Resident macrophage-dependent immune cell scaffolds drive anti-bacterial defense in the peritoneal cavity.

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.

Inflammatory Ly6Chigh Monocytes Protect against Candidiasis through IL-15-Driven NK Cell/Neutrophil Activation.

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 Ly6Chigh 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 Ly6Chigh 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.

Interferon-ß production via Dectin-1-Syk-IRF5 signaling in dendritic cells is crucial for immunity to C. albicans.

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-ß) 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-ß production after fungal infection was recently reported, although the underlying mechanism remains controversial. Here we describe that IFN-ß production by dendritic cells (DCs) induced by Candida albicans is largely dependent on Dectin-1- and Dectin-2-mediated signaling. Dectin-1-induced IFN-ß 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-ß production by renal infiltrating leukocytes was severely reduced in C. albicans-infected mice with Syk-deficient DCs. These data indicate that Dectin-induced IFN-ß production by renal DCs is crucial for defense against C. albicans infection.

IVIg Promote Cross-Tolerance against Inflammatory Stimuli In Vitro and In Vivo.

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.

Rewiring monocyte glucose metabolism via C-type lectin signaling protects against disseminated candidiasis.

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.

In vivo induction of immune responses to pathogens by conventional dendritic cells.

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.

CD81 controls immunity to Listeria infection through rac-dependent inhibition of proinflammatory mediator release and activation of cytotoxic T cells.

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.

CCL2 shapes macrophage polarization by GM-CSF and M-CSF: identification of CCL2/CCR2-dependent gene expression profile.

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.

p38γ and p38δ kinases regulate the Toll-like receptor 4 (TLR4)-induced cytokine production by controlling ERK1/2 protein kinase pathway activation.

On the basis mainly of pharmacological experiments, the p38α MAP kinase isoform has been established as an important regulator of immune and inflammatory responses. However, the role of the related p38γ and p38δ kinases has remained unclear. Here, we show that deletion of p38γ and p38δ impaired the innate immune response to lipopolysaccharide (LPS), a Toll-like receptor 4 (TLR4) ligand, by blocking the extracellular signal-regulated kinase 1/2 (ERK1/2) activation in macrophages and dendritic cells. p38γ and p38δ were necessary to maintain steady-state levels of tumor progression locus 2 (TPL2), the MKK kinase that mediates ERK1/2 activation after TLR4 stimulation. TNFα, IL-1ß, and IL-10 production were reduced in LPS-stimulated macrophages from p38γ/δ-null mice, whereas IL-12 and IFNß production increased, in accordance with the known effects of TPL2/ERK1/2 signaling on the induction of these cytokines. Furthermore, p38γ/δ-deficient mice were less sensitive than controls to LPS-induced septic shock, showing lower TNFα and IL-1ß levels after challenge. Together, our results establish p38γ and p38δ as key components in innate immune responses.

Origin, precursors and differentiation of mouse dendritic cells.

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.

IL-4 blocks TH1-polarizing/inflammatory cytokine gene expression during monocyte-derived dendritic cell differentiation through histone hypoacetylation.

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.

Differentiation and function of mouse monocyte-derived dendritic cells in steady state and inflammation.

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.

Monocytes as effector cells: activated Ly-6C(high) mouse monocytes migrate to the lymph nodes through the lymph and cross-present antigens to CD8+ T cells.

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.

Mouse Tissue-Resident Peritoneal Macrophages in Homeostasis, Repair, Infection, and Tumor Metastasis.

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.

Whole-mount immunofluorescence imaging and isolation of mesothelium-bound immune cell aggregates during mouse peritoneal inflammation.

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.

Airway allergy causes alveolar macrophage death, profound alveolar disorganization and surfactant dysfunction.

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.

Statins inhibit iNOS-mediated microbicidal potential of activated monocyte-derived dendritic cells by an IFN-ß-dependent mechanism.

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-ß production. In contrast, lovastatin promoted negative regulation of IFN-ß-mediated autocrine signaling through the IFN-αß 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.

Trained immunity induction by the inactivated mucosal vaccine MV130 protects against experimental viral respiratory infections.

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.

Imaging of plasmacytoid dendritic cell interactions with T cells.

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.

Statins induce regulatory T cell recruitment via a CCL1 dependent pathway.

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.