C-C Chemokine Receptor 7 Promotes T-Cell Acute Lymphoblastic Leukemia Invasion of the Central Nervous System via ß2-Integrins.

C-C Chemokine Receptor 7 (CCR7) mediates T-cell acute lymphoblastic leukemia (T-ALL) invasion of the central nervous system (CNS) mediated by chemotactic migration to C-C chemokine ligand 19 (CCL19). To determine if a CCL19 antagonist, CCL198-83, could inhibit CCR7-induced chemotaxis and signaling via CCL19 but not CCL21, we used transwell migration and Ca2+ mobilization signaling assays. We found that in response to CCL19, human T-ALL cells employ ß2 integrins to invade human brain microvascular endothelial cell monolayers. In vivo, using an inducible mouse model of T-ALL, we found that we were able to increase the survival of the mice treated with CCL198-83 when compared to non-treated controls. Overall, our results describe a targetable cell surface receptor, CCR7, which can be inhibited to prevent ß2-integrin-mediated T-ALL invasion of the CNS and potentially provides a platform for the pharmacological inhibition of T-ALL cell entry into the CNS.

Profiling migration of human monocytes in response to chemotactic and barotactic guidance cues.

Monocytes are critical to innate immunity, participating in chemotaxis during tissue injury, infection, and inflammatory conditions. However, the migration dynamics of human monocytes under different guidance cues are not well characterized. Here, we developed a microfluidic device to profile the migration characteristics of human monocytes under chemotactic and barotactic guidance cues while also assessing the effects of age and cytokine stimulation. Human monocytes preferentially migrated toward the CCL2 gradient through confined microchannels, regardless of donor age and migration pathway. Stimulation with interferon (IFN)-γ, but not granulocyte-macrophage colony-stimulating factor (GM-CSF), disrupted monocyte navigation through complex paths and decreased monocyte CCL2 chemotaxis, velocity, and CCR2 expression. Additionally, monocytes exhibited a bias toward low-hydraulic-resistance pathways in asymmetric environments, which remained consistent across donor ages, cytokine stimulation, and chemoattractants. This microfluidic system provides insights into the unique migratory behaviors of human monocytes and is a valuable tool for studying peripheral immune cell migration in health and disease.

Volatile Sex Pheromone Extraction and Chemoattraction Assay in Caenorhabditis elegans.

Chemical communication is vital in organismal health, reproduction, and overall well-being. Understanding the molecular pathways, neural processes, and computations governing these signals remains an active area of research. The nematode Caenorhabditis elegans provides a powerful model for studying these processes as it produces a volatile sex pheromone. This pheromone is synthesized by virgin females or sperm-depleted hermaphrodites and serves as an attractant for males. This protocol describes a detailed method for isolating the volatile sex pheromone from several C. elegans strains (WT strain N2, daf-22, and fog-2) and C. remanei. We also provide a protocol for quantifying the male chemotaxis response to the volatile sex pheromone. Our analysis utilizes measurements such as chemotaxis index (C.I.), arrival time (A.T.), and a trajectory plot to compare male responses under various conditions accurately. This method allows for robust comparisons between males of different genetic backgrounds or developmental stages. Furthermore, the experimental setup outlined here is adaptable to investigating other chemoattraction chemicals.

Chemotaxis Assay of Bone Marrow-Derived Macrophages.

Immune responses rely on efficient and coordinated migration of immune cells to the site of infection or injury. To reach the site of immunological threat often requires long-range navigation of immune cells through complex tissue and vascular networks. Chemotaxis, cell migration steered by gradients of cell-attractive chemicals that bind sensory receptors, is central to this response. Chemoattractant receptors mostly belong to the G-protein-coupled receptor (GPCR) family, but the way attractant-receptor signaling directs cell migration is not fully understood. Direct-viewing chemotaxis chambers combined with time-lapse microscopy give a powerful tool to study the dynamic details of cells' responses to different attractant landscapes. Here, we describe the application of one such chamber (the Dunn chamber) to study bone marrow-derived macrophage chemotaxis to gradients of complement C5a.

Calycosin inhibited MIF-mediated inflammatory chemotaxis of macrophages to ameliorate ischemia reperfusion-induced acute kidney injury.

BACKGROUND: Inflammatory macrophage infiltration plays a critical role in acute kidney disease induced by ischemia-reperfusion (IRI-AKI). Calycosin is a natural flavone with multiple bioactivities. This study aimed to investigate the therapeutic role of calycosin in IRI-AKI and its underlying mechanism. METHODS: The renoprotective and anti-inflammatory effects of calycosin were analyzed in C57BL/6 mice with IRI-AKI and lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. RNA-seq was used for mechanism investigation. The molecular target of calycosin was screened by in silico methods and validated by surface plasmon resonance (SPR). Macrophage chemotaxis was analyzed using Transwell and agarose gel spot assays. RESULTS: Calycosin treatment significantly reduced serum creatinine and urea nitrogen and attenuated tubular destruction in IRI-AKI mice. Additionally, calycosin markedly suppressed NF-κB signaling activation and the expression of inflammatory mediators IL-1ß and TNF-α in IRI-AKI kidneys and LPS-stimulated RAW 264.7 cells. Interestingly, RNA-seq revealed calycosin remarkably downregulated chemotaxis-related pathways in RAW 264.7 cells. Among the differentially expressed genes, Ccl2/MCP-1, a critical chemokine mediating macrophage inflammatory chemotaxis, was downregulated in both LPS-stimulated RAW 264.7 cells and IRI-AKI kidneys. Consistently, calycosin treatment attenuated macrophage infiltration in the IRI-AKI kidneys. Importantly, in silico target prediction, molecular docking, and SPR assay demonstrated that calycosin directly binds to macrophage migration inhibitory factor (MIF). Functionally, calycosin abrogated MIF-stimulated NF-κB signaling activation and Ccl2 expression and MIF-mediated chemotaxis in RAW 264.7 cells. CONCLUSIONS: In summary, calycosin attenuates IRI-AKI by inhibiting MIF-mediated macrophage inflammatory chemotaxis, suggesting it could be a promising therapeutic agent for the treatment of IRI-AKI.

Artificial Neutrophils Against Vascular Graft Infection.

Efficient neutrophil migration to infection sites plays a vital role in the body's defense against bacterial infections and natural immune responses. Neutrophils have a short lifespan and cannot be mass-cultured in vitro. Therefore, developing more stable artificial neutrophils (AN) in a controllable manner has become a research focus. However, existing AN lack chemotaxis, which is the ability to migrate toward high-signal-concentration positions in a dynamic blood- flow environment. Supplying AN with chemotaxis is key to designing AN that are more similar to natural neutrophils in terms of morphology and function. In this study, micrometer-sized, spherical, biocompatible AN are developed. These AN consist of zeolitic imidazolate framework-8 nanoparticles encapsulating two enzymes, coacervate droplet frameworks, and outer phospholipid bilayers carrying enzymes. The AN exhibit responsiveness to elevated hydrogen peroxide levels at inflammation sites, actively chemotaxing toward these sites along concentration gradients. They also demonstrate effective combat against Staphylococcus aureus infections. The capabilities of the AN are further validated through in vitro experiments and in vivo evaluations using vascular graft infection models. This study replicates natural neutrophils in terms of chemical composition, functionality, and physiological impact. It introduces new ideas for advancing the development of advanced artificial cells.

Synthetic silica fibers of different length, diameter and shape: synthesis and interaction with rat (NR8383) and human (THP-1) macrophages in vitro, including chemotaxis and gene expression profile.

BACKGROUND: Inhalation of biopersistent fibers like asbestos can cause strong chronic inflammatory effects, often resulting in fibrosis or even cancer. The interplay between fiber shape, fiber size and the resulting biological effects is still poorly understood due to the lack of reference materials. RESULTS: We investigated how length, diameter, aspect ratio, and shape of synthetic silica fibers influence inflammatory effects at doses up to 250 µg cm-2. Silica nanofibers were prepared with different diameter and shape. Straight (length ca. 6 to 8 µm, thickness ca. 0.25 to 0.35 µm, aspect ratio ca. 17:1 to 32:1) and curly fibers (length ca. 9 µm, thickness ca. 0.13 µm, radius of curvature ca. 0.5 µm, aspect ratio ca. 70:1) were dispersed in water with no apparent change in the fiber shape during up to 28 days. Upon immersion in aqueous saline (DPBS), the fibers released about 5 wt% silica after 7 days irrespectively of their shape. The uptake of the fibers by macrophages (human THP-1 and rat NR8383) was studied by scanning electron microscopy and confocal laser scanning microscopy. Some fibers were completely taken up whereas others were only partially internalized, leading to visual damage of the cell wall. The biological effects were assessed by determining cell toxicity, particle-induced chemotaxis, and the induction of gene expression of inflammatory mediators. CONCLUSIONS: Straight fibers were only slightly cytotoxic and caused weak cell migration, regardless of their thickness, while the curly fibers were more toxic and caused significantly stronger chemotaxis. Curly fibers also had the strongest effect on the expression of cytokines and chemokines. This may be due to the different aspect ratio or its twisted shape.

An automatic measurement method for the response of Caenorhabditis elegans to chemicals.

BACKGROUND: Caenorhabditis elegans is a widely used model animal. Chemotaxis assay is one of the experiments that study the effects of different chemicals on nematodes. It is mainly used to study the effects of different chemicals on the perception behavior of nematodes. By conducting this experiment, not only can the neurotoxicity of chemicals be reflected, but also the impact of chemicals on physiological functions regulated by the nervous system, such as nematode feeding behavior and basic motor ability. OBJECTIVE: The experiment of detecting the response of nematode to chemicals is also a common method of chemical toxicity testing based on nematode models. In the analysis of worm tendency behavior, manual operations are generally used. Manually processing a large number of worms under a microscope is very time-consuming and labor-intensive. The current quantitative methods for nematode chemotaxis experiments are not only time-consuming and labor-intensive, but also biased in experimental results due to differences in judgment standards among experimenters. The automatic and efficient quantification method for nematode chemotaxis experiments is a very important technical difficulty in the field of nematode experiments. METHODS: Here, we have designed an automatic quantification method for nematode chemotaxis experiments by incorporating image acquisition and processing techniques into the nematode experiment. RESULTS: The experimental results show that the Pearson correlation coefficient between manual and automatic counting results is 0.978. CONCLUSION: This proves the effectiveness of our method. Applying the automatic measurement method to replace manual counting by the experimenter can improve work efficiency, and reduce errors in human counting operations.

Chinese medicine Di-long (Pheretima vulgaris) and its active fraction exhibit anti-rheumatoid arthritis effects by inhibiting CXCL10/CXCR3 chemotaxis in synovium.

ETHNOPHARMACOLOGICAL RELEVANCE: Di-Long (Pheretima vulgaris) is a classic animal sourced traditional Chinese medicine. It has been used for the treatment of joint inflammation and arthralgia for over two thousand years due to its effects of Tong-Luo-Zhi-Tong (dredging collaterals and alleviating pain). Our previous study showed that Chinese medicine Di-Long has significant anti-rheumatoid arthritis (RA) effects. AIM OF THE STUDY: Considering Di-Long as a potential source of active compounds with specific anti-RA therapeutic effects, this research was to obtain the anti-RA target-specific active fraction from Di-Long extracts (DL), and to further explore the chemical basis and verify the anti-RA mechanism of this active fraction. MATERIALS AND METHODS: Transcriptomic was applied to obtain the main anti-RA targets of DL on human RA fibroblast-like synoviocytes (FLS) and validated by qPCR. The target-corresponding active fraction was isolated from DL by ethanol precipitation and gel chromatography, and analyzed by nanoliter chromatography-mass spectrometry. Anti-RA effects of this active fraction was investigated by collagen-induced arthritis (CIA) in mice, and anti-RA mechanisms were verified in cocultured model of rat FLS and peripheral blood lymphocytes. RESULTS: We confirmed that CXCL10/CXCR3 was the main anti-RA target of DL. The active fraction - A (2182 - 890 Da) was isolated from DL based on its CXCL10 inhibiting effects in RA-FLS. Fraction A contains 195 peptides (192 were newly discovered), 26 of which might be bioactive and were considered to be the chemical basis of its anti-RA effects. Fraction A significantly ameliorated the joint destruction and overall inflammation in CIA mice, and downregulated CXCR3 expression in mice joint. Fraction A inhibited the chemotaxis of Th-cells in rat peripheral blood lymphocytes towards the TNF-α-induced rat FLS through CXCL10/CXCR3 pathway. CONCLUSIONS: Our work indicated that active fraction from DL containing small peptides exhibits promising therapeutic effects for RA through inhibiting CXCL10/CXCR3 chemotaxis.

Bortezomib restrains M2 polarization and reduces CXCL16-associated CXCR6+CD4 T cell chemotaxis in bleomycin-induced pulmonary fibrosis.

BACKGROUND: The development of pulmonary fibrosis involves a cascade of events, in which inflammation mediated by immune cells plays a pivotal role. Chemotherapeutic drugs have been shown to have dual effects on fibrosis, with bleomycin exacerbating pulmonary fibrosis and bortezomib alleviating tissue fibrotic processes. Understanding the intricate interplay between chemotherapeutic drugs, immune responses, and pulmonary fibrosis is likely to serve as the foundation for crafting tailored therapeutic strategies. METHODS: A model of bleomycin-induced pulmonary fibrosis was established, followed by treatment with bortezomib. Tissue samples were collected for analysis of immune cell subsets and functional assessment by flow cytometry and in vitro cell experiments. Additionally, multi-omics analysis was conducted to further elucidate the expression of chemokines and chemokine receptors, as well as the characteristics of cell populations. RESULTS: Here, we observed that the expression of CXCL16 and CXCR6 was elevated in the lung tissue of a pulmonary fibrosis model. In the context of pulmonary fibrosis or TGF-ß1 stimulation in vitro, macrophages exhibited an M2-polarized phenotype and secreted more CXCL16 than those of the control group. Moreover, flow cytometry revealed increased expression levels of CD69 and CXCR6 in pulmonary CD4 T cells during fibrosis progression. The administration of bortezomib alleviated bleomycin-induced pulmonary fibrosis, accompanied by reduced ratio of M2-polarized macrophages and decreased accumulation of CD4 T cells expressing CXCR6. CONCLUSIONS: Our findings provide insights into the key immune players involved in bleomycin-induced pulmonary fibrosis and offer preclinical evidence supporting the repurposing strategy and combination approaches to reduce lung fibrosis.

Novel artesunate and isatin hybrid CT3-1 suppresses collagen-induced arthritis through abrogating dendritic cell chemotaxis-induced by CCR5.

BACKGROUND: Chemotaxis and trafficking of dendritic cells (DCs) induced by cytokine receptors are crucial steps in rheumatoid arthritis (RA) pathogenesis. C-C chemokine receptor type 5 (CCR5) plays a key role in DC movement and has been implicated in multitudinous inflammatory and immunology diseases. Thus, targeting CCR5 to suppress DC chemotaxis is considered as a potential strategy for the management of RA. METHODS: Herein, we first synthesized a new hybrid named CT3-1 which based on artesunate and isatin. Besides, we studied the regulating effectiveness of CT3-1 on bone marrow-derived DCs (BMDCs) and on collagen-induced arthritis (CIA) through RNA-seq analysis, cell function experiments in vitro and mice model in vivo. RESULTS: The results shown that CT3-1 mainly reduced CCR5 expression of immature BMDCs and importantly inhibited immature BMDC migration induced by CCR5 in vitro, with no or minor influence on other functions of DCs, such as phagocytosis and maturation. In the mouse model, CT3-1 relieved arthritis severity and inhibited CIA development. Furthermore, CT3-1 intervention decreased the expression of CCR5 in DCs and reduced the proportion of DCs in the peripheral blood of CIA mice. CONCLUSIONS: Our findings suggest that CCR5-induced chemotaxis and trafficking of immature DCs are important in RA. Targeting CCR5 and inhibiting immature DC chemotaxis may provide a novel choice for the treatment of RA and other similar autoimmune diseases. Moreover, we synthesized a new hybrid compound CT3-1 that could inhibit immature DC trafficking and effectively relieve RA by directly reducing the CCR5 expression of immature DCs.

Open-Top Patterned Hydrogel-Laden 3D Glioma Cell Cultures for Creation of Dynamic Chemotactic Gradients to Direct Cell Migration.

The laminar flow profiles in microfluidic systems coupled to rapid diffusion at flow streamlines have been widely utilized to create well-controlled chemical gradients in cell cultures for spatially directing cell migration. However, within hydrogel-based closed microfluidic systems of limited depth (≤0.1 mm), the biomechanical cues for the cell culture are dominated by cell interactions with channel surfaces rather than with the hydrogel microenvironment. Also, leaching of poly(dimethylsiloxane) (PDMS) constituents in closed systems and the adsorption of small molecules to PDMS alter chemotactic profiles. To address these limitations, we present the patterning and integration of a PDMS-free open fluidic system, wherein the cell-laden hydrogel directly adjoins longitudinal channels that are designed to create chemotactic gradients across the 3D culture width, while maintaining uniformity across its ∼1 mm depth to enhance cell-biomaterial interactions. This hydrogel-based open fluidic system is assessed for its ability to direct migration of U87 glioma cells using a hybrid hydrogel that includes hyaluronic acid (HA) to mimic the brain tumor microenvironment and gelatin methacrylate (GelMA) to offer the adhesion motifs for promoting cell migration. Chemotactic gradients to induce cell migration across the hydrogel width are assessed using the chemokine CXCL12, and its inhibition by AMD3100 is validated. This open-top hydrogel-based fluidic system to deliver chemoattractant cues over square-centimeter-scale areas and millimeter-scale depths can potentially serve as a robust screening platform to assess emerging glioma models and chemotherapeutic agents to eradicate them.

Targeting myeloid chemotaxis to reverse prostate cancer therapy resistance.

Inflammation is a hallmark of cancer1. In patients with cancer, peripheral blood myeloid expansion, indicated by a high neutrophil-to-lymphocyte ratio, associates with shorter survival and treatment resistance across malignancies and therapeutic modalities2-5. Whether myeloid inflammation drives progression of prostate cancer in humans remain unclear. Here we show that inhibition of myeloid chemotaxis can reduce tumour-elicited myeloid inflammation and reverse therapy resistance in a subset of patients with metastatic castration-resistant prostate cancer (CRPC). We show that a higher blood neutrophil-to-lymphocyte ratio reflects tumour myeloid infiltration and tumour expression of senescence-associated mRNA species, including those that encode myeloid-chemoattracting CXCR2 ligands. To determine whether myeloid cells fuel resistance to androgen receptor signalling inhibitors, and whether inhibiting CXCR2 to block myeloid chemotaxis reverses this, we conducted an investigator-initiated, proof-of-concept clinical trial of a CXCR2 inhibitor (AZD5069) plus enzalutamide in patients with metastatic CRPC that is resistant to androgen receptor signalling inhibitors. This combination was well tolerated without dose-limiting toxicity and it decreased circulating neutrophil levels, reduced intratumour CD11b+HLA-DRloCD15+CD14- myeloid cell infiltration and imparted durable clinical benefit with biochemical and radiological responses in a subset of patients with metastatic CRPC. This study provides clinical evidence that senescence-associated myeloid inflammation can fuel metastatic CRPC progression and resistance to androgen receptor blockade. Targeting myeloid chemotaxis merits broader evaluation in other cancers.

Pamoic acid is an inhibitor of HMGB1·CXCL12 elicited chemotaxis and reduces inflammation in murine models of Pseudomonas aeruginosa pneumonia.

BACKGROUND: High-mobility group box 1 protein (HMGB1) is an ubiquitous nuclear protein that once released in the extracellular space acts as a Damage Associated Molecular Pattern and promotes inflammation. HMGB1 is significantly elevated during Pseudomonas aeruginosa infections and has a clinical relevance in respiratory diseases such as Cystic Fibrosis (CF). Salicylates are HMGB1 inhibitors. To address pharmacological inhibition of HMGB1 with small molecules, we explored the therapeutic potential of pamoic acid (PAM), a salicylate with limited ability to cross epithelial barriers. METHODS: PAM binding to HMGB1 and CXCL12 was tested by Nuclear Magnetic Resonance Spectroscopy using chemical shift perturbation methods, and inhibition of HMGB1·CXCL12-dependent chemotaxis was investigated by cell migration experiments. Aerosol delivery of PAM, with single or repeated administrations, was tested in murine models of acute and chronic P. aeruginosa pulmonary infection in C57Bl/6NCrlBR mice. PAM efficacy was evaluated by read-outs including weight loss, bacterial load and inflammatory response in lung and bronco-alveolar lavage fluid. RESULTS: Our data and three-dimensional models show that PAM is a direct ligand of both HMGB1 and CXCL12. We also showed that PAM is able to interfere with heterocomplex formation and the related chemotaxis in vitro. Importantly, PAM treatment by aerosol was effective in reducing acute and chronic airway murine inflammation and damage induced by P. aeruginosa. The results indicated that PAM reduces leukocyte recruitment in the airways, in particular neutrophils, suggesting an impaired in vivo chemotaxis. This was associated with decreased myeloperoxidase and neutrophil elastase levels. Modestly increased bacterial burdens were recorded with single administration of PAM in acute infection; however, repeated administration in chronic infection did not affect bacterial burdens, indicating that the interference of PAM with the immune system has a limited risk of pulmonary exacerbation. CONCLUSIONS: This work established the efficacy of treating inflammation in chronic respiratory diseases, including bacterial infections, by topical delivery in the lung of PAM, an inhibitor of HMGB1.

Artemisinin inhibits neutrophil and macrophage chemotaxis, cytokine production and NET release.

Immune cell chemotaxis to the sites of pathogen invasion is critical for fighting infection, but in life-threatening conditions such as sepsis and Covid-19, excess activation of the innate immune system is thought to cause a damaging invasion of immune cells into tissues and a consequent excessive release of cytokines, chemokines and neutrophil extracellular traps (NETs). In these circumstances, tempering excessive activation of the innate immune system may, paradoxically, promote recovery. Here we identify the antimalarial compound artemisinin as a potent and selective inhibitor of neutrophil and macrophage chemotaxis induced by a range of chemotactic agents. Artemisinin released calcium from intracellular stores in a similar way to thapsigargin, a known inhibitor of the Sarco/Endoplasmic Reticulum Calcium ATPase pump (SERCA), but unlike thapsigargin, artemisinin blocks only the SERCA3 isoform. Inhibition of SERCA3 by artemisinin was irreversible and was inhibited by iron chelation, suggesting iron-catalysed alkylation of a specific cysteine residue in SERCA3 as the mechanism by which artemisinin inhibits neutrophil motility. In murine infection models, artemisinin potently suppressed neutrophil invasion into both peritoneum and lung in vivo and inhibited the release of cytokines/chemokines and NETs. This work suggests that artemisinin may have value as a therapy in conditions such as sepsis and Covid-19 in which over-activation of the innate immune system causes tissue injury that can lead to death.

Mimic Nature Using Chemotaxis of Ionic Liquid Microdroplets for Drug Delivery Purposes.

Due to the growing prevalence of incurable diseases, such as cancer, worldwide, nowadays, the development of smart drug delivery systems is an inevitable necessity. Chemotaxis-driven movement of ionic liquid microdroplets containing therapeutic compounds is a well-known example of a smart drug delivery system. This review aims to classify, summarize, and compare ionic liquid-based chemotaxis systems in an easily understandable article. Chemotaxis is the basis of the movement of cells and microorganisms in biological environments, which is the cause of many vital biochemical and biological processes. This review attempts to summarize the available literature on single-component biomimetic and self-propelling microdroplet systems based on ionic liquids, which exhibit chemotaxis and spontaneously move in a determined direction by an external gradient, particularly a chemical change. It also aims to review artificial ionic liquid-based chemotaxis systems that can be used as drug carriers for medical purposes. The various ionic liquids used for this purpose are discussed, and different forms of chemical gradients and mechanisms that cause movement in microfluidic channels will be reviewed.

Influence of glucose on swarming and quorum sensing of Dickeya solani.

Dickeya solani is a pathogen most frequently responsible for infecting potato plants in Europe. As in the case of most plant pathogens, its ability to colonize and invade the host depends on chemotaxis and motility. The coordinated movement of Dickeya over solid surfaces is governed by a quorum sensing mechanism. In D. solani motility is regulated by ExpI-ExpR proteins, homologous to luxI-luxR system from Vibrio fisheri, in which N-acyl-homoserine lactones (AHLs) serve as signaling molecules. Moreover, in many Gram-negative bacteria motility is coupled with central metabolism via carbon catabolite repression. This enables them to reach more nutrient-efficient niches. The aim of this study was to analyze the swarming motility of D. solani depending on the volume of the medium in the cultivation plate and glucose content. We show that the ability of this bacterium to move is strictly dependent on both these factors. Moreover, we analyze the production of AHLs and show that the quorum sensing mechanism in D. solani is also influenced by the availability of glucose in the medium and that the distribution of these signaling molecules are different depending on the volume of the medium in the plate.

Isoflurane attenuates sepsis-associated lung injury.

Acute lung injury is one of major complications associated with sepsis, responsible for morbidity and mortality. Patients who suffer from acute lung injury often require respiratory support under sedations, and it would be important to know the role of sedatives in lung injury. We examined volatile anesthetic isoflurane, which is commonly used in surgical setting, but also used as an alternative sedative in intensive care settings in European countries and Canada. We found that isoflurane exposure attenuated neutrophil recruitment to the lungs in mice suffering from experimental polymicrobial abdominal sepsis. We found that isoflurane attenuated one of major neutrophil chemoattractants LTB4 mediated response via its receptor BLT1 in neutrophils. Furthermore, we have shown that isoflurane directly bound to BLT1 by a competition assay using newly developed labeled BLT1 antagonist, suggesting that isoflurane would be a BLT1 antagonist.

Transcriptional regulation of chemokine network by biologic monotherapy in ileum of patients with Crohn's disease.

BACKGROUND: Crohn's disease (CD) exacerbation is marked by an intense cellular trafficking. We set out to determine the specific impact of biologic therapies on regulating chemokine network gene expression in healthy, mildly and severely inflamed tissue of CD patients. METHODS: Twenty CD patients on biologics (adalimumab, ustekinumab, vedolizumab) or untreated undergoing colonoscopy due to clinical symptoms of flare. Healthy, mildly and severely inflamed ileum biopsies from each patient were collected. Chemokines and receptors gene expression was analyzed and a STRING analysis for functional enrichment was performed. RESULTS: The chemokine network exhibited wide transcriptional differences among tissues in active untreated patients, whereas all biologic treatments reduced these differences and homogenized their transcriptional activity. In mildly inflamed tissue, all treatments showed gene upregulation while ustekinumab additionally maintained the downregulation of genes such as CCL2, CCL3, CCL17 or CCL23, involved in T cell chemotaxis, inflammatory monocyte and NK trafficking. In severely inflamed tissue, all treatments shared a downregulatory effect on chemokines controlling T cell response (i.e. CXCL16, CXCR3). Adalimumab and vedolizumab significantly reduced the expression of genes promoting antigen presentation by DCs and the initiation of leukocyte extravasation (i.e. CXCL12, CCL25, CCR7). Ustekinumab significantly reduced genes positively regulating Th1 cytokine production and IL-8 mediated signaling (i.e. IL1B, XCL1, CXCR1, CXCR2). CONCLUSION: Biologic therapies differentially target the chemokine network gene expression profile in the ileal tissue of active CD patients. These results may contribute to better understanding cell homing and to defining future personalized therapeutic strategies for CD patients.

P17 induces chemotaxis and differentiation of monocytes via MRGPRX2-mediated mast cell-line activation.

BACKGROUND: P17, a peptide isolated from Tetramorium bicarinatum ant venom, is known to induce an alternative phenotype of human monocyte-derived macrophages via activation of an unknown G protein-coupled receptor (GPCR). OBJECTIVE: We sought to investigate the mechanism of action and the immunomodulatory effects of P17 mediated through MRGPRX2 (Mas-related G protein-coupled receptor X2). METHODS: To identify the GPCR for P17, we screened 314 GPCRs. Upon identification of MRGPRX2, a battery of in silico, in vitro, ex vivo, and in vivo assays along with the receptor mutation studies were performed. In particular, to investigate the immunomodulatory actions, we used ß-hexosaminidase release assay, cytokine releases, quantification of mRNA expression, cell migration and differentiation assays, immunohistochemical labeling, hematoxylin and eosin, and immunofluorescence staining. RESULTS: P17 activated MRGPRX2 in a dose-dependent manner in ß-arrestin recruitment assay. In LAD2 cells, P17 induced calcium and ß-hexosaminidase release. Quercetin- and short hairpin RNA-mediated knockdown of MRGPRX2 reduced P17-evoked ß-hexosaminidase release. In silico and in vitro mutagenesis studies showed that residue Lys8 of P17 formed a cation-π interaction with the Phe172 of MRGPRX2 and [Ala8]P17 lost its activity partially. P17 activated LAD2 cells to recruit THP-1 and human monocytes in Transwell migration assay, whereas MRGPRX2-impaired LAD2 cells cannot. In addition, P17-treated LAD2 cells stimulated differentiation of THP-1 and human monocytes, as indicated by the enhanced expression of macrophage markers cluster of differentiation 11b and TNF-α by quantitative RT-PCR. Immunohistochemical and immunofluorescent staining suggested monocyte recruitment in mice ears injected with P17. CONCLUSIONS: Our data provide novel structural information regarding the interaction of P17 with MRGPRX2 and intracellular pathways for its immunomodulatory action.