Decay-Accelerating Factor 1 Deficiency Exacerbates Leptospiral-Induced Murine Chronic Nephritis and Renal Fibrosis

Leptospirosis is a global zoonosis caused by pathogenicLeptospira, which can colonize the proximal renal tubules andpersist for long periods in the kidneys of infected hosts. Here, we characterized the infection of C57BL/6J wild-type andDaf12/2mice, which have an enhanced host response, with a virulentLeptospira interrogansstrain at 14 days post-infection,its persistence in the kidney, and its link to kidney fibrosis at 90 days post-infection. We found thatLeptospira interroganscan induce acute moderate nephritis in wild-type mice and is able to persist in some animals, inducing fibrosis in theabsence of mortality. In contrast, Daf12/2mice showed acute mortality, with a higher bacterial burden. At the chronic stage,Daf12/2mice showed greater inflammation and fibrosis than at 14 days post-infection and higher levels at all times thanthe wild-type counterpart. Compared with uninfected mice, infected wild-type mice showed higher levels of IL-4, IL-10 andIL-13, with similar levels ofa-smooth muscle actin, galectin-3, TGF-b1, IL-17, IFN-c, and lower IL-12 levels at 90 days post-infection. In contrast, fibrosis in Daf12/2mice was accompanied by high expression ofa-smooth muscle actin, galectin-3, IL-10, IL-13, and IFN-c, similar levels of TGF-b1, IL-12, and IL-17 and lower IL-4 levels. This study demonstrates the link betweenLeptospira-induced murine chronic nephritis with renal fibrosis and shows a protective role of Daf1.Fil: Ferrer, Maria Florencia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Biotecnología y Biología Molecular. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Biotecnología y Biología Molecular; ArgentinaFil: Scharrig Fernandez, Maria Emilia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Biotecnología y Biología Molecular. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Biotecnología y Biología Molecular; ArgentinaFil: Alberdi, Maria Lucrecia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Biotecnología y Biología Molecular. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Biotecnología y Biología Molecular; ArgentinaFil: Cédola, Maia Tatiana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Biotecnología y Biología Molecular. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Biotecnología y Biología Molecular; ArgentinaFil: Prêtre, Gabriela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Biotecnología y Biología Molecular. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Biotecnología y Biología Molecular; ArgentinaFil: Drut, Ricardo. Universidad Nacional de la Plata. Facultad de Cs.médicas. Cátedra de Patología Ii; ArgentinaFil: Song, Wen-Chao. University of Pennsylvania; Estados UnidosFil: Gomez, Ricardo Martin. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Biotecnología y Biología Molecular. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Biotecnología y Biología Molecular; Argentin

Association of Toll-like receptor 2Arg 753Gln and Toll-like receptor 1Ile 602Ser single-nucleotide polymorphisms with leptospirosis in anargentine population

Toll-like receptor 2 (TLR2), a member of the Toll-like receptor family, plays an50 important role in the recognition of and subsequent immune response activation51 against leptospirosis in humans. The genetic polymorphism in TLR2 of an arginine to52 glutamine substitution at residue 753 (Arg753Gln) has been associated with a53 negative influence on TLR2 function, which may, in turn, determine the innate host54 response to Leptospira spp. This bacterium signals through TLR2/TLR155 heterodimers in human cells. The aim of the present study was to investigate the56 Arg753Gln single-nucleotide polymorphism (SNP) of the TLR2 gene, and the57 isoleucine to serine transversion at position 602 (Ile602Ser) of the TLR1 gene58 (previously associated with Lyme disease), in leptospirosis patients compared to59 healthy controls, carrying out a retrospective case/control study. The TLR260 polymorphism adenine (A) allele was observed in 7.3% of leptospirosis patients but61 was not found in the control group, whereas the guanine (G) allele of the TLR162 polymorphism was found in 63.6% of patients and 41.6% of controls. Susceptibility to63 leptospirosis disease was increased 10.57-fold for carriers of the TLR2 G/A64 genotype (P=0.0493) and 3.85-fold for carriers of the TLR1 G/G genotype65 (P=0.0428). Furthermore, the risk of developing hepatic insufficiency and jaundice66 was increased 18.86- and 27.60-fold for TLR2 G/A carriers, respectively. Similarly,67 the risk of developing jaundice was increased 12.67-fold for TLR1 G allele carriers68 (G/G and T/G genotypes). In conclusion, the present data suggest that the TLR269 Arg753Gln and TLR1 Ile602Ser SNPs influence the risk of developing leptospirosis70 and its severity.Fil: Cédola, Maia Tatiana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Biotecnología y Biología Molecular. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Biotecnología y Biología Molecular; ArgentinaFil: Chiani, Yosena. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud “Dr. C. G. Malbrán”; Argentina. Universidad Nacional del Litoral; ArgentinaFil: Prêtre, Gabriela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Biotecnología y Biología Molecular. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Biotecnología y Biología Molecular; ArgentinaFil: Alberdi, Maria Lucrecia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Biotecnología y Biología Molecular. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Biotecnología y Biología Molecular; ArgentinaFil: Vanasco, Norma Bibiana. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud “Dr. C. G. Malbrán”; Argentina. Universidad Nacional del Litoral; ArgentinaFil: Gomez, Ricardo Martin. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Biotecnología y Biología Molecular. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Biotecnología y Biología Molecular; Argentin

Neutrophil extracellular traps are involved in the innate immune response to infection with Leptospira

NETosis is a process by which neutrophils extrude their DNA together with bactericidal proteins that trap and/or kill pathogens. In the present study, we evaluated the ability of Leptospira spp. to induce NETosis using human ex vivo and murine in vivo models. Microscopy and fluorometric studies showed that incubation of human neutrophils with Leptospira interrogans serovar Copenhageni strain Fiocruz L1-130 (LIC) resulted in the release of DNA extracellular traps (NETs). The bacteria number, pathogenicity and viability were relevant factors for induction of NETs, but bacteria motility was not. Entrapment of LIC in the NETs resulted in LIC death; however, pathogenic but not saprophytic Leptospira sp. exerted nuclease activity and degraded DNA. Mice infected with LIC showed circulating NETs after 2 days post-infection (dpi). Depletion of neutrophils with mAb1A8 significantly reduced the amount of intravascular NETs in LIC-infected mice, increasing bacteremia at 3 dpi. Although there was a low bacterial burden, scarce neutrophils and an absence of inflammation in the early stages of infection in the kidney and liver, at the beginning of the leptospiruric phase, the bacterial burden was significantly higher in kidneys of neutrophil-depleted-mice compared to non-depleted and infected mice. Surprisingly, interstitial nephritis was of similar intensity in both groups of infected mice. Taken together, these data suggest that LIC triggers NETs, and that the intravascular formation of these DNA traps appears to be critical not only to prevent early leptospiral dissemination but also to preclude further bacterial burden.Fil: Scharrig Fernandez, Maria Emilia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Biotecnología y Biología Molecular. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Biotecnología y Biología Molecular; ArgentinaFil: Carestia, Agostina. Academia Nacional de Medicina de Buenos Aires. Instituto de Invest. Hematológicas ; ArgentinaFil: Ferrer, Maria Florencia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Biotecnología y Biología Molecular. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Biotecnología y Biología Molecular; ArgentinaFil: Cédola, Maia Tatiana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Biotecnología y Biología Molecular. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Biotecnología y Biología Molecular; ArgentinaFil: Prêtre, Gabriela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Biotecnología y Biología Molecular. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Biotecnología y Biología Molecular; ArgentinaFil: Drut, Ricardo. Universidad Nacional de la Plata. Facultad de Cs.médicas. Cátedra de Patología Ii; ArgentinaFil: Picardeau, Mathieu. Universite Louis Pasteur; FranciaFil: Schattner, Mirta Ana. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Gomez, Ricardo Martin. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Biotecnología y Biología Molecular. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Biotecnología y Biología Molecular; Argentin

Pathogenic but not saprophyte <i>Leptospira</i> spp. degrade DNA.

<p>Representative analysis of DNA digestion by gel electrophoresis. From left to right: plasmid DNA (100 ng/μL) after incubation with PBS (negative control), DNase I (positive control), and live <i>Leptospira interrogans</i> serovar Copenhageni (LIC) or <i>Leptospira biflexa</i> serovar Patoc (Patoc) (1x10⁸/mL) after 60 minutes of incubation at 37°C.</p

NETs kill <i>Leptospira</i> sp.

<p><b>(A)</b> Percentage viability after 180 minutes of <i>Leptospira interrogans</i> serovar Copenhageni (LIC) (MOI = 50) alone, after being incubated with human neutrophils (Neu) (2x10⁵/mL), or in the presence of DNase (0.25 U/mL). <b>(B)</b> Percentage viability of LIC (MOI = 50) after 60 minutes of incubation with different concentrations of recombinant histone H4. Bars represent standard error of the mean (SEM) of assays from five independent assays; *<i>p</i> <0.05, **<i>p</i> <0.01, ****<i>p</i> <0.0001.</p

Early role of NETs in murine experimental model.

<p><b>(A)</b> Granulocytes were quantified in blood samples taken from C57BL/6J mice treated or not with mAb1A8 antibody with a veterinarian hematology counter. Bars represent the mean ± SEM. * p <0.05. <b>(B)</b> C57BL/6J male weanlings were inoculated intraperitoneally with 200 μL of 1x10⁷/mL pathogenic <i>Leptospira interrogans</i> serovar Copenhageni (LIC) and 0.25 mg of non-immune rat IgG or purified anti-Ly6G rat mAb1A8 every 48 h. Blood was collected by retro-orbital venous puncture and circulating nucleosomes were measured by ELISA *p <0.05, ***p <0.001, ****p <0.0001. <b>(C)</b> Blood, kidney, and liver samples of non-depleted and mAb1A8-depleted LIC-infected mice were collected at 3 days post-infection. Leptospiral DNA was quantified by real-time PCR and normalized to host cell number. Bars represent standard error of the mean (SEM) of assays from two independent assays; *p <0.05. <b>(D)</b> Kidney, and liver tissues samples of non-depleted animals were immunostained for neutrophils using anti-Ly6G rat mAb1A8 at 3 days post-infection. Representative hematoxylin and eosin (H&E) stained kidney and liver tissues samples of non-depleted animals at 3 days post-infection showing histology <b>(E)</b> and inflammation score <b>(F)</b> do not showed any alteration after analysis (n = 6–10 mice). Scale bar indicates 50 μm.</p

Pathogenicity and viability are relevant factors for NET formation.

<p><b>(A)</b> Human neutrophils (2x10⁵/mL) were incubated with live or inactivated with 4% PF or by heat <i>Leptospira interrogans</i> serovar Copenhageni (LIC) or <i>Leptospira biflexa</i> serovar Patoc (Patoc) (MOI = 50) for 180 min and then fixed (PF 4%), stained with propidium iodide (red) or with the specific marker anti-neutrophil elastase (green), and analyzed by fluorescence microscopy (n = 10). Scale bar indicates 50 μm. <b>(B)</b> Quantification of NETs released by fluorometry in the same conditions as in <b>(A)</b>. Bars represent standard error of the mean (SEM) of assays from ten independent assays; *<i>p</i> <0.05, ** <i>p</i> <0.01.</p

<i>Leptospira interrogans</i> induce NETs in a concentration-dependent manner.

<p>Human neutrophils (2x10⁵/mL) were incubated with <i>Leptospira interrogans</i> serovar Copenhageni (LIC) (MOI = 50) for 180 min and (A) visualized with a Nikon E200 photomicroscope or (B) fixed (PF 4%) and stained with propidium iodide (red) or with the specific marker anti-neutrophil elastase (green), and analyzed by fluorescence microscopy (n = 10). Scale bar indicates 50 μm. (C) DNA or nucleosomes were measured by fluorometry (black bars) or ELISA kit (white bars) respectively in supernatants of LICa (bacteria alone) or unstimulated neutrophils (None) (500/μL) (negative control), stimulated with PMA (50 ng/mL) (positive control), or with LIC (MOI 50, 5 or 1) for 180 min Bars represent standard error of the mean (SEM) of assays from 3–10 independent assays; ***<i>p</i> <0.0001 vs. None. ##p<0.01 and ####p<0.0001 vs. LIC 50.</p

Renal histopathology at the beginning of the leptospiruric phase.

<p>C57BL/6J male weanlings received 0.25 mg of non-immune rat IgG or purified anti-Ly6G rat mAb1A8 every 48 h and 200 μL 1x10⁷/mL pathogenic <i>Leptospira interrogans</i> serovar Copenhageni (LIC). Representative hematoxylin and eosin (H&E) stained kidney sections from 14 days post-infection showing histology <b>(A)</b> and inflammation score <b>(B)</b> from uninfected mAb1A8-treated mice, rat IgG-treated LIC-infected mice, and mAb1A8-treated LIC-infected mice (n = 6–10 mice). Scale bar indicates 50 μm.</p