A LTB4/CD11b self-amplifying loop drives pyogranuloma formation in chronic granulomatous disease.

Sterile pyogranulomas and heightened cytokine production are hyperinflammatory hallmarks of Chronic Granulomatous Disease (CGD). Using peritoneal cells of zymosan-treated CGD (gp91phox-/-) versus wild-type (WT) mice, an ex vivo system of pyogranuloma formation was developed to determine factors involved in and consequences of recruitment of neutrophils and monocyte-derived macrophages (MoMacs). Whereas WT cells failed to aggregate, CGD cells formed aggregates containing neutrophils initially, and MoMacs recruited secondarily. LTB4 was key, as antagonizing BLT1 blocked neutrophil aggregation, but acted only indirectly on MoMac recruitment. LTB4 upregulated CD11b expression on CGD neutrophils, and the absence/blockade of CD11b inhibited LTB4 production and cell aggregation. Neutrophil-dependent MoMac recruitment was independent of MoMac Nox2 status, BLT1, CCR1, CCR2, CCR5, CXCR2, and CXCR6. As proof of concept, CD11b-deficient CGD mice developed disrupted pyogranulomas with poorly organized neutrophils and diminished recruitment of MoMacs. Importantly, the disruption of cell aggregation and pyogranuloma formation markedly reduced proinflammatory cytokine production.

TNFα: TNFR1 signaling inhibits maturation and maintains the pro-inflammatory programming of monocyte-derived macrophages in murine chronic granulomatous disease.

Introduction: Loss of NADPH oxidase activity results in proinflammatory macrophages that contribute to hyperinflammation in Chronic Granulomatous Disease (CGD). Previously, it was shown in a zymosan-induced peritonitis model that gp91phox-/- (CGD) monocyte-derived macrophages (MoMacs) fail to phenotypically mature into pro-resolving MoMacs characteristic of wild type (WT) but retain the ability to do so when placed in the WT milieu. Accordingly, it was hypothesized that soluble factor(s) in the CGD milieu thwart appropriate programming. Methods: We sought to identify key constituents using ex vivo culture of peritoneal inflammatory leukocytes and their conditioned media. MoMac phenotyping was performed via flow cytometry, measurement of efferocytic capacity and multiplex analysis of secreted cytokines. Addition of exogenous TNFα, TNFα neutralizing antibody and TNFR1-/- MoMacs were used to study the role of TNFα: TNFR1 signaling in MoMac maturation. Results: More extensive phenotyping defined normal MoMac maturation and demonstrated failure of maturation of CGD MoMacs both ex vivo and in vivo. Protein components, and specifically TNFα, produced and released by CGD neutrophils and MoMacs into conditioned media was identified as critical to preventing maturation. Exogenous addition of TNFα inhibited WT MoMac maturation, and its neutralization allowed maturation of cultured CGD MoMacs. TNFα neutralization also reduced production of IL-1ß, IL-6 and CXCL1 by CGD cells though these cytokines played no role in MoMac programming. MoMacs lacking TNFR1 matured more normally in the CGD milieu both ex vivo and following adoptive transfer in vivo. Discussion: These data lend mechanistic insights into the utility of TNFα blockade in CGD and to other diseases where such therapy has been shown to be beneficial.

CCL5-producing migratory dendritic cells guide CCR5+ monocytes into the draining lymph nodes.

Dendritic cells (DCs) and monocytes capture, transport, and present antigen to cognate T cells in the draining lymph nodes (LNs) in a CCR7-dependent manner. Since only migratory DCs express this chemokine receptor, it is unclear how monocytes reach the LN. In steady-state and following inhalation of several PAMPs, scRNA-seq identified LN mononuclear phagocytes as monocytes, resident, or migratory type 1 and type 2 conventional (c)DCs, despite the downregulation of Xcr1, Clec9a, H2-Ab1, Sirpa, and Clec10a transcripts on migratory cDCs. Migratory cDCs, however, upregulated Ccr7, Ccl17, Ccl22, and Ccl5. Migratory monocytes expressed Ccr5, a high-affinity receptor for Ccl5. Using two tracking methods, we observed that both CD88hiCD26lomonocytes and CD88-CD26hi cDCs captured inhaled antigens in the lung and migrated to LNs. Antigen exposure in mixed-chimeric Ccl5-, Ccr2-, Ccr5-, Ccr7-, and Batf3-deficient mice demonstrated that while antigen-bearing DCs use CCR7 to reach the LN, monocytes use CCR5 to follow CCL5-secreting migratory cDCs into the LN, where they regulate DC-mediated immunity.

Mapping Macrophage Polarization and Origin during the Progression of the Foreign Body Response to a Poly(ethylene glycol) Hydrogel Implant.

Poly(ethylene glycol) (PEG) hydrogels hold promise for in vivo applications but induce a foreign body response (FBR). While macrophages are key in the FBR, many questions remain. This study investigates temporal changes in the transcriptome of implant-associated monocytes and macrophages. Proinflammatory pathways are upregulated in monocytes compared to control monocytes but subside by day 28. Macrophages are initially proinflammatory but shift to a profibrotic state by day 14, coinciding with fibrous capsule emergence. Next, this study assesses the origin of macrophages responsible for fibrous encapsulation using wildtype, C-C Motif Chemokine Receptor 2 (CCR2)-/- mice that lack recruited macrophages, and Macrophage Fas-Induced Apoptosis (MaFIA) mice that enable macrophage ablation. Subpopulations of recruited and tissue-resident macrophages are identified. Fibrous encapsulation proceeds in CCR2-/- mice similar to wildtype mice. However, studies in MaFIA mice indicate that macrophages are necessary for fibrous capsule formation. These findings suggest that macrophage origin impacts the FBR progression and provides evidence that tissue-resident macrophages and not the recruited macrophages may drive fibrosis in the FBR to PEG hydrogels. This study demonstrates that implant-associated monocytes and macrophages have temporally distinct transcriptomes in the FBR and that profibrotic pathways associated with macrophages may be enriched in tissue-resident macrophages.

Heightened turnover and failed maturation of monocyte-derived macrophages in murine chronic granulomatous disease.

Loss of NADPH oxidase activity leads to altered phagocyte responses and exaggerated inflammation in chronic granulomatous disease (CGD). We sought to assess the effects of Nox2 absence on monocyte-derived macrophages (MoMacs) in gp91phox-/y mice during zymosan-induced peritonitis. MoMacs from CGD and wild-type (WT) peritonea were characterized over time after zymosan injection. Although numbers lavaged from both genotypes were virtually identical, there were marked differences in maturation: newly recruited WT MoMacs rapidly enlarged and matured, losing Ly6C and gaining MHCII, CD206, and CD36, whereas CGD MoMacs remained small and were mostly Ly6C+MHCII-. RNA-sequencing analyses showed few intrinsic differences between genotypes in newly recruited MoMacs but significant differences with time. WT MoMacs displayed changes in metabolism, adhesion, and reparative functions, whereas CGD MoMacs remained inflammatory. PKH dye labeling revealed that although WT MoMacs were mostly recruited within the first 24 hours and remained in the peritoneum while maturing and enlarging, CGD monocytes streamed into the peritoneum for days, with many migrating to the diaphragm where they were found in fibrin(ogen) clots surrounding clusters of neutrophils in nascent pyogranulomata. Importantly, these observations seemed to be driven by milieu: adoptive transfer of CGD MoMacs into inflamed peritonea of WT mice resulted in immunophenotypic maturation and normal behavior, whereas altered maturation/behavior of WT MoMacs resulted from transfer into inflamed peritonea of CGD mice. In addition, Nox2-deficient MoMacs behaved similarly to their Nox2-sufficient counterparts within the largely WT milieu of mixed bone marrow chimeras. These data show persistent recruitment with fundamental failure of MoMac maturation in CGD.

LN Monocytes Limit DC-Poly I:C Induced Cytotoxic T Cell Response via IL-10 and Induction of Suppressor CD4 T Cells.

Every immune response has accelerators and brakes. Depending on the pathogen or injury, monocytes can play either role, promoting or resolving immunity. Poly I:C, a potent TLR3 ligand, licenses cross-presenting dendritic cells (DC1) to accelerate a robust cytotoxic T cells response against a foreign antigen. Poly I:C thus has promise as an adjuvant in cancer immunotherapy and viral subunit vaccines. Like DC1s, monocytes are also abundant in the LNs. They may act as either immune accelerators or brakes, depending on the inflammatory mediator they encounter. However, little is known about their contribution to adaptive immunity in the context of antigen and Poly I:C. Using monocyte-deficient and chimeric mice, we demonstrate that LN monocytes indirectly dampen a Poly I:C induced antigen-specific cytotoxic T cell response, exerting a "braking" function. This effect is mediated by IL-10 production and induction of suppressor CD4+ T cells. In a metastatic melanoma model, we show that a triple-combination prophylactic treatment consisting of anti-IL-10, tumor peptides and Poly I:C works because removing IL-10 counteracts the monocytic brake, resulting in significantly fewer tumors compared to mice treated with tumor peptides and Poly I:C alone. Finally, in human LN tissue, we observed that monocytes (unlike DCs) express high levels of IL-10, suggesting that anti-IL-10 may be an important addition to treatments. Overall, our data demonstrates that LN monocytes regulate the induction of a robust DC1-mediated immune response. Neutralization of either IL-10 or monocytes can augment Poly I:C-based treatments and enhance T cell cytotoxicity.

Human and Mouse Transcriptome Profiling Identifies Cross-Species Homology in Pulmonary and Lymph Node Mononuclear Phagocytes.

The mononuclear phagocyte (MP) system consists of macrophages, monocytes, and dendritic cells (DCs). MP subtypes play distinct functional roles in steady-state and inflammatory conditions. Although murine MPs are well characterized, their pulmonary and lymph node (LN) human homologs remain poorly understood. To address this gap, we have created a gene expression compendium across 24 distinct human and murine lung and LN MPs, along with human blood and murine spleen MPs, to serve as validation datasets. In-depth RNA sequencing identifies corresponding human-mouse MP subtypes and determines marker genes shared and divergent across species. Unexpectedly, only 13%-23% of the top 1,000 marker genes (i.e., genes not shared across species-specific MP subtypes) overlap in corresponding human-mouse MP counterparts. Lastly, CD88 in both species helps distinguish monocytes/macrophages from DCs. Our cross-species expression compendium serves as a resource for future translational studies to investigate beforehand whether pursuing specific MP subtypes or genes will prove fruitful.

Localization of Macrophages in the Human Lung via Design-based Stereology.

Rationale: Interstitial macrophages (IMs) and airspace macrophages (AMs) play critical roles in lung homeostasis and host defense, and are central to the pathogenesis of a number of lung diseases. However, the absolute numbers of macrophages and the precise anatomic locations they occupy in the healthy human lung have not been quantified.Objectives: To determine the precise number and anatomic location of human pulmonary macrophages in nondiseased lungs and to quantify how this is altered in chronic cigarette smokers.Methods: Whole right upper lobes from 12 human donors without pulmonary disease (6 smokers and 6 nonsmokers) were evaluated using design-based stereology. CD206 (cluster of differentiation 206)-positive/CD43+ AMs and CD206+/CD43- IMs were counted in five distinct anatomical locations using the optical disector probe.Measurements and Main Results: An average of 2.1 × 109 IMs and 1.4 × 109 AMs were estimated per right upper lobe. Of the AMs, 95% were contained in diffusing airspaces and 5% in airways. Of the IMs, 78% were located within the alveolar septa, 14% around small vessels, and 7% around the airways. The local density of IMs was greater in the alveolar septa than in the connective tissue surrounding the airways or vessels. The total number and density of IMs was 36% to 56% greater in the lungs of cigarette smokers versus nonsmokers.Conclusions: The precise locations occupied by pulmonary macrophages were defined in nondiseased human lungs from smokers and nonsmokers. IM density was greatest in the alveolar septa. Lungs from chronic smokers had increased IM numbers and overall density, supporting a role for IMs in smoking-related disease.

Isolation and Characterization of Mononuclear Phagocytes in the Mouse Lung and Lymph Nodes.

There is a diverse population of mononuclear phagocytes (MPs) in the lungs, comprised of macrophages, dendritic cells (DCs), and monocytes. The existence of these various cell types suggests that there is a clear division of labor and delicate balance between the MPs under steady-state and inflammatory conditions. Here we describe how to identify pulmonary MPs using flow cytometry and how to isolate them via cell sorting. In steady-state conditions, murine lungs contain a uniform population of alveolar macrophages (AMs), three distinct interstitial macrophage (IM) populations, three DC subtypes, and a small number of tissue-trafficking monocytes. During an inflammatory response, the monocyte population is more abundant and complex since it acquires either macrophage-like or DC-like features. All in all, studying how these cell types interact with each other, structural cells, and other leukocytes within the environment will be important to understanding their role in maintaining homeostasis and during the development of disease.

Immune Surveillance by Natural IgM Is Required for Early Neoantigen Recognition and Initiation of Adaptive Immunity.

Early recognition of neoantigen-expressing cells is complex, involving multiple immune cell types. In this study, in vivo, we examined how antigen-presenting cell subtypes coordinate and induce an immunological response against neoantigen-expressing cells, particularly in the absence of a pathogen-associated molecular pattern, which is normally required to license antigen-presenting cells to present foreign or self-antigens as immunogens. Using two reductionist models of neoantigen-expressing cells and two cancer models, we demonstrated that natural IgM is essential for the recognition and initiation of adaptive immunity against neoantigen-expressing cells. Natural IgM antibodies form a cellular immune complex with the neoantigen-expressing cells. This immune complex licenses surveying monocytes to present neoantigens as immunogens to CD4+ T cells. CD4+ T helper cells, in turn, use CD40L to license cross-presenting CD40+ Batf3+ dendritic cells to elicit a cytotoxic T cell response against neoantigen-expressing cells. Any break along this immunological chain reaction results in the escape of neoantigen-expressing cells. This study demonstrates the surprising, essential role of natural IgM as the initiator of a sequential signaling cascade involving multiple immune cell subtypes. This sequence is required to coordinate an adaptive immune response against neoantigen-expressing cells.

A Consistent Method to Identify and Isolate Mononuclear Phagocytes from Human Lung and Lymph Nodes.

Mononuclear phagocytes (MP) consist of macrophages, dendritic cells (DCs), and monocytes. In all organs, including the lung, there are multiple subtypes within these categories. The existence of all these cell types suggest that there is a clear division of labor and delicate balance between the MPs under steady state and inflammatory conditions. Although great strides have been made to understand MPs in the mouse lung, and human blood, little is known about the MPs that exist in the human lung and lung-draining lymph nodes (LNs), and even less is known about their functional roles, studies of which will require a large number of sorted cells. We have comprehensively examined cell surface markers previously used in a variety of organs to identify human pulmonary MPs. In the lung, we consistently identify five extravascular pulmonary MPs and three LN MPs. These MPs were present in over 100 lungs regardless of age or gender. Notably, the human blood CD141+ DCs, as described in the literature, were not observed in non-diseased lungs or their draining LNs. In the lung and draining LNs, expression of CD141 was only observed on HLADR+ CD11c+ CD14+ extravascular monocytes (often confused in the LN as resident DCs based on the level of HLADR expression and mouse LN data). In the human lung and LNs there are at least two DC subtypes expressing HLADR, DEC205 and CD1c, along with circulating monocytes that behave as either antigen-presenting cells or macrophages. Furthermore, we demonstrate how to distinguish between alveolar macrophages and interstitial macrophage subtypes. It still remains unclear how the human pulmonary MPs identified here align with mouse MPs. Clearly, we are now past the stage of cell surface marker characterization, and future studies will need to move toward understanding what these cell types are and how they function. Our hope is that the strategy described here can help the pulmonary community take this next step.

Isolation and Identification of Interstitial Macrophages from the Lungs Using Different Digestion Enzymes and Staining Strategies.

Interstitial macrophages (IMs) are present in multiple organs. Although there is limited knowledge of the unique functional role IM subtypes play, macrophages, in general, are known for their contribution in homeostatic tissue maintenance and inflammation such as clearing pathogens and debris and secreting inflammatory mediators and growth factors. IM subtypes have been identified in the heart, skin, and gut, and more recently we identified three distinct IMs in the lung. IMs express on their surface high levels of MerTK, CD64, and CD11b, with differences in CD11c, CD206, and MHC II expression, and referred to the three pulmonary IM subtypes as IM1 (CD11cloCD206+MHCIIlo), IM2 (CD11cloCD206+MHCIIhi), and IM3 (CD11chiCD206loMHCIIhi). In this chapter, we highlight how to extract IMs from the lung using three different digestion enzymes: elastase, collagenase D, and Liberase TM. Of these three commonly used enzymes, Liberase TM was the most effective at IM extraction, particularly IM3. Furthermore, alternative staining strategies to identify IMs were examined, which included CD64, MerTK, F4/80, and Tim4. Thus, future studies highlighting the functional role of IM subtypes will help further our understanding of how tissue homeostasis is maintained and inflammatory conditions are induced and resolved.

Deletion of c-FLIP from CD11bhi Macrophages Prevents Development of Bleomycin-induced Lung Fibrosis.

Idiopathic pulmonary fibrosis is a progressive lung disease with complex pathophysiology and fatal prognosis. Macrophages (MΦ) contribute to the development of lung fibrosis; however, the underlying mechanisms and specific MΦ subsets involved remain unclear. During lung injury, two subsets of lung MΦ coexist: Siglec-Fhi resident alveolar MΦ and a mixed population of CD11bhi MΦ that primarily mature from immigrating monocytes. Using a novel inducible transgenic system driven by a fragment of the human CD68 promoter, we targeted deletion of the antiapoptotic protein cellular FADD-like IL-1ß-converting enzyme-inhibitory protein (c-FLIP) to CD11bhi MΦ. Upon loss of c-FLIP, CD11bhi MΦ became susceptible to cell death. Using this system, we were able to show that eliminating CD11bhi MΦ present 7-14 days after bleomycin injury was sufficient to protect mice from fibrosis. RNA-seq analysis of lung MΦ present during this time showed that CD11bhi MΦ, but not Siglec-Fhi MΦ, expressed high levels of profibrotic chemokines and growth factors. Human MΦ from patients with idiopathic pulmonary fibrosis expressed many of the same profibrotic chemokines identified in murine CD11bhi MΦ. Elimination of monocyte-derived MΦ may help in the treatment of fibrosis. We identify c-FLIP and the associated extrinsic cell death program as a potential pathway through which these profibrotic MΦ may be pharmacologically targeted.

Three Unique Interstitial Macrophages in the Murine Lung at Steady State.

The current paradigm in macrophage biology is that some tissues mainly contain macrophages from embryonic origin, such as microglia in the brain, whereas other tissues contain postnatal-derived macrophages, such as the gut. However, in the lung and in other organs, such as the skin, there are both embryonic and postnatal-derived macrophages. In this study, we demonstrate in the steady-state lung that the mononuclear phagocyte system is comprised of three newly identified interstitial macrophages (IMs), alveolar macrophages, dendritic cells, and few extravascular monocytes. We focused on similarities and differences between the three IM subtypes, specifically, their phenotype, location, transcriptional signature, phagocytic capacity, turnover, and lack of survival dependency on fractalkine receptor, CX3CR1. Pulmonary IMs were located in the bronchial interstitium but not the alveolar interstitium. At the transcriptional level, all three IMs displayed a macrophage signature and phenotype. All IMs expressed MER proto-oncogene, tyrosine kinase, CD64, CD11b, and CX3CR1, and were further distinguished by differences in cell surface protein expression of CD206, Lyve-1, CD11c, CCR2, and MHC class II, along with the absence of Ly6C, Ly6G, and Siglec F. Most intriguingly, in addition to the lung, similar phenotypic populations of IMs were observed in other nonlymphoid organs, perhaps highlighting conserved functions throughout the body. These findings promote future research to track four distinct pulmonary macrophages and decipher the division of labor that exists between them.

Flow Cytometric Analysis of Mononuclear Phagocytes in Nondiseased Human Lung and Lung-Draining Lymph Nodes.

RATIONALE: The pulmonary mononuclear phagocyte system is a critical host defense mechanism composed of macrophages, monocytes, monocyte-derived cells, and dendritic cells. However, our current characterization of these cells is limited because it is derived largely from animal studies and analysis of human mononuclear phagocytes from blood and small tissue resections around tumors. OBJECTIVES: Phenotypic and morphologic characterization of mononuclear phagocytes that potentially access inhaled antigens in human lungs. METHODS: We acquired and analyzed pulmonary mononuclear phagocytes from fully intact nondiseased human lungs (including the major blood vessels and draining lymph nodes) obtained en bloc from 72 individual donors. Differential labeling of hematopoietic cells via intrabronchial and intravenous administration of antibodies within the same lobe was used to identify extravascular tissue-resident mononuclear phagocytes and exclude cells within the vascular lumen. Multiparameter flow cytometry was used to identify mononuclear phagocyte populations among cells labeled by each route of antibody delivery. MEASUREMENTS AND MAIN RESULTS: We performed a phenotypic analysis of pulmonary mononuclear phagocytes isolated from whole nondiseased human lungs and lung-draining lymph nodes. Five pulmonary mononuclear phagocytes were observed, including macrophages, monocyte-derived cells, and dendritic cells that were phenotypically distinct from cell populations found in blood. CONCLUSIONS: Different mononuclear phagocytes, particularly dendritic cells, were labeled by intravascular and intrabronchial antibody delivery, countering the notion that tissue and blood mononuclear phagocytes are equivalent systems. Phenotypic descriptions of the mononuclear phagocytes in nondiseased lungs provide a precedent for comparative studies in diseased lungs and potential targets for therapeutics.

Transcriptome analysis highlights the conserved difference between embryonic and postnatal-derived alveolar macrophages.

Alveolar macrophages (AMs) reside on the luminal surfaces of the airways and alveoli where they maintain host defense and promote alveolar homeostasis by ingesting inhaled particulates and regulating inflammatory responses. Recent studies have demonstrated that AMs populate the lungs during embryogenesis and self-renew throughout life with minimal replacement by circulating monocytes, except under extreme conditions of depletion or radiation injury. Here we demonstrate that on a global scale, environment appears to dictate AM development and function. Indeed, transcriptome analysis of embryonic host-derived and postnatal donor-derived AMs coexisting within the same mouse demonstrated >98% correlation and overall functional analyses were similar. However, we also identified several genes whose expression was dictated by origin rather than environment. The most differentially expressed gene not altered by environment was Marco, a gene recently demonstrated to have enhancer activity in embryonic-derived but not postnatal-derived tissue macrophages. Overall, we show that under homeostatic conditions, the environment largely dictates the programming and function of AMs, whereas the expression of a small number of genes remains linked to the origin of the cell.

Cutting Edge: Roles for Batf3-Dependent APCs in the Rejection of Minor Histocompatibility Antigen-Mismatched Grafts.

In transplantation, a major obstacle for graft acceptance in MHC-matched individuals is the mismatch of minor histocompatibility Ags. Minor histocompatibility Ags are peptides derived from polymorphic proteins that can be presented by APCs on MHC molecules. The APC subtype uniquely responsible for the rejection of minor Ag-mismatched grafts has not yet been identified. In this study, we examined graft rejection in three mouse models: 1) mismatch of male-specific minor Ags, 2) mismatch of minor Ags distinct from male-specific minor Ags, and 3) skin transplant. This study demonstrates that in the absence of pathogen-associated molecular patterns, Batf3-dependent dendritic cells elicit the rejection of cells and grafts expressing mismatched minor Ags. The implication of our findings in clinical transplantation may be significant, as minor Ag reactivity has been implicated in the pathogenesis of multiple allograft tissues.

Dendritic cell subsets require cis-activation for cytotoxic CD8 T-cell induction.

Dendritic cells (DCs) are required for the induction of cytotoxic T cells (CTL). In most tissues, including the lung, the resident DCs fall into two types expressing the integrin markers CD103 and CD11b. The current supposition is that DC function is predetermined by lineage, designating the CD103(+) DC as the major cross-presenting DC able to induce CTL. Here we show that Poly I:C (TLR3 agonist) or R848 (TLR7 agonist) do not activate all endogenous DCs. CD11b(+) DCs can orchestrate a CTL response in vivo in the presence of a TLR7 agonist but not a TLR3 agonist, whereas CD103(+) DCs require ligation of TLR3 for this purpose. This selectivity does not extend to antigen cross-presentation for T-cell proliferation but is required for induction of cytotoxicity. Thus, we demonstrate that the ability of DCs to induce functional CTLs is specific to the nature of the pathogen-associated molecular pattern (PAMP) encountered by endogenous DC.

Minimal differentiation of classical monocytes as they survey steady-state tissues and transport antigen to lymph nodes.

It is thought that monocytes rapidly differentiate to macrophages or dendritic cells (DCs) upon leaving blood. Here we have shown that Ly-6C⁺ monocytes constitutively trafficked into skin, lung, and lymph nodes (LNs). Entry was unaffected in gnotobiotic mice. Monocytes in resting lung and LN had similar gene expression profiles to blood monocytes but elevated transcripts of a limited number of genes including cyclo-oxygenase-2 (COX-2) and major histocompatibility complex class II (MHCII), induced by monocyte interaction with endothelium. Parabiosis, bromodoxyuridine (BrdU) pulse-chase analysis, and intranasal instillation of tracers indicated that instead of contributing to resident macrophages in the lung, recruited endogenous monocytes acquired antigen for carriage to draining LNs, a function redundant with DCs though differentiation to DCs did not occur. Thus, monocytes can enter steady-state nonlymphoid organs and recirculate to LNs without differentiation to macrophages or DCs, revising a long-held view that monocytes become tissue-resident macrophages by default.