Organoid-based in vitro systems to model Cryptosporidium parvum infection in 2D and 3D.

Typical cancer cell-based culture systems cannot support the full life cycle of Cryptosporidium parvum, despite its monoxenous life cycle which is completed in the small intestine of a single host. There is a block to fertilization and zygote formation in vitro. In this paper, we adapted a 2D organoid derived monolayer system and a 3D inverted enteroid system for use in C. parvum culture. 3D inverted enteroids were successfully infected by C. parvum without the need for microinjection and supported subculture of C. parvum. Using the 2D organoid derived monolayer (ODM) system, the infection can be maintained for at least 3 weeks with new oocyst production throughout. Fertilization was confirmed based on successful mating of two strains of C. parvum. We demonstrated that the apparent block to fertilization in typical cell culture is overcome using ODMs.

Characterizing Biofilm Interactions between Ralstonia insidiosa and Chryseobacterium gleum.

Ralstonia insidiosa and Chryseobacterium gleum are bacterial species commonly found in potable water systems, and these two species contribute to the robustness of biofilm formation in a model six-species community from the International Space Station (ISS) potable water system. Here, we set about characterizing the interaction between these two ISS-derived strains and examining the extent to which this interaction extends to other strains and species in these two genera. The enhanced biofilm formation between the ISS strains of R. insidiosa and C. gleum is robust to starting inoculum and temperature and occurs in some but not all tested growth media, and evidence does not support a soluble mediator or coaggregation mechanism. These findings shed light on the ISS R. insidiosa and C. gleum interaction, though such enhancement is not common between these species based on our examination of other R. insidiosa and C. gleum strains, as well as other species of Ralstonia and Chryseobacterium. Thus, while the findings presented here increase our understanding of the ISS potable water model system, not all our findings are broadly extrapolatable to strains found outside of the ISS. IMPORTANCE Biofilms present in drinking water systems and terminal fixtures are important for human health, pipe corrosion, and water taste. Here, we examine the enhanced biofilm of cocultures for two very common bacteria from potable water systems: Ralstonia insidiosa and Chryseobacterium gleum. While strains originally isolated on the International Space Station show enhanced dual-species biofilm formation, terrestrial strains do not show the same interaction properties. This study contributes to our understanding of these two species in both dual-culture and monoculture biofilm formation.

Conjugated bile acids attenuate allergen-induced airway inflammation and hyperresponsiveness by inhibiting UPR transducers.

Conjugated bile acids (CBAs), such as tauroursodeoxycholic acid (TUDCA), are known to resolve the inflammatory and unfolded protein response (UPR) in inflammatory diseases, such as asthma. Whether CBAs exert their beneficial effects on allergic airway responses via 1 arm or several arms of the UPR, or alternatively through the signaling pathways for conserved bile acid receptor, remains largely unknown. We used a house dust mite-induced (HDM-induced) murine model of asthma to evaluate and compare the effects of 5 CBAs and 1 unconjugated bile acid in attenuating allergen-induced UPR and airway responses. Expression of UPR-associated transcripts was assessed in airway brushings from human patients with asthma and healthy subjects. Here we show that CBAs, such as alanyl ß-muricholic acid (AßM) and TUDCA, significantly decreased inflammatory, immune, and cytokine responses; mucus metaplasia; and airway hyperresponsiveness, as compared with other CBAs in a model of allergic airway disease. CBAs predominantly bind to activating transcription factor 6α (ATF6α) compared with the other canonical transducers of the UPR, subsequently decreasing allergen-induced UPR activation and resolving allergic airway disease, without significant activation of the bile acid receptors. TUDCA and AßM also attenuated other HDM-induced ER stress markers in the lungs of allergic mice. Quantitative mRNA analysis of airway epithelial brushings from human subjects demonstrated that several ATF6α-related transcripts were significantly upregulated in patients with asthma compared with healthy subjects. Collectively, these results demonstrate that CBA-based therapy potently inhibits the allergen-induced UPR and allergic airway disease in mice via preferential binding of the canonical transducer of the UPR, ATF6α. These results potentially suggest a novel avenue to treat allergic asthma using select CBAs.

Lung epithelial protein disulfide isomerase A3 (PDIA3) plays an important role in influenza infection, inflammation, and airway mechanics.

Protein disulfide isomerases (PDI) are a family of redox chaperones that catalyze formation or isomerization of disulfide bonds in proteins. Previous studies have shown that one member, PDIA3, interacts with influenza A virus (IAV) hemagglutinin (HA), and this interaction is required for efficient oxidative folding of HA in vitro. However, it is unknown whether these host-viral protein interactions occur during active infection and whether such interactions represent a putative target for the treatment of influenza infection. Here we show that PDIA3 is specifically upregulated in IAV-infected mouse or human lung epithelial cells and PDIA3 directly interacts with IAV-HA. Treatment with a PDI inhibitor, LOC14 inhibited PDIA3 activity in lung epithelial cells, decreased intramolecular disulfide bonds and subsequent oligomerization (maturation) of HA in both H1N1 (A/PR8/34) and H3N2 (X31, A/Aichi/68) infected lung epithelial cells. These reduced disulfide bond formation significantly decreased viral burden, and also pro-inflammatory responses from lung epithelial cells. Lung epithelial-specific deletion of PDIA3 in mice resulted in a significant decrease in viral burden and lung inflammatory-immune markers upon IAV infection, as well as significantly improved airway mechanics. Taken together, these results indicate that PDIA3 is required for effective influenza pathogenesis in vivo, and pharmacological inhibition of PDIs represents a promising new anti-influenza therapeutic strategy during pandemic and severe influenza seasons.

Effect of a chemical chaperone, tauroursodeoxycholic acid, on HDM-induced allergic airway disease.

Endoplasmic reticulum (ER) stress-induced unfolded protein response plays a critical role in inflammatory diseases, including allergic airway disease. However, the benefits of inhibiting ER stress in the treatment of allergic airway disease are not well known. Herein, we tested the therapeutic potential of a chemical chaperone, tauroursodeoxycholic acid (TUDCA), in combating allergic asthma, using a mouse model of house dust mite (HDM)-induced allergic airway disease. TUDCA was administered during the HDM-challenge phase (preventive regimen), after the HDM-challenge phase (therapeutic regimen), or therapeutically during a subsequent HDM rechallenge (rechallenge regimen). In the preventive regimen, TUDCA significantly decreased HDM-induced inflammation, markers of ER stress, airway hyperresponsiveness (AHR), and fibrosis. Similarly, in the therapeutic regimen, TUDCA administration efficiently decreased HDM-induced airway inflammation, mucus metaplasia, ER stress markers, and AHR, but not airway remodeling. Interestingly, TUDCA administered therapeutically in the HDM rechallenge regimen markedly attenuated HDM-induced airway inflammation, mucus metaplasia, ER stress markers, methacholine-induced AHR, and airway fibrotic remodeling. These results indicate that the inhibition of ER stress in the lungs through the administration of chemical chaperones could be a valuable strategy in the treatment of allergic airway diseases.