Levels of peripheral blood polymorphonuclear myeloid-derived suppressor cells and selected cytokines are potentially prognostic of disease progression for patients with non-small cell lung cancer.

Polymorphonuclear-MDSC (PMN-MDSC) have emerged as an independent prognostic factor for survival in NSCLC. Similarly, cytokine profiles have been used to identify subgroups of NSCLC patients with different clinical outcomes. This prospective study investigated whether the percentage of circulating PMN-MDSC, in conjunction with the levels of plasma cytokines, was more informative of disease progression than the analysis of either factor alone. We analyzed the phenotypic and functional profile of peripheral blood T-cell subsets (CD3+, CD3+CD4+ and CD3+CD8+), neutrophils (CD66b+) and polymorphonuclear-MDSC (PMN-MDSC; CD66b+CD11b+CD15+CD14-) as well as the concentration of 14 plasma cytokines (IL-1ß, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12 p70, IL-17A, IL-27, IL-29, IL-31, and IL-33, TNF-α, IFN-γ) in 90 treatment-naïve NSCLC patients and 25 healthy donors (HD). In contrast to HD, NSCLC patients had a higher percentage of PMN-MDSC and neutrophils (P < 0.0001) but a lower percentage of CD3+, CD3+CD4+ and CD3+CD8+ cells. PMN-MDSC% negatively correlated with the levels of IL1-ß, IL-2, IL-27 and IL-29. Two groups of patients were identified according to the percentage of circulating PMN-MDSC. Patients with low PMN-MDSC (≤ 8%) had a better OS (22.1 months [95% CI 4.3-739.7]) than patients with high PMN-MDSC (9.3 months [95% CI 0-18.8]). OS was significantly different among groups of patients stratified by both PMN-MDSC% and cytokine levels. In sum, our findings provide evidence suggesting that PMN-MDSC% in conjunction with the levels IL-1ß, IL-27, and IL-29 could be a useful strategy to identify groups of patients with potentially unfavorable prognoses.

Expression of PD-1/PD-L1 and PD-L2 in peripheral T-cells from non-small cell lung cancer patients.

Binding of programmed death-1 (PD-1) with its ligands (PD-L1/2) transmits a co-inhibitory signal in activated T-cells that promotes T-cell exhaustion, leading to tumor immune evasion. The efficacy of antibodies targeting PD-1 and PD-L1 has led to a paradigm shift in lung cancer treatment but the prognostic and predictive value of tumor PD-L1 expression remains controversial. Evaluating PD-1, PD-L1/2 expression in peripheral blood cells may serve as a potential biomarker for prognosis and response to therapy. In this prospective observational study, plasma cytokine levels and PD-1, PD-L1 and PD-L2 expression was evaluated in circulating CD3+, CD3+CD4+ and CD3+CD8+ cells from 70 treatment-naïve patients with advanced NSCLC (Stage IIIB and IV) and from 10 healthy donors. The primary objective was to assess OS according to PD-1, PD-L1, PD-L2 expression status on PBMCs and lymphocyte subsets. Our results indicate that the percentage of PD-L1+CD3+, PD-L1+CD3+CD8+ PD-L2+PBMCs, PD-L2+CD3+, PD-L2+CD3+CD4+ cells was higher in patients than in healthy donors. Survival was decreased among patients with a high percentage of either PD-1+PBMCs, PD-1+CD3+, PD-L1+CD3+, PD-L1+CD3+CD8+, PD-L2+CD3+, PD-L2+CD3+CD4+, or PD-L2+CD3+CD8+ cells. IL-2 and TNF-α showed the strongest association with PD-L1 and PD-L2 expression on specific subsets of T-lymphocytes. Our findings suggest that increased PD-1/PD-L1/PDL-2 expression in PBMCs, particularly in T-cells, may be an additional mechanism leading to tumor escape from immune control. This study is registered with ClinicalTrials.gov, number NCT02758314.

CD47 overexpression is associated with decreased neutrophil apoptosis/phagocytosis and poor prognosis in non-small-cell lung cancer patients.

BACKGROUND: Non-small-cell lung cancer (NSCLC) patients often exhibit neutrophilia, which has been associated with poor clinical outcomes. However, the mechanisms that lead to neutrophilia have not been fully established. CD47 is an antiphagocytic molecule that promotes neutrophil recruitment. METHODS: Blood was collected from 50 treatment-naive patients with advanced NSCLC and from 25 healthy subjects. The frequency of CD66b+ cells and the expression of CD47 were determined by flow cytometry. Neutrophil apoptosis was determined by 7-amino-actinomycin D/Annexin V-APC staining. Phagocytosis was assessed by flow cytometry. Reactive oxygen species production after phorbol 12-myristate 13-acetate treatment was quantified by 2',7'-dichlorofluorescein fluorescence. Pro-inflammatory plasma cytokines were quantified using a cytometric bead array assay. RESULTS: The percentage of circulating neutrophils was significantly higher in patients than in controls (P<0.001). Patient-derived neutrophils had a higher oxidative potential than those of controls (P=0.0286). The number of neutrophils in late apoptosis/necrosis was lower in patients than in controls (P=0.0317). Caspase 3/7 activation was also lower in patients than in controls (P=0.0079). CD47 expression in whole-blood samples and in the neutrophil fraction was higher in NSCLC patients than in controls (P=0.0408 and P<0.001). Patient-derived neutrophils were phagocytosed at a lower rate than those of controls (P=0.0445). CD47 expression in neutrophils negatively correlated with their ingestion by macrophages (P=0.0039). High CD47 expression was associated with a lower overall survival. CONCLUSIONS: Increased CD47 expression on the surface of neutrophils was associated with a delay in neutrophil apoptosis and with an impairment in their phagocytic clearance by macrophages, suggesting that CD47 overexpression may be one of the underlying mechanisms leading to neutrophilia in NSCLC patients.

Differential Expression of Adhesion-Related Proteins and MAPK Pathways Lead to Suitable Osteoblast Differentiation of Human Mesenchymal Stem Cells Subpopulations.

Cellular adhesion enables communication between cells and their environment. Adhesion can be achieved throughout focal adhesions and its components influence osteoblast differentiation of human mesenchymal stem cells (hMSCs). Because cell adhesion and osteoblast differentiation are closely related, this article aimed to analyze the expression profiles of adhesion-related proteins during osteoblastic differentiation of two hMSCs subpopulations (CD105(+) and CD105(-)) and propose a strategy for assembling bone grafts based on its adhesion ability. In vitro experiments of osteogenic differentiation in CD105(-) cells showed superior adhesion efficiency and 2-fold increase of α-actinin expression compared with CD105(+) cells at the maturation stage. Interestingly, levels of activated ß1-integrin increased in CD105(-) cells during the process. Additionally, the CD105(-) subpopulation showed 3-fold increase of phosphorylated FAK(Y397) compared to CD105(+) cells. Results also indicate that ERK1/2 was activated during CD105(-) bone differentiation and participation of mitogen-activated protein kinase (MAPK)-p38 in CD105(+) differentiation through a focal adhesion kinase (FAK)-independent pathway. In vivo trial demonstrated that grafts containing CD105(-) showed osteocytes embedded in a mineralized matrix, promoted adequate graft integration, increased host vascular infiltration, and efficient intramembranous repairing. In contrast, grafts containing CD105(+) showed deficient endochondral ossification and fibrocartilaginous tissue. Based on the expression of α-actinin, FAKy,(397) and ERK1/2 activation, we define maturation stage as critical for bone graft assembling. By in vitro assays, CD105(-) subpopulation showed superior adhesion efficiency compared to CD105(+) cells. Considering in vitro and in vivo assays, this study suggests that integration of a scaffold with CD105(-) subpopulation at the maturation stage represents an attractive strategy for clinical use in orthopedic bioengineering.

Dehydroepiandrosterone has strong antifibrotic effects and is decreased in idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is an ageing-related lung disorder characterised by expansion of the myofibroblast population and aberrant lung remodelling. Dehydroepiandrosterone (DHEA), a steroid pro-hormone, decreases with age but an exaggerated decline has been associated with chronic degenerative diseases. We quantified the plasma levels of DHEA and its sulfated form (DHEA-S) in 137 IPF patients and 58 controls and examined the effects of DHEA on human lung fibroblasts. Plasma DHEA/DHEA-S was significantly decreased in male IPF patients (median (range) DHEA: 4.4 (0.2-29.2) versus 6.7 (2.1-15.2) ng · mL(-1), p<0.01; DHEA-S: 47 (15.0-211) versus 85.2 (37.6-247.0) µg · dL(-1), p<0.001), while in females only DHEA-S was significantly decreased (32.6 (15.0-303.0) versus 68.3 (16.4-171) µg · dL(-1), p<0.001). DHEA caused a decrease in fibroblast proliferation and an approximately two-fold increase in fibroblast apoptosis, probably through the intrinsic pathway with activation of caspase-9. This effect was accompanied by upregulation of several pro-apoptotic proteins (Bax and cyclin-dependent kinase-inhibitor CDNK1A) and downregulation of anti-apoptotic proteins, such as cellular inhibitor of apoptosis (c-IAP)1 and c-IAP2. DHEA also caused a significant decrease of transforming growth factor-ß1-induced collagen production and fibroblast to myofibroblast differentiation, and inhibited platelet-derived growth factor-induced fibroblast migration. These findings demonstrate a disproportionate decrease of DHEA/DHEA-S in IPF patients and indicate that this molecule has multiple antifibrotic properties.

Characterization of mesenchymal stem cell subpopulations from human amniotic membrane with dissimilar osteoblastic potential.

Human fetal mesenchymal stem cells can be isolated from the amniotic membrane (AM-hMSCs) by enzymatic digestion. The biological properties of this cell population have been characterized; however, few studies have focused on the presence of stem cell subpopulations and their differentiation potential. The aim of the present study was to isolate homogeneous AM-hMSC subpopulations based on the coexpression of surface markers. In addition, we aimed to characterize stem cell subpopulations through the detection of typical stem cell markers and its differentiation potential. In this study, fluorescence-activated cell sorting (FACS) was used to positively select for the surface markers CD44, CD73, and CD105. Two subpopulations were isolated: CD44+ / CD73+ / CD105+ (CD105+), and CD44+ / CD73+ / CD105- (CD105-). To characterize the cell subpopulations, the expression of pluripotency-associated markers was analyzed by reverse transcriptase-polymerase chain reaction and immunofluorescence. Our results showed positive expression of SOX2, SOX3, PAX6, OCT3/4, and NANOG in the CD105+ and CD105(-) cell subpopulations. In contrast, we did not detect expression of SSEA4 or FOXD3 in either subpopulation. Immunophenotypes, such as mesenchymal and hematopoietic markers, were studied by FACS analyses. Our data revealed the expression of the CD49a, CD49d, CD29, integrin α9ß1, CD44, CD73, and CD105 antigens in both subpopulations. In contrast, CD90, CD45, CD34, CD14, and HLA-DR expression was not detected. The ability of both subpopulations to differentiate into osteoblasts, adipocytes, and chondrocytes was evidenced using Alizarin red, Oil-Red, and Alcian blue staining, respectively. Furthermore, neuronal differentiation was demonstrated by the expression of GFAP and NEURO-D. Interestingly, we observed a dissimilar osteoblastic differentiation potential between the subpopulations. CD105- cells showed stronger expression of secreted protein acidic and rich in cysteine (SPARC) and osteonectin, which was associated with more effective calcium deposition, than CD105+ cells. In conclusion, we described a systematic method for the isolation of hMSCs that was highly reproducible and generated homogeneous cultures for osteoblast differentiation with an efficient capacity for mineralization.

The Tim3-galectin 9 pathway induces antibacterial activity in human macrophages infected with Mycobacterium tuberculosis.

T cell Ig and mucin domain 3 (Tim3) is an inhibitory molecule involved in immune tolerance, autoimmune responses, and antiviral immune evasion. However, we recently demonstrated that Tim3 and Galectin-9 (Gal9) interaction induces a program of macrophage activation that results in killing of Mycobacterium tuberculosis in the mouse model of infection. In this study, we sought to determine whether the Tim3-Gal9 pathway plays a similar role in human pulmonary TB. We identified that pulmonary TB patients have reduced expression of Tim3 on CD14(+) monocytes in vivo. By blocking Tim3 and Gal9 interaction in vitro, we show that these molecules contribute to the control of intracellular bacterial replication in human macrophages. The antimicrobial effect was partially dependent on the production of IL-1ß. Our results establish that Tim3-Gal9 interaction activates human M. tuberculosis -infected macrophages and leads to the control of bacterial growth through the production of the proinflammatory cytokine IL-1ß. Data presented in this study suggest that one of the potential pathways activated by Tim3/Gal9 is the secretion of IL-1ß, which plays a crucial role in antimicrobial immunity by modulating innate inflammatory networks.

Role of Sonic Hedgehog in idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a progressive and lethal disease of unknown etiology and uncertain pathogenic mechanisms. Recent studies indicate that the pathogenesis of the disease may involve the abnormal expression of certain developmental pathways. Here we evaluated the expression of Sonic Hedgehog (SHH), Patched-1, Smoothened, and transcription factors glioma-associated oncogene homolog (GLI)1 and GLI2 by RT-PCR, as well as their localization in IPF and normal lungs by immunohistochemistry. The effects of SHH on fibroblast proliferation, migration, collagen and fibronectin production, and apoptosis were analyzed by WST-1, Boyden chamber chemotaxis, RT-PCR, Sircol, and annexin V-propidium iodide binding assays, respectively. Our results showed that all the main components of the Sonic signaling pathway were overexpressed in IPF lungs. With the exception of Smoothened, they were also upregulated in IPF fibroblasts. SHH and GLI2 localized to epithelial cells, whereas Patched-1, Smoothened, and GLI1 were observed mainly in fibroblasts and inflammatory cells. No staining was detected in normal lungs. Recombinant SHH increased fibroblast proliferation (P < 0.05), collagen synthesis, (2.5 ± 0.2 vs. 4.5 ± 1.0 µg of collagen/ml; P < 0.05), fibronectin expression (2-3-fold over control), and migration (190.3 ± 12.4% over control, P < 0.05). No effect was observed on α-smooth muscle actin expression. SHH protected lung fibroblasts from TNF-α/IFN-γ/Fas-induced apoptosis (14.5 ± 3.2% vs. 37.3 ± 7.2%, P < 0.0001). This protection was accompanied by modifications in several apoptosis-related proteins, including increased expression of X-linked inhibitor of apoptosis. These findings indicate that the SHH pathway is activated in IPF lungs and that SHH may contribute to IPF pathogenesis by increasing the proliferation, migration, extracellular matrix production, and survival of fibroblasts.

Absence of Thy-1 results in TGF-ß induced MMP-9 expression and confers a profibrotic phenotype to human lung fibroblasts.

Fibroblasts differ in a variety of phenotypic features, including the expression of Thy-1 a glycophosphatidylinositol-linked glycoprotein. Fibroblasts in idiopathic pulmonary fibrosis (IPF) are Thy-1 negative, whereas most fibroblasts from normal lungs are Thy-1 positive. However, the functional consequences of the absence of Thy-1 are not fully understood. We analyzed the expression of Thy-1 in several primary fibroblasts lines derived from IPF, hypersensitivity pneumonitis (HP), and normal human lungs. We found that a high proportion, independently of their origin, expressed Thy-1 in vitro. We identified a primary culture of HP fibroblasts, which did not express Thy-1, and compared several functional activities between Thy-1 (-) and Thy-1 (+) fibroblasts. Thy-1 (-) fibroblasts were smaller (length: 41.3±20.8 µ versus 83.1±40 µ), showed increased proliferative capacity and enhanced PDGF-induced transmigration through collagen I (59.9% versus 42.2% over control under basal conditions, P<0.01). Likewise, Thy-1 (-) fibroblasts either spontaneously or after TGF-ß stimulation demonstrated stronger contraction of collagen matrices (eg, 0.17±0.03 versus 0.6±0.05 cm² after TGF-ß stimulation at 24 h; P<0.01). Thy-1 (-) lung fibroblasts stimulated with TGF-ß1 expressed MMP-9, an enzyme that is usually not produced by lung fibroblasts. TGFß-induced MMP-9 expression was reversible upon re-expression of Thy-1 after transfection with full-length Thy-1. ß-glycan, a TGF-ß receptor antagonist abolished MMP-9 expression. TGF-ß1-induced MMP-9 in Thy-1 (-) fibroblasts depended on the activation of ERK1/2 signaling pathway. Finally, we demonstrated that fibroblasts from IPF fibroblastic foci, which do not express Thy-1 exhibit strong staining for immunoreactive MMP-9 protein in vivo. These findings indicate that loss of Thy-1 in human lung fibroblasts induces a fibrogenic phenotype.

[Familial pulmonary fibrosis in 2 Mexican sisters with Hermansky-Pudlak syndrome]. / Fibrosis pulmonar familiar en 2 hermanas mexicanas con síndrome de Hermansky-Pudlak.

Hermansky-Pudlak syndrome is an autosomal recessive disorder commonly found in individuals of Puerto Rican ancestry. We present 2 cases of familial pulmonary fibrosis in 2 Mexican sisters with Hermansky-Pudlak syndrome. Pulmonary fibrosis was biopsy-proven in 1 of the patients. This report shows that Hermansky-Pudlak syndrome may occur in individuals of Mexican ancestry.

MICA polymorphisms and decreased expression of the MICA receptor NKG2D contribute to idiopathic pulmonary fibrosis susceptibility.

Idiopathic pulmonary fibrosis (IPF) is a progressive and lethal lung disorder of unknown etiology. IPF is likely the result of complex interrelationships between environmental and host factors, although the genetic risk factors are presently uncertain. Because we have found that some MHC polymorphisms confer susceptibility to IPF, in the present study we aimed to evaluate the role of the MHC class I chain-related gene A (MICA) in the risk of developing the disease. MICA molecular typing was done by reference strand mediated conformation analysis in a cohort of 80 IPF patients and 201 controls. In addition, the lung cellular source of the protein was examined by immunohistochemistry, the expression of the MICA receptor NKG2D in lung cells by flow cytometry and soluble MICA by ELISA. A significant increase of MICA*001 was observed in the IPF cohort (OR = 2.91, 95% CI = 1.04-8.25; pC = 0.03). Likewise, the frequency of the MICA*001/*00201 genotype was significantly increased in patients with IPF compared with the healthy controls (OR = 4.72, 95% CI = 1.15-22.51; pC = 0.01). Strong immunoreactive MICA staining was localized in alveolar epithelial cells and fibroblasts from IPF lungs while control lungs were negative. Soluble MICA was detected in 35% of IPF patients compared with 12% of control subjects (P = 0.0007). The expression of NKG2D was significantly decreased in gammadelta T cells and natural killer cells obtained from IPF lungs. These findings indicate that MICA polymorphisms and abnormal expression of the MICA receptor NKG2D might contribute to IPF susceptibility.

Gene expression profiles distinguish idiopathic pulmonary fibrosis from hypersensitivity pneumonitis.

RATIONALE: Many of the interstitial lung diseases represent a diagnostic and therapeutic challenge because their clinical and even histologic features are often nonspecific. Likewise, the transcriptional signatures of most of them are unknown. OBJECTIVE: To compare the gene expression patterns from patients with idiopathic pulmonary fibrosis (IPF) hypersensitivity pneumonitis (HP), and nonspecific interstitial pneumonia (NSIP) using custom oligonucleotide microarrays. METHODS: We profiled lung biopsies from 15 patients with IPF, 12 with HP, and eight with NSIP. Labeled complementary ribonucleic acid was hybridized to a custom Affymetrix oligonucleotide DNA microarray using standard Affymetrix protocols. The custom array, Hu03, contained 59,619 probe sets representing an estimated 46,000 gene clusters. RESULTS: We identified statistically significant gene expression signatures that characterize HP and IPF. The HP gene expression signature was enriched for genes that are functionally associated with inflammation, T-cell activation, and immune responses, whereas the IPF signature was characterized by the expression of tissue remodeling, epithelial, and myofibroblast genes. We then compared these gene expression signatures to classify NSIP, a histologic pattern that is often difficult to differentiate consistently from HP and IPF. Two cases exhibited an IPF-like gene expression, another one could be more properly classified as HP, whereas others did not resemble HP or IPF, suggesting that they may represent idiopathic NSIP. CONCLUSIONS: Our results underscore the value of gene expression signatures to classify the interstitial lung diseases and to understand pathogenic mechanisms, and suggest new ways to improve the diagnosis and treatment of patients with these diseases.

Up-regulation of L-selectin and E-selectin in hypersensitivity pneumonitis.

BACKGROUND: Selectins are adhesion molecules that contribute to leukocyte recruitment into the tissue after an injury. Hypersensitivity pneumonitis (HP) is a lymphocytic alveolitis, and we hypothesized that the overexpression of selectins could play a role in this process. PATIENTS AND MEASUREMENTS: We studied 16 patients with HP and 7 healthy control subjects (HCs). Sera and BAL selectins and tumor necrosis factor-alpha were determined by enzyme-linked immunosorbent assay, and cellular lung localization was determined by immunohistochemistry. Additionally, BAL L-selectin, and L-selectin-bearing T-lymphocytes analyzed by flow cytometry were evaluated in HP patients and in exposed but asymptomatic subjects (EAS). SETTING: Tertiary referral center and immunohistochemistry laboratory. RESULTS: Raised levels of E-selectin (mean [+/- SD], 178.9 +/- 30.5 vs 59.4 +/- 4.7 ng/mL, respectively; p < 0.001) and P-selectin (mean, 232.6 +/- 29.9 vs 67.6 +/- 14.2 ng/mL, respectively; p < 0.001) were detected in HP patient sera compared to control subjects, while L-selectin levels showed no differences between groups. Conversely, HP patients displayed a significant increase in levels of L-selectin found in BAL fluid compared with both HCs and EAS (11.0 +/- 1.7 vs 6.9 +/- 0.43 and 3.1 +/- 0.5 ng/mL, respectively; p < 0.05). The levels of E-selectin found in BAL fluid were similar in patients from both groups, and P-selectin was not detected. Percentage of CD3+CD62 L+ lymphocytes was lower in HP patients compared with EAS (2.33 +/- 0.8 vs 4.31 +/- 2.4, respectively; p = 0.05). By immunohistochemistry, L-selectin was detected in interstitial macrophages and polymorphonuclear cells, and E-selectin was detected in endothelial cells. CONCLUSION: These findings demonstrate that L-selectin and E-selectin are up-regulated during the development of HP, suggesting that they may contribute to the increased traffic of lung inflammatory cells.