Chicken intestinal microbiota modulation of resistance to nephropathogenic infectious bronchitis virus infection through IFN-I

Abstract

Background: Mammalian intestinal microbiomes are necessary for antagonizing systemic viral infections. However, very few studies have identified whether poultry commensal bacteria play a crucial role in protecting against systemic viral infections. Nephropathogenic infectious bronchitis virus (IBV) is a pathogenic coronavirus that causes high morbidity and multiorgan infection tropism in chickens.

Results: In this study, we used broad-spectrum oral antibiotics (ABX) to treat specific pathogen free (SPF) chickens to deplete the microbiota before infection with nephropathogenic IBV to analyze the impact of microbiota on IBV infections in vivo. Depletion of the SPF chicken microbiota increases pathogenicity and viral burden following IBV infection. The gnotobiotic chicken infection model further demonstrated that intestinal microbes are resistant to nephropathogenic IBV infection. In addition, ABX-treated chickens showed a severe reduction in macrophage activation, impaired type I IFN production, and IFN-stimulated gene expression in peripheral blood mononuclear cells and the spleen. Lactobacillus isolated from SPF chickens could restore microbiota-depleted chicken macrophage activation and the IFNAR-dependent type I IFN response to limit IBV infection. Furthermore, exopolysaccharide metabolites of Lactobacillus spp. could induce IFN-β.

Conclusions: This study revealed the resistance mechanism of SPF chicken intestinal microbiota to nephropathogenic IBV infection, providing new ideas for preventing and controlling nephropathogenic IBV. Video abstract.

Keywords: IFN; Intestinal microbiota; Lactobacillus; Nephropathogenic infectious bronchitis virus; SPF chicken.

Fig. 1

Antibiotic treatment for microbiota depletion. The magnitude by which the ABX (ampicillin, neomycin, metronidazole, and vancomycin) depleted the specific pathogen-free (SPF) chicken (n = 6) gut microbiota was assessed by a Chao1, b Shannon, c Venn diagram, and d PCoA plot based on unweighted Unifrac analysis. Data are from two independent experiments. Biological replicates for each sample are three. P values were determined using unpaired, two-tailed Student’s t tests. **P < 0.01

Fig. 2

Microbiota depletion accelerated nephropathogenic IBV pathogenesis and viral replication in vivo. a Trachea, peripheral blood, and kidney of SPF or ABX chicken were harvested and tested for viral loads at 3 and 5 dpi using qRT-PCR (n = 6). b Immunohistochemical anti-N staining of hematoxylin-counterstained tracheal or kidney sections (representative images of 4 or 5 per group). Scale bar, 200 μm. c Histopathological analysis of IBV infection in SPF and ABX chickens. Histopathological analysis was performed on tracheal or kidney samples from noninfected or IBV-infected chickens (SPF or ABX chickens). Histological scoring of hematoxylin and eosin stain-colored tracheal or kidney sections (n = 7 or 8). d Consequences of ABX treatment on IBV virus replication in embryonated chicken eggs. Eleven-day-old embryonated chicken eggs (n = 6) were injected with ABX or PBS. Viral growth was analyzed by quantifying viral RNA using qRT-PCR from RNA extracted from allantoic fluids collected at 72 h post-infection. e Kidney and peripheral blood of gnotobiotic chicken (n = 6) were harvested and tested for viral loads at 3 and 5 dpi using qRT-PCR. Data are from three independent experiments. Biological replicates for each sample are three. P values were determined by an unpaired two-tailed Student’s t test. ns, not significant ; **P < 0.01

Fig. 3

Innate immune responses were diminished in ABX chicken post-IBV infection. a Frequency of a panel of macrophages in PBMC of mock or infected SPF or ABX chicken (n = 6) at 5 dpi. b Expression of MHC-II on PBMC macrophages from mock or infected SPF or ABX chicken (n = 6) at 5 dpi. c Depletion of splenic macrophages following administration of clodronate liposomes. Histograms showing KUL01+ cells isolated from the spleen of chicken (n = 6) treated with PBS liposomes or clodronate liposomes. d Blood and kidney viral titers of SPF or ABX chicken (n = 6) treated with PBS liposomes or clodronate liposomes at 5 dpi. Data are from three independent experiments. Biological replicates for each sample are three. P values were determined by an unpaired two-tailed Student’s t test. ns, not significant ; **P < 0.01

Fig. 4

Microbiota depletion results in significantly impaired systemic type I IFN responses to IBV. a IFN-β levels in the serum at 5 dpi following IBV infection detected by ELISA (n = 6). b Fold change of IFN-β and ISG expression in PBMC and spleen of SPF or ABX chicken (n = 6) at 5 dpi following IBV infection relative to respective mock controls. c IFN-β levels in the serum at 5 dpi from infected IFNAR antibody-treated SPF or ABX chicken (n = 6). d Viral load in blood or kidney collected from infected IFNAR antibody-treated SPF or ABX chicken (n = 6). Data are from three independent experiments. Biological replicates for each sample are three. P values were determined by an unpaired two-tailed Student’s t test. ns, not significant ; **P < 0.01

Fig. 5

L. murinus promotes macrophage activation and IFN-I responses to IBV infection. a Frequency of a panel of macrophages in PBMC of mock or infected SPF or ABX chicken (n = 6), gavaged with L. murinus at 5dpi following IBV inoculation. b Expression of MHC-II on PBMC macrophages from mock or infected SPF or ABX chicken, gavaged with L. murinus or control at 5 dpi following IBV inoculation. c Peripheral blood of ABX chicken (n = 6) gavaged with Lactobacillus was harvested and tested for viral loads at 5 dpi using qRT-PCR. d Viral loads of peripheral blood of infected L. murinus-gavaged ABX chicken (n= 6) injected with clodronate liposomes. e IFN-β levels in the serum of ABX chicken gavaged with L. murinus gavaged at 5 dpi following IBV infection as detected by ELISA. f Fold change of IFN-β and ISG expression in PBMC and the spleen of SPF or ABX chicken (n = 6) gavaged with L. murinus at 5 dpi following IBV infection relative to respective mock controls. g IFN-β levels in the serum of ABX chicken gavaged with L. murinus ABX chicken (n = 6) injected with IFNAR antibody. h Viral loads of peripheral blood of ABX chicken (n = 6) gavaged with L. murinus injected with IFNAR antibody. i Viral loads of peripheral blood or kidney of gnotobiotic chicken (n = 6) gavaged with L. murinus injected with IFNAR antibody. Data are from three independent experiments. Biological replicates for each sample are three. P values were determined using a one-way analysis of variance; ns, not significant; **P < 0.01

Fig. 6

Exopolysaccharide induces IFN-β expression in macrophages to resistance to IBV infection. a IFN-β levels in the monocytes/macrophages supernatant from PBMC of ABX chickens (n = 6), treated with 50 mg/mL EPS complex and CTRL in vitro. b Fold change of IFN-β and ISG expression in PBMC of ABX chickens (n = 6), treated with 50 mg/mL EPS following IBV infection relative to respective mock controls. c IBV loads of macrophages from PBMC of ABX chickens (n = 6), treated with 50 mg/mL EPS complex. d IFN-β levels in the monocytes/macrophages supernatant from PBMC of ABX chicken (n = 6) injected with IFNAR antibody. e IFN-β levels in the serum of ABX chicken (n = 6) gavaged with EPS orally starting 7 days prior to and continuing to the day of infection with IBV. f Viral loads of peripheral blood of ABX chicken (n = 6) gavaged with EPS orally starting 7 days prior to and continuing to the day of infection with IBV. Data are from three independent experiments. Biological replicates for each sample are three. P values were determined using a one-way analysis of variance; ns, not significant; **P < 0.01

Fig. 7

IFN-β induction is a shared function of Lactobacillus spp. metabolites EPS. a Macrophages were differentiated from SPF chicken (n = 6) or ABX chicken (n = 5) mice and treated with 50 mg/mL EPS for 6 h. IFN-β in the supernatants was measured by ELISA and normalized by subtracting vehicle control. b, c qRT-PCR analysis of ISG expression was performed on the PBMC and spleen harvested from SPF chicken (n = 6), SPF chicken after 2 weeks of metronidazole (Met) treatment. Fold change gene expression in the PBMC (b) or spleen (c) was calculated compared to SPF chicken. Data are from three independent experiments. Biological replicates for each sample are three. P values were determined using a one-way analysis of variance; ns, not significant; **P < 0.01

Fig. 8

Schematic summary: EPS from Lactobacillus of SPF chicken intestinal microbes can activate macrophage to secrete IFN-β, engine subsequently systemic ISG expression to resistance to nephropathogenic IBV infection