Liver Fetuin-A at Initiation of Insulin Resistance.

Hepatokines (liver secreted proteins with possible distant action) are emerging potential players in insulin resistance in type 2 diabetic patients. Here, we explored the effect of a high-fat diet on the expression of fetuin-A, one of those candidate liver proteins, and its relationship with liver macrophage activation. Mice were fed a normal diet or a high-fat diet for 3 days, known to initiate steatosis and liver insulin resistance. A preventive liver macrophage depletion was obtained by intravenous injection of clodronate-loaded liposomes. The mRNA and protein expression of fetuin-A was evaluated by qPCR, Western blot and immunofluorescence on different insulin-sensitive tissues (liver, adipose tissue, and muscle). Short-term high-fat diet-induced steatosis, liver macrophage activation, and hepatic insulin resistance together with a significantly increased expression of liver AHSG (α2-HS glycoprotein/fetuin-A) mRNA and serum fetuin-A concentration. On immunofluorescence, fetuin-A was mostly expressed in centrilobular hepatocytes. This increase in fetuin-A under high-fat diet was not evidenced in other peripheral insulin-sensitive tissues (skeletal muscle and adipose tissue). The mRNA expression of α2-HS glycoprotein was 800 times higher within the liver compared with the adipose tissue or the muscle. Liver macrophage depletion that significantly ameliorated insulin sensitivity was associated with a significant decrease in α2-HS glycoprotein mRNA expression. In conclusion, this study demonstrated liver fetuin-A overexpression at the initiation of high-fat diet feeding, concurrent with hepatic steatosis and insulin resistance. Targeting liver macrophages in this setting reduced liver α2-HS glycoprotein expression suggesting that fetuin-A acts as an hepatokine with proinsulin resistance effects.

Microglia/Macrophages and CD4+CD25+ T Cells Enhance the Ability of Injury-Activated Lymphocytes to Reduce Traumatic Optic Neuropathy In Vitro.

Inflammation after acute CNS injury plays a dual role. The interplay between immune cells and inflammatory mediators is critical to the outcome of injured neurons. Microglia/macrophages are the first sensors and regulators of the immune response. We previously found that the enhancement of macrophages on neuron survival does not persist in thymectomized rats. How T lymphocytes and macrophages interact and benefit neuron survival is not fully elucidated. To this point, we introduce and characterize a cell-retina co-culture model that mimics the recruitment of peripheral lymphocytes at the injury site. Three-day post-optic nerve transection (ONT) in Fischer 344 rats, transected retinas were co-cultured with either peripheral lymph node-derived lymphocytes (injury-activated) or from intact rats as the control. The injury-activated lymphocytes preserved retinal ganglion cells (RGCs) and caused extensive retina microglial/macrophage infiltration. CD4+CD25+ T cells were upregulated in the injury-activated lymphocytes and increased RGC survival, suggesting that CD4+CD25+ T cells suppressed the cytotoxicity of control lymphocytes. When microglia/macrophages were depleted by clodronate, neuron loss was more extensive, the cytotoxicity of control lymphocytes on RGCs was alleviated, and the neuroprotective effect of injury-activated lymphocytes remain unchanged Cytokine detection showed an increase in IL-6 and TNF-α levels that were reduced with microglia/macrophage depletion. Our results suggest that microglial/macrophage infiltration into axotomized retinas promotes RGC survival by secreting cytokines to induce CD4+CD25+ T cells and suppress T cell-mediated RGC toxicity. These findings reveal a specific role for microglia/macrophage and CD4+CD25+ T cells in inflammation after CNS injury, thereby adding to the mechanistic basis for the development of microglial/macrophage modulation therapy for traumatic CNS injury.

Pioglitazone-Mediated Peroxisome Proliferator-Activated Receptor γ Activation Aggravates Murine Immune-Mediated Hepatitis.

The nuclear receptor peroxisome proliferator-activated receptor gamma (PPARγ) regulates target gene expression upon ligand binding. Apart from its effects on metabolism, PPARγ activity can inhibit the production of pro-inflammatory cytokines by several immune cells, including dendritic cells and macrophages. In chronic inflammatory disease models, PPARγ activation delays the onset and ameliorates disease severity. Here, we investigated the effect of PPARγ activation by the agonist Pioglitazone on the function of hepatic immune cells and its effect in a murine model of immune-mediated hepatitis. Cytokine production by both liver sinusoidal endothelial cells (IL-6) and in T cells ex vivo (IFNγ) was decreased in cells from Pioglitazone-treated mice. However, PPARγ activation did not decrease pro-inflammatory tumor necrosis factor alpha TNFα production by Kupffer cells after Toll-like receptor (TLR) stimulation ex vivo. Most interestingly, although PPARγ activation was shown to ameliorate chronic inflammatory diseases, it did not improve hepatic injury in a model of immune-mediated hepatitis. In contrast, Pioglitazone-induced PPARγ activation exacerbated D-galactosamine (GalN)/lipopolysaccharide (LPS) hepatitis associated with an increased production of TNFα by Kupffer cells and increased sensitivity of hepatocytes towards TNFα after in vivo Pioglitazone administration. These results unravel liver-specific effects of Pioglitazone that fail to attenuate liver inflammation but rather exacerbate liver injury in an experimental hepatitis model.

Alveolar Macrophage Apoptosis-associated Bacterial Killing Helps Prevent Murine Pneumonia.

Rationale: Antimicrobial resistance challenges therapy of pneumonia. Enhancing macrophage microbicidal responses would combat this problem but is limited by our understanding of how alveolar macrophages (AMs) kill bacteria. Objectives: To define the role and mechanism of AM apoptosis-associated bacterial killing in the lung. Methods: We generated a unique CD68.hMcl-1 transgenic mouse with macrophage-specific overexpression of the human antiapoptotic Mcl-1 protein, a factor upregulated in AMs from patients at increased risk of community-acquired pneumonia, to address the requirement for apoptosis-associated killing. Measurements and Main Results: Wild-type and transgenic macrophages demonstrated comparable ingestion and initial phagolysosomal killing of bacteria. Continued ingestion (for ≥12 h) overwhelmed initial killing, and a second, late-phase microbicidal response killed viable bacteria in wild-type macrophages, but this response was blunted in CD68.hMcl-1 transgenic macrophages. The late phase of bacterial killing required both caspase-induced generation of mitochondrial reactive oxygen species and nitric oxide, the peak generation of which coincided with the late phase of killing. The CD68.hMcl-1 transgene prevented mitochondrial reactive oxygen species but not nitric oxide generation. Apoptosis-associated killing enhanced pulmonary clearance of Streptococcus pneumoniae and Haemophilus influenzae in wild-type mice but not CD68.hMcl-1 transgenic mice. Bacterial clearance was enhanced in vivo in CD68.hMcl-1 transgenic mice by reconstitution of apoptosis with BH3 mimetics or clodronate-encapsulated liposomes. Apoptosis-associated killing was not activated during Staphylococcus aureus lung infection. Conclusions: Mcl-1 upregulation prevents macrophage apoptosis-associated killing and establishes that apoptosis-associated killing is required to allow AMs to clear ingested bacteria. Engagement of macrophage apoptosis should be investigated as a novel, host-based antimicrobial strategy.

TGFß inhibition restores a regenerative response in acute liver injury by suppressing paracrine senescence.

Liver injury results in rapid regeneration through hepatocyte proliferation and hypertrophy. However, after acute severe injury, such as acetaminophen poisoning, effective regeneration may fail. We investigated how senescence may underlie this regenerative failure. In human acute liver disease, and murine models, p21-dependent hepatocellular senescence was proportionate to disease severity and was associated with impaired regeneration. In an acetaminophen injury mouse model, a transcriptional signature associated with the induction of paracrine senescence was observed within 24 hours and was followed by one of impaired proliferation. In mouse genetic models of hepatocyte injury and senescence, we observed transmission of senescence to local uninjured hepatocytes. Spread of senescence depended on macrophage-derived transforming growth factor-ß1 (TGFß1) ligand. In acetaminophen poisoning, inhibition of TGFß receptor 1 (TGFßR1) improved mouse survival. TGFßR1 inhibition reduced senescence and enhanced liver regeneration even when delivered beyond the therapeutic window for treating acetaminophen poisoning. This mechanism, in which injury-induced senescence impairs liver regeneration, is an attractive therapeutic target for developing treatments for acute liver failure.

Casein kinase-II inhibition promotes retinal ganglion cell survival and axonal regeneration.

Neuron survival is critical for the maintenance of central nervous system physiology upon diseases or injury. We previously demonstrated that the blockage of phosphatidylinositol 3-kinase/Akt and Janus kinase/STAT3 pathways promotes retinal ganglion cell (RGC) survival and axonal regeneration via macrophage activation; yet, the complexity of the inflammatory regulation for neural repair indicates the involvement of additional unresolved signaling pathways. Here we report the effects and underlying mechanism of casein kinase-II (CK2) inhibition on RGC survival and axonal regeneration in rats after optic nerve (ON) injury. Adult rats received intravitreal injection of CK2 inhibitors, TBB (4,5,6,7-Tetrabromo-2-azabenzimidazole) and DMAT (2-Dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole), after ON transection and peripheral nerve (PN) grafting. Intravitreal application of TBB and DAMT effectively suppressed the CK2 phosphorylation activity in the retina, and enhanced RGC survival and axonal regeneration in vivo. Meanwhile, the numbers of infiltrating macrophages were increased. Removal of macrophages by clodronate liposomes significantly abolished the CK2 inhibition-induced RGC survival and axonal regeneration. Clodronate liposomes also weakened the RGC protective effects by TBB and DMAT in vitro. In summary, this study revealed that inhibition of CK2 enhances RGC survival and axonal regeneration via macrophage activation in rats. CK2 could be a therapeutic target for RGC protection after ON injury.

Malt1 blocks IL-1ß production by macrophages in vitro and limits dextran sodium sulfate-induced intestinal inflammation in vivo.

This study tested the hypothesis that Malt1 deficiency in macrophages contributes to dextran sodium sulfate (DSS)-induced intestinal inflammation in Malt1-deficient mice. In people, combined immunodeficiency caused by a homozygous mutation in the MALT1 gene is associated with increased susceptibility to bacterial infections and chronic inflammation, including severe inflammation along the gastrointestinal tract. The consequences of Malt1 deficiency have largely been attributed to its role in lymphocytes, but Malt1 is also expressed in macrophages, where it is activated downstream of TLR4 and dectin-1. The effect of Malt1 deficiency in murine macrophages and its contribution to DSS-induced colitis have not been investigated. Our objectives were to compare the susceptibility of Malt1+/+ and Malt1-/- mice to DSS-induced colitis, to determine the contribution of macrophages to DSS-induced colitis in Malt1-/- mice, and to assess the effect of innate immune stimuli on Malt1-/- macrophage inflammatory responses. We found that Malt1 deficiency exacerbates DSS-induced colitis in mice, accompanied by higher levels of IL-1ß, and that macrophages and IL-1 signaling contribute to pathology in Malt1-/- mice. Malt1-/- macrophages produce more IL-1ß in response to either TLR4 or dectin-1 ligation, whereas inhibition of Malt1 proteolytic (paracaspase) activity blocked IL-1ß production. TLR4 or dectin-1 stimulation induced Malt1 protein levels but decreased its paracaspase activity. Taken together, these data support the hypothesis that Malt1-/- macrophages contribute to increased susceptibility of Malt1-/- mice to DSS-induced colitis, which is dependent on IL-1 signaling. Increased IL-1ß production by MALT1-deficient macrophages may also contribute to chronic inflammation in people deficient in MALT1.

TRAIL-Dependent Resolution of Pulmonary Fibrosis.

Idiopathic pulmonary fibrosis (IPF) is the most common form of interstitial lung disease characterized by the persistence of activated myofibroblasts resulting in excessive deposition of extracellular matrix proteins and profound tissue remodeling. In the present study, the expression of tumor necrosis factor- (TNF-) related apoptosis-inducing ligand (TRAIL) was key to the resolution of bleomycin-induced pulmonary fibrosis. Both in vivo and in vitro studies demonstrated that Gr-1+TRAIL+ bone marrow-derived myeloid cells blocked the activation of lung myofibroblasts. Although soluble TRAIL was increased in plasma from IPF patients, the presence of TRAIL+ myeloid cells was markedly reduced in IPF lung biopsies, and primary lung fibroblasts from this patient group expressed little of the TRAIL receptor-2 (DR5) when compared with appropriate normal samples. IL-13 was a potent inhibitor of DR5 expression in normal fibroblasts. Together, these results identified TRAIL+ myeloid cells as a critical mechanism in the resolution of pulmonary fibrosis, and strategies directed at promoting its function might have therapeutic potential in IPF.

Interleukin-4 activated macrophages mediate immunity to filarial helminth infection by sustaining CCR3-dependent eosinophilia.

Eosinophils are effectors in immunity to tissue helminths but also induce allergic immunopathology. Mechanisms of eosinophilia in non-mucosal tissues during infection remain unresolved. Here we identify a pivotal function of tissue macrophages (Mϕ) in eosinophil anti-helminth immunity using a BALB/c mouse intra-peritoneal Brugia malayi filarial infection model. Eosinophilia, via C-C motif chemokine receptor (CCR)3, was necessary for immunity as CCR3 and eosinophil impairments rendered mice susceptible to chronic filarial infection. Post-infection, peritoneal Mϕ populations proliferated and became alternatively-activated (AAMϕ). Filarial AAMϕ development required adaptive immunity and interleukin-4 receptor-alpha. Depletion of Mϕ prior to infection suppressed eosinophilia and facilitated worm survival. Add back of filarial AAMϕ in Mϕ-depleted mice recapitulated a vigorous eosinophilia. Transfer of filarial AAMϕ into Severe-Combined Immune Deficient mice mediated immunological resistance in an eosinophil-dependent manner. Exogenous IL-4 delivery recapitulated tissue AAMϕ expansions, sustained eosinophilia and mediated immunological resistance in Mϕ-intact SCID mice. Co-culturing Brugia with filarial AAMϕ and/or filarial-recruited eosinophils confirmed eosinophils as the larvicidal cell type. Our data demonstrates that IL-4/IL-4Rα activated AAMϕ orchestrate eosinophil immunity to filarial tissue helminth infection.

Macrophage-dependent impairment of α2-adrenergic autoreceptor inhibition of Ca2+ channels in sympathetic neurons from DOCA-salt but not high-fat diet-induced hypertensive rats.

DOCA-salt and obesity-related hypertension are associated with inflammation and sympathetic nervous system hyperactivity. Prejunctional α2-adrenergic receptors (α2ARs) provide negative feedback to norepinephrine release from sympathetic nerves through inhibition of N-type Ca2+ channels. Increased neuronal norepinephrine release in DOCA-salt and obesity-related hypertension occurs through impaired α2AR signaling; however, the mechanisms involved are unclear. Mesenteric arteries are resistance arteries that receive sympathetic innervation from the superior mesenteric and celiac ganglia (SMCG). We tested the hypothesis that macrophages impair α2AR-mediated inhibition of Ca2+ channels in SMCG neurons from DOCA-salt and high-fat diet (HFD)-induced hypertensive rats. Whole cell patch-clamp methods were used to record Ca2+ currents from SMCG neurons maintained in primary culture. We found that DOCA-salt, but not HFD-induced, hypertension caused macrophage accumulation in mesenteric arteries, increased SMCG mRNA levels of monocyte chemoattractant protein-1 and tumor necrosis factor-α, and impaired α2AR-mediated inhibition of Ca2+ currents in SMCG neurons. α2AR dysfunction did not involve changes in α2AR expression, desensitization, or downstream signaling factors. Oxidative stress impaired α2AR-mediated inhibition of Ca2+ currents in SMCG neurons and resulted in receptor internalization in human embryonic kidney-293T cells. Systemic clodronate-induced macrophage depletion preserved α2AR function and lowered blood pressure in DOCA-salt rats. HFD caused hypertension without obesity in Sprague-Dawley rats and hypertension with obesity in Dahl salt-sensitive rats. HFD-induced hypertension was not associated with inflammation in SMCG and mesenteric arteries or α2AR dysfunction in SMCG neurons. These results suggest that macrophage-mediated α2AR dysfunction in the mesenteric circulation may only be relevant to mineralocorticoid-salt excess. NEW & NOTEWORTHY Here, we identify a contribution of macrophages to hypertension development through impaired α2-adrenergic receptor (α2AR)-mediated inhibition of sympathetic nerve terminal Ca2+ channels in DOCA-salt hypertensive rats. Impaired α2AR function may involve oxidative stress-induced receptor internalization. α2AR dysfunction may be unique to mineralocorticoid-salt excess, as it does not occur in obesity-related hypertension.

Tumor Necrosis Factor-Mediated Survival of CD169+ Cells Promotes Immune Activation during Vesicular Stomatitis Virus Infection.

Innate immune activation is essential to mount an effective antiviral response and to prime adaptive immunity. Although a crucial role of CD169+ cells during vesicular stomatitis virus (VSV) infections is increasingly recognized, factors regulating CD169+ cells during viral infections remain unclear. Here, we show that tumor necrosis factor is produced by CD11b+ Ly6C+ Ly6G+ cells following infection with VSV. The absence of TNF or TNF receptor 1 (TNFR1) resulted in reduced numbers of CD169+ cells and in reduced type I interferon (IFN-I) production during VSV infection, with a severe disease outcome. Specifically, TNF triggered RelA translocation into the nuclei of CD169+ cells; this translocation was inhibited when the paracaspase MALT-1 was absent. Consequently, MALT1 deficiency resulted in reduced VSV replication, defective innate immune activation, and development of severe disease. These findings indicate that TNF mediates the maintenance of CD169+ cells and innate and adaptive immune activation during VSV infection.IMPORTANCE Over the last decade, strategically placed CD169+ metallophilic macrophages in the marginal zone of the murine spleen and lymph nodes (LN) have been shown to play a very important role in host defense against viral pathogens. CD169+ macrophages have been shown to activate innate and adaptive immunity via "enforced virus replication," a controlled amplification of virus particles. However, the factors regulating the CD169+ macrophages remain to be studied. In this paper, we show that after vesicular stomatitis virus infection, phagocytes produce tumor necrosis factor (TNF), which signals via TNFR1, and promote enforced virus replication in CD169+ macrophages. Consequently, lack of TNF or TNFR1 resulted in defective immune activation and VSV clearance.

Macrophages in bone fracture healing: Their essential role in endochondral ossification.

In fracture healing, skeletal and immune system are closely interacting through common cell precursors and molecular mediators. It is thought that the initial inflammatory reaction, which involves migration of macrophages into the fracture area, has a major impact on the long term outcome of bone repair. Interestingly, macrophages reside during all stages of fracture healing. Thus, we hypothesized a critical role for macrophages in the subsequent phases of bone regeneration. This study examined the impact of in vivo induced macrophage reduction, using clodronate liposomes, on the different healing phases of bone repair in a murine model of a standard closed femoral fracture. A reduction in macrophages had no obvious effect on the early fracture healing phase, but resulted in a delayed hard callus formation, thus severely altering endochondral ossification. Clodronate treated animals clearly showed delayed bony consolidation of cartilage and enhanced periosteal bone formation. Therefore, we decided to backtrack macrophage distribution during fracture healing in non-treated mice, focusing on the identification of the M1 and M2 subsets. We observed that M2 macrophages were clearly prevalent during the ossification phase. Therefore enhancement of M2 phenotype in macrophages was investigated as a way to further bone healing. Induction of M2 macrophages through interleukin 4 and 13 significantly enhanced bone formation during the 3week investigation period. These cumulative data illustrate their so far unreported highly important role in endochondral ossification and the necessity of a fine balance in M1/M2 macrophage function, which appears mandatory to fracture healing and successful regeneration.

A systemic macrophage response is required to contain a peripheral poxvirus infection.

The goal of the innate immune system is to reduce pathogen spread prior to the initiation of an effective adaptive immune response. Following an infection at a peripheral site, virus typically drains through the lymph to the lymph node prior to entering the blood stream and being systemically disseminated. Therefore, there are three distinct spatial checkpoints at which intervention to prevent systemic spread of virus can occur, namely: 1) the site of infection, 2) the draining lymph node via filtration of lymph or 3) the systemic level via organs that filter the blood. We have previously shown that systemic depletion of phagocytic cells allows viral spread after dermal infection with Vaccinia virus (VACV), which infects naturally through the skin. Here we use multiple depletion methodologies to define both the spatial checkpoint and the identity of the cells that prevent systemic spread of VACV. Subcapsular sinus macrophages of the draining lymph node have been implicated as critical effectors in clearance of lymph borne viruses following peripheral infection. We find that monocyte populations recruited to the site of VACV infection play a critical role in control of local pathogenesis and tissue damage, but do not prevent dissemination of virus. Following infection with virulent VACV, the subcapsular sinus macrophages within the draining lymph node become infected, but are not exclusively required to prevent systemic spread. Rather, small doses of VACV enter the bloodstream and the function of systemic macrophages, but not dendritic cells, is required to prevent further spread. The results illustrate that a systemic innate response to a peripheral virus infection may be required to prevent widespread infection and pathology following infection with virulent viruses, such as poxviruses.

Inflammatory monocytes require type I interferon receptor signaling to activate NK cells via IL-18 during a mucosal viral infection.

The requirement of type I interferon (IFN) for natural killer (NK) cell activation in response to viral infection is known, but the underlying mechanism remains unclear. Here, we demonstrate that type I IFN signaling in inflammatory monocytes, but not in dendritic cells (DCs) or NK cells, is essential for NK cell function in response to a mucosal herpes simplex virus type 2 (HSV-2) infection. Mice deficient in type I IFN signaling, Ifnar-/- and Irf9-/- mice, had significantly lower levels of inflammatory monocytes, were deficient in IL-18 production, and lacked NK cell-derived IFN-γ. Depletion of inflammatory monocytes, but not DCs or other myeloid cells, resulted in lower levels of IL-18 and a complete abrogation of NK cell function in HSV-2 infection. Moreover, this resulted in higher susceptibility to HSV-2 infection. Although Il18-/- mice had normal levels of inflammatory monocytes, their NK cells were unresponsive to HSV-2 challenge. This study highlights the importance of type I IFN signaling in inflammatory monocytes and the induction of the early innate antiviral response.

Mechanisms of anaphylaxis in human low-affinity IgG receptor locus knock-in mice.

BACKGROUND: Anaphylaxis can proceed through distinct IgE- or IgG-dependent pathways, which have been investigated in various mouse models. We developed a novel mouse strain in which the human low-affinity IgG receptor locus, comprising both activating (hFcγRIIA, hFcγRIIIA, and hFcγRIIIB) and inhibitory (hFcγRIIB) hFcγR genes, has been inserted into the equivalent murine locus, corresponding to a locus swap. OBJECTIVE: We sought to determine the capabilities of hFcγRs to induce systemic anaphylaxis and identify the cell types and mediators involved. METHODS: hFcγR expression on mouse and human cells was compared to validate the model. Passive systemic anaphylaxis was induced by injection of heat-aggregated human intravenous immunoglobulin and active systemic anaphylaxis after immunization and challenge. Anaphylaxis severity was evaluated based on hypothermia and mortality. The contribution of receptors, mediators, or cell types was assessed based on receptor blockade or depletion. RESULTS: The human-to-mouse low-affinity FcγR locus swap engendered hFcγRIIA/IIB/IIIA/IIIB expression in mice comparable with that seen in human subjects. Knock-in mice were susceptible to passive and active anaphylaxis, accompanied by downregulation of both activating and inhibitory hFcγR expression on specific myeloid cells. The contribution of hFcγRIIA was predominant. Depletion of neutrophils protected against hypothermia and mortality. Basophils contributed to a lesser extent. Anaphylaxis was inhibited by platelet-activating factor receptor or histamine receptor 1 blockade. CONCLUSION: Low-affinity FcγR locus-switched mice represent an unprecedented model of cognate hFcγR expression. Importantly, IgG-related anaphylaxis proceeds within a native context of activating and inhibitory hFcγRs, indicating that, despite robust hFcγRIIB expression, activating signals can dominate to initiate a severe anaphylactic reaction.

IgG subclasses determine pathways of anaphylaxis in mice.

BACKGROUND: Animal models have demonstrated that allergen-specific IgG confers sensitivity to systemic anaphylaxis that relies on IgG Fc receptors (FcγRs). Mouse IgG2a and IgG2b bind activating FcγRI, FcγRIII, and FcγRIV and inhibitory FcγRIIB; mouse IgG1 binds only FcγRIII and FcγRIIB. Although these interactions are of strikingly different affinities, these 3 IgG subclasses have been shown to enable induction of systemic anaphylaxis. OBJECTIVE: We sought to determine which pathways control the induction of IgG1-, IgG2a-, and IgG2b-dependent passive systemic anaphylaxis. METHODS: Mice were sensitized with IgG1, IgG2a, or IgG2b anti-trinitrophenyl mAbs and challenged with trinitrophenyl-BSA intravenously to induce systemic anaphylaxis that was monitored by using rectal temperature. Anaphylaxis was evaluated in mice deficient for FcγRs injected with mediator antagonists or in which basophils, monocytes/macrophages, or neutrophils had been depleted. FcγR expression was evaluated on these cells before and after anaphylaxis. RESULTS: Activating FcγRIII is the receptor primarily responsible for all 3 models of anaphylaxis, and subsequent downregulation of this receptor was observed. These models differentially relied on histamine release and the contribution of mast cells, basophils, macrophages, and neutrophils. Strikingly, basophil contribution and histamine predominance in mice with IgG1- and IgG2b-induced anaphylaxis correlated with the ability of inhibitory FcγRIIB to negatively regulate these models of anaphylaxis. CONCLUSION: We propose that the differential expression of inhibitory FcγRIIB on myeloid cells and its differential binding of IgG subclasses controls the contributions of mast cells, basophils, neutrophils, and macrophages to IgG subclass-dependent anaphylaxis. Collectively, our results unravel novel complexities in the involvement and regulation of cell populations in IgG-dependent reactions in vivo.

Depletion of myeloid cells exacerbates hepatitis and induces an aberrant increase in histone H3 in mouse serum.

Tissue-resident macrophages and bone marrow (BM)-derived monocytes play a crucial role in the maintenance of tissue homeostasis; however, their contribution to recovery from acute tissue injury is not fully understood. To address this issue, we generated an acute murine liver injury model using hepatocyte-specific Cflar-deficient (CflarHep-low ) mice. Cellular FLICE-inhibitory protein expression was down-regulated in Cflar-deficient hepatocytes, which thereby increased susceptibility of hepatocytes to death receptor-induced apoptosis. CflarHep-low mice developed acute hepatitis and recovered with clearance of apoptotic hepatocytes at 24 hours after injection of low doses of tumor necrosis factor α (TNFα), which could not induce hepatitis in wild-type (WT) mice. Depletion of Kupffer cells (KCs) by clodronate liposomes did not impair clearance of dying hepatocytes or exacerbate hepatitis in CflarHep-low mice. To elucidate the roles of BM-derived monocytes and neutrophils in clearance of apoptotic hepatocytes, we examined the effect of depletion of these cells on TNFα-induced hepatitis in CflarHep-low mice. We reconstituted CflarHep-low mice with BM cells from transgenic mice in which human diphtheria toxin receptor (DTR) was expressed under control of the lysozyme M (LysM) promoter. TNFα-induced infiltration of myeloid cells, including monocytes and neutrophils, was completely ablated in LysM-DTR BM-reconstituted CflarHep-low mice pretreated with diphtheria toxin, whereas KCs remained present in the livers. Under these experimental conditions, LysM-DTR BM-reconstituted CflarHep-low mice rapidly developed severe hepatitis and succumbed within several hours of TNFα injection. We found that serum interleukin-6 (IL-6), TNFα, and histone H3 were aberrantly increased in LysM-DTR BM-reconstituted, but not in WT BM-reconstituted, CflarHep-low mice following TNFα injection. CONCLUSION: These findings indicate an unexpected role of myeloid cells in decreasing serum IL-6, TNFα, and histone H3 levels via the suppression of TNFα-induced hepatocyte apoptosis. (Hepatology 2017;65:237-252).

Perivascular macrophages mediate the neurovascular and cognitive dysfunction associated with hypertension.

Hypertension is a leading risk factor for dementia, but the mechanisms underlying its damaging effects on the brain are poorly understood. Due to a lack of energy reserves, the brain relies on continuous delivery of blood flow to its active regions in accordance with their dynamic metabolic needs. Hypertension disrupts these vital regulatory mechanisms, leading to the neuronal dysfunction and damage underlying cognitive impairment. Elucidating the cellular bases of these impairments is essential for developing new therapies. Perivascular macrophages (PVMs) represent a distinct population of resident brain macrophages that serves key homeostatic roles but also has the potential to generate large amounts of reactive oxygen species (ROS). Here, we report that PVMs are critical in driving the alterations in neurovascular regulation and attendant cognitive impairment in mouse models of hypertension. This effect was mediated by an increase in blood-brain barrier permeability that allowed angiotensin II to enter the perivascular space and activate angiotensin type 1 receptors in PVMs, leading to production of ROS through the superoxide-producing enzyme NOX2. These findings unveil a pathogenic role of PVMs in the neurovascular and cognitive dysfunction associated with hypertension and identify these cells as a putative therapeutic target for diseases associated with cerebrovascular oxidative stress.

F4/80+ Host Macrophages Are a Barrier to Murine Embryonic Stem Cell-Derived Hematopoietic Progenitor Engraftment In Vivo.

Understanding how embryonic stem cells and their derivatives interact with the adult host immune system is critical to developing their therapeutic potential. Murine embryonic stem cell-derived hematopoietic progenitors (ESHPs) were generated via coculture with the bone marrow stromal cell line, OP9, and then transplanted into NOD.SCID.Common Gamma Chain (NSG) knockout mice, which lack B, T, and natural killer cells. Compared to control mice transplanted with adult lineage-negative bone marrow (Lin- BM) progenitors, ESHP-transplanted mice attained a low but significant level of donor hematopoietic chimerism. Based on our previous studies, we hypothesized that macrophages might contribute to the low engraftment of ESHPs in vivo. Enlarged spleens were observed in ESHP-transplanted mice and found to contain higher numbers of host F4/80+ macrophages compared to BM-transplanted controls. In vivo depletion of host macrophages using clodronate-loaded liposomes improved the ESHP-derived hematopoietic chimerism in the spleen but not in the BM. F4/80+ macrophages demonstrated a striking propensity to phagocytose ESHP targets in vitro. Taken together, these results suggest that macrophages are a barrier to both syngeneic and allogeneic ESHP engraftment in vivo.

Targeting Macrophages Sensitizes Chronic Lymphocytic Leukemia to Apoptosis and Inhibits Disease Progression.

The role of monocytes/macrophages in the development and progression of chronic lymphocytic leukemia (CLL) is poorly understood. Transcriptomic analyses show that monocytes/macrophages and leukemic cells cross talk during CLL progression. Macrophage depletion impairs CLL engraftment, drastically reduces leukemic growth, and favorably impacts mouse survival. Targeting of macrophages by either CSF1R signaling blockade or clodrolip-mediated cell killing has marked inhibitory effects on established leukemia also. Macrophage killing induces leukemic cell death mainly via the TNF pathway and reprograms the tumor microenvironment toward an antitumoral phenotype. CSF1R inhibition reduces leukemic cell load, especially in the bone marrow, and increases circulating CD20(+) leukemic cells. Accordingly, co-targeting TAMs and CD20-expressing leukemic cells provides a survival benefit in the mice. These results establish the important role of macrophages in CLL and suggest therapeutic strategies based on interfering with leukemia-macrophage interactions.