CsA increases susceptibility to renal retrograde infection by UPEC

WT mice treated with CsA (15 mg/kg) or its vehicle for 5 days were then infected by transurethral inoculation with the UPEC HT7 strain isolated from the urine of a woman with acute pyelonephritis [4], [25], to test whether CsA affected renal antibacterial defenses. 24 h after the inoculation of live UPEC, the bacterial burden and E. coli positive immunostaining were greater in CsA-treated mice than vehicle-treated mice (Figure 1A and B). As a control, we checked that CsA did not modify the growth rate of UPEC (not shown), thus excluding any direct effect of the calcineurin inhibitor on the bacteria. CsA also increased renal bacterial loads in kidneys from Rag2^−/− mice, which lack mature lymphocytes, to almost the same extent as in WT kidneys (Figure 1A), suggesting that CsA promotes UPEC infection independently of its inhibitory effect on the adaptive immune system. The amount of secreted chemokines MIP-2/CXCL2 and KC/CXCL1, which play key roles in the chemoattraction of neutrophils during experimental UTI [23], [26]–[28], was also significantly lower in kidneys from CsA-treated mice than vehicle-treated mice (Figure 1C). Fewer Ly-6G⁺ neutrophils were detected in the kidney medulla and in the urinary space in the CsA-treated mice than in untreated mice (Figure 1D). Quantification of neutrophils assessed by measuring myeloperoxidase (MPO) activity also revealed significant lower MPO activity in the 24 h post-infected kidney homogenates from CsA-treated mice than vehicle-treated mice (Figure 1E).

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Figure 1. CsA decreases renal host resistance to UPEC.

(A) Bacterial loads in kidneys from vehicle- (-) and CsA-treated (+) WT mice, and vehicle or CsA-treated Rag2^−/− mice (n = 6–8 per group) 24 h after the transurethral inoculation of the UPEC strain HT7. Horizontal bars are the mean value of each group. (B) Illustrations of the intrarenal E. coli immunostaining in the infected kidneys from vehicle- and CsA-treated mice. Bars, 100 µm. (C) Production of CXCL2 and CXCL1 in the 24 h post-infected kidneys from vehicle-treated (−) and CsA-treated (+) mice. (D) Illustrations of neutrophils infiltrates (arrowheads) in the papilla from post-infected vehicle- and CsA-treated mice kidneys. Bars, 100 µm. (E) MPO activity in the 24 h post-infected kidney homogenates from vehicle- and CsA-treated mice. Values are presented as mean ± SE from 6–8 determinations per group. *, p<0.05 (Two-tailed, unpaired Student's t test).

https://doi.org/10.1371/journal.ppat.1003152.g001

CsA impairs the migration of neutrophils and phagocytic killing of UPEC

Flow cytometry (FACS) analysis revealed that the CD45⁺ leukocyte population detected in the 24 h post-infected kidneys was essentially composed of F4/80⁺ CD11b^LO Gr1^−/LO MHC-II⁺ CD11c⁺ renal dendritic cells (DCs), which have been shown to form a contiguous network in the renal tubulointerstitium [29], F4/80⁻ CD11b⁺ Gr1^HI MHC-II⁻ CD11c⁻ neutrophils, and to a lesser extent F4/80⁺ CD11b⁺ Gr1^INT MHC-II⁻ CD11c⁻ inflammatory monocytes/macrophages. FACS analysis revealed a significant decrease in the proportion of neutrophils over the total CD45⁺ renal cell population detected in the 24 h post-infected kidneys from CsA-treated mice compared to vehicle-treated mice kidneys (Figure S1 and Figure 2A and B). In contrast, CsA only slightly, and non-significantly reduced the number of monocytes/macrophages or DCs present in the 24 h post-infected kidneys (Figure S1). This suggests that CsA preferentially impairs the migration capacity of neutrophils in the UPEC-infected kidneys. In vitro experiments using the Boyden chamber method also revealed that the migration capacity of neutrophils isolated from the blood of the 5-day CsA-treated mice and stimulated by the neutrophil activating agent N-formyl-methionyl leucyl-phenylalanine (fMLP) or by CXCL2 was significantly decreased compared to that of neutrophils from vehicle-treated WT mice (Figure S2A and B). CsA also altered the bacterial phagocytic killing capacity by neutrophils. In contrast to neutrophil-enriched peritoneal cells (NPCs) isolated from vehicle-treated mice, NPCs collected from CsA-treated mice exhibited significantly lower ex vivo capacity to internalize Texas red-coupled E. coli and kill serum-opsonized E. coli (Figure 2C and D).

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Figure 2. CsA impairs the migration of neutrophils and macrophages in UPEC-infected kidneys and neutrophil bacterial phagocytic killing capacity.

(A and B) Flow cytometry analysis of the CD45⁺ leukocyte population infiltrating kidneys from CsA- and vehicle-treated mice, 24 h after UPEC inoculation. Representative dot-plots of CD45⁺ F4/80⁻ renal cells (A) and percentages (A, upper right insets and B) of F4/80⁻ CD11b⁺ Gr1^HI neutrophils infiltrating the infected CsA- and vehicle-treated mice kidneys. The bars indicate the mean percentage of neutrophils over total CD45⁺ leukocytes (n = 4 mice per group). (C) Illustrations and quantification of Texas red-coupled E. coli internalized by peritoneal neutrophils from vehicle-treated (−) and CsA-treated mice. Values are represented as mean ± SE of the mean count values (3–5 per condition) from three independent experiments. Cell membranes were stained with CD11b-FITC. Bars, 10 µm. (D) Killing of serum-opsonized E. coli by neutrophils from untreated or CsA-treated WT mice. Bacterial viability (n = 5–7 determinations from three experiments) was expressed relative to a control assay performed without neutrophils. (E) Variation in the levels of Tlr2, Tlr4, Tlr5, Tlr9, Nod1, and Nod2 mRNAs measured by quantitative real time PCR in kidneys from CsA-treated (+) and vehicle-treated mice 24 h after UPEC inoculation. (n = 6–8 determinations from 3–4 experiments). (F) Immunoblot analysis of the Tlr4, Nod1, and Nod2 proteins and the corresponding ß-actin in kidney homogenates from vehicle-treated and CsA-treated mice 24 h after the inoculation of UPEC. Values are presented as mean ± SE. *, p<0.05 (B–D, Two-tailed, unpaired Student's t test; E, Mann-Whitney test).

https://doi.org/10.1371/journal.ppat.1003152.g002

Nod1^−/− neutrophils were shown to exhibit deficient capacity of bacterial phagocytic killing and lower migration capacity than WT neutrophils [20], [30]. Given that neutrophil migration and their phagocytic killing capacities were markedly reduced in CsA-treated mice, we tested whether CsA directly alters intrarenal expression of Nod1. Quantitative real-time PCR revealed that the levels of Nod1 mRNA expression and, to a lesser extent those of Tlr4, but not of Nod2, were markedly decreased in the 24 h post-infected CsA-treated mice kidneys compared to those of the infected vehicle-treated mice (Figure 2E). In accordance with these findings, the amount of the immunodetected Nod2 protein remained equivalent in the infected kidneys from CsA-and vehicle-treated mice, whereas the amounts of Nod1 and Tlr4 proteins were ∼50% and ∼30%, respectively, lower in the infected kidneys from CsA-treated mice than in those from their vehicle-treated counterparts (Figure 2F). Despite the decrease in Nod1 expression, the level of phosphorylated over total RIP-2, which is involved in the control of Nod-mediated NF-κB activation [12], was similar in the 24 h post-infected kidneys from CsA-treated mice and vehicle-treated mice (not shown). Collectively, these findings suggest that, in addition to an inhibitory effect on Tlr4 mRNA and protein expression, CsA impairs the recruitment and functions of neutrophils in the inflamed kidneys by a mechanism possibly linked to downregulation of Nod1 expression.

Nod1 is involved in host renal defenses against UPEC

We next investigated the consequence of Nod1 deficiency in host renal bacterial defenses. The renal bacterial burden and the number of immunodetected UPEC were significantly greater in the kidneys of Nod1^−/− mice than in those of WT, 24 h after the inoculation of UPEC (Figure 3A and B). Less Ly-6G⁺ neutrophils were detected in the urinary space from post-infected Nod1^−/− mice than from post-infected WT or Nod2^−/− mice (Figure 3C), and, like in UPEC-infected CsA-treated mice, the MPO activity also remained significantly lower in the post-infected Nod1^−/− than in post-infected WT or Nod2^−/− kidneys (Figure 3D). FACS also showed that the proportion of polymorphonuclear neutrophils infiltrating the 24 h post-infected Nod1^−/− mice kidneys was lower than in the WT kidneys (Figure 3E and F). In accordance with the findings of Dharancy et al. [30], in vitro experiments using a Boyden chamber revealed that the migration capacity of neutrophils isolated from naive Nod1^−/− mice and activated by fMLP (or with CXCL2, not shown) was significantly lower than that of WT neutrophils (Figure S2C and D). These findings also suggested that Nod1 is implicated in the migration capacity of neutrophils. Consistently with a role for Nod1 in the bacterial phagocytic killing capacity of neutrophils [20] NPCs collected from Nod1^−/− mice, but not those isolated from WT or Nod2^−/− mice, were unable to internalize Texas red-coupled E. coli (Figure S3A and B). A significant reduction in the killing of serum-opsonized UPEC was also observed in Nod1^−/− neutrophils compared to WT or Nod2^−/− neutrophils (Figure S3C). We then analyzed the effects of CsA on the renal susceptibility of Nod1^−/− mice to UPEC. Administration of CsA slightly, but not significantly, increased the renal bacterial loads in kidneys from Nod1^−/− mice compared to those from untreated Nod1^−/− mice (Figure 3G), further suggesting that CsA impairs Nod1-mediated antibacterial defenses to UPEC.

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Figure 3. Nod1^−/− mice display increased susceptibility to renal retrograde UPEC infection.

(A) Bacterial loads in kidneys from WT, Nod1^−/− and Nod2^−/− mice (n = 8 per group) 24 h after the transurethral inoculation of UPEC strain HT7. Horizontal bars are the mean value of each group. (B) Illustrations of the E. coli immunostaining (arrowheads) in the infected WT and Nod1^−/− mice kidneys. Bars = 100 µm. (C) Illustrations of neutrophils infiltrates (arrowheads) in the urinary space from post-infected WT, Nod1^−/−, and Nod2^−/− kidneys. Bars = 100 µm. (D) Levels of MPO activity in kidney homogenates from post-infected WT, Nod1^−/−, and Nod2^−/− mice. (E and F) Flow cytometry analysis of the CD45⁺ leukocyte population infiltrating the infected kidneys from WT and Nod1^−/− mice 24 h after UPEC inoculation. Representative dot-plots of CD45⁺ F4/80⁻ renal cells (E) and percentages (E, upper right insets and F) of F4/80⁻ CD11b⁺ Gr1^HI neutrophils infiltrating the infected WT and Nod1^−/− mice kidneys. The bars indicate the mean percentage of neutrophils over total CD45⁺ leukocytes (n = 3 mice per group. (G) Bacterial loads in kidneys from untreated (–) and day-5 Nod1^−/− mice (n = 8 per group) 24 h after the transurethral inoculation of the UPEC strain HT7. Horizontal bars are the mean value of each group. Values are presented as mean ± SE. *, p<0.05 (A, D, Mann-Whitney test; F, G, Two-tailed, unpaired Student's t test).

https://doi.org/10.1371/journal.ppat.1003152.g003

CsA differently alters TLR4- and Nod-mediated signaling in renal tubule cells and neutrophils, macrophages, or renal dendritic cells activated by UPEC

Previous studies had demonstrated that bladder epithelial cells and renal epithelial tubule cells are actively involved, together with bone marrow-derived cells, in the chemoattraction of neutrophils to the site of inflammation in experimental models of ascending UTI [31], [32]. We also showed that UPEC preferentially binds to the apical side of epithelial cells constituting the terminal collecting duct (Figure 4A) [33]. Activation of TLR4 signaling in the urinary tract system infected by UPEC plays a key role in this process [23], [34]. Because renal tubule cells also express the Nod1 and Nod2 receptors, that can be activated in inflamed kidneys [35], [36], experiments were carried out to analyze the effects of CsA on both Tlr4, and Nod1 or Nod2 signaling in renal tubule cells and bone marrow-derived cells activated by LPS, Nod ligands or UPEC. We checked that the renal medullary collecting duct (MCD) cells did express Tlr4, Nod1, and Nod2 mRNAs (Figure 4B). LPS, and to a much lesser extent the Nod1 agonist FK156 and the Nod2 agonist MDP, stimulate the production of CXCL2 in cultured WT MCD cells (Figure 4C). CsA inhibited in a dose-dependent manner the Tlr4 mRNA and protein expressions without altering Nod1 and Nod2 expressions (Figure 4D and E). We then tested whether 100 nM (corresponding to 120 ng/ml) CsA alters the TLR4- and/or Nod-mediated cellular response in MCD cells. The CXCL2 production caused by UPEC significantly decreased in untreated Tlr4^−/− MCD cells compared to WT, Nod1^−/−, or Nod2^−/− MCD cells, and also decreased to almost the same extent in CsA-treated WT, Nod1^−/−, or Nod2^−/− MCD cells than in CsA-treated Tlr4^−/− MCD cells (Figure 4F, upper panel). As controls, similar profiles of CXCL2 production were obtained by incubating WT, Nod1^−/−, and Nod2^−/− MCD cells with LPS, and no significant production of CXCL2 was detected in Tlr4^−/− MCD cells (Figure 4F, lower panel). These findings thus suggest that CsA mainly affects the predominant TLR4-mediated production of CXCL2 and has only a minor effect on epithelial Nod1- and Nod2-mediated renal tubule cell activation caused by UPEC. CsA also significantly reduced the ability of LPS-activated confluent WT MCD cells, which developed high electrical transepithelial resistance (∼4500 Ω. cm²), to stimulate the in vitro migration capacity of neutrophils as compared to untreated WT MCD cells incubated with LPS (Figure S4A to C). As a control, Tlr4^−/− MCD cells challenged with LPS did not stimulate the migration of neutrophils (Figure S4B and C).

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Figure 4. CsA impairs Tlr4-mediated chemokine production in renal medullary collecting duct cells.

(A) Illustration showing UPEC (in blue) adhering to the luminal surface of cells from collecting duct sections (arrows), identified by the AQP-2 positive staining of the collecting duct principal cells (in dark red). Bar = 20 µm. (B) Identification by reverse transcription PCR of amplified products of Tlr4 (311 bp), Nod1 (525 bp), and Nod2 (507 bp) mRNAs in a confluent culture of medullary collecting ducts (MCDs) dissected from the kidney of a WT mouse. (C) CXCL2 production in cultured WT MCD cells incubated with or without LPS, FK156, or MDP for 6 h at 37°C. (D) Relative levels of Tlr4, Nod1 and Nod2 mRNAs measured by quantitative real time PCR in confluent cultures of WT MCD cells incubated with various concentrations of CsA for 48 h and compared to untreated MCD cells. (E) Immunoblot analysis of Tlr4 and Nod1, and the corresponding β-actin in WT MCD cells incubated with or without various concentrations of CsA for 48 h. (F) CXCL2 production in cultured MCD cells dissected from WT, Tlr4^−/−, Nod1^−/−, or Nod2^−/− kidneys incubated with or without CsA for 48 h, then with or without 10⁴ UPEC (upper panel, n = 3–5 determinations from 3 independent experiments) or 10 ng/ml LPS (lower panel, values from 4 separate cultures of MCD cells dissected from a single mouse in each group) for 3 h or 6 h, respectively. Values are presented as mean ± SE. *, p<0.05 (C, E, Mann-Whitney test; D, Two-tailed, unpaired Student's t test).

https://doi.org/10.1371/journal.ppat.1003152.g004

We next analyzed the effects of CsA on Tlr4 and Nod mRNAs expression in neutrophils, macrophages, and renal DCs. Incubating primary bone marrow neutrophils with CsA for 8 h or bone marrow macrophages (BMMs) with CsA for longer times (48 h) significantly decreased the relative levels of Nod1 mRNA and protein expressions, and to a lesser extent reduced the expression of Nod2 mRNA, but not that of Tlr4 mRNA (Figure 5A, B, D and E). Renal DCs expressing Nod1 and Nod2 [37], were shown to produce substantial amounts of CXCL2, which is involved in the recruitment of neutrophils in the kidneys following UPEC challenge [26], [27], [38]. Incubating highly-enriched CD11c⁺ cells isolated from WT kidneys by gradient centrifugation and magnetic beads separation [39] with 100 nM CsA for 48 h significantly reduced Nod1 mRNA expression without affecting the expression of Tlr4 or Nod2 (Figure 5G). However, the small number of purified renal CD11c⁺ cells obtained did not permit reliable Western blot analysis of the Nod1 protein. Consistent with an inhibitory action of CsA on Nod1, the production of CCL5, which has been shown to be highly sensitive to Nod agonist stimulation [40], when stimulated by the Nod1 agonist FK156 was significantly lower in CsA-treated than in untreated neutrophils and macrophages (Figure 5C and F). CsA only slightly, and non-significantly, reduced the FK156-stimulated production of CCL5 in renal DCs (Figure 5H). Given that CXCL2 plays an essential role in the recruitment of neutrophils, we went on to test the effects of CsA on CXCL2 production in neutrophils, macrophages, and renal DCs. Unlike renal MCD cells, CsA only slightly reduced the CXCL2 production stimulated by LPS in neutrophils, macrophages, and renal DCs (Figure 5C, F and H). Taken as a whole, these findings indicate that CsA globally impairs the functional expression of Nod1 in neutrophils, macrophages, and renal DCs.

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Figure 5. CsA preferentially impairs Nod1 expression in neutrophils, macrophages, and renal dendritic cells.

(A, D, G) Fold variations of Tlr4, Nod1 and Nod2 mRNAs expression measured by quantitative real-time PCR in WT bone marrow neutrophils (A), macrophages (D), and renal CD11c⁺ DCs (G) incubated with CsA for 8 h (neutrophils) or 36–48 h (macrophages, DCs) at 37°C, and compared to corresponding untreated cells. (B and E). Immunodetection of the Nod1 protein in untreated (− CsA) and CsA-treated (+ CsA) neutrophils and BMMs. (C, F, H) Effects of 100 nM CsA on CCL5 and CXCL2 production [mean values of mean individual values (n = 3–4) from 3 to 4 separate experiments] in supernatants from neutrophils (C) and macrophages (F) (10⁶ cells/well, or renal DCs (10⁵ cells/well) sequentially incubated without or with CsA for 8 h (neutrophils) or 36–48 h (macrophages, renal DCs), then with CsA and 10 ng/ml LPS, or 1 µM FK156 or MDP for a further 6 h at 37°C. Values are presented as mean ± SE. *, p<0.05 between groups or versus untreated or CsA-treated cells (Two-tailed, unpaired Student's t test).

https://doi.org/10.1371/journal.ppat.1003152.g005

Since Nod1 senses a number of invasive Gram-negative bacteria, we tested whether UPEC can directly activate Nod1 mRNA expression in macrophages and whether CsA impairs the UPEC-induced activation of Nod1. Incubating WT BMMs with UPEC for 3 h had almost no stimulatory effect on Tlr4 mRNA expression, but in contrast induced a significant increase in Nod1 and Nod2 mRNAs expression (Figure S5). Pre-incubating BMMs with CsA impairs the increase in Nod1 mRNA expression, and to a much lesser extent that of Nod2, caused by the subsequent incubation with UPEC for additional 3 h (Figure S5). These findings further suggest that CsA preferentially alters the activation of Nod1 induced by UPEC in phagocytic cells.

Silencing NFATc1 mRNA expression and the 11R-VIVIT peptide inhibitor of NFATs markedly inhibit Nod1 mRNA expression in macrophages

CsA inhibits the nuclear translocation of NFATs, which in turn inhibit the transcription of T cell effector cytokines [8]. Because CsA preferentially alters Nod1 expression in phagocytic cells, experiments were carried out to test whether the downregulation of the NFATc1 isoform, which is highly expressed in both murine and human neutrophils and macrophages [41], [42], impairs mRNA expression of Nods. Because neutrophils have a limited life-span, experiments were carried out on mouse BMMs. Knockdown of NFATc1 mRNA expression using a multiple set of NFATc1 siRNAs (referred to as NFATc1_a–d siRNA) in WT BMMs resulted in the almost complete inhibition of the expression of NFATc1 mRNA when compared to non-transfected WT BMMs or cells transfected with a control siRNA (Figure 6A). Silencing NFATc1 by the set of NFATc1 siRNAs markedly inhibited the relative level of Nod1 mRNA expression, but had almost no effect on Nod2 mRNA expression (Figure 6B). To further assess the inhibitory action of CsA on Nod mRNAs expression, experiments were performed on WT BMMs incubated with 11R-VIVIT, a cell-permeable peptide that specifically inhibits the calcineurin-NFATs interaction without affecting calcineurin phosphatase activity [43]. Incubating WT BMMs with 1 µM 11R-VIVIT for 48 h also markedly inhibited the relative levels of Nod1 mRNA expression, and, to a lesser extent, that of Nod2 mRNA (Figure 6C). In contrast, knock-down of NFATc1 mRNA expression or incubation of BMMs with the 11R-VIVIT had no effect on Tlr4 mRNA expression (Figure S6A and B). Given that inhibition of NFATc1 can affect the transcriptional expression of many proteins, the possibility that a contrario in vitro activation of NFATs could specifically stimulate the expression of Nod1 was investigated. NFATs are activated by increased intracellular calcium concentration during T-cell activation [7]. Calcium mobilization induces the dephosphorylation of cytosolic NFATs which translocate into the nucleus [44]. Ionomycin (2 µM, 60 min) induced the translocation of NFATc1 from the cytosol into nuclei from WT BMMs, whereas the pre-incubation of WT BMMs with CsA or 11R-VIVIT totally or almost totally impaired the nuclear translocation of NFATc1 caused by subsequent addition of ionomycin (Figure 6D). Ionomycin also significantly stimulated Nod1, but failed to stimulate Nod2 and Tlr4 mRNAs expression in WT BMMs compared to untreated cells, or to cells pre-treated with CsA or 11R-VIVIT (Figure 6E and Figure S6C). Collectively, these data strongly suggest a role for NFATc1 as a transcriptional activator of Nod1.

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Figure 6. NFATc1 controls Nod1 mRNA expression in macrophages.

(A) Representative reverse transcription-PCR of the inhibition of NFATc1 in BMMs transfected with a multiple set (n = 4) of NFATc1_a–d siRNAs compared to non-transfected BMMS or with cells transfected with a negative control (Control) siRNA. GAPDH mRNA expression was used as internal control. (B and C) Fold variation of Nod1 and Nod2 mRNAs measured by quantitative real time PCR in BMMs transfected with the set of NFATc1_a–d siRNAs, or with a negative control (Control) siRNA and compared to non-transfected BMMs (B), and in BMMs incubated with or without 1 µM 11R-VIVIT for 48 h (C) (n = 3 independent experiments in each condition tested). (D) Nucleo-cytoplasmic distribution of the NFATc1 immunostaining (shown in red) (left panel) and percentage of NFATc1 immunostaining co-localized with Sytox green nuclear acid staining (n = 7–13 nuclei analyzed for each condition from two separate experiments) (right panel) in WT BMMs sequentially incubated without or with 11R-VIVIT or 10⁻⁷ M CsA for 48 h, then without or with 2 µM ionomycin for additional 60 min. Bar = 10 µm. (E) Fold variation of Nod1 and Nod2 mRNAs expression over corresponding β-actin mRNA measured by quantitative real-time PCR in WT BMMs incubated or not with 11R-VIVIT or CsA for 48 h, then with or without 2 µM ionomycin. (n = 3 independent experiments). *, p<0.05 between groups (Two-tailed, unpaired Student's t test).

https://doi.org/10.1371/journal.ppat.1003152.g006

In vivo administration of 11R-VIVIT impairs neutrophil bacterial phagocytic killing capacity of UPEC and increases renal susceptibility to UPEC

Given that the 11R-VIVIT inhibited Nod1 mRNA expression, 11R-VIVIT should also impair the Nod1-mediated bacterial phagocytic function and decrease the renal defense against UPEC. NPCs isolated from WT mice given daily intraperitoneal injections of 10 µg/kg 11R-VIVIT for 48 h exhibited a significant lower ex vivo capacity to internalize E. coli and lower phagocytic killing of serum-opsonized UPEC than untreated NPCs (Figure 7A and B). 24 h after the inoculation of UPEC, 11R-VIVIT-treated mice exhibited reduced intrarenal MPO activity and reduced amount of immunodetected Nod1 protein, and significant greater renal bacterial burden than in non-treated WT mice (Figure 7C to E).

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Figure 7. The 11R-VIVIT peptide inhibitor of NFATs decreases bacterial phagocytic killing capacity by neutrophils and increases renal susceptibility to UPEC.

WT mice were given daily intraperitoneal injections of 10 µg/kg 11R-VIVIT for 48 h before the transurethral inoculation of UPEC. (A) Illustrations and quantification of internalized Texas red-coupled E. coli by peritoneal neutrophils from untreated (−) and 11R-VIVIT-treated (+) mice. Values are represented as mean ± SE of the mean count values (2–4 per condition) from three independent experiments. Cell membranes were stained with CD11b-FITC. Bars = 10 µm. (B) Killing of serum-opsonized E. coli by peritoneal neutrophils from untreated and 11R-VIVIT-treated mice. Bacterial viability was expressed relative to control assay performed without neutrophils (n = 3–5 determinations from 3 experiments). (C to E) MPO activity (C), representative immunoblot analysis of Nod1 and corresponding ß-actin (D), and bacterial counts (E) in kidneys from untreated and 11R-VIVIT-treated WT mice 24 h after UPEC inoculation. The numbers indicate the ratio of Nod1 over ß-actin densitometric values from 4 experiments and the horizontal bars show the mean of bacterial counts of each group (n = 6 determinations in each group). Values are presented as mean ± SE. *, p<0.05 between groups (Two-tailed, unpaired Student's t test).

https://doi.org/10.1371/journal.ppat.1003152.g007

Nod1 stimulating agents partially restore the CsA-induced downregulation of Nod1 and renal resistance to UPEC

The fact that the stimulation of Nod1 can enhance systemic innate immunity [20] and that the administration of Nod1 peptide agonists to mice confer resistance against several pathogens [45], led us to test whether the stimulation of Nod1 by synthetic Nod1-stimulating agonists could reinforce renal defense against UPEC. The cell-permeable Nod1 activating agonist C12-iEDAP (50 µg/ml for 24 h) induced a significant increase in Nod1 mRNA expression, which overcome the inhibition of Nod1 mRNA expression caused by CsA alone (not shown). We then tested whether the in vivo administration of synthetic Nod1 agonists can reactivate Nod1-mediated phagocytic function and reinforce renal resistance of CsA-treated mice to UPEC. The capacity of neutrophils to internalize Texas red-coupled E. coli was greater in neutrophils from CsA-treated mice that had been treated with C12-iEDAP than in those collected from CsA-treated mice which had not received C12-iEDAP (Figure 8A). Furthermore, intra-peritoneal injection of C12-iEDAP or FK156 (80 µg/mouse) one day before the transurethral inoculation of UPEC to CsA-treated WT mice, induced significant reduction in the renal bacterial burden when compared to CsA-treated mice which had not received C12-iEDAP or FK156, or CsA-treated mice which had received the Nod2 agonist MDP (Figure 8B). The observed decrease in renal bacterial burden was associated with greater MPO activity in the infected kidneys from CsA-treated mice pre-treated with the Nod1 agonists (Figure 8C). Figure 8D illustrates the greater amount of Nod1 protein in the infected kidneys from CsA-treated mice which had been pre-treated with FK156. The amounts of CXCL2 and CXCL1 secreted were not significantly different in the 24-h, post-infected kidneys from CsA-treated mice and the vehicle-treated mice (Figure 8E), suggesting that the administration of Nod1 agonists does not induce any major renal inflammatory response. Collectively, these findings are consistent with the restimulation of Nod1-mediated host protective functions in CsA-treated mice.

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Figure 8. The Nod1 stimulating agonists C12-iEDAP and FK156 stimulate neutrophil bacterial phagocytosis impaired by CsA and increase renal defenses against UPEC.

(A) Illustrations and quantification of Texas red-coupled E. coli internalized by circulating neutrophils collected from WT mice treated with CsA for 5 days then with CsA plus C12-iEDAP (80 µg per mouse) for a further 5 h. Neutrophils were isolated from the blood of 3 mice under each of the conditions tested. Representative values (mean ± SE) of the mean counts (9–15 per condition) from 3 independent experiments. Cell membranes were stained with WGA Alexa Fluor 647 conjugate. Bars, 10 µm. (B and C) Bacterial counts (B) and MPO activity (C) in kidneys of CsA-treated WT mice pre-treated or not with C12-iEDAP (n = 6), FK156 (n = 9), or MDP (n = 7) (80 µg per mouse), and then challenged 24 h later with E. coli HT7. (D) Immunoblot analysis of Nod1 and the corresponding ß-actin in the post-infected kidney homogenates from vehicle- and CsA-treated mice which had received FK156 12 h before UPEC inoculation. (E) Production of CXCL2 and CXCL1 in the 24 h post-infected kidneys from vehicle- or CsA-treated WT mice pre-treated or not with C12-iEDAP or FK156 (n = 6–9 per group). *, p<0.05 (A, C, E, Two-tailed, unpaired Student's t test; B, Mann-Whitney test).

https://doi.org/10.1371/journal.ppat.1003152.g008

Human transplant recipients treated with CsA exhibit downregulated expression of NOD1

Investigations were performed on blood samples from random renal transplant recipients treated with CsA (n = 25) to test whether human renal transplant recipients exhibit similar decrease in NOD1 expression and defective NOD1-mediated bacterial phagocytosis capacity. The demographic characteristics of renal transplant recipients are summarized in Table S1. Transplant recipients all received CsA and additional immunosuppressive drugs, including prednisolone and mycophenolate mofetil, which is a selective inhibitor of the de novo synthesis of guanosine nucleotides in T and B lymphocytes [46]. For comparison, investigations were also performed on healthy volunteers (n = 10) used as controls. The production of IL-8 was first measured in whole blood samples incubated with various TLR and NOD agonists. The levels of IL-8 triggered by all TLR agonists tested did not significantly differ in blood samples from transplant recipients and normal healthy controls (Figure 9A). In contrast, the levels of IL-8 production stimulated by the human NOD1 synthetic agonist M-TriDAP was significantly less in blood samples from the transplant recipients treated with CsA than in normal controls (Figure 9A and B). The levels of NOD1 mRNA, but not those of NOD2, TLR2, or TLR4 mRNAs extracted from whole blood samples, were also significantly lower in transplant recipients than in normal controls (Figure 9C). Flow cytometry analysis of phagocytosis of Texas red-coupled E. coli and quantification of NOD1 in human neutrophils also revealed that the low levels of NOD1 in the neutrophils from CsA-treated renal transplant recipients were closely correlated with their capacity to phagocyte E. coli, which was significantly lower than in the neutrophils from normal healthy controls (Figure 9D). Moreover, three of the transplant recipients analyzed who exhibited low NOD1 expression and low capacity of E. coli phagocytosis by neutrophils had a previous history of UTI/APN. These findings suggest that the NFATs-dependent inhibitory mechanism of Nod1-mediated innate immune response identified in the mouse also occurs in human transplant recipients treated with CsA.

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Figure 9. Functional downexpression of NOD1 in leukocytes from CsA-treated renal transplant recipients.

(A) IL-8 production in blood samples from healthy volunteers (Controls, n = 10) and renal transplant recipients treated with CsA (n = 25) incubated without (None) or with 1 ng/ml Pam3CSK4 or LPS, 1 µg/ml flagellin, 50 µg/ml CpG-DNA, 1 µM M-TriDAP or MDP. (B) IL-8 production in blood samples from Controls and renal transplant recipients incubated with increasing concentrations of M-TriDAP. (C) Relative fold variation of TLR2, TLR4, NOD1 and NOD2 mRNAs in blood samples from renal transplant recipients (n = 25) compared to that found in Controls (n = 10). (D) Flow cytometry analyses of phagocytosed E. coli (expressed as fluorescence intensity of internalized E. coli) and mean fluorescence intensity (MFI) of NOD1 expression in neutrophils from healthy volunteers (n = 10) and renal transplant recipients treated with CsA (n = 25). * Transplant recipients who experienced UTI/APN. Values are presented as mean ± SE from n determinations per condition tested. *, p<0.05 (A, B, Two-tailed, unpaired t test; C, Mann-Whitney test).

https://doi.org/10.1371/journal.ppat.1003152.g009

Ethics statement

All animal experiments were approved by and conducted in accordance with guidelines of the French Agricultural Office and in compliance with the French and European regulations on Animal Welfare (Service de la protection et Santé animale; Approval Number 75–687, revised 2008) and with Public Health Service recommendations. All the efforts were made to minimize suffering of mice.

Blood samples were obtained from transplant recipients and healthy volunteers after being informed and given oral consent, according to French law for non interventional studies using a leftover or a small additional blood sample (Public Health Code, article L1121-1, revised in May 2009). All samples were anonymized. Human and animal studies were approved by the Institutional Ethics Committee (Comité de Protection des Personnes (CCP #5) affiliated to the Tenon Hospital (AP-HP)-University Paris 6 (Approval CCP-0612/2011). All experiments were conducted in accordance with the principles expressed in the Declaration of Helsinski.

Renal retrograde infection in mice

Adult female (8–10 week old) WT mice (supplied by the Centre d'Elevage Janvier, Le Genest-Saint-Isle, France), Rag2^−/− mice, and Nod1^−/− and Nod2^−/− mice from the same C57BL/6 genetic background were used. Mice were infected with the uropathogenic E. coli strain HT7 (10⁸ bacteria in 50 µl sterile PBS) introduced via the transurethral route into the bladder as described [4], [25]. 100 µl CsA (Neoral, Novartis International Pharmaceutical Ltd, 15 mg/kg), or its vehicle (castor oil) were administered sub-cutaneously to mice for 5 days before the inoculation of UPEC. Bacterial loads (CFU) in kidneys were determined 24 h after infection by plating. Kidney sections were stained using anti-E. coli antibody (Interchim), anti-Ly6-G antibody (BD Biosciences), or aquaporin-2 (AQP-2) as described [23].

Cultured collecting duct cells

Primary cultures of medullary collecting duct (MCD) isolated from WT, Nod1^−/−, Nod2^−/−, and Tlr4^−/− mice kidneys were grown as described [23]. Experiments were carried out on confluent cells two weeks after seeding.

Macrophage, neutrophil, and renal dendritic cell isolation

Bone marrow neutrophils and circulating blood neutrophils were isolated by gradient density centrifugation using Ficoll-Paque PREMIUM (GE Healthcare) as described elsewhere [20]. Bone marrow-derived macrophages (BMMs) were isolated and grown as described [40]. Indirect immunofluorescence studies were performed on WT BMMs using a mouse anti-NFATc1 monoclonal antibody (Thermo Scientific Pierce Antibodies) and Sytox green nucleic acid stain (Invitrogen). Renal dendritic CD11c⁺ cells were isolated as previously described with slight modifications [39], [65]. For each cell preparation both kidneys from 5 naïve WT mice were used. Briefly, the kidneys of each mouse were minced and then digested for 45 min at 37°C with 1 mg/ml collagenase (Roche Diagnostics, Meylan, France) and 10 µg/ml DNAse I in RPMI 1640-Glutamax medium (Life Technologies) supplemented with 10% heat-inactivated fetal calf serum, 10 mM HEPES, 100 U penicillin, and 0.1 mg/ml streptomycin. Kidney homogenates from each mouse were then filtered through 70 µm nylon mesh, washed with PBS, centrifuged (250 g, 5 min), resuspended in 3 ml of 0.01 M ethylenediaminetetracetic acid (EDTA) in FCS and layered on top of 3 ml Histopaque-1077 (Sigma). Density centrifugation (400 g, 30 min) was performed at room temperature. The interphase cells were then harvested, washed, and resuspended in 600 µl MACS buffer (Miltenyi Biotec.). CD11c⁺ cells were then isolated using microbead-labeled specific monoclonal antibody (clone N418, Miltenyi Biotec.), and separated using magnetic beads according to the manufacturer's instructions. The enriched- CD11c⁺ cell suspension obtained from 5 mice were then pooled and used for the cytokine assay.

In vitro neutrophil migration assay

The migration capacity of neutrophils isolated from untreated or CsA-treated WT mice or Nod1^−/− mice was analyzed using the Boyden chamber technique as previously described [30]. After the lysis of red blood cells, blood samples from vehicle- and CsA-treated WT were laid on the top of a Ficoll-Paque PREMIUM (GE Healthcare, Uppsala, Sweden) density gradient, then centrifuged (400 g, 30 min at 4°C), and the bottom layer containing the neutrophil-enriched fraction was collected. 10⁶ neutrophils were then resuspended in 200 µl Hank's buffered salt solution (HBSS) containing 0.5% bovine serum albumin and added to the upper compartment of a Transwell Clear membrane insert (3 µm pore size, Corning Inc., Lowel, MA). The lower compartment (600 µl) of the chamber contained either HBSS alone or supplemented with fMLP (10⁻⁷ M) or CXCL2 (200 ng/ml). Incubations were performed at 37°C for 40 min in a 5% CO₂/95% air atmosphere. A neutrophil migration assay was also carried out using isolated WT and Tlr4^−/− MCDs seeded and grown to confluence in defined DMEM/Hams'F12 culture medium [23] on the apical side of the filters. Confluent WT MCD cells were then incubated with or without 100 ng/ml CsA for 48 h, then with or without (10 ng/ml) LPS, which was added when required to the upper compartment of the chamber for 4 h in a 5% CO₂/95% air atmosphere. The lower compartment contained 10⁶ WT neutrophils resuspended in 600 µl defined culture medium. In all cases, the filters were rinsed, then fixed in methanol and stained using the RAL 555 Kit (Réactifs RAL, Martillac, France). The neutrophils (stained deep purple) detected in the filters were counted by microscopic observation. MCD cells (stained pale red) were also stained using the RAL kit containing eosin. In parallel, the transepithelial electrical resistance (R_T) was measured using dual silver/silver chloride (Ag/AgCl) electrodes connected to a Millicel-ERS voltohmmeter (Millipore, Billerica, MA).

E. coli phagocytosis and bacterial killing assay

Enriched-neutrophil peritoneal cells collected by peritoneal lavages 3 h after a single intraperitoneal injection of 1.5 ml of thioglycollate (Bio-Rad Laboratories) were incubated with Texas red-coupled E. coli (10⁴ bacteria/10⁷ cells) for 30 min at 37°C, and then stained with CD11b-FITC or Wheat Germ Agglutinin (WGA)-Alexa Fluor 647 (Invitrogen) to delineate cell peripheries. The internalization of E. coli was determined by measuring the intracellular red fluorescence intensity using confocal microscopy analysis as described elsewhere [66]. For the ex vivo bacterial killing assay, E. coli were mixed without or with peritoneal neutrophils (10³ bacteria/10⁶ neutrophils) following the same procedure as described elsewhere [20].

Renal transplant recipients

Blood samples from 25 renal transplant recipients with a functioning graft during the first three years after surgery and exposed to CsA (Table S1) were randomly taken during the regular routine consultations at Tenon hospital (Assistance Publique-Hôpitaux de Paris, France). In all cases, the blood samples were taken at least 6 months after surgery. In addition, ten healthy volunteers served as normal controls.

Real-time PCR and reverse transcription PCR

Total RNA from mouse kidneys, neutrophils, or macrophages was purified with RNAble (Eurobio laboratories) and reverse transcribed using Moloney Murine Leukemia Virus reverse transcriptase (Invitrogen). cDNA was subjected to quantitative real-time PCR using a Chromo IV sequence detector (MJ Research). The mouse Tlr2, Tlr4, Nod1, Nod2 and ß-actin primers used and the corresponding Taqman probes are listed in Table S2. PCR data were reported as the relative increase in mRNA transcripts versus that found in uninfected kidneys or vehicle-treated neutrophils or macrophages cells and corrected using the respective levels of ß-actin mRNA. Quantitative real-time PCR was also performed on RNA extracted from blood samples of renal transplant recipients using human TLR2, TLR4, NOD1, NOD2, and ß-ACTIN primers and corresponding Taqman probes (listed in Table S2). PCR data were reported as the relative increase in mRNA transcripts versus that found in a pool of RNA of untreated leukocytes from healthy volunteers. For reverse transcription PCR, cDNA and non-reverse transcribed RNA (250 ng) from cultured mouse MCD cells or BMMs were amplified for 35 cycles in 35 µl of Platinum Blue PCR SuperMix (Invitrogen) containing 10 pmol of mouse NFATc1, Tlr4, Nod1, Nod2, or GAPDH primers (described in Table S2). Amplification products were run on a 2% agarose gel containing SYBR Safe DNA gel stain (Invitrogen) and photographed.

Small interfering RNA

Experiments were performed using different predesigned HP GenomeWide (Qiagen, Courtaboeuf, France) siRNAs (referred to as NFATc1a, b, c, and d) for the murine NFATc1 gene target DNA sequence. NFATc1_a DNA sequence: 5′-TCGGATCGAGGTGCAGCCCAA-3′; sense: 5′-GGAUCGAGGUGCAGCCCAATT-3′; antisense: 5′-UUGGGCUGCACCUCGAUCCGA-3′; NFATc1_b DNA sequence: 5′-CACGGTTACTTGGAGAATGAA-3′; sense: 5′-CGGUUACUUGGAGAAUGAATT-3′; antisense: 5′-UUCAUUCUCCAAGUAACCGTG-3′; NFATc1_c DNA sequence: 5′-CCCGTCCAAGTCAGTTTCTAT-3′; sense: 5′-CGUCCAAGUCAGUUUCUAUTT-3′; antisense: 5′-AUAGAAACUGACUUGGACGGG-3′; NFATc1_d DNA sequence: 5′-CCGGGACCTGTGCAAGCCAAA-3′; sense: 5′-GGGACCUGUGCAAGCCAAATT-3′; antisense: 5′-UUUGGCUUGCACAGGUCCCGG-3′. A universal negative control siRNA (target DNA sequence: 5′-AATTCTCCGAACGTGTCACGT-3′; sense: 5′-UUCUCCGAACGUGUCACGUdTdT-3′; antisense: 5′ ACGUGACACGUUCGGAGAAdTdT-3′) was also used (Qiagen). Single strand sense and antisense RNA nucleotides were annealed to generate a RNA duplex according to the Manufacturer's instructions. WT BMMs were seeded in 6-well plates and incubated with 10 nM of each siRNA tested and 2 µl of Lipofectamine RNAiMAX Reagent (Invitrogen) for 48 h at 37°C before use. As a control, we checked that each of the NFATc1 (a to d) siRNAs inhibited NFATc1 mRNA expression in macrophages using reverse-transcription PCR (not shown).

Western blotting

Mouse kidney homogenates and BMMs were lysed and processed for Western blotting using mouse anti-TLR4 [25]), anti-Nod1 (Cell Signaling) or anti-Nod2 (eBiosciences) antibodies, and phospho-RIP-2 (Ser 176), and total RIP-2 (Ozyme) antibodies. Protein bands were revealed using horse raddish peroxidase-conjugated goat anti-rabbit IgG (Jackson Immunoresearch), and analyzed by Western Blotting.

ELISA

Cytokine production was measured in mouse kidney homogenates, or cell supernatants using DuoSet mouse ELISA kits (R&D Systems, Minneapolis, MN). Neutrophils, macrophages, or cultured MCD cells were incubated either with LPS (Escherichia coli 0111:B4 LPS Ultra-Pure, InvivoGen, Toulouse, France), 50 µg/ml C12-iEDAP (InvivoGen), 1 µM FK156 (provided by Nami Kawano, Astellas Pharma Inc., Osaka, Japan), or 1 µM MDP (InvivoGen, Toulouse, France) for 8 to 18 h at 37°C. For FK156 and MDP stimulations, mouse macrophages were pre-treated with 1 µM cytochalasin D (Calbiochem, Darmstadt, Germany) for 30 min to allow efficient internalization of the synthetic Nod activating agonists as described elsewhere [40]. Human blood samples (10 µl) were incubated in 500 µl RPMI culture medium (Invitrogen) at 37°C alone or with 1 ng/ml Pam3CSK4 (InvivoGen) or LPS, 1 µg/ml flagellin (InvivoGen); 50 µg/ml unmethylated CpG-DNA (HyCult Biotechnology, Uden, The Netherlands), 50 nM MDP or various concentrations (0.05–2 µM) of M-TriDAP (InvivoGen) for 18 h. IL-8 production was measured using a DuoSet human ELISA kit (R&D Systems, Lille, France). All the reagents used were tested negative for endotoxin contamination using the Limulus amoebocyte assay according to the Manufacturer's recommendations (QCL-1000, Biowhittaker, Buckinghamshire, UK). MPO activity was measured using HyCult Biotechnology ELISA kit.

Flow cytometry analysis

The cell populations infiltrating the infected mouse kidneys were analyzed by flow cytometry. 24 h after UPEC infection, kidneys were carefully rinsed with PBS to remove the remaining circulating blood cells. The kidneys were then minced and digested for 45 min at 37°C with 1 mg/ml collagenase (Roche Diagnostics) and 10 µg/ml DNAse I in the same RPMI 1640-Glutamax medium (Life Technologies) as described above for the isolation of renal DCs. After rinsing, kidney homogenates were then passed through a 70 µm pore sized nylon Cell Strainer (BD Biosciences) with 15 ml PBS. The resulting cell suspension was centrifuged (1600 rpm, 10 min) again and then resuspended (10×10⁶ total cells/ml) in FACS buffer containing 2% BSA and 0.05% sodium azide. Non-specific binding of antibody to Fc receptors was blocked by incubating the cell suspension with the anti-mouse CD16/32 (2.4G2) antibody (10 µg/ml) and ChromePure rat IgG (100 µg/ml, Jackson Immunoresearch) for 30 min at 4°C. Cells were then incubated in pre-determined optimal concentrations of fluorochrome-conjugated antibodies to cell surface antigens or matching isotype control antibodies for 30 min at 4°C. APC anti-mouse Ly-6G/Ly-6C (Gr1; RB6-8C5), Pe anti-mouse F4/80 (BM8), PerCP/Cy5.5 anti-mouse CD11b (M1/70), Pacific Blue anti-mouse (MHC-II/IA/IE, M5/114.15.2) and PeCy7 anti-mouse CD45 (RA3-6B2), and matching fluorophores-conjugated antibodies isotypes were purchased from Biolegend. PeCy7 anti-mouse CD11c (HL3) and V500 anti mouse CD45.2 (104) were purchased from BD Pharmingen. Fluorescent measurements were conducted with identical settings on at least 100,000 CD45⁺ cells per kidney per experiment using a BD FACSCanto II cytometer operating BD FACSDiva software v6.1.3 (BD Biosciences, Erembodegem, Belgium), and FlowJo v7.6.5 (Tree Star. Inc., Ashland, OR). Analyses of NOD1 expression and bacterial phagocytosis capacity by human neutrophils were also analyzed by flow cytometry. Human whole blood samples (1 ml) were incubated with Texas red-coupled E. coli (10⁷ bacteria) (Molecular Probes) with gentle stirring for 30 min at 37°C, while negative control samples were kept on ice before analysis. Red blood cells were then lyzed by adding 10 ml NH₄Cl 0.8% wt/vol for 15 min. Trypan blue (0.05 mg/ml) was added to the samples to reduce the quenching of surface-bound fluorescence. Samples were centrifuged (400 g, 10 min) at 4°C to remove cell debris, and pelleted leukocytes were then rinsed in PBS. In parallel, aliquots of NH₄Cl-treated blood samples were permeabilized with methanol, and then incubated with an anti-human NOD1 antibody (Imgenex Corp.). All fluorescence measurements were conducted with identical settings and forward and side-scatter parameters to identify the neutrophil population and to gate out other cells and debris [67].

Statistical analysis

Statistical analysis was performed using the GraphPad Prism program. The unpaired t test, (two-tailed p values) was used to compare two groups. The distribution of three or more groups was analyzed by One-Way ANOVA and the Kruskal-Wallis test. The Mann-Whitney test was used to compare the group with one another. A p value<0.05 was considered significant.

Accession numbers

The mouse gene accession numbers (GenBank) are as follows: ß-actin, NM_007393.3; GAPDH, AK144690; NFATc1/NFAT2, NM_016791.4; Nod1, NM_172729; Nod2, NM_145857; RipK2, NM_138952.3; Tlr2, NM_011905.3; Tlr4, NM_021297, Tlr5, AF186107.1; Tlr9, AF314224.

The human gene accession numbers (GenBank) are as follows: GAPDH, NM_002046.3; NOD1, NM_006092.2; NOD2, NM_022162.1; TLR2, NM_003264.3; TLR4, NM_138554.3.

The mouse protein accession number (UniProtKB/Swiss-Prot) are as follows: ß-actin, P60710; CCL5, P30882; CD11b/integrin alpha-M/beta-2; P05555; CXCL1; P12850; CXCL2, P10889; NFATc1/NFAT2, 088942; Nod1, Q8BHB0; Nod2, Q8K3Z0; RipK2, P58801; Tlr2, Q9QUN7; Tlr4, Q9QUK6, Tlr5, Q9JLF7; Tlr9, Q9EQU3.

The human protein accession numbers (UniProtKB/Swiss-Prot) are as follows: IL-8, P10145; NOD1, Q9Y239.