Department of Microbiology and Immunology

Permanent URI for this community

https://hdl.handle.net/1805/6746

Browse

Browsing Department of Microbiology and Immunology by Title

Now showing 1 - 10 of 553
  • Thumbnail Image
    Item
    4-Ethylguaiacol Modulates Neuroinflammation and Promotes Heme Oxygenase-1 Expression to Ameliorate Brain Injury in Ischemic Stroke
    (Frontiers, 2022-07) Weng, Wen-Tsan; Kuo, Ping-Chang; Scofield, Barbara A.; Paraiso, Hallel C.; Brown, Dennis A.; Yu, I-Chen; Yen, Jui-Hung; Microbiology and Immunology, School of Medicine
    Ischemic stroke is caused by a sudden reduction in cerebral blood flow that subsequently induces a complex cascade of pathophysiological responses, leading to brain inflammation and irreversible infarction. 4-ethylguaiacol (4-EG) is reported to suppress inflammatory immune responses. However, whether 4-EG exerts anti-inflammatory effects in ischemic stroke remains unexplored. We evaluated the therapeutic potential of 4-EG and examined the cellular and molecular mechanisms underlying the protective effects of 4-EG in ischemic stroke. The effect of 4-EG in ischemic stroke was determined by using a transient middle cerebral artery occlusion (MCAO) animal model followed by exploring the infarct size, neurological deficits, microglia activation, inflammatory cytokine production, blood–brain barrier (BBB) disruption, brain endothelial cell adhesion molecule expression, and microglial heme oxygenase-1 (HO-1) expression. Nrf2-/- and HO-1 inhibitor ZnPP-treated mice were also subjected to MCAO to evaluate the role of the Nrf2/HO-1 pathway in 4-EG-mediated protection in ischemic stroke. We found that 4-EG attenuated infarct size and neurological deficits, and lessened BBB disruption in ischemic stroke. Further investigation revealed that 4-EG suppressed microglial activation, peripheral inflammatory immune cell infiltration, and brain endothelial cell adhesion molecule upregulation in the ischemic brain. Finally, we identified that the protective effect of 4-EG in ischemic stroke was abolished in Nrf2-/– and ZnPP-treated MCAO mice. Our results identified that 4-EG confers protection against ischemic stroke and reveal that the protective effect of 4-EG in ischemic stroke is mediated through the induction of the Nrf2/HO1 pathway. Thus, our findings suggest that 4-EG could be developed as a novel therapeutic agent for the treatment of ischemic stroke.
  • Thumbnail Image
    Item
    4-Ethylguaiacol Modulates Neuroinflammation and Promotes Heme Oxygenase-1 Expression to Ameliorate Brain Injury in Ischemic Stroke
    (Frontiers Media, 2022-07-01) Weng, Wen-Tsan; Kuo, Ping-Chang; Scofield, Barbara A.; Paraiso, Hallel C.; Brown, Dennis A.; Yu, I-Chen; Yen, Jui-Hung; Microbiology and Immunology, School of Medicine
    Ischemic stroke is caused by a sudden reduction in cerebral blood flow that subsequently induces a complex cascade of pathophysiological responses, leading to brain inflammation and irreversible infarction. 4-ethylguaiacol (4-EG) is reported to suppress inflammatory immune responses. However, whether 4-EG exerts anti-inflammatory effects in ischemic stroke remains unexplored. We evaluated the therapeutic potential of 4-EG and examined the cellular and molecular mechanisms underlying the protective effects of 4-EG in ischemic stroke. The effect of 4-EG in ischemic stroke was determined by using a transient middle cerebral artery occlusion (MCAO) animal model followed by exploring the infarct size, neurological deficits, microglia activation, inflammatory cytokine production, blood-brain barrier (BBB) disruption, brain endothelial cell adhesion molecule expression, and microglial heme oxygenase-1 (HO-1) expression. Nrf2-/- and HO-1 inhibitor ZnPP-treated mice were also subjected to MCAO to evaluate the role of the Nrf2/HO-1 pathway in 4-EG-mediated protection in ischemic stroke. We found that 4-EG attenuated infarct size and neurological deficits, and lessened BBB disruption in ischemic stroke. Further investigation revealed that 4-EG suppressed microglial activation, peripheral inflammatory immune cell infiltration, and brain endothelial cell adhesion molecule upregulation in the ischemic brain. Finally, we identified that the protective effect of 4-EG in ischemic stroke was abolished in Nrf2-/- and ZnPP-treated MCAO mice. Our results identified that 4-EG confers protection against ischemic stroke and reveal that the protective effect of 4-EG in ischemic stroke is mediated through the induction of the Nrf2/HO1 pathway. Thus, our findings suggest that 4-EG could be developed as a novel therapeutic agent for the treatment of ischemic stroke.
  • Thumbnail Image
    Item
    A Fur family protein BosR is a novel RNA-binding protein that controls rpoS RNA stability in the Lyme disease pathogen
    (Oxford University Press, 2024) Raghunandanan, Sajith; Priya, Raj; Alanazi, Fuad; Lybecker, Meghan C.; Schlax, Paula Jean; Yang, X. Frank; Microbiology and Immunology, School of Medicine
    2´-O-methylation (Nm) is one of the most abundant modifications found in both mRNAs and noncoding RNAs. It contributes to many biological processes, such as the normal functioning of tRNA, the protection of mRNA against degradation by the decapping and exoribonuclease (DXO) protein, and the biogenesis and specificity of rRNA. Recent advancements in single-molecule sequencing techniques for long read RNA sequencing data offered by Oxford Nanopore technologies have enabled the direct detection of RNA modifications from sequencing data. In this study, we propose a bio-computational framework, Nm-Nano, for predicting the presence of Nm sites in direct RNA sequencing data generated from two human cell lines. The Nm-Nano framework integrates two supervised machine learning (ML) models for predicting Nm sites: Extreme Gradient Boosting (XGBoost) and Random Forest (RF) with K-mer embedding. Evaluation on benchmark datasets from direct RNA sequecing of HeLa and HEK293 cell lines, demonstrates high accuracy (99% with XGBoost and 92% with RF) in identifying Nm sites. Deploying Nm-Nano on HeLa and HEK293 cell lines reveals genes that are frequently modified with Nm. In HeLa cell lines, 125 genes are identified as frequently Nm-modified, showing enrichment in 30 ontologies related to immune response and cellular processes. In HEK293 cell lines, 61 genes are identified as frequently Nm-modified, with enrichment in processes like glycolysis and protein localization. These findings underscore the diverse regulatory roles of Nm modifications in metabolic pathways, protein degradation, and cellular processes. The source code of Nm-Nano can be freely accessed at https://github.com/Janga-Lab/Nm-Nano.
  • Thumbnail Image
    Item
    A Member of an Ancient Family of Bacterial Amino Acids Transporters Contributes to Chlamydia Nutritional Virulence and Immune Evasion
    (American Society for Microbiology, 2023) Banerjee, Arkaprabha; Sun, Yuan; Muramatsu, Matthew K.; Toh, Evelyn; Nelson, David E.; Microbiology and Immunology, School of Medicine
    Many obligate intracellular bacteria, including members of the genus Chlamydia, cannot synthesize a variety of amino acids de novo and acquire these from host cells via largely unknown mechanisms. Previously, we determined that a missense mutation in ctl0225, a conserved Chlamydia open reading frame of unknown function, mediated sensitivity to interferon gamma. Here, we show evidence that CTL0225 is a member of the SnatA family of neutral amino acid transporters that contributes to the import of several amino acids into Chlamydia cells. Further, we show that CTL0225 orthologs from two other distantly related obligate intracellular pathogens (Coxiella burnetii and Buchnera aphidicola) are sufficient to import valine into Escherichia coli. We also show that chlamydia infection and interferon exposure have opposing effects on amino acid metabolism, potentially explaining the relationship between CTL0225 and interferon sensitivity. Overall, we show that phylogenetically diverse intracellular pathogens use an ancient family of amino acid transporters to acquire host amino acids and provide another example of how nutritional virulence and immune evasion can be linked in obligate intracellular pathogens.
  • Thumbnail Image
    Item
    A novel Gardnerella, Prevotella, and Lactobacillus standard that improves accuracy in quantifying bacterial burden in vaginal microbial communities
    (Frontiers Media, 2023-06-19) Elnaggar, Jacob H.; Ardizzone, Caleb M.; Cerca, Nuno; Toh, Evelyn; Łaniewski, Paweł; Lillis, Rebecca A.; Herbst-Kralovetz, Melissa M.; Quayle, Alison J.; Muzny, Christina A.; Taylor, Christopher M.; Microbiology and Immunology, School of Medicine
    Bacterial vaginosis (BV) is the most common vaginal dysbiosis. In this condition, a polymicrobial biofilm develops on vaginal epithelial cells. Accurately quantifying the bacterial burden of the BV biofilm is necessary to further our understanding of BV pathogenesis. Historically, the standard for calculating total bacterial burden of the BV biofilm has been based on quantifying Escherichia coli 16S rRNA gene copy number. However, E. coli is improper for measuring the bacterial burden of this unique micro-environment. Here, we propose a novel qPCR standard to quantify bacterial burden in vaginal microbial communities, from an optimal state to a mature BV biofilm. These standards consist of different combinations of vaginal bacteria including three common BV-associated bacteria (BVAB) Gardnerella spp. (G), Prevotella spp. (P), and Fannyhessea spp. (F) and commensal Lactobacillus spp. (L) using the 16S rRNA gene (G:P:F:L, G:P:F, G:P:L and 1G:9L). We compared these standards to the traditional E. coli (E) reference standard using known quantities of mock vaginal communities and 16 vaginal samples from women. The E standard significantly underestimated the copy numbers of the mock communities, and this underestimation was significantly greater at lower copy numbers of these communities. The G:P:L standard was the most accurate across all mock communities and when compared to other mixed vaginal standards. Mixed vaginal standards were further validated with vaginal samples. This new G:P:L standard can be used in BV pathogenesis research to enhance reproducibility and reliability in quantitative measurements of BVAB, spanning from the optimal to non-optimal (including BV) vaginal microbiota.
  • Thumbnail Image
    Item
    A systemic approach to identify non-abundant immunogenic proteins in Lyme disease pathogens
    (American Society for Microbiology, 2024) Yaş, Ozlem Buyuktanir; Coleman, Adam S.; Lipman, Rachel M.; Sharma, Kavita; Raghunandanan, Sajith; Alanazi, Fuad; Rana, Vipin S.; Kitsou, Chrysoula; Yang, Xiuli; Pal, Utpal; Microbiology and Immunology, School of Medicine
    Borrelia burgdorferi, the pathogen of Lyme disease, differentially produces many outer surface proteins (Osp), some of which represent the most abundant membrane proteins, such as OspA, OspB, and OspC. In cultured bacteria, these proteins can account for a substantial fraction of the total cellular or membrane proteins, posing challenges to the identification and analysis of non-abundant proteins, which could serve as novel pathogen detection markers or as vaccine candidates. Herein, we introduced serial mutations to remove these abundant Osps and generated a B. burgdorferi mutant deficient in OspA, OspB, and OspC in an infectious 297-isolate background, designated as OspABC- mutant. Compared to parental isolate, the mutant did not reflect growth defects in the cultured medium but showed differential mRNA expression of representative tested genes, in addition to gross changes in cellular and membrane protein profiles. The analysis of differentially detectable protein contents of the OspABC- mutant, as compared to the wild type, by two-dimensional gel electrophoresis followed by liquid chromatography-mass spectrometry, identified several spirochete proteins that are dominated by proteins of unknown functions, as well as membrane transporters, chaperons, and metabolic enzymes. We produced recombinant forms of two of these represented proteins, BBA34 and BB0238, and showed that these proteins are detectable during spirochete infection in the tick-borne murine model of Lyme borreliosis and thus serve as potential antigenic markers of the infection. IMPORTANCE: The present manuscript employed a systemic approach to identify non-abundant proteins in cultured Borrelia burgdorferi that are otherwise masked or hidden due to the overwhelming presence of abundant Osps like OspA, OspB, and OspC. As these Osps are either absent or transiently expressed in mammals, we performed a proof-of-concept study in which their removal allowed the analysis of otherwise less abundant antigens in OspABC-deficient mutants and identified several immunogenic proteins, including BBA34 and BB0238. These antigens could serve as novel vaccine candidates and/or genetic markers of Lyme borreliosis, promoting new research in the clinical diagnosis and prevention of Lyme disease.
  • Thumbnail Image
    Item
    A TLR7-nanoparticle adjuvant promotes a broad immune response against heterologous strains of influenza and SARS-CoV-2
    (Springer Nature, 2023) Yin, Qian; Luo, Wei; Mallajosyula, Vamsee; Bo, Yang; Guo, Jing; Xie, Jinghang; Sun, Meng; Verma, Rohit; Li, Chunfeng; Constantz, Christian M.; Wagar, Lisa E.; Li, Jing; Sola, Elsa; Gupta, Neha; Wang, Chunlin; Kask, Oliver; Chen, Xin; Yuan, Xue; Wu, Nicholas C.; Rao, Jianghong; Chien, Yueh-hsiu; Cheng, Jianjun; Pulendran, Bali; Davis, Mark M.; Microbiology and Immunology, School of Medicine
    The ideal vaccine against viruses such as influenza and SARS-CoV-2 must provide a robust, durable and broad immune protection against multiple viral variants. However, antibody responses to current vaccines often lack robust cross-reactivity. Here we describe a polymeric Toll-like receptor 7 agonist nanoparticle (TLR7-NP) adjuvant, which enhances lymph node targeting, and leads to persistent activation of immune cells and broad immune responses. When mixed with alum-adsorbed antigens, this TLR7-NP adjuvant elicits cross-reactive antibodies for both dominant and subdominant epitopes and antigen-specific CD8+ T-cell responses in mice. This TLR7-NP-adjuvanted influenza subunit vaccine successfully protects mice against viral challenge of a different strain. This strategy also enhances the antibody response to a SARS-CoV-2 subunit vaccine against multiple viral variants that have emerged. Moreover, this TLR7-NP augments antigen-specific responses in human tonsil organoids. Overall, we describe a nanoparticle adjuvant to improve immune responses to viral antigens, with promising implications for developing broadly protective vaccines.
  • Thumbnail Image
    Item
    A viral-specific CD4+ T cell response protects female mice from Coxsackievirus B3 infection
    (Frontiers Media, 2024-01-11) Pattnaik, Aryamav; Dhalech, Adeeba H.; Condotta, Stephanie A.; Corn, Caleb; Richer, Martin J.; Snell, Laura M.; Robinson, Christopher M.; Microbiology and Immunology, School of Medicine
    Background: Biological sex plays an integral role in the immune response to various pathogens. The underlying basis for these sex differences is still not well defined. Here, we show that Coxsackievirus B3 (CVB3) induces a viral-specific CD4+ T cell response that can protect female mice from mortality. Methods: We inoculated C57BL/6 Ifnar-/- mice with CVB3. We investigated the T cell response in the spleen and mesenteric lymph nodes in male and female mice following infection. Results: We found that CVB3 can induce expansion of CD62Llo CD4+ T cells in the mesenteric lymph node and spleen of female but not male mice as early as 5 days post-inoculation, indicative of activation. Using a recombinant CVB3 virus expressing a model CD4+ T cell epitope, we found that this response is due to viral antigen and not bystander activation. Finally, the depletion of CD4+ T cells before infection increased mortality in female mice, indicating that CD4+ T cells play a protective role against CVB3 in our model. Conclusions: Overall, these data demonstrated that CVB3 can induce an early CD4 response in female but not male mice and further emphasize how sex differences in immune responses to pathogens affect disease.
  • Thumbnail Image
    Item
    Abstract 16: Insights into Highly Engraftable Hematopoietic Cells from 27-Year Cryopreserved Umbilical Cord Blood
    (Oxford University Press, 2023-09-04) Broxmeyer, Hal; Luchsinger, Larry; Weinberg, Rona; Jimenez, Alexandra; Masson Frenet, Emeline; van't Hof, Wouter; Capitano, Maegan; Hillyer, Christopher; Kaplan, Mark; Cooper, Scott; Ropa, James; Microbiology and Immunology, School of Medicine
    Introduction: Cord blood banking has consistently outpaced the utilization of cord blood units (CBUs). Thus, the average duration of cryopreservation among banked CBUs will likely continue to increase. It remains unclear how long cryopreserved CBUs remain functional, and it is critical to determine whether duration of cryopreservation should be used as an exclusionary criterion during selection for clinical use or if alternative post-thaw metrics can identify potent cryopreserved CBUs regardless of age. Objectives: Our goal was to determine whether long-term (27-year) cryopreserved CBUs retain viable and functional hematopoietic stem (HSCs) and progenitor cells (HPCs). We further sought to leverage differences in HSC/HPC function (measured by in vivo engraftment) to demonstrate the utility of using omics approaches to identify candidate genes for use as molecular potency markers. Methods: We performed comprehensive ex vivo, in vivo, and molecular analyses on the numbers, viability, and function of three 27-year cryopreserved CBUs using 3-year cryopreserved and fresh CBUs for comparison. Assays included viability staining, immunophenotyping by flow cytometry, primary and secondary colony forming unit (CFU) assays, ex vivo expansion of immunophenotypic HSCs/HPCs/CFUs, limiting dilution transplantations into immune-deficient mice, secondary transplantations, and RNA-sequencing of sorted HSCs and multipotent progenitor cells. Results: Compared to fresh and recently cryopreserved CBU controls, long-term cryopreserved CBUs yield statistically similar numbers of viable immunophenotypic HSCs, multipotent HPCs, and committed myeloid and lymphoid HPCs. They retain highly functional cells, demonstrating similar primary and secondary CFU numbers and expansion capacity compared to controls, as well as robust engraftment, SCID repopulating cell frequency, and secondary engraftment capacity in mouse models of transplantation. Transcriptomic modelling revealed 18 genes, including MALT1 and MAP2K1, and several gene programs, including lineage determination programs and oxidative stress responses, that are strongly enriched in high engrafting HSCs/HPCs. Discussion: CBUs cryopreserved for up to 27 years retain highly functional HSCs/HPCs. Thus, duration of cryopreservation alone is not an ideal exclusionary criteria for selection of CBUs. Preserving older CBUs may help to maintain a large and diverse pool of donors for clinical selection. Further, transcriptomics can identify candidate genes associated with engraftment for elucidation of possible CBU potency markers regardless of the duration of cryopreservation.
  • Thumbnail Image
    Item
    Abstract 26: The Role of Oxygen in Cord Blood Hematopoietic Stem and Progenitor Cell Expansion and Engraftment
    (Oxford University Press, 2023-09-04) Ropa, James; Gutch, Sarah; Beasley, Lindsay; Van't Hof, Wouter; Kaplan, Mark; Capitan, Maegan; Microbiology and Immunology, School of Medicine
    Introduction: Hematopoietic stem (HSC) and progenitor cells (HPCs) are exposed to differing oxygen tensions ranging from <1% to 21% as they reside in/move through different tissues or are harvested for clinical utility. Functional changes in HSCs/HPCs are induced by acute changes in oxygen tension (e.g., a change in percent of cells in cycle). Objectives: We sought to determine if variable oxygen levels affect expansion and/or functional properties of cord blood (CB) HSCs/HPCs ex vivo and in vivo. Methods: Human CB CD34+ cells were grown in expansion culture +/-UM171, an agonist of HSC self-renewal that expands transplantable CB HSCs, in five oxygen tensions: 1%O2, 3%O2, 5%O2, 14%O2, and 21%O2. HSCs/HPCs were enumerated by flow cytometry. Functional HPCs were enumerated by plating in semi solid media for colony forming unit assays (CFU). Cell cycle and reactive oxygen species (ROS) were measured by flow cytometry. Ability of expanded cells to engraft was determined by transplantation in non-lethally irradiated NSG mice. Results: Immunophenotypic HPCs and functional HPC CFUs expanded significantly more after 7 days of growth in higher oxygen tensions (5%O2-21%O2) compared to lower (1%O2-3%O2), while immunophenotypic HSCs expanded best at 5% O2. HSCs/HPCs grown in low oxygen tensions had significantly lower ROS levels, significantly higher percentage of cells in G0, and were slightly but reproducibly smaller/less granular than those grown in high oxygen levels. HSC/HPC numbers were reduced in high oxygen tensions 1-2 days after plating but were better maintained in low, suggesting cells undergo a culture shock/stress after plating that is mitigated by reduced oxygen. In the presence of UM171, HSCs expanded significantly better at higher oxygen levels, but HPCs are better maintained in 5%O2. Ex vivo CD34+ expansions maintained under physiological O2 levels (1-14%O2) demonstrated significantly better/faster neutrophil recovery following transplantation compared to cells expanded at 21%O2 or input. Discussion: HSCs/HPCs proliferate rapidly in high oxygen but have fewer quiescent cells, higher ROS, and are larger and more granular which are all characteristics associated with exhaustion. While high oxygen allows for faster growth, low tensions may mitigate cell stress and allow for prolonged growth (i.e., HSC/HPC expansion) while maintaining functional properties.