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.

Single-cell RNA sequencing reveals unique monocyte-derived interstitial macrophage subsets during lipopolysaccharide-induced acute lung inflammation.

Interstitial macrophages (IMs) reside in the lung tissue surrounding key structures including airways, vessels, and alveoli. Recent work has described IM heterogeneity during homeostasis, however, there are limited data on IMs during inflammation. We sought to characterize IM origin, subsets, and transcriptomic profiles during homeostasis and lipopolysaccharide (LPS) induced acute lung inflammation. During homeostasis, we used three complementary methods, spectral flow cytometry, single-cell RNA-sequencing, and gene regulatory network enrichment, to demonstrate that IMs can be divided into two core subsets distinguished by surface and transcriptional expression of folate receptor ß (Folr2/FRß). These subsets inhabited distinct niches within the lung interstitium. Within FRß+ IMs we identified a subpopulation marked by coexpression of LYVE1. During acute LPS-induced inflammation, lung IM numbers expand. Lineage tracing revealed IM expansion was due to recruitment of monocyte-derived IMs. At the peak of inflammation, recruited IMs were comprised two unique subsets defined by expression of genes associated with interferon signaling and glycolytic pathways. As recruited IMs matured, they adopted the overall transcriptional state of FRß- resident IMs but retained expression in several origin-specific genes, such as IL-1ß. FRß+ IMs were of near-pure resident origin. Taken together our data show that during LPS-induced inflammation, there are distinct populations of IMs that likely have unique functions. FRΒ+ IMs comprise a stable, resident population, whereas FRß- ΙΜs represent a mixed population of resident and recruited IMs.

Cigarette smoke-induced airspace disease in mice develops independently of HIF-1α signaling in leukocytes.

The pathogenesis of chronic obstructive pulmonary disease (COPD), a prevalent disease primarily caused by cigarette smoke exposure, is incompletely elucidated. Studies in humans and mice have suggested that hypoxia-inducible factor-1α (HIF-1α) may play a role. Reduced lung levels of HIF-1α are associated with decreased vascular density, whereas increased leukocyte HIF-1α may be responsible for increased inflammation. To elucidate the specific role of leukocyte HIF-1α in COPD, we exposed transgenic mice with conditional deletion or overexpression of HIF-1α in leukocytes to cigarette smoke for 7 mo. Outcomes included pulmonary physiology, aerated lung volumes via microcomputed tomography, lung morphometry and histology, and cardiopulmonary hemodynamics. On aggregate, cigarette smoke increased the aerated lung volume, quasi-static lung compliance, inspiratory capacity of all strains while reducing the total alveolar septal volume. Independent of smoke exposure, mice with leukocyte-specific HIF-1α overexpression had increased quasi-static compliance, inspiratory capacity, and alveolar septal volume compared with mice with leukocyte-specific HIF-1α deletion. However, the overall development of cigarette smoke-induced lung disease did not vary relative to control mice for either of the conditional strains. This suggests that the development of murine cigarette smoke-induced airspace disease occurs independently of leukocyte HIF-1α signaling.

Polyamine import and accumulation causes immunomodulation in macrophages engulfing apoptotic cells.

Phagocytosis of apoptotic cells, termed efferocytosis, is critical for tissue homeostasis and drives anti-inflammatory programming in engulfing macrophages. Here, we assess metabolites in naive and inflammatory macrophages following engulfment of multiple cellular and non-cellular targets. Efferocytosis leads to increases in the arginine-derived polyamines, spermidine and spermine, in vitro and in vivo. Surprisingly, polyamine accumulation after efferocytosis does not arise from retention of apoptotic cell metabolites or de novo synthesis but from enhanced polyamine import that is dependent on Rac1, actin, and PI3 kinase. Blocking polyamine import prevents efferocytosis from suppressing macrophage interleukin (IL)-1ß or IL-6. This identifies efferocytosis as a trigger for polyamine import and accumulation, and imported polyamines as mediators of efferocytosis-induced immune reprogramming.

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.

Airspace Macrophages and Monocytes Exist in Transcriptionally Distinct Subsets in Healthy Adults.

Rationale: Macrophages are the most abundant immune cell in the alveoli and small airways and are traditionally viewed as a homogeneous population during health. Whether distinct subsets of airspace macrophages are present in healthy humans is unknown. Single-cell RNA sequencing allows for examination of transcriptional heterogeneity between cells and between individuals. Understanding the conserved repertoire of airspace macrophages during health is essential to understanding cellular programing during disease.Objectives: We sought to determine the transcriptional heterogeneity of human cells obtained from BAL of healthy adults.Methods: Ten subjects underwent bronchoscopy with BAL. Cells from lavage were subjected to single-cell RNA sequencing. Unique cell populations and putative functions were identified. Transcriptional profiles were compared across individuals.Measurements and Main Results: We identify two novel subgroups of resident airspace macrophages-defined by proinflammatory and metallothionein gene expression profiles. We define subsets of monocyte-like cells and compare them with peripheral blood mononuclear cells. Finally, we compare global macrophage and monocyte programing between males and females.Conclusions: Healthy human airspaces contain multiple populations of myeloid cells that are highly conserved between individuals and between sexes. Resident macrophages make up the largest population and include novel subsets defined by inflammatory and metal-binding profiles. Monocyte-like cells within the airspaces are transcriptionally aligned with circulating blood cells and include a rare population defined by expression of cell-matrix interaction genes. This study is the first to delineate the conserved heterogeneity of airspace immune cells during health and identifies two previously unrecognized macrophage subsets.

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.

Single cell RNA sequencing identifies unique inflammatory airspace macrophage subsets.

Macrophages are well recognized for their dual roles in orchestrating inflammatory responses and regulating tissue repair. In almost all acutely inflamed tissues, 2 main subclasses of macrophages coexist. These include embryonically derived resident tissue macrophages and BM-derived recruited macrophages. While it is clear that macrophage subsets categorized in this fashion display distinct transcriptional and functional profiles, whether all cells within these categories and in the same inflammatory microenvironment share similar functions or whether further specialization exists has not been determined. To investigate inflammatory macrophage heterogeneity on a more granular level, we induced acute lung inflammation in mice and performed single cell RNA sequencing of macrophages isolated from the airspaces during health, peak inflammation, and resolution of inflammation. In doing so, we confirm that cell origin is the major determinant of alveolar macrophage (AM) programing, and, to our knowledge, we describe 2 previously uncharacterized, transcriptionally distinct subdivisions of AMs based on proliferative capacity and inflammatory programing.

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.

Phagocytosis of microparticles by alveolar macrophages during acute lung injury requires MerTK.

Microparticles are a newly recognized class of mediators in the pathophysiology of lung inflammation and injury, but little is known about the factors that regulate their accumulation and clearance. The primary objective of our study was to determine whether alveolar macrophages engulf microparticles and to elucidate the mechanisms by which this occurs. Alveolar microparticles were quantified in bronchoalveolar fluid of mice with lung injury induced by LPS and hydrochloric acid. Microparticle numbers were greatest at the peak of inflammation and declined as inflammation resolved. Isolated, fluorescently labeled particles were placed in culture with macrophages to evaluate ingestion in the presence of endocytosis inhibitors. Ingestion was blocked with cytochalasin D and wortmannin, consistent with a phagocytic process. In separate experiments, mice were treated intratracheally with labeled microparticles, and their uptake was assessed though microscopy and flow cytometry. Resident alveolar macrophages, not recruited macrophages, were the primary cell-ingesting microparticles in the alveolus during lung injury. In vitro, microparticles promoted inflammatory signaling in LPS primed epithelial cells, signifying the importance of microparticle clearance in resolving lung injury. Microparticles were found to have phosphatidylserine exposed on their surfaces. Accordingly, we measured expression of phosphatidylserine receptors on macrophages and found high expression of MerTK and Axl in the resident macrophage population. Endocytosis of microparticles was markedly reduced in MerTK-deficient macrophages in vitro and in vivo. In conclusion, microparticles are released during acute lung injury and peak in number at the height of inflammation. Resident alveolar macrophages efficiently clear these microparticles through MerTK-mediated phagocytosis.

Neutrophil transfer of miR-223 to lung epithelial cells dampens acute lung injury in mice.

Intercellular transfer of microRNAs can mediate communication between critical effector cells. We hypothesized that transfer of neutrophil-derived microRNAs to pulmonary epithelial cells could alter mucosal gene expression during acute lung injury. Pulmonary-epithelial microRNA profiling during coculture of alveolar epithelial cells with polymorphonuclear neutrophils (PMNs) revealed a selective increase in lung epithelial cell expression of microRNA-223 (miR-223). Analysis of PMN-derived supernatants showed activation-dependent release of miR-223 and subsequent transfer to alveolar epithelial cells during coculture in vitro or after ventilator-induced acute lung injury in mice. Genetic studies indicated that miR-223 deficiency was associated with severe lung inflammation, whereas pulmonary overexpression of miR-223 in mice resulted in protection during acute lung injury induced by mechanical ventilation or by infection with Staphylococcus aureus Studies of putative miR-223 gene targets implicated repression of poly(adenosine diphosphate-ribose) polymerase-1 (PARP-1) in the miR-223-dependent attenuation of lung inflammation. Together, these findings suggest that intercellular transfer of miR-223 from neutrophils to pulmonary epithelial cells may dampen acute lung injury through repression of PARP-1.

Cell Removal: Efferocytosis.

In metazoans, removal of cells in situ is involved in larval maturation, metamorphosis, and embryonic development. In adults, such cell removal plays a role in the homeostatic maintenance of cell numbers and tissue integrity as well as in the response to cell injury and damage. This removal involves uptake of the whole or fragmented target cells into phagocytes. Depending on the organism, these latter may be near-neighbor tissue cells and/or professional phagocytes such as, in vertebrates, members of the myeloid family of cells, especially macrophages. The uptake processes appear to involve specialized and highly conserved recognition ligands and receptors, intracellular signaling in the phagocytes, and mechanisms for ingestion. The recognition of cells destined for this form of removal is critical and, significantly, is distinguished for the most part from the recognition of foreign materials and organisms by the innate and adaptive immune systems. In keeping with the key role of cell removal in maintaining tissue homeostasis, constant cell removal is normally silent, i.e., does not initiate a local tissue reaction. This article discusses these complex and wide-ranging processes in general terms as well as the implications when these processes are disrupted in inflammation, immunity, and disease.

Unbiased Quantitation of Alveolar Type II to Alveolar Type I Cell Transdifferentiation during Repair after Lung Injury in Mice.

The alveolar epithelium consists of squamous alveolar type (AT) I and cuboidal ATII cells. ATI cells cover 95-98% of the alveolar surface, thereby playing a critical role in barrier integrity, and are extremely thin, thus permitting efficient gas exchange. During lung injury, ATI cells die, resulting in increased epithelial permeability. ATII cells re-epithelialize the alveolar surface via proliferation and transdifferentiation into ATI cells. Transdifferentiation is characterized by down-regulation of ATII cell markers, up-regulation of ATI cell markers, and cell spreading, resulting in a change in morphology from cuboidal to squamous, thus restoring normal alveolar architecture and function. The mechanisms underlying ATII to ATI cell transdifferentiation have not been well studied in vivo. A prerequisite for mechanistic investigation is a rigorous, unbiased method to quantitate this process. Here, we used SPCCreERT2;mTmG mice, in which ATII cells and their progeny express green fluorescent protein (GFP), and applied stereologic techniques to measure transdifferentiation during repair after injury induced by LPS. Transdifferentiation was quantitated as the percent of alveolar surface area covered by ATII-derived (GFP+) cells expressing ATI, but not ATII, cell markers. Using this methodology, the time course and magnitude of transdifferentiation during repair was determined. We found that ATI cell loss and epithelial permeability occurred by Day 4, and ATII to ATI cell transdifferentiation began by Day 7 and continued until Day 16. Notably, transdifferentiation and barrier restoration are temporally correlated. This methodology can be applied to investigate the molecular mechanisms underlying transdifferentiation, ultimately revealing novel therapeutic targets to accelerate repair after lung injury.

Monocyte differentiation and antigen-presenting functions.

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.

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.

Myeloid Cell Turnover and Clearance.

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.

G2A Signaling Dampens Colitic Inflammation via Production of IFN-γ.

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 Ly6C(hi)MHCII(-) 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 Ly6C(hi)MHCII(-) to Ly6C(int)MHCII(+) 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.