Macrophages are prominent in the stromal compartment of virtually all types of malignancy. These highly versatile cells respond to the presence of stimuli in different parts of tumors with the release of a distinct repertoire of growth factors, cytokines, chemokines, and enzymes that regulate tumor growth, angiogenesis, invasion, and/or metastasis. The distinct microenvironments where tumor-associated macrophages (TAM) act include areas of invasion where TAMs promote cancer cell motility, stromal and perivascular areas where TAMs promote metastasis, and avascular and perinecrotic areas where hypoxic TAMs stimulate angiogenesis. This review will discuss the evidence for differential regulation of TAMs in these microenvironments and provide an overview of current attempts to target or use TAMs for therapeutic purposes. (Cancer Res 2006; 66(2): 605-12)

https://cancerres.aacrjournals.org/content/canres/66/2/605.full.pdf

Distinct Role of Macrophages in Different Tumor Microenvironments

Gene-encoded anti-microbial peptides (AMPs) are widespread in nature, as they are synthesized by microorganisms as well as by multicellular organisms from both the vegetal and the animal kingdoms. These naturally occurring AMPs form a first line of host defense against pathogens and are involved in innate immunity. Depending on their tissue distribution, AMPs ensure either a systemic or a local protection of the organism against environmental pathogens. They are classified into three major groups: (i) peptides with an alpha-helical conformation (insect cecropins, magainins, etc.), (ii) cyclic and open-ended cyclic peptides with pairs of cysteine residues (defensins, protegrin, etc.), and (iii) peptides with an over-representation of some amino acids (proline rich, histidine rich, etc.). Most AMPs display hydrophobic and cationic properties, have a molecular mass below 25-30 kDa, and adopt an amphipathic structure (alpha-helix, beta-hairpin-like beta-sheet, beta-sheet, or alpha-helix/beta-sheet mixed structures) that is believed to be essential to their anti-microbial action. Interestingly, in recent years, a series of novel AMPs have been discovered as processed forms of large proteins. Despite the extreme diversity in their primary and secondary structures, all natural AMPs have the in vitro particularity to affect a large number of microorganisms (bacteria, fungi, yeast, virus, etc.) with identical or complementary activity spectra. This review focuses on AMPs forming alpha-helices, beta-hairpin-like beta-sheets, beta-sheets, or alpha-helix/beta-sheet mixed structures from invertebrate and vertebrate origins. These molecules show some promise for therapeutic use.

Anti-microbial peptides: from invertebrates to vertebrates

In this review, antimicrobials from a range of plant, animal, and microbial sources are reviewed along with their potential applications in food systems. Chemical and biochemical antimicrobial compounds derived from these natural sources and their activity against a range of pathogenic and spoilage microorganisms pertinent to food, together with their effects on food organoleptic properties, are outlined. Factors influencing the antimicrobial activity of such agents are discussed including extraction methods, molecular weight, and agent origin. These issues are considered in conjunction with the latest developments in the quantification of the minimum inhibitory (and noninhibitory) concentration of antimicrobials and/or their components. Natural antimicrobials can be used alone or in combination with other novel preservation technologies to facilitate the replacement of traditional approaches. Research priorities and future trends focusing on the impact of product formulation, intrinsic product parameters, and extrinsic storage parameters on the design of efficient food preservation systems are also presented.

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Application of Natural Antimicrobials for Food Preservation

Lactate generated from pyruvate fuels production of intracellular NAD(+) as an end result of the glycolytic process in tumors. Elevated lactate concentration represents a good indicator of the metabolic adaptation of tumors and is actually correlated to clinical outcome in a variety of human cancers. In this study, we investigated whether lactate could directly modulate the endothelial phenotype and thereby tumor vascular morphogenesis and perfusion. We found that lactate could enter endothelial cells through the monocarboxylate transporter MCT-1, trigger the phosphorylation/degradation of IκBα, and then stimulate an autocrine NF-κB/IL-8 (CXCL8) pathway driving cell migration and tube formation. These effects were prevented by 2-oxoglutarate and reactive oxygen species (ROS) inhibitors, pointing to a role for prolyl-hydroxylase and ROS in the integration of lactate signaling in endothelial cells. PHD2 silencing in endothelial cells recapitulated the proangiogenic effects of lactate, whereas a blocking IL-8 antibody or IL-8-targeting siRNA prevented them. Finally, we documented in mouse xenograft models of human colorectal and breast cancer that lactate release from tumor cells through the MCT4 (and not MCT1) transporter is sufficient to stimulate IL-8-dependent angiogenesis and tumor growth. In conclusion, our findings establish a signaling role for lactate in endothelial cells and they identify the lactate/NF-κB/IL-8 pathway as an important link between tumor metabolism and angiogenesis.

https://cancerres.aacrjournals.org/content/canres/71/7/2550.full.pdf

Lactate influx through the endothelial cell monocarboxylate transporter MCT1 supports an NF-κB/IL-8 pathway that drives tumor angiogenesis.

Objective
Macrophages show extreme heterogeneity and different subsets have been characterized by their activation route and their function. For instance, macrophage subsets are distinct by acting differently under pathophysiological conditions such as inflammation and cancer. Macrophages also contribute to angiogenesis, but the role of various specific subsets in angiogenesis has not been thoroughly investigated.

Anti-inflammatory M2, but not pro-inflammatory M1 macrophages promote angiogenesis in vivo

The respiratory epithelium maintains an effective antimicrobial environment to prevent colonization by microorganisms in inspired air. In addition to constitutively present host defenses which include antimicrobial peptides and proteins, the epithelial cells respond to the presence of microbes by the induction two complementary parts of an innate immune response. The first response is the increased production of antimicrobial agents, and the second is the induction of a signal network to recruit phagocytic cells to contain the infection. Inflammatory mediators released by the recruited cells as well as from the epithelium itself further induce the expression of the antimicrobial agents. The result is an effective prevention of microbial colonization. The epithelial cells recognize the pathogen-associated patterns on microbes by surface receptors such as CD14 and Toll-like receptors. Subsequent signal transduction pathways have been identified which result in the increased transcription of host defense response genes. Diseases such as cystic fibrosis, or environmental exposures such as the inhalation of air pollution particles, may create an environment that impairs the expression or activity of the host defenses in the airway. This can lead to increased susceptibility to airway infections.

The innate immune response of the respiratory epithelium

Murine thioglycolate-induced peritoneal macrophages (MPMs) and the murine RAW264.7 macrophage-like cell line (RAW cells) constitutively produce vascular endothelial growth factor (VEGF). VEGF production is increased under hypoxic conditions or after cell activation with interferon-gamma (IFNgamma) and endotoxin (lipopolysaccharide, LPS). In contrast, tumor necrosis factor-alpha is produced only by IFNgamma/LPS-activated cells. Lactate (25 mmol/L) does not increase VEGF production by these cells. However, hypoxia, lactate, and IFNgamma/LPS-activated MPMs express angiogenic activity, whereas normoxic, nonactivated MPMs do not. Lack of angiogenic activity is not due to an antiangiogenic factor(s) in the medium of these cells. Angiogenic activity produced by hypoxia and lactate-treated MPMs is neutralized by anti-VEGF antibody, which also neutralizes most of the angiogenic activity produced by IFNgamma/LPS-activated MPMs. The inducible nitric oxide synthase inhibitors Ng-nitro-L-arginine-methyl ester (1.5 mmol/L) and aminoguanidine (1 mmol/L) block production of angiogenic activity by MPMs and RAW cells. In RAW cells, Ng-nitro-L-arginine-methyl ester and AG block IFNgamma/LPS-activated, but not constitutive, VEGF production, whereas in MPMs, neither constitutive nor IFNgamma/LPS-activated VEGF synthesis is affected. Synthesis of tumor necrosis factor-alpha is also unaffected. In contrast to normoxic, nonactivated MPMs, inducible nitric oxide synthase-inhibited, IFNgamma/LPS-activated MPMs produce an antiangiogenic factor(s). We conclude that VEGF is a major contributor to macrophage-derived angiogenic activity, and that activation by hypoxia, lactate, or IFNgamma/LPS switches macrophage-derived VEGF from a nonangiogenic to an angiogenic state. This switch may involve a posttranslational modification of VEGF, possibly by the process of ADP-ribosylation. ADP-ribosylation by MPM cytosolic extracts or by cholera toxin switches rVEGF165 from an angiogenic to a nonangiogenic state. In IFNgamma/LPS-activated MPMs, the inducible nitric oxide synthase-dependent pathway also regulates the expression of an antiangiogenic factor(s) that antagonizes the bioactivity of VEGF and provides an additional regulatory pathway controlling the angiogenic phenotype of macrophages.

Production of Vascular Endothelial Growth Factor by Murine Macrophages: Regulation by Hypoxia, Lactate, and the Inducible Nitric Oxide Synthase Pathway

Antimicrobial peptides are proposed to act as the first line of mucosal host defense by exerting broad-spectrum microbicidal activity against pathogenic microbes. Pleurocidin, a new 25-residue linear antimicrobial peptide, was recently isolated from the skin secretions of winter flounder (Pleuronectes americanus). The present study identifies the cDNA and gene encoding pleurocidin. The pleurocidin gene comprises four exons. Its upstream region demonstrates consensus binding sequences for transcription factors found in host defense genes in mammals, including sequences identical to the NF-IL6 and alpha and gamma interferon response elements. Pleurocidin is predicted to exist as a 68-residue prepropeptide that undergoes proteolytic cleavage of its amino-terminal signal and carboxy-terminal anionic propiece to form the active, mature peptide. Transmission electron microscopy localized pleurocidin to the mucin granules of skin and intestinal goblet cells. Significant synergy was shown to occur between pleurocidin and D-cycloserine targeting Mycobacterium smegmatis. Pleurocidin was functionally active at physiologic concentrations of magnesium and calcium; however, high concentrations of these divalent cations ablated pleurocidin's activity against a standard test strain, Escherichia coli D31. Pleurocidin was tested against bacterial and fungal clinical isolates and showed broad-spectrum antimicrobial activity. Together, these data support the hypothesis that pleurocidin participates in innate mucosal immunity, and it may prove to be a beneficial therapeutic agent.

Characterization of a fish antimicrobial peptide: gene expression, subcellular localization, and spectrum of activity.

The iron-binding protein lactoferrin is a ubiquitous and abundant constituent of human exocrine secretions. Lactoferrin inhibits bacterial growth by sequestering essential iron and also exhibits non-iron-dependent antibacterial, antifungal, antiviral, antitumor, anti-inflammatory, and immunoregulatory activities. All of these non-iron-dependent activities are mediated by the highly charged N terminus of lactoferrin. In this study we characterized a Lys/Arg polymorphism at position 29 in the N-terminal region of human lactoferrin that results from a single nucleotide polymorphism in exon 1 of the human lactoferrin gene. We expressed cDNAs encoding both lactoferrin variants in insect cells and purified the two proteins by ion exchange chromatography. The two lactoferrin variants exhibited nearly identical iron-binding and iron-releasing activities and equivalent bactericidal activities against a strain of the gram-negative bacterium Actinobacillus actinomycetemcomitans. When tested against the gram-positive species Streptococcus mutans and Streptococcus mitis, however, lactoferrin containing Lys at position 29 exhibited significantly greater bactericidal activity than did lactoferrin containing Arg. In addition, the Lys-containing lactoferrin stimulated bovine tracheal epithelial cells to synthesize much higher levels of tracheal antimicrobial peptide mRNA than did the Arg-containing variant. A genotyping assay that distinguished between the two alleles based on a polymorphic EarI restriction site showed that the Lys and Arg alleles had frequencies of 24% and 76%, respectively, among 17 healthy human subjects, and 72% and 28%, respectively, among nine patients with localized juvenile periodontitis. Our findings suggest that these two lactoferrin variants are functionally different and that these differences may contribute to the pathogenesis of localized juvenile periodontitis.

https://iai.asm.org/content/iai/71/11/6141.full.pdf

One of two human lactoferrin variants exhibits increased antibacterial and transcriptional activation activities and is associated with localized juvenile periodontitis.

Expression of innate immune genes such as beta-defensins is induced in airway epithelium by bacterial components via activation of NF-kappaB. We show here that live Gram-negative bacteria can similarly stimulate this pathway, resulting in upregulation of the beta-defensin tracheal antimicrobial peptide (TAP) in primary cultures of bovine tracheal epithelial cells (TECs), by a Toll-like receptor 4 (TLR4)-mediated pathway. The Gram-negative airway pathogen Bordetella bronchiseptica possesses a type III secretion system previously suggested to inhibit the nuclear translocation of NF-kappaB in a cell line by immunohistochemistry. We therefore hypothesized that this pathogen might interfere in the innate immune response of the epithelium. Exposure of TECs to wild-type B. bronchiseptica suppressed the activation of NF-kappaB and the subsequent induction of TAP mRNA levels, whereas a type III secretion-defective strain did not. These results suggest a mechanism for bacterial evasion of the innate immune response in the airway, which could allow for the observed persistent colonization of this pathogen.

Diana Legarda

Papers

The innate immune response of the respiratory epithelium

Production of Vascular Endothelial Growth Factor by Murine Macrophages: Regulation by Hypoxia, Lactate, and the Inducible Nitric Oxide Synthase Pathway

Characterization of a fish antimicrobial peptide: gene expression, subcellular localization, and spectrum of activity.

One of two human lactoferrin variants exhibits increased antibacterial and transcriptional activation activities and is associated with localized juvenile periodontitis.

Suppression of NF-kappaB-mediated beta-defensin gene expression in the mammalian airway by the Bordetella type III secretion system.