LPS-elicited TSLPR Expression Enriches a Functionally Discrete Subset of Human CD14+ CD1c+ Monocytes

Thymic stromal lymphopoietin (TSLP) is a cytokine produced mainly by epithelial cells in response to inflammatory or microbial stimuli and binds to the TSLP receptor complex, a heterodimer composed of TSLP receptor (TSLPR) and IL-7 receptor α (CD127). TSLP activates multiple immune cell subsets expressing the TSLP receptor complex and plays a role in several models of disease. Although human monocytes express TSLPR and CD127 mRNAs in response to the TLR4 agonist LPS, their responsiveness to TSLP is ill-defined. We demonstrate that TSLP enhances human CD14+ monocyte CCL17 production in response to LPS and IL-4. Surprisingly, only a subset of CD14+ CD16- monocytes (TSLPR+ mono) expresses TSLP receptor complex upon LPS stimulation in an NF-κB- and p38-dependent manner. Phenotypic, functional and transcriptomic analysis revealed specific features of TSLPR+ mono, including higher CCL17 and IL-10 production and increased expression of genes with important immune functions (i.e. GAS6, ALOX15B, FCGR2B, LAIR1). Strikingly, TSLPR+ mono express higher levels of the dendritic cell marker CD1c. This evidence led us to identify a subset of peripheral blood CD14+ CD1c+ cells that expresses the highest levels of TSLPR upon LPS stimulation. The translational relevance of these findings is highlighted by the higher expression of TSLPR and CD127 mRNAs in monocytes isolated from patients with Gram-negative sepsis compared to healthy controls. Our results emphasize a phenotypic and functional heterogeneity in an apparently homogeneous population of human CD14+ CD16- monocytes and prompt further ontogenetic and functional analysis of CD14+ CD1c+ and LPS-activated CD14+ CD1c+ TSLPR+ monocytes

Group V Secreted Phospholipase A2 Induces the Release of Proangiogenic and Antiangiogenic Factors by Human Neutrophils

Secreted phospholipases A2 (sPLA2s) are extracellular enzymes that catalyze the release of free fatty acids and lysophospholipids from membrane phospholipids and also bind to different receptors (e.g., PLA2R1 or integrins). To date, 12 mammalian sPLA2s have been identified, which play a critical role in pathophysiological processes including inflammation and cancer. sPLA2s activate immune cells such as human neutrophils (PMNs) by enzymatic activity- or receptor-mediated mechanisms. In addition, human PMNs synthesize and store human group V (hGV) and human group X (hGX) sPLA2s in their granules, but only the former is released upon cellular activation. We investigated the effects of sPLA2s on the release of proangiogenic and antiangiogenic factors by PMNs. We found that exogenous hGV and hGX sPLA2s induce the release of vascular endothelial growth factor (VEGF)-A, angiopoietin 1 (Ang1), and CXCL8/IL-8. Only hGV induces the secretion of the antiangiogenic isoform of VEGF-A, namely, VEGF-A165b. While the release of VEGF-A, Ang1, and CXCL8/IL-8 was likely mediated by hGV enzymatic activity and/or binding to PLA2R1 and heparan sulfate proteoglycans, the release of VEGF-A165b requires the interaction with αVβ3 and α4β1 integrins. We also provide evidence that endogenous hGV released by N-formyl-met-leu-phe (fMLF)-activated PMNs is involved in the release of angiogenic factors. The translational relevance of these data is supported by our findings that hGV expression is increased in human samples of lung cancer which are infiltrated by PMNs. Overall, our results suggest that the hGV-neutrophil axis may play a relevant role in the modulation of cancer-related inflammation and angiogenesis

Influences of base excision repair defects on the lethality and mutagenicity induced by Me-lex, a sequence-selective N3-adenine methylating agent.

Due to its minor groove selectivity, Me-lex preferentially generates N3-methyladenine (3-MeA) adducts in double-stranded DNA. We undertook a genetic approach in yeast to establish the influence of base excision repair (BER) defects on the processing of Me-lex lesions on plasmid DNA that harbors the p53 cDNA as target. We constructed a panel of isogenic strains containing a reporter gene to test p53 function and the following gene deletions: deltamag1, deltaapn1apn2, and deltaapn1apn2mag1. When compared with the wild-type strain, a decrease in survival was observed in deltamag1, deltaapn1apn2, and deltaapn1apn2mag1. The Me-lex-induced mutation frequency increased in the following order: wild typedeltamag1deltaapn1apn2 = deltaapn1apn2mag1. A total of 77 mutants (23 in wild type, 31 in deltamag1, and 23 in deltaapn1apn2) were sequenced. Eighty-one independent mutations (24 in wild type, 34 in deltamag1, and 23 in deltaapn1apn2) were detected. The majority of base pair substitutions were AT-targeted in all strains (14/23, 61% in wild type; 20/34, 59%, in deltamag1; and 14/23, 61%, in deltaapn1apn2). The Mag1 deletion was associated with a significant decrease of GCAT transitions when compared with both the wild-type and the AP endonuclease mutants. This is the first time that the impact of Mag1 and/or AP endonuclease defects on the mutational spectra caused by 3-MeA has been determined. The results suggest that 3-MeA is critical for Me-lex cytotoxicity and that its mutagenicity is slightly elevated in the absence of Mag1 glycosylase activity but significantly higher in the absence of AP endonuclease activity

COVID-19 in rheumatic diseases in Italy: first results from the Italian registry of the Italian Society for Rheumatology (CONTROL-19)

OBJECTIVES: Italy was one of the first countries significantly affected by the coronavirus disease 2019 (COVID-19) epidemic. The Italian Society for Rheumatology promptly launched a retrospective and anonymised data collection to monitor COVID-19 in patients with rheumatic and musculoskeletal diseases (RMDs), the CONTROL-19 surveillance database, which is part of the COVID-19 Global Rheumatology Alliance. METHODS: CONTROL-19 includes patients with RMDs and proven severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) updated until May 3rd 2020. In this analysis, only molecular diagnoses were included. The data collection covered demographic data, medical history (general and RMD-related), treatments and COVID-19 related features, treatments, and outcome. In this paper, we report the first descriptive data from the CONTROL-19 registry. RESULTS: The population of the first 232 patients (36% males) consisted mainly of elderly patients (mean age 62.2 years), who used corticosteroids (51.7%), and suffered from multi-morbidity (median comorbidities 2). Rheumatoid arthritis was the most frequent disease (34.1%), followed by spondyloarthritis (26.3%), connective tissue disease (21.1%) and vasculitis (11.2%). Most cases had an active disease (69.4%). Clinical presentation of COVID-19 was typical, with systemic symptoms (fever and asthenia) and respiratory symptoms. The overall outcome was severe, with high frequencies of hospitalisation (69.8%), respiratory support oxygen (55.7%), non-invasive ventilation (20.9%) or mechanical ventilation (7.5%), and 19% of deaths. Male patients typically manifested a worse prognosis. Immunomodulatory treatments were not significantly associated with an increased risk of intensive care unit admission/mechanical ventilation/death. CONCLUSIONS: Although the report mainly includes the most severe cases, its temporal and spatial trend supports the validity of the national surveillance system. More complete data are being acquired in order to both test the hypothesis that RMD patients may have a different outcome from that of the general population and determine the safety of immunomodulatory treatments

Economic consequences of investing in anti-HCV antiviral treatment from the Italian NHS perspective : a real-world-based analysis of PITER data

OBJECTIVE: We estimated the cost consequence of Italian National Health System (NHS) investment in direct-acting antiviral (DAA) therapy according to hepatitis C virus (HCV) treatment access policies in Italy. METHODS: A multistate, 20-year time horizon Markov model of HCV liver disease progression was developed. Fibrosis stage, age and genotype distributions were derived from the Italian Platform for the Study of Viral Hepatitis Therapies (PITER) cohort. The treatment efficacy, disease progression probabilities and direct costs in each health state were obtained from the literature. The break-even point in time (BPT) was defined as the period of time required for the cumulative costs saved to recover the Italian NHS investment in DAA treatment. Three different PITER enrolment periods, which covered the full DAA access evolution in Italy, were considered. RESULTS: The disease stages of 2657 patients who consecutively underwent DAA therapy from January 2015 to December 2017 at 30 PITER clinical centres were standardized for 1000 patients. The investment in DAAs was considered to equal €25 million, €15 million, and €9 million in 2015, 2016, and 2017, respectively. For patients treated in 2015, the BPT was not achieved, because of the disease severity of the treated patients and high DAA prices. For 2016 and 2017, the estimated BPTs were 6.6 and 6.2 years, respectively. The total cost savings after 20 years were €50.13 and €55.50 million for 1000 patients treated in 2016 and 2017, respectively. CONCLUSIONS: This study may be a useful tool for public decision makers to understand how HCV clinical and epidemiological profiles influence the economic burden of HCV

Adenosine limits the therapeutic effectiveness of anti-CTLA4 mAb in a mouse melanoma model.

Combination therapies for melanoma that target immune-regulatory networks are entering clinical practice, and more are under investigation in preclinical or clinical studies. Adenosine plays a key role in regulating melanoma progression. We investigated the effectiveness of cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) antibody (mAb) in combination with either modulators of adenosine receptors (AR) activation or an inhibitor of adenosine production in a murine model of melanoma. We found that treatment with APCP, selective inhibitor of the adenosine-generating nucleotidase CD73, enhanced the activity of anti-CTLA4 mAb, by improving tumor immune response. Blockade of the adenosine A2a receptor (A2aR), which plays a critical role in the regulation of T-cell functions, significantly reduced melanoma growth. Most importantly, combination therapy including an A2aR antagonist with anti-CTLA4 mAb markedly inhibited tumor growth and enhanced anti-tumor immune responses. Targeting A3R and CTLA4 was not as effective in limiting melanoma growth as targeting A2aR. These data suggest that the efficacy of anti-CTLA4 melanoma therapy may be improved by targeting multiple mechanisms of immune suppression within tumor tissue, including CD73 or A2a receptor

Blockade of A2b Adenosine Receptor Reduces Tumor Growth and Immune Suppression Mediated by Myeloid-Derived Suppressor Cells in a Mouse Model of Melanoma

The A2b receptor (A2bR) belongs to the adenosine receptor family. Emerging evidence suggest that A2bR is implicated in tumor progression in some murine tumor models, but the therapeutic potential of targeting A2bR in melanoma has not been examined. This study first shows that melanoma-bearing mice treated with Bay 60-6583, a selective A2bR agonist, had increased melanoma growth. This effect was associated with higher levels of immune regulatory mediators interleukin-10 (IL-10) and monocyte chemoattractant protein 1 (MCP-1) and accumulation of tumor-associated CD11b positive Gr1 positive cells (CD11b+Gr1+) myeloid-derived suppressor cells (MDSCs). Depletion of CD11b+Gr1+ cells completely reversed the protumor activity of Bay 60-6583. Conversely, pharmacological blockade of A2bR with PSB1115 reversed immune suppression in the tumor microenvironment, leading to a significant melanoma growth delay. PSB1115 treatment reduced both levels of IL-10 and MCP-1 and CD11b+Gr1+ cell number in melanoma lesions. These effects were associated with higher frequency of tumor-infiltrating CD8 positive (CD8+) T cells and natural killer T (NKT) cells and increased levels of T helper 1 (Th1)-like cytokines. Adoptive transfer of CD11b+Gr1+ cells abrogated the antitumor activity of PSB1115. These data suggest that the antitumor activity of PSB1115 relies on its ability to lower accumulation of tumor-infiltrating MDSCs and restore an efficient antitumor T cell response. The antitumor effect of PSB1115 was not observed in melanoma-bearing nude mice. Furthermore, PSB1115 enhanced the antitumor efficacy of dacarbazine. These data indicate that A2bR antagonists such as PSB1115 should be investigated as adjuvants in the treatment of melanoma

Blockade of A2b adenosine receptor reduces tumor growth and immune suppression mediated by myeloid-derived suppressor cells in a mouse model of melanoma.

The A2b receptor (A2bR) belongs to the adenosine receptor family. Emerging evidence suggest that A2bR is implicated in tumor progression in some murine tumor models, but the therapeutic potential of targeting A2bR in melanoma has not been examined. This study first shows that melanoma-bearing mice treated with Bay 60-6583, a selective A2bR agonist, had increased melanoma growth. This effect was associated with higher levels of immune regulatory mediators interleukin-10 (IL-10) and monocyte chemoattractant protein 1 (MCP-1) and accumulation of tumorassociated CD11b positive Gr1 positive cells (CD11b+Gr1+)myeloid-derived suppressor cells (MDSCs). Depletion of CD11b+Gr1+ cells completely reversed the protumor activity of Bay 60-6583. Conversely, pharmacological blockade of A2bR with PSB1115 reversed immune suppression in the tumor microenvironment, leading to a significant melanoma growth delay. PSB1115 treatment reduced both levels of IL-10 and MCP-1 and CD11b+Gr1+ cell number in melanoma lesions. These effects were associated with higher frequency of tumor-infiltrating CD8 positive (CD8+) T cells and natural killer T (NKT) cells and increased levels of T helper 1 (Th1)-like cytokines. Adoptive transfer of CD11b+Gr1+ cells abrogated the antitumor activity of PSB1115. These data suggest that the antitumor activity of PSB1115 relies on its ability to lower accumulation of tumor-infiltrating MDSCs and restore an efficient antitumor T cell response. The antitumor effect of PSB1115 was not observed in melanoma-bearing nude mice. Furthermore, PSB1115 enhanced the antitumor efficacy of dacarbazine. These data indicate that A2bR antagonists such as PSB1115 should be investigated as adjuvants in the treatment of melanoma

NOTCH signaling promotes the survival of irradiated basal airway stem cells

Radiation-induced lung injury to normal airway epithelium is a frequent side-effect and dose-limiting factor in radiotherapy of tumors in the thoracic cavity. NOTCH signaling plays key roles in self-renewal and differentiation of upper airway basal lung stem cells during development, and the NOTCH pathway is frequently deregulated in lung cancer. In preclinical lung cancer models, NOTCH inhibition was shown to improve the radiotherapy response by targeting tumor stem cells, but the effects in combination with irradiation on normal lung stem cells are unknown. NOTCH/gamma-secretase inhibitors are potent clinical candidates to block NOTCH function in tumors, but their clinical implementation has been hampered by normal tissue side-effects. Here we show that NOTCH signaling is active in primary human-and murine-derived airway epithelial stem cell models and when combined with radiation NOTCH inhibition provokes a decrease in S-phase and increase in G(1)-phase arrest. We show that NOTCH inhibition in irradiated lung basal stem cells leads to a more potent activation of the DNA damage checkpoint kinases pATM and pCHK2 and results in an increased level of residual 53BP1 foci in irradiated lung basal stem cells reducing their capacity for self-renewal. The effects are recapitulated in ex vivo cultured lung basal stem cells after in vivo whole thorax irradiation and NOTCH inhibition. These results highlight the importance of studying normal tissue effects that may counteract the therapeutic benefit in the use of NOTCH/gamma-secretase inhibitors in combination with radiation for antitumor treatment