Inhibition of hyaluronan synthesis prevents ß-cell loss in obesity-associated type 2 diabetes.

Pancreatic ß-cell dysfunction and death are central to the pathogenesis of type 2 diabetes (T2D). We identified a novel role for the inflammatory extracellular matrix polymer hyaluronan (HA) in this pathophysiology. Low concentrations of HA were present in healthy pancreatic islets. However, HA substantially accumulated in cadaveric islets of T2D patients and islets of the db/db mouse model of T2D in response to hyperglycemia. Treatment with 4-methylumbelliferone (4-MU), an inhibitor of HA synthesis, or the deletion of the main HA receptor CD44, preserved glycemic control and insulin concentrations in db/db mice despite ongoing weight gain, indicating a critical role for this pathway in T2D pathogenesis. 4-MU treatment and the deletion of CD44 likewise preserved glycemic control in other settings of ß-cell injury including streptozotocin treatment and islet transplantation. Mechanistically, we found that 4-MU increased the expression of the apoptosis inhibitor survivin, a downstream transcriptional target of CD44 dependent on HA/CD44 signaling, on ß-cells such that caspase 3 activation did not result in ß-cell apoptosis. These data indicated a role for HA accumulation in diabetes pathogenesis and suggested that it may be a viable target to ameliorate ß-cell loss in T2D. These data are particularly exciting, because 4-MU is already an approved drug (also known as hymecromone), which could accelerate translation of these findings to clinical studies.

Reprogramming Cancer into Antigen-Presenting Cells as a Novel Immunotherapy.

Therapeutic cancer vaccination seeks to elicit activation of tumor-reactive T cells capable of recognizing tumor-associated antigens (TAA) and eradicating malignant cells. Here, we present a cancer vaccination approach utilizing myeloid-lineage reprogramming to directly convert cancer cells into tumor-reprogrammed antigen-presenting cells (TR-APC). Using syngeneic murine leukemia models, we demonstrate that TR-APCs acquire both myeloid phenotype and function, process and present endogenous TAAs, and potently stimulate TAA-specific CD4+ and CD8+ T cells. In vivo TR-APC induction elicits clonal expansion of cancer-specific T cells, establishes cancer-specific immune memory, and ultimately promotes leukemia eradication. We further show that both hematologic cancers and solid tumors, including sarcomas and carcinomas, are amenable to myeloid-lineage reprogramming into TR-APCs. Finally, we demonstrate the clinical applicability of this approach by generating TR-APCs from primary clinical specimens and stimulating autologous patient-derived T cells. Thus, TR-APCs represent a cancer vaccination therapeutic strategy with broad implications for clinical immuno-oncology. SIGNIFICANCE: Despite recent advances, the clinical benefit provided by cancer vaccination remains limited. We present a cancer vaccination approach leveraging myeloid-lineage reprogramming of cancer cells into APCs, which subsequently activate anticancer immunity through presentation of self-derived cancer antigens. Both hematologic and solid malignancies derive significant therapeutic benefit from reprogramming-based immunotherapy. This article is highlighted in the In This Issue feature, p. 1027.

Inhibition of hyaluronan synthesis prevents ß-cell loss in obesity-associated type 2 diabetes.

Pancreatic ß-cell dysfunction and death are central to the pathogenesis of type 2 diabetes (T2D). We have identified a novel role for the inflammatory extracellular matrix polymer hyaluronan (HA) in this pathophysiology. Low levels of HA are present in healthy pancreatic islets. However, HA substantially accumulates in cadaveric islets of human T2D and islets of the db/db mouse model of T2D in response to hyperglycemia. Treatment with 4-methylumbelliferone (4-MU), an inhibitor of HA synthesis, or the deletion of the major HA receptor CD44, preserve glycemic control and insulin levels in db/db mice despite ongoing weight gain, indicating a critical role for this pathway in T2D pathogenesis. 4-MU treatment and the deletion of CD44 likewise preserve glycemic control in other settings of ß-cell injury including streptozotocin treatment and islet transplantation. Mechanistically, we find that 4-MU increases the expression of the apoptosis inhibitor survivin, a downstream transcriptional target of CD44 dependent on HA/CD44 signaling, on ß-cells such that caspase 3 activation does not result in ß-cell apoptosis. These data indicate a role for HA accumulation in diabetes pathogenesis and suggest that it may be a viable target to ameliorate ß-cell loss in T2D. These data are particularly exciting, because 4-MU is already an approved drug (also known as hymecromone), which could accelerate translation of these findings to clinical studies.

Anti-GD2 synergizes with CD47 blockade to mediate tumor eradication.

The disialoganglioside GD2 is overexpressed on several solid tumors, and monoclonal antibodies targeting GD2 have substantially improved outcomes for children with high-risk neuroblastoma. However, approximately 40% of patients with neuroblastoma still relapse, and anti-GD2 has not mediated significant clinical activity in any other GD2+ malignancy. Macrophages are important mediators of anti-tumor immunity, but tumors resist macrophage phagocytosis through expression of the checkpoint molecule CD47, a so-called 'Don't eat me' signal. In this study, we establish potent synergy for the combination of anti-GD2 and anti-CD47 in syngeneic and xenograft mouse models of neuroblastoma, where the combination eradicates tumors, as well as osteosarcoma and small-cell lung cancer, where the combination significantly reduces tumor burden and extends survival. This synergy is driven by two GD2-specific factors that reorient the balance of macrophage activity. Ligation of GD2 on tumor cells (a) causes upregulation of surface calreticulin, a pro-phagocytic 'Eat me' signal that primes cells for removal and (b) interrupts the interaction of GD2 with its newly identified ligand, the inhibitory immunoreceptor Siglec-7. This work credentials the combination of anti-GD2 and anti-CD47 for clinical translation and suggests that CD47 blockade will be most efficacious in combination with monoclonal antibodies that alter additional pro- and anti-phagocytic signals within the tumor microenvironment.

Hyaluronan synthesis inhibition impairs antigen presentation and delays transplantation rejection.

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.

Bacteriophage trigger antiviral immunity and prevent clearance of bacterial infection.

Bacteriophage are abundant at sites of bacterial infection, but their effects on mammalian hosts are unclear. We have identified pathogenic roles for filamentous Pf bacteriophage produced by Pseudomonas aeruginosa (Pa) in suppression of immunity against bacterial infection. Pf promote Pa wound infection in mice and are associated with chronic human Pa wound infections. Murine and human leukocytes endocytose Pf, and internalization of this single-stranded DNA virus results in phage RNA production. This triggers Toll-like receptor 3 (TLR3)- and TIR domain-containing adapter-inducing interferon-ß (TRIF)-dependent type I interferon production, inhibition of tumor necrosis factor (TNF), and the suppression of phagocytosis. Conversely, immunization of mice against Pf prevents Pa wound infection. Thus, Pf triggers maladaptive innate viral pattern-recognition responses, which impair bacterial clearance. Vaccination against phage virions represents a potential strategy to prevent bacterial infection.

4-Methylumbelliferyl glucuronide contributes to hyaluronan synthesis inhibition.

4-Methylumbelliferone (4-MU) inhibits hyaluronan (HA) synthesis and is an approved drug used for managing biliary spasm. However, rapid and efficient glucuronidation is thought to limit its utility for systemically inhibiting HA synthesis. In particular, 4-MU in mice has a short half-life, causing most of the drug to be present as the metabolite 4-methylumbelliferyl glucuronide (4-MUG), which makes it remarkable that 4-MU is effective at all. We report here that 4-MUG contributes to HA synthesis inhibition. We observed that oral administration of 4-MUG to mice inhibits HA synthesis, promotes FoxP3+ regulatory T-cell expansion, and prevents autoimmune diabetes. Mice fed either 4-MUG or 4-MU had equivalent 4-MU:4-MUG ratios in serum, liver, and pancreas, indicating that 4-MU and 4-MUG reach an equilibrium in these tissues. LC-tandem MS experiments revealed that 4-MUG is hydrolyzed to 4-MU in serum, thereby greatly increasing the effective bioavailability of 4-MU. Moreover, using intravital 2-photon microscopy, we found that 4-MUG (a nonfluorescent molecule) undergoes conversion into 4-MU (a fluorescent molecule) and that 4-MU is extensively tissue bound in the liver, fat, muscle, and pancreas of treated mice. 4-MUG also suppressed HA synthesis independently of its conversion into 4-MU and without depletion of the HA precursor UDP-glucuronic acid (GlcUA). Together, these results indicate that 4-MUG both directly and indirectly inhibits HA synthesis and that the effective bioavailability of 4-MU is higher than previously thought. These findings greatly alter the experimental and therapeutic possibilities for HA synthesis inhibition.

Hyaluronan in immune dysregulation and autoimmune diseases.

The tissue microenvironment contributes to local immunity and to the pathogenesis of autoimmune diseases - a diverse set of conditions characterized by sterile inflammation, immunity against self-antigens, and destruction of tissues. However, the specific factors within the tissue microenvironment that contribute to local immune dysregulation in autoimmunity are poorly understood. One particular tissue component implicated in multiple autoimmune diseases is hyaluronan (HA), an extracellular matrix (ECM) polymer. HA is abundant in settings of chronic inflammation and contributes to lymphocyte activation, polarization, and migration. Here, we first describe what is known about the size, amount, and distribution of HA at sites of autoimmunity and in associated lymphoid structures in type 1 diabetes, multiple sclerosis, and rheumatoid arthritis. Next, we examine the recent literature on HA and its impact on adaptive immunity, particularly in regards to the biology of lymphocytes and Foxp3+ regulatory T-cells (Treg), a T-cell subset that maintains immune tolerance in healthy individuals. We propose that HA accumulation at sites of chronic inflammation creates a permissive environment for autoimmunity, characterized by CD44-mediated inhibition of Treg expansion. Finally, we address potential tools and strategies for targeting HA and its receptor CD44 in chronic inflammation and autoimmunity.