NLRX1 is not involved in the host defense against Escherichia coli induced pyelonephritis

Animals

NLRX1 KO mice with a C57BL6/J background were generated as described previously¹⁴ and bred at the animal facility of the Academic Medical Center (AMC) in Amsterdam, The Netherlands. Age- and gender-matched C57BL6/J WT mice were obtained from Charles River (Maastricht, The Netherlands). The mice were allowed to acclimatize for a week in the same room and conditions as the transgenic animals before starting the experimental procedures. Animals were housed in individual ventilated cages (IVCs) with bedding and cage enrichment that were kept under standard environmental conditions (temperature, humidity, ventilation, light/dark cycle) and under specific pathogen-free conditions (SPF) with ad libitum access to water and food.

Animal experimental procedures

The in vivo and ex vivo experiments were carried out once and the data showed in the article is based on biological replicates. The in vivo study was performed with 2 experimental groups: 1) WT (n=8) and 2) NLRX1-KO (n=8) and 2 sham/control groups: 3) WT (n=4) 4) NLRX1-KO (n=4). Each experimental group was subjected for two time points (24h and 48h) to UTI as described previously¹⁵ and briefly explained later. The total number of mice per in vivo experimental group was 16 and the total number per sham/control group was 4. For the data obtained in Suplementary Figure 3 only WT mice (n=6-8 per time point) were subjected to UTI according the protocol described below, mice were sacrificed 4h, 8h, 24h and 48h post inoculation. To be able to reach a statistical significant effect of NLRX1 deficiency the number of 8 mice per experimental group was assessed with an unpaired t-test based on a variation coefficient of 15%, a minimal relative effect of 30%, a P value of 5% and a power of 80%, that were based on previous studies done in our group^15–17. For both experimental and sham/control groups 11–12 week old female mice (median weights: WT; 19,6 and NLRX1-KO; 21,3 grams) were used. Each experimental group was divided in 2 cages of 3 and 5 animals and in the sham/control group 4 animals per cage were kept. For the experimental groups uropathogenic E. coli 1677, isolated from an uroseptic patient, was cultured in the laboratory in sterile Tryptic Soy Broth (TSB) overnight at 37°C, 5% CO₂. The next day, in the morning this suspension was diluted 1:100 in fresh TSB and in 2–3h cultured to optical density OD620_nm = 1 was reached (measured with a spectrophotometer (DU640, Beckman, USA)). Subsequently bacteria were spun down for 14 min at 4000 rpm at 4°C, washed three times and resuspended in 10 mL sterile PBS. The same day, in the animal facility, under general anaesthesia (10 µl/1 g body weight of FFM mixture, containing 1.25 mg/ml midazolam (Dormicum^®, Roche, Woerden, The Netherlands), 0.08 mg/ml fentanyl citrate/2.5 mg/ml fluanisone (Hypnorm, Veta Pharma Ltd., Leeds, UK)) that was given intraperitoneal, mice were via the urethra intravesically inoculated with 8*10⁸ CFU in a 100µl volume. Mice in the sham/control group underwent the same procedure with administration of 100µl sterile PBS. CFU concentrations in the inoculum were determined by plating 10-fold serial dilutions on blood-agar plates at 37°C, 5% CO₂ overnight. Mice were sacrificed 24 and 48 hour post-inoculation by cardiac puncture under 4% isoflurane/O₂ followed by cervical dislocation. Blood was collected in lithium-heparin containing tubes and kidneys and bladders were collected for further analysis. In the WT 24h group one animal reached, because of signs of severe sepsis, the humane end point and was excluded from further data analysis. The animals used to study leukocyte composition and ex vivo granulocyte and monocyte functioning contained 2 experimental groups: 1) WT (n=6) and 2) NLRX1-KO (n=6) and were sacrificed as described earlier for the in vivo experiments. The total number of mice per ex vivo experimental group. To be able to reach a statistical significant effect of NLRX1 deficiency the number of 6 mice per experimental group was assessed with an unpaired t-test based on a variation coefficient of 10%, a minimal relative effect of 30%, a significance of 5% and a power of 80%. Mice used for the ex vivo experimental groups were 13–14 week old female mice (median weights: WT;26.5 and NLRX1-KO;26.6 grams). In the WT group one sample was excluded from further data analysis due to low blood gain.

Ethics statement

All animal procedures were ethically approved under DPA 25 AB-1 by the Animal Care and Use Committee of the Academic Medical Center Amsterdam and were conducted in compliance with the ARRIVE guidelines (NC3Rs).

Bacterial outgrowth determination

Bladder 20% (w/v) and left kidney 10% (w/v) tissues were homogenized in sterile PBS with a tissue homogenizer (Polytron PT1300D homogenizer, Kinematica AG). To determine bacterial loads 10-fold serial dilutions of bladder- and kidney-homogenates were plated onto blood agar plates. Colonies were counted 16h after incubation at 37°C.

Leukocyte composition and count

Absolute leukocyte number in blood was measured with a Coulter Counter (Beckmann Coulter Inc., Fullerton, CA). To assess relative leukocyte composition, 100 µl whole blood erythrocytes were lysed by adding 2 ml lysis buffer (8.3g/L NH₄Cl, 1.0g/L KHCO₃, 0.1 mM EDTA, pH 7.4) for 15 min at RT. The remaining cells, leukocytes, were washed once, centrifuged and resuspended in FACS buffer (0.5% BSA, 0.01% NaN₃, 0.35mM EDTA in PBS) and were measured by flow cytometry on a FACS Canto (BD Biosciences, San Jose, CA, USA) and analyzed with FlowJo version 10 software. We applied a forward versus side scatter (FSC vs SSC) gating strategy on living cells as displayed in Supplemental Figure 2 to discriminate between lymphocytes, granulocytes and monocytes, although adding specific markers would have given a better discrimination of the different populations.

Whole blood stimulation

Whole blood of WT and NLRX1 KO mice was incubated at 37°C, 5% CO₂ in 10ng/mL LPS (cat no. l4391, Sigma-Aldrich, Zwijndrecht, The Netherlands) conditioned RPMI medium (Thermo fisher Scientific, Waltham, MA, USA) containing 10% FCS (Invitrogen, Carlsbad, CA, USA) with 2 mM L-glutamine and 100 U/mL penicillin/streptomycin (all from Thermo fisher Scientific, Waltham, MA, USA). After 14h, cells were spun down (5 min, 4000 rpm) and supernatants were collected and stored at -20°C prior to use for cytokine measurements.

Phagocytosis assay

Phagocytosis by granulocytes and monocytes was measured with the PHAGOTEST (cat no. 10–0100, Glycotope Biotechnology, Heidelberg, Germany) according to the manufacturer’s instructions. Briefly, 100 µl of heparinized whole blood was incubated with opsonized FITC- labeled E. coli for 10 minutes in a 37°C water bath, whereas the negative controls remained on ice. After phagocytosis was stopped the surface signal was quenched. After lysing the erythrocytes and fixation of the leukocytes, phagocytic capacity was measured by flow cytometry on a FACS Canto (BD Biosciences, San Jose, CA, USA) and analyzed with FlowJo version 10 software according the gating strategy displayed in Supplementary Figure 2¹⁸

Enzyme-linked immunoabsorbance assay (ELISA)

Bladder 5% (w/v) and kidney 10% (w/v) tissues from 24h and 48h UTI subjected WT and NLRX1 KO mice were homogenized with a tissue homogenizer (Polytron PT1300D homogenizer, Kinematica AG) in Greenberger lysis buffer (GLB) (300mM NaCl, 30mM Tris, 2mM MgCl₂, 2mM CaCl₂, 1% (v/v) Triton X-100, pH set at 7.4, supplemented with Protease Inhibitor Cocktail II (cat no. p8340, Sigma-Aldrich, Sigma-Aldrich, Zwijndrecht, The Netherlands). Levels of keratinocyte-derived chemokine (KC), macrophage inflammatory protein-2 (MIP-2), monocyte chemoattractant protein 1 (MCP-1), interleukin 1β (IL1β), interleukin 6 (IL6), tumor necrosis factor alpha (TNFα) and mouse myeloid peroxidase (MPO) were determined in bladder and kidney homogenates and whole blood plasma (KC, TNFα and IL6 only) by duo set ELISAs (cat no. MKC00B, MM200, MJE00, MLB00C, M6000B, MTA00B, DY3667, R&D Systems, Abingdon, UK), performed according to the supplied protocols. ELISA data measured in bladder and kidney homogenates was adjusted for total protein concentration as determined by BCA protein assay (cat no. B9643, Sigma-Aldrich, Sigma-Aldrich, Zwijndrecht, The Netherlands) developed with 4% CuSO₄.

Plasma biochemical analysis

From hepanarized blood, plasma was obtained from the upper phase after spinning the tube for 10 minutes at 2000 rpm. Urea and creatinine levels in plasma were determined at room temperature by colorimetric enzyme reactions involving creatinase and urease and analyzed on the Cobas c702 Chemistry Analyzer (Roche Diagnostics, Indianapolis, IN, USA) according to standard diagnostic procedures performed by the department of Clinical Chemistry of the Academic Medical Center Amsterdam.

RNA isolation and real time quantitative PCR (RT-qPCR)

Total RNA was extracted from snap-frozen -80°C stored bladder and kidney using TriReagent (cat no. T9424, Sigma-Aldrich, Zwijndrecht, The Netherlands) followed by chloroform phase separation to obtain the aqueous RNA containing upper phase and isopropanol precipitation according to the manufacturer protocol procedure description and converted to cDNA. cDNA was synthesized using M-MLV reverse transcriptase according the procedure described in the manufacturer protocol (cat no. 28025, Thermo Scientific). Transcription was analyzed by RT-qPCR on a Roche LightCycler 480 using 2.5 µl sensiFAST SYBR master mix (cat no. bio-98020, Bioline reagents, London, UK), 0.20 µl forward primer, 0.20 µl reverse primer (Table 1), 2.10 µl distilled H₂O and 1µl cDNA per reaction. qPCR primers were synthesized by Eurogentec (Maastricht, The Netherlands) and described in the list below. qPCR data was analyzed based on linear regression using the LinRegPCR program, that is freely available^19,20. Briefly, the LinRegPCR program imports non-baseline corrected qPCR data, performs a baseline correction on each sample separately, determines a window-of-linearity and then uses linear regression analysis to determine the PCR efficiency per sample from the slope of the regression line. The mean PCR efficiency per amplicon and the Cq value per sample are used to calculate a starting concentration per sample, expressed in arbitrary fluorescence units (au)²⁰.

Statistical analysis

Data are expressed as mean ± standard error of the mean (SEM), bacterial outgrowth data are expressed on a logarithmic scale as median scatterplot. The non-parametric Mann Whitney U test was performed for two group comparison. For all analyses, values of P ≤ 0.05 were considered significant. All statistical analyses were performed using GraphPad Prism 5 (GraphPad Software, San Diego, CA, USA).

Local NLRX1 expression in the bladder and kidney during experimental UTI

To determine whether NLRX1 expression is modulated in the murine bladder and kidney during urinary tract infection (UTI), wild-type (WT) mice were intravesically inoculated with uropathogenic E. coli and sacrificed at 24h and 48h after infection. Non-infected sham mice were used as controls. Real-time quantitative PCR revealed that Nlrx1 transcript levels were constitutively present in the bladder and kidney (Figure 1A and B). Nlrx1 transcript levels show a non-significant trend towards increased levels in the bladder after 24h, while levels are returned towards baseline sham levels at 48h (Figure 1A). In the kidney Nlrx1 transcript levels remained at baseline level 24h after infection while after 48h levels were significantly increased (Figure 1B). Together, these data show that in response to UTI, local Nlrx1 expression is increased upon E. coli infection.

Figure 1. NLRX1 expression in bladder and kidney during experimental urinary tract infection (UTI).

Nlrx1 mRNA transcript levels in wild-type (WT) (A) bladder and (B) kidney from sham, and uropathogenic E. coli-inoculated mice. All data are expressed as mean ± SEM, n=4 (sham) and n=7-8 (24h and 48h) animals per group. Statistical significance was determined by non-parametric Mann Whitney U test, *P<0.05.

NLRX1 deficiency enhances early bacterial burden from the bladder while it does not influence bacterial burden in the kidney during experimental UTI

To investigate whether NLRX1 plays a role in bladder and kidney during lower and upper UTI, we examined bacterial loads in these organs from WT and NLRX1 knock-out (KO) mice 24h and 48 h after inoculation with uropathogenic E. coli. This revealed that the bacterial outgrowth, as measured by colony forming units (CFU), in bladder tissue from NLRX1 KO mice was significantly higher at 24h after infection compared to WT while at 48h no differences in bacterial burden were found (Figure 2A). NLRX1 deficient mice had more improved bacterial clearance from the bladder at 48h as compared to 24h (Figure 2A). No differences in the amount of CFU were found between kidneys from WT and NLRX1 KO mice at both time points (Figure 2B). To monitor the local inflammatory response during infection, we next measured the levels of KC, MIP-2, MCP-1, IL-1β, IL-6 and TNFα in kidney and bladder homogenates (Figure 2C and D). The production of MIP-2 in the bladder was in NLRX1 KO mice reduced at both 24h and 48h after infection compared to WT animals (Figure 2C). In addition, NLRX1 KO bladders show reduced levels of MCP-1 and TNFα compared to WT at 48h, while no differences were found in KC, IL-1β and IL6 levels (Figure 2C). We identified an increase in renal KC levels at 24h in NLRX1 KO mice compared WT mice, whereas at 48h KC levels were similar between both groups (Figure 2D). At both 24h and 48h no differences were found in renal MIP-2, MCP-1, IL-1β, IL-6 and TNFα levels between WT and NLRX1 KO mice (Figure 2D). In addition, upper UTI and NLRX1 have no significant influence on renal function as reflected by similar plasma levels of urea and creatinine between all mice (Supplementary Figure 1A and B²¹). Accordingly, this indicates that in the bladder the lack of NLRX1 together with an impaired pro-inflammatory cytokine response is associated with an early impaired ability to clear uropathogenic E. coli. No differences were found in bacterial burden and cytokine response in the kidney when mice were deficient for NLRX1.

Figure 2. Bacterial outgrowth and inflammatory response in WT and NLRX1 KO bladder and kidney during experimental urinary tract infection (UTI).

Outgrowth of uropathogenic E. coli expressed in colony forming units (CFU) in (A) bladder and (B) kidney homogenates from wild-type (WT) and NLRX1 knock-out (KO) mice 24h and 48h after inoculation. Detection limit: 10 CFU/ml. Levels of KC, MIP-2, MCP-1, IL-1β, IL6 and TNFα in (C) bladder and (D) kidney homogenates from the E. coli inoculated WT (white bars) and NLRX1 KO (black bars) mice. Data at A and B are expressed on a logarithmic scale as median scatterplot. Data at C and D are expressed as mean ± SEM. For all data n=7-8 animals per group and statistical significance between WT and NLRX1 KO was determined by non-parametric Mann Whitney U test, *P<0.05 and **P<0.01.

NLRX1 does not affect circulating leukocyte number and local presence of activated neutrophils during experimental UTI

By analyzing inflammatory cells in the circulation we observed that the numbers of granulocytes and monocytes were equal between uninfected WT and NLRX1 KO mice (Figure 3A). A non-significant trend towards an increased presence of lymphocytes in NLRX1 KO compared to WT is shown (Figure 3A). We observed that WT and NLRX1 KO mice 24h and 48h after inoculation, have similar numbers of circulating leukocytes (Figure 3B). Recruitment of neutrophils in the kidney and bladder are essential for the host defense against uropathogenic E. coli²². Therefore, we determined active neutrophil presence by measuring kidney and bladder myeloperoxidase (MPO) concentrations. No differences in MPO levels were found in bladder and kidney between WT and NLRX1 KO mice at 24h and 48h (Figure 3C and D), indicating a similar number of activated neutrophils.

Figure 3. Circulating and local leukocyte dynamics in naïve WT and NLRX1 KO mice and upon experimental urinary tract infection (UTI).

(A) Total circulating leukocyte, lymphocyte, granulocyte and monocyte cell counts in blood from uninfected wild-type (WT) (white bars) and NLRX1 knock-out (KO) (black bars) mice n=5–6 animals per group. (B) Circulating leukocytes in blood from WT (white bars) and NLRX1 KO (black bars) mice 24h and 48h after uropathogenic E. coli inoculation. MPO levels in (C) bladder and (D) kidney homogenates from 24h and 48h inoculated WT (white bars) and NLRX1 KO mice (black bars). All data are expressed as mean ± SEM, n=7–8 (B–D) animals per group. Statistical significance between WT and NLRX1 KO was determined by non-parametric Mann Whitney U test.

NLRX1 is not essential for pro-inflammatory cytokine secretion in response to LPS and phagocytosis of E. coli by granulocytes and monocytes

Since we observed differences in bacterial outgrowth in the bladder while the number of local neutrophils after infection is equal between WT and NLRX1 KO, we investigated if NLRX1 absence causes functional changes to granulocytes and monocytes. This revealed that NLRX1 is not critical for the secretion of the pro-inflammatory cytokines KC, TNFα and IL6 after ex vivo whole blood LPS stimulation (Figure 4A, B and C). To investigate if NLRX1 is important for the phagocytic activity of granulocytes and monocytes, leukocytes from WT and NLRX1 KO mice were ex vivo incubated with fluorescein labelled opsonized E. coli, and phagocytosis was analyzed using flow cytometry. Granulocytes and monocytes from both WT and NLRX1 KO mice show increased phagocytic activity responses when challenged with E. coli at 37°C compared to 0°C (Figure 4D and E). However, no differences were observed in the percentage of granulocytes and monocytes that undergo phagocytosis between WT and NLRX1 KO (Figure 4E and F). Together, these results suggest that the early decreased bacterial clearance in the bladders from NLRX1 KO mice cannot be explained by an impaired granulocyte or monocyte response.

Figure 4. Cytokine secretion in response to LPS and E. coli phagocytosis by WT and NLRX1 KO granulocytes and monocytes.

Levels of (A) KC (B) TNFα and (C) IL6 after 14h ex vivo LPS whole blood stimulation from wild-type (WT) (white bars) and NLRX1 knock-out (KO) (black bars). ND = not detectable. Phagocytic activity responses of (D) granulocytes and (E) monocytes from WT (white bars) and NLRX1 KO (black bars) mice ex vivo challenged with E. coli at 0°C and 37°C. All data are expressed as mean ± SEM, n=5–6 animals per group. Statistical significance between all columns was determined by non-parametric Mann Whitney U test, *P<0.05 and **P<0.01.

Underlying data

Dataset 1: NLRX1 expression data – This file contains the data underlying the analysis of the NLRX1 expression in bladder and kidney as shown in Figure 1. https://doi.org/10.6084/m9.figshare.9879635.v1³⁶

Dataset 2: In vivo mice bladder and kidney colony forming units (CFU)-, cytokine-, general marker-data and leukocyte counts in sham and after 24h and 48h of infection – This file contains the data underlying the analysis of the data used in Figure 2, Figure 3 and Supplementary Figure 1. https://doi.org/10.6084/m9.figshare.9879632.v1³⁷

Dataset 3: Ex vivo cytokine data – This file contains the data underlying the cytokine determination in LPS stimulated whole blood as shown in Figure 4. https://doi.org/10.6084/m9.figshare.9879629.v1³⁸

Dataset 4: Ex vivo FACS output data - This file contains the FACS data underlying the leukocyte composition analysis as shown in Figure 3. https://doi.org/10.6084/m9.figshare.9879620.v1³⁹

Dataset 5: Ex vivo FACS output data on granuocyte and monocytes phagocytosis - This file contains the FACS data underlying the granuocytes and monocytes phagocytosis as shown in Figure 4. https://doi.org/10.6084/m9.figshare.9879611.v1⁴⁰

Dataset 6: In vivo mice bladder colony forming units (CFU) data of WT mice 4h, 8h, 24h and 48h of infection – This file contains the data underlying the analysis of the data used in Supplementary Figure 3²⁵. https://doi.org/10.6084/m9.figshare.9879455.v1⁴¹

Extended data

Supplementary Figure 1. Renal function of wild-type (WT) and NLRX1 knock-out (KO) mice during experimental urinary tract infections (UTI).

Plasma levels of renal function markers (A) urea and (B) creatinine from sham, 24h and 48h uropathogenic Escherichia coli inoculated WT (white bars) and NLRX1 KO (black bars) mice. All data are expressed as mean ± SEM, n=3–4 (sham) and n=7–8 (24h and 48h) animals per group. Statistical significance was determined by non-parametric Mann Whitney U test.

https://doi.org/10.6084/m9.figshare.9879662.v1²¹

Supplementary Figure 2. Gating strategy of circulating leukocytes.

Of total leukocytes, living cells, granulocyte (population 1), monocyte (population 2) and lymphocyte (population 3) populations were determined based on forward-scattered light (FSC)/side-scattered light (SCC). For the phagocytosis capacity experiments FSC/FITC gates were set based on the negative control and Escherichia coli FITC positive cells were identified in population 1 and population 2.

https://doi.org/10.6084/m9.figshare.9879677.v1¹⁸

Supplementary Figure 3. Bacterial outgrowth bladder.

Outgrowth of uropathogenic E. coli expressed in colony forming units (CFU) in bladder homogenates from wild-type (WT) mice 4h, 8h, 24h and 48h after inoculation. Detection limit: 10 CFU/ml.

https://doi.org/10.6084/m9.figshare.9879683.v1²⁵