Angiogenesis, apoptosis and re-epithelialization at the foci of recent injury in usual interstitial pneumonia and bronchiolitis obliterans organizing pneumonia

Abstract Idiopathic usual interstitial pneumonia (UIP) and bronchiolitis obliterans organizing pneumonia (BOOP) are fibrous pulmonary disorders in both of which there is newly formed connective tissue in distal air spaces. UIP is a progressive and usually fatal lung disease without any efficient treatment, while the prognosis of BOOP is good. In both diseases, an injury of the alveolar epithelium and its basement membrane (BM) leads to migration of fibroblasts and myofibroblasts into air spaces and production of extracellular matrix by these cells. In UIP, the newly formed intraluminal connective tissue lesions cause fusion of alveolar structures and interstitial remodeling, while in BOOP the newly formed connective tissue may resolve completely. One of the major aims of the research on pulmonary fibrosis is to define the mechanisms that lead to persistence of the newly formed connective tissue and thus to irreversible fibrosis in UIP. The aim of the present study was to compare the extent of capillarization, apoptotic activity and re-epithelialization of the newly formed connective tissue in BOOP and UIP. The number of capillaries per tissue surface area was measured. Furthermore, the expression of angiogenic growth factors vascular endothelial growth factor-A (VEGF-A) and basic fibroblast growth factor (bFGF) was evaluated in the same areas, in addition to the expression of Flt-1 and Flk-1, which serve as receptors for VEGF. Apoptotic activity was analyzed using TUNEL-method, and the immunohistochemical expression of apoptosis regulating proteins bcl-2, mcl-1, and bax was studied. Finally, the extent of re-epithelialization was studied with the immunohistochemical and ultrastructural localization of laminin-5 γ2 chain, and the sites of synthesis of laminin-5 γ2 chain mRNA. In BOOP, an efficient repair process with good capillarization along with high expression of VEGF and bFGF, and orderly re-epithelialization of the newly formed connective tissue takes place after lung injury. The apoptotic activity of the newly formed connective tissue is also high, presumably leading to resolution of the intraluminal connective tissue in BOOP. In UIP, the newly formed connective tissue showed poor capillarization, inadequate re-epithelialization and low apoptotic activity. The results suggest disturbed or delayed repair process in UIP, contributing to irreversible interstitial fibrosis and remodeling

Smoking effect on the ultrastructural properties of cultured lung myofibroblasts

Abstract This study aimed at an ultrastructural characterization of myofibroblasts cultured from different compartments of lung from never-smokers and smokers with or without COPD. In addition, we evaluated the expression of alpha smooth muscle actin (α-SMA), a marker for myofibroblasts, and contractile properties. Stromal cells cultured from central and corresponding peripheral or only from peripheral lung of never-smokers, smokers without COPD and COPD patients were analyzed by transmission electron microscopy (TEM), immunoelectron microscopy (IEM), Western analysis and/or by collagen gel contraction assay. TEM revealed that myofibroblasts cultured from smokers and COPD had less prominent intracellular actin filaments. We also examined fibronexus (FNX), which is a typical ultrastructural feature of myofibroblasts, and observed that patients with COPD more frequently had tandem-like FNX as compared to other samples. Western analysis showed that the samples derived from the central lung of never-smokers expressed higher levels of α-SMA than those of smokers and COPD patients. Cells from central lung were less contractile than those from peripheral lung. We conclude that myofibroblasts have variable ultrastructural and functional properties based on their localization in the lung and, moreover, these properties are affected by both smoking history and COPD

The prognostic and predictive roles of plasma C-reactive protein and PD-L1 in non-small cell lung cancer

Abstract Background: Anti-PD-(L)1 agents have revolutionized the treatment paradigms of non-small cell lung cancer (NSCLC), while predictive biomarkers are limited. It has been previously shown that systemic inflammation, indicated by elevated C-reactive protein (CRP) level, is associated with a poor prognosis in anti-PD-(L)1 treated. The aim of the study was to analyze the prognostic and predictive value of CRP in addition to traditional prognostic and predictive markers and tumor PD-L1 score. Methods: We identified all NSCLC patients (n = 329) who had undergone PD-L1 tumor proportion score (TPS) analysis at Oulu University Hospital 2015–22. CRP levels, treatment history, immune checkpoint inhibitor (ICI) therapy details, and survival were collected. The patients were categorized based on CRP levels (≤10 vs. >10) and PD-L1 TPS scores (<50 vs. ≥50). Results: In the whole cohort (n = 329), CRP level of ≤10 mg/L was associated with improved survival in univariate (HR 0.30, Cl 95% 0.22–0.41) and multivariate analyzes (HR 0.44, CI 95% 0.28–0.68). With ICI treated (n = 70), both CRP of ≤10 and PD-L1 TPS of ≥50 were associated with improved progression-free survival (PFS) in univariate (HR 0.51, CI 95% 0.27–0.96; HR 0.54, CI 95% 0.28–1.02) and multivariate (HR 0.48, CI 95% 0.26–0.90; HR 0.50, CI 95% 0.26–0.95) analyzes. The combination (PD-L1 TPS ≥50 and CRP >10) carried a high negative predictive value with a median PFS of 4.11 months (CI 95% 0.00–9.63), which was similar to patients with low PD-L1 (4.11 months, CI 95% 2.61–5.60). Conclusions: Adding plasma CRP levels to PD-L1 TPS significantly increased the predictive value of sole PD-L1. Furthermore, patients with high CRP beard little benefit from anti-PD-(L)1 therapies independent of PD-L1 score. The study highlights the combined evaluation of plasma CRP and PD-L1 TPS as a negative predictive marker for ICI therapies

Interleukin-26 production in human primary bronchial epithelial cells in response to viral stimulation:modulation by Th17 cytokines

Abstract Interleukin (IL)-26 is abundant in human airways and this cytokine is involved in the local immune response to a bacterial stimulus in vivo. Specifically, local exposure to the toll-like receptor (TLR) 4 agonist endotoxin does increase IL-26 in human airways and this cytokine potentiates chemotactic responses in human neutrophils. In addition to T-helper (Th) 17 cells, alveolar macrophages can produce IL-26, but it remains unknown whether this cytokine can also be produced in the airway mucosa per se in response to a viral stimulus. Here, we evaluated whether this is the case using primary bronchial epithelial cells from the airway epithelium in vitro and explored the signaling mechanisms involved, including the modulatory effects of additional Th17 cytokines. Finally, we assessed IL-26 and its archetype signaling responses in healthy human airways in vivo. We found increased transcription and release of IL-26 protein after stimulation with the viral-related double stranded (ds) RNA polyinosinic-polycytidylic acid (poly-IC) and showed that this IL-26 release involved mitogen-activated protein (MAP) kinases and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB). The release of IL-26 in response to a viral stimulus was modulated by additional Th17 cytokines. Moreover, there was transcription of IL26 mRNA and expression of the protein in epithelial cells of bronchial brush and tissue biopsies respectively after harvest in vivo. In addition, the extracellular IL-26 protein concentrations in bronchoalveolar lavage (BAL) samples did correlate with increased epithelial cell transcription of an archetype intracellular signaling molecule downstream of the IL-26-receptor complex, STAT1, in the bronchial brush biopsies. Thus, our study suggests that viral stimulation causes the production of IL-26 in lining epithelial cells of human airways, structural cells that constitute a critical immune barrier and that this production is modulated by Th17 cytokines

Smoking-associated increase in mucins 1 and 4 in human airways

Abstract Rationale: Smoking-related chronic obstructive pulmonary disease (COPD) is associated with dysregulated production of mucus. Mucins (MUC) are important both for mucus secretion and epithelial defense. We have examined the distribution of MUC1 and MUC4 in the airway epithelial cells of never-smokers and smokers with and without COPD. Methods: Mucosal biopsies and bronchial wash samples were obtained by bronchoscopy from age- and sex-matched COPD-patients (n = 38; GOLD I-II/A-B), healthy never-smokers (n = 40) and current smokers with normal lung function (n = 40) from the Karolinska COSMIC cohort (NCT02627872). Cell-specific expressions of MUC1, MUC4 and regulating factors, i.e., epithelial growth factor receptor (EGFR) 1 and 2, were analyzed by immunohistochemistry. Soluble MUC1 was measured by quantitative immunodetection on slot blot. Results: The levels of cell-bound MUC1 expression in basal cells and in soluble MUC1 in bronchial wash were increased in smokers, regardless of airway obstruction. Patients with chronic bronchitis had higher MUC1 expression. The expression of MUC4 in cells with goblet cell phenotype was increased in smokers. The expression of EGFR2, but not that of EGFR1, was higher in never-smokers than in smokers. Conclusions: Smoking history and the presence of chronic bronchitis, regardless of airway obstruction, affect both cellular and soluble MUC1 in human airways. Therefore, MUC1 may be a novel marker for smoking- associated airway disease

High NHLRC2 expression is associated with shortened survival in lung adenocarcinoma

Abstract Background: Certain variants of NHL repeat (named after NCL-1, HT2A and LIN-41)-containing protein 2 (NHLRC2) gene have been linked to severe fibrotic interstitial lung disease in children. The aim of the current study was to evaluate the expression of NHLRC2 in lung cell and tissue samples from patients with lung adenocarcinoma (ADC) and squamous cell carcinoma (SCC). Methods: The expression of NHLRC2 in lung tissue samples was studied by immunohistochemistry (102 ADC, 111 SCC), mRNA in situ hybridization (4 ADC, 3 SCC), and Western blot analysis (3 ADC, 2 SCC). The immunohistochemical NHLRC2 expression was measured by image analysis software and the percentage of NHLRC2-positive cancer cells was evaluated by semiquantitative analysis. The immunohistochemical results of NHLRC2 were compared with the clinical and histological characteristics of the patients. NHLRC2 protein levels in primary stromal and epithelial lung cancer cell lines were measured by Western blot analysis. Results: NHLRC2 was mainly expressed in cancer cells and inflammatory cells within the tumor. The NHLRC2 expression evaluated by image analysis method was significantly higher in ADC compared with that in SCC (P<0.001). High NHLRC2 expression was associated with reduced disease specific survival (P=0.002), overall survival (P=0.001), and high mitotic activity (P=0.042) in ADC. Additionally, the proportion of NHLRC2-positive cancer cells analyzed by the semiquantitative method was significantly higher in ADC than in SCC (P<0.001). Conclusions: NHLRC2 expression was higher in lung ADC than in SCC and its expression was associated with poor survival in ADC patients. Further studies are required to clarify the pathogenetic role of NHLRC2 in lung cancer

The neutrophil-mobilizing cytokine interleukin-26 in the airways of long-term tobacco smokers

Abstract Long-term tobacco smokers with chronic obstructive pulmonary disease (COPD) or chronic bronchitis display an excessive accumulation of neutrophils in the airways; an inflammation that responds poorly to established therapy. Thus, there is a need to identify new molecular targets for the development of effective therapy. Here, we hypothesized that the neutrophil-mobilizing cytokine interleukin (IL)-26 (IL-26) is involved in airway inflammation amongst long-term tobacco smokers with or without COPD, chronic bronchitis or colonization by pathogenic bacteria. By analyzing bronchoalveolar lavage (BAL), bronchail wash (BW) and induced sputum (IS) samples, we found increased extracellular IL-26 protein in the airways of long-term smokers in vivo without further increase amongst those with clinically stable COPD. In human alveolar macrophages (AM) in vitro, the exposure to water-soluble tobacco smoke components (WTC) enhanced IL-26 gene and protein. In this cell model, the same exposure increased gene expression of the IL-26 receptor complex (IL10R2 and IL20R1) and nuclear factor κ B (NF-κB); a proven regulator of IL-26 production. In the same cell model, recombinant human IL-26 in vitro caused a concentration-dependent increase in the gene expression of NF-κB and several pro-inflammatory cytokines. In the long-term smokers, we also observed that extracellular IL-26 protein in BAL samples correlates with measures of lung function, tobacco load, and several markers of neutrophil accumulation. Extracellular IL-26 was further increased in long-term smokers with exacerbations of COPD (IS samples), with chronic bronchitis (BAL samples ) or with colonization by pathogenic bacteria (IS and BW samples). Thus, IL-26 in the airways emerges as a promising target for improving the understanding of the pathogenic mechanisms behind several pulmonary morbidities in long-term tobacco smokers

Mucins 3A and 3B are expressed in the epithelium of human large airway

Abstract Aberrant mucus secretion is a hallmark of chronic obstructive pulmonary disease (COPD). Expression of the membrane-tethered mucins 3A and 3B (MUC3A, MUC3B) in human lung is largely unknown. In this observational cross-sectional study, we recruited subjects 45–65 years old from the general population of Stockholm, Sweden, during the years 2007–2011. Bronchial mucosal biopsies, bronchial brushings, and bronchoalveolar lavage fluid (BALF) were retrieved from COPD patients (n = 38), healthy never-smokers (n = 40), and smokers with normal lung function (n = 40). Protein expression of MUC3A and MUC3B in bronchial mucosal biopsies was assessed by immunohistochemical staining. In a subgroup of subjects (n = 28), MUC3A and MUC3B mRNAs were quantified in bronchial brushings using microarray. Non-parametric tests were used to perform correlation and group comparison analyses. A value of p %lt; 0.05 was considered statistically significant. MUC3A and MUC3B immunohistochemical expression was localized to ciliated cells. MUC3B was also expressed in basal cells. MUC3A and MUC3B immunohistochemical expression was equal in all study groups but subjects with emphysema had higher MUC3A expression, compared to those without emphysema. Smokers had higher mRNA levels of MUC3A and MUC3B than non-smokers. MUC3A and MUC3B mRNA were higher in male subjects and correlated negatively with expiratory air flows. MUC3B mRNA correlated positively with total cell concentration and macrophage percentage, and negatively with CD4/CD8 T cell ratio in BALF. We concluded that MUC3A and MUC3B in large airways may be a marker of disease or may play a role in the pathophysiology of airway obstruction