Search

In murine peritoneal and other serous cavities, Gata6 drives identity of the major resident cavity macrophage population, with a smaller subset of cavity macrophages dependent upon Irf4. Here we show that Gata6+ macrophages in the human peritoneum are rare, regardless of age. Instead, many aligned with CD206+LYVE1+ mouse cavity macrophages from a differentiation stage just preceding expression of Gata6. The comparatively low abundance of CD206+ converting macrophages in healthy C57BL/6J mice was sustained after high-fat diet feeding or in mice capture from the wild, suggesting that the major resident macrophages between the species share overlapping differentiation programs but differing predispositions to distinct differentiation stages. Irf4-dependent mouse serous cavity macrophages aligned closely with CD1c+CD14+CD64+ human peritoneal dendritic cells (DCs) that, in turn, resembled CD1c+CD14-CD64- peritoneal cDC2. Peritoneal DC1 and plasmacytoid DCs were rare in both species. This cross-species resource comparing mouse and human serous cavity immune cells will facilitate translational research.

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. ...

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.

Myeloid, T, and NK cells are key players in the elimination phase of cancer immunoediting, also referred to as cancer immunosurveillance. However, the role of B cells and NAbs, which are present prior to the encounter with cognate antigens, has been overlooked. One reason is due to the popular use of a single B cell-deficient mouse model, muMT mice. Cancer models using muMT mice display a similar tumor burden as their wild-type (WT) counterparts. Empirically, we observe what others have previously reported with muMT mice. However, using two other B cell-deficient mouse models (IgHELMD4 and CD19creDTA), we show a three- to fivefold increase in tumor burden relative to WT mice. In addition, using an unconventional, non-cancer-related, immune neoantigen model where hypoxic conditions and cell clustering are absent, we provide evidence that B cells and their innate, natural antibodies (NAbs) are critical for the detection and elimination of neoantigen-expressing cells. Finally, we find that muMT mice display anti-tumor immunity because of an unexpected compensatory mechanism consisting of significantly enhanced type 1 interferon (IFN)-producing plasmacytoid dendritic cells (pDCs), which recruit a substantial number of NK cells to the tumor microenvironment compared to WT mice. Diminishing this compensatory pDC-IFN-NK cell mechanism revealed that muMT mice develop a three- to fivefold increase in tumor burden compared to WT mice. In summary, our findings suggest that NAbs are part of an early defense against not only microorganisms and dying cells, but precancerous cells as well.

Macrophages have a key role in shaping the tumour microenvironment (TME), tumour immunity and response to immunotherapy, which makes them an important target for cancer treatment(1,2). However, modulating macrophages has proved extremely difficult, as we still lack a complete understanding of the molecular and functional diversity of the tumour macrophage compartment. Macrophages arise from two distinct lineages. Tissue-resident macrophages self-renew locally, independent of adult haematopoiesis(3–5), whereas short-lived monocyte-derived macrophages arise from adult haematopoietic stem cells, and accumulate mostly in inflamed lesions(1). How these macrophage lineages contribute to the TME and cancer progression remains unclear. To explore the diversity of the macrophage compartment in human non-small cell lung carcinoma (NSCLC) lesions, here we performed single-cell RNA sequencing of tumour-associated leukocytes. We identified distinct populations of macrophages that were enriched in human and mouse lung tumours. Using lineage tracing, we discovered that these macrophage populations differ in origin and have a distinct temporal and spatial distribution in the TME. Tissue-resident macrophages accumulate close to tumour cells early during tumour formation to promote epithelial–mesenchymal transition and invasiveness in tumour cells, and they also induce a potent regulatory T cell response that protects tumour cells from adaptive immunity. Depletion of tissue-resident macrophages reduced the numbers and altered the phenotype of regulatory T cells, promoted the accumulation of CD8(+) T cells and reduced tumour invasiveness and growth. During tumour growth, tissue-resident macrophages became redistributed at the periphery of the TME, which becomes dominated by monocyte-derived macrophages in both mouse and human NSCLC. This study identifies the contribution of tissue-resident macrophages to early lung cancer and establishes them as a target for the prevention and treatment of early lung cancer lesions.

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.

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.

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.

It is commonly believed that B cells play no role in cancer immune surveillance. However, this conclusion is based on studies using only a single B cell-deficient mouse strain, muMT mice, which does not show increased tumorigenesis compared with wild-type (WT) mice. In this study, we demonstrate a critical role for B cells in anti-tumor immunity and identify the cellular mechanisms that make muMT mice a special case. First, we replicate previous findings using a melanoma model in muMT mice. Then, we show that in muMT mice, B-cell deficiency is compensated for by a significant increase in another potent anti-tumor cell type, Type-1 interferon (IFN I)-producing plasmacytoid DCs (pDCs), resulting in normative anti-tumor responses. Depleting pDCs in muMT mice resulted in a significant increase in tumor size and burden. Conversely, adoptive transfer of antibodies from naïve WT serum into pDC-depleted muMT mice significantly decreased the tumor load to WT levels. Additionally, a B cell antibody repertoire-deficient mouse strain, IghelMD4 mice, showed a 3-fold increase in tumors relative to WT mice. Overall, these findings indicate the need for a diverse antibody repertoire for early neoplastic cell recognition and the critical role B cells play in anti-cancer immunity.

We dissect metabolic variability of mononuclear phagocyte (MNP) subpopulations across different tissues through integrative analysis of three large scale datasets. Specifically, we introduce ImmGen MNP Open Source dataset that profiled 337 samples and extended previous ImmGen effort which included 202 samples of mononuclear phagocytes and their progenitors. Next, we analysed Tabula Muris Senis dataset to extract data for 51,364 myeloid cells from 18 tissues. Taken together, a compendium of data assembled in this work covers phagocytic populations found across 38 different tissues. To analyse common metabolic features, we developed novel network-based computational approach for unbiased identification of key metabolic subnetworks based on cellular transcriptional profiles in large-scale datasets. Using ImmGen MNP Open Source dataset as baseline, we define 9 metabolic subnetworks that encapsulate the metabolic differences within mononuclear phagocytes, and demonstrate that these features are robustly found across all three datasets, including lipid metabolism, cholesterol biosynthesis, glycolysis, and a set of fatty acid related metabolic pathways, as well as nucleotide and folate metabolism. We systematically define major features specific to macrophage and dendritic cell subpopulations. Among other things, we find that cholesterol synthesis appears particularly active within the migratory dendritic cells. We demonstrate that interference with this pathway through statins administration diminishes migratory capacity of the dendritic cells in vivo. This result demonstrates the power of our approach and highlights importance of metabolic diversity among mononuclear phagocytes.

SUMMARY 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., Graphical Abstract, In Brief Understanding the homology of mononuclear phagocytes between humans and mice is essential for translating murine studies into human medicine. Leach et al. use in-depth RNA sequencing to define highly discriminatory marker genes for 15 human and 9 mouse MPs that align cross-species homologs and define conserved and divergent marker genes.

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-1β-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.

Dendritic cells (DCs) are highly specialized immune cells that capture antigens and then migrate to lymphoid tissue and present antigen to T cells. This critical function of DCs is well defined, and recent studies further demonstrate that DCs are also key regulators of several innate immune responses. Studies focused on the roles of DCs in the pathogenesis of common lung diseases, such as asthma, infection, and cancer, have traditionally driven our mechanistic understanding of pulmonary DC biology. The emerging development of novel DC reagents, techniques, and genetically modified animal models has provided abundant data revealing distinct populations of DCs in the lung, and allow us to examine mechanisms of DC development, migration, and function in pulmonary disease with unprecedented detail. This enhanced understanding of DCs permits the examination of the potential role of DCs in diseases with known or suspected immunological underpinnings. Recent advances in the study of rare lung diseases, including pulmonary Langerhans cell histiocytosis, sarcoidosis, hypersensitivity pneumonitis, and pulmonary fibrosis, reveal expanding potential pathogenic roles for DCs. Here, we provide a review of DC development, trafficking, and effector functions in the lung, and discuss how alterations in these DC pathways contribute to the pathogenesis of rare lung diseases.

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.

Monocytes develop in the bone marrow and represent the primary type of mononuclear phagocyte found in the blood. They were long thought of as a source for tissue macrophages, but recent studies indicate more complex roles for monocytes, both within the circulation and after their migration into tissues and lymphoid organs. In this Review, we discuss the newer concepts underlying the maturation of emigrating monocytes into different classes of tissue macrophages, as well as their potential functions, as monocyte-derived cells, in the tissues. In addition, we consider the emerging roles for monocytes in adaptive immunity as antigen-presenting cells.

Proinflammatory consequences have been described for lysophosphatidylcholine, a lipid product of cellular injury, signaling via the G protein–coupled receptor G2A on myeloid and lymphoid inflammatory cells. This prompted the hypothesis that genetic deletion of G2A would limit intestinal inflammation in a mouse model of colitis induced by dextran sodium sulfate. Surprisingly, G2A−/− mice exhibited significantly worsened colitis compared with wild-type mice, as demonstrated by disease activity, colon shortening, histology, and elevated IL-6 and IL-5 in colon tissues. Investigation of inflammatory cells recruited to inflamed G2A−/− colons showed significantly more TNF-α+ and Ly6ChiMHCII− proinflammatory monocytes and eosinophils than in wild-type colons. Both monocytes and eosinophils were pathogenic as their depletion abolished the excess inflammation in G2A−/− mice. G2A−/− mice also had less IFN-γ in inflamed colon tissues than wild-type mice. Fewer CD4+ lymphocytes were recruited to inflamed G2A−/− colons, and fewer colonic lymphocytes produced IFN-γ upon ex vivo stimulation. Administration of IFN-γ to G2A−/− mice during dextran sodium sulfate exposure abolished the excess colitic inflammation and reduced colonic IL-5 and eosinophil numbers to levels seen in wild-type mice. Furthermore, IFN-γ reduced the numbers of TNF-α+ monocyte and enhanced their maturation from Ly6ChiMHCII− to Ly6CintMHCII+. Taken together, the data suggest that G2A signaling serves to dampen intestinal inflammation via the production of IFN-γ, which, in turn, enhances monocyte maturation to a less inflammatory program and ultimately reduces eosinophil-induced injury of colonic tissues.

Recently it was shown that circulating Ly6C(+) monocytes traffic from tissue to the draining lymph nodes (LNs) with minimal alteration in their overall phenotype. Furthermore, in the steady state, Ly6C(+) monocytes are as abundant as classical dendritic cells (DCs) within the draining LNs, and even more abundant during inflammation. However, little is known about the functional roles of constitutively trafficking Ly6C(+) monocytes. In this study we investigated whether Ly6C(+) monocytes can efferocytose (acquire dying cells) and cross-present cell-associated antigen, a functional property particularly attributed to Batf3(+) DCs. We demonstrated that Ly6C(+) monocytes intrinsically efferocytose and cross-present cell-associated antigen to CD8(+) T cells. In addition, efferocytosis was enhanced upon direct activation of the Ly6C(+) monocytes through its corresponding TLRs, TLR4 and TLR7. However, only ligation of TLR7, and not TLR4, enhanced cross-presentation by Ly6C(+) monocytes. Overall, this study outlines two functional roles, among others, that Ly6C(+) monocytes have during an adaptive immune response.

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.

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.

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.

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.

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.

Changes made in the 8th edition of the Guide for the Care and Use of Laboratory Animals included new recommendations for the amount of space for breeding female mice. Adopting the new recommendations required, in essence, the elimination of trio breeding practices for all institutions. Both public opinion and published data did not readily support the new recommendations. In response, the National Jewish Health Institutional Animal Care and Use Committee established a program to directly compare the effects of breeding format on mouse pup survival and growth. Our study showed an overall parity between trio and pairwise breeding formats on the survival and growth of the litters, suggesting that the housing recommendations for breeding female mice as stated in the current Guide for the Care and Use of Laboratory Animals should be reconsidered.

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.

Given the dual and intrinsically contradictory roles of myeloid cells in both protective and yet also damaging effects of inflammatory and immunological processes, we suggest that it is important to consider the mechanisms and circumstances by which these cells are removed, either in the normal unchallenged state or during inflammation or disease. In this essay we address these subjects from a conceptual perspective, focusing as examples on four main myeloid cell types (neutrophils, monocytes, macrophages, and myeloid dendritic cells) and their clearance from the circulation or from naive and inflamed tissues. While the primary clearance process appears to involve endocytic uptake into macrophages, various tissue cell types can also recognize and remove dying cells, though their overall quantitative contribution is unclear. In fact, surprisingly, given the wealth of study in this area over the last 30 years, our conclusion is that we are still challenged with a substantial lack of mechanistic and regulatory understanding of when, how, and by what mechanisms migratory myeloid cells come to die and are recognized as needing to be removed, and indeed the precise processes of uptake of either the intact or fragmented cells. This reflects the extreme complexity and inherent redundancy of the clearance processes and argues for substantial investigative effort in this arena. In addition, it leads us to a sense that approaches to significant therapeutic modulation of selective myeloid clearance are still a long way off.

We re-examined the observation that γδ T cells, when transferred from mice tolerized to an inhaled conventional Ag, suppress the allergic IgE response to this Ag specifically. Using OVA and hen egg lysozyme in crisscross fashion, we confirmed the Ag-specific IgE-regulatory effect of the γδ T cells. Although only Vγ4+ γδ T cells are regulators, the Ag specificity does not stem from specificity of their γδ TCRs. Instead, the Vγ4+ γδ T cells failed to respond to either Ag, but rapidly acquired Ag-specific regulatory function in vivo following i.v. injection of non-T cells derived from the spleen of Ag-tolerized mice. This correlated with their in vivo Ag acquisition from i.v. injected Ag-loaded splenic non-T cells, and in vivo transfer of membrane label provided evidence for direct contact between the injected splenic non-T cells and the Vγ4+ γδ T cells. Together, our data suggest that Ag itself, when acquired by γδ T cells, directs the specificity of their IgE suppression.

SummaryIt 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.

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.Phenotypic and morphologic characterization of mononuclear phagocytes that potentially access inhaled antigens in human lungs.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.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.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.

During acute lung injury (ALI) the macrophage pool expands markedly as inflammatory monocytes migrate from the circulation to the airspaces. As inflammation resolves, macrophage numbers return to preinjury levels and normal tissue structure and function are restored.To determine the fate of resident and recruited macrophages during the resolution of ALI in mice and to elucidate the mechanisms responsible for macrophage removal.ALI was induced in mice using influenza A (H1N1; PR8) infection and LPS instillation. Dye labeling techniques, bone marrow transplantation, and surface immunophenotyping were used to distinguish resident and recruited macrophages during inflammation and to study the role of Fas in determining macrophage fate during resolving ALI.During acute and resolving lung injury from influenza A and LPS, a high proportion of the original resident alveolar macrophages persisted. In contrast, recruited macrophages exhibited robust accumulation in early inflammation, followed by a progressive decline in their number. This decline was mediated by apoptosis with local phagocytic clearance. Recruited macrophages expressed high levels of the death receptor Fas and were rapidly depleted from the airspaces by Fas-activating antibodies. In contrast, macrophage depletion was inhibited in mice treated with Fas-blocking antibodies and in chimeras with Fas-deficient bone marrow. Caspase-8 inhibition prevented macrophage apoptosis and delayed the resolution of ALI.These findings indicate that Fas-induced apoptosis of recruited macrophages is essential for complete resolution of ALI.

CD103-expressing dendritic cells in the lungs preferentially take up and cross-present antigen from apoptotic cells., Cells undergoing programmed cell death (apoptosis) are removed in situ by macrophages and dendritic cells (DCs) through a specialized form of phagocytosis (efferocytosis). In the lung, there are two primary DC subsets with the potential to migrate to the local lymph nodes (LNs) and initiate adaptive immune responses. In this study, we show that only CD103+ DCs were able to acquire and transport apoptotic cells to the draining LNs and cross present apoptotic cell–associated antigen to CD8 T cells. In contrast, both the CD11bhi and the CD103+ DCs were able to ingest and traffic latex beads or soluble antigen. CD103+ DCs selectively exhibited high expression of TLR3, and ligation of this receptor led to enhanced in vivo cytotoxic T cell responses to apoptotic cell–associated antigen. The selective role for CD103+ DCs was confirmed in Batf3−/− mice, which lack this DC subtype. Our findings suggest that CD103+ DCs are the DC subset in the lung that captures and presents apoptotic cell–associated antigen under homeostatic and inflammatory conditions and raise the possibility for more focused immunological targeting to CD8 T cell responses.

Monocytes are central mediators in the advance of atherosclerotic plaque, making them a natural therapeutic target for reducing disease burden. Here, we highlight recent advances in our current understanding of monocyte heterogeneity and its relevance to regulation of monocyte accumulation and function within atherosclerotic plaques. Differences that distinguish monocyte subsets include differential expression of chemokine receptors, especially CCR2 and CX3CR1. Ablation of expression of these 2 receptors (or their ligands) in mice has an additive inhibition on monocyte recruitment to atherosclerotic plaques. Moreover, simultaneously interfering with 3 key pathways—CCR2, CX3CR1, and CCR5—essentially abolishes atherosclerosis in mice. Here, we discuss how these chemokine receptors act at multiple points on at least 1 monocyte subset, regulating their mobilization from bone marrow, survival, or recruitment to plaques. Finally, we discuss how this knowledge may be useful clinically, emphasizing that CX3CR1 may in particular be a viable target for therapeutic manipulation of monocyte-derived cell fate in cardiovascular disease.

Observations that dendritic cells (DCs) constitutively enter afferent lymphatic vessels in many organs and that DCs in some tissues, such as the lung, turnover rapidly in the steady state have led to the concept that a major fraction of lymph node DCs are derived from migratory DCs that enter the lymph node through upstream afferent lymphatic vessels. We used the lysozyme M–Cre reporter mouse strain to assess the relationship of lymph node and nonlymphoid organ DCs. Our findings challenge the idea that a substantial proportion of lymph node DCs derive from the upstream tissue during homeostasis. Instead, our analysis suggests that nonlymphoid organ DCs comprise a major population of DCs within lymph nodes only after introduction of an inflammatory stimulus.

Dendritic cell migration from the airway to lymph nodes is a key event in the development of airway immunity during infection, allergy, and vaccination. To identify the best approaches to investigate DC migration to lung-draining lymph nodes, we directly compared three methods previously used to track DC migration: airway administration of fluorescent OVA, latex beads, or carboxyfluorescein succinimidyl ester (CFSE). We show that two of the methods employed in optimal conditions-administration of fluorescent OVA or latex particles-have broadly relevant utility in studies of pulmonary DC migration, both in the presence and absence of inflammatory mediators. However, CFSE was of limited value because it induced a robust airway inflammatory response upon instillation. Unexpectedly, antigen-loaded tracers with distinct physical properties differently affected the populations that acquired the tracers and the overall T cell response. Specifically, soluble OVA and OVA formulated as a particulate after conjugation to latex beads were acquired in different proportions in vivo by the two characterized subsets of pulmonary DCs: CD11b(hi)CD103(-) and CD11b(lo)CD103(+)langerin(+) DC populations. Consequently, and in line with recent studies that these two subsets of DCs respectively activate CD4(+) and CD8(+) lymphocyte populations, the physical nature of the antigen delivery vehicle strongly influenced the degree of CD4(+) versus CD8(+) OVA-specific T cell activation. This finding suggests that changes in the physical presentation of the same antigen delivered to the airway during natural immune responses or vaccinations may markedly affect the character of the T cell response that ensues.

There are two major myeloid pulmonary dendritic cell (DC) populations: CD103+ DCs and CD11bhigh DCs. In this study, we investigated in detail the origins of both myeloid DC pools using multiple experimental approaches. We show that, in resting lung, Ly-6ChighCCR2high monocytes repopulated CD103+ DCs using a CCR2-dependent mechanism, and these DCs preferentially retained residual CCR2 in the lung, whereas, conversely, Ly-6ClowCCR2low monocytes repopulated CD11bhigh DCs. CX3CR1 was required to generate normal numbers of pulmonary CD11bhigh DCs, possibly because Ly-6Clow monocytes in the circulation, which normally express high levels of CX3CR1, failed to express bcl-2 and may have diminished survival in the circulation in the absence of CX3CR1. Overall, these data demonstrate that the two circulating subsets of monocytes give rise to distinct tissue DC populations.

Idiopathic pulmonary fibrosis (IPF)/usual interstitial pneumonia (UIP) is the severest form of idiopathic interstitial pneumonia for which therapeutic targets are needed. Surgical lung biopsy specimens from IPF/UIP patients exhibit focal expression of CC chemokine receptor (CCR) 7, but the identity of these CCR7-positive cells is unknown. The purpose of the present study was to examine the functional and signalling significance of CCR7 expression of primary fibroblasts grown from IPF/UIP and normal surgical lung biopsy specimens. Primary fibroblasts were cultured from surgical lung biopsy specimens from IPF/UIP and normal patients. Fibroblasts treated with or without CC chemokine ligand (CCL) 21 were analysed for functional, transcriptional and proteomic differences using immunocytochemical analysis, gene arrays, Taqman real-time PCR, and migration, proliferation and Western blot assays. CCR7 was expressed by IPF/UIP fibroblasts, but not normal fibroblasts. IPF/UIP fibroblasts, but not normal fibroblasts, showed significant migratory and proliferative responses when exposed to CCL21, which were inhibited by pertussis toxin or neutralising antibodies to CCR7. Exposure of IPF/UIP fibroblasts to CCL21 altered the phosphorylation status of mitogen-activated protein kinase kinase 1/2, extracellular signal-regulated kinase 1/2 and ribosomal S6 kinase (90 kDa) in these cells; this was abrogated by pertussis toxin or CCR7-specific small interfering RNA. Together, these data demonstrate that CC chemokine ligand 21 modulates the functional properties of idiopathic pulmonary fibrosis/usual interstitial pneumonia fibroblasts, but not normal fibroblasts.

Monocytes participate critically in atherosclerosis. There are 2 major subsets expressing different chemokine receptor patterns: CCR2(+)CX3CR1(+)Ly-6C(hi) and CCR2(-)CX3CR1(++)Ly-6C(lo) monocytes. Both C-C motif chemokine receptor 2 (CCR2) and C-X(3)-C motif chemokine receptor 1 (CX3CR1) are linked to progression of atherosclerotic plaques. Here, we analyzed mouse monocyte subsets in apoE-deficient mice and traced their differentiation and chemokine receptor usage as they accumulated within atherosclerotic plaques. Blood monocyte counts were elevated in apoE(-/-) mice and skewed toward an increased frequency of CCR2(+)Ly-6C(hi) monocytes in apoE(-/-) mice fed a high-fat diet. CCR2(+)Ly-6C(hi) monocytes efficiently accumulated in plaques, whereas CCR2(-)Ly-6C(lo) monocytes entered less frequently but were more prone to developing into plaque cells expressing the dendritic cell-associated marker CD11c, indicating that phagocyte heterogeneity in plaques is linked to distinct types of entering monocytes. CCR2(-) monocytes did not rely on CX3CR1 to enter plaques. Instead, they were partially dependent upon CCR5, which they selectively upregulated in apoE(-/-) mice. By comparison, CCR2(+)Ly-6C(hi) monocytes unexpectedly required CX3CR1 in addition to CCR2 and CCR5 to accumulate within plaques. In many other inflammatory settings, these monocytes utilize CCR2, but not CX3CR1, for trafficking. Thus, antagonizing CX3CR1 may be effective therapeutically in ameliorating CCR2(+) monocyte recruitment to plaques without impairing their CCR2-dependent responses to inflammation overall.

Monocytes are circulating precursors for tissue macrophages and dendritic cells (DCs) but are not recognized to directly participate in antigen presentation. We developed techniques to label mouse monocyte subsets with particulate tracers in vivo. Gr-1lo but not Gr-1hi monocytes were stably labeled by intravenous injection of 0.5-μm microspheres. Gr-1hi monocytes could be labeled when the microspheres were injected after systemic depletion of blood monocytes and spleen macrophages. In this condition, the phagocytic tracer was transferred to immature bone marrow monocytes by neutrophils and B cells that first carried the particles to the bone marrow. Moreover, antigens from B cells or proteins conjugated to the tracer particles were processed for presentation by monocytes and could induce T cell responses in the periphery. Cell-associated antigen taken up by bone marrow monocytes was retained intracellularly for presentation of the antigen days later when monocyte-derived DCs migrated to lymph nodes or in vitro after differentiation with granulocyte/macrophage colony-stimulating factor. These data reveal that immature monocytes unexpectedly sample antigen from the bone marrow environment and that they can present these antigens after they leave the bone marrow.

Chemokines are increased and may exert effects on both inflammatory and remodeling events in idiopathic pulmonary pneumonia (IIP). Accordingly, we examined the concomitant expression of inflammatory CC chemotactic cytokines or chemokines and their corresponding receptors in surgical lung biopsies obtained at the time of disease diagnosis and pulmonary fibroblasts grown from these biopsies. By gene array analysis, upper and lower lobe biopsies and primary fibroblast lines from patients with usual interstitial pneumonia (UIP), nonspecific interstitial pneumonia, and respiratory bronchiolitis-interstitial lung disease, but not patients without IIP, exhibited CCL7 gene expression. TAQMAN, immunohistochemical, and ELISA analyses confirmed that CCL7 was expressed at significantly higher levels in UIP lung biopsies compared with biopsies from patients with nonspecific interstitial pneumonia, respiratory bronchiolitis-interstitial lung disease, and from patients without IIP. Higher levels of CCL7 were present in cultures of IIP fibroblasts compared with non-IIP fibroblasts, and CCL5, a CCR5 agonist, significantly increased the synthesis of CCL7 by UIP fibroblasts. Together, these data suggest that CCL7 is highly expressed in biopsies and pulmonary fibroblast lines obtained from patients with UIP relative to patients with other IIP and patients without IIP, and that this CC chemokine may have a major role in the progression of fibrosis in this IIP patient group.

Background: Some idiopathic interstitial pneumonias (IIPs) are characterised by fibroproliferation and deposition of extracellular matrix. Because efficacious treatment options are limited, research has been directed towards understanding the cytokine networks that may affect fibroblast activation and, hence, the progression of certain IIPs. Aims: To examine the expression of interleukin 4 (IL-4), IL-13, and their corresponding receptor subunits in the various forms of IIP and normal patient groups. Methods: Molecular and immunohistochemical analysis of IL-4, interferon γ (IFNγ), IL-13, IL-4 receptor (IL-R), and IL-13 receptor subunits in surgical lung biopsies (SLBs) from 39 patients (21 usual interstitial pneumonia (UIP), six non-specific interstitial pneumonia (NSIP), eight respiratory bronchiolitic interstitial lung disease (RBILD), and five normal controls). Results: Molecular analysis demonstrated that IL-13Rα2, IL-13Rα1, and IL-4Rα were present in a greater proportion of upper and lower lobe biopsies from patients with UIP than patients with NSIP and RBILD. Immunohistochemical analysis of patients with UIP, NSIP, and RBILD revealed interstitial staining for all three receptor subunits, whereas such staining was only seen in mononuclear cells present in normal SLBs. Fibroblastic foci in patients with UIP strongly stained for IL-4Rα and IL-13Rα2. Localised expression of IL-4Rα was also seen in SLBs from patients with NSIP but not in other groups. Conclusion: Some histological subtypes of IIP are associated with increased pulmonary expression of receptor subunits responsive to IL-4 and IL-13. These findings may be of particular importance in understanding the pathogenesis of IIP and, more importantly, may provide important novel therapeutic targets.

Intratracheal injection of FITC results in acute lung injury and progresses to fibrosis by day 21 postchallenge. In response to FITC, BALB/c mice produce IL-4 and IL-13 in the lung. To investigate whether IL-4 and/or IL-13 were important profibrotic mediators in this model, we examined the fibrotic response to FITC in mice that were genetically deficient in IL-4 (IL-4−/−), IL-13 (IL-13−/−), or IL-4 and IL-13 combined (IL-4/13−/−). Baseline levels of collagen were similar in all mice. In response to FITC, both BALB/c and IL-4−/− mice developed fibrosis, whereas the IL-13−/− and IL-4/13−/− mice were significantly protected, as measured by total lung collagen levels and histology. Total leukocyte recruitment to the lung was similar in all four strains of mice when measured on days 7, 14, and 21 post-FITC. BALB/c mice showed prominent eosinophilia on day 7 that was absent in IL-4−/−, IL-13−/−, and IL-4/13−/− mice, suggesting that eosinophilia is not necessary for development of a fibrotic response. There were no significant differences in the percentages of any other leukocytes analyzed between the genotypes. Similarly, protection in IL-13−/− mice was not associated with alterations in cytokine or eicosanoid profiles. Interestingly, TGF-β1 production was not reduced in IL-13−/− mice. Analyses of fibroblasts isolated from the four genotypes demonstrated that although there were similar numbers of fibroblasts present in cultures of lung minces, fibroblasts from IL-13-deficient strains have reduced basal and stimulated levels of collagen production. IL-13Rα1 expression increases on fibroblasts during fibrotic responses in vivo, and IL-13 increases collagen synthesis in fibroblasts. Thus, IL-13 mediates its profibrotic actions through direct effects on fibroblast production of extracellular matrix.

Severe forms of idiopathic interstitial pneumonia (IIP), such as usual interstitial pneumonia, can be impervious to modern steroid and immunosuppressive treatment regimens, thereby emphasizing the need for novel effective therapies. Consequently, research attention has been directed toward understanding the cytokine networks that may affect fibroblast activation and, hence, the progression of certain IIPs. This led us to investigate whether the specific targeting of resident lung cells responsive to IL-4 and IL-13 exerted a therapeutic effect in an experimental model of IIP, namely the bleomycin-induced model of pulmonary fibrosis. IL-4, IL-13, and their corresponding receptor subunits, IL-4Rα, IL-13Rα1, and IL-13Rα2, were maximally expressed at the mRNA and protein levels in whole lung samples on day 21 or 28 after an intratracheal bleomycin challenge. The intranasal administration of an IL-13 immunotoxin chimeric molecule (IL13-PE) from days 21–28, but not for 1-wk periods at earlier times, after bleomycin challenge had a significant therapeutic effect on histological and biochemical parameters of bleomycin-induced pulmonary fibrosis compared with the control group. The intranasal IL13-PE therapy significantly reduced the numbers of IL-4 and IL-13 receptor-positive mononuclear cells and macrophages and the levels of profibrotic cytokine and chemokine in the lungs of bleomycin-challenged mice on day 28. Thus, this study demonstrates that IL-4- and/or IL-13-binding cells are required for the maintenance of pulmonary fibrosis induced by bleomycin and highlights the importance of further investigation of antifibrotic therapeutics that target these cells during pulmonary fibrosis.

Interleukin-13 (IL-13) has emerged as a major cytokine mediator of fibroblast activation and pulmonary fibrosis. Normal (from noninflamed lung), Th1-type (induced by the pulmonary embolization of purified peptide derivative-coated beads in mice sensitized to purified peptide derivative), and Th2-type (induced by the pulmonary embolization of Schistosoma mansoni egg antigen-coated beads in mice sensitized with S. mansoni eggs) primary fibroblast cell lines all exhibited constitutive gene expression of two receptor chains that bind and signal IL-13-mediated cellular events: IL-4Ralpha and IL-13Ralpha1. However, all three fibroblast cell lines exhibited divergent synthetic and proliferative responses to the exogenous addition of either recombinant IL-13 or a chimeric protein comprised of IL-13 and a truncated version of Pseudomonas exotoxin (IL13-PE), which targets and kills IL-13 receptor overexpressing cells. The exogenous addition of IL-13 to Th1-type and Th2-type fibroblast cultures significantly increased the cellular expression of IL-13Ralpha2, which may function as an IL-13 decoy receptor. After a 24-hour exposure to IL-13, the total collagen generation and cellular proliferation by Th2-type fibroblasts were significantly higher than that observed in similar numbers of normal and Th1-type fibroblasts. In addition IL13-PE, which binds with highest affinity to IL-13Ralpha2, exhibited down-regulatory effects on proliferation and matrix generation expression by Th1- and Th2-type, but not normal, fibroblasts. Thus, these data demonstrate that fibroblasts derived from murine pulmonary granulomas exhibit divergent expression of functional IL-13 receptor and this expression dictates the responsiveness and susceptibility to recombinant IL-13 and IL-13 immunotoxin, respectively.

Signal transducer and activator of transcription 6 (Stat6) is critical for Th2-mediated responses during allergic airway disease. To investigate the role of Stat6 in fungus-induced airway hyperresponsiveness and remodeling, Stat6-deficient (Stat6−/−) and Stat6-wildtype (Stat6+/+) mice were sensitized to Aspergillus fumigatus and airway disease was subsequently assessed in both groups at days 21, 30, 38, and 44 after an intratracheal challenge with live A. fumigatus conidia. At all times after conidia, histological analysis revealed an absence of goblet cell hyperplasia and markedly diminished peribronchial inflammation in Stat6−/− mice in contrast to Stat6+/+ mice. Airway hyperresponsiveness and peribronchial fibrosis in Stat6−/− mice were significantly reduced at day 21 after conidia compared with Stat6+/+ mice, but both groups exhibited significant, similar increases in these parameters at all subsequent times after conidia. In separate experiments, IL-13-responsive cells in Stat6−/− mice were targeted via the daily intranasal administration of 200 ng of IL-13-PE38QQR (IL13-PE), comprised of human IL-13 and a derivative of Pseudomonas exotoxin, from days 38 to 44 after the conidia challenge. IL13-PE treatment abolished airway hyperresponsiveness, but not peribronchial fibrosis in Stat6−/− mice. Taken together, these data demonstrate that the chronic development of airway hyperresponsiveness during fungal asthma is IL-13-dependent but Stat6-independent.