Monocyte-derived macrophages aggravate pulmonary vasculitis via cGAS/STING/IFN-mediated nucleic acid sensing

Kessler et al. identify aberrant DNA recognition by cGAS/STING and IFN-I production by inflammatory macrophages as a driver of severe ANCA-associated vasculitis. Pharmacological interventions blocking this pathway ameliorate disease and accelerate recovery, identifying potential targets for therapeutic intervention in patients. Autoimmune vasculitis is a group of life-threatening diseases, whose underlying pathogenic mechanisms are incompletely understood, hampering development of targeted therapies. Here, we demonstrate that patients suffering from anti-neutrophil cytoplasmic antibodies (ANCA)-associated vasculitis (AAV) showed increased levels of cGAMP and enhanced IFN-I signature. To identify disease mechanisms and potential therapeutic targets, we developed a mouse model for pulmonary AAV that mimics severe disease in patients. Immunogenic DNA accumulated during disease onset, triggering cGAS/STING/IRF3-dependent IFN-I release that promoted endothelial damage, pulmonary hemorrhages, and lung dysfunction. Macrophage subsets played dichotomic roles in disease. While recruited monocyte-derived macrophages were major disease drivers by producing most IFN-beta, resident alveolar macrophages contributed to tissue homeostasis by clearing red blood cells and limiting infiltration of IFN-beta-producing macrophages. Moreover, pharmacological inhibition of STING, IFNAR-I, or its downstream JAK/STAT signaling reduced disease severity and accelerated recovery. Our study unveils the importance of STING/IFN-I axis in promoting pulmonary AAV progression and identifies cellular and molecular targets to ameliorate disease outcomes.

Engineering Lymphangiogenesis

Manuel Andreas Fankhauser

Melanoma causes the vast majority of skin cancer deaths, and there is currently no conventional treatment available that is able to cure metastatic melanoma patients. However, a novel treatment modality has recently emerged: cancer immunotherapy. Cancer immunotherapy aims at raising potent, tumor-specific T cell responses that are able to recognize and eliminate cancerous cells. Despite the fact that for the first time in history some metastatic melanoma patients can be cured, it is currently unclear why a large fraction of patients does not respond to immunotherapy. Thus, the overarching goal of this thesis was to gain a better understanding of the molecular mechanisms dictating the outcome to immunotherapy. Pre-existing inflammation within the tumor microenvironment is associated with good clinical prognosis following immunotherapy. Because our lab has previously shown that tumor associated lymphatic vessels (LVs) can modulate tumor inflammation, we hypothesized that LVs might be involved in modulating the outcome of cancer immunotherapy. To explore this hypothesis, we characterized two mouse models that recapitulate primary human lymphangiogenic melanoma. Using these models, we found that tumor-associated lymphatics secrete CCL21 to actively attract naïve T cells into the tumor microenvironment. Interestingly, we found that antigen-specific immunotherapy induced activation of these naïve T cell infiltrates, which not only resulted in eradication of the primary melanoma tumors, but also conferred the mice with long-term protection against tumor re-challenge. We thus demonstrate that LVs increase the quantity and quality of tumor inflammation, thereby establishing a microenvironment that is able to potentiate antigen-specific immunotherapy. We then show that controlled release of VEGFC from injectable hydrogels leads to lymphangiogenesis in the mouse dermis. Similarly to what we found in lymphangiogenic tumors, engineered lymphangiogenic skin sites displayed increased expression of CCL21, and increased infiltrations of CD4+ and CD8+ T cells. Delivery of the model antigen ovalbumin into lymphangiogenic sites led to enhanced release of the effector cytokine interferon-¿ upon antigen-restimulation, suggesting that engineered lymphangiogenic sites could be exploited for therapeutic immunomodulation. Finally, to better understand mechanisms underlying successful cancer immunotherapy, we established a tool for the observation of anti-tumor immune responses in the context of the native tumor microenvironment. By combining existing methods in a novel way, we developed an intravital microscopy method based on immunofluorescence that allows simultaneous, high resolution and dynamic visualization of the tumor microenvironment including immune cells and extracellular matrix proteins. In conclusion, this thesis demonstrates that lymphatic endothelial cells (LECs) orchestrate immune cell recruitment into peripheral tissues by secretion of CCL21 chemokine, both in steady-state and disease. Our findings add more evidence to the hypothesis that LECs might play an underappreciated role in driving the formation of peripheral sites of immunomodulation. Our findings have translational potential, as immune cell infiltrates attracted by local lymphangiogenesis can be exploited for therapeutic immune regulation, such as to improving the efficacy of cancer immunotherapy.

EPFL2016

Molecular profiling and functional analysis of macrophage-derived tumour extracellular vesicles

Tim Beltraminelli

Extracellular vesicles (EVs) are nanosized vesicles released by virtually all cell types. Increasing data suggest that EVs function in the regulation of intercellular communication both in physiological and pathological states. In cancer biology, mounting evidence suggests that cancer cell-derived EVs directly modulate several aspects of tumour progression by acting on both cancer cells and tumour-associated stromal cells. In addition to cancer cells, the tumour microenvironment (TME) also features an intertwined ensemble of recruited host-derived cells. Tumour-associated macrophages (TAMs) often infiltrate tumours in high numbers, thus representing one of the major host-derived cell populations. The involvement of TAMs in cancer progression has been extensively documented. It is well established that according to their polarisation state, TAMs can exert both pro- and anti-tumoural functions. However, under the influence of cancer cell-derived factors TAMs often acquire a phenotype that fosters tumour progression by supporting angiogenesis, metastasis and immunosuppression. While the role of secreted soluble factors contributing to the pro- and anti-tumoural functions of TAMs is well characterised, the molecular composition, properties, and effects of TAM-derived EVs (TAM-EVs) in the TME are poorly understood. In this thesis, I studied TAM-EVs produced in intact mouse tumours. We developed a methodology for the enrichment, quantification, and selective isolation of TAM-EVs produced in the TME. Furthermore, we performed comprehensive proteomic and lipidomic analyses of tumour-derived EVs and identified molecular signatures indicative of a role for TAM-EVs in immune response and lipid metabolism. Whereas source TAMs display a phenotype consistent with immunosuppressive functions, TAM-EVs have a pro-inflammatory and immune-stimulatory molecular signature. Indeed, TAM-EV-enriched preparations promoted T-cell proliferation and activation ex vivo and modulated arachidonic acid metabolism of cancer cells by inhibiting the production of the immunosuppressive PGE2 while favouring the secretion of pro-inflammatory thromboxane. Therefore, TAM-EVs might have the potential to stimulate, rather than limit, anti-tumour immunity. I also showed that while portraying some similarities with EVs of in vitro-polarised macrophages, TAM-EVs isolated from the TME present distinctive molecular profiles, suggesting that simplistic in vitro models do not faithfully recapitulate the complexity of the TME dynamics. Overall, this work emphasises the importance of studying TAM-EVs in their physiological context and identifies a potential role for TAM-EVs in favouring the development of an anti-tumoural immune microenvironment. Findings from this work extend our knowledge of the multifaceted role of TAMs in tumour biology.

EPFL2020

Molecular profiling and functional analysis of macrophage-derived tumour extracellular vesicles

Tim Beltraminelli

EPFL2020

Engineering Lymphangiogenesis

Manuel Andreas Fankhauser

EPFL2016