Kelly L. Brown

Modulation of the TLR-Mediated Inflammatory Response by the Endogenous Human Host Defense Peptide LL-37

The sole human cathelicidin peptide, LL-37, has been demonstrated to protect animals against endotoxemia/sepsis. Low, physiological concentrations of LL-37 (≤1 μg/ml) were able to modulate inflammatory responses by inhibiting the release of the proinflammatory cytokine TNF-α in LPS-stimulated human monocytic cells. Microarray studies established a temporal transcriptional profile and identified differentially expressed genes in LPS-stimulated monocytes in the presence or absence of LL-37. LL-37 significantly inhibited the expression of specific proinflammatory genes up-regulated by NF-κB in the presence of LPS, including NFκB1 (p105/p50) and TNF-α-induced protein 2 (TNFAIP2). In contrast, LL-37 did not significantly inhibit LPS-induced genes that antagonize inflammation, such as TNF-α-induced protein 3 (TNFAIP3) and the NF-κB inhibitor, NFκBIA, or certain chemokine genes that are classically considered proinflammatory. Nuclear translocation, in LPS-treated cells, of the NF-κB subunits p50 and p65 was reduced ≥50% in the presence of LL-37, demonstrating that the peptide altered gene expression in part by acting directly on the TLR-to-NF-κB pathway. LL-37 almost completely prevented the release of TNF-α and other cytokines by human PBMC following stimulation with LPS and other TLR2/4 and TLR9 agonists, but not with cytokines TNF-α or IL-1β. Biochemical and inhibitor studies were consistent with a model whereby LL-37 modulated the inflammatory response to LPS/endotoxin and other agonists of TLR by a complex mechanism involving multiple points of intervention. We propose that the natural human host defense peptide LL-37 plays roles in the delicate balancing of inflammatory responses in homeostasis as well as in combating sepsis induced by certain TLR agonists.

Intracellular generation of superoxide by the phagocyte NADPH oxidase: How, where, and what for?

Professional phagocytes increase their consumption of molecular oxygen during the phagocytosis of microbes or when encountering a variety of nonparticulate stimuli. In these circumstances, oxygen is reduced by the phagocyte NADPH oxidase, and reactive oxygen species (ROS), which are important for the microbicidal activity of the cells, are generated. The structure and function of the NADPH oxidase have been resolved in part by studying cells from patients with chronic granulomatous disease (CGD), a condition characterized by the inability of phagocytes to assemble a functional NADPH oxidase and thus to produce ROS. As a result, patients with CGD have a predisposition to infections as well as a variety of inflammatory symptoms. A long-standing paradigm has been that NADPH oxidase assembly occurs exclusively in the plasma membrane or invaginations thereof (phagosomes). A growing body of evidence points to the possibility that phagocytes are capable of NADPH oxidase assembly in nonphagosomal intracellular membranes, resulting in ROS generation within intracellular organelles also in the absence of phagocytosis. The exact nature of these ROS-producing organelles is yet to be determined, but granules are prime suspects. Recent clinical findings indicate that the generation of intracellular ROS by NADPH oxidase activation is important for limiting inflammatory reactions and that intracellular and extracellular ROS production are regulated differently. Here we discuss the accumulating knowledge of intracellular ROS production in phagocytes and speculate on the precise role of these oxidants in regulating the inflammatory process.

Bovine and human cathelicidin cationic host defense peptides similarly suppress transcriptional responses to bacterial lipopolysaccharide

Genomic approaches can be exploited to expose the complexities and conservation of biological systems such as the immune network across various mammalian species. In this study, temporal transcriptional expression profiles were analyzed in human and bovine monocytic cells in response to the TLR‐4 agonist, LPS, in the presence or absence of their respective host defense peptides. The cathelicidin peptides, human LL‐37 and bovine myeloid antimicrobial peptide‐27 (BMAP‐27), are homologs, yet they have diverged notably in terms of sequence similarity. In spite of their low sequence similarities, both of these cathelicidin peptides demonstrated potent, antiendotoxin activity in monocytic cells at low, physiologically relevant concentrations. Microarray studies indicated that 10 ng/ml LPS led to the up‐regulation of 125 genes in human monocytes, 106 of which were suppressed in the presence of 5 μg/ml of the human peptide LL‐37. To confirm and extend these data, temporal transcriptional responses to LPS were assessed in the presence or absence of the species‐specific host defense peptides by quantitative real‐time PCR. The transcriptional trends of 20 LPS‐induced genes were analyzed in bovine and human monocytic cells. These studies demonstrated conserved trends of gene responses in that both peptides were able to profoundly suppress many LPS‐induced genes. Consistent with this, the human and bovine peptides suppressed LPS‐induced translocation of NF‐κB subunits p50 and p65 into the nucleus of monocytic cells. However, there were also distinct differences in responses to LPS and the peptides; for example, treatment with 5 μg/ml BMAP‐27 alone tended to influence gene expression (RELA, TNF‐α‐induced protein 2, MAPK phosphatase 1/dual specificity phosphatase 1, IκBκB, NFκBIL1, TNF receptor‐associated factor 2) to a greater extent than did the same amount of human LL‐37. We hypothesize that the immunomodulatory effects of the species‐specific host defense peptides play a critical role in regulating inflammation and represent an evolutionarily conserved mechanism for maintaining homeostasis, although the sequence divergence of these peptides is substantial.

Host Defense Peptide LL-37 Selectively Reduces Proinflammatory Macrophage Responses

The human cathelicidin peptide, LL-37, is a host defense peptide with a wide range of immunomodulatory activities and modest direct antimicrobial properties. LL-37 can exert both pro- and anti-inflammatory effects and can modulate the proinflammatory responses of human peripheral blood monocytes and epithelial cells. In this study, we evaluated the effect of LL-37 on mouse bone marrow-derived macrophages (BMDM) and tissue macrophages in vitro and in vivo. LL-37 dramatically reduced TNF-α and NO levels produced by LPS and IFN-γ–polarized M1-BMDM and slightly reduced reactive oxygen species production by these cells. LL-37 did not affect the ability of IL-4–polarized M2-BMDM to upregulate arginase activity, although it did inhibit LPS-induced TNF-α secretion in these cells. LL-37 did not compromise the ability of M1-polarized BMDM to phagocytose and kill bacteria and did not affect the uptake of apoptotic neutrophils by M2-polarized BMDM. However, LL-37-treated M1-BMDM were more efficient at suppressing tumor growth in vitro. LL-37 significantly reduced LPS-induced TNF-α secretion in ex vivo alveolar macrophages, whereas its effect on peritoneal macrophages was much less dramatic. Effective inhibition of LPS-induced TNF-α secretion by alveolar macrophages also occurred in vivo when LL-37 was administered by intratracheal injection. This demonstrates a selective ability of LL-37 to decrease M1-BMDM, M2-BMDM, and tissue macrophage production of the proinflammatory cytokine TNF-α in response to LPS while leaving other crucial anti-inflammatory M1 and M2 macrophage functions unaltered.

A Role for the Cell Adhesion Molecule CD44 and Sulfation in Leukocyte-Endothelial Cell Adhesion during an Inflammatory Response?

CD44 is a widely expressed cell adhesion molecule that has been implicated in a variety of biological processes including lymphopoiesis, angiogenesis, wound healing, leukocyte extravasation at inflammatory sites, and tumor metastasis. The adhesive function of CD44, like other molecules involved in inducible adhesion, is tightly regulated. Post-translational modifications, isoform expression, aggregation state, and protein associations all can affect the ligand binding properties of CD44, and these can vary depending on the cell type and the activation state of the cell. The most extensively characterized ligand for CD44 is hyaluronan, a component of the extracellular matrix. Interactions between CD44 and hyaluronan can mediate both cell-cell and cell-extracellular matrix adhesion. In the immune system, both the selectin molecules and CD44 have been implicated in the initial binding of leukocytes to endothelial cells at an inflammatory site. Sulfation is required for selectin-mediated leukocyte-endothelial cell interactions, and, recently, inducible sulfation also was shown to regulate CD44-mediated leukocyte adhesion to endothelial cells. Sulfation, therefore, may be important in the regulation of cell adhesion at inflammatory sites. In this commentary we have reviewed the molecular aspects of CD44 and the mechanisms that regulate its binding to hyaluronan. In addition, we have summarized the role of CD44 and hyaluronan in mediating leukocyte-endothelial cell interactions and have discussed how this interaction may be regulated. Finally, we examined the potential role of sulfation as an inducible means to regulate CD44-mediated leukocyte adhesion and as a more general mechanism to regulate leukocyte-endothelial cell interactions.

Galectin-3 functions as an opsonin and enhances the macrophage clearance of apoptotic neutrophils.

Galectin-3, a beta-galactoside binding, endogenous lectin, takes part in various inflammatory events and is produced in substantial amounts at inflammatory foci. We investigated whether extracellular galectin-3 could participate in the phagocytic clearance of apoptotic neutrophils by macrophages, a process of crucial importance for termination of acute inflammation. Using human leukocytes, we show that exogenously added galectin-3 increased the uptake of apoptotic neutrophils by monocyte-derived macrophages (MDM). Both the proportion of MDM that engulfed apoptotic prey and the number of apoptotic neutrophils that each MDM engulfed were enhanced in the presence of galectin-3. The effect was lactose-inhibitable and required galectin-3 affinity for N-acetyllactosamine, a saccharide typically found on cell surface glycoproteins, since a mutant lacking this activity was without effect. The enhanced uptake relied on the presence of galectin-3 during the cellular interaction and was paralleled by lectin binding to apoptotic cells as well as MDM in a lactose-dependent manner. These findings suggest that galectin-3 functions as a bridging molecule between phagocyte and apoptotic prey, acting as an opsonin. The process of clearance, whereby apoptotic neutrophils are removed by macrophages, is crucial for the resolution of acute inflammation and our data imply that the increased levels of galectin-3 often found at inflammatory sites could potently affect this process.

Enhanced inflammatory responses of chronic granulomatous disease leukocytes involve ROS-independent activation of NF-κB

Reactive oxygen species (ROS) generated by the cellular NADPH‐oxidase are crucial for phagocytic killing of ingested microbes and have been implicated as signaling molecules in various processes. For example, ROS are thought to be involved in activation of the transcription factor NF‐κB, central for mediating production of proinflammatory cytokines in response to inflammatory stimuli. Several studies have demonstrated that inhibitors of the NADPH‐oxidase interfere with NF‐κB activation and production of proinflammatory cytokines. Curiously, patients with chronic granulomatous disease (CGD), an immunodeficiency characterized by an inability to produce ROS, are not only predisposed to severe infections, but also frequently develop various inflammatory complications indicative of exaggerated inflammatory responses. Here, we show that human CGD leukocytes display a hyperinflammatory phenotype with increased production of proinflammatory cytokines in response to stimulation with Toll‐like receptor agonists. The hyperinflammatory phenotype was also evident in mononuclear cells from CGD mice (gp91phox–/–), but not in control cells in the presence of NADPH‐oxidase inhibitor diphenyleneiodonium, probably reflecting NADPH‐oxidase‐independent effects of the inhibitor. Furthermore, we show that the major steps involved in NF‐κB activation were intact in human CGD cells. These data indicate that ROS were nonessential for activation of NF‐κB and their production may even attenuate inflammation.

Galectin 3 aggravates joint inflammation and destruction in antigen-induced arthritis

OBJECTIVE Galectin 3, an endogenous β-galactoside-binding lectin, plays an important role in the modulation of immune responses. The finding that galectin 3 is present in the inflamed synovium in patients with rheumatoid arthritis suggests that the protein is associated with the pathogenesis of this disease. We undertook this study to investigate the influence of galectin 3 deficiency in a murine model of arthritis. METHODS Wild-type (WT) and galectin 3-deficient (galectin 3(-/-) ) mice were subjected to antigen-induced arthritis (AIA) through immunization with methylated bovine serum albumin. The concentration of serum cytokines (interleukin-6 [IL-6] and tumor necrosis factor α [TNFα]) and antigen-specific antibodies was evaluated using a cytometric bead array platform and enzyme-linked immunosorbent assay (ELISA). Cellular IL-17 responses were examined by flow cytometry, ELISA, and enzyme-linked immunospot assay. RESULTS The joint inflammation and bone erosion of AIA were markedly suppressed in galectin 3(-/-) mice as compared with WT mice. The reduced arthritis in galectin 3(-/-) mice was accompanied by decreased levels of antigen-specific IgG and proinflammatory cytokines. The frequency of IL-17-producing cells in the spleen was reduced in galectin 3(-/-) mice as compared with WT mice. Exogenously added recombinant galectin 3 could partially restore the reduced arthritis and cytokines in galectin 3(-/-) mice. CONCLUSION Our findings show that galectin 3 plays a pathogenic role in the development and progression of AIA and that the disease severity is accompanied by alterations of antigen-specific IgG levels, systemic levels of TNFα and IL-6, and frequency of IL-17-producing T cells. To our knowledge, this is the first report of in vivo evidence that galectin 3 plays a crucial role in the development of arthritis.

ROS-deficient monocytes have aberrant gene expression that correlates with inflammatory disorders of chronic granulomatous disease

Chronic granulomatous disease is an immunodeficiency caused by an inability to produce reactive oxygen species. While the mechanism of hyper-sensitivity to infection is well understood in CGD, the basis for debilitating inflammatory disorders that arise in the absence of evident infection has not been fully explained. Herein it is demonstrated that resting and TLR-activated monocytes from individuals with CGD expressed significantly higher levels of inflammatory mediators than control cells; the expression in CGD cells resembled normal cells stimulated with lipopolysaccharide. The lack of acute illness, infection or circulating endotoxin in the blood of the CGD patients at the time of sampling was consistent with infection-free inflammation. The enhanced expression of inflammatory mediators correlated with elevated expression of NF-kappaB and was dependent on ERK1/2 signalling. The results are consistent with the hypothesis that ROS are anti-inflammatory mediators that control gene expression and potentially limit the development of sterile inflammatory disorders.

Novel anti-infectives: is host defence the answer?

Resistance to antimicrobial agents and the limited development of novel agents are threatening to worsen the burden of infections that are already a leading cause of morbidity and mortality. This has increased interest in the development of novel strategies such as selective modulation of our natural immune defences. Innate immunity is a complex, evolutionarily conserved, multi-facetted response to defeating infection that is naturally stimulated by pathogenic organisms through pattern recognition receptors on host cells. It is amplifiable and broad spectrum but if overstimulated can lead to the potential for harmful inflammatory responses. A broad variety of therapies are already available or increasingly under development, to stimulate protective innate immunity without overtly stimulating harmful inflammation or even suppressing such damaging pro-inflammatory responses.