1. Volume 19, Issue 5, Pages 651-663 (May 2016)
Defensive Mutualism Rescues NADPH Oxidase Inactivation in Gut Infection 
Gratiela Pircalabioru, Gabriella Aviello, Malgorzata Kubica, Alexander Zhdanov, Marie-Helene Paclet, Lorraine Brennan, Rosanne Hertzberger, Dmitri Papkovsky, Billy Bourke, Ulla G. Knaus 
Cell Host & Microbe 
Volume 19, Issue 5, Pages (May 2016)
DOI: /j.chom
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2. Cell Host & Microbe 2016 19, 651-663DOI: (10.1016/j.chom.2016.04.007)
Copyright © 2016 Elsevier Inc. Terms and Conditions

3. Figure 1
Sequential NADPH Oxidase Activation in Colon, NOX1 Regulates DUOX2 Expression
(A) Designations of mice, not functional-NF and whole body without IEC-WB.
(B) Basal O2•− generation of colon tissue in WT and p22ΔIEC mice by Hydro-Cy3 fluorescence, n = 3.
(C) O2•− production measured in WT and p22 KO colon crypts using PMA (1 μg/ml).
(D) H2O2 release from WT and p22 KO derived IEC (± 1 μM thapsigargin). The inhibitors were DPI (1 μM) or BAPTA-AM (30 nM).
(E) Duox2 expression in colons of WT1, Nox2 KO, and p22 KO mice at day 0 or 6 dpi with C. rodentium, n = 6–8.
(F) Duox2 expression in colons of WT3 and p22ΔIEC mice, n = 5–8.
(G and H) Immunoblot of colon crypt lysates before and after C. rodentium infection in vivo (G) or after 2 hr ex vivo stimulation with 106 C. rodentium (H). p22phox and total p38 served as controls, n = 3.
(I) Duox2 expression in colons of WT and Nox1 KO mice, n = 6.
(J) Anti-DUOX and anti-phospho p38 immunoblot of colon crypt lysates derived from WT2 and Nox1 KO mice before and after C. rodentium infection at 6 dpi; total p38 as loading control; and n = 2–3.
Means ± SEM (B), ∗p < 0.05 and independent t test (C–F and I), ∗∗∗p < 0.001, ∗∗p < 0.01, and 1-way ANOVA post hoc Bonferroni test. See also Figure S1.
Cell Host & Microbe  , DOI: ( /j.chom )
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4. Figure 2
Loss of Mucosal NOX Activity Is Protective in C. rodentium Infection
(A) Weight change for WT, Nox2 KO, p22 KO, and p22ΔIEC mice during C. rodentium infection, n = 8–10.
(B) Disease scores at 6 dpi, n = 10.
(C) Pathological changes of colon at 6 dpi, n = 6–10.
(D) Cecal C. rodentium colonization at 6 dpi (CFU), n = 11–21.
(E–H) Expression of Cxcl1 (E), Ifng (F), Il17a (G), and pro-Il1b (H) in colon of uninfected and infected mice; Hprt was used for normalization; and n = 6–9.
(I) Cecum colonization 24 hr post infection with L. monocytogenes, n = 7.
Means ± SEM (A–C and E–H), ∗∗∗p < 0.001, ∗∗p < 0.01, and ∗p < 0.05 by 1-way ANOVA post hoc Bonferroni test. Geometric means (D and I) (95% CI), ∗∗∗p < 0.001, and ∗∗p < 0.01 by Kruskal-Wallis post hoc Dunn’s test.
See also Figure S2.
Cell Host & Microbe  , DOI: ( /j.chom )
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5. Figure 3
Antibiotics or WD Render p22phox-Deficient Mice Susceptible to C. rodentium Colitis
(A–C) Weight (A), survival curves (B), and disease scores (C, 3 dpi) for antibiotic (Abx)-pretreated WT, Nox2 KO, p22 KO, and p22ΔIEC mice during C. rodentium infection, n = 6–8.
(D) Hematoxylin and eosin (H&E) staining of distal colons derived from microbiota-depleted, C. rodentium infected mice. A magnified section is shown (right). The scale bar represents 200 μm.
(E) IL-6 levels in colon homogenates from Abx-pretreated WT and p22ΔIEC mice, n = 5–8.
(F) Cecal colonization of C. rodentium in Abx-treated mice, n = 6–8.
(G) C. rodentium dissemination to spleen, liver, and cardiac blood after Abx and C. rodentium infection, n = 5–10.
(H) Experimental design for WD administration.
(I) Weight and disease scores for WD-fed WT and p22ΔIEC mice during C. rodentium infection, n = 6.
(J) Cecum and spleen colonization of C. rodentium at 6 dpi in WD-fed mice, n = 6.
Means ± SEM (A, C, E, and I), ∗∗∗p < 0.001, ∗∗p < 0.01, and ∗p < 0.05 by 1-way ANOVA post hoc Bonferroni test. p = (∗∗) by Gehan-Breslow-Wilcoxon Test for (B). Geometric means (F, G, and J) (95% CI) and ∗∗∗p < by Kruskal-Wallis post hoc Dunn’s test. See also Figure S3.
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6. Figure 4
Microbiota Analysis of p22phox-Deficient Mice under Standard or WD Conditions
(A) Cecal microbiota analysis of WT1, p22 KO, and p22ΔIEC mice. The relative abundance plot at phyla level (16S rDNA sequencing); the squares represent individual mice; and n = 8–10.
(B) PCoA plot for the unweighted UniFrac analysis of cecal samples from indicated mice on standard versus WD; the colors indicate strains and the dots indicate individual mice.
(C) Percentage of anaerobic and facultative anaerobic bacteria out of total sequences identified by sequencing in cecal DNA of mice fed a standard or WD, n = 8–10.
(D, E, H, and I) Relative abundance at the genus level for Escherichia (D), Streptococcus (E), Bifidobacterium (H), and Butyricicoccus (I), n = 8–10.
(F and G) Mean relative abundance of Lactobacillus (F) and L. reuteri (G) as determined by qPCR of cecal DNA extracts from WT and p22ΔIEC mice fed conventional or WD. The eubacteria 16S rDNA was used for normalization, n = 6.
Means ± SEM, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and unpaired t test. See also Figure S4.
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7. Figure 5
Protection of Nox2 KO and WT Mice by Lactobacilli or Microbiota Association
(A and B) Scheme for mono-association of WT and Nox2 KO mice with L. reuteri (or L. murinus) and weight (A) or cecal colonization (B) in mono-associated Abx mice during C. rodentium infection, n = 6.
(C) C. rodentium dissemination to spleen and liver of Abx mice (for comparison, data points taken from Figure 3H) and L. reuteri mono-associated Abx WT and Nox2 KO mice, n = 5–7.
(D) Scheme for cecal matter transplant (CMT).
(E) H&E staining of colon sections of C. rodentium infected WT mice with WT microbiota (WT1→WT1), p22 KO (p22 KO→WT1), or p22ΔIEC microbiota (p22ΔIEC →WT3); the arrow indicates hyperplasia; and the scale bar represents 200 μm.
(F) Disease scores for mice in (E) at 6 dpi, n = 5–10.
(G) Cecal C. rodentium colonization in mice receiving CMT before infection. The data pooled from two infections, n = 5–12.
(H) Mean relative abundance of Lactobacillus sp. by qPCR analysis of cecal DNA extracts from mice receiving CMT at 6 dpi. The eubacteria 16S rDNA was used for normalization, n = 4–6.
Means ± SEM (A, F, and H), ∗p < 0.05, ∗∗∗p < 0.001, and unpaired t test. Geometric means (B, C, and G) (95% CI), ∗∗∗p < 0.001, and ∗p < 0.05 by Kruskal-Wallis post hoc Dunn’s test. See also Figure S5.
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8. Figure 6
Lactobacilli-Derived H2O2 Downregulates C. rodentium Virulence Genes
(A) Fecal IgA levels in WT and p22ΔIEC mice, n = 5.
(B) Fecal shedding of C. rodentium at 4, 6, and 10 hr pi, and cecal C. rodentium colonization 24 hr pi, n = 4.
(C) Ileal intestinal motility in WT and p22ΔIEC mice; atropine (2 mg/kg) served as control; and n = 6–12.
(D) L. reuteri growth on Abx-pre-treated biopsies derived from WT and p22ΔIEC mice, n = 5.
(E) C. rodentium growth after co-culture with L. reuteri or exposed to L. reuteri o/n culture supernatant (CFS). The controls are C. rodentium incubated with MRS broth (MRS) and catalase, n = 4.
(F) H2O2 release by L. rhamnosus, L. murinus, and L. reuteri. Catalase (CAT; 25 U/ml) was used for H2O2 decomposition, n = 3.
(G) ler expression in C. rodentium co-cultured with L. reuteri CFS, n = 4.
(H) ler expression in C. rodentium after exposure to Cos-p22 or Cos-NOX4 cells (4 hr, DMEM) with or without NaHCO3, n = 4.
(I and J) ler and escN expression in C. rodentium isolated from the lumen (I) or mucus (J) of infected mice (6 dpi), n = 5.
(K) H2O2 release by L. johnsonii WT NCC 533 and mutant NCC 9359, n = 3.
(L) ler expression in C. rodentium co-cultured with NCC 533 or NCC 9359 CFS, n = 5.
(M) Cecal C. rodentium colonization (6 dpi) in WT3 mice treated with NCC 533 or NCC 9359, n = 4–5.
(N) ler and escN expression in C. rodentium isolated from the lumen of infected WT3 mice treated with NCC 533 or NCC 9359, n = 4–5.
gfp expression was used for normalization for (G)–(J), (L), and (N).
Geometric means (B, D, E, and M) (95% CI), ∗p < 0.05, ∗∗p < 0.005, ∗∗∗p < 0.001, and Mann-Whitney test.
Means ± SEM (A, C, F–L, and N), ∗p < 0.05, ∗∗p < 0.005, ∗∗∗p < 0.001, and unpaired t test.
See also Figure S6.
Cell Host & Microbe  , DOI: ( /j.chom )
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