Transmissible gastroenteritis virus induces inflammatory responses via RIG-I/NF-κB/HIF-1α/glycolysis axis in intestinal organoids and in vivo.

Transmissible gastroenteritis virus (TGEV)-induced enteritis is characterized by watery diarrhea, vomiting, and dehydration, and has high mortality in newborn piglets, resulting in significant economic losses in the pig industry worldwide. Conventional cell lines have been used for many years to investigate inflammation induced by TGEV, but these cell lines may not mimic the actual intestinal environment, making it difficult to obtain accurate results. In this study, apical-out porcine intestinal organoids were employed to study TEGV-induced inflammation. We found that apical-out organoids were susceptible to TGEV infection, and the expression of representative inflammatory cytokines was significantly upregulated upon TGEV infection. In addition, retinoic acid-inducible gene I (RIG-I) and the nuclear factor-kappa B (NF-κB) pathway were responsible for the expression of inflammatory cytokines induced by TGEV infection. We also discovered that the transcription factor hypoxia-inducible factor-1α (HIF-1α) positively regulated TGEV-induced inflammation by activating glycolysis in apical-out organoids, and pig experiments identified the same molecular mechanism as the ex vivo results. Collectively, we unveiled that the inflammatory responses induced by TGEV were modulated via the RIG-I/NF-κB/HIF-1α/glycolysis axis ex vivo and in vivo. This study provides novel insights into TGEV-induced enteritis and verifies intestinal organoids as a reliable model for investigating virus-induced inflammation. IMPORTANCE: Intestinal organoids are a newly developed culture system for investigating immune responses to virus infection. This culture model better represents the physiological environment compared with well-established cell lines. In this study, we discovered that inflammatory responses induced by TGEV infection were regulated by the RIG-I/NF-κB/HIF-1α/glycolysis axis in apical-out porcine organoids and in pigs. Our findings contribute to understanding the mechanism of intestinal inflammation upon viral infection and highlight apical-out organoids as a physiological model to mimic virus-induced inflammation.

Hypoxia inducible factor-1α facilitates transmissible gastroenteritis virus replication by inhibiting type I and type III interferon production.

Transmissible gastroenteritis virus (TGEV) is characterized by watery diarrhea, vomiting, and dehydration and is associated with high mortality especially in newborn piglets, causing significant economic losses to the global pig industry. Hypoxia inducible factor-1α (HIF-1α) has been identified as a key regulator of TGEV-induced inflammation, but understanding of the effect of HIF-1α on TGEV infection remains limited. This study found that TGEV infection was associated with a marked increase in HIF-1α expression in ST cells and an intestinal organoid epithelial monolayer. Furthermore, HIF-1α was shown to facilitate TGEV infection by targeting viral replication, which was achieved by restraining type I and type III interferon (IFN) production. In vivo experiments in piglets demonstrated that the HIF-1α inhibitor BAY87-2243 significantly reduced HIF-1α expression and inhibited TGEV replication and pathogenesis by activating IFN production. In summary, we unveiled that HIF-1α facilitates TGEV replication by restraining type I and type III IFN production in vitro, ex vivo, and in vivo. The findings from this study suggest that HIF-1α could be a novel antiviral target and candidate drug against TGEV infection.

PEDV promotes the differentiation of CD4+T cells towards Th1, Tfh, and Treg cells via CD103+DCs.

Porcine epidemic diarrhea virus (PEDV) can infect all ages of pigs, particularly newborn piglets with a mortality almost reaching to 80-100%, causing significant economic losses to the global pig industry. The mucosal immune response is crucial for PEDV prevention, in which specific dendritic cells (DCs) and differentiated T cells play vital roles. In this study, CD103+DCs were differentiated successfully with retinoic acid (RA) treatment in vitro. PEDV could not replicate efficiently in differentiated CD103+DCs but could promote maturation of CD103+DCs by up-regulating the expression of SLA-DR, CD1a, CD86, and cytokines of IL-1ß and IL-10. In addition, PEDV-infected CD103+DCs and CD4+T cells were co-cultured, and the results showed that the differentiation of CD4+T cells toward Th1, Tfh, and Treg, but not Th2. These results demonstrate that PEDV-infected CD103+DCs could promote the differentiation of CD4+T cells, which provided the basis for further study of mucosal response induced by PEDV via CD103+DCs.

Transmissible Gastroenteritis Virus Infection Promotes the Self-Renewal of Porcine Intestinal Stem Cells via Wnt/ß-Catenin Pathway.

Intestinal stem cells (ISCs) play an important role in tissue repair after injury. A recent report delineates the effect of transmissible gastroenteritis virus (TGEV) infection on the small intestine of recovered pigs. However, the mechanism behind the epithelium regeneration upon TGEV infection remains unclear. To address this, we established a TGEV infection model based on the porcine intestinal organoid monolayer. The results illustrated that the porcine intestinal organoid monolayer was susceptible to TGEV. In addition, the TGEV infection initiated the interferon and inflammatory responses following the loss of absorptive enterocytes and goblet cells. However, TGEV infection did not disturb epithelial integrity but induced the proliferation of ISCs. Furthermore, TGEV infection activated the Wnt/ß-catenin pathway by upregulating the accumulation and nuclear translocation of ß-catenin, as well as promoting the expression of Wnt target genes, such as C-myc, Cyclin D1, Mmp7, Lgr5, and Sox9, which were associated with the self-renewal of ISCs. Collectively, these data demonstrated that the TGEV infection activated the Wnt/ß-catenin pathway to promote the self-renewal of ISCs and resulted in intestinal epithelium regeneration. IMPORTANCE The intestinal epithelium is a physical barrier to enteric viruses and commensal bacteria. It plays an essential role in maintaining the balance between the host and intestinal microenvironment. In addition, intestinal stem cells (ISCs) are responsible for tissue repair after injury. Therefore, prompt self-renewal of intestinal epithelium will facilitate the rebuilding of the physical barrier and maintain gut health. In the manuscript, we found that the transmissible gastroenteritis virus (TGEV) infection did not disturb epithelial integrity but induced the proliferation of ISCs and facilitated epithelium regeneration. Detailed mechanism investigations revealed that the TGEV infection activated the Wnt/ß-catenin pathway to promote the self-renewal of ISCs and resulted in intestinal epithelium regeneration. These findings will contribute to understanding the mechanism of intestinal epithelial regeneration and reparation upon viral infection.

Basophils balance healing after myocardial infarction via IL-4/IL-13.

The inflammatory response after myocardial infarction (MI) is a precisely regulated process that greatly affects subsequent remodeling. Here, we show that basophil granulocytes infiltrated infarcted murine hearts, with a peak occurring between days 3 and 7. Antibody-mediated and genetic depletion of basophils deteriorated cardiac function and resulted in enhanced scar thinning after MI. Mechanistically, we found that basophil depletion was associated with a shift from reparative Ly6Clo macrophages toward increased numbers of inflammatory Ly6Chi monocytes in the infarcted myocardium. Restoration of basophils in basophil-deficient mice by adoptive transfer reversed this proinflammatory phenotype. Cellular alterations in the absence of basophils were accompanied by lower cardiac levels of IL-4 and IL-13, two major cytokines secreted by basophils. Mice with basophil-specific IL-4/IL-13 deficiency exhibited a similarly altered myeloid response with an increased fraction of Ly6Chi monocytes and aggravated cardiac function after MI. In contrast, IL-4 induction in basophils via administration of the glycoprotein IPSE/α-1 led to improved post-MI healing. These results in mice were corroborated by the finding that initially low counts of blood basophils in patients with acute MI were associated with a worse cardiac outcome after 1 year, characterized by a larger scar size. In conclusion, we show that basophils promoted tissue repair after MI by increasing cardiac IL-4 and IL-13 levels.

Cholesterol 25-hydroxylase negatively regulates porcine intestinal coronavirus replication by the production of 25-hydroxycholesterol.

Cholesterol 25-hydroxylase (CH25H) has been shown lately to be a host restriction factor that encodes an enzyme, which catalyzes the oxidized form of cholesterol to 25-hydroxycholesterol (25HC). A series of studies have shown that 25HC activity in hosts plays a vital role in inhibiting viral infection. In this study, we explored the antiviral effect of CH25H and 25HC on porcine epidemic diarrhea virus (PEDV), which causes high mortality rates in newborn piglets with severe diarrhea, and considerable financial loss in the swine industry worldwide. Our results showed that PEDV infection downregulated the expression of CH25H in Vero cells. An overexpression and knockdown assay indicated that CH25H has significant antiviral action against PEDV, and a CH25H mutant (CH25H-M) that lacks hydroxylase activity also retains antiviral activity to a lesser extent. Furthermore, 25HC had a broad-spectrum antiviral effect against different PEDV strains by blocking viral entry. In addition, CH25H and 25HC inhibited the replication of porcine transmissible gastroenteritis virus (TGEV). Taken together, CH25H as a natural host restriction factor could inhibit PEDV and TGEV infection.

Physiological Correlation of Airway Pressure and Transpulmonary Pressure Stress Index on Respiratory Mechanics in Acute Respiratory Failure.

BACKGROUND: Stress index at post-recruitment maneuvers could be a method of positive end-expiratory pressure (PEEP) titration in acute respiratory distress syndrome (ARDS) patients. However, airway pressure (Paw) stress index may not reflect lung mechanics in the patients with high chest wall elastance. This study was to evaluate the Pawstress index on lung mechanics and the correlation between Pawstress index and transpulmonary pressure (PL) stress index in acute respiratory failure (ARF) patients. METHODS: Twenty-four ARF patients with mechanical ventilation (MV) were consecutively recruited from July 2011 to April 2013 in Zhongda Hospital, Nanjing, China and Ospedale S. Giovanni Battista-Molinette Hospital, Turin, Italy. All patients underwent MV with volume control (tidal volume 6 ml/kg) for 20 min. PEEP was set according to the ARDSnet study protocol. The patients were divided into two groups according to the chest wall elastance/respiratory system elastance ratio. The high elastance group (H group, n = 14) had a ratio ≥30%, and the low elastance group (L group, n = 10) had a ratio <30%. Respiratory elastance, gas-exchange, Pawstress index, and PLstress index were measured. Student's t-test, regression analysis, and Bland-Altman analysis were used for statistical analysis. RESULTS: Pneumonia was the major cause of respiratory failure (71.0%). Compared with the L group, PEEP was lower in the H group (5.7 ± 1.7 cmH2O vs. 9.0 ± 2.3 cmH2O, P < 0.01). Compared with the H group, lung elastance was higher (20.0 ± 7.8 cmH2O/L vs. 11.6 ± 3.6 cmH2O/L, P < 0.01), and stress was higher in the L group (7.0 ± 1.9 vs. 4.9 ± 1.9, P = 0.02). A linear relationship was observed between the Pawstress index and the PLstress index in H group (R2 = 0.56, P < 0.01) and L group (R2 = 0.85, P < 0.01). CONCLUSION: In the ARF patients with MV, Pawstress index can substitute for PLto guide ventilator settings. TRIAL REGISTRATION: ClinicalTrials.gov NCT02196870 (https://clinicaltrials.gov/ct2/show/NCT02196870).

[The value of nitrogen washout/washin method in assessing alveolar recruitment volume in acute lung injury patients].

OBJECTIVE: To evaluate the precision and feasibility of nitrogen washout/washin method in assessing lung recruitment of acute lung injury (ALI) patients. METHODS: Fifteen ALI patients underwent mechanical ventilation were involved. Two positive end-expiratory pressure (PEEP) levels (high and low) were adjusted according to ARDSnet recommendations or measurement of transpulmonary pressure, each for 30 minutes. Tidal volume (Vt), plateau of airway pressure (Pplat), other respiratory mechanics, gas-exchange and hemodynamics were measured. End expiration lung volume (EELV) was measured at different PEEP levels through nitrogen washout/washin method, and formula (EELVhighPEEP-EELVlowPEEP)-VtlowPEEP/(PplatlowPEEP-PEEPlowPEEP)×(PEEPhigh-PEEPlow) was used as recruitment (Rec-N2). Alveolar recruitment was measured using pressure-volume (P-V) curves (Rec-mes). Correlation and consistency of Rec-N2 and Rec-mes were compared by correlation analysis and Bland-Altman technique. RESULTS: PEEP titrated by ARDSnet recommendations or transpulmonary pressure were (7 ± 2) cm H2O (1 cm H2O = 0.098 kPa) vs (14 ± 5) cm H2O (P = 0.008); and there were significant differences in peak pressure (23 ± 5) cm H2O vs (28 ± 6) cm H2O, plateau of airway pressure (17 ± 4)cm H2O vs (22 ± 6) cm H2O, esophageal pressure, transpulmonary pressure and other respiratory mechanics between the two PEEP levels (P < 0.05). The P-V curve technique gave Rec-mes a value of 100 (-25 ∼ 185)ml. The nitrogen washout/washin technique gave Rec-N2 a value of 180 (-19 ∼ 255) ml, which showed a good correlation with a bias of 46 (8 ∼ 80) ml (R(2) = 0.755, P < 0.0001). CONCLUSION: Nitrogen washout/washin technique can be used to determine lung recruitment volume of ALI patients.