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Abstract Background Myeloid cells play an essential role in cancer metastasis. The phenotypic diversity of these cells during cancer development has attracted great interest; however, their functional heterogeneity and plasticity have limited their role as prognostic markers and therapeutic targets. Methods To identify markers associated with myeloid cells in metastatic tumours, we compared transcriptomic data from immune cells sorted from metastatic and non-metastatic mammary tumours grown in BALB/cJ mice. To assess the translational relevance of our in vivo findings, we assessed human breast cancer biopsies and evaluated the association between arginase 1 protein expression in breast cancer tissues with tumour characteristics and patient outcomes. Results Among the differentially expressed genes, arginase 1 (ARG1) showed a unique expression pattern in tumour-infiltrating myeloid cells that correlated with the metastatic capacity of the tumour. Even though ARG1-positive cells were found almost exclusively inside the metastatic tumour, ARG1 protein was also present in the plasma. In human breast cancer biopsies, the presence of ARG1-positive cells was strongly correlated with high-grade proliferating tumours, poor prognosis, and low survival. Conclusion Our findings highlight the potential use of ARG1-positive myeloid cells as an independent prognostic marker to evaluate the risk of metastasis in breast cancer patients. Graphical Abstract

Abstract Elevated activity of bone-degrading osteoclasts (OC) contributes to pathological bone degradation in diseases such as multiple myeloma. Several proinflammatory cytokines, including TNF, contribute to osteoclastogenesis. The receptor-interacting protein kinase 1 (RIPK1) regulates inflammation and cell death. It is recruited to the TNF-receptor complex, where it is ubiquitinated, and activates transcription factor NF-κB and mitogen-activated protein kinases (MAPK). Smac-mimetics (SM) is a group of drugs that block RIPK1 ubiquitination and shifts RIPK1 to activation of apoptosis or necroptosis. In this manuscript, we show that the two SM birinapant and LCL-161 reduced the number and viability of primary human OC, and induced TNF-dependent cell death in OC precursors (pre-OC). Birinapant was more cytotoxic than LCL-161 and induced predominantly apoptosis and to some degree necroptosis. Both inhibitors restrained osteoclastogenesis induced by myeloma patient bone-marrow aspirates. SM has gained attention as novel treatment strategies both for cancer and chronic inflammatory pathologies, but limited information has been available on interactions with primary human immune cells. As LCL-161 is in phase 2 clinical studies for multiple myeloma, we propose that SM might possess additional benefits in reducing bone degradation in myeloma patients. Taken together, we show that SM reduces human osteoclastogenesis, and that these compounds may represent promising drug candidates for pathological bone degradation.

Abstract Background The majority of patients with advanced cancer develop cachexia, a weight loss syndrome that severely reduces quality of life and limits survival. Our understanding of the underlying mechanisms that cause the condition is limited, and there are currently no treatment options that can completely reverse cachexia. Several tumour‐derived factors and inflammatory mediators have been suggested to contribute to weight loss in cachectic patients. However, inconsistencies between studies are recurrent. Activin A and interleukin 6 (IL‐6) are among the best studied factors that seem to be important, and several studies support their individual role in cachexia development. Methods We investigated the interplay between activin A and IL‐6 in the cachexia‐inducing TOV21G cell line, both in culture and in tumours in mice. We previously found that the human TOV21G cells secrete IL‐6 that induces autophagy in reporter cells and cachexia in mice. Using this established cachexia cell model, we targeted autocrine activin A by genetic, chemical, and biological approaches. The secretion of IL‐6 from the cancer cells was determined in both culture and tumour‐bearing mice by a species‐specific ELISA. Autophagy reporter cells were used to monitor the culture medium for autophagy‐inducing activities, and muscle mass changes were evaluated in tumour‐bearing mice. Results We show that activin A acts in an autocrine manner to promote the synthesis and secretion of IL‐6 from cancer cells. By inhibiting activin A signalling, the production of IL‐6 from the cancer cells is reduced by 40–50% (up to 42% reduction on protein level, P = 0.0048, and 48% reduction on mRNA level, P = 0.0308). Significantly reduced IL‐6 secretion (P < 0.05) from the cancer cells is consistently observed when using biological, chemical, and genetic approaches to interfere with the autocrine activin A loop. Inhibiting activin signalling also reduces the ability of the cancer cells to accelerate autophagy in non‐cancerous cells (up to 43% reduced autophagy flux, P = 0.0006). Coherent to the in vitro data, the use of an anti‐activin receptor 2 antibody in cachectic tumour‐bearing mice reduces serum levels of cancer cell‐derived IL‐6 by 62% (from 417 to 159 pg/mL, P = 0.03), and, importantly, it reverses cachexia and counteracts loss of all measured muscle groups (P < 0.0005). Conclusions Our data support a functional link between activin A and IL‐6 signalling pathways and indicate that interference with activin A‐induced IL‐6 secretion from the tumour has therapeutic potential for cancer‐induced cachexia.

Abstract The majority of cancer patients with advanced disease experience weight loss, including loss of lean body mass. Severe weight loss is characteristic for cancer cachexia, a condition that significantly impairs functional status and survival. The underlying causes of cachexia are incompletely understood, and currently no therapeutic approach can completely reverse the condition. Autophagy coordinates lysosomal destruction of cytosolic constituents and is systemically induced by starvation. We hypothesized that starvation-mimicking signaling compounds secreted from tumor cells may cause a systemic acceleration of autophagy during cachexia. We found that IL-6 secreted by tumor cells accelerates autophagy in myotubes when complexed with soluble IL-6 receptor (trans-signaling). In lung cancer patients, were cachexia is prevalent, there was a significant correlation between elevated IL-6 expression in the tumor and poor prognosis of the patients. We found evidence for an autophagy-inducing bioactivity in serum from cancer patients and that this is clearly associated with weight loss. Importantly, the autophagy-inducing bioactivity was reduced by interference with IL-6 trans-signaling. Together, our findings suggest that IL-6 trans-signaling may be targeted in cancer cachexia.

Background: Myeloid cells play an essential role in cancer metastasis. The phenotypic diversity of these cells during cancer development has attracted great interest; however, their functional heterogeneity and plasticity have limited their role as prognostic markers and therapeutic targets. Methods: To identify markers associated with myeloid cells in metastatic tumours, we compared transcriptomic data from immune cells sorted from metastatic and non-metastatic mammary tumours grown in BALB/cJ mice. To assess the translational relevance of our in vivo findings, we assessed human breast cancer biopsies and evaluated the association between arginase 1 protein expression in breast cancer tissues with tumour characteristics and patient outcomes. Results: Among the differentially expressed genes, arginase 1 (ARG1) showed a unique expression pattern in tumour-infiltrating myeloid cells that correlated with the metastatic capacity of the tumour. Even though ARG1-positive cells were found almost exclusively inside the metastatic tumour, ARG1 protein was also present in the plasma. In human breast cancer biopsies, the presence of ARG1-positive cells was strongly correlated with high-grade proliferating tumours, poor prognosis, and low survival. Conclusion: Our findings highlight the potential use of ARG1-positive myeloid cells as an independent prognostic marker to evaluate the risk of metastasis in breast cancer patients. [ABSTRACT FROM AUTHOR]

Mycobacterium tuberculosis is a global health problem in part as a result of extensive cytotoxicity caused by the infection. Here, we show how M. tuberculosis causes caspase-1/NLRP3/gasdermin D-mediated pyroptosis of human monocytes and macrophages. A type VII secretion system (ESX-1) mediated, contact-induced plasma membrane damage response occurs during phagocytosis of bacteria. Alternatively, this can occur from the cytosolic side of the plasma membrane after phagosomal rupture in infected macrophages. This damage causes K+ efflux and activation of NLRP3-dependent IL-1β release and pyroptosis, facilitating the spread of bacteria to neighbouring cells. A dynamic interplay of pyroptosis with ESCRT-mediated plasma membrane repair also occurs. This dual plasma membrane damage seems to be a common mechanism for NLRP3 activators that function through lysosomal damage. Inflammasome activation is a response to bacterial infection but can cause damage and spread infection. Here, the authors use live single-cell imaging to show two mechanisms by which M. tuberculosis causes damage to human macrophage cell plasma membranes, resulting in activation of the NLRP3 inflammasome, pyroptosis and release of infectious particles. [ABSTRACT FROM AUTHOR]

Background: The majority of patients with advanced cancer develop cachexia, a weight loss syndrome that severely reduces quality of life and limits survival. Our understanding of the underlying mechanisms that cause the condition is limited, and there are currently no treatment options that can completely reverse cachexia. Several tumour‐derived factors and inflammatory mediators have been suggested to contribute to weight loss in cachectic patients. However, inconsistencies between studies are recurrent. Activin A and interleukin 6 (IL‐6) are among the best studied factors that seem to be important, and several studies support their individual role in cachexia development. Methods: We investigated the interplay between activin A and IL‐6 in the cachexia‐inducing TOV21G cell line, both in culture and in tumours in mice. We previously found that the human TOV21G cells secrete IL‐6 that induces autophagy in reporter cells and cachexia in mice. Using this established cachexia cell model, we targeted autocrine activin A by genetic, chemical, and biological approaches. The secretion of IL‐6 from the cancer cells was determined in both culture and tumour‐bearing mice by a species‐specific ELISA. Autophagy reporter cells were used to monitor the culture medium for autophagy‐inducing activities, and muscle mass changes were evaluated in tumour‐bearing mice. Results: We show that activin A acts in an autocrine manner to promote the synthesis and secretion of IL‐6 from cancer cells. By inhibiting activin A signalling, the production of IL‐6 from the cancer cells is reduced by 40–50% (up to 42% reduction on protein level, P = 0.0048, and 48% reduction on mRNA level, P = 0.0308). Significantly reduced IL‐6 secretion (P < 0.05) from the cancer cells is consistently observed when using biological, chemical, and genetic approaches to interfere with the autocrine activin A loop. Inhibiting activin signalling also reduces the ability of the cancer cells to accelerate autophagy in non‐cancerous cells (up to 43% reduced autophagy flux, P = 0.0006). Coherent to the in vitro data, the use of an anti‐activin receptor 2 antibody in cachectic tumour‐bearing mice reduces serum levels of cancer cell‐derived IL‐6 by 62% (from 417 to 159 pg/mL, P = 0.03), and, importantly, it reverses cachexia and counteracts loss of all measured muscle groups (P < 0.0005). Conclusions: Our data support a functional link between activin A and IL‐6 signalling pathways and indicate that interference with activin A‐induced IL‐6 secretion from the tumour has therapeutic potential for cancer‐induced cachexia. [ABSTRACT FROM AUTHOR]

Phagocytosis is a complex process that eliminates microbes and is performed by specialised cells such as macrophages. Toll-like receptor 4 (TLR4) is expressed on the surface of macrophages and recognizes Gram-negative bacteria. Moreover, TLR4 has been suggested to play a role in the phagocytosis of Gram-negative bacteria, but the mechanisms remain unclear. Here we have used primary human macrophages and engineered THP-1 monocytes to show that the TLR4 sorting adapter, TRAM, is instrumental for phagocytosis of Escherichia coli as well as Staphylococcus aureus. We find that TRAM forms a complex with Rab11 family interacting protein 2 (FIP2) that is recruited to the phagocytic cups of E. coli. This promotes activation of the actin-regulatory GTPases Rac1 and Cdc42. Our results show that FIP2 guided TRAM recruitment orchestrates actin remodelling and IRF3 activation, two events that are both required for phagocytosis of Gram-negative bacteria. [ABSTRACT FROM AUTHOR]

ROR family of nuclear receptor transcription factors forms nodes connecting metabolic and inflammatory signaling pathways. The RORα members of the family have intrinsic transcriptional activity and they are involved in both activation and repression of a wide range of genes. The role of RORα in control of inflammation has been extensively studied using animal models but its function in human cells is not as well understood. To address this shortcoming, we analyzed how RORα is shaping the inflammatory state of human macrophages. Using CRISPR-Cas9 system, we deleted RORA in THP-1 human monocytic cell line. In mutant cells we observed a dramatic increase in basal expression of a subset of NF-κB regulated genes, including TNF, IL-1β and IL-6, at both transcriptional and translational levels. Furthermore, RORA-deletion cells produced notable amounts of pro-IL-1β even in the absence of LPS stimulation. Subsequent LPS stimulation induced cleavage of pro-IL-1β to mature form. Our RNAseq analysis further confirmed the key role of RORA in setting the inflammatory state of macrophages and defined the set of differentially regulated genes. Overall, our data provides evidence supporting the anti-inflammatory function of RORα in human macrophages. [ABSTRACT FROM AUTHOR]

Toll-like receptor 4 ( TLR4) is responsible for the immediate response to Gram-negative bacteria and signals via two main pathways by recruitment of distinct pairs of adaptor proteins. Mal- MyD88 [Mal (MyD88-adaptor-like) - MYD88 (Myeloid differentiation primary response gene (88))] is recruited to the plasma membrane to initiate the signaling cascade leading to production of pro-inflammatory cytokines while TRAM-TRIF [TRAM (TRIF-related adaptor molecule)-TRIF (TIR-domain-containing adapter-inducing interferon-β)] is recruited to early endosomes to initiate the subsequent production of type I interferons. We have investigated the dynamics of TLR4 and TRAM during lipopolysaccharide ( LPS) stimulation. We found that LPS induced a CD14-dependent immobile fraction of TLR4 in the plasma membrane. Total internal reflection fluorescence microscopy ( TIRF) revealed that LPS stimulation induced clustering of TLR4 into small punctate structures in the plasma membrane containing CD14/ LPS and clathrin, both in HEK293 cells and the macrophage model cell line U373-CD14. These results suggest that laterally immobilized TLR4 receptor complexes are being formed and prepared for endocytosis. RAB11A was found to be involved in localizing TRAM to the endocytic recycling compartment ( ERC) and to early sorting endosomes. Moreover, CD14/ LPS but not TRAM was immobilized on RAB11A-positive endosomes, which indicates that TRAM and CD14/ LPS can independently be recruited to endosomes. [ABSTRACT FROM AUTHOR]

Anti-microbial peptides might influence the pathogenesis and course of inflammatory bowel disease ( IBD). We sought to clarify the role of the anti-microbial glycoprotein lipocalin 2 ( LCN2) in the colon by determining its localization and regulation in IBD. Following a microarray gene expression study of colonic biopsies from a large IBD population ( n = 133), LCN2 was localized using immunohistochemistry and in-situ hybridization. Moreover, we examined the regulation of LCN2 in HT-29 cells with a panel of pattern recognition receptors ( PRRs) and sought evidence by immunohistochemistry that the most relevant PRR, the Toll-like receptor ( TLR)-3, was indeed expressed in colonic epithelium in IBD. LCN2 was among the 10 most up-regulated genes in both active ulcerative colitis ( UCa) and active Crohn's disease ( CDa) versus healthy controls. LCN2 protein was found in both epithelial cells and infiltrating neutrophils, while mRNA synthesis was located solely to epithelial cells, indicating that de-novo synthesis and thus regulation of LCN2 as measured in the gene expression analysis takes place in the mucosal epithelial cells. LCN2 is a putative biomarker in faeces for intestinal inflammation, different from calprotectin due to its epithelial site of synthesis. LCN2 release from the colonic epithelial cell line HT-29 was enhanced by both interleukin ( IL)-1β and the TLR-3 ligand poly( I: C), and TLR-3 was shown to be expressed constitutively in colonic epithelial cells and markedly increased during inflammation. [ABSTRACT FROM AUTHOR]

Presents several abstracts on immunology. "Antibody Response to Pneumococcal Polysaccharide Vaccine in T Lymphocyte Deficient Mice," by Ingeborg S. Aaberge, Martinus Lovik and Else-Carin Groeng; "Collagen Receptor on Lymphocytes," by Ignacio Arencibia and Karl-Gösta Sundqvist; "SLE-Like Syndrome Induced by Allogenic Lymphocytes Injection," by Lin Chen, Xia Ya-Li, Na Bao-Li; "Characterization of Two Classes of Fe Receptor on Mink Phagocytes by Fluorescence-Activated Flow Cytometry," by Jesper Christensen, Graham Leslie and Anne Frahm; Others.

In the present study the authors elucidated the involvement of the two TNF receptors (TNFR) in discriminating TNF and lymphotoxin-α (LT-α) effects in human SW480-βGal and KYM-1 cell lines. A non-competitive p55 TNFR monoclonal antibody (MoAb) 44E strongly enhanced LT-α-mediated gene regulation and cytotoxicity up to the level of the responses caused by TNF. TNF-induced biological responses were only weakly influenced by 44E. 44E did not affect both binding and the rate of dissociation of the cytokines. The combination of the two p55 TNFR MoAb 44E and Htr5 elicited strong TNF responses, while none of them were agonistic alone. When the p75 TNFR was blocked with Utr1, LT-α was still less potent than TNF in mediating CMV promoter activation and cytotoxicity. However, the addition of 44E in the presence of Utr1 merged the LT-α dose-response curves with those obtained with TNF plus Utr1. Using antagonistic TNFR MoAb, the authors further showed that TNF functions through both TNFR types while LT-α mediates its effects largely via the p55 TNFR. These data suggest that LT-α is less potent than TNF due to its lower ability to properly trigger the p55 TNFR and because of its lack of signalling through the p75 TNFR. [ABSTRACT FROM AUTHOR]

Simple Summary: Lung cancer is the leading cause of cancer-related death worldwide, accounting for nearly one-fifth of all cancer-related deaths. Immunotherapy with immune checkpoint inhibitors has become one of the most promising approaches in the treatment of advanced lung cancer, although beneficial responses are seen only in a proportion of patients. To improve immunotherapy treatment responses in lung cancer, we need to identify which immunosuppression mechanisms are activated in the tumor microenvironment. In this study, we investigated gene expression profiles in intra-tumoral immune cells in lung cancer, focusing on tumor-associated macrophages, and interactions with CD4+ and CD8+ T cells. Our data highlight two newly described immunosuppressive pathways, which may represent novel innate immune checkpoints dampening the anti-tumor T cell immune response in lung cancer. Our results substantiate the importance of tumor-associated macrophages as a mediator of immunosuppression and a promising target for immunotherapy. Non-small cell lung carcinoma (NSCLC) is one of the most commonly diagnosed cancers and a leading cause of cancer-related deaths. Immunotherapy with immune checkpoint inhibitors shows beneficial responses, but only in a proportion of patients. To improve immunotherapy in NSCLC, we need to map the immune checkpoints that contribute immunosuppression in NSCLC-associated immune cells and to identify novel pathways that regulate immunosuppression. Here, we investigated the gene expression profiles of intra-tumoral immune cells isolated from NSCLC patients and compared them to the expression profiles of their counterparts in adjacent healthy tissue. Transcriptome analysis was performed on macrophages, CD4+ and CD8+ T cells. The data was subjected to Gene Ontology (GO) term enrichment and weighted correlation network analysis in order to identify mediators of immunosuppression in the tumor microenvironment in NSCLC. Immune cells from NSCLC revealed a consistent differential expression of genes involved in interactions between myeloid cells and lymphocytes. We further identified several immunosuppressive molecules and pathways that may be activated in tumor-associated macrophages in NSCLC. Importantly, we report novel data on immune cell expression of the newly described CD200/CD200R1 pathway, and the leukocyte immunoglobulin-like receptors (LILRs), which may represent novel innate immune checkpoints, dampening the anti-tumor T cell immune response in NSCLC. Our study substantiates the importance of tumor-associated macrophages as a mediator of immunosuppression and a promising target for immunotherapy. [ABSTRACT FROM AUTHOR]

The chemokine C-C motif ligand 20 (CCL20) is increased in the colonic mucosa during active inflammatory bowel disease (IBD) and can be found both in the epithelium and immune cells in the lamina propria. The present study investigated CCL20 and C-C motif Chemokine Receptor 6 (CCR6) in peripheral blood mononuclear cells (PBMCs) (n = 40) from IBD patients and healthy controls, to identify inductors of CCL20 release encountered in a local proinflammatory environment. CCL20 release from PBMCs was increased when activating TLR2/1 or NOD2, suggesting that CCL20 is part of a first line response to danger-associated molecular patterns also in immune cells. Overall, ulcerative colitis (UC) had a significantly stronger CCL20 release than Crohn's disease (CD) (+242%, p < 0.01), indicating that the CCL20-CCR6 axis may be more involved in UC. The CCL20 receptor CCR6 is essential for the chemotactic function of CCL20. UC with active inflammation had significantly decreased CCR6 expression and a reduction in CCR6+ cells in circulation, indicating chemoattraction of CCR6+ cells from circulation towards peripheral tissues. We further examined CCL20 induced release of cytokines from PBMCs. Stimulation with CCL20 combined with TNF increased IL-1β release from PBMCs. By attracting additional immune cells, as well as inducing proinflammatory IL-1β release from immune cells, CCL20 may protract the inflammatory response in ulcerative colitis. [ABSTRACT FROM AUTHOR]