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ABSTRACT Murine macrophages are activated by interferon-γ (IFN-γ) and/or Toll-like receptor (TLR) agonists such as bacterial endotoxin (lipopolysaccharide [LPS]) to express an inflammatory (M1) phenotype characterized by the expression of nitric oxide synthase-2 (iNOS) and inflammatory cytokines such as tumor necrosis factor-α (TNF-α) and interleukin (IL)-12. In contrast, Th2 cytokines IL-4 and IL-13 activate macrophages by inducing the expression of arginase-1 and the anti-inflammatory cytokine IL-10 in an IL-4 receptor-α (IL-4Rα)-dependent manner. Macrophages activated in this way are designated as “alternatively activated” (M2a) macrophages. We have shown previously that adenosine A 2A receptor (A 2A R) agonists act synergistically with TLR2, TLR4, TLR7, and TLR9 agonists to switch macrophages into an “M2-like” phenotype that we have termed “M2d.” Adenosine signaling suppresses the TLR-dependent expression of TNF-α, IL-12, IFN-γ, and several other inflammatory cytokines by macrophages and induces the expression of vascular endothelial growth factor (VEGF) and IL-10. We show here using mice lacking a functional IL-4Rα gene (IL-4Rα −/− mice) that this adenosine-mediated switch does not require IL-4Rα-dependent signaling. M2d macrophages express high levels of VEGF, IL-10, and iNOS, low levels of TNF-α and IL-12, and mildly elevated levels of arginase-1. In contrast, M2d macrophages do not express Ym1, Fizz1 (RELM-α), or CD206 at levels greater than those induced by LPS, and dectin-1 expression is suppressed. The use of these markers in vivo to identify “M2” macrophages thus provides an incomplete picture of macrophage functional status and should be viewed with caution.

Psoriasis is an autoinflammatory skin disease of unknown etiology. Topical application of Aldara cream containing the Toll-like receptor (TLR)7 agonist Imiquimod (IMQ) onto patients induces flares of psoriasis. Likewise, in mice IMQ triggers pathological changes closely resembling psoriatic plaque formation. Key cytokines like IL-23 and type-I IFN (IFN-I), both being produced mainly by dendritic cells (DCs), have been implicated in psoriasis. Although plasmacytoid DCs (pDCs) are the main source of IFNα and thought to initiate disease, conventional DCs (cDCs) appear to maintain the psoriatic lesions. Any role of cDCs during lesion formation remains elusive. Here, we report that selective activation of TLR7 signaling specifically in CD11c(+) DCs was sufficient to induce psoriasiform skin disease in mice. Intriguingly, both pDCs and the IFN-I pathway were dispensable for the development of local skin inflammation. Selective TLR7 triggering of Langerin(+) DCs resulted in attenuated disease, whereas their depletion did not alter the severity of skin lesions. Moreover, after IMQ-painting, IL-23 was exclusively produced by Langerin(neg) DCs in vivo. In conclusion, TLR7-activated Langerin(neg) cDCs trigger psoriatic plaque formation via IL-23-mediated activation of innate IL-17/IL-22-producing lymphocytes, independently of pDCs or IFN-I. These results suggest therapeutic targeting of IL-23 production by cDCs to refine current treatment strategies for psoriasis.

Key Points In contrast to most dendritic-cell populations, Langerhans cells repopulate locally throughout life in the steady state, independently of any input from the blood circulation. In contrast to quiescent skin, in major inflammatory skin injuries (such as exposure to ultraviolet B radiation) Langerhans cells are replaced by circulating monocytes. Langerhans cells repopulate locally after a lethal dose of radiation and remain of host origin following congenic bone-marrow transplantation. By contrast, following allogeneic bone-marrow transplantation, cutaneous graft-versus-host disease occurs and leads to the elimination of recipient Langerhans cells and their replacement with donor-derived cells. Langerhans cells are absent in mice that lack the macrophage colony-stimulating factor (M-CSF) receptor but remain unaffected in mice that lack the receptor for FMS-like-tyrosine-kinase 3 ligand (FLT3). Langerin is not uniquely expressed by Langerhans cells in the skin, but is also expressed by dendritic cells in stratified epithelial surfaces and by a subset of dendritic cells that is present in most connective tissues, including the dermis, lung, kidney and liver. Langerin + dendritic cells can be distinguished from Langerhans cells based on the expression of the integrin CD103 and the low expression of CD11b. Our understanding of the origin, phenotype and function of epidermal Langerhans cells and langerin-expressing dendritic cells has expanded in recent years, details of which, as well as the challenges that remain, are discussed in this Review. Langerhans cells (LCs) are a specialized subset of dendritic cells (DCs) that populate the epidermal layer of the skin. Langerin is a lectin that serves as a valuable marker for LCs in mice and humans. In recent years, new mouse models have led to the identification of other langerin + DC subsets that are not present in the epidermis, including a subset of DCs that is found in most non-lymphoid tissues. In this Review we describe new developments in the understanding of the biology of LCs and other langerin + DCs and discuss the challenges that remain in identifying the role of different DC subsets in tissue immunity.

Objective Delayed thrombolytic therapy with recombinant tissue plasminogen activator (tPA) may exacerbate blood‐brain barrier (BBB) breakdown after ischemic stroke and lead to catastrophic hemorrhagic transformation (HT). Rosiglitazone(RSG), a widely used antidiabetic drug that activates peroxisome proliferator‐activated receptor‐γ (PPAR‐γ), has been shown to protect against cerebral ischemia through promoting poststroke microglial polarization toward the beneficial anti‐inflammatory phenotype. However, whether RSG can alleviate HT after delayed tPA treatment remains unknown. In this study, we sort to examine the role of RSG on tPA‐induced HT after stroke. Methods and results We used the murine suture middle cerebral artery occlusion (MCAO) models of stroke followed by delayed administration of tPA (10 mg/kg, 2 hours after suture occlusion) to investigate the therapeutic potential of RSG against tPA‐induced HT. When RSG(6 mg/kg) was intraperitoneally administered 1 hour before MCAO in tPA‐treated MCAO mice, HT in the ischemic territory was significantly attenuated 1 day after stroke. In the tPA‐treated MCAO mice, we found RSG significantly mitigated BBB disruption and hemorrhage development compared to tPA‐alone‐treated stroke mice. Using flow cytometry and immunostaining, we confirmed that the expression of CD206 was significantly upregulated while the expression of iNOS was down‐regulated in microglia of the RSG‐treated mice. We further found that the expression of Arg‐1 was also upregulated in those tPA and RSG‐treated stroke mice and the protection against tPA‐induced HT and BBB disruption in these mice were abolished in the presence of PPAR‐γ antagonist GW9662 (4 mg/kg, 1 hour before dMCAO through intraperitoneal injection). Conclusions RSG treatment protects against BBB damage and ameliorates HT in delayed tPA‐treated stroke mice by activating PPAR‐γ and favoring microglial polarization toward anti‐inflammatory phenotype.

Production of recombinant protein bio-insecticides on a commercial scale can only be cost effective if host strains with very high expression levels are available. A recombinant fusion protein containing an arthropod toxin, ω-hexatoxin-Hv1a, (from funnel web spider Hadronyche versuta) linked to snowdrop lectin (Galanthus nivalis agglutinin; GNA) is an effective oral insecticide and candidate biopesticide. However, the fusion protein was vulnerable to proteolysis during production in the yeast Pichia pastoris. To prevent proteolysis, the Hv1a/GNA fusion expression construct was modified by site-directed mutagenesis to remove a potential Kex2 cleavage site at the C-terminus of the Hv1a peptide. To obtain a high expressing clone of P. pastoris to produce recombinant Hv1a/GNA, a straightforward method was used to produce multi-copy expression plasmids, which does not require multiple integrations to give clones of P. pastoris containing high copy numbers of the introduced gene. Removal of the Kex2 site resulted in increased levels of intact fusion protein expressed in wild-type P. pastoris strains, improving levels of intact recombinant protein recoverable. Incorporation of a C-terminal (His)₆ tag enabled single step purification of the fusion protein. These modifications did not affect the insecticidal activity of the recombinant toxin towards lepidopteran larvae. Introduction of multiple expression cassettes increased the amount of secreted recombinant fusion protein in a laboratory scale fermentation by almost tenfold on a per litre of culture basis. Simple modifications in the expression construct can be advantageous for the generation of high expressing P. pastoris strains for production of a recombinant protein, without altering its functional properties.

Galectin-2 is a monocyte-expressed carbohydrate-binding lectin, for which increased expression is genetically determined and associated with decreased collateral arteriogenesis in obstructive coronary artery disease patients. The inhibiting effect of galectin-2 on arteriogenesis was confirmed in vivo, but the mechanism is largely unknown. In this study we aimed to explore the effects of galectin-2 on monocyte/macrophage phenotype in vitro and vivo, and to identify the receptor by which galectin-2 exerts these effects. We now show that the binding of galectin-2 to different circulating human monocyte subsets is dependent on monocyte surface expression levels of CD14. The high affinity binding is blocked by an anti-CD14 antibody but not by carbohydrates, indicating a specific protein-protein interaction. Galectin-2 binding to human monocytes modulated their transcriptome by inducing proinflammatory cytokines and inhibiting pro-arteriogenic factors, while attenuating monocyte migration. Using specific knock-out mice, we show that galectin-2 acts through the CD14/toll-like receptor (TLR)-4 pathway. Furthermore, galectin-2 skews human macrophages to a M1-like proinflammatory phenotype, characterized by a reduced motility and expression of an anti-arteriogenic cytokine/growth factor repertoire. This is accompanied by a switch in surface protein expression to CD40-high and CD206-low (M1). In a murine model we show that galectin-2 administration, known to attenuate arteriogenesis, leads to increased numbers of CD40-positive (M1) and reduced numbers of CD206-positive (M2) macrophages surrounding actively remodeling collateral arteries. In conclusion galectin-2 is the first endogenous CD14/TLR4 ligand that induces a proinflammatory, non-arteriogenic phenotype in monocytes/macrophages. Interference with CD14-Galectin-2 interaction may provide a new intervention strategy to stimulate growth of collateral arteries in genetically compromised cardiovascular patients.

Inflammation plays a critical role in the pathogenesis of ischemic stroke. This process depends, in part, upon proinflammatory factors released by activated resident central nervous system (CNS) microglia (MG). Previous studies demonstrated that transfer of IL-10 + B-cells reduced infarct volumes in male C57BL/6 J recipient mice when given 24 h prior to or therapeutically at 4 or 24 h after experimental stroke induced by 60 min middle cerebral artery occlusion (MCAO). The present study assesses possible sex differences in immunoregulation by IL-10 + B-cells on primary male vs. female MG cultured from naïve and ischemic stroke-induced mice. Thus, MG cultures were treated with recombinant (r)IL-10, rIL-4 or IL-10 + B-cells after lipopolysaccharide (LPS) activation and evaluated by flow cytometry for production of proinflammatory and anti-inflammatory factors. We found that IL-10 + B-cells significantly reduced MG production of TNF-α, IL-1β and CCL3 post-MCAO and increased their expression of the anti-inflammatory M2 marker, CD206, by cell-cell interactions. Moreover, MG from female vs. male mice had higher expression of IL-4 and IL-10 receptors and increased production of IL-4, especially after treatment with IL-10 + B-cells. These findings indicate that IL-10-producing B-cells play a crucial role in regulating MG activation, proinflammatory cytokine release and M2 phenotype induction, post-MCAO, with heightened sensitivity of female MG to IL-4 and IL-10. This study, coupled with our previous demonstration of increased numbers of transferred IL-10 + B-cells in the ischemic hemisphere, provide a mechanistic basis for local regulation by secreted IL-10 and IL-4 as well as direct B-cell/MG interactions that promote M2-MG.

The absence of tumor necrosis factor (TNF) causes lethal infection by in normally resistant C57BL/6J (B6.WT) mice. The underlying pathogenic mechanism of this fatal disease has so far remained elusive. We found that B6.WT mice deficient for the gene (B6.TNF ) displayed not only a non-healing cutaneous lesion but also a serious infection of the liver upon inoculation. Infected B6.TNF mice developed an enlarged liver that showed increased inflammation. Furthermore, we detected an accumulating monocyte-derived macrophage population (CD45 F4/80 CD11b Ly6C ) that displayed a M2 macrophage phenotype with high expression of CD206, arginase-1, and IL-6, supporting the notion that IL-6 could be involved in M2 differentiation. In experiments, we demonstrated that IL-6 upregulated M-CSF receptor expression and skewed monocyte differentiation from dendritic cells to macrophages. This was countered by the addition of TNF. Furthermore, TNF interfered with the activation of IL-6-induced gp130-signal transducer and activator of transcription (STAT) 3 and IL-4-STAT6 signaling, thereby abrogating IL-6-facilitated M2 macrophage polarization. Therefore, our results support the notion of a general role of TNF in the inflammatory activation of macrophages and define a new role of IL-6 signaling in macrophage polarization downstream of TNF.

Background Interleukin-33 is a member of the IL-1 cytokine family whose functions are mediated and modulated by the ST2 receptor. IL-33-ST2 expression and interactions have been explored in mouse macrophages but little is known about the effect of IL-33 on human macrophages. The expression of ST2 transcript and protein levels, and IL-33-mediated effects on M1 (i.e. classical activation) and M2 (i.e. alternative activation) chemokine marker expression in human bone marrow-derived macrophages were examined. Results Human macrophages constitutively expressed the membrane-associated (i.e. ST2L) and the soluble (i.e. sST2) ST2 receptors. M2 (IL-4 + IL-13) skewing stimuli markedly increased the expression of ST2L, but neither polarizing cytokine treatment promoted the release of sST2 from these cells. When added to naïve macrophages alone, IL-33 directly enhanced the expression of CCL3. In combination with LPS, IL-33 blocked the expression of the M2 chemokine marker CCL18, but did not alter CCL3 expression in these naive cells. The addition of IL-33 to M1 macrophages markedly increased the expression of CCL18 above that detected in untreated M1 macrophages. Similarly, alternatively activated human macrophages treated with IL-33 exhibited enhanced expression of CCL18 and the M2 marker mannose receptor above that detected in M2 macrophages alone. Conclusions Together, these data suggest that primary responses to IL-33 in bone marrow derived human macrophages favors M1 chemokine generation while its addition to polarized human macrophages promotes or amplifies M2 chemokine expression.

The aim of this study is to investigate macrophages polarization induced by methionine enkephalin (MENK) that promotes tumoricidal responses in vivo and in vitro. Both phenotypic and functional activities of macrophages were assessed by the quantitative analysis of key surface molecules on macrophages with flow cytometry, immunofluorescent staining, and the production of cytokines with enzyme-linked immunosorbent assay and reverse transcriptase-polymerase chain reaction. Our results showed that MENK could down-regulate the expression of CD206 and the production of arginase-1 (the markers of alternatively activated (M2) macrophage) in tumor-associated macrophages in vivo, meanwhile it could significantly up-regulate the expression of CD64, MHC-II, and the production of induced nitric oxide synthase (the markers of classically activated (M1) macrophages). Furthermore, the studies on bone marrow-derived macrophages treated with MENK (10 −12  M) in vitro had demonstrated that MENK could markedly increase tumoricidal activity. MENK could also enhance the release of reactive oxidant species and the production of interleukin-12p40, tumor necrosis factor-α, while decrease the production of interleukin-10. In conclusion, MENK could effectively induce M2 macrophages polarizing to M1 macrophages, sequentially to modulate the Th1 responses of the host immune system. Our results suggest that MENK might have great potential as a new therapeutic agent for cancer.