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1.
bioRxiv ; 2024 May 10.
Article in English | MEDLINE | ID: mdl-38766200

ABSTRACT

Bacteriophages (phages), viruses that specifically target and kill bacteria, represent a promising strategy to combat multidrug-resistant (MDR) pathogens such as Pseudomonas aeruginosa (Pa). However, delivering sufficient concentrations of active phages directly to the infection site remains challenging, with current methods having variable success. Here we present "HydroPhage", an innovative hydrogel system for the sustained release of high-titer phages to effectively treat infections caused by MDR pathogens. Our injectable hydrogels, featuring dual-crosslinking of hyaluronic acid and PEG-based hydrogels through static covalent thioether bonds and dynamic covalent hemithioacetal crosslinks (DCC), encapsulate phages at concentration up to 1011 PFU/mL, and achieves controlled release of 109 PFU daily over a week, surpassing levels of current clinical dosages, with more than 60% total phage recovery. In a preclinical mouse model of extended wound infection, compared to intravenous treatment, we demonstrate enhanced bacterial clearance by localized, high-dose, and repeated phage dosing despite the emergence of bacterial resistance to phages. This work advances the development of clinically practical wound dressings tailored for resistant infections.

2.
Nat Commun ; 15(1): 1564, 2024 Feb 20.
Article in English | MEDLINE | ID: mdl-38378682

ABSTRACT

Although FOXP3+ regulatory T cells (Treg) depend on IL-2 produced by other cells for their survival and function, the levels of IL-2 in inflamed tissue are low, making it unclear how Treg access this critical resource. Here, we show that Treg use heparanase (HPSE) to access IL-2 sequestered by heparan sulfate (HS) within the extracellular matrix (ECM) of inflamed central nervous system tissue. HPSE expression distinguishes human and murine Treg from conventional T cells and is regulated by the availability of IL-2. HPSE-/- Treg have impaired stability and function in vivo, including in the experimental autoimmune encephalomyelitis (EAE) mouse model of multiple sclerosis. Conversely, endowing monoclonal antibody-directed chimeric antigen receptor (mAbCAR) Treg with HPSE enhances their ability to access HS-sequestered IL-2 and their ability to suppress neuroinflammation in vivo. Together, these data identify a role for HPSE and the ECM in immune tolerance, providing new avenues for improving Treg-based therapy of autoimmunity.


Subject(s)
Encephalomyelitis, Autoimmune, Experimental , T-Lymphocytes, Regulatory , Mice , Animals , Humans , Interleukin-2/metabolism , Glucuronidase/genetics , Glucuronidase/metabolism , Extracellular Matrix/metabolism , Heparitin Sulfate/metabolism
3.
bioRxiv ; 2023 Feb 27.
Article in English | MEDLINE | ID: mdl-36909599

ABSTRACT

FOXP3+ regulatory T cells (Treg) depend on exogenous IL-2 for their survival and function, but circulating levels of IL-2 are low, making it unclear how Treg access this critical resource in vivo. Here, we show that Treg use heparanase (HPSE) to access IL-2 sequestered by heparan sulfate (HS) within the extracellular matrix (ECM) of inflamed central nervous system tissue. HPSE expression distinguishes human and murine Treg from conventional T cells and is regulated by the availability of IL-2. HPSE-/- Treg have impaired stability and function in vivo, including the experimental autoimmune encephalomyelitis (EAE) mouse model of multiple sclerosis. Conversely, endowing Treg with HPSE enhances their ability to access HS-sequestered IL-2 and their tolerogenic function in vivo. Together, these data identify novel roles for HPSE and the ECM in immune tolerance, providing new avenues for improving Treg-based therapy of autoimmunity.

4.
Matrix Biol ; 116: 49-66, 2023 02.
Article in English | MEDLINE | ID: mdl-36750167

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) recently emerged as the cause of a global pandemic. Infection with SARS-CoV-2 can result in COVID-19 with both acute and chronic disease manifestations that continue to impact many patients long after the resolution of viral replication. There is therefore great interest in understanding the host factors that contribute to COVID-19 pathogenesis. In this review, we address the role of hyaluronan (HA), an extracellular matrix polymer with roles in inflammation and cellular metabolism, in COVID-19 and critically evaluate the hypothesis that HA promotes COVID-19 pathogenesis. We first provide a brief overview of COVID-19 infection. Then we briefly summarize the known roles of HA in airway inflammation and immunity. We then address what is known about HA and the pathogenesis of COVID-19 acute respiratory distress syndrome (COVID-19 ARDS). Next, we examine potential roles for HA in post-acute SARS-CoV-2 infection (PASC), also known as "long COVID" as well as in COVID-associated fibrosis. Finally, we discuss the potential therapeutics that target HA as a means to treat COVID-19, including the repurposed drug hymecromone (4-methylumbelliferone). We conclude that HA is a promising potential therapeutic target for the treatment of COVID-19.


Subject(s)
COVID-19 , Humans , SARS-CoV-2 , Hyaluronic Acid , Inflammation/pathology , Post-Acute COVID-19 Syndrome
5.
Matrix Biol ; 96: 69-86, 2021 02.
Article in English | MEDLINE | ID: mdl-33290836

ABSTRACT

A coat of pericellular hyaluronan surrounds mature dendritic cells (DC) and contributes to cell-cell interactions. We asked whether 4-methylumbelliferone (4MU), an oral inhibitor of HA synthesis, could inhibit antigen presentation. We find that 4MU treatment reduces pericellular hyaluronan, destabilizes interactions between DC and T-cells, and prevents T-cell proliferation in vitro and in vivo. These effects were observed only when 4MU was added prior to initial antigen presentation but not later, consistent with 4MU-mediated inhibition of de novo antigenic responses. Building on these findings, we find that 4MU delays rejection of allogeneic pancreatic islet transplant and allogeneic cardiac transplants in mice and suppresses allogeneic T-cell activation in human mixed lymphocyte reactions. We conclude that 4MU, an approved drug, may have benefit as an adjunctive agent to delay transplantation rejection.


Subject(s)
Dendritic Cells/cytology , Graft Rejection/prevention & control , Hyaluronic Acid/biosynthesis , Hymecromone/administration & dosage , T-Lymphocytes, Regulatory/cytology , Animals , Antigen Presentation/drug effects , Cell Proliferation/drug effects , Cells, Cultured , Dendritic Cells/drug effects , Dendritic Cells/metabolism , Disease Models, Animal , Graft Rejection/immunology , Heart Transplantation/adverse effects , Humans , Hymecromone/pharmacology , Leukocytes/cytology , Leukocytes/drug effects , Leukocytes/immunology , Mice , Pancreas Transplantation/adverse effects , T-Lymphocytes, Regulatory/drug effects , T-Lymphocytes, Regulatory/metabolism , Transplantation, Homologous
6.
J Biol Chem ; 293(14): 5236-5246, 2018 04 06.
Article in English | MEDLINE | ID: mdl-29414784

ABSTRACT

For nearly 30 years, coenzyme M (CoM) was assumed to be present solely in methanogenic archaea. In the late 1990s, CoM was reported to play a role in bacterial propene metabolism, but no biosynthetic pathway for CoM has yet been identified in bacteria. Here, using bioinformatics and proteomic approaches in the metabolically versatile bacterium Xanthobacter autotrophicus Py2, we identified four putative CoM biosynthetic enzymes encoded by the xcbB1, C1, D1, and E1 genes. Only XcbB1 was homologous to a known CoM biosynthetic enzyme (ComA), indicating that CoM biosynthesis in bacteria involves enzymes different from those in archaea. We verified that the ComA homolog produces phosphosulfolactate from phosphoenolpyruvate (PEP), demonstrating that bacterial CoM biosynthesis is initiated similarly as the phosphoenolpyruvate-dependent methanogenic archaeal pathway. The bioinformatics analysis revealed that XcbC1 and D1 are members of the aspartase/fumarase superfamily (AFS) and that XcbE1 is a pyridoxal 5'-phosphate-containing enzyme with homology to d-cysteine desulfhydrases. Known AFS members catalyze ß-elimination reactions of succinyl-containing substrates, yielding fumarate as the common unsaturated elimination product. Unexpectedly, we found that XcbC1 catalyzes ß-elimination on phosphosulfolactate, yielding inorganic phosphate and a novel metabolite, sulfoacrylic acid. Phosphate-releasing ß-elimination reactions are unprecedented among the AFS, indicating that XcbC1 is an unusual phosphatase. Direct demonstration of phosphosulfolactate synthase activity for XcbB1 and phosphate ß-elimination activity for XcbC1 strengthened their hypothetical assignment to a CoM biosynthetic pathway and suggested functions also for XcbD1 and E1. Our results represent a critical first step toward elucidating the CoM pathway in bacteria.


Subject(s)
Mesna/metabolism , Phosphates/metabolism , Xanthobacter/metabolism , Aspartate Ammonia-Lyase/metabolism , Bacteria/metabolism , Computational Biology/methods , Crystallography, X-Ray , Fumarate Hydratase/metabolism , Fumarates , Phosphoenolpyruvate/metabolism , Phosphoric Acids , Phosphoric Monoester Hydrolases , Proteomics , Pyridoxal Phosphate
7.
Nat Commun ; 7: 13848, 2016 12 15.
Article in English | MEDLINE | ID: mdl-27976744

ABSTRACT

A robust primary immune response has been correlated with the precursor number of antigen-specific T cells, as identified using peptide MHCII tetramers. However, these tetramers identify only the highest-affinity T cells. Here we show the entire CD4+ T-cell repertoire, inclusive of low-affinity T cells missed by tetramers, using a T-cell receptor (TCR) signalling reporter and micropipette assay to quantify naive precursors and expanded populations. In vivo limiting dilution assays reveal hundreds more precursor T cells than previously thought, with higher-affinity tetramer-positive T cells, comprising only 5-30% of the total antigen-specific naive repertoire. Lower-affinity T cells maintain their predominance as the primary immune response progresses, with no enhancement of survival of T cells with high-affinity TCRs. These findings demonstrate that affinity for antigen does not control CD4+ T-cell entry into the primary immune response, as a diverse range in affinity is maintained from precursor through peak of T-cell expansion.


Subject(s)
Antigens/immunology , CD4-Positive T-Lymphocytes/immunology , Precursor Cells, T-Lymphoid/immunology , Adjuvants, Immunologic , Adoptive Transfer , Animals , Freund's Adjuvant , Green Fluorescent Proteins/genetics , Mice , Mice, Knockout , Mycobacterium tuberculosis/immunology , Nuclear Receptor Subfamily 4, Group A, Member 1/genetics , Pertussis Toxin/immunology , Receptors, Antigen, T-Cell, alpha-beta/genetics , Spleen/cytology , Up-Regulation
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