Neutrophil-activating Chemokines Research Articles

386 The Adaptive Response of Old ABCB5+ MSCs is Changed Upon Exposure to LPS

Mesenchymal stem cells (MSCs) are endowed with the unique capacity to raise an adaptive response to environmental cues. This allows MSCs to control their direct neighbourhood and endogenous tissue niche. Upon exposure of MSCs with infection mimicking lipopolysaccharide (LPS), MSCs completely shift their transcriptome with the release of neutrophil activating chemokines. The LPS induced transcriptomic shift resulted in a significant increase in neutrophil expulsed DNA traps (NETs) and proteolytic enzymes. This adaptive response guarantees the defense from bacterial attack. Wound healing decreases with age and propensity for infection increases. Therefore, we addressed the question whether MSCs from old healthy donors (> 65 years) unlike young healthy donors (< 30 years) may change their adaptive response upon LPS exposure towards a reduced microbicidal response. Cultured ABCB5+ MSCs from young and old donors treated with LPS revealed differences in the time kinetic of NF-κB translocation from the cytoplasm to the nucleus. By contrast to ABCB5+ MSCs from young donors, ABCB5+ MSCs from old donors depicted a significantly delayed back regulation of nuclear NF-κB translocation, supported by an overshooting increase of p-p65. This correlates with a higher and longer persisting expression of NF-κB target genes like IL-6 in MSCs of elderly individuals compared to young individuals. Notably, ABCB5+ MSCs from young donors depict higher expression levels of IL-8 and IL-10 compared to old donors. Of note, LPS primed ABCB5+ MSCs from young donors can activate neutrophils by producing NETs to a higher level in comparison to old donors. NE activity indicative for microbicidal NET formation from co-cultures of LPS primed ABCB5+ MSC from young donors with PMA stimulated neutrophils is significantly higher and LPS-concentration-dependent compared to old donors. Collectively, MSCs from old individuals reveal a dysregulated anti-bacterial response which is supported by an impressively reduced killing ability of gram negative bacteria and at least in part explained higher susceptibility for severe bacterial infections in elderly.

The Candida albicans toxin candidalysin mediates distinct epithelial inflammatory responses through p38 and EGFR-ERK pathways.

The fungal pathogen Candida albicans secretes the peptide toxin candidalysin, which damages epithelial cells and drives an innate inflammatory response mediated by the epidermal growth factor receptor (EGFR) and mitogen-activated protein kinase (MAPK) pathways and the transcription factor c-Fos. In cultured oral epithelial cells (OECs), candidalysin activated the p38 MAPK signaling pathway, which resulted in heat shock protein 27 (Hsp27) activation, IL-6 release, and EGFR phosphorylation without influencing the induction of c-Fos. p38 activation was not triggered by EGFR but by two non-redundant pathways involving MAPK kinases (MKKs) and the kinase Src, which differentially controlled p38 signaling outputs. Whereas MKKs mainly promoted p38-dependent release of IL-6, Src promoted p38-mediated phosphorylation of EGFR in a ligand-independent fashion. In parallel, candidalysin also activated the EGFR-ERK pathway in a manner that depended on EGFR ligands, resulting in c-Fos activation and release of the neutrophil-activating chemokines G-CSF and GM-CSF. In mice, p38 was important for the early clearance events of oral C. albicans infection, but c-Fos was not. These findings delineate how candidalysin activates the p38 and ERK MAPK pathways that differentially contribute to immune activation during C. albicans infection.

Abstract P048: Steatosis promote liver cancer development by inducing chemokine production from Kupffer cells

Abstract Liver cancer is hallmarked with chronic inflammation resulting from underlying liver diseases such as liver steatosis. Steatotic liver injury recruits inflammatory cells and establishes the tumor immune environment that can promote the development of liver cancer. Previously, we showed that macrophages produce growth factors to promote liver cancer growth in a genetic model where steatosis and liver tumors were induced by the deletion of tumor suppressor Pten (phosphatase and tensin homologue deleted on chromosome 10). In this model, we showed that attenuating steatosis and depletion of macrophages leads to inhibition of liver cancer development. To investigate the recruitment and functions of immune cells in liver cancer, we analyzed the expression profiles of these tumors as well as tumors from other mouse models together with human liver samples. Our analysis of the inflammatory cytokines identified one chemokine that was commonly upregulated in all tumor model and attenuated in non-tumor models. CXCL5 is a member of the neutrophil-activating chemokines. In the Pten deleted liver tumors, CXCL5 is significantly upregulated whereas its expression is inhibited when steatosis is attenuated via caloric restriction or deletion of a metabolic kinase, Akt2. We explored the cell types that may produce CXCL5. To our surprise, the upregulation of CXCL5 is concurrent with increases in macrophage but not neutrophil populations. We further observed significant upregulation of CXCL5 mRNA expression in Kupffer cells, the liver resident macrophages isolated from the Pten deleted mice. We explored the hypothesis that macrophages secrets CXCL5 to establish the tumor environment and promote tumor growth. Our data showed that CXCL5 treatment increased mouse hepatocytes and human HepG2 cells proliferation. This effect is blocked by inhibition of CXCR2, the receptor for CXCL5. Additionally, we explored the mechanisms of CXCL5 upregulation in Kupffer cells and discovered that lipopolysaccharide (LPS) induces the expression of CXCL5 by nearly 20-fold in Kupffer cells. This is in contrast with the lack of induction in murine macrophage cell lines and primary peritoneal macrophages. This unique response of Kupffer cells suggests that the source of CXCL5 in the Pten deletion mouse model is likely Kupffer cells. This data is collaborated by bioinformatic analysis of datasets where steatotic liver injury is induced by diet feeding. Furthermore, analysis of patient proteomic data detected CXCL5 as the primary chemokine that is induced in tumors among other neutrophil-activating peptides. In summary, our data identified CXCL5 as a novel chemokine produced by Kupffer cells that plays key roles in steatosis driven liver cancer development. Chronic liver diseases such as steatohepatitis can establish tumorigenesis environment through accumulation of LPS stimulated CXCL5 release from Kupffer cells. Citation Format: Bangyan L. Stiles, Taojian Tu, Lina He, Mario Alba. Steatosis promote liver cancer development by inducing chemokine production from Kupffer cells [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2021 Oct 5-6. Philadelphia (PA): AACR; Cancer Immunol Res 2022;10(1 Suppl):Abstract nr P048.

367 The Adaptive Response of Old ABCB5+ MSCs is Changed Upon Exposure to LPS

Mesenchymal Stem Cells (MSC) can control their direct neighborhood and endogenous tissue niche and raise an adaptive response to environmental cues. Upon exposure of MSCs with lipopolysaccharide (LPS), MSCs completely shift their transcriptome with the release of neutrophil activating chemokines. The LPS induced transcriptomic shift resulted in a significant increase in neutrophil expulsed DNA traps (NETs) and proteolytic enzymes. This adaptive response guarantees the defense from bacterial attack.

Structural basis of chemokine interactions with heparan sulfate, chondroitin sulfate, and dermatan sulfate

Chemokines play diverse roles in human pathophysiology, ranging from trafficking leukocytes and immunosurveillance to the regulation of metabolism and neural function. Chemokine function is intimately coupled to binding tissue glycosaminoglycans (GAGs), heparan sulfate (HS), chondroitin sulfate (CS), and dermatan sulfate (DS). Currently, very little is known about how the structural features and sequences of a given chemokine, the structure and sulfation pattern of a given GAG, and structural differences among GAGs and among chemokines impact binding interactions. In this study, we used solution NMR spectroscopy to characterize the binding interactions of two related neutrophil-activating chemokines, CXCL1 and CXCL5, with HS, CS, and DS. For both chemokines, the dimer bound all three GAGs with higher affinity than did the monomer, and affinities of the chemokines for CS and DS were lower than for HS. NMR-based structural models reveal diverse binding geometries and show that the binding surfaces for each of the three GAGs were different between the two chemokines. However, a given chemokine had similar binding interactions with CS and DS that were different from HS. Considering the fact that CXCL1 and CXCL5 activate the same CXCR2 receptor, we conclude that GAG interactions play a role in determining the nature of chemokine gradients, levels of free chemokine available for receptor activation, how chemokines bind their receptors, and that differences in these interactions determine chemokine-specific function.

Candidalysin activates innate epithelial immune responses via epidermal growth factor receptor

Candida albicans is a fungal pathobiont, able to cause epithelial cell damage and immune activation. These functions have been attributed to its secreted toxin, candidalysin, though the molecular mechanisms are poorly understood. Here, we identify epidermal growth factor receptor (EGFR) as a critical component of candidalysin-triggered immune responses. We find that both C. albicans and candidalysin activate human epithelial EGFR receptors and candidalysin-deficient fungal mutants poorly induce EGFR phosphorylation during murine oropharyngeal candidiasis. Furthermore, inhibition of EGFR impairs candidalysin-triggered MAPK signalling and release of neutrophil activating chemokines in vitro, and diminishes neutrophil recruitment, causing significant mortality in an EGFR-inhibited zebrafish swimbladder model of infection. Investigation into the mechanism of EGFR activation revealed the requirement of matrix metalloproteinases (MMPs), EGFR ligands and calcium. We thus identify a PAMP-independent mechanism of immune stimulation and highlight candidalysin and EGFR signalling components as potential targets for prophylactic and therapeutic intervention of mucosal candidiasis.

Glycosaminoglycan Recognition of Neutrophil‐Activating Chemokines

Neutrophil‐activating chemokines are known for their fundamental and diverse role in recruiting immune cells to the site of inflammation/infection. Extracellular glycosaminoglycans (GAGs), especially heparan sulfate, sequester chemokines and engineer a concentration gradient to regulate the immune cell trafficking. Not much is known about the structural and dynamical aspects of chemokine – GAG interactions. We have studied the interaction of several human and mouse chemokines, including CXCL1, CXCL2, and CXCL5 with heparin/heparan sulfate oligosaccharides using computational (modeling and dynamics) and biophysical (NMR &amp; ITC) techniques. These studies have led to fine atomistic level understanding of GAG recognition of chemokines. First, our studies show that even though these chemokines are structurally homologous, they exhibit striking differences in their recognition of GAGs. Diverse binding geometries have been observed in which a GAG chain may bind each monomer present in a chemokine dimer or a GAG chain may bind two non‐overlapping sites by spanning across the dimer interface. Second, molecular dynamics simulations show large concerted motions of the Lys/Arg side chains bound to sulfate/carboxylates of GAGs, which imply translational motion of a GAG chain over the binding interface leading to considerable plasticity in binding. At the same time, a GAG chain may have restricted motion arising from specific interactions with a group of core amino acid residues. Third, a large number of hydrogen bond contributing residues occur in chemokines, e.g., in N‐loop, 40s turn, β3 strand and C‐helix, which provide strong/weak interactions to stabilize the complex. Interestingly, GAG recognition of chemokines is also brought about by water‐mediated interactions between amino acid side chains and sulfates/carboxylates, which introduce additional stability to GAG's dynamical motion. Fourth, relative importance of individual amino acid residues correlate well with experimental observations, thus implying that computational trajectories can be reliably used to understand GAG–chemokine systems. In addition, the computational trajectories can lead to identification of preferred chain length, mode of binding, and dynamical motions of GAGs in recognition of neutrophil‐activating chemokines. Our work contributes fundamental understanding on the structural biology of GAG–chemokine interactions.Support or Funding InformationThis work was supported in part by grants from the NIH including HL107152, HL090586 and HL128639This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Lysines and Arginines play non-redundant roles in mediating chemokine-glycosaminoglycan interactions

Glycosaminoglycans (GAGs) bind a large array of proteins and mediate fundamental and diverse roles in human physiology. Ion pair interactions between protein lysines/arginines and GAG sulfates/carboxylates mediate binding. Neutrophil-activating chemokines (NAC) are GAG-binding proteins, and their sequences reveal high selectivity for lysines over arginines indicating they are functionally not equivalent. NAC binding to GAGs impacts gradient formation, receptor functions, and endothelial activation, which together regulate different components of neutrophil migration. We characterized the consequence of mutating lysine to arginine in NAC CXCL8, a well-characterized GAG-binding protein. We chose three lysines — two highly conserved lysines (K20 and K64) and a CXCL8-specific lysine (K67). Interestingly, the double K64R/K20R and K64R/K67R mutants are highly impaired in recruiting neutrophils in a mouse model. Further, both the mutants bind GAG heparin with higher affinity but show similar receptor activity. NMR and MD studies indicate that the structures are essentially identical to the WT, but the mutations alter the network of intramolecular ion pair interactions. These observations collectively indicate that the reduced in vivo recruitment is due to altered GAG interactions, higher GAG binding affinity can be detrimental, and specificity of lysines fine-tunes in vivo GAG interactions and function.

Glycosaminoglycan Interactions Fine-Tune Chemokine-Mediated Neutrophil Trafficking: Structural Insights and Molecular Mechanisms.

Circulating neutrophils, rapidly recruited in response to microbial infection, form the first line in host defense. Humans express ~50 chemokines, of which a subset of seven chemokines, characterized by the conserved "Glu-Leu-Arg" motif, mediate neutrophil recruitment. Neutrophil-activating chemokines (NACs) share similar structures, exist as monomers and dimers, activate the CXCR2 receptor on neutrophils, and interact with tissue glycosaminoglycans (GAGs). Considering cellular assays have shown that NACs have similar CXCR2 activity, the question has been and remains, why do humans express so many NACs? In this review, we make the case that NACs are not redundant and that distinct GAG interactions determine chemokine-specific in vivo functions. Structural studies have shown that the GAG-binding interactions of NACs are distinctly different, and that conserved and specific residues in the context of structure determine geometries that could not have been predicted from sequences alone. Animal studies indicate recruitment profiles of monomers and dimers are distinctly different, monomer-dimer equilibrium regulates recruitment, and that recruitment profiles vary between chemokines and between tissues, providing evidence that GAG interactions orchestrate neutrophil recruitment. We propose in vivo GAG interactions impact several chemokine properties including gradients and lifetime, and that these interactions fine-tune and define the functional response of each chemokine that can vary between different cell and tissue types for successful resolution of inflammation.

Mechanistic insights into molecular evolution of species-specific differential glycosaminoglycan binding surfaces in growth-related oncogene chemokines.

Chemokines are chemotactic cytokines involved in leucocyte trafficking to infected tissue. Growth-related oncogene (GRO) chemokines namely CXCL1, CXCL2 and CXCL3 are neutrophil activating chemokines sharing a conserved three-dimensional structure, but encompassing functional diversity due to gene duplication and evolutionary events. However, the evolutionary mechanisms including selection pressures involved in diversification of GRO genes have not yet been characterized. Here, we performed comprehensive evolutionary analysis of GRO genes among different mammalian species. Phylogenetic analysis illustrated a species-specific evolution pattern. Selection analysis evidenced that these genes have undergone concerted evolution. Seventeen positively selected sites were obtained, although the majority of the protein is under purifying selection. Interestingly, these positively selected sites are more concentrated on the C-terminal/glycosaminoglycan (GAG) binding and dimerization segment compared to receptor binding domain. Substitution rate analysis confirmed the C-terminal domain of GRO genes as the highest substituted segment. Further, structural analysis established that the nucleotide alterations in the GAG binding domain are the source of surface charge modulation, thus generating the differential GAG binding surfaces and multiple binding sites as per evolutionary pressure, although the helical surface is primordial for GAG binding. Indeed, such variable electrostatic surfaces are crucial to regulate chemokine gradient formation during a host's defence against pathogens and also explain the significance of chemokine promiscuity.

Generation of inflammatory cytokines and chemokines from peripheral blood mononuclear cells by HLA Class II antibody–containing plasma unit that was associated with severe nonhemolytic transfusion reactions

HLA Class II antibodies are thought to be involved in severe transfusion reactions including transfusion-related acute lung injury (TRALI). The activation of monocytes by HLA Class II antibody may play an important role in the etiology of TRALI. An 81-year-old man with non-Hodgkin's lymphoma (Clinical Stage IIIA) received a plateletpheresis unit containing at least 4 x 10(11) platelets because of thrombocytopenia and a bleeding tendency. Approximately 30 minutes after the start of transfusion, he developed chills, tachycardia, dyspnea, lumber, and abdominal pain and then a fever (40.3 degrees C). His SaO(2) dropped to 70 percent. The transfusion was discontinued immediately. His symptoms disappeared after treatment with oxygen and the administration of corticosteroid and aminophyrine. A chest X-ray showed no sign of pulmonary edema. The donor serum sample had HLA-DR antibodies against multiple DR antigens including DR13, the recipient's HLA-DR type. The cross-match between the patient's lymphocytes and the donor serum was positive. The treatment of peripheral blood mononuclear cells from healthy subjects bearing DR13 antigen with the donor plasma caused the secretion of inflammatory cytokines (i.e., interleukin [IL]-1beta, IL-6, and tumor necrosis factor-alpha) and neutrophil-activating chemokines (i.e., IL-8 and CXCL1/GRO-alpha) in a cognate antigen-antibody relationship. In addition, the secretion of inflammatory cytokines appeared to require the involvement of CD32 and/or CD16. HLA-DR antibodies, detected in this case, had biologic functions to induce production of not only inflammatory cytokines but also neutrophil-attractant chemokines in vitro, which could contribute to the etiology of severe nonhemolytic transfusion reactions.

Circulating Inflammatory Endothelial Cells Contribute to Endothelial Progenitor Cell Dysfunction in Patients with Vasculitis and Kidney Involvement

Impaired angiogenic function has been reported in patients with kidney failure. During vascular damage, endothelial cells may detach from the site of inflammation and be released into the peripheral blood. With the use of Wegener's granulomatosis as a study model, whether circulating inflammatory endothelial cells (IEC) can (1) be used as a disease activity marker and (2) contribute to sustained vascular damage by inducing endothelial progenitor cell (EPC) dysfunction were examined. IEC-defined as endothelial cells that express the two inflammatory-associated markers vascular-adhesion protein-1 (VAP-1) and MHC class I-related chain A (MICA)-were increased significantly in patients with active disease as compared with those in remission. IEC expressed high levels of inducible nitric oxide synthase and neutrophil-activating chemokines, such as macrophage inflammatory protein-1alpha, growth-related oncogene-alpha, epithelial neutrophil activating peptide-78, and IL-8, and induced increased neutrophil migration. IEC levels significantly correlated with C-reactive protein and extent of organ involvement. Patients with active disease had decreased numbers of EPC colony-forming units and a high expression of VAP-1 and MICA in kidney endothelium. EPC did not express VAP-1 or MICA. IEC significantly inhibited proliferation, migration, and endothelial nitric oxide synthase expression in EPC. Thus, apart from being a new disease activity marker, IEC may contribute to vascular damage by impairing the functional capacity for repair by EPC. IEC may provide a unique in vitro system to study pathogenesis of kidney and vascular diseases.

Helicobacter pylori lipopolysaccharide binds to CD14 and stimulates release of interleukin-8, epithelial neutrophil-activating peptide 78, and monocyte chemotactic protein 1 by human monocytes.

Helicobacter pylori gastritis is characterized by leukocyte infiltration of the gastric mucosa. The aims of this study were to determine whether H. pylori-derived factors stimulate chemokine release from human monocytes and to ascertain whether H. pylori lipopolysaccharide (LPS) may be responsible for this effect. Human peripheral blood monocytes were exposed to an H. pylori water extract (HPE) or to purified H. pylori LPS. Levels of the chemokines interleukin-8 (IL-8), epithelial neutrophil-activating peptide 78 (ENA-78), and monocyte chemotactic protein 1 (MCP-1) were measured by enzyme-linked immunosorbent assay. The contribution of H. pylori LPS to monocyte activation was determined by using the LPS antagonist Rhodobacter sphaeroides lipid A (RSLA) and a blocking monoclonal antibody to CD14 (60bca). HPE increased monocyte secretion of IL-8, ENA-78, and MCP-1. Heat treatment of HPE did not reduce its ability to activate monocytes. Purified H. pylori LPS also stimulated monocyte chemokine production but was 1,000-fold less potent than Salmonella minnesota lipid A. RSLA blocked H. pylori LPS-induced monocyte IL-8 release in a dose-dependent fashion (maximal inhibition 82%, P < 0.001). RSLA also inhibited HPE-induced IL-8 release (by 93%, P < 0.001). The anti-CD14 monoclonal antibody 60bca substantially inhibited IL-8 release from HPE-stimulated monocytes (by 88%, P < 0.01), whereas the nonblocking anti-CD14 monoclonal antibody did not. These experiments with potent and specific LPS inhibitors indicate that the main monocyte-stimulating factor in HPE is LPS. H. pylori LPS, acting through CD14, stimulates human monocytes to release the neutrophil-activating chemokines IL-8 and ENA-78 and the monocyte-activating chemokine MCP-1. Despite its low relative potency, H. pylori LPS may play an important role in the pathogenesis of H. pylori gastritis.

Neutrophil-activating chemokines in psoriasis

Neutrophil-activating chemokines in psoriasis