Metabolic homeostasis of tissue macrophages across the lifespan.

Macrophages are present in almost all organs. Apart from being immune sentinels, tissue-resident macrophages (TRMs) have organ-specific functions that require a specialized cellular metabolism to maintain homeostasis. In addition, organ-dependent metabolic adaptations of TRMs appear to be fundamentally distinct in homeostasis and in response to a challenge, such as infection or injury. Moreover, TRM function becomes aberrant with advancing age, contributing to inflammaging and organ deterioration, and a metabolic imbalance may underlie TRM immunosenescence. Here, we outline current understanding of the particular metabolic states of TRMs across organs and the relevance for their function. Moreover, we discuss the concomitant aging-related decline in metabolic plasticity and functions of TRMs, highlighting potential novel therapeutic avenues to promote healthy aging.

Dendritic cells as orchestrators of anticancer immunity and immunotherapy.

Dendritic cells (DCs) are a heterogeneous group of antigen-presenting innate immune cells that regulate adaptive immunity, including against cancer. Therefore, understanding the precise activities of DCs in tumours and patients with cancer is important. The classification of DC subsets has historically been based on ontogeny; however, single-cell analyses are now additionally revealing a diversity of functional states of DCs in cancer. DCs can promote the activation of potent antitumour T cells and immune responses via numerous mechanisms, although they can also be hijacked by tumour-mediated factors to contribute to immune tolerance and cancer progression. Consequently, DC activities are often key determinants of the efficacy of immunotherapies, including immune-checkpoint inhibitors. Potentiating the antitumour functions of DCs or using them as tools to orchestrate short-term and long-term anticancer immunity has immense but as-yet underexploited therapeutic potential. In this Review, we outline the nature and emerging complexity of DC states as well as their functions in regulating adaptive immunity across different cancer types. We also describe how DCs are required for the success of current immunotherapies and explore the inherent potential of targeting DCs for cancer therapy. We focus on novel insights on DCs derived from patients with different cancers, single-cell studies of DCs and their relevance to therapeutic strategies.

Expression of HMGCS2 in intestinal epithelial cells is downregulated in inflammatory bowel disease associated with endoplasmic reticulum stress.

Introduction: The Unfolded Protein Response, a mechanism triggered by the cell in response to Endoplasmic reticulum stress, is linked to inflammatory responses. Our aim was to identify novel Unfolded Protein Response-mechanisms that might be involved in triggering or perpetuating the inflammatory response carried out by the Intestinal Epithelial Cells in the context of Inflammatory Bowel Disease. Methods: We analyzed the transcriptional profile of human Intestinal Epithelial Cell lines treated with an Endoplasmic Reticulum stress inducer (thapsigargin) and/or proinflammatory stimuli. Several genes were further analyzed in colonic biopsies from Ulcerative Colitis patients and healthy controls. Lastly, we generated Caco-2 cells lacking HMGCS2 by CRISPR Cas-9 and analyzed the functional implications of its absence in Intestinal Epithelial Cells. Results: Exposure to a TLR ligand after thapsigargin treatment resulted in a powerful synergistic modulation of gene expression, which led us to identify new genes and pathways that could be involved in inflammatory responses linked to the Unfolded Protein Response. Key differentially expressed genes in the array also exhibited transcriptional alterations in colonic biopsies from active Ulcerative Colitis patients, including NKG2D ligands and the enzyme HMGCS2. Moreover, functional studies showed altered metabolic responses and epithelial barrier integrity in HMGCS2 deficient cell lines. Conclusion: We have identified new genes and pathways that are regulated by the Unfolded Protein Response in the context of Inflammatory Bowel Disease including HMGCS2, a gene involved in the metabolism of Short Chain Fatty Acids that may have an important role in intestinal inflammation linked to Endoplasmic Reticulum stress and the resolution of the epithelial damage.

Immunometabolism at the crossroads of obesity and cancer-a Keystone Symposia report.

Immunometabolism considers the relationship between metabolism and immunity. Typically, researchers focus on either the metabolic pathways within immune cells that affect their function or the impact of immune cells on systemic metabolism. A more holistic approach that considers both these viewpoints is needed. On September 5-8, 2022, experts in the field of immunometabolism met for the Keystone symposium "Immunometabolism at the Crossroads of Obesity and Cancer" to present recent research across the field of immunometabolism, with the setting of obesity and cancer as an ideal example of the complex interplay between metabolism, immunity, and cancer. Speakers highlighted new insights on the metabolic links between tumor cells and immune cells, with a focus on leveraging unique metabolic vulnerabilities of different cell types in the tumor microenvironment as therapeutic targets and demonstrated the effects of diet, the microbiome, and obesity on immune system function and cancer pathogenesis and therapy. Finally, speakers presented new technologies to interrogate the immune system and uncover novel metabolic pathways important for immunity.

Oxidative phosphorylation selectively orchestrates tissue macrophage homeostasis.

In vitro studies have associated oxidative phosphorylation (OXPHOS) with anti-inflammatory macrophages, whereas pro-inflammatory macrophages rely on glycolysis. However, the metabolic needs of macrophages in tissues (TMFs) to fulfill their homeostatic activities are incompletely understood. Here, we identified OXPHOS as the highest discriminating process among TMFs from different organs in homeostasis by analysis of RNA-seq data in both humans and mice. Impairing OXPHOS in TMFs via Tfam deletion differentially affected TMF populations. Tfam deletion resulted in reduction of alveolar macrophages (AMs) due to impaired lipid-handling capacity, leading to increased cholesterol content and cellular stress, causing cell-cycle arrest in vivo. In obesity, Tfam depletion selectively ablated pro-inflammatory lipid-handling white adipose tissue macrophages (WAT-MFs), thus preventing insulin resistance and hepatosteatosis. Hence, OXPHOS, rather than glycolysis, distinguishes TMF populations and is critical for the maintenance of TMFs with a high lipid-handling activity, including pro-inflammatory WAT-MFs. This could provide a selective therapeutic targeting tool.

Metabolism of tissue macrophages in homeostasis and pathology.

Cellular metabolism orchestrates the intricate use of tissue fuels for catabolism and anabolism to generate cellular energy and structural components. The emerging field of immunometabolism highlights the importance of cellular metabolism for the maintenance and activities of immune cells. Macrophages are embryo- or adult bone marrow-derived leukocytes that are key for healthy tissue homeostasis but can also contribute to pathologies such as metabolic syndrome, atherosclerosis, fibrosis or cancer. Macrophage metabolism has largely been studied in vitro. However, different organs contain diverse macrophage populations that specialize in distinct and often tissue-specific functions. This context specificity creates diverging metabolic challenges for tissue macrophage populations to fulfill their homeostatic roles in their particular microenvironment and conditions their response in pathological conditions. Here, we outline current knowledge on the metabolic requirements and adaptations of macrophages located in tissues during homeostasis and selected diseases.

Tumor stem cells fuse with monocytes to form highly invasive tumor-hybrid cells.

The 'cancer cell fusion' theory is controversial due to the lack of methods available to identify hybrid cells and to follow the phenomenon in patients. However, it seems to be one of the best explanations for both the origin and metastasis of primary tumors. Herein, we co-cultured lung cancer stem cells with human monocytes and analyzed the dynamics and properties of tumor-hybrid cells (THC), as well as the molecular mechanisms beneath this fusion process by several techniques: electron-microscopy, karyotyping, CRISPR-Cas9, RNA-seq, immunostaining, signaling blockage, among others. Moreover, mice models were assessed for in vivo characterization of hybrids colonization and invasiveness. Then, the presence of THCs in bloodstream and samples from primary and metastatic lesions were detected by FACS and immunofluorescence protocols, and their correlations with TNM stages established. Our data indicate that the generation of THCs depends on the expression of CD36 on tumor stem cells and the oxidative state and polarization of monocytes, the latter being strongly influenced by microenvironmental fluctuations. Highly oxidized M2-like monocytes show the strongest affinity to fuse with tumor stem cells. THCs are able to proliferate, colonize and invade organs. THC-specific cell surface signature CD36+CD14+PANK+ allows identifying them in matched primary tumor tissues and metastases as well as in bloodstream from patients with lung cancer, thus functioning as a biomarker. THCs levels in circulation correlate with TNM classification. Our results suggest that THCs are involved in both origin and spread of metastatic cells. Furthermore, they might set the bases for future therapies to avoid or eradicate lung cancer metastasis.

Early Neutrophil Responses to Chemical Carcinogenesis Shape Long-Term Lung Cancer Susceptibility.

Neoplastic transformation causing cancer is a key problem in tumor biology and can be triggered by exposure to environmental substances. We investigated whether the cellular composition of a tissue contributes to its predisposition to cancer upon a specific carcinogen. Neutrophils are important immune components involved in cancer progression, but their contribution to generation of transformed cells is elusive. Yet, neutrophil-released reactive oxygen species (ROS) can cause tissue damage, which potentially favors tumorigenesis. Here, we show that neutrophils contribute directly to neoplastic transformation by amplifying the genotoxicity of urethane in lung cells via ROS. Neutrophil-driven ROS-dependent DNA damage is timely restricted to urethane exposure and notably uncoupled from broad tissue damage or inflammation. Neutropenic granulocyte colony-stimulating factor (Gcsf)-knockout mice show reduced lung tumorigenesis, and forcing neutrophil recruitment only during urethane exposure rescues cancer incidence months later. This study shows that the time-restricted neutrophil response to carcinogens can impact the long-term tissue susceptibility to cancer.

Dendritic cells in cancer immunology and immunotherapy.

Dendritic cells (DCs) are a diverse group of specialized antigen-presenting cells with key roles in the initiation and regulation of innate and adaptive immune responses. As such, there is currently much interest in modulating DC function to improve cancer immunotherapy. Many strategies have been developed to target DCs in cancer, such as the administration of antigens with immunomodulators that mobilize and activate endogenous DCs, as well as the generation of DC-based vaccines. A better understanding of the diversity and functions of DC subsets and of how these are shaped by the tumour microenvironment could lead to improved therapies for cancer. Here we will outline how different DC subsets influence immunity and tolerance in cancer settings and discuss the implications for both established cancer treatments and novel immunotherapy strategies.

Author Correction: Neutrophils support lung colonization of metastasis-initiating breast cancer cells.

Change history: In this Letter, the final two pages of the Supplementary Information, which included two tables listing primer and antibodies, respectively, were missing; see accompanying Amendment for the missing Supplementary Tables. This Letter has not been corrected online.

Metabolic Control of Dendritic Cell Functions: Digesting Information.

Dendritic cells (DCs) control innate and adaptive immunity by patrolling tissues to gather antigens and danger signals derived from microbes and tissue. Subsequently, DCs integrate those environmental cues, orchestrate immunity or tolerance, and regulate tissue homeostasis. Recent advances in the field of immunometabolism highlight the notion that immune cells markedly alter cellular metabolic pathways during differentiation or upon activation, which has important implications on their functionality. Previous studies showed that active oxidative phosphorylation in mitochondria is associated with immature or tolerogenic DCs, while increased glycolysis upon pathogen sensing can promote immunogenic DC functions. However, new results in the last years suggest that regulation of DC metabolism in steady state, after immunogenic activation and during tolerance in different pathophysiological settings, may be more complex. Moreover, ontogenically distinct DC subsets show different functional specializations to control T cell responses. It is, thus, relevant how metabolism influences DC differentiation and plasticity, and what potential metabolic differences exist among DC subsets. Better understanding of the emerging connection between metabolic adaptions and functional DC specification will likely allow the development of therapeutic strategies to manipulate immune responses.

The clinical application of cancer immunotherapy based on naturally circulating dendritic cells.

Dendritic cells (DCs) can initiate and direct adaptive immune responses. This ability is exploitable in DC vaccination strategies, in which DCs are educated ex vivo to present tumor antigens and are administered into the patient with the aim to induce a tumor-specific immune response. DC vaccination remains a promising approach with the potential to further improve cancer immunotherapy with little or no evidence of treatment-limiting toxicity. However, evidence for objective clinical antitumor activity of DC vaccination is currently limited, hampering the clinical implementation. One possible explanation for this is that the most commonly used monocyte-derived DCs may not be the best source for DC-based immunotherapy. The novel approach to use naturally circulating DCs may be an attractive alternative. In contrast to monocyte-derived DCs, naturally circulating DCs are relatively scarce but do not require extensive culture periods. Thereby, their functional capabilities are preserved, the reproducibility of clinical applications is increased, and the cells are not dysfunctional before injection. In human blood, at least three DC subsets can be distinguished, plasmacytoid DCs, CD141+ and CD1c+ myeloid/conventional DCs, each with distinct functional characteristics. In completed clinical trials, either CD1c+ myeloid DCs or plasmacytoid DCs were administered and showed encouraging immunological and clinical outcomes. Currently, also the combination of CD1c+ myeloid and plasmacytoid DCs as well as the intratumoral use of CD1c+ myeloid DCs is under investigation in the clinic. Isolation and culture strategies for CD141+ myeloid DCs are being developed. Here, we summarize and discuss recent clinical developments and future prospects of natural DC-based immunotherapy.

Effective cancer immunotherapy by natural mouse conventional type-1 dendritic cells bearing dead tumor antigen.

BACKGROUND: The manipulation of dendritic cells (DCs) for cancer vaccination has not reached its full potential, despite the revolution in cancer immunotherapy. DCs are fundamental for CD8+ T cell activation, which relies on cross-presentation of exogenous antigen on MHC-I and can be fostered by immunogenic cancer cell death. Translational and clinical research has focused on in vitro-generated monocyte-derived DCs, while the vaccination efficacy of natural conventional type 1 DCs (cDC1s), which are associated with improved anti-tumor immunity and specialize on antigen cross-presentation, remains unknown. METHODS: We isolated primary spleen mouse cDC1s and established a protocol for fast ex vivo activation and antigen-loading with lysates of tumor cells that underwent immunogenic cell death by UV irradiation. Natural tumor antigen-loaded cDC1s were transferred and their potential for induction of endogenous CD8+ and CD4+ T cell responses in vivo, cancer prevention and therapy were assessed in three grafted cancer models. Further, we tested the efficacy of natural cDC1 vaccination in combination and comparison with anti-PD-1 treatment in two "wildtype" tumor models not expressing exogenous antigens. RESULTS: Herein, we reveal that primary mouse cDC1s ex vivo loaded with dead tumor cell-derived antigen are activated and induce strong CD8+ T cell responses from the endogenous repertoire upon adoptive transfer in vivo through tumor antigen cross-presentation. Notably, cDC1-based vaccines enhance tumor infiltration by cancer-reactive CD8+ and CD4+ T cells and halt progression of engrafted cancer models, including tumors that are refractory to anti-PD-1 treatment. Moreover, combined tumor antigen-loaded primary cDC1 and anti-PD-1 therapy had strong synergistic effects in a PD-1 checkpoint inhibition susceptible cancer model. CONCLUSIONS: This preclinical proof-of-principle study is first to support the therapeutic efficacy of cancer immunotherapy with syngeneic dead tumor cell antigen-loaded mouse cDC1s, the equivalents of the human dendritic cell subset that correlates with beneficial prognosis of cancer patients. Our data pave the way for translation of cDC1-based cancer treatments into the clinic when isolation of natural human cDC1s becomes feasible.

DNGR-1 in dendritic cells limits tissue damage by dampening neutrophil recruitment.

Host injury triggers feedback mechanisms that limit tissue damage. Conventional type 1 dendritic cells (cDC1s) express dendritic cell natural killer lectin group receptor-1 (DNGR-1), encoded by the gene Clec9a, which senses tissue damage and favors cross-presentation of dead-cell material to CD8+ T cells. Here we find that DNGR-1 additionally reduces host-damaging inflammatory responses induced by sterile and infectious tissue injury in mice. DNGR-1 deficiency leads to exacerbated caerulein-induced necrotizing pancreatitis and increased pathology during systemic Candida albicans infection without affecting fungal burden. This effect is B and T cell-independent and attributable to increased neutrophilia in DNGR-1-deficient settings. Mechanistically, DNGR-1 engagement activates SHP-1 and inhibits MIP-2 (encoded by Cxcl2) production by cDC1s during Candida infection. This consequently restrains neutrophil recruitment and promotes disease tolerance. Thus, DNGR-1-mediated sensing of injury by cDC1s serves as a rheostat for the control of tissue damage, innate immunity, and immunopathology.

Integrin signalling regulates YAP and TAZ to control skin homeostasis.

The skin is a squamous epithelium that is continuously renewed by a population of basal layer stem/progenitor cells and can heal wounds. Here, we show that the transcription regulators YAP and TAZ localise to the nucleus in the basal layer of skin and are elevated upon wound healing. Skin-specific deletion of both YAP and TAZ in adult mice slows proliferation of basal layer cells, leads to hair loss and impairs regeneration after wounding. Contact with the basal extracellular matrix and consequent integrin-Src signalling is a key determinant of the nuclear localisation of YAP/TAZ in basal layer cells and in skin tumours. Contact with the basement membrane is lost in differentiating daughter cells, where YAP and TAZ become mostly cytoplasmic. In other types of squamous epithelia and squamous cell carcinomas, a similar control mechanism is present. By contrast, columnar epithelia differentiate an apical domain that recruits CRB3, Merlin (also known as NF2), KIBRA (also known as WWC1) and SAV1 to induce Hippo signalling and retain YAP/TAZ in the cytoplasm despite contact with the basal layer extracellular matrix. When columnar epithelial tumours lose their apical domain and become invasive, YAP/TAZ becomes nuclear and tumour growth becomes sensitive to the Src inhibitor Dasatinib.

Neutrophils support lung colonization of metastasis-initiating breast cancer cells.

Despite progress in the development of drugs that efficiently target cancer cells, treatments for metastatic tumours are often ineffective. The now well-established dependency of cancer cells on their microenvironment suggests that targeting the non-cancer-cell component of the tumour might form a basis for the development of novel therapeutic approaches. However, the as-yet poorly characterized contribution of host responses during tumour growth and metastatic progression represents a limitation to exploiting this approach. Here we identify neutrophils as the main component and driver of metastatic establishment within the (pre-)metastatic lung microenvironment in mouse breast cancer models. Neutrophils have a fundamental role in inflammatory responses and their contribution to tumorigenesis is still controversial. Using various strategies to block neutrophil recruitment to the pre-metastatic site, we demonstrate that neutrophils specifically support metastatic initiation. Importantly, we find that neutrophil-derived leukotrienes aid the colonization of distant tissues by selectively expanding the sub-pool of cancer cells that retain high tumorigenic potential. Genetic or pharmacological inhibition of the leukotriene-generating enzyme arachidonate 5-lipoxygenase (Alox5) abrogates neutrophil pro-metastatic activity and consequently reduces metastasis. Our results reveal the efficacy of using targeted therapy against a specific tumour microenvironment component and indicate that neutrophil Alox5 inhibition may limit metastatic progression.

EGFR has a tumour-promoting role in liver macrophages during hepatocellular carcinoma formation.

Hepatocellular carcinoma (HCC) is a frequent cancer with limited treatment options and poor prognosis. Tumorigenesis has been linked with macrophage-mediated chronic inflammation and diverse signalling pathways, including the epidermal growth factor receptor (EGFR) pathway. The precise role of EGFR in HCC is unknown, and EGFR inhibitors have shown disappointing clinical results. Here we discover that EGFR is expressed in liver macrophages in both human HCC and in a mouse HCC model. Mice lacking EGFR in macrophages show impaired hepatocarcinogenesis, whereas mice lacking EGFR in hepatocytes unexpectedly develop more HCC owing to increased hepatocyte damage and compensatory proliferation. Mechanistically, following interleukin-1 stimulation, EGFR is required in liver macrophages to transcriptionally induce interleukin-6, which triggers hepatocyte proliferation and HCC. Importantly, the presence of EGFR-positive liver macrophages in HCC patients is associated with poor survival. This study demonstrates a tumour-promoting mechanism for EGFR in non-tumour cells, which could lead to more effective precision medicine strategies.