Increased Regulatory T Cells and Decreased Myeloid-Derived Suppressor Cells Induced by High CCL17 Levels May Account for Normal Incidence of Cancers among Patients with Atopic Dermatitis.

The incidence of cancers in atopic dermatitis (AD) is not increased, although the Th2-dominant environment is known to downregulate tumor immunity. To gain mechanistic insights regarding tumor immunity in AD, we utilized CCL17 transgenic (TG) mice overexpressing CCL17, which is a key chemokine in AD. Tumor formation and lung metastasis were accelerated in CCL17 TG mice when melanoma cells were injected subcutaneously or intravenously. Flow cytometric analysis showed increases in regulatory T cells (Tregs) in lymph nodes in CCL17 TG mice with high mRNA levels of IL-10 and Foxp3 in tumors, suggesting that Tregs attenuated tumor immunity. The frequency of myeloid-derived suppressor cells (MDSCs), however, was significantly decreased in tumors of CCL17 TG mice, suggesting that decreased MDSCs might promote tumor immunity. Expression of CXCL17, a chemoattractant of MDSCs, was decreased in tumors of CCL17 TG mice. Depletion of Tregs by the anti-CD25 antibody markedly reduced tumor volumes in CCL17 TG mice, suggesting that tumor immunity was accelerated by the decrease in MDSCs in the absence of Tregs. Thus, CCL17 attenuates tumor immunity by increasing Tregs and Th2 cells, while it decreases MDSCs through reductions in CXCL17, which may work as a "safety-net" to reduce the risk of malignant tumors in the Th2-dominant environment.

TLR2 Deficiency Exacerbates Imiquimod-Induced Psoriasis-Like Skin Inflammation through Decrease in Regulatory T Cells and Impaired IL-10 Production.

Emerging evidence has demonstrated that Toll-like receptors (TLRs) are associated with autoimmune diseases. In this study, we investigated the role of TLR2 in psoriasis using imiquimod-induced psoriasis-like dermatitis. Although TLR2 signaling is known to play a critical role in the induction of proinflammatory cytokines by immune cells, such as dendritic cells (DCs), macrophages, and monocytes, TLR2 deficiency unexpectedly exacerbated psoriasiform skin inflammation. Importantly, messenger RNA (mRNA) levels of Foxp-3 and IL-10 in the lesional skin were significantly decreased in TLR2 KO mice compared with wild-type mice. Furthermore, flow cytometric analysis of the lymph nodes revealed that the frequency of regulatory T cells (Tregs) among CD4-positive cells was decreased. Notably, stimulation with Pam3CSK4 (TLR2/1 ligand) or Pam2CSK4 (TLR2/6 ligand) increased IL-10 production from Tregs and DCs and the proliferation of Tregs. Finally, adoptive transfer of Tregs from wild-type mice reduced imiquimod-induced skin inflammation in TLR2 KO mice. Taken together, our results suggest that TLR2 signaling directly enhances Treg proliferation and IL-10 production by Tregs and DCs, suppressing imiquimod-induced psoriasis-like skin inflammation. Enhancement of TLR2 signaling may be a new therapeutic strategy for psoriasis.

CX3CR1 Deficiency Attenuates DNFB-Induced Contact Hypersensitivity Through Skewed Polarization Towards M2 Phenotype in Macrophages.

CX3CL1 can function as both an adhesion molecule and a chemokine for CX3CR1+ cells, such as T cells, monocytes, and NK cells. Recent studies have demonstrated that CX3CL1-CX3CR1 interaction is associated with the development of various inflammatory skin diseases. In this study, we examined CX3CR1 involvement in 2,4-dinitrofluorobenzene (DNFB)-induced contact hypersensitivity using CX3CR1-/- mice. Ear swelling and dermal edema were attenuated after DNFB challenge in CX3CR1-/- mice. Expression of TNF-α, IL-6, and M1 macrophage markers was decreased in the ears of CX3CR1-/- mice, whereas expression of M2 macrophage markers including arginase-1 was increased. Decreased TNF-α and IL-6 expression and increased arginase-1 expression were found in peritoneal macrophages from CX3CR1-/- mice. Furthermore, ear swelling was attenuated by depleting dermal macrophages in wild-type mice to a similar level to CX3CR1-/- mice. These results suggest that CX3CR1 deficiency could induce skewed polarization towards M2 phenotype in macrophages, resulting in attenuation of contact hypersensitivity response.

Interaction between CX3CL1 and CX3CR1 regulates vasculitis induced by immune complex deposition.

A type III hypersensitivity reaction induced by an immune complex, such as leukocytoclastic vasculitis, is mediated by inflammatory cell infiltration that is highly regulated by multiple adhesion molecules. CX3CL1, a ligand for CX3C chemokine receptor 1 (CX3CR1), has recently been identified as a key mediator of leukocyte adhesion that functions without the recruitment of integrins or selectin-mediated rolling. To elucidate the role of CX3CL1 and CX3CR1 in the development of leukocytoclastic vasculitis, the cutaneous and peritoneal reverse Arthus reactions, classic experimental models for immune complex-mediated tissue injury, were examined in mice lacking CX3CR1. CX3CL1 expression in sera and lesional skin of patients with polyarteritis nodosa (PN) and healthy controls was also examined. Edema and hemorrhage were significantly reduced in CX3CR1(-/-) mice compared with wild-type mice. Infiltration of neutrophils and mast cells and expression of IL-6 and tumor necrosis factor-α were also decreased in CX3CR1(-/-) mice. CX3CL1 was expressed in endothelial cells during the cutaneous reverse Arthus reactions. Furthermore, serum CX3CL1 levels were significantly higher in patients with PN than in healthy controls. Endothelial cells in lesional skin of patients with PN strongly expressed CX3CL1. These results suggest that interactions between CX3CL1 and CX3CR1 may contribute to the development of leukocytoclastic vasculitis by regulating neutrophil and mast cell recruitment and cytokine expression.

Whole-Exome Sequencing Revealed a Pathogenic Germline Variant in the Fumarate Hydratase Gene, Leading to the Diagnosis of Hereditary Leiomyomatosis and Renal Cell Cancer.

The diagnosis of hereditary skin tumors is difficult for "old" diagnostic tools such as immunohistochemistry. Whole-exome sequencing analysis as a "new" diagnostic tool enables us to make a final diagnosis in spite of unknown hereditary diseases in the past. Hereditary leiomyomatosis and renal cell cancer are autosomal dominant hereditary cancer syndromes characterized by uterine myomas, cutaneous leiomyomas, and aggressive renal cell cancer. The syndrome is associated with pathogenic germline variants in the fumarate hydratase gene. Herein, we demonstrate a pathogenic germline variant of the fumarate hydratase gene in a 60-year-old woman with multiple cutaneous leiomyomas, leading to the diagnosis of hereditary leiomyomatosis and renal cell cancer. Whole-exome sequencing analysis using genomic DNA extracted from peripheral blood leukocytes revealed one germline variant in the FH gene on chromosome 1 (c.290G>A, p.Gly97Asp). She received total hysterectomy due to uterine myoma, which strongly supported the diagnosis. No tumor was detected in her kidney by computed tomography and ultrasound examination. Genetic examination for the mutation of the fumarate hydratase gene is important in order to reach the correct diagnosis and to detect renal cancer at its early stage.

Secondary Malignant Tumors Arising in Nevus Sebaceus: Two Case Reports.

Nevus sebaceus is a benign tumor that is present at birth and is often seen on the scalp or face. Secondary malignant tumors sometimes occur in nevus sebaceus in adulthood. Herein, we present two malignant tumors arose from nevus sebaceus. One is basal cell carcinoma on the face and the other is sebaceus carcinoma on the lower back, where nevus sebaceus rarely occurs. Basal cell carcinoma sometimes develops in sebaceus nevus after a few decades, seen usually on the scalp or face. Sebaceus carcinoma is a rare malignant tumor that arises in nevus sebaceus.

EpCAM (CD326) Regulates Intestinal Epithelial Integrity and Stem Cells via Rho-Associated Kinase.

Humans with biallelic inactivating mutations in Epithelial Cell Adhesion Molecule (EpCAM) develop congenital tufting enteropathy (CTE). To gain mechanistic insights regarding EpCAM function in this disorder, we prepared intestinal epithelial cell (IEC) organoids and spheroids. IEC organoids and spheroids were generated from ROSA-CreERT2 EpCAMfl/fl mice. Proliferation, tight junctions, cell polarity and epithelial integrity were assessed in tamoxifen-induced EpCAM-deficient organoids via confocal immunofluorescence microscopy and Western blotting. Olfm4-expressing stem cells were assessed in IEC cells in vitro and in vivo via fluorescence in situ hybridization. To determine if existing drugs could ameliorate effects of EpCAM deficiency in IEC cells, a variety of pharmacologic inhibitors were screened. Deletion of EpCAM resulted in increased apoptosis and attenuated growth of organoids and spheroids. Selected claudins were destabilized and epithelial integrity was severely compromised. Epithelial integrity was improved by treatment with Rho-associated coiled-coil kinase (ROCK) inhibitors without restoration of claudin expression. Correspondingly, enhanced phosphorylation of myosin light chain, a serine/threonine ROCK substrate, was observed in EpCAM-deficient organoids. Strikingly, frequencies of Olfm4-expressing stem cells in EpCAM-deficient IEC cells in vitro and in vivo were decreased. Treatment with ROCK inhibitors increased numbers of stem cells in EpCAM-deficient organoids and spheroids. Thus, EpCAM regulates intestinal epithelial homeostasis via a signaling pathway that includes ROCK.

Amelioration of Congenital Tufting Enteropathy in EpCAM (TROP1)-Deficient Mice via Heterotopic Expression of TROP2 in Intestinal Epithelial Cells.

TROP1 (EpCAM) and TROP2 are homologous cell surface proteins that are widely expressed, and often co-expressed, in developing and adult epithelia. Various functions have been ascribed to EpCAM and TROP2, but responsible mechanisms are incompletely characterized and functional equivalence has not been examined. Adult intestinal epithelial cells (IEC) express high levels of EpCAM, while TROP2 is not expressed. EpCAM deficiency causes congenital tufting enteropathy (CTE) in humans and a corresponding lethal condition in mice. We expressed TROP2 and EpCAM in the IEC of EpCAM-deficient mice utilizing a villin promoter to assess EpCAM and TROP2 function. Expression of EpCAM or TROP2 in the IEC of EpCAM knockout mice prevented CTE. TROP2 rescue (T2R) mice were smaller than controls, while EpCAM rescue (EpR) mice were not. Abnormalities were observed in the diameters and histology of T2R small intestine, and Paneth and stem cell markers were decreased. T2R mice also exhibited enlarged mesenteric lymph nodes, enhanced permeability to 4 kDa FITC-dextran and increased sensitivity to detergent-induced colitis, consistent with compromised barrier function. Studies of IEC organoids and spheroids revealed that stem cell function was also compromised in T2R mice. We conclude that EpCAM and TROP2 exhibit functional redundancy, but they are not equivalent.

IRF-2 haploinsufficiency causes enhanced imiquimod-induced psoriasis-like skin inflammation.

BACKGROUNDS: IFN regulatory factor (IRF)-2 is one of the potential susceptibility genes for psoriasis, but how this gene influences psoriasis pathogenesis is unclear. Topical application of imiquimod (IMQ), a TLR7 ligand, induces psoriasis-like skin lesions in mice. OBJECTIVE: The aim of this study was to investigate whether IRF-2 gene status would influence severity of skin disease in IMQ-treated mice. METHODS: Imiquimod-induced psoriasis-like skin inflammation was assessed by clinical findings, histology, and cytokine expression. The effects of imiquimod or IFN on peritoneal macrophages were analyzed in vitro. RESULTS: IMQ-induced skin inflammation assessed by clinical findings and histology was more severe in IRF-2+/- mice than in wild-type mice. In inflamed skin, mRNA expression levels of tumor necrosis factor (TNF)-α, IL-12/23p40, IL-17A, and IL-22 were significantly elevated in IRF-2+/- mice compared to wild-type mice. Stimulation of peritoneal macrophages by IMQ significantly increased mRNA levels of TNF-α, IL-12/23p40, IL-23p19, IL-12p35, and IL-36. Interestingly, macrophages from IRF-2+/- mice expressed higher levels of TNF-α, IL-12/23p40, and IL-36 compared to those from wild-type mice 24 h after stimulation, while they expressed similar levels of IL-12p35 and IL-23p19. Moreover, elevated mRNA expression of inducible nitric oxide synthase was observed only in IMQ-stimulated macrophages derived from IRF-2+/- mice, which correlated with angiogenesis in IMQ-treated ears of IRF-2+/- mice. CONCLUSIONS: These results suggest that IRF-2 haploinsufficiency creates heightened biologic responses to IFN-α that phenotypically lead to enhanced angiogenesis and psoriasis-like inflammation within skin.

Exacerbated Immune Complex-Mediated Vascular Injury in Mice with Heterozygous Deficiency of Aryl Hydrocarbon Receptor through Upregulation of Fcγ Receptor III Expression on Macrophages.

Aryl hydrocarbon receptor (AhR), which was discovered as a receptor for environmental concomitants, plays an important role widely in the immune system. In this study, we assessed AhR involvement in immune-complex-mediated vascular injury by examining the reverse-passive Arthus reaction in AhR heterozygous knockout (AhR+/-) mice. In the cutaneous Arthus reaction, dermal edema was severer in AhR+/- mice than in wild-type mice. The number of infiltrating neutrophils and mRNA expression levels of CXC chemokine ligand 1 and IL-6 were also increased in AhR+/- mice. Similarly, in the peritoneal Arthus reaction, infiltration of neutrophils was increased in AhR+/- mice. Peritoneal macrophages from AhR+/- mice expressed higher levels of Fcγ receptor III and produced higher levels of CXC chemokine ligand 1 and IL-6 after immune complex treatment. In addition, AhR occupied the promoter regions of Fcγ receptor III gene in peritoneal macrophages in a ligand-dependent manner. Depletion of macrophages reduced the cutaneous Arthus reaction in AhR+/- mice, and adoptive transfer of AhR+/- mice macrophages into wild-type mice exacerbated the peritoneal Arthus reaction. Furthermore, AhR expression was decreased and Fcγ receptor III expression was increased in CD14+ monocytes in peripheral blood from patients with immune-complex-mediated vasculitis compared with those from healthy controls. These results suggest that downregulation of AhR is associated with the exacerbation of immune-complex-mediated vascular injury.

Matriptase-mediated cleavage of EpCAM destabilizes claudins and dysregulates intestinal epithelial homeostasis.

Congenital tufting enteropathy (CTE) is a severe autosomal recessive human diarrheal disorder with characteristic intestinal epithelial dysplasia. CTE can be caused by mutations in genes encoding EpCAM, a putative adhesion molecule, and HAI-2, a cell surface protease inhibitor. A similar phenotype occurs in mice whose intestinal epithelial cells (IECs) fail to express the tight junction-associated protein claudin-7. EpCAM stabilizes claudin-7 in IECs, and HAI-2 regulates the cell surface serine protease matriptase, a known modifier of intestinal epithelial physiology. Therefore, we hypothesized that HAI-2, matriptase, EpCAM, and claudin-7 were functionally linked. Herein we have demonstrated that active matriptase cleaves EpCAM after Arg80 and that loss of HAI-2 in IECs led to unrestrained matriptase activity and efficient cleavage of EpCAM. Cleavage of EpCAM decreased its ability to associate with claudin-7 and targeted it for internalization and lysosomal degradation in conjunction with claudin-7. CTE-associated HAI-2 mutant proteins exhibited reduced ability to inhibit matriptase and also failed to efficiently stabilize claudin-7 in IECs. These results identify EpCAM as a substrate of matriptase and link HAI-2, matriptase, EpCAM, and claudin-7 in a functionally important pathway that causes disease when it is dysregulated.