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Human adenoviruses from multiple species bind to coagulation factor X (FX), yet the importance of this interaction in adenovirus dissemination is unknown. Upon contact with blood, vectors based on adenovirus serotype 5 (Ad5) binds to FX via the hexon protein with nanomolar affinity, leading to selective uptake of the complex into the liver and spleen. The Ad5:FX complex putatively targets heparan sulfate proteoglycans (HSPGs). The aim of this study was to elucidate the specific requirements for Ad5:FX-mediated cellular uptake in this high-affinity pathway, specifically the HSPG receptor requirements as well as the role of penton base-mediated integrin engagement in subsequent internalisation. Removal of HS sidechains by enzymatic digestion or competition with highly-sulfated heparins/heparan sulfates significantly decreased FX-mediated Ad5 cell binding in vitro and ex vivo. Removal of N-linked and, in particular, O-linked sulfate groups significantly attenuated the inhibitory capabilities of heparin, while the chemical inhibition of endogenous HSPG sulfation dose-dependently reduced FX-mediated Ad5 cellular uptake. Unlike native heparin, modified heparins lacking O- or N-linked sulfate groups were unable to inhibit Ad5 accumulation in the liver 1h after intravascular administration of adenovirus. Similar results were observed in vitro using Ad5 vectors possessing mutations ablating CAR- and/or αv integrin binding, demonstrating that attachment of the Ad5:FX complex to the cell surface involves HSPG sulfation. Interestingly, Ad5 vectors ablated for αv integrin binding showed markedly delayed cell entry, highlighting the need for an efficient post-attachment internalisation signal for optimal Ad5 uptake and transport following surface binding mediated through FX. This study therefore integrates the established model of αv integrin-dependent adenoviral infection with the high-affinity FX-mediated pathway. This has important implications for mechanisms that define organ targeting following contact of human adenoviruses with blood., Author Summary Adenoviruses can infect many cell types and cause a range of illnesses in humans, including respiratory, ocular and gastrointestinal disorders. These illnesses are rarely fatal; however, in immunocompromised individuals, especially young children, disseminated adenovirus infections can cause serious and life-threatening complications. Studies have shown that several adenoviruses including vectors based on adenovirus serotype 5 (Ad5) bind to coagulation factor X (FX) in the bloodstream. Ad5 uses the high-affinity interaction with FX to putatively bind to heparan sulfate proteoglycans (HSPGs). However, very little is known about this infection pathway. Here we demonstrate that interaction of Ad5:FX with HSPGs is solely via the HS sidechains of these ubiquitously-expressed molecules. We further show that this interaction is dependent on HS sulfation, in particular O-sulfation. Although attachment of Ad5:FX to HSPGs is independent of the coxsackievirus and adenovirus receptor (CAR) or αv integrins, efficient and rapid intracellular transport of Ad5 retains a dependence on engagement of αv integrins via the penton base protein. This is the first study to characterise the receptor requirements for cell uptake via the recently-identified, FX-mediated infection pathway, which may be of significance for the development of therapies against disseminated adenoviral disease.

Invariant natural killer T cells (iNKT cells) are involved in the host defense against microbial infection. Although it is known that iNKT cells recognize glycolipids presented by CD1d, how and where they encounter antigen in vivo remains unclear. Here we used multiphoton microscopy to visualize the dynamics and activation of iNKT cells in lymph nodes. After antigen administration, iNKT cells became confined in a CD1d-dependent manner in close proximity to subcapsular sinus CD169(+) macrophages. These macrophages retained, internalized and presented lipid antigen and were required for iNKT cell activation, cytokine production and population expansion. Thus, CD169(+) macrophages can act as true antigen-presenting cells controlling early iNKT cell activation and favoring the fast initiation of immune responses.

Dichloromethylene diphosphonate (Cl2MDP) enclosed in liposomes, when injected intravenously, selectively eliminates all phagocytizing macrophages that are in direct contact with the blood circulation of the spleen and the liver. We examined whether Cl2MDP containing liposomes affect, in addition, rat monocytes and bone marrow macrophage precursors (BMMP's). In addition to Phosphatidylcholine-Cholesterol liposomes (PC liposomes), we also used mannosylated PC liposomes (PCMAN liposomes), which are reported to bind more efficiently to macrophages. Monocytes appeared to be affected 24 h after injections of 2 ml of Cl2MDP containing PC as well as PCMAN liposomes. In addition, almost no macrophages could be detected in one week cultures of blood leukocytes isolated from these animals; cultures from control animals contained +/- 50% macrophages. Bone marrow macrophage precursors did not appear to be affected.

Abstract 723 The anti-CD20 monoclonal antibody rituximab exerts its anti-tumor effects against B cell non-Hodgkin's lymphoma (NHL) via antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity, and apoptosis induction. Toll-like receptor 9 agonist CpG oligodeoxynucleotides (CpG ODN) are potent activators of ADCC and T cell immunity, and have been studied for anti-NHL effects when administered by systemic or intratumoral (i.t.) routes. We have used an aggressive human CD20-expressing syngeneic murine B cell lymphoma (38C13-huCD20) that is resistant to single agent rituximab to study techniques for improving antibody efficacy. We recently reported that combining rituximab with i.t. (but not systemic) CpG promotes eradication of 7-day established rituximab-resistant 38C13-huCD20 lymphoma from 42% of mice (D. Betting et al, J. Immunotherapy 2009). Six doses of i.t. CpG (50 μg days 7, 8, and 9 and 25 μg days 11, 13, and 15) were required to achieve tumor eradication in this setting. Both natural killer cells and complement participated in the cure of tumors by rituximab plus i.t CpG, by increasing tumor cell sensitivity to ADCC and complement lysis, and by augmenting the cytotoxicity of ADCC effectors. To overcome the need for repeated i.t. injections, eliminate the need for an accessible tumor site, and allow for more direct targeting of CpG to tumor cells, we chemically linked CpG to rituximab using a cleavable linker. The conjugate retained both antibody binding activity to human CD20 and potent CpG biologic activity, demonstrated via upregulation of costimulatory molecules on cultured lymphoma cells and activation of bone marrow-derived dendritic cells. Just 2 injections of rituximab-CpG conjugate (containing 50 μg CpG, days 7 and 9) achieved eradication of 7-day established tumors from 100% of mice, while injection of rituximab-control ODN or trastuzumab-CpG conjugates had no therapeutic effects. Treatment with equivalent doses of rituximab plus i.v. or i.t. CpG prolonged survival slightly, but did not result in tumor eradication. In vivo depletion of NK cells or complement nearly eliminated tumor eradication effects. In contrast, in vivo depletion of macrophages, or CD4+ or CD8+ T cells had no significant effects on tumor eradication by rituximab-CpG, indicating that adaptive T cell immunity did not contribute to elimination of tumor during rituximab-CpG conjugate therapy. As CpGs can stimulate leukocytes to secrete alpha interferon (αaIFN), which can inhibit the growth of B cell lymphomas, we sought to determine whether αaIFN played a significant role in tumor control after rituximab-CpG therapy. 38C13-huCD20 tumor cells rendered deficient in αaIFN receptor (IFNAR) by lentiviral transduction with shRNA were nearly as sensitive to rituximab-CpG as wild-type IFNAR+ 38C13-huCD20 cells, indicating no role for direct αaIFN tumor cytotoxicity in eradication of 38C13-huCD20. In conclusion, natural killer cells and complement, but not T cells, macrophages, or αaIFN, are critical to the efficacy of rituximab-CpG conjugate in efficiently eradicating an established human CD20+ B cell lymphoma that is fully resistant to single-agent rituximab. Further pre-clinical studies of anti-CD20-CpG conjugates against B cell lymphomas are thus warranted. Anti-CD20-CpG conjugates may represent a novel treatment modality for human NHL. Disclosures: No relevant conflicts of interest to declare.

When a pathogen escapes innate host responses at the site of entry, it has the potential to spread systemically by accessing tissue-draining lymph vessels that channel interstitial fluid to the venous circulation. Before reaching the blood, the tissue-derived lymph must first pass through draining lymph nodes (LNs). LN act as filter stations preventing pathogens from reaching the systemic circulation and they are the staging grounds for adaptive immune responses, providing a specialized environment for the presentation of antigens to B and T cells. It is unclear how viruses are cleared from afferent lymph and how antiviral immune responses are generated. We have previously identified a population of CD11b+CD169+ macrophages on the floor of the subcapsular sinus (SCS) and in the medulla of LNs that capture viral particles within minutes after subcutaneous (sc) injection. Using VSV, an acutely cytopathic RNA virus closely related to rabies virus, we show here that these macrophages are critical to prevent viral access to nerve termini located in the SCS. Indeed, mice depleted of SCS macrophages develop an ascending CNS paralysis that leads to death in 50% of the animals 6-10 days after sc infection. Macrophage-depleted mice die despite mounting an increased neutralizing antibody (nAb) response. The notion that nAb are dispensable for protection against a sc infection (the natural route of transmission of this virus) is further demonstrated by the fact that antibody-deficient mice, which die upon intravenous infection, survive a sc VSV infection as long as SCS macrophages are present. While nAb are unnecessary for survival, B cells are required, and their protective role presumably relies on maintenance of LN architecture and normal macrophage phenotype.

International audience; BACKGROUND: Macrophages, key regulators of healing/regeneration processes, strongly infiltrate ischemic tissues from patients suffering from critical limb ischemia (CLI). However pro-inflammatory markers correlate with disease progression and risk of amputation, suggesting that modulating macrophage activation state might be beneficial. We previously reported that thrombospondin-1 (TSP-1) is highly expressed in ischemic tissues during CLI in humans. TSP-1 is a matricellular protein that displays well-known angiostatic properties in cancer, and regulates inflammation in vivo and macrophages properties in vitro. We therefore sought to investigate its function in a mouse model of CLI. METHODS AND FINDINGS: Using a genetic model of tsp-1(-/-) mice subjected to femoral artery excision, we report that tsp-1(-/-) mice were clinically and histologically protected from necrosis compared to controls. Tissue protection was associated with increased postischemic angiogenesis and muscle regeneration. We next showed that macrophages present in ischemic tissues exhibited distinct phenotypes in tsp-1(-/-) and wt mice. A strong reduction of necrotic myofibers phagocytosis was observed in tsp-1(-/-) mice. We next demonstrated that phagocytosis of muscle cell debris is a potent pro-inflammatory signal for macrophages in vitro. Consistently with these findings, macrophages that infiltrated ischemic tissues exhibited a reduced postischemic pro-inflammatory activation state in tsp-1(-/-) mice, characterized by a reduced Ly-6C expression and a less pro-inflammatory cytokine expression profile. Finally, we showed that monocyte depletion reversed clinical and histological protection from necrosis observed in tsp-1(-/-) mice, thereby demonstrating that macrophages mediated tissue protection in these mice. CONCLUSION: This study defines targeting postischemic macrophage activation state as a new potential therapeutic approach to protect tissues from necrosis and promote tissue repair during CLI. Furthermore, our data suggest that phagocytosis plays a crucial role in promoting a deleterious intra-tissular pro-inflammatory macrophage activation state during critical injuries. Finally, our results describe TSP-1 as a new relevant physiological target during critical leg ischemia.

In this study we present a new method for the elimination of mononuclear phagocytic cells from cell suspensions. By making use of liposome-encapsulated dichloromethylene diphosphonate we were able to effectively remove macrophages from spleen cell suspensions. This effect was not observed when using the free drug or control (PBS) liposomes. The use of this procedure has no effect on other cell types, as measured by growth, protein production, antigen presentation and antigen specific T cell proliferation, though PBS liposomes in very high doses were able to inhibit antigen presentation. The finding that lymphocytes are not affected by the liposome encapsulated drug suggests that the observed loss of lymphocytes in vivo, after intravenous dichloromethylene diphosphonate liposome treatment, may be due to damage inflicted by lysosomal enzymes released from dying macrophages. This method permits the removal of both macrophages and monocytes from heterogeneous cell populations (i.e., blood, lymphoid tissue suspension) in vitro with a very high rate of reliability. With the concentrations and incubation time used, no negative effects on other cell types were observed.

To study the role of macrophages in the in situ immune response to particulate antigens in spleen and popliteal lymph nodes (PLN), mice were injected with dichloromethylene diphosphonate (C12MDP)-containing liposomes to eliminate macrophages, followed by immunization with trinitrophenylated sheep red blood cells (TNP-SRBC). Depletion of macrophages in the spleen caused a strong decrease in the number of antibodyforming cells (AFC), which develop after intravenous (i.v.) injection of the antigen. These results strongly suggested the involvement of splenic macrophages in the processing of TNPSRBC. In particular, the populations of marginal zone macrophages may be involved in the inductive phase of an antibody response to particulate antigens. These macrophages are strategically positioned at the end of the white pulp capillaries in the marginal zone of the spleen and they have their cell processes between the marginal zone-B cells. Elimination of macrophages in PLN had no effect on the number of AFC, which develop after subcutaneous (s.c.) injection of the antigen in the hind footpads. This indicates that the macrophages are not essential for the induction of a local immune response to the particulate antigen TNP-SRBC. After depletion of lymph node macrophages, the number of AFC developing in the spleen after s.c. footpad injection of the antigen increased and the anti-TNP serum titers were elevated. This may well be caused by the fact that more of the antigen reaches the circulation and subsequently stimulates the spleen.

Recombinant adenoviruses are among the most extensively used vectors in gene therapy studies. To optimize adenovirus-mediated liver transduction we have studied the effect of the route of administration using first and third generation adenovirus vectors. We compared the local delivery of those vectors, using direct intrahepatic injection (IH), and the systemic administration via tail vein (IV). IH injection of first generation adenovirus resulted in a three-fold increase transgene expression when a high dose of virus was administered. More importantly when a relatively low dose of virus was used, IV administration resulted in no detectable protein expression while IH administration resulted in high levels of protein in serum. Significant differences in the kinetic and location of protein expression were observed depending on the route, IH injection resulted in a faster protein expression which localized to the site of injection, compared to widespread liver expression following IV administration. A 3 fold increase in the level of protein expression was also obtained after IH injection of a helper-dependent adenovirus expressing hIL-12 (HD-hIL12). Furthermore, IH injection using a HD-Ad carrying murine IL12 (HD-mIL12) correlated with the improvement of its antitumoral efficacy in a murine tumor model. Macrophage depletion experiments using clodronate loaded-liposomes showed that IH injection partially circumvent macrophage phagocytic activity. Interestingly, the administration of empty liposomes increases transgene expression without macrophage depletion regardless of the route of administration. In summary, our study describes two ways to improve liver transduction using adenovirus-based vectors: 1, direct liver injection of the adenovirus 2, administration of empty liposomes prior to virus injection.

Glycogen storage disease type II (GSD-II) is a fatal genetic muscle disorder caused by a massive accumulation of glycogen in multiple muscle groups, and is caused by the deficiency of intralysosomal acid alpha glucosidase (GAA). Two approaches to the treatment of GSD-II are currently being pursued in our research group: enzyme replacement therapy and gene therapy. Relative to gene therapy, our previous work demonstrated that a single intravenous administration of advanced generation, [E1-,pol-] adenoviral (Ad) based vectors encoding human GAA (hGAA) resulted in efficient hepatic transduction and secretion of high levels of hGAA, consequently, the correction of all affected muscles in a GAA-KO mouse model as well a quail model of GSD-II occurred. The long-term persistence of the improved vector in liver tissues facilitated significant glycogen reduction in multiple muscles (though, with variable responses in extent and duration among the individual muscle group analyzed) for at least 6 months, despite the fact that hGAA is perceived as a foreign antigen in these animal models. Unfortunately, our most recent work suggests that very high doses of Ad vectors must be intravenously injected to facilitate the large amounts of hepatic hGAA secretion required to correct both cardiac and skeletal muscles in these animal models. To determine if this problem can be overcome, we have begun attempts to pre-emptively suppress the innate immune systems of immunocompetent GAA-KO mice, prior to injection with our viral vectors. Macrophages, in particular hepatic Kupffer cells, play a central role in sequestering Ad vectors, as well in triggering early innate immune responses, and hence have a profound impact on the adaptive immune responses following adenoviral vector administration. In this study we evaluated the effect of selective and transient depletion of Kupffer cells (via pretreatment of mice with clodronate liposomes-dichloromethylene-bisphosphonate) one day prior to vector injection. We intravenously injected into the clodronate liposome pretreated GAA-KO mice different dosages of the vector, to determine how removal of Kupffer cells impacted upon the known efficacy of this approach. We found that clodronate pretreatment allowed lower doses of the vector to show greater efficacy. For example, the GAA activities in the clodronate-treated mice were significantly elevated both in plasma and in tissues such as liver, heart, diaphram and quadriceps, as compared to the non-Kupffer cell depleted mice injected with identical doses of the [E1-,pol-]AdhGAA vector. As a result, the pathogenic glycogen accumulations in clodronate liposome pretreated GAA-KO mice were significantly reduced at lower doses of the Ad vector (10–100 fold less) as compared to Ad treated GAA-KO mice that had not been pretreated with clodronate containing liposomes. Our data also suggests however, that liver toxicities were also now apparent at lower vector dosages, suggesting that Kupffer cells act to prevent liver damage by sequestering Ad particles. Whether or not other “Ad induced” toxicities are prevented by Kupffer cell depletion are studies currently ongoing in our laboratories.

[no abstract available]

Rabbits were intravenously primed and boosted with trinitrophenyl-keyhole limpet hemocyanin (TNP-KLH) and human serum albumin (HSA); both antigens were injected simultaneously. The localization of anti-TNP-antibodyforming cells (AFCs) and anti-HSA-AFCs was determined in various lymphoid organs of the rabbit. In all lymphoid organs of primed rabbits anti-TNP-AFCs outnumbered anti-HSA-AFCs, with the exception of the thymus, in which neither of them was encountered. In the spleen the antibody-forming cells were mainly situated in the periphery of the periarteriolar lymphocyte sheaths (PALS) and in the coaxial sheaths of lymphoid tissue surrounding the terminal arterioles. In the lymphoepithelial organs AFCs were almost exclusively situated in the interfollicular areas, and in the lymph nodes largely in the medulla. An intravenous booster injection led to a secondary immune response (i.e., increase of AFCs) in the spleen. No visible change in the number of specific AFCs was observed in the lymphoepithelial organs. However, in the mesenteric and popliteal lymph node the number of anti-TNP-AFCs had increased tremendously.

Mice were i.v. immunized with various 2,4,6-trinitrophenyl (TNP)-carrier conjugates, and the development and localization of specific (i.e. anti-TNP) antibody-forming cells (AFC) in the spleen was studied. The nature of the carrier used, thymus dependent (TD) or thymus independent type 1 or type 2 (TI-1, TI-2), had marked effects on the number and time of appearance of AFC. However, no influence of the different carriers on the localization of specific AFC in the spleen was observed. By using a double immunocytochemical technique we could simultaneously determine specificity (anti-TNP) and isotype (class and subclass) of the AFC. The TNP-carrier conjugates evoked (intracellular) immunoglobulins with carrier-characteristic isotype distribution. No carrier-dependent localization pattern, in the lymphoid compartments of the spleen, of these AFC was found. These results show for the first time the actual in situ effects of various TI and TD carriers on the development of specific AFC. Based on the present findings, no direct evidence for a special role of the marginal zone in the immune response against TI-2 antigens could be demonstrated. We suggest that this new method for in vivo investigation, in combination with existing standard enzyme- and immunohistochemical techniques, can provide more insight into the precise role of lymphoid and nonlymphoid cells in TD and TI immune responses.

Summary Attempts were made to study the localization of intravenously injected liposomes and enclosed labelled antigens in the spleen of mice, using autoradiography. A more than hundred fold increased uptake of antigen by the spleen was obtained when liposome entrapped antigen was compared with free antigen or antigen-antibody complexes which have been used in earlier studies. No marked accumulation of the labelled antigenwas found in the follicles of the spleen, although specific antibodies to the injected antigen have been injected simultaneously with the liposome entrapped antigen. It is argued that the bulk of the labelled antigen in the spleen 0.5 to 2 h after injection was still enclosed in the liposomes, whereas part of the label, arranged in patches in the red pulp and marginal zone, corresponds with labelled antigen in ;macrophages, released after digestion of the liposomal membranes. The present techniques did not enable the detection of the liposomes themselves.

SummaryThere is widespread interest in macrophages as a therapeutic target in cancer. Here, we demonstrate that trabectedin, a recently approved chemotherapeutic agent, induces rapid apoptosis exclusively in mononuclear phagocytes. In four mouse tumor models, trabectedin caused selective depletion of monocytes/macrophages in blood, spleens, and tumors, with an associated reduction of angiogenesis. By using trabectedin-resistant tumor cells and myeloid cell transfer or depletion experiments, we demonstrate that cytotoxicity on mononuclear phagocytes is a key component of its antitumor activity. Monocyte depletion, including tumor-associated macrophages, was observed in treated tumor patients. Trabectedin activates caspase-8-dependent apoptosis; selectivity for monocytes versus neutrophils and lymphocytes is due to differential expression of signaling and decoy TRAIL receptors. This unexpected property may be exploited in different therapeutic strategies.

Novel antiangiogenic strategies with complementary mechanisms are needed to maximize efficacy and minimize resistance to current angiogenesis inhibitors. We explored the therapeutic potential and mechanisms of alphaPlGF, an antibody against placental growth factor (PlGF), a VEGF homolog, which regulates the angiogenic switch in disease, but not in health. alphaPlGF inhibited growth and metastasis of various tumors, including those resistant to VEGF(R) inhibitors (VEGF(R)Is), and enhanced the efficacy of chemotherapy and VEGF(R)Is. alphaPlGF inhibited angiogenesis, lymphangiogenesis, and tumor cell motility. Distinct from VEGF(R)Is, alphaPlGF prevented infiltration of angiogenic macrophages and severe tumor hypoxia, and thus, did not switch on the angiogenic rescue program responsible for resistance to VEGF(R)Is. Moreover, it did not cause or enhance VEGF(R)I-related side effects. The efficacy and safety of alphaPlGF, its pleiotropic and complementary mechanism to VEGF(R)Is, and the negligible induction of an angiogenic rescue program suggest that alphaPlGF may constitute a novel approach for cancer treatment.

The use of adenovirus serotype 5 (Ad5) vectors in the clinical setting is severely hampered by the profound liver tropism observed after intravascular delivery coupled with the pronounced inflammatory and innate immune response elicited by these vectors. Liver transduction by circulating Ad5 virions is mediated by a high-affinity interaction between the capsid hexon protein and blood coagulation factor X (FX), whilst penton–αvintegrin interactions are thought to contribute to the induction of anti-Ad5 inflammatory and innate immune responses. To overcome these limitations, we sought to develop and characterise for the first time novel Ad5 vectors possessing mutations ablating both hexon:FX and penton:integrin interactions. As expected, intravascular administration of the FX binding-ablated Ad5HVR5*HVR7*E451Q vector (AdT*) resulted in significantly reduced liver transduction in vivo compared to Ad5. In macrophage-depleted mice, increased spleen uptake of AdT* was accompanied by an elevation in the levels of several inflammatory mediators. However ablation of the penton RGD motif in the AdT* vector background (AdT*RGE) resulted in a significant 5-fold reduction in spleen uptake and attenuated the antiviral inflammatory response. A reduction in spleen uptake and inflammatory activation was also observed in animals after intravascular administration of Ad5RGE compared to the parental Ad5 vector, with reduced co-localisation of the viral beta-galactosidase transgene with MAdCAM-1 + sinus-lining endothelial cells. Our detailed assessment of these novel adenoviruses indicates that penton base RGE mutation in combination with FX binding-ablation may be a viable strategy to attenuate the undesired liver uptake and pro-inflammatory responses to Ad5 vectors after intravascular delivery., Graphical abstract

Cyclooxygenases (COXs) play important roles during inflammation. While reports on COX-2 function in inflammation preceded those on COX-1, it is now well established that both isoforms participate in this process. During inflammation, COX expression was reported in inflammatory leukocytes, but much less is known about their presence in tissue- resident leukocytes. The aim was thus to verify the expression and activity of the COX isoforms in resident peritoneal mast cells and macrophages during acute peritonitis.Zymosan peritoneal inflammation was induced in C57BL/6J mice and COX-1 and COX-2 expression was evaluated by RT-PCR (mRNA level) and immunocytochemistry (protein level). COX activity was assessed by a specific assay and prostaglandin production by ELISA. Furthermore, some mice were selectively depleted of either peritoneal mast cells or macrophages and then COX activity was determined.The study revealed that both COXs are expressed/active at the peak of inflammation, but COX-2 predominates during resolution. The expressions of the COXs were detectable in both populations of resident peritoneal leukocytes. In peritoneal macrophages both isoforms were active even during the late phases of peritonitis and the cells significantly contributed to PGE(2) and PGD(2) synthesis. The most striking observation was that resident macrophages are critical for PGD(2) production during the resolution of inflammation.This study documents that both COX isoforms participate in all stages of acute inflammation and that tissue-resident leukocytes, especially macrophages, are important sites of COX-1/COX-2 expression and prostaglandin synthesis.

PMCID: PMC4128329.-- et al., Unique among leukocytes, neutrophils follow daily cycles of release from and migration back into the bone marrow, where they are eliminated. Because removal of dying cells generates homeostatic signals, we explored whether neutrophil elimination triggers circadian events in the steady state. Here, we report that the homeostatic clearance of neutrophils provides cues that modulate the physiology of the bone marrow. We identify a population of CD62L LO CXCR4HI neutrophils that have >aged> in the circulation and are eliminated at the end of the resting period in mice. Aged neutrophils infiltrate the bone marrow and promote reductions in the size and function of the hematopoietic niche. Modulation of the niche depends on macrophages and activation of cholesterol-sensing nuclear receptors and is essential for the rhythmic egress of hematopoietic progenitors into the circulation. Our results unveil a process that synchronizes immune and hematopoietic rhythms and expand the ascribed functions of neutrophils beyond inflammation. PaperFlick © 2013 Elsevier Inc., This study was supported by Deutsche Forschungsgemeinschaft (DFG WE1913/11-2 and SFB914-B8) to C.W.; R01-DK056638 and R01-HL69438 to P.S.F.; grants SAF2008-00057 and SAF2011-29244 to A.C.; Ramón y Cajal Fellowship (RYC-2007-00697) and SAF2009-11037 from MINECO, S2010/BMD-2314 from Comunidad de Madrid, and FP7-People-IRG Program (246655) to A.H.; C.N-A. is funded by a Human Frontiers in Science Program long-term fellowship 00194/2008. M.C-A. is supported by a FPI fellowship from the Spanish Ministry of Economy and Competitivity. The Centro Nacional de Investigaciones Cardiovasculares is supported by the Spanish Ministry of Economy and Competitivity and the Pro-CNIC Foundation.

Macrophages in the spleen can be eliminated by treatment with liposome encapsulated dichloromethylene diphosphonate (DMDP; reviewed in ref. 1. These macrophages disappeared during the first day after intravenous injection with liposome-encapsulated DMDP, remained absent for one week and reappeared gradually in the next weeks, as studied with the aid of enzyme histochemical techniques (2). Macrophages in different compartments of the mouse spleen can now be characterized by various monoclonal antibodies (3–6).

Naturally occurring (autoimmune) antibodies reactive with phospholipids are found in normal rabbit and human sera. In this short article, Nico van Rooijen postulates that bacterial endotoxins trigger the production of these autoimmune antiphospholipid antibodies by CD5+ (Ly-1+) B cells. According to this postulate, the endotoxins, or their lipid-A-containing fractions, become inserted in cell membranes and induce the production of autoimmune IgM antibodies against surface antigens, such as phospholipids, of such cells.

A large variety of antigens may initiate a series of events, finally leading to the production of specific antibodies by defined cells belonging to the B cell lineage of the immune system. The immunogenicity of these antigens may depend on different cellular requirements. Many antigens (e.g. proteins and erythrocytes) need the help of T-lymphocytes to start an immune response. These are referred to as thymus-dependent (TD) antigens which evoke a TD immune response. A number of other antigens have the capability to start an immune response without the help of T-lymphocytes. These are the thymus-independent (TI) antigens which evoke a TI immune response. Apart from T-lymphocytes and B-lymphocytes, most antigens require the participation of cells of the mononuclear phagocyte system (dendritic cells or macrophages). Although many immunologists nowadays seem to consider the immune response as a purely in vitro phenomenon, the immune system belongs in the body (Marx, 1985), where antigens, cells and their products have the opportunity to meet each other under optimum conditions. Evidence is growing that it is the microenvironment in a lymphoid organ, for which there is no in vitro equivalent, that creates the optimum conditions for the cells of the immune system to cooperate either directly or through their products. The possible cellular interactions during an immune response in the spleen and the localization and migration of the participating cells in defined splenic compartments have been recently reviewed (Van Rooijen et al, 1986). Herein we also postulated that there is a single differentiation pathway for all antibody-forming cells in the spleen, which is independent of the antigen and the type of immune response. We believe that this pathway is running along all of the different cells which may be required for any particular type of response.

The macrophages in the marginal zones in mice spleens were eliminated by i.v. injection of dichloromethylene diphosphonate encapsulated in liposomes. After immunization with a thymus-dependent (trinitrophenylated keyhole limpet hemocyanin; TNP-KLH) or thymus-independent type 1 (TNP-lipopolysaccharide) antigen, no differences in antibody responses in treated and untreated mice were observed. However, immunization with a thymus-independent type 2 (TI-2) antigen (TNP-Ficoll) resulted in a strong decrease of the antibody response in macrophage-depleted animals. Anti-TNP serum levels dropped 10-fold and the number of antibody-forming cells in the spleen 30-60-fold. These results confirm the special role of the marginal zone macrophages in the processing of TI-2 antigens, as earlier suggested in splenectomy studies.

Introduction Macrophages in the mammary gland are essential for morphogenesis of the ductal epithelial tree and have been implicated in promoting breast tumor metastasis. Although it is well established that macrophages influence normal mammopoiesis, the mammary cell types that these accessory cells influence have not been determined. Here we have explored a role for macrophages in regulating mammary stem cell (MaSC) activity, by assessing the ability of MaSCs to reconstitute a mammary gland in a macrophage-depleted fat pad. Methods Two different in vivo models were used to deplete macrophages from the mouse mammary fat pad, allowing us to examine the effect of macrophage deficiency on the mammary repopulating activity of MaSCs. Both the Csf1(op/op) mice and clodronate liposome-mediated ablation models entailed transplantation studies using the MaSC-enriched population. Results We show that mammary repopulating ability is severely compromised when the wild-type MaSC-enriched subpopulation is transplanted into Csf1(op/op) fat pads. In reciprocal experiments, the MaSC-enriched subpopulation from Csf1(op/op) glands had reduced regenerative capacity in a wildtype environment. Utilizing an alternative strategy for selective depletion of macrophages from the mammary gland, we demonstrate that co-implantation of the MaSC-enriched subpopulation with clodronate-liposomes leads to a marked decrease in repopulating frequency and outgrowth potential. Conclusions Our data reveal a key role for mammary gland macrophages in supporting stem/progenitor cell function and suggest that MaSCs require macrophage-derived factors to be fully functional. Macrophages may therefore constitute part of the mammary stem cell niche

Immunization with irradiated sporozoites is currently the most effective vaccination strategy against liver stages of malaria parasites, yet the mechanisms underpinning the success of this approach are unknown. Here we show that the complete development of protective CD8+ T cell responses requires prolonged antigen presentation. Using TCR transgenic cells specific for the malaria circumsporozoite protein, a leading vaccine candidate, we found that sporozoite antigen persists for over 8 weeks after immunization—a remarkable finding since irradiated sporozoites are incapable of replication and do not differentiate beyond early liver stages. Persisting antigen was detected in lymphoid organs and depends on the presence of CD11c+ cells. Prolonged antigen presentation enhanced the magnitude of the CD8+ T cell response in a number of ways. Firstly, reducing the time primed CD8+ T cells were exposed to antigen in vivo severely reduced the final size of the developing memory population. Secondly, fully developed memory cells expanded in previously immunized mice but not when transferred to naïve animals. Finally, persisting antigen was able to prime naïve cells, including recent thymic emigrants, to become functional effector cells capable of eliminating parasites in the liver. Together these data show that the optimal development of protective CD8+ T cell immunity against malaria liver stages is dependent upon the prolonged presentation of sporozoite-derived antigen., Author Summary Malaria causes the deaths of 0.5–2 million people each year, mainly in Africa. A safe and effective vaccine is likely needed for the control or eradication of this disease. It has been shown that if people are bitten by malaria-infected mosquitoes that have been irradiated they are protected against fully infectious bites. Radiation makes the parasites in the mosquito (called sporozoites) incapable of dividing; however, the irradiated parasites are still capable of stimulating the immune system to provide protection against fully infectious parasites. Many trial vaccines are based on trying to simulate the immune response induced by irradiated sporozoites in a safe and cost-effective way. We want to know how sporozoites are able to efficiently induce immune responses. We found that sprozoite molecules are retained by the immune system for months after immunization. These molecules continue to stimulate the cells of the immune system such that they divide more and thus probably protect better. The ability of the immune system to retain parasite material long after the parasites themselves have disappeared is remarkable and gives new insights into how effective immune responses can be generated by vaccines.

Background There is increasing interest in astrocyte biology because astrocytes have been demonstrated to play prominent roles in physiological and pathological conditions of the central nervous system, including neuroinflammation. To understand astrocyte biology, primary astrocyte cultures are most commonly used because of the direct accessibility of astrocytes in this system. However, this advantage can be hindered by microglial contamination. Although several authors have warned regarding microglial contamination in this system, complete microglial elimination has never been achieved. Methods The number and proliferative potential of contaminating microglia in primary astrocyte cultures were quantitatively assessed by immunocytologic and flow cytometric analyses. To examine the utility of clodronate for microglial elimination, primary astrocyte cultures or MG-5 cells were exposed to liposomal or free clodronate, and then immunocytologic, flow cytometric, and gene expression analyses were performed. The gene expression profiles of microglia-eliminated and microglia-contaminated cultures were compared after interleukin-6 (IL-6) stimulation. Results The percentage of contaminating microglia exceeded 15% and continued to increase because of their high proliferative activity in conventional primary astrocyte cultures. These contaminating microglia were selectively eliminated low concentration of liposomal clodronate. Although primary microglia and MG-5 cells were killed by both liposomal and free clodronate, free clodronate significantly affected the viability of astrocytes. In contrast, liposomal clodronate selectively eliminated microglia without affecting the viability, proliferation or activation of astrocytes. The efficacy of liposomal clodronate was much higher than that of previously reported methods used for decreasing microglial contamination. Furthermore, we observed rapid tumor necrosis factor-α and IL-1b gene induction in conventional primary astrocyte cultures after IL-6 stimulation, which was due to the activation of the Janus kinase/signal transducer and activator of the transcription pathway in contaminating microglia. Conclusions Because contaminating microglia could result in erroneous data regarding the pro-inflammatory properties of astrocytes, astrocyte biology should be studied in the absence of microglial contamination. Our simple method will be widely applicable to experimental studies of astrocyte biology and provide clues for understanding the role of astrocytes in neural development, function and disease.

Abstract 81 We investigated the contribution of macrophages to physiological and pathological conditions in which erythropoietic activity is enhanced. We utilized mouse models of a) anemia by phlebotomy-induced stress erythropoiesis (SE); b) increased erythropoiesis by erythropoietin (Epo) administration; c) Polycythemia Vera (Jak2V617F/+ or PV) and d) beta-thalassemia intermedia (Hbbth3/+ or BTI) in which macrophages were chemically depleted by injection of liposome-clodronate (LC). While chronic injection (up to 3 months) of LC in normal mice had little effect on steady state erythropoiesis, depletion of macrophages severely impaired recovery from anemia following phlebotomy and significantly limited the increase in hematocrit (Htc) in animals treated with Epo. To exclude that this effect was mediated by decreased serum iron parameters, we used mice iron overloaded by dietary means or affected by hemochromatosis (Hfe-KO and Hamp-KO). In these mice, recovery from anemia was still impaired following macrophage depletion, even though serum iron and transferrin saturation levels were elevated and unaffected by LC administration. In vitro studies using both mouse and human primary erythroblasts (EBs) indicated that EBs in S-phase were twice as many compared to EBs cultured in absence of macrophages. The numbers of terminally mature erythroid cells were up to six fold higher in co-culture conditions. Experiments using transwells indicate that direct contact between EBs and macrophages was required to generate this effect. Since our data highlighted an important role of macrophages in enhancing erythropoiesis, we investigated two disorders in which the pool of erythroid progenitor cells is expanded, such as PV and BTI. Chronic administration of LC in PV mice completely reversed splenomegaly and the Htc (P Our observations indicate that macrophages directly modulate stress- and pathological erythropoiesis. Several adhesion molecules participate in the formation of interactions within the erythroblastic islands, including integrins. Interestingly, βeta1integrin and its associated protein, focal adhesion kinase-1 (Fak1), were reported to be necessary for the compensatory response to anemia, suggesting that this pathway might be involved in the macrophage-EB cross-talk. More EBs co-cultured with macrophages retained cell surface expression of βeta1integrin molecule during the last stage of cell differentiation compared to EBs cultured alone, even though other differentiation markers did not shown any variation. Fak1 phosphorylation in EBs was induced by co-culturing them with splenic macrophages, suggesting that Fak1 signaling is one of the pathways activated in EBs through contact with macrophages. Administration of a FAK1 inhibitor (FAK1i) decreased proliferation of EB co-cultured with macrophages, while delayed recovery from anemia and decreased the spleen size in phlebotomized animals (40% decrease compared to phlebotomized control animals at day 4; P=0.032). Finally, short-term administration of FAK1i to BTI animals rapidly reverted splenomegaly with a concurrent reduction of erythroid expansion in both BM and spleen and led to amelioration of anemia, supported by increased RBCs count. Our data indicate that, while macrophages allow proper erythroid response under conditions of induced anemia or increased erythropoiesis in wt mice, they contribute to the pathological progression of PV and BTI. Activation of Fak1 promotes erythroid proliferation and pathological development, while its inhibition limits ineffective erythropoiesis and splenomegaly in BTI. In conclusion, we identified a new mechanism contributing to the pathophysiology of these disorders, which we believe will have critical scientific and therapeutic implications in the near future. Disclosures: Levine: Agios Pharmaceuticals: Research Funding. Rivella:Novartis Pharmaceuticals: Consultancy; Biomarin: Consultancy; Merganser Biotech: Consultancy, Equity Ownership, Research Funding; Isis Pharma: Consultancy, Research Funding.

SWISS mice, edible frogs and goldfish i.p. injected with zymosan (Z groups) develop peritoneal inflammation connected with a massive intraperitoneal accumulation of leukocytes, which is significantly diminished in mice and fish (but not frogs) by supplementation of zymosan with morphine (ZM groups). In order to check the putative role of resident peritoneal macrophages in morphine-modulated zymosan-induced peritonitis, some animals were depleted of resident macrophages by repeated i.p. injections of clodronate-liposomes (CL) followed by Z or ZM injection. In SWISS mice such CL-induced removal ofMac-3-positive cells (macrophages) resulted in an enhanced influx and prolonged accumulation of polymorphonuclear leukocytes (PMNs) in CL-Z and CL-ZM groups in comparison with their counterparts with intact macrophages. Nevertheless, supplementation of zymosan with morphine inhibited the early stages of peritonitis in CL-treated animals as it did in untreated mice. This indicates that intact peritoneal macrophages of SWISS mice are important for limiting PMN accumulation, perhaps mainly through the release of IL-10, but are not critical for the induction of anti-inflammatory effects of morphine during the early stages of peritonitis. Unexpectedly, macrophage depletion in CL-treated frogs and fish resulted in a lack of a typical peritonitis in both Z and ZM groups of these ectothermic animals.

Background Sequential accumulation of M1 and M2 macrophages is critical for skeletal muscle recovery after an acute injury. While M1 accumulation is believed to rely on monocyte infiltration, the mechanisms of M2 accumulation remain controversial, but could involve an infiltrating precursor. Yet, strong depletion of monocytes only partially impairs skeletal muscle healing, supporting the existence of alternative mechanisms to palliate the loss of infiltrating macrophage progenitors. The aims of this study are thus to investigate if proliferation occurs in macrophage subsets within injured skeletal muscles; and to determine if monocyte depletion leads to increased proliferation of macrophages after injury. Methods Injury was induced by bupivacaine injection in the tibialis anterior muscle of rats. Blood monocytes were depleted by daily intravenous injections of liposome-encapsulated clodronate, starting 24 h prior to injury. In separate experiments, irradiation of hind limb was also performed to prevent resident cell proliferation. Upon euthanasia, blood and muscles were collected for flow cytometric analyses of macrophage/monocyte subsets. Results Clodronate induced a 80%-90% depletion of monocyte but only led to 57% and 41% decrease of M1 and M2 macrophage accumulation, respectively, 2 d following injury. Conversely, the number of M1 macrophages in monocyte-depleted rats was 2.4-fold higher than in non-depleted rats 4 d after injury. This was associated with a 16-fold increase in the number of proliferative M1 macrophages, which was reduced by 46% in irradiated animals. Proliferation of M2 macrophages was increased tenfold by clodronate treatment 4 d post injury. The accumulation of M2 macrophages was partially impaired by irradiation, regardless of monocyte depletion. Conclusions M1 and M2 subsets proliferate after skeletal muscle injury and their proliferation is enhanced under condition of monocyte depletion. Our study supports the conclusion that both infiltrating and resident precursors could contribute to M1 or M2 macrophage accumulation in muscle injury.

In contrast to peripheral lymphoid organs, in the liver a high proportion of T cells are CD4+NKT cells. We have previously reported that LFA-1 plays a pivotal role in the homing of thymic CD4+NKT cells to the liver. In the present study, we further assessed which cell type participates in the homing of thymic CD4+NKT cells to the liver. The accumulation of donor thymocyte-derived CD4+NKT cells in the liver of SCID mice that had been reconstituted with thymocytes from C57BL/6 mice was severely impaired by in vivo depletion of NK cells, but not Kupffer cells in recipients. These results suggest that NK cells participate in the homing of thymic CD4+NKT cells to the liver. We assume that LFA-1 expressed on NK cells is involved in this mechanism.