An agonistic anti-signal regulatory protein α antibody for chronic inflammatory diseases.

Signal regulatory protein (SIRPα) is an immune inhibitory receptor expressed by myeloid cells to inhibit immune cell phagocytosis, migration, and activation. Despite the progress of SIRPα and CD47 antagonist antibodies to promote anti-cancer immunity, it is not yet known whether SIRPα receptor agonism could restrain excessive autoimmune tissue inflammation. Here, we report that neutrophil- and monocyte-associated genes including SIRPA are increased in inflamed tissue biopsies from patients with rheumatoid arthritis and inflammatory bowel diseases, and elevated SIRPA is associated with treatment-refractory ulcerative colitis. We next identify an agonistic anti-SIRPα antibody that exhibits potent anti-inflammatory effects in reducing neutrophil and monocyte chemotaxis and tissue infiltration. In preclinical models of arthritis and colitis, anti-SIRPα agonistic antibody ameliorates autoimmune joint inflammation and inflammatory colitis by reducing neutrophils and monocytes in tissues. Our work provides a proof of concept for SIRPα receptor agonism for suppressing excessive innate immune activation and chronic inflammatory disease treatment.

LOXL4, but not LOXL2, is the critical determinant of pathological collagen cross-linking and fibrosis in the lung.

Idiopathic pulmonary fibrosis is a progressive fibrotic disease characterized by excessive deposition of (myo)fibroblast produced collagen fibrils in alveolar areas of the lung. Lysyl oxidases (LOXs) have been proposed to be the central enzymes that catalyze the cross-linking of collagen fibers. Here, we report that, while its expression is increased in fibrotic lungs, genetic ablation of LOXL2 only leads to a modest reduction of pathological collagen cross-linking but not fibrosis in the lung. On the other hand, loss of another LOX family member, LOXL4, markedly disrupts pathological collagen cross-linking and fibrosis in the lung. Furthermore, knockout of both Loxl2 and Loxl4 does not offer any additive antifibrotic effects when compared to Loxl4 deletion only, as LOXL4 deficiency decreases the expression of other LOX family members including Loxl2. On the basis of these results, we propose that LOXL4 is the main LOX activity underlying pathological collagen cross-linking and lung fibrosis.

Obligate role for Rock1 and Rock2 in adult stem cell viability and function.

The ability of stem cells to rapidly proliferate and differentiate is integral to the steady-state maintenance of tissues with high turnover such as the blood and intestine. Mutations that alter these processes can cause primary immunodeficiencies, malignancies and defects in barrier function. The Rho-kinases, Rock1 and Rock2, regulate cell shape and cytoskeletal rearrangement, activities essential to mitosis. Here, we use inducible gene targeting to ablate Rock1 and Rock2 in adult mice, and identify an obligate requirement for these enzymes in the preservation of the hematopoietic and gastrointestinal systems. Hematopoietic cell progenitors devoid of Rho-kinases display cell cycle arrest, blocking the differentiation to mature blood lineages. Similarly, these mice exhibit impaired epithelial cell renewal in the small intestine, which is ultimately fatal. Our data reveal a novel role for these kinases in the proliferation and viability of stem cells and their progenitors, which is vital to maintaining the steady-state integrity of these organ systems.

A WISP1 antibody inhibits MRTF signaling to prevent the progression of established liver fibrosis.

Fibrosis is the major risk factor associated with morbidity and mortality in patients with non-alcoholic steatohepatitis (NASH)-driven chronic liver disease. Although numerous efforts have been made to identify the mediators of the initiation of liver fibrosis, the molecular underpinnings of fibrosis progression remain poorly understood, and therapies to arrest liver fibrosis progression are elusive. Here, we identify a pathway involving WNT1-inducible signaling pathway protein 1 (WISP1) and myocardin-related transcription factor (MRTF) as a central mechanism driving liver fibrosis progression through the integrin-dependent transcriptional reprogramming of myofibroblast cytoskeleton and motility. In mice, WISP1 deficiency protects against fibrosis progression, but not fibrosis onset. Moreover, the therapeutic administration of a novel antibody blocking WISP1 halted the progression of existing liver fibrosis in NASH models. These findings implicate the WISP1-MRTF axis as a crucial determinant of liver fibrosis progression and support targeting this pathway by antibody-based therapy for the treatment of NASH fibrosis.

BMP1 is not required for lung fibrosis in mice.

Bone morphogenetic protein 1 (BMP1) belongs to the astacin/BMP1/tolloid-like family of zinc metalloproteinases, which play a fundamental role in the development and formation of extracellular matrix (ECM). BMP1 mediates the cleavage of carboxyl terminal (C-term) propeptides from procollagens, a crucial step in fibrillar collagen fiber formation. Blocking BMP1 by small molecule or antibody inhibitors has been linked to anti-fibrotic activity in the preclinical models of skin, kidney and liver fibrosis. Therefore, we reason that BMP1 may be important for the pathogenesis of lung fibrosis and BMP1 could be a potential therapeutic target for progressive fibrotic disease such as idiopathic pulmonary fibrosis (IPF). Here, we observed the increased expression of BMP1 in both human IPF lungs and mouse fibrotic lungs induced by bleomycin. Furthermore, we developed an inducible Bmp1 conditional knockout (cKO) mouse strain. We found that Bmp1 deletion does not protect mice from lung fibrosis triggered by bleomycin. Moreover, we found no significant impact of BMP1 deficiency upon C-term propeptide of type I procollagen (CICP) production in the fibrotic mouse lungs. Based on these results, we propose that BMP1 is not required for lung fibrosis in mice and BMP1 may not be considered a candidate therapeutic target for IPF.

TGFß2 and TGFß3 isoforms drive fibrotic disease pathogenesis.

Transforming growth factor-ß (TGFß) is a key driver of fibrogenesis. Three TGFß isoforms (TGFß1, TGFß2, and TGFß3) in mammals have distinct functions in embryonic development; however, the postnatal pathological roles and activation mechanisms of TGFß2 and TGFß3 have not been well characterized. Here, we show that the latent forms of TGFß2 and TGFß3 can be activated by integrin-independent mechanisms and have lower activation thresholds compared to TGFß1. Unlike TGFB1, TGFB2 and TGFB3 expression is increased in human lung and liver fibrotic tissues compared to healthy control tissues. Thus, TGFß2 and TGFß3 may play a pathological role in fibrosis. Inducible conditional knockout mice and anti-TGFß isoform-selective antibodies demonstrated that TGFß2 and TGFß3 are independently involved in mouse fibrosis models in vivo, and selective TGFß2 and TGFß3 inhibition does not lead to the increased inflammation observed with pan-TGFß isoform inhibition. A cocrystal structure of a TGFß2-anti-TGFß2/3 antibody complex reveals an allosteric isoform-selective inhibitory mechanism. Therefore, inhibiting TGFß2 and/or TGFß3 while sparing TGFß1 may alleviate fibrosis without toxicity concerns associated with pan-TGFß blockade.

Function of CSF1 and IL34 in Macrophage Homeostasis, Inflammation, and Cancer.

Colony-stimulating factor 1 (CSF1) and interleukin 34 (IL34) signal via the CSF1 receptor to regulate macrophage differentiation. Studies in IL34- or CSF1-deficient mice have revealed that IL34 function is limited to the central nervous system and skin during development. However, the roles of IL34 and CSF1 at homeostasis or in the context of inflammatory diseases or cancer in wild-type mice have not been clarified in vivo. By neutralizing CSF1 and/or IL34 in adult mice, we identified that they play important roles in macrophage differentiation, specifically in steady-state microglia, Langerhans cells, and kidney macrophages. In several inflammatory models, neutralization of both CSF1 and IL34 contributed to maximal disease protection. However, in a myeloid cell-rich tumor model, CSF1 but not IL34 was required for tumor-associated macrophage accumulation and immune homeostasis. Analysis of human inflammatory conditions reveals IL34 upregulation that may account for the protection requirement of IL34 blockade. Furthermore, evaluation of IL34 and CSF1 blockade treatment during Listeria infection reveals no substantial safety concerns. Thus, IL34 and CSF1 play non-redundant roles in macrophage differentiation, and therapeutic intervention targeting IL34 and/or CSF1 may provide an effective treatment in macrophage-driven immune-pathologies.

IL-17A is associated with the breakdown of the blood-brain barrier in relapsing-remitting multiple sclerosis.

IL-17 has been implicated in the pathogenesis of multiple sclerosis (MS). Here, we show that blockade of IL-17A, but not IL-17F, attenuated experimental autoimmune encephalomyelitis (EAE). We further show that IL-17A levels were elevated in the CSF of relapsing-remitting MS (RRMS) patients and that they correlated with the CSF/serum albumin quotient (Qalb), a measure of blood-brain barrier (BBB) dysfunction. We then demonstrated that the combination of IL-17A and IL-6 reduced the expression of tight junction (TJ)-associated genes and disrupted monolayer integrity in the BBB cell line hCMEC/D3. However, unlike IL-17A, IL-6 in the CSF from RRMS patients did not correlate with Qalb. These data highlight the potential importance of targeting IL-17A in preserving BBB integrity in RRMS.

Disruption of XIAP-RIP2 Association Blocks NOD2-Mediated Inflammatory Signaling.

Inflammatory responses mediated by NOD2 rely on RIP2 kinase and ubiquitin ligase XIAP for the activation of nuclear factor κB (NF-κB), mitogen-activated protein kinases (MAPKs), and cytokine production. Herein, we demonstrate that selective XIAP antagonism blocks NOD2-mediated inflammatory signaling and cytokine production by interfering with XIAP-RIP2 binding, which removes XIAP from its ubiquitination substrate RIP2. We also establish that the kinase activity of RIP2 is dispensable for NOD2 signaling. Rather, the conformation of the RIP2 kinase domain functions to regulate binding to the XIAP-BIR2 domain. Effective RIP2 kinase inhibitors block NOD2 signaling by disrupting RIP2-XIAP interaction. Finally, we identify NOD2 signaling and XIAP-dependent ubiquitination sites on RIP2 and show that mutating these lysine residues adversely affects NOD2 pathway signaling. Overall, these results reveal a critical role for the XIAP-RIP2 interaction in NOD2 inflammatory signaling and provide a molecular basis for the design of innovative therapeutic strategies based on XIAP antagonists and RIP2 kinase inhibitors.

The kinase TPL2 activates ERK and p38 signaling to promote neutrophilic inflammation.

Tumor progression locus 2 (TPL2; also known as MAP3K8) is a mitogen-activated protein kinase (MAPK) kinase kinase (MAP3K) that phosphorylates the MAPK kinases MEK1 and MEK2 (MEK1/2), which, in turn, activate the MAPKs extracellular signal-regulated kinase 1 (ERK1) and ERK2 (ERK1/2) in macrophages stimulated through the interleukin-1 receptor (IL-1R), Toll-like receptors (TLRs), or the tumor necrosis factor receptor (TNFR). We describe a conserved and critical role for TPL2 in mediating the effector functions of neutrophils through the activation of the p38 MAPK signaling pathway. Gene expression profiling and functional studies of neutrophils and monocytes revealed a MEK1/2-independent branch point downstream of TPL2 in neutrophils. Biochemical analyses identified the MAPK kinases MEK3 and MEK6 and the MAPKs p38α and p38δ as downstream effectors of TPL2 in these cells. Genetic ablation of the catalytic activity of TPL2 or therapeutic intervention with a TPL2-specific inhibitor reduced the production of inflammatory mediators by neutrophils in response to stimulation with the TLR4 agonist lipopolysaccharide (LPS) in vitro, as well as in rodent models of inflammatory disease. Together, these data suggest that TPL2 is a drug target that activates not only MEK1/2-dependent but also MEK3/6-dependent signaling to promote inflammatory responses.

Potassium channels Kv1.3 and KCa3.1 cooperatively and compensatorily regulate antigen-specific memory T cell functions.

Voltage-gated Kv1.3 and Ca2+-dependent KCa3.1 are the most prevalent K+ channels expressed by human and rat T cells. Despite the preferential upregulation of Kv1.3 over KCa3.1 on autoantigen-experienced effector memory T cells, whether Kv1.3 is required for their induction and function is unclear. Here we show, using Kv1.3-deficient rats, that Kv1.3 is involved in the development of chronically activated antigen-specific T cells. Several immune responses are normal in Kv1.3 knockout (KO) rats, suggesting that KCa3.1 can compensate for the absence of Kv1.3 under these specific settings. However, experiments with Kv1.3 KO rats and Kv1.3 siRNA knockdown or channel-specific inhibition of human T cells show that maximal T-cell responses against autoantigen or repeated tetanus toxoid stimulations require both Kv1.3 and KCa3.1. Finally, our data also suggest that T-cell dependency on Kv1.3 or KCa3.1 might be irreversibly modulated by antigen exposure.

Inhibition of the kinase ITK in a mouse model of asthma reduces cell death and fails to inhibit the inflammatory response.

Interleukin-2 (IL-2)-inducible T cell kinase (ITK) mediates T cell receptor (TCR) signaling primarily to stimulate the production of cytokines, such as IL-4, IL-5, and IL-13, from T helper 2 (TH2) cells. Compared to wild-type mice, ITK knockout mice are resistant to asthma and exhibit reduced lung inflammation and decreased amounts of TH2-type cytokines in the bronchoalveolar lavage fluid. We found that a small-molecule selective inhibitor of ITK blocked TCR-mediated signaling in cultured TH2 cells, including the tyrosine phosphorylation of phospholipase C-γ1 (PLC-γ1) and the secretion of IL-2 and TH2-type cytokines. Unexpectedly, inhibition of the kinase activity of ITK during or after antigen rechallenge in an ovalbumin-induced mouse model of asthma failed to reduce airway hyperresponsiveness and inflammation. Rather, in mice, pharmacological inhibition of ITK resulted in T cell hyperplasia and the increased production of TH2-type cytokines. Thus, our studies predict that inhibition of the kinase activity of ITK may not be therapeutic in patients with asthma.

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.

Dendritic cells require NIK for CD40-dependent cross-priming of CD8+ T cells.

Dendritic cells (DCs) link innate and adaptive immunity and use a host of innate immune and inflammatory receptors to respond to pathogens and inflammatory stimuli. Although DC maturation via canonical NF-κB signaling is critical for many of these functions, the role of noncanonical NF-κB signaling via the serine/threonine kinase NIK (NF-κB-inducing kinase) remains unclear. Because NIK-deficient mice lack secondary lymphoid organs, we generated transgenic mice with targeted NIK deletion in CD11c(+) cells. Although these mice exhibited normal lymphoid organs, they were defective in cross-priming naive CD8(+) T cells following vaccination, even in the presence of anti-CD40 or polyinosinic:polycytidylic acid to induce DC maturation. This impairment reflected two intrinsic defects observed in splenic CD8(+) DCs in vitro, namely antigen cross-presentation to CD8(+) T cells and secretion of IL-12p40, a cytokine known to promote cross-priming in vivo. In contrast, antigen presentation to CD4(+) T cells was not affected. These findings reveal that NIK, and thus probably the noncanonical NF-κB pathway, is critical to allow DCs to acquire the capacity to cross-present antigen and prime CD8 T cells after exposure to licensing stimuli, such as an agonistic anti-CD40 antibody or Toll-like receptor 3 ligand.

Lack of Widespread BBB Disruption in Alzheimer's Disease Models: Focus on Therapeutic Antibodies.

The blood-brain barrier (BBB) limits brain uptake of therapeutic antibodies. It is believed that the BBB is disrupted in Alzheimer's disease (AD), potentially increasing drug permeability de facto. Here we compared active versus passive brain uptake of systemically dosed antibodies (anti-transferrin receptor [TfR] bispecific versus control antibody) in mouse models of AD. We first confirmed BBB disruption in a mouse model of multiple sclerosis as a positive control. Importantly, we found that BBB permeability was vastly spared in mouse models of AD, including PS2-APP, Tau transgenics, and APOE4 knockin mice. Brain levels of TfR in mouse models or in human cases of AD resembled controls, suggesting target engagement of TfR bispecific is not limited. Furthermore, infarcts from human AD brain showed similar occurrences compared to age-matched controls. These results question the widely held view that the BBB is largely disrupted in AD, raising concern about assumptions of drug permeability in disease.

An entirely automated method to score DSS-induced colitis in mice by digital image analysis of pathology slides.

The DSS (dextran sulfate sodium) model of colitis is a mouse model of inflammatory bowel disease. Microscopic symptoms include loss of crypt cells from the gut lining and infiltration of inflammatory cells into the colon. An experienced pathologist requires several hours per study to score histological changes in selected regions of the mouse gut. In order to increase the efficiency of scoring, Definiens Developer software was used to devise an entirely automated method to quantify histological changes in the whole H&E slide. When the algorithm was applied to slides from historical drug-discovery studies, automated scores classified 88% of drug candidates in the same way as pathologists' scores. In addition, another automated image analysis method was developed to quantify colon-infiltrating macrophages, neutrophils, B cells and T cells in immunohistochemical stains of serial sections of the H&E slides. The timing of neutrophil and macrophage infiltration had the highest correlation to pathological changes, whereas T and B cell infiltration occurred later. Thus, automated image analysis enables quantitative comparisons between tissue morphology changes and cell-infiltration dynamics.

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 T(H)17 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.

Antibodies specific for a segment of human membrane IgE deplete IgE-producing B cells in humanized mice.

IgE-mediated hypersensitivity is central to the pathogenesis of asthma and other allergic diseases. Although neutralization of serum IgE with IgE-specific antibodies is in general an efficacious treatment for allergic asthma, one limitation of this approach is its lack of effect on IgE production. Here, we have developed a strategy to disrupt IgE production by generating monoclonal antibodies that target a segment of membrane IgE on human IgE-switched B cells that is not present in serum IgE. This segment is known as the M1' domain, and using genetically modified mice that contain the human M1' domain inserted into the mouse IgE locus, we demonstrated that M1'-specific antibodies reduced serum IgE and IgE-producing plasma cells in vivo, without affecting other immunoglobulin isotypes. M1'-specific antibodies were effective when delivered prophylactically and therapeutically in mouse models of immunization, allergic asthma, and Nippostrongylus brasiliensis infection, likely by inducing apoptosis of IgE-producing B cells. In addition, we generated a humanized M1'-specific antibody that was active on primary human cells in vivo, as determined by its reduction of serum IgE levels and IgE plasma cell numbers in a human PBMC-SCID mouse model. Thus, targeting of human IgE-producing B cells with apoptosis-inducing M1'-specific antibodies may be a novel treatment for asthma and allergy.

A human antibody that inhibits factor IX/IXa function potently inhibits arterial thrombosis without increasing bleeding.

10C12, a human antibody F(ab')2, which specifically binds to the gamma-carboxyglutamic acid domain of factor IX/factor IXa (F.IX/IXa), interferes with all known coagulation processes in which F.IX/IXa is involved. In a rabbit model of carotid artery injury, intravenous administration of 10C12 or heparin decreased thrombosis dose dependently. The dose that resulted in a 90% reduction of thrombus mass (ED90) was a 30-microg/kg bolus of 10C12 or a 100-U/kg bolus plus 1.0 U x kg(-1) x min(-1) infusion of heparin. Heparin, at and below the ED90, significantly prolonged coagulation times and cuticle bleeding times. In contrast, 10C12 had no effect on coagulation or bleeding times at doses up to 4 times the ED90. To further evaluate the effect of 10C12 on bleeding, it was compared with heparin in a novel model of blood loss. At the ED90 of heparin, blood loss induced by a standardized injury to the vasculature of the rabbit tibia increased to more than 2 times that of saline controls. In contrast, the dose of 10C12 required to produce a similar increase in blood loss was more than 30 times the ED90. The antithrombotic potency and relative safety of this fully human antibody suggests that it may have therapeutic value for treatment of thrombotic disorders.