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Programmed cell death-1 (PD-1) and its ligand (PD-L1) are significant mediators of immune suppression in the tumor microenvironment. We focused on the immunological impact of PD-1/PD-L1 signaling during tumor progression in colorectal carcinoma (CRC) and its association with resistance to neoadjuvant chemoradiotherapy (NCRT) in locally advanced rectal carcinoma (LAd-RC). Histopathological and immunohistochemical analyses of 100 CRC cases (including 34 RC) without NCRT and 109 NCRT-treated LAd-RC cases were performed. Membranous tumoral PD-L1 expression was identified in 9 of 100 (9%) CRC cases, including 1 of 34 (2.9%) RC cases, but PD-L1 immunopositivity was not associated with any clinicopathological factors, with the exception of deficient mismatch repair (dMMR) status. In contrast, stromal PD-L1+ immune cells, which frequently exhibited coexpression of PD-1 and CD8 markers, were significantly correlated with tumor vessel invasion, nuclear β-catenin+ tumor budding cancer stem cell (CSC)-like features, and unfavorable prognosis. In the LAd-RC cases, stromal CD8+ (but not PD-L1+) immune cell infiltration in pretreatment-biopsied samples was significantly and positively associated with therapeutic efficacy. After NCRT, tumoral PD-L1 expression was observed in only 2 of 83 (2.4%) tumors, independent of dMMR status, whereas high stromal PD-L1+ and tumoral nuclear β-catenin positivity were significantly linked to a poor response to NCRT and high tumor budding features. In addition, high stromal PD-L1 immunoreactivity was significantly associated with poorer overall survival. In conclusion, a combination of stromal PD-L1+ immune cells and nuclear β-catenin+ tumor budding may contribute to tumor progression in CRC and resistance to NCRT in LAd-RC, through formation of niche-like lesions that exhibit immune resistance and CSC properties., (© 2022 The Authors. The Journal of Pathology: Clinical Research published by The Pathological Society of Great Britain and Ireland and John Wiley & Sons Ltd.)

Immune checkpoint blockade (ICB) therapy is an important treatment option for individuals with cancer, but it has certain limitations. Identifying a better target that can overcome tumor immune escape and stimulate T cell activity is critical. This research aimed to delve into the molecular mechanism underlying the immunoregulatory function of metadherin (MTDH), which is a novel and potential therapeutic target in hepatocellular cancer (HCC). A small interfering RNA library was screened using the luciferase reporter assay and PD-L1 promoter. The Cancer Genome Atlas database and HCC tissues were used to investigate the relationship between MTDH and PD-L1. The association between MTDH and β-catenin/lymphoid enhancer binding factor (LEF-1) was discovered by co-immunoprecipitation. The chromatin immunoprecipitation assay was used to investigate the interaction of MTDH with the PD-L1 promoter when LEF-1 expression was silenced. Locked nucleic acid antisense oligonucleotides (ASOs) were used to inhibit MTDH. We utilized in vitro co-cultures and in vivo syngeneic tumor development experiments to confirm the effectiveness of MTDH ASO combined with PD-1 monoclonal antibody (mAb). MTDH was demonstrated to be a PD-L1 modulator. MTDH increased PD-L1 expression and upregulated PD-L1 transcriptional activity through β-catenin/LEF-1 signaling. More importantly, MTDH ASO improved the anti-PD-1 response and increased cytotoxic T-cell infiltration in PD-1 mAb-treated malignancies. MTDH effectively predicts the therapeutic efficacy of ICB therapy. Our results imply that combining MTDH ASO with PD-1 mAb could be a promising therapeutic strategy for HCC. In addition, MTDH is a potential novel biomarker for predicting the effectiveness of immune checkpoint inhibitor treatment., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Background: Understanding the mechanisms of non-T cell inflamed tumor microenvironment (TME) and their modulation are important to improve cancer immunotherapies such as immune checkpoint inhibitors. The involvement of various immunometabolisms has recently been indicated in the formation of immunosuppressive TME. In this study, we investigated the immunological roles of stearoyl-CoA desaturase 1 (SCD1), which is essential for fatty acid metabolism, in the cancer immune response., Methods: We investigated the roles of SCD1 by inhibition with the chemical inhibitor or genetic manipulation in antitumor T cell responses and the therapeutic effect of anti-programmed cell death protein 1 (anti-PD-1) antibody using various mouse tumor models, and their cellular and molecular mechanisms. The roles of SCD1 in human cancers were also investigated by gene expression analyses of colon cancer tissues and by evaluating the related free fatty acids in sera obtained from patients with non-small cell lung cancer who were treated with anti-PD-1 antibody., Results: Systemic administration of a SCD1 inhibitor in mouse tumor models enhanced production of CCL4 by cancer cells through reduction of Wnt/β-catenin signaling and by CD8 + effector T cells through reduction of endoplasmic reticulum stress. It in turn promoted recruitment of dendritic cells (DCs) into the tumors and enhanced the subsequent induction and tumor accumulation of antitumor CD8 + T cells. SCD1 inhibitor was also found to directly stimulate DCs and CD8 + T cells. Administration of SCD1 inhibitor or SCD1 knockout in mice synergized with an anti-PD-1 antibody for its antitumor effects in mouse tumor models. High SCD1 expression was observed in one of the non-T cell-inflamed subtypes in human colon cancer, and serum SCD1 related fatty acids were correlated with response rates and prognosis of patients with non-small lung cancer following anti-PD-1 antibody treatment., Conclusions: SCD1 expressed in cancer cells and immune cells causes immunoresistant conditions, and its inhibition augments antitumor T cells and therapeutic effects of anti-PD-1 antibody. Therefore, SCD1 is an attractive target for the development of new diagnostic and therapeutic strategies to improve current cancer immunotherapies including immune checkpoint inhibitors., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY. Published by BMJ.)

The mechanically activated Piezo channel plays a versatile role in conferring mechanosensitivity to various cell types. However, how it incorporates its intrinsic mechanosensitivity and cellular components to effectively sense long-range mechanical perturbation across a cell remains elusive. Here we show that Piezo channels are biochemically and functionally tethered to the actin cytoskeleton via the cadherin-β-catenin mechanotransduction complex, whose perturbation significantly impairs Piezo-mediated responses. Mechanistically, the adhesive extracellular domain of E-cadherin interacts with the cap domain of Piezo1, which controls the transmembrane gate, while its cytosolic tail might interact with the cytosolic domains of Piezo1, which are in close proximity to its intracellular gates, allowing a direct focus of adhesion-cytoskeleton-transmitted force for gating. Specific disruption of the intermolecular interactions prevents cytoskeleton-dependent gating of Piezo1. Thus, we propose a force-from-filament model to complement the previously suggested force-from-lipids model for mechanogating of Piezo channels, enabling them to serve as versatile and tunable mechanotransducers., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

The signaling pathways activated following interaction between dendritic cells (DCs) and a pathogen determine the polarization of effector T-cell and regulatory T-cell (Treg) responses to the infection. Several recent studies, mostly in the context of bacterial infections, have shown that the Wnt/β-catenin pathway plays a major role in imparting tolerogenic features in DCs and in promotion of Treg responses. However, the significance of the Wnt/β-catenin pathway's involvement in regulating the immune response to the fungal species is not known. Using Aspergillus fumigatus, a ubiquitous airborne opportunistic fungal species, we show here that fungi activate the Wnt/β-catenin pathway in human DCs and are critical for mediating the immunosuppressive Treg responses. Pharmacological inhibition of this pathway in DCs led to inhibition of maturation-associated molecules and interleukin 10 (IL-10) secretion without affecting the majority of the inflammatory cytokines. Furthermore, blockade of Wnt signaling in DCs suppressed DC-mediated Treg responses in CD4 + T cells and downregulated both tumor necrosis factor alpha (TNF-α) and IL-10 responses in CD8 + T cells. Mechanistically, induction of β-catenin pathway by A. fumigatus required C-type lectin receptors and promoted Treg polarization via the induction of programmed death-ligand 1 on DCs. Further investigation on the identity of fungal molecular patterns has revealed that the cell wall polysaccharides β-(1, 3)-glucan and α-(1, 3)-glucan, but not chitin, possess the capacity to activate the β-catenin pathway. Our data suggest that the Wnt/β-catenin pathway is a potential therapeutic target to selectively suppress the Treg response and to sustain the protective Th1 response in the context of invasive aspergillosis caused by A. fumigatus. IMPORTANCE The balance between effector CD4 + T-cell and immunosuppressive regulatory T-cell (Treg) responses determines the outcome of an infectious disease. The signaling pathways that regulate human CD4 + T-effector versus Treg responses to the fungi are not completely understood. By using Aspergillus fumigatus, a ubiquitous opportunistic fungal species, we show that fungi activate the Wnt/β-catenin pathway in human dendritic cells (DCs) that promotes Treg responses via induction of immune checkpoint molecule programmed death ligand 1 on DCs. Blockade of the Wnt/β-catenin pathway in DCs led to the selective inhibition of Treg without affecting the Th1 response. Dissection of the identity of A. fumigatus pathogen-associated molecular patterns (PAMPs) revealed that cell wall polysaccharides exhibit selectivity in their capacity to activate the β-catenin pathway in DCs. Our data thus provide a pointer that Wnt/β-catenin pathway represents potential therapeutic target to selectively suppress Treg responses and to sustain protective a Th1 response against invasive fungal diseases.

β-catenin is a structural protein that makes the cell-cell connection in adherence junctions. Besides the structural functions, it also plays a role as a central transducer of the canonical Wnt signaling cascade, regulating nearly four hundred genes related to various cellular processes. Recently the immune functions of β-catenin during pathogenic invasion have gained more attention. In the present study, we elucidated the immune function of fish β-catenin by identifying and characterizing the β-catenin homolog (PhCatβ) from redlip mullet, Planiliza haematocheila. The complete open reading frame of PhCatβ consists of 2352 bp, which encodes a putative β-catenin homolog (molecular weight: 85.7 kDa). Multiple sequence alignment analysis revealed that β-catenin is highly conserved in vertebrates. Phylogenetic reconstruction demonstrated the close evolutionary relationship between PhCatβ and other fish β-catenin counterparts. The tissue distribution analysis showed the highest mRNA expression of PhCatβ in heart tissues of the redlip mullet under normal physiological conditions. While in response to pathogenic stress, the PhCatβ transcription level was dramatically increased in the spleen and gill tissues. The overexpression of PhCatβ stimulated M2 polarization and cell proliferation of murine RAW 264.7 macrophage. In fish cells, the overexpression of PhCatβ resulted in a significant upregulation of antiviral gene transcription and vice versa. Moreover, the overexpression of PhCatβ could inhibit the replication of VHSV in FHM cells. Our results strongly suggest that PhCatβ plays a role in macrophage activation and antiviral immune response in redlip mullet., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Background: The emergence of immune checkpoint inhibitors (ICIs) marks the beginning of a new era of immunotherapy for hepatocellular carcinoma (HCC), however, not all patients respond successfully to this treatment. A major challenge for HCC immunotherapy is the development of ways to screen for those patients that would benefit from this type of treatment and determine the optimal treatment plan for individual patients. Therefore, it is important to find a biomarker which allows for the stratification of HCC patients, which distinguishes responders from non-responders, thereby further improving the clinical benefits for those undergoing immunotherapy., Methods: We used univariate and multivariate Cox risk proportional regression models to evaluate the relationship between non-synonymous mutations with a mutation frequency greater than 10%. We made a prognosis of an immunotherapy HCC cohort using mutation and prognosis data. An additional three HCC queues from the cbioportal webtool were used for further verification. The CIBERSORT, IPS, quanTIseq, and MCPcounter algorithms were used to evaluate the immune cells. PCA and z-score algorithm were used to calculate immune-related signature with published gene sets. Gene set enrichment analysis (GSEA) was used to compare the differences in the pathway-based enrichment scores of candidate genes between mutant and wild types., Results: Univariate and multivariate Cox results showed that only CTNNB1-Mutant(CTNNB1-MUT) was associated with progression-free survival (PFS) of HCC patients in the immunotherapy cohort. After excluding the potential bias introduced by other clinical features, it was found that CTNNB1-MUT served as an independent predictor of the prognosis of HCC patients after immunotherapy (P < 0.05; HR > 1). The results of the tumor immune microenvironment (TIME) analysis showed that patients with CTNNB1-MUT had significantly reduced activated immune cells [such as T cells, B cells, M1-type macrophages, and dendritic cells (DCs)], significantly increased M2-type macrophages, a significantly decreased expression of immunostimulating molecules, low activity of the immune activation pathways (cytokine pathway, immune cell activation and recruitment) and highly active immune depletion pathways (fatty acid metabolism, cholesterol metabolism, and Wnt pathway)., Conclusions: In this study, we found CTNNB1-MUT to be a potential biomarker for HCC immunotherapy patients, because it identified those patients are less likely to benefit from ICIs., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Chen, Zhou, Liu and Zhang.)

Reversible cellular senescence was demonstrated previously to constitute colon cancer cell response to methotrexate. The current study presents a comparison of two senescent states of colon cancer cells, arrested and reversing, resulting from respectively, 120 h exposure to the drug, and 48 h exposure followed by 96 h regrowth in drug-free media. The upregulation of immunoproteasome subunit-coding genes and the increase in human leukocyte antigen HLA-A/B/C membrane level indicated MHC-I-restricted antigen presentation as common to both senescent states. Nuclear factor NF-κB p65 level decreased and activating protein AP-1: c-Jun, Fra2 and JunB nuclear levels increased in both senescent cell populations. Notably, the increase in AP-1- dependent transcription occurred after 48 h exposure to methotrexate. β-catenin nuclear level increased after 48 h exposure to the drug and remained as such only in senescence-arrested cells. β-catenin level was found uncoupled from the protein phosphorylation status indicating the deregulation of β-catenin signaling in colon cancer cells employed in the study. These findings carry implications for both, a general mechanism of senescence establishment and putative advantages for colon cancer treatment., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Desmoid-type fibromatosis (DF) is a locally aggressive but non-metastatic (myo)fibroblastic neoplasm. A hallmark of the tumor is nuclear positivity for beta-catenin in immunohistochemistry due mostly to CTNNB1 mutations. However, a recent study has reported that even beta-catenin 'nuclear-negative' DFs can harbor CTNNB1 mutations and that the positive ratio of nuclear beta-catenin in DF is different among antibodies. Here, we reviewed soft tissue lesions for which the possibility of DF was considered and compared the sensitivity and specificity of nuclear beta-catenin for the diagnosis of DF among commonly used anti-beta-catenin antibodies, i.e., clone beta-catenin 1, 17C2 and 14. We analyzed 26 cases of DF, 28 cases of benign fibroblastic lesions, and 27 cases of other soft tissue tumors. The sensitivity and specificity of nuclear beta-catenin for the diagnosis of DF were different among antibodies; 54% and 98% in clone beta-catenin 1, 85% and 84% in 17C2, and 96% and 62% in 14. IHC of LEF1 showed comparable results with IHC of beta-catenin, with a sensitivity of 88% and specificity of 76%. Additionally, when beta-catenin 1 was used, DFs showed characteristic dotted cytoplasmic staining, often appearing as rings. Our results might be helpful for making a correct diagnosis of DF., (© 2021 Japanese Society of Pathology and John Wiley & Sons Australia, Ltd.)

Background: Periodontitis is a chronic infection initiated by oral bacterial and their virulence factors, yet the severity of periodontitis is largely determined by the dysregulated host immuno-inflammatory response. Baicalein is a flavonoid extracted from Scutellaria baicalensis with promising anti-inflammatory properties. This study aims to clarify the anti-inflammatory and osteogenic effects of baicalein in periodontal ligament cells (PDLCs) treated with lipopolysaccharides (LPS)., Methods: Human PDLCs were incubated with baicalein (0-100 μM) for 2 h prior to LPS challenge for 24 h. MTT analysis was adopted to assess the cytoxicity of baicalein. The mRNA and protein expression of inflammatory and osteogenic markers were measured by real-time polymerase chain reaction (PCR), western blot and enzyme-linked immunosorbent assay (ELISA) as appropriate. Alkaline phosphatase (ALP) and Alizarin red S (ARS) staining were performed to evaluate the osteogenic differentiation of PDLCs. The expression of Wnt/β-catenin and mitogen-activated protein kinase (MAPK) signaling related proteins was assessed by western blot., Results: MTT results showed that baicalein up to 100 μM had no cytotoxicity on PDLCs. Baicalein significantly attenuated the inflammatory factors induced by LPS, including interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), matrix metalloprotein-1 (MMP-1), MMP-2 and monocyte chemoattractant protein 1 (MCP-1) at both mRNA and protein level. Moreover, MAPK signaling (ERK, JNK and p38) was significantly inhibited by baicalein, which may account for the mitigated inflammatory response. Next, we found that baicalein effectively restored the osteogenic differentiation of LPS-treated PDLCs, as shown by the increased ALP and ARS staining. Accordingly, the protein and gene expression of osteogenic markers, namely runt-related transcription factor 2 (RUNX2), collagen-I, and osterix were markedly upregulated. Importantly, baicalein could function as the Wnt/β-catenin signaling activator, which may lead to the increased osteoblastic differentiation of PDLCs., Conclusions: With the limitation of the study, we provide in vitro evidence that baicalein ameliorates inflammatory response and restores osteogenesis in PDLCs challenged with LPS, indicating its potential use as the host response modulator for the management of periodontitis.

Introduction: An immunohistochemical study has occasionally been performed to diagnose anaplastic thyroid carcinoma (ATC). However, antibodies to confirm the undifferentiated nature of ATC have not yet been evaluated. The aim of this study was to evaluate E-cadherin and β-catenin expressions in immunoreactivity to determine undifferentiated carcinoma cells in the diagnosis of ATC., Methods: We immunohistochemically examined 29 ATCs, 30 poorly differentiated thyroid carcinomas (PDTCs), 22 well-differentiated thyroid carcinomas (WDTCs), and 3 squamous cell carcinomas. Antibodies for thyroid transcription factor-1 (TTF-1), paired-box gene 8 (PAX8), β-catenin, and E-cadherin were used., Results: All WDTCs tested positive for TTF-1, PAX8, and E-cadherin. The positive rates of TTF-1, PAX8, and E-cadherin were 93.3, 93.3, and 100%, respectively, in PDTCs and 17.2, 51.7, and 10.3%, respectively, in ATCs. WDTC expressed the lateral cell membrane staining for β-catenin and E-cadherin, whereas PDTC showed circumferential cell membranous expression (fishnet pattern). β-catenin cell membrane expression in ATCs is lost or discontinuous. Carcinoma cells with β-catenin nuclear expression without cell membranous expression were scattered in 72.4% of ATCs but were not observed in the other carcinomas., Conclusion: We propose 3 immunohistochemical findings to determine undifferentiated carcinoma cells in the diagnosis of ATC: (1) β-catenin nuclear expression with no or reduced cell membranous expression, (2) the loss or discontinuous pattern of E-cadherin expression, and (3) the loss of PAX8 nuclear expression., (© 2021 S. Karger AG, Basel.)

Loss of immune tolerance to gut microflora is inextricably linked to chronic intestinal inflammation and colitis-associated colorectal cancer (CAC). The LRP5/6 signaling cascade in APCs contributes to immune homeostasis in the gut, but whether this pathway in APCs protects against CAC is not known. In the current study, using a mouse model of CAC, we show that the LRP5/6-β-catenin-IL-10 signaling axis in intestinal CD11c + APCs protects mice from CAC by regulating the expression of tumor-promoting inflammatory factors in response to commensal flora. Genetic deletion of LRP5/6 in CD11c + APCs in mice (LRP5/6 ΔCD11c ) resulted in enhanced susceptibility to CAC. This is due to a microbiota-dependent increased expression of proinflammatory factors and decreased expression of the immunosuppressive cytokine IL-10. This condition could be improved in LRP5/6 ΔCD11c mice by depleting the gut flora, indicating the importance of LRP5/6 in mediating immune tolerance to the gut flora. Moreover, mechanistic studies show that LRP5/6 suppresses the expression of tumor-promoting inflammatory factors in CD11c + APCs via the β-catenin-IL-10 axis. Accordingly, conditional activation of β-catenin specifically in CD11c + APCs or in vivo administration of IL-10 protected LRP5/6 ΔCD11c mice from CAC by suppressing the expression of inflammatory factors. In summary, in this study, we identify a key role for the LRP5/6-β-catenin-IL-10 signaling pathway in intestinal APCs in resolving chronic intestinal inflammation and protecting against CAC in response to the commensal flora., (Copyright © 2020 by The American Association of Immunologists, Inc.)

BACKGROUND Immunotherapy is one of the research hotspots in the field of hepatocellular carcinoma (HCC). Successive clinical trials have shown that patients with CTNNB1 mutations are resistant to immunotherapy, but the mechanism is still unclear. MATERIAL AND METHODS We identified differentially expressed immune genes (DEIGs) in patients with and without CTNNB1 mutations in the Cancer Genome Atlas (TCGA) database and then paired them to explore any correlation with prognosis. Univariate Cox regression analysis and Lasso regression analysis were used to develop the prognostic model. We first divided the TCGA cohort into 29 subgroups for internal validation and then used the International Cancer Genome Consortium (ICGC) cohort to conduct external validation. We also used a CIBERSORT algorithm to quantify immune infiltration of the different risk groups. RESULTS The novel prognostic model consisted of 45 immune-gene pairs with general applicability. It was more accurate than the traditional prognostic signature, which is based on gene expression by comparison of area under the receiver operating characteristic curve (AUC) values. The infiltration proportion of B cells, CD8 T lymphocytes, activated natural killer cells, and M1 macrophages in the low-risk group was greater in the high-risk group, while the infiltration proportion of M0 and M2 macrophages was greater in the high-risk group. CONCLUSIONS In this study, a novel approach was proposed for evaluating HCC prognosis, which may be useful in evaluatingthe intensity of the immune response in the HCC microenvironment.

Dysregulation of Wnt/β-catenin signaling is frequently observed in human gastric cancer. Elucidation of the tumor immune microenvironment is essential for understanding tumorigenesis and for the development of immunotherapeutic strategies. However, it remains unclear how β-catenin signaling regulates the tumor immune microenvironment in the stomach. Here, we identify CCL28 as a direct transcriptional target gene of β-catenin/T-cell factor (TCF). Protein levels of β-catenin and CCL28 positively correlated in human gastric adenocarcinoma. β-Catenin-activated CCL28 recruited regulatory T (Treg) cells in a transwell migration assay. In a clinically relevant mouse gastric cancer model established by Helicobacter ( H .) felis infection and N -methyl- N -nitrosourea (MNU) treatment, inhibition of β-catenin/TCF activity by a pharmacologic inhibitor iCRT14 suppressed CCL28 expression and Treg cell infiltration in the stomach. Moreover, an anti-CCL28 antibody attenuated Treg cell infiltration and tumor progression in H. felis /MNU mouse models. Diphtheria toxin-induced Treg cell ablation restrained gastric cancer progression in H. felis /MNU-treated DEREG (Foxp3-DTR) mice, clarifying the tumor-promoting role of Treg cells. Thus, the β-catenin-CCL28-Treg cell axis may serve as an important mechanism for immunosuppression of the stomach tumor microenvironment. Our findings reveal an immunoregulatory role of β-catenin signaling in stomach tumors and highlight the therapeutic potential of CCL28 blockade for the treatment of gastric cancer. SIGNIFICANCE: These findings demonstrate an immunosuppressive role of tumor-intrinsic β-catenin signaling and the therapeutic potential of CCL28 blockade in gastric cancer., (©2020 American Association for Cancer Research.)

Despite antiretroviral therapy human immunodeficiency virus type-1 (HIV-1) infection results in neuroinflammation of the central nervous system that can cause HIV-associated neurocognitive disorders (HAND). The molecular mechanisms involved in the development of HAND are unclear, however, they are likely due to both direct and indirect consequences of HIV-1 infection and inflammation of the central nervous system. Additionally, opioid abuse in infected individuals has the potential to exacerbate HIV-comorbidities, such as HAND. Although restricted for productive HIV replication, astrocytes (comprising 40-70% of all brain cells) likely play a significant role in neuropathogenesis in infected individuals due to the production and response of viral proteins. The HIV-1 protein Tat is critical for viral transcription, causes neuroinflammation, and can be secreted from infected cells to affect uninfected bystander cells. The Wnt/β-catenin signaling cascade plays an integral role in restricting HIV-1 infection in part by negatively regulating HIV-1 Tat function. Conversely, Tat can overcome this negative regulation and inhibit β-catenin signaling by sequestering the critical transcription factor TCF-4 from binding to β-catenin. Here, we aimed to explore how opiate exposure affects Tat-mediated suppression of β-catenin in astrocytes and the downstream modulation of neuroinflammatory genes. We observed that morphine can potentiate Tat suppression of β-catenin activity in human astrocytes. In contrast, Tat mutants deficient in secretion, and lacking neurotoxic effects, do not affect β-catenin activity in the presence or absence of morphine. Finally, morphine treatment of astrocytes was sufficient to reduce the expression of genes involved in neuroinflammation. Examining the molecular mechanisms of how HIV-1 infection and opiate exposure exacerbate neuroinflammation may help us inform or predict disease progression prior to HAND development., Competing Interests: The authors have declared that no competing interests exist.

Dendritic cells (DCs) control the strength and quality of antigen-specific adaptive immune responses. This is critical for launching a robust immunity against invading pathogens while maintaining a state of tolerance to self-antigens. However, this also represents a fundamental barrier to anti-tumor immune responses and cancer immunotherapy. DCs in the tumor microenvironment (TME) play a key role in this process. The factors in the TME and signaling networks that program DCs to a regulatory state are not fully understood. Recent advances point to novel mechanisms by which the canonical Wnt signaling cascade in DCs regulates immune suppression, and the same pathway in tumors is associated with the evasion of anti-tumor immunity. Here, we review these recent advances in the context of the pleiotropic effects of the Wnts in shaping anti-tumor immune responses by modulating DC functions. In addition, we will discuss how Wnt/β-catenin pathway in DCs can be targeted for successful cancer immunotherapy., (Copyright © 2020 Suryawanshi, Hussein, Prasad and Manicassamy.)

The diversity of the naïve T cell repertoire drives the replenishment potential and capacity of memory T cells to respond to immune challenges. Attrition of the immune system is associated with an increased prevalence of pathologies in aged individuals, but whether stem cell memory T lymphocytes (T SCM ) contribute to such attrition is still unclear. Using single cells RNA sequencing and high-dimensional flow cytometry, we demonstrate that T SCM heterogeneity results from differential engagement of Wnt signaling. In humans, aging is associated with the coupled loss of Wnt/β-catenin signature in CD4 T SCM and systemic increase in the levels of Dickkopf-related protein 1, a natural inhibitor of the Wnt/β-catenin pathway. Functional assays support recent thymic emigrants as the precursors of CD4 T SCM . Our data thus hint that reversing T SCM defects by metabolic targeting of the Wnt/β-catenin pathway may be a viable approach to restore and preserve immune homeostasis in the context of immunological history.

WNT/β-catenin signaling is involved in many physiological processes. Its implication in embryonic development, cell migration, and polarization has been shown. Nevertheless, alterations in this signaling have also been related with pathological events such as sustaining and proliferating the cancer stem cell (CSC) subset present in the tumor bulk. Related with this, WNT signaling has been associated with the maintenance, expansion, and epithelial-mesenchymal transition of stem cells, and furthermore with two distinctive features of this tumor population: therapeutic resistance (MDR, multidrug resistance) and immune escape. These mechanisms are developed and maintained by WNT activation through the transcriptional control of the genes involved in such processes. This review focuses on the description of the best known WNT pathways and the molecules involved in them. Special attention is given to the WNT cascade proteins deregulated in tumors, which have a decisive role in tumor survival. Some of these proteins function as extrusion pumps that, in the course of chemotherapy, expel the drugs from the cells; others help the tumoral cells hide from the immune effector mechanisms. Among the WNT targets involved in drug resistance, the drug extrusion pump MDR-1 (P-GP, ABCB1) and the cell adhesion molecules from the CD44 family are highlighted. The chemokine CCL4 and the immune checkpoint proteins CD47 and PD-L1 are included in the list of WNT target molecules with a role in immunity escape. This pathway should be a main target in cancer therapy as WNT signaling activation is essential for tumor progression and survival, even in the presence of the anti-tumoral immune response and/or antineoplastic drugs. The appropriate design and combination of anti-tumoral strategies, based on the modulation of WNT mediators and/or protein targets, could negatively affect the growth of tumoral cells, improving the efficacy of these types of therapies., (Copyright © 2019 Martin-Orozco, Sanchez-Fernandez, Ortiz-Parra and Ayala-San Nicolas.)

The major obstacle to human immunodeficiency type 1 virus (HIV-1) eradication is a reservoir of latently infected cells that persists despite long-term antiretroviral therapy (ART) and is maintained through cellular proliferation. Long-lived memory CD4 + T cells with high self-renewal capacity, such as central memory (CM) T cells and stem cell memory (SCM) T cells, are major contributors to the viral reservoir in HIV-infected individuals on ART. The Wnt/β-catenin signaling pathway regulates the balance between self-renewal and differentiation of SCM and CM T cells, and pharmacological manipulation of this pathway offers an opportunity to interfere with the proliferation of latently infected cells. Here, we evaluated in vivo a novel approach to inhibit self-renewal of SCM and CM CD4 + T cells in the rhesus macaque (RM) model of simian immunodeficiency (SIV) infection. We used an inhibitor of the Wnt/β-catenin pathway, PRI-724, that blocks the interaction between the coactivator CREB-binding protein (CBP) and β-catenin, resulting in the cell fate decision to differentiate rather than proliferate. Our study shows that PRI-724 treatment of ART-suppressed SIV mac251 -infected RMs resulted in decreased proliferation of SCM and CM T cells and modified the SCM and CM CD4 + T cell transcriptome toward a profile of more differentiated memory T cells. However, short-term treatment with PRI-724 alone did not significantly reduce the size of the viral reservoir. This work demonstrates for the first time that stemness pathways of long-lived memory CD4 + T cells can be pharmacologically modulated in vivo , thus establishing a novel strategy to target HIV persistence. IMPORTANCE Long-lasting CD4 + T cell subsets, such as central memory and stem cell memory CD4 + T cells, represent critical reservoirs for human immunodeficiency virus (HIV) persistence despite suppressive antiretroviral therapy. These cells possess stem cell-like properties of enhanced self-renewal/proliferation, and proliferation of latently infected memory CD4 + T cells plays a key role in maintaining the reservoir over time. Here, we evaluated an innovative strategy targeting the proliferation of long-lived memory CD4 + T cells to reduce viral reservoir stability. Using the rhesus macaque model, we tested a pharmacological inhibitor of the Wnt/β-catenin signaling pathway that regulates T cell proliferation. Our study shows that administration of the inhibitor PRI-724 decreased the proliferation of SCM and CM CD4 + T cells and promoted a transcriptome enriched in differentiation genes. Although the viral reservoir size was not significantly reduced by PRI-724 treatment alone, we demonstrate the potential to pharmacologically modulate the proliferation of memory CD4 + T cells as a strategy to limit HIV persistence., (Copyright © 2019 Mavigner et al.)

Mast cells constitutively express ß-catenin and expand in solid tumors such as colon and skin cancer. However, the role of ß-catenin signaling in mast cells and the cause or effect of mast cell expansion and tumor growth has yet to be established. In earlier studies we used mast cell depletion and protease staining approaches, to provide evidence for a causative role of mast cells in small bowel polyposis, and related specific phenotypes and distributions of tumor infiltrating mast cells to stages of tumor growth. Here we report that, stabilization of ß-catenin expands mast cells to promote high incidence of colon polyposis and infrequent small bowel polyps and skin cancer. Expression of a dominant acting ß-catenin in mast cells (5CreCAT) stimulated maturation and expression of granule stored proteases. Both mucosal and connective tissue type mast cells accumulated in colonic small bowel polyps independent of gender, and mice developed chronic systemic inflammation with splenomegaly. Reconstitution of polyposis-prone mice with bone marrow from 5CreCAT mice resulted in focal expansion of connective tissue like mast cells, which are normally rare in benign polyps and characteristically expand during adenoma-to-carcinoma transition. Our findings highlight a hitherto unknown contribution of ß-catenin signaling in mast cells to their maturation and to increased risk of colon cancer., (Copyright © 2019 Saadalla, Lima, Tsai, Osman, Singh, Linden, Dennis, Haeryfar, Gurish, Gounari and Khazaie.)

Background: Clinical response to MAPK inhibitors in metastatic melanoma patients is heterogeneous for reasons still needing to be elucidated. As the patient immune activity contributes to treatment clinical benefit, the pre-existing level of immunity at tumor site may provide biomarkers of disease outcome to therapy. Here we investigated whether assessing the density and spatial tissue distribution of key immune cells in the tumor microenvironment could identify patients predisposed to respond to MAPK inhibitors., Methods: Pretreatment tumor biopsies from a total of 213 patients (158 for the training set and 55 for the validation set) treated with BRAF or BRAF/MEK inhibitors within the Italian Melanoma Intergroup were stained with selected immune markers (CD8, CD163, β-catenin, PD-L1, PD-L2). Results, obtained by blinded immunohistochemical scoring and digital image analysis, were correlated with clinical response and outcome by multivariate logistic models on response to treatment and clinical outcome, adjusted for American Joint Committee on Cancer stage, performance status, lactate dehydrogenase and treatment received., Results: Patients with high intratumoral, but not peritumoral, CD8 + T cells and concomitantly low CD163 + myeloid cells displayed higher probability of response (OR 9.91, 95% CI 2.23-44.0, p = 0.003) and longer overall survival (HR 0.34, 95% CI 0.16-0.72, p = 0.005) compared to those with intratumoral low CD8 + T cells and high CD163 + myeloid cells. The latter phenotype was instead associated with a shorter progression free survival (p = 0.010). In contrast, PD-L1 and PD-L2 did not correlate with clinical outcome while tumor β-catenin overexpression showed association with lower probability of response (OR 0.48, 95% CI 0.21-1.06, p = 0.068)., Conclusions: Analysis of the spatially constrained distribution of CD8 + and CD163 + cells, representative of the opposite circuits of antitumor vs protumor immunity, respectively, may assist in identifying melanoma patients with improved response and better outcome upon treatment with MAPK inhibitors. These data underline the role of endogenous immune microenvironment in predisposing metastatic melanoma patients to benefit from therapies targeting driver-oncogenic pathways.

The Wnt/β-catenin signaling pathway is crucial to regulate cell proliferation and polarity, cell determination, and tissue homeostasis. The activation of Wnt/β-catenin signaling is based on the interaction between Wnt glycoproteins and seven transmembrane receptors-Frizzled (Fzd). This binding promotes recruitment of the scaffolding protein Disheveled (Dvl), which results in the phosphorylation of the co-receptor LRP5/6. The resultant molecular complex Wnt-Fzd-LRP5/6-Dvl forms a structural region for Axin interaction that disrupts Axin-mediated phosphorylation/degradation of the transcriptional co-activator β-catenin, thereby allowing it to stabilize and accumulate in the nucleus where it activates the expression of Wnt-dependent genes. Due to the prominent physiological function, the Wnt/β-catenin signaling must be strictly controlled because its dysregulation, which is caused by different stimuli, may lead to alterations in cell proliferation, apoptosis, and inflammation-associated cancer. The virulence factors from pathogenic bacteria such as Salmonella enterica sv Typhimurium, Helicobacter pylori, Mycobacterium tuberculosis, Pseudomonas aeruginosa, Citrobacter rodentium, Clostridium difficile, Bacteroides fragilis, Escherichia coli, Haemophilus parasuis, Lawsonia intracellularis, Shigella dysenteriae , and Staphylococcus epidermidis employ a variety of molecular strategies to alter the appropriate functioning of diverse signaling pathways. Among these, Wnt/β-catenin has recently emerged as an important target of several virulence factors produced by bacteria. The mechanisms used by these factors to interfere with the activity of Wnt/β-catenin is diverse and include the repression of Wnt inhibitors' expression by the epigenetic modification of histones, blocking Wnt-Fzd ligand binding, activation or inhibition of β-catenin nuclear translocation, down- or up-regulation of Wnt family members, and inhibition of Axin-1 expression that promotes β-catenin activity. Such a variety of mechanisms illustrate an evolutionary co-adaptation of eukaryotic molecular signaling to a battery of soluble or structural components synthesized by pathogenic bacteria. This review gathers the recent efforts to elucidate the mechanistic details through which bacterial virulence factors modulate Wnt/β-catenin signaling and its physiological consequences concerning the inflammatory response and cancer., (Copyright © 2019 Silva-García, Valdez-Alarcón and Baizabal-Aguirre.)

Immunotherapy with checkpoint inhibitors has greatly prolonged the overall survival of cancer patients in melanoma and many other cancer types. However, only a subset of patients shows clinical responses from these interventions, which was predicated by the T cell-inflamed tumor microenvironment. T cell-inflamed phenotype is characterized by the infiltration of CD8 + T cells, CD8α/CD103-lineage dendritic cells (DCs), as well as high density of forkhead box P3 (FoxP3) + regulatory T cells (Tregs) that are associated with the efficacy of immune checkpoint blockade. A number of regulators has been associated with T cell-inflammation in the tumor microenvironment, and WNT/β-catenin signaling is one of the best characterized. The tumor-intrinsic WNT/β-catenin signaling activation is frequently associated with poor spontaneous T cell infiltration across most human cancers. In this article, we review the essential roles of WNT/β-catenin signaling in the T cell-inflamed and non-T cell-inflamed tumor microenvironment, including the development and function of immune cells, activation of immune exclusion of tumor cells, and cancer immunosurveillance. We also discuss the impact of this pathway in driving the non-T cell-inflamed tumor microenvironment in other tumor types. To improve immunotherapy efficacy, we argue that targeting Wnt/β-catenin signaling should be a high priority for combinational cancer therapy to restore T cell infiltration., (Copyright © 2019 Li, Xiang, Li, Yin, Li and Ke.)

Ethnopharmacological Relevance: Sishen Wan (SSW) is a commercial and frequently used Chinese patent medicine listed in the Chinese Pharmacopeia, which is usually used to treat chronic colitis., Aim of the Study: We explored the pharmacological mechanism of Sishen Wan attenuated experimental chronic colitis by inhibiting Wnt/β-catenin pathway., Materials and Methods: Experimental chronic colitis was induced by trinitrobenzene sulfonic acid (TNBS). The therapeutic effect of SSW were analyzed by index of colonic weight, colonic length, pathological score. Cytokines expression were analyzed by ELISA, while the apoptosis level was checked by TUNEL staining. These proteins of Wnt/β-catenin signaling pathway was analyzed by Western blot assay., Results: Rats with TNBS-induced chronic colitis were treated by SSW for 10 days. The efficacy of SSW was demonstrated by improved macroscopic and microscopic colonic damage. SSW increased the level of ATP in colonic mucosa, while SSW inhibited β-catenin, ubiquitination of Nemo-like-kinase-associated ring finger protein and T-cell factor, and expression of Wnt/β-catenin downstream proteins (including c-Myc, cyclo-oxygenase-2, cyclin D1, survivin, signal transducer and activator of transcription 3 and zipper-interacting protein kinase), and improved lymphoid enhancer factor ubiquitination and β-TrCP activity, followed by excessive apoptosis of colonic epithelial cells., Conclusions: SSW effectively attenuated experimental chronic colitis induced by TNBS, which was realized by inhibition of the Wnt/β-catenin signaling pathway., (Copyright © 2019. Published by Elsevier B.V.)

β-catenin is a multifunctional protein that participates in a variety of physiological activities, including immune regulation, sex determination, nervous system development and, cell differentiation. However, the function of β-catenin in freshwater mussel Hyriopsis cumingii remains unclear. Herein, the gene encoding β-catenin from H. cumingii (Hc-β-catenin) was cloned and characterised. The full-length 5544 bp gene includes an open reading frame (ORF) of 2463 bp encoding a putative protein of 820 amino acids residues containing 12 armadillo (ARM) repeats. After injecting H. cumingii with Aeromonas hydrophila or lipopolysaccharides, Hc-β-catenin transcription was induced in hemocytes and gills, and the greatest responses occurred at 24 h after bacterial challenge, confirming an important role in immune responses. Quantitative real-time PCR analysis showed that Hc-β-catenin mRNA was distributed in the gill, foot, liver, kidney, mantle, adductor muscle and gonad of male and female mussels. In gonad, Hc-β-catenin expression was markedly higher in females than males. During the embryonic period, Hc-β-catenin expression was highest at 3 day. In 1-, 2- and 3-year-old mature mussels, Hc-β-catenin expression in female gonad tissue was notably higher than in males. In situ hybridisation revealed a significant hybridisation signal in female gonads, indicating that Hc-β-catenin is a pro-ovarian, anti-testis gene. Our findings demonstrate that Hc-β-catenin is important in immune regulation and sex determination in freshwater mussel., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Macrophages are often viewed through the lens of their core functions, but recent transcriptomic studies reveal them to be largely distinct across tissue types. While these differences appear to be shaped by their local environment, the key signals that drive these transcriptional differences remain unclear. Since Wnt signaling plays established roles in cell fate decisions, and tissue patterning during development and tissue repair after injury, we consider evidence that Wnt signals both target and are affected by macrophage functions. We propose that the Wnt gradients present in developing and adult tissues effectively shape macrophage fates and phenotypes. We also highlight evidence that macrophages, through an ability to dispatch Wnt signals, may couple tissue debridement and matrix remodeling with stem cell activation and tissue repair.

Osteosarcoma and Ewing sarcoma are the most common malignant primary bone tumors mainly occurring in children, adolescents and young adults. Current standard therapy includes multidrug chemotherapy and/or radiation specifically for Ewing sarcoma, associated with tumor resection. However, patient survival has not evolved for the past decade and remains closely related to the response of tumor cells to chemotherapy, reaching around 75% at 5 years for patients with localized forms of osteosarcoma or Ewing sarcoma but less than 30% in metastatic diseases and patients resistant to initial chemotherapy. Despite Ewing sarcoma being characterized by specific EWSR1-ETS gene fusions resulting in oncogenic transcription factors, currently, no targeted therapy could be implemented. It seems even more difficult to develop a targeted therapeutic strategy in osteosarcoma which is characterized by high complexity and heterogeneity in genomic alterations. Nevertheless, the common point between these different bone tumors is their ability to deregulate bone homeostasis and remodeling and divert them to their benefit. Therefore, targeting different actors of the bone tumor microenvironment has been hypothesized to develop new therapeutic strategies. In this context, it is well known that the Wnt/β-catenin signaling pathway plays a key role in cancer development, including osteosarcoma and Ewing sarcoma as well as in bone remodeling. Moreover, recent studies highlight the implication of the Wnt/β-catenin pathway in angiogenesis and immuno-surveillance, two key mechanisms involved in metastatic dissemination. This review focuses on the role played by this signaling pathway in the development of primary bone tumors and the modulation of their specific microenvironment., Competing Interests: The authors declare no conflict of interest.

Whether conventional dendritic cells (cDC) acquire subset identity under direction of Wnt family glycoproteins is unknown. We demonstrate that Wnt4, a β-catenin-independent Wnt ligand, is produced by both hematopoietic and nonhematopoietic cells and is both necessary and sufficient for preconventional DC1/cDC1 maintenance. Whereas bone marrow cDC precursors undergo phosphoJNK/c-Jun activation upon Wnt4 treatment, loss of cDC Wnt4 in CD11c Cre Wnt4 flox/flox mice impaired differentiation of CD24 + , Clec9A + , CD103 + cDC1 compared with CD11c Cre controls. Conversely, single-cell RNA sequencing analysis of bone marrow revealed a 2-fold increase in cDC2 gene signature genes, and flow cytometry demonstrated increased numbers of SIRP-α + cDC2 amid lack of Wnt4. Increased cDC2 numbers due to CD11c-restricted Wnt4 deficiency increased IL-5 production, group 2 innate lymphoid cell expansion, and host resistance to the hookworm parasite Nippostrongylus brasiliensis Collectively, these data uncover a novel and unexpected role for Wnt4 in cDC subset differentiation and type 2 immunity., (Copyright © 2019 by The American Association of Immunologists, Inc.)

Aim: In this study, we report the anti-inflammatory activity of XAV939, a modulator of the Wnt/β-catenin pathway. Methods: WNT/β-catenin pathway and NF-κB signaling pathway were examined in LPS-stimulated human bronchial epithelial cells and effects of XAV939 on these pathways were analyzed. The effect of XAV939 was confirmed in human umbilical vein endothelial cells. Results: LPS-induced expressions of pro-inflammatory genes IL-6, IL-8, TNF-α, IL-1β, MCP-1, MMP-9, iNOS and COX-2 were significantly and dose-dependently suppressed by XAV939. LPS-induced NF-κB signaling, such as IκB phosphorylation and degradation as well as nuclear translocation of NF-κB, was also suppressed by XAV939. Target DNA binding of NF-κB was significantly and dose-dependently suppressed by XAV939 during LPS-induced inflammatory response. The suppressive effects of XAV939 on NF-κB signaling, target DNA binding of NF-κB and pro-inflammatory gene expression were all rescued by over expression of β-catenin, which shows that the anti-inflammatory effect of XAV939 is mediated by the modulation of β-catenin, a central component of the WNT/β-catenin pathway. Conclusion: The findings of this study showed that XAV939 exerts anti-inflammatory effects through the modulation of the Wnt/β-catenin pathway.

Immune checkpoint blockade therapy requires a preestablished activated immune landscape. Understanding tumor-intrinsic mechanisms that lead to T-cell desertification is key to resensitizing them to such therapies. The WNT/β-catenin tumor-intrinsic signaling is emerging as an immune exclusion pathway that holds high promise to counteract resistance to immunotherapy. See related article by Luke et al., p. 3074 ., (©2019 American Association for Cancer Research.)

Intestinal stem cells (ISCs) are maintained by stemness signaling for precise modulation of self-renewal and differentiation under homeostasis. However, the way in which intestinal immune cells regulate the self-renewal of ISCs remains elusive. Here we found that mouse and human Lgr5 + ISCs showed high expression of the immune cell-associated circular RNA circPan3 (originating from the Pan3 gene transcript). Deletion of circPan3 in Lgr5 + ISCs impaired their self-renewal capacity and the regeneration of gut epithelium in a manner dependent on immune cells. circPan3 bound mRNA encoding the cytokine IL-13 receptor subunit IL-13Rα1 (Il13ra1) in ISCs to increase its stability, which led to the expression of IL-13Rα1 in ISCs. IL-13 produced by group 2 innate lymphoid cells in the crypt niche engaged IL-13Rα1 on crypt ISCs and activated signaling mediated by IL-13‒IL-13R, which in turn initiated expression of the transcription factor Foxp1. Foxp1 is associated with β-catenin in rendering its nuclear translocation, which caused activation of the β-catenin pathway and the maintenance of Lgr5 + ISCs.

Foxp3 + regulatory T cells (T reg cells) are the central component of peripheral immune tolerance. Whereas a dysregulated T reg cytokine signature has been observed in autoimmune diseases, the regulatory mechanisms underlying pro- and anti-inflammatory cytokine production are elusive. Here, we identify an imbalance between the cytokines IFN-γ and IL-10 as a shared T reg signature present in patients with multiple sclerosis and under high-salt conditions. RNA-sequencing analysis on human T reg subpopulations revealed β-catenin as a key regulator of IFN-γ and IL-10 expression. The activated β-catenin signature was enriched in human IFN-γ + T reg cells, as confirmed in vivo with T reg -specific β-catenin-stabilized mice exhibiting lethal autoimmunity with a dysfunctional T reg phenotype. Moreover, we identified prostaglandin E receptor 2 (PTGER2) as a regulator of IFN-γ and IL-10 production under a high-salt environment, with skewed activation of the β-catenin-SGK1-Foxo axis. Our findings reveal a novel PTGER2-β-catenin loop in T reg cells linking environmental high-salt conditions to autoimmunity.

Despite the dramatic antitumor efficiency of certain immune checkpoint inhibitors, immunotherapy has met a bottleneck regarding the response rate and resistance in cancer patients. Increasing evidence indicates that Wnt/β-catenin signaling, one of the best-characterized cancer drivers, promotes cancer progression by regulating the tumor-immune cycle in most of the nodes, including dendritic cells, T cells, and tumor cells. Specifically, abnormal Wnt/β-catenin signaling directly alters a number of regulators critical for the antitumor activities of T cells, especially effector T cells, T helper cells, and regulatory T cells. We propose that targeting Wnt/β-catenin signaling would potentially improve clinical outcomes of cancer patients by overcoming the primary, adaptive, and acquired resistance to immunotherapy., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

This work aims to discuss the possibility that disordered CUL4B was involved in the pathogenesis of adjuvant-induced arthritis (AIA) in rats. Synovium and FLS from AIA rats both showed increased CUL4B and β-catenin, and up-regulated CUL4B enhanced the canonical Wnt signaling by targeting the GSK3β. Increased CUL4B promoted the FLS abnormal proliferation, activated the secretion of IL-1β and IL-8, and promoted the production of AIA pathology gene MMP3 and fibronectin. Furthermore, miR-101-3p was significantly down-regulated in AIA rats compared with controls, and transfection of AIA FLS with miR-101-3p mimics significantly down-regulated the CUL4B expression, whereas transfection with miR-101-3p inhibitors resulted in an opposite observation. The dual-luciferase reporter assay confirmed that the CUL4B was a direct target of miR-101-3p, and further analysis suggested that lowly expressed miR-101-3p contributed to disordered CUL4B activating the canonical Wnt signaling pathway and further promoting the development of AIA rats. Thus clarification of the CUL4B roles in the pathogenesis of AIA rats and corresponding mechanisms will contribute to the disease diagnosis and treatment for rheumatoid arthritis (RA) patients., Key Messages: CUL4B expression is up-regulated in synovium and FLS from AIA rats. Increased CUL4B promotes the canonical Wnt signaling. Increased CUL4B promotes the pathogenesis of AIA rats. Decreased miR-101-3p contributes to disordered CUL4B.

Enriched PD-L1 expression in cancer stem-like cells (CSCs) contributes to CSC immune evasion. However, the mechanisms underlying PD-L1 enrichment in CSCs remain unclear. Here, we demonstrate that epithelial-mesenchymal transition (EMT) enriches PD-L1 in CSCs by the EMT/β-catenin/STT3/PD-L1 signaling axis, in which EMT transcriptionally induces N-glycosyltransferase STT3 through β-catenin, and subsequent STT3-dependent PD-L1 N-glycosylation stabilizes and upregulates PD-L1. The axis is also utilized by the general cancer cell population, but it has much more profound effect on CSCs as EMT induces more STT3 in CSCs than in non-CSCs. We further identify a non-canonical mesenchymal-epithelial transition (MET) activity of etoposide, which suppresses the EMT/β-catenin/STT3/PD-L1 axis through TOP2B degradation-dependent nuclear β-catenin reduction, leading to PD-L1 downregulation of CSCs and non-CSCs and sensitization of cancer cells to anti-Tim-3 therapy. Together, our results link MET to PD-L1 stabilization through glycosylation regulation and reveal it as a potential strategy to enhance cancer immunotherapy efficacy.

Immunotherapy prolongs survival in only a subset of melanoma patients, highlighting the need to better understand the driver tumor microenvironment. We conducted bioinformatic analyses of 703 transcriptomes to probe the immune landscape of primary cutaneous melanomas in a population-ascertained cohort. We identified and validated 6 immunologically distinct subgroups, with the largest having the lowest immune scores and the poorest survival. This poor-prognosis subgroup exhibited expression profiles consistent with β-catenin-mediated failure to recruit CD141+ DCs. A second subgroup displayed an equally bad prognosis when histopathological factors were adjusted for, while 4 others maintained comparable survival profiles. The 6 subgroups were replicated in The Cancer Genome Atlas (TCGA) melanomas, where β-catenin signaling was also associated with low immune scores predominantly related to hypomethylation. The survival benefit of high immune scores was strongest in patients with double-WT tumors for BRAF and NRAS, less strong in BRAF-V600 mutants, and absent in NRAS (codons 12, 13, 61) mutants. In summary, we report evidence for a β-catenin-mediated immune evasion in 42% of melanoma primaries overall and in 73% of those with the worst outcome. We further report evidence for an interaction between oncogenic mutations and host response to melanoma, suggesting that patient stratification will improve immunotherapeutic outcomes.

Natural killer T (NKT) cells are prominent innate-like lymphocytes in the liver with critical roles in immune responses during infection, cancer, and autoimmunity. Interferon gamma (IFN-γ) and IL-4 are key cytokines rapidly produced by NKT cells upon recognition of glycolipid antigens presented by antigen-presenting cells (APCs). It has previously been reported that the transcriptional coactivator β-catenin regulates NKT cell differentiation and functionally biases NKT cell responses toward IL-4, at the expense of IFN-γ production. β-Catenin is not only a central effector of Wnt signaling but also contributes to other signaling networks. It is currently unknown whether Wnt ligands regulate NKT cell functions. We thus investigated how Wnt ligands and β-catenin activity shape liver NKT cell functions in vivo in response to the glycolipid antigen, α-galactosylceramide (α-GalCer) using a mouse model. Pharmacologic targeting of β-catenin activity with ICG001, as well as myeloid-specific genetic ablation of Wntless (Wls) , to specifically target Wnt protein release by APCs, enhanced early IFN-γ responses. By contrast, within several hours of α-GalCer challenge, myeloid-specific Wls deficiency, as well as pharmacologic targeting of Wnt release using the small molecule inhibitor IWP-2 impaired α-GalCer-induced IFN-γ responses, independent of β-catenin activity. These data suggest that myeloid cell-derived Wnt ligands drive early Wnt/β-catenin signaling that curbs IFN-γ responses, but that, subsequently, Wnt ligands sustain IFN-γ expression independent of β-catenin activity. Our analyses in ICG001-treated mice confirmed a role for β-catenin activity in driving early IL-4 responses by liver NKT cells. However, neither pharmacologic nor genetic perturbation of Wnt production affected the IL-4 response, suggesting that IL-4 production by NKT cells in response to α-GalCer is not driven by released Wnt ligands. Collectively, these data reveal complex temporal roles of Wnt ligands and β-catenin signaling in the regulation of liver NKT cell activation, and highlight Wnt-dependent and -independent contributions of β-catenin to NKT cell functions.

Objective: The purpose of this study was to compare the immunohistochemical expression of BRCA1, Ki67, and β-catenin in women with low-grade (LGSOC) and high-grade serous ovarian carcinomas (HGSOC) and their relationship with clinicopathological features, response to platinum-based chemotherapy, and survival., Methods: For this study, 21 LGSOC and 85 HGSOC stage I to IV cases, diagnosed and treated from 1996 to 2013 and followed-up until December 2016, were included. BRCA1, Ki67, and β-catenin expression was assessed using tissue microarray-based immunohistochemistry., Results: Women with HGSOC were significantly more likely to have advanced-stage disease (P < 0.001), higher CA125 levels (P < 0.001), postsurgery residual disease (P < 0.01), and higher rates of disease progression and recurrence (P = 0.001). The percentage of women with HGSOC whose tumors expressed Ki67 was significantly higher compared with women with LGSOC (P < 0.001). The expression of BRCA1 and β-catenin did not differ between LGSOC and HGSOC (P = 0.12 and P = 1.00, respectively). The clinicopathological features and the response to platinum-based chemotherapy did not differ according to the BRCA1, Ki67, and β-catenin expression in either group. In HGSOC, only International Federation of Gynecology and Obstetrics stage was independently associated with poor survival (PFS and OS)., Conclusions: Ki67 expression was significantly higher in HGSOC. BRCA1 and β-catenin expression did not differ between LGSOC and HGSOC samples. BRCA1, Ki67, and β-catenin expression was neither related to clinicopathological features, response to platinum-based chemotherapy, nor survival. Only International Federation of Gynecology and Obstetrics stage remained associated with poor survival in women with HGSOC.

T helper cell 17 (Th17), one type of CD4 + T cell, plays an important role in regulating the acute lung injury (ALI) inflammatory response. Recent studies showed that Wnt/β-catenin pathway could modulate the differentiation and the function of CD4 + T cell. However, whether Wnt/β-catenin could regulate the differentiation and function of Th17 in the development and progress of ALI induced by lipopolysaccharide (LPS) is still unknown. To test this, we used dickkopf1 (Dkk-1) to block the Wnt/β-catenin pathway and LiCl to activate the Wnt/β-catenin pathway by instillation to the murine model of ALI. Our results revealed that activation of Wnt/β-catenin pathway significantly aggravated the LPS-induced lung inflammation. Meanwhile, we observed that activation of Wnt/β-catenin pathway promoted Th17 response by analyzing CD4 + T cells and the related cytokines secretions. Enhanced Th17 response was responsible for the further neutrophils infiltration and pro-inflammatory cytokines production. In addition, activation of Wnt/β-catenin pathway resulted in induced expression of retinoic acid related orphan receptor-γt (RORγt) via histone acetyltransferase p300. These data suggested that Wnt/β-catenin pathway might be a potential target to treat the LPS-induced inflammation in ALI., (Copyright © 2017 Elsevier Inc. All rights reserved.)

β-Catenin is essential for embryonic development and required for cell renewal/regeneration in adult life. Cellular β-catenin exists in three different pools: membranous, cytoplasmic and nuclear. In this review, we focus on functions of the nuclear pool in relation to tumorigenesis. In the nucleus, beta-catenin functions as both activator and repressor of transcription in a context-dependent manner. It promotes cell proliferation and supports tumour growth by enhancing angiogenesis. β-Catenin-mediated signalling regulates cancer cell metabolism and is associated with tumour-initiating cells in multiple malignancies. In addition, it functions as both pro- and anti-apoptotic factor besides acting to inhibit recruitment of inflammatory anti-tumour T-cells. Thus, β-catenin appears to possess a multifaceted nuclear function that may significantly impact tumour initiation and progression.

Mycotoxin T-2 exerts a causative role in Kashin-Beck disease (KBD) suffering chondrocyte apoptosis and cartilage matrix homeostasis disruption. Recent research corroborated the aberrant levels of pro-inflammatory cytokine IL-1ß in KBD patients and mycotoxin environment. In the present study, we investigated the relevance of IL-1ß in T-2 toxin-evoked chondrocyte cytotoxic injury and aberrant catabolism. High levels of IL-1ß were detected in serum and cartilages from KBD patients and in T-2-stimulated chondrocytes. Moreover, knockdown of IL-1ß antagonized the adverse effects of T-2 on cytotoxic injury by enhancing cell viability and inhibiting apoptosis. However, exogenous supplementation of IL-1β further aggravated cell damage in response to T-2. Additionally, cessation of IL-1β rescued T-2-elicited tilt of matrix homeostasis toward catabolism by elevating the transcription of collagen II and aggrecan, promoting release of sulphated glycosaminoglycans (sGAG) and TIMP1, and suppressing matrix metalloproteinases production including MMP-1, MMP-3 and MMP-13. Conversely, IL-1β stimulation deteriorated T-2-induced disruption of matrix metabolism balance toward catabolism. Mechanistic analysis found the high activation of Wnt/β-catenin in KBD patients and chondrocytes upon T-2. Furthermore, this activation was mitigated after IL-1β inhibition, but further enhanced following IL-1β precondition. Importantly, blocking this pathway by transfection with β-catenin alleviated the adverse roles of IL-1β on cytotoxic injury and metabolism disorders under T-2 conditioning. Together, this study elucidates a new insight into how T-2 deteriorates the pathological progression of KBD by regulating inflammation-related pathways, indicating a promising anti-inflammation strategy for KBD therapy., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

The mechanisms leading to autoimmune and inflammatory diseases in the CNS have not been elucidated. The environmental triggers of the aberrant presence of CD4 + T cells in the CNS are not known. In this article, we report that abnormal β-catenin expression in T cells drives a fatal neuroinflammatory disease in mice that is characterized by CNS infiltration of T cells, glial activation, and progressive loss of motor function. We show that enhanced β-catenin expression in T cells leads to aberrant and Th1-biased T cell activation, enhanced expression of integrin α4β1, and infiltration of activated T cells into the spinal cord, without affecting regulatory T cell function. Importantly, expression of β-catenin in mature naive T cells was sufficient to drive integrin α4β1 expression and CNS migration, whereas pharmacologic inhibition of integrin α4β1 reduced the abnormal T cell presence in the CNS of β-catenin-expressing mice. Together, these results implicate deregulation of the Wnt/β-catenin pathway in CNS inflammation and suggest novel therapeutic strategies for neuroinflammatory disorders., (Copyright © 2017 by The American Association of Immunologists, Inc.)

CD4 + Foxp3 + regulatory T (Treg) cells include differentiated populations of effector Treg cells characterized by the expression of specific transcription factors. Tumours, including intestinal malignancies, often present with local accumulation of Treg cells that can prevent tumour clearance, but how tumour progression leads to Treg cell accumulation is incompletely understood. Here using genetically modified mouse models we show that ablation of E-cadherin, a process associated with epithelial to mesenchymal transition and tumour progression, promotes the accumulation of intestinal Treg cells by the specific accumulation of the KLRG1 + GATA3 + Treg subset. Epithelial E-cadherin ablation activates the β-catenin pathway, and we find that increasing β-catenin signals in intestinal epithelial cells also boosts Treg cell frequencies through local accumulation of KLRG1 + GATA3 + Treg cells. Both E-cadherin ablation and increased β-catenin signals resulted in epithelial cells with higher levels of interleukin-33, a cytokine that preferentially expands KLRG1 + GATA3 + Treg cells. Tumours often present reduced E-cadherin expression and increased β-catenin signalling and interleukin-33 production. Accordingly, Treg cell accumulation in intestinal tumours from APC min/+ mice was exclusively due to the increase in KLRG1 + GATA3 + Treg cells. Our data identify a novel axis through which epithelial cells control local Treg cell subsets, which may be activated during intestinal tumorigenesis., (© 2017 John Wiley & Sons Ltd.)

Background: Immunohistochemical staining with conventional anti-β-catenin antibody has been applied as a diagnostic tool for desmoid-type fibromatosis (DF). This study aimed to evaluate the diagnostic and prognostic value of immunohistochemical staining with anti-non-phospho β-catenin antibody, which might more accurately reflect the aggressiveness of DF, in comparison to the conventional anti-β-catenin antibody., Methods: Between 2003 and 2015, 40 patients with extra-peritoneal sporadic DF were prospectively treated with meloxicam or celecoxib, a COX-2 inhibitor, therapy. The efficacy of this treatment was evaluated according to Response Evaluation Criteria in Solid Tumors (RECIST). Immunohistochemical staining was performed on formalin-fixed material to evaluate the expression of β-catenin and non-phospho β-catenin, and the positivity was grouped as negative, weak, moderate, and strong. DNA was isolated from frozen tissue or formalin-fixed materials, and the CTNNB1 mutation status was determined by direct sequencing., Results: Of the 40 patients receiving COX-2 inhibitor treatment, there was one with complete remission, 12 with partial remission, 7 with stable disease, and 20 with progressive disease. The mutation sites in CTNNB1 were detected in 22 (55%) of the 40 cases: T41A (17 cases), S45F (3 cases), and T41I and S45P (1 each). The positive nuclear expression of non-phospho β-catenin showed a significant correlation with positive CTNNB1 mutation status detected by Sanger method (p = 0.025), and poor outcome in COX-2 inhibitor therapy (p = 0.022). In contrast, nuclear expression of β-catenin did not show a significant correlation with either CTNNB1 mutation status (p = 0.43) or outcome of COX-2 inhibitor therapy (p = 0.38)., Conclusions: Nuclear expression of non-phospho β-catenin might more appropriately reflect the biological behavior of DF, and immunohistochemical staining with non-phospho β-catenin could serve as a more useful diagnostic and prognostic tool of COX-2 inhibitor therapy for patients with DF.

The etiology determines quality and extent of the immune response after udder infection (mastitis). Infections with Gram negative bacteria (e.g. Escherichia coli) will quickly elicit strong inflammation of the udder, fully activate its immune defence via pathogen receptor driven activation of IκB/NF-κB signaling. This often eradicates the pathogen. In contrast, Gram-positive bacteria (e.g. Staphylococcus aureus) will slowly elicit a much weaker inflammation and immune response, frequently resulting in chronic infections. However, it was unclear which immune regulatory pathways are specifically triggered by S. aureus causing this partial immune subversion. We therefore compared in first lactating cows the earliest (1-3 h) udder responses against infection with mastitis causing pathogens of either species. Global transcriptome profiling, bioinformatics analysis and experimental validation of key aspects revealed as S. aureus infection specific features the (i) failure to activating IκB/NF-κB signaling; (ii) activation of the wnt/β-catenin cascade resulting in active suppression of NF-κB signaling and (iii) rearrangement of the actin-cytoskeleton through modulating Rho GTPase regulated pathways. This facilitates invasion of pathogens into host cells. Hence, S. aureus mastitis is characterized by eliciting unbalanced immune suppression rather than inflammation and invasion of S. aureus into the epithelial cells of the host causing sustained infection.

Objective To investigate β-catenin-mediated bacterial elimination during Pseudomonas aeruginosa infection of macrophage-like RAW264.7 cells. Methods Cell viability and catenin beta 1 ( CTNNB1) expression in RAW264.7 cells following P. aeruginosa infection versus uninfected cells, were detected by cell counting kit-8 assay and β-catenin Western blots. RAW264.7 cells with CTNNB1 overexpression were established with β-catenin lentivirus using flow cytometry and clonogenic limiting dilution assays. Bacterial killing was measured by plate counts; phagocytosis and nitric oxide (NO) were measured by flow cytometry; and reactive oxygen species (ROS) were measured using Griess reaction. Autophagy was determined by microtubule-associated protein 1 light chain 3 alpha-phosphatidylethanolamine conjugate (LC3-II) protein levels and formation of LC3 puncta, using Western blot and immunofluorescence staining. Results Following P. aeruginosa infection, RAW264.7 cell β-catenin levels were reduced in a time- and multiplicity of infection-dependent manner. CTNNB1 overexpression was associated with increased P. aeruginosa elimination, but had no effect on RAW264.7 cell phagocytosis, ROS and NO. CTNNB1 overexpression reduced LC3-II levels and formation of LC3 puncta, suggesting autophagy inhibition. Rapamycin/starvation-induced autophagy resulted in reduced bacterial killing following P. aeruginosa infection. Conclusion β-catenin may promote bacterial killing via suppression of P. aeruginosa-induced macrophage autophagy.

Cephalochordates, commonly called amphioxus or lancelets, are widely regarded as a useful proxy for the chordate ancestor. In recent decades, expression patterns of important developmental genes have been used extensively to infer homologies between cephalochordate and vertebrate embryos. Such comparisons provided important insight into cephalochordate biology and the origin of vertebrate traits. Most of the developmental expression data are collected using whole-mount in situ hybridization that allows the distributions of specific transcripts to be detected in fixed embryos. Here, we describe an experimental pipeline for production of small amounts of functional antibodies directed against amphioxus antigens for use in immunohistochemical labelling. In this pilot study, we generated antibodies against β-catenin and the transcription factors FoxA, Lhx1, Lhx3 and Pax6. We demonstrate the usefulness of antibodies by performing immunostainings on fixed specimens of B. lanceolatum and B. floridae. We anticipate that amphioxus-specific antibodies will provide a useful tool for high-resolution labelling of individual cells within the embryo and for determining the subcellular localization of the corresponding proteins.

Intestinal epithelial cells form a single layer separating the intestinal lumen containing nutriments and microbiota from the underlying sterile tissue and therefore play a key role in maintaining homeostasis. We investigated the factors contributing to the alteration of the epithelial barrier function during Cryptosporidium parvum infection. Infected polarized epithelial cell monolayers exhibit a drop in transepithelial resistance associated with a delocalization of E-cadherin and β-catenin from their intercellular area of contact, the adherens junction complex. In neonatal mice infected by C. parvum, the increased permeability is correlated with parasite development and with an important recruitment of Ly6c + inflammatory monocytes to the subepithelial space. TNFα and IL-1β produced by inflammatory monocytes play a key role in the loss of barrier function. Our findings demonstrate for the first time that both the parasite and inflammatory monocytes contribute to the loss of intestinal barrier function during cryptosporidiosis., (© 2016 John Wiley & Sons Ltd.)

A characteristic feature of gastrointestinal tract inflammatory disorders, such as inflammatory bowel disease, is polymorphonuclear neutrophil (PMN) transepithelial migration (TEM) and accumulation in the gut lumen. PMN accumulation within the intestinal mucosa contributes to tissue injury. Although epithelial infiltration by large numbers of PMNs results in mucosal injury, we found that PMN interactions with luminal epithelial membrane receptors may also play a role in wound healing. Intercellular adhesion molecule-1 (ICAM-1) is a PMN ligand that is upregulated on apical surfaces of intestinal epithelial cells under inflammatory conditions. In our study, increased expression of ICAM-1 resulted in enhanced PMN binding to the apical epithelium, which was associated with reduced PMN apoptosis. Following TEM, PMN adhesion to ICAM-1 resulted in activation of Akt and β-catenin signaling, increased epithelial-cell proliferation, and wound healing. Such responses were ICAM-1 dependent as engagement of epithelial ICAM-1 by antibody-mediated cross-linking yielded similar results. Furthermore, using an in-vivo biopsy-based, colonic-mucosal-injury model, we demonstrated epithelial ICAM-1 has an important role in activation of epithelial Akt and β-catenin signaling and wound healing. These findings suggest that post-migrated PMNs within the intestinal lumen can regulate epithelial homeostasis, thereby identifying ICAM-1 as a potential therapeutic target for promoting mucosal wound healing.