Coordinated chemokine expression defines macrophage subsets across tissues.

Lung-resident macrophages, which include alveolar macrophages and interstitial macrophages (IMs), exhibit a high degree of diversity, generally attributed to different activation states, and often complicated by the influx of monocytes into the pool of tissue-resident macrophages. To gain a deeper insight into the functional diversity of IMs, here we perform comprehensive transcriptional profiling of resident IMs and reveal ten distinct chemokine-expressing IM subsets at steady state and during inflammation. Similar IM subsets that exhibited coordinated chemokine signatures and differentially expressed genes were observed across various tissues and species, indicating conserved specialized functional roles. Other macrophage types shared specific IM chemokine profiles, while also presenting their own unique chemokine signatures. Depletion of CD206hi IMs in Pf4creR26EYFP+DTR and Pf4creR26EYFPCx3cr1DTR mice led to diminished inflammatory cell recruitment, reduced tertiary lymphoid structure formation and fewer germinal center B cells in models of allergen- and infection-driven inflammation. These observations highlight the specialized roles of IMs, defined by their coordinated chemokine production, in regulating immune cell influx and organizing tertiary lymphoid tissue architecture.

Human serous cavity macrophages and dendritic cells possess counterparts in the mouse with a distinct distribution between species.

In mouse peritoneal and other serous cavities, the transcription factor GATA6 drives the identity of the major cavity resident population of macrophages, with a smaller subset of cavity-resident macrophages dependent on the transcription factor IRF4. Here we showed that GATA6+ macrophages in the human peritoneum were rare, regardless of age. Instead, more human peritoneal macrophages aligned with mouse CD206+ LYVE1+ cavity macrophages that represent a differentiation stage just preceding expression of GATA6. A low abundance of CD206+ macrophages was retained in C57BL/6J mice fed a high-fat diet and in wild-captured mice, suggesting that differences between serous cavity-resident macrophages in humans and mice were not environmental. IRF4-dependent mouse serous cavity macrophages aligned closely with human CD1c+CD14+CD64+ peritoneal cells, which, in turn, resembled human peritoneal CD1c+CD14-CD64- cDC2. Thus, major populations of serous cavity-resident mononuclear phagocytes in humans and mice shared common features, but the proportions of different macrophage differentiation stages greatly differ between the two species, and dendritic cell (DC2)-like cells were especially prominent in humans.

Macrophages show up in style when Th2 lymphocytes organize their homecoming.

Monocytes can differentiate into tissue-resident pleural macrophages, but the mechanisms underlying this process are not yet fully understood. In this issue of Immunity, Finlay et al.1 show that Th2 cytokines promote this differentiation in resistant mice infected with Litomosoides sigmodontis.

Biology of lung macrophages in health and disease.

Tissue-resident alveolar and interstitial macrophages and recruited macrophages are critical players in innate immunity and maintenance of lung homeostasis. Until recently, assessing the differential functional contributions of tissue-resident versus recruited macrophages has been challenging because they share overlapping cell surface markers, making it difficult to separate them using conventional methods. This review describes how scRNA-seq and spatial transcriptomics can separate these subpopulations and help unravel the complexity of macrophage biology in homeostasis and disease. First, we provide a guide to identifying and distinguishing lung macrophages from other mononuclear phagocytes in humans and mice. Second, we outline emerging concepts related to the development and function of the various lung macrophages in the alveolar, perivascular, and interstitial niches. Finally, we describe how different tissue states profoundly alter their functions, including acute and chronic lung disease, cancer, and aging.

Immunogenicity Threshold in Allogeneic Cells Impacts CTL Response to Nondominant Congenic Antigens.

Transplantation and cancer expose the immune system to neoantigens, including immunogenic (dominant and subdominant) and nonimmunogenic Ags with varying quantities and affinities of immunodominant peptides. Conceptually, immunity is believed to mainly target dominant Ags when subdominant or nondominant Ags are linked within the same cell due to T cell interference. This phenomenon is called immunodominance. However, our previous study in mice showed that linked nonimmunogenic Ags (OVA and GFP) containing immunodominant peptides mount immunity irrespective of the MHC-matched allogeneic cell's immunogenicity. Consequently, we further explored 1) under what circumstances does the congenic marker CD45.1 provoke immunity in CD45.2 mice, and 2) whether linking two dominant or subdominant Ags can instigate an immune response. Our observations showed that CD45.1 (or CD45.2), when connected to low-immunogenic cell types is presented as an immunogen, which contrasts with its outcome when linked to high-immunogenic cell types. Moreover, we found that both dominant and subdominant Ags are presented as immunogens when linked in environments with lower immunogenic thresholds. These findings challenge the existing perception that immunity is predominantly elicited against dominant Ags when linked to subdominant or nondominant Ags. This study takes a fundamental step toward understanding the nuanced relationship between immunogenic and nonimmunogenic Ags, potentially opening new avenues for comprehending cancer immunoediting and enhancing the conversion of cold tumors with low immunogenicity into responsive hot tumors.

Natural Antibodies Alert the Adaptive Immune System of the Presence of Transformed Cells in Early Tumorigenesis.

Recent studies have revealed a critical role for natural Abs (NAbs) in antitumor immune responses. However, the role of NAbs in cancer immunosurveillance remains unexplored, mainly because of the lack of in vivo models that mimic the early recognition and elimination of transforming cells. In this article, we propose a role for NAbs in alerting the immune system against precancerous neoantigen-expressing cells immediately after they escape intrinsic tumor suppression mechanisms. We identify four distinct reproducible, trackable, MHC-matched neoantigen-expressing cell models that do not form tumors as the end point. This amplified readout in the critical window prior to tumor formation allows investigation of new mediators of cancer immunosurveillance. We found that neoantigen-expressing cells adoptively transferred in NAb-deficient mice persisted, whereas they were eliminated in wild-type mice, indicating that the circulating NAb repertoire alerts the immune system to the presence of transformed cells. Moreover, immunity is mounted against immunogenic and nonimmunogenic neoantigens contained in the NAb-tagged cells, regardless of whether the NAb directly recognizes the neoantigens. Beyond these neoantigen-expressing model systems, we observed a significantly greater tumor burden in chemically and virally induced tumor models in NAb-deficient mice compared with wild-type mice. Restoration of the NAb repertoire in NAb-deficient mice elicited the recognition and elimination of neoantigen-expressing cells and cancer. These data show that NAbs are required and sufficient for elimination of transformed cells early in tumorigenesis. These models can now be used to investigate how NAbs stimulate immunity via recognition receptors to eliminate precancerous cells.

Advancing Lung Immunology Research: An Official American Thoracic Society Workshop Report.

The mammalian airways and lungs are exposed to a myriad of inhaled particulate matter, allergens, and pathogens. The immune system plays an essential role in protecting the host from respiratory pathogens, but a dysregulated immune response during respiratory infection can impair pathogen clearance and lead to immunopathology. Furthermore, inappropriate immunity to inhaled antigens can lead to pulmonary diseases. A complex network of epithelial, neural, stromal, and immune cells has evolved to sense and respond to inhaled antigens, including the decision to promote tolerance versus a rapid, robust, and targeted immune response. Although there has been great progress in understanding the mechanisms governing immunity to respiratory pathogens and aeroantigens, we are only beginning to develop an integrated understanding of the cellular networks governing tissue immunity within the lungs and how it changes after inflammation and over the human life course. An integrated model of airway and lung immunity will be necessary to improve mucosal vaccine design as well as prevent and treat acute and chronic inflammatory pulmonary diseases. Given the importance of immunology in pulmonary research, the American Thoracic Society convened a working group to highlight central areas of investigation to advance the science of lung immunology and improve human health.

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.

Dietary Lipids Differentially Shape Nonalcoholic Steatohepatitis Progression and the Transcriptome of Kupffer Cells and Infiltrating Macrophages.

A crucial component of nonalcoholic fatty liver disease (NAFLD) pathogenesis is lipid stress, which may contribute to hepatic inflammation and activation of innate immunity in the liver. However, little is known regarding how dietary lipids, including fat and cholesterol, may facilitate innate immune activation in vivo. We hypothesized that dietary fat and cholesterol drive NAFLD progression to steatohepatitis and hepatic fibrosis by altering the transcription and phenotype of hepatic macrophages. This hypothesis was tested by using RNA-sequencing methods to characterize and analyze sort-purified hepatic macrophage populations that were isolated from mice fed diets with varying amounts of fat and cholesterol. The addition of cholesterol to a high-fat diet triggered hepatic pathology reminiscent of advanced nonalcoholic steatohepatitis (NASH) in humans characterized by signs of cholesterol dysregulation, generation of oxidized low-density lipoprotein, increased recruitment of hepatic macrophages, and significant fibrosis. RNA-sequencing analyses of hepatic macrophages in this model revealed that dietary cholesterol induced a tissue repair and regeneration phenotype in Kupffer cells (KCs) and recruited infiltrating macrophages to a greater degree than fat. Furthermore, comparison of diseased KCs and infiltrating macrophages revealed that these two macrophage subsets are transcriptionally diverse. Finally, direct stimulation of murine and human macrophages with oxidized low-density lipoprotein recapitulated some of the transcriptional changes observed in the RNA-sequencing study. These findings indicate that fat and cholesterol synergize to alter macrophage phenotype, and they also challenge the dogma that KCs are purely proinflammatory in NASH. Conclusion: This comprehensive view of macrophage populations in NASH indicates mechanisms by which cholesterol contributes to NASH progression and identifies potential therapeutic targets for this common disease.

Improving the Quality and Reproducibility of Flow Cytometry in the Lung. An Official American Thoracic Society Workshop Report.

Defining responses of the structural and immune cells in biologic systems is critically important to understanding disease states and responses to injury. This requires accurate and sensitive methods to define cell types in organ systems. The principal method to delineate the cell populations involved in these processes is flow cytometry. Although researchers increasingly use flow cytometry, technical challenges can affect its accuracy and reproducibility, thus significantly limiting scientific advancements. This challenge is particularly critical to lung immunology, as the lung is readily accessible and therefore used in preclinical and clinical studies to define potential therapeutics. Given the importance of flow cytometry in pulmonary research, the American Thoracic Society convened a working group to highlight issues and technical challenges to the performance of high-quality pulmonary flow cytometry, with a goal of improving its quality and reproducibility.

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.

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.

Cell Origin Dictates Programming of Resident versus Recruited Macrophages during Acute Lung Injury.

Two populations of alveolar macrophages (AMs) coexist in the inflamed lung: resident AMs that arise during embryogenesis, and recruited AMs that originate postnatally from circulating monocytes. The objective of this study was to determine whether origin or environment dictates the transcriptional, metabolic, and functional programming of these two ontologically distinct populations over the time course of acute inflammation. RNA sequencing demonstrated marked transcriptional differences between resident and recruited AMs affecting three main areas: proliferation, inflammatory signaling, and metabolism. Functional assays and metabolomic studies confirmed these differences and demonstrated that resident AMs proliferate locally and are governed by increased tricarboxylic acid cycle and amino acid metabolism. Conversely, recruited AMs produce inflammatory cytokines in association with increased glycolytic and arginine metabolism. Collectively, the data show that even though they coexist in the same environment, inflammatory macrophage subsets have distinct immunometabolic programs and perform specialized functions during inflammation that are associated with their cellular origin.

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.

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.

Collecting lymphatic vessel permeability facilitates adipose tissue inflammation and distribution of antigen to lymph node-homing adipose tissue dendritic cells.

Collecting lymphatic vessels (CLVs), surrounded by fat and endowed with contractile muscle and valves, transport lymph from tissues after it is absorbed into lymphatic capillaries. CLVs are not known to participate in immune responses. In this study, we observed that the inherent permeability of CLVs allowed broad distribution of lymph components within surrounding fat for uptake by adjacent macrophages and dendritic cells (DCs) that actively interacted with CLVs. Endocytosis of lymph-derived Ags by these cells supported recall T cell responses in the fat and also generated Ag-bearing DCs for emigration into adjacent lymph nodes (LNs). Enhanced recruitment of DCs to inflammation-reactive LNs significantly relied on adipose tissue DCs to maintain sufficient numbers of Ag-bearing DCs as the LN expanded. Thus, CLVs coordinate inflammation and immunity within adipose depots and foster the generation of an unexpected pool of APCs for Ag transport into the adjacent LN.

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.