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.

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.

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.

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.

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.

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.

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.

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.

Mechanism of microglia neuroprotection: Involvement of P2X7, TNFα, and valproic acid.

Recently, we have demonstrated that ramified microglia are neuroprotective in N-methyl-D-aspartate (NMDA)-induced excitotoxicity in organotypic hippocampal slice cultures (OHSCs). The present study aimed to elucidate the underlying neuron-glia communication mechanism. It is shown here that pretreatment of OHSC with high concentrations of adenosine 5'-triphosphate (ATP) reduced NMDA-induced neuronal death only in presence of microglia. Specific agonists and antagonists identified the P2X7 receptor as neuroprotective receptor which was confirmed by absence of ATP-dependent neuroprotection in P2X7-deficient OHSC. Microglia replenished chimeric OHSC consisting of wild-type tissue replenished with P2X7-deficient microglia confirmed the involvement of microglial P2X7 receptor in neuroprotection. Stimulation of P2X7 in primary microglia induced tumor necrosis factor α (TNFα) release and blocking TNFα by a neutralizing antibody in OHSC abolished neuroprotection by ATP. OHSC from TNFα-deficient mice show increased exicitoxicity and activation of P2X7 did not rescue neuronal survival in the absence of TNFα. The neuroprotective effect of valproic acid (VPA) was strictly dependent on the presence of microglia and was mediated by upregulation of P2X7 in the cells. The present study demonstrates that microglia-mediated neuroprotection depends on ATP-activated purine receptor P2X7 and induction of TNFα release. This neuroprotective pathway was strengthened by VPA elucidating a novel mechanism for the neuroprotective function of VPA.

Activity of SHIP, Which Prevents Expression of Interleukin 1ß, Is Reduced in Patients With Crohn's Disease.

BACKGROUND & AIMS: Crohn's disease (CD) is associated with a dysregulated immune response to commensal micro-organisms in the intestine. Mice deficient in inositol polyphosphate 5'-phosphatase D (INPP5D, also known as SHIP) develop intestinal inflammation resembling that of patients with CD. SHIP is a negative regulator of PI3Kp110α activity. We investigated mechanisms of intestinal inflammation in Inpp5d(-/-) mice (SHIP-null mice), and SHIP levels and activity in intestinal tissues of subjects with CD. METHODS: We collected intestines from SHIP-null mice, as well as Inpp5d(+/+) mice (controls), and measured levels of cytokines of the interleukin 1 (IL1) family (IL1α, IL1ß, IL1ra, and IL6) by enzyme-linked immunosorbent assay. Macrophages were isolated from lamina propria cells of mice, IL1ß production was measured, and mechanisms of increased IL1ß production were investigated. Macrophages were incubated with pan-phosphatidylinositol 3-kinase inhibitors or PI3Kp110α-specific inhibitors. Some mice were given an antagonist of the IL1 receptor; macrophages were depleted from ilea of mice using clodronate-containing liposomes. We obtained ileal biopsies from sites of inflammation and peripheral blood mononuclear cells (PBMCs) from treatment-naïve subjects with CD or without CD (controls), and measured SHIP levels and activity. PBMCs were incubated with lipopolysaccharide and adenosine triphosphate, and levels of IL1ß production were measured. RESULTS: Inflamed intestinal tissues and intestinal macrophages from SHIP-null mice produced higher levels of IL1B and IL18 than intestinal tissues from control mice. We found PI3Kp110α to be required for macrophage transcription of Il1b. Macrophage depletion or injection of an IL1 receptor antagonist reduced ileal inflammation in SHIP-null mice. Inflamed ileal tissues and PBMCs from patients with CD had lower levels of SHIP protein than controls (P < .0001 and P < .0002, respectively). There was an inverse correlation between levels of SHIP activity in PBMCs and induction of IL1ß production by lipopolysaccharide and adenosine triphosphate (R(2) = .88). CONCLUSIONS: Macrophages from SHIP-deficient mice have increased PI3Kp110α-mediated transcription of Il1b, which contributes to spontaneous ileal inflammation. SHIP levels and activity are lower in intestinal tissues and peripheral blood samples from patients with CD than controls. There is an inverse correlation between SHIP activity and induction of IL1ß production by lipopolysaccharide and adenosine triphosphate in PBMCs. Strategies to reduce IL1B might be developed to treat patients with CD found to have low SHIP activity.

Hyperglycemia and PPARγ Antagonistically Influence Macrophage Polarization and Infarct Healing After Ischemic Stroke.

BACKGROUND AND PURPOSE: Secondary intracerebral hemorrhage (sICH) is a potentially serious complication of ischemic stroke, in particular under concomitant oral anticoagulation. Previous studies in murine stroke models defined a novel vascular repair function of hematogenous monocytes/macrophages (MO/MP), which proved essential for the prevention of oral anticoagulation-associated sICH. Here, we addressed the question whether hyperglycemia as a clinically relevant prohemorrhagic risk factor and peroxisome proliferator-activated receptor gamma (PPARγ) activation affect MO/MP differentiation and the risk of sICH after ischemic stroke. METHODS: Oral anticoagulation-associated sICH was induced by phenprocoumon feeding to mice undergoing transient middle cerebral artery occlusion. Hyperglycemia was induced by streptozotocin treatment. The role of PPARγ-dependent MO/MP differentiation was addressed in mice with myeloid cell-specific PPARγ-knockout (LysM-PPARγ(KO)). Pharmacological PPARγ activation via pioglitazone was tested as a treatment option. RESULTS: Hyperglycemic mice and normoglycemic LysM-PPARγ(KO) mice exhibited abnormal proinflammatory skewing of their hematogenous MO/MP response and abnormal vascular remodeling in the infarct border zone, leading to an increased rate of oral anticoagulation-associated sICH. Pharmacological PPARγ activation in hyperglycemic mice corrected the inflammatory response toward an anti-inflammatory profile, stabilized neovessels in the infarct border zone, and reduced the rate of sICH. This preventive effect was dependent on the presence of macrophages, but independent from effects on blood glucose levels. CONCLUSIONS: Hyperglycemia and macrophage-specific PPARγ activation exert opposing effects on MO/MP polarization in ischemic stroke lesions and, thereby, critically determine the risk of hemorrhagic infarct transformation.

Macrophage depletion lowers blood pressure and restores sympathetic nerve α2-adrenergic receptor function in mesenteric arteries of DOCA-salt hypertensive rats.

We tested the hypothesis that vascular macrophage infiltration and O2 (-) release impairs sympathetic nerve α2-adrenergic autoreceptor (α2AR) function in mesenteric arteries (MAs) of DOCA-salt hypertensive rats. Male rats were uninephrectomized or sham operated (sham). DOCA pellets were implanted subcutaneously in uninephrectomized rats who were provided high-salt drinking water or high-salt water with apocynin. Sham rats received tap water. Blood pressure was measured using radiotelemetry. Treatment of sham and DOCA-salt rats with liposome-encapsulated clodronate was used to deplete macrophages. After 3-5, 10-13, and 18-21 days of DOCA-salt treatment, MAs and peritoneal fluid were harvested from euthanized rats. Norepinephrine (NE) release from periarterial sympathetic nerves was measured in vitro using amperometry with microelectrodes. Macrophage infiltration into MAs as well as TNF-α and p22(phox) were measured using immunohistochemistry. Peritoneal macrophage activation was measured by flow cytometry. O2 (-) was measured using dihydroethidium staining. Hypertension developed over 28 days, and apocynin reduced blood pressure on days 18-21. O2 (-) and macrophage infiltration were greater in DOCA-salt MAs compared with sham MAs after day 10. Peritoneal macrophage activation occurred after day 10 in DOCA-salt rats. Macrophages expressing TNF-α and p22(phox) were localized near sympathetic nerves. Impaired α2AR function and increased NE release from sympathetic nerves occurred in MAs from DOCA-salt rats after day 18. Macrophage depletion reduced blood pressure and vascular O2 (-) while restoring α2AR function in DOCA-salt rats. Macrophage infiltration into the vascular adventitia contributes to increased blood pressure in DOCA-salt rats by releasing O2 (-), which disrupts α2AR function, causing enhanced NE release from sympathetic nerves.