Tumor-Infiltrating T Cells in Skin Basal Cell Carcinomas and Squamous Cell Carcinomas: Global Th1 Preponderance with Th17 Enrichment-A Cross-Sectional Study.

Basal cell carcinomas (BCCs) and squamous cell carcinomas (SCCs) are high-incidence, non-melanoma skin cancers (NMSCs). The success of immune-targeted therapies in advanced NMSCs led us to anticipate that NMSCs harbored significant populations of tumor-infiltrating lymphocytes with potential anti-tumor activity. The main aim of this study was to characterize T cells infiltrating NMSCs. Flow cytometry and immunohistochemistry were used to assess, respectively, the proportions and densities of T cell subpopulations in BCCs (n = 118), SCCs (n = 33), and normal skin (NS, n = 30). CD8+ T cells, CD4+ T cell subsets, namely, Th1, Th2, Th17, Th9, and regulatory T cells (Tregs), CD8+ and CD4+ memory T cells, and γδ T cells were compared between NMSCs and NS samples. Remarkably, both BCCs and SCCs featured a significantly higher Th1/Th2 ratio (~four-fold) and an enrichment for Th17 cells. NMSCs also showed a significant enrichment for IFN-γ-producing CD8+T cells, and a depletion of γδ T cells. Using immunohistochemistry, NMSCs featured denser T cell infiltrates (CD4+, CD8+, and Tregs) than NS. Overall, these data favor a Th1-predominant response in BCCs and SCCs, providing support for immune-based treatments in NMSCs. Th17-mediated inflammation may play a role in the progression of NMSCs and thus become a potential therapeutic target in NMSCs.

CLOCK gene circannual expression in cluster headache.

BACKGROUND: Cluster headache is a primary headache disorder characterized by bouts with circadian and circannual patterns. The CLOCK gene has a central role in regulating circadian rhythms. Here, we investigate the circannual CLOCK expression in a population of cluster headache patients in comparison to matched controls. METHODS: Patients with cluster headache were sampled two to four times over at least one year, both in or outside bouts, one week after each solstice and equinox. The expression of CLOCK was measured by quantitative real-time polymerase chain reaction (RT-PCR) in the peripheral blood. RESULTS: This study included 50 patients and 58 matched controls. Among the patient population, composed of 42/50 males (84%) with an average age of 44.6 years, 45/50 (90%) suffered from episodic cluster headache. Two to four samples were collected from each patient adding up to 161 samples, 36 (22.3%) of which were collected within a bout. CLOCK expression for cluster headache patients was considerably different from that of the control population in winter (p-value mean = 0.006283), spring (p-value mean = 0.000006) and summer (p-value mean = 0.000064), but not in autumn (p-value mean = 0.262272). For each season transition, the variations in CLOCK expression were more pronounced in the control group than in the cluster headache population. No statistically significant differences were found between bout and non-bout samples. No individual factors (age, sex, circadian chronotype, smoking and coffee habits or history of migraine) were related to CLOCK expression. CONCLUSIONS: We observed that CLOCK expression in cluster headache patients fluctuates less throughout the year than in the control population. Bout activity and lifestyle factors do not seem to influence CLOCK expression.

MCL1 alternative polyadenylation is essential for cell survival and mitochondria morphology.

Alternative polyadenylation in the 3' UTR (3' UTR-APA) is a mode of gene expression regulation, fundamental for mRNA stability, translation and localization. In the immune system, it was shown that upon T cell activation, there is an increase in the relative expression of mRNA isoforms with short 3' UTRs resulting from 3' UTR-APA. However, the functional significance of 3' UTR-APA remains largely unknown. Here, we studied the physiological function of 3' UTR-APA in the regulation of Myeloid Cell Leukemia 1 (MCL1), an anti-apoptotic member of the Bcl-2 family essential for T cell survival. We found that T cells produce two MCL1 mRNA isoforms (pA1 and pA2) by 3' UTR-APA. We show that upon T cell activation, there is an increase in both the shorter pA1 mRNA isoform and MCL1 protein levels. Moreover, the less efficiently translated pA2 isoform is downregulated by miR-17, which is also more expressed upon T cell activation. Therefore, by increasing the expression of the more efficiently translated pA1 mRNA isoform, which escapes regulation by miR-17, 3' UTR-APA fine tunes MCL1 protein levels, critical for activated T cells' survival. Furthermore, using CRISPR/Cas9-edited cells, we show that depletion of either pA1 or pA2 mRNA isoforms causes severe defects in mitochondria morphology, increases apoptosis and impacts cell proliferation. Collectively, our results show that MCL1 alternative polyadenylation has a key role in the regulation of MCL1 protein levels upon T cell activation and reveal an essential function for MCL1 3' UTR-APA in cell viability and mitochondria dynamics.

Combined glucocorticoid resistance and hyperlactatemia contributes to lethal shock in sepsis.

Sepsis is a potentially lethal syndrome resulting from a maladaptive response to infection. Upon infection, glucocorticoids are produced as a part of the compensatory response to tolerate sepsis. This tolerance is, however, mitigated in sepsis due to a quickly induced glucocorticoid resistance at the level of the glucocorticoid receptor. Here, we show that defects in the glucocorticoid receptor signaling pathway aggravate sepsis pathophysiology by lowering lactate clearance and sensitizing mice to lactate-induced toxicity. The latter is exerted via an uncontrolled production of vascular endothelial growth factor, resulting in vascular leakage and collapse with severe hypotension, organ damage, and death, all being typical features of a lethal form of sepsis. In conclusion, sepsis leads to glucocorticoid receptor failure and hyperlactatemia, which collectively leads to a lethal vascular collapse.

Tetracycline Antibiotics Induce Host-Dependent Disease Tolerance to Infection.

Several classes of antibiotics have long been known to have beneficial effects that cannot be explained strictly on the basis of their capacity to control the infectious agent. Here, we report that tetracycline antibiotics, which target the mitoribosome, protected against sepsis without affecting the pathogen load. Mechanistically, we found that mitochondrial inhibition of protein synthesis perturbed the electron transport chain (ETC) decreasing tissue damage in the lung and increasing fatty acid oxidation and glucocorticoid sensitivity in the liver. Using a liver-specific partial and acute deletion of Crif1, a critical mitoribosomal component for protein synthesis, we found that mice were protected against sepsis, an observation that was phenocopied by the transient inhibition of complex I of the ETC by phenformin. Together, we demonstrate that mitoribosome-targeting antibiotics are beneficial beyond their antibacterial activity and that mitochondrial protein synthesis inhibition leading to ETC perturbation is a mechanism for the induction of disease tolerance.

Deletion of iRhom2 protects against diet-induced obesity by increasing thermogenesis.

OBJECTIVE: Obesity is the result of positive energy balance. It can be caused by excessive energy consumption but also by decreased energy dissipation, which occurs under several conditions including when the development or activation of brown adipose tissue (BAT) is impaired. Here we evaluated whether iRhom2, the essential cofactor for the Tumour Necrosis Factor (TNF) sheddase ADAM17/TACE, plays a role in the pathophysiology of metabolic syndrome. METHODS: We challenged WT versus iRhom2 KO mice to positive energy balance by chronic exposure to a high fat diet and then compared their metabolic phenotypes. We also carried out ex vivo assays with primary and immortalized mouse brown adipocytes to establish the autonomy of the effect of loss of iRhom2 on thermogenesis and respiration. RESULTS: Deletion of iRhom2 protected mice from weight gain, dyslipidemia, adipose tissue inflammation, and hepatic steatosis and improved insulin sensitivity when challenged by a high fat diet. Crucially, the loss of iRhom2 promotes thermogenesis via BAT activation and beige adipocyte recruitment, enabling iRhom2 KO mice to dissipate excess energy more efficiently than WT animals. This effect on enhanced thermogenesis is cell-autonomous in brown adipocytes as iRhom2 KOs exhibit elevated UCP1 levels and increased mitochondrial proton leak. CONCLUSION: Our data suggest that iRhom2 is a negative regulator of thermogenesis and plays a role in the control of adipose tissue homeostasis during metabolic disease.

Celastrol: A Spectrum of Treatment Opportunities in Chronic Diseases.

The identification of new bioactive compounds derived from medicinal plants with significant therapeutic properties has attracted considerable interest in recent years. Such is the case of the Tripterygium wilfordii (TW), an herb used in Chinese medicine. Clinical trials performed so far using its root extracts have shown impressive therapeutic properties but also revealed substantial gastrointestinal side effects. The most promising bioactive compound obtained from TW is celastrol. During the last decade, an increasing number of studies were published highlighting the medicinal usefulness of celastrol in diverse clinical areas. Here we systematically review the mechanism of action and the therapeutic properties of celastrol in inflammatory diseases, namely, rheumatoid arthritis, systemic lupus erythematosus, inflammatory bowel diseases, osteoarthritis and allergy, as well as in cancer, neurodegenerative disorders and other diseases, such as diabetes, obesity, atherosclerosis, and hearing loss. We will also focus in the toxicological profile and limitations of celastrol formulation, namely, solubility, bioavailability, and dosage issues that still limit its further clinical application and usefulness.

CD11b activation suppresses TLR-dependent inflammation and autoimmunity in systemic lupus erythematosus.

Genetic variations in the ITGAM gene (encoding CD11b) strongly associate with risk for systemic lupus erythematosus (SLE). Here we have shown that 3 nonsynonymous ITGAM variants that produce defective CD11b associate with elevated levels of type I interferon (IFN-I) in lupus, suggesting a direct link between reduced CD11b activity and the chronically increased inflammatory status in patients. Treatment with the small-molecule CD11b agonist LA1 led to partial integrin activation, reduced IFN-I responses in WT but not CD11b-deficient mice, and protected lupus-prone MRL/Lpr mice from end-organ injury. CD11b activation reduced TLR-dependent proinflammatory signaling in leukocytes and suppressed IFN-I signaling via an AKT/FOXO3/IFN regulatory factor 3/7 pathway. TLR-stimulated macrophages from CD11B SNP carriers showed increased basal expression of IFN regulatory factor 7 (IRF7) and IFN-ß, as well as increased nuclear exclusion of FOXO3, which was suppressed by LA1-dependent activation of CD11b. This suggests that pharmacologic activation of CD11b could be a potential mechanism for developing SLE therapeutics.

Absence of miR-146a in Podocytes Increases Risk of Diabetic Glomerulopathy via Up-regulation of ErbB4 and Notch-1.

Podocyte injury is an early event in diabetic kidney disease and is a hallmark of glomerulopathy. MicroRNA-146a (miR-146a) is highly expressed in many cell types under homeostatic conditions, and plays an important anti-inflammatory role in myeloid cells. However, its role in podocytes is unclear. Here, we show that miR-146a expression levels decrease in the glomeruli of patients with type 2 diabetes (T2D), which correlates with increased albuminuria and glomerular damage. miR-146a levels are also significantly reduced in the glomeruli of albuminuric BTBR ob/ob mice, indicating its significant role in maintaining podocyte health. miR-146a-deficient mice (miR-146a-/-) showed accelerated development of glomerulopathy and albuminuria upon streptozotocin (STZ)-induced hyperglycemia. The miR-146a targets, Notch-1 and ErbB4, were also significantly up-regulated in the glomeruli of diabetic patients and mice, suggesting induction of the downstream TGFß signaling. Treatment with a pan-ErbB kinase inhibitor erlotinib with nanomolar activity against ErbB4 significantly suppressed diabetic glomerular injury and albuminuria in both WT and miR-146a-/- animals. Treatment of podocytes in vitro with TGF-ß1 resulted in increased expression of Notch-1, ErbB4, pErbB4, and pEGFR, the heterodimerization partner of ErbB4, suggesting increased ErbB4/EGFR signaling. TGF-ß1 also increased levels of inflammatory cytokine monocyte chemoattractant protein-1 (MCP-1) and MCP-1 induced protein-1 (MCPIP1), a suppressor of miR-146a, suggesting an autocrine loop. Inhibition of ErbB4/EGFR with erlotinib co-treatment of podocytes suppressed this signaling. Our findings suggest a novel role for miR-146a in protecting against diabetic glomerulopathy and podocyte injury. They also point to ErbB4/EGFR as a novel, druggable target for therapeutic intervention, especially because several pan-ErbB inhibitors are clinically available.

Decrease of CD68 Synovial Macrophages in Celastrol Treated Arthritic Rats.

BACKGROUND: Rheumatoid arthritis (RA) is a chronic immune-mediated inflammatory disease characterized by cellular infiltration into the joints, hyperproliferation of synovial cells and bone damage. Available treatments for RA only induce remission in around 30% of the patients, have important adverse effects and its use is limited by their high cost. Therefore, compounds that can control arthritis, with an acceptable safety profile and low production costs are still an unmet need. We have shown, in vitro, that celastrol inhibits both IL-1ß and TNF, which play an important role in RA, and, in vivo, that celastrol has significant anti-inflammatory properties. Our main goal in this work was to test the effect of celastrol in the number of sublining CD68 macrophages (a biomarker of therapeutic response for novel RA treatments) and on the overall synovial tissue cellularity and joint structure in the adjuvant-induced rat model of arthritis (AIA). METHODS: Celastrol was administered to AIA rats both in the early (4 days after disease induction) and late (11 days after disease induction) phases of arthritis development. The inflammatory score, ankle perimeter and body weight were evaluated during treatment period. Rats were sacrificed after 22 days of disease progression and blood, internal organs and paw samples were collected for toxicological blood parameters and serum proinflammatory cytokine quantification, as well as histopathological and immunohistochemical evaluation, respectively. RESULTS: Here we report that celastrol significantly decreases the number of sublining CD68 macrophages and the overall synovial inflammatory cellularity, and halted joint destruction without side effects. CONCLUSIONS: Our results validate celastrol as a promising compound for the treatment of arthritis.

Anthracyclines induce DNA damage response-mediated protection against severe sepsis.

Severe sepsis remains a poorly understood systemic inflammatory condition with high mortality rates and limited therapeutic options in addition to organ support measures. Here we show that the clinically approved group of anthracyclines acts therapeutically at a low dose regimen to confer robust protection against severe sepsis in mice. This salutary effect is strictly dependent on the activation of DNA damage response and autophagy pathways in the lung, as demonstrated by deletion of the ataxia telangiectasia mutated (Atm) or the autophagy-related protein 7 (Atg7) specifically in this organ. The protective effect of anthracyclines occurs irrespectively of pathogen burden, conferring disease tolerance to severe sepsis. These findings demonstrate that DNA damage responses, including the ATM and Fanconi Anemia pathways, are important modulators of immune responses and might be exploited to confer protection to inflammation-driven conditions, including severe sepsis.

Analysis of ESCRT functions in exosome biogenesis, composition and secretion highlights the heterogeneity of extracellular vesicles.

Exosomes are extracellular vesicles (EVs) secreted upon fusion of endosomal multivesicular bodies (MVBs) with the plasma membrane. The mechanisms involved in their biogenesis have not yet been fully identified although they could be used to modulate exosome formation and therefore are a promising tool in understanding exosome functions. We have performed an RNA interference screen targeting 23 components of the endosomal sorting complex required for transport (ESCRT) machinery and associated proteins in MHC class II (MHC II)-expressing HeLa-CIITA cells. Silencing of HRS, STAM1 or TSG101 reduced the secretion of EV-associated CD63 and MHC II but each gene altered differently the size and/or protein composition of secreted EVs, as quantified by immuno-electron microscopy. By contrast, depletion of VPS4B augmented this secretion while not altering the features of EVs. For several other ESCRT subunits, it was not possible to draw any conclusions about their involvement in exosome biogenesis from the screen. Interestingly, silencing of ALIX increased MHC II exosomal secretion, as a result of an overall increase in intracellular MHC II protein and mRNA levels. In human dendritic cells (DCs), ALIX depletion also increased MHC II in the cells, but not in the released CD63-positive EVs. Such differences could be attributed to a greater heterogeneity in size, and higher MHC II and lower CD63 levels in vesicles recovered from DCs as compared with HeLa-CIITA. The results reveal a role for selected ESCRT components and accessory proteins in exosome secretion and composition by HeLa-CIITA. They also highlight biogenetic differences in vesicles secreted by a tumour cell line and primary DCs.

SAMHD1-dependent retroviral control and escape in mice.

SAMHD1 is a host restriction factor for human immunodeficiency virus 1 (HIV-1) in cultured human cells. SAMHD1 mutations cause autoimmune Aicardi-Goutières syndrome and are found in cancers including chronic lymphocytic leukaemia. SAMHD1 is a triphosphohydrolase that depletes the cellular pool of deoxynucleoside triphosphates, thereby preventing reverse transcription of retroviral genomes. However, in vivo evidence for SAMHD1's antiviral activity has been lacking. We generated Samhd1 null mice that do not develop autoimmune disease despite displaying a type I interferon signature in spleen, macrophages and fibroblasts. Samhd1(-/-) cells have elevated deoxynucleoside triphosphate (dNTP) levels but, surprisingly, SAMHD1 deficiency did not lead to increased infection with VSV-G-pseudotyped HIV-1 vectors. The lack of restriction is likely attributable to the fact that dNTP concentrations in SAMHD1-sufficient mouse cells are higher than the KM of HIV-1 reverse transcriptase (RT). Consistent with this notion, an HIV-1 vector mutant bearing an RT with lower affinity for dNTPs was sensitive to SAMHD1-dependent restriction in cultured cells and in mice. This shows that SAMHD1 can restrict lentiviruses in vivo and that nucleotide starvation is an evolutionarily conserved antiviral mechanism.

TET1 is a negative transcriptional regulator of IL-1ß in the THP-1 cell line.

TET1 is a member of the recently identified family of epigenetic regulators, TET1-3 which catalyze the enzymatic conversion of the methyl mark on cytosine (methylcytosine, mC) to the hydroxymethyl mark (hmC). The functions of hmC are required for stem cell maintenance and for controlling differentiation and reprogramming. So far, no roles for TET proteins have been identified in cells of the immune system. Here we show that TET1 is a negative regulator of IL-1ß transcription following an inflammatory stimulus and negatively modulates IL-1ß secretion in THP-1 cells. In addition, TET1 expression is regulated during inflammation both in THP-1 and in primary dendritic cells. Importantly, other highly induced pro-inflammatory genes are also regulated by TET1, including cytokines, chemokines and adhesion molecules. The other member of the TET family with known roles in stem cell regulation, TET2, is also regulated in THP-1 cells following the inflammatory stimulus and may also participate in IL-1ß regulation, according to our observations. Our results suggest a TET1-dependent anti-inflammatory pathway, which may include TET2. In particular, IL-1ß transcriptional regulation is likely to depend on TET1-regulated chromatin domains. This work highlights the contribution of epigenetic mechanisms to the efficient organization of inflammatory responses.

Rab27a supports exosome-dependent and -independent mechanisms that modify the tumor microenvironment and can promote tumor progression.

During progression from single cancer cells to a tumor mass and metastases, tumor cells send signals that can subvert their tissue microenvironment. These signals involve soluble molecules and various extracellular vesicles, including a particular type termed exosomes. The specific roles of exosomes secreted in the tumor microenvironment, however, is unclear. The small GTPases RAB27A and RAB27B regulate exocytosis of multivesicular endosomes, which lead to exosome secretion, in human HeLa cells. Here, we used mouse models to show that Rab27a blockade in mammary carcinoma cells decreased secretion of exosomes characterized by endocytic markers, but also of matrix metalloproteinase 9, which is not associated with exosomes. Rab27a blockade resulted in decreased primary tumor growth and lung dissemination of a metastatic carcinoma (4T1), but not of a nonmetastatic carcinoma (TS/A). Local growth of 4T1 tumors required mobilization of a population of neutrophil immune cells induced by Rab27a-dependent secretion of exosomes together with a specific combination of cytokines and/or metalloproteinases. Our findings offer in vivo validation of the concept that exosome secretion can exert key pathophysiologic roles during tumor formation and progression, but they also highlight the idiosyncratic character of the tumor context.

RNAi screen for kinases and phosphatases that play a role in antigen presentation by dendritic cells.

Effective CD8(+) T-cell responses against tumor or microbial antigens that are not directly expressed in antigen-presenting cells (APCs) depend on the cross-presentation of these antigens on MHC class I in APCs. To identify signaling molecules that regulate cross-presentation, we used lentiviral-based RNA interference to test the roles of hundreds of kinases and phosphatases in this process. Our study uncovered eight previously unknown genes, consisting of one positive and seven negative regulators of antigen cross-presentation. Depletion of Acvr1c, a type I receptor for TGF-ß family of signaling molecules, led to an increase in CD80 and CD86 co-stimulator surface expression and secreted IL-12 in mouse bone marrow-derived DCs, as well as antigen-specific T-cell proliferation.

Highly dynamic host actin reorganization around developing Plasmodium inside hepatocytes.

Plasmodium sporozoites are transmitted by Anopheles mosquitoes and infect hepatocytes, where a single sporozoite replicates into thousands of merozoites inside a parasitophorous vacuole. The nature of the Plasmodium-host cell interface, as well as the interactions occurring between these two organisms, remains largely unknown. Here we show that highly dynamic hepatocyte actin reorganization events occur around developing Plasmodium berghei parasites inside human hepatoma cells. Actin reorganization is most prominent between 10 to 16 hours post infection and depends on the actin severing and capping protein, gelsolin. Live cell imaging studies also suggest that the hepatocyte cytoskeleton may contribute to parasite elimination during Plasmodium development in the liver.

Sec22b regulates phagosomal maturation and antigen crosspresentation by dendritic cells.

Antigen (Ag) crosspresentation by dendritic cells (DCs) involves the presentation of internalized Ags on MHC class I molecules to initiate CD8+ T cell-mediated immunity in response to certain pathogens and tumor cells. Here, we identify the SNARE Sec22b as a specific regulator of Ag crosspresentation. Sec22b localizes to the ER-Golgi intermediate compartment (ERGIC) and pairs to the plasma membrane SNARE syntaxin 4, which is present in phagosomes (Phgs). Depletion of Sec22b inhibits the recruitment of ER-resident proteins to Phgs and to the vacuole containing the Toxoplasma gondii parasite. In Sec22b-deficient DCs, crosspresentation is compromised after Ag phagocytosis or endocytosis and after invasion by T. gondii. Sec22b silencing inhibited Ag export to the cytosol and increased phagosomal degradation by accelerating lysosomal recruitment. Our findings provide insight into an intracellular traffic pathway required for crosspresentation and show that Sec22b-dependent recruitment of ER proteins to Phgs critically influences phagosomal functions in DCs.