<i>Leptospira</i> infection triggers inflammation and fibrosis in the mouse kidney.

<p>(A) Light microscopy of nodular infiltrates stained with hematoxylin-eosin (HE), infiltrating CD11b⁺ macrophages and CD3⁺ T cells and collagen deposition stained with Red Sirius (RS) in kidneys from C57BL/6J mice 30 days (D30) after the inoculation of 2×10⁸<i>L. interrogans</i> strain Fiocruz. As controls, mice were injected with PBS. Magnification, ×100. (B) Score of kidney inflammation of interstitial nodular infiltrates per surface areas from five different renal tissue sections in control (PBS) and <i>L. interrogans</i> strain Fiocruz infected mice (n = 8 per group). (C) Quantification of the number of CD11b⁺ macrophages, Gr1⁺ neutrophils and CD3⁺ T Lymphocytes per surface area in kidneys from day-3 (D3) and day-30 post-infected mice. (D) Fibrosis quantification by Red Sirius morphometry, expressed as percent of surface area and (E) Inflammation evaluation by mRNA expression of proinflammatory mediators in kidneys of 10 infected mice sacrificed at different time points. Values are means ± SD of counts (C and D) and mRNA quantification (E) from 5 different tissue sections from n = 2 separate mice in each group tested. ***P<0.001.</p

TLRs and NLRs are not involved in <i>Leptospira</i>-induced fibrosis.

<p>WT and Myd88ko mice infected with 2×10⁶<i>L. interrogans</i> Fiocruz were sacrificed at 15 days p.i. (A) Bacterial loads in 100 µl of urine. (B) Inflammation score in kidneys. (C) Red Sirius staining (C, left panel) and mRNA expression of ACTA-2, fibronectin and Mmp2, in the infected WT and Myd88ko mice kidneys (C, right panel). Data are means ± SD from WT mice (n = 3) and MyD88ko mice (n = 5). The images in C are representative of three separate experiments. (D) Percentage of Red Sirius staining in kidneys from WT mice, TLR3ko, Casp1ko and double Nod1/2ko mice infected with 2×10⁸<i>L. interrogans</i> Fiocruz and sacrificed at day-15 p.i. (n = 4 per genotype group). Three naïve kidneys of each genotype were used as controls. *P<0.05; **P<0.01 between groups.</p

Main characteristics of the <i>E. coli</i> isolates used in this study.

1<p>Determined by the triplex PCR method of Clermont <i>et al.</i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046547#pone.0046547-Clermont4" target="_blank">[53]</a>.</p>2<p>According to Wirth <i>et al.</i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046547#pone.0046547-Wirth1" target="_blank">[55]</a>.</p>3<p>According to Le Gall <i>et al.</i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046547#pone.0046547-LeGall1" target="_blank">[33]</a>.</p

T and B cells are not involved in <i>Leptospira</i>-induced renal fibrosis.

<p>(A) Quantification of the number of infiltrating CD3⁺ cells in <i>Leptospira</i>-infected kidneys from WT C57BL/6J mice at different time points post p.i.. Values are means ± SD from five kidney sections per surface area from (n = 2) mice at each different time point tested. (B) Percentage of Red Sirius labeling per surface area in kidney sections from WT and CD3ko mice at day-15 p.i. (D15) and in naive mice. The bars represent the mean value in each group. (C) Electron microscopy (magnification ×100) of kidneys sections from a naïve mouse and CD3ko mouse at 4 months p.i. (D120) showing fibrosis. (D) Survival curves and (E) images of renal Red Sirius staining from B cell deficient naïve μMT mice or infected with 10⁷<i>L. interrogans</i> strain Fiocruz, rescued by immune serum from <i>Leptospira</i>-infected WT mice at day-15 p.i. (D15). *P<0.05; ***P<0.001 between groups.</p

Intestinal colonization.

<p>Intestinal competition tests were performed in streptomycin-pre-treated CD1 mice. Competitive indexes (CIs) are given for day 1 and day 7 post-inoculation. The CI index is a ratio of ratios, in which the ratio between resistant (TN03) and sensitive (K-12 MG1655, IAI1 or ED1a) strains at post-inoculation time points is divided by this same ratio at the initial inoculation time. Horizontal bars represent median log₁₀ CI ratios and compared to a ratio of null effect (0, that is log₁₀ 1.0) using a Wilcoxon signed-rank test.</p

iNOS is involved in <i>Leptospira</i>-induced renal fibrosis.

<p>NO2⁻ production in supernatants from bone marrow macrophages derived from C57BL/6J mice, stimulated for 24 h with live (Lepto) or heat-killed (HKLepto) <i>L. interrogans</i> strain Fiocruz at a multiplicity of infection of 1 (1∶1) 10 (10∶1) and 100 (100∶1), as measured by the Griess reaction. Values are means +SD from 3 separate experiments. (B–E) Bacterial loads in urine (B), renal inflammatory scores (C), percentage of Red Sirius staining (D) and mRNA expression of ACTA-2, fibronectin and Mmp2 (E, upper panel) in kidneys from WT and iNOSko mice infected with 2×10⁸<i>L. interrogans</i> Fiocruz and sacrificed at day-15 p.i.. Values are means ± SD from WT (n = 3) and iNOSko (n = 5) mice. (E, lower panel), comparison of mRNA expression levels of fibrosis markers in kidneys of naïve WT and iNOSko mice, measured as Δ^CT compared to HPRT. Values are means ± SD from WT (n = 3) and iNOSko (n = 3) mice. *P<0.05; **P<0.01 between groups.</p

TLR2 and TLR4 are not involved in <i>Leptospira</i>-induced fibrosis.

<p>(A–D) Renal fibrosis, levels of inflammatory mediators, bacterial loads, and serum creatinine levels in WT or TLR2 and/or TLR4 deficient mice infected with 2×10⁶<i>L. interrogans</i> Fiocruz at 3 months p.i. (D90) (A) Red Sirius staining. (B) Fibrosis and inflammation evaluation by mRNA expression of fibronectin, Mmp2, ACTA-2 and RANTES, in kidneys of WT, TLR2ko, TLR4ko and TLR2/4dko mice at 3 months p.i. Values are means ± SD from n = 5 mice per genotype group. No statistical difference between genotypes was found for the different markers by One-Way Anova. (C) Bacterial loads in 100 µl of urine from mice at 3 months p.i. The bars represent the mean values in each group tested. (D) Serum creatinine levels in naïve and <i>Leptospira</i>-infected mice at 3 months p.i. *P<0.05; **P<0.01; ***P<0.001 between groups.</p

Expression of pro-inflammatory mediators in kidneys infected by UPEC.

<p>Expression of MIP-2, KC, IL-1ß, IL-6, MCP-1, RANTES and TNF-α in the day 2 and day 7 post-infection kidneys following the transurethral inoculation of the CFT073 and TN03 UPEC isolates. mRNA expression of each inflammatory marker was calculated using the −ΔΔCT method. Values are expressed as the relative fold increase in kidneys of each mRNA level of proinflammatory mediator in comparison with that measured in naive mice. Horizontal bars represent the median values. Statistical differences between the strains were determined at each time point using the Kruskal-Wallis equality-of-populations rank test.</p

Effects of antibiotic treatment on <i>Leptospira</i>-infected mice.

<p>C57BL/6J mice were infected with 10⁷<i>L. interrogans</i> serovar Manilae (n = 3) or PBS (n = 1) and injected (IP) daily for 5 days with penicillin G (Pen) from day-1 p.i. until day-5 (D1–D5) or from day-3 p.i. until day-7 (D3–D7). Thereafter, mice were sacrificed at day-24 p.i. (A) Leptospiral loads in 100 µl urine determined by qRT-PCR, and imaging of the LipL32 leptospiral major antigen immunostaining in kidney sections. (B) Inflammation score (left panel) and pro-inflammatory RANTES mRNA expression (right panel). (C) Quantification (left panel) and microscopy (right panels) of fibrosis by Red Sirius staining. Values are counts (The bars represent the mean value in each group) or means of mRNA quantification ± SD from n = 3 mice per group and are from one representative experiment of two. *P<0.05; ***P<0.001 between groups.</p

Kidney phenotype at 30 weeks of age.

<p>Values are means ± SEM; n, number of mice;</p>*<p>P<0.05 between diabetic and control animals.</p