Blockade of interleukin-6 effects on cytokine profiles and macrophage activation after spinal cord injury in mice

The objective of this study was to clarify the effects of a temporal blockade of IL-6/IL-6 receptor (IL-6R) engagement, using an anti-mouse IL-6R monoclonal antibody (MR16-1), on macrophage activation and the inflammatory response in the acute phase after spinal cord injury (SCI) in mice. MR16-1 antibodies versus isotype control antibodies or saline alone was administered immediately after thoracic SCI in mice. MR16-1-treated group samples showed increased neuronal regeneration and locomotor recovery compared with controls. Immunoblot analysis of the MR16-1-treated samples identified downregulation of Th1 and upregulation of Th2 cytokines. MR16-1 treatment promoted arginase-1-positive, CD206-positive M2 macrophages, with preferential localization of these cells at the injury site and enhanced positivity for Mac-2 and Mac-3, suggestive of increased phagocytic behavior. The results suggest that temporal blockade of IL-6 signaling after SCI abrogates damaging inflammatory activity and promotes functional recovery by promoting the formation of alternatively activated M2 macrophages.

Exaggerated inflammation and monocytosis associate with diastolic dysfunction in heart failure with preserved ejection fraction: evidence of M2 macrophage activation in disease pathogenesis

BACKGROUND: Heart failure with preserved ejection fraction (HFPEF) is a major health problem associated with myocardial leukocyte infiltration, inflammation, and fibrosis. Monocyte and macrophage subsets play a role in HFPEF but have not been studied. We analyzed peripheral blood monocyte phenotype and plasma markers of monocyte activation in patients with HFPEF, asymptomatic LV diastolic dysfunction (aLVDD), and asymptomatic hypertension (aHTN).

METHODS AND RESULTS: Peripheral blood was collected from 23 aHTN, 30 aLVDD, and 30 HFPEF patients. Peripheral cytokines of classic/pro-inflammatory (tumor necrosis factor alpha, interleukin (IL) 12, IL-6, monocyte chemoattractant protein 1, C-X-C motif chemokine 10) and alternative/anti-inflammatory monocytes (chemokine-C-C motif ligand (CCL) 17, CCL-18, soluble CD163) were increased in aLVDD and HFPEF. Peripheral blood mononuclear cells and monocytes were purified and surface-stained for CD14, CD16, CD163, and CD206. Peripheral monocyte percentage was increased in aLVDD and HFPEF and correlated with echocardiographic LVDD indices. Classic/pro-inflammatory monocyte numbers were increased in aLVDD and HFPEF, and alternative/anti-inflammatory monocyte numbers were increased in HFPEF. CD163 M2-macrophage receptor was reduced in HFPEF. Culture of healthy donor monocytes (n = 3) with HFPEF patient-derived sera (n = 6) promoted M2 macrophage features as evidenced by altered morphology and genes (CD206, IL-10).

CONCLUSIONS: Increased peripheral inflammation, monocytosis, and monocyte differentiation to anti-inflammatory/profibrotic M2 macrophages likely associate with HFPEF and its precedent asymptomatic LVDD phase.

Granulocyte-macrophage precursor cell and colony-stimulating factor responses of mice infected with Salmonella typhimurium

The response of granulocyte-macrophage progenitor cells (in vitro colony-forming cells) and of colony-stimulating (CS) factor in serum were studied in mice infected intraperitoneally with 10(3) viable Salmonella typhimurium. Increases in the number of colony-forming cells in marrow and spleen and increases in the serum level of CS factor occurred during the infection. There was no evidence to suggest that progressive infection was associated with failure of macrophage production. Medium rich in CS factor increased the bactericidal activity of macrophages in vitro and it was suggested that CS factor could be involved in macrophage activation.

EFFECT OF MACROPHAGE BLOCKADE ON THE RESISTANCE OF INBRED MICE TO PARACOCCIDIOIDES-BRASILIENSIS INFECTION

The effect of macrophage blockade on the natural resistance and on the adaptative immune response of susceptible (B10.D2/oSn) and resistant (A/Sn) mice to Paracoccidioides brasiliensis infection was investigated. B10.D2/oSn and A/Sn mice previously injected with colloidal carbon were infected ip with yeast cells to determine the 50% lethal dose, and to evaluate the anatomy and histopathology, macrophage activation, antibody production and DTH reactions. Macrophage blockade rendered both resistant and susceptible mice considerably more susceptible to infection, as evidenced by increased mortality and many disseminated lesions. P. brasiliensis infection and/or carbon treatment increased the ability of macrophages from resistant mice to spread up to 25 days after treatment. In susceptible mice the enhanced spreading capacity induced by carbon treatment was impaired at ail assayed periods except at 1 week after infection. Macrophage blockade enhanced DTH reactions in resistant mice, but did not alter these reactions in susceptible mice, which remained anergic. To the contrary, macrophage blockade enhanced specific antibody production by susceptible mice, but did nor affect the low levels produced by resistant mice. The effect of macrophage blockade confirms the natural tendency of resistant animals to mount DTH reactions in the course of the disease and the preferential antibody response developed by susceptible mice after P. brasiliensis infection. on the whole, macrophage functions appear to play a fundamental role in the natural and acquired resistance mechanisms to P. brasiliensis infection.

Macrophage activity and histopathology of the lymphohematopoietic organs in male Wistar rats orally exposed to single or mixed pesticides

The noxious effects of low or effective dose exposure to single or mixed pesticides on macrophage activity and the lymphohematopoietic organs were investigated. Male Wistar rats were orally exposed to dichlorvos, dicofol, endosulfan, dieldrin and permethrin, either as single or combined mixtures during a 28-day study containing eight groups: one group received a semipurified diet (non-treated); two groups received a semipurified diet containing low dose mixture (dieldrin 0.025 mg/kg, endosulfan, 0.6 mg/kg, dicofol 0.22 mg/kg, dichlorvos 0.23 mg/kg, permethrin 5 mg/kg) or an effective dose mixture (dichlorvos 2.3 mg/kg, dicofol 2.5 mg/kg, endosulfan 2.9 mg/kg, dieldrin 0.05 mg/kg and permethrin 25.0 mg/kg), respectively; the other five groups received a semipurified diet containing each single pesticide in effective doses. At sacrifice, the thymus, spleen, mesenteric lymph nodes, Payer's patches and bone marrow were removed for histological analysis. Peritoneal macrophages were obtained to determine the phagocytosis and spreading indexes and tumoral necrosis factor alpha (TNF-α), nitric oxide (NO) and H2O2 production. Exposure to pesticide mixtures did not alter the percentage of macrophage phagocytosis and spreading, TNF-α production or the NO and H2O2 release when compared to the non-treated group. Neither was there any apparent evidence that a pesticide mixture at low or effective doses altered the histological structure of the lymphohematopoietic organs. The findings indicate that short-term treatment with pesticide mixtures did not induce an apparent immunotoxic effect in male Wistar rats. © 2013 Copyright Taylor and Francis Group, LLC.

Pathogenic Mycobacterium bovis strains differ in their ability to modulate the proinflammatory activation phenotype of macrophages

Background: Tuberculosis, caused by Mycobacterium tuberculosis or Mycobacterium bovis, remains one of the leading infectious diseases worldwide. The ability of mycobacteria to rapidly grow in host macrophages is a factor contributing to enhanced virulence of the bacteria and disease progression. Bactericidal functions of phagocytes are strictly dependent on activation status of these cells, regulated by the infecting agent and cytokines. Pathogenic mycobacteria can survive the hostile environment of the phagosome through interference with activation of bactericidal responses. To study the mechanisms employed by highly virulent mycobacteria to promote their intracellular survival, we investigated modulating effects of two pathogenic M. bovis isolates and a reference M. tuberculosis H37Rv strain, differing in their ability to multiply in macrophages, on activation phenotypes of the cells primed with major cytokines regulating proinflammatory macrophage activity. Results: Bone marrow- derived macrophages obtained from C57BL/6 mice were infected by mycobacteria after a period of cell incubation with or without treatment with IFN-gamma, inducing proinflammatory type-1 macrophages (M1), or IL-10, inducing anti-inflammatory type-2 cells (M2). Phenotypic profiling of M1 and M2 was then evaluated. The M. bovis strain MP287/03 was able to grow more efficiently in the untreated macrophages, compared with the strains B2 or H37Rv. This strain induced weaker secretion of proinflammatory cytokines, coinciding with higher expression of M2 cell markers, mannose receptor (MR) and arginase-1 (Arg-1). Treatment of macrophages with IFN-gamma and infection by the strains B2 and H37Rv synergistically induced M1 polarization, leading to high levels of inducible nitric oxide synthase (iNOS) expression, and reduced expression of the Arg-1. In contrast, the cells infected with the strain MP287/03 expressed high levels of Arg-1 which competed with iNOS for the common substrate arginine, leading to lower levels of NO production. Conclusions: The data obtained demonstrated that the strain, characterized by increased growth in macrophages, down- modulated classical macrophage activation, through induction of an atypical mixed M1/M2 phenotype.

Paracoccidioides brasiliensis lipids modulate macrophage activity via Toll-dependent or independent mechanisms

The macrophages are the first host cells that interact with the fungus Paracoccidioides brasiliensis, but the main mechanisms that regulate this interaction are not well understood. Because the role played by P. brasiliensis lipids in macrophage activation was not previously investigated, we aimed to assess the influence of diverse lipid fractions from P. brasiliensis yeasts in this process. The possible participation of TLR2 and TLR4 signaling was also evaluated using TLR2- and TLR4-defective macrophages. Four lipid-rich fractions were studied as follows: F1, composed by membrane phospholipids and neutral lipids, F2 by glycolipids of short chain, F3a by membrane glycoproteins anchored by glycosylphosphatidylinositol (GPI) groups, and F3b by glycolipids of long chain. All assayed lipid fractions were able to activate peritoneal macrophages and induce nitric oxide (NO) production. Importantly, the F1 and F3a fractions exerted opposite effects in the control of P. brasiliensis uptake and killing, but both fractions inhibited cytokines production. Furthermore, the increased NO production and expression of costimulatory molecules induced by F3a was shown to be TLR2 dependent although F1 used Toll-independent mechanisms. In conclusion, our work suggests that lipid components may play a role in the innate immunity against P. brasiliensis infection using Toll-dependent and independent mechanisms to control macrophage activation.

Effects of asbestos and silica on superoxide anion production in the guinea pig alveolar macrophage

Asbestos and silica are important industrial hazards. Exposure to these dusts can result in pulmonary fibrosis and, in the case of asbestos, cancer. Although the hazards of asbestos and silica exposure have long been known, the pathogenesis of dust-related disease is not well understood. Both silica and asbestos are thought to alter the function of the alveolar macrophage, but the nature of the biochemical alteration is unknown. Therefore, this study examined the effect of asbestos and silica on the activation pathway of the guinea pig alveolar macrophage. Activation of macrophages by physiological agents results in stimulation of phospholipase C causing phosphatidyl inositol turnover and intracellular calcium mobilization. Phosphatidyl inositol turnover produces diacylglycerol which activates protein kinase C causing superoxide anion production.^ Chrysotile stimulated alveolar macrophages to produce superoxide anion. This stimulation proceeded via phospholipase C, since chrysotile stimulated phosphatidyl inositol turnover and intracellular calcium mobilization. The possible involvement of a coupling protein was evaluated by pretreating cells with pertussis toxin. Pertussis toxin pretreatment partially inhibited chrysotile stimulation, suggesting that chrysotile activates a coupling protein in an non-classical manner. Potential binding sites for chrysotile stimulation were examined using a series of nine lectins. Chrysotile-stimulated superoxide anion production was blocked by pretreatment with lectins which bound to N-acetylglucosamine, but not by lectins which bound to mannose, fucose, or N-acetylgalactosamine. In addition, incubation with the N-acetylglucosamine polymer, chitin, inhibited chrysotile-stimulated superoxide anion production, suggesting that chrysotile stimulated superoxide anion production by binding to N-acetylglucosamine residues.^ On the other hand, silica did not stimulate superoxide anion production. The effect of silica on agonist stimulation of this pathway was examined using two stimulants of superoxide anion production, N-formyl-nle-leu-phe (FNLP, which stimulates through phospholipase C) and phorbol-12,13-dibutyrate (which directly activates protein kinase C). Sublethal doses of silica inhibited FNLP-stimulated superoxide anion production, but did not affect phorbol-12,13-dibutyrate-stimulated superoxide anion production, suggesting that the site of inhibition precedes protein kinase C. This inhibition was not due to cell membrane damage, since cell permeability to calcium-45 and rubidium-86 was not increased. It is concluded that chrysotile binds to N-acetylglucosamine residues on macrophage surface glycoproteins to stimulate the physiological pathway resulting in superoxide anion production. In contrast, silica does not stimulate superoxide anion production, but it did inhibit FNLP-stimulated superoxide anion production. ^

MIC-1, a novel macrophage inhibitory cytokine, is a divergent member of the TGF-β superfamily

Macrophages play a key role in both normal and pathological processes involving immune and inflammatory responses, to a large extent through their capacity to secrete a wide range of biologically active molecules. To identify some of these as yet not characterized molecules, we have used a subtraction cloning approach designed to identify genes expressed in association with macrophage activation. One of these genes, designated macrophage inhibitory cytokine 1 (MIC-1), encodes a protein that bears the structural characteristics of a transforming growth factor β (TGF-β) superfamily cytokine. Although it belongs to this superfamily, it has no strong homology to existing families, indicating that it is a divergent member that may represent the first of a new family within this grouping. Expression of MIC-1 mRNA in monocytoid cells is up-regulated by a variety of stimuli associated with activation, including interleukin 1β, tumor necrosis factor α (TNF-α), interleukin 2, and macrophage colony-stimulating factor but not interferon γ, or lipopolysaccharide (LPS). Its expression is also increased by TGF-β. Expression of MIC-1 in CHO cells results in the proteolytic cleavage of the propeptide and secretion of a cysteine-rich dimeric protein of Mr 25 kDa. Purified recombinant MIC-1 is able to inhibit lipopolysaccharide -induced macrophage TNF-α production, suggesting that MIC-1 acts in macrophages as an autocrine regulatory molecule. Its production in response to secreted proinflammatory cytokines and TGF-β may serve to limit the later phases of macrophage activation.

Distinct Modes of Macrophage Recognition for Apoptotic and Necrotic Cells Are Not Specified Exclusively by Phosphatidylserine Exposure

The distinction between physiological (apoptotic) and pathological (necrotic) cell deaths reflects mechanistic differences in cellular disintegration and is of functional significance with respect to the outcomes that are triggered by the cell corpses. Mechanistically, apoptotic cells die via an active and ordered pathway; necrotic deaths, conversely, are chaotic and passive. Macrophages and other phagocytic cells recognize and engulf these dead cells. This clearance is believed to reveal an innate immunity, associated with inflammation in cases of pathological but not physiological cell deaths. Using objective and quantitative measures to assess these processes, we find that macrophages bind and engulf native apoptotic and necrotic cells to similar extents and with similar kinetics. However, recognition of these two classes of dying cells occurs via distinct and noncompeting mechanisms. Phosphatidylserine, which is externalized on both apoptotic and necrotic cells, is not a specific ligand for the recognition of either one. The distinct modes of recognition for these different corpses are linked to opposing responses from engulfing macrophages. Necrotic cells, when recognized, enhance proinflammatory responses of activated macrophages, although they are not sufficient to trigger macrophage activation. In marked contrast, apoptotic cells profoundly inhibit phlogistic macrophage responses; this represents a cell-associated, dominant-acting anti-inflammatory signaling activity acquired posttranslationally during the process of physiological cell death.

CNI-1493 inhibits monocyte/macrophage tumor necrosis factor by suppression of translation efficiency.

Tumor necrosis factor (TNF) mediates a wide variety of disease states including septic shock, acute and chronic inflammation, and cachexia. Recently, a multivalent guanylhydrazone (CNI-1493) developed as an inhibitor of macrophage activation was shown to suppress TNF production and protect against tissue inflammation and endotoxin lethality [Bianchi, M., Ulrich, P., Bloom, O., Meistrell, M., Zimmerman, G. A., Schmidtmayerova, H., Bukrinsky, M., Donnelley, T., Bucala, R., Sherry, B., Manogue, K. R., Tortolani, A. J., Cerami, A. & Tracey, K. J. (1995) Mol. Med. 1, 254-266, and Bianchi, M., Bloom, O., Raabe, T., Cohen, P. S., Chesney, J., Sherry, B., Schmidtmayerova, H., Zhang, X., Bukrinsky, M., Ulrich, P., Cerami, A. & Tracey, J. (1996) J. Exp. Med., in press]. We have now elucidated the mechanism by which CNI-1493 inhibits macrophage TNF synthesis and show here that it acts through suppression of TNF translation efficiency. CNI-1493 blocked neither the lipopolysaccharide (LPS)-induced increases in the expression of TNF mRNA nor the translocation of nuclear factor NF-kappa B to the nucleus in macrophages activated by 15 min of LPS stimulation, indicating that CNI-1493 does not interfere with early NF-kappa B-mediated transcriptional regulation of TNF. However, synthesis of the 26-kDa membrane form of TNF was effectively blocked by CNI-1493. Further evidence for the translational suppression of TNF is given by experiments using chloram-phenicol acetyltransferase (CAT) constructs containing elements of the TNF gene that are involved in TNF translational regulation. Both the 5' and 3' untranslated regions of the TNF gene were required to elicit maximal translational suppression by CNI-1493. Identification of the molecular target through which CNI-1493 inhibits TNF translation should provide insight into the regulation of macrophage activation and mechanisms of inflammation.

Cytokine secretion via cholesterol-rich lipid raft-associated SNAREs at the phagocytic cup

Lipopolysaccharide-activated macrophages rapidly synthesize and secrete tumor necrosis factor α (TNFα) to prime the immune system. Surface delivery of membrane carrying newly synthesized TNFα is controlled and limited by the level of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins syntaxin 4 and SNAP-23. Many functions in immune cells are coordinated from lipid rafts in the plasmamembrane, and we investigated a possible role for lipid rafts in TNFα trafficking and secretion. TNFα surface delivery and secretion were found to be cholesterol- dependent. Upon macrophage activation, syntaxin 4 was recruited to cholesterol-dependent lipid rafts, whereas its regulatory protein, Munc18c, was excluded from the rafts. Syntaxin 4 in activated macrophages localized to discrete cholesterol-dependent puncta on the plasmamembrane, particularly on filopodia. Imaging the early stages of TNFα surface distribution revealed these puncta to be the initial points of TNFα delivery. During the early stages of phagocytosis, syntaxin 4 was recruited to the phagocytic cup in a cholesterol dependent manner. Insertion of VAMP3-positive recycling endosome membrane is required for efficient ingestion of a pathogen. Without this recruitment of syntaxin 4, it is not incorporated into the plasma membrane, and phagocytosis is greatly reduced. Thus, relocation of syntaxin 4 into lipid rafts in macrophages is a critical and rate-limiting step in initiating an effective immune response.

Flightless, secreted through a late endosome/lysosome pathway, binds LPS and dampens cytokine secretion.

Flightless (Flii) is upregulated in response to wounding and has been shown to function in wound closure and scarring. In macrophages intracellular Flii negatively modulates TLR signalling and dampens cytokine production. We now show that Flii is constitutively secreted from macrophages and fibroblasts and is present in human plasma. Secretion from fibroblasts is upregulated in response to scratch wounding and LPS-activated macrophages also temporally upregulate their secretion of Flii. Using siRNA, wild-type and mutant proteins we show that Flii is secreted via a late endosomal/lysosomal pathway that is regulated by Rab7 and Stx11. Flii contains 11 leucine rich repeat (LRR) domains in its N-terminus that have nearly 50% similarity to those in the extracellular pathogen binding portion of Toll-like receptor 4 (TLR4). We show secreted Flii can also bind LPS and has the ability to alter macrophage activation. LPS activation of macrophages in Flii depleted conditioned media leads to enhanced macrophage activation and increased TNF secretion compared to cells activated in the presence of Flii. These results show secreted Flii binds to LPS and in doing so alters macrophage activation and cytokine secretion, suggesting that like the intracellular pool of Flii, secreted Flii also has the ability to alter inflammation.

An acute growth factor treatment that preserves function after spinal cord contusion injury

Inflammation of the spinal cord after traumatic spinal cord injury leads to destruction of healthy tissue. This “secondary degeneration” is more damaging than the initial physical damage and is the major contributor to permanent loss of functions. In our previous study we showed that combined delivery of two growth factors, vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF), significantly reduced secondary degeneration after hemi-section injury of the spinal cord in the rat. Growth factor treatment reduced the size of the lesion cavity at 30d compared to control animals and further reduced the cavity at 90d in treated animals while in control animals the lesion cavity continued to increase in size. Growth factor treatment also reduced astrogliosis and reduced macroglia/macrophage activation around the injury site. Treatment with individual growth factors alone had similar effects to control treatments. The present study investigated whether growth factor treatment would improve locomotor behaviour after spinal contusion injury, a more relevant preclinical model of spinal cord injury. The growth factors were delivered for the first 7d to the injury site via osmotic minipump. Locomotor behaviour was monitored at 1-28d after injury using the BBB score and at 30d using automated gait analysis. Treated animals had BBB scores of 18; Control animals scored 10. Treated animals had significantly reduced lesion cavities and reduced macroglia/macrophage activation around the injury site. We conclude that growth factor treatment preserved spinal cord tissues after contusion injury, thereby allowing functional recovery. This treatment has the potential to significantly reduce the severity of human spinal cord injuries.

Identification of 15 new psoriasis susceptibility loci highlights the role of innate immunity

To gain further insight into the genetic architecture of psoriasis, we conducted a meta-analysis of 3 genome-wide association studies (GWAS) and 2 independent data sets genotyped on the Immunochip, including 10,588 cases and 22,806 controls. We identified 15 new susceptibility loci, increasing to 36 the number associated with psoriasis in European individuals. We also identified, using conditional analyses, five independent signals within previously known loci. The newly identified loci shared with other autoimmune diseases include candidate genes with roles in regulating T-cell function (such as RUNX3, TAGAP and STAT3). Notably, they included candidate genes whose products are involved in innate host defense, including interferon-mediated antiviral responses (DDX58), macrophage activation (ZC3H12C) and nuclear factor (NF)-κB signaling (CARD14 and CARM1). These results portend a better understanding of shared and distinctive genetic determinants of immune-mediated inflammatory disorders and emphasize the importance of the skin in innate and acquired host defense. © 2012 Nature America, Inc. All rights reserved.