Mercedesz Balazs

Blood dendritic cells interact with splenic marginal zone B cells to initiate T-independent immune responses.

Marginal zone (MZ) and B1 B lymphocytes participate jointly in the early immune response against T-independent (TI) particulate antigens. Here we show that blood-derived neutrophil granulocytes and CD11c(lo) immature dendritic cells (DC) are the primary cells that efficiently capture and transport particulate bacteria to the spleen. In a systemic infection, CD11c(lo) DC, but not neutrophils, provide critical survival signals, which can be inhibited by TACI-Fc, to antigen-specific MZ B cells and promote their differentiation into IgM-secreting plasmablasts. In a local TI response, peritoneal cavity macrophages provide similar support to B1 B-derived Ag-specific blasts. In the absence of soluble TACI ligands, Ag-activated MZ- and B1-derived blasts lack survival signals and undergo apoptosis, resulting in severely impaired antibody responses.

IL-17C regulates the innate immune function of epithelial cells in an autocrine manner

Interleukin 17C (IL-17C) is a member of the IL-17 family that is selectively induced in epithelia by bacterial challenge and inflammatory stimuli. Here we show that IL-17C functioned in a unique autocrine manner, binding to a receptor complex consisting of the receptors IL-17RA and IL-17RE, which was preferentially expressed on tissue epithelial cells. IL-17C stimulated epithelial inflammatory responses, including the expression of proinflammatory cytokines, chemokines and antimicrobial peptides, which were similar to those induced by IL-17A and IL-17F. However, IL-17C was produced by distinct cellular sources, such as epithelial cells, in contrast to IL-17A, which was produced mainly by leukocytes, especially those of the TH17 subset of helper T cells. Whereas IL-17C promoted inflammation in an imiquimod-induced skin-inflammation model, it exerted protective functions in dextran sodium sulfate–induced colitis. Thus, IL-17C is an essential autocrine cytokine that regulates innate epithelial immune responses.

Loss of the Tumor Suppressor BAP1 Causes Myeloid Transformation

Identifying BAP1 Targets Inactivating mutations in the deubiquitinating enzyme BAP1 have been associated with cancer. Dey et al. (p. 1541, published online 9 August; see the Perspective by White and Harper) reveal molecular targets of the enzyme and show evidence for a role in leukemia. Mice specifically lacking the target of BAP1, HCF-1, in the bone marrow developed myeloid leukemia. BAP1 appears to be part of a complex that regulates modification of histones and gene expression important for normal hematopoiesis and tumor suppression. The deubiquitinating enzyme BAP1 is implicated in myelodysplastic syndrome. De-ubiquitinating enzyme BAP1 is mutated in a hereditary cancer syndrome with increased risk of mesothelioma and uveal melanoma. Somatic BAP1 mutations occur in various malignancies. We show that mouse Bap1 gene deletion is lethal during embryogenesis, but systemic or hematopoietic-restricted deletion in adults recapitulates features of human myelodysplastic syndrome (MDS). Knockin mice expressing BAP1 with a 3xFlag tag revealed that BAP1 interacts with host cell factor–1 (HCF-1), O-linked N-acetylglucosamine transferase (OGT), and the polycomb group proteins ASXL1 and ASXL2 in vivo. OGT and HCF-1 levels were decreased by Bap1 deletion, indicating a critical role for BAP1 in stabilizing these epigenetic regulators. Human ASXL1 is mutated frequently in chronic myelomonocytic leukemia (CMML) so an ASXL/BAP1 complex may suppress CMML. A BAP1 catalytic mutation found in a MDS patient implies that BAP1 loss of function has similar consequences in mice and humans.

Specific Btk inhibition suppresses B cell– and myeloid cell–mediated arthritis

Brutons tyrosine kinase (Btk) is a therapeutic target for rheumatoid arthritis, but the cellular and molecular mechanisms by which Btk mediates inflammation are poorly understood. Here we describe the discovery of CGI1746, a small-molecule Btk inhibitor chemotype with a new binding mode that stabilizes an inactive nonphosphorylated enzyme conformation. CGI1746 has exquisite selectivity for Btk and inhibits both auto- and transphosphorylation steps necessary for enzyme activation. Using CGI1746, we demonstrate that Btk regulates inflammatory arthritis by two distinct mechanisms. CGI1746 blocks B cell receptor-dependent B cell proliferation and in prophylactic regimens reduces autoantibody levels in collagen-induced arthritis. In macrophages, Btk inhibition abolishes FcγRIII-induced TNFα, IL-1β and IL-6 production. Accordingly, in myeloid- and FcγR-dependent autoantibody-induced arthritis, CGI1746 decreases cytokine levels within joints and ameliorates disease. These results provide new understanding of the function of Btk in both B cell- or myeloid cell-driven disease processes and provide a compelling rationale for targeting Btk in rheumatoid arthritis.

Targeted depletion of lymphotoxin-alpha-expressing TH1 and TH17 cells inhibits autoimmune disease.

Uncontrolled T helper type 1 (TH1) and TH17 cells are associated with autoimmune responses. We identify surface lymphotoxin-α (LT-α) as common to TH0, TH1 and TH17 cells and employ a unique strategy to target these subsets using a depleting monoclonal antibody (mAb) directed to surface LT-α. Depleting LT-α–specific mAb inhibited T cell–mediated models of delayed-type hypersensitivity and experimental autoimmune encephalomyelitis. In collagen-induced arthritis (CIA), preventive and therapeutic administration of LT-α–specific mAb inhibited disease, and immunoablated T cells expressing interleukin-17 (IL-17), interferon-γ and tumor necrosis factor-α (TNF-α), whereas decoy lymphotoxin-β receptor (LT-βR) fusion protein had no effect. A mutation in the Fc tail, rendering the antibody incapable of Fcγ receptor binding and antibody-dependent cellular cytotoxicity activity, abolished all in vivo effects. Efficacy in CIA was preceded by a loss of rheumatoid-associated cytokines IL-6, IL-1β and TNF-α within joints. These data indicate that depleting LT-α–expressing lymphocytes with LT-α–specific mAb may be beneficial in the treatment of autoimmune disease.

Addressing safety liabilities of TfR bispecific antibodies that cross the blood-brain barrier.

The safety of therapeutic bispecific antibodies that use TfR for delivery to the brain can be improved by reducing affinity for TfR and eliminating antibody effector function. Averting Roadblocks En Route to the Brain The blood-brain barrier represents a formidable blockade preventing therapeutic antibody delivery into the brain. Bispecific antibodies using the transferrin receptor (TfR) have shown promise for boosting therapeutic antibody uptake into the brain. Although TfR can act as a molecular lift to promote brain uptake, little is known about the safety ramifications of this approach. Building on a pair of studies published in Science Translational Medicine, Couch and colleagues now report that when mice were dosed with therapeutic TfR antibodies, the animals showed acute clinical reactions and a reduction in immature red blood cells, known as reticulocytes. TfR bispecific antibodies engineered to lack Fc interactions with immune cells eliminated adverse acute clinical reactions and reduced reticulocyte loss; the extent of reticulocyte loss was also influenced by binding to TfR and interaction with the complement cascade. Because reticulocytes express high levels of TfR, other cell types that express high levels of TfR were also investigated. The authors observed, for example, that the blood-brain barrier remained completely intact after TfR antibodies were administered to mice, despite the high expression of TfR in brain endothelial cells. Finally, multiple doses of TfR/BACE1 bispecific antibodies reduced amyloid-β, a toxic protein implicated in Alzheimer’s disease, with minimal sustained toxicity. Investigation of monkey and human TfR levels in circulating reticulocytes suggested that loss of these cells may be less likely to occur in primates than in mice. The translational implications of these discoveries suggest that the blood-brain barrier is not the only obstacle to surmount on the way to the brain, at least when using TfR as a molecular lift. Bispecific antibodies using the transferrin receptor (TfR) have shown promise for boosting antibody uptake in brain. Nevertheless, there are limited data on the therapeutic properties including safety liabilities that will enable successful development of TfR-based therapeutics. We evaluate TfR/BACE1 bispecific antibody variants in mouse and show that reducing TfR binding affinity improves not only brain uptake but also peripheral exposure and the safety profile of these antibodies. We identify and seek to address liabilities of targeting TfR with antibodies, namely, acute clinical signs and decreased circulating reticulocytes observed after dosing. By eliminating Fc effector function, we ameliorated the acute clinical signs and partially rescued a reduction in reticulocytes. Furthermore, we show that complement mediates a residual decrease in reticulocytes observed after Fc effector function is eliminated. These data raise important safety concerns and potential mitigation strategies for the development of TfR-based therapies that are designed to cross the blood-brain barrier.

Phosphorylation and linear ubiquitin direct A20 inhibition of inflammation

Inactivation of the TNFAIP3 gene, encoding the A20 protein, is associated with critical inflammatory diseases including multiple sclerosis, rheumatoid arthritis and Crohn’s disease. However, the role of A20 in attenuating inflammatory signalling is unclear owing to paradoxical in vitro and in vivo findings. Here we utilize genetically engineered mice bearing mutations in the A20 ovarian tumour (OTU)-type deubiquitinase domain or in the zinc finger-4 (ZnF4) ubiquitin-binding motif to investigate these discrepancies. We find that phosphorylation of A20 promotes cleavage of Lys63-linked polyubiquitin chains by the OTU domain and enhances ZnF4-mediated substrate ubiquitination. Additionally, levels of linear ubiquitination dictate whether A20-deficient cells die in response to tumour necrosis factor. Mechanistically, linear ubiquitin chains preserve the architecture of the TNFR1 signalling complex by blocking A20-mediated disassembly of Lys63-linked polyubiquitin scaffolds. Collectively, our studies reveal molecular mechanisms whereby A20 deubiquitinase activity and ubiquitin binding, linear ubiquitination, and cellular kinases cooperate to regulate inflammation and cell death.

Abrogation of growth hormone secretion rescues fatty liver in mice with hepatocyte-specific deletion of JAK2

Non-alcoholic fatty liver disease is associated with multiple comorbid conditions, including diabetes, obesity, infection, and malnutrition. Mice with hepatocyte-specific disruption of growth hormone (GH) signaling develop fatty liver (FL), although the precise mechanism underlying this finding remains unknown. Because GH signals through JAK2, we developed mice bearing hepatocyte-specific deletion of JAK2 (referred to herein as JAK2L mice). These mice were lean, but displayed markedly elevated levels of GH, liver triglycerides (TGs), and plasma FFAs. Because GH is known to promote lipolysis, we crossed GH-deficient little mice to JAK2L mice, and this rescued the FL phenotype. Expression of the fatty acid transporter CD36 was dramatically increased in livers of JAK2L mice, as was expression of Pparg. Since GH signaling represses PPARγ expression and Cd36 is a known transcriptional target of PPARγ, we treated JAK2L mice with the PPARγ-specific antagonist GW9662. This resulted in reduced expression of liver Cd36 and decreased liver TG content. These results provide a mechanism for the FL observed in mice with liver-specific disruption in GH signaling and suggest that the development of FL depends on both GH-dependent increases in plasma FFA and increased hepatic uptake of FFA, likely mediated by increased expression of CD36.

Equilibrative Nucleoside Transporter 3 Deficiency Perturbs Lysosome Function and Macrophage Homeostasis

From Nucleoside Recycling to Histiocytosis Macrophages remove billions of apoptotic cells daily, releasing their nucleic acid material through lysosomal degradation, which allows the resulting nucleosides to be recycled. Hsu et al. (p. 89, published online 15 December) found that the nucleoside transporter, equilibrative nucleoside transporter 3 (ENT3), was highly expressed in macrophages and showed that mice deficient in this transporter develop histiocytosis and features of lysosomal storage disease. When exposed to apoptotic cells, macrophages carrying human ENT3 mutations accumulated adenosine and increased their lysosomal pH. These changes contributed to an enhanced signaling through macrophage colony-stimulating factor (M-CSF) receptor and, ultimately, to M-CSF–driven myeloproliferative disease. Lack of the transporter critical for recycling of nucleosides after phagocytosis results in a fatal expansion of macrophages. Lysosomal storage diseases (LSDs) are a group of heterogeneous disorders caused by defects in lysosomal enzymes or transporters, resulting in accumulation of undegraded macromolecules or metabolites. Macrophage numbers are expanded in several LSDs, leading to histiocytosis of unknown pathophysiology. Here, we found that mice lacking the equilibrative nucleoside transporter 3 (ENT3) developed a spontaneous and progressive macrophage-dominated histiocytosis. In the absence of ENT3, defective apoptotic cell clearance led to lysosomal nucleoside buildup, elevated intralysosomal pH, and altered macrophage function. The macrophage accumulation was partly due to increased macrophage colony-stimulating factor and receptor expression and signaling secondary to the lysosomal defects. These studies suggest a cellular and molecular basis for the development of histiocytosis in several human syndromes associated with ENT3 mutations and potentially other LSDs.

A humanized monoclonal antibody targeting the β7 integrin selectively blocks intestinal homing of T lymphocytes

BACKGROUND AND PURPOSE rhuMAb Beta7 is a humanized anti‐human β7 monoclonal antibody currently in phase I in inflammatory bowel disease. rhuMAb Beta7 binds the β7 subunit of the integrins α4β7 and αEβ7, blocking interaction with their ligands. These integrins play key roles in immune cell homing to and retention in mucosal sites, and are associated with chronic inflammatory diseases of the gastrointestinal tract. The goal of this study was to evaluate the mucosal specificity of rhuMAb Beta7.