Daiko Wakita

IL-1 Signaling Is Critically Required in Stromal Cells in Kawasaki Disease Vasculitis Mouse Model

Objective—Kawasaki disease (KD) is the most common cause of acute vasculitis and acquired cardiac disease among US children. We have previously shown that both TLR2/MyD88 and interleukin (IL)-1&bgr; signaling are required for the Lactobacillus casei cell wall extract–induced KD vasculitis mouse model. The objectives of this study were to investigate the cellular origins of IL-1 production, the role of CD11c+ dendritic cells and macrophages, and the relative contribution of hematopoietic and stromal cells for IL-1 responsive cells, as well the MyD88 signaling, in Lactobacillus casei cell wall extract–induced KD mouse model of vasculitis. Approach and Results—Using mouse knockout models and antibody depletion, we found that both IL-1&agr; and IL-1&bgr; were required for Lactobacillus casei cell wall extract–induced KD. Both dendritic cells and macrophages were necessary, and we found that MyD88 signaling was required in both hematopoietic and stromal cells. However, IL-1 response and signaling were critically required in nonendothelial stromal cells, but not in hematopoietic cells. Conclusions—Our results suggest that IL-1&agr; and IL-1&bgr;, as well as CD11c+ dendritic cells and macrophages, are essential for the development of KD vasculitis and coronary arteritis in this mouse model. Bone marrow chimera experiments suggest that MyD88 signaling is important in both hematopoietic and stromal cells, whereas IL-1 signaling and response are required only in stromal cells, but not in endothelial cells. Determining the role of IL-1&agr; and IL-1&bgr; and of specific cell types in the KD vasculitis mouse model may have important implications for the design of more targeted therapies and understanding of the molecular mechanisms of KD immunopathologies.

Quercetin Inhibits Inflammasome Activation by Interfering with ASC Oligomerization and Prevents Interleukin-1 Mediated Mouse Vasculitis

Interleukin-1β (IL-1β) is a highly inflammatory cytokine that significantly contributes to both acute and chronic inflammatory diseases. The secretion of IL-1β requires a unique protease, caspase-1, which is activated by various protein platforms called inflammasomes. Data suggests a key role for mitochondrial reactive oxygen species for inflammasome activation. Flavonoids constitute a group of naturally occurring polyphenolic molecules with many biological activities, including antioxidant effects. In this study, we investigated the effect of three flavonoids, quercetin (QUC), naringenin, and silymarim on inflammasome activation. We found that QUC inhibits IL-1β secretion by both the NLRP3 and AIM2 inflammasome in a dose dependent manner, but not the NLRC4 inflammasome. QUC inhibition of the inflammasome was still observed in Atg16l1 knockout macrophages, indicating that QUC’s effect was autophagy independent. Since QUC inhibited both NLRP3 and AIM2 inflammasomes but not NLRC4, we assessed ASC speck formation. QUC reduced ASC speck formation and ASC oligomerization compared with controls. Additionally, QUC inhibited IL-1β in Cryopyrin-Associated Periodic Syndromes (CAPS) macrophages, where NLRP3 inflammasome is constitutively activated. In conclusion, QUC inhibits both the NLRP3 and AIM2 inflammasome by preventing ASC oligomerization and may be a potential therapeutic candidate for Kawasaki disease vasculitis and other IL-1 mediated inflammatory diseases.

Role of Interleukin-1 Signaling in a Mouse Model of Kawasaki Disease–Associated Abdominal Aortic Aneurysm

Objective—Kawasaki disease (KD) is the most common cause of acquired cardiac disease in US children. In addition to coronary artery abnormalities and aneurysms, it can be associated with systemic arterial aneurysms. We evaluated the development of systemic arterial dilatation and aneurysms, including abdominal aortic aneurysm (AAA) in the Lactobacillus casei cell-wall extract (LCWE)–induced KD vasculitis mouse model. Methods and Results—We discovered that in addition to aortitis, coronary arteritis and myocarditis, the LCWE-induced KD mouse model is also associated with abdominal aorta dilatation and AAA, as well as renal and iliac artery aneurysms. AAA induced in KD mice was exclusively infrarenal, both fusiform and saccular, with intimal proliferation, myofibroblastic proliferation, break in the elastin layer, vascular smooth muscle cell loss, and inflammatory cell accumulation in the media and adventitia. Il1r−/−, Il1a−/−, and Il1b−/− mice were protected from KD associated AAA. Infiltrating CD11c+ macrophages produced active caspase-1, and caspase-1 or NLRP3 deficiency inhibited AAA formation. Treatment with interleukin (IL)-1R antagonist (Anakinra), anti–IL-1&agr;, or anti–IL-1&bgr; mAb blocked LCWE-induced AAA formation. Conclusions—Similar to clinical KD, the LCWE-induced KD vasculitis mouse model can also be accompanied by AAA formation. Both IL-1&agr; and IL-1&bgr; play a key role, and use of an IL-1R blocking agent that inhibits both pathways may be a promising therapeutic target not only for KD coronary arteritis, but also for the other systemic arterial aneurysms including AAA that maybe seen in severe cases of KD. The LCWE-induced vasculitis model may also represent an alternative model for AAA disease.

IL-1 Signaling Is Critically Required in Stromal Cells in Kawasaki Disease Vasculitis Mouse Model Role of Both IL-1α and IL-1β

Objective—Kawasaki disease (KD) is the most common cause of acute vasculitis and acquired cardiac disease among US children. We have previously shown that both TLR2/MyD88 and interleukin (IL)-1&bgr; signaling are required for the Lactobacillus casei cell wall extract–induced KD vasculitis mouse model. The objectives of this study were to investigate the cellular origins of IL-1 production, the role of CD11c+ dendritic cells and macrophages, and the relative contribution of hematopoietic and stromal cells for IL-1 responsive cells, as well the MyD88 signaling, in Lactobacillus casei cell wall extract–induced KD mouse model of vasculitis. Approach and Results—Using mouse knockout models and antibody depletion, we found that both IL-1&agr; and IL-1&bgr; were required for Lactobacillus casei cell wall extract–induced KD. Both dendritic cells and macrophages were necessary, and we found that MyD88 signaling was required in both hematopoietic and stromal cells. However, IL-1 response and signaling were critically required in nonendothelial stromal cells, but not in hematopoietic cells. Conclusions—Our results suggest that IL-1&agr; and IL-1&bgr;, as well as CD11c+ dendritic cells and macrophages, are essential for the development of KD vasculitis and coronary arteritis in this mouse model. Bone marrow chimera experiments suggest that MyD88 signaling is important in both hematopoietic and stromal cells, whereas IL-1 signaling and response are required only in stromal cells, but not in endothelial cells. Determining the role of IL-1&agr; and IL-1&bgr; and of specific cell types in the KD vasculitis mouse model may have important implications for the design of more targeted therapies and understanding of the molecular mechanisms of KD immunopathologies.

CD8+ T cells contribute to the development of coronary arteritis in the Lactobacillus casei extract-induced murine model of Kawasaki Disease

Kawasaki disease (KD) is the leading cause of acquired heart disease among children in developed countries. Coronary lesions in KD in humans are characterized by an increased presence of infiltrating CD3+ T cells; however, the specific contributions of the different T cell subpopulations in coronary arteritis development remain unknown. Therefore, we sought to investigate the function of CD4+ and CD8+ T cells, Treg cells, and natural killer (NK) T cells in the pathogenesis of KD.

T Cell-Intrinsic Receptor Interacting Protein 2 Regulates Pathogenic T Helper 17 Cell Differentiation

&NA; Receptor interacting protein 2 (RIP2) plays a role in sensing intracellular pathogens, but its function in T cells is unclear. We show that RIP2 deficiency in CD4+ T cells resulted in chronic and severe interleukin‐17A‐mediated inflammation during Chlamydia pneumoniae lung infection, increased T helper 17 (Th17) cell formation in lungs of infected mice, accelerated atherosclerosis, and more severe experimental autoimmune encephalomyelitis. While RIP2 deficiency resulted in reduced conventional Th17 cell differentiation, it led to significantly enhanced differentiation of pathogenic (p)Th17 cells, which was dependent on ROR&agr; transcription factor and interleukin‐1 but independent of nucleotide oligomerization domain (NOD) 1 and 2. Overexpression of RIP2 resulted in suppression of pTh17 cell differentiation, an effect mediated by its CARD domain, and phenocopied by a cell‐permeable RIP2 CARD peptide. Our data suggest that RIP2 has a T cell‐intrinsic role in determining the balance between homeostatic and pathogenic Th17 cell responses. Graphical Abstract Figure. No caption available. HighlightsRIP2 deficiency in CD4+ T cells leads to severe IL‐17A‐mediated diseasesRIP2 suppresses pathogenic Th17 cell and supports conventional Th17 cell polarizationRIP2 suppression of pathogenic Th17 cell differentiation is ROR&agr; and IL‐1 dependentRIP2 CARD domain regulates pathogenic Th17 cell differentiation in CD4+ T cells &NA; RIP2 is the key adaptor molecule for NOD1‐ and NOD2‐mediated intracellular signaling to sense pathogens and cell activation in myeloid cells. Shimada, Porritt, and colleagues demonstrate a previously unappreciated role for RIP2 in Th17 cell regulation and differentiation in a T cell‐intrinsic manner.

Abstract 1459: Revealing the underlying causes of the gender disparity in melanoma: Role of testosterone

Introduction: At advanced age, men have increasingly higher incidence of melanoma compared with women, as well as continuously decreasing levels of testosterone. With the low rate of survival for distant metastatic patients with melanoma, especially in men, studies aimed at elucidating the immunological and hormonal mechanisms underlying the gender disparity are warranted. Procedures: To investigate the gender disparity observed between men and women affected by melanoma, we used an experimental murine model where B16 melanoma cells were injected i.v. into C57BL/6 mice and lung colonization was assessed at day 14. The roles of neutrophils and NK cells were then investigated by specific cell depletion using anti-Ly6G(1A8) and anti-NK1.1 monoclonal antibodies, respectively. Depleting antibodies or control IgG were injected every 3 days, starting one day before melanoma cell injection. To elucidate the role of hormones, ovariectomy or castration surgeries were performed 4 weeks before B16 injection and 60 day slow release pellets were inserted at the time of surgery. Findings were reproduced with a second murine melanoma cell line (YUMM1.7) derived from a genetically engineered mouse harboring human mutations in BrafV600, as well as loss of Cdkn2a and PTEN. Data: Compared with males, naive female mice injected with melanoma (B16 or YUMM1.7) cells have higher lung tumor burden, reduced neutrophil infiltration, and decreased NK cell activation. Ovariectomy of female mice did not affect lung tumor burden, indicating that estrogen and progesterone are not protective. Interestingly, castrated males revealed increased lung metastasis and worse survival rates compared with sham male mice, suggesting a protective role for testosterone. Further, testosterone replacement in castrated mice significantly reduced the elevated lung tumor burden to equal levels observed in sham males. Depletion of NK cells greatly increased tumor burden in both male and female mice in a gender independent manner. In contrast, neutrophil depletion increased lung tumor burden only in male mice and had no significant affect in female mice, indicating a potential gender difference in neutrophils. Moreover, neutrophil depletion in male mice also reduced NK activation and IFN-γ production, suggesting a stimulatory relationship between neutrophils and NK cells. Conclusion: Our data indicates that both neutrophils and NK cells are important for the initial response against melanoma colonization of the lung. Neutrophil depletion, as well as our castration data, indicate that there is a gender specific differential response, which may contribute to the gender disparity seen in human melanoma incidence and survival. Further, testosterone replacement at physiological levels may benefit a subset of melanoma patients. Citation Format: Janet Markman, Daiko Wakita, Timothy Crother, Moshe Arditi. Revealing the underlying causes of the gender disparity in melanoma: Role of testosterone. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1459.

Abstract 4057: Gender and the immune system in lung and liver colonization of murine melanoma in a B16 metastatic model

Introduction: Until age 45, incidence rates of melanoma are higher in woman than men, but by age 60, rates in men are more than double and by age 80, they are almost triple. With the low rate of survival for distant metastatic patients with melanoma, especially in men, studies aimed at elucidating the mechanisms underlying metastasis and the gender disparity are warranted. The purpose of this study is to identify the immune cells and cellular interactions that are crucial for initial colonization of metastatic melanoma in a gender dependent manner. Experimental Procedures: We used a B16 melanoma metastatic model in which 2×105 cells are injected via the tail vein of B6 mice and allowed to colonize for 14 days. Positive organs are analyzed by visual counting of the metastases, extraction of the melanin pigment and measurement by spectrophotometry, and by flow cytometry. To investigate the role of innate immunity, we injected ip either anti-Ly6G, 1A8 (neutrophil depletion), anti NK1.1, PK136 (natural killer [NK] cell depletion) or control IgG every 3 days, starting 1 day before tumor injection. For hormone ablation studies, an LHRH antagonist was injected. Data: Naive mice injected with B16 cells revealed that females have higher lung tumor burden than males. Previous castration studies indicate that this may be dependent on androgens. Intriguingly, neutrophil depletion increases tumor burden only in male mice when compared to male IgG and does not significantly affect tumor burden in female mice, indicating a potential gender difference in neutrophils. Neutrophil depletion in male mice also reduced NK activation and IFN-γ production, indicating a stimulatory relationship between neutrophils and NK cells. A subsequent NK depletion study revealed significantly increased tumor burden in both male and female NK-depleted mice when compared to the gender-matched controls. However, the increased tumor burden normally seen in lungs of females compared to males was still apparent when comparing lungs of NK depleted male mice to NK depleted female mice. This study also revealed that NK cells are crucial for the prevention of liver metastasis as both genders of NK depleted mice revealed multiple melanoma tumor nodules on the surface of the liver. Conclusion: Our data indicates that both neutrophils, and to a greater extent, NK cells are important for the initial response against B16 melanoma colonization of the lung and may be necessary to prevent metastasis. For the liver, NK cells were absolutely crucial for the prevention of melanoma colonization in this organ. Neutrophil depletion, as well as our previous castration data, indicated that there is a gender specific differential response of these cells, which may contribute to the gender disparity seen in human incidence and survival. Further, the importance of NK cells in killing of tumor cells indicates a population of patients that may benefit from ex vivo NK expansion therapy currently in clinical trials. Citation Format: Janet L. Markman, Daiko Wakita, Timothy R. Crother, Moshe Arditi. Gender and the immune system in lung and liver colonization of murine melanoma in a B16 metastatic model. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4057. doi:10.1158/1538-7445.AM2015-4057

IL-1 Signaling Is Critically Required in Stromal Cells in Kawasaki Disease Vasculitis Mouse ModelSignificance

Objective—Kawasaki disease (KD) is the most common cause of acute vasculitis and acquired cardiac disease among US children. We have previously shown that both TLR2/MyD88 and interleukin (IL)-1&bgr; signaling are required for the Lactobacillus casei cell wall extract–induced KD vasculitis mouse model. The objectives of this study were to investigate the cellular origins of IL-1 production, the role of CD11c+ dendritic cells and macrophages, and the relative contribution of hematopoietic and stromal cells for IL-1 responsive cells, as well the MyD88 signaling, in Lactobacillus casei cell wall extract–induced KD mouse model of vasculitis. Approach and Results—Using mouse knockout models and antibody depletion, we found that both IL-1&agr; and IL-1&bgr; were required for Lactobacillus casei cell wall extract–induced KD. Both dendritic cells and macrophages were necessary, and we found that MyD88 signaling was required in both hematopoietic and stromal cells. However, IL-1 response and signaling were critically required in nonendothelial stromal cells, but not in hematopoietic cells. Conclusions—Our results suggest that IL-1&agr; and IL-1&bgr;, as well as CD11c+ dendritic cells and macrophages, are essential for the development of KD vasculitis and coronary arteritis in this mouse model. Bone marrow chimera experiments suggest that MyD88 signaling is important in both hematopoietic and stromal cells, whereas IL-1 signaling and response are required only in stromal cells, but not in endothelial cells. Determining the role of IL-1&agr; and IL-1&bgr; and of specific cell types in the KD vasculitis mouse model may have important implications for the design of more targeted therapies and understanding of the molecular mechanisms of KD immunopathologies.

Interaction of NK cells with bacteria

Publisher Summary Natural killer (NK) cells produce many chemokines and inflammatory cytokines in response to, and exhibit cytotoxic activity against, pathogen-infected cells. They contribute to the prevention of various infectious diseases, especially during the early phases of infection, before CD8+ cytotoxic T lymphocyte (CTL) induction. Natural killer (NK) cells, including NKT cells, are components of the innate immune system and contribute significantly to the clearance of pathogen-infected or malignant cells. Recently, it has been demonstrated that quite diverse pattern-recognition receptors expressed on dendritic cells (DCs) and macrophages (M) recognize pathogen-specific components and subsequently initiate NK cell activation through two distinct signals: soluble factors and cell-to-cell contact. Crosstalk between NK cells and DCs plays a pivotal role in bridging innate and acquired immunity. Most pathogens and some lactic acid bacteria can modulate the immune balance towards a Th1- predominance. Therefore, bacteria themselves or their components are used to improve the disrupted immune balance or to induce Th1 responses critical for the prevention of infectious diseases, cancers and allergies. The cellular and molecular mechanisms of regulating the innate and acquired immune responses by bacterial stimuli and NKT cell ligands via reciprocal interactions among DCs, NK and NKT cells has been reviewed. Sustaining healthy conditions in mice and humans require precise control of immune responses towards exogenous antigens because disruption of immune homeostasis causes various immune-associated diseases, such as allergy, autoimmune disease and cancer.