Biology subjects

Bunnell, S. C.

Publications and source records attributed to Bunnell, S. C..

5 recordsLinked to original sources

bioRxiv2026

Neutrophil degranulation and extracellular ROS production are inactivated by Yersinia pseudotuberculosis YopE through a SKAP2 independent pathway

Upon sensing Yersinia pseudotuberculosis (Yptb), receptor-mediated pathways are stimulated to trigger polymorphonuclear (PMN) antimicrobial responses. Yptb injects multiple Type 3 secreted effector proteins, Yops (Yersinia outer proteins), that possess distinct biochemical functions, into PMNs to inhibit PMN responses. Here, we show that several Yops, YopE, YopH, and YopO, each partially interfered with CD63 mobilization to the plasma membrane, a marker for primary degranulation. The host pathways involved in CD63 mobilization are complex and it is not completely understood how Yops collaborate to inactivate this process. Here, CRISPR/Cas9 technology was used in an immortalized system of myeloid progenitor cells (Cas9-ER-HoxB8) to generate a panel of knockout PMN cell lines. To probe the impact of different Yops on the neutrophil pathways activated upon encountering Yptb, we interrogated the panel of genetically modified neutrophils with genetically modified bacteria. This approach of targeted gene deletion to inactivate specific pathways/proteins uncovered host pathways that synergize to induce CD63 mobilization that are distinctly targeted by YopE and YopH. YopE specifically inhibited CD63 mobilization in the absence of SKAP2, a YopH target, whereas YopH inhibited CD63 mobilization in the absence of RhoG, a YopE target, indicating that these Yops inactivate distinct signaling pathways contributing to CD63 mobilization. Furthermore, the SKAP2-independent pathway inactivated by YopE is involved in primary granule release and ROS production. Overall, this work highlights the diverse Yop-mediated mechanisms that WT-Yptb employs to effectively disarm PMN responses and provides an avenue to untangle neutrophil signaling pathways targeted by pathogens using Cas9-ER-HoxB8 cells. Author SummaryWhen sensing invading bacteria, neutrophils become activated through multiple receptors that trigger signal-transduction cascades resulting in the generation antimicrobial responses. The enteric pathogen, Yersinia pseudotuberculosis (Yptb), is equipped to effectively inhibit these responses using its collection of effector proteins (Yops). Here, we developed a system to overcome the limitations of performing genetic manipulations in neutrophils by implementing CRISPR/Cas9 technology in an engineered system of myeloid progenitor cells (Cas9-ER-HoxB8) that can be induced to differentiate into neutrophils. By infecting genetically modified neutrophils with Yptb strains expressing individual Yops, we identified distinct host signaling pathways that synergize to induce neutrophil antimicrobial responses. Our findings provide insight into several signaling events triggered by Yptb infection and show how YopE and YopH target distinct pathways to block vesicle trafficking and extracellular ROS production. This powerful genetic system can be applied to other pathogens to dissect the intricacies of neutrophil-pathogen interactions.

microbiology↗

bioRxiv2024

Differentiating Peromyscus leucopus bone marrow-derived macrophages for characterization of responses to Borrelia burgdorferi and lipopolysaccharide

Currently, most tools utilized in host-pathogen interaction studies depend on the use of human or mouse (Mus musculus) cells and tissues. While these species have led to countless breakthroughs in our understanding of infectious disease, there are undoubtably important biological processes that are missed by limiting studies to these two vertebrate species. For instance, it is well-established that a common North American deermouse, Peromyscus leucopus, has unique interactions with microbes, which likely shape its ability to serve as a critical reservoir for numerous zoonotic pathogens, including a Lyme disease spirochete, Borrelia burgdorferi. In this work, we expand the immunological toolkit to study P. leucopus biology by performing the first differentiation of deermouse bone marrow to macrophages using P. leucopus M-CSF producing HEK293T cells. We find that P. leucopus BMDMs generated through this method behave broadly very similarly to C57BL/6J macrophages generated with L-929 supernatant, though RNA sequencing revealed modest differences in transcriptomic responses to B. burgdorferi and lipopolysaccharide. In particular, differences in Il10 induction and caspase expression were observed between the species.

immunology↗

bioRxiv2024

STING promotes homeostatic maintenance of tissues and confers longevity with aging

Local immune processes within aging tissues are a significant driver of aging associated dysfunction, but tissue-autonomous pathways and cell types that modulate these responses remain poorly characterized. The cytosolic DNA sensing pathway, acting through cyclic GMP-AMP synthase (cGAS) and Stimulator of Interferon Genes (STING), is broadly expressed in tissues, and is poised to regulate local type I interferon (IFN-I)-dependent and independent inflammatory processes within tissues. Recent studies suggest that the cGAS/STING pathway may drive pathology in various in vitro and in vivo models of accelerated aging. To date, however, the role of the cGAS/STING pathway in physiological aging processes, in the absence of genetic drivers, has remained unexplored. This remains a relevant gap, as STING is ubiquitously expressed, implicated in multitudinous disorders, and loss of function polymorphisms of STING are highly prevalent in the human population (>50%). Here we reveal that, during physiological aging, STING-deficiency leads to a significant shortening of murine lifespan, increased pro-inflammatory serum cytokines and tissue infiltrates, as well as salient changes in histological composition and organization. We note that aging hearts, livers, and kidneys express distinct subsets of inflammatory, interferon-stimulated gene (ISG), and senescence genes, collectively comprising an immune fingerprint for each tissue. These distinctive patterns are largely imprinted by tissue-specific stromal and myeloid cells. Using cellular interaction network analyses, immunofluorescence, and histopathology data, we show that these immune fingerprints shape the tissue architecture and the landscape of cell-cell interactions in aging tissues. These age-associated immune fingerprints are grossly dysregulated with STING-deficiency, with key genes that define aging STING- sufficient tissues greatly diminished in the absence of STING. Changes in immune signatures are concomitant with a restructuring of the stromal and myeloid fractions, whereby cell:cell interactions are grossly altered and resulting in disorganization of tissue architecture in STING-deficient organs. This altered homeostasis in aging STING-deficient tissues is associated with a cross-tissue loss of homeostatic tissue-resident macrophage (TRM) populations in these tissues. Ex vivo analyses reveal that basal STING- signaling limits the susceptibility of TRMs to death-inducing stimuli and determines their in situ localization in tissue niches, thereby promoting tissue homeostasis. Collectively, these data upend the paradigm that cGAS/STING signaling is primarily pathological in aging and instead indicate that basal STING signaling sustains tissue function and supports organismal longevity. Critically, our study urges caution in the indiscriminate targeting of these pathways, which may result in unpredictable and pathological consequences for health during aging. HIGHLIGHTSO_LIAging tissues are associated with tissue-autonomous immune fingerprints, primarily driven by interactions of tissue stromal and myeloid populations. C_LIO_LISTING shapes these immune fingerprints of aging tissues in unexpected ways. C_LIO_LILoss of STING alters the location, numbers, and viability of tissue resident macrophages. C_LIO_LISTING signaling is critical for longer lifespans and maintenance of tissue architecture. C_LI

immunology↗

bioRxiv2021

Neutrophils require SKAP2 for reactive oxygen species production following C-type lectin and Candida stimulation

Signaling cascades that convert the recognition of pathogens to efficient inflammatory responses by immune cells, specifically neutrophils, are critical for host survival. SKAP2, an adaptor protein, is required for reactive oxygen species (ROS) generation following stimulation by integrins, formyl peptide receptors and gram-negative bacteria Klebsiella pneumoniae and Yersinia pseudotuberculosis in vitro (Nguyen et al., 2020, Shaban et al., 2020, Boras et al., 2017). SKAP2 is also required for the host defense against K. pneumoniae and{Delta} yopH Y. pseudotuberculosis infection in vivo in mouse models (Shaban et al., 2020, Nguyen et al., 2020). Another class of pattern recognition receptors (PRR) is the C-type lectin receptors (CLR), such as Dectin-1, Dectin-2 and Mincle, that are critical to trigger innate immune responses. Using neutrophils from murine HoxB8-immortalized progenitors, we show that SKAP2 is crucial for maximal ROS response to purified CLR agonists and to the fungal pathogens Candida glabrata and C. albicans, as well as for robust killing of C. glabrata. Skap2-/- murine neutrophils failed to generate ROS and exhibited reduced cellular adhesion in response to trehalose-6,6-dibehenate (TDB), furfurman, and curdlan, Mincle, Dectin-2, and Dectin-1 agonists, respectively. TDB, furfurman, and curdlan stimulation also led to SKAP2-independent integrin conformational changes, showing that inside-out signaling by these CLRs to integrin occurs in the absence of SKAP2. Pyk2 phosphorylation was significantly reduced after infection with C. glabrata in Skap2-/- neutrophils, while Syk phosphorylation was unaffected by the loss of SKAP2. These data strengthen the importance of SKAP2 in the activation of neutrophil ROS production by PRRs to include CLRs and extend the role of SKAP2 in host defense beyond antibacterial immunity to include Candida species.

immunology↗

bioRxiv2019

Polyubiquitin-dependent recruitment of NEMO/IKKγ into T cell receptor signaling microclusters

The NF-{kappa}B essential modulator protein (NEMO) is required for activation of canonical NF-{kappa}B by the T cell antigen receptor (TCR). However, the subcellular localization of NEMO during this process is not well understood. By dynamically imaging fluorescent NEMO chimeras in live human T cells, we demonstrate that NEMO is rapidly recruited into TCR microclusters via domains previously implicated in the recognition of linear and K63-linked polyubiquitin. The recruitment of NEMO into TCR microclusters requires the activities of the tyrosine kinases Lck and ZAP-70, but not the adaptor proteins LAT or SLP-76. Thus, our findings reveal that the pathways leading from TCR to NF-{kappa}B bifurcate downstream of ZAP-70 to independently control the recruitment and activation of NEMO.

immunology↗