Functional insights into human macrophage response against Scedosporium apiospermum and Scedosporium dehoogii.

Fungal infections caused by Scedosporium species are rising among immunocompromised and immunocompetent patients. Within the immunocompetent group, patients with cystic fibrosis (pwCF) are at high risk of developing a chronic airway colonization by these molds. While S. apiospermum is one of the major species encountered in the lungs of pwCF, S. dehoogii has rarely been reported. The innate immune response is believed to be critical for host defense against fungal infections. However, its role has only recently been elucidated and the immune mechanisms against Scedosporium species are currently unknown. In this context, we undertook a comparative investigation of macrophage-mediated immune responses toward S. apiospermum and S. dehoogii conidia. Our data showed that S. apiospermum and S. dehoogii conidia strongly stimulated the expression of a set of pro-inflammatory cytokines and chemokines such as IL-1ß, IL-8, IL-6 and TNFα. We demonstrated that S. dehoogii was more potent in stimulating the early release of pro-inflammatory cytokines and chemokines while S. apiospermum induced a late inflammatory response at a higher level. Flow cytometry analysis showed that M1-like macrophages were able to internalize both S. apiospermum and S. dehoogii conidia, with a similar intracellular killing rate for both species. In conclusion, these results suggest that M1-like macrophages can rapidly initiate a strong immune response against both S. apiospermum and S. dehoogii. This response is characterized by a similar killing of internalized conidia, but a different time course of cytokine production.

Clusterin Neutralizes the Inflammatory and Cytotoxic Properties of Extracellular Histones in Sepsis.

Rationale: Extracellular histones, released into the surrounding environment during extensive cell death, promote inflammation and cell death, and these deleterious roles have been well documented in sepsis. Clusterin (CLU) is a ubiquitous extracellular protein that chaperones misfolded proteins and promotes their removal. Objectives: We investigated whether CLU could protect against the deleterious properties of histones. Methods: We assessed CLU and histone expression in patients with sepsis and evaluated the protective role of CLU against histones in in vitro assays and in vivo models of experimental sepsis. Measurements and Main Results: We show that CLU binds to circulating histones and reduces their inflammatory, thrombotic, and cytotoxic properties. We observed that plasma CLU levels decreased in patients with sepsis and that the decrease was greater and more durable in nonsurvivors than in survivors. Accordingly, CLU deficiency was associated with increased mortality in mouse models of sepsis and endotoxemia. Finally, CLU supplementation improved mouse survival in a sepsis model. Conclusions: This study identifies CLU as a central endogenous histone-neutralizing molecule and suggests that, in pathologies with extensive cell death, CLU supplementation may improve disease tolerance and host survival.

Acetoacetate protects macrophages from lactic acidosis-induced mitochondrial dysfunction by metabolic reprograming.

Lactic acidosis, the extracellular accumulation of lactate and protons, is a consequence of increased glycolysis triggered by insufficient oxygen supply to tissues. Macrophages are able to differentiate from monocytes under such acidotic conditions, and remain active in order to resolve the underlying injury. Here we show that, in lactic acidosis, human monocytes differentiating into macrophages are characterized by depolarized mitochondria, transient reduction of mitochondrial mass due to mitophagy, and a significant decrease in nutrient absorption. These metabolic changes, resembling pseudostarvation, result from the low extracellular pH rather than from the lactosis component, and render these cells dependent on autophagy for survival. Meanwhile, acetoacetate, a natural metabolite produced by the liver, is utilized by monocytes/macrophages as an alternative fuel to mitigate lactic acidosis-induced pseudostarvation, as evidenced by retained mitochondrial integrity and function, retained nutrient uptake, and survival without the need of autophagy. Our results thus show that acetoacetate may increase tissue tolerance to sustained lactic acidosis.

Carrot Genotypes Contrasted by Root Color and Grown under Different Conditions Displayed Differential Pharmacological Profiles in Vascular and Metabolic Cells.

Carrots' genotype and growing conditions influence their potential properties to fight against cardiovascular and metabolic diseases. The present study evaluated the influence of carrot genotypes contrasted by root color (Bolero, Presto, Karotan, Deep Purple, Kintoki and Blanche des Vosges) growing under standard, water-restricted, biotic stress (Alternaria dauci inoculation), and combined stress conditions (water restriction and A.dauci inoculation). The effect of carrots' polyphenol and carotenoid content was assessed on endothelial and smooth muscle cells, hepatocytes, adipocytes and macrophages functions (oxidative stress, apoptosis, proliferation, lipid accumulation and inflammation). Independently of varieties or growing conditions, all carrot extracts affected vascular cells' oxidative stress and apoptosis, and metabolic cells' oxidative stress and lipid accumulation. Three clusters were revealed and displayed beneficial properties mostly for adipocytes function, smooth muscle cells and hepatocytes, and endothelial cells and hepatocytes, respectively. Karotan and Presto varieties exhibited endothelial tropism while Blanche des Vosges targeted adipocytes. Carrots under biotic stress are more efficient in inducing beneficial effects, with the Bolero variety being the most effective. However, extracts from carrots which grew under combined stress conditions had limited beneficial effects. This report underscores the use of certain carrot extracts as potential effective nutraceutical supplements for metabolic diseases.

Lactic Acidosis Together with GM-CSF and M-CSF Induces Human Macrophages toward an Inflammatory Protumor Phenotype.

In established tumors, tumor-associated macrophages (TAM) orchestrate nonresolving cancer-related inflammation and produce mediators favoring tumor growth, metastasis, and angiogenesis. However, the factors conferring inflammatory and protumor properties on human macrophages remain largely unknown. Most solid tumors have high lactate content. We therefore analyzed the impact of lactate on human monocyte differentiation. We report that prolonged lactic acidosis induces the differentiation of monocytes into macrophages with a phenotype including protumor and inflammatory characteristics. These cells produce tumor growth factors, inflammatory cytokines, and chemokines as well as low amounts of IL10. These effects of lactate require its metabolism and are associated with hypoxia-inducible factor-1α stabilization. The expression of some lactate-induced genes is dependent on autocrine M-CSF consumption. Finally, TAMs with protumor and inflammatory characteristics (VEGFhigh CXCL8+ IL1ß+) are found in solid ovarian tumors. These results show that tumor-derived lactate links the protumor features of TAMs with their inflammatory properties. Treatments that reduce tumor glycolysis or tumor-associated acidosis may help combat cancer.

Screening of ordinary commercial varieties of apple fruits under different storage conditions for their potential vascular and metabolic protective properties.

Epidemiological studies reported that apple consumption is associated with a decrease of cardiovascular and metabolic dysfunction, probably due to the polyphenols and fibers present in this fruit. The storage conditions and genetic origin of apples have been reported to influence their content and, as a consequence, their pharmacological properties. The present study evaluated the influence of varieties and storage conditions of traditional and highly appreciated apples including Gala, Golden Delicious, Granny Smith and Pink Lady varieties after harvest and storage under classic cold conditions, under a controlled atmosphere, or under extreme ultra-low oxygen conditions. Thus, a multi-parametric screening on cell models associated with vascular and metabolic dysfunctions - such as endothelial and smooth muscle cells, hepatocytes, adipocytes and macrophages - in relation to the apple polyphenol content has been developed. This strategy demonstrated that, overall, peeled apple samples exhibited a vascular tropism and acted mainly on proliferation and oxidative stress in endothelial and smooth muscle cells. Apple extracts appeared to be less effective on adipocytes and macrophages, but they exhibited antioxidant properties in hepatocytes. Among the varieties, Gala and Golden Delicious were the most efficient against the processes involved in the development of atherosclerosis. Concerning storage conditions, most of the apple varieties were more efficient under harvest conditions, while they could not be discriminated under all other cold conditions and the concentration used, except for the Gala samples. Interestingly, pharmacological properties were associated with the polyphenol profiles of freeze dried apple flesh powder. The present report revealed the potential use of some apple extracts as effective food supplements or nutraceuticals for the prevention and/or management of cardiovascular and metabolic diseases.

Detection of Anti-Pentraxin-3 Autoantibodies in ANCA-Associated Vasculitis.

OBJECTIVES: Pentraxin 3 (PTX3), in common with myeloperoxidase and proteinase 3, is stored in human neutrophil granules and is expressed on apoptotic neutrophil surface. We therefore investigated the presence of anti-PTX3 autoantibodies (aAbs) in the sera of antineutrophil cytoplasmic antibodies (ANCA)-associated vasculitis (AAV) patients. METHODS: Presence of anti-PTX3 autoantibodies was analysed by a specific enzyme-linked immunosorbent assay in sera from 150 patients with microscopic polyangiitis (MPA), granulomatosis with polyangiitis (GPA), and eosinophilic granulomatosis with polyangiitis (EGPA), and in sera of 227 healthy subjects (HS), 40 systemic sclerosis (SSc) patients, and 25 giant cell arteritis patients (GCA). Using indirect immunofluorescence on fixed human neutrophils, we also analyzed the staining pattern associated with the presence of anti-PTX3 aAbs. RESULTS: Anti-PTX3 aAbs were detected in 56 of 150 (37.3%) of the AAV patients (versus 12 of 227 (5.3%) of HS, p<0.001) and, interestingly, in 7 of 14 MPO and PR3 ANCA negative AAV patients. Moreover, by indirect immunofluorescence on fixed neutrophils, anti-PTX3 aAbs gave rise to a specific cytoplasmic fluorescence pattern distinct from the classical cytoplasmic (c-ANCA), perinuclear (p-ANCA), and atypical (a-ANCA) pattern. Anti-PTX3 aAbs levels were higher in patients with active AAV as compared to patients with inactive disease. CONCLUSION: Our work suggests that PTX3 is as a novel ANCA antigen. Anti-PTX3 aAbs appear thus as a promising novel biomarker in the diagnosis of AAV, including in patients without detectable MPO and PR3 ANCA.

Differential expression of the immunosuppressive enzyme IL4I1 in human induced Aiolos+, but not natural Helios+, FOXP3+ Treg cells.

IL4I1 encodes an L-phenylalanine oxidase that inhibits T-cell proliferation. It has been recently reported that IL4I1 is expressed in TH17 cells as part of a mechanism that limits their pathogenicity. We have previously identified a population of human FOXP3(+) Treg cells that secrete IL-17 ex vivo; here, we addressed the expression of IL4I1 in that Treg-cell population. We found that in ex vivo isolated circulating Treg cells, IL4I1 expression is induced by activation. Moreover, IL4I1 expression is restricted to cells that do not express Helios, a transcription factor that characterizes natural Treg cells, but that express Aiolos, which is involved in the differentiation of TH17 and induced Treg cells. We also showed that conversion of Treg cells under inflammatory conditions increases IL4I1 expression, likely as part of a regulatory loop that attempts to limit the pathogenicity resulting from their conversion into TH17. The specific expression of IL4I1 in TH17 and iTreg cells may provide insights into approaches that aim at modulating these populations in different pathological conditions involving inflammation-mediated immunosuppression.

Expression of MAGE-A3/6 in primary breast cancer is associated with hormone receptor negative status, high histologic grade, and poor survival.

The cancer testis antigen (CTA), melanoma-associated antigen A3/6 (MAGE-A3/6), is expressed in human cancers of different histologic types, to variable extents, and is an important target for immunotherapy. In this study, to address the potential of MAGE-A3/6 as an immunotherapeutic target in breast cancer (BC), we assessed MAGE-A3/6 expression by PCR in a cohort of 362 primary BC tumors and analyzed the correlation between MAGE-A3/6 expression, tumors hormone receptor (HR) status, and other clinicopathologic features. We found expression of MAGE-A3/6 in 10% of primary BC tumors. MAGE-A3/6 expression was significantly correlated with estrogen receptor (ER) and progesterone receptor (PR) negative status and was frequent in ER (29%) and in PR (24%) tumors. MAGE-A3/6 expression was also significantly associated with high histologic grade but not with patients age, tumor size, tumor type, lymph-node invasion, and human epidermal growth factor receptor 2 (HER2) overexpression. Consistent with the associated poor clinicopathologic features, patients with MAGE-A3/6-expressing tumors had a worse disease-specific survival as compared with patients with MAGE-A3/6 tumors. The frequent expression of MAGE-A3/6 in tumors of patients with primary HR BC, who have, for a large part, limited therapeutic options, encourages the selection of BC patients bearing MAGE-A3/6-expressing tumors for targeted immunotherapy.

CD4+ T effectors specific for the tumor antigen NY-ESO-1 are highly enriched at ovarian cancer sites and coexist with, but are distinct from, tumor-associated Treg.

Whereas tumor infiltration by T effectors is generally associated with a more favorable prognosis, the accumulation of CD4(+) regulatory T cells (Treg) within tumors is instead often associated with poor disease outcome. Because approaches to improve antitumor immunity aim, on one hand, at expanding tumor antigen-specific T cells and, on the other, at eliminating or inactivating Treg, an outstanding question is whether, and to what extent, tumor antigen-specific CD4(+) T effectors present at tumor sites overlap with tumor-associated Treg. Here, we used MHC class II/peptide tetramers incorporating an immunodominant peptide from the human tumor-specific antigen NY-ESO-1 to assess antigen-specific CD4(+) T cells among conventional CD4(+) T effectors and Treg at sites of ovarian cancer. We found that, in patients who spontaneously respond to the antigen, the frequency of NY-ESO-1 tetramer(+) cells detected ex vivo was highly enriched in tumors as compared with the periphery. At tumor sites, NY-ESO-1 tetramer(+) cells were detected concomitantly with high proportions of Treg but were distinct from the latter and displayed characteristics of TH1 effectors. Thus, even in the presence of high proportions of Treg, tumor antigen-specific CD4(+) T cells can accumulate in ovarian tumors and maintain an effector phenotype.

CXCR3+ T regulatory cells selectively accumulate in human ovarian carcinomas to limit type I immunity.

Antitumor type I T-cell responses involving IFN-γ production are critical to control cancer, but the efficacy of this response is limited by a variety of immunosuppressive mechanisms that promote tumoral immune escape. One critical mechanism involves the accumulation of FOXP3(+) T regulatory cells (Treg), a class of suppressive T cells that prevent excessive tissue destruction caused by unchecked immune responses. Recent studies have revealed that FOXP3(+) Treg include distinct subsets specifically controlling over the corresponding effector subset. In particular, CXCR3(+) Treg have been described as a subset specialized in the control of type I T-cell responses in vivo. Here, we show that CXCR3(+) Treg are highly enriched in human ovarian carcinomas, particularly in solid tumor masses, where they represent the majority of Treg. Tumor-associated CXCR3(+.) Treg coexpress T-bet but do not secrete IFN-γ ex vivo and suppress proliferation and IFN-γ secretion of T effectors. In addition, they coexpress Helios, suggesting that they originate from natural Treg. Finally, we show that the proportion of CXCR3(+) Treg at tumor sites is directly correlated with that of CXCR3(+) T effectors, consistent with expression of CXCR3 ligands. Together, our findings support the concept that natural CXCR3(+) T-bet(+) Treg selectively accumulate in ovarian tumors to control type I T-cell responses, resulting in the collateral limitation of efficient antitumor immunity.

Human T(H)17 immune cells specific for the tumor antigen MAGE-A3 convert to IFN-γ-secreting cells as they differentiate into effector T cells in vivo.

The role of T(H)17 cells in cancer is being investigated, but the existence of tumor antigen-specific T(H)17 cells has yet to be ascertained. Here, we report the first description of a spontaneous T(H)17 (IL-17(+)) response to the important tumor antigen MAGE-A3, which occurred concurrently with a T(H)1 (IFN-γ(+)) response in a lung cancer patient. MAGE-A3-specific interleukin (IL)-17(+) T cells were mainly CCR7(+) central memory T cells, whereas IFN-γ(+) cells were enriched for CCR7(-) effector memory T cells. An assessment of the fine specificity of antigen recognition by these T cells indicated that the CCR6(+)CCR4(+) and CCR6(+)CXCR3(+) fractions contained the same T(H)17/T(H)1 population at early and late differentiation stages, respectively, whereas the CCR6(-)CXCR3(+) fraction contained a distinct T(H)1 population. These findings are important because they suggest a differentiation model in which tumor antigen-specific CD4(+) T cells that are primed under T(H)17 polarizing conditions will progressively convert into IFN-γ-secreting cells in vivo as they differentiate into effector T cells that can effectively attack tumors.

Antibody responses to NY-ESO-1 in primary breast cancer identify a subtype target for immunotherapy.

The highly immunogenic human tumor antigen NY-ESO-1 (ESO) is a target of choice for anti-cancer immune therapy. In this study, we assessed spontaneous antibody (Ab) responses to ESO in a large cohort of patients with primary breast cancer (BC) and addressed the correlation between the presence of anti-ESO Ab, the expression of ESO in the tumors and their characteristics. We found detectable Ab responses to ESO in 1% of the patients. Tumors from patients with circulating Ab to ESO exhibited common characteristics, being mainly hormone receptor (HR)⁻ invasive ductal carcinomas of high grade, including both HER2⁻ and HER2⁺ tumors. In line with these results, we detected ESO expression in 20% of primary HR⁻ BC, including both ESO Ab⁺ and Ab⁻ patients, but not in HR⁺ BC. Interestingly, whereas expression levels in ESO⁺ BC were not significantly different between ESO Ab⁺ and Ab⁻ patients, the former had, in average, significantly higher numbers of tumor-infiltrated lymph nodes, indicating that lymph node invasion may be required for the development of spontaneous anti-tumor immune responses. Thus, the presence of ESO Ab identifies a tumor subtype of HR⁻ (HER2⁻ or HER2⁺) primary BC with frequent ESO expression and, together with the assessment of antigen expression in the tumor, may be instrumental for the selection of patients for whom ESO-based immunotherapy may complement standard therapy.

Assessment of vaccine-induced CD4 T cell responses to the 119-143 immunodominant region of the tumor-specific antigen NY-ESO-1 using DRB1*0101 tetramers.

PURPOSE: NY-ESO-1 (ESO), a tumor-specific antigen of the cancer/testis group, is presently viewed as an important model antigen for the development of generic anticancer vaccines. The ESO(119-143) region is immunodominant following immunization with a recombinant ESO vaccine. In this study, we generated DRB1*0101/ESO(119-143) tetramers and used them to assess CD4 T-cell responses in vaccinated patients expressing DRB1*0101 (DR1). EXPERIMENTAL DESIGN: We generated tetramers of DRB1*0101 incorporating peptide ESO(119-143) using a previously described strategy. We assessed ESO(119-143)-specific CD4 T cells in peptide-stimulated postvaccine cultures using the tetramers. We isolated DR1/ESO(119-143) tetramer(+) cells by cell sorting and characterized them functionally. We assessed vaccine-induced CD4(+) DR1/ESO(119-143) tetramer(+) T cells ex vivo and characterized them phenotypically. RESULTS: Staining of cultures from vaccinated patients with DR1/ESO(119-143) tetramers identified vaccine-induced CD4 T cells. Tetramer(+) cells isolated by cell sorting were of T(H)1 type and efficiently recognized full-length ESO. We identified ESO(123-137) as the minimal optimal epitope recognized by DR1-restricted ESO-specific CD4 T cells. By assessing DR1/ESO(119-143) tetramer(+) cells using T cell receptor (TCR) ß chain variable region (Vß)-specific antibodies, we identified several frequently used Vß. Finally, direct ex vivo staining of patients' CD4 T cells with tetramers allowed the direct quantification and phenotyping of vaccine-induced ESO-specific CD4 T cells. CONCLUSIONS: The development of DR1/ESO(119-143) tetramers, allowing the direct visualization, isolation, and characterization of ESO-specific CD4 T cells, will be instrumental for the evaluation of spontaneous and vaccine-induced immune responses to this important tumor antigen in DR1-expressing patients.

Monitoring of NY-ESO-1 specific CD4+ T cells using molecularly defined MHC class II/His-tag-peptide tetramers.

MHC-peptide tetramers have become essential tools for T-cell analysis, but few MHC class II tetramers incorporating peptides from human tumor and self-antigens have been developed. Among limiting factors are the high polymorphism of class II molecules and the low binding capacity of the peptides. Here, we report the generation of molecularly defined tetramers using His-tagged peptides and isolation of folded MHC/peptide monomers by affinity purification. Using this strategy we generated tetramers of DR52b (DRB3*0202), an allele expressed by approximately half of Caucasians, incorporating an epitope from the tumor antigen NY-ESO-1. Molecularly defined tetramers avidly and stably bound to specific CD4(+) T cells with negligible background on nonspecific cells. Using molecularly defined DR52b/NY-ESO-1 tetramers, we could demonstrate that in DR52b(+) cancer patients immunized with a recombinant NY-ESO-1 vaccine, vaccine-induced tetramer-positive cells represent ex vivo in average 1:5,000 circulating CD4(+) T cells, include central and transitional memory polyfunctional populations, and do not include CD4(+)CD25(+)CD127(-) regulatory T cells. This approach may significantly accelerate the development of reliable MHC class II tetramers to monitor immune responses to tumor and self-antigens.