TLR2‐activated human langerhans cells promote Th17 polarization via IL‐1β, TGF‐β and IL‐23

The cytokines IL‐6, IL‐1β, TGF‐β, and IL‐23 are considered to promote Th17 commitment. Langerhans cells (LC) represent DC in the outer skin layers of the epidermis, an environment extensively exposed to pathogenic attack. The question whether organ‐resident DC like LC can evoke Th17 immune response is still open. Our results show that upon stimulation by bacterial agonists, epidermal LC and LC‐like cells TLR2‐dependently acquire the capacity to polarize Th17 cells. In Th17 cells, expression of retinoid orphan receptor γβ was detected. To clarify if IL‐17⁺cells could arise per se by stimulated LC we did not repress Th1/Th2 driving pathways by antibodies inhibiting differentiation. In CD1c⁺/langerin⁺ monocyte‐derived LC‐like cells (MoLC), macrophage‐activating lipopeptide 2, and peptidoglycan (PGN) induced the release of the cytokines IL‐6, IL‐1β, and IL‐23. TGF‐β, a cytokine required for LC differentiation and survival, was found to be secreted constitutively. Anti‐TLR2 inhibited secretion of IL‐6, IL‐1β, and IL‐23 by MoLC, while TGF‐β was unaffected. The amount of IL‐17 and the ratio of IL‐17 to IFN‐γ expression was higher in MoLC‐ than in monocyte‐derived DC‐cocultured Th cells. Anti‐IL‐1β, ‐TGF‐β and ‐IL‐23 decreased the induction of Th17 cells. Interestingly, blockage of TLR2 on PGN‐stimulated MoLC prevented polarization of Th cells into Th17 cells. Thus, our findings indicate a role of TLR2 in eliciting Th17 immune responses in inflamed skin.     

Suppressed Expression of Autophagosomal Protein LC3 in Cortical Tubers of Tuberous Sclerosis Complex

Tuberous sclerosis complex (TSC) is characterized by benign tumors and hamartomas, including cortical tubers. Hamartin and tuberin, encoded by the TSC 1 and 2 genes, respectively, constitute a functional complex that negatively regulates the mammalian target of rapamycin (mTOR) signaling pathway, eventually promoting the induction of autophagy. In the present study, we assessed the induction of autophagy in cortical tubers surgically removed from seven patients with TSC in comparison with five controls of cortical tissue taken from non‐TSC patients with epilepsy. Immunoblotting demonstrated a marked reduction of LC3B‐I and LC3B‐II in tubers relative to the controls. In tubers, strong, diffuse and dot‐like immunoreactivity (IR) for LC3B was observed in dysmorphic neurons and balloon cells, but LC3B‐IR in other neurons with normal morphology was significantly weaker than that in neurons in the controls. Immunoelectron microscopy revealed diffuse distribution of LC3B‐IR within the cytoplasm of balloon cells. The dot‐like pattern may correspond to abnormal aggregation bodies involving LC3. In an autopsy patient with TSC, we observed that LC3B‐IR in neurons located outside of the tubers was preserved. Thus, autophagy is suppressed in tubers presumably through the mTOR pathway, and possibly a pathological autophagy reaction occurs in the dysmorphic neurons and balloon cells.     

Extracellular 2′5′‐oligoadenylate synthetase 2 mediates T‐cell receptor CD3‐ζ chain down‐regulation via caspase‐3 activation in oral cancer

Decreased expression of CD3‐ζ chain, an adaptor protein associated with T‐cell signalling, is well documented in patients with oral cancer, but the mechanistic justifications are fragmentary. Previous studies in patients with oral cancer have shown that decreased expression of CD3‐ζ chain was associated with decreased responsiveness of T cells. Tumours are known to induce localized as well as systemic immune suppression. This study provides evidence that oral tumour‐derived factors promote immune suppression by down‐regulating CD3‐ζ chain expression. 2′5′‐Oligoadenylate synthetase 2 (OAS2) was identified by the proteomic approach and our results established a causative link between CD3‐ζ chain down‐regulation and OAS2 stimulation. The surrogate situation was established by over‐expressing OAS2 in a HEK293 cell line and cell‐free supernatant was collected. These supernatants when incubated with T cells resulted in down‐regulation of CD3‐ζ chain, which shows that the secreted OAS2 is capable of regulating CD3‐ζ chain expression. Incubation of T cells with cell‐free supernatants of oral tumours or recombinant human OAS2 (rh‐OAS2) induced caspase‐3 activation, which resulted in CD3‐ζ chain down‐regulation. Caspase‐3 inhibition/down‐regulation using pharmacological inhibitor or small interfering RNA restored down‐regulated CD3‐ζ chain expression in T cells induced by cell‐free tumour supernatant or rh‐OAS2. Collectively these results show that OAS2 leads to impairment in CD3‐ζ chain expression, so offering an explanation that might be applicable to the CD3‐ζ chain deficiency observed in cancer and diverse disease conditions.     

Liquid Crystal Alignment Properties of Poly‐(3‐thiopheneacetate)/Dialkyldimethylammonium Complexes

Liquid crystal alignment properties were investigated in liquid crystal (LC) cells fabricated with poly(3‐thiopheneacetate)/dialkyldimethylammonium (PTA‐Cn) complexes, where n is the number of carbon atoms in the alkyl groups in dialkyldimethylammonium bromide. Homeotropic LC alignment was observed in PTA‐Cn LC cells containing long alkyl groups (n = 14, 16, 18) with high pretilt angles close to 90°. Homeotropic LC alignment was observed from prepared LC cell made from PTA‐C12 film, while the homeotropic LC alignment was gradually changed to random alignment within 10 min. LC cells fabricated from PTA‐C10 always showed random alignment behavior. The surface orientational order parameters of the alkyl groups of PTA‐Cn films were estimated by near edge X‐ray absorption fine structure (NEXAFS) spectroscopy, producing values of 0.040, 0.111, 0.153, 0.151, and 0.170 for PTA‐C10, PTA‐12, PTA‐14, PTA‐16, and PTA‐18, respectively. Therefore, homeotropic LC alignment behavior correlated well with the order of alkyl groups on the complex surface.

     

High expression of Mcl‐1L via the MEK‐ERK‐phospho‐STAT3 (Ser727) pathway protects melanocytes and melanoma from UVB‐induced apoptosis

Ultraviolet (UV) B is a major factor in melanomagenesis. This fact is linked to the resistance of melanocytes to UVB‐induced apoptosis. In this study, we characterized the involvement of Mcl‐1L in the regulation of UVB‐induced apoptosis in melanocytes and in melanoma cells. In melanocytes, apoptosis was not evident at 24 h after UVB irradiation. The Mcl‐1L expression increased after UVB irradiation, and the high Mcl‐1L expression continued for at least 24 h. This UVB‐dependent increase in Mcl‐1L was mediated by the MEK‐ERK‐pS‐STAT3 (STAT3 phosphorylated at Ser727) pathway. The Ser727 phosphorylation facilitated nuclear localization of STAT3. In melanoma cells, the expression levels of Mcl‐1L varied depending on the cell line. WM39 melanoma cells expressed high levels of Mcl‐1L via the MEK‐ERK‐pS‐STAT3 pathway and were resistant to UVB‐induced apoptosis without up‐regulation of Mcl‐1L. In melanocytes and in WM39 cells, transfection with Mcl‐1 siRNA promoted UVB‐induced apoptosis. Immunohistochemical studies showed that melanoma cells in in situ lesions expressed high amounts of Mcl‐1L. These results indicate that the high expression of Mcl‐1L mediated by the MEK‐ERK‐pS‐STAT3 pathway protects melanocytes and melanoma cells from UVB‐induced apoptosis.     

Membrane‐bound Dickkopf‐1 in Foxp3+ regulatory T cells suppresses T‐cell‐mediated autoimmune colitis

Induction of tolerance is a key mechanism to maintain or to restore immunological homeostasis. Here we show that Foxp3⁺ regulatory T (Treg) cells use Dickkopf‐1 (DKK‐1) to regulate T‐cell‐mediated tolerance in the T‐cell‐mediated autoimmune colitis model. Treg cells from DKK‐1 hypomorphic doubleridge mice failed to control CD4⁺ T‐cell proliferation, resulting in CD4 T‐cell‐mediated autoimmune colitis. Thymus‐derived Treg cells showed a robust expression of DKK‐1 but not in naive or effector CD4 T cells. DKK‐1 expression in Foxp3⁺ Treg cells was further increased upon T‐cell receptor stimulation in vitro and in vivo. Interestingly, Foxp3⁺ Treg cells expressed DKK‐1 in the cell membrane and the functional inhibition of DKK‐1 using DKK‐1 monoclonal antibody abrogated the suppressor function of Foxp3⁺ Treg cells. DKK‐1 expression was dependent on de novo protein synthesis and regulated by the mitogen‐activated protein kinase pathway but not by the canonical Wnt pathway. Taken together, our results highlight membrane‐bound DKK‐1 as a novel Treg‐derived mediator to maintain immunological tolerance in T‐cell‐mediated autoimmune colitis.     

LC3A‐Positive “Stone‐Like” Structures Predict an Adverse Prognosis of Gastric Cancer

Microtubule‐associated protein light chain 3 (LC3A) is a reliable marker of autophagy that displays three distinct patterns of immunohistochemical staining in solid tumors: diffuse cytoplasmic staining, juxtanuclear staining, and staining of “stone‐like” structures. These three patterns have a different prognostic significance in many solid tumors, but little is known about their influence in gastric cancer (GC). This study was a retrospective analysis of 188 GC patients from stages I to IV. The pattern of LC3A expression was examined in tumor and nontumor tissues by immunohistochemistry. Then, the association between the pattern of LC3A expression in GC and the prognosis was investigated by Kaplan‐Meier analysis and the Cox proportional hazards model. Two distinct patterns of LC3A immunostaining (diffuse cytoplasmic expression and “stone‐like” structures) were observed in GC tissues. LC3A‐positive “stone‐like” structures were found only in the tumors, and the number of such structures was correlated with both the tumor type and tumor stage. In addition, a high number of LC3A‐positive “stone‐like” structures was closely associated with an increased risk of recurrence after radical resection of stages I–III cancer (P < 0.001; HR = 0.205) and was associated with a lower overall survival rate for stage IV cancer (P < 0.001; HR = 0.364). Taken together, our data demonstrate that LC3A‐positive “stone‐like” structures can be used as an independent biomarker for an adverse prognosis of GC, suggesting that “stone‐like” structures are correlated with the malignancy of this disease. Anat Rec, 297:653–662, 2014. © 2014 The Authors The Anatomical Record: Advances in Integrative Anatomy and Evolutionary Biology Published by Wiley Periodicals, Inc.     

PIAS1 and STAT‐3 impair the tumoricidal potential of IFN‐γ‐stimulated mouse dendritic cells generated with IL‐15

Primarily defined by their antigen‐presenting property, dendritic cells (DCs) are being implemented as cancer vaccines in immunotherapeutic interventions. DCs can also function as direct tumor cell killers. How DC cytotoxic activity can be efficiently harnessed and the mechanisms controlling this nonconventional property are not fully understood. We report here that the tumoricidal potential of mouse DCs generated from myeloid precursors with GM‐CSF and IL‐15 (IL‐15 DCs) can be triggered with the Toll‐like receptor (TLR) 4 ligand lipopolysaccharide to a similar extent compared with that of their counterparts, conventionally generated with IL‐4 (IL‐4 DCs). The mechanism of tumor cell killing depends on the induction of iNOS expression by DCs. In contrast, interferon (IFN)‐γ induces the cytotoxic activity of IL‐4 but not IL‐15 DCs. Although the IFN‐γ‐STAT‐1 signaling pathway is overall functional in IL‐15 DCs, IFN‐γ fails to induce iNOS expression in these cells. iNOS expression is negatively controlled in IFN‐γ‐stimulated IL‐15 DCs by the cooperation between the E3 SUMO ligase PIAS1 and STAT‐3, and can be partially restored with PIAS1 siRNA and STAT‐3 inhibitors.     

Loss of N‐WASP drives early progression in an Apc model of intestinal tumourigenesis

N‐WASP (WASL) is a widely expressed cytoskeletal signalling and scaffold protein also implicated in regulation of Wnt signalling and homeostatic maintenance of skin epithelial architecture. N‐WASP mediates invasion of cancer cells in vitro and its depletion reduces invasion and metastatic dissemination of breast cancer. Given this role in cancer invasion and universal expression in the gastrointestinal tract, we explored a role for N‐WASP in the initiation and progression of colorectal cancer. While deletion of N‐wasp is not detectably harmful in the murine intestinal tract, numbers of Paneth cells increased, indicating potential changes in the stem cell niche, and migration up the crypt–villus axis was enhanced. Loss of N‐wasp promoted adenoma formation in an adenomatous polyposis coli (Apc) deletion model of intestinal tumourigenesis. Thus, we establish a tumour suppressive role of N‐WASP in early intestinal carcinogenesis despite its later pro‐invasive role in other cancers. Our study highlights that while the actin cytoskeletal machinery promotes invasion of cancer cells, it also maintains normal epithelial tissue function and thus may have tumour suppressive roles in pre‐neoplastic tissues. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.     

Peroxin Pex14p is the key component for coordinated autophagic degradation of mammalian peroxisomes by direct binding to LC3‐II

Pexophagy can be experimentally induced in mammalian cells by removing the culture serum. Pex14p, a peroxisomal membrane protein essential for matrix protein import in docking of soluble receptor Pex5p, is involved in the mammalian autophagic degradation of peroxisomes and interacts with the lipidated form of LC3, termed LC3‐II, an essential factor for autophagosome formation, under the starvation condition in CHO‐K1 cells. However, molecular mechanisms underlying the Pex14p‐LC3‐II interaction remain largely unknown. To verify whether Pex14p directly binds LC3‐II, we reconstituted an in vitro conjugation system for synthesis of LC3‐II. We show here that Pex14p directly interacts with LC3‐II via the transmembrane domain of Pex14p. Pex5p competitively inhibited this interaction, implying that Pex14p preferentially binds to Pex5p under the nutrient‐rich condition. Moreover, a Pex5p mutant defective in PTS1‐protein import lost its affinity for Pex14p under the condition of nutrient deprivation, thereby more likely explaining why Pex14p prefers to interact with LC3‐II under the starvation condition in vivo. Together, these results suggest that Pex14p is a unique factor that functions in the dual processes in peroxisomal biogenesis and degradation with the coordination of Pex5p in response to the environmental changes.     

Direct quantitative estimation of Paneth and total cell populations in the jejunal glands of Lieberkühn

Direct morphometric estimation of the number of intestinal crypts per 10 mm length of jejunum, the total number of cells per crypt, and the number of Paneth cells per crypt was performed using Zenker-fixed, serially sectioned small intestines from adult BALB/c mice. The mean volume of a crypt cell was estimated to be in the order of 6.6 ± 0.1 × 10^?4 mm³, while the total volume of all cells in a single crypt was 1.833 ± 0.02 × 10^?1 mm³. The cell population per crypt was 278 ± 7 cells, of which 44 ± 10 were Paneth cells. It was estimated further that 265 ± 30 crypts per 10 mm length of jejunum contained 7.4 ± 0.1 × 10⁴ cells, including 1.2 ± 0.24 × 10³ Paneth cells. The volume of all crypt and Paneth cells per 10 mm length of jejunum was in the order of 49 ± 1.2 and 8 ± 1.8 mm³, respectively. The volume of the tunica muscularis and the surface area of the crypt's lumina were estimated to be 33.5 ± 2.2 mm³ and 118 ± 19 mm², respectively.     

2‐(Pro‐1‐ynyl)‐5‐(5,6‐dihydroxypenta‐1,3‐diynyl) Thiophene Induces Apoptosis Through Reactive Oxygen Species‐Mediated JNK Activation in Human Colon Cancer SW620 Cells

2‐(Pro‐1‐ynyl)?5‐(5,6‐dihydroxypenta‐1,3‐diynyl) thiophene (PYDDT) is a naturally occurring thiophene isolated from the roots of Echinops grijsii, a Chinese herbal medicine used to treat colon cancer, breast cancer, and lung cancer. There are many reports on the clinical use of Echinops grijsii alone or in combination with other herbs to treat malignant tumors. We previously reported that the expression and activity of phase II enzymes including GSTs and NQO1 could be induced through the activation of Keap1‐Nrf2 pathway by the treatment of PYDDT. In this study, we reported the anticancer effect and mechanism of PYDDT against human colon cancer SW620 cells. Our results demonstrate that treatment of SW620 cells with PYDDT leads to induction of mitochondrial‐mediated apoptosis, which is characterized by the cleavage of PARP, activation of caspase 9 and caspase 3, release of cytochrome c from mitochondria, loss of mitochondrial membrane potential, down‐regulation of Bcl‐2, and mitochondrial translocation of Bax. The PYDDT treatment caused the production of reactive oxygen species (ROS), and the activation of JNK but not p38 mitogen‐activated protein kinases and ERK1/2. Specific JNK inhibitor SP600125 prevented the PYDDT‐induced down‐regulation of Bcl‐2, mitochondrial translocation of Bax, activation of caspase 3, and apoptosis of SW620 cells. Moreover, PYDDT‐induced apoptosis as well as activation of JNK was abrogated by the pretreatment with antioxidant N‐acetylcysteine. Taken together, these findings suggest that PYDDT induces apoptosis in SW620 cells through a ROS/JNK‐mediated mitochondrial pathway. Anat Rec, 298:376–385, 2015. © 2014 Wiley Periodicals, Inc.     

Immunolocalization of G‐Protein Alpha Subunits in the Olfactory System of the Cartilaginous Fish Scyliorhinus Canicula

In the olfactory and vomeronasal systems of vertebrates, the morphology of the receptor neurons, the receptor gene family they express, the G‐protein coupled with the receptor (in particular the G‐protein alpha subunit), and their projection to the olfactory bulb are correlated. Much information about this complicated system have been collected in different groups, but nothing is known about Chondrichthyes. In this work, the presence and distribution of immunoreactivity for different types of G‐protein alpha subunit (Gα_o, Gα_q and Gα_s/olf) were investigated in the olfactory mucosa and olfactory bulb of the shark Scyliorhinus canicula. Only Gα_o‐like immunoreactivity was detected in the olfactory mucosa and bulb, both in tissues and homogenates. Its distribution was partially similar to that found in other vertebrates: it was localized in the microvillous receptor neurons, in numerous axon bundles of the fila olfactoria, in the stratum nervosum and in the most of glomeruli in the stratum glomerulosum. No immunoreactivity was instead observed in the crypt neurons, the second type of olfactory neurons present in cartilaginous fish. The projections of crypt neurons to olfactory bulb probably correspond to the few ventrally‐located glomeruli which were negative to the antiserum against Gα_o. These data suggest, in S. canicula, different olfactory neuron types send projections to the olfactory bulb with a segregated distribution, as observed in other vertebrates. Anat Rec, 2009. © 2009 Wiley‐Liss, Inc.