CEBP-ß and PLK1 as Potential Mediators of the Breast Cancer/Obesity Crosstalk: In Vitro and In Silico Analyses.

Over the last two decades, obesity has reached pandemic proportions in several countries, and expanding evidence is showing its contribution to several types of malignancies, including breast cancer (BC). The conditioned medium (CM) from mature adipocytes contains a complex of secretes that may mimic the obesity condition in studies on BC cell lines conducted in vitro. Here, we report a transcriptomic analysis on MCF-7 BC cells exposed to adipocyte-derived CM and focus on the predictive functional relevance that CM-affected pathways/processes and related biomarkers (BMs) may have in BC response to obesity. CM was demonstrated to increase cell proliferation, motility and invasion as well as broadly alter the transcript profiles of MCF-7 cells by significantly modulating 364 genes. Bioinformatic functional analyses unraveled the presence of five highly relevant central hubs in the direct interaction networks (DIN), and Kaplan-Meier analysis sorted the CCAAT/enhancer binding protein beta (CEBP-ß) and serine/threonine-protein kinase PLK1 (PLK1) as clinically significant biomarkers in BC. Indeed, CEBP-ß and PLK1 negatively correlated with BC overall survival and were up-regulated by adipocyte-derived CM. In addition to their known involvement in cell proliferation and tumor progression, our work suggests them as a possible "deus ex machina" in BC response to fat tissue humoral products in obese women.

Smooth Muscle Cell Phenotypic Switch Induced by Traditional Cigarette Smoke Condensate: A Holistic Overview.

Cigarette smoke (CS) is a risk factor for inflammatory diseases, such as atherosclerosis. CS condensate (CSC) contains lipophilic components that may represent a systemic cardiac risk factor. To better understand CSC effects, we incubated mouse and human aortic smooth muscle cells (SMCs) with CSC. We evaluated specific markers for contractile [i.e., actin, aortic smooth muscle (ACTA2), calponin-1 (CNN1), the Kruppel-like factor 4 (KLF4), and myocardin (MYOCD) genes] and inflammatory [i.e., IL-1ß, and IL-6, IL-8, and galectin-3 (LGALS-3) genes] phenotypes. CSC increased the expression of inflammatory markers and reduced the contractile ones in both cell types, with KLF4 modulating the SMC phenotypic switch. Next, we performed a mass spectrometry-based differential proteomic approach on human SMCs and could show 11 proteins were significantly affected by exposition to CSC (FC ≥ 2.7, p ≤ 0.05). These proteins are active in signaling pathways related to expression of pro-inflammatory cytokines and IFN, inflammasome assembly and activation, cytoskeleton regulation and SMC contraction, mitochondrial integrity and cellular response to oxidative stress, proteostasis control via ubiquitination, and cell proliferation and epithelial-to-mesenchymal transition. Through specific bioinformatics resources, we showed their tight functional correlation in a close interaction niche mainly orchestrated by the interferon-induced double-stranded RNA-activated protein kinase (alternative name: protein kinase RNA-activated; PKR) (EIF2AK2/PKR). Finally, by combining gene expression and protein abundance data we obtained a hybrid network showing reciprocal integration of the CSC-deregulated factors and indicating KLF4 and PKR as the most relevant factors.

BAL Proteomic Signature of Lung Adenocarcinoma in IPF Patients and Its Transposition in Serum Samples for Less Invasive Diagnostic Procedures.

Idiopathic pulmonary fibrosis (IPF) is a form of chronic and irreversible fibrosing interstitial pneumonia of unknown etiology. Although antifibrotic treatments have shown a reduction of lung function decline and a slow disease progression, IPF is characterize by a very high mortality. Emerging evidence suggests that IPF increases the risk of lung carcinogenesis. Both diseases show similarities in terms of risk factors, such as history of smoking, concomitant emphysema, and viral infections, besides sharing similar pathogenic pathways. Lung cancer (LC) diagnosis is often difficult in IPF patients because of the diffuse lung injuries and abnormalities due to the underlying fibrosis. This is reflected in the lack of optimal therapeutic strategies for patients with both diseases. For this purpose, we performed a proteomic study on bronchoalveolar lavage fluid (BALF) samples from IPF, LC associated with IPF (LC-IPF) patients, and healthy controls (CTRL). Molecular pathways involved in inflammation, immune response, lipid metabolism, and cell adhesion were found for the dysregulated proteins in LC-IPF, such as TTHY, APOA1, S10A9, RET4, GDIR1, and PROF1. The correlation test revealed a relationship between inflammation- and lipid metabolism-related proteins. PROF1 and S10A9, related to inflammation, were up-regulated in LC-IPF BAL and serum, while APOA1 and APOE linked to lipid metabolism, were highly abundant in IPF BAL and low abundant in IPF serum. Given the properties of cytokine/adipokine of the nicotinamide phosphoribosyltransferase, we also evaluated its serum abundance, highlighting its down-regulation in LC-IPF. Our retrospective analyses of BAL samples extrapolated some potential biomarkers of LC-IPF useful to improve the management of these contemporary pathologies. Their differential abundance in serum samples permits the measurement of these potential biomarkers with a less invasive procedure.

Neurodegenerative Disorder Risk in Krabbe Disease Carriers.

Krabbe disease (KD) is a rare autosomal recessive disorder caused by mutations in the galactocerebrosidase gene (GALC). Defective GALC causes aberrant metabolism of galactolipids present almost exclusively in myelin, with consequent demyelinization and neurodegeneration of the central and peripheral nervous system (NS). KD shares some similar features with other neuropathies and heterozygous carriers of GALC mutations are emerging with an increased risk in developing NS disorders. In this work, we set out to identify possible variations in the proteomic profile of KD-carrier brain to identify altered pathways that may imbalance its homeostasis and that may be associated with neurological disorders. The differential analysis performed on whole brains from 33-day-old twitcher (galc -/-), heterozygous (galc +/-), and wild-type mice highlighted the dysregulation of several multifunctional factors in both heterozygous and twitcher mice. Notably, the KD-carrier mouse, despite its normal phenotype, presents the deregulation of vimentin, receptor of activated protein C kinase 1 (RACK1), myelin basic protein (MBP), 2',3'-cyclic-nucleotide 3'-phosphodiesterase (CNP), transitional endoplasmic reticulum ATPase (VCP), and N-myc downstream regulated gene 1 protein (NDRG1) as well as changes in the ubiquitinated-protein pattern. Our findings suggest the carrier may be affected by dysfunctions classically associated with neurodegeneration: (i) alteration of (mechano) signaling and intracellular trafficking, (ii) a generalized affection of proteostasis and lipid metabolism, with possible defects in myelin composition and turnover, and (iii) mitochondrion and energy supply dysfunctions.

Changes in serum metabolomics in idiopathic pulmonary fibrosis and effect of approved antifibrotic medication.

Idiopathic pulmonary fibrosis (IPF) is a progressive disease with significant mortality and morbidity. Approval of antifibrotic therapy has ameliorated disease progression, but therapy response is heterogeneous and to date, adequate biomarkers predicting therapy response are lacking. In recent years metabolomic technology has improved and is broadly applied in cancer research thus enabling its use in other fields. Recently both aberrant metabolic and lipidomic pathways have been described to influence profibrotic responses. We thus aimed to characterize the metabolomic and lipidomic changes between IPF and healthy volunteers (HV) and analyze metabolomic changes following treatment with nintedanib and pirfenidone. We collected serial serum samples from two IPF cohorts from Germany (n = 122) and Spain (n = 21) and additionally age-matched healthy volunteers (n = 16). Metabolomic analysis of 630 metabolites covering 14 small molecule and 12 different lipid classes was carried out using flow injection analysis tandem mass spectrometry for lipids and liquid chromatography tandem mass spectrometry for small molecules. Levels were correlated with survival and disease severity. We identified 109 deregulated analytes in IPF compared to HV in cohort 1 and 112 deregulated analytes in cohort 2. Metabolites which were up-regulated in both cohorts were mainly triglycerides while the main class of down-regulated metabolites were phosphatidylcholines. Only a minority of de-regulated analytes were small molecules. Triglyceride subclasses were inversely correlated with baseline disease severity (GAP-score) and a clinical compound endpoint of lung function decline or death. No changes in the metabolic profiles were observed following treatment with pirfenidone. Nintedanib treatment induced up-regulation of triglycerides and phosphatidylcholines. Patients in whom an increase in these metabolites was observed showed a trend towards better survival using the 2-years composite endpoint (HR 2.46, p = 0.06). In conclusion, we report major changes in metabolites in two independent cohorts testing a large number of patients. Specific lipidic metabolite signatures may serve as biomarkers for disease progression or favorable treatment response to nintedanib.

A Novel Ex Vivo Approach Based on Proteomics and Biomarkers to Evaluate the Effects of Chrysene, MEHP, and PBDE-47 on Loggerhead Sea Turtles (Caretta caretta).

The principal aim of the present study was to develop and apply novel ex vivo tests as an alternative to cell cultures able to evaluate the possible effects of emerging and legacy contaminants in Caretta caretta. To this end, we performed ex vivo experiments on non-invasively collected whole-blood and skin-biopsy slices treated with chrysene, MEHP, or PBDE-47. Blood samples were tested by oxidative stress (TAS), immune system (respiratory burst, lysozyme, and complement system), and genotoxicity (ENA assay) biomarkers, and genotoxic and immune system effects were observed. Skin slices were analyzed by applying a 2D-PAGE/MS proteomic approach, and specific contaminant signatures were delineated on the skin proteomic profile. These reflect biochemical effects induced by each treatment and allowed to identify glutathione S-transferase P, peptidyl-prolyl cis-trans isomerase A, mimecan, and protein S100-A6 as potential biomarkers of the health-threatening impact the texted toxicants have on C. caretta. Obtained results confirm the suitability of the ex vivo system and indicate the potential risk the loggerhead sea turtle is undergoing in the natural environment. In conclusion, this work proved the relevance that the applied ex vivo models may have in testing the toxicity of other compounds and mixtures and in biomarker discovery.

Serum Proteomic Profile of Asthmatic Patients after Six Months of Benralizumab and Mepolizumab Treatment.

Severe eosinophilic asthma is characterized by chronic airway inflammation, oxidative stress, and elevated proinflammatory cytokines, especially IL-5. Mepolizumab and benralizumab are both humanized IgG antibodies directed against IL-5 signaling, directly acting on eosinophils count. Together with the complexity of severe asthma classification and patient selection for the targeted treatment, there is also the urgency to clarify the follow-up of therapy to identify biomarkers, in addition to eosinophils, for the optimal duration of treatment, persistence of effectiveness, and safety. To this purpose, here we performed a follow-up study using differential proteomic analysis on serum samples after 1 and 6 months of both therapies and sera from healthy patients. Statistical analysis by PCA and heatmap analyses were performed, and identified proteins were used for enrichment analysis by MetaCore software. The analysis highlighted 82 differences among all considered conditions. In particular, 30 referred to benralizumab time point (T0, T1B, T6B) and 24 to mepolizumab time point (T0, T1M, T6M) analyses. t-SNE and heatmap analyses evidence that the differential serum protein profile at 6 months of both treatments is more similar to that of the healthy subjects. Among the identified proteins, APOAI, APOC-II, and APOC-III are upregulated principally after 6 months of benralizumab treatment, plasminogen is upregulated after 6 months of both treatments and ceruloplasmin, upregulated already after 1 month of benralizumab, becoming higher after 6 months of mepolizumab. Using enrichment analysis, identified proteins were related to lipid metabolism and transport, blood coagulation, and ECM remodeling.

Alteration of Serum Proteome in Levo-Thyroxine-Euthyroid Thyroidectomized Patients.

The monotherapy with levo-thyroxine (LT4) is the treatment of choice for patients with hypothyroidism after thyroidectomy. However, many athyreotic LT4-treated patients with thyroid hormones in the physiological range experience hypothyroid-like symptoms, showing post-operative, statistically significant lower FT3 levels with respect to that before total thyroidectomy. Since we hypothesized that the lower plasmatic FT3 levels observed in this subgroup could be associated with tissue hypothyroidism, here we compared, by a preliminary proteomic analysis, eight sera of patients with reduced post-surgical FT3 to eight sera from patients with FT3 levels similar to pre-surgery levels, and six healthy controls. Proteomic analysis highlights a different serum protein profile among the considered conditions. By enrichment analysis, differential proteins are involved in coagulation processes (PLMN-1.61, -1.98 in reduced vs. stable FT3, p < 0.02; A1AT fragmentation), complement system activation (CFAH + 1.83, CFAB + 1.5, C1Qb + 1.6, C1S + 7.79 in reduced vs. stable FT3, p < 0.01) and in lipoprotein particles remodeling (APOAI fragmentation; APOAIV + 2.13, p < 0.003), potentially leading to a pro-inflammatory response. This study suggests that LT4 replacement therapy might restore biochemical euthyroid conditions in thyroidectomized patients, but in some cases without re-establishing body tissue euthyroidism. Since our results, this condition is reflected by the serum protein profile.

Neutrophil-to-lymphocyte ratio in bronchoalveolar lavage from IPF patients: a novel prognostic biomarker?

BACKGROUND: THIS is the first time that a bronchoalveolar lavage (BAL) neutrophil-to-lymphocyte ratio (NL-ratio) has been demonstrated in sarcoidosis and chronic hypersensitivity pneumonitis (cHP) than in idiopathic pulmonary fibrosis (IPF) patients. METHODS: Consecutive BAL samples from the 167 interstitial lung disease (ILD) patients were retrospectively enrolled in the study and clustered into three diagnostic categories: IPF, cHP and sarcoidosis. RESULTS: NL-ratio which proved higher in IPF (mean±SD, 2.1±3.8) than sarcoidosis (mean±SD 0.7±1.9; P<1E-04) and cHP patients (mean±SD 1.6±3.1; P=7.7E-03). ROC curve analysis to discriminate between Sarcoidosis and other ILDs showed an area under the curve (AUC) of 83.7%, (56% sensitivity and 96% specificity) while IPF and the other ILD were discriminated with AUC of 73% using a NL-ratio threshold value of 0.48 (73% sensitivity and 63% specificity). Interestingly, the NL-ratio was significantly correlated with other prognostic parameters: it was inversely correlated with forced vital capacity (FVC) (r=-0.3; P=2.5E-02) and forced expiration volume in 1 second (FEV1) (r=-0.3; P=2E-02) percentages and directly correlated with composite pulmonary index (CPI) score (r=0.3; P=3.2E-02). A decision-tree statistical algorithm was applied. CONCLUSIONS: This is the first time that a lower NL-ratio has been demonstrated in sarcoidosis and cHP than in IPF patients. The present preliminary report indicates a relationship between BAL NL-ratio and lung function parameters in patients with IPF: this ratio may help to optimize management of IPF patients and to improve follow-up and outcome.

Harnessing the Role of HDAC6 in Idiopathic Pulmonary Fibrosis: Design, Synthesis, Structural Analysis, and Biological Evaluation of Potent Inhibitors.

Idiopathic pulmonary fibrosis (IPF) is an interstitial lung disease characterized by a progressive-fibrosing phenotype. IPF has been associated with aberrant HDAC activities confirmed by our immunohistochemistry studies on HDAC6 overexpression in IPF lung tissues. We herein developed a series of novel hHDAC6 inhibitors, having low inhibitory potency over hHDAC1 and hHDAC8, as potential pharmacological tools for IPF treatment. Their inhibitory potency was combined with low in vitro and in vivo toxicity. Structural analysis of 6h and structure-activity relationship studies contributed to the optimization of the binding mode of the new molecules. The best-performing analogues were tested for their efficacy in inhibiting fibrotic sphere formation and cell viability, proving their capability in reverting the IPF phenotype. The efficacy of analogue 6h was also determined in a validated human lung model of TGF-ß1-dependent fibrogenesis. The results highlighted in this manuscript may pave the way for the identification of first-in-class molecules for the treatment of IPF.

Proteome Characterization of BALF Extracellular Vesicles in Idiopathic Pulmonary Fibrosis: Unveiling Undercover Molecular Pathways.

In the longtime challenge of identifying specific, easily detectable and reliable biomarkers of IPF, BALF proteomics is providing interesting new insights into its pathogenesis. To the best of our knowledge, the present study is the first shotgun proteomic investigation of EVs isolated from BALF of IPF patients. Our main aim was to characterize the proteome of the vesicular component of BALF and to explore its individual impact on the pathogenesis of IPF. To this purpose, ultracentrifugation was chosen as the EVs isolation technique, and their purification was assessed by TEM, 2DE and LC-MS/MS. Our 2DE data and scatter plots showed considerable differences between the proteome of EVs and that of whole BALF and of its fluid component. Analysis of protein content and protein functions evidenced that EV proteins are predominantly involved in cytoskeleton remodeling, adenosine signaling, adrenergic signaling, C-peptide signaling and lipid metabolism. Our findings may suggest a wider system involvement in the disease pathogenesis and support the importance of pre-fractioning of complex samples, such as BALF, in order to let low-abundant proteins-mediated pathways emerge.

First Attempt to Couple Proteomics with the AhR Reporter Gene Bioassay in Soil Pollution Monitoring and Assessment.

A topsoil sample obtained from a highly industrialized area (Taranto, Italy) was tested on the DR-CALUX® cell line and the exposed cells processed with proteomic and bioinformatics analyses. The presence of polyhalogenated compounds in the topsoil extracts was confirmed by GC-MS/MS analysis. Proteomic analysis of the cells exposed to the topsoil extracts identified 43 differential proteins. Enrichment analysis highlighted biological processes, such as the cellular response to a chemical stimulus, stress, and inorganic substances; regulation of translation; regulation of apoptotic process; and the response to organonitrogen compounds in light of particular drugs and compounds, extrapolated by bioinformatics all linked to the identified protein modifications. Our results confirm and reflect the complex epidemiological situation occurring among Taranto inhabitants and underline the need to further investigate the presence and sources of inferred chemicals in soils. The combination of bioassays and proteomics reveals a more complex scenario of chemicals able to affect cellular pathways and leading to toxicities rather than those identified by only bioassays and related chemical analysis. This combined approach turns out to be a promising tool for soil risk assessment and deserves further investigation and developments for soil monitoring and risk assessment.

Antithrombin III as predictive indicator of survival in idiopathic pulmonary fibrosis (IPF) patients treated with nintedanib: a preliminary study.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a progressive lung disease often managed with nintedanib, a tyrosine kinase inhibitor targeting several profibrotic pathways. Although clotting processes are involved in wound healing and repair in the lung, there are no data on the role of antithrombin III (ATIII) in IPF patients treated with nintedanib. A previous proteomic analysis of serum of IPF patients before and after 1 year of nintedanib treatment showed differential protein expression of ATIII. AIMS: Here we used quantitative methods to evaluate differential ATIII concentrations in IPF patients before and after 1 year of nintedanib treatment and to assess the potential of ATIII as a prognostic biomarker in IPF patients. METHODS: Serum levels of ATIII were measured by enzyme-linked immunosorbent assay in 14 IPF patients before and after 1 year of nintedanib treatment. RESULTS: A statistically significant inverse correlation was found between serum ATIII concentrations and pulmonary function test parameters in all patients at baseline and follow up. Baseline serum ATIII and bronchoalveolar lavage (BAL) neutrophils proved to be reliable predictors of poor prognosis. A baseline ATIII threshold of 126.5 µg/mL discriminated survivors from non-survivors. CONCLUSIONS: After 12 months of antifibrotic treatment, IPF patients with high serum ATIII concentrations and high BAL neutrophil percentages had a poor prognosis and increased survival risk. The results of this preliminary study suggest that ATIII has potential as a biomarker of IPF severity and in predicting response to nintedanib therapy. As a marker, ATIII showed several advantages over BAL neutrophil percentage.

Ceruloplasmin and oxidative stress in severe eosinophilic asthma patients treated with Mepolizumab and Benralizumab.

INTRODUCTION: Severe eosinophilic asthma has been associated with Th2 airway inflammation and elevated proinflammatory cytokines and chemokines, such as IL-5. Precision therapies have recently been shown to improve asthma symptoms with a steroid-sparing effect. Two such therapies, Benralizumab and Mepolizumab, humanized IgG antibodies directed against the IL-5 receptor and IL-5, have been approved for severe eosinophilic asthma. METHODS: Here we used a differential proteomic approach to analyse serum from patients with severe eosinophilic asthma treated with Benralizumab and Mepolizumab in a search for differential molecular modifications responsible of their effects. Enrichment analysis of differential proteins was performed for the two treatments. RESULTS AND DISCUSSION: After one month of Benralizumab treatment we detected up-regulation of certain protein species of the antioxidant ceruloplasmin. To investigate oxidative stress, we performed redox proteomics which detected lower oxidative burst after one month of Benralizumab treatment than in the pre-treatment phase or after one month of Mepolizumab therapy.

Specificity of serum amyloid A as a biomarker of idiopathic pulmonary fibrosis.

Serum amyloid A (SAA) is an apo-lipoprotein produced by the liver in response to proinflammatory cytokines. Few data are available on SAA levels in patients with idiopathic pulmonary fibrosis (IPF), the most common idiopathic form of interstitial pneumonitis (ILD). This study compared SAA concentration in IPF patients to other ILD groups to explore its potential use as a clinical biomarker.

Idiopathic Pulmonary Fibrosis Serum proteomic analysis before and after nintedanib therapy.

Idiopathic pulmonary fibrosis (IPF) is a fatal progressive disease with a median survival of 2-5 years. Nintedanib is a small tyrosine kinase inhibitor that reduces IPF progression, significantly slowing the annual decline in Forced Vital Capacity (FVC). Very little data is available on the molecular mechanisms of this treatment in IPF, despite a growing interest in the definition of IPF pathogenesis and target therapy. A functional proteomic approach was applied to the analysis of serum samples from IPF patients in order to highlight differential proteins potentially indicative of drug-induced molecular pathways modifications and response to therapy. Twelve serum samples were collected from six IPF patients in care at Siena Regional Referral Center for Interstitial Lung Diseases (ILDs) and treated with nintedanib for one year. Serum samples were analyzed at baseline (T0 before starting therapy) and after one year of treatment (T1) and underwent differential proteomic and bioinformatic analysis. Proteomic analysis revealed 13 protein species that were significantly increased after one year of treatment. When the targets of nintedanib (VEGFR, FGFR and PDGFR) were added, enrichment analysis extracted molecular pathways and process networks involved in cell differentiation (haptoglobin and albumin), coagulation (antithrombin III), epithelial mesenchymal transition, cell proliferation and transmigration. PI3K and MAPK induced up-regulation of apolipoprotein C3. Proteomic study found 13 protein species up-regulated in IPF patients after one year of nintedanib treatment. Haptoglobin, a central hub of our analysis was validated by 2D-WB and ELISA as theranostic marker in a more numerous populations of patients.

Proteomic characterization of idiopathic pulmonary fibrosis patients: stable versus acute exacerbation.

Acute exacerbations (AEs) are among the main causes of death in idiopathic pulmonary fibrosis (IPF) patients. In this study proteomic comparative analysis of bronchoalveolar lavage (BAL) fluid samples was performed in stable IPF patients versus AEs IPF group to identify AE pathogenetic mechanisms and novel potential predictive biomarkers. A functional proteomic analysis of BAL fluid samples from stable and AE-IPF patients was conducted in a population of 27 IPF patients. Fifty-one differentially abundant spots were observed and identified by mass spectrometry. Enrichment analysis found proteins of interest involved in the regulation of macrophages and lipid metabolism receptors. In acute exacerbation IPF group, differentially abundant proteins were involved in propagation of the ß-catenin WNT transduction signal, and proteins up-regulated in lung carcinogenesis (IGKC, S100A9, PEDF, IGHG1, ALDOA, A1AT, HPT, CO3 and PIGR) and acute phase proteins involved in protease-antiprotease imbalance (such as A1AT fragments). Dot-blot analysis of A1AT C-36 peptide allowed validating our findings, confirming up-regulation in AE IPF patients and suggesting its potential pathogenetic role. A crucial role of protease/antiprotease imbalance, clathrin-mediated endocytosis signalling and carcinogenesis emerged in IPF patients developing acute exacerbations.

Correction to: BAL biomarkers' panel for differential diagnosis of interstitial lung diseases.

The original version of this article unfortunately contained a mistake. First and last names of the authors were interchanged.

BAL biomarkers' panel for differential diagnosis of interstitial lung diseases.

Bronchoalveolar lavage (BAL) is a useful procedure for differential diagnosis of interstitial lung diseases (ILDs) and for identification of granulomatous lung diseases. We investigated a panel of biomarkers from BAL fluid of ILD patients to evaluate their utility in differentiating ILDs. Bronchoscopy with BAL was performed in 100 consecutive patients with suspected ILD (41 sarcoidosis, 11 cHP and 24 other ILDs); the 24 patients negative for ILD diagnosis were included as control group. BAL phenotypes and cell profiles (CD4+/CD8+ ratio, NK and CD103+ cell counts, chitotriosidase and KL-6 levels in BAL) were determined by flow cytometry. A decision-tree statistical algorithm was applied. Sarcoidosis was discriminated by a higher BAL CD4+/CD8+ ratio (p = 5.8E-05), a lower BAL CD103+CD4+ count (p = 5.0E-02) and lower BAL NK percentages (p = 8.8E-03) than the other groups. BAL KL-6 concentrations were higher in sarcoidosis than in other ILDs (p = 1.5E-02) and were directly correlated with CD4+/CD8+ ratio. We used decision-tree statistical analysis to combine our biomarkers into two diagnostic algorithms for differential diagnosis of ILDs. A panel of BAL biomarkers for diagnosis of ILDs is proposed; CD4+/CD8+ ratio, KL-6 concentrations, and NK and CD103+CD4+ cell percentages in BAL could improve the identification and differential diagnosis of sarcoidosis.