Regeneration of Planarian Auricles and Reestablishment of Chemotactic Ability

Detection of chemical stimuli is crucial for living systems and also contributes to quality of life in humans. Since loss of olfaction becomes more prevalent with aging, longer life expectancies have fueled interest in understanding the molecular mechanisms behind the development and maintenance of chemical sensing. Planarian flatworms possess an unsurpassed ability for stem cell-driven regeneration that allows them to restore any damaged or removed part of their bodies. This includes anteriorly-positioned lateral flaps known as auricles, which have long been thought to play a central role in chemotaxis. The contribution of auricles to the detection of positive chemical stimuli was tested in this study using Girardia dorotocephala, a North American planarian species known for its morphologically prominent auricles. Behavioral experiments staged under laboratory conditions revealed that removal of auricles by amputation leads to a significant decrease in the ability of planarians to find food. However, full chemotactic capacity is observed as early as 2 days post-amputation, which is days prior from restoration of auricle morphology, but correlative with accumulation of ciliated cells in the position of auricle regeneration. Planarians subjected to x-ray irradiation prior to auricle amputation were unable to restore auricle morphology, but were still able to restore chemotactic capacity. These results indicate that although regeneration of auricle morphology requires stem cells, some restoration of chemotactic ability can still be achieved in the absence of normal auricle morphology, corroborating with the initial observation that chemotactic success is reestablished 2-days post-amputation in our assays. Transcriptome profiles of excised auricles were obtained to facilitate molecular characterization of these structures, as well as the identification of genes that contribute to chemotaxis and auricle development. A significant overlap was found between genes with preferential expression in auricles of G. dorotocephala and genes with reduced expression upon SoxB1 knockdown in Schmidtea mediterranea, suggesting that SoxB1 has a conserved role in regulating auricle development and function. Models that distinguish between possible contributions to chemotactic behavior obtained from cellular composition, as compared to anatomical morphology of the auricles, are discussed.

Therapeutic Effects of CXCL9-overexpressing Human Umbilical Cord Mesenchymal Stem Cells on Liver Fibrosis in Rats

Background: Umbilical cord mesenchymal stem cells (UC-MSCs) transplantation have become a promising treatment for liver fibrosis. However, UC-MSCs have limited anti-fibrosis ability, and their homing ability of UC-MSCs to the injured liver sites appears to be poor. In this study, we aimed to determining if overexpression of CXCL9 could have the synergistic anti-fibrosis effect with UC-MSCs, and whether it can promote the homing ability of UC-MSCs.Methods: Overexpression of CXCL9 in UC-MSCs (CXCL9-UC-MSCs) was attained by transfection of naive UC-MSCs with the lenti-CXCL9-mCherry. The impact of transplanted CXCL9-UC-MSCs on both repairing of liver fibrosis and homing was evaluated and compared with lenti-mCherry empty vector transfected UC-MSCs (control UC-MSCs).Results: After puromycin screening, UC-MSCs could stably express CXCL9 without affecting the stem and differentiation ability of UC-MSCs. In addition, biochemical analysis showed that the liver function of CXCL9-UC-MSCs was significantly increased in comparison with that of control UC-MSCs (P < 0.05). Futhermore, histopathology after 4 weeks of cell therapy demonstrated that the content of collagen fibers decreased obviously, the pseudo-lobules almost disappeared, and the morphology of hepatic lobules was basically normal. Frozen sections were performed 24 hours and 4 weeks after the cell injection. It can be seen that the fluorescence expression of the CXCL9-UC-MSCs group was significantly higher than that of the control UC-MSCs group, which proved that CXCL9-UC-MSCs have a stronger chemotactic ability, and can stay longer than control UC-MSCs in the injured liver.Conclusion: Overexpression of CXCL9 improves the efficacy of UC-MSC therapy for liver fibrosis repair, thereby promoting the homing and staying of UC-MSCs to injured hepatic sites in a rat model of liver fibrosis.

Invasion of homogeneous and polyploid populations in nutrient-limiting environments

Breast cancer progresses in a multistep process from primary tumor growth and stroma invasion to metastasis. Progression is accompanied by a switch to an invasive cell phenotype. Nutrient-limiting environments promote chemotaxis with aggressive morphologies characteristic of invasion. It is unknown how co-existing cells differ in their response to nutrient limitations and how this impacts invasion of the metapopulation as a whole. We integrate mathematical modeling with microenvironmental perturbation-data to investigate invasion in nutrient-limiting environments inhabited by one or two cancer cell subpopulations. Hereby, subpopulations are defined by their energy efficiency and chemotactic ability. We estimate the invasion-distance traveled by a homogeneous population. For heterogeneous populations, our results suggest that an imbalance between nutrient efficacy and chemotactic superiority accelerates invasion. Such imbalance will spatially segregate the two populations and only one type will dominate at the invasion front. Only if these two phenotypes are balanced do the two subpopulations compete for the same space, which decelerates invasion. We investigate ploidy as a candidate biomarker of this phenotypic heterogeneity to discern circumstances when inhibiting chemotaxis amplifies internal competition and decelerates tumor progression, from circumstances that render clinical consequences of chemotactic inhibition unfavorable.SignificanceA better understanding of the nature of the double-edged sword of high ploidy is a prerequisite to personalize combination-therapies with cytotoxic drugs and inhibitors of signal transduction pathways such as MTOR-Is.

Platelet-Dependent Neutrophil Function Is Dysregulated by M Protein from Streptococcus pyogenes

Platelets are rapidly responsive sentinel cells that patrol the bloodstream and contribute to the host response to infection. Platelets have been reported to form heterotypic aggregates with leukocytes and may modulate their function. Here, we have investigated platelet-neutrophil complex formation and neutrophil function in response to distinct agonists. The endogenous platelet activator thrombin gave rise to platelet-dependent neutrophil activation, resulting in enhanced phagocytosis and bacterial killing.Streptococcus pyogenesis an important causative agent of severe infectious disease, which can manifest as sepsis and septic shock. M1 protein fromS. pyogenesalso mediated platelet-neutrophil complex formation; however, these neutrophils were dysfunctional and exhibited diminished chemotactic ability and bacterial killing. This reveals an important agonist-dependent neutrophil dysfunction during platelet-neutrophil complex formation and highlights the role of platelets during the immune response to streptococcal infection.

A Gain-of-Function CCR4 Mutations in Adult T-Cell Leukemia/Lymphoma (ATLL) Enhance the Chemotactic Abilities and PI3K/AKT Activation

High expression of CC chemokine receptor 4 (CCR4) has been identified as a hallmark gene in ATLL, an aggressive peripheral T-cell neoplasm. CCR4 is a chemokine receptor, which has a critical role in immune cell trafficking including Th2, T-reg and skin-homing memory T-cells. CCR4 ligands, CCL17 and CCL22, were produced in lymph nodes and skin from dendritic cells, macrophages and Langerhans cells. Most ATLL cases express surface CCR4 (90%) and infiltrate to lymph nodes and skin. These observations suggest that CCR4 could have a role in ATLL biology, but it is still unclear whether dysregulation of CCR4 function contributes to ATLL pathogenesis. We performed RNA-Seq for two primary ATLL cases and discovered recurrent non-sense mutations in CCR4. Though an extended analysis using Sanger sequencing, CCR4 mutations were detected in 14/53 ATLL samples (26%) and consisted exclusively of nonsense or frameshift mutations that truncated the coding region at C329, Q330 or Y331 in the carboxy-terminus. We noticed that the location of the CCR4 mutations in ATLL were reminiscent of mutations affecting the chemokine receptor CXCR4 in the WHIM syndrome, a human immunodeficiency disease. Most CXCR4 mutations in the WHIM syndrome are nonsense or frameshift, resulting in carboxy-terminal truncation of the protein and conferring a gain-of-function phenotype with respect to chemotaxis towards the CXCR4 ligand SDF1. We therefore hypothesized that mutant CCR4 isoforms might enhance chemotaxis of the affected cells to CCR4 ligands. Chemotaxic assay using 32Db, a mouse myeloid cell line, and ED40515(+), an ATLL cell line, clarified that the ectopic expression of CCR4-Q330* enhanced the chemotactic ability of the transduced cells toward CCL17 and CCL22 rather than CCR4-WT transduced cells with statistical significance. To understand the mechanism of this enhanced chemotactic ability, we studied the change in surface CCR4 levels after CCL22 exposure in CCR4-WT-and CCR4-Q330*-reconstituted ED40515(+) cells. Compared with CCR4-WT, CCR4 internalization in CCR4-Q330*-reconstituted cells was significantly impaired. Thus, the ATLL CCR4 mutants impair desensitization by ligand, which likely contributes to the enhanced chemotaxis of cells bearing these mutants. We explored the influence of the ATLL CCR4 mutants on PI(3) kinase (PI3K)-dependent activation of AKT since it has been reported that binding of CCL22 to CCR4 activates AKT in CEM leukemic T-cells and in human Th2 cells. CCR4-Q330*-reconstituted ED40515(+) showed strong activation of AKT with CCL22 ligation compared with CCR4-WT-reconstituted cell. The AKT activation was cancelled with pan-PI3K inhibitor, BKM120, indicating that CCR4-mediated AKT activation was PI3K dependent. Lastly, we tested whether the acquisition of CCR4 mutations by ATLL cells imparts a selective growth advantage relative to cells with wild type CCR4. CCR4-Q330*-reconstituted cells had a selective growth advantage in the presence of CCL22, supporting at least in part the hypothesis that CCR4 mutation are able to provide the affected cells a positive selection pressure through CCL22 ligation and contributes to ATLL pathogenesis. We discovered for the first time recurrent somatic mutations in CCR4 in ATLL. CCR4 mutations were detected in 14/53 ATLL samples (26%) and consisted exclusively of nonsense or frameshift mutations that truncated the coding region at C329, Q330 or Y331 in the carboxy-terminus. Functionally, the CCR4-Q330* was a gain-of-function since it increased cell migration towards the CCR4 ligands CCL17 and CCL22, in part by impairing receptor internalization. This mutant enhanced PI(3) kinase/AKT activation following receptor engagement by CCL22 in ATLL cells, and conferred a growth advantage in in vitro cultures. Our findings provide a rationale to test whether inhibition of CCR4 signaling might have a therapeutic potential for patients with ATLL. Disclosures No relevant conflicts of interest to declare.

Chemotactic Signaling by an Escherichia coli CheA Mutant That Lacks the Binding Domain for Phosphoacceptor Partners

ABSTRACT CheA is a multidomain histidine kinase for chemotaxis in Escherichia coli. CheA autophosphorylates through interaction of its N-terminal phosphorylation site domain (P1) with its central dimerization (P3) and ATP-binding (P4) domains. This activity is modulated through the C-terminal P5 domain, which couples CheA to chemoreceptor control. CheA phosphoryl groups are donated to two response regulators, CheB and CheY, to control swimming behavior. The phosphorylated forms of CheB and CheY turn over rapidly, enabling receptor signaling complexes to elicit fast behavioral responses by regulating the production and transmission of phosphoryl groups from CheA. To promote rapid phosphotransfer reactions, CheA contains a phosphoacceptor-binding domain (P2) that serves to increase CheB and CheY concentrations in the vicinity of the adjacent P1 phosphodonor domain. To determine whether the P2 domain is crucial to CheA's signaling specificity, we constructed CheAΔP2 deletion mutants and examined their signaling properties in vitro and in vivo. We found that CheAΔP2 autophosphorylated and responded to receptor control normally but had reduced rates of phosphotransfer to CheB and CheY. This defect lowered the frequency of tumbling episodes during swimming and impaired chemotactic ability. However, expression of additional P1 domains in the CheAΔP2 mutant raised tumbling frequency, presumably by buffering the irreversible loss of CheAΔP2-generated phosphoryl groups from CheB and CheY, and greatly improved its chemotactic ability. These findings suggest that P2 is not crucial for CheA signaling specificity and that the principal determinants that favor appropriate phosphoacceptor partners, or exclude inappropriate ones, most likely reside in the P1 domain.