Pulmonary surfactant (PS) exhibits unique composition and biophysical properties to transport different type of therapeutic molecules. This has motivated its study as an efficient delivery vehicle for various drugs, especially molecules with hydrophobic character. PS is synthesized and secreted to the alveolar space by type II pneumocytes, where it spontaneously adsorbs into the air-liquid interface to reduce the surface tension. The adsorbed surfactant film is constituted by an interfacial monolayer with multi-layered membranes associated to it. Surfactant proteins SP-B and SP-C are responsible for maintaining the contact between these reservoirs and the interfacial layer. In order to study whether the multi-layered and multi-vesicular structures spread along the interface together with the interfacial monolayer, potentially serving as a vehicle for drugs, we have designed a novel vehiculization surface balance that connects a donor with a recipient compartment through a long interfacial pathway. Typically, the vehiculization of drugs mediated by PS has been evaluated in vitro by setups that combine different compartments through interfacial paper bridges. By incorporating a lipid fluorescent probe and the model hydrophobic drug Budesonide (BUD) into a porcine derived PS or into proteo-lipid vesicles, we determined that the amount of material transferred from a donor to a recipient compartment was substantially reduced due to the barrier imposed by the paper bridge. The bridge reduced the spreading to a simpler interfacial layer, while our novel setup reveals that a larger three-dimensional layer of liquid may facilitate the spreading of associated membranes and surfactant/drug complexes. We have determined that the three-dimensional structures associated to the interfacial layer spread along the air-liquid interface and entail an important contribution to the transport of lipids and drugs in surfactant-mediated delivery.

Several reports have suggested a reduction in the lung level of Dipalmitoylphosphatidylcholine(DPPC) upon COVID-19-related-Acute Respiratory Distress Syndrome(cvARDS). Thus, to gain a deeper understanding of the lung lipid profile upon cvARDS, we conducted a comprehensive lipidomic analysis by liquid chromatography–high-resolution mass spectrometry of bronchoalveolar lavage(BAL) samples from 6 cvARDS patients of the first wave of the pandemic, 6 non-COVID-19-patients with lung inflammation(LIP) and a control group of 12 subjects with no lung diseases(NLD). Results were normalized to the total amount of lipids. There was a significant increase in the levels of total cholesterol in cvARDS patients compared to both LIP and NLD groups [30.31(17.75-53.41)% vs 24.50(19.01-42.37)% and 15.011(12.54-18.39)% (p=0.038)] along with a tendency to increase the free cholesterol/DPPC ratio (p=0.080). Moreover, when lipid subclasses were analysed, we detected several significant changes in cvARDS when compared to the other groups: a decrease in PC32:1 [(6.08(2.08-7.73)% vs 10.78(7.14-13.53)% and 14.48(11.58-17.73)% (p=0.001)], PC34:0 [2.35(1.75-4.21)% vs 3.83(1.84-4.59)% and 5.19(4.12-5.96)% (p=0.020)], PG32:0 [5.96(2.24-8.55)% vs 10.58(8.08-41.59)% and 9.65(7.16-11.70)% (p=0.047)] and PG36:1 [(6.76(2.63-13.52)% vs 13.23(8.30-16.37)% and 15.53(13.99-18.64) (p=0.005)]. A significant increase in polyunsaturated PC36:3 was also observed [3.28(2.77-4.38)% vs 1.36(0.97-2.68)% and 1.25(0.78-2.02)% (p=0.005)]. Our findings provide new insights into the role of lipids in the respiratory response to SARS-CoV-2 infection, suggesting how perturbations in the lung lipid profile may have been involved in the pathogenesis of cvARDS.

A significant number of cancer patients do not benefit from PD-L1/PD-1 blockade immunotherapies. PD-1 and LAG-3 co-upregulation in T-cells is one of the major mechanisms of resistance by establishing a highly dysfunctional state in T-cells. A high-throughput screening was performed of PD-1/LAG-3 multiomic expression profiles in public databases of human cancer biopsies, to establish relationships between infiltrating tumor-infiltrating PD-1/LAG-3 T cells with biomarkers both in T cells and within the tumour microenvironment. These results were validated in engineered T-cell lines with constitutively active PD-1, LAG-3 pathways, and their combination, analysed by high-throughput quantitative proteomics and validated by conventional molecular techniques on primary T cells from NSCLC patients. The high-throughput multiomic human cancers screening and the experimental proteomic T cell lines uncovered a strong PD-1/LAG-3 dysfunctionality signature that was found which regulated immune, proteomic, metabolic, genetic, and epigenetic pathways. These mainly relied on differential regulation of E3 ubiquitin ligases CBL-B and C-CBL. Notably, LAG-3 expression has never been associated to CBL ubiquitin ligases before. PD-1/LAG-3 co-signaling profile uncovers a highly dysfunctional regulated programme. Co-blockade with a bispecific drug but not with a combination of anti-PD-1/anti-LAG-3 antibodies achieved both CBL-B and C-CBL inhibition, reverting T-cell dysfunctionality in lung cancer patients resistant to PD-L1/PD- 1 blockade. These results will help identifying the mechanisms of intrinsic resistance to PD-1 blockade mediated by LAG-3 co-signaling.

Pulmonary surfactant (PS) has been proposed as an efficient drug delivery vehicle for inhaled therapies. Its ability to adsorb and spread interfacially and transport different drugs associated with it has been studied mainly by different surface balance designs, typically interconnecting various compartments by interfacial paper bridges, mimicking in vitro the respiratory air–liquid interface. It has been demonstrated that only a monomolecular surface layer of PS/drug is able to cross this bridge. However, surfactant films are typically organized as multi-layered structures associated with the interface. The aim of this work was to explore the contribution of surface-associated structures to the spreading of PS and the transport of drugs. We have designed a novel vehiculization balance in which donor and recipient compartments are connected by a whole three-dimensional layer of liquid and not only by an interfacial bridge. By combining different surfactant formulations and liposomes with a fluorescent lipid dye and a model hydrophobic drug, budesonide (BUD), we observed that the use of the bridge significantly reduced the transfer of lipids and drug through the air–liquid interface in comparison to what can be spread through a fully open interfacial liquid layer. We conclude that three-dimensional structures connected to the surfactant interfacial film can provide an important additional contribution to interfacial delivery, as they are able to transport significant amounts of lipids and drugs during surfactant spreading.

Tumor microenvironment (TME) remodeling is one of the major research subjects in oncology. Several strategies can be implemented to modulate the tumor microenvironment, particularly in reprogramming myeloid cells to stimulate their anti-cancer activities. Indeed, myeloid cells constitute the major component of TME. Hence, it is important to identify the molecular signatures associated to cancer- promoting myeloid cells. Here, we defined the phenotype and proteome of tumor associated myeloid cells. Moreover, we identified the relationships between myeloid-derived suppressor cells (MDSCs) and tumor associated macrophages (TAM). The proteomic atlas of tumor-associated cells revealed important routes to reprogram cancer-associated myeloid cells. We used an ex vivo differentiation system for MDSCs and TAM by from C57BL/6J mouse bone marrow cells in cancer-polarized conditioning medium. We also differentiated resting macrophages (M0) as controls using standard techniques. Flow cytometry and microscopy confirmed their phenotype by assessing their morphology and presence of characteristic lineage markers. Three global experiments of quantitative mass spectrometry (shotgun proteomics) were performed. Construction of functional interactomes maps from up- or down-regulated proteins was conducted with the Ingenuity Pathway Analysis (IPA) Tool from Quiagen. We evaluated the effect at differentiation, maturation and immunosuppressive level in MDSCs and TAMs of several compounds. Markers were evaluated by cytometry and western blot. We confirmed morphological and phenotypic differences in ex vivo differentiated myeloid populations. High-throughput proteomics uncovered protein expression patterns characteristic of populations modelling tumor-infiltrating subsets, as a result of cancer-derived factors. Therefore, we evaluated several compounds for reprogramming tumor-associated cells. A resemblance to activated myeloid cells and a greater rise of macrophages and DC were observed. Moreover, the immunosuppressive functions of MDSCs decreased which led to enhanced CD4 cells proliferation and increased CD4 ability to release more IFN-gamma and IL2. In the present study, we identified differences in proteomic signatures between M-MDSCs, G-MDSC and TAMs related to lineage, and cancer-driven polarization. Moreover, these result permit us develop strategies to reprogram myeloid cells cancer associated.

A contribution of Lung Surfactant (LS) inactivation to COVID-19-related ARDS (cvARDS) has been argued, but not been clearly demonstrated to date. In the present study, we have characterised the extent of lung neutrophil infiltration along with the surface-active properties and protein composition of LS in bronchoalveolar lavages (BALs) collected from 12 cvARDS patients. A control group of 9 subjects without respiratory diseases was also enrolled. BAL cell sorting was performed by flow cytometry. The adsorption of LS at the air-liquid interface was assessed by Surfactant Adsorption Test (SAT), whereas the level of surfactant hydrophobic proteins was measured by Western Blot analysis. Results were normalised by phosphatidylcholine (PC) total amount. Significant increase in neutrophil [61.3(47.5-84.6)% vs 1.6(0.9-4.9)%, p<0.0001] and decrease in macrophage percentages [13.6(6.1-28.9)% vs 90.8(87.1-92-6)%, p<0.0001] of total BAL cells were detected in cvARDS patients. A lower overtime LS adsorption/accumulation at the air-liquid interface was also observed in those patients compared to the control group from 60min onward [14003(10232-19736) vs 24501(16386-28489) RFU, p=0.0471]. Moreover, cvARDS patients under the acute phase showed the lowest surfactant activity at the end of SAT (12191(11588-20159) RFU, p=0.048). An increase in both SP-B and SP-C/PC was also evident in cvARDS BALs. Here, we report for the first time on the reduction of LS surface-active properties during the acute period and even under the recovery phases of cvARDS. This may confirm how LS inactivation may be involved in both early and late consequences of severe cvARDS.

Resistance to PD-1 monoblockade immunotherapies is frequent in cancer patients and associated to the up-regulation of other immune checkpoint molecules in T cells such as LAG-3. Experimental evidence suggests thad PD-1 and LAG-3 cooperatively establish a strong dysfunctional estate in T cells through co-signaling.

Single agent immunotherapy (IO) has been accepted as standard frontline treatment for NSCLC with high PD-L1 expression, however most of the patients do not respond and some of them could benefit from chemo-immunotherapy (CT+IT). Low-density neutrophils (LDNs) are cells with immunosuppresive activities enriched in peripheral blood of cancer patients. We have studied the association between baseline LDNs and response to IO in NSCLC. PBMCs from 31 patients treated with IO and 21 treated with CT + IT were purified from fresh peripheral blood. Baseline LDNs were quantified through flow cytometry, and the proportions were correlated with clinical outcomes. Plasma from patients was compared through quantitative proteomics. Elevated baseline LDNs predict primary resistance to IO monotherapy. ROC analysis established a threshold of 7.09% (AUC 0.895), over which ORR was 0% and mPFS of 6.1 wk. No association was found between LDN levels and resistance to CT+IT (AUC 0.471), with an ORR of 57.1% when LDNs were > 7.09% and mPFS not reached (NR). A depletion of LDNs was observed in patients who responded to CT + IT. Ex vivo cocultures demonstrated that soluble factors present in plasma from high LDN patients prevented anticancer cytotoxicity. Comparative quantitative proteomics revealed the key role of the HGF/c-MET pathway. High baseline LDN levels are associated with primary resistance to IO monotherapy in patients with NSCLC. However, these patients can respond to CT+IT, thus identifying a subgroup whom should be offered this treatment regardless of high PD-L1 tumor expression. The upregulation of HGF/c-MET suggests that that targeting this pathway could have synergistic effect.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Surfactant protein D (SP-D) is a glycoprotein part of the pulmonary surfactant (PS) system that carries out important roles in the innate immune defense of the lungs and participates in surfactant homeostasis. Pulmonary surfactant (PS) is a lipid-protein complex, synthesized and secreted to the alveolar space by type II pneumocytes. It spontaneously adsorbs and spreads over the air-liquid interface, reducing the surface tension and enabling the process of breathing. PS has been recently proposed as a drug delivery carrier due to its unique surface-active properties and its peculiar composition. The anti-inflammatory and immunomodulatory features of SP-D make it a good candidate as a therapy to alleviate lung injuries associated with inflammation or infectious processes. In this work, we used a recombinant human SP-D (rhSP-D) and in vitro surface balances to evaluate the possibility of the protein to be transported through the air-liquid interface using PS as a carrier. We have analyzed the interfacial properties of the protein alone or in combination with PS by using a custom-built double-surface balance setup. rhSP-D showed low adsorption and spreading capabilities by itself, but this was improved upon combination with PS or by the mere presence of a surfactant film at the air-liquid interface. The interaction of the protein with surfactant membranes was investigated by using different PS/rhSP-D preparations and modes of combination. The transport of SP-D over long distances by its association with PS membranes suggests the possibility to formulate SP-D/PS combinations to ameliorate different lung pathologies.

Reducing temperature to values close to lung surfactant melting point may improve the dynamic interfacial properties of the system. in this work, we have analysed the temperature-mediated enhancement of the interfacial performance of purified porcine surfactant (PS) under plasma inhibition in a Constrained Drop Surfactometer. We have also explored the temperature-related rescue of surfactant performance. To do so, the drop interface was previously covered by plasma and PS until impairing surfactant dynamic properties. Synthetic lipid/protein mixtures or Poractant alfa were then dispensed at the air-liquid interface during compression-expansion cycles of the drop. All experiments were performed at both physiological (37 °C) and therapeutic hypothermia (33 °C) temperatures.The lower the concentration of tested material, the greater the capability of surfactant to reduce surface tension at 33 °C compared with 37 °C. At therapeutic hypothermia temperature, surfactant resistance to plasma inhibition is improved and restoration therapies are more effective regardless of the applied material. These in vitro results help to explain the good respiratory outcomes of cooled patients with direct acute respiratory distress syndrome and suggest new treatments for acute lung injury.

Mucopolysaccharidosis IIIA (MPS IIIA) is a lysosomal storage disease with significant neurological and skeletal pathologies. Respiratory dysfunction is a secondary pathology contributing to mortality in MPS IIIA patients. Pulmonary surfactant is crucial to optimal lung function and has not been investigated in MPS IIIA. We measured heparan sulphate (HS), lipids and surfactant proteins (SP) in pulmonary tissue and bronchoalveolar lavage fluid (BALF), and surfactant activity in healthy and diseased mice (20 weeks of age). Heparan sulphate, ganglioside GM3 and bis(monoacylglycero)phosphate (BMP) were increased in MPS IIIA lung tissue. There was an increase in HS and a decrease in BMP and cholesteryl esters (CE) in MPS IIIA BALF. Phospholipid composition remained unchanged, but BALF total phospholipids were reduced (49.70%) in MPS IIIA. There was a reduction in SP-A, -C and -D mRNA, SP-D protein in tissue and SP-A, -C and -D protein in BALF of MPS IIIA mice. Captive bubble surfactometry showed an increase in minimum and maximum surface tension and percent surface area compression, as well as a higher compressibility and hysteresis in MPS IIIA surfactant upon dynamic cycling. Collectively these biochemical and biophysical changes in alveolar surfactant are likely to be detrimental to lung function in MPS IIIA.