Organoid and enteroid modeling of Salmonella Infection

Salmonella are Gram-negative rod-shaped facultative anaerobic bacteria that are comprised of over 2,000 serovars. They cause gastroenteritis (salmonellosis) with headache, abdominal pain and diarrhea clinical symptoms. Salmonellosis brings a heavy burden for the public health in both developing and developed countries. Antibiotics are usually effective in treating the infected patients with severe gastroenteritis, although antibiotic resistance is on the rise. Understanding the molecular mechanisms of Salmonella infection is vital to combat the disease. In vitro immortalized 2-D cell lines, ex vivo tissues/organs and several animal models have been successfully utilized to study Salmonella infections. Although these infection models have contributed to uncovering the molecular virulence mechanisms, some intrinsic shortcomings have limited their wider applications. Notably, cell lines only contain a single cell type, which cannot reproduce some of the hallmarks of natural infections. While ex vivo tissues/organs alleviate some of these concerns, they are more difficult to maintain, in particular for long term experiments. In addition, non-human animal models are known to reflect only part of the human disease process. Enteroids and induced intestinal organoids are emerging as effective infection models due to their closeness in mimicking the infected tissues/organs. Induced intestinal organoids are derived from iPSCs and contain mesenchymal cells whereas enteroids are derive from intestinal stem cells and are comprised of epithelial cells only. Both enteroids and induced intestinal organoids mimic the villus and crypt domains comparable to the architectures of the in vivo intestine. We review here that enteroids and induced intestinal organoids are emerging as desired infection models to study bacterial-host interactions of Salmonella

Modeling infection and antiviral therapy of enteric viruses using primary intestinal organoids

In this thesis, we first highlighted important clinical complications associated with rotavirus infection in setting of orthotopic organ transplantation, I then analyzed the incidence of rotavirus infection, its diagnosis, its pathogenesis, how to rationally use of immunosuppressive agents in patients at risk for rotavirus infection, especially by studying the interactions of rotavirus with specific immunosuppressants, and also how to manage rotavirus infection in organ recipients. I also found 3% incidence of rotavirus infections among 6176 transplantations. I conclude that rotavirus infections occurring in transplantation patients remain clinically largely not diagnosed, and thus more attention should be paid to this pathogen. The situation might be improved through the development of new superior model systems allowing new directions of research. I demonstrated that primary intestinal organoids can support infection with both laboratory rotavirus strains and with patient-derived rotavirus isolates. Thus, the organoid model might become exceedingly useful for obtaining new scientific insights but may also become important for individualized assessment of the efficacy of different antivirals in a particular patient, next to their potential for developing new and effective medicines against rotavirus. In support for this notion I found that the broadly used antivirals including interferon a and ribavirin inhibit rotavirus replication utilizing the organoid model. I thus propose that organoids provide a promising novel avenue for investigating rotavirus-host interactions and the evaluation of medicines against rotavirus. I profiled common used immunosuppressive medicines on enteric rotavirus and norovirus infections. I found that CsA moderately inhibits rotavirus and norovirus infections, and that MPA is very in this respect for both viruses and also with high barrier towards the development of d.rug resistance. Mechanistically, the antiviral effects of MPA are mediated through inhibition of its canonical cellular target IMPDH and depend on the resulting guanosine nucleotide depletion. I also found that 6-TG potently inhibits rotavirus via blocking the formation of the active form of Rael {GTP-Racl), again with high barrier towards the development of drug resistance. I hope my findings will become an important reference for clinicians to design optimal immunosuppressive therapy for rotavirus infected transplantation patients and aid the development of novel antiviral medicines against rotavirus. I also demonstrated that Pl3K-Akt-mTOR signaling pathway sustains rotavirus infection and that the clinically used mTOR inhibitor rapamycin significantly inhibits rotavirus infection. Rapamycin induces autophagy via 4E-BP1, and induction of autophagy exerted antiviral effect on rotavirus, suggesting that this is the mechanistic explanation of this finding. Although I perceive that the findings presented in this thesis contributed

Modeling infection and antiviral therapy of enteric viruses using primary intestinal organoids

In this thesis, we first highlighted important clinical complications associated with rotavirus infection in setting of orthotopic organ transplantation, I then analyzed the incidence of rotavirus infection, its diagnosis, its pathogenesis, how to rationally use of immunosuppressive agents in patients at risk for rotavirus infection, especially by studying the interactions of rotavirus with specific immunosuppressants, and also how to manage rotavirus infection in organ recipients. I also found 3% incidence of rotavirus infections among 6176 transplantations. I conclude that rotavirus infections occurring in transplantation patients remain clinically largely not diagnosed, and thus more attention should be paid to this pathogen. The situation might be improved through the development of new superior model systems allowing new directions of research. I demonstrated that primary intestinal organoids can support infection with both laboratory rotavirus strains and with patient-derived rotavirus isolates. Thus, the organoid model might become exceedingly useful for obtaining new scientific insights but may also become important for individualized assessment of the efficacy of different antivirals in a particular patient, next to their potential for developing new and effective medicines against rotavirus. In support for this notion I found that the broadly used antivirals including interferon a and ribavirin inhibit rotavirus replication utilizing the organoid model. I thus propose that organoids provide a promising novel avenue for investigating rotavirus-host interactions and the evaluation of medicines against rotavirus. I profiled common used immunosuppressive medicines on enteric rotavirus and norovirus infections. I found that CsA moderately inhibits rotavirus and norovirus infections, and that MPA is very in this respect for both viruses and also with high barrier towards the development of d.rug resistance. Mechanistically, the antiviral effects of MPA are mediated through inhibition of its canonical cellular target IMPDH and depend on the resulting guanosine nucleotide depletion. I also found that 6-TG potently inhibits rotavirus via blocking the formation of the active form of Rael {GTP-Racl), again with high barrier towards the development of drug resistance. I hope my findings will become an important reference for clinicians to design optimal immunosuppressive therapy for rotavirus infected transplantation patients and aid the development of novel antiviral medicines against rotavirus. I also demonstrated that Pl3K-Akt-mTOR signaling pathway sustains rotavirus infection and that the clinically used mTOR inhibitor rapamycin significantly inhibits rotavirus infection. Rapamycin induces autophagy via 4E-BP1, and induction of autophagy exerted antiviral effect on rotavirus, suggesting that this is the mechanistic explanation of this finding. Although I perceive that the findings presented in this thesis contributed

Suppression of pyrimidine biosynthesis by targeting DHODH enzyme robustly inhibits rotavirus replication

Rotavirus infection remains a great health burden worldwide especially in some developing countries. It causes severe dehydrating diarrhea in infants, young children, as well as immunocompromised and organ transplanted patients. Viral replication heavily relies on the host to supply nucleosides. Thus, host enzymes involved in nucleotide biosynthesis represent potential targets for antiviral development. Dihydroorotate dehydrogenase (DHODH) is the rate-limiting enzyme in the de novo biosynthesis pathway of pyrimidines. In this study, we demonstrated that two specific DHODH enzyme inhibitors, brequinar (BQR) and leflunomide (LFM) robustly inhibited rotavirus replication in conven

PI3K-Akt-mTOR axis sustains rotavirus infection via the 4E-BP1 mediated autophagy pathway and represents an antiviral target

Rotavirus infection is a major cause of severe dehydrating diarrhea in infants younger than 5 y old and in particular cases of immunocompromised patients irrespective to the age of the patients. Although vaccines have been developed, an

Convergent Transcription of Interferon-stimulated Genes by TNF-α and IFN-α Augments Antiviral Activity against HCV and HEV

IFN-α has been used for decades to treat chronic hepatitis B and C, and as an off-label treatment for

Basal interferon signaling and therapeutic use of interferons in controlling rotavirus infection in human intestinal cells and organoids

Rotavirus (RV) primarily infects enterocytes and results in severe diarrhea, particularly in children. It is known that the host immune responses determine the outcome of viral infections. Following infections, interferons (IFNs) are produced as the first and the main anti-viral cytokines to combat the virus. Here we showed that RV predominantly induced type III IFNs (IFN-λ1), and to a less extent, type I IFNs (IFN-α and IFN-β) in human intestinal cells. However, it did not produce detectable IFN proteins and thus, was not sufficient to inhibit RV replication. In contrast, we revealed the essential roles of the basal IFN signaling in limiting RV replication by silencing STAT1, STAT2 and IRF9 genes. In addition, exogenous IFN treatment demonstrated that RV replication was able to be inhibited by all types of IFNs, both in human intestinal Caco2 cell line and in primary intestinal organoids. In these models, IFNs significantly upregulated a panel of well-known anti-viral IFN-stimulated genes (ISGs). Importantly, inhibition of the JAK-STAT cascade abrogated ISG induction and the anti-RV effects of IFNs. Thus, our study shall contribute to better understanding of the complex RV-host interactions and provide rationale for therapeutic development of IFN-based treatment against RV infection

TNF-α exerts potent anti-rotavirus effects via the activation of classical NF-κB pathway

Active virus-host interactions determine the outcome of pathogen invasions. It has been shown that in isolated dendritic cells (DCs), rotavirus can induce the expression of tumor necrosis factor α (TNF-α), a vital cytokine mediating host immune responses. However, the role of TNF-α in rotavirus infection is unknown. In this study, we demonstrated that TNF-α has potent anti-rotavirus effects, independent of type I interferon production. Blocking of TNF-α by infliximab, a clinically available TNFα antibody, totally abrogated this effect. Mechanistic studies revealed that the anti-rotavirus effect of TNF-α was achieved by NFκB-regulated genes via the activation of classical nuclear factor κB (NF-κB) signaling. Our study reveals the pivotal role and the mechanism-of-actions of TNF-α in the host defense against rotavirus. Thus, this knowledge may contribute to the better understanding of the complexity of rotavirus-host interactions

6-Thioguanine inhibits rotavirus replication through suppression of Rac1 GDP/GTP cycling

Rotavirus infection has emerged as an important cause of complications in organ transplantation recipients and might play a role in the pathogenesis of inflammatory bowel disease (IBD). 6-Thioguanine (6-TG) has been widely used as an immunosuppressive drug for organ recipients and treatment of IBD in the clinic. This study aims to investigate the effects and mode-of-action of 6-TG on rotavirus replication. Human intestinal Caco2 cell line, 3D model of human primary intestinal organoids, laboratory rotavirus strain (SA11) and patient-derived rotavirus isolates were used. We have demonstrated that 6-TG significantly inhibits rotavirus replication in these intestinal epithelium models. Importantly, gene knockdown or knockout of Rac1, the cellular target of 6-TG, significantly inhibited rotavirus replication, indicating the supportive role of Rac1 for rotavirus infection. We have further demonstrated that 6-TG can effectively inhibit the active form of Rac1 (GTP-Rac1), which essentially mediates the anti-rotavirus effect of 6-TG. Consistently, ectopic over-expression of GTP-Rac1 facilitates but an inactive Rac1 (N17) or a specific Rac1 inhibitor (NSC23766) inhibits rotavirus replication. In conclusion, we have identified 6-TG as an effective inhibitor of rotavirus replication via the inhibition of Rac1 activation. Thus, for transplantation patients or IBD patients infected with rotavirus or at risk of rotavirus infection, the choice of 6-TG as a treatment appears rational

Drug screening identifies gemcitabine inhibiting rotavirus through alteration of pyrimidine nucleotide synthesis pathway

Although rotavirus infection is usually acute and self-limiting, it can cause chronic infection with severe diseases in immunocompromised patients, including organ transplantation recipients and cancer patients irrespective of pediatric or adult patients. Since no approved medication against rotavirus infection is available, this study screened a library of safe-in-man broad-spectrum antivirals. We identified gemcitabine, a widely used anti-cancer drug, as a potent inhibitor of rotavirus infection. We confirmed this effect in 2D cell cultures and 3D cultured human intestinal organoids with both laboratory-adapted rotavirus strains and five clinical isolates. Supplementation of UTP or uridine largely abolished the anti-rotavirus activity of gemcitabine, suggesting its function through inhibition of pyrimidine biosynthesis pathway. Our results support repositioning of gemcitabine for treating rotavirus infection, especially for infected cancer patients