Heparan sulfate-dependent phase separation of CCL5 and its chemotactic activity.

Secreted chemokines form concentration gradients in target tissues to control migratory directions and patterns of immune cells in response to inflammatory stimulation; however, how the gradients are formed is much debated. Heparan sulfate (HS) binds to chemokines and modulates their activities. In this study, we investigated the roles of HS in the gradient formation and chemoattractant activity of CCL5 that is known to bind to HS. CCL5 and heparin underwent liquid-liquid phase separation and formed gradient, which was confirmed using CCL5 immobilized on heparin-beads. The biological implication of HS in CCL5 gradient formation was established in CHO-K1 (wild-type) and CHO-677 (lacking HS) cells by Transwell assay. The effect of HS on CCL5 chemoattractant activity was further proved by Transwell assay of human peripheral blood cells. Finally, peritoneal injection of the chemokines into mice showed reduced recruitment of inflammatory cells either by mutant CCL5 (lacking heparin-binding sequence) or by addition of heparin to wild-type CCL5. Our experimental data propose that co-phase separation of CCL5 with HS establishes a specific chemokine concentration gradient to trigger directional cell migration. The results warrant further investigation on other heparin-binding chemokines and allows for a more elaborate insight into disease process and new treatment strategies.

Learning optimal integration of spatial and temporal information in noisy chemotaxis.

We investigate the boundary between chemotaxis driven by spatial estimation of gradients and chemotaxis driven by temporal estimation. While it is well known that spatial chemotaxis becomes disadvantageous for small organisms at high noise levels, it is unclear whether there is a discontinuous switch of optimal strategies or a continuous transition exists. Here, we employ deep reinforcement learning to study the possible integration of spatial and temporal information in an a priori unconstrained manner. We parameterize such a combined chemotactic policy by a recurrent neural network and evaluate it using a minimal theoretical model of a chemotactic cell. By comparing with constrained variants of the policy, we show that it converges to purely temporal and spatial strategies at small and large cell sizes, respectively. We find that the transition between the regimes is continuous, with the combined strategy outperforming in the transition region both the constrained variants as well as models that explicitly integrate spatial and temporal information. Finally, by utilizing the attribution method of integrated gradients, we show that the policy relies on a nontrivial combination of spatially and temporally derived gradient information in a ratio that varies dynamically during the chemotactic trajectories.

Quantitative Monitoring of GPCR-Mediated Spatiotemporal IP3 Dynamics Using Confocal Fluorescence Microscopy.

Activation of G protein-coupled receptors upon chemoattractant stimulation induces activation of multiple signaling pathways. To fully understand how these signaling pathway coordinates to achieve directional migration of neutrophils, it is essential to determine the dynamics of the spatiotemporal activation profile of signaling components at the level of single living cells. Here, we describe a detailed methodology for monitoring and quantitatively analyzing the spatiotemporal dynamics of 1,4,5-inositol trisphosphate (IP3) in neutrophil-like HL60 cells in response to various chemoattractant fields by applying Förster resonance energy transfer (FRET) fluorescence microscopy.

Photosystems and photoreceptors in cyanobacterial phototaxis and photophobotaxis.

Cyanobacteria move by gliding motility on surfaces toward the light or away from it. It is as yet unclear how the light direction is sensed on the molecular level. Diverse photoreceptor knockout mutants have a stronger response toward the light than the wild type. Either the light direction is sensed by multiple photoreceptors or by photosystems. In a study on photophobotaxis of the filamentous cyanobacterium Phormidium lacuna, broad spectral sensitivity, inhibition by 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), and a highly sensitive response speaks for photosystems as light direction sensors. Here, it is discussed whether the photosystem theory could hold for phototaxis of other cyanobacteria.

Distinctive chemotactic responses of three marine herbivore protists to DMSP and related compounds.

Marine planktonic predator-prey interactions occur in microscale seascapes, where diffusing chemicals may act either as chemotactic cues that enhance or arrest predation, or as elemental resources that are complementary to prey ingestion. The phytoplankton osmolyte dimethylsulfoniopropionate (DMSP) and its degradation products dimethylsulfide (DMS) and acrylate are pervasive compounds with high chemotactic potential, but there is a longstanding controversy over whether they act as grazing enhancers or deterrents. Here, we investigated the chemotactic responses of three herbivorous dinoflagellates to point-sourced, microscale gradients of dissolved DMSP, DMS, and acrylate. We found no evidence for acrylate being a chemotactic repellent and observed a weak attractor role of DMS. DMSP behaved as a strong chemoattractor whose potential for grazing facilitation through effects on swimming patterns and aggregation depends on the grazer's feeding mode and ability to incorporate DMSP. Our study reveals that predation models will fail to predict grazing impacts unless they incorporate chemotaxis-driven searching and finding of prey.

Gene expression in heart, kidney, and liver identifies possible mechanisms underpinning fetal resistance and susceptibility to in utero PRRSV infection.

Porcine reproductive and respiratory syndrome (PRRS) is one of the costliest diseases to pork producers worldwide. We tested samples from the pregnant gilt model (PGM) to better understand the fetal response to in-utero PRRS virus (PRRSV) infection. Our goal was to identify critical tissues and genes associated with fetal resilience or susceptibility. Pregnant gilts (N=22) were infected with PRRSV on day 86 of gestation. At 21 days post maternal infection, the gilts and fetuses were euthanized, and fetal tissues collected. Fetuses were characterized for PRRS viral load in fetal serum and thymus, and preservation status (viable or meconium stained: VIA or MEC). Fetuses (N=10 per group) were compared: uninfected (UNIF; <1 log/µL PRRSV RNA), resilient (HV_VIA, >5 log virus/µL but viable), and susceptible (HV_MEC, >5 log virus/µL with MEC). Gene expression in fetal heart, kidney, and liver was investigated using NanoString transcriptomics. Gene categories investigated were hypothesized to be involved in fetal response to PRRSV infection: renin- angiotensin-aldosterone, inflammatory, transporter and metabolic systems. Following PRRSV infection, CCL5 increased expression in heart and kidney, and ACE2 decreased expression in kidney, each associated with fetal PRRS susceptibility. Liver revealed the most significant differential gene expression: CXCL10 decreased and IL10 increased indicative of immune suppression. Increased liver gene expression indicated potential associations with fetal PRRS susceptibility on several systems including blood pressure regulation (AGTR1), energy metabolism (SLC16A1 and SLC16A7), tissue specific responses (KL) and growth modulation (TGFB1). Overall, analyses of non-lymphoid tissues provided clues to mechanisms of fetal compromise following maternal PRRSV infection.

A Cautionary Perspective on Hydrogel-Induced Concentration Gradient Generation for Studying Chemotaxis.

The achievement of consistent and static chemical gradients is critically important in the study of diffusion and chemotaxis at the micro- and nanoscales. In this context, a number of groups have reported on hydrogel-based systems for generating concentration gradients. Here, we analyze the behavior of agarose and gelatin-based hydrogels in hybridization chambers of different heights. Our focus is on the issues that are caused by the presence of robust bulk fluid flows in such systems due to the solutes present in the hydrogel and/or the surrounding fluid. We describe the key insights derived from these experiments, offering practical guidelines for establishing gradients using hydrogel-based systems and make the community aware of different variables that can make the experiments nonreproducible and prone to misinterpretations.

Surface-programmed microbiome assembly in phycosphere to microplastics contamination.

Recalcitrance in microplastics accounts for ubiquitous white pollution. Of special interest are the capabilities of microorganisms to accelerate their degradation sustainably. Compared to the well-studied pure cultures in degrading natural polymers, the algal-bacterial symbiotic system is considered as a promising candidate for microplastics removal, cascading bottom-up impacts on ecosystem-scale processes. This study selected and enriched the algae-associated microbial communities hosted by the indigenous isolation Desmodesmus sp. in wastewater treatment plants with micro-polyvinyl chloride, polyethylene terephthalate, polyethylene, and polystyrene contamination. Results elaborated that multiple settled and specific affiliates were recruited by the uniform algae protagonist from the biosphere under manifold microplastic stress. Alteration of distinct chemical functionalities and deformation of polymers provide direct evidence of degradation in phycosphere under illumination. Microplastic-induced phycosphere-derived DOM created spatial gradients of aromatic protein, fulvic and humic acid-like and tryptophan components to expanded niche-width. Surface thermodynamic analysis was conducted to simulate the reciprocal and reversible interaction on algal-bacterial and phycosphere-microplastic interface, revealing the enhancement of transition to stable and irreversible aggregation for functional microbiota colonization and microplastics capture. Furthermore, pangenomic analysis disclosed the genes related to the chemotaxis and the proposed microplastics biodegradation pathway in enriched algal-bacterial microbiome, orchestrating the evidence for common synthetic polymer particles and ultimately to confirm the effectiveness and potential. The present study emphasizes the necessity for future endeavors aimed at fully leveraging the potential of algal-bacterial mutualistic systems within sustainable bioremediation strategies targeting the eradication of microplastic waste.

Signal integration and adaptive sensory diversity tuning in Escherichia coli chemotaxis.

In uncertain environments, phenotypic diversity can be advantageous for survival. However, as the environmental uncertainty decreases, the relative advantage of having diverse phenotypes decreases. Here, we show how populations of E. coli integrate multiple chemical signals to adjust sensory diversity in response to changes in the prevalence of each ligand in the environment. Measuring kinase activity in single cells, we quantified the sensitivity distribution to various chemoattractants in different mixtures of background stimuli. We found that when ligands bind uncompetitively, the population tunes sensory diversity to each signal independently, decreasing diversity when the signal's ambient concentration increases. However, among competitive ligands, the population can only decrease sensory diversity one ligand at a time. Mathematical modeling suggests that sensory diversity tuning benefits E. coli populations by modulating how many cells are committed to tracking each signal proportionally as their prevalence changes.

Self-extinguishing relay waves enable homeostatic control of human neutrophil swarming.

Neutrophils collectively migrate to sites of injury and infection. How these swarms are coordinated to ensure the proper level of recruitment is unknown. Using an ex vivo model of infection, we show that human neutrophil swarming is organized by multiple pulsatile chemoattractant waves. These waves propagate through active relay in which stimulated neutrophils trigger their neighbors to release additional swarming cues. Unlike canonical active relays, we find these waves to be self-terminating, limiting the spatial range of cell recruitment. We identify an NADPH-oxidase-based negative feedback loop that is needed for this self-terminating behavior. We observe near-constant levels of neutrophil recruitment over a wide range of starting conditions, revealing surprising robustness in the swarming process. This homeostatic control is achieved by larger and more numerous swarming waves at lower cell densities. We link defective wave termination to a broken recruitment homeostat in the context of human chronic granulomatous disease.

LAIR1 promotes hepatocellular carcinoma cell metastasis and induces M2-macrophage infiltration through activating AKT-IKKß-p65 axis.

LAIR1, a receptor found on immune cells, is capable of binding to collagen and is involved in immune-related diseases. However, the precise contribution of LAIR1 expressed on hepatocellular carcinoma (HCC) cells to tumor microenvironment is still unclear. In our study, bioinformatics analysis and immunofluorescence were employed to study the correlation between LAIR1 levels and clinical indicators. Transwell and scratch tests were used to evaluate how LAIR1 affected the migration and invasion of HCC cells. The chemotactic capacity and alternative activation of macrophages were investigated using RT-qPCR, transwell, and immunofluorescence. To investigate the molecular mechanisms, transcriptome sequencing analysis, Western blot, nucleus/cytoplasm fractionation, ELISA, and cytokine microarray were employed. We revealed a significant correlation between the presence of LAIR1 and an unfavorable outcome in HCC. We indicated that LAIR1 promoted migration and invasion of HCC cells through the AKT-IKKß-p65 axis. Additionally, the alternative activation and infiltration of tumor-associated macrophages induced by LAIR1 were reliant on the upregulation of IL6 and CCL5 within this axis, respectively. In conclusion, blocking LAIR1 was found to be an effective approach in combating the cancerous advancement of HCC.

Alpha-synuclein shapes monocyte and macrophage cell biology and functions by bridging alterations of autophagy and inflammatory pathways.

Introduction: Abnormal spreading of alpha-synuclein (αS), a hallmark of Parkinson's disease, is known to promote peripheral inflammation, which occurs in part via functional alterations in monocytes/macrophages. However, underlying intracellular mechanisms remain unclear. Methods: Herein we investigate the subcellular, molecular, and functional effects of excess αS in human THP-1 monocytic cell line, THP-1-derived macrophages, and at least preliminarily, in primary monocyte-derived macrophages (MDMs). In cells cultured w/wo recombinant αS (1 µM) for 4 h and 24 h, by Confocal microscopy, Western Blot, RT-qPCR, Elisa, and Flow Cytometry we assessed: i) αS internalization; ii) cytokine/chemokine expression/secretion, and C-C motif chemokine receptor 2 (CCR2) levels; iii) autophagy (LC3II/I, LAMP1/LysoTracker, p62, pS6/total S6); and iv) lipid droplets (LDs) accumulation, and cholesterol pathway gene expression. Transwell migration assay was employed to measure THP-1 cell migration/chemotaxis, while FITC-IgG-bead assay was used to analyze phagocytic capacity, and the fate of phagocytosed cargo in THP-1-derived macrophages. Results: Extracellular αS was internalized by THP-1 cells, THP-1-derived macrophages, and MDMs. In THP1 cells, αS induced a general pro-inflammatory profile and conditioned media from αS-exposed THP-1 cells potently attracted unstimulated cells. However, CCL2 secretion peaked at 4 h αS, consistent with early internalization of its receptor CCR2, while this was blunted at 24 h αS exposure, when CCR2 recycled back to the plasma membrane. Again, 4 h αS-exposed THP-1 cells showed increased spontaneous migration, while 24 h αS-exposed cells showed reduced chemotaxis. This occurred in the absence of cell toxicity and was associated with upregulation of autophagy/lysosomal markers, suggesting a pro-survival/tolerance mechanism against stress-related inflammation. Instead, in THP-1-derived macrophages, αS time-dependently potentiated the intracellular accumulation, and release of pro-inflammatory mediators. This was accompanied by mild toxicity, reduced autophagy-lysosomal markers, defective LDs formation, as well as impaired phagocytosis, and the appearance of stagnant lysosomes engulfed with phagocytosed cargo, suggesting a status of macrophage exhaustion reminiscent of hypophagia. Discussion: In summary, despite an apparently similar pro-inflammatory phenotype, monocytes and macrophages respond differently to intracellular αS accumulation in terms of cell survival, metabolism, and functions. Our results suggest that in periphery, αS exerts cell- and context-specific biological effects bridging alterations of autophagy, lipid dynamics, and inflammatory pathways.

Genome-wide screen of genetic determinants that govern Escherichia coli growth and persistence in lake water.

Although enteric bacteria normally reside within the animal intestine, the ability to persist extraintestinally is an essential part of their overall lifestyle, and it might contribute to transmission between hosts. Despite this potential importance, few genetic determinants of extraintestinal growth and survival have been identified, even for the best-studied model, Escherichia coli. In this work, we thus used a genome-wide library of barcoded transposon insertions to systematically identify functional clusters of genes that are crucial for E. coli fitness in lake water. Our results revealed that inactivation of pathways involved in maintaining outer membrane integrity, nucleotide biosynthesis, and chemotaxis negatively affected E. coli growth or survival in this extraintestinal environment. In contrast, inactivation of another group of genes apparently benefited E. coli growth or persistence in filtered lake water, resulting in higher abundance of these mutants. This group included rpoS, which encodes the general stress response sigma factor, as well as genes encoding several other global transcriptional regulators and RNA chaperones, along with several poorly annotated genes. Based on this co-enrichment, we identified these gene products as novel positive regulators of RpoS activity. We further observed that, despite their enhanced growth, E. coli mutants with inactive RpoS had reduced viability in lake water, and they were not enriched in the presence of the autochthonous microbiota. This highlights the duality of the general stress response pathway for E. coli growth outside the host.

Potassium-mediated bacterial chemotactic response.

Bacteria in biofilms secrete potassium ions to attract free swimming cells. However, the basis of chemotaxis to potassium remains poorly understood. Here, using a microfluidic device, we found that Escherichia coli can rapidly accumulate in regions of high potassium concentration on the order of millimoles. Using a bead assay, we measured the dynamic response of individual flagellar motors to stepwise changes in potassium concentration, finding that the response resulted from the chemotaxis signaling pathway. To characterize the chemotactic response to potassium, we measured the dose-response curve and adaptation kinetics via an Förster resonance energy transfer (FRET) assay, finding that the chemotaxis pathway exhibited a sensitive response and fast adaptation to potassium. We further found that the two major chemoreceptors Tar and Tsr respond differently to potassium. Tar receptors exhibit a biphasic response, whereas Tsr receptors respond to potassium as an attractant. These different responses were consistent with the responses of the two receptors to intracellular pH changes. The sensitive response and fast adaptation allow bacteria to sense and localize small changes in potassium concentration. The differential responses of Tar and Tsr receptors to potassium suggest that cells at different growth stages respond differently to potassium and may have different requirements for potassium.

Mechanisms of Tripterygium wilfordii Hook F on treating rheumatoid arthritis explored by network pharmacology analysis and molecular docking.

Background: Rheumatoid arthritis (RA) is a chronic inflammatory and disabling disease that imposes significant economic and social costs. Tripterygium wilfordii Hook F (TwHF) has a long history of use in traditional Chinese medicine for treating joint disorders, and it has been shown to be cost-effective in treating RA, but its exact mechanism is unknown. Objective: The goal of the network pharmacology analysis and molecular docking was to investigate the potential active compounds and associated anti-RA mechanisms of TwHF. Methods: TCMSP and UniProt databases were searched for active compounds and related targets of TwHF. PharmGKB, DrugBank, OMIM, TTD, and the Human Gene Databases were used to identify RA-related targets. The intersected RA and TwHF targets were entered into the STRING database to create a protein-protein interaction network. R software was used for gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses. Molecular docking technology was used to analyze the optimal effective components from TwHF for docking with the selected target gene. Results: Following screening and duplicate removal, a total of 51 active compounds and 96 potential targets were chosen. The PPI network revealed that the target proteins are CXCL8, CXCL6, STAT3, STAT1, JUN, PPARG, TP53, IL14, MMP9, VEGFA, RELA, CASP3, PTGS2, IFNG, AKT1, FOS, ICAM1, and MAPK14. The results of the GO enrichment analysis focused primarily on the response to lipopolysaccharide, the response to molecules of bacterial origin, and the response to drugs. The KEGG results indicated that the mechanisms were closely related to lipid and atherosclerosis, chemical carcinogenesis-receptor activation, Kaposi sarcoma-associated, herpesvirus infection, hepatitis B, fluid shear stress and atherosclerosis, IL-17 signaling pathways, Th17-cell differentiation, and so on, all of which are involved in angiogenesis, immune cell chemotaxis, and inflammatory responses. Molecular docking results suggested that triptolide was the appropriate PTGS1, PTGS2, and TNF inhibitors. Conclusion: Our findings provide an essential role and basis for further immune inflammatory studies into the molecular mechanisms of TwHF and PTGS1, PTGS2, and TNF inhibitor development in RA.

A multiscale spatial modeling framework for the germinal center response.

The germinal center response or reaction (GCR) is a hallmark event of adaptive humoral immunity. Unfolding in the B cell follicles of the secondary lymphoid organs, a GC culminates in the production of high-affinity antibody-secreting plasma cells along with memory B cells. By interacting with follicular dendritic cells (FDC) and T follicular helper (Tfh) cells, GC B cells exhibit complex spatiotemporal dynamics. Driving the B cell dynamics are the intracellular signal transduction and gene regulatory network that responds to cell surface signaling molecules, cytokines, and chemokines. As our knowledge of the GC continues to expand in depth and in scope, mathematical modeling has become an important tool to help disentangle the intricacy of the GCR and inform novel mechanistic and clinical insights. While the GC has been modeled at different granularities, a multiscale spatial simulation framework - integrating molecular, cellular, and tissue-level responses - is still rare. Here, we report our recent progress toward this end with a hybrid stochastic GC framework developed on the Cellular Potts Model-based CompuCell3D platform. Tellurium is used to simulate the B cell intracellular molecular network comprising NF-κB, FOXO1, MYC, AP4, CXCR4, and BLIMP1 that responds to B cell receptor (BCR) and CD40-mediated signaling. The molecular outputs of the network drive the spatiotemporal behaviors of B cells, including cyclic migration between the dark zone (DZ) and light zone (LZ) via chemotaxis; clonal proliferative bursts, somatic hypermutation, and DNA damage-induced apoptosis in the DZ; and positive selection, apoptosis via a death timer, and emergence of plasma cells in the LZ. Our simulations are able to recapitulate key molecular, cellular, and morphological GC events, including B cell population growth, affinity maturation, and clonal dominance. This novel modeling framework provides an open-source, customizable, and multiscale virtual GC simulation platform that enables qualitative and quantitative in silico investigations of a range of mechanistic and applied research questions on the adaptive humoral immune response in the future.

Ly6Chi Monocytes Are Metabolically Reprogrammed in the Blood during Inflammatory Stimulation and Require Intact OxPhos for Chemotaxis and Monocyte to Macrophage Differentiation.

Acute inflammation is a rapid and dynamic process involving the recruitment and activation of multiple cell types in a coordinated and precise manner. Here, we investigate the origin and transcriptional reprogramming of monocytes using a model of acute inflammation, zymosan-induced peritonitis. Monocyte trafficking and adoptive transfer experiments confirmed that monocytes undergo rapid phenotypic change as they exit the blood and give rise to monocyte-derived macrophages that persist during the resolution of inflammation. Single-cell transcriptomics revealed significant heterogeneity within the surface marker-defined CD11b+Ly6G-Ly6Chi monocyte populations within the blood and at the site of inflammation. We show that two major transcriptional reprogramming events occur during the initial six hours of Ly6Chi monocyte mobilisation, one in the blood priming monocytes for migration and a second at the site of inflammation. Pathway analysis revealed an important role for oxidative phosphorylation (OxPhos) during both these reprogramming events. Experimentally, we demonstrate that OxPhos via the intact mitochondrial electron transport chain is essential for murine and human monocyte chemotaxis. Moreover, OxPhos is needed for monocyte-to-macrophage differentiation and macrophage M(IL-4) polarisation. These new findings from transcriptional profiling open up the possibility that shifting monocyte metabolic capacity towards OxPhos could facilitate enhanced macrophage M2-like polarisation to aid inflammation resolution and tissue repair.

Rac1 plays a crucial role in MCP-1-induced monocyte adhesion and migration.

Monocyte migration is an important process in inflammation and atherogenesis. Identification of the key signalling pathways that regulate monocyte migration can provide prospective targets for prophylactic treatments in inflammatory diseases. Previous research showed that the focal adhesion kinase Pyk2, Src kinase and MAP kinases play an important role in MCP-1-induced monocyte migration. In this study, we demonstrate that MCP-1 induces iPLA2 activity, which is regulated by PKCß and affects downstream activation of Rac1 and Pyk2. Rac1 interacts directly with iPLA2 and Pyk2, and plays a crucial role in MCP-1-mediated monocyte migration by modulating downstream Pyk2 and p38 MAPK activation. Furthermore, Rac1 is necessary for cell spreading and F-actin polymerization during monocyte adhesion to fibronectin. Finally, we provide evidence that Rac1 controls the secretion of inflammatory mediator vimentin from MCP-1-stimulated monocytes. Altogether, this study demonstrates that the PKCß/iPLA2/Rac1/Pyk2/p38 MAPK signalling cascade is essential for MCP-1-induced monocyte adhesion and migration.

Methods and computational tools to study eukaryotic cell migration in vitro.

Cellular movement is essential for many vital biological functions where it plays a pivotal role both at the single cell level, such as during division or differentiation, and at the macroscopic level within tissues, where coordinated migration is crucial for proper morphogenesis. It also has an impact on various pathological processes, one for all, cancer spreading. Cell migration is a complex phenomenon and diverse experimental methods have been developed aimed at dissecting and analysing its distinct facets independently. In parallel, corresponding analytical procedures and tools have been devised to gain deep insight and interpret experimental results. Here we review established experimental techniques designed to investigate specific aspects of cell migration and present a broad collection of historical as well as cutting-edge computational tools used in quantitative analysis of cell motion.

Benzofuran-2-Carboxamide Derivatives as Immunomodulatory Agents Blocking the CCL20-Induced Chemotaxis and Colon Cancer Growth.

The correlation between the CCL20/CCR6 axis and autoimmune and non-autoimmune disorders is widely recognized. Inhibition of the CCL20-dependent cell migration represents therefore a promising approach for the treatment of many diseases, such as inflammatory bowel diseases and colorectal cancer. We report herein our efforts to explore the biologically relevant chemical space around the benzofuran scaffold of MR120, a modulator of the CCL20/CCR6 axis previously discovered by our group. A functional screening allowed us to identify C4 and C5-substituted derivatives as the most effective inhibitors of the CCL20-induced chemotaxis of human peripheral blood mononuclear cells (PBMC). Moreover, selected compounds (16e and 24b) also proved to potently inhibit the growth of different colon cancer cell lines, with cytotoxic/cytostatic and antiproliferative activity.