The NLRP3 Inflammasome Has a Critical Role in Peritoneal Dialysis-Related Peritonitis

Abstract

Bacterial peritonitis remains the main cause of technique failure in peritoneal dialysis (PD). During peritonitis, the peritoneal membrane undergoes structural and functional alterations that are mediated by IL-1β. The NLRP3 inflammasome is a caspase-1–activating multiprotein complex that links sensing of microbial and stress products to activation of proinflammatory cytokines, including IL-1β. The potential roles of the NLRP3 inflammasome and IL-1β in the peritoneal membrane during acute peritonitis have not been investigated. Here, we show that the NLRP3 inflammasome is activated during acute bacterial peritonitis in patients on PD, and this activation associates with the release of IL-1β in the dialysate. In mice, lipopolysaccharide- or Escherichia coli-induced peritonitis led to IL-1β release in the peritoneal membrane. The genetic deletion of Nalp3, which encodes NLRP3, abrogated defects in solute transport during acute peritonitis and restored ultrafiltration. In human umbilical vein endothelial cells, IL-1β treatment directly enhanced endothelial cell proliferation and increased microvascular permeability. These in vitro effects require endothelial IL-1 receptors, shown by immunofluorescence to be expressed in peritoneal capillaries in mice. Furthermore, administration of the IL-1β receptor antagonist, anakinra, efficiently decreased nitric oxide production and vascular proliferation and restored peritoneal function in mouse models of peritonitis, even in mice treated with standard-of-care antibiotherapy. These data demonstrate that NLRP3 activation and IL-1β release have a critical role in solute transport defects and tissue remodeling during PD-related peritonitis. Blockade of the NLRP3/IL-1β axis offers a novel method for rescuing morphologic alterations and transport defects during acute peritonitis.

Peritoneal dialysis (PD) is used by >200,000 patients with ESRD worldwide, a prevalence that is rapidly growing in many parts of the world.¹ Peritonitis constitutes a major complication of PD, representing the main cause (approximately 20%) of technique failure and being associated with an increased risk of death, either as a primary or a contributing factor.^2–4 Patients experiencing peritonitis show an enlarged effective vascular surface area in the peritoneal membrane, with increased transport of small solutes and glucose, loss of proteins into the dialysate, and dissipation of the osmotic gradient, leading to ultrafiltration failure.^5,6 Our previous studies have demonstrated the critical role of vasoactive substances released during peritoneal inflammation and the role of specific isoforms of nitric oxide (NO) synthase in altered microvascular permeability during peritonitis.^7–9

At a structural level, peritonitis is characterized by an infiltration of leukocytes into the peritoneal membrane. The latter starts with a rapid accumulation of neutrophils, which are progressively cleared and replaced by a population of mononuclear cells, monocytes and/or macrophages, and lymphocytes. These cells contribute to the release of proinflammatory cytokines, which drive tissue remodeling in the membrane.^6,10,11 Studies using adenoviral-mediated gene transfer in rat models identified IL-1β as driving the release of growth factors, such as vascular endothelial growth factor and TGF-β, to promote peritoneal fibrosis and angiogenesis.¹² Recent analysis of immunologic signatures in patients on PD presenting with peritonitis showed that IL-1β levels can be used as biomarker to discriminate various types of peritonitis, and to select the appropriate antibiotic treatment.¹³

The NLRP3 inflammasome is a multiprotein complex expressed in the cytosol of immune cells (macrophages and neutrophils), which links alarming signals to activation of innate immunity.¹⁴ In response to bacterial toxins, exogenous structures on microorganisms (pathogen-associated molecular patterns), or other stimuli such as crystals (e.g., urate, cholesterol), activation of the NLRP3 inflammasome leads to the release of IL-1β via the autocatalysis of caspase-1, which in turn enables the cleavage of pro–IL-1β into its active form, IL-1β.¹⁵ An increasing body of evidence has demonstrated the activation and role of NLRP3 inflammasome in the pathogenesis of a large variety of diseases, including gout, atherosclerosis, crystallopathies, and infectious diseases.^16,17 Mutations in genes encoding for proteins involved in regulation of NLRP3 inflammasome machinery and IL-1β processing have been associated with cryopyrinopathies, a heterogenous group of diseases characterized by recurrent inflammatory episodes of serosal membranes and aseptic peritonitis.¹⁸ Despite the association of such genetic disorders of the NLRP3/IL-1β pathway and the peritoneal membrane, the activation and potential role of NLRP3 inflammasome in IL-1β processing and maturation during PD-related acute peritonitis have not been investigated.

In this study, we demonstrate that the NLRP3 inflammasome is activated during peritonitis in patients on PD and in mouse models of peritonitis, and that activated NLRP3 is directly involved in the deleterious inflammatory response, leading to structural and functional impairment in the peritoneal membrane. Genetic and pharmacologic blockade of the NLRP3/IL-1β axis rescued morphologic alterations and transport defects during acute peritonitis, revealing novel therapeutic perspectives for this severe complication of PD.

Results

Acute Peritonitis Activates NLRP3 Inflammasome and Induces IL-1β Release

We first tested the hypothesis that NLRP3 inflammasome is activated during acute peritonitis, causing the release of IL-1β in the peritoneal effluent. We observed a strong upregulation of the NLRP3 inflammasome components (mRNA level) in the dialysate leukocytes from patients on PD with peritonitis compared with controls (Figure 1A), matched with the release of large amounts of mature IL-1β in the dialysate (Table 1). The morphologic counterpart is illustrated in a peritoneal biopsy specimen showing NALP3 expression in mononuclear cells infiltrating the peritoneal membrane of a patient experiencing acute, PD-related peritonitis (Figure 1, B and C).

Figure 1.

Activation of NLRP3 inflammasome in patients on PD with acute peritonitis. (A) Quantitative RT-PCR to measure the mRNA expression of NLRP3 inflammasome components ASC, NLRP3, caspase-1, pyrin, and pro–IL-1β in total leukocytes from patients on PD with peritonitis (n=5) compared with controls (n=3, hatched bar). **P<0.01; ***P<0.001 versus controls. (B) Morphology of the visceral peritoneum stained by Masson trichrome in a PD control patient and a patient on PD with peritonitis. The peritonitis is characterized by an inflammatory infiltrate composed of mononuclear cells. Scale bar, 50 µm; original magnification, ×40. (C) Representative pictures of double immunostaining with anti-CD11b (red channel) and anti-NLRP3 (green channel) antibodies in peritoneal sections from a PD control and a patient on PD with acute peritonitis (same patients as in B). The latter illustrates the expression of NALP3 in CD11b⁺ cells infiltrating the membrane. Scale bar, 50 µm; original magnification, ×20. m, mesothelium.

Table 1.

Clinical and inflammatory parameters in patients on PD

Sex	Age, yr	PD Duration, mo	Acute Peritonitis	Dialysate WBC, n/µl	Neutrophils, %	Bacteriology	Serum CRP, mg/dl	Dialysate IL-1β, pg/ml
W	50	40	+	3700	92	Proteus mirabilis	16.8	103.5
W	84	11	+	15,500	89	Streptococcus bovis	9.7	85.3
W	50	42	+	1100	96	Citrobacter freundii	14.4	71.9
M	77	0.1	+	1000	56	Negative	17.6	42.9
W	69	56	+	1000	80	Negative	39.5	44.0
M	65	34	−	<100	14	Negative	0.5	0.0
W	68	42	−	<100	7	Negative	1.0	0.0

WBC, white blood cells; CRP, C-reactive protein; W, woman; +, present; M, man; −, absent.

We substantiated the activation of NLRP3 in a well established mouse model of acute peritonitis induced by lipopolysaccharide (LPS). A single intraperitoneal (i.p.) injection (10 mg/kg body wt) of Escherichia coli-derived LPS induced a major increase in Nalp3 and Il-1β mRNA expression in the membrane, with large amounts of IL-1β released in the peritoneal effluent (Figure 2, A and B). The activation of NLRP3 and IL-1β was reflected by an increased proportion of neutrophils in dialysate (Figure 2C), higher levels of NO metabolites in the peritoneal effluent (Figure 2D), and increased peritoneal solute transport rate with a loss of ultrafiltration because of faster dissipation of the osmotic gradient across the membrane (Table 2). In this model, defects in peritoneal transport occurred in the absence of any significant change in vascular density in the membrane (Figure 2, E and F).

Figure 2.

Activation of NLRP3/IL-1β during LPS-induced peritonitis in mice. (A) Quantitative RT-PCR to measure the mRNA expression of Nalp3 and Il-1β in visceral peritoneum from control mice and mice treated by LPS (10 mg/kg). Increase of mature IL-1β release (B), neutrophilic recruitment (C), and total NO metabolites (NOx) levels (D) in the dialysate of PD-related peritonitis mice. (n=5 mice per group). (E) The counting of vessels was performed after immunohistochemistry against CD31 (ten fields per mouse, n=5 mice per group) and do not show vascular proliferation in the visceral peritoneum of mice with LPS peritonitis. (F) Morphology of the visceral peritoneum stained by Masson trichrome in control mice and mice with LPS-induced peritonitis. Acute peritonitis induces a slight inflammatory recruitment in the peritoneum. Scale bars, 50 µm; original magnification, ×20. Immunoreactivity for CD31 is located in the endothelium lining peritoneal vessels and capillaries. Scale bars, 25 µm; original magnification, ×40. *P<0.05; **P<0.01; ***P<0.001. WBC, white blood cells.

Table 2.

Time course of PD transport parameters during LPS or E. coli-related peritonitis

Groups	MTAC Urea, µl/min	Dialysate Albumin, mg/ml	Net UF/Body Wt, µl/g
Vehicle	20.3±0.8	2.6±0.2	56.1±1.0
LPS 6 h	39.3±3.3a	3.4±0.1a	35.1±3.8a
LPS 24 h	37.0±0.8a	3.6±0.3a	31.9±2.3a
LPS 48 h	35.3±2.0a	3.5±0.3a	23.7±3.0a
Vehicle	16.1±0.9	2.4±0.2	56.1±1.9
E. coli 2 d	40.2±2.6a	3.2±0.3a	19.3±1.2a
E. coli 5 d	36.3±1.8a	3.5±0.1a	18.0±1.6a
E. coli 6 d	36.5±2.3a	3.6±0.2a	8.3±2.0a

n=5 (LPS groups), n=4 (E. coli groups). MTAC, mass transfer area coefficient; UF, ultrafiltration.

^aP<0.001 versus vehicle.

We next investigated these parameters in a mouse model of acute peritonitis induced by E. coli during 6 days (Figure 3), i.e., closer to the clinical situation of acute peritonitis induced by Gram-negative rods.⁸ Repeated i.p. injections of E. coli led to sustained inflammation, reflected by the upregulation of Nalp3 and Il-1β, the release of IL-1β in the dialysate (Figure 3, A and B), increased levels of NO metabolites (Figure 3C), and increased solute transport rate with loss of ultrafiltration (Table 2). Compared with the LPS model, the E. coli exposure model induced a stronger leukocyte infiltration in the peritoneal membrane, an increase in the number of white blood cells in the drained dialysate (Figure 3D), and a significant vascular proliferation (80.2±5.7 vessels/field versus 8.1±0.9 vessels/field after 6 days of E. coli exposure and after 48 hours of LPS, respectively; P<0.001; n=5) (Figure 3, E and F). Of note, AQP1 expression was unchanged in both models, ruling out water channels as the cause of impaired osmotic water transport (data not shown).

Figure 3.

Activation of NLRP3/IL-1β during E. coli-induced peritonitis in mice. (A) Quantitative RT-PCR to measure the mRNA expression of Nalp3 and Il-1β in visceral peritoneum from control mice and mice exposed to E. coli (10⁹/ml E. coli diluted in 2 ml dialysate). Increase of mature IL-1β release (B), total NO metabolites (NOx) levels (C), and white blood cells (WBC) recruitment (D) in the dialysate of peritonitis mice. (E) The counting of vessels was performed after immunohistochemistry against CD31 (ten fields per mouse, n=4 mice) and shows a significant vascular proliferation in the visceral peritoneum of mice with bacterial peritonitis (n=4 mice per group). (F) Morphology of the visceral peritoneum stained by Masson trichrome or immunohistochemistry for CD31 in control mice and mice with E. coli-induced peritonitis. Peritonitis is reflected by a massive infiltrate of mononuclear cells in visceral peritoneum. Immunoreactivity for CD31 is located in the endothelium lining peritoneal vessels and capillaries. E. coli-exposed mice are associated with an increased density of CD31⁺ blood vessels and capillaries. Scale bars, 50 µm; magnification, ×20. *P<0.05; **P<0.01; ***P<0.001.

Altogether, these data demonstrated that NLRP3 inflammasome is activated and associated with the release of mature IL-1β during peritonitis, both in patients on PD and in acute/subacute mouse models of LPS- or E. coli-induced peritonitis.

Deletion of Nalp3 Prevents IL-1β Release and Rescues Transport Defects in Peritonitis

To cast light on the functional role of NLRP3 during acute peritonitis, we tested the effect of LPS exposure in Nalp3 knockout (KO) mice versus control littermates (Figure 4). An i.p. injection of LPS strongly upregulated Nalp3 and Il-1β in the peritoneal membrane of Nalp3^+/+ mice (Figure 4A), with release of IL-1β (Figure 4B), increased proportion of neutrophils in the drained dialysate (Figure 4C), selective upregulation of Inos (Figures 4, D and E), increased dialysate levels of NO metabolites (Figure 4F), as well as transport changes such as increased protein loss, faster solute transport, and impaired ultrafiltration (Table 3). All of the changes induced by LPS exposure were significantly reduced in Nalp3^−/− mice, which showed a complete abolition of IL-1β release in the peritoneal cavity, and were protected against the deleterious effects of acute peritonitis on membrane inflammation and function (Figure 4). These data evidenced that NLRP3 inflammasome plays a critical role in the maturation process of IL-1β during acute peritonitis, thereby contributing to increased vascular permeability, faster solute transport, and loss of ultrafiltration capacity.

Figure 4.

Genetic deletion of Nalp3 prevents IL-1β maturation induced by LPS. (A) Quantitative RT-PCR to measure the mRNA expression of Nalp3 and Il-1β in visceral peritoneum from control mice and mice treated by LPS. Nalp3 and Il-1β are upregulated by LPS-induced peritonitis. The upregulation of Il-1β is blunted in Nalp3^−/− mice. (B) Increase of mature IL-1β release induced by LPS in peritoneal effluent of Nalp3^+/+ mice is abolished by Nalp3 deletion. (C) The LPS-induced increase of neutrophils in the dialysate of Nalp3^+/+ mice is reduced by Nalp3 deletion. (D) Quantitative RT-PCR to measure the mRNA expression of Inos and Enos in visceral peritoneum. LPS-induced peritonitis is reflected by a major upregulation of Inos whereas Enos is unchanged. The upregulation of Inos is reduced in Nalp3^−/− mice. (E) Representative immunoblots and densitometry analyses for eNOS, iNOS, and NALP3 in the visceral peritoneum of Nalp3^+/+ and Nalp3^−/− mice treated by LPS. Nalp3^+/+ mice treated by LPS show a significant upregulation of iNOS and NALP3. Nalp3 deletion reduces this upregulation, whereas the expression of eNOS remains unchanged (20 μg proteins in each lane). (F) Increase of total NO metabolites (NOx) level induced by LPS in peritoneal effluent of Nalp3^+/+ mice is reduced by Nalp3 deletion. (n=4 mice in control group, n=6 in LPS treated mice group) *P<0.05; **P<0.01; ***P<0.001. eNOS, endothelial nitric oxide synthase; iNOS, inducible nitric oxide synthase; WBC, white blood cells.

Table 3.

PD transport parameters during LPS-related peritonitis in Nalp3^−/− mice

Groups	MTAC Urea, µl/min	Dialysate Albumin, mg/ml	Net UF/Body Wt, µl/g
Nalp3+/++vehicle	16.0±0.9	2.5±0.0	57.9±0.5
Nalp3+/++LPS	33.3±0.7a	3.5±0.2a	29.2±3.2a
Nalp3−/−+vehicle	18.7±0.8	2.6±0.1	57.2±3.2
Nalp3−/−+LPS	28.6±0.8b,c	2.9±0.0b	42.2±0.3b,c

n=4 (vehicle groups), n=6 (LPS groups). MTAC, mass transfer area coefficient; UF, ultrafiltration.

^aP<0.05 Nalp3^+/+ + LPS versus Nalp3^+/+ + vehicle.

^bP<0.05 Nalp3^−/− + LPS versus Nalp3^+/+ + LPS.

^cP<0.05 Nalp3^−/− + LPS versus Nalp3^−/− + vehicle.

The Deleterious Effects of IL-1β Result from Vascular Proliferation and Increased Vascular Permeability through Endothelial IL-1β Receptor

We next investigated the mechanisms by which IL-1β may lead to peritoneal transport defects in peritonitis, by monitoring endothelial cell proliferation, angiogenesis, and microvascular permeability in vivo and in vitro (Figure 5). The IL-1β receptor (IL-1R1) was located on CD31⁺ peritoneal capillaries, where its expression closely followed the increase in vascular density during E. coli-induced peritonitis, and on CD31⁻ mononuclear inflammatory cells (Figure 5A; Supplemental Figure 1). To investigate the effect of IL-1β on endothelial cells, we exposed human umbilical vein endothelial cells (HUVECs) to IL-1β (10 ng/ml) for up to 72 hours. Exposure to IL-1β induced a time-dependent proliferation of HUVECs, with a maximum after 72 hours (Figure 5B; Supplemental Figure 2), and a strong increase in vascular permeability as assessed by FITC dextran leakage (Figure 5C). Exposure to IL-1β also enhanced the formation of capillary-like endothelial tubes by HUVECs plated on a growth factor–reduced Matrigel, with increased cumulative tube length and number of tube junctions (Figure 5D). Cotreatment of HUVECs with anakinra, a selective antagonist of IL-1β receptor used for the treatment of rheumatoid arthritis or cryopyrin-associated periodic syndromes,¹⁹ prevented the increase in tube formation and abolished vascular permeability and proliferation induced by IL-1β (Figure 5, B and D). It must be noted that lower IL-1β concentrations (1 ng/ml) had no effect on vascular proliferation or tube formation in this system (data not shown).

Figure 5.

Deleterious effects of IL-1β result from endothelial cell proliferation, angiogenesis, and increased vascular permeability through endothelial IL-1β receptor. (A) Representative immunostaining for the IL-1β receptor IL-1R1 (green channel) and CD31 (red channel) in the visceral peritoneum of control (vehicle) and E. coli-induced peritonitis mice. IL-1R1 is expressed in mononuclear inflammatory cells (CD31⁻) (arrowheads) and endothelial cells (CD31⁺) lining capillaries and small vessels (arrows) in the peritoneal membrane. Bacterial peritonitis is reflected by an increase in both IL-1R1⁺ and CD31⁺ areas in the membrane, suggesting vascular proliferation. Scale bar, 25 µm; original magnification, ×63; inset, ×200. (B) Effect of IL-1β and anakinra on HUVEC proliferation. IL-1β (10 ng/ml) induces a time-dependent increase of endothelial cells proliferation, which is decreased by anakinra treatment (1 mg/ml; n=6 per group). (C) Microvascular permeability was quantified by measuring the rate of FITC-dextran transport across endothelial cell monolayers on semipermeable membrane. The permeability of HUVEC monolayers is increased by IL-1β (100 ng/ml), and rescued by anakinra treatment (1 mg/ml; n=4 per group). (D) Representative structure of endothelial tubes forming on Matrigel after 6 hours of incubation with IL-1β (10 ng/ml) and anakinra (1 mg/ml). Quantification is matched to the length of tubes network and the number of junctions between tubes. IL-1β stimulation induces an increase of tubular network and junctions between tubes whereas anakinra inhibits these effects (n=10 per group). Scale bar, 100 µm; original magnification, ×40. *P<0.05; **P<0.01; ***P<0.001.

These data demonstrated that IL-1β induces endothelial cell proliferation and increases microvascular permeability through activation of its endothelial IL-1β receptor. They also suggested that pharmacologic blockade of this receptor might prevent deleterious structural and functional changes related to IL-1β exposure.

Pharmacologic Blockade of IL-1β Receptor Rescues Peritoneal Damage In Vivo

On the basis of the effects of anakinra on angiogenesis and microvascular permeability, we next tested whether anakinra could prevent peritoneal damage in mouse models of LPS- and E. coli-induced peritonitis (Figures 6 and 7, Table 4).

Figure 6.

Blockade of IL-1β receptor by anakinra prevents alterations during LPS-induced peritonitis. (A) Quantitative RT-PCR for the mRNA expression of Nalp3 and Il-1β in visceral peritoneum from control mice and mice treated by LPS (10 mg/kg) and LPS+anakinra (daily injection, 50 mg/kg). Nalp3 and Il-1β are upregulated by LPS-induced peritonitis, with a partial rescue observed with anakinra. (B) Increase of neutrophils induced by LPS in the dialysate is reduced by anakinra treatment. (C) Representative immunoblots and densitometry analysis for eNOS and iNOS in the visceral peritoneum of control mice and mice treated by LPS and LPS+anakinra. Mice treated by LPS show a significant upregulation of eNOS and iNOS. Anakinra totally prevents the iNOS upregulation, contrasting with a nonsignificant effect on eNOS (20 μg proteins loaded in each lane). (D) Increase of total NO metabolites (NOx) level induced by LPS in peritoneal effluent is reduced by anakinra injections (n=4 mice per group). (E) Effect of LPS-induced peritonitis and anakinra on the structure of peritoneum. The mild infiltrate induced by LPS is reduced by anakinra treatment, as shown by Masson trichrome staining. Treatment with LPS does not increase the density of CD31⁺ blood vessels and capillaries in the visceral peritoneum. Scale bars, 50 µm; original magnification, ×20. Anakinra treatment is also reflected by a decrease of IL-1β and a reduction of NALP3⁺ cells in the peritoneum. Scale bars, 50 µm; original magnification, ×40. *P<0.05; **P<0.01; ***P<0.001. eNOS, endothelial nitric oxide synthase; iNOS, inducible nitric oxide synthase.

Figure 7.

Anakinra prevents functional and structural alterations during E. coli-induced peritonitis in mice. (A) Quantitative RT-PCR for the mRNA expression of Nalp3 and Il-1β in visceral peritoneum from control (vehicle) mice and mice treated by E. coli (10⁹/ml E. coli diluted in 2 ml of dialysate) and E. coli+anakinra (daily injection, 50 mg/kg). Nalp3 and Il-1β are upregulated by E. coli-induced peritonitis. Anakinra reduces the upregulation of Nalp3 and Il-1β expression. (B) Representative immunoblots and densitometry showing levels of eNOS and iNOS in the visceral peritoneum of control mice and mice treated by E. coli and E. coli+anakinra. The induction of eNOS and iNOS expression observed in mice infected by E. coli is significantly reduced by anakinra (20 μg proteins loaded in each lane). (C) Increase of total NO metabolites (NOx) level induced by E. coli in peritoneal effluent is reduced by anakinra. (D) Effect of E. coli-induced peritonitis and anakinra on the structure of visceral peritoneum. Treatment with anakinra is reflected by a decrease in the mononuclear cell infiltrate (Masson trichrome staining); in the CD31⁺ blood vessels and capillaries; in the IL-1β⁺ inflammatory cells; and in the NALP3⁺ infiltrate in the visceral peritoneum of mice exposed to E. coli. Scale bars, 50 µm; original magnification, ×40. (E) Density of CD31⁺ vessels in the visceral peritoneum, with a vascular proliferation in mice with bacterial peritonitis that is significantly reduced by anakinra (ten fields per mouse, n=4 mice). (F) Quantification of white blood cells (WBC) in the dialysate, with induction by E. coli and rescue by anakinra. (G) Kaplan–Meier curve analysis shows that E. coli induces a high mortality, an effect that is significantly reduced by anakinra treatment (n=11 mice per group). (H) Bacterial clearance in the peritoneal cavity determined by CFU count. Anakinra treatment does not affect significantly peritoneal bacterial clearance. *P<0.05; **P<0.01; ***P<0.001. eNOS, endothelial nitric oxide synthase; iNOS, inducible nitric oxide synthase.

Table 4.

PD transport parameters during LPS or E. coli-related peritonitis

Groups	MTAC Urea, µl/min	Dialysate Albumin, mg/ml	Net UF/Body Wt, µl/g
Vehicle	20.3±0.8	2.6±0.1	56.1±1.5
LPS+vehicle	36.0±2.8a	3.4±0.2a	23.7±2.6a
LPS+anakinra	20.3±0.8b	2.7±0.2b	50.3±2.6b
Vehicle	16.5±0.8	2.3±0.1	53.0±3.4
E. coli+vehicle	35.3±2.4	3.5±0.1	8.8±2.0
E. coli+vehicle+ceftazidime	31.9±0.7c	3.0±0.2c	41.1±0.5c
E. coli+anakinra	19.8±0.9d	2.9±0.2e	32.7±3.0d
E. coli+anakinra+ceftazidime	23.5±2.0g	2.6±0.2	45.9±1.6f,g

n=4 in all groups. MTAC, mass transfer area coefficient; UF, ultrafiltration.

^aP<0.05 LPS+vehicle versus vehicle.

^bP<0.05 LPS+anakinra versus LPS+vehicle.

^cP<0.05 E. coli+vehicle+ceftazidime versus E. coli+vehicle.

^dP<0.01, ^eP<0.05 E. coli+anakinra versus E. coli+vehicle.

^fP<0.05 E. coli+anakinra+ceftazidime versus E. coli+anakinra.

^gP<0.05 E. coli+anakinra+ceftazidime versus E. coli+vehicle+ceftazidime.

Following LPS exposure, treatment with anakinra significantly attenuated the upregulation of Nalp3 and Il-1β at the mRNA and protein levels in the peritoneal membrane (Figure 6, A and E). Compared with vehicle-treated mice, treatment with anakinra significantly reduced the proportion of neutrophils in the peritoneal effluent (Figure 6B), the upregulation of inducible NOS in the peritoneal membrane (Figure 6C), and the levels of NO metabolites in the dialysate (Figure 6D). Anakinra also corrected the increase in peritoneal solute transport rate and the loss of ultrafiltration induced by LPS exposure (Table 4).

Anakinra was associated with similar protection in mice exposed to E. coli, with decreased expression of Nalp3 and Il-1β at mRNA and protein levels, decreased induction of both inducible NOS, endothelial NOS, and NO levels, as well as a significant reduction of the leukocyte infiltrate and vascular proliferation in the peritoneal membrane (Figure 7). Also, treatment with anakinra significantly improved survival of mice exposed to E. coli, with a 35% reduction in mortality rate after 48 hours, as compared with vehicle-treated mice (Figure 7G). This reduction of mortality was not related to changes in the bactericidal clearance of E. coli in this model (number of CFU [×10⁶] in the peritoneal effluent on day 6: 1.2±0.2 versus 0.9±0.1 in E. coli+vehicle and E. coli+anakinra groups, respectively; P=0.2) (Figure 7H). It must be noted that delayed anakinra initiation (24 hours after E. coli injection) still had a beneficial effect on the effects of peritonitis, as evidenced by a significant reduction in the peritoneal solute transport rate and protein losses, and a significant increase in ultrafiltration capacity, as compared with vehicle-treated mice (Supplemental Table 1).

To mimic what happens in clinical practice, mice exposed to E. coli were treated with i.p. ceftazidime (40 mg/kg per day, day 2 to day 5) or vehicle, 24 hours after the bacterial challenge, in addition to anakinra administration (Figure 8, Table 4). Compared with mice treated with vehicle, treatment with ceftazidime alone significantly improved the peritoneal transport parameters (decreased MTAC for urea and dialysate albumin levels, increased ultrafiltration). These changes were significantly amplified when mice were treated with ceftazidime and anakinra (Table 4). Compared with treatment with ceftazidime alone, the addition of anakinra in this model of E. coli-induced peritonitis resulted in (1) a significant decrease in the expression of Nalp3 and Il-1β at the mRNA (Figure 8A) and protein (Figure 8D) levels; (2) a significant reduction in the number of white blood cells (Figure 8B) and E. coli (number of CFU; Figure 8C) in the peritoneal effluent; (3) a significant decrease in the vascular density in the peritoneal membrane (Figure 8, D and E); and (4) a significant improvement of the ultrafiltration capacity paralleled by a decreased MTAC for urea (Figure 8F, Table 4).

Figure 8.

Effect of anakinra in addition to ceftazidime during E. coli-induced peritonitis in mice. (A) Quantitative RT-PCR for the mRNA expression of Nalp3 and Il-1β in visceral peritoneum from control (vehicle) mice and mice treated by E. coli + ceftazidime (10⁹/ml E. coli diluted in 2 ml of dialysate) and E. coli+anakinra+ceftazidime (anakinra, 50 mg/kg per day; ceftazidime 40 mg/kg per day). Compared with treatment with ceftazidime alone, the addition of anakinra significantly attenuates the upregulation of Nalp3 and Il-1β that is driven by E. coli-induced peritonitis. (B) Quantification of white blood cells (WBC) in the dialysate, with induction by E. coli and additive effect of ceftazidime and anakinra. (C) Bacterial clearance in the peritoneal cavity determined by number of colony forming unit (CFU) in the peritoneal effluent. Combination of anakinra and ceftazidime treatment reduces the number of peritoneal E. coli compared with mice treated with ceftazidime alone, thus increasing significantly the bactericidal clearance. (D) Effect of anakinra and ceftazidime on the peritoneum structure in E. coli-induced peritonitis. Peritonitis is reflected by a mononuclear cell infiltrate, as evidenced by Masson trichrome staining. The association of anakinra and ceftazidime induces a remarkable decrease in the infiltrate, in the density of CD31⁺ blood vessels and capillaries (scale bars, 50 µm; original magnification, ×20), and in the number of IL-1β⁺ and of NALP3⁺ infiltrating cells (scale bars, 50 µm; original magnification, ×40). (E) Density of CD31⁺ vessels in the visceral peritoneum: the vascular proliferation associated with E. coli peritonitis is significantly reduced by anakinra in addition to ceftazidime (ten fields per mouse, n=4 mice). *P<0.05; **P<0.01; ***P<0.001. (F) Effect of anakinra in addition to ceftazidime on peritoneal transport parameters (MTAC for urea, albumin in dialysate, and net ultrafiltration; 2 hour exchange with 3.86% glucose dialysate, n=4). Anakinra significantly improves the transport parameters compared with mice treated with ceftazidime alone. *P<0.05; **P<0.01; ***P<0.001. MTAC, mass transfer area coefficient; UF, ultrafiltration; BW, body weight.

These data reveal that the IL-1β receptor antagonist, anakinra, efficiently reduced IL-1β–mediated changes in peritoneal morphology and function, and improved survival in an experimental model of E. coli-induced peritonitis, on top of standard-of-care antibiotherapy.

Additional Effects of CKD and Peritonitis on Systemic but not Peritoneal Cytokine Levels

As uremia is typically associated with persistent low-grade inflammation and elevated levels of cytokines, we tested the potential additive effects of renal impairment and PD-related peritonitis on inflammatory biomarkers in established mouse models of CKD with superimposed LPS-induced peritonitis. Both in adenine-induced or uromodulin-associated kidney disease (Supplemental Figure 3), the combination of an acute i.p. inflammatory challenge and uremia synergistically enhanced the systemic inflammatory response, with plasma levels of IL-1β, IL-6, and TNF-α that were superior to the sum of those observed during kidney disease and peritonitis alone (Table 5). Altogether, these data demonstrate that uremia and PD-related peritonitis have additional effects on the systemic inflammatory status.

Table 5.

Additional effects of CKD and peritonitis on systemic but not peritoneal cytokine levels in mice

Groups	Characteristics				Systemic Levels			Peritoneal Levels
	n	Body Wt, g	Kidney Weight, mg/g Body Wt	BUN, mg/dl	IL-1β, pg/ml	IL-6, pg/ml	TNF-α, pg/ml	IL-1β, pg/ml	IL-6, pg/ml	TNF-α, pg/ml
Sham/vehicle	4	25.4±3.5	12.9±0.2	42.0±6.6	0.0±0.0	9.8±3.0	4.3±1.7	0.0±0.0	6.0±0.6	2.0±0.1
Adenine/vehicle	4	24.8±3.5	18.7±0.6a	138±26.7a	2.0±0.1b	26.0±4.8b	28.8±1.3a	N.D.	7.5±0.9	2.3±0.5
Sham/LPS	4	22.5±1.0	12.8±1.6	62.2±3.2	265±33.9	5703±100.2	711±28.0	151±31.5	550±61.2	302±30.2
Adenine/LPS	4	21.3±0.8	18.0±0.2c	129±25.1c	492±54.7c	6085±230.4	902±99.5	160±22.2	600±58.1	311±29.4
Umodwt/wt/vehicle	3	25.5±3.6	13.3±0.8	35.2±0.8	0.0±0.0	4.4±1.0	0.7±0.2	0.0±0.0	24.7±4.5	4.4±1.0
Umodmut/mut/vehicle	3	25.5±2.3	13.4±0.4	66.8±8.4d	4.1±1.5d	25.5±5.6d	13.2±3.6d	0.0±0.0	22.1±3.5	5.6±0.8
Umodwt/wt/LPS	3	24.6±4.2	14.1±1.1	60.4±3.0	202±28.0	5345±159.3	155±25.3	128±15.6	522±41.4	106±14.5
Umodmut/mut/LPS	3	24.0±2.1	15.4±0.2	98.4±11.9e	436±78.7e	5655±259.3	378±35.7f	119±20.3	512±28.8	120±8.2

N.D., not determined.

^aP<0.01,

^bP<0.05 adenine/vehicle versus sham/vehicle.

^cP<0.01 adenine/LPS versus sham/LPS.

^dP<0.05 Umod^mut/mut/vehicle versus Umod^wt/wt/vehicle.

^eP<0.05,

^fP<0.01 Umod^mut/mut/LPS versus Umod^wt/wt/LPS.

Discussion

In this study, we combined clinical, molecular, and functional data in mouse and humans to demonstrate that PD-related peritonitis activates the NLRP3 inflammasome, which in turn mediates the processing and release of IL-1β, causing infiltration by inflammatory cells in the peritoneal membrane, changes in microvascular permeability and proliferation, and functional alterations leading to ultrafiltration failure. Genetic invalidation and pharmacologic blockade of the NLRP3/IL-1β axis significantly prevented, and even restored structural and functional defects of NLRP3 activation. These data reveal novel perspectives to limit the deleterious effects of acute peritonitis on peritoneal structure and transport properties.

Inflammation is a protective response of the innate immune system triggered by stimuli including pathogens, dead cells, or other dangers. The host response must be tightly regulated: insufficient inflammation can lead to persistent infection, whereas excessive inflammation can cause tissue remodeling as well as chronic or systemic inflammatory diseases. The NLRP3 inflammasome is part of the innate immune system, acting as a sensor of a large range of stimuli like pathogen-associated molecular patterns to promote IL-1β release and induce inflammation in response to pathogens and various molecules.²⁰ Using human samples and well established mouse models, our studies reveal the critical role of NLRP3 inflammasome in structural and functional changes in the peritoneal membrane during acute inflammation associated with peritonitis. Following exposure to a single injection of LPS or to repeated exposure to E. coli, components of NLRP3 inflammasome were strongly and rapidly activated in the peritoneal membrane, leading to the release of IL-1β in the peritoneal effluent and to the production of NO. These events led to an increased microvascular permeability, accelerated dissipation of the osmotic gradient, and loss of ultrafiltration capacity (Figure 9).

Figure 9.

Role of NLRP3 and IL-1β on vascular changes during PD-related peritonitis. PD-related peritonitis induced by LPS or E. coli activates the NLRP3 inflammasome and the production of IL-1β by mononuclear cells including macrophages. The NF-κB activation and the autocrine effect of IL-1β lead to an increase of iNOS expression and the production of NO in the peritoneal cavity. The paracrine effect of IL-1β on endothelial cells and the release of NO induce VEGF and upregulate eNOS, amplifying the production of NO. The latter enhances microvascular permeability, which leads to increased small solute transport, loss of albumin, and faster dissipation of the osmotic gradient, causing ultrafiltration failure. The increased microvascular permeability is accompanied by recruitment of inflammatory cells and vascular proliferation, which further aggravates the transport defects. eNOS, endothelial nitric oxide synthase; iNOS, inducible nitric oxide synthase; NLRP3, NOD-like receptor containing pyrin domain 3; TLR4, Toll-like receptor 4; ROS, reactive oxygen species; VEGF, vascular endothelial growth factor.

Several lines of evidence demonstrate the direct role of NLRP3 activation in structural and functional defects associated with acute peritonitis. The genetic deletion of Nalp3, which had no influence on baseline parameters, led to the suppression of IL-1β release in the peritoneal effluent, a three-fold reduction in dialysate levels of NO, and a restored ultrafiltration capacity in the mouse model of LPS-induced peritonitis. In the mouse model of E. coli-induced peritonitis, pharmacologic blockade of the NLRP3/IL-1β axis with anakinra was associated with a 50% decrease in NO levels, decreased loss of proteins into the dialysate, and a restoration in peritoneal function. Also, anakinra efficiently prevented tissue remodeling by reducing vascular proliferation and inflammatory infiltration in the peritoneal membrane.

On the basis of human and mouse peritoneum samples, we show that upregulation of the NLRP3 inflammasome occurs predominantly in resident peritoneal monocytes and macrophages. The recognition of LPS or other components of the bacterial wall by toll-like receptors on the surface of white blood cells activates the transcription factor NF-κB, leading to generation of NLRP3 components and pro–IL-1β.^21,22 The cytosolic formation of NLRP3 inflammasome is then triggered either by pathogens or by factors released by damaged host cells.²³ Once activated, NLRP3 mediates the processing and release of IL-1β by immune cells to initiate and propagate the inflammatory response.²⁴

In the peritoneal membrane, the IL-1β receptor, IL-1R1, is predominantly expressed in CD31⁺ cells, including endothelial cells. We show that IL-1β directly acts on the endothelium lining peritoneal capillaries to promote vascular permeability and proliferation. These observations are in line with numerous studies linking IL-1β, inflammation, and tumor-mediated angiogenesis.^25–27 The release of IL-1β initiates and enhances inflammation, with increased vascular permeability and endothelial cell mitotic activity, remodeling of capillaries and, ultimately, increased capillary density.²⁸ Accordingly, deletion of the Il-1β gene in experimental models of aggressive cancers reduced angiogenesis, as compared with controls, thereby improving survival in these models.²⁵ These observations support the use of drugs targeting the NLRP3/IL-1β axis to regulate the inflammatory response and prevent deleterious microvascular remodeling and permeability changes.

We previously demonstrated the important role of inducible and endothelial NO synthases mediating the production of large amounts of NO during acute peritonitis.⁶ Inducible NOS is transcriptionally induced in macrophages and monocytes during infection, releasing large quantities of NO, a process that has been associated with circulatory failure and organ damage.²⁹ Endothelial NOS is constitutively expressed in endothelial cells of the peritoneal microvasculature, and upregulated in response to peritonitis.⁹ Induction of both inducible NOS and endothelial NOS is associated with increased permeability for small solutes and proteins, and loss of ultrafiltration capacity observed in mice with catheter-induced peritonitis.^8,30 These permeability changes are explained by disruption of the interendothelial junction, induced by increased NO level.³¹ Here, we show that genetic and pharmacologic blockade of the NLRP3/IL-1β axis prevents the upregulation of inducible NOS and endothelial NOS isoforms and the subsequent release of NO during acute peritonitis, supporting the view that NO acts as a downstream effector of NLRP3 and IL-1β.³² These data confirm the central role of IL-1β and NO in functional and structural changes during acute peritonitis, and demonstrate that, at least in some models, NLRP3 mediates activation of both inducible and endothelial NOS isoforms.

Because a dysregulated activity of NLRP3/IL-1β plays a role in diseases ranging from rare cryopyrinopathies to gout, type 2 diabetes, cancer, and infections,^20,32 several compounds targeting the inflammasome products have been developed and validated. These include neutralizing IL-1β antibody canakinumab, soluble decoy IL-1 receptor rilonacept, and recombinant IL-1R antagonist anakinra.³³ Our results support the translational potential of anakinra for the treatment of acute PD-related peritonitis, as suggested by its beneficial effects on peritoneal membrane structure and function in both models of peritonitis, with an approximately 40% decrease in mortality of mice with E. coli peritonitis. Its potential use in this indication is further supported by its excellent safety profile in patients with ESRD.^34,35 The early administration of anakinra in addition to appropriate empirical antibiotherapy may effectively reduce peritoneal damage and improve outcome in patients on PD with severe peritonitis. The fact that the delayed administration of anakinra still improved the functional parameters strengthens the translational potential of pharmacologic blockade of the NALP3/IL-1β axis.

Lastly, we confirmed activation of NLRP3 inflammasome in kidney diseases with the systemic release of IL-1β, and showed the additional effects of uremia and PD-related peritonitis on the systemic inflammatory status.^36,37 Conversely, uremia alone does not affect the i.p. level of cytokines, in line with recent data from the Global Fluid Study showing that i.p. and systemic inflammations are largely independent and reflect distinct processes and consequences.³⁸

In summary, PD-related peritonitis induces the activation of the NLRP3 inflammasome, which in turn mediates the processing and release of IL-1β, the production of NO, and deleterious structural and functional changes in the peritoneal membrane, causing ultrafiltration failure. The pharmacologic blockade of NLRP3/IL-1β by anakinra may have translational potential to improve outcome of patients with severe PD-related peritonitis.

Concise Methods

Detailed methods for mouse models, FACS, real-time RT-PCR, ELISA, determination of nitrite/nitrate, vascular permeability and tube formation assays, as well as antibodies and reagents are provided in Supplemental Material.

Human Peritonitis and Samples Collection

Peritoneal effluent was collected in patients on PD with peritonitis (defined as cloudy dialysate containing >100 white blood cells/mm³ and at least 50% of polymorphonuclear neutrophils) and PD controls.³⁹ Peritoneal biopsies were collected at the time of catheter withdrawal in a patient with refractory peritonitis, and in PD controls at the time of transplantation or transfer to hemodialysis. All patients gave informed consent for the process of tissues and samples obtained during routine procedures. The use of human biopsy samples and dialysate was approved by the Ethical Review Board of Saint-Luc Academic Hospital (Brussels, Belgium).

Experimental Animals and Peritonitis

Experiments were conducted using male C57BL/6J (Charles Rivers, Brussels, Belgium), Nalp3 and Umod mutant mice (Umod^mut/mut) with appropriate controls. The experiments were conducted in accordance with the National Research Council Guide for the Care and Use of Laboratory Animals and approved by the Ethics Committee of the Université catholique de Louvain.

Peritoneal Transport Studies and Tissue Sampling

Transport of water and solutes across the peritoneal membrane was investigated in a well established mouse model of PD.^8,9,40 At the end of the dwell, mice were euthanized by exsanguination and samples were processed for mRNA and protein studies.

Cell Culture

HUVECs were routinely cultured in endothelial cell growth medium (Cell Applications) and used between passages two and five.

The proliferation of endothelial cells was evaluated using PrestoBlue reagent (Invitrogen, Carlsbad, CA). Permeability was quantified by incubating monolayers of endothelial cells exposed to FITC-dextran (Merck Millipore, Darmstadt, Germany).

Tissue Staining, Immunoblotting, Immunohistochemistry, Immunofluorescence, and Vessels Counting

SDS-PAGE, immunoblotting, immunohistochemistry, and staining on human and mice peritoneum sections, and immunofluorescence were performed as previously described.^30,41 Vessels counting was performed after immunohistochemistry for the endothelial cell marker CD31. The counts were obtained from ten fields at 40× magnification (n=5 mice per group).

Data Analyses

Data are given as mean±SEM. Differences between groups were assessed by unpaired t test or one-way ANOVA, followed by Bonferroni multiple comparisons test (GraphPad Software, San Diego, CA), as appropriate.

Disclosures

None.