Anti-CD99 Antibody Therapy Triggers Macrophage-Dependent Ewing Cell Death In Vitro and Myeloid Cell Recruitment In Vivo.

BACKGROUND: Ewing sarcoma is a rare tumor of the bone or soft tissues characterized by diffuse membranous staining for CD99. As this tumor remains incurable in the metastatic, relapsed, and refractory settings, we explored the downstream immune implications of targeting CD99. METHODS: We discovered a human anti-CD99 antibody (NOA2) by phagemid panning and investigated NOA2 immune cell-mediated cytotoxicity in vitro and in vivo focusing on the myeloid cell compartment, given that M2 macrophages are present in human tumors and associated with a poor prognosis. RESULTS: NOA2 is capable of inducing immune effector cell-mediated Ewing death in vitro via engagement of macrophages. Mice with metastatic Ewing tumors, treated with NOA2, experience tumor growth arrest and an associated increase in intratumoral macrophages. Further, incubation of macrophages and Ewing cells with NOA2, in conjunction with anti-PILRα antibody blockade in vitro, results in the reactivation of previously dormant macrophages possibly due to interrupted binding of Ewing CD99 to macrophage PILRα. CONCLUSIONS: These studies are the first to demonstrate the role of human immune effector cells in anti-CD99-mediated Ewing tumor death. We propose that the engagement of CD99 by NOA2 results in the recruitment of intratumoral macrophages. In addition, interruption of the CD99:PILRα checkpoint axis may be a relevant therapeutic approach to activate tumor-associated macrophages.

Immune-restoring CAR-T cells display antitumor activity and reverse immunosuppressive TME in a humanized ccRCC mouse model.

One of the major barriers that have restricted successful use of chimeric antigen receptor (CAR) T cells in the treatment of solid tumors is an unfavorable tumor microenvironment (TME). We engineered CAR-T cells targeting carbonic anhydrase IX (CAIX) to secrete anti-PD-L1 monoclonal antibody (mAb), termed immune-restoring (IR) CAR G36-PDL1. We tested CAR-T cells in a humanized clear cell renal cell carcinoma (ccRCC) orthotopic mouse model with reconstituted human leukocyte antigen (HLA) partially matched human leukocytes derived from fetal CD34+ hematopoietic stem cells (HSCs) and bearing human ccRCC skrc-59 cells under the kidney capsule. G36-PDL1 CAR-T cells, haploidentical to the tumor cells, had a potent antitumor effect compared to those without immune-restoring effect. Analysis of the TME revealed that G36-PDL1 CAR-T cells restored active antitumor immunity by promoting tumor-killing cytotoxicity, reducing immunosuppressive cell components such as M2 macrophages and exhausted CD8+ T cells, and enhancing T follicular helper (Tfh)-B cell crosstalk.

Alcohol consumption increases susceptibility to pneumococcal pneumonia in a humanized murine HIV model mediated by intestinal dysbiosis.

Alcohol use in persons living with HIV (PLWH) worsens the severity of bacterial pneumonia. However, the exact mechanism(s) by which this occurs remain ill-defined. We hypothesized that alcohol in the setting of HIV infection decreases Streptococcus pneumoniae clearance from the lung through mechanisms mediated by the gut microbiota. Humanized BLT (bone marrow, liver, thymus) mice were infected with 1 × 104 TCID50 of HIV (BAL and JRCSF strains) via intraperitoneal (i.p.) injection. One week post-HIV infection, animals were switched to a Lieber-DeCarli 5% ethanol diet or an isocaloric control diet for 10 days. Alcohol-fed animals were also given two binges of 2 g/kg ethanol on days 5 and 10. Feces were also collected, banked, and the community structures were analyzed. Mice were then infected with 1 × 105 CFU (colony-forming units) of S. pneumoniae and were sacrificed 48 h later. HIV-infected mice had viral loads of ∼2 × 104 copies/mL of blood 1 week post-infection, and exhibited an ∼57% decrease in the number of circulating CD4+ T cells at the time of sacrifice. Fecal microbial community structure was significantly different in each of the feeding groups, as well as with HIV infection. Alcohol-fed mice had a significantly higher burden of S. pneumoniae 48 h post-infection, regardless of HIV status. In follow-up experiments, female C57BL/6 mice were treated with a cocktail of antibiotics daily for 2 weeks and recolonized by gavage with intestinal microbiota from HIV+ ethanol-fed, HIV+ pair-fed, HIV- ethanol-fed, or HIV- pair-fed mice. Recolonized mice were then infected with S. pneumoniae and were sacrificed 48 h later. The intestinal microbiota from alcohol-fed mice (regardless of HIV status) significantly impaired clearance of S. pneumoniae. Collectively, these data indicate that alcohol feeding, as well as alcohol-associated intestinal dysbiosis, compromise pulmonary host defenses against pneumococcal pneumonia. Determining whether HIV infection acts synergistically with alcohol use in impairing pulmonary host defenses will require additional study.

Chimeric antigen receptor T cells secreting anti-PD-L1 antibodies more effectively regress renal cell carcinoma in a humanized mouse model.

Advances in the treatment of metastatic clear cell renal cell carcinoma (ccRCC) have led to improved progression-free survival of many patients; however the therapies are toxic, rarely achieve durable long-term complete responses and are not curative. Herein we used a single bicistronic lentiviral vector to develop a new combination immunotherapy that consists of human anti-carbonic anhydrase IX (CAIX)-targeted chimeric antigen receptor (CAR) T cells engineered to secrete human anti-programmed death ligand 1 (PD-L1) antibodies at the tumor site. The local antibody delivery led to marked immune checkpoint blockade. Tumor growth diminished 5 times and tumor weight reduced 50-80% when compared with the anti-CAIX CAR T cells alone in a humanized mice model of ccRCC. The expression of PD-L1 and Ki67 in the tumors decreased and an increase in granzyme B levels was found in CAR T cells. The anti-PD-L1 IgG1 isotype, which is capable of mediating ADCC, was also able to recruit human NK cells to the tumor site in vivo. These armed second-generation CAR T cells empowered to secrete human anti-PD-L1 antibodies in the ccRCC milieu to combat T cell exhaustion is an innovation in this field that should provide renewed potential for CAR T cell immunotherapy of solid tumors where limited efficacy is currently seen.

Anti-CCR4 monoclonal antibody enhances antitumor immunity by modulating tumor-infiltrating Tregs in an ovarian cancer xenograft humanized mouse model.

Recent studies have demonstrated that regulatory T cells (Tregs) are recruited to tumor sites where they can suppress antitumor immunity. The chemokine receptor CCR4 is expressed at high levels on functional CD4+CD25+FoxP3+ Tregs and production of the CCR4 ligand CCL22 by tumor cells and tumor-associated macrophages is associated with Treg recruitment to the tumor site. Here, we tested IgG1 and IgG4 isotypes of human anti-CCR4 mAb2-3 for their in vitro activity and in vivo capacity in a NSG mouse model bearing CCL22-secreting ovarian cancer (OvCA) xenograft to modulate Tregs and restore antitumor activity. Both mAb2-3 isotypes blocked in vitro chemoattraction of Tregs to CCL22-secreting OvCA cells. However, they differed in their in vivo mode of action with IgG1 causing Treg depletion and IgG4 blocking migration to the tumors. Primary T cells that were primed with OvCA-pulsed dendritic cells (DCs) demonstrated INFγ secretion that could be enhanced through Treg depletion by mAb2-3. Humanized mice reconstructed with allogeneic tumor-primed T cells (TP-T) were used to evaluate the restoration of OvCA immunity by depletion or blockade of Tregs with mAb2-3. We observed that IgG1 was more potent than IgG4 in inhibiting tumor growth. Mechanism studies demonstrated that mAb2-3 treatment lead to inhibition of IL-2 binding to its receptor. Further studies showed that mAb2-3 induced CD25 shedding (sCD25) from Tregs which lead to a decrease in IL-2-dependent survival. Together, the results demonstrate that mAb2-3 is an agonist antibody that can restore anti-OvCA immunity through modulation of Treg activity.

Humanized mouse G6 anti-idiotypic monoclonal antibody has therapeutic potential against IGHV1-69 germline gene-based B-CLL.

In 10-20% of the cases of chronic lymphocytic leukemia of B-cell phenotype (B-CLL), the IGHV1-69 germline is utilized as VH gene of the B cell receptor (BCR). Mouse G6 (MuG6) is an anti-idiotypic monoclonal antibody discovered in a screen against rheumatoid factors (RFs) that binds with high affinity to an idiotope expressed on the 51p1 alleles of IGHV1-69 germline gene encoded antibodies (G6-id(+)). The finding that unmutated IGHV1-69 encoded BCRs are frequently expressed on B-CLL cells provides an opportunity for anti-idiotype monoclonal antibody immunotherapy. In this study, we first showed that MuG6 can deplete B cells encoding IGHV1-69 BCRs using a novel humanized GTL mouse model. Next, we humanized MuG6 and demonstrated that the humanized antibodies (HuG6s), especially HuG6.3, displayed ∼2-fold higher binding affinity for G6-id(+) antibody compared to the parental MuG6. Additional studies showed that HuG6.3 was able to kill G6-id(+) BCR expressing cells and patient B-CLL cells through antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). Finally, both MuG6 and HuG6.3 mediate in vivo depletion of B-CLL cells in NSG mice. These data suggest that HuG6.3 may provide a new precision medicine to selectively kill IGHV1-69-encoding G6-id(+) B-CLL cells.

Pancreatic cancer cell-derived IGFBP-3 contributes to muscle wasting.

BACKGROUND: Progressive loss of skeletal muscle, termed muscle wasting, is a hallmark of cancer cachexia and contributes to weakness, reduced quality of life, as well as poor response to therapy. Previous studies have indicated that systemic host inflammatory response regarding tumor development results in muscle wasting. However, how tumor directly regulates muscle wasting via tumor-derived secreted proteins is still largely unknown. METHODS: In this study, we performed bioinformatics analysis in two datasets of pancreatic ductal adenocarcinoma, which causes cancer cachexia and muscle wasting with the highest prevalence, and uncovered that IGFBP3, which encodes IGF-binding protein-3 (IGFBP-3), is dramatically up-regulated in pancreatic tumor samples. We also verified the wasting effect of IGFBP-3 on C2C12 muscle cells with biochemical and genetic assays. RESULTS: IGFBP-3 potently leads to impaired myogenesis and enhanced muscle protein degradation, the major features of muscle wasting, via IGF signaling inhibition. Moreover, conditioned medium from Capan-1 pancreatic cancer cells, which contains abundant IGFBP-3, significantly induces muscle cell wasting. This wasting effect is potently alleviated by IGFBP3 knockdown in Capan-1 cells or IGFBP-3 antibody neutralization. Strikingly, compared to muscle cells, IGF signaling and proliferation rate of Capan-1 cells were rarely affected by IGFBP-3 treatment. CONCLUSIONS: Our results demonstrated that pancreatic cancer cells induce muscle wasting via IGFBP-3 production.

Human anti-CAIX antibodies mediate immune cell inhibition of renal cell carcinoma in vitro and in a humanized mouse model in vivo.

BACKGROUND: Carbonic anhydrase (CA) IX is a surface-expressed protein that is upregulated by the hypoxia inducible factor (HIF) and represents a prototypic tumor-associated antigen that is overexpressed on renal cell carcinoma (RCC). Therapeutic approaches targeting CAIX have focused on the development of CAIX inhibitors and specific immunotherapies including monoclonal antibodies (mAbs). However, current in vivo mouse models used to characterize the anti-tumor properties of fully human anti-CAIX mAbs have significant limitations since the role of human effector cells in tumor cell killing in vivo is not directly evaluated. METHODS: The role of human anti-CAIX mAbs on CAIX(+) RCC tumor cell killing by immunocytes or complement was tested in vitro by antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC) and antibody-dependent cellular phagocytosis (ADCP) as well as on CAIX(+) RCC cellular motility, wound healing, migration and proliferation. The in vivo therapeutic activity mediated by anti-CAIX mAbs was determined by using a novel orthotopic RCC xenograft humanized animal model and analyzed by histology and FACS staining. RESULTS: Our studies demonstrate the capacity of human anti-CAIX mAbs that inhibit CA enzymatic activity to result in immune-mediated killing of RCC, including nature killer (NK) cell-mediated ADCC, CDC, and macrophage-mediated ADCP. The killing activity correlated positively with the level of CAIX expression on RCC tumor cell lines. In addition, Fc engineering of anti-CAIX mAbs was shown to enhance the ADCC activity against RCC. We also demonstrate that these anti-CAIX mAbs inhibit migration of RCC cells in vitro. Finally, through the implementation of a novel orthotopic RCC model utilizing allogeneic human peripheral blood mononuclear cells in NOD/SCID/IL2Rγ(-/-) mice, we show that anti-CAIX mAbs are capable of mediating human immune response in vivo including tumor infiltration of NK cells and activation of T cells, resulting in inhibition of CAIX(+) tumor growth. CONCLUSIONS: Our findings demonstrate that these novel human anti-CAIX mAbs have therapeutic potential in the unmet medical need of targeted killing of HIF-driven CAIX(+)RCC. The orthotopic tumor xenografted humanized mouse provides an improved model to evaluate the in vivo anti-tumor capabilities of fully human mAbs for RCC therapy.

Fatal autoimmunity in mice reconstituted with human hematopoietic stem cells encoding defective FOXP3.

Mice reconstituted with a human immune system provide a tractable in vivo model to assess human immune cell function. To date, reconstitution of murine strains with human hematopoietic stem cells (HSCs) from patients with monogenic immune disorders have not been reported. One obstacle precluding the development of immune-disease specific "humanized" mice is that optimal adaptive immune responses in current strains have required implantation of autologous human thymic tissue. To address this issue, we developed a mouse strain that lacks murine major histocompatibility complex class II (MHC II) and instead expresses human leukocyte antigen DR1 (HLA-DR1). These mice displayed improved adaptive immune responses when reconstituted with human HSCs including enhanced T-cell reconstitution, delayed-type hypersensitivity responses, and class-switch recombination. Following immune reconstitution of this novel strain with HSCs from a patient with immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome, associated with aberrant FOXP3 function, mice developed a lethal inflammatory disorder with multiorgan involvement and autoantibody production mimicking the pathology seen in affected humans. This humanized mouse model permits in vivo evaluation of immune responses associated with genetically altered HSCs, including primary immunodeficiencies, and should facilitate the study of human immune pathobiology and the development of targeted therapeutics.

Chemokine (C-C motif) receptor 2 mediates mast cell migration to abdominal aortic aneurysm lesions in mice.

AIMS: Mast cells participate importantly in abdominal aortic aneurysms (AAAs) by releasing inflammatory cytokines to promote vascular cell protease expression and arterial wall remodelling. Mast cells accumulate in AAA lesions during disease progression, but the exact chemokines by which mast cells migrate to the site of vascular inflammation remain unknown. This study tested the hypothesis that mast cells use chemokine (C-C motif) receptor 2 (CCR2) for their accumulation in experimental mouse AAA lesions. METHODS AND RESULTS: We generated mast cell and apolipoprotein E double-deficient (Apoe(-/-)Kit(W-sh/W-sh)) mice and found that they were protected from angiotensin II (Ang II) chronic infusion-induced AAAs compared with Apoe(-/-) littermates. Using bone-marrow derived mast cells (BMMC) from Apoe(-/-) mice and CCR2 double-deficient (Apoe(-/-)Ccr2(-/-)) mice, we demonstrated that Apoe(-/-)Kit(W-sh/W-sh) mice receiving BMMC from Apoe(-/-)Ccr2(-/-) mice, but not those from Apoe(-/-) mice, remained protected from AAA formation. Adoptive transfer of BMMC from Apoe(-/-) mice into Apoe(-/-)Kit(W-sh/W-sh) mice also increased lesion content of macrophages, T cells, and MHC class II-positive cells; there was also increased apoptosis, angiogenesis, cell proliferation, elastin fragmentation, and medial smooth muscle cell loss. In contrast, adoptive transfer of BMMC from Apoe(-/-)Ccr2(-/-) mice into Apoe(-/-)Kit(W-sh/W-sh) mice did not affect these variables. CONCLUSIONS: The increased AAA formation and associated lesion characteristics in Apoe(-/-)Kit(W-sh/W-sh) mice after receiving BMMC from Apoe(-/-) mice, but not from Apoe(-/-)Ccr2(-/-) mice, suggests that mast cells use CCR2 as the chemokine receptor for their recruitment in Ang II-induced mouse AAA lesions.

Cathepsin K deficiency reduces elastase perfusion-induced abdominal aortic aneurysms in mice.

OBJECTIVE: Cathepsin K (CatK) is one of the most potent mammalian elastases. We have previously shown increased expression of CatK in human abdominal aortic aneurysm (AAA) lesions. Whether this protease participates directly in AAA formation, however, remains unknown. METHODS AND RESULTS: Mouse experimental AAA was induced with aortic perfusion of a porcine pancreatic elastase. Using this experimental model, we demonstrated that absence of CatK prevented AAA formation in mice 14 days postperfusion. CatK deficiency significantly reduced lesion CD4(+) T-cell content, total lesion and medial cell proliferation and apoptosis, medial smooth muscle cell (SMC) loss, elastinolytic CatL and CatS expression, and elastin fragmentation, but it did not affect AAA lesion Mac-3(+) macrophage accumulation or CD31(+) microvessel numbers. In vitro studies revealed that CatK contributed importantly to CD4(+) T-cell proliferation, SMC apoptosis, and other cysteinyl cathepsin and matrix metalloproteinase expression and activities in SMCs and endothelial cells but played negligible roles in microvessel growth and monocyte migration. AAA lesions from CatK-deficient mice showed reduced elastinolytic cathepsin activities compared with those from wild-type control mice. CONCLUSIONS: This study demonstrates that CatK plays an essential role in AAA formation by promoting T-cell proliferation, vascular SMC apoptosis, and elastin degradation and by affecting vascular cell protease expression and activities.

Cathepsin L activity is essential to elastase perfusion-induced abdominal aortic aneurysms in mice.

OBJECTIVE: The development of abdominal aortic aneurysms (AAA) requires extensive aortic wall matrix degradation. Human AAA lesions express high levels of cathepsin L (CatL), one of the most potent mammalian elastases. Whether this protease participates directly in AAA pathogenesis, however, is unknown. METHODS AND RESULTS: We generated experimental AAA with aortic elastase perfusion in mice and established an essential role of CatL in AAA formation. After 14 days postperfusion, most wild-type (Ctsl(+/+)) mice developed AAA, but none of the CatL-deficient (Ctsl(-/-)) mice did. AAA lesion macrophage contents, CD4(+) T cell numbers, CD31(+) and laminin-5 angiogenic fragment γ2(+) microvessel numbers, and elastin fragmentation were all significantly lower in Ctsl(-/-) mice than in Ctsl(+/+) mice. While lesions from Ctsl(-/-) mice contained fewer Ki67(+) proliferating cells than did Ctsl(+/+) mice, the absence of CatL did not affect lesion apoptotic cell contents or medial smooth-muscle cell loss significantly. Mechanistic studies indicated that the absence of CatL reduced lesion chemokine monocyte chemotactic protein-1 content, macrophage and T-cell in vitro transmigration, and angiogenesis, and altered the expression and activities of matrix metalloproteinases and other cysteinyl cathepsins in inflammatory cells, vascular cells, and AAA lesions. CONCLUSION: CatL contributes to AAA formation by promoting lesion inflammatory cell accumulation, angiogenesis, and protease expression.

Mast cell tryptase deficiency attenuates mouse abdominal aortic aneurysm formation.

RATIONALE: Mast cells (MCs) contribute to the formation of abdominal aortic aneurysms (AAAs) by producing biologically active mediators. Tryptase is the most abundant MC granule protein and participates in MC activation, protease maturation, leukocyte recruitment, and angiogenesis-all processes critical to AAA pathogenesis. OBJECTIVE: To test the hypothesis that tryptase participates directly in AAA formation. METHODS AND RESULTS: Immunohistochemistry demonstrated enhanced tryptase staining in media and adventitia of human and mouse AAA lesions. Serum tryptase levels correlated significantly with the annual expansion rate of AAA before (r = 0.30, P = 0.003) and after (r = 0.29, P = 0.005) adjustment for common AAA risk factors in a patient follow-up study, and associated with risks for later surgical repair or overall mortality before (P = 0.009, P = 0.065) and after (P = 0.004, P = 0.001) the adjustment. Using MC protease-6-deficient mice (Mcpt6(-/-)) and aortic elastase perfusion-induced experimental AAAs, we proved a direct role of this tryptase in AAA pathogenesis. Whereas all wild-type (WT) mice developed AAA at 14 or 56 days postperfusion, Mcpt6(-/-) mice were fully protected. AAA lesions from Mcpt6(-/-) mice had fewer inflammatory and apoptotic cells, and lower chemokine levels, than did those from WT mice. MC from WT mice restored reduced AAA lesions and lesion inflammatory cell content in MC-deficient Kit(W-sh/W-sh) mice, but those prepared from Mcpt6(-/-) mice did not. Mechanistic studies demonstrated that tryptase deficiency affected endothelial cell (EC) chemokine and cytokine expression, monocyte transmigration, smooth-muscle cell apoptosis, and MC and AAA lesion cysteinyl cathepsin expression and activities. CONCLUSIONS: This study establishes the direct participation of MC tryptase in the pathogenesis of experimental AAAs, and suggests that levels of this protease can serve as a novel biomarker for abdominal aortic expansion.

Regulation of endothelial cell adhesion molecule expression by mast cells, macrophages, and neutrophils.

BACKGROUND: Leukocyte adhesion to the vascular endothelium and subsequent transendothelial migration play essential roles in the pathogenesis of cardiovascular diseases such as atherosclerosis. The leukocyte adhesion is mediated by localized activation of the endothelium through the action of inflammatory cytokines. The exact proinflammatory factors, however, that activate the endothelium and their cellular sources remain incompletely defined. METHODS AND RESULTS: Using bone marrow-derived mast cells from wild-type, Tnf(-/-), Ifng(-/-), Il6(-/-) mice, we demonstrated that all three of these pro-inflammatory cytokines from mast cells induced the expression of vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), P-selectin, and E-selectin in murine heart endothelial cells (MHEC) at both mRNA and protein levels. Compared with TNF-α and IL6, IFN-γ appeared weaker in the induction of the mRNA levels, but at protein levels, both IL6 and IFN-γ were weaker inducers than TNF-α. Under physiological shear flow conditions, mast cell-derived TNF-α and IL6 were more potent than IFN-γ in activating MHEC and in promoting neutrophil adhesion. Similar observations were made when neutrophils or macrophages were used. Neutrophils and macrophages produced the same sets of pro-inflammatory cytokines as did mast cells to induce MHEC adhesion molecule expression, with the exception that macrophage-derived IFN-γ showed negligible effect in inducing VCAM-1 expression in MHEC. CONCLUSION: Mast cells, neutrophils, and macrophages release pro-inflammatory cytokines such as TNF-α, IFN-γ, and IL6 that induce expression of adhesion molecules in endothelium and recruit of leukocytes, which is essential to the pathogenesis of vascular inflammatory diseases.

Cystatin C deficiency promotes inflammation in angiotensin II-induced abdominal aortic aneurisms in atherosclerotic mice.

An imbalance between cysteinyl cathepsins and their principal endogenous inhibitor cystatin C (CystC) may favor proteolysis in the pathogenesis of human abdominal aortic aneurysms (AAA), yet a direct role of CystC in AAA remains unproven. This study used CystC and apolipoprotein E (ApoE) compound mutant (CystC(-/-)ApoE(-/-)) mice to examine directly the role of cysteine protease/protease inhibitor imbalance in AAA formation in angiotensin II-induced AAA. CystC-deficiency increased lumenal diameter and lesion size compared with control mice. CystC(-/-) ApoE(-/-) lesions also demonstrated enhanced inflammatory cell accumulation, more severe elastin fragmentation, and fewer smooth muscle cells in the tunica media. Macrophage content, measured as percent positive area (23.2 +/- 1.4% versus 11.2 +/- 1.4%; P = 0.0003) and number of the CD4(+) T cells (ninefold; P = 0.048), increased significantly in CystC(-/-)ApoE(-/-) lesions. CystC deficiency increased cathepsin activity (5.5 fold; P = 0.001) in AAA, yielding greater elastin degradation and proangiogenic laminin-5 gamma2 peptide production, which may account for increased microvascularization in CystC(-/-)ApoE(-/-) compared with ApoE(-/-) lesions. Increased leukocyte adhesion molecule VCAM-1 expression and leukocyte proliferation might also promote inflammation in CystC-deficient AAA. These data indicate that CystC contributes to experimental AAA pathogenesis and that enhanced cysteine protease activity, due to the lack of CystC, favors inflammation in AAA lesions induced in atherosclerotic mice by promoting microvascularization and smooth muscle cell apoptosis as well as leukocytes adhesion and proliferation.

Critical role of mast cell chymase in mouse abdominal aortic aneurysm formation.

BACKGROUND: Mast cell chymase may participate in the pathogenesis of human abdominal aortic aneurysm (AAA), yet a direct contribution of this serine protease to AAA formation remains unknown. METHODS AND RESULTS: Human AAA lesions had high numbers of chymase-immunoreactive mast cells. Serum chymase level correlated with AAA growth rate (P=0.009) in a prospective clinical study. In experimental AAA produced by aortic elastase perfusion in wild-type (WT) mice or those deficient in the chymase ortholog mouse mast cell protease-4 (mMCP-4) or deficient in mMCP-5 (Mcpt4(-/-), Mcpt5(-/-)), Mcpt4(-/-) but not Mcpt5(-/-) had reduced AAA formation 14 days after elastase perfusion. Even 8 weeks after perfusion, aortic expansion in Mcpt4(-/-) mice fell by 50% compared with that of the WT mice (P=0.0003). AAA lesions in Mcpt4(-/-) mice had fewer inflammatory cells and less apoptosis, angiogenesis, and elastin fragmentation than those of WT mice. Although Kit(W-sh/W-sh) mice had protection from AAA formation, reconstitution with mast cells from WT mice, but not those from Mcpt4(-/-) mice, partially restored the AAA phenotype. Mechanistic studies suggested that mMCP-4 regulates expression and activation of cysteine protease cathepsins, elastin degradation, angiogenesis, and vascular cell apoptosis. CONCLUSIONS: High chymase-positive mast cell content in human AAA lesions, greatly reduced AAA formation in Mcpt4(-/-) mice, and significant correlation of serum chymase levels with human AAA expansion rate suggests participation of mast cell chymase in the progression of human and mouse AAA.

Genetic deficiency and pharmacological stabilization of mast cells reduce diet-induced obesity and diabetes in mice.

Although mast cell functions have classically been related to allergic responses, recent studies indicate that these cells contribute to other common diseases such as multiple sclerosis, rheumatoid arthritis, atherosclerosis, aortic aneurysm and cancer. This study presents evidence that mast cells also contribute to diet-induced obesity and diabetes. For example, white adipose tissue (WAT) from obese humans and mice contain more mast cells than WAT from their lean counterparts. Furthermore, in the context of mice on a Western diet, genetically induced deficiency of mast cells, or their pharmacological stabilization, reduces body weight gain and levels of inflammatory cytokines, chemokines and proteases in serum and WAT, in concert with improved glucose homeostasis and energy expenditure. Mechanistic studies reveal that mast cells contribute to WAT and muscle angiogenesis and associated cell apoptosis and cathepsin activity. Adoptive transfer experiments of cytokine-deficient mast cells show that these cells, by producing interleukin-6 (IL-6) and interferon-gamma (IFN-gamma), contribute to mouse adipose tissue cysteine protease cathepsin expression, apoptosis and angiogenesis, thereby promoting diet-induced obesity and glucose intolerance. Our results showing reduced obesity and diabetes in mice treated with clinically available mast cell-stabilizing agents suggest the potential of developing new therapies for these common human metabolic disorders.

Deficiency and inhibition of cathepsin K reduce body weight gain and increase glucose metabolism in mice.

OBJECTIVE: Previous studies demonstrated increased levels of cysteine proteases cathepsins in serum and adipose tissues from obese patients. We now provide evidence from a mouse model of obesity to suggest a direct participation of cathepsin K (CatK) in mouse body weight gain and glucose metabolism. METHODS AND RESULTS: Using real-time polymerase chain reaction, we detected 12-fold increase in CatK transcripts after adipogenesis of human preadipocytes. Using an immunohistology analysis, we consistently observed high levels of CatK expression in adipose tissues from obese humans and mice. Selective inhibition of CatK activity blocked the lipid accumulation in human and mouse preadipocytes. In mice, CatK deficiency reduced significantly diet-induced body weight gain and serum glucose and insulin levels. Similar results were obtained in diet-induced and genetically created (ob/ob) obese mice after animals were treated with a CatK-selective inhibitor. Mechanistic study demonstrated a role for CatK in degrading fibronectin, a matrix protein that controls adipogenesis. Deficiency or inhibition of CatK leads to fibronectin accumulation in muscle and adipose tissues. CONCLUSIONS: This study demonstrates an essential role of CatK in adipogenesis and mouse body weight gain, possibly via degradation of fibronectin, thus suggesting a novel therapeutic strategy for the control of obesity by regulating CatK activity.

Mast cells modulate the pathogenesis of elastase-induced abdominal aortic aneurysms in mice.

Abdominal aortic aneurysm (AAA), an inflammatory disease, involves leukocyte recruitment, immune responses, inflammatory cytokine production, vascular remodeling, neovascularization, and vascular cell apoptosis, all of which contribute to aortic dilatation. This study demonstrates that mast cells, key participants in human allergic immunity, participate in AAA pathogenesis in mice. Mast cells were found to accumulate in murine AAA lesions. Mast cell-deficient KitW-sh/KitW-sh mice failed to develop AAA elicited by elastase perfusion or periaortic chemical injury. KitW-sh/KitW-sh mice had reduced aortic expansion and internal elastic lamina degradation; decreased numbers of macrophages, CD3+ T lymphocytes, SMCs, apoptotic cells, and CD31+ microvessels; and decreased levels of aortic tissue IL-6 and IFN-gamma. Activation of mast cells in WT mice via C48/80 injection resulted in enhanced AAA growth while mast cell stabilization with disodium cromoglycate diminished AAA formation. Mechanistic studies demonstrated that mast cells participated in angiogenesis, aortic SMC apoptosis, and matrix-degrading protease expression. Reconstitution of KitW-sh/KitW-sh mice with bone marrow-derived mast cells from WT or TNF-alpha-/- mice, but not from IL-6-/- or IFN-gamma-/- mice, caused susceptibility to AAA formation to be regained. These results demonstrate that mast cells participate in AAA pathogenesis in mice by releasing proinflammatory cytokines IL-6 and IFN-gamma, which may induce aortic SMC apoptosis, matrix-degrading protease expression, and vascular wall remodeling, important hallmarks of arterial aneurysms.