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. 2014 Feb;10(2):331-8.
doi: 10.4161/auto.27196. Epub 2013 Dec 18.

NOD2 Is Dispensable for ATG16L1 Deficiency-Mediated Resistance to Urinary Tract Infection

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Free PMC article

NOD2 Is Dispensable for ATG16L1 Deficiency-Mediated Resistance to Urinary Tract Infection

Caihong Wang et al. Autophagy. .
Free PMC article

Abstract

NOD2 (nucleotide-binding oligomerization domain containing 2) functions as a pathogen sensor and is involved in development of Crohn disease, a form of inflammatory bowel disease. NOD2 functions in concert with the autophagy protein ATG16L1, which is also implicated in Crohn disease. Recently, we identified a novel protective role of ATG16L1 deficiency in uropathogenic Escherichia coli-induced urinary tract infections (UTIs), which are common infectious diseases in humans. Given the known roles of NOD2 in recruiting ATG16L1 to the bacterial entry site, autophagy induction, and Crohn disease, we hypothesized that NOD2 may also play an important role in UTI pathogenesis. Instead, we found evidence that NOD2 is dispensable in the pathogenesis of UTIs in mice and humans. First, loss of Nod2 did not affect the clearance of bacteriuria and the recruitment of innate immune cells to the bladder. Second, we showed that, although nod2(-/-) mice display increased kidney abscesses in the upper urinary tract, there were no increased bacterial loads or persistence in this niche. Third, although a previous study indicates that loss of Nod2 reverses the protection from intestinal infection afforded by loss of ATG16L1 in mice, we found NOD2 deficiency did not reverse the ATG16L1-deficiency-induced protection from UTI. Finally, a population-based study of a cohort of 1819 patients did not reveal any association of NOD2 polymorphisms with UTI incidence. Together, our data indicated that NOD2 is dispensable for UTI pathogenesis in both mice and humans and does not contribute to ATG16L1-deficiency-induced resistance to UTI in mice.

Keywords: Crohn disease; G908R; L1007finsC; NLR; R702W; bladder; kidney; pyelonephritis; uropathogenic E. coli.

Figures

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Figure 1. Loss of Nod2 does not affect the course of UTI. (A) CFU counts of bacteriuria plotted as mean ± SEM of the Log10 value at 1–14 dpi with UTI89. *P < 0.05 by 2-way ANOVA with Bonferroni post-test. n = 5 to 15 mice/timepoint/genotype; n = 5 experiments. (B) Neutrophil counts in the urine. Bars represent mean ± SEM; n = 4 or 5 mice/time point; n = 2 experiments. (C) Monocyte counts in the urine. Bars represent mean ± SEM; n = 4 to 5 mice/time point; n = 2 experiments. (D) Quantification of IBCs in bladders from control vs. nod2−/− mice at 6 hpi. (E) Quantification of QIRs in bladders from control vs. nod2−/− mice at 14 dpi. n = 8 sections/bladder; n = 32 and 20 bladders from C57BL/6 and nod2−/− mice, respectively. (F) TEM of control and nod2−/− superficial cell ultrastructure. Panels representative of 10- to 15-sq µm regions were examined in n = 3 mice. Scale bar: 1 µm. (G and H) Quantification of MVBs (G) and lysosomes (H) in TEM images. Bars represent mean ± SEM (I) H&E stained bladders from control and nod2−/− mice at 14 dpi. Scale bar: 10 µm. (J) IF imaging analysis of control and nod2−/− urothelium stained with an antibody to UPK3 (red), and biz-benzimide to highlight nuclei (blue). Scale bar: 10 µm.
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Figure 2. Nod2 deficiency is associated with kidney abscess formation during UTI. (A) Quantification of the percentage of mice with kidney abscesses at 14 dpi. (B) Inflammation scores of kidneys from control and nod2−/− mice. n = 20 for control; n = 14 for nod2−/−; **P < 0.01. (C and D) H&E staining of kidneys from control (C) and nod2−/− (D) mice. Arrow indicates a region of neutrophil influx. Scale bar: 63 ìm. (E–H) Immunofluorescence staining of UPEC (green), E-cadherin (cyan), and nuclei (blue) in kidneys of control (E and G) and nod2−/− (F and H) mice. Arrow indicates clumps of bacteria and neutrophils inside renal tubules. (I) CFU counts of bacterial load in the kidney plotted as mean ± SEM of the Log10 value at 1 at 14 dpi, n = 4 to 9 mice/group, n = 3 experiments.
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Figure 3. Nod2 deficiency does not reverse the ATG16L1-deficiency-induced protection from UTI. CFU counts of bacteriuria plotted as mean ± SEM of the Log10 value at 1–14 dpi with UTI89. *P < 0.05 by 2-way ANOVA with Bonferroni post-test. n = 5 to 15 mice/timepoint/genotype; n = 5 experiments.
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Figure 4. NOD2 polymorphisms are not associated with the incidence of UTI. (A) Percentage of patients with regular bladder infections among NOD2 SNP variant groups. (B) Percentage of patients with UTIs treated with antibiotics among NOD2 SNP variant groups. Person Chi-Square test and Fisher Exact Test indicated that none of the differences were statistically significant.

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