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Item The Rrp2-RpoN-RpoS pathway plays an important role in the blood-brain barrier transmigration of the Lyme disease pathogen(American Society for Microbiology, 2023) Alanazi, Fuad; Raghunandanan, Sajith; Priya, Raj; Yang, X. Frank; Microbiology and Immunology, School of MedicineLyme disease, caused by Borrelia (or Borreliella) burgdorferi, is a complex multisystemic disorder that includes Lyme neuroborreliosis resulting from the invasion of both the central and peripheral nervous systems. However, factors that enable the pathogen to cross the blood-brain barrier (BBB) and invade the central nervous system (CNS) are still not well understood. The objective of this study was to identify the B. burgdorferi factors required for BBB transmigration. We utilized a transwell BBB model based on human brain-microvascular endothelial cells and focused on investigating the Rrp2-RpoN-RpoS pathway, a central regulatory pathway that is essential for mammalian infection by B. burgdorferi. Our results demonstrated that the Rrp2-RpoN-RpoS pathway is crucial for BBB transmigration. Furthermore, we identified OspC, a major surface lipoprotein controlled by the Rrp2-RpoN-RpoS pathway, as a significant contributor to BBB transmigration. Constitutive production of OspC in a mutant defective in the Rrp2-RpoN-RpoS pathway did not rescue the impairment in BBB transmigration, indicating that this pathway controls additional factors for this process. Two other major surface lipoproteins controlled by this pathway, DbpA/B and BBK32, appeared to be dispensable for BBB transmigration. In addition, both the surface lipoprotein OspA and the Rrp1 pathway, which are required B. burgdorferi colonization in the tick vector, were found not required for BBB transmigration. Collectively, our findings using in vitro transwell assays uncover another potential role of the Rrp2-RpoN-RpoS pathway in BBB transmigration of B. burgdorferi and invasion into the CNS.Item Molecular, metabolic, and functional CD4 T cell paralysis in the lymph node impedes tumor control(Elsevier, 2023) Guo, Mengdi; Abd-Rabbo, Diala; Bertol, Bruna C.; Carew, Madeleine; Lukhele, Sabelo; Snell, Laura M.; Xu, Wenxi; Boukhaled, Giselle M.; Elsaesser, Heidi; Halaby, Marie Jo; Hirano, Naoto; McGaha, Tracy L.; Brooks, David G.; Microbiology and Immunology, School of MedicineCD4 T cells are central effectors of anti-cancer immunity and immunotherapy, yet the regulation of CD4 tumor-specific T (TTS) cells is unclear. We demonstrate that CD4 TTS cells are quickly primed and begin to divide following tumor initiation. However, unlike CD8 TTS cells or exhaustion programming, CD4 TTS cell proliferation is rapidly frozen in place by a functional interplay of regulatory T cells and CTLA4. Together these mechanisms paralyze CD4 TTS cell differentiation, redirecting metabolic circuits, and reducing their accumulation in the tumor. The paralyzed state is actively maintained throughout cancer progression and CD4 TTS cells rapidly resume proliferation and functional differentiation when the suppressive constraints are alleviated. Overcoming their paralysis established long-term tumor control, demonstrating the importance of rapidly crippling CD4 TTS cells for tumor progression and their potential restoration as therapeutic targets.Item The neurotransmitter receptor Gabbr1 regulates proliferation and function of hematopoietic stem and progenitor cell(American Society of Hematology, 2021) Shao, Lijian; Elujoba-Bridenstine, Adedamola; Zink, Katherine E.; Sanchez, Laura M.; Cox, Brian J.; Pollok, Karen E.; Sinn, Anthony L.; Bailey, Barbara J.; Sims, Emily C.; Cooper, Scott H.; Broxmeyer, Hal E.; Pajcini, Kostandin V.; Tamplin, Owen J.; Microbiology and Immunology, School of MedicineHematopoietic and nervous systems are linked via innervation of bone marrow (BM) niche cells. Hematopoietic stem/progenitor cells (HSPCs) express neurotransmitter receptors, such as the γ-aminobutyric acid (GABA) type B receptor subunit 1 (GABBR1), suggesting that HSPCs could be directly regulated by neurotransmitters like GABA that directly bind to GABBR1. We performed imaging mass spectrometry and found that the endogenous GABA molecule is regionally localized and concentrated near the endosteum of the BM niche. To better understand the role of GABBR1 in regulating HSPCs, we generated a constitutive Gabbr1-knockout mouse model. Analysis revealed that HSPC numbers were significantly reduced in the BM compared with wild-type littermates. Moreover, Gabbr1-null hematopoietic stem cells had diminished capacity to reconstitute irradiated recipients in a competitive transplantation model. Gabbr1-null HSPCs were less proliferative under steady-state conditions and upon stress. Colony-forming unit assays demonstrated that almost all Gabbr1-null HSPCs were in a slow or noncycling state. In vitro differentiation of Gabbr1-null HSPCs in cocultures produced fewer overall cell numbers with significant defects in differentiation and expansion of the B-cell lineage. To determine whether a GABBR1 agonist could stimulate human umbilical cord blood (UCB) HSPCs, we performed brief ex vivo treatment prior to transplant into immunodeficient mice, with significant increases in long-term engraftment of HSPCs compared with GABBR1 antagonist or vehicle treatments. Our results indicate a direct role for GABBR1 in HSPC proliferation, and identify a potential target to improve HSPC engraftment in clinical transplantation.Item Regulation of Bacterial Two-Component Systems by Cardiolipin(American Society for Microbiology, 2023) Yeo, Won-Sik; Dyzenhaus, Sophie; Torres, Victor J.; Brinsmade, Shaun R.; Bae, Taeok; Microbiology and Immunology, School of MedicineThe regulation of membrane protein activity for cellular functions is critically dependent on the composition of phospholipid membranes. Cardiolipin, a unique phospholipid found in bacterial membranes and mitochondrial membranes of eukaryotes, plays a crucial role in stabilizing membrane proteins and maintaining their function. In the human pathogen Staphylococcus aureus, the SaeRS two-component system (TCS) controls the expression of key virulence factors essential for the bacterium’s virulence. The SaeS sensor kinase activates the SaeR response regulator via phosphoryl transfer to bind its gene target promoters. In this study, we report that cardiolipin is critical for sustaining the full activity of SaeRS and other TCSs in S. aureus. The sensor kinase protein SaeS binds directly to cardiolipin and phosphatidylglycerol, enabling SaeS activity. Elimination of cardiolipin from the membrane reduces SaeS kinase activity, indicating that bacterial cardiolipin is necessary for modulating the kinase activities of SaeS and other sensor kinases during infection. Moreover, the deletion of cardiolipin synthase genes cls1 and cls2 leads to reduced cytotoxicity to human neutrophils and lower virulence in a mouse model of infection. These findings suggest a model where cardiolipin modulates the kinase activity of SaeS and other sensor kinases after infection to adapt to the hostile environment of the host and expand our knowledge of how phospholipids contribute to membrane protein function.Item Interleukin-9 promotes mast cell progenitor proliferation and CCR2-dependent mast cell migration in allergic airway inflammation(Elsevier, 2023) Pajulas, Abigail; Fu, Yongyao; Cheung, Cherry C. L.; Chu, Michelle; Cannon, Anthony; Alakhras, Nada; Zhang, Jilu; Ulrich, Benjamin J.; Nelson, Andrew S.; Zhou, Baohua; Kaplan, Mark H.; Microbiology and Immunology, School of MedicineAllergic asthma is a chronic lung disease characterized by airway hyperresponsiveness and cellular infiltration that is exacerbated by immunoglobulin E-dependent mast cell (MC) activation. Interleukin-9 (IL-9) promotes MC expansion during allergic inflammation but precisely how IL-9 expands tissue MCs and promotes MC function is unclear. In this report, using multiple models of allergic airway inflammation, we show that both mature MCs (mMCs) and MC progenitors (MCp) express IL-9R and respond to IL-9 during allergic inflammation. IL-9 acts on MCp in the bone marrow and lungs to enhance proliferative capacity. Furthermore, IL-9 in the lung stimulates the mobilization of CCR2+ mMC from the bone marrow and recruitment to the allergic lung. Mixed bone marrow chimeras demonstrate that these are intrinsic effects in the MCp and mMC populations. IL-9-producing T cells are both necessary and sufficient to increase MC numbers in the lung in the context of allergic inflammation. Importantly, T cell IL-9-mediated MC expansion is required for the development of antigen-induced and MC-dependent airway hyperreactivity. Collectively, these data demonstrate that T cell IL-9 induces lung MC expansion and migration by direct effects on the proliferation of MCp and the migration of mMC to mediate airway hyperreactivity.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 MedicineIntroduction: 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.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 MedicineIntroduction: 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.Item Formate production is dispensable for Haemophilus ducreyi virulence in human volunteers(American Society for Microbiology, 2023) Brothwell, Julie A.; Fortney, Kate R.; Williams, Jalan S.; Batteiger, Teresa A.; Duplantier, Rory; Grounds, Danielle; Jannasch, Amber S.; Katz, Barry P.; Spinola, Stanley M.; Microbiology and Immunology, School of MedicineHaemophilus ducreyi is a causative agent of cutaneous ulcers in children who live in the tropics and of the genital ulcer disease chancroid in sexually active persons. In the anaerobic environment of abscesses and ulcers, anaerobic respiration and mixed acid fermentation (MAF) can be used to provide cellular energy. In Escherichia coli, MAF produces formate, acetate, lactate, succinate, and ethanol; however, MAF has not been studied in H. ducreyi. In human challenge experiments with H. ducreyi 35000HP, transcripts of the formate transporter FocA and pyruvate formate lyase (PflB) were upregulated in pustules compared to the inocula. We made single and double mutants of focA and pflB in 35000HP. Growth of 35000HPΔfocA was similar to 35000HP, but 35000HPΔpflB and 35000HPΔfocA-pflB had growth defects during both aerobic and anaerobic growth. Mutants lacking pflB did not secrete formate into the media. However, formate was secreted into the media by 35000HPΔfocA, indicating that H. ducreyi has alternative formate transporters. The pH of the media during anaerobic growth decreased for 35000HP and 35000HPΔfocA, but not for 35000HPΔpflB or 35000HPΔfocA-pflB, indicating that pflB is the main contributor to media acidification during anaerobic growth. We tested whether formate production and transport were required for virulence in seven human volunteers in a mutant versus parent trial between 35000HPΔfocA-pflB and 35000HP. The pustule formation rate was similar for 35000HP (42.9%)- and 35000HPΔfocA-pflB (62%)-inoculated sites. Although formate production occurs during in vitro growth and focA-pflB transcripts are upregulated during human infection, focA and pflB are not required for virulence in humans.Item Interactions between B cells and T follicular regulatory cells enhance susceptibility to Brucella infection independent of the anti-Brucella humoral response(Public Library of Science, 2023-09-18) Dadelahi, Alexis S.; Abushahba, Mostafa F. N.; Ponzilacqua-Silva, Bárbara; Chambers, Catherine A.; Moley, Charles R.; Lacey, Carolyn A.; Dent, Alexander L.; Skyberg, Jerod A.; Microbiology and Immunology, School of MedicineBrucellosis, caused by facultative, intracellular Brucella spp., often results in chronic and/or lifelong infection. Therefore, Brucella must employ mechanisms to subvert adaptive immunity to cause chronic infection. B lymphocytes enhance susceptibility to infection with Brucella spp. though the mechanisms remain unclear. Here we investigated the role of antibody secretion, B cell receptor (BCR) specificity, and B cell antigen presentation on susceptibility to B. melitensis. We report that mice unable to secrete antibody do not display altered resistance to Brucella. However, animals with B cells that are unable to recognize Brucella through their BCR are resistant to infection. In addition, B cell MHCII expression enhances susceptibility to infection in a CD4+ T cell-dependent manner, and we found that follicular B cells are sufficient to inhibit CD4+ T cell-mediated immunity against Brucella. B cells promote development of T follicular helper (TFH) and T follicular regulatory (TFR) cells during Brucella infection. Inhibition of B cell and CD4+ T cell interaction via CD40L blockade enhances resistance to Brucella in a B cell dependent manner concomitant with suppression of TFH and TFR differentiation. Conversely, PD-1 blockade increases Brucella burdens in a B and CD4+ T cell dependent manner while augmenting T regulatory (TReg) and TFR responses. Intriguingly, TFR deficiency enhances resistance to Brucella via a B cell dependent, but antibody independent mechanism. Collectively, these results demonstrate B cells support TFR responses that promote susceptibility to Brucella infection independent of the antibody response.Item Regulation of lung inflammation by adiponectin(Frontiers Media, 2023-09-01) Lim, Joo-Yeon; Templeton, Steven P.; Microbiology and Immunology, School of MedicineAdiponectin is an insulin sensitizing hormone that also plays a role in the regulation of inflammation. Although adiponectin can exert pro-inflammatory effects, more studies have reported anti-inflammatory effects, even in non-adipose tissues such as the lung. Obesity is considered an inflammatory disease, is a risk factor for lung diseases, and is associated with decreased levels of plasma adiponectin. The results of recent studies have suggested that adiponectin exerts anti-inflammatory activity in chronic obstructive pulmonary disease, asthma and invasive fungal infection. The signaling receptors of adiponectin, AdipoR1 and AdipoR2, are expressed by epithelial cells, endothelial cells, and immune cells in the lung. In this mini-review, we discuss the anti-inflammatory mechanisms of adiponectin in lung cells and tissues.