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Background/objectives Adipose tissue macrophages (ATMs) exist in either the M1 or M2 form. The anti-inflammatory M2 ATMs accumulate in lean individuals, whereas the pro-inflammatory M1 ATMs accumulate in obese individuals. Bee venom phospholipase A2 (bvPLA2), a major component in honeybee (Apis mellifera) venom, exerts potent anti-inflammatory effects via interactions with regulatory T cells (Treg) and macrophages. This study investigated the effects of bvPLA2 on a high-fat diet (HFD)-induced obesity in mice. Subjects/methods For in vivo experiments, male C57BL/6, CD206-deficient, and Treg-depleted mice models were fed either a normal diet 41.86 kJ (ND, 10 kcal% fat) or high-fat diet 251.16 kJ (HFD, 60 kcal% fat). Each group was i.p. injected with PBS or bvPLA2 (0.5 mg/kg) every 3 days for 11 weeks. Body weight and food intake were measured weekly. Histological changes in the white adipose tissue (WAT), liver, and kidney as well as the immune phenotypes of the WAT were examined. Immune cells, cytokines, and lipid profiles were also evaluated. The direct effects of bvPLA2 on 3T3-L1 pre-adipocytes and bone marrow-derived macrophages were measured in vitro. Results bvPLA2 markedly decreased bodyweight in HFD-fed mice. bvPLA2 treatment also decreased lipid accumulation in the liver and reduced kidney inflammation in the mice. It was confirmed that bvPLA2 exerted immunomodulatory effects through the CD206 receptor. In addition, bvPLA2 decreased M1 ATM and alleviated the M1/M2 imbalance in vivo. However, bvPLA2 did not directly inhibit adipogenesis in the 3T3-L1 adipose cells in vitro. Conclusions bvPLA2 is a potential therapeutic strategy for the management of obesity by regulating adipose tissue macrophage homeostasis.

Mitochondria, in addition to cellular energy production, are now being recognized as regulators of the innate immune response of phagocytes. Here we found that down regulation of the mitochondria associated enzyme, Cytidine Monophosphate Kinase 2 (CMPK2), results in a significant de-regulation of resting immune homeostasis of macrophages, presenting as an enhanced expression of the pro-inflammatory genes- IL1β, TNFα and IL8 in these cells associated with enhanced mitochondrial ROS and altered mitochondrial shape. Contrary to expectation, these phenotypic changes were also preserved in cells with constitutive over expression of CMPK2 thereby implicating an important role for this gene in the regulation of inflammatory balance of macrophages. Interestingly, long term modulation of CMPK2 expression resulted in increased glycolytic flux in both the silenced and over-expressing cells akin to the altered physiological state of activated M1 macrophages. While infection induced inflammation for restricting pathogens is regulated, our observation of a total dysregulation of basal inflammation by bidirectional alteration of CMPK2 expression, only highlights a critical role of this gene in mitochondria mediated control of inflammation.

Macrophages are dynamic cells that play critical roles in the induction and resolution of sterile inflammation. In this review, we will compile and interpret recent findings on the plasticity of macrophages and how these cells contribute to the development of non-infectious inflammatory diseases, with a particular focus on allergic and autoimmune disorders. The critical roles of macrophages in the resolution of inflammation will then be examined, emphasizing the ability of macrophages to clear apoptotic immune cells. Rheumatoid arthritis (RA) is a chronic autoimmune-driven spectrum of diseases where persistent inflammation results in synovial hyperplasia and excessive immune cell accumulation, leading to remodeling and reduced function in affected joints. Macrophages are central to the pathophysiology of RA, driving episodic cycles of chronic inflammation and tissue destruction. RA patients have increased numbers of active M1 polarized pro-inflammatory macrophages and few or inactive M2 type cells. This imbalance in macrophage homeostasis is a main contributor to pro-inflammatory mediators in RA, resulting in continual activation of immune and stromal populations and accelerated tissue remodeling. Modulation of macrophage phenotype and function remains a key therapeutic goal for the treatment of this disease. Intriguingly, therapeutic intervention with glucocorticoids or other DMARDs promotes the re-polarization of M1 macrophages to an anti-inflammatory M2 phenotype; this reprogramming is dependent on metabolic changes to promote phenotypic switching. Allergic asthma is associated with Th2-polarised airway inflammation, structural remodeling of the large airways, and airway hyperresponsiveness. Macrophage polarization has a profound impact on asthma pathogenesis, as the response to allergen exposure is regulated by an intricate interplay between local immune factors including cytokines, chemokines and danger signals from neighboring cells. In the Th2-polarized environment characteristic of allergic asthma, high levels of IL-4 produced by locally infiltrating innate lymphoid cells and helper T cells promote the acquisition of an alternatively activated M2a phenotype in macrophages, with myriad effects on the local immune response and airway structure. Targeting regulators of macrophage plasticity is currently being pursued in the treatment of allergic asthma and other allergic diseases. Macrophages promote the re-balancing of pro-inflammatory responses towards pro-resolution responses and are thus central to the success of an inflammatory response. It has long been established that apoptosis supports monocyte and macrophage recruitment to sites of inflammation, facilitating subsequent corpse clearance. This drives resolution responses and mediates a phenotypic switch in the polarity of macrophages. However, the role of apoptotic cell-derived extracellular vesicles (ACdEV) in the recruitment and control of macrophage phenotype has received remarkably little attention. ACdEV are powerful mediators of intercellular communication, carrying a wealth of lipid and protein mediators that may modulate macrophage phenotype, including a cargo of active immune-modulating enzymes. The impact of such interactions may result in repair or disease in different contexts. In this review, we will discuss the origin, characterization, and activity of macrophages in sterile inflammatory diseases and the underlying mechanisms of macrophage polarizationviaACdEV and apoptotic cell clearance, in order to provide new insights into therapeutic strategies that could exploit the capabilities of these agile and responsive cells.

Insulin resistance is a key event in type 2 diabetes onset and a major comorbidity of obesity. It results from a combination of fat excess-triggered defects, including lipotoxicity and metaflammation, but the causal mechanisms remain difficult to identify. Here, we report that hyperactivation of the tyrosine phosphatase SHP2 found in Noonan syndrome (NS) led to an unsuspected insulin resistance profile uncoupled from altered lipid management (for example, obesity or ectopic lipid deposits) in both patients and mice. Functional exploration of an NS mouse model revealed this insulin resistance phenotype correlated with constitutive inflammation of tissues involved in the regulation of glucose metabolism. Bone marrow transplantation and macrophage depletion improved glucose homeostasis and decreased metaflammation in the mice, highlighting a key role of macrophages. In-depth analysis of bone marrow-derived macrophages in vitro and liver macrophages showed that hyperactive SHP2 promoted a proinflammatory phenotype, modified resident macrophage homeostasis, and triggered monocyte infiltration. Consistent with a role of SHP2 in promoting inflammation-driven insulin resistance, pharmaceutical SHP2 inhibition in obese diabetic mice improved insulin sensitivity even better than conventional antidiabetic molecules by specifically reducing metaflammation and alleviating macrophage activation. Together, these results reveal that SHP2 hyperactivation leads to inflammation-triggered metabolic impairments and highlight the therapeutical potential of SHP2 inhibition to ameliorate insulin resistance.

Phenolic compounds of red wine powder (RWP) extracted from the Italian red wine Aglianico del Vulture have been investigated for the potential immunomodulatory and anti-inflammatory capacity on human macrophages. These compounds reduce the secretion of IL-1β, IL-6, and TNF-α proinflammatory cytokines and increase the release of IL-10 anti-inflammatory cytokine induced by lipopolysaccharide (LPS). In addition, RWP restores Annexin A1 levels, thus involving activation of proresolutive pathways. Noteworthy, RWP lowers NF-κB protein levels, promoter activity, and nuclear translocation. As a consequence of NF-κB inhibition, reduced promoter activities of SLC25A1—encoding the mitochondrial citrate carrier (CIC)—and ATP citrate lyase (ACLY) metabolic genes have been observed. CIC, ACLY, and citrate are components of the citrate pathway: in LPS-activated macrophages, the mitochondrial citrate is exported by CIC into the cytosol where it is cleaved by ACLY in oxaloacetate and acetyl-CoA, precursors for ROS, NO⋅, and PGE2 inflammatory mediators. We identify the citrate pathway as a RWP target in carrying out its anti-inflammatory activity since RWP reduces CIC and ACLY protein levels, ACLY enzymatic activity, the cytosolic citrate concentration, and in turn ROS, NO⋅, PGE2, and histone acetylation levels. Overall findings suggest that RWP potentially restores macrophage homeostasis by suppressing inflammatory pathways and activating proresolutive processes.

Macrophages are the most abundant immune cells in the ovary. In addition to their roles in the innate immune system, these heterogeneous tissue-resident cells are responsive to tissue-derived signals, adapt to their local tissue environment, and specialize in unique functions to maintain tissue homeostasis. Research in the past decades has established a strong link between macrophages and various aspects of ovarian physiology, indicating a pivotal role of macrophages in ovarian health. However, unlike other intensively studied organs, the knowledge of ovarian macrophages dates back to the time when the heterogeneity of ontogeny, phenotype, and function of macrophages was not fully understood. In this review, we discuss the evolving understanding of the biology of ovarian tissue-resident macrophages, highlight their regulatory roles in normal ovarian functions, review the association between certain ovarian pathologies and disturbed macrophage homeostasis, and finally, discuss the technologies that are essential for addressing key questions in the field.

Traumatic brain injury (TBI) is a leading cause of long-term neurological disability. Currently there is no effective pharmacological treatment for patients suffering from the long-lasting symptoms of TBI. We recently discovered that colony stimulating factor 1 (CSF1), an essential regulator of macrophage homeostasis, is neuroprotective and reduces neuroinflammation in two models of neurological disease in mice. Here we used a mouse model of repetitive mild TBI (mTBI) to examine whether CSF1 would attenuate cognitive deficits and improve pathological outcomes in two paradigms. In the acute paradigm, a single bolus treatment of CSF1 administered 24 h after injury significantly reduces memory impairment and astrocyte reactivity assessed 3 months later. In the chronic paradigm, the mice were tested 3 months after mTBI when they showed cognitive deficits. The mice were then randomly assigned to receive CSF1 or PBS (as control) treatment. After one month of treatment, the PBS-treated mice remained cognitively impaired, but the CSF1-treated showed significant improvements in cognitive function. RNA-seq and Ingenuity Pathway Analysis reveals CSF1 treatment alters cognition- and memory-related transcriptomic changes and pathways. The results of this study show that acute as well as delayed CSF1 treatment attenuate chronically impaired cognitive functions and improve pathological outcomes long after mTBI. The wide therapeutic time window of CSF1, together with the fact that CSF1 is approved for human use in clinical trials, strongly supports the potential clinical usefulness of this treatment in patients with mTBI.

Macrophages are critically involved in not only immune and inflammatory responses but also maintenance of metabolic fitness of organisms. Combined genetic deficiency of three clusters in the miR-17~92 family drastically shifted macrophage phenotypes toward the inflammatory spectrum characterized by heightened production of pro-inflammatory mediator TNF and diminished expression of anti-inflammatory cytokine IL-10. Consequently, macrophages residing in the adipose tissues from myeloid-specific miRNA triple knockout mice spontaneously developed inflammatory phenotypes and displayed alterations of overall physiological conditions as evidenced by obesity and compromised glucose tolerance. Mechanistically, miR-17~92 family miRNAs sustained IL-10 production by promoting transcription of the Fos gene, which is secondary to downregulation of Fos by transcription factor YY1, a direct target of miR-17~92 family miRNAs. Together, these results identified miR-17~92 family miRNAs as crucial regulators of the balance between pro- and anti-inflammatory cytokines and exemplified how macrophage-intrinsic regulatory circuit exerted impactful influence on general physiology.

International audience; Macrophages are specialized cells that control tissue homeostasis. They include non-resident and tissue-resident macrophage populations which are characterized by the expression of particular cell surface markers and the secretion of molecules with a wide range of biological functions. The differentiation and polarization of macrophages relies on specific growth factors and their receptors. Macrophage-colony stimulating factor (CSF-1) and interleukine-34 (IL-34), also known as "twin" cytokines, are part of this regluatory landscape. CSF-1 and IL-34 share a common receptor, the macrophage-colony stimulating factor receptor (CSF-1R), which is activated in a similar way by both factors and turns on identical signaling pathways. However, there is some discrete differential activation leading to specific activities. In this review, we disscuss recent progress in understanding of the role of the twin cytokines in macrophage differentiation, from their interaction with CSF-1R and the activation of signaling pathways, to their implication in macrophage polarization of non-resident and tissue-resident macrophages. A special focus on IL-34, its involvement in pathophsyiological contexts, and its potential as a theranostic target for macrophage therapy will be proposed.

Problem The state of self-renewal and self-maintain of decidual macrophages would be important for immune homeostasis at the maternal-fetal interface. The roles of interleukin (IL)-24 derived from decidual stromal cells (DSCs) on decidual macrophages have not been explored. Method of study IL-24 expression in DSCs was interfered by lentivirus, and the transcription levels of IL-24 in DSCs were verified by real time (RT)-PCR. The levels of IL-24 receptors were determined by flow cytometry assays. The effect of recombination human IL-24 (rhIL-24) on the differentiation and apoptosis of macrophages was analyzed by flow cytometry in vitro. The viability of macrophages was detected by Cell Counting Kit-8 assays. Results The growth of DSCs was not affected obviously only by IL-24 knockdown while the growth of knockdown DSCs was inhibited significantly after co-cultured with decidual macrophages. The levels of IL-24 receptors (IL-20R1 and IL-22R1) were moderately to highly expressed on decidual macrophages and human macrophage cell line U937. The differentiation of decidual macrophages treated by rhIL-24 or co-cultured with IL-24 knockdown DSCs was not affected. Both apoptosis and viability of U937 cells were promoted by rhIL-24. The ratio of Bcl-2/Bax was down-regulated and Ki-67 was up-regulated by IL-24 treatment. The expression of Bcl-2/Bax was up-regulated while Ki-67 was down-regulated in U937 cells after co-cultured by IL-24 knockdown DSCs. Conclusion IL-24 secreted by DSCs promotes the renewal and homeostasis of decidual macrophages possibly via down-regulating the ratio of Bcl-2/Bax and up-regulating of the expression of Ki-67 in early pregnancy.

SHIP-1 is an inositol phosphatase that hydrolyzes phosphatidylinositol 3-kinase (PI3K) products and negatively regulates protein kinase B (Akt) activity, thereby modulating a variety of cellular processes in mammals. However, the role of SHIP-1 in bacterial-induced sepsis is largely unknown. Here, we show that SHIP-1 regulates inflammatory responses during Gram-negative bacterium Pseudomonas aeruginosa infection. We found that infected-SHIP-1−/− mice exhibited decreased survival rates, increased inflammatory responses, and susceptibility owing to elevated expression of PI3K than wild-type (WT) mice. Inhibiting SHIP-1 via siRNA silencing resulted in lipid raft aggregates, aggravated oxidative damage, and bacterial burden in macrophages after PAO1 infection. Mechanistically, SHIP-1 deficiency augmented phosphorylation of PI3K and nuclear transcription of signal transducer and activator of transcription 5 (STAT5) to induce the expression of Trib1, which is critical for differentiation of M2 but not M1 macrophages. These findings reveal a previously unrecognized role of SHIP-1 in inflammatory responses and macrophage homeostasis during P. aeruginosa infection through a PI3K/Akt–STAT5–Trib1 axis.

Summary Pattern recognition receptors, such as Toll-like receptors (TLRs), play an important role in the host defense against invading microbial pathogens. Their activation must be precisely regulated, as inappropriate activation or overactivation of TLR signaling pathways may result in inflammatory disorders, such as septic shock or autoimmune diseases. TMEM106A is a type II transmembrane protein constitutively expressed in macrophages. Our current study demonstrated that TMEM106A levels were increased in macrophages upon lipopolysaccharide (LPS) stimulation, as well as in the peripheral monocytes of patients with sepsis. Tmem106a knockout mice were more sensitive to lipopolysaccharide (LPS)-induced septic shock than wild-type mice. Further experiments indicated that Tmem106a ablation enhanced the expression of CD80, CD86 and major histocompatibility complex (MHC)-II in mouse macrophages upon LPS stimulation, accompanied with up-regulation of tumor necrosis factor (TNF)-α, interleukin (IL)-6, interferon (IFN)-β and inducible nitric oxide synthase (iNOS), indicating the activation of macrophages and polarization towards the M1 inflammatory phenotype. Moreover, elevated mitogen-activated protein kinase (MAPK) and nuclear factor kappa B (NF-κB) signaling were found to be involved in the LPS-induced inflammatory response in Tmem106a−/− macrophages. However, this effect was largely abrogated by macrophage deletion in Tmem106a−/− mice. Therefore, deficiency of Tmem106a in macrophages may enhance the M1 polarization in mice, resulting in inflammation. This suggests that TMEM106A plays an important regulatory role in maintaining macrophage homeostasis.

Monocytes and macrophages are major cellular components of the innate immunity that play essential roles in tissue homeostasis. The contribution of different subsets of monocytes/macrophages to periodontal health and disease has not been fully elucidated. Type 2 diabetes mellitus (T2DM) is a risk factor for periodontitis. We hypothesized that the monocyte/macrophage signaling is perturbed in periodontitis-affected sites versus periodontally-healthy sites and that this perturbation plays a critical role in the pathogenesis of periodontitis. Pairs of gingival tissue samples (each from a periodontally healthy and a periodontitis-affected site of the same patient) were harvested from 27 periodontitis patients, with and without T2DM. Each sample was processed to form a single-cell suspension, and a flow-cytometry panel was designed and validated to study monocyte and macrophage phenotypes. In separate experiments, the transcriptional changes associated with a pro-inflammatory phenotype were also examined in monocyte/macrophage subsets obtained from peripheral blood of patients with T2DM versus diabetes-free controls. A significantly higher proportion of intermediate (CD14+CD16+) monocytes was observed in periodontitis-affected tissues compared to healthy tissues. These monocytes overexpressed HLA-DR and PDL1 molecules, suggesting their activated inflammatory status. PDL1 increase was specific to intermediate monocytes. The ratio of M1/M2 macrophages was also significantly higher in periodontally-affected sites, signifying an imbalance between inflammatory and repair mechanisms. We found a significantly higher expression of PDL1 in overall monocytes and M1 macrophages in periodontitis-affected sites compared to controls. Importantly, we identified a subpopulation of M1 macrophages present in periodontally-affected tissues which expressed high levels of CD47, a glycoprotein of the immunoglobulin family that plays a critical role in self-recognition and impairment of phagocytosis. Analysis of the transcriptional landscape of monocytes/macrophages in gingival tissue of T2DM patients with periodontitis revealed a significant disruption in homeostasis towards a proinflammatory phenotype, elevation of pro-inflammatory transcription factors STAT1 and IRF1, and repression of anti-inflammatory JMJD3 in circulating monocytes. Taken together, our results demonstrate disruption of myeloid-derived cell homeostasis in periodontitis, with or without T2DM, and highlight a potentially significant role of these cell types in its pathogenesis. The impact of macrophage and monocyte signaling pathways on the pathobiology of periodontitis should be further evaluated.

Monocytes and macrophages are critical effectors and regulators of innate immune response. They not only play crucial and distinctive roles in homeostasis, but also contribute to some pathologic processes. The heterogeneity of the macrophage lineage has been widely recognized and, in part, is a result of the specialization of resident macrophages in particular tissue microenvironments. Monocytes are usually known to originate in the bone marrow from a common myeloid progenitor that is shared with neutrophils, and they are then released into the peripheral blood. However, the origin of tissue-resident macrophages, crucial for homeostasis and immunity, has remained controversial until recently. During embryonic organogenesis, macrophages derived from yolk sac and fetal liver precursors are seeded throughout tissues, persisting in the adulthood as resident, self-maintaining populations. After birth, bone marrow-derived monocytes can replenish tissue resident macrophages following injury, infection and inflammation. In this review, we will mainly summarize our current understanding on the origin, ontogeny and fates of tissue macrophages and will briefly discuss the molecular regulation of resident macrophage homeostasis in physiological situation.

The coordination of macrophage polarization is essential for the robust regenerative potential of skeletal muscle. Repair begins with a phase mediated by inflammatory monocytes (IM) and proinflammatory macrophages (M1), followed by polarization to a proregenerative macrophage (M2) phenotype. Recently, regulatory T cells (Tregs) were described as necessary for this M1 to M2 transition. We report that chronic infection with the protozoan parasite Toxoplasma gondii causes a nonresolving Th1 myositis with prolonged tissue damage associated with persistent M1 accumulation. Surprisingly, Treg ablation during chronic infection rescues macrophage homeostasis and skeletal muscle fiber regeneration, showing that Tregs can directly contribute to muscle damage. This study provides evidence that the tissue environment established by the parasite could lead to a paradoxical pathogenic role for Tregs. As such, these findings should be considered when tailoring therapies directed at Tregs in inflammatory settings.

Objective Skeletal muscle is an important secretory organ, producing and releasing numerous myokines, which may be involved in mediating beneficial health effects of physical activity. More than 100 myokines have been identified by different proteomics approaches, but these techniques may not detect all myokines. We used mRNA sequencing as an untargeted approach to study gene expression of secreted proteins in skeletal muscle upon acute as well as long-term exercise. Methods Twenty-six middle-aged, sedentary men underwent combined endurance and strength training for 12 weeks. Skeletal muscle biopsies from m. vastus lateralis and blood samples were taken before and after an acute bicycle test, performed at baseline as well as after 12 weeks of training intervention. We identified transcripts encoding secretory proteins that were changed more than 1.5-fold in muscle after exercise. Secretory proteins were defined based on either curated UniProt annotations or predictions made by multiple bioinformatics methods. Results This approach led to the identification of 161 candidate secretory transcripts that were up-regulated after acute exercise and 99 that where increased after 12 weeks exercise training. Furthermore, 92 secretory transcripts were decreased after acute and/or long-term physical activity. From these responsive transcripts, we selected 17 candidate myokines sensitive to short- and/or long-term exercise that have not been described as myokines before. The expression of these transcripts was confirmed in primary human skeletal muscle cells during in vitro differentiation and electrical pulse stimulation (EPS). One of the candidates we identified was macrophage colony-stimulating factor-1 (CSF1), which influences macrophage homeostasis. CSF1 mRNA increased in skeletal muscle after acute and long-term exercise, which was accompanied by a rise in circulating CSF1 protein. In cultured muscle cells, EPS promoted a significant increase in the expression and secretion of CSF1. Conclusion We identified 17 new, exercise-responsive transcripts encoding secretory proteins. We further identified CSF1 as a novel myokine, which is secreted from cultured muscle cells and up-regulated in muscle and plasma after acute exercise., Highlights • Numerous transcripts were identified that were regulated in human skeletal muscle after acute and/or long-term exercise. • These transcripts encode potential myokines, which may play key roles in local and systemic adaptations to exercise. • CSF1 was identified as a novel myokine. CSF1 was increased after acute exercise, and secreted from cultured human myotubes in response to EPS.

Colony-stimulating factor 1 (CSF1) and interleukin 34 (IL34) signal via the CSF1 receptor to regulate macrophage differentiation. Studies in IL34- or CSF1-deficient mice have revealed that IL34 function is limited to the central nervous system and skin during development. However, the roles of IL34 and CSF1 at homeostasis or in the context of inflammatory diseases or cancer in wild-type mice have not been clarified in vivo. By neutralizing CSF1 and/or IL34 in adult mice, we identified that they play important roles in macrophage differentiation, specifically in steady-state microglia, Langerhans cells, and kidney macrophages. In several inflammatory models, neutralization of both CSF1 and IL34 contributed to maximal disease protection. However, in a myeloid cell-rich tumor model, CSF1 but not IL34 was required for tumor-associated macrophage accumulation and immune homeostasis. Analysis of human inflammatory conditions reveals IL34 upregulation that may account for the protection requirement of IL34 blockade. Furthermore, evaluation of IL34 and CSF1 blockade treatment during Listeria infection reveals no substantial safety concerns. Thus, IL34 and CSF1 play non-redundant roles in macrophage differentiation, and therapeutic intervention targeting IL34 and/or CSF1 may provide an effective treatment in macrophage-driven immune-pathologies.

Titanium surface mediated immunomodulation may address compromised post-implantation bone healing in diabetes mellitus. To assess in vitro phenotypic changes, M1 and M2 polarised Type 2 diabetic rat (Goto Kakizaki, GK) macrophages were cultured on micro-rough (SLA) or hydrophilic nanostructured SLA (modSLA) titanium. The in vivo effects of the SLA and modSLA surfaces on macrophage phenotype, wound-associated protein expression and bone formation were investigated using a critical-sized calvarial defect model. Compared to healthy macrophages, GK M2 macrophage function was compromised, secreting significantly lower levels of the anti-inflammatory cytokine IL-10. The modSLA surface attenuated the pro-inflammatory cellular environment, reducing pro-inflammatory cytokine production and promoting M2 macrophage phenotype differentiation. ModSLA also suppressed gene expression associated with macrophage multinucleation and giant cell formation and stimulated pro-osteogenic genes in co-cultured osteoblasts. In vivo, modSLA enhanced osteogenesis compared to SLA in GK rats. During early healing, proteomic analysis of both surface adherent and wound exudate material showed that modSLA promoted an immunomodulatory pro-reparative environment. The modSLA surface therefore successfully compensated for the compromised M2 macrophage function in Type 2 diabetes by attenuating the pro-inflammatory response and promoting M2 macrophage activity, thus restoring macrophage homeostasis and resulting in a cellular environment favourable for enhanced osseous healing.

Macrophages are pleiotropic cells capable of performing a broad spectrum of functions. Macrophage phenotypes are classified along a continuum between the extremes of proinflammatory M1 macrophages and anti-inflammatory M2 macrophages. The seemingly opposing functions of M1 and M2 macrophages must be tightly regulated for an effective and proper response to foreign molecules or damaged tissue. Excessive activation of either M1 or M2 macrophages contributes to the pathology of many diseases. Emodin is a Chinese herb-derived compound and has shown potential to inhibit inflammation in various settings. In this study, we tested the ability of emodin to modulate the macrophage response to both M1 and M2 stimuli. Primary mouse macrophages were stimulated with LPS/IFNγ or IL4 with or without emodin, and the effects of emodin on gene transcription, cell signaling pathways, and histone modifications were examined by a variety of approaches, including microarray, quantitative real-time PCR, Western blotting, chromatin immunoprecipitation, and functional assays. We found that emodin bidirectionally tunes the induction of LPS/IFNγ- and IL4-responsive genes through inhibiting NFκB/IRF5/STAT1 signaling and IRF4/STAT6 signaling, respectively. Thereby, emodin modulates macrophage phagocytosis, migration, and NO production. Furthermore, emodin inhibited the removal of H3K27 trimethylation (H3K27m3) marks and the addition of H3K27 acetylation (H3K27ac) marks on genes required for M1 or M2 polarization of macrophages. In conclusion, our data suggest that emodin is uniquely able to suppress the excessive response of macrophages to both M1 and M2 stimuli and therefore has the potential to restore macrophage homeostasis in various pathologies.

MicroRNAs (miRNAs) are short noncoding RNAs that regulate a broad spectrum of biological processes, including immune responses. Although the contributions of miRNAs to the function of immune cells are beginning to emerge, their specific roles remain largely unknown. IL-33 plays an important role in macrophage activation for innate host defense and proinflammatory responses. In this study, we report that miR-487b can suppress the levels of mRNA and protein for IL-33 during the differentiation of bone marrow–derived macrophages (BMDMs). This results in inhibition of IL-33–induced expression of Ag-presenting and costimulatory molecules and proinflammatory mediators. A luciferase assay showed that miR-487b binds to the IL-33 3′-untranslated region. We also confirmed that IL-33 directly promotes the activation of BMDMs by increasing the expression of MHC class I, MHC class II, CD80/CD86, and inducible NO synthase (iNOS) in a dose-dependent manner. Exposure of BMDMs to the TLR4 ligand, LPS, decreased miR-487b expression, increased IL-33 transcript levels, and induced the production of proinflammatory mediators (e.g., iNOS, IL-1β, IL-6, and TNF-α). Treatment with a specific inhibitor of miR-487b function also resulted in increased levels of IL-33 mRNA, which augmented LPS-induced expression of these inflammatory mediators in macrophages. Collectively, our results indicate that miR-487b plays a negative regulatory role in macrophages by controlling the levels of IL-33 transcript and protein to fine-tune innate immune host defense and proinflammatory responses of these cells. Thus, miR-487b plays an important role in the regulation of macrophage homeostasis and activation by targeting IL-33 transcripts.

International audience; Innate lymphoid cells (ILCs) residing in adipose tissue participate in the pathogenesis of obesity, but their contribution toward adipose tissue homeostasis in the lean state is unclear. Boulenouar et al. (2017) now report that heterogenous type 1 ILCs in adipose tissues regulate macrophage homeostasis through cytotoxicity.

Macrophages in the healthy intestine are highly specialized and usually respond to the gut microbiota without provoking an inflammatory response. A breakdown in this tolerance leads to inflammatory bowel disease (IBD), but the mechanisms by which intestinal macrophages normally become conditioned to promote microbial tolerance are unclear. Strong epidemiological evidence linking disruption of the gut microbiota by antibiotic use early in life to IBD indicates an important role for the gut microbiota in modulating intestinal immunity. Here, we show that antibiotic use causes intestinal macrophages to become hyperresponsive to bacterial stimulation, producing excess inflammatory cytokines. Re-exposure of antibiotic-treated mice to conventional microbiota induced a long-term, macrophage-dependent increase in inflammatory T helper 1 (TH1) responses in the colon and sustained dysbiosis. The consequences of this dysregulated macrophage activity for T cell function were demonstrated by increased susceptibility to infections requiring TH17 and TH2 responses for clearance (bacterial Citrobacter rodentium and helminth Trichuris muris infections), corresponding with increased inflammation. Short-chain fatty acids (SCFAs) were depleted during antibiotic administration; supplementation of antibiotics with the SCFA butyrate restored the characteristic hyporesponsiveness of intestinal macrophages and prevented T cell dysfunction. Butyrate altered the metabolic behavior of macrophages to increase oxidative phosphorylation and also promoted alternative macrophage activation. In summary, the gut microbiota is essential to maintain macrophage-dependent intestinal immune homeostasis, mediated by SCFA-dependent pathways. Oral antibiotics disrupt this process to promote sustained T cell-mediated dysfunction and increased susceptibility to infections, highlighting important implications of repeated broad-spectrum antibiotic use.

The increasing incidence of obesity and its socio-economical impact is a global health issue due to its associated co-morbidities, namely diabetes and cardiovascular disease [1-5]. Obesity is characterized by an increase in adipose tissue, which promotes the recruitment of immune cells resulting in low-grade inflammation and dysfunctional metabolism. Macrophages are the most abundant immune cells in the adipose tissue of mice and humans. The adipose tissue also contains other myeloid cells (dendritic cells (DC) and neutrophils) and to a lesser extent lymphocyte populations, including T cells, B cells, Natural Killer (NK) and Natural Killer T (NKT) cells. While the majority of studies have linked adipose tissue macrophages (ATM) to the development of low-grade inflammation and co-morbidities associated with obesity, emerging evidence suggests for a role of other immune cells within the adipose tissue that may act in part by supporting macrophage homeostasis. In this review, we summarize the current knowledge of the functions ATMs, DCs and B cells possess during steady-state and obesity.

We would like to thank Prof. Ira Tabas (Columbia University, New York, NY, USA) for providing the L929 cell line, Prof. Herbert Virgin (Washington University, St. Louis, MO, USA) for providing p62-KO mice legs, Dr. H. Girao (CNC. IBILI, Univ. of Coimbra) for the E1-inhibitor and T. Pereira for technical assistance with microscopy. This work was supported by the Foundation for Science and Technology of the Portuguese Ministry of Science and Higher Education [HMSP-ICT/0024/2010, co-founded by the European Union (FEDER - Fundo Europeu de Desenvolvimento Regional) through COMPETE - Programa Operacional Factores de Competitividade and QREN - Quadro de Referencia Estrategico], iNOVA4Health - UID/Multi/04462/2013, a program financially supported by FCT through national funds and co-funded by FEDER under the PT2020 Partnership Agreement and FCT to OVV. PhD fellowships SFRH/BD/62197/2009, SFRH/BD/90258/2012 and SFRH/BD/51877/2012 and SFRH/BD/52293/2013. PTDC/SAU-TOX/116627/2010, HMSP-ICT/0022/2010, European Community 7th Framework Grant ERC-2011-AdG 294709-DAMAGECONTROL to MPS. Editor Erythrophagocytosis, the phagocytic removal of damaged red blood cells (RBC), and subsequent phagolysosome biogenesis are important processes in iron/heme metabolism and homeostasis. Phagolysosome biogenesis implies the interaction of nascent phagosomes with endocytic compartments and also autophagy effectors. Here, we report that besides recruitment of microtubule-associated protein-1-light chain 3 (LC3), additional autophagy machinery such as sequestosome 1 (p62) is also acquired by single-membrane phagosomes at very early stages of the phagocytic process and that its acquisition is very important to the outcome of the process. In bone marrow-derived macrophages (BMDM) silenced for p62, RBC degradation is inhibited. P62, is also required for nuclear translocation and activation of the transcription factor Nuclear factor E2-related Factor 2 (NRF2) during erythrophagocytosis. Deletion of the Nrf2 allele reduces p62 expression and compromises RBC degradation. In conclusion, we reveal that erythrophagocytosis relies on an interplay between p62 and NRF2, potentially acting as protective mechanism to maintain reactive oxygen species at basal levels and preserve macrophage homeostasis. © 2017 The Author(s). publishersversion published

Adipose tissue has a dynamic immune system that adapts to changes in diet and maintains homeostatic tissue remodeling. Adipose type 1 innate lymphoid cells (AT1-ILCs) promote pro-inflammatory macrophages in obesity, but little is known about their functions at steady state. Here we found that human and murine adipose tissue harbor heterogeneous populations of AT1-ILCs. Experiments using parabiotic mice fed a high-fat diet (HFD) showed differential trafficking of AT1-ILCs, particularly in response to short- and long-term HFD and diet restriction. At steady state, AT1-ILCs displayed cytotoxic activity toward adipose tissue macrophages (ATMs). Depletion of AT1-ILCs and perforin deficiency resulted in alterations in the ratio of inflammatory to anti-inflammatory ATMs, and adoptive transfer of AT1-ILCs exacerbated metabolic disorder. Diet-induced obesity impaired AT1-ILC killing ability. Our findings reveal a role for AT1-ILCs in regulating ATM homeostasis through cytotoxicity and suggest that this function is relevant in both homeostasis and metabolic disease.

A single nucleotide polymorphism of PRDM1, the gene encoding Blimp-1, is strongly associated with inflammatory bowel disease. Here, we demonstrate that Blimp-1 in CD103(+) dendritic cells (DCs) critically contributes to the regulation of macrophage homeostasis in the colon. Dextran sodium sulfate (DSS)-exposed Blimp-1(cko) mice with a deletion of Blimp-1 in CD103(+) DCs and CD11c(hi) macrophages exhibited severe inflammatory symptoms, pronounced weight loss, high mortality, robust infiltration of neutrophils in epithelial regions of the colon, an increased expression of proinflammatory cytokines and a significant decrease in CD103(+) DCs in the colon compared with DSS exposed wild-type (WT) mice. Purified colonic macrophages from Blimp-1(cko) mice expressed increased levels of matrix metalloproteinase 8, 9 and 12 mRNA. WT macrophages cocultured with colonic DCs but not bone marrow-derived DCs from Blimp-1(cko) produced increased matrix metalloproteinases in an interleukin (IL)-1β- and IL-6-dependent manner. Treatment of Blimp-1(cko) mice with anti-IL-1β and anti-IL-6 abrogated the exaggerated clinical response. Overall, these data demonstrate that Blimp-1 expression in DCs can alter an innate inflammatory response by modulating the activation of myeloid cells. This is a novel mechanism of contribution of Blimp-1 for the pathogenesis of inflammatory bowel diseases, implicating another therapeutic target for the development of inflammatory bowel disease.

Erythroid function and development is intimately linked to macrophages. The primary function of erythrocytes is oxygen delivery, which is mediated by iron-containing hemoglobin. The major source of this iron is a recycling pathway where macrophages scavenge old and damaged erythrocytes to release iron contained within the heme moiety. Macrophages also promote erythropoiesis by providing a supportive niche in the bone marrow as an integral component of “erythorblastic islands.” Importantly, inflammation leads to alterations in iron handling by macrophages with significant impact on iron homeostasis and erythropoiesis. The importance of macrophages in erythropoiesis and iron homeostasis is well established and has been extensively reviewed. However, this developmental relationship is not one way, and erythrocytes can also regulate macrophage development and function. Erythrocyte-derived heme can induce the development of iron-recycling macrophages from monocytes, engage pattern recognition receptors to activate macrophages, and act as ligand for specific nuclear receptors to modulate macrophage function. Here, we discuss the role of heme as a signaling molecule impacting macrophage homeostasis. We will review these actions of heme within the framework of our current understanding of the role of micro-environmental factors in macrophage development and function.

Mer tyrosine kinase (MerTK) is a major macrophage apoptotic cell (AC) receptor. Its functional impairment promotes autoimmunity and atherosclerosis, whereas overexpression correlates with poor prognosis in cancer. However, little is known about mechanisms regulating MerTK expression in humans. We found that MerTK expression is heterogenous among macrophage subsets, being mostly restricted to anti-inflammatory M2c (CD14+CD16+CD163+CD204+CD206+CD209−) cells, differentiated by M-CSF or glucocorticoids. Small numbers of MerTK+ “M2c-like” cells are also detectable among circulating CD14brightCD16+ monocytes. MerTK expression levels adapt to changing immunologic environment, being suppressed in M1 and M2a macrophages and in dendritic cells. Remarkably, although glucocorticoid-induced differentiation is IL-10 independent, M-CSF–driven M2c polarization and related MerTK upregulation require IL-10. However, neither IL-10 alone nor TGF-β are sufficient to fully differentiate M2c (CD16+CD163+MerTK+) macrophages. M-CSF and IL-10, both released by T lymphocytes, may thus be required together to promote regulatory T cell–mediated induction of anti-inflammatory monocytes-macrophages. MerTK enables M2c macrophages to clear early ACs more efficiently than other macrophage subsets, and it mediates AC clearance by CD14brightCD16+ monocytes. Moreover, M2c cells release Gas6, which in turn amplifies IL-10 secretion via MerTK. IL-10–dependent induction of the Gas6/MerTK pathway may, therefore, constitute a positive loop for M2c macrophage homeostasis and a critical checkpoint for maintenance of anti-inflammatory conditions. Our findings give new insight into human macrophage polarization and favor a central role for MerTK in regulation of macrophage functions. Eliciting M2c polarization can have therapeutic utility for diseases such as lupus, in which a defective AC clearance contributes to initiate and perpetuate the pathological process.

From Nucleoside Recycling to Histiocytosis Macrophages remove billions of apoptotic cells daily, releasing their nucleic acid material through lysosomal degradation, which allows the resulting nucleosides to be recycled. Hsu et al. (p. 89 , published online 15 December) found that the nucleoside transporter, equilibrative nucleoside transporter 3 (ENT3), was highly expressed in macrophages and showed that mice deficient in this transporter develop histiocytosis and features of lysosomal storage disease. When exposed to apoptotic cells, macrophages carrying human ENT3 mutations accumulated adenosine and increased their lysosomal pH. These changes contributed to an enhanced signaling through macrophage colony-stimulating factor (M-CSF) receptor and, ultimately, to M-CSF–driven myeloproliferative disease.

The development of the mononuclear phagocyte system requires macrophage colony-stimulating factor (CSF-1) signaling through the CSF-1 receptor (CSF1R, CD115). We examined the effect of an antibody against CSF1R on macrophage homeostasis and function using the MacGreen transgenic mouse (csf1r-enhanced green fluorescent protein) as a reporter. The administration of a novel CSF1R blocking antibody selectively reduced the CD115+Gr-1neg monocyte precursor of resident tissue macrophages. CD115+Gr-1+ inflammatory monocytes were correspondingly increased, supporting the view that monocytes are a developmental series. Within tissue, the antibody almost completely depleted resident macrophage populations in the peritoneum, gastrointestinal tract, liver, kidney, and skin, but not in the lung or female reproductive organs. CSF1R blockade reduced the numbers of tumor-associated macrophages in syngeneic tumor models, suggesting that these cells are resident type macrophages. Conversely, it had no effect on inflammatory monocyte recruitment in models, including lipopolysaccharide-induced lung inflammation, wound healing, peritonitis, and severe acute graft-versus-host disease. Depletion of resident tissue macrophages from bone marrow transplantation recipients actually resulted in accelerated pathology and exaggerated donor T-cell activation. The data indicate that CSF1R signaling is required only for the maturation and replacement of resident-type monocytes and tissue macrophages, and is not required for monocyte production or inflammatory function.

Nijmegen breakage syndrome 1 (NBS1) is a component of the MRE11 complex, which is a sensor of DNA double-strand breaks and plays a crucial role in the DNA damage response. Because activated macrophages produce large amounts of reactive oxygen species (ROS) that can cause DNA lesions, we examined the role of NBS1 in macrophage functional activity. Proliferative and proinflammatory (interferon gamma [IFN-γ] and lipopolysaccharide [LPS]) stimuli led to increased NBS1 levels in macrophages. In mice expressing a hypomorphic allele of Nbs1, Nbs1(∆B/∆B), macrophage activation-induced ROS caused increased levels of DNA damage that were associated with defects in proliferation, delayed differentiation, and increased senescence. Furthermore, upon stimulation, Nbs1(∆B/∆B) macrophages exhibited increased expression of proinflammatory cytokines. In the in vivo 2,4-dinitrofluorobenzene model of inflammation, Nbs1(∆B/∆B) animals showed increased weight and ear thickness. By using the sterile inflammation by zymosan injection, we found that macrophage proliferation was drastically decreased in the peritoneal cavity of Nbs1(∆B/∆B) mice. Our findings show that NBS1 is crucial for macrophage function during normal aging. These results have implications for understanding the immune defects observed in patients with NBS and related disorders.

To ensure invading pathogens are eliminated with minimal damage to host tissues it is essential that macrophage activation be tightly regulated. Previously we demonstrated that a subset of gammadelta T cells (Vgamma1(+)) contributes to resolving pathogen-induced immune responses by killing activated macrophages. However, the exaggerated macrophage response seen in infected Vgamma1(+) T cell-deficient mice suggests that gammadelta T cells play a broader role in macrophage homeostasis and other subsets might promote macrophage activation. Using a macrophage:gammadelta T cell co-culture system we have shown that gammadelta T cells increase the activity of macrophages activated in vivo by Listeria monocytogenes infection. In a dose-dependent manner, gammadelta T cells up-regulated production of cytokines (TNF-alpha, IL-6, IL-10) and chemokines (MIP-1alpha, MIP-1beta) by Listeria-elicited macrophages. The ability to increase macrophage cytokine production was prominent among Vgamma4(+) gammadelta T cells. Reciprocally, Vgamma4(+) gammadelta T cells were activated by Listeria-elicited macrophages, resulting in production of the anti-inflammatory cytokine, IL-10. gammadelta T cell adoptive transfer experiments showed that Vgamma4(+) T cells protected TCRdelta(-/-) mice against Listeria-induced liver injury and necrosis. These findings identify distinct and non-overlapping roles for gammadelta T cell subsets in regulating macrophage function during pathogen-induced immune responses.

Human alveolar macrophages play a critical role in host defense and in the development of inflammation and fibrosis in the lung. Unlike their precursor cells, blood monocytes, alveolar macrophages are long-lived and tend to be resistant to apoptotic stimuli. In this study, we examined the role of differentiation in altering baseline phosphatidylinositol (PI) 3-kinase/Akt activity. We found that differentiation increased activity of pro-survival PI 3-kinase/Akt while decreasing amounts of the negative PI 3-kinase regulator, PTEN. PTEN is a lipid phosphatase with activity against phosphatidylinositol 3,4,5-trisphosphate (PI3,4,5P3), the major bioactive product of PI 3-kinase. Examining in vivo differentiation of alveolar macrophages (by comparing blood monocytes to alveolar macrophages from single donors), we found that differentiation resulted in increased baseline reactive oxygen species (ROS) in the alveolar macrophages. This led to a deficiency in PTEN, increased activity of Akt, and prolonged survival of alveolar macrophages. These data support the hypothesis that alterations in ROS levels contribute to macrophage homeostasis by altering the balance between PI 3-kinase/Akt and the phosphatase, PTEN.

Macrophages play essential roles in immunity and homeostasis. As professional scavengers, macrophages phagocytose microbes and apoptotic and necrotic cells and take up modified lipoprotein particles. These functions require tightly regulated mechanisms for the processing and disposal of cellular lipids. Under pathological conditions, arterial wall macrophages become foam cells by accumulating large amounts of cholesterol, contributing to the development of atherosclerosis. Peroxisome proliferator–activated receptors (PPARs) and liver X receptors (LXRs) are members of the nuclear receptor superfamily of transcription factors that have emerged as key regulators of macrophage homeostasis. PPARs and LXRs control transcriptional programs involved in processes of lipid uptake and efflux, lipogenesis, and lipoprotein metabolism. In addition, PPARs and LXRs negatively regulate transcriptional programs involved in the development of inflammatory responses. This review summarizes recent efforts to decode the differential and overlapping roles of PPARs and LXRs in the context of macrophage lipid homeostasis and the control of inflammation.

Macrophages provide a bridge linking innate and adaptive immunity. An increased frequency of macrophages and other myeloid cells paired with excessive cytokine production is commonly seen in the aging immune system, known as ‘inflamm-aging’. It is presently unclear how healthy macrophages are maintained throughout life and what connects inflammation with myeloid dysfunction during aging. Autophagy, an intracellular degradation mechanism, has known links with aging and lifespan extension. Here, we show for the first time that autophagy regulates the acquisition of major aging features in macrophages. In the absence of the essential autophagy gene Atg7, macrophage populations are increased and key functions such as phagocytosis and nitrite burst are reduced, while the inflammatory cytokine response is significantly increased – a phenotype also observed in aged macrophages. Furthermore, reduced autophagy decreases surface antigen expression and skews macrophage metabolism toward glycolysis. We show that macrophages from aged mice exhibit significantly reduced autophagic flux compared to young mice. These data demonstrate that autophagy plays a critical role in the maintenance of macrophage homeostasis and function, regulating inflammation and metabolism and thereby preventing immunosenescence. Thus, autophagy modulation may prevent excess inflammation and preserve macrophage function during aging, improving immune responses and reducing the morbidity and mortality associated with inflamm-aging.

Macrophages play diverse roles in tissue homeostasis and immunity, and canonically activated macrophages are critically associated with acute inflammatory responses. It is known that activated macrophages undergo cell death after transient activation in some settings, and the viability of macrophages impacts on inflammatory status. Here we report that TGFβ- activated kinase (TAK1) activators, TAK1-binding protein 1 (TAB1) and TAK1-binding protein 2 (TAB2), are critical molecules in the regulation of activated macrophage survival. While deletion of Tak1 induced cell death in bone marrow derived macrophages even without activation, Tab1 or Tab2 deletion alone did not profoundly affect survival of naive macrophages. However, in lipopolysaccharide (LPS)-activated macrophages, even single deletion of Tab1 or Tab2 resulted in macrophage death with both necrotic and apoptotic features. We show that TAB1 and TAB2 were redundantly involved in LPS-induced TAK1 activation in macrophages. These results demonstrate that TAK1 activity is the key to activated macrophage survival. Finally, in an in vivo setting, Tab1 deficiency impaired increase of peritoneal macrophages upon LPS challenge, suggesting that TAK1 complex regulation of macrophages may participate in in vivo macrophage homeostasis. Our results demonstrate that TAB1 and TAB2 are required for activated macrophages, making TAB1 and TAB2 effective targets to control inflammation by modulating macrophage survival.

The aim of our research is to determine the biological function of gamma delta lymphocytes and in particular the role they play in microbial immunity. Although evidence of gamma delta T-cell activation and expansion has been obtained from numerous infectious diseases, how they contribute to pathogen-induced immune responses is still not clear. Based upon extensive studies of gamma delta T-cell involvement in the immune response to viral and bacterial pathogens in both mice and humans, we have uncovered evidence of their direct involvement in terminating host immune responses to infection and preventing chronic disease. We have identified an interaction between peripheral gamma delta T cells and a population of activated, proinflammatory macrophages elicited by infection that occurs late in the course of infection during or after pathogen clearance. As a result of this interaction, activated gamma delta T cells acquire cytotoxic activity and kill the stimulatory macrophages, leading us to propose a model for gamma delta T-cell-macrophage interactions that contributes to macrophage homeostasis, the resolution of inflammatory immune responses, and prevention of chronic inflammatory disease.

Anthrax lethal toxin (LeTx), consisting of protective antigen (PA) and lethal factor (LF), rapidly kills primary mouse macrophages and macrophage-like cell lines such as RAW 264.7. LF is translocated by PA into the cytosol of target cells, where it acts as a metalloprotease to cleave mitogen-activated protein kinase kinase 1 (MEK1) and possibly other proteins. In this study, we show that proteasome inhibitors such as acetyl-Leu-Leu-norleucinal, MG132, and lactacystin efficiently block LeTx cytotoxicity, whereas other protease inhibitors do not. The inhibitor concentrations that block LF cytotoxicity are similar to those that inhibit the proteasome-dependent IκB-α degradation induced by lipopolysaccharide. The inhibitors did not interfere with the proteolytic cleavage of MEK1 in LeTx-treated cells, indicating that they do not directly block the proteolytic activity of LF. However, the proteasome inhibitors did prevent ATP depletion, an early effect of LeTx. No overall activation of the proteasome by LeTx was detected, as shown by the cleavage of fluorogenic substrates of the proteasome. All of these results suggest that the proteasome mediates a toxic process initiated by LF in the cell cytosol. This process probably involves degradation of unidentified molecules that are essential for macrophage homeostasis. Moreover, this proteasome-dependent process is an early step in LeTx intoxication, but it is downstream of the cleavage by LF of MEK1 or other putative substrates.

The gut mucosal immune system is considered to play an important role in counteracting potential adverse effects of food-derived antigens including nanovesicles. Whether nanovesicles naturally released from edible fruit work in a coordinated manner with gut immune cells to maintain the gut in a noninflammatory status is not known. Here, as proof of concept, we demonstrate that grapefruit-derived nanovesicles (GDNs) are selectively taken up by intestinal macrophages and ameliorate dextran sulfate sodium (DSS)-induced mouse colitis. These effects were mediated by upregulating the expression of heme oxygenase-1 (HO-1) and inhibiting the production of IL-1β and TNF-α in intestinal macrophages. The inherent biocompatibility and biodegradability, stability at wide ranges of pH values, and targeting of intestinal macrophages led us to further develop a novel GDN-based oral delivery system. Incorporating methotrexate (MTX), an anti-inflammatory drug, into GDNs and delivering the MTX-GDNs to mice significantly lowered the MTX toxicity when compared with free MTX, and remarkably increased its therapeutic effects in DSS-induced mouse colitis. These findings demonstrate that GDNs can serve as immune modulators in the intestine, maintain intestinal macrophage homeostasis, and can be developed for oral delivery of small molecule drugs to attenuate inflammatory responses in human disease.

To prevent excessive inflammatory responses to commensal microbes, intestinal macrophages, unlike their systemic counterparts, do not produce inflammatory cytokines in response to enteric bacteria. Consequently, loss of macrophage tolerance to the enteric microbiota plays a central role in the pathogenesis of inflammatory bowel diseases. Therefore, we examined whether the hyporesponsive phenotype of intestinal macrophages is programmed by prior exposure to the microbiota. IL-10, but not in vivo exposure to the microbiota, programs intestinal macrophage tolerance, because wild-type (WT) colonic macrophages from germ-free and specific pathogen-free (SPF)-derived mice produce IL-10, but not IL-12 p40, when activated with enteric bacteria. Basal and activated IL-10 expression is mediated through a MyD88-dependent pathway. Conversely, colonic macrophages from germ-free and SPF-derived colitis-prone Il10−/− mice demonstrated robust production of IL-12 p40. Next, mechanisms through which IL-10 inhibits Il12b expression were investigated. Although Il12b mRNA was transiently induced in LPS-activated WT bone marrow-derived macrophages (BMDMs), expression persisted in Il10−/− BMDMs. There were no differences in nucleosome remodeling, mRNA stability, NF-κB activation, or MAPK signaling to explain prolonged transcription of Il12b in Il10−/− BMDMs. However, acetylated histone H4 transiently associated with the Il12b promoter in WT BMDMs, whereas association of these factors was prolonged in Il10−/− BMDMs. Experiments using histone deacetylase (HDAC) inhibitors and HDAC3 short hairpin RNA indicate that HDAC3 is involved in histone deacetylation of the Il12b promoter by IL-10. These results suggest that histone deacetylation on the Il12b promoter by HDAC3 mediates homeostatic effects of IL-10 in macrophages.

Macrophages remove billions of apoptotic cells daily, releasing their nucleic acid material through lysosomal degradation, which allows the resulting nucleosides to be recycled. Hsu et al . found that the nucleoside transporter equilibrative nucleoside transporter 3 (ENT3) was highly expressed in macrophages and showed that mice deficient in this transporter develop histiocytosis and features of lysosomal storage disease. When exposed to apoptotic cells, macrophages carrying human ENT3 mutations accumulated adenosine and increased their lysosomal pH. These changes contributed to an enhanced signaling through macrophage colony-stimulating factor (M-CSF) receptor and, ultimately, to M-CSF–driven myeloproliferative disease. C.-L. Hsu, W. Lin, D. Seshasayee, Y.-H. Chen, X. Ding, Z. Lin, E. Suto, Z. Huang, W. P. Lee, H. Park, M. Xu, M. Sun, L. Rangell, J. L. Lutman, S. Ulufatu, E. Stefanich, C. Chalouni, M. Sagolla, L. Diehl, P. Fielder, B. Dean, M. Balazs, F. Martin, Equilibrative nucleoside transporter 3 deficiency perturbs lysosome function and macrophage homeostasis. Science 335 , 89–92 (2012). [Abstract][Full Text]

CD200 receptor 1 is an inhibitory receptor expressed by myeloid cells which has inhibitory effects on macrophage function after binding its ubiquitously expressed ligand CD200. Recent evidence suggests that this is important in controlling inflammatory reactions in the lung and here we have explored if the CD200R1-CD200 axis plays a similar role in other mucosal surfaces such as the intestine. We show for the first time that CD200R1 is expressed selectively by resident macrophages in normal mouse colon and that CD200 is present on many haematopoietic and non-haematopoietic cells in the intestine. Although acute colitis induced by feeding dextran sodium sulphate is associated with an influx of CD200R1(neg) macrophages, CD200R1 KO mice have normal macrophage function in the colon and they do not develop spontaneous intestinal inflammation, nor are they more susceptible to DSS colitis. CD200 KO mice also develop experimental colitis normally and we conclude that CD200R1 does not play an essential role in macrophage homeostasis in the colon, indicating that these molecules may have distinct functions in different mucosal tissues.

The two most common forms of inflammatory bowel disease (IBD), Crohn's disease and ulcerative colitis, affect approximately 1 million people in the United States. Uncontrolled APC reactivity toward commensal bacteria is implicated in the pathogenesis of the disease. A number of functionally distinct APC populations exist in the mucosal lamina propria (LP) below the intestinal epithelium, but their relative contributions to inflammation remain unclear. Here, we demonstrate in mice important roles for the chemokine receptor CX3CR1 in maintaining LP macrophage populations, preventing translocation of commensal bacteria to mesenteric lymph nodes (mLNs), and limiting colitogenic Th17 responses. CX3CR1 was found to be expressed in resident LP macrophages (defined as CD11b(+)F4/80(+)) but not DCs (defined as CD11c(+)CD103(+)). LP macrophage frequency and number were decreased in two strains of CX3CR1-knockout mice and in mice deficient in the CX3CR1 ligand CX3CL1. All these knockout strains displayed markedly increased translocation of commensal bacteria to mLNs. Additionally, the severity of DSS-induced colitis was dramatically enhanced in the knockout mice as compared with controls. Disease severity could be limited by either administration of neutralizing IL-17A antibodies or transfer of CX3CR1-sufficient macrophages. Our data thus suggest key roles for the CX3CR1/CX3CL1 axis in the intestinal mucosa; further clarification of CX3CR1 function will likely direct efforts toward therapeutic intervention for mucosal inflammatory disorders such as IBD.

Macrophages contribute to both initiation and resolution of inflammatory processes. These cells of diverse functional activity participate as both effectors and affectors in host tissue development, homeostasis and response to injury. Survival and function of the macrophage is dependent on colony stimulating factors granulocyte colony stimulating factor (GM-CSF) and macrophage colony stimulating factor (M-CSF). These colony stimulating factors play a significant role in defining the phenotype and activity of macrophages through regulating the expression of transcription factors PU.1 and peroxisome proliferator activator receptor gamma (PPARγ). Recent data suggests that alveolar macrophages are not passive participants in immune responses and that the status of “macrophage activation” defines the phenotype of the ensuing innate and adaptive responses. The status of alveolar macrophage activation, including cytokine production, phagocytosis, and antigen presentation, orchestrates the intensity and duration of the immune response. This chapter summarizes the perspectives of colony stimulating factor regulation of macrophage activity in immunity and pulmonary homeostasis.

Alveolar macrophages (AMs) and peribronchial/perivascular macrophages are probably involved in lung allograft damage. We investigate leukocyte infiltration into graft tissue and address the question whether proliferation in situ contributes to macrophage homeostasis and accumulation. Lung transplantation was performed in the Lewis (LEW)-to-LEW and in the Dark Agouti-to-LEW rat strain combination. Graft infiltration by ED1+ and ED2+ (CD163) macrophages was analyzed by immunohistochemistry (IHC) and compared with infiltration by lymphocytes. Cells in the S-phase of the cell cycle were pulse-labeled with BrdU and detected immunohistochemically. Finally, the donor or recipient origin of AMs was determined by IHC and in situ hybridization. ED1+ AMs in allogeneic transplants increased by more than 25-fold from Days 1 to 5. In addition, large, peribronchial/perivascular infiltrates developed containing numerous ED1+ cells. Although AMs in normal rat lungs are CD163−, AMs up-regulated CD163 between Days 4 and 5, reaching maximum values on Day 6. Lymphocytes were less numerous than macrophages. About 16% of the AMs and 10% of the peribronchial/perivascular macrophages were in the S-phase of the cell cycle on Day 2 post-transplantation. No differences in the frequency of BrdU+ macrophages were obvious between isografts and allografts. AMs of donor origin increased in number considerably during allograft rejection. In conclusion, the cellular infiltrate in lung allografts is dominated by macrophages, which exhibit an unusual phenotype and a strong capacity for mitotic self-renewal.

Over 1 billion monocytes are produced daily, with a small percentage differentiating into macrophages, suggesting that excess monocytes are deleted through a tightly regulated process. Although the in vivo mechanism governing monocyte/macrophage homeostasis is unknown, deletion of monocytes in culture is mediated by the Fas death pathway and is blocked by M-CSF. To determine the in vivo significance of Fas in monocyte development, mice lacking Fas (lpr/lpr) and mice deficient in Fas and M-CSF were examined. Compared with congenic control C57BL/6 (B6) mice, lpr/lpr mice displayed increased numbers of circulating monocytes. The lack of Fas in M-CSF-deficient mice resulted in an enhanced percentage, but not total numbers, of monocytes. Fas deficiency led to an increase in myeloid bone marrow progenitor potential only in M-CSF-intact mice. Although lpr/lpr and B6 mice had similar numbers of tissue macrophages, the loss of Fas in M-CSF-deficient mice was sufficient to increase the number of macrophages in a subset of tissues. Additionally, after stimulation with thioglycolate, lpr/lpr and B6 mice showed equivalent numbers of peritoneal macrophages. However, Fas-deficient peritoneal macrophages displayed a marked increase in spontaneous and LPS-induced proinflammatory molecule production. Moreover, Fas-deficient mice showed enhanced systemic inflammatory arthritis associated with up-regulation of IL-1β and CCL2 secretion, elevated numbers of inflammatory monocytes, and increased numbers of tissue macrophages. Collectively, these data suggest that Fas may be required for maintaining circulating monocytes and for suppressing macrophage activation and recruitment that are stimulus dependent.

Nuclear hormone receptors comprise a superfamily of ligand-dependent transcription factors that regulate diverse aspects of development and homeostasis. Several members of this superfamily play important roles in the regulation of inflammatory responses and lipid homeostasis in macrophages. These include the glucocorticoid receptor, which acts to inhibit inflammatory programs of gene expression in response to natural corticosteroids and synthetic anti-inflammatory agents such as dexamethasone, peroxisome proliferator-activated receptors (PPARs) that regulate fatty acid homeostasis and inflammation in response to endogenous eicosanoids, and liver X receptors (LXRs) that regulate cholesterol efflux in response to endogenous oxysterols. Recent progress in defining the physiological roles of these receptor systems in macrophages and understanding their mechanisms of action suggest that they may be important targets for the development of new classes of pharmaceuticals that will be useful for treating human diseases in which macrophages play critical pathogenic roles, such as atherosclerosis and arthritis.

The human marrow produces ∼1010 monocytes daily, and this production must be balanced by a similar rate of destruction. Monocytes/macrophages can undergo apoptosis after activating CD4+ T cells, suggesting one mechanism that may contribute to macrophage homeostasis. Previous reports indicate that Fas-Fas ligand interactions are the principle molecules mediating this response. However, D10, an Iak-restricted cloned Th2 line, will similarly induce apoptosis in Ag-presenting macrophages, and D10 cells lack Fas ligand. To confirm that D10 cells kill macrophages through Fas-independent pathways, D10 cells were shown to kill MRL lpr/lpr (Iak) macrophages in an Ag-dependent fashion, indicating additional mechanisms. Recent reports demonstrate that TNF-related apoptosis-inducing ligand (TRAIL), interacting with Apo2, and TNF-like weak inducer of apoptosis (TWEAK), interacting with Apo3, will induce apoptosis in some cells. Using Abs to TRAIL and an Apo3-IgG Fc fusion protein, we demonstrated that D10 cells express both TRAIL and TWEAK. The Apo3 fusion protein, but not human IgG, inhibited D10-induced macrophage apoptosis, as did anti-TRAIL. Further studies demonstrated that AE7, a cloned Th1 line, and splenic T cells express TWEAK, TRAIL, and Fas ligand, and inhibiting these molecules also inhibited macrophage killing. These results indicate that D10 cells induce macrophage apoptosis through TRAIL- and TWEAK-dependent pathways. Because normal T cells also express these molecules, these results support the concept that T cells have multiple pathways by which to induce macrophage apoptosis. These pathways may be important in immune processes such as macrophage homeostasis as well as in down-regulation of immune responses and elimination of macrophages infected with intracellular organisms.