Epithelial responses to CFTR modulators are improved by inflammatory cytokines and impaired by anti-inflammatory drugs.

Cystic fibrosis (CF) is a genetic disorder that disrupts CF transmembrane conductance regulator (CFTR) anion channels and impairs airway host defenses. Airway inflammation is ubiquitous in CF and suppressing it has generally been considered to improve outcomes. However, the role of inflammation in people taking CFTR modulators, small-molecule drugs that restore CFTR function, is not well-understood. We previously showed that inflammation enhances the efficacy of CFTR modulators. To further elucidate this relationship, we treated human ∆F508-CF epithelia with TNFα and IL-17, two inflammatory cytokines that are elevated in CF airways. TNFα+IL-17 enhanced CFTR modulator-evoked anion secretion through mechanisms that raise intracellular Cl- (Na+/K+/2Cl- co-transport) and HCO3- (carbonic anhydrases and Na+/HCO3- co-transport). This enhancement required p38 MAPK signaling. Importantly, CFTR modulators did not affect CF airway surface liquid viscosity under control conditions, but prevented the rise in viscosity in epithelia treated with TNFα+IL-17. Lastly, anti-inflammatory drugs limited CFTR modulator responses in TNFα+IL-17-treated epithelia. These results provide critical insights into mechanisms by which inflammation increases responses to CFTR modulators. They also suggest an equipoise between potential benefits versus limitations of suppressing inflammation in people taking modulators, call into question current treatment approaches, and highlight a need for additional studies.

Arteriovenous metabolomics in pigs reveals CFTR regulation of metabolism in multiple organs.

Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene cause cystic fibrosis (CF), a multiorgan disease that is characterized by diverse metabolic defects. However, other than specific CFTR mutations, the factors that influence disease progression and severity remain poorly understood. Aberrant metabolite levels have been reported, but whether CFTR loss itself or secondary abnormalities (infection, inflammation, malnutrition, and various treatments) drive metabolic defects is uncertain. Here, we implemented comprehensive arteriovenous metabolomics in newborn CF pigs, and the results revealed CFTR as a bona fide regulator of metabolism. CFTR loss impaired metabolite exchange across organs, including disruption of lung uptake of fatty acids, yet enhancement of uptake of arachidonic acid, a precursor of proinflammatory cytokines. CFTR loss also impaired kidney reabsorption of amino acids and lactate and abolished renal glucose homeostasis. These and additional unexpected metabolic defects prior to disease manifestations reveal a fundamental role for CFTR in controlling multiorgan metabolism. Such discovery informs a basic understanding of CF, provides a foundation for future investigation, and has implications for developing therapies targeting only a single tissue.

Mitochondrial uncoupling proteins protect human airway epithelial ciliated cells from oxidative damage.

Apical cilia on epithelial cells defend the lung by propelling pathogens and particulates out of the respiratory airways. Ciliated cells produce ATP that powers cilia beating by densely grouping mitochondria just beneath the apical membrane. However, this efficient localization comes at a cost because electrons leaked during oxidative phosphorylation react with molecular oxygen to form superoxide, and thus, the cluster of mitochondria creates a hotspot for oxidant production. The relatively high oxygen concentration overlying airway epithelia further intensifies the risk of generating superoxide. Thus, airway ciliated cells face a unique challenge of producing harmful levels of oxidants. However, surprisingly, highly ciliated epithelia produce less reactive oxygen species (ROS) than epithelia with few ciliated cells. Compared to other airway cell types, ciliated cells express high levels of mitochondrial uncoupling proteins, UCP2 and UCP5. These proteins decrease mitochondrial protonmotive force and thereby reduce production of ROS. As a result, lipid peroxidation, a marker of oxidant injury, decreases. However, mitochondrial uncoupling proteins exact a price for decreasing oxidant production; they decrease the fraction of mitochondrial respiration that generates ATP. These findings indicate that ciliated cells sacrifice mitochondrial efficiency in exchange for safety from damaging oxidation. Employing uncoupling proteins to prevent oxidant production, instead of relying solely on antioxidants to decrease postproduction oxidant levels, may offer an advantage for targeting a local area of intense ROS generation.

Injectable long-acting ivacaftor-loaded poly (lactide-co-glycolide) microparticle formulations for the treatment of cystic fibrosis: In vitro characterization and in vivo pharmacokinetics in mice.

Optimizing a sustained-release drug delivery system for the treatment of cystic fibrosis (CF) is crucial for decreasing the dosing frequency and improving patients' compliance with the treatment regimen. In the current work, we developed an injectable poly(D,L-lactide-co-glycolide) (PLGA) microparticle formulation loaded with ivacaftor, a cystic fibrosis transmembrane conductance regulator (CFTR) potentiator that increases the open probability of the CFTR anion channel, using a single emulsion solvent evaporation technique. We aimed to study the effect of different parameters on the characteristics of the prepared formulations to select an optimized microparticle formulation to be used in an in vivo pharmacokinetic study in mice. First, a suite of ivacaftor-loaded microparticles were prepared using different formulation parameters in order to study the effect of varying these parameters on microparticle size, morphology, drug loading, encapsulation efficiency, and in vitro release profiles. Prepared microparticles were spherical with diameters ranging from 1.91-6.93 µm, percent drug loading (% DL) of 3.91-10.3%, percent encapsulation efficiencies (% EE) of 26.6-100%, and an overall slow cumulative release profile. We selected the formulation that demonstrated optimal combined % DL and % EE values (8.25 and 90.7%, respectively) for further studies. These microparticles had an average particle size of 6.83 µm and a slow tri-phasic in vitro release profile (up to 6 weeks). In vivo pharmacokinetic studies in mice showed that the subcutaneously injected microparticles resulted in steady plasma levels of ivacaftor over a period of 28 days, and a 6-fold increase in AUC 0 - t (71.6 µg/mL*h) compared to the intravenously injected soluble ivacaftor (12.3 µg/mL*h). Our results suggest that this novel ivacaftor-loaded microparticle formulation could potentially eliminate the need for the frequent daily administration of ivacaftor to people with CF thus improving their compliance and ensuring successful treatment outcomes.

CFTR-rich ionocytes mediate chloride absorption across airway epithelia.

The volume and composition of a thin layer of liquid covering the airway surface defend the lung from inhaled pathogens and debris. Airway epithelia secrete Cl- into the airway surface liquid through cystic fibrosis transmembrane conductance regulator (CFTR) channels, thereby increasing the volume of airway surface liquid. The discovery that pulmonary ionocytes contain high levels of CFTR led us to predict that ionocytes drive secretion. However, we found the opposite. Elevating ionocyte abundance increased liquid absorption, whereas reducing ionocyte abundance increased secretion. In contrast to other airway epithelial cells, ionocytes contained barttin/Cl- channels in their basolateral membrane. Disrupting barttin/Cl- channel function impaired liquid absorption, and overexpressing barttin/Cl- channels increased absorption. Together, apical CFTR and basolateral barttin/Cl- channels provide an electrically conductive pathway for Cl- flow through ionocytes, and the transepithelial voltage generated by apical Na+ channels drives absorption. These findings indicate that ionocytes mediate liquid absorption, and secretory cells mediate liquid secretion. Segregating these counteracting activities to distinct cell types enables epithelia to precisely control the airway surface. Moreover, the divergent role of CFTR in ionocytes and secretory cells suggests that cystic fibrosis disrupts both liquid secretion and absorption.

Inflammation as a Regulator of the Airway Surface Liquid pH in Cystic Fibrosis.

The airway surface liquid (ASL) is a thin sheet of fluid that covers the luminal aspect of the airway epithelium. The ASL is a site of several first-line host defenses, and its composition is a key factor that determines respiratory fitness. Specifically, the acid-base balance of ASL has a major influence on the vital respiratory defense processes of mucociliary clearance and antimicrobial peptide activity against inhaled pathogens. In the inherited disorder cystic fibrosis (CF), loss of cystic fibrosis transmembrane conductance regulator (CFTR) anion channel function reduces HCO3- secretion, lowers the pH of ASL (pHASL), and impairs host defenses. These abnormalities initiate a pathologic process whose hallmarks are chronic infection, inflammation, mucus obstruction, and bronchiectasis. Inflammation is particularly relevant as it develops early in CF and persists despite highly effective CFTR modulator therapy. Recent studies show that inflammation may alter HCO3- and H+ secretion across the airway epithelia and thus regulate pHASL. Moreover, inflammation may enhance the restoration of CFTR channel function in CF epithelia exposed to clinically approved modulators. This review focuses on the complex relationships between acid-base secretion, airway inflammation, pHASL regulation, and therapeutic responses to CFTR modulators. These factors have important implications for defining optimal ways of tackling CF airway inflammation in the post-modulator era.

Pulmonary neuroendocrine cells sense succinate to stimulate myoepithelial cell contraction.

Pulmonary neuroendocrine cells (PNECs) are rare airway cells with potential sensory capacity linked to vagal neurons and immune cells. How PNECs sense and respond to external stimuli remains poorly understood. We discovered PNECs located within pig and human submucosal glands, a tissue that produces much of the mucus that defends the lung. These PNECs sense succinate, an inflammatory molecule in liquid lining the airway surface. The results indicate that succinate migrates down the submucosal gland duct to the acinus, where it triggers apical succinate receptors, causing PNECs to release ATP. The short-range ATP signal stimulates the contraction of myoepithelial cells wrapped tightly around the submucosal glands. Succinate-triggered gland contraction may complement the action of neurotransmitters that induce mucus release but not gland contraction to promote mucus ejection onto the airway surface. These findings identify a local circuit in which rare PNECs within submucosal glands sense an environmental cue to orchestrate the function of airway glands.

Use of Glycolysis-Enhancing Drugs and Risk of Parkinson's Disease.

BACKGROUND: Terazosin (TZ) and closely related α1-adrenergic receptor antagonists (doxazosin [DZ] and alfuzosin [AZ]) enhance glycolysis and reduce neurodegeneration in animal models. Observational evidence in humans from several databases supports this finding; however, a recent study has suggested that tamsulosin, the comparator medication, increases the risk of Parkinson's disease. AIMS: We consider a different comparison group of men taking 5α-reductase inhibitors (5ARIs) as a new, independent comparison allowing us to both obtain new estimates of the association between TZ/DZ/AZ and Parkinson's disease outcomes and validate tamsulosin as an active comparator. METHODS: Using the Truven Health Analytics Marketscan database, we identified men without Parkinson's disease, newly started on TZ/DZ/AZ, tamsulosin, or 5ARIs. We followed these matched cohorts to compare the hazard of developing Parkinson's disease. We conducted sensitivity analyses using variable duration of lead-in to mitigate biases introduced by prodromal disease. RESULTS: We found that men taking TZ/DZ/AZ had a lower hazard of Parkinson's disease than men taking tamsulosin (hazard ratio (HR) = 0.71, 95% CI [confidence interval]: 0.65-0.77, n = 239,888) and lower than men taking 5ARIs (HR = 0.84, 95% CI: 0.75-0.94, n = 129,116). We found the TZ/DZ/AZ versus tamsulosin HR to be essentially unchanged with up to 5 years of lead-in time; however, the TZ/DZ/AZ versus 5ARI effect became attenuated with longer lead-in durations. CONCLUSIONS: These data suggest that men using TZ/DZ/AZ have a somewhat lower risk of developing Parkinson's disease than those using tamsulosin and a slightly lower risk than those using 5ARIs. © 2022 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

WNK Inhibition Increases Surface Liquid pH and Host Defense in Cystic Fibrosis Airway Epithelia.

In cystic fibrosis (CF), reduced HCO3- secretion acidifies the airway surface liquid (ASL), and the acidic pH disrupts host defenses. Thus, understanding the control of ASL pH (pHASL) in CF may help identify novel targets and facilitate therapeutic development. In diverse epithelia, the WNK (with-no-lysine [K]) kinases coordinate HCO3- and Cl- transport, but their functions in airway epithelia are poorly understood. Here, we tested the hypothesis that WNK kinases regulate CF pHASL. In primary cultures of differentiated human airway epithelia, inhibiting WNK kinases acutely increased both CF and non-CF pHASL. This response was HCO3- dependent and involved downstream SPAK/OSR1 (Ste20/SPS1-related proline-alanine-rich protein kinase/oxidative stress responsive 1 kinase). Importantly, WNK inhibition enhanced key host defenses otherwise impaired in CF. Human airway epithelia expressed two WNK isoforms in secretory cells and ionocytes, and knockdown of either WNK1 or WNK2 increased CF pHASL. WNK inhibition decreased Cl- secretion and the response to bumetanide, an NKCC1 (sodium-potassium-chloride cotransporter 1) inhibitor. Surprisingly, bumetanide alone or basolateral Cl- substitution also alkalinized CF pHASL. These data suggest that WNK kinases influence the balance between transepithelial Cl- versus HCO3- secretion. Moreover, reducing basolateral Cl- entry may increase HCO3- secretion and raise pHASL, thereby improving CF host defenses.

A Single-Cell Atlas of Large and Small Airways at Birth in a Porcine Model of Cystic Fibrosis.

Lack of CFTR (cystic fibrosis transmembrane conductance regulator) affects the transcriptome, composition, and function of large and small airway epithelia in people with advanced cystic fibrosis (CF); however, whether lack of CFTR causes cell-intrinsic abnormalities present at birth versus inflammation-dependent abnormalities is unclear. We performed a single-cell RNA-sequencing census of microdissected small airways from newborn CF pigs, which recapitulate CF host defense defects and pathology over time. Lack of CFTR minimally affected the transcriptome of large and small airways at birth, suggesting that infection and inflammation drive transcriptomic abnormalities in advanced CF. Importantly, common small airway epithelial cell types expressed a markedly different transcriptome than corresponding large airway cell types. Quantitative immunohistochemistry and electrophysiology of small airway epithelia demonstrated basal cells that reach the apical surface and a water and ion transport advantage. This single cell atlas highlights the archetypal nature of airway epithelial cells with location-dependent gene expression and function.

Elastic mucus strands impair mucociliary clearance in cystic fibrosis pigs.

SignificanceIn many lung diseases, increased amounts of and/or abnormal mucus impair mucociliary clearance, a key defense against inhaled and aspirated material. Submucosal glands lining cartilaginous airways secrete mucus strands that are pulled by cilia until they break free from the duct and sweep upward toward the larynx, carrying particulates. In cystic fibrosis (CF) pigs, progressive clearance of insufflated microdisks was repeatedly interrupted as microdisks abruptly recoiled. Aerosolizing a reducing agent to break disulfide bonds linking mucins ruptured mucus strands, freeing them from submucosal gland ducts and allowing cilia to propel them up the airways. These findings highlight the abnormally increased elasticity of CF mucus and suggest that agents that break disulfide bonds might have value in lung diseases with increased mucus.

Perspectives on Vascular Regulation of Mechanisms Controlling Selective Immune Cell Function in the Tumor Immune Response.

The vasculature plays a major role in regulating the tumor immune cell response although the underlying mechanisms explaining such effects remain poorly understood. This review discusses current knowledge on known vascular functions with a viewpoint on how they may yield distinct immune responses. The vasculature might directly influence selective immune cell infiltration into tumors by its cell surface expression of cell adhesion molecules, expression of cytokines, cell junction properties, focal adhesions, cytoskeleton and functional capacity. This will alter the tumor microenvironment and unleash a plethora of responses that will influence the tumor's immune status. Despite our current knowledge of numerous mechanisms operating, the field is underexplored in that few functions providing a high degree of specificity have yet been provided in relation to the enormous divergence of responses apparent in human cancers. Further exploration of this field is much warranted.

Cellular and molecular architecture of submucosal glands in wild-type and cystic fibrosis pigs.

Submucosal glands (SMGs) protect lungs but can also contribute to disease. For example, in cystic fibrosis (CF), SMGs produce abnormal mucus that disrupts mucociliary transport. CF is an ion transport disease, yet knowledge of the ion transporters expressed by SMG acini, which produce mucus, and SMG ducts that carry it to the airway lumen is limited. Therefore, we isolated SMGs from newborn pigs and used single-cell messenger RNA sequencing, immunohistochemistry, and in situ hybridization to identify cell types, gene expression, and spatial distribution. Cell types and transcript levels were the same in non-CF and CF SMGs, suggesting that loss of epithelial anion secretion rather than an intrinsic cell defect causes CF mucus abnormalities. Gene signatures of acinar mucous and acinar serous cells revealed specialized functions in producing mucins and antimicrobials, respectively. However, surprisingly, these two cell types expressed the same ion transporters and neurohumoral receptors, suggesting the importance of balancing mucin and liquid secretion to produce optimal mucus properties. SMG duct cell transcripts suggest that they secrete HCO3- and Cl-, and thus have some similarity to pancreatic ducts that are also defective in CF. These and additional findings suggest the functions of the SMG acinus and duct and provide a baseline for understanding how environmental and genetic challenges impact their contribution to lung disease.

Tyrosine-protein kinase Yes controls endothelial junctional plasticity and barrier integrity by regulating VE-cadherin phosphorylation and endocytosis.

Vascular endothelial (VE)-cadherin in endothelial adherens junctions is an essential component of the vascular barrier, critical for tissue homeostasis and implicated in diseases such as cancer and retinopathies. Inhibitors of Src cytoplasmic tyrosine kinase have been applied to suppress VE-cadherin tyrosine phosphorylation and prevent excessive leakage, edema and high interstitial pressure. Here we show that the Src-related Yes tyrosine kinase, rather than Src, is localized at endothelial cell (EC) junctions where it becomes activated in a flow-dependent manner. EC-specific Yes1 deletion suppresses VE-cadherin phosphorylation and arrests VE-cadherin at EC junctions. This is accompanied by loss of EC collective migration and exaggerated agonist-induced macromolecular leakage. Overexpression of Yes1 causes ectopic VE-cadherin phosphorylation, while vascular leakage is unaffected. In contrast, in EC-specific Src-deficiency, VE-cadherin internalization is maintained, and leakage is suppressed. In conclusion, Yes-mediated phosphorylation regulates constitutive VE-cadherin turnover, thereby maintaining endothelial junction plasticity and vascular integrity.

A pilot to assess target engagement of terazosin in Parkinson's disease.

BACKGROUND: Impaired brain energy metabolism is a key feature of Parkinson's disease (PD). Terazosin (TZ) binds phosphoglycerate kinase 1 and stimulates its activity, which enhances glycolysis and increases ATP levels. Preclinical and epidemiologic data suggest that TZ may be neuroprotective in PD. We aimed to assess target engagement and safety of TZ in people with PD. METHODS: We performed a 12-week pilot study in people with PD. Participants were randomized to receive 5 mg TZ or placebo. Participants and study personnel were blinded. We assessed TZ target engagement by measuring brain ATP with 31P-magnetic resonance spectroscopy (MRS) and whole blood ATP with a luminescence assay. Robust linear regression models compared changes between groups controlling for baseline brain and blood ATP levels, respectively. We also assessed clinical measures of PD and adverse events. RESULTS: Thirteen participants were randomized. Mild dizziness/lightheadedness was more common in the TZ group, and three participants taking TZ dropped out because of dizziness and/or orthostatic hypotension. Compared to the placebo group, the TZ group had a significant increase in the ratio of ßATP to inorganic phosphate in the brain. The TZ group also had a significant increase in blood ATP levels compared to the placebo group (p < 0.01). CONCLUSIONS: This pilot study suggests that TZ may engage its target and change ATP levels in the brain and blood of people with PD. Further studies may be warranted to test the disease-modifying potential of TZ.

Combining Ivacaftor and Intensive Antibiotics Achieves Limited Clearance of Cystic Fibrosis Infections.

Drugs called CFTR modulators improve the physiologic defect underlying cystic fibrosis (CF) and alleviate many disease manifestations. However, studies to date indicate that chronic lung infections that are responsible for most disease-related mortality generally persist. Here, we investigated whether combining the CFTR modulator ivacaftor with an intensive 3.5-month antibiotic course could clear chronic Pseudomonas aeruginosa or Staphylococcus aureus lung infections in subjects with R117H-CFTR, who are highly ivacaftor-responsive. Ivacaftor alone improved CFTR activity, and lung function and inflammation within 48 h, and reduced P. aeruginosa and S. aureus pathogen density by ∼10-fold within a week. Antibiotics produced an additional ∼10-fold reduction in pathogen density, but this reduction was transient in subjects who remained infected. Only 1/5 P. aeruginosa-infected and 1/7 S. aureus-infected subjects became persistently culture-negative after the combined treatment. Subjects appearing to clear infection did not have particularly favorable baseline lung function or inflammation, pathogen density or antibiotic susceptibility, or bronchiectasis scores on CT scans, but they did have remarkably low sweat chloride values before and after ivacaftor. All persistently P. aeruginosa-positive subjects remained infected by their pretreatment strain, whereas subjects persistently S. aureus-positive frequently lost and gained strains. This work suggests chronic CF infections may resist eradication despite marked and rapid modulator-induced improvements in lung infection and inflammation parameters and aggressive antibiotic treatment. IMPORTANCE Recent work shows that people with CF and chronic lung infections generally remain persistently infected after treatment with drugs that target the CF physiological defect (called CFTR modulators). However, changes produced by modulators could increase antibiotic efficacy. We tested the approach of combining modulators and intensive antibiotics in rapid succession and found that while few subjects cleared their infections, combined treatment appeared most effective in subjects with the highest CFTR activity. These findings highlight challenges that remain to improve the health of people with CF.

Mouse Breast Carcinoma Monocytic/Macrophagic Myeloid-Derived Suppressor Cell Infiltration as a Consequence of Endothelial Dysfunction in Shb-Deficient Endothelial Cells Increases Tumor Lung Metastasis.

Metastasis reflects both the inherent properties of tumor cells and the response of the stroma to the presence of the tumor. Vascular barrier properties, either due to endothelial cell (EC) or pericyte function, play an important role in metastasis in addition to the contribution of the immune system. The Shb gene encodes the Src homology-2 domain protein B that operates downstream of tyrosine kinases in both vascular and immune cells. We have investigated E0771.lmb breast carcinoma metastasis in mice with conditional deletion of the Shb gene using the Cdh5-CreERt2 transgene, resulting in inactivation of the Shb-gene in EC and some hematopoietic cell populations. Lung metastasis from orthotopic tumors, tumor vascular and immune cell characteristics, and immune cell gene expression profiles were determined. We found no increase in vascular leakage that could explain the observed increase in metastasis upon the loss of Shb expression. Instead, Shb deficiency in EC promoted the recruitment of monocytic/macrophagic myeloid-derived suppressor cells (mMDSC), an immune cell type that confers a suppressive immune response, thus enhancing lung metastasis. An MDSC-promoting cytokine/chemokine profile was simultaneously observed in tumors grown in mice with EC-specific Shb deficiency, providing an explanation for the expanded mMDSC population. The results demonstrate an intricate interplay between tumor EC and immune cells that pivots between pro-tumoral and anti-tumoral properties, depending on relevant genetic and/or environmental factors operating in the microenvironment.

The Felicitous Success of the Subsection Molecular Oncology of International Journal of Molecular Sciences.

The evolvement of the newly started subsection IJMS molecular oncology is discussed. The breadth and depth of the journal articles is alluded to. A bright future for this subsection is anticipated, developing into a top tier cancer journal.

Inflammatory cytokines TNF-α and IL-17 enhance the efficacy of cystic fibrosis transmembrane conductance regulator modulators.

Without cystic fibrosis transmembrane conductance regulator-mediated (CFTR-mediated) HCO3- secretion, airway epithelia of newborns with cystic fibrosis (CF) produce an abnormally acidic airway surface liquid (ASL), and the decreased pH impairs respiratory host defenses. However, within a few months of birth, ASL pH increases to match that in non-CF airways. Although the physiological basis for the increase is unknown, this time course matches the development of inflammation in CF airways. To learn whether inflammation alters CF ASL pH, we treated CF epithelia with TNF-α and IL-17 (TNF-α+IL-17), 2 inflammatory cytokines that are elevated in CF airways. TNF-α+IL-17 markedly increased ASL pH by upregulating pendrin, an apical Cl-/HCO3- exchanger. Moreover, when CF epithelia were exposed to TNF-α+IL-17, clinically approved CFTR modulators further alkalinized ASL pH. As predicted by these results, in vivo data revealed a positive correlation between airway inflammation and CFTR modulator-induced improvement in lung function. These findings suggest that inflammation is a key regulator of HCO3- secretion in CF airways. Thus, they explain earlier observations that ASL pH increases after birth and indicate that, for similar levels of inflammation, the pH of CF ASL is abnormally acidic. These results also suggest that a non-cell-autonomous mechanism, airway inflammation, is an important determinant of the response to CFTR modulators.

In vitro evaluation of novel (nanoparticle) oral delivery systems allow selection of gut immunomodulatory formulations.

Oral delivery is the most convenient way to vaccinate cultured fish, however it is still problematic, primarily due to a lack of a commercially valid vaccine vehicle to protect the antigen against gastric degradation and ensure its uptake from the intestine. With the goal of advancing the potential to vaccinate orally, this study evaluates a novel silicon nanoparticle-based vehicle (VacSaf carrier). Aeromonas salmonicida antigens were formulated with the VacSaf carrier using different preparation methods to generate dry powder and liquid formulations. Twelve formulations were first subjected to an in vitro evaluation where the A. salmonicida bacterin conjugated to VacSaf carriers were found superior at inducing pro-inflammatory cytokine expression in primary leucocyte cultures and the macrophage/monocyte cell line RTS-11 compared with A. salmonicida bacterin alone. This was especially apparent after exposure to acid conditions to mimic stomach processing. One formulation (FD1) was taken forward to oral delivery using two doses and two administration schedules (5 days vs 10 days, the latter 5 days on, 5 days off, 5 days on), and the transcript changes of immune genes in the intestine (pyloric caeca, midgut and hindgut) and spleen were evaluated by qPCR and serum IgM was measured by ELISA. The VacSaf carrier alone was shown to be safe for use in vivo, in that no side-effects were seen, but it did induce expression of some cytokines, and may have value as an oral adjuvant candidate. The FD1 bacterin formulation was effective at inducing a range of cytokines associated with innate and adaptive immunity, mainly in the pyloric caeca, compared to A. salmonicida bacterin alone (which had almost no effect), and confirms the immune competence of this gut region following appropriate oral vaccination. These results reveal that in vitro screening of formulations for oral delivery has value and can be used to assess the most promising formulations to test further.