https://orcid.org/0000-0003-4309-8450
Figures & data
Figure 1. MNoV_S99 aggravates inflammation in DSS-induced colitis. a)- in vivo experimental design, C57BL/6J mice were orally gavaged with PBS (200 µL) or MNoV_S99 (5×107 TCID50/mL), 7 d later, all the mice were given DSS in drinking water for a week. Mice were weighed daily for weight loss comparison. b)- 3% DSS-induced colitis ± MNoV_S99 (n = 6). c)- 5% DSS-induced colitis ± MNoV_S99 (n = 6). In b) and c), statistical differences were determined by two-way ANOVA test, *p < 0.05, **p < 0.01, ***p < 0.001. d)- Representative images from H&E staining of colon sections from mock-treated (control) or 3% DSS ± MNoV_S99 treated mice. Insets showing higher magnifications from each image are presented next to each image. e)- muscle wall thickness was determined with image J from colon section images. Percentages of increase are plotted after baseline subtraction relative to the values obtained from control mice (n = 6). f)- histological intestinal epithelial inflammation scores comparison between control, 3% or 5% DSS ± MNoV_S99 treated mice (n = 6). g)- colon length comparison between control and 5% DSS ± MNoV_S99 treated mice h)- IL6 secretion levels from colon explants from mock or 5% DSS ± MNoV_S99 treated mice (n = 6). Statistical differences were determined with Student’s t-test in E, F, G and H.
Figure 2. MNoV_S99-associated inflammation is NOD2 dependent weight loss comparison in a)- 3% DSS-induced colitis ± MNoV_S99 in WT mice (n = 6) and b)- in Nod2-/- mice (n = 6), statistical differences were determined by two-way ANOVA test, *p < 0,05, **p < 0,01, ***p < 0,001. c)- Representative images of H&E staining of colon sections from mice in a) and b). Insets showing higher magnifications are presented below each image. d)- Percentages of increase of muscle wall thickness from colon section images are plotted after baseline subtraction relatively to the values obtained from WT mice treated only with DSS (n = 6), statistical differences were determined with Student’s t-test.
Figure 3. MNoV_S99 associated pro-inflammatory signaling is NOD2 and MAVS-dependent in myeloid lineage cells. a)- Representative image showing an intestinal villus from a Nod2GFP mouse. NOD2-GFP is shown in green, actin is stained with Phalloidin in gray and nuclei were stained with DAPI in blue; scale bare represents 50 µm. b)- Representative western blot showing STAT1 signaling pathway activation after 1 h infection (Moi 1) with the mentioned MNoV strains in BMDM. Below are shown quantification of pSTAT1 bands intensities normalized to β-actin and relative to mock-infected cells (n = 3, mean ± SEM), statistical differences were determined by one-way ANOVA test *p < 0,05, **p < 0,005. c)- STAT1 activation in response to MNoV_S99 (Moi 1 or 5) in BMDM from WT vs Nod2-/- infected for 30 or 60 minu. The lower panel shows quantification of pSTAT1, bands intensities normalized to β- actin and relative to mock-infected cells (n = 3, mean ± SEM). Statistical differences were determined by two-way ANOVA test *p < 0,05. d)- STAT1 activation in response to MNoV_S99 (Moi 0.1) in WT or Nod2-/- BMDM infected for 24 h. Relative quantification of pSTAT1 (n = 3, mean ± SEM) is shown in the lower panel, statistical differences were determined by two-way ANOVA test ***p < 0,0005. e)- STAT1 activation in response to MNoV_S99 (Moi 0.1) in BMDM from WT vs Mavs-/- infected for 60 min. Relative quantification of pSTAT1 (n = 4, mean ± SEM) are shown below. Statistical differences were determined by two-way ANOVA test *p < 0,05.
Figure 4. NOD2-dependent noroviral load. a)- WT, Nod2-/- and Atg16l1HM mice (n = 5 each) were treated with 3% DSS and infected with MNoV_CR6 (3×107 PFU). Inflammation was measured as percentage of increase in muscle thickness at the anal-rectal junction in colonic section images plotted after baseline subtraction relatively to the values obtained from WT mice. b)- the viral load measured in stools from WT, Nod2-/- and Atg16l1HM mice at days 3, 5, and 7 post-infection with MNoV_CR6 (3×107 PFU) (n = 8). c)- the viral load measured from indicated tissue samples from MNoV_CR6 infected (3×107 PFU) WT, Nod2-/- and Atg16l1HM mice that were treated for 1 week with D55 3% (n = 5). d)- MNoV_S99 genome quantification from WT and Nod2−/− BMDM cells infected with MNoV_S99 (Moi 0.1 for 24 h) (n = 3). All the statistical differences were measured with Student’s t-test.
Figure 5. NOD2-dependent pro-inflammatory signaling associated with MNoV_S99 infection and bacterial MDP. Quantification of Nod2 mRNA levels measured by RT-qPCR in a)- in Raw264.7 cells infected with MNoV_S99 (Moi 0.1 or 1, for 24 h) and in b)- in BMDM from WT mice (Moi 0.1, for 24 h) normalized to ActB and relative to mock-infected cells (n = 3). c)- Representative western blot showing STAT1 and IκBα signaling pathways modulation in cell lysates from monocytes that were either infected with MNoV_S99 (Moi 5, 2 h), treated with MDP (10 ng/mL, 2 h), or a combination of both. d)- quantification and comparison of pSTAT1 and pIκbα signals between MNoV_S99 infected alone cells or in combination with MDP (10 ng/mL, 2 h), (n = 3), with the band intensity being normalized to β-ACTIN and relative to mock-treated cells. e) TNFα production by BMDC subjected to MNoV_S99’s ssRNA (10 µg/mL) alone, or supplemented with either MDP (1 µg/mL) or MDP-DD (1 µg/mL) overnight. f) TNFα production by BMDM cells infected with MNoV_S99 (Moi 0.1 or 1) for 6 h, prior to being treated with MDP (10 µg/mL) overnight. g) Net production of MNoV_S99 in Raw264.7 cells infected with MNoV_S99 (Moi 1 for 2 h before MDP treatment (100 ng/mL) for 24 h). Statistical differences were analyzed with Student’s t test.
Table 1. Antibodies used for western blotting.
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The data that support the findings of this study are available from the corresponding authors, GM and MC, upon request on HAL. https://hal.science/hal-04101382.