ETV3 and ETV6 enable monocyte differentiation into dendritic cells by repressing macrophage fate commitment.

In inflamed tissues, monocytes differentiate into macrophages (mo-Macs) or dendritic cells (mo-DCs). In chronic nonresolving inflammation, mo-DCs are major drivers of pathogenic events. Manipulating monocyte differentiation would therefore be an attractive therapeutic strategy. However, how the balance of mo-DC versus mo-Mac fate commitment is regulated is not clear. In the present study, we show that the transcriptional repressors ETV3 and ETV6 control human monocyte differentiation into mo-DCs. ETV3 and ETV6 inhibit interferon (IFN)-stimulated genes; however, their action on monocyte differentiation is independent of IFN signaling. Instead, we find that ETV3 and ETV6 directly repress mo-Mac development by controlling MAFB expression. Mice deficient for Etv6 in monocytes have spontaneous expression of IFN-stimulated genes, confirming that Etv6 regulates IFN responses in vivo. Furthermore, these mice have impaired mo-DC differentiation during inflammation and reduced pathology in an experimental autoimmune encephalomyelitis model. These findings provide information about the molecular control of monocyte fate decision and identify ETV6 as a therapeutic target to redirect monocyte differentiation in inflammatory disorders.

The More, the Merrier: DC3s Join the Human Dendritic Cell Family.

Recent studies have reported additional subpopulations of human dendritic cells (DCs), but whether they are distinct subsets has been unclear. In this issue of Immunity, Cytlak et al. and Bourdely et al. show that DC3s possess a specific precursor and represent a separate DC lineage.

Monocyte differentiation within tissues: a renewed outlook.

When recruited to mammalian tissues, monocytes differentiate into macrophages or dendritic cells (DCs). In the past few years, the existence of monocyte-derived DCs (moDCs) was questioned by the discovery of new DC populations with overlapping phenotypes. Here, we critically review the evidence for monocyte differentiation into DCs in tissues and highlight their specific functions. Recent studies have shown that monocyte-derived macrophages (moMacs) with distinct life cycles coexist in tissues, both at steady state and upon inflammation. Integrating studies in mice and humans, we highlight specific features of moMacs during inflammation and tissue repair. We also discuss the notion of monocyte differentiation occurring via a binary fate decision. Deciphering monocyte-derived cell properties is essential for understanding their role in nonresolving inflammation and how they might be targeted for therapies.

Monocytes differentiate along two alternative pathways during sterile inflammation.

During inflammation, monocytes differentiate within tissues into macrophages (mo-Mac) or dendritic cells (mo-DC). Whether these two populations derive from alternative differentiation pathways or represent different stages along a continuum remains unclear. Here, we address this question using temporal single-cell RNA sequencing in an in vitro model, allowing the simultaneous differentiation of human mo-Mac and mo-DC. We find divergent differentiation paths, with a fate decision occurring within the first 24 h and confirm this result in vivo using a mouse model of sterile peritonitis. Using a computational approach, we identify candidate transcription factors potentially involved in monocyte fate commitment. We demonstrate that IRF1 is necessary for mo-Mac differentiation, independently of its role in regulating transcription of interferon-stimulated genes. In addition, we describe the transcription factors ZNF366 and MAFF as regulators of mo-DC development. Our results indicate that mo-Macs and mo-DCs represent two alternative cell fates requiring distinct transcription factors for their differentiation.

Expression of Hv1 proton channels in myeloid-derived suppressor cells (MDSC) and its potential role in T cell regulation.

Myeloid-derived suppressor cells (MDSC) are a heterogeneous cell population with high immunosuppressive activity that proliferates in infections, inflammation, and tumor microenvironments. In tumors, MDSC exert immunosuppression mainly by producing reactive oxygen species (ROS), a process triggered by the NADPH oxidase 2 (NOX2) activity. NOX2 is functionally coupled with the Hv1 proton channel in certain immune cells to support sustained free-radical production. However, a functional expression of the Hv1 channel in MDSC has not yet been reported. Here, we demonstrate that mouse MDSC express functional Hv1 proton channel by immunofluorescence microscopy, flow cytometry, and Western blot, besides performing a biophysical characterization of its macroscopic currents via patch-clamp technique. Our results show that the immunosuppression by MDSC is conditional to their ability to decrease the proton concentration elevated by the NOX2 activity, rendering Hv1 a potential drug target for cancer treatment.

Decoding the Heterogeneity of Human Dendritic Cell Subsets.

Dendritic cells (DCs) have been classified into distinct subsets based on phenotype and ontogeny. In the past few years, high throughput single-cell approaches have revealed further heterogeneity of human DCs, in particular at the transcriptomic level. Herein examined, are recent studies describing new human DC populations based on single-cell RNA-seq analysis, and a unified view of these emerging DC populations is presented. Also assessed are the features that define bona fide DC lineages, as opposed to cell states of the same lineage. Finally, where these newly described DC populations fit in the ontogeny-based classification of human DCs is examined.

C-type lectin receptors MR and DC-SIGN are involved in recognition of hemocyanins, shaping their immunostimulatory effects on human dendritic cells.

Hemocyanins are used as immunomodulators in clinical applications because they induce a strong Th1-biased cell-mediated immunity, which has beneficial effects. They are multiligand glycosylated molecules with abundant and complex mannose-rich structures. It remains unclear whether these structures influence hemocyanin-induced immunostimulatory processes in human APCs. We have previously shown that hemocyanin glycans from Concholepas concholepas (CCH), Fissurella latimarginata (FLH), and Megathura crenulata (KLH), participate in their immune recognition and immunogenicity in mice, interacting with murine C-type lectin receptors (CLRs). Here, we studied the interactions of these hemocyanins with two major mannose-binding CLRs on monocyte-derived human DCs: MR (mannose receptor) and DC-SIGN (DC-specific ICAM-3-grabbing nonintegrin). Diverse analyses showed that hemocyanins are internalized by a mannose-sensitive mechanism. This process was calcium dependent. Moreover, hemocyanins colocalized with MR and DC-SIGN, and were partly internalized through clathrin-mediated endocytosis. The hemocyanin-mediated proinflammatory cytokine response was impaired when using deglycosylated FLH and KLH compared to CCH. We further showed that hemocyanins bind to human MR and DC-SIGN in a carbohydrate-dependent manner with affinity constants in the physiological concentration range. Overall, we showed that these three clinically valuable hemocyanins interact with human mannose-sensitive CLRs, initiating an immune response and promoting a Th1 cell-driving potential.

N-Glycosylation of mollusk hemocyanins contributes to their structural stability and immunomodulatory properties in mammals.

Hemocyanins are widely used as carriers, adjuvants, and nonspecific immunostimulants in cancer because they promote Th1 immunity in mammals. Hemocyanins also interact with glycan-recognizing innate immune receptors on antigen-presenting cells, such as the C-type lectin immune receptors mannose receptor (MR), macrophage galactose lectin (MGL), and the Toll-like receptors (TLRs), stimulating proinflammatory cytokine secretion. However, the role of N-linked oligosaccharides on the structural and immunological properties of hemocyanin is unclear. Mollusk hemocyanins, such as Concholepas concholepas (CCH), Fissurella latimarginata (FLH), and Megathura crenulata (KLH), are oligomeric glycoproteins with complex dodecameric quaternary structures and heterogeneous glycosylation patterns, primarily consisting of mannose-rich N-glycans. Here, we report that enzyme-catalyzed N-deglycosylation of CCH, FLH, and KLH disrupts their quaternary structure and impairs their immunogenic effects. Biochemical analyses revealed that the deglycosylation does not change hemocyanin secondary structure but alters their refolding mechanism and dodecameric structure. Immunochemical analyses indicated decreased binding of N-deglycosylated hemocyanins to the MR and MGL receptors and TLR4 and reduced endocytosis concomitant with an impaired production of tumor necrosis factor α, and interleukins 6 and 12 (IL-6 and IL-12p40, respectively) in macrophages. Evaluating the function of N-deglycosylated hemocyanins in the humoral immune response and their nonspecific antitumor effects in the B16F10 melanoma model, we found that compared with native hemocyanins N-deglycosylated hemocyanins elicited reduced antibody titers, as well as partially diminished antitumor effects and altered carrier activities. In conclusion, the glycan content of hemocyanins is, among other structural characteristics, critically required for their immunological activities and should be considered in biomedical applications.

Hemocyanins Stimulate Innate Immunity by Inducing Different Temporal Patterns of Proinflammatory Cytokine Expression in Macrophages.

Hemocyanins induce a potent Th1-dominant immune response with beneficial clinical outcomes when used as a carrier/adjuvant in vaccines and nonspecific immunostimulant in cancer. However, the mechanisms by which hemocyanins trigger innate immune responses, leading to beneficial adaptive immune responses, are unknown. This response is triggered by a proinflammatory signal from various components, of which macrophages are an essential part. To understand how these proteins influence macrophage response, we investigated the effects of mollusks hemocyanins with varying structural and immunological properties, including hemocyanins from Concholepas concholepas, Fissurella latimarginata, and Megathura crenulata (keyhole limpet hemocyanin), on cultures of peritoneal macrophages. Hemocyanins were phagocytosed and slowly processed. Analysis of this process showed differential gene expression along with protein levels of proinflammatory markers, including IL-1ß, IL-6, IL-12p40, and TNF-α. An extended expression analysis of 84 cytokines during a 24-h period showed a robust proinflammatory response for F. latimarginata hemocyanin in comparison with keyhole limpet hemocyanin and C. concholepas hemocyanin, which was characterized by an increase in the transcript levels of M1 cytokines involved in leukocyte recruitment. These cytokine genes included chemokines (Cxcl1, Cxcl3, Cxcl5, Ccl2, and Ccl3), ILs (Il1b and Ifng), growth factors (Csf2 and Csf3), and TNF family members (Cd40lg). The protein levels of certain cytokines were increased. However, every hemocyanin maintains downregulated key M2 cytokine genes, including Il4 and Il5 Collectively, our data demonstrate that hemocyanins are able to trigger the release of proinflammatory factors with different patterns of cytokine expression, suggesting differential signaling pathways and transcriptional network mechanisms that lead to the activation of M1-polarized macrophages.

Culture System Allowing the Simultaneous Differentiation of Human Monocytes into Dendritic Cells and Macrophages Using M-CSF, IL-4, and TNF-α.

Monocytes circulate in the blood and infiltrate tissues where they differentiate into either macrophages or dendritic cells, in particular during inflammation. In vivo, monocytes are exposed to various signals that modulate their commitment toward macrophage or dendritic cell fate. Classical culture systems for human monocyte differentiation yield either macrophages or dendritic cells, but not both populations in the same culture. In addition, monocyte-derived dendritic cells obtained with such methods do not closely mimic dendritic cells that are present in clinical samples. Here, we describe a protocol to simultaneously differentiate human monocytes into macrophages and dendritic cells that resemble their in vivo counterparts from inflammatory fluids.

Functional specialization of short-lived and long-lived macrophage subsets in human tonsils.

Macrophages play a central role in tissue homeostasis and host defense. However, the properties of human macrophages in non-diseased tissues remain poorly understood. Here, we characterized human tonsil macrophages and identified three subsets with distinct phenotype, transcriptome, life cycle, and function. CD36hi macrophages were related to monocytes, while CD36lo macrophages showed features of embryonic origin and CD36int macrophages had a mixed profile. scRNA-seq on non-human primate tonsils showed that monocyte recruitment did not pre-exist an immune challenge. Functionally, CD36hi macrophages were specialized for stimulating T follicular helper cells, by producing Activin A. Combining reconstruction of ligand-receptor interactions and functional assays, we identified stromal cell-derived TNF-α as an inducer of Activin A secretion. However, only CD36hi macrophages were primed for Activin A expression, via the activity of IRF1. Our results provide insight into the heterogeneity of human lymphoid organ macrophages and show that tonsil CD36hi macrophage specialization is the result of both intrinsic features and interaction with stromal cells.

Ubiquitin-like protein 3 (UBL3) is required for MARCH ubiquitination of major histocompatibility complex class II and CD86.

The MARCH E3 ubiquitin (Ub) ligase MARCH1 regulates trafficking of major histocompatibility complex class II (MHC II) and CD86, molecules of critical importance to immunity. Here we show, using a genome-wide CRISPR knockout screen, that ubiquitin-like protein 3 (UBL3) is a necessary component of ubiquitination-mediated trafficking of these molecules in mice and in humans. Ubl3-deficient mice have elevated MHC II and CD86 expression on the surface of professional and atypical antigen presenting cells. UBL3 also regulates MHC II and CD86 in human dendritic cells (DCs) and macrophages. UBL3 impacts ubiquitination of MARCH1 substrates, a mechanism that requires UBL3 plasma membrane anchoring via prenylation. Loss of UBL3 alters adaptive immunity with impaired development of thymic regulatory T cells, loss of conventional type 1 DCs, increased number of trogocytic marginal zone B cells, and defective in vivo MHC II and MHC I antigen presentation. In summary, we identify UBL3 as a conserved, critical factor in MARCH1-mediated ubiquitination with important roles in immune responses.

Recent advances towards deciphering human dendritic cell development.

Dendritic cell (DC) populations are the orchestrators of immune responses and arise from hematopoietic progenitors. Studies to unravel DC ontogeny have been conducted mainly in mice due to historical and practical reasons. However, understanding DC development in humans is a prerequisite for manipulating this process for therapeutic design. Here, we review the advantages and limitations of methods used to study human DC development in vitro and in vivo. In particular, we examine the in vitro culture systems that support the differentiation of all or some DC subpopulations. We also review recent discoveries regarding human DC precursors and factors that regulate their differentiation.

Immunotherapeutic Potential of Mollusk Hemocyanins in Combination with Human Vaccine Adjuvants in Murine Models of Oral Cancer.

Mollusk hemocyanins have been used for decades in immunological and clinical applications as natural, nontoxic, nonpathogenic, and nonspecific immunostimulants for the treatment of superficial bladder cancer, as carriers/adjuvants of tumor-associated antigens in cancer vaccine development and as adjuvants to dendritic cell-based immunotherapy, because these glycoproteins induce a bias towards Th1 immunity. Here, we analyzed the preclinical therapeutic potential of the traditional keyhole limpet hemocyanin (KLH) and two new hemocyanins from Concholepas concholepas (CCH) and Fissurella latimarginata (FLH) in mouse models of oral squamous cell carcinoma. Due to the aggressiveness and deadly malignant potential of this cancer, the hemocyanins were applied in combination with adjuvants, such as alum, AddaVax, and QS-21, which have been shown to be safe and effective in human vaccines, to potentiate their antitumor activity. The immunogenic performance of the hemocyanins in combination with the adjuvants was compared, and the best formulation was evaluated for its antitumor effects in two murine models of oral cancer: MOC7 cells implanted in the flank (heterotopic) and bioluminescent AT-84 E7 Luc cells implanted in the floor of the mouth (orthotopic). The results demonstrated that the hemocyanins in combination with QS-21 showed the greatest immunogenicity, as reflected by a robust, specific humoral response predominantly characterized by IgG2a antibodies and a sustained cellular response manifesting as a delayed hypersensitivity reaction. The KLH- and FLH-QS-21 formulations showed reduced tumor development and greater overall survival. Hemocyanins, as opposed to QS-21, had no cytotoxic effect on either oral cancer cell line cultured in vitro, supporting the idea that the antitumor effects of hemocyanins are associated with their modulation of the immune response. Therefore, hemocyanin utilization would allow a lower QS-21 dosage to achieve therapeutic results. Overall, our study opens a new door to further investigation of the use of hemocyanins plus adjuvants for the development of immunotherapies against oral carcinoma.

TLR4, but Neither Dectin-1 nor Dectin-2, Participates in the Mollusk Hemocyanin-Induced Proinflammatory Effects in Antigen-Presenting Cells From Mammals.

Mollusk hemocyanins have biomedical uses as carriers/adjuvants and nonspecific immunostimulants with beneficial clinical outcomes by triggering the production of proinflammatory cytokines in antigen-presenting cells (APCs) and driving immune responses toward type 1 T helper (Th1) polarization. Significant structural features of hemocyanins as a model antigen are their glycosylation patterns. Indeed, hemocyanins have a multivalent nature as highly mannosylated antigens. We have previously shown that hemocyanins are internalized by APCs through receptor-mediated endocytosis with proteins that contain C-type lectin domains, such as mannose receptor (MR). However, the contribution of other innate immune receptors to the proinflammatory signaling pathway triggered by hemocyanins is unknown. Thus, we studied the roles of Dectin-1, Dectin-2, and Toll-like receptor 4 (TLR4) in the hemocyanin activation of murine APCs, both in dendritic cells (DCs) and macrophages, using hemocyanins from Megathura crenulata (KLH), Concholepas concholepas (CCH) and Fissurella latimarginata (FLH). The results showed that these hemocyanins bound to chimeric Dectin-1 and Dectin-2 receptors in vitro; which significantly decreased when the glycoproteins were deglycosylated. However, hemocyanin-induced proinflammatory effects in APCs from Dectin-1 knock-out (KO) and Dectin-2 KO mice were independent of both receptors. Moreover, when wild-type APCs were cultured in the presence of hemocyanins, phosphorylation of Syk kinase was not detected. We further showed that KLH and FLH induced ERK1/2 phosphorylation, a key event involved in the TLR signaling pathway. We confirmed a glycan-dependent binding of hemocyanins to chimeric TLR4 in vitro. Moreover, DCs from mice deficient for MyD88-adapter-like (Mal), a downstream adapter molecule of TLR4, were partially activated by FLH, suggesting a role of the TLR pathway in hemocyanin recognition to activate APCs. The participation of TLR4 was confirmed through a decrease in IL-12p40 and IL-6 secretion induced by FLH when a TLR4 blocking antibody was used; a reduction was also observed in DCs from C3H/HeJ mice, a mouse strain with a nonfunctional mutation for this receptor. Moreover, IL-6 secretion induced by FLH was abolished in macrophages deficient for TLR4. Our data showed the involvement of TLR4 in the hemocyanin-mediated proinflammatory response in APCs, which could cooperate with MR in innate immune recognition of these glycoproteins.

Prácticas y experiencias de violencia obstétrica y gineco-obstétrica como violencia de género en Chile

Objetivo Describir la experiencia de mujeres que han sufrido violencia obstétrica y gineco-obstétrica en Chile. Métodos Estudio cualitativo en el que participaron 34 mujeres entre 18 y 60 años que experimentaron algún tipo de violencia (obstétrica o gineco-obstétrica) en Chile. Se emplearon entrevistas en profundidad. El análisis se realizó siguiendo los procedimientos del análisis temático. Resultados La violencia obstétrica es un proceso que marca las relaciones de poder médico en distintos niveles: desde lo procedimental hasta el trato que se tiene con las propias mujeres. Esto puede provocar en ellas diversos estados emocionales y psicológicos catalogados como negativos y traumáticos. Discusión La violencia obstétrica y gineco-obstétrica es una dimensión de la violencia de género, pues representa cómo la mujer ha sido instrumentalizada en los procedimientos y atenciones médicas. Se evidencia la necesidad de profundizar en las relaciones de poder médico y en las prácticas gineco-obstétricas para la generación de políticas que protejan los derechos reproductivos y sexuales de las mujeres.

Objective To describe the experience of women who have suffered obstetric and gynecological violence in Chile. Methods Using a qualitative methodology, this study involved the participation of 34 women aged between 18 and 60 years who have experienced some form of violence (obstetric or gynecological violence) in Chile. In-depth interviews were conducted, and subsequently, the analysis was carried out following the procedures of thematic analysis. Results Obstetric violence is a process that marks medical power relations at different levels, from the procedural to the treatment of the women themselves, provoking diverse emotional and psychological states that are associated as negative and traumatic. Discussion Obstetric and gynecological violence is a dimension of gender-based violence as far as it represents how women have been instrumentalized in medical procedures and care. It is evident that there is a need to go deeper into medical power relations and gynecological-obstetric practices in order to generate policies that protect women's reproductive and sexual rights.