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Respiratory disorders caused by allergy have been associated to bronchiolar inflammation leading to life-threatening airway narrowing. However, whether airway allergy causes alveolar dysfunction contributing to the pathology of allergic asthma remains unaddressed. To explore whether airway allergy causes alveolar dysfunction that might contribute to the pathology of allergic asthma, alveolar structural and functional alterations were analyzed during house dust mite (HDM)-induced airway allergy in mice, by flow cytometry, light and electron microscopy, monocyte transfer experiments, assessment of intra-alveolarly-located cells, analysis of alveolar macrophage regeneration in Cx3cr1 cre : R26-yfp chimeras, analysis of surfactant-associated proteins, and study of lung surfactant biophysical properties by captive bubble surfactometry. Our results demonstrate that HDM-induced airway allergic reactions caused severe alveolar dysfunction, leading to alveolar macrophage death, pneumocyte hypertrophy and surfactant dysfunction. SP-B/C proteins were reduced in allergic lung surfactant, that displayed a reduced efficiency to form surface-active films, increasing the risk of atelectasis. Original alveolar macrophages were replaced by monocyte-derived alveolar macrophages, that persisted at least two months after the resolution of allergy. Monocyte to alveolar macrophage transition occurred through an intermediate stage of pre-alveolar macrophage and was paralleled with translocation into the alveolar space, Siglec-F upregulation, and downregulation of CX3CR1. These data support that the severe respiratory disorders caused by asthmatic reactions not only result from bronchiolar inflammation, but additionally from alveolar dysfunction compromising an efficient gas exchange., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Feo-Lucas, Godio, Minguito de la Escalera, Alvarez-Ladrón, Villarrubia, Vega-Pérez, González-Cintado, Domínguez-Andrés, García-Fojeda, Montero-Fernández, Casals, Autilio, Pérez-Gil, Crainiciuc, Hidalgo, López-Bravo and Ardavín.)

Large peritoneal macrophages (LPMs) are long-lived, tissue-resident macrophages, formed during embryonic life, developmentally and functionally confined to the peritoneal cavity. LPMs provide the first line of defense against life-threatening pathologies of the peritoneal cavity, such as abdominal sepsis, peritoneal metastatic tumor growth, or peritoneal injuries caused by trauma, or abdominal surgery. Apart from their primary phagocytic function, reminiscent of primitive defense mechanisms sustained by coelomocytes in the coelomic cavity of invertebrates, LPMs fulfill an essential homeostatic function by achieving an efficient clearance of apoptotic, that is crucial for the maintenance of self-tolerance. Research performed over the last few years, in mice, has unveiled the mechanisms by which LPMs fulfill a crucial role in repairing peritoneal injuries and controlling microbial and parasitic infections, reflecting that the GATA6-driven LPM transcriptional program can be modulated by extracellular signals associated with pathological conditions. In contrast, recent experimental evidence supports that peritoneal tumors can subvert LPM metabolism and function, leading to the acquisition of a tumor-promoting potential. The remarkable functional plasticity of LPMs can be nevertheless exploited to revert tumor-induced LPM protumor potential, providing the basis for the development of novel immunotherapeutic approaches against peritoneal tumor metastasis based on macrophage reprogramming., (© 2023 The Authors. Advanced Science published by Wiley-VCH GmbH.)

Resident peritoneal macrophages (resMØs) are crucial for repairing peritoneal injuries and controlling infections by forming mesothelium-bound resMØ-aggregates in the peritoneal wall and omentum. Here we present a protocol to analyze these structures in mouse models of peritoneal inflammation. We describe the dissection, fixation, immunofluorescent staining, and mounting of whole peritoneal wall and omentum samples and subsequent confocal microscopy imaging of resMØ-aggregates. We also detail the steps to isolate resMØ-aggregates for additional studies, including flow cytometry and electron-microscopy-based analysis. For complete details on the use and execution of this protocol, please refer to Vega-Pérez et al. (2021). 1 ., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

MV130 is an inactivated polybacterial mucosal vaccine that confers protection to patients against recurrent respiratory infections, including those of viral etiology. However, its mechanism of action remains poorly understood. Here, we find that intranasal prophylaxis with MV130 modulates the lung immune landscape and provides long-term heterologous protection against viral respiratory infections in mice. Intranasal administration of MV130 provides protection against systemic candidiasis in wild-type and Rag1-deficient mice lacking functional lymphocytes, indicative of innate immune-mediated protection. Moreover, pharmacological inhibition of trained immunity with metformin abrogates the protection conferred by MV130 against influenza A virus respiratory infection. MV130 induces reprogramming of both mouse bone marrow progenitor cells and in vitro human monocytes, promoting an enhanced cytokine production that relies on a metabolic shift. Our results unveil that the mucosal administration of a fully inactivated bacterial vaccine provides protection against viral infections by a mechanism associated with the induction of trained immunity., Competing Interests: Declaration of interests J.L.S., M.C., L.C., S. M.-C., and P.S.-L. were employees of Inmunotek S.L. at the time of the work. The D.S. lab receives funds from a collaboration agreement between CNIC and Inmunotek. The other authors declare no competing interests., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Peritoneal immune cells reside unanchored within the peritoneal fluid in homeostasis. Here, we examined the mechanisms that control bacterial infection in the peritoneum using a mouse model of abdominal sepsis following intraperitoneal Escherichia coli infection. Whole-mount immunofluorescence and confocal microscopy of the peritoneal wall and omentum revealed that large peritoneal macrophages (LPMs) rapidly cleared bacteria and adhered to the mesothelium, forming multilayered cellular aggregates composed by sequentially recruited LPMs, B1 cells, neutrophils, and monocyte-derived cells (moCs). The formation of resident macrophage aggregates (resMφ-aggregates) required LPMs and thrombin-dependent fibrin polymerization. E. coli infection triggered LPM pyroptosis and release of inflammatory mediators. Resolution of these potentially inflammatory aggregates required LPM-mediated recruitment of moCs, which were essential for fibrinolysis-mediated resMφ-aggregate disaggregation and the prevention of peritoneal overt inflammation. Thus, resMφ-aggregates provide a physical scaffold that enables the efficient control of peritoneal infection, with implications for antimicrobial immunity in other body cavities, such as the pleural cavity or brain ventricles., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

IVIg is an approved therapy for immunodeficiency and for several autoimmune and inflammatory diseases. However, the molecular basis for the IVIg anti-inflammatory activity remains to be fully explained and cannot be extrapolated from studies on animal models of disease. We now report that IVIg impairs the generation of human monocyte-derived anti-inflammatory macrophages by inducing JNK activation and activin A production and limits proinflammatory macrophage differentiation by inhibiting GM-CSF-driven STAT5 activation. In vivo, IVIg provokes a rapid increase in peripheral blood activin A, CCL2, and IL-6 levels, an effect that can be recapitulated in vitro on human monocytes. On differentiating monocytes, IVIg promotes the acquisition of altered transcriptional and cytokine profiles, reduces TLR expression and signaling, and upregulates negative regulators of TLR-initiated intracellular signaling. In line with these effects, in vivo IVIg infusion induces a state tolerant toward subsequent stimuli that results in reduced inflammatory cytokine production after LPS challenge in human peripheral blood and significant protection from LPS-induced death in mice. Therefore, IVIg conditions human macrophages toward the acquisition of a state of cross-tolerance against inflammatory stimuli, an effect that correlates with the net anti-inflammatory action of IVIg in vivo., (Copyright © 2018 by The American Association of Immunologists, Inc.)

Candida albicans is a frequent aetiologic agent of sepsis associated with high mortality in immunocompromised patients. Developing new antifungal therapies is a medical need due to the low efficiency and resistance to current antifungal drugs. Here, we show that p38γ and p38δ regulate the innate immune response to C. albicans We describe a new TAK1-TPL2-MKK1-ERK1/2 pathway in macrophages, which is activated by Dectin-1 engagement and positively regulated by p38γ/p38δ. In mice, p38γ/p38δ deficiency protects against C. albicans infection by increasing ROS and iNOS production and thus the antifungal capacity of neutrophils and macrophages, and by decreasing the hyper-inflammation that leads to severe host damage. Leucocyte recruitment to infected kidneys and production of inflammatory mediators are decreased in p38γ/δ-null mice, reducing septic shock. p38γ/p38δ in myeloid cells are critical for this effect. Moreover, pharmacological inhibition of p38γ/p38δ in mice reduces fungal burden, revealing that these p38MAPKs may be therapeutic targets for treating C. albicans infection in humans., (© 2018 The Authors. Published under the terms of the CC BY 4.0 license.)

GM-CSF promotes the functional maturation of lung alveolar macrophages (A-MØ), whose differentiation is dependent on the peroxisome proliferator-activated receptor gamma (PPARγ) transcription factor. In fact, blockade of GM-CSF-initiated signaling or deletion of the PPARγ-encoding gene PPARG leads to functionally defective A-MØ and the onset of pulmonary alveolar proteinosis. In vitro , macrophages generated in the presence of GM-CSF display potent proinflammatory, immunogenic and tumor growth-limiting activities. Since GM-CSF upregulates PPARγ expression, we hypothesized that PPARγ might contribute to the gene signature and functional profile of human GM-CSF-conditioned macrophages. To verify this hypothesis, PPARγ expression and activity was assessed in human monocyte-derived macrophages generated in the presence of GM-CSF [proinflammatory GM-CSF-conditioned human monocyte-derived macrophages (GM-MØ)] or M-CSF (anti-inflammatory M-MØ), as well as in ex vivo isolated human A-MØ. GM-MØ showed higher PPARγ expression than M-MØ, and the expression of PPARγ in GM-MØ was found to largely depend on activin A. Ligand-induced activation of PPARγ also resulted in distinct transcriptional and functional outcomes in GM-MØ and M-MØ. Moreover, and in the absence of exogenous activating ligands, PPARγ knockdown significantly altered the GM-MØ transcriptome, causing a global upregulation of proinflammatory genes and significantly modulating the expression of genes involved in cell proliferation and migration. Similar effects were observed in ex vivo isolated human A-MØ, where PPARγ silencing led to enhanced expression of genes coding for growth factors and chemokines and downregulation of cell surface pathogen receptors. Therefore, PPARγ shapes the transcriptome of GM-CSF-dependent human macrophages ( in vitro derived GM-MØ and ex vivo isolated A-MØ) in the absence of exogenous activating ligands, and its expression is primarily regulated by activin A. These results suggest that activin A, through enhancement of PPARγ expression, help macrophages to switch from a proinflammatory to an anti-inflammatory polarization state, thus contributing to limit tissue damage and restore homeostasis.

Monocytes are innate immune cells that play a pivotal role in antifungal immunity, but little is known regarding the cellular metabolic events that regulate their function during infection. Using complementary transcriptomic and immunological studies in human primary monocytes, we show that activation of monocytes by Candida albicans yeast and hyphae was accompanied by metabolic rewiring induced through C-type lectin-signaling pathways. We describe that the innate immune responses against Candida yeast are energy-demanding processes that lead to the mobilization of intracellular metabolite pools and require induction of glucose metabolism, oxidative phosphorylation and glutaminolysis, while responses to hyphae primarily rely on glycolysis. Experimental models of systemic candidiasis models validated a central role for glucose metabolism in anti-Candida immunity, as the impairment of glycolysis led to increased susceptibility in mice. Collectively, these data highlight the importance of understanding the complex network of metabolic responses triggered during infections, and unveil new potential targets for therapeutic approaches against fungal diseases.

Neutrophils play a crucial role in defense against systemic candidiasis, a disease associated with a high mortality rate in patients receiving immunosuppressive therapy, although the early immune mechanisms that boost the candidacidal activity of neutrophils remain to be defined in depth. Here, we used a murine model of systemic candidiasis to explore the role of inflammatory Ly6C high monocytes in NK cell-mediated neutrophil activation during the innate immune response against C. albicans. We found that efficient anti-Candida immunity required a collaborative response between the spleen and kidney, which relied on type I interferon-dependent IL-15 production by spleen inflammatory Ly6C high monocytes to drive efficient activation and GM-CSF release by spleen NK cells; this in turn was necessary to boost the Candida killing potential of kidney neutrophils. Our findings unveil a role for IL-15 as a critical mediator in defense against systemic candidiasis and hold promise for the design of IL-15-based antifungal immunotherapies., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Modern subunit vaccines require the development of new adjuvant strategies. Recently, we showed that CpG-ODN formulated with a liquid crystal nanostructure formed by self-assembly of 6-O-ascorbyl palmitate (Coa-ASC16) is an attractive system for promoting an antigen-specific immune response to weak antigens. Here, we showed that after subcutaneous injection of mice with near-infrared fluorescent dye-labeled OVA antigen formulated with Coa-ASC16, the dye-OVA was retained at the injection site for a longer period than when soluble dye-OVA was administered. Coa-ASC16 alone elicited a local inflammation, but how this material triggers this response has not been described yet. Although it is known that some materials used as a platform are not immunologically inert, very few studies have directly focused on this topic. In this study, we explored the underlying mechanisms concerning the interaction between Coa-ASC16 and the immune system and we found that the whole inflammatory response elicited by Coa-ASC16 (leukocyte recruitment and IL-1β, IL-6 and IL-12 production) was dependent on the MyD88 protein. TLR2, TLR4, TLR7 and NLRP3-inflammasome signaling were not required for induction of this inflammatory response. Coa-ASC16 induced local release of self-DNA, and in TLR9-deficient mice IL-6 production was absent. In addition, Coa-ASC16 revealed an intrinsic adjuvant activity which was affected by MyD88 and IL-6 absence. Taken together these results indicate that Coa-ASC16 used as a vaccine platform is effective due to the combination of the controlled release of antigen and its intrinsic pro-inflammatory activity. Understanding how Coa-ASC16 works might have significant implications for rational vaccine design., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Despite recent evidence on the involvement of CD81 in pathogen binding and Ag presentation by dendritic cells (DCs), the molecular mechanism of how CD81 regulates immunity during infection remains to be elucidated. To investigate the role of CD81 in the regulation of defense mechanisms against microbial infections, we have used the Listeria monocytogenes infection model to explore the impact of CD81 deficiency in the innate and adaptive immune response against this pathogenic bacteria. We show that CD81(-/-) mice are less susceptible than wild-type mice to systemic Listeria infection, which correlates with increased numbers of inflammatory monocytes and DCs in CD81(-/-) spleens, the main subsets controlling early bacterial burden. Additionally, our data reveal that CD81 inhibits Rac/STAT-1 activation, leading to a negative regulation of the production of TNF-α and NO by inflammatory DCs and the activation of cytotoxic T cells by splenic CD8α(+) DCs. In conclusion, this study demonstrates that CD81-Rac interaction exerts an important regulatory role on the innate and adaptive immunity against bacterial infection and suggests a role for CD81 in the development of novel therapeutic targets during infectious diseases., (Copyright © 2015 by The American Association of Immunologists, Inc.)

The CCL2 chemokine mediates monocyte egress from bone marrow and recruitment into inflamed tissues through interaction with the CCR2 chemokine receptor, and its expression is upregulated by proinflammatory cytokines. Analysis of the gene expression profile in GM-CSF- and M-CSF-polarized macrophages revealed that a high CCL2 expression characterizes macrophages generated under the influence of M-CSF, whereas CCR2 is expressed only by GM-CSF-polarized macrophages. Analysis of the factors responsible for this differential expression identified activin A as a critical factor controlling the expression of the CCL2/CCR2 pair in macrophages, as activin A increased CCR2 expression but inhibited the acquisition of CCL2 expression by M-CSF-polarized macrophages. CCL2 and CCR2 were found to determine the extent of macrophage polarization because CCL2 enhances the LPS-induced production of IL-10, whereas CCL2 blockade leads to enhanced expression of M1 polarization-associated genes and cytokines, and diminished expression of M2-associated markers in human macrophages. Along the same line, Ccr2-deficient bone marrow-derived murine macrophages displayed an M1-skewed polarization profile at the transcriptomic level and exhibited a significantly higher expression of proinflammatory cytokines (TNF-α, IL-6) in response to LPS. Therefore, the CCL2-CCR2 axis regulates macrophage polarization by influencing the expression of functionally relevant and polarization-associated genes and downmodulating proinflammatory cytokine production.

Background: Whereas recent research has characterized the mechanism by which dendritic cells (DCs) induce T(H)1/T(H)17 responses, the functional specialization enabling DCs to polarize T(H)2 responses remains undefined. Because IL-4 is essential during T(H)2 responses not only by acting on CD4(+) T cells through the activation of GATA-3 but also by regulating IgE class-switching, epithelial cell permeability, and muscle contractility, we hypothesized that IL-4 could also have a role in the conditioning of DCs during T(H)2 responses., Objective: We sought to analyze whether IL-4 exerts an immunomodulatory function on DCs during their differentiation, leading to their functional specialization for the induction of T(H)2 responses., Methods: Monocyte-derived DCs (moDCs) conditioned by IL-4 during their differentiation (IL-4-conditioned moDCs [IL-4-moDCs]) were analyzed for T(H)1-polarizing/inflammatory cytokine production in response to Toll-like receptor stimulation. The acetylation level of the promoters of the genes encoding these cytokines was analyzed by using chromatin immunoprecipitation. Gene expression profiling of IL-4-moDCs was defined by using mouse genome microarrays. IL-4-moDCs were tested for their capacity to induce house dust mite-mediated allergic reactions., Results: Our data suggest that IL-4 inhibits T(H)1-polarizing/inflammatory cytokine gene expression on IL-4-moDCs through the deacetylation of the promoters of these genes, leading to their transcriptional repression. Microarray analyses confirmed that IL-4 upregulated T(H)2-related genes as eosinophil-associated ribonucleases, eosinophil/basophil chemokines, and M2 genes. IL-4 licensed moDCs for the induction of T(H)2 responses, causing house dust mite-mediated allergic airway inflammation., Conclusion: This study describes a new role for IL-4 by demonstrating that moDCs are conditioned by IL-4 for the induction of T(H)2 responses by blocking T(H)1-polarizing/inflammatory cytokine production through histone hypoacetylation and upregulating T(H)2-related genes., (Copyright © 2013 American Academy of Allergy, Asthma & Immunology. Published by Mosby, Inc. All rights reserved.)

Type I interferon (IFN) is crucial during infection through its antiviral properties and by coordinating the immunocompetent cells involved in antiviral or antibacterial immunity. Type I IFN (IFN-α and IFN-β) is produced after virus or bacteria recognition by cytosolic receptors or membrane-bound TLR receptors following the activation of the transcription factors IRF3 or IRF7. IFN-β production after fungal infection was recently reported, although the underlying mechanism remains controversial. Here we describe that IFN-β production by dendritic cells (DCs) induced by Candida albicans is largely dependent on Dectin-1- and Dectin-2-mediated signaling. Dectin-1-induced IFN-β production required the tyrosine kinase Syk and the transcription factor IRF5. Type I IFN receptor-deficient mice had a lower survival after C. albicans infection, paralleled by defective renal neutrophil infiltration. IFN-β production by renal infiltrating leukocytes was severely reduced in C. albicans-infected mice with Syk-deficient DCs. These data indicate that Dectin-induced IFN-β production by renal DCs is crucial for defense against C. albicans infection., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Monocytes have the capacity to differentiate into macrophages or dendritic cells (DCs) after extravasation into lymphoid and nonlymphoid tissues. They have thus been consequently considered as precursors, but not effector cells, recirculating exclusively through the blood. In this report, we demonstrate for the first time that, after subcutaneous injection, activated monocytes migrate through the lymphatics from the dermis into the draining lymph nodes by a CCR7-dependent mechanism. LPS-activated monocytes were less efficient than DCs in stimulating CD4(+) T cells, but unexpectedly, they were highly efficient in inducing antigen-specific CD8(+) T-cell proliferation by cross-presentation, both in vitro and in vivo. Interestingly, CD8(+) T cells stimulated in vivo by activated monocytes expressed a high level of CD62L, suggesting that they had undergone an unconventional activation process. In conclusion, our data strongly support the concept that monocytes can behave not only as precursor cells for macrophages and DCs, but also as effector cells with the capacity to migrate from the periphery to the lymph nodes through the lymph and to cross-present antigens to CD8(+) T cells. These results suggest that monocytes can play an important role in the induction and regulation of CD4(+) and CD8(+) T-cell responses., (© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Cervical cancer is caused by persistent high-risk human papillomavirus (HR-HPV) infection and represents the second most frequent gynecological malignancy in the world. The HPV-16 type accounts for up to 55% of all cervical cancers. The HPV-16 oncoproteins E6 and E7 are necessary for induction and maintenance of malignant transformation and represent tumor-specific antigens for targeted cytotoxic T lymphocyte-mediated immunotherapy. Therapeutic cancer vaccines have become a challenging area of oncology research in recent decades. Among current cancer immunotherapy strategies, virus-like particle (VLP)-based vaccines have emerged as a potent and safe approach. We generated a vaccine (VLP-E7) incorporating a long C-terminal fragment of HPV-16 E7 protein into the infectious bursal disease virus VLP and tested its therapeutic potential in HLA-A2 humanized transgenic mice grafted with TC1/A2 tumor cells. We performed a series of tumor challenge experiments demonstrating a strong immune response against already-formed tumors (complete eradication). Remarkably, therapeutic efficacy was obtained with a single dose without adjuvant and against two injections of tumor cells, indicating a potent and long-lasting immune response.

Statins are prescribed to 25 million people worldwide for treating hypercholesterolemia and reducing the risk of cardiovascular diseases. However, the side effects of statins on immunity, and particularly on DC immunobiology, have not been analyzed in-depth. Here, we have investigated the impact of lovastatin treatment during monocyte differentiation into DCs on the responsiveness of the resulting monocyte-derived DCs (moDCs) to TLR-mediated activation. Lovastatin positively regulated TLR4 signaling in LPS-stimulated moDCs, leading to strong activation of p38 MAP-kinase paralleled by increased proinflammatory cytokine and IFN-β production. In contrast, lovastatin promoted negative regulation of IFN-β-mediated autocrine signaling through the IFN-αβ receptor, paralleled by low expression of the transcription factor IRF-1, leading to the inhibition of the enzymes iNOS and HO-1. Defective activation of iNOS/HO-1 resulted in limited cytoprotective capacity against ROS and reduced microbicidal potential. These data were validated using an in vivo model of Listeria monocytogenes infection, which revealed that iNOS activation by splenic inflammatory moDCs, specialized in NO and TNF-α production, was strongly reduced in lovastatin-treated, Listeria-infected mice. Statin treatment could have severe implications in immunity against pathogens due to defective iNOS/HO-1 metabolism activation in inflammatory moDCs that might lead to immune failure., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Although monocytes were originally described as precursors to all the different subpopulations of macrophages found in the steady state and formed under inflammatory and infectious conditions, recent data have demonstrated conclusively that monocytes can also differentiate into dendritic cells (DCs). Monocytes are the precursors to different subsets of DCs, such as Langerhans cells and DCs found in the lamina propria of the gastrointestinal, respiratory, and urogenital tracts. In addition, monocyte-derived DCs (moDCs), newly formed during inflammatory reactions, appear to fulfill an essential role in defense mechanisms against pathogens by participating in the induction of both adaptive and innate immune responses. In this regard, moDCs have the capacity to activate antigen-specific CD4(+) T-cell responses and to cross-prime CD8(+) T cells, during viral, bacterial, and parasitic infections. In addition, monocytes have been recently described as the precursors to a subset of DCs specialized in innate immunity against pathogens, named TipDCs [for TNF-alpha (tumor necrosis factor-alpha)-iNOS (inducible nitric oxide synthase)-producing DCs] that display a remarkable microbicidal activity and also provide iNOS-dependent help for antibody production by B cells. Importantly, in contrast to DCs developing in the steady state, moDCs formed during inflammatory and infectious processes are subjected to diverse soluble mediators that determine the multiple functional specificities displayed by moDCs, as a result of the remarkable developmental plasticity of monocytes. In this review, we discuss recent findings dealing with the differentiation and functional relevance of moDCs that have widened the frontiers of DC immunobiology in relation to innate and adaptive immunity and the etiology of chronic inflammatory diseases.

Plasmacytoid dendritic cells (pDCs) efficiently produce type I interferon and participate in adaptive immune responses, although the molecular interactions between pDCs and antigen-specific T cells remain unknown. This study examines immune synapse (IS) formation between murine pDCs and CD4(+) T cells. Mature pDCs formed canonical ISs, involving relocation to the contact site of the microtubule-organizing center, F-actin, protein kinase C-, and pVav, and activation of early signaling molecules in T cells. However, immature pDCs were less efficient at forming conjugates with T cells and inducing IS formation, microtubule-organizing center translocation, and T-cell signaling and activation. Time-lapse videomicroscopy and 2-photon in vivo imaging of pDC-T-cell interactions revealed that immature pDCs preferentially mediated transient interactions, whereas mature pDCs promoted more stable contacts. Our data indicate that, under steady-state conditions, pDCs preferentially establish transient contacts with naive T cells and show a very modest immunogenic capability, whereas on maturation, pDCs are able to form long-lived contacts with T cells and significantly enhance their capacity to activate these lymphocytes.

Specific defense mechanisms against pathogens are fulfilled by different subsets of nonmucosal conventional dendritic cells (DCs), including migratory Langerhans cells (LCs), dermal DCs, and resident CD8(+) and CD8(-) DCs found in lymphoid organs. Dermal DCs capture antigens in the skin and migrate to lymph nodes, where they can transfer the antigens to CD8(+) DCs and activate CD4(+) T cells. Differential antigen-processing machinery grants CD8(+) DCs a high efficiency in activating CD8(+) T cells through crosspresentation, whereas CD8(-) DCs preferentially trigger CD4(+) T cell responses. Recent findings have revealed the important role played by monocyte-derived DCs (mo-DCs), newly formed during infection, in activating CD4(+) and CD8(+) T cells, regulating immunoglobulin production, and killing pathogens. However, a number of controversial issues regarding the function of different DC subsets during viral, bacterial, and parasitic infections remain to be resolved.

The statins, a group of inhibitors of the 3-hydroxy-3-methylglutaryl coenzyme A reductase, are reported to influence a variety of immune system activities through 3-hydroxy-3-methylglutaryl coenzyme A reductase-dependent and -independent mechanisms. How statin treatment regulates immune system function in vivo nonetheless remains to be fully defined. We analyzed the immunomodulatory effects of lovastatin in a Candida albicans-induced delayed-type hypersensitivity reaction in mice. In this model, lovastatin administration reduced the acute inflammatory response elicited by C. albicans challenge. This anti-inflammatory activity of lovastatin was associated with a shift from a Th1 to a Th2 immune response, as well as an increase in the percentage of regulatory T cells at the inflammation site and in the regional draining lymph node. The lovastatin-induced increase in regulatory T cells in the inflamed skin was dependent on expression of CCL1, a chemokine that is locally up-regulated by statin administration. The anti-inflammatory effect of lovastatin was abrogated in CCL1-deficient mice. These results suggest that local regulation of chemokine expression may be an important process in statin-induced modulation of the immune system.

Monocytes have been classically considered essential elements in relation with innate immune responses against pathogens, and inflammatory processes caused by external aggressions, infection and autoimmune disease. However, although their potential to differentiate into dendritic cells (DCs) was discovered 14 years ago, their functional relevance with regard to adaptive immune responses has only been uncovered very recently. Studies performed over the last years have revealed that monocyte-derived DCs play an important role in innate and adaptive immunity, due to their microbicidal potential, capacity to stimulate CD4(+) and CD8(+) T-cell responses and ability to regulate Immunoglobulin production by B cells. In addition, monocyte-derived DCs not only constitute a subset of DCs formed at inflammatory foci, as previously thought, but also comprise different subsets of DCs located in antigen capture areas, such as the skin and the intestinal, respiratory and reproductive tracts.

Monocyte recruitment and differentiation into dendritic cells or macrophages play a critical role in defense mechanisms against pathogens and in inflammatory and autoimmune diseases. Important contributions have been made on the molecular events controlling neutrophil and lymphocyte extravasation under steady state or inflammation. However, the molecules involved in monocyte rolling during their migration to antigen capture areas and lymphoid organs during infection remain undefined. Here we have analyzed the homing molecules controlling mouse monocyte rolling in an experimental model of Leishmania major infection. Monocyte migration through inflamed dermal venules was dependent on interactions of PSGL-1 with P- and E-selectins, and of L-selectin with PNAd, whereas migration through lymph node high endothelial venules relied essentially on L-selectin-PNAd interactions. These results might have important implications regarding the induction of immune responses against pathogens and future immunotherapeutic protocols of inflammatory and autoimmune diseases, based on selective inhibition of monocyte migration to specific inflammatory foci.

Improvement of the strategy to target tumor Ags to dendritic cells (DCs) for immunotherapy requires the identification of the most appropriate ligand/receptor pairing. We screened a library of Ab fragments on mouse DCs to isolate new potential Abs capable of inducing protective immune responses. The screening identified a high-affinity Ab against CD36, a multi-ligand scavenger receptor primarily expressed by the CD8alpha+ subset of conventional DCs. The Ab variable regions were genetically linked to the model Ag OVA and tested in Ag presentation assays in vitro and in vivo. Anti-CD36-OVA was capable of delivering exogenous Ags to the MHC class I and MHC class II processing pathways. In vivo, immunization with anti-CD36-OVA induced robust activation of naive CD4+ and CD8+ Ag-specific T lymphocytes and the differentiation of primed CD8+ T cells into long-term effector CTLs. Vaccination with anti-CD36-OVA elicited humoral and cell-mediated protection from the growth of an Ag-specific tumor. Notably, the relative efficacy of targeting CD11c/CD8alpha+ via CD36 or DEC205 was qualitatively different. Anti-DEC205-OVA was more efficient than anti-CD36-OVA in inducing early events of naive CD8+ T cell activation. In contrast, long-term persistence of effector CTLs was stronger following immunization with anti-CD36-OVA and did not require the addition of exogenous maturation stimuli. The results identify CD36 as a novel potential target for immunotherapy and indicate that the outcome of the immune responses vary by targeting different receptors on CD8alpha+ DCs.

Infection-induced inflammatory reactions involve a strong increase in dendritic cells (DCs) at the infection site and draining lymph nodes (dLNs). Whether inflammatory DCs are recruited to these locations or differentiate locally, and what their functional relevance is, remain unclear. Here we showed that during Leishmania infection, monocytes were recruited to the dermis and differentiated into "dermal monocyte-derived DCs," which subsequently migrated into the dLNs. In addition, monocyte recruitment to the dLNs resulted in the differentiation into "LN monocyte-derived DCs." Analysis of the kinetics of monocyte differentiation into DCs, susceptibility to infection, IL-12 production, and L. major-specific T cell stimulation potential suggest that dermal monocyte-derived DCs controlled the induction of protective T helper 1 responses against Leishmania. Thus, the demonstration of monocyte differentiation potential into DCs during in vivo infection and of local DC differentiation in inflammatory foci suggests that de novo formed monocyte-derived DCs are essential in T cell immunity against pathogens.

Expression of the physiological cellular prion protein (PrP(C)) is remarkably regulated during differentiation and activation of cells of the immune system. Among these, dendritic cells (DCs) display particularly high levels of membrane PrP(C), which increase upon maturation, in parallel with that of molecules involved in Ag presentation to T cells. Freshly isolated mouse Langerhans cells, dermal DCs, and DCs from thymus, spleen, and mesenteric lymph nodes expressed low to intermediate levels of PrP(C). Highest levels of both PrP(C) and MHC class II molecules were displayed by lymph node CD8alpha(int) DCs, which represent fully mature cells having migrated from peripheral tissues. Maturation induced by overnight culture resulted in increased levels of surface PrP(C), as did in vivo DC activation by bacterial LPS. Studies on Fms-like tyrosine kinase 3 ligand bone marrow-differentiated B220(-) DCs confirmed that PrP(C) expression followed that of MHC class II and costimulatory molecules, and correlated with IL-12 production in response to TLR-9 engagement by CpG. However, at variance with conventional DCs, B220(+) plasmacytoid DCs isolated from the spleen, or in vitro differentiated, did not significantly express PrP(C), both before and after activation by TLR-9 engagement. PrP knockout mice displayed higher numbers of spleen CD8alpha(+) DCs, but no significant differences in their maturation response to stimulation through TLR-4 and TLR-9 were noticed. Results are discussed in relation to the functional relevance of PrP(C) expression by DCs in the induction of T cell responses, and to the pathophysiology of prion diseases.

CD36 is a member of the scavenger receptor type B family implicated in the binding of lipoproteins, phosphatidylserine, thrombospondin-1, and the uptake of long-chain fatty acids. On mononuclear phagocytes, recognition of apoptotic cells by CD36 contributes to peripheral tolerance and prevention of autoimmunity by impairing dendritic cell (DC) maturation. Besides, CD36 acts as a coreceptor with TLR2/6 for sensing microbial diacylglycerides, and its deficiency leads to increased susceptibility to Staphylococcus aureus infections. The RUNX3 transcription factor participates in reprogramming DC transcription after pathogen recognition, and its defective expression leads to abnormally accelerated DC maturation. We present evidence that CD36 expression is negatively regulated by the RUNX3 transcription factor during myeloid cell differentiation and activation. In molecular terms, RUNX3 impairs the activity of the proximal regulatory region of the CD36 gene in myeloid cells through in vitro recognition of two functional RUNX-binding elements. Moreover, RUNX3 occupies the CD36 gene proximal regulatory region in vivo, and its overexpression in myeloid cells results in drastically diminished CD36 expression. The down-regulation of CD36 expression by RUNX3 implies that this transcription factor could impair harmful autoimmune responses by contributing to the loss of pathogen- and apoptotic cell-recognition capabilities by mature DCs.

It has been recently demonstrated that, in addition to function as macrophage precursors, monocytes have the capacity to differentiate into dendritic cells (DCs), and therefore they play an essential role in both the innate and adaptive immunity. Monocytes display a remarkable functional diversity, allowing them to perform multiple defense functions, from pathogen elimination by phagocytosis, to the induction of antigen-specific T cell responses. This functional potential relies essentially in their developmental plasticity, permitting monocytes to differentiate into different subsets of macrophages and DCs. Although recent data suggest that the acquisition of functional specialization by monocytes is controlled by chemotactic, activation and differentiation factors, how monocyte differentiation occurs under physiological conditions remains largely unknown.

The monocyte capacity to differentiate into dendritic cells (DCs) was originally demonstrated by human in vitro DC differentiation assays that have subsequently become the essential methodologic approach for the production of DCs to be used in DC-mediated cancer immunotherapy protocols. In addition, in vitro DC generation from monocytes is a powerful tool to study DC differentiation and maturation. However, whether DC differentiation from monocytes occurs in vivo remains controversial, and the physiologic counterparts of in vitro monocyte-derived DCs are unknown. In addition, information on murine monocytes and monocyte-derived DCs is scarce. Here we show that mouse bone marrow monocytes can be differentiated in vitro into DCs using similar conditions as those defined in humans, including in vitro cultures with granulocyte-macrophage colony-stimulating factor and interleukin 4 and reverse transendothelial migration assays. Importantly, we demonstrate that after in vivo transfer monocytes generate CD8- and CD8+ DCs in the spleen, but differentiate into macrophages on migration to the thoracic cavity. In conclusion, we support the hypothesis that monocytes generate DCs not only on entry into the lymph and migration to the lymph nodes as proposed, but also on extravasation from blood and homing to the spleen, suggesting that monocytes represent immediate precursors of lymphoid organ DCs.

A recent workshop on "Dendritic Cells: Biology and Therapeutic Applications," sponsored by the Juan March Foundation, brought together basic and clinical research scientists to discuss the mechanisms underlying the control of immune responses and tolerance by dendritic cells (DCs), as well as recent research in cancer immunotherapy based on DC vaccination. Particular emphasis was placed on antigen processing and presentation by DCs, C-type lectin antigen receptors, DC maturation and polarization of T cell responses, the control of immunity versus tolerance by DCs, the developmental origin of DCs, and the use of DCs in cancer immunotherapy.

Functional specialization allows defined dendritic-cell (DC) subsets to induce efficient defence mechanisms against pathogens and tumour cells, and maintain T-cell tolerance by inducing the inactivation of autoreactive T cells. A crucial question, which has important implications for both our understanding of the induction and control of immunity by DCs, as well as the use of DCs for immunotherapy, is whether the functional diversity of DCs results from the existence of developmentally independent DC subpopulations, or whether DC subsets that share a common differentiation origin acquire specific functions in response to environmental signals. This review discusses recent findings on mouse DC development.

We describe a new B220+ subpopulation of immaturelike dendritic cells (B220+ DCs) with low levels of expression of major histocompatibility complex (MHC) and costimulatory molecules and markedly reduced T-cell stimulatory potential, located in the thymus, bone marrow, spleen, and lymph nodes. B220+ DCs display ultrastructural characteristics resembling those of human plasmacytoid cells and accordingly produce interferon-alpha after virus stimulation. B220+ DCs acquired a strong antigen-presenting cell capacity on incubation with CpG oligodeoxynucleotides, concomitant with a remarkable up-regulation of MHC and costimulatory molecules and the production of interleukin-12 (IL-12) and IL-10. Importantly, our data suggest that nonstimulated B220+ DCs represent a subset of physiological tolerogenic DCs endowed with the capacity to induce a nonanergic state of T-cell unresponsiveness, involving the differentiation of T regulatory cells capable of suppressing antigen-specific T-cell proliferation. In conclusion, our data support the hypothesis that B220+ DCs represent a lymphoid organ subset of immature DCs with a dual role in the immune system-exerting a tolerogenic function in steady state but differentiating on microbial stimulation into potent antigen-presenting cells with type 1 interferon production capacity.

Dendritic cells (DCs) are essential for the establishment of immune responses against pathogens and tumour cells, and thus have great potential as tools for vaccination and cancer immunotherapy trials. Experimental evidence has led to a dual DC differentiation model, which involves the existence of both myeloid- and lymphoid-derived DCs. But this concept has been challenged by recent reports demonstrating that both CD8- and CD8+ DCs, considered in mice as archetypes of myeloid and lymphoid DCs respectively, can be generated from either lymphoid or myeloid progenitors. The issue of DC physiological derivation therefore remains an open question. Here we report the characterization of a DC-committed precursor population, which has the capacity to generate all the DC subpopulations present in mouse lymphoid organs---including CD8- and CD8+ DCs, as well as the B220+ DC subset---but which is devoid of myeloid or lymphoid differentiation potential. These data support an alternative model of DC development, in which there is an independent, common DC differentiation pathway.

Despite the information dealing with the differential phenotype and function of the main mouse dendritic cell (DC) subpopulations, namely, CD8alpha(-) and CD8alpha(+) DCs, their origin and involvement in antiviral immune responses in vivo are still largely unknown. To address these issues, this study used the changes occurring in DC subpopulations during the experimental infection by the Swiss (SW) strain of the mouse mammary tumor virus (MMTV). MMTV(SW) induced an 18-fold increase in lymph node DCs, which can be blocked by anti-CD62L treatment, concomitant with the presence of high numbers of DCs in the outer cortex, in close association with high endothelial venules. These data suggest that the DC increase caused by MMTV(SW) infection results from the recruitment of blood-borne DCs via high endothelial venules, by a CD62L-dependent mechanism. In addition, skin sensitization assays indicate that MMTV(SW) infection inhibits epidermal Langerhans cell migration to the draining lymph node. Moreover, data on the kinetics of MMTV(SW)-induced expansion of the different DC subsets support the hypothesis that CD8(-) and CD8(+) DCs represent different maturation stages of the same DC population, rather than myeloid- and lymphoid-derived DCs, respectively, as previously proposed. Finally, the fact that DCs were infected by MMTV(SW) suggests their participation in the early phases of infection.

CD8alpha+ and CD8alpha- dendritic cells (DCs) have been considered as independent DC subpopulations both ontogenetically and functionally during recent years. However, it has been demonstrated that both DC subsets can be generated from a single precursor population, supporting the concept that they do not represent separate DC lineages. By using highly purified splenic CD8alpha- DCs, which were injected intravenously and traced by means of an Ly5.1/Ly5.2 transfer system, this study shows that CD8alpha- DCs acquired the phenotypic characteristics of CD8alpha+ DCs, by a differentiation process involving CD8alpha, DEC-205, and CD24 up-regulation, paralleled by the down-regulation of CD11b, F4/80, and CD4. These data demonstrate that CD8alpha+ DCs derive from CD8alpha- DCs, and strongly support that CD8alpha- and CD8alpha+ DCs represent different maturation or differentiation stages of the same DC population. Therefore, CD8alpha+ DCs would represent the last stage of DC differentiation, playing an essential role in the induction of T-cell responses, due to their antigen-presenting potential, cross-priming ability, and capacity to secrete large amounts of key cytokines such as interferon gamma and interleukin-12.