cancer progression are less understood. Using a p53-/- syngeneic model of premalignant progression to invasive cancer, we identified distinct subpopulations of macrophages that resemble both normal tissue resident macrophages and tumor-associated macrophages. Interestingly, most of these macrophage subsets highly express C/EBPβ, suggesting a global function of C/EBPβ in macrophages. C/EBPβ is a transcription factor that is overexpressed in breast cancer tissues and is important for monocyte differentiation and myeloid-derived suppressor cell function. To address the function of macrophage C/EBPβ during tumorigenesis, Cebpb-floxed mice were bred to iCsf1r-Cre mice to generate mice (CebpbΔM) where Cebpb is selectively deleted in macrophages (CSF1R-expressing myeloid cells). Genetic ablation of macrophage C/EBPβ in premalignant lesions results in a significant decrease in progression to invasive cancer. In established claudin-low tumors, deletion of macrophage C/EBPβ results in decreased tumor latency and the re-expression of cytokeratin 8+E-cadherin+ cells, reminiscent of luminal-like tumors. This phenoytpe was recapitulated in an ex vivo 3D co-culture model, where uniformly round structures that lack mesenchymal projections form in the presence of CebpbΔM BMDMs. These data suggest that macrophage C/EBPβ may help maintain a mesenchymal phenotype in claudin-low tumors. Ongoing studies are aimed at identifying the Cebpb-induced gene networks in macrophages that mediate tumor progression. These studies will have critical implications for whether inhibition of C/EBPβ-regulated genes may revert claudin-low tumors to a luminal-like phenotype, rendering tumors responsive to current therapies. This work is supported by NIH R01CA212518 (H.L.M.). Citation Format: Alexa M. Dow, Michelle D. Rojo, Scout M. Treadwell, Heather L. Machado. Macrophage-derived C/EBPb promotes breast tumorigenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 933.

ADAP restraint of STAT1 signaling regulates macrophage phagocytosis in immune thrombocytopenia.

Heightened platelet phagocytosis by macrophages accompanied by an increase in IFN-γ play key roles in the etiology of immune thrombocytopenia (ITP); however, it remains elusive how macrophage-mediated platelet clearance is regulated in ITP. Here, we report that adhesion and degranulation-protein adaptor protein (ADAP) restrains platelet phagocytosis by macrophages in ITP via modulation of signal transducer and activator of transcription 1 (STAT1)-FcγR signaling. We show that ITP was associated with the underexpression of ADAP in splenic macrophages. Furthermore, macrophages from Adap−/− mice exhibited elevated platelet phagocytosis and upregulated proinflammatory signaling, and thrombocytopenia in Adap−/− mice was mitigated by the depletion of macrophages. Mechanistically, ADAP interacted and competed with STAT1 binding to importin α5. ADAP deficiency potentiated STAT1 nuclear entry, leading to a selective enhancement of FcγRI/IV transcription in macrophages. Moreover, pharmacological inhibition of STAT1 or disruption of the STAT1-importin α5 interaction relieved thrombocytopenia in Adap−/− mice. Thus, our findings not only reveal a critical role for ADAP as an intracellular immune checkpoint for shaping macrophage phagocytosis in ITP but also identify the ADAP-STAT1-importin α5 module as a promising therapeutic target in the treatment of ITP.

The Lung Microenvironment Instructs Gene Transcription in Neonatal and Adult Alveolar Macrophages.

Immaturity of alveolar macrophages (AMs) around birth contributes to the susceptibility of newborns to lung disease. However, the molecular features differentiating neonatal and mature, adult AMs are poorly understood. In this study, we identify the unique transcriptomes and enhancer landscapes of neonatal and adult AMs in mice. Although the core AM signature was similar, murine adult AMs expressed higher levels of genes involved in lipid metabolism, whereas neonatal AMs expressed a largely proinflammatory gene profile. Open enhancer regions identified by an assay for transposase-accessible chromatin followed by high-throughput sequencing (ATAC-seq) contained motifs for nuclear receptors, MITF, and STAT in adult AMs and AP-1 and NF-κB in neonatal AMs. Intranasal LPS activated a similar innate immune response in both neonatal and adult mice, with higher basal expression of inflammatory genes in neonates. The lung microenvironment drove many of the distinguishing gene expression and open chromatin characteristics of neonatal and adult AMs. Neonatal mouse AMs retained high expression of some proinflammatory genes, suggesting that the differences in neonatal AMs result from both inherent cell properties and environmental influences.

Selective bromodomain and extra-terminal bromodomain inhibitor inactivates macrophages and hepatic stellate cells to inhibit liver inflammation and fibrosis

ABSTRACT Liver fibrosis occurs following inflammation triggered by the integrated actions of activated liver-resident macrophages (Kupffer cells) and hepatic stellate cells (HSCs), and the multiplicity of these mechanisms complicates drug therapy. Here, we demonstrate that the selective bromodomain and extra-terminal (BET) bromodomain inhibitor compound38 can block both the Janus kinase-signal transducer and activator of transcription and mitogen-activated protein kinase signaling pathways in macrophages, which decreased their secretion of proinflammatory cytokines in a dose-dependent manner. The inactivation of macrophages attenuated lipopolysaccharide-induced injurious inflammation concurrent with a reduction in F4/80+ cells, proinflammatory cytokine levels, and neutrophil infiltration. Moreover, compound 38 inhibited the Wnt/β-catenin and transforming growth factor-beta/SMAD signaling pathways to abolish the activation of HSCs. In vivo, compound 38 significantly decreased the collagen deposition and fibrotic area of a CCl4-induced liver fibrosis model, and restored the deficiency of activated HSCs and the upregulation of liver inflammation. These results highlight the potential role of compound 38 in treating liver fibrosis considering its simultaneous inhibitory effects on liver inflammation and related fibrosis.

Inhibition of Histone H3 Lysine-27 Demethylase Activity Relieves Rheumatoid Arthritis Symptoms via Repression of IL6 Transcription in Macrophages

Rheumatoid arthritis (RA) occurs in about 5 per 1,000 people and can lead to severe joint damage and disability. However, the knowledge of pathogenesis and treatment for RA remains limited. Here, we found that histone demethylase inhibitor GSK-J4 relieved collagen induced arthritis (CIA) symptom in experimental mice model, and the underlying mechanism is related to epigenetic transcriptional regulation in macrophages. The role of epigenetic regulation has been introduced in the process of macrophage polarization and the pathogenesis of inflammatory diseases. As a repressive epigenetic marker, tri-methylation of lysine 27 on histone H3 (H3K27me3) was shown to be important for transcriptional gene expression regulation. Here, we comprehensively analyzed H3K27me3 binding promoter and corresponding genes function by RNA sequencing in two differentially polarized macrophage populations. The results revealed that H3K27me3 binds on the promoter regions of multiple critical cytokine genes and suppressed their transcription, such as IL6, specifically in M-CSF derived macrophages but not GM-CSF derived counterparts. Our results may provide a new approach for the treatment of inflammatory and autoimmune disorders.

HIV-1 provirus transcription and translation in macrophages differs from pre-integrated cDNA complexes and requires E2F transcriptional programs

ABSTRACT HIV-1 cDNA pre-integration complexes persist for weeks in macrophages and remain transcriptionally active. While previous work has focused on the transcription of HIV-1 genes; our understanding of the cellular milieu that accompanies viral production is incomplete. We have used an in vitro system to model HIV-1 infection of macrophages, and single-cell RNA sequencing (scRNA-seq) to compare the transcriptomes of uninfected cells, cells harboring pre-integration complexes (PIC), and those containing integrated provirus and making late HIV proteins. scRNA-seq can distinguish between provirus and PIC cells because their background transcriptomes vary dramatically. PIC cell transcriptomes are characterized by NFkB and AP-1 promoted transcription, while transcriptomes of cells transcribing from provirus are characterized by E2F family transcription products. We also find that the transcriptomes of PIC cells and Bystander cells (defined as cells not producing any HIV transcript and thus presumably not infected) are indistinguishable except for the presence of HIV-1 transcripts. Furthermore, the presence of pathogen alters the transcriptome of the uninfected Bystander cells, so that they are distinguishable from true control cells (cells not exposed to any pathogen). Therefore, a single cell comparison of transcriptomes from provirus and PIC cells provides a new understanding of the transcriptional changes that accompany HIV-1 integration.

TPL-2 Inhibits IFN-β Expression via an ERK1/2-TCF-FOS Axis in TLR4-Stimulated Macrophages.

TPL-2 kinase plays an important role in innate immunity, activating ERK1/2 MAPKs in myeloid cells following TLR stimulation. We investigated how TPL-2 controls transcription in TLR4-stimulated mouse macrophages. TPL-2 activation of ERK1/2 regulated expression of genes encoding transcription factors, cytokines, chemokines, and signaling regulators. Bioinformatics analysis of gene clusters most rapidly induced by TPL-2 suggested that their transcription was mediated by the ternary complex factor (TCF) and FOS transcription factor families. Consistently, TPL-2 induced ERK1/2 phosphorylation of the ELK1 TCF and the expression of TCF target genes. Furthermore, transcriptomic analysis of TCF-deficient macrophages demonstrated that TCFs mediate approximately half of the transcriptional output of TPL-2 signaling, partially via induced expression of secondary transcription factors. TPL-2 signaling and TCFs were required for maximal TLR4-induced FOS expression. Comparative analysis of the transcriptome of TLR4-stimulated Fos -/- macrophages indicated that TPL-2 regulated a significant fraction of genes by controlling FOS expression levels. A key function of this ERK1/2-TCF-FOS pathway was to mediate TPL-2 suppression of type I IFN signaling, which is essential for host resistance against intracellular bacterial infection.

Avian Macrophage Responses to Virulent and Avirulent Clostridium perfringens.

The present study evaluated the avian macrophage responses against Clostridium perfringens that varied in their ability to cause necrotic enteritis in chickens. Strains CP5 (avirulent-netB+), CP1 (virulent-netB+), and CP26 (highly virulent-netB+tpeL+) were used to evaluate their effect on macrophages (MQ-NCSU cells) and primary splenic and cecal tonsil mononuclear cells. The bacilli (whole cells) or their secretory products from all three strains induced a significant increase in the macrophage transcription of Toll-like receptor (TLR)21, TLR2, interleukin (IL)-1β, inducible nitric oxide synthase (iNOS), and CD80 genes as well as their nitric oxide (NO) production and major histocompatibility complex (MHC)-II surface expression compared to an unstimulated control. The CP1 and CP26-induced expression of interferon (IFN)γ, IL-6, CD40 genes, MHC-II upregulation, and NO production was significantly higher than that of CP5 and control groups. Furthermore, splenocytes and cecal tonsillocytes stimulated with bacilli or secretory products from all the strains showed a significant increase in the frequency of macrophages, their surface expression of MHC-II and NO production, while CP26-induced responses were significantly higher for the rest of the groups. In summary, macrophage interaction with C. perfringens can lead to cellular activation and, the ability of this pathogen to induce macrophage responses may depend on its level of virulence.

Aging disrupts circadian gene regulation and function in macrophages.

Aging is characterized by an increased vulnerability to infection and the development of inflammatory diseases, such as atherosclerosis, frailty, cancer and neurodegeneration. Here, we find that aging is associated with the loss of diurnally rhythmic innate immune responses, including monocyte trafficking from bone marrow to blood, response to lipopolysaccharide and phagocytosis. This decline in homeostatic immune responses was associated with a striking disappearance of circadian gene transcription in aged compared to young tissue macrophages. Chromatin accessibility was significantly greater in young macrophages than in aged macrophages; however, this difference did not explain the loss of rhythmic gene transcription in aged macrophages. Rather, diurnal expression of Kruppel-like factor 4 (Klf4), a transcription factor (TF) well established in regulating cell differentiation and reprogramming, was selectively diminished in aged macrophages. Ablation of Klf4 expression abolished diurnal rhythms in phagocytic activity, recapitulating the effect of aging on macrophage phagocytosis. Examination of individuals harboring genetic variants of KLF4 revealed an association with age-dependent susceptibility to death caused by bacterial infection. Our results indicate that loss of rhythmic Klf4 expression in aged macrophages is associated with disruption of circadian innate immune homeostasis, a mechanism that may underlie age-associated loss of protective immune responses.

Microvascular Inflammation: An Incremental Path to Refining the Diagnosis of Antibody-mediated Rejection in Heart Transplantation

Microvascular Inflammation: An Incremental Path to Refining the Diagnosis of Antibody-mediated Rejection in Heart Transplantation

MicroRNA-mediated calcineurin signaling activation induces CCL2, CCL3, CCL5, IL8, and chemotactic activities in 4,4′-methylene diphenyl diisocyanate exposed macrophages

Occupational exposure to 4,4′-methylene diphenyl diisocyanate (MDI), the most widely used monomeric diisocyanate, is one of the leading causes of occupational asthma (OA). Previously, we identified microRNA (miR)-206-3p/miR-381-3p-mediated PPP3CA/calcineurin signalling regulated iNOS transcription in macrophages and bronchoalveolar lavage cells (BALCs) after acute MDI exposure; however, whether PPP3CA/calcineurin signalling participates in regulation of other asthma-associated mediators secreted by macrophages/BALCs after MDI exposure is unknown. Several asthma-associated, macrophage-secreted mediator mRNAs from MDI exposed murine BALCs and MDI-glutathione (GSH) conjugate treated differentiated THP-1 macrophages were analysed using RT-qPCR. Endogenous IL1B, TNF, CCL2, CCL3, CCL5, and TGFB1 were upregulated in MDI or MDI-GSH conjugate exposed BALCs and macrophages, respectively. Calcineurin inhibitor tacrolimus (FK506) attenuated the MDI-GSH conjugate-mediated induction of CCL2, CCL3, CCL5, and CXCL8/IL8 but not others. Transfection of either miR-inhibitor-206-3p or miR-inhibitor-381-3p in macrophages induced chemokine CCL2, CCL3, CCL5, and CXCL8 transcription, whereas FK506 attenuated the miR-inhibitor-206-3p or miR-inhibitor-381-3p-mediated effects. Finally, MDI-GSH conjugate treated macrophages showed increased chemotactic ability to various immune cells, which may be attenuated by FK506. In conclusion, these results indicate that MDI exposure to macrophages/BALCs may recruit immune cells into the airway via induction of chemokines by miR-206-3p and miR-381-3p-mediated calcineurin signalling activation.

Rationale for an anti‐inflammatory insulin sensitizer in a phase 3 Alzheimer's disease trial

AbstractBackgroundNE3107 (3α‐ethynyl‐androst‐5‐ene‐3β,7β,17β‐triol), shows safety, antiinflammatory and insulin‐sensitizing activities in vitro, in animals, and humans. NE3107 binds ERK1/2 and inhibits inflammatory, but not homeostatic NFkB/ERK signaling in vitro, in animals and in humans. NE3107 is blood‐brain permeable, and does not affect neurotransmitter receptors. NE3107 inhibits TNFα‐stimulated NFκB transcription in human macrophages. It also inhibits NFκB nuclear translocation and LPS‐stimulated inflammatory signal transduction. NE3107 prevented IκBα proteolysis in murine macrophages. NE3107 decreased EAE optic nerve microglia, and increased nuclear NFκB in collicular and retinal neurons (homeostatic), but decreased optic nerve head glial nuclear NFκB (inflammatory) in a glaucoma model. NE3107 preserved dopaminergic neurons in MPTP‐treated mice and marmosets. In obese impaired glucose tolerance (IGT) humans, NE3107 decreased CRP, which is involved in AD inflammation and tissue damage, and increased antiinflammatory HDL, which decreases risk of AD pathogenesis. NE3107 increased adiponectin, which suppresses insulin resistance, inflammation, and AD pathology. In obese, inflamed IGT and type 2 diabetes (T2D) subjects, NE3107 decreased insulin resistance, which drives neurodegeneration and AD. Importantly, in obese inflamed T2D subjects, NE3107 decreased inflammation‐driven systems deregulation, known to contribute to AD progression.MethodSafety and activity data from preclinical, toxicology, human clinical trials, and a protocol synopsis for a pivotal trial in AD were submitted, and the FDA allowed NeurMedix to proceed with the NM101 trial.ResultA Phase 3, Double Blind, Randomized, Placebo Controlled, Parallel Group, Multicenter Study of NE3107 in Subjects Who Have Mild to Moderate Alzheimer's Disease (NCT04669028) will evaluate safety and efficacy of oral 20 mg BID NE3107 vs. placebo in 316 subjects with mild to moderate AD. Two coprimary outcome measures ADAS Cog12 and ADCS CGIC will be evaluated as the change from Baseline to Week 30. Secondary endpoints include measures of neuropsychological deficits, functional performance, glycemic control and resource utilization. A subset of patients may volunteer for exploratory vMRI and FDG‐PET scans at baseline and week 30.ConclusionInflammation and insulin resistance drive AD. NE3107 decreases these insults that drive systems dysregulation in AD. This trial is well‐positioned to examine the effects of NE3107 on progression of AD dementia, affecting more than 40 million people globally.

Human nasal wash RNA-Seq reveals distinct cell-specific innate immune responses in influenza versus SARS-CoV-2.

BACKGROUNDInfluenza A virus (IAV) and SARS-CoV-2 are pandemic viruses causing millions of deaths, yet their clinical manifestations are distinctly different.METHODSWith the hypothesis that upper airway immune and epithelial cell responses are also distinct, we performed single-cell RNA sequencing (scRNA-Seq) on nasal wash cells freshly collected from adults with either acute COVID-19 or influenza or from healthy controls. We focused on major cell types and subtypes in a subset of donor samples.ResultsNasal wash cells were enriched for macrophages and neutrophils for both individuals with influenza and those with COVID-19 compared with healthy controls. Hillock-like epithelial cells, M2-like macrophages, and age-dependent B cells were enriched in COVID-19 samples. A global decrease in IFN-associated transcripts in neutrophils, macrophages, and epithelial cells was apparent in COVID-19 samples compared with influenza samples. The innate immune response to SARS-CoV-2 appears to be maintained in macrophages, despite evidence for limited epithelial cell immune sensing. Cell-to-cell interaction analyses revealed a decrease in epithelial cell interactions in COVID-19 and highlighted differences in macrophage-macrophage interactions for COVID-19 and influenza.ConclusionsOur study demonstrates that scRNA-Seq can define host and viral transcriptional activity at the site of infection and reveal distinct local epithelial and immune cell responses for COVID-19 and influenza that may contribute to their divergent disease courses.FundingMassachusetts Consortium on Pathogen Readiness, the Mathers Foundation, and the Department of Defense (W81XWH2110029) “COVID-19 Expansion for AIRe Program.”

Identification and characterization of novel infection associated transcripts in macrophages

ABSTRACT By analysis of lncRNA expression profiles of macrophages in response to Mycobacterium tuberculosis (Mtb) infection, we identified novel highly expressed transcripts, unique in encompassing a protein coding transcript- Cytidine Monophosphate Kinase 2 (CMPK2) and a previously identified lncRNA- Negative Regulator of Interferon Response (NRIR). While these transcripts (TILT1, 2,3 – TLR4 and Infection induced Long Transcript) are induced by virulent Mtb as well as lipopolysaccharide (LPS) early, lack of/delayed expression in non-viable Mtb/BCG infected cells, respectively, suggest an important role in macrophage responses. The elevated expression by 3 hr in response to fast growing bacteria further emphasizes the importance of these RNAs in the macrophage infection response. Overall, we provide evidence for the presence of multiple transcripts that form a part of the early infection response programme of macrophages. Abbreviations: IFN: Interferon; NRIR: negative regulator of interferon response; CMPK2: cytidine/ uridine monophosphate kinase; LPS: lipopolysaccharide; LAM: Lipoarabinomannan; PIMs: Phosphatidylinositol Mannosides; TILT1, 2,3: TLR4 and Infection induced Long Transcript; TLR4: Toll-like receptor 4; Mtb: Mycobacterium tuberculosis; BCG: Mycobacterium bovis BCG; MDMs: human monocyte derived macrophages

The unfolded protein response links tumor aneuploidy to local immune dysregulation

Aneuploidy is a chromosomal abnormality associated with poor prognosis in many cancer types. Here, we tested the hypothesis that the unfolded protein response (UPR) mechanistically links aneuploidy and local immune dysregulation. Using a single somatic copy number alteration (SCNA) score inclusive of whole-chromosome, chromosome arm, and focal alterations in a pan-cancer analysis of 9,375 samples in The Cancer Genome Atlas (TCGA) database, we found an inverse correlation with a cytotoxicity (CYT) score across disease stages. Co-expression patterns of UPR genes changed substantially between SCNAlow and SCNAhigh groups. Pathway activity scores showed increased activity of multiple branches of the UPR in response to aneuploidy. The PERK branch showed the strongest association with a reduced CYT score. The conditioned medium of aneuploid cells transmitted XBP1 splicing and caused IL-6 and arginase 1 transcription in receiver bone marrow-derived macrophages and markedly diminished the production of IFN-γ and granzyme B in activated human T cells. We propose the UPR as a mechanistic link between aneuploidy and immune dysregulation in the tumor microenvironment.

Leishmania Parasites Differently Regulate Antioxidant Genes in Macrophages Derived From Resistant and Susceptible Mice.

Macrophage–Leishmania interactions are central to parasite growth and disease outcome. Macrophages have developed various strategies to fight invaders, including oxidative burst. While some microorganisms seem to survive and even thrive in an oxidative environment, others are susceptible and get killed. To counter oxidative stress, macrophages switch the expressions of cytoprotective and detoxifying enzymes, which are downstream targets of the nuclear factor erythroid 2-related factor 2 (Nrf2), to enhance cell survival. We have explored the transcription of NRF2 and of its target genes and compared the effect of the parasite on their transcription in bone marrow-derived macrophages (BMdMs) from Leishmania-resistant and Leishmania-susceptible mice. While heme oxygenase 1 (HO-1) transcription is independent of the genetic background, the transcription of glutathione reductase (Gsr) and of cysteine/glutamate exchange transporter (Slc7a11), involved in glutathione accumulation, was differentially regulated in BMdMs from both mouse strains. We also show that, except for HO-1, known to favor the survival of the parasite, the transcription of the selected genes, including Gsr, CD36, and catalase (CAT), was actively repressed, if not at all time points at least at the later ones, by the parasite, especially in Balb/c BMdMs. Consistent with these results, we found that the silencing of NRF2 in this study increases the survival and multiplication of the parasite.

The Role of Copper in the Regulation of Ferroportin Expression in Macrophages.

The critical function of ferroportin (Fpn) in maintaining iron homeostasis requires complex and multilevel control of its expression. Besides iron-dependent cellular and systemic control of Fpn expression, other metals also seem to be involved in regulating the Fpn gene. Here, we found that copper loading significantly enhanced Fpn transcription in an Nrf2-dependent manner in primary bone-marrow-derived macrophages (BMDMs). However, prolonged copper loading resulted in decreased Fpn protein abundance. Moreover, CuCl2 treatment induced Fpn expression in RAW 264.7 macrophages at both the mRNA and protein level. These data suggest that cell-type-specific regulations have an impact on Fpn protein stability after copper loading. Transcriptional suppression of Fpn after lipopolysaccharide (LPS) treatment contributes to increased iron storage inside macrophages and may result in anemia of inflammation. Here, we observed that in both primary BMDMs and RAW 264.7 macrophages, LPS treatment significantly decreased Fpn mRNA levels, but concomitant CuCl2 stimulation counteracted the transcriptional suppression of Fpn and restored its expression to the control level. Overall, we show that copper loading significantly enhances Fpn transcription in macrophages, while Fpn protein abundance in response to CuCl2 treatment, depending on macrophage type and factors specific to the macrophage population, can influence Fpn regulation in response to copper loading.

Transcriptome Profiling of Atlantic Salmon Adherent Head Kidney Leukocytes Reveals That Macrophages Are Selectively Enriched During Culture

The Atlantic salmon (Salmo salar) is an economically important fish, both in aquaculture and in the wild. In vertebrates, macrophages are some of the first cell types to respond to pathogen infection and disease. While macrophage biology has been characterized in mammals, less is known in fish. Our previous work identified changes in the morphology, phagocytic ability, and miRNA profile of Atlantic salmon adherent head kidney leukocytes (HKLs) from predominantly “monocyte-like” at Day 1 of in vitro culture to predominantly “macrophage-like” at Day 5 of culture. Therefore, to further characterize these two cell populations, we examined the mRNA transcriptome profile in Day 1 and Day 5 HKLs using a 44K oligonucleotide microarray. Large changes in the transcriptome were revealed, including changes in the expression of macrophage and immune-related transcripts (e.g. csf1r, arg1, tnfa, mx2), lipid-related transcripts (e.g. fasn, dhcr7, fabp6), and transcription factors involved in macrophage differentiation and function (e.g. klf2, klf9, irf7, irf8, stat1). The in silico target prediction analysis of differentially expressed genes (DEGs) using miRNAs known to change expression in Day 5 HKLs, followed by gene pathway enrichment analysis, supported that these miRNAs may be involved in macrophage maturation by targeting specific DEGs. Elucidating how immune cells, such as macrophages, develop and function is a key step in understanding the Atlantic salmon immune system. Overall, the results indicate that, without the addition of exogenous factors, the adherent HKL cell population differentiates in vitro to become macrophage-like.