RATIONALE:Early-life lung function trajectories predict long-term respiratory health, including COPD risk. Club Cell protein 16 (CC16) is a key determinant of lung health, with low levels associated with impaired lung development, reduced lung function, and COPD. Cigarette smoking lowers CC16, but it is unknown whether maternal smoking leads to persistent CC16 deficiency from early life, thereby disrupting lung development and predisposing to COPD risk and progression. METHODS:CC16 expression was analyzed across 4 human cohorts, in plasma samples (COPDGene [n = 1062] and ECLIPSE [n = 2164]), nasal brushings (ALLIANCE [n = 63]), and peripheral lung sections (LTRC [n = 44]) from participants with and without a history of maternal smoking exposure. Lung histology and respiratory mechanics were assessed in WT and Cc16-/- mice with and without maternal smoking exposure. Recombinant human (rh)CC16 effects on lung maturation were assessed in embryonic murine lung explants. RESULTS:Maternal smoking was linked to reduced circulating and airway CC16 in COPD patients, controls, and a preclinical murine COPD model. In human adults, lower CC16 correlated with accelerated lung function decline and emphysema progression, while in children it was associated with obstructive physiology and early small airway impairment. In both mice and humans, maternal smoking-induced CC16 reduction was accompanied by greater epithelial injury (fibrosis, inflammation, apoptosis, and oxidative stress). In murine explants, smoking impaired lung branching, whereas rhCC16 restored branching via α2-integrin binding. CONCLUSIONS:Maternal smoking reduces CC16 levels, disrupting lung development in ways that predispose to lifelong impairment of lung function and worse COPD outcomes. Defining the mechanisms by which CC16 regulates lung maturation is essential for establishing reliable outcome measures and designing trials aimed at preventing early COPD.

Low Club Cell 16 kDa protein (CC16) plasma levels are linked to accelerated lung function decline in patients with chronic obstructive pulmonary disease (COPD). Cigarette smoke-exposed (CS-exposed) Cc16-/- mice have exaggerated COPD-like disease associated with increased NF-κB activation in their lungs. It is unclear whether CC16 augmentation can reverse exaggerated COPD in CS-exposed Cc16-/- mice and whether increased NF-κB activation contributes to the exaggerated COPD in CS-exposed Cc16-/- lungs. CS-exposed WT and Cc16-/- mice were treated with recombinant human CC16 (rhCC16) or an NF-κB inhibitor versus vehicle beginning at the midpoint of the exposures. COPD-like disease and NF-κB activation were measured in the lungs. RhCC16 limited the progression of emphysema, small airway fibrosis, and chronic bronchitis-like disease in WT and Cc16-/- mice partly by reducing pulmonary inflammation (reducing myeloid leukocytes and/or increasing regulatory T and/or B cells) and alveolar septal cell apoptosis, reducing NF-κB activation in CS-exposed Cc16-/- lungs, and rescuing the reduced Foxj1 expression in CS-exposed Cc16-/- lungs. IMD0354 treatment reduced exaggerated lung inflammation and rescued the reduced Foxj1 expression in CS-exposed Cc16-/- mice. RhCC16 treatment reduced NF-κB activation in luciferase reporter A549 cells. Thus, rhCC16 treatment limits COPD progression in CS-exposed Cc16-/- mice partly by inhibiting NF-κB activation and represents a potentially novel therapeutic approach for COPD.

Secretoglobin (SCGB) 3A2 was first identified in 2001 as a protein exhibiting similarities in amino acid sequence and gene structure to SCGB1A1, a multi-functional cytokine-like molecule highly expressed in airway epithelial Club cells that was the first identified and extensively studied member of the SCGB gene superfamily. SCGB3A2 is a small secretory protein of ~10 kDa that forms a dimer and a tetramer. SCGB3A2 is predominantly expressed in airway epithelial Club cells, and has anti-inflammatory, growth factor, anti-fibrotic, and anti-cancer activities that influence various lung diseases. This review summarizes the current understanding of SCGB3A2 biological functions and its role in human diseases with emphasis on its mechanisms of actions and signaling pathway.

Non-canonical inflammasome activation that recognizes intracellular lipopolysaccharide (LPS) causes pyroptosis, the inflammatory death of innate immune cells. The role of pyroptosis in innate immune cells is to rapidly eliminate pathogen-infected cells and limit the replication niche in the host body. Whether this rapid cell elimination process of pyroptosis plays a role in elimination of cancer cells is largely unknown. Our earlier study demonstrated that a multi-functional secreted protein, secretoglobin (SCGB) 3A2, chaperones LPS to cytosol, and activates caspase-11 and the non-canonical inflammasome pathway, leading to pyroptosis. Here we show that SCGB3A2 exhibits marked anti-cancer activity against 5 out of 11 of human non-small cell lung cancer cell lines in mouse xenographs, while no effect was observed in 6 of 6 small cell lung cancer cell lines examined. All SCGB3A2-LPS-sensitive cells express syndecan 1 (SDC1), a SCGB3A2 cell surface receptor, and caspase-4 (CASP4), a critical component of the non-canonical inflammasome pathway. Two epithelial-derived colon cancer cell lines expressing SDC1 and CASP4 were also susceptible to SCGB3A2-LPS treatment. TCGA analysis revealed that lung adenocarcinoma patients with higher SCGB3A2 mRNA levels exhibited better survival. These data suggest that SCGB3A2 uses the machinery of pyroptosis for the elimination of human cancer cells via the non-canonical inflammasome pathway, and that SCGB3A2 may serve as a novel therapeutic to treat cancer, perhaps in combination with immuno and/or targeted therapies.

Rationale CC16 (club cell secretory protein) is a pneumoprotein produced predominantly by pulmonary club cells. Circulating CC16 is associated with protection from the inception and progression of the two most common obstructive lung diseases (asthma and chronic obstructive pulmonary disease). Objectives Although exact mechanisms remain elusive, studies consistently suggest a causal role of CC16 in mediating antiinflammatory and antioxidant functions in the lung. We sought to determine any novel receptor systems that could participate in CC16's role in obstructive lung diseases. Methods Protein alignment of CC16 across species led to the discovery of a highly conserved sequence of amino acids, leucine-valine-aspartic acid (LVD), a known integrin-binding motif. Recombinant CC16 was generated with and without the putative integrin-binding site. A Mycoplasma pneumoniae mouse model and a fluorescent cellular adhesion assay were used to determine the impact of the LVD site regarding CC16 function during live infection and on cellular adhesion during inflammatory conditions. Measurements and Main Results CC16 bound to integrin α4β1), also known as the adhesion molecule VLA-4 (very late antigen 4), dependent on the presence of the LVD integrin-binding motif. During infection, recombinant CC16 rescued lung function parameters both when administered to the lung and intravenously but only when the LVD integrin-binding site was intact; likewise, neutrophil recruitment during infection and leukocyte adhesion were both impacted by the loss of the LVD site. Conclusions We discovered a novel receptor for CC16, VLA-4, which has important mechanistic implications for the role of CC16 in circulation as well as in the lung compartment.

Background Preterm neonates can develop chronic pulmonary insufficiency of prematurity (CPIP) later in infancy. Recombinant human CC10 protein (rhCC10) is an anti-inflammatory agent that could potentially prevent CPIP. Methods The safety and efficacy of a single intratracheal dose of rhCC10 in reducing CPIP at 12 months corrected gestational age (CGA) was evaluated in a Phase II double-blind, randomized, placebo-controlled, multisite clinical trial. Eighty-eight neonates were randomized: 22 to placebo and 22 to 1.5 mg/kg rhCC10 in the first cohort and 21 to placebo and 23 to 5 mg/kg rhCC10 in the second cohort. Neonates were followed to 12 months CGA. Results With CPIP defined as signs/symptoms, medical visits, hospital readmissions, and use of medications for respiratory complications at 12 months CGA, no significant differences were observed between rhCC10 or placebo groups. Only 5% of neonates had no evidence of CPIP at 12 months CGA. Conclusions A single dose of rhCC10 was not effective in reducing CPIP at 12 CGA. Since most neonates had evidence of CPIP using these exploratory endpoints, it is essential to develop more robust outcome measures for clinical trials of respiratory medications in high-risk premature neonates.

ABSTRACT Objectives: To evaluate the dose effects of Recombinant human Club cell 10-kDa protein (rhCC10) on lung function in a well-characterized ovine model of acute respiratory distress syndrome (ARDS) induced by smoke inhalation injury (SII); specifically, the potential of rhCC10 protein to control the inflammatory response and protect pulmonary tissue and function following SII. Design: Randomized, controlled, prospective, and large animal translational studies. Setting: University large animal intensive care unit. Subjects: Thirty-six adult female sheep were surgically prepared and allocated into five groups (Sham (no SII), n = 6; 1 mg/kg/d CC10, n = 8; 3 mg/kg/d CC10, n = 7; 10 mg/kg/d CC10, n = 8; Control SII, n = 7). Interventions: All groups except the sham group were subjected to SII with cooled cotton smoke. Then, the animals were placed on a ventilator, treated with 1, 3, and 10 mg/kg/d of intravenous rhCC10 or vehicle, divided evenly into two administrations per day every 12 h, fluid resuscitated, and monitored for 48 h in a conscious state. Measurements and main results: The group treated with 10 mg/kg/d rhCC10 attenuated changes in the following variables: PaO2/FiO2 ratio, oxygenation index, and peak inspiratory pressure; neutrophil content in the airway and myeloperoxidase levels; obstruction of the large and small airways; systemic leakage of fluid and proteins, and pulmonary edema. Conclusions: In this study, high-dose rhCC10 significantly attenuated ARDS progression and lung dysfunction and significantly reduced systemic extravasation of fluid and proteins, normalizing fluid balance. Based on these results, rhCC10 may be considered a novel therapeutic option for the treatment of SII-induced ARDS.

Intracellular lipopolysaccharide (LPS) triggers the non-canonical inflammasome pathway, resulting in pyroptosis of innate immune cells. In addition to its well-known proinflammatory role, LPS can directly cause regression of some tumors, although the underlying mechanism has remained unknown. Here we show that secretoglobin(SCGB)3A2, a small protein predominantly secreted in airways, chaperones LPS to the cytosol through the cell surface receptor syndecan-1; this leads to pyroptotic cell death driven by caspase-11. SCGB3A2 and LPS co-treatment significantly induced pyroptosis of macrophage RAW264.7 cells and decreased cancer cell proliferation in vitro, while SCGB3A2 treatment resulted in reduced progression of xenograft tumors in mice. These data suggest a conserved function for SCGB3A2 in the innate immune system and cancer cells. These findings demonstrate a critical role for SCGB3A2 as an LPS delivery vehicle; they reveal one mechanism whereby LPS enters innate immune cells leading to pyroptosis, and they clarify the direct effect of LPS on cancer cells.

BACKGROUND:The club cell 10-kDa protein (CC10) is a homeostatic protein that is produced in the lung, diffuses into the blood, and is then excreted into the urine and stool. CC10 is known to have anti-inflammatory properties and to have lower endogenous production in preterm infants.OBJECTIVES:As recombinant human CC10 (rhCC10) is being studied in preterm infants to reduce lung injury, understanding CC10 levels in term infants with normal lungs is needed to establish appropriate target dosing ranges.METHODS:Serum, urine, and stool samples were collected from 24 healthy full-term infants, and CC10 levels were measured. Levels in term infants were then compared to those in preterm infants who were examined in our previous studies.RESULTS:The mean gestational age and birth weight of the term infants were 38.8 ±1.1 weeks and 3,257 ± 513 g, respectively. The mean gestational age of the preterm infants was 26.8 ± 1.4 weeks. The median serum [CC10] levels with minimum and maximum values in term infants (214.2 ng/mL [34.1, 428.1]) were >7-fold higher than in preterm infants (27.5 ng/mL [8.0, 760.0]; p < 0.05). A significant correlation was found between [CC10] in urine and stool as well as between gestational age and stool [CC10] (p < 0.05).CONCLUSIONS:CC10 is detectable in serum, urine, and stool in healthy term infants, with levels significantly higher than in preterm infants. This provides important data for ongoing therapeutic intervention trials with rhCC10 in high-risk preterm infants.