Background: Mesenchymal stem cells (MSCs)-derived extracellular vesicles (EVs) have been proposed as an alternative to live-cell administration for Acute Respiratory Distress Syndrome (ARDS). MSC-EVs can be chiefly influenced by the environment to which the MSCs are exposed. Here, lipopolysaccharide (LPS) priming of MSCs was used as a strategy to boost the natural therapeutic potential of the EVs in acute lung injury (ALI). Methods: The regenerative and immunemodulatory effect of LPS-primed MSC-EVs (LPS-EVs) and non-primed MSC-EVs (C-EVs) were evaluated in vitro on alveolar epithelial cells and macrophage-like THP-1 cells. In vivo, ALI was induced in adult male rats by the intrapulmonary instillation of HCl and LPS. Rats (n = 8 to 22/group) were randomized to receive a single bolus (1 × 10⁸ particles) of LPS-EVs, C-EVs, or saline. Lung injury severity was assessed at 72 h in lung tissue and bronchoalveolar lavage. Results: In vitro, LPS-EVs improved wound regeneration and attenuated the inflammatory response triggered by the P. aeruginosa infection, enhancing the M2 macrophage phenotype. In in vivo studies, LPS-EVs, but not C-EVs, significantly decreased the neutrophilic infiltration and myeloperoxidase (MPO) activity in lung tissue. Alveolar macrophages from LPS-EVs-treated animals exhibited a reduced expression of CXCL-1, a key neutrophil chemoattractant. However, both C-EVs and LPS-EVs reduced alveolar epithelial and endothelial permeability, mitigating lung damage. Conclusions: EVs from LPS-primed MSCs resulted in a better resolution of ALI, achieving a greater balance in neutrophil infiltration and activation, while avoiding the complete disruption of the alveolar barrier. This opens new avenues, paving the way for the clinical implementation of cell-based therapies.

BACKGROUND: Ventilator-associated pneumonia (VAP) is a severe condition. Early and adequate antibiotic treatment is the most important strategy for improving prognosis. Pancreatic Stone Protein (PSP) has been described as a biomarker that increases values 3–4 days before the clinical diagnosis of nosocomial sepsis in different clinical settings. We hypothesized that serial measures of PSP and its kinetics allow for an early diagnosis of VAP. METHODS: The BioVAP study was a prospective observational study designed to evaluate the role of biomarker dynamics in the diagnosis of VAP. To determine the association between repeatedly measured PSP and the risk of VAP, we used joint models for longitudinal and time-to-event data. RESULTS: Of 209 patients, 43 (20.6%) patients developed VAP, with a median time of 4 days. Multivariate joint models with PSP, CRP, and PCT did not show an association between biomarkers and VAP for the daily absolute value, with a hazard ratio (HR) for PSP of 1.01 (95% credible interval: 0.97 to 1.05), for CRP of 1.00 (0.83 to 1.22), and for PCT of 0.95 (0.82 to 1.08). The daily change of biomarkers provided similar results, with an HR for PSP of 1.15 (0.94 to 1.41), for CRP of 0.76 (0.35 to 1.58), and for PCT of 0.77 (0.40 to 1.45). CONCLUSION: Neither absolute PSP values nor PSP kinetics alone nor in combination with other biomarkers were useful in improving the prediction diagnosis accuracy in patients with VAP. Clinical Trial Registration: Registered retrospectively on August 3rd, 2012. NCT02078999.

Objective: To evaluate the inflammatory response of macrophage-like THP-1 as a result of infection with Pseudomonas aeruginosa (PA) or Streptococcus pneumoniae (SP) under different CO₂ concentrations, and determine the impact of macrophage response in bacterial survival. Methods: Cultures of macrophage-like THP-1 cells were separately infected with PA or SP for 1h at 37°C under normocapnic (5% CO₂) or hypercapnic (15% CO₂) conditions. Extracellular and intracellular bacterial survival post-infection was evaluated through seeding in blood agar plates. 24h after infection, at 37ºC and 5% or 15% CO₂, cell culture supernatant was analysed through ELISA technique for inflammatory cytokines (IL-1β, IFN-γ, CCL-2, IL-8 and IL-10). Results: Under hypercapnic conditions, IL-1β secretion is enhanced in both infections, IL-10 decreases only in PA infection and chemokines are decreased (Fig 1A-E). In PA-infected macrophages the secretion of chemokines is inhibited, in SP infection macrophages could play a pivotal role as recruiters (Fig 1D-E). No differences in post-infection bacterial extracellular survival were determined (Fig 1F). PA could be establishing intracellular niches within macrophages since intracellular growth is detected 24h post-infection (Fig 1G). erj;64/suppl_68/PA5323/F1 F1 F1 Conclusion: Macrophages perform different defensive responses against PA and SP infection. Hypercapnic condition on bacterial-infection response could play a detrimental role.

Pneumonia frequently develops under hypercapnic conditions. While hypercapnia is known to inhibit canonical NF-κB signaling, previous work by our group demonstrated that elevated CO₂ levels paradoxically increase IL-1β secretion during bacterial infection. Objective: To evaluate the impact of hypercapnia on the expression of key inflammation-related genes and bacterial persistence in co-cultures of macrophages (THP-1) and alveolar epithelial cells (HPAEpiC) infected with Pseudomonas aeruginosa (PA) or Streptococcus pneumoniae (SP). Methods: THP-1/HPAEpiC co-cultures were infected with PA or SP for 1 hour and maintained for 24 hours under normocapnic or hypercapnic conditions. The expression of IL1B, NLRP3, IRAK1, and RIPK1 was quantified using RT-qPCR. Extracellular and intracellular bacterial survival were also assessed. Results: Hypercapnia increased IL1B expression in infected cultures, with a marked upregulation of NLRP3 specifically during PA infection. IRAK1 expression was consistently inhibited under hypercapnia, while RIPK1 was upregulated. PA was recovered from cell lysates 24 hours post-infection, suggesting the establishment of intracellular niches, whereas SP showed no intracellular persistence. erj;68/suppl_70/PA5468/F1 F1 F1 Conclusions: Hypercapnia amplifies pro-inflammatory responses to bacterial infection through pathways that bypass canonical NF-κB signaling. Furthermore, PA may exploit hypercapnic environments to persist intracellularly, linking CO₂-dependent immune modulation with pathogen survival strategies.

Objective. To evaluate the bacterial survival and biological response of the co-culture of alveolar epithelial cells (HPAEpiC) and macrophages as a result of infection with different types of bacteria, Pseudomonas aeruginosa (PA) and Streptococcus pneumoniae (SPNE), under different CO₂ concentrations. Methods. Co-cultures of HPAEpiC cells and THP-1 cells differentiated to macrophages were separately infected with PA or SPNE for 1h at 37°C under normocapnic (5% CO₂) or hypercapnic (15% CO₂) conditions. Extracellular and intracellular bacterial survival were assessed after infection. 24h after infection intracellular and extracellular proteins were analyzed by ELISA for inflammatory mediators (IL-1β, IL-8, CCL2). Cell binding ZO-1 protein was measured 1h and 24h after infection. Apoptosis was also evaluated by cytometry and TUNEL assay. Results. Infection with PA or SPNE in a culture of HPAEpiC and THP-1 cells under hypercapnia increases inflammation, decreases phagocyte chemotaxis, and enhances ZO-1 production after infection but not after 24h (Fig1A-C). Moreover, PA-infected THP-1 cells show decreased apoptosis under hypercapnia (Fig1D-E). Regarding bacterial survival, hypercapnia could allow the intracellular establishment of PA and SPNE (Fig1H-I). Conclusion. The hypercapnic condition on bacterial-infection response could play a detrimental role on PA and SPNE infection.

Sepsis is a complex condition with time⍰dependent evolution, and longitudinal biomarker dynamics may improve its characterisation. We hypothesised that biomarker kinetics are associated with sepsis, the intensity of organ dysfunction, and patient survival. This single⍰centre, prospective, observational study included adult patients presenting to the Emergency Department with suspected infection and a National Early Warning Score 2 (NEWS2) ≥3. Blood samples were collected at baseline, 4 and 24 hours. Linear mixed models assessed associations between biomarker trajectories, sepsis diagnosis, and organ dysfunction severity. Joint models evaluated the predictive value of biomarker kinetics during the first 24 hours for in⍰hospital mortality. Of 214 screened patients, 173 were analysed and 137(79%) developed sepsis. Linear mixed models showed significant time⍰dependent decreases in IL10(β −0.016, 95%CI −0.028 to −0.004), IL1RN(β −0.014, 95%CI −0.024 to −0.004), and IL6(β −0.012, 95%CI −0.024 to 0.00). Sepsis diagnosis was associated with higher IL1RN(β 0.378, 95%CI 0.153– 0.603) and TNFRSF1A(β 0.40, 95%CI 0.21–0.58); a significant interaction between sepsis and time was observed only for IL6. Increasing SOFA scores correlated with elevated IL10(β 0.048), IL1RN(β 0.044), CCL2(β 0.046), TNFRSF1A(β 0.050), and procalcitonin(PCT; β 2.63), all p<0.01. The SOFA–time interaction was significant only for IL6 (β −0.003, 95%CI −0.005 to −0.001). Joint survival models adjusted for age and maximum SOFA identified IL8 (HR 0.655, 95%CrI 0.582– 0.728), TNFRSF1A (HR 0.505, 95%CrI 0.419–0.682), and PCT (HR 1.004, 95%CrI 1.001– 1.008) as outcome predictors. In conclusion, sepsis and organ dysfunction severity are associated with distinct inflammatory biomarker levels and kinetics. IL6 showed a unique time⍰dependent relationship, while TNFRSF1A, IL8 and PCT dynamics were associated with survival.