Depletion Of Macrophages In Mice Research Articles

In vivo evolution of a Klebsiella pneumoniae capsule defect with wcaJ mutation promotes complement-mediated opsono-phagocytosis during recurrent infection.

Klebsiella pneumoniae carbapenemase-producing K. pneumoniae (KPC-Kp) bloodstream infections are associated with high mortality. We studied clinical bloodstream KPC-Kp isolates to investigate mechanisms of resistance to complement, a key host defense against bloodstream infection. We tested growth of KPC-Kp isolates in human serum. In serial isolates from a single patient, we performed whole genome sequencing and tested for complement resistance and binding by mixing study, direct ELISA, flow cytometry, and electron microscopy. We utilized an isogenic deletion mutant in phagocytosis assays and an acute lung infection model. We found serum resistance in 16 of 59 (27%) KPC-Kp clinical bloodstream isolates. In five genetically-related bloodstream isolates from a single patient, we noted a loss-of-function mutation in the capsule biosynthesis gene, wcaJ. Disruption of wcaJ was associated with decreased polysaccharide capsule, resistance to complement-mediated killing, and surprisingly, increased binding of complement proteins. Furthermore, an isogenic wcaJ deletion mutant exhibited increased opsono-phagocytosis in vitro and impaired in vivo control in the lung after airspace macrophage depletion in mice. Loss of function in wcaJ led to increased complement resistance, complement binding, and opsono-phagocytosis, which may promote KPC-Kp persistence by enabling co-existence of increased bloodstream fitness and reduced tissue virulence.

SGK1 aggravates idiopathic pulmonary fibrosis by triggering H3k27ac-mediated macrophage reprogramming and disturbing immune homeostasis.

Idiopathic pulmonary fibrosis (IPF) is characterized by fibrotic matrix deposition and irreversible aberrant tissue remodeling. Their mechanisms of action are associated with the activation of macrophages and a disturbed immune environment. We aim to determine how these activated macrophages influenced the pathogenesis of pulmonary fibrosis. We found the fibrotic areas of IPF patients contained more serum and glucocorticoid-induced kinase 1 (SGK1)-positive and M2-type macrophages. Similarly, bleomycin (BLM)+LPS significantly triggered high expression of SGK1 in the IPF mice, accompanied by destroyed lung structure and function, increased fibrosis markers and disturbed immune microenvironment. Mechanistically, SGK1 markedly promoted the reprogramming of M2-type macrophages in fibrotic lungs by triggering glycogen synthase kinase 3beta (GSK3β)-tat-interacting protein 60 (TIP60)- histone-3 lysine-27 acetylation (H3K27ac) signalings, which further released chemokine (C-C motif) ligand 9 (CCL9) to attract Th17 cells and delivered TGF-β to fibroblasts for synergistically destroying immune microenvironment, which was largely reversed by macrophage depletion in mice. We took macrophages as the entry point to deeply analyze IPF pathogenesis and further provided insights for the development of novel drugs represented by SGK1.

Macrophages polarize to the pro-inflammatory phenotype and delay neutrophil efferocytosis to augment Aggregatibacter actinomycetemcomitance JP2 clearance.

The study examines how neutrophils cross-talk with macrophages during JP2 Aggregatibacter actinomycetemcomitance infection and factors that are involved in inflammatory resolution and efferocytosis. Although sub-gingival bacteria constitute the primary initiating factor in the pathogenesis of molar-incisor pattern periodontitis (MIPP), the non-resolved host response has a major role in tissue destruction. While evidence links neutrophils to MIPP pathogenesis, their clearance during inflammatory resolution, governed by macrophages, is poorly understood. Human neutrophils (differentiated from HL60 cells) and macrophages (differentiated from THP1 cells) were inoculated with JP2. The supernatants were collected and exposed to naïve neutrophils or macrophages with or without exposure to JP2. Reactive oxygen species (ROS) were measured with 2'-7'-dichlorofluorescein-diacetate and a fluorescent plate reader. Immunofluorescence labeling of CD47 and cell vitality were examined using flow cytometry. Macrophage polarization was tested by immunofluorescence staining for CD163 and CD68 and a fluorescent microscope, and TNFα and IL-10 secretion was tested using ELISA and RT-PCR. Efferocytosis was examined by pHrodo and carboxyfluorescein succinimidyl ester staining and fluorescent microscopy. In vivo, macrophages were depleted from C57Bl/6 mice and neutrophil CD47 levels were tested using the subcutaneous chamber model. Neutrophils exposed to macrophage supernatant show increased ROS, mainly extracellularly, that increased during JP2 infection. Macrophages showed pro-inflammatory M1 phenotype polarization during JP2 infection, and their supernatants prolonged neutrophil survival by inhibiting CD47 down-expression and reducing neutrophil necrosis and apoptosis. Also, the macrophages delay neutrophil efferocytosis during JP2 infection which, in turn, enhanced JP2 clearance. Depletion of macrophages in mice mildly prevented neutrophils CD47 reduction and reduced JP2 clearance. The JP2 infection in mice also led to macrophage M1 polarization similar to the in vitro results. As shown in this study, neutrophil efferocytosis potentially may be reduced during JP2 infection, promoting JP2 clearance, which may contribute to the inflammatory-mediated periodontal tissue damage.

Macrophages promote survival of granulosa cells

Abstract The ovary contains numerous macrophages, and macrophage depletion in mice leads to dysfunctional ovulation. Macrophage function in the ovary is presumed to be related efferocytosis, however, the absence of macrophages also disrupts follicular development and corpus luteum development. Granulosa cells (GC) are critical to ovarian follicle development and survival, and we hypothesized that macrophage-GC crosstalk promoted GC function. To determine whether macrophage-GC interaction has an anti-apoptotic effect on GCs, BMDM were polarized to proinflammatory phenotype with LPS (M1), alternative activation phenotype with IL-4 (M2), or not stimulated (M0). After removing stimuli, primary mouse GCs were co-cultured with macrophages and serum-starvation was used to induce apoptosis of GCs. The cells were analyzed by flow cytometry, assessing viability by exclusion of propidium iodide and Annexin V. Co-culture with macrophages protected GCs from serum-starvation induced cell death. In co-cultures with quiescent BMDM, 60% of GCs were late apoptotic/early necrotic compared to 81% without co-culture, (p=0.0031). Unexpectedly, LPS-treated macrophages promoted survival of GC slightly more effectively than IL-4-treated or quiescent macrophages (45% versus 60% and 53% apoptotic, respectively p=0.0230). Both quiescent and inflammatory macrophages promote survival of GCs. This result is unexpected, as LPS-treated macrophages secrete TNF-α, a cytokine that is known to induce death in GCs. This study elucidates the role of macrophages in ovarian function by supporting GC survival and may also have implications for the promotion of GC tumors by tumor-associated macrophages. Supported by grant from the NIH 3P30GM118228-05S3

Duodenal macrophages control dietary iron absorption via local degradation of transferrin

AbstractIron is an essential cellular metal that is important for many physiological functions including erythropoiesis and host defense. It is absorbed from the diet in the duodenum and loaded onto transferrin (Tf), the main iron transport protein. Inefficient dietary iron uptake promotes many diseases, but mechanisms regulating iron absorption remain poorly understood. By assessing mice that harbor a macrophage-specific deletion of the tuberous sclerosis complex 2 (Tsc2), a negative regulator of mechanistic target of rapamycin complex 1 (mTORC1), we found that these mice possessed various defects in iron metabolism, including defective steady-state erythropoiesis and a reduced saturation of Tf with iron. This iron deficiency phenotype was associated with an iron import block from the duodenal epithelial cells into the circulation. Activation of mTORC1 in villous duodenal CD68+ macrophages induced serine protease expression and promoted local degradation of Tf, whereas the depletion of macrophages in mice increased Tf levels. Inhibition of mTORC1 with everolimus or serine protease activity with nafamostat restored Tf levels and Tf saturation in the Tsc2-deficient mice. Physiologically, Tf levels were regulated in the duodenum during the prandial process and Citrobacter rodentium infection. These data suggest that duodenal macrophages determine iron transfer to the circulation by controlling Tf availability in the lamina propria villi.

SARS-CoV-2 N Protein Triggers Acute Lung Injury via Modulating Macrophage Activation and Infiltration in in vitro and in vivo.

SARS-CoV-2-induced acute lung injury but its nucleocapsid (N) and/or Spike (S) protein involvements in the disease pathology remain elusive. In vitro, the cultured THP-1 macrophages were stimulated with alive SARS-CoV-2 virus at different loading dose, N protein or S protein with/without TICAM2-siRNA, TIRAP-siRNA or MyD88-siRNA. The TICAM2, TIRAP and MyD88 expression in the THP-1 cells after N protein stimulation were determined. In vivo, naïve mice or mice with depletion macrophages were injected with N protein or dead SARS-CoV-2. The macrophages in the lung were analyzed with flow cytometry, and lung sections were stained with H&E or immunohistochemistry. Culture supernatants and serum were harvested for cytokines measurements with cytometric bead array. Alive SARS-CoV-2 virus or N protein but not S protein induced high cytokine releases from macrophages in a time or virus loading dependent manner. MyD88 and TIRAP but not TICAM2 were highly involved in macrophage activation triggered by N protein whilst both inhibited with siRNA decreased inflammatory responses. Moreover, N protein and dead SARS-CoV-2 caused systemic inflammation, macrophage accumulation and acute lung injury in mice. Macrophage depletion in mice decreased cytokines in response to N protein. SARS-CoV-2 and its N protein but not S protein induced acute lung injury and systemic inflammation, which was closely related to macrophage activation, infiltration and release cytokines.

Small extracellular vesicles from Ptpn1-deficient macrophages alleviate intestinal inflammation by reprogramming macrophage polarization via lactadherin enrichment

Tyrosine-protein phosphatase non-receptor type 1 (Ptpn1) is known to be involved in macrophage polarization. However, whether and how Ptpn1 regulates macrophage phenotype to affect intestinal epithelial barrier function remains largely unexplored. Herein, we investigated the impact of Ptpn1 and macrophage-derived small extracellular vesicles (sEVs) on macrophage-intestinal epithelial cell (IEC) interactions in the context of intestinal inflammation. We found that Ptpn1 knockdown shifts macrophages toward the anti-inflammatory M2 phenotype, thereby promoting intestinal barrier integrity and suppressing inflammatory response in the macrophage-IEC co-culture model. We further revealed that conditioned medium or sEVs isolated from Ptp1b knockdown macrophages are the primary factor driving the beneficial outcomes. Consistently, administration of the sEVs from Ptpn1-knockdown macrophages reduced disease severity and ameliorated intestinal inflammation in LPS-challenged mice. Furthermore, depletion of macrophages in mice abrogated the protective effect of Ptpn1-knockdown macrophage sEVs against Salmonella Typhimurium infection. Importantly, we found lactadherin to be highly enriched in the sEVs of Ptpn1-knockdown macrophages. Administration of recombinant lactadherin alleviated intestinal inflammation and barrier dysfunction by inducing macrophage M2 polarization. Interestingly, sEVs lactadherin was also internalized by macrophages and IECs, leading to macrophage M2 polarization and enhanced intestinal barrier integrity. Mechanistically, the anti-inflammatory and barrier-enhancing effect of lactadherin was achieved by reducing TNF-α and NF-κB activation. Thus, we demonstrated that sEVs from Ptpn1-knockdown macrophages mediate the communication between IECs and macrophages through enrichment of lactadherin. The outcome could potentially lead to the development of novel therapies for intestinal inflammatory disorders.

MiR-182 targeting reprograms tumor-associated macrophages and limits breast cancer progression.

The protumor roles of alternatively activated (M2) tumor-associated macrophages (TAMs) have been well established, and macrophage reprogramming is an important therapeutic goal. However, the mechanisms of TAM polarization remain incompletely understood, and effective strategies for macrophage targeting are lacking. Here, we show that miR-182 in macrophages mediates tumor-induced M2 polarization and can be targeted for therapeutic macrophage reprogramming. Constitutive miR-182 knockout in host mice and conditional knockout in macrophages impair M2-like TAMs and breast tumor development. Targeted depletion of macrophages in mice blocks the effect of miR-182 deficiency in tumor progression while reconstitution of miR-182-expressing macrophages promotes tumor growth. Mechanistically, cancer cells induce miR-182 expression in macrophages by TGFβ signaling, and miR-182 directly suppresses TLR4, leading to NFκb inactivation and M2 polarization of TAMs. Importantly, therapeutic delivery of antagomiR-182 with cationized mannan-modified extracellular vesicles effectively targets macrophages, leading to miR-182 inhibition, macrophage reprogramming, and tumor suppression in multiple breast cancer models of mice. Overall, our findings reveal a crucial TGFβ/miR-182/TLR4 axis for TAM polarization and provide rationale for RNA-based therapeutics of TAM targeting in cancer.

The Proresolving Lipid Mediator Maresin1 Alleviates Experimental Pancreatitis via Switching Macrophage Polarization.

Method Repeated caerulein injection was used to induce AP and chronic pancreatitis (CP) models in mice. The histopathological and serological changes were examined for evaluating the severity of the AP model, and flow cytometry was used for detecting macrophage phagocytosis and phenotype. Meanwhile, clodronate liposomes were used for macrophage depletion in mice. Finally, the CP model was adopted to further observe the protective effect of MaR1. Result MaR1 administration manifested the improved histopathological changes and the lower serum levels of amylase and lipase. However, MaR1 played no protective role in the pancreatic acinar cell line in vitro. It obviously reduced the macrophage infiltration in the injured pancreas, especially M1-type macrophages. After macrophage clearance, MaR1 showed no further protection in vivo. This study also demonstrated that MaR1 could alleviate fibrosis to limit AP progression in the CP model. Conclusion Our data suggests that MaR1 was a therapeutic and preventive target for AP in mice, likely operating through its effects on decreased macrophage infiltration and phenotype switch.

Abstract 5651: JNJ-61186372, an Fc enhanced EGFR/cMet bispecific antibody, mediates EGFR and cMet downmodulation and therapeutic efficacy preclinically through monocyte / macrophage mediated trogocytosis

Abstract Small molecule inhibitors targeting EGFR are now standard of care in NSCLC patients harboring EGFR mutations, but acquired resistance invariably develops through secondary mutations within EGFR and/or through activation of compensatory pathways such as cMet. JNJ-61186372 (JNJ-372) is an anti-EGFR and cMet bispecific antibody with enhanced binding to immune cell Fcγ receptors, designed to target tumors with activated EGFR and cMet signaling through a distinct mechanism of action. Ongoing first-in-human study in patients with advanced, treatment refractory EGFR mutant NSCLC revealed JNJ-372 to have clinical activity in patients with diverse EGFR-mutated NSCLC, including Exon 20 mutations, TKI resistance mutations (T790M, C797S), and resistance due to MET amplification. However preclinically, despite potent anti-tumor activity in NSCLC xenograft models, only modest anti-proliferative effects were observed with JNJ-372 in cell lines in vitro. Interestingly, the addition of isolated human immune cells (PBMCs) to the in vitro assays enhanced JNJ-372-mediated EGFR and cMet downregulation, and dose-dependent tumor cell killing. Through depletion or enrichment of individual immune cell types, we demonstrated that monocytes and/or macrophages are necessary for JNJ-372 Fc interaction-mediated EGFR/cMet downmodulation. Depletion of macrophages in mice showed that they are required for JNJ-372 anti-tumor efficacy. Finally, we showed that the down-modulation of EGFR and cMet receptors occurs through monocyte or macrophage-mediated trogocytosis. Collectively, these results demonstrate a novel Fc-dependent mechanism of action for JNJ-372 and support its continued clinical development in patients with aberrant EGFR and cMet signaling. Citation Format: Smruthi Vijayaraghavan, Lorraine Lipfert, Barbara Bushey, Kristen Chevalier, Benjamin Henley, Ryan Lenhart, Marilda Beqiri, Hillary J. Millar, Kathryn Packman, Matthew V. Lorenzi, Sylvie Laquerre, Sheri Moores. JNJ-61186372, an Fc enhanced EGFR/cMet bispecific antibody, mediates EGFR and cMet downmodulation and therapeutic efficacy preclinically through monocyte / macrophage mediated trogocytosis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5651.

Chemokine receptor 5 blockade modulates macrophage trafficking in renal ischaemic-reperfusion injury.

Chemokine receptor 5 (CCR5) is a pivotal regulator of macrophage trafficking in the kidneys in response to an inflammatory cascade. We investigated the role of CCR5 in experimental ischaemic‐reperfusion injury (IRI) pathogenesis. To establish IRI, we clamped the bilateral renal artery pedicle for 30 min and then reperfused the kidney. We performed adoptive transfer of lipopolysaccharide (LPS)‐treated RAW 264.7 macrophages following macrophage depletion in mice. B6.CCR5−/− mice showed less severe IRI based on tubular epithelial cell apoptosis than did wild‐type mice. CXCR3 expression in CD11b+ cells and inducible nitric oxide synthase levels were more attenuated in B6.CCR5−/− mice. B6.CCR5−/− mice showed increased arginase‐1 and CD206 expression. Macrophage‐depleted wild‐type mice showed more injury than B6.CCR5−/− mice after M1 macrophage transfer. Adoptive transfer of LPS‐treated RAW 264.7 macrophages reversed the protection against IRI in wild‐type, but not B6.CCR5−/− mice. Upon knocking out CCR5 in macrophages, migration of bone marrow‐derived macrophages from wild‐type mice towards primary tubular epithelial cells with recombinant CCR5 increased. Phospho‐CCR5 expression in renal tissues of patients with acute tubular necrosis was increased, showing a positive correlation with tubular inflammation. In conclusion, CCR5 deficiency favours M2 macrophage activation, and blocking CCR5 might aid in treating acute kidney injury.

Involvement of M1 Macrophage Polarization in Endosomal Toll-Like Receptors Activated Psoriatic Inflammation.

Psoriasis is a chronic inflammatory skin disorder that affects ~2%–3% of the worldwide population. Inappropriate and excessive activation of endosomal Toll-like receptors 7, 8, and 9 (TLRs 7–9) at the psoriatic site has been shown to play a pathogenic role in the onset of psoriasis. Macrophage is a major inflammatory cell type that can be differentiated into phenotypes M1 and M2. M1 macrophages produce proinflammatory cytokines, and M2 macrophages produce anti-inflammatory cytokines. The balance between these two types of macrophages determines the progression of various inflammatory diseases; however, whether macrophage polarization plays a role in psoriatic inflammation activated by endosomal TLRs has not been investigated. In this study, we investigated the function and mechanism of macrophages related to the pathogenic role of TLRs 7–9 in the progression of psoriasis. Analysis of clinical data in database revealed significantly increased expression of macrophage markers and inflammatory cytokines in psoriatic tissues over those in normal tissues. In animal studies, depletion of macrophages in mice ameliorated imiquimod, a TLR 7 agonist-induced psoriatic response. Imiquimod induced expression of genes and cytokines that are signature of M1 macrophage in the psoriatic lesions. In addition, treatment with this TLR 7 agonist shifted macrophages in the psoriatic lesions to a higher M1/M2 ratio. Both of the exogenous and endogenous TLR 7–9 ligands activated M1 macrophage polarization. M1 macrophages expressed higher levels of proinflammatory cytokines and TLRs 7–9 than M2 macrophages. These results suggest that by rendering macrophages into a more inflammatory status and capable of response to their ligands in the psoriatic sites, TLR 7–9 activation drives them to participate in endosomal TLR-activated psoriatic inflammation, resulting in an amplified inflammatory response. Our results also suggest that blocking M1 macrophage polarization could be a strategy which enables inhibition of psoriatic inflammation activated by these TLRs.

Abstract 2888: Metabolically activated macrophages in obesity associated TNBC

Abstract Triple negative breast cancer (TNBC) accounts for about 20% of all breast cancers. TNBC patients have an extremely poor prognosis due to their high metastatic potential and lack of targeted drug therapies. Emerging epidemiological data suggest that obesity is strongly linked to the incidence and severity of TNBC; obese women have a 35% higher risk of developing TNBC and 46% higher risk of developing distant metastases. Thus, understanding the biological processes that link obesity and TNBC has important clinical applications for prognosis and treatment. Mechanisms by which obesity worsens TNBC prognosis are incompletely understood. One clue to its action is that obesity causes chronic inflammation, and macrophage infiltration into adipose tissue is a key mediator of this inflammation. Recent studies demonstrated that macrophages are enriched in breast adipose tissue of obese humans and mice. Moreover, depletion of macrophages in mice diminished the effects of obesity on TNBC growth and metastasis. Although anti-inflammatory ‘M2-like’ TAMs are key effector cells that promote tumorigenesis, it is well accepted that obesity stimulates macrophages to adopt a pro-inflammatory ‘M1-like’ state. However, M1 macrophages exhibit potent anti-tumor functions. From these discordant observations an important paradox emerges: How can obesity promote breast cancer if it elicits an M1, anti-tumor macrophage phenotype? Using a combination of proteomics, immunology, and cell biology, we recently identified a novel metabolically activated (MMe) macrophage phenotype produced by exposure to high levels of insulin, glucose, and palmitate, conditions characteristic of obese and diabetic patients. Here we show that gene expression patterns in MMe (but not M1) macrophages are strongly associated with pathways involved in breast cancer. Moreover, pre-treating BM1 cells with conditioned media derived from MMe macrophages promotes TNBC cell invasion by 2.3 fold, suggesting that MMe macrophages potentiate metastasis. We further show that breast adipose tissue macrophages isolated from obese women express cell surface markers of MMe (CD36, ABCA1), but not M1 (CD38, CD319, CD274), macrophages. Furthermore, treating naïve macrophages with media conditioned by human mammary adipose tissue from an obese subject (BMI = 37), but not a lean subject (BMI = 19), induced genes diagnostic of the MMe phenotype. Thus, mammary adipose tissue from obese women supports metabolic activation of macrophages. Together, these observations suggest that obesity-induced changes to adipose tissue reprogram macrophages to an MMe phenotype that potentiates TNBC. A comprehensive understanding of signaling mechanisms involved in metabolic activation would enable development of directed therapies towards this specific pro-tumorigenic macrophage phenotype, thereby leaving the immune system of cancer patients intact. Citation Format: Payal Tiwari, Kelly Schoenfelt, Swati Kulkarni, Marsha Rosner, Lev Becker. Metabolically activated macrophages in obesity associated TNBC. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2888. doi:10.1158/1538-7445.AM2015-2888

Depletion and Reconstitution of Macrophages in Mice

Macrophages are critical players in the innate immune response to infectious challenge or injury, initiating the innate immune response and directing the acquired immune response. Macrophage dysfunction can lead to an inability to mount an appropriate immune response and as such, has been implicated in many disease processes, including inflammatory bowel diseases. Macrophages display polarized phenotypes that are broadly divided into two categories. Classically activated macrophages, activated by stimulation with IFNγ or LPS, play an essential role in response to bacterial challenge whereas alternatively activated macrophages, activated by IL-4 or IL-13, participate in debris scavenging and tissue remodeling and have been implicated in the resolution phase of inflammation. During an inflammatory response in vivo, macrophages are found amid a complex mixture of infiltrating immune cells and may participate by exacerbating or resolving inflammation. To define the role of macrophages in situ in a whole animal model, it is necessary to examine the effect of depleting macrophages from the complex environment. To ask questions about the role of macrophage phenotype in situ, phenotypically defined polarized macrophages can be derived ex vivo, from bone marrow aspirates and added back to mice, with or without prior depletion of macrophages. In the protocol presented here clodronate-containing liposomes, versus PBS injected controls, were used to deplete colonic macrophages during dextran sodium sulfate (DSS)-induced colitis in mice. In addition, polarized macrophages were derived ex vivo and transferred to mice by intravenous injection. A caveat to this approach is that clodronate-containing liposomes deplete all professional phagocytes, including both dendritic cells and macrophages so to ensure the effect observed by depletion is macrophage-specific, reconstitution of phenotype by adoptive transfer of macrophages is necessary. Systemic macrophage depletion in mice can also be achieved by backcrossing mice onto a CD11b-DTR background, which is an excellent complementary approach. The advantage of clodronate-containing liposome-mediated depletion is that it does not require the time and expense involved in backcrossing mice and it can be used in mice regardless of the background of the mice (C57BL/6, BALB/c, or mixed background).

Depletion and Reconstitution of Macrophages in Mice

Macrophages are critical players in the innate immune response to infectious challenge or injury, initiating the innate immune response and directing the acquired immune response. Macrophage dysfunction can lead to an inability to mount an appropriate immune response and as such, has been implicated in many disease processes, including inflammatory bowel diseases. Macrophages display polarized phenotypes that are broadly divided into two categories. Classically activated macrophages, activated by stimulation with IFNγ or LPS, play an essential role in response to bacterial challenge whereas alternatively activated macrophages, activated by IL-4 or IL-13, participate in debris scavenging and tissue remodeling and have been implicated in the resolution phase of inflammation. During an inflammatory response in vivo, macrophages are found amid a complex mixture of infiltrating immune cells and may participate by exacerbating or resolving inflammation. To define the role of macrophages in situ in a whole animal model, it is necessary to examine the effect of depleting macrophages from the complex environment. To ask questions about the role of macrophage phenotype in situ, phenotypically defined polarized macrophages can be derived ex vivo, from bone marrow aspirates and added back to mice, with or without prior depletion of macrophages. In the protocol presented here clodronate-containing liposomes, versus PBS injected controls, were used to deplete colonic macrophages during dextran sodium sulfate (DSS)-induced colitis in mice. In addition, polarized macrophages were derived ex vivo and transferred to mice by intravenous injection. A caveat to this approach is that clodronate-containing liposomes deplete all professional phagocytes, including both dendritic cells and macrophages so to ensure the effect observed by depletion is macrophage-specific, reconstitution of phenotype by adoptive transfer of macrophages is necessary. Systemic macrophage depletion in mice can also be achieved by backcrossing mice onto a CD11b-DTR background, which is an excellent complementary approach. The advantage of clodronate-containing liposome-mediated depletion is that it does not require the time and expense involved in backcrossing mice and it can be used in mice regardless of the background of the mice (C57BL/6, BALB/c, or mixed background).

Depletion of macrophages in mice results in higher dengue virus titers and highlights the role of macrophages for virus control

Monocytes and macrophages are target cells for dengue infection. Besides their potential role for virus replication, activated monocytes/macrophages produce cytokines that may be critical for dengue pathology. To study the in vivo role of monocytes and macrophages for virus replication, we depleted monocytes and macrophages in IFN-alphabetagammaR knockout mice with clodronate liposomes before dengue infection. Although less virus was first recovered in the draining LN in the absence of macrophages, monocyte/macrophage depletion eventually resulted in a ten-fold higher systemic viral titer. A massive infiltration of CD11b(+)CD11c(low)Ly6C(low) monocytes into infected organs was observed in parallel with increasing virus titers before viremia was controlled. Depletion of monocytes in the blood before or after local infection had no impact on virus titers, suggesting that monocytes are not required as "virus-shuttles". Our data provide evidence that systemic viremia is established independently of tissue macrophages present at the site of infection and blood monocytes. Instead, we demonstrate the importance of monocytes/macrophages for the control of dengue virus.

Role of Sialic Acid for Platelet Lifespan and Function

Although sialic acid is considered a key determinant for the survival of circulating blood cells and glycoproteins, its role in platelet half-life is not fully clarified. We and others have previously provided evidence that thrombocytopenia in mice deficient in the ST3Gal-IV sialyltransferase gene (ST3Gal-IV−/− mice) is caused by rapid clearance of the platelets due to recognition of surface galactose by asialoglycoprotein receptor-expressing scavenger cells. Here we report new insight into clearance mechanisms, activation and production of sialic acid deficient platelets. Immunofluorescence staining of organ specimens harvested shortly after platelet transfusion demonstrated the predominant clearance of ST3Gal-IV−/− platelets by liver macrophages and, as previously reported, hepatocytes. The differential cellular clearance pathways were further explored following macrophage depletion in mice using clodronate encapsulated liposomes, a procedure that restored ST3Gal-IV−/− platelet circulation to approximately 40% of normal values, confirming that macrophages play a major role in the clearance of sialic acid deficient platelets and that other clearance pathways are equally important. Ingestion of ST3Gal-IV−/− platelets by hepatocytes was confirmed by in vitro HepG2 phagocytosis assays. We next investigated the function of desialylated platelets. Platelet binding of von Willebrand factor (vWf) upon botrocetin stimulation was 3 fold higher for ST3Gal-IV−/− platelet rich plasma compared to ST3Gal- IV+/+ platelet rich plasma. The circulation time of wild-type platelets transfused into ST3Gal-IV−/− mice was 20% reduced compared to control platelets, indicating that platelet removal could be accelerated due to binding of desialylated vWf to platelets. Loss of sialic acid did not affect platelet production in vitro and in vivo as cultivated megakaryocytes was found to produce proplatelets normally and measurements of reticulated platelets by flow cytometry showed a 3-fold increased thrombocytopoietic activity in the ST3Gal- IV−/− mice compared to wild-type mice. Interestingly, thiazole orange staining revealed a significant negative correlation between platelet size and platelet age for both genotypes, In conclusion, depletion of the ST3Gal-IV gene promotes binding of vWf to platelets, exposes platelet surface galactose residues to the lectin domain of asialoglycoprotein receptors on both hepatocytes and liver macrophages, resulting in rapid platelet clearance from the circulation and supporting previous findings that platelets decrease in size while aging in circulation.

Virulizin, a novel immunotherapy agent, activates NK cells through induction of IL-12 expression in macrophages.

Virulizin, a novel biological response modifier, has demonstrated significant antitumor efficacy in a variety of human tumor xenograft models including melanoma, pancreatic cancer, breast cancer, ovarian cancer and prostate cancer. The significant role of macrophages and NK (Natural killer) cells was implicated in the antitumor mechanism of Virulizin where expansion as well as increased activity of macrophages and NK cells were observed in mice treated with Virulizin. Depletion of macrophages compromised Virulizin-induced NK1.1+ cell infiltration into xenografted tumors and was accompanied by reduced antitumor efficacy. In the present study, involvement of macrophages in NK cell activation was investigated further. We found that depletion of NK cells in CD-1 nude mice by anti-ASGM1 antibody significantly compromised the antitumor activity of Virulizin. Cytotoxicity of NK cells isolated from Virulizin-treated mice was enhanced against NK-sensitive YAC-1 cells and C8161 human melanoma cells, but not against NK-insensitive P815 cells. An increased level of IL-12beta was observed in the serum of mice treated with Virulizin. IL-12 mRNA and protein levels were also increased in peritoneal macrophages isolated from Virulizin-treated mice. Moreover, Virulizin-induced cytotoxic activity of NK cells isolated from the spleen was abolished when an IL-12 neutralizing antibody was co-administered. In addition, depletion of macrophages in mice significantly impaired Virulizin-induced NK cell cytotoxicty. Taken together, the results suggest that Virulizin induces macrophage IL-12 production, which in turn stimulates NK cell-mediated antitumor activity.

West Nile virus neuroinvasion and encephalitis induced by macrophage depletion in mice.

The encephalitic West Nile virus and its nonneuroinvasive variant, WN-25, were used to study the effect of macrophage depletion on viral invasion of the central nervous system. The in vivo elimination of macrophages was achieved by use of liposome-encapsulated drug dichloromethylene diphosphonate. Depletion of macrophages had an exacerbating effect on the course of the viral infection, exhibited by higher and extended viremia and accelerated development of encephalitis and death. Using a low dose of West Nile virus (5 PFU/mouse), an increase in mortality (from 50% to 100%) due to macrophage depletion was demonstrated. Furthermore, the attenuated noninvasive variant WN-25 showed high and prolonged viremia in the macrophage depleted mice (approximately 5 log 10 PFU/ml versus 2 in control mice), that allowed the penetration of the virus into the central nervous system. The mortality rate caused by the attenuated virus in the macrophage-depleted mice was 70-75%, as compared to complete survival in the control inoculated mice. These results indicate a significant role of macrophages in the non-specific immediate defence system of the organism in case of viral infection.