The Wnt Antagonist Dickkopf-1 Promotes Pathological Type 2 Cell-Mediated Inflammation.

Exposure to a plethora of environmental challenges commonly triggers pathological type 2 cell-mediated inflammation. Here we report the pathological role of the Wnt antagonist Dickkopf-1 (Dkk-1) upon allergen challenge or non-healing parasitic infection. The increased circulating amounts of Dkk-1 polarized T cells to T helper 2 (Th2) cells, stimulating a marked simultaneous induction of the transcription factors c-Maf and Gata-3, mediated by the kinases p38 MAPK and SGK-1, resulting in Th2 cell cytokine production. Circulating Dkk-1 was primarily from platelets, and the increase of Dkk-1 resulted in formation of leukocyte-platelet aggregates (LPA) that facilitated leukocyte infiltration to the affected tissue. Functional inhibition of Dkk-1 impaired Th2 cell cytokine production and leukocyte infiltration, protecting mice from house dust mite (HDM)-induced asthma or Leishmania major infection. These results highlight that Dkk-1 from thrombocytes is an important regulator of leukocyte infiltration and polarization of immune responses in pathological type 2 cell-mediated inflammation.

Colon cancer cells acquire immune regulatory molecules from tumor-infiltrating lymphocytes by trogocytosis.

Cancer cells can develop an immunosuppressive tumor microenvironment to control tumor-infiltrating lymphocytes. The underlying mechanisms still remain unclear. Here, we report that mouse and human colon cancer cells acquire lymphocyte membrane proteins including cellular markers such as CD4 and CD45. We observed cell populations harboring both a tumor-specific marker and CD4 in the tumor microenvironment. Sorted cells from these populations were capable of forming organoids, identifying them as cancer cells. Live imaging analysis revealed that lymphocyte membrane proteins were transferred to cancer cells via trogocytosis. As a result of the transfer in vivo, cancer cells also acquired immune regulatory surface proteins such as CTLA4 and Tim3, which suppress activation of immune cells [T. L. Walunas et al, Immunity 1, 405-413 (1994) and L. Monney et al., Nature 415, 536-541 (2002)]. RNA sequencing analysis of ex vivo-cocultured splenocytes with trogocytic cancer cells showed reductions in Th1 activation and natural killer cell signaling pathways compared with the nontrogocytic control. Cancer cell trogocytosis was confirmed in the patient-derived xenograft models of colorectal cancer and head and neck cancer. These findings suggest that cancer cells utilize membrane proteins expressed in lymphocytes, which in turn contribute to the development of the immunosuppressive tumor microenvironment.

Positive conversion of negative signaling of CTLA4 potentiates antitumor efficacy of adoptive T-cell therapy in murine tumor models.

Cytotoxic T lymphocyte-associated antigen 4 (CTLA4) has been known to be a strong tolerance-inducing inhibitory receptor on T-cell surface. Systemic blocking of CTLA4 function with blocking antibodies has been regarded as an attractive strategy to enhance antitumor immunity. However, this strategy accompanies systemic autoimmune side effects that are sometimes problematic. Therefore, we developed a novel CTLA4 mutant that could be expressed in tumor antigen-specific T cells to enhance antitumor effect without systemic autoimmunity. This mutant, named CTLA4-CD28 chimera, consists of extracellular and transmembrane domains of CTLA4, linked with cytoplasmic CD28 domain. Overexpression of CTLA4-CD28 chimera in T cells delivered stimulatory signals rather than inhibitory signals of CTLA4 and significantly enhanced T-cell reactivity. Although this effect was observed in both CD4 and CD8 T cells, the effect on CD4 T cells was predominant. CTLA4-CD28 chimera gene modification of CD4 T cells significantly enhanced antitumor effect of unmodified CD8 T cells. Nonetheless, the gene modification of CD8 T cells along with CD4 T cells further maximized antitumor effect of T cells in 2 different murine tumor models. Thus, CTLA4-CD28 chimera gene modification of both tumor antigen-specific CD4 and CD8 T cells would be an ideal way of modulating CTLA4 function to enhance tumor-specific T-cell reactivity.

Adenovirus expressing both thymidine kinase and soluble PD1 enhances antitumor immunity by strengthening CD8 T-cell response.

Adenoviruses harboring the herpes simplex virus thymidine kinase (HSVtk) gene under the regulation of a trans-splicing ribozyme targeting human telomerase reverse transcriptase (hTERT-TR) show marked and specific antitumor activity. In addition to inducing tumor cell death by direct cytotoxicity, it is becoming clear that HSVtk also induces antitumor immunity. Programmed death ligand 1 (PD-L1) expressed on tumor cell surfaces mediates tumor-induced immunoresistance by inhibiting PD1-expressing tumor-infiltrating T cells. Here, we explored whether a soluble form of PD1 (sPD1-Ig), which blocks PD-L1, could synergize with TERT-TR-regulated HSVtk to enhance the adenoviral therapeutic efficacy by boosting antitumor immunity. Tumor antigen released by HSVtk-transduced tumors successfully primed tumor antigen-specific CD8 T cells via dendritic cells (DC). Regression of murine tumors was markedly enhanced when sPD1-Ig was incorporated into the adenovirus as compared with a single-module adenovirus expressing only HSVtk. This effect was abolished by CD8 T-cell depletion. Consistent with this, following adoptive transfer of tumor antigen-specific CD8 T cells into tumor-bearing Rag1(-/-) mice, dual-module adenovirus significantly enhanced CD8 T cell-mediated tumor rejection. In addition, secondary tumor challenge at a distal site was completely suppressed in mice treated with a dual-module adenovirus. These results suggest that a dual-targeting strategy to elicit both tumor antigen priming and tumor-induced immunoresistance enhances CD8 T cell-mediated antitumor immunity.

Intercellular Transfer of Immune Regulatory Molecules Via Trogocytosis.

Trogocytosis, an active cellular process involving the transfer of plasma membrane and attached cytosol during cell-to-cell contact, has been observed prominently in CD4 T cells interacting with antigen-presenting cells carrying antigen-loaded major histocompatibility complex (MHC) class II molecules. Despite the inherent absence of MHC class II molecules in CD4 T cells, they actively acquire these molecules from encountered antigen-presenting cells, leading to the formation of antigen-loaded MHC class II molecules-dressed CD4 T cells. Subsequently, these dressed CD4 T cells engage in antigen presentation to other CD4 T cells, revealing a dynamic mechanism of immune communication. The transferred membrane proteins through trogocytosis retain their surface localization, thereby altering cellular functions. Concurrently, the donor cells experience a loss of membrane proteins, resulting in functional changes due to the altered membrane properties. This chapter provides a focused exploration into trogocytosis-mediated transfer of immune regulatory molecules and its consequential impact on diverse immune responses.

Metastasis of colon cancer requires Dickkopf-2 to generate cancer cells with Paneth cell properties.

Metastasis is the leading cause of cancer-related mortality. Paneth cells provide stem cell niche factors in homeostatic conditions, but the underlying mechanisms of cancer stem cell niche development are unclear. Here we report that Dickkopf-2 (DKK2) is essential for the generation of cancer cells with Paneth cell properties during colon cancer metastasis. Splenic injection of Dkk2-knockout (KO) cancer organoids into C57BL/6 mice resulted in a significant reduction of liver metastases. Transcriptome analysis showed reduction of Paneth cell markers such as lysozymes in KO organoids. Single cell RNA sequencing analyses of murine metastasized colon cancer cells and patient samples identified the presence of lysozyme positive cells with Paneth cell properties including enhanced glycolysis. Further analyses of transcriptome and chromatin accessibility suggested Hepatocyte nuclear factor 4-alpha (HNF4A) as a downstream target of DKK2. Chromatin immunoprecipitation followed by sequencing analysis revealed that HNF4A binds to the promoter region of Sox9, a well-known transcription factor for Paneth cell differentiation. In the liver metastatic foci, DKK2 knockout rescued HNF4A protein levels followed by reduction of lysozyme positive cancer cells. Taken together, DKK2-mediated reduction of HNF4A protein promotes the generation of lysozyme positive cancer cells with Paneth cell properties in the metastasized colon cancers.

Cell-permeable Foxp3 protein alleviates autoimmune disease associated with inflammatory bowel disease and allergic airway inflammation.

Foxp3 is a key transcription factor for differentiation and function of regulatory T (Treg) cells that is critical for maintaining immunological self-tolerance. Therefore, increasing Treg function by Foxp3 transduction to regulate an inflammatory immune response is an important goal for the treatment of autoimmune and allergic diseases. Here we have generated a cell-permeable Foxp3 protein by fusion with the unique human HHph-1-PTD (protein transduction domain), examined its regulatory function in T cells, and characterized its therapeutic effect in autoimmune and allergic disease models. HHph-1-Foxp3 was rapidly and effectively transduced into cells within 30 min and conferred suppressor function to CD4(+)CD25(-) T cells as well as directly inhibiting T-cell activation and proliferation. Systemic delivery of HHph-1 Foxp3 remarkably inhibited the autoimmune symptoms of scurfy mice and the development of colitis induced by scurfy or wild-type CD4 T cells. Moreover, intranasal delivery of HHph-1-Foxp3 strongly suppressed ovalbumin-induced allergic airway inflammation. These results demonstrate the clinical potential of the cell-permeable recombinant HHph-1-Foxp3 protein in autoimmune and hypersensitive allergic diseases.

Fear of COVID-19 and its Differential Effect on Attitudes and Intentions towards Online and Face-to-Face Counseling.

The present study examines the impact of fear of COVID-19 on attitudes and intentions towards online and face-to-face counseling. A total of 526 adults participated in this study. The path analysis results indicated that attitude towards online and face-to-face counseling differentially mediated the relationship between COVID-19 fear and both counseling intentions, even when controlling for other covariates. Specifically, fear of COVID-19 predicted positive attitudes towards online counseling (value of counseling), which in turn, predicted online counseling intention. On the other hand, COVID-19 fear affected negative attitudes towards face-to-face counseling (discomfort with counseling), resulting in a decrease in face-to-face counseling intention.

Flow goes forward and cells step backward: endothelial migration.

Systemic and pulmonary circulations constitute a complex organ that serves multiple important biological functions. Consequently, any pathological processing affecting the vasculature can have profound systemic ramifications. Endothelial and smooth muscle are the two principal cell types composing blood vessels. Critically, endothelial proliferation and migration are central to the formation and expansion of the vasculature both during embryonic development and in adult tissues. Endothelial populations are quite heterogeneous and are both vasculature type- and organ-specific. There are profound molecular, functional, and phenotypic differences between arterial, venular and capillary endothelial cells and endothelial cells in different organs. Given this endothelial cell population diversity, it has been challenging to determine the origin of endothelial cells responsible for the angiogenic expansion of the vasculature. Recent technical advances, such as precise cell fate mapping, time-lapse imaging, genome editing, and single-cell RNA sequencing, have shed new light on the role of venous endothelial cells in angiogenesis under both normal and pathological conditions. Emerging data indicate that venous endothelial cells are unique in their ability to serve as the primary source of endothelial cellular mass during both developmental and pathological angiogenesis. Here, we review recent studies that have improved our understanding of angiogenesis and suggest an updated model of this process.

Dickkopf proteins in pathological inflammatory diseases.

The human body encounters various challenges. Tissue repair and regeneration processes are augmented after tissue injury to reinstate tissue homeostasis. The Wnt pathway plays a crucial role in tissue repair since it induces target genes required for cell proliferation and differentiation. Since tissue injury causes inflammatory immune responses, it has become increasingly clear that the Wnt ligands can function as immunomodulators while critical for tissue homeostasis. The Wnt pathway and Wnt ligands have been studied extensively in cancer biology and developmental biology. While the Wnt ligands are being studied actively, how the Wnt antagonists and their regulatory mechanisms can modulate immune responses during chronic pathological inflammation remain elusive. This review summarizes DKK family proteins as immunomodulators, aiming to provide an overarching picture for tissue injury and repair. To this end, we first review the Wnt pathway components and DKK family proteins. Next, we will review DKK family proteins (DKK1, 2, and 3) as a new class of immunomodulatory protein in cancer and other chronic inflammatory diseases. Taken together, DKK family proteins and their immunomodulatory functions in chronic inflammatory disorders provide novel insights to understand immune diseases and make them attractive molecular targets for therapeutic intervention.

MAL2 mediates the formation of stable HER2 signaling complexes within lipid raft-rich membrane protrusions in breast cancer cells.

The lipid raft-resident protein, MAL2, has been implicated as contributing to the pathogenesis of several malignancies, including breast cancer, but the underlying mechanism for its effects on tumorigenesis is unknown. Here, we show that MAL2-mediated lipid raft formation leads to HER2 plasma membrane retention and enhanced HER2 signaling in breast cancer cells. We demonstrate physical interactions between HER2 and MAL2 in lipid rafts using proximity ligation assays. Super-resolution structured illumination microscopy imaging displays the structural organization of the HER2/Ezrin/NHERF1/PMCA2 protein complex. Formation of this protein complex maintains low intracellular calcium concentrations in the vicinity of the plasma membrane. HER2/MAL2 protein interactions in lipid rafts are enhanced in trastuzumab-resistant breast cancer cells. Our findings suggest that MAL2 is crucial for lipid raft formation, HER2 signaling, and HER2 membrane stability in breast cancer cells, suggesting MAL2 as a potential therapeutic target.

Dickkopf-2 regulates the stem cell marker LGR5 in colorectal cancer via HNF4α1.

Enhanced stemness in colorectal cancer has been reported and it contributes to aggressive progression, but the underlying mechanisms remain unclear. Here we report a Wnt ligand, Dickkopf-2 (DKK2) is essential for developing colorectal cancer stemness. Genetic depletion of DKK2 in intestinal epithelial or stem cells reduced tumorigenesis and expression of the stem cell marker genes including LGR5 in a model of colitis-associated cancer. Sequential mutations in APC, KRAS, TP53, and SMAD4 genes in colonic organoids revealed a significant increase of DKK2 expression by APC knockout and further increased by additional KRAS and TP53 mutations. Moreover, DKK2 activates proto-oncogene tyrosine-protein kinse Src followed by increased LGR5 expressing cells in colorectal cancer through degradation of HNF4α1 protein. These findings suggest that DKK2 is required for colonic epithelial cells to enhance LGR5 expression during the progression of colorectal cancer.

Virilizing adrenocortical oncocytoma in a child: a case report.

Functioning adrenocortical oncocytomas are extremely rare and most reported patients are 40-60 yr of age. To our knowledge, only 2 cases of functioning adrenocortical oncocytomas have been reported in childhood. We report a case of functioning adrenocortical oncocytoma in a 14-yr-old female child presenting with virilization. She presented with deepening of the voice and excessive hair growth, and elevation of plasma testosterone and dehydroepiandrosterone sulfate. She had an adrenalectomy. The completely resected tumor composed predominantly of oncocytes without atypical mitosis and necrosis. A discussion of this case and a review of the literature on this entity are presented.

Genetic risk score combining six genetic variants associated with the cellular NRF2 expression levels correlates with Type 2 diabetes in the human population.

BACKGROUND: Type 2 diabetes (T2D) is known as an inflammatory disease. NRF2 (Nuclear Factor Erythroid 2 Like2) encodes a transcription factor that binds to antioxidant response elements (AREs) and regulates the expression of genes involved in many antioxidant responses. OBJECTIVE: This study aimed to gain insight into individual anti-inflammatory activity to prevent T2D development in humans. METHODS: We performed a genome-wide association study (GWAS) to identify genetic variants influencing NRF2 expression in LCLs (lymphoblastoid cell lines) generated from 74 different individuals. Association analyses between T2D or its related traits and genetic risk score (GRS) calculated by combining genetic variants detected from GWAS for cellular NRF2 expression were performed using data from 8715 subjects. The T2D prediction model using GRS was evaluated by measuring the area under the curve (AUC) of the receiver operating characteristics (ROC) curve. RESULTS: Our GWAS identified six genetic variants (SNP) showing suggestive evidence of associations with cellular NRF2 expression (P < 10- 6). Logistic regression analysis demonstrated that GRS was associated with an increased risk of T2D (P value = 0.003, OR = 1.13). In addition, linear regression analyses showed positive associations between GRS and fasting glucose (P value = 0.028, ß = 0.62), 2-h glucose (P value = 0.0004, ß = 1.13) and HbA1C (P value = 0.033, ß = 0.03). In the T2D prediction model using GRS, the AUC of the ROC curve was 0.69. CONCLUSION: This study highlights genetic variants associated with cellular NRF2 expression and suggests that the GRS of NRF2 expression-associated variants is likely to be a useful indicator of T2D development in the human population.

Enhanced osteogenesis of ß-tricalcium phosphate reinforced silk fibroin scaffold for bone tissue biofabrication.

Scaffolds, used for tissue regeneration are important to preserve their function and morphology during tissue healing. Especially, scaffolds for bone tissue engineering should have high mechanical properties to endure load of bone. Silk fibroin (SF) from Bombyx mori silk cocoon has potency as a type of biomaterials in the tissue engineering. ß-tricalcium phosphate (ß-TCP) as a type of bioceramics is also critical as biomaterials for bone regeneration because of its biocompatibility, osteoconductivity, and mechanical strength. The aim of this study was to fabricate three-dimensional SF/ß-TCP scaffolds and access its availability for bone grafts through in vitro and in vivo test. The scaffolds were fabricated in each different ratios of SF and ß-TCP (100:0, 75:25, 50:50, 25:75). The characterizations of scaffolds were conducted by FT-IR, compressive strength, porosity, and SEM. The in vitro and in vivo tests were carried out by MTT, ALP, RT-PCR, SEM, µ-CT, and histological staining. We found that the SF/ß-TCP scaffolds have high mechanical strength and appropriate porosity for bone tissue engineering. The study showed that SF/ß-TCP (75:25) scaffold exhibited the highest osteogenesis compared with other scaffolds. The results suggested that SF/ß-TCP (75:25) scaffold can be applied as one of potential bone grafts for bone tissue engineering.

Enhanced Anti-tumor Reactivity of Cytotoxic T Lymphocytes Expressing PD-1 Decoy.

Programmed death-1 (PD-1) is a strong negative regulator of T lymphocytes in tumor-microenvironment. By engaging PD-1 ligand (PD-L1) on tumor cells, PD-1 on T cell surface inhibits anti-tumor reactivity of tumor-infiltrating T cells. Systemic blockade of PD-1 function using blocking antibodies has shown significant therapeutic efficacy in clinical trials. However, approximately 10 to 15% of treated patients exhibited serious autoimmune responses due to the activation of self-reactive lymphocytes. To achieve selective activation of tumor-specific T cells, we generated T cells expressing a dominant-negative deletion mutant of PD-1 (PD-1 decoy) via retroviral transduction. PD-1 decoy increased IFN-γ secretion of antigen-specific T cells in response to tumor cells expressing the cognate antigen. Adoptive transfer of PD-1 decoy-expressing T cells into tumor-bearing mice potentiated T cell-mediated tumor regression. Thus, T cell-specific blockade of PD-1 could be a useful strategy for enhancing both efficacy and safety of anti-tumor T cell therapy.

Ndrg1 is a T-cell clonal anergy factor negatively regulated by CD28 costimulation and interleukin-2.

Induction of T-cell clonal anergy involves serial activation of transcription factors, including NFAT and Egr2/3. However, downstream effector mechanisms of these transcription factors are not fully understood yet. Here we identify Ndrg1 as an anergy factor induced by Egr2. Ndrg1 is upregulated by anergic signalling and maintained at high levels in resting anergic T cells. Overexpression of Ndrg1 mimics the anergic state and knockout of the gene prevents anergy induction. Interestingly, Ndrg1 is phosphorylated and degraded by CD28 signalling in a proteasome-dependent manner, explaining the costimulation dependence of anergy prevention. Similarly, IL-2 treatment of anergic T cells, under conditions that lead to the reversal of anergy, also induces Ndrg1 phosphorylation and degradation. Finally, older Ndrg1-deficient mice show T-cell hyperresponsiveness and Ndrg1-deficient T cells aggravate inducible autoimmune inflammation. Thus, Ndrg1 contributes to the maintenance of clonal anergy and inhibition of T-cell-mediated inflammation.