Jul 2026· Journal of Immunology· Vol 215· 0 citations
TL;DR
The findings suggest that S. aureus alters activation of the canonical type I IFN-mediated Jak-STAT pathway through suppression of STAT1 activation and altering chromatin remodeling and may alter the host response to this pathogen in a context-dependent manner and contribute to difficulty in clearing S. aureus infection.
Abstract
Staphylococcus aureus is a Gram-positive pathogen that is a major cause of soft tissue and bloodstream infections in humans. Previous studies have shown that S. aureus triggers myeloid cell-driven innate immune activation including inflammatory signaling and type I interferon (IFN) responses; however, the role type I IFNs remains controversial.
Primary human CD14+ monocytes were purified from PBMCs of healthy donors using anti-CD14 magnetic beads. Monocytes were infected with various doses of S. aureus and harvested for RT-qPCR, Western blotting, and integrated bulk RNA sequencing, ATAC-seq and CUT&RUN.
We observed a dose-dependent dichotomous regulation of the IFN response, which was induced by low doses and suppressed at high doses of S. aureus. Only a subset of interferon-stimulated genes (ISGs) was induced despite intact IFNβ production following S. aureus infection. Intriguingly, S. aureus selectively abrogated the activation and nuclear translocation of STAT1, while activation and nuclear translocation of the other ISGF3 components STAT2 and IRF9 were preserved. Furthermore, RNA profiling revealed a disparate regulation of gene expression, with a distinct subset of genes exhibiting significantly enhanced expression upon combined S. aureus and IFNβ treatment. Epigenomic profiling showed a complete repression of chromatin accessibility as well as STAT1 binding at a subset of ISG loci upon S. aureus infection, whereas another subset of ISGs displayed increased chromatin accessibility and transcriptional activation upon infection.
Our findings suggest that S. aureus alters activation of the canonical type I IFN-mediated Jak-STAT pathway through suppression of STAT1 activation and altering chromatin remodeling . This change in signaling is associated with gene-specific reprogramming of the type I IFN response by S. aureus that may alter the host response to this pathogen in a context-dependent manner and contribute to difficulty in clearing S. aureus infection.
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Innate Immune Responses and Host Defense: Molecular Mechanisms (INM)
Staphylococcus aureus can express a large arsenal of virulence factors, readily acquire antibiotic resistance, and survive harsh environmental conditions through biofilm formation. These traits facilitate evasion of host immunity and antibiotic killing, leading to chronic infections. A better understanding of pathogenicity and immune evasion are critical for more effective treatments. In a mouse model of S. aureus biofilm infection, we uncovered a critical role for triggering receptor expressed on myeloid cells-2 (TREM2) in exacerbating infection severity via anti-inflammatory responses to pathogen (PAMPs) and damage-associated molecular patterns (DAMPs). Interestingly, the role for TREM2 was specific to females, suggesting a role for estrogens. Further studies with bone marrow-derived macrophages showed that TREM2 loss enhanced surface expression of Toll-like receptor 2 (TLR2), a key immune receptor that senses bacterial lipoprotein and lipoteichoic acid for proinflammatory activation. Cross-regulation between TREM2, TLR2, and estrogens in the context of infection has not been described.
Mouse background (8-10 week old C57BL/6) and S. aureus (USA300 LAC-13C) strains were identical for all studies. Craniotomy infection was induced by inoculating bone flaps with 103 CFU. In vitro studies used primary bone marrow-derived macrophages. Flow cytometry (surface markers, metabolism, ROS/RNS), Cytometric Bead Array (mediator production), and Western blot (protein quantification) were also used.
Macrophages with individual knockouts of TREM2 and TLR2, and a double knockout of TREM2/TLR2, showed cross-regulatory effects from these receptors and 17β-estradiol on inflammatory surface markers, mediator production, metabolism, and reactive oxygen/nitrogen species production.
TREM2, TLR2, and estrogen appear to have an interconnected role in driving immune responses, which could be exploited for moderating pro- vs anti-inflammatory immune activation during S. aureus infection.
Preliminary work was supported by NIH F32NS126302 to Lee Korshoj and R01NS107369 and R01AI169788 to Tammy Kielian. Current work is supported by startup funds from the University of Nebraska Medical Center to Lee Korshoj.
Microbial, Parasitic, and Fungal Immunology (MPF)
Rachael Urquhart, Lee E. Korshoj· Journal of Immunology· 0 citations
Influenza is often complicated by bacterial coinfections. Among the most common is methicillin-resistant S. aureus (MRSA). Viruses and bacteria are recognized by pattern recognition receptors (PRRs) such as Toll-like receptor 9 (TLR9). Most studies have focused on TLR9 function in immune cells. In contrast, its role in structural cells remains less understood. Our laboratory found that TLR9 knockout mice exhibit improved outcomes during influenza and MRSA coinfection, prompting us to explore the role of TLR9 in structural cells during coinfection.
To study TLR9 in structural cells, we generated mice that lack TLR9 in lung alveolar type II epithelial cells or in mouse lung fibroblasts. We measured bacterial burden and viral load following coinfection with influenza virus and MRSA. We performed primary isolation of alveolar type II cells and alveolar macrophages for in vitro supernatant studies and phagocytosis of MRSA.
Influenza infection upregulates TLR9 through a MyD88-dependent pathway in fibroblasts, and we are investigating the signaling cascade that leads to TLR9 upregulation in epithelial cells. Epithelial TLR9 expression is detrimental during coinfection: mice lacking epithelial TLR9 have reduced bacterial burden without changes in immune recruitment or lung injury. Lack of TLR9 in alveolar epithelial type II cells does not affect responses to single infections with influenza virus or MRSA. Additionally, alveolar macrophage phagocytosis of MRSA was significantly enhanced by supernatant from influenza-infected TLR9-knockout epithelial type II cells compared with supernatant from influenza-infected wild type epithelial type II cells. We are further exploring epithelial-derived factors that modulate macrophage function.
These novel findings reveal that epithelial TLR9 regulates antibacterial immunity and highlight its potential as a target for improving coinfection outcome.
Beth Moore: NHLBI R35HL144481 Helen Rich: NHLBI T32HL007517, Michigan Postdoctoral Pioneer Program Francina Gonzalez De Los Santos: Immunology Graduate Program AI007413 T32, University of Michigan; Herman & Dorothy Miller Award, Immunology Program, University of Michigan
Viral Immunology (VIR)
F. G. de Los Santos, Helen E Rich, S. Gurczynski et al.· Journal of Immunology· 0 citations
Macrophages, first responders to bacterial infection, integrate pattern recognition receptor (PRR) and interferon signaling to induce the production of reactive oxygen and nitrogen intermediates, which destroy intracellular bacteria. We have previously demonstrated that these genotoxic intermediates also damage macrophage genomic DNA, activating a DNA damage response (DDR) that is coordinated by kinases ATM and DNA-PKcs. This DDR modulates macrophage transcriptional responses during the innate immune response to infection. Though ATM has an established role in regulating cell-type-specific immune responses downstream of DNA damage, a potential role for DNA-PKcs in this regard has not been well-characterized. Thus, we asked whether or not DNA-PKcs regulates macrophage transcriptional responses during bacterial infection.
We compared the transcriptomes of wildtype and DNA-PKcs-deficient macrophages, after exposure to bacterial stimuli, using bulk RNA-sequencing. We also compared the transcriptomes of activated macrophages that were treated with a small molecule inhibitor of DNA-PKcs, AZD7648, relative to vehicle-only controls. Among the aforementioned experimental conditions, we assessed expression of a cohort of pro-inflammatory mediators using quantitative real-time PCR and ELISA. We also addressed relative activation of PRR- and interferon-regulated signaling cascades, among our experimental conditions, using immunoblotting.
We find that DNA-PKcs signaling modulates the macrophage transcriptome of activated macrophages. Additionally, DNA-PKcs kinase activity is required for optimal STING-dependent and -independent type I interferon production in activated macrophages. Of note, we find reduced expression of some, but not all, interferon-stimulated genes (ISGs) in macrophages in which DNA-PKcs kinase activity is abrogated.
Taken together, these data suggest a potential role for DNA-PKcs in modulating ISG expression beyond its regulation of type I interferon production.
National Institutes of Health (1R16GM159931-01)
Innate Immune Responses and Host Defense: Molecular Mechanisms (INM)
Abigail J Morales, Nikko Sacramento, Aniqua Mansoor et al.· Journal of Immunology· 0 citations
ABSTRACT Cells detect invading viruses and produce type I interferons (IFNs) to stimulate an innate antiviral effector response. However, IFN levels must be fine-tuned to achieve antiviral efficacy while limiting hyperinflammatory and tissue-damaging effects. Here, we report that NAT10, a histone and cytidine acetyltransferase, regulates the production of type I IFNs and RNA virus infections. Depletion of NAT10 increased the expression of IFN-β and IFN-stimulated genes, and correspondingly impaired viral replication. Mechanistically, NAT10 dynamically associated with the IFN-β promoter and also negatively regulated IRF3’s chromatin associations through modulation of long noncoding RNAs that inhibit IRF3. Treatment of cells with Remodelin, a NAT10 inhibitor, similarly increased IFN-β expression and inhibited viral infections. Overall, our findings reveal NAT10 is a potential host-directed target for antiviral treatment via regulation of type I IFN. IMPORTANCE Type I interferons (IFNs) signaling pathway is critical to cellular defense and innate immunity against evading pathogens, including viruses. However, induction of type I IFNs is fine-tuned to achieve the antiviral consequence while maintaining host cellular homeostasis. This paper presents a novel mechanism for the NAT10 protein to silence IFN-β induction through modulation of IRF3 activity at the promoter of IFN-β, and further demonstrates the therapeutic potential of the NAT10 inhibitor Remodelin to restrict viral infection while inducing IFN-β. Type I interferons (IFNs) signaling pathway is critical to cellular defense and innate immunity against evading pathogens, including viruses. However, induction of type I IFNs is fine-tuned to achieve the antiviral consequence while maintaining host cellular homeostasis. This paper presents a novel mechanism for the NAT10 protein to silence IFN-β induction through modulation of IRF3 activity at the promoter of IFN-β, and further demonstrates the therapeutic potential of the NAT10 inhibitor Remodelin to restrict viral infection while inducing IFN-β.
Youngmin Park, Yan Liu, Jinshan He et al.· mBio· 0 citations
Severe infections often cause life-threatening inflammation, multi-organ failure, and death. During infection, pathogens carry multiple pathogen-associated molecular patterns (PAMPs) such as LPS, flagellin, nucleic acids, and lipoproteins which activate more than one pattern-recognition receptor (PRR). These PRRs trigger cytokine secretion and inflammatory cell death to control pathogen load; however, a dysregulated immune response can lead to excessive inflammation and lethality. While earlier studies have primarily focused on single PAMP responses, cells in vivo are exposed to multiple microbial ligands simultaneously.
Primary bone marrow-derived macrophages (BMDMs) were stimulated with PAMPs, and real-time imaging of cell death was performed using the IncuCyte system. Microarray analysis was conducted to examine differential gene expression.
We found that specific PAMP combinations induce lytic cell death, while other combinations do not. Toll-like receptors (TLRs) and their adaptor molecules, MyD88 and TRIF, functioned as upstream regulators that activated inflammatory cell death. In addition, signaling through the interferon-α/β receptor (IFNAR) and interferon regulatory factor 1 (IRF1) was essential for the induction of inflammatory cell death. We also performed microarray analysis to understand the upregulated and downregulated genes in PAMP combinations compared with the untreated control. Through this analysis, we identified significant differences in specific molecules, and we performed genetic, functional validation to show that these molecules directly modulated the induction of inflammatory cell death.
These findings identify key mediators of lytic cell death and suggest that targeting these pathways can be used to mitigate inflammation and lethality during infections.
This research was supported by NIH grants AI101935, AI124346, AI160179, AR056296, and CA253095 and the American Lebanese Syrian Associated Charities to Dr. Kanneganti.
Innate Immune Responses and Host Defense: Molecular Mechanisms (INM)
Balamurugan Sundaram, T. Kanneganti· Journal of Immunology· 0 citations
Aspergillus fumigatus (Af) is the main cause of invasive pulmonary aspergillosis (IA), responsible for over 90% of cases, and has been identified by the World Health Organization (WHO) as the only mold among its list of critical fungal pathogens.Myeloid cells, CCR2+ inflammatory monocytes (CCR2+Mo), and neutrophils are necessary to control pulmonary Af infection.CCR2+Mo gives rise to monocyte-derived dendritic cells (mo-DCs) and alveolar macrophages (AMs), key effector populations within the CD11c+ cell compartment.Our lab found that mice with defective expression of the type I interferon receptor (IFNAR1) had increased mortality and fungal burden.Furthermore, mice with a deficiency in STAT1, a key transcription factor activated by IFNs, on CCR2+Mo and CD11c+cells had increased mortality, reduced ROS formation, and diminished control of fungal burden.Therefore, we hypothesize that type I IFNs regulate antifungal immunity through CCR2+Mo and CD11c+cells, enhancing host defense against Af infections.
Using CCR2creIFNAR1fl/fl and CD11ccreIFNAR1fl/fl mice, we evaluated the immune cell recruitment in bronchoalveolar lavage fluid and lung tissue.Antifungal responses were examined by ROS production and visualization of conidia-killing using the Af-FLARE strain.Furthermore, monocytes were sorted and analyzed for bioenergetic function using the Seahorse Analyzer and fungal burden by colony-forming units.
Mice deficient in type I IFNs in CCR2+Mo and CD11c+cells succumbed to IA with impaired ROS production and increased fungal burden.Af viability was significantly increased in CCR2creIFNAR1fl/fl mice, indicating decreased fungal clearance.The seahorse analysis demonstrated decreased oxygen consumption rate, spare respiratory capacity, and extracellular acidification rate in monocytes from CCR2creIFNAR1fl/fl.
These findings can reveal new immunological mechanisms to enhance host defense against Af and could also help improve outcomes in patients at risk for aspergillosis.
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Microbial, Parasitic, and Fungal Immunology (MPF)
Angelica Gonzalez-Martinez, V. Espinosa, A. Rivera· Journal of Immunology· 0 citations
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