Cell Polarity Research Articles

Unlocking the secrets of Hofbauer cells in placental (patho-) physiology: Isolation and quality assessment in human term placenta

IntroductionHofbauer cells (HBCs) are macrophages of fetal origin that reside in the villous tissue. They are the only immune cells within healthy villi. While HBCs perform innate immune functions such as phagocytosis and antigen presentation, they are increasingly recognized for their diverse roles in placental physiology e.g. vascular functionality, tissue homeostasis, tolerance. Consequently, HBCs are of utmost interest in a variety of non-physiological placental conditions. IsolationVillous tissue is collected freshly after delivery and finely minced. The resulting tissue is digested in a two-step process, using Trypsin/DNase to separate cytotrophoblasts and collagenase/DNase to penetrate deeper into the villous stroma, containing HBCs, and obtain a single cell suspension. After a density gradient centrifugation, the corresponding cell layer is collected and subjected to negative immune selection of HBCs, yielding unaffected cells that have not been activated during the isolation process. Quality controlIn addition to a classical immunocytochemistry (ICC) approach including macrophage markers, and markers for potentially contaminating cell types (e.g. fibroblasts, muscle, mesenchymal cells), we have developed a multi-color flow cytometry (FC) panel. This panel assesses Hofbauer cell purity and polarization states more accurately and comprehensively than qualitative ICC, using percentage analysis of parent cells to estimate the expression levels of specific markers. DiscussionThe presented protocol allows us to isolate HBCs in significant numbers and high purity, even from placentae compromised by preeclampsia (PE) with limited placental volume. We have successfully developed and implemented this protocol to study healthy, diabetic and PE macrophages, aiding a better understanding of the underlying placental pathophysiology at the cellular level.

Spatio-Temporal Regulation of Notch Activation in Asymmetrically Dividing Sensory Organ Precursor Cells in Drosophila melanogaster Epithelium.

The Notch communication pathway, discovered in Drosophila over 100 years ago, regulates a wide range of intra-lineage decisions in metazoans. The division of the Drosophila mechanosensory organ precursor is the archetype of asymmetric cell division in which differential Notch activation takes place at cytokinesis. Here, we review the molecular mechanisms by which epithelial cell polarity, cell cycle and intracellular trafficking participate in controlling the directionality, subcellular localization and temporality of mechanosensitive Notch receptor activation in cytokinesis.

Suppressor of Cytokine Signaling Proteins 3 and 5 Potentially Delineate Polarization of Th cells in Chronic Rhinosinusitis.

Background: Chronic rhinosinusitis (CRS) is an inflammatory condition classified into chronic rhinosinusitis with nasal polyps (CRSwNP) and chronic rhinosinusitis without nasal polyps (CRSsNP). Th cells manage inflammatory cells in CRS. Suppressor of Cytokine Signaling (SOCS) proteins regulate Janus kinase (JAK)-signal transducer and activator of transcription (STAT) pathway in Th cells by polarizing toward Th1, Th2, and Th17 cells. This study evaluated the levels of SOCS1,3,5 in CRS patients to find associations with Th cells. Methods: In this cross-sectional study, 20 CRSwNP patients, 12 CRSsNP patients, and 12 controls participated. The infiltration of CD4+ T cells was determined using immunohistochemistry. The expression of specific transcription factors and SOCS proteins was assessed using real-time PCR. Cytokine levels were evaluated using ELISA. SOCS protein levels were investigated using western blot analysis. Results: The expression of SOCS3 increased in the CRSwNP group compared to CRSsNP and control groups (p <0.001). SOCS3 protein levels increased in the CRSwNP group compared to CRSsNP (p <0.05) and control (p <0.001) groups. Although there was a significant difference in SOCS5 expression between CRSsNP and control groups, SOCS5 protein levels were significantly different between CRSsNP and control (p <0.001) and CRSwNP (p <0.05) groups. Conclusions: Targeted therapies may be suggested for CRS by modulating SOCS3 and SOCS5 proteins that are responsible for polarization of Th cells toward Th2 or Th1 cells, respectively. JAK-STAT pathway targeting, which encompasses numerous cells, can be limited to SOCS proteins to more effectively orchestrate Th cell differentiation.

PIN2-mediated self-organizing transient auxin flow contributes to auxin maxima at the tip of Arabidopsis cotyledons.

Directional auxin transport and formation of auxin maxima are critical for embryogenesis, organogenesis, pattern formation, and growth coordination in plants, but the mechanisms underpinning the initiation and establishment of these auxin dynamics are not fully understood. Here we show that a self-initiating and -terminating transient auxin flow along the marginal cells (MCs) contributes to the formation of an auxin maximum at the tip of Arabidopsis cotyledon that globally coordinates the interdigitation of puzzle-shaped pavement cells in the cotyledon epidermis. Prior to the interdigitation, indole butyric acid (IBA) is converted to indole acetic acid (IAA) to induce PIN2 accumulation and polarization in the marginal cells, leading to auxin flow toward and accumulation at the cotyledon tip. When IAA levels at the cotyledon tip reaches a maximum, it activates pavement cell interdigitation as well as the accumulation of the IBA transporter TOB1 in MCs, which sequesters IBA to the vacuole and reduces IBA availability and IAA levels. The reduction of IAA levels results in PIN2 down-regulation and cessation of the auxin flow. Hence, our results elucidate a self-activating and self-terminating transient polar auxin transport system in cotyledons, contributing to the formation of localized auxin maxima that spatiotemporally coordinate pavement cell interdigitation.

Intestinal mRNA expression profiles associated with mucosal healing in ustekinumab-treated Crohn's disease patients: bioinformatics analysis and prospective cohort validation

BackgroundVariations exist in the response of patients with Crohn’s disease (CD) to ustekinumab (UST) treatment, but the underlying cause remains unknown. Our objective was to investigate the involvement of immune cells and identify potential biomarkers that could predict the response to interleukin (IL) 12/23 inhibitors in patients with CD.MethodsThe GSE207022 dataset, which consisted of 54 non-responders and 9 responders to UST in a CD cohort, was analyzed. Differentially expressed genes (DEGs) were identified and subjected to Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses. Least absolute shrinkage and selection operator (LASSO) regression was used to screen the most powerful hub genes. Receiver operating characteristic (ROC) curve analysis was performed to evaluate the predictive performances of these genes. Single-sample Gene Set Enrichment Analysis (ssGSEA) was used to estimate the proportions of immune cell types. These significantly altered genes were subjected to cluster analysis into immune cell-related infiltration. To validate the reliability of the candidates, patients prescribed UST as a first-line biologic in a prospective cohort were included as an independent validation dataset.ResultsA total of 99 DEGs were identified in the integrated dataset. GO and KEGG analyses revealed significant enrichment of immune response pathways in patients with CD. Thirteen genes (SOCS3, CD55, KDM5D, IGFBP5, LCN2, SLC15A1, XPNPEP2, HLA-DQA2, HMGCS2, DDX3Y, ITGB2, CDKN2B and HLA-DQA1), which were primarily associated with the response versus nonresponse patients, were identified and included in the LASSO analysis. These genes accurately predicted treatment response, with an area under the curve (AUC) of 0.938. T helper cell type 1 (Th1) cell polarization was comparatively strong in nonresponse individuals. Positive connections were observed between Th1 cells and the LCN2 and KDM5D genes. Furthermore, we employed an independent validation dataset and early experimental verification to validate the LCN2 and KDM5D genes as effective predictive markers.ConclusionsTh1 cell polarization is an important cause of nonresponse to UST therapy in patients with CD. LCN2 and KDM5D can be used as predictive markers to effectively identify nonresponse patients.Trial registration: Trial registration number: NCT05542459; Date of registration: 2022-09-14; URL: https://www.clinicaltrials.gov.

A 3D micro-printed single cell micro-niche with asymmetric niche signals – An in vitro model for asymmetric cell division study

Intricate microenvironment signals orchestrate to affect cell behavior and fate during tissue morphogenesis. However, the underlying mechanisms on how specific local niche signals influence cell behavior and fate are not fully understood, owing to the lack of in vitro platform able to precisely, quantitatively, spatially, and independently manipulate individual niche signals. Here, microarrays of protein-based 3D single cell micro-niche (3D-SCμN), with precisely engineered biophysical and biochemical niche signals, are micro-printed by a multiphoton microfabrication and micropatterning technology. Mouse embryonic stem cell (mESC) is used as the model cell to study how local niche signals affect stem cell behavior and fate. By precisely engineering the internal microstructures of the 3D SCμNs, we demonstrate that the cell division direction can be controlled by the biophysical niche signals, in a cell shape-independent manner. After confining the cell division direction to a dominating axis, single mESCs are exposed to asymmetric biochemical niche signals, specifically, cell-cell adhesion molecule on one side and extracellular matrix on the other side. We demonstrate that, symmetry-breaking (asymmetric) niche signals successfully trigger cell polarity formation and bias the orientation of asymmetric cell division, the mitosis process resulting in two daughter cells with differential fates, in mESCs.

Identification of diagnostic biomarkers and potential therapeutic targets for biliary atresia via WGCNA and machine learning methods.

Biliary atresia (BA) is a severe and progressive biliary obstructive disease in infants that requires early diagnosis and new therapeutic targets. This study employed bioinformatics methods to identify diagnostic biomarkers and potential therapeutic targets for BA. Our analysis of mRNA expression from Gene Expression Omnibus datasets revealed 3,273 differentially expressed genes between patients with BA and those without BA (nBA). Weighted gene coexpression network analysis determined that the turquoise gene coexpression module, consisting of 298 genes, is predominantly associated with BA. The machine learning method then filtered out the top 2 important genes, CXCL8 and TMSB10, from the turquoise module. The area under receiver operating characteristic curves for TMSB10 and CXCL8 were 0.961 and 0.927 in the training group and 0.819 and 0.791 in the testing group, which indicated a high diagnostic value. Besides, combining TMSB10 and CXCL8, a nomogram with better diagnostic performance was built for clinical translation. Several studies have highlighted the potential of CXCL8 as a therapeutic target for BA, while TMSB10 has been shown to regulate cell polarity, which was related to BA progression. Our analysis with qRT PCR and immunohistochemistry also confirmed the upregulation of TMSB10 at mRNA and protein levels in BA liver samples. These findings highlight the sensitivity of CXCL8 and TMSB10 as diagnostic biomarkers and their potential as therapeutic targets for BA.

Dual-reporter fluorescent probe for precise identification of liver cancer by sequentially responding to carboxylesterase and polarity

Early treatment significantly improves the survival rate of liver cancer patients, so the development of early diagnostic methods for liver cancer is urgent. Liver cancer can develop from viral hepatitis, alcoholic liver, and fatty liver, thus making the above diseases share common features such as elevated viscosity, reactive oxygen species, and reactive nitrogen species. Therefore, accurate differentiation between other liver diseases and liver cancer is both a paramount practical need and challenging. Numerous fluorescent probes have been reported for the diagnosis of liver cancer by detecting a single biomarker, but these probes lack specificity for liver cancer in complex biological systems. Obviously, using multiple liver cancer biomarkers as the basis for judgment can dramatically improve diagnostic accuracy. Herein, we report the first fluorescent probe, LD-TCE, that sequentially detects carboxylesterase (CE) and lipid droplet polarity in liver cancer cells with high sensitivity and selectivity, with linear detection of CE in the range of 0–6 U/mL and a 65-fold fluorescence enhancement in response to polarity. The probe first reacts with CE and releases weak fluorescence, which is then dramatically enhanced due to the decrease in lipid droplet polarity in liver cancer cells. This approach allows the probe to enable specific imaging of liver cancer with higher contrast and accuracy. The probe successfully achieved the screening of liver cancer cells and the precise identification of liver cancer in mice. More importantly, it is not disturbed by liver fibrosis, which is a common pathological feature of many liver diseases. We believe that the LD-TCE is expected to be a powerful tool for early diagnosis of liver cancer.

The Development of a 3D PET Fibrous Scaffold Modified with an Umbilical Cord dECM for Liver Tissue Engineering.

Organ and tissue dysfunction represents a clinically significant condition. By integrating cell biology with materials science, tissue engineering enables the reconstruction and restoration of damaged tissues or organs, offering a noninvasive repair approach. In our study, we replicated the cellular growth environment by utilizing a human umbilical cord-derived decellularized extracellular matrix (dECM) as a modifying agent for the polyethylene terephthalate (PET) polymeric fiber scaffold. This allowed us to create a dECM-coated polyester fiber-based scaffold, PET-dECM, tailored for liver tissue engineering purposes. We effectively produced a decellularized human umbilical cord-derived ECM through a combined decellularization process involving trypsin/EDTA, TritonX-100, and sodium deoxycholate. The application of the dECM coating onto the PET material was accomplished through several steps, such as ester hydrolysis, EDC/NHS-activated crosslinking, and dECM conjugation. The biological performance of the PET-dECM was validated using RG cell culture assays. Notably, the dECM coating significantly improved PET's hydrophilicity and biocompatibility, thereby aiding cell adhesion, proliferation, and functional differentiation (p < 0.05). It was further found that the hepatocyte function of HepaRG was significantly enhanced on the PET-dECM, which may be attributed to the dECM's ability to facilitate the restoration of cell polarity. The PET-dECM holds promise as an effective hepatocyte culture carrier and could potentially find application in liver tissue engineering.

Pathogenic variants in autism gene KATNAL2 cause hydrocephalus and disrupt neuronal connectivity by impairing ciliary microtubule dynamics

Enlargement of the cerebrospinal fluid (CSF)-filled brain ventricles (cerebral ventriculomegaly), the cardinal feature of congenital hydrocephalus (CH), is increasingly recognized among patients with autism spectrum disorders (ASD). KATNAL2, a member of Katanin family microtubule-severing ATPases, is a known ASD risk gene, but its roles in human brain development remain unclear. Here, we show that nonsense truncation of Katnal2 (Katnal2Δ17) in mice results in classic ciliopathy phenotypes, including impaired spermatogenesis and cerebral ventriculomegaly. In both humans and mice, KATNAL2 is highly expressed in ciliated radial glia of the fetal ventricular-subventricular zone as well as in their postnatal ependymal and neuronal progeny. The ventriculomegaly observed in Katnal2Δ17 mice is associated with disrupted primary cilia and ependymal planar cell polarity that results in impaired cilia-generated CSF flow. Further, prefrontal pyramidal neurons in ventriculomegalic Katnal2Δ17 mice exhibit decreased excitatory drive and reduced high-frequency firing. Consistent with these findings in mice, we identified rare, damaging heterozygous germline variants in KATNAL2 in five unrelated patients with neurosurgically treated CH and comorbid ASD or other neurodevelopmental disorders. Mice engineered with the orthologous ASD-associated KATNAL2 F244L missense variant recapitulated the ventriculomegaly found in human patients. Together, these data suggest KATNAL2 pathogenic variants alter intraventricular CSF homeostasis and parenchymal neuronal connectivity by disrupting microtubule dynamics in fetal radial glia and their postnatal ependymal and neuronal descendants. The results identify a molecular mechanism underlying the development of ventriculomegaly in a genetic subset of patients with ASD and may explain persistence of neurodevelopmental phenotypes in some patients with CH despite neurosurgical CSF shunting.

Actin-organizing protein palladin modulates C2C12 cell fate determination

BackgroundCell confluency and serum deprivation promote the transition of C2C12 myoblasts into myocytes and subsequence fusion into myotubes. However, despite all myoblasts undergoing the same serum deprivation trigger, their responses vary: whether they become founder myocytes, remain proliferative, or evolve into fusion-competent myocytes remains unclear. We have previously shown that depletion of the scaffolding protein palladin in myoblasts inhibits cell migration and promotes premature muscle differentiation, pointing to its potential significance in muscle development and the necessity for a more in-depth examination of its function in cellular heterogeneity. Methods and resultsHere, we showed that the subcellular localization of palladin might contribute to founder-fate cell decision in the early differentiation process. Depleting palladin in C2C12 myoblasts depleted integrin-β3 plasma membrane localization of and focal adhesion formation at the early stage of myogenesis, decreased kindlin-2 and metavinculin expression during the myotube maturation process, leading to the inability of myocytes to fuse into preexisting mature myotubes. This aligns with previous findings where early differentiation into nascent myotubes occurred but compromised maturation. In contrast, wildtype C2C12 overexpressing the 140-kDa palladin isoform developed a polarized morphology with star-like structures toward other myoblasts. However, this behaviour was not observed in palladin-depleted cells, where the 140-kDa palladin overexpression could not recover cell migration capacity, suggesting other palladin isoforms are also needed to establish cell polarity. ConclusionOur study identifies a counter-intuitive role for palladin in regulating myoblast-to-myocyte cell fate decisions and impacting their ability to form mature multinucleated myotubes by influencing cell signalling pathways and cytoskeletal organization, necessary for skeletal muscle regeneration and repair studies.

Using adjusted local assortativity with Molecular Pixelation unveils colocalization of membrane proteins with immunological significance.

Advances in spatial proteomics and protein colocalization are a driving force in the understanding of cellular mechanisms and their influence on biological processes. New methods in the field of spatial proteomics call for the development of algorithms and open up new avenues of research. The newly introduced Molecular Pixelation (MPX) provides spatial information on surface proteins and their relationship with each other in single cells. This allows for in silico representation of neighborhoods of membrane proteins as graphs. In order to analyze this new data modality, we adapted local assortativity in networks of MPX single-cell graphs and created a method that is able to capture detailed information on the spatial relationships of proteins. The introduced method can evaluate the pairwise colocalization of proteins and access higher-order similarity to investigate the colocalization of multiple proteins at the same time. We evaluated the method using publicly available MPX datasets where T cells were treated with a chemokine to study uropod formation. We demonstrate that adjusted local assortativity detects the effects of the stimuli at both single- and multiple-marker levels, which enhances our understanding of the uropod formation. We also applied our method to treating cancerous B-cell lines using a therapeutic antibody. With the adjusted local assortativity, we recapitulated the effect of rituximab on the polarity of CD20. Our computational method together with MPX improves our understanding of not only the formation of cell polarity and protein colocalization under stimuli but also advancing the overall insight into immune reaction and reorganization of cell surface proteins, which in turn allows the design of novel therapies. We foresee its applicability to other types of biological spatial data when represented as undirected graphs.

Role of tissue fluidization and topological defects in epithelial tubulogenesis

Cellular rearrangements, as primary sources of tissue fluidization, facilitate topological transitions during tissue morphogenesis. We study the role of intrinsic cell properties such as cell polarity and cell-cell adhesion in shaping epithelial tissues using a minimal model of interacting polarized cells. The presence of a vortex in the cell polarization poses the topological constraint that induces an inwards migration with the formation of a conical shape. Local rearrangements at the tip of the cone lead to the onset of tube formation. Switching between collective migration and structural rearrangements is key for balancing the contrasting tendencies, such as the tissue rigidity needed to preserve shape and the tissue fluidity allowing for topological transitions during tissue morphogenesis. Published by the American Physical Society 2024

Swaying the advantage: multifaceted functions of inflammasomes in adaptive immunity.

Eukaryotic cells are equipped with cytoplasmic sensors that recognize diverse pathogen- or danger-associated molecular patterns. In cells of the myeloid lineage, activation of these sensors leads to the assembly of a multimeric protein complex, called the inflammasome, that culminates in the production of inflammatory cytokines and pyroptosis. Recently, investigation of the inflammasomes in lymphocytes led to the discovery of functional pathways that were initially believed to be confined to the innate arm of the immune system. Thus, the adapter protein apoptosis-associated speck-like protein containing a CARD (ASC) was documented to play a critical role in antigen uptake by dendritic cells, and regulation of T- and B-cell motility at several stages, and absent in melanoma 2 (AIM2) was found to act as a modulator of regulatory T-cell differentiation. Remarkably, NLRP3 was demonstrated to act as a transcription factor that controls Th2 cell polarization, and as a negative regulator of regulatory T-cell differentiation by limiting Foxp3 expression. In B lymphocytes, NLRP3 plays a role in the transcriptional network that regulates B-cell development and homing, and its activation is essential for germinal center formation and maturation of high-affinity antibody responses. Such recently discovered inflammasome-mediated functions in T and B lymphocytes offer multiple cross-talk opportunities for the innate and adaptive arms of the immune system. A better understanding of the dialog between inflammasomes and intracellular components could be beneficial for therapeutic purposes in restoring immune homeostasis and mitigating inflammation in a wide range of disorders.

A CUC1/auxin genetic module links cell polarity to patterned tissue growth and leaf shape diversity in crucifer plants

How tissue-level information encoded by fields of regulatory gene activity is translated into the patterns of cell polarity and growth that generate the diverse shapes of different species remains poorly understood. Here, we investigate this problem in the case of leaf shape differences between Arabidopsis thaliana, which has simple leaves, and its relative Cardamine hirsuta that has complex leaves divided into leaflets. We show that patterned expression of the transcription factor CUP-SHAPED COTYLEDON1 in C. hirsuta (ChCUC1) is a key determinant of leaf shape differences between the two species. Through inducible genetic perturbations, time-lapse imaging of growth, and computational modeling, we find that ChCUC1 provides instructive input into auxin-based leaf margin patterning. This input arises via transcriptional regulation of multiple auxin homeostasis components, including direct activation of WAG kinases that are known to regulate the polarity of PIN-FORMED auxin transporters. Thus, we have uncovered a mechanism that bridges biological scales by linking spatially distributed and species-specific transcription factor expression to cell-level polarity and growth, to shape diverse leaf forms.

Optimized PAR-2 RING dimerization mediates cooperative and selective membrane binding for robust cell polarity

Cell polarity networks are defined by quantitative features of their constituent feedback circuits, which must be tuned to enable robust and stable polarization, while also ensuring that networks remain responsive to dynamically changing cellular states and/or spatial cues during development. Using the PAR polarity network as a model, we demonstrate that these features are enabled by the dimerization of the polarity protein PAR-2 via its N-terminal RING domain. Combining theory and experiment, we show that dimer affinity is optimized to achieve dynamic, selective, and cooperative binding of PAR-2 to the plasma membrane during polarization. Reducing dimerization compromises positive feedback and robustness of polarization. Conversely, enhanced dimerization renders the network less responsive due to kinetic trapping of PAR-2 on internal membranes and reduced sensitivity of PAR-2 to the anterior polarity kinase, aPKC/PKC-3. Thus, our data reveal a key role for a dynamically oligomeric RING domain in optimizing interaction affinities to support a robust and responsive cell polarity network, and highlight how optimization of oligomerization kinetics can serve as a strategy for dynamic and cooperative intracellular targeting.

THP-1 Monocytic Cells Are Polarized to More Antitumorigenic Macrophages by Serial Treatment with Phorbol-12-Myristate-13-Acetate and PD98059.

Background and Objectives: As modulators of the tumor microenvironment, macrophages have been extensively studied for their potential in developing anticancer strategies, particularly in regulating macrophage polarization towards an antitumorigenic (M1) phenotype rather than a protumorigenic (M2) one in various experimental models. Here, we evaluated the effect of PD98059, a mitogen-activated protein kinase kinase MAPKK MEK1-linked pathway inhibitor, on the differentiation and polarization of THP-1 monocytes in response to phorbol-12-myristate-13-acetate (PMA) under various culture conditions for tumor microenvironmental application. Materials and Methods: Differentiation and polarization of THP-1 were analyzed by flow cytometry and RT-PCR. Polarized THP-1 subsets with different treatment were compared by motility, phagocytosis, and so on. Results: Clearly, PMA induced THP-1 differentiation occurs in adherent culture conditions more than nonadherent culture conditions by increasing CD11b expression up to 90%, which was not affected by PD98059 when cells were exposed to PMA first (post-PD) but inhibited when PD98059 was treated prior to PMA treatment (pre-PD). CD11bhigh THP-1 cells treated with PMA and PMA-post-PD were categorized into M0 (HLA-DRlow and CD206low), M1 (HLA-DRhigh and CD206low), and M2 (HLA-DRlow and CD206high), resulting in an increased population of M1 macrophages. The transcription levels of markers of macrophage differentiation and polarization confirmed the increased M1 polarization of THP-1 cells with post-PD treatment rather than with PMA-only treatment. The motility and cytotoxicity of THP-1 cells with post-PD treatment were higher than THP-1 cells with PMA, suggesting that post-PD treatment enhanced the anti-tumorigenicity of THP-1 cells. Confocal microscopy and flow cytometry showed the effect of post-PD treatment on phagocytosis by THP-1 cells. Conclusions: We have developed an experimental model of macrophage polarization with THP-1 cells which will be useful for further studies related to the tumor microenvironment.

Lactate drives epithelial-mesenchymal transition in diabetic kidney disease via the H3K14la/KLF5 pathway

High levels of urinary lactate are an increased risk of progression in patients with diabetic kidney disease (DKD). However, it is still unveiled how lactate drive DKD. Epithelial-mesenchymal transition (EMT), which is characterized by the loss of epithelial cells polarity and cell-cell adhesion, and the acquisition of mesenchymal-like phenotypes, is widely recognized a critical contributor to DKD. Here, we found a switch from oxidative phosphorylation (OXPHOS) toward glycolysis in AGEs-induced renal tubular epithelial cells, thus leading to elevated levels of renal lactic acid. We demonstrated that reducing the lactate levels markedly delayed EMT progression and improved renal tubular fibrosis in DKD. Mechanically, we observed lactate increased the levels of histone H3 lysine 14 lactylation (H3K14la) in DKD. ChIP-seq & RNA-seq results showed histone lactylation contributed to EMT process by facilitating KLF5 expression. Moreover, KLF5 recognized the promotor of cdh1 and inhibited its transcription, which accelerated EMT of DKD. Additionally, nephro-specific knockdown and pharmacological inhibition of KLF5 diminished EMT development and attenuated DKD fibrosis. Thus, our study provides better understanding of epigenetic regulation of DKD pathogenesis, and new therapeutic strategy for DKD by disruption of the lactate-drived H3K14la/KLF5 pathway.

Hierarchical global and local auxin signals coordinate cellular interdigitation in Arabidopsis.

The development of multicellular tissues requires both local and global coordination of cell polarization, however, the mechanisms underlying their interplay are poorly understood. In Arabidopsis, leaf epidermal pavement cells (PC) develop a puzzle-piece shape locally coordinated through apoplastic auxin signaling. Here we show auxin also globally coordinates interdigitation by activating the TIR1/AFB-dependent nuclear signaling pathway. This pathway promotes a transient maximum of auxin at the cotyledon tip, which then moves across the leaf activating local PC polarization, as demonstrated by locally uncaged auxin globally rescuing defects in tir1;afb1;afb2;afb4;afb5 mutant but not in tmk1;tmk2;tmk3;tmk4 mutants. Our findings show that hierarchically integrated global and local auxin signaling systems, which respectively depend on TIR1/AFB-dependent gene transcription in the nucleus and TMK-mediated rapid activation of ROP GTPases at the cell surface, control PC interdigitation patterns in Arabidopsis cotyledons, revealing a mechanism for coordinating a local cellular process with the development of whole tissues.

IMMU-03. DELETION OF RASA2 IN CAR T CELLS OVERCOMES DYSFUNCTIONAL IMMUNE SYNAPSE FORMATION AGAINST DIPGS, ENHANCING IN VITRO AND IN VIVO ANTITUMOR RESPONSE

Abstract BACKGROUND Diffuse intrinsic pontine gliomas (DIPGs) are highly lethal pediatric brain tumors and a leading cause of cancer-related death in children. There is currently no cure for DIPG, highlighting an urgent need for novel therapeutics. Chimeric antigen receptor (CAR)-T-cell therapy offers great promise for DIPG treatment; however, several limitations need to be addressed. The goal of this study is to identify why CAR T cells are ineffective against DIPG and overcome this roadblock by improving the immune synapse (IS), or the interaction between tumor and CAR T cells. METHODS In this study, we targeted the tumor associated antigen B7-H3 by generating B7-H3 specific CAR T cells with CD28z signaling domain. We compared in vitro and in vivo antitumor response of CAR T cells against patient derived DIPGs and non-DIPG brain tumors. We contrasted the IS quality in DIPGs against other brain tumors by confocal microscopy. We further generated IS-enhanced CAR T cells by knocking out RASA2 and compared their performance against control KO in terms of IS-quality, killing kinetics, expansion, cytokine secretion, and in vivo tumor control. RESULTS Our data shows that CAR T cells have potent in vitro antitumor activity but fail in vivo against DIPGs which correlates with the formation of deficient IS, reflected by small synapses and poor calcium flux and cell polarity. RASA2 deletion in CAR T cells resulted in improved IS quality evidenced by bigger synapses, increased calcium flux, and CAR clustering upon antigen activation. In concordance, RASA2 KO CAR T cells elicited increased expansion, cytokine secretion, and in vivo antitumor activity against DIPGs compared to control KO. CONCLUSIONS This study shows that poor IS formation contributes to CAR T cell failure against DIPGs. Enhancing the IS formation in CAR T cells improves antitumor activity against DIPGs and should be considered for clinical translation.