Intrarenal Toll-like receptor 4 and Toll-like receptor 2 expression correlates with injury in antineutrophil cytoplasmic antibody-associated vasculitis

Abstract

In antineutrophil cytoplasmic antibody-associated vasculitis (AAV), Toll-like receptors (TLRs) may be engaged by infection-associated patterns and by endogenous danger signals, linking infection and innate inflammation with this autoimmune disease. This study examined intrarenal TLR2, TLR4, and TLR9 expression and renal injury in AAV, testing the hypothesis that increased TLR expression correlates with renal injury. Patients with AAV exhibited both glomerular and tubulointerstitial expression of TLR2, TLR4, and TLR9, with TLR4 being the most prominent in both compartments. Glomerular TLR4 expression correlated with glomerular segmental necrosis and cellular crescents, with TLR2 expression correlating with glomerular segmental necrosis. The extent and intensity of glomerular and tubulointerstitial TLR4 expression and the intensity of glomerular TLR2 expression inversely correlated with the presenting estimated glomerular filtration rate. Although myeloid cells within the kidney expressed TLR2, TLR4, and TLR9, TLR2 and TLR4 colocalized with endothelial cells and podocytes, whereas TLR9 was expressed predominantly by podocytes. The functional relevance of intrarenal TLR expression was further supported by the colocalization of TLRs with their endogenous ligands high-mobility group box 1 and fibrinogen. Therefore, in AAV, the extent of intrarenal TLR4 and TLR2 expression and their correlation with renal injury indicates that TLR4, and to a lesser degree TLR2, may be potential therapeutic targets in this disease.

INTRODUCTION

Antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) often involves the kidney, with necrotizing and crescentic glomerulonephritis (GN) that can result in progressive renal failure. Proteinase 3 (PR3) and myeloperoxidase (MPO) are the recognized dominant autoantigens in this disease. Whereas the pathogenesis of AAV is not yet fully understood, ANCA have been shown to be pathogenic in vitro and in vivo (42, 62). Like many autoimmune diseases, AAV is likely to result from a combination of genetic susceptibility elements and multiple environmental influences, with infections being linked both to disease onset and to relapse (31, 46, 53). Toll-like receptors are innate pattern recognition receptors (PRRs), located on the cell surface or intracellularly within endosomes (21), that recognize pathogen-associated molecular patterns (PAMPS) on microbes, and also endogenous danger-associated molecular patterns (DAMPs). Although Toll-like receptors (TLRs) play a key role in host defense, they may also link infection, danger, and autoimmunity (21).

Several lines of evidence implicate TLRs in the pathogenesis of AAV. Seasonal variations in the incidence of AAV and relapses, with a peak in winter, suggest an association with microbial infection (53), with prophylactic antibiotic therapy having the capacity to avert relapses in some patients (45). In vitro studies using human ANCA and leukocytes have demonstrated the capacity of TLRs to prime neutrophils and to promote ANCA production (17, 18), whereas in mouse models of anti-MPO GN, TLR engagement is pathogenic (19, 47). In the current studies, we chose TLR2, TLR4, and TLR9 because of their relevance in studies of peripheral leukocytes in human AAV and their pathogenicity in experimental anti-MPO GN (17, 47, 49, 50). TLR2 and TLR4 are expressed on the cell surface and recognize a range of PAMPs. These include, for TLR2, recognizing lipotechoic acids, peptidoglycans, zymosan, and lipomannan (21) and for TLR4 lipopolysaccharides (LPS) and viral glycoproteins (43). In contrast, TLR9 is intracellularly located within endosomes, where it recognizes hypomethylated CpG motifs derived from microbial and from self DNA (20, 27). TLRs have a number of endogenous ligands, including extracellular matrix proteins and heat shock proteins, and self- and altered self-nucleic acids. The nuclear protein high-mobility group box 1 (HMGB1) can bind to TLR2, TLR4, and TLR9 to induce the release of proinflammatory cytokines (39). Fibrinogen binds to both TLR4 and TLR2, which induces chemokine production by macrophages (43). TLR9 endogenous ligands include endogenous hypomethylated DNA, self DNA from necrotic cells and via neutrophil extracellular traps (NETs), and HMGB1 (20, 22, 26–28, 38).

Although several human AAV studies have examined the expression of TLRs in circulating leukocytes (17, 25, 50), only one has assessed the distribution of TLR2, TLR4, and TLR9 in kidneys from patients with AAV (58). Somewhat counterintuitively, Wang et al. found that intrarenal TLR4 (and to some degree TLR2) expression negatively correlated with histological and functional renal injury, whereas significant intrarenal TLR9 expression was not detected. The current studies aimed to characterize the cellular distribution and extent of TLR2, TLR4, and TLR9 expression in human ANCA-associated GN, to establish whether intrarenal TLRs colocalized with the endogenous ligands HMGB1 and fibrinogen, and to determine whether TLR expression correlated with histological and functional injury.

METHODS

Patient cohort and biopsy specimens.

A total of 38 patients presenting with AAV and GN (30 MPO-AAV and 8 PR3-AAV) were included in this study. Patients from this cohort were a subset of the cohort described in a previous report (38). Patients who were both ANCA and anti-glomerular basement membrane antibody (Ab) positive, or those who were MPO-ANCA positive with features of systemic lupus erythematosus were excluded from the study. No patient had a urinary tract infection or bacterial sepsis at the time of renal biopsy. Data on whether patients had evidence of a localized bacterial infection at another site, or evidence of viral infection at biopsy, were not collected. For disease comparisons, eight biopsies from patients with minimal change disease (MCD) and eight with lupus nephritis (LN) International Society of Nephrology/Renal Pathology Society (ISN/RPS) Class IV served as control tissue [2 biopsies were Class IV-G (A), 3 Class IV-G (A/C), 1 Class IV-S (A), 1 Class IV S A/C, and 1 Class IV/V-S (A/C)]. Biopsies from consented patients with ethics approval (application no. 08216B) were collected between 2001 and 2013 at Monash Medical Centre (Clayton, Victoria, Australia). Three independent pathologists examined and classified the AAV biopsies according to the classification scheme of Berden et al. (5). Of the MPO-AAV cohort, 23% were focal, 37% crescentic, 30% mixed, and 10% exhibited a sclerotic pattern. The patterns of the PR3-AAV cohort were 25% focal, 25% crescentic, 40% mixed, and 13% sclerotic. One pathologist scored the biopsies according to the proportions of normal glomeruli and those exhibiting segmental necrosis, cellular crescents, or both segmental necrosis and cellular crescents. Clinical and laboratory data were obtained from hospital records. ANCA testing was performed by both indirect immunofluorescence and enzyme-linked immunosorbent assay at presentation.

Confocal microscopy.

Sections (2 μm) of formalin-fixed paraffin-embedded tissue specimens were mounted on Superfrost Plus slides (Menzel, Braunschweig, Germany), dewaxed, rehydrated, and pretreated with antigen retrieval solution Tris-EDTA, pH 9, in a pressure cooker for 10 min, blocked (30 min) in 10% chicken sera (or donkey serum) in 5% bovine serum albumin/phosphate-buffered saline, and probed with previously validated and published antibodies against TLR2, TLR4, TLR9, CD34 (endothelial cell marker), nephrin (podocyte marker), neutrophil elastase (neutrophil marker), CD68 (macrophage marker), HMGB1, and fibrinogen (Table 1) in 1% bovine serum albumin/phosphate-buffered saline for 16 h (4°C). Fluorescent detection was achieved by incubation with either Alexa Fluor 594-conjugated chicken anti-rabbit IgG, Alexa Fluor 488-conjugated chicken anti-mouse IgG, Alexa Fluor 647-conjugated donkey anti-sheep IgG, or Alexa fluor 488-conjugated chicken anti-goat IgG (1:200, 40 min, room temperature; all from Molecular Probes). To quench tissue autofluorescence, slides were incubated with Sudan Black (0.1% in 70% ethanol, 30 min; Sigma-Aldrich, St. Louis, MO), washed in phosphate-buffered saline, and coverslipped in 4′,6-diamidino-2-phenylindole (DAPI) prolong gold (Molecular Probes). Negative controls for each TLR included isotype-matched monoclonal Abs. Positive controls included the titrating out of anti-TLR Ab on human spleen tissue during the process of optimizing staining protocols. For TLR9, three different primary Abs were used and were positive for TLR9 staining on the same section (data not shown). Fluorescent images were acquired using a NIKON C1 confocal laser-scanning head attached to a Nikon Ti-E inverted microscope (Coherent Scientific, SA, Australia); 405-, 488-, 561-, and 647-nm lasers were used to specifically excite DAPI, Alexa 488, Alexa 594, and Alexa 647. Confocal images were captured as single plane 512 × 512 × 12 bit files, converted from ND2 grayscale files to colorized RGB TIFFs using Image J (National Institutes of Health, Bethesda, MD), resized and cropped in Adobe Photoshop, and assembled using Adobe Illustrator (Adobe Systems).

Table 1.

Primary antibodies used for immunofluorescence and colocalization studies

Antibody	Specificity	Catalog No.	Dilution	Source	Ref. No.
Mouse anti-human TLR2	TLR2	ab16894	5 µg/ml	Abcam*	37, 55
Mouse anti-human TLR4	TLR4	ab22048	5 µg/ml	Abcam	14, 15
Mouse anti-human TLR9	TLR9	ab12121	5 µg/ml	Abcam	54, 65
Rabbit anti-human TLR2	TLR2	sc10739	5 µg/ml	Santa Cruz†	3, 51
Rabbit anti-human TLR4	TLR4	ab13556	1:50	Abcam	44, 67
Rabbit anti human TLR9	TLR9	ab37154	5 µg/ml	Abcam	29, 30
Goat anti-human TLR9	TLR9	ab53396	1:100	Abcam	52
Rabbit anti-human CD34	Endothelial cells	ab81289	1:50	Abcam	16, 64
Mouse anti-human CD34	Endothelial cells	QBend 10	1/50	Dako‡	40, 41
Sheep anti-human nephrin	Podocytes	LS-C150012	5 µg/ml	LifeSpan Bioscience¶	38
Sheep anti-human NE	Neutrophils	LS-B4244	1:100	LifeSpan Biosciences	23, 38
Mouse anti-human CD68	Macrophages	PG-M1	1:40	Dako	38, 59
Mouse anti human HMGB1	HMGB1	ab184532§	1:100	Abcam	8, 34
Goat anti-human fibrinogen	Fibrinogen	ab6666§	1:10,000	Abcam	7, 33

HMGB1, high-mobility group box 1; NE, neutrophil elastase, TLR2, Toll-like receptor 2; TLR4, Toll-like receptor 4; TLR9, Toll-like receptor 9.

^*Cambridge, UK;

^†Dallas, TX;

^‡Glostrup, Denmark;

^¶Seattle, MA

^§These antibodies have been discontinued by Abcam and replaced with ab77302 and ab118488 (respectively).

Analysis of TLR expression.

The expression of TLRs was measured using Image J (National Institutes of Health). Relative levels of TLR expression were quantitated as a product between TLR signal area multiplied by the TLR signal intensity. Analyses used the following formula applied to every single pixel in an image signal (TLR)_total − signal (TLR)_background = signal(TLR) expressed in arbitrary units (AU). For the measurement of glomerular TLR signal a region of interest (ROI) was drawn using the pencil tool, and the pixels were processed using the formula above. Tubulointerstitial TLR expression was assessed in 10 high-powered fields (×400), measured in Image J, and expressed as the mean per high-powered field, in AU and percent area.

Statistical analyses.

Statistical analysis for nonparametric data was performed using Mann-Whitney U-test for two groups and the Kruskal-Wallis test for three or more groups. Correlation between two independent continuous variables was analyzed using the nonparametric Spearman’s rho. Significance was set at P < 0.05, using SPSS (IBM, Armonk, NY) or GraphPad (La Jolla, CA) statistical software.

RESULTS

Patient characteristics.

A total of 38 renal biopsies from patients with a first presentation of AAV were used in this study (30 MPO-AAV and 8 PR3-AAV; Table 2). Both cohorts presented with significant renal disease, with an estimated glomerular filtration rate (eGFR) of 21 ± 3 ml·min⁻¹·1.73 m⁻² in MPO-AAV and 13 ± 3 ml·min⁻¹·1.73 m⁻² in PR3-AAV patients (P = 0.253).

Table 2.

Clinical and histological features of patients with ANCA-associated glomerulonephritis

	All AAV	MPO-AAV	PR3-AAV
Patient characteristics
n	38	30	8
Age at biopsy, yr	65 ± 3	66 ± 3	59 ± 3
Sex (F/M)	17/29	9/21	4/4
No. of glomeruli	19 ± 1	21 ± 2	14 ± 4
Laboratory values
ANCA titer, U/ml	NA	128 ± 17	142 ± 29
eGFR, ml·min−1·1.73 m−2	19 ± 3	21 ± 3	13 ± 3
ESR, mm/h	80 ± 7	72 ± 8	101 ± 7
CRP, mmol/l	94 ± 20	77 ± 22	150 ± 49
UTP-24 h, g/day	1.72 ± 0.4	1.60 ± 0.6 (n = 13)	1.88 ± 1.0 (n = 5)
Red blood cells, cells/HPF	698 ± 131	636 ± 146	899 ± 301
Extrarenal involvement	13/38	10/30	3/8
Biopsy histology, %
Normal glomeruli	34 ± 5	31 ± 5	46 ± 10
Cellular crescents	27 ± 2	26 ± 5	30 ± 8
Segmental necrosis	11 ± 4	12 ± 3	8. ± 3
Cellular crescents and segmental necrosis	7 ± 1	7 ± 2	4 ± 3

n, No. of patients; AAV, antibody-associated vasculitis; F, females; M, males; ANCA, antineutrophil cytoplasmic antibody; CRP, C-reactive protein; eGFR, estimated glomerular filtration rate; ESR, erythrocyte sedimentation rate; MPO, myeloperoxidase; HPF, high-powered field; NA not applicable; PR3, proteinase 3; UTP-24 h, 24-h urinary protein excretion.

TLR4 is more prominently expressed in glomeruli compared with TLR2 and TLR9.

To examine the relative expression of TLR2, TLR4, and TLR9, serial sections from AAV patient biopsies were probed with Ab for each TLR. Two methods were used, in separate analyses, to control for differences that may exist between the Ab used for each TLR. Both the mean intensity of staining (Fig. 1A) and the proportion of each glomerular cross section (GCS) staining positive (Fig. 1B) were assessed for each TLR, using the Image J software program. TLR2, TLR4, and TLR9 were each present in glomeruli of patients with AAV (Fig. 1C). However, the predominant TLR expressed within glomeruli was TLR4, with both the mean signal intensity (Fig. 1A) and extent of staining (Fig. 1B) being approximately six times that of TLR2 and threefold that of TLR9 (Fig. 1, A–C).

Fig. 1.

Toll-like receptor (TLR) 4 is the predominant TLR expressed within glomeruli of patients with antineutrophil cytoplasmic antibody-associated glomerulonephritis. A: semiquantitative assessment and comparison of the intensity of intraglomerular TLR2, TLR4, and TLR9 expression in antibody-associated vasculitis (AAV) patients demonstrates that, compared with TLR2 and TLR9, TLR4 is most highly expressed in glomeruli. AU, arbitrary units; GCS, glomerular cross section. B: semiquantitative assessment and comparison of the proportion of the glomerulus positive for TLR2, TLR4, or TLR9 shows a similar TLR4-dominant pattern. C: representative images showing serial sections stained with anti-TLR2, -TLR4, and -TLR9 antibodies showing that TLR4 is prominently expressed with glomeruli of AAV patients. Data are expressed as means ± SE from 38 biopsies, analyzed by the Kruskal-Wallis test. **P < 0.005 and ****P < 0.0001. Original magnification ×600, scale bar = 20 µm.

In AAV, TLR2 and TLR4 associate with both endothelial cells and podocytes, whereas TLR9 is more strongly podocyte associated.

To determine whether TLRs were differentially associated with glomerular endothelial cells or podocytes, serial sections stained for each TLR were then probed for CD34 (endothelial cells) and nephrin (podocytes). TLR4 expression was frequently associated with both endothelial cells and podocytes (Fig. 2). Expression was more prominent in areas where CD34 and nephrin had been shed (a sign of cellular damage and/or progression), and in cellular crescents (38), but less prominent in more preserved glomeruli. Like TLR4, TLR2 was also present on both endothelial cells and podocytes (Fig. 2) and was more prominent in segmental areas lacking CD34 or nephrin staining, but TLR2 was also found in glomeruli with minimal histological pathology (intact CD34 and nephrin staining, and without crescent formation). TLR9, although expressed on endothelial cells, was more prominent on podocytes and was also observed in areas of segmental necrosis and cellular crescents (Fig. 2). TLR2, TLR4, and TLR9 were also observed in areas characteristic of mesangial cells but not examined via colocalization because of the lack of specific mesangial cell markers.

Fig. 2.

In antineutrophil cytoplasmic antibody-associated glomerulonephritis, Toll-like receptor (TLR) 2 and TLR4 associate with endothelial cells and podocytes, whereas TLR9 associates largely with podocytes. Representative examples of the pattern of TLR expression with reference to glomerular endothelial cells and podocytes, with staining on the same section for each TLR (green), CD34 (red, denoting endothelial cells), nephrin (gray, denoting podocytes), and 4′,6-diamidino-2-phenylindole (DAPI, blue, nuclear marker). Insets demonstrate TLR-positive cells colocalizing with podocytes (white, nephrin) and endothelial cells (red, CD34). White arrows indicate areas of intense staining for TLR4 where markers of endothelial cells (CD34) and podocytes (nephrin) are lost because of damaged cells. Yellow arrows indicate intensely stained TLR9-positive cells (green) colocalizing with podocytes (white). Original magnification ×800, scale bar = 20 µm.

Glomerular TLR4 expression correlates with severe glomerular lesions and inversely correlates with presenting eGFR.

To determine the relationship between glomerular TLR expression and glomerular injury, correlations between the percentage of glomeruli affected with acute and severe glomerular lesions were examined as a continuous variable (Fig. 3 and Table 3). TLR4 expression reflected histological and functional injury. The intensity and extent of glomerular TLR4 expression correlated with glomeruli exhibiting both segmental necrosis and cellular crescent formation (Fig. 3, A and B). Concordant with these findings, a significant negative correlation with presenting eGFR was observed for both the intensity and the proportion of glomerular cross sections positive for TLR4 staining (Fig. 3, C and D). TLR2 expression also correlated with some parameters of disease severity (Tables 3 and 4). Glomerular expression of TLR2 correlated with the proportion of glomeruli affected by segmental necrosis, with both the intensity of staining and relative proportion of the glomerulus. Whereas the intensity of TLR2 staining alone inversely correlated with eGFR, TLR9 expression did not correlate with glomerular lesions or presenting eGFR (Tables 3 and 4). Glomerular TLR expression was evaluated in the context of the Berden classification of glomerular histopathology in AAV (5) that treats lesion type as a categorical variable. In these analyses, the relatively low number of biopsies in each category did not permit conclusive statistical results (data not shown).

Fig. 3.

Glomerular Toll-like receptor (TLR) 4 expression is associated with more severe glomerular disease, and glomerular infiltrating myeloid cells express TLR2, TLR4, and TLR9. The proportion of glomeruli with the most active glomerular lesions containing both cellular crescents and segmental necrosis correlates with both the intensity of TLR4 staining (A) and the percentage area of the glomeruli positive for staining (B). Both the intensity (C) and area (D) of TLR4 staining correlate inversely with estimated glomerular filtration rate (eGFR) at presentation. AU, arbitrary units; GCS, glomerular cross section. A–D: data are means ± SE from the 38 antibody-associated vasculitis patients’ biopsies analyzed by the nonparametric Spearman’s correlation. Kidney biopsies from n = 10 patients, stained for TLR2, TLR4, or TLR9 (green), 4′,6-diamidino-2-phenylindole (DAPI, blue, nuclear marker), and CD68 (macrophages, red, E) or neutrophil elastase (neutrophils, red, F). White arrows indicate double-positive cells for TLR and macrophages/neutrophils. Areas with asterisks denote nonmacrophage/neutrophil-positive cells staining positive for TLRs. Original magnification ×600, scale bar = 20 µm.

Table 3.

Correlation of glomerular expression of TLR2, TLR4, and TLR9 with glomeruli injury and renal function

	TLR2		TLR4		TLR9
	Intensity	Area, %	Intensity	Area, %	Intensity	Area, %
Spearman's	r	r	r	r	r	r
Normal glomeruli, %	−0.115	−0.155	−0.254	0.141	−0.239	−0.278
Cellular crescents, %	0.287	0.160	0.374*	0.229	−0.059	0.032
Segmental necrosis, %	0.354*	0.343*	0.453**	0.200	0.280	0.174
Segmental necrosis and cellular crescents, %	0.258	0.241	0.572**	0.420*	0.363	0.206

Intensity refers to the intensity of fluorescent staining measured in Image J. TLR2, Toll-like receptor 2; TLR4, Toll-like receptor 4; TLR9, Toll-like receptor 9.

^*Correlation is significant at 0.05 level (2 tailed).

^**Correlation is significant at 0.01 level (2 tailed).

Table 4.

Correlations between glomerular and tubulointerstitial TLR expression and presenting eGFR

	TLR2		TLR4		TLR9
eGFR	Intensity	Area, %	Intensity	Area, %	Intensity	Area, %
Spearman's	r	r	r	r	r	r
Glomerular	−0.389*	−0.235	−0.392*	−0.443**	−0.131	−0.188
Tubulointerstitial	−0.144	−0.012	−0.372*	−0.468**	−0.074	−0.061

Intensity refers to the intensity of fluorescent staining measured in Image J. %Area refers to the percentage area of the glomerulus with staining. TLR2, Toll-like receptor 2; TLR4, Toll-like receptor 4; TLR9, Toll-like receptor 9; eGFR, estimated glomerular filtration rate.

^*Correlation is significant at 0.05 level (2 tailed).

^**Correlation is significant at 0.005 level (2 tailed).

TLR2, TLR4, and TLR9 are expressed by glomerular infiltrating cells.

Based on studies implicating monocyte/macrophage and neutrophil TLRs in AAV, we sought evidence of TLR expression in vivo on intraglomerular infiltrating leukocytes. A subset of MPO-AAV biopsies (n = 10) was stained for TLR expression on myeloid cells, using CD68 as a marker for macrophages and neutrophil elastase (NE) as a marker for neutrophils (Fig. 3, E and F). TLR expression was prevalent in glomerular macrophages and neutrophils: TLR2 staining was evident in 41% of macrophages and 31% of neutrophils, TLR4 in 54% of macrophages and 58% of neutrophils, and TLR9 in 53% of macrophages and 81% of neutrophils. Thus, although there were infiltrating cells positive for TLRs within glomeruli, most TLR2, TLR4, and TLR9 expression related to intrinsic glomerular cells (endothelial cells and podocytes, Fig. 2).

HMGB1 and fibrinogen, endogenous TLR ligands, colocalize with glomerular TLRs.

Because the endogenous TLR ligands HMGB1 and fibrinogen have been implicated in AAV (10, 24, 32), we sought evidence of colocalization of these endogenous ligands with TLRs. HMGB1, a ligand for TLR2, TLR4, and TLR9, was present in most affected glomeruli (Fig. 4A) and colocalized most prominently with TLR4, and to a lesser degree with TLR2 and TLR9. Thus, although HMGB1 is a ligand for each of TLR2, TLR4, and TLR9, TLR4 may act as the dominant TLR for HMGB1. Fibrinogen, known to be present in segmental lesions and in glomerular crescents in AAV, and a known ligand for TLR2 and TLR4, was also present in diseased glomeruli (Fig. 4B). As for HMGB1, colocalization was most prominent in the case of TLR4. In some instances HMGB1, fibrinogen, and a TLR could be colocalized in the same cell. Colocalization of either HMGB1 or fibrinogen was not present in biopsies of patients with the nonproliferative glomerular lesion, minimal change disease (data not shown).

Fig. 4.

Colocalization of the endogenous Toll-like receptor (TLR) ligands high-mobility group box 1 (HMGB1) and fibrinogen with TLRs in glomeruli of patients with antibody-associated vasculitis (AAV). A: TLR2, TLR4, or TLR9 (green) is colocalized with the endogenous TLR ligand HMGB1 (red) within glomeruli of patient biopsies with AAV. Insets show higher-power magnification of cells positive for both HMGB1 and the relevant TLR. DAPI, 4′,6-diamidino-2-phenylindole. B: TLR2, TLR4, or TLR9 (green) is colocalized with the endogenous TLR2 and TLR4 ligand fibrinogen (red) within glomeruli of patient biopsies with AAV. Insets show higher-power magnification of colocalization of fibrinogen and the relevant TLR. Original magnification ×600, scale bar = 20 µm.

Tubulointerstitial TLR4 expression is prominent and correlates inversely with renal function.

TLR2, TLR4, and TLR9 expression was also assessed in the tubulointerstitium in AAV. Similar to the approach employed in assessing glomeruli, TLR staining in serial sections (Fig. 5A) was assessed both for mean fluorescence intensity (Fig. 5B) and the percentage area of tubulointerstitial staining in 10 high-powered fields (Fig. 5C). As for glomerular TLR expression, TLR4 was overall the most highly expressed TLR. Although there was no correlation between presenting eGFR and tubulointerstitial TLR2 or TLR9 (Table 4), there was, similar to glomerular TLR4, an inverse correlation between both TLR4 staining intensity and TLR4-positive tubulointerstitial area and the presenting eGFR (Fig. 5, D and E).

Fig. 5.

Tubulointerstitial Toll-like receptor (TLR) 2, TLR4, and TLR9 expression in antibody-associated vasculitis (AAV) patients. A: representative TLR2, TLR4, and TLR9 staining often in and around the same tubules and infiltrating cells. AU, arbitrary units; GCS, glomerular cross section. Semiquantitative assessment and comparison of the intensity (B) and the proportion (C) of tubulointerstitial high-powered field (mean of 10) positive for TLR2, TLR4, or TLR9 expression in AAV patients demonstrating a TLR4-dominant pattern. Tubulointerstitial TLR4 intensity (D) and extent (E) are negatively correlated with presenting estimated glomerular filtration rate (eGFR). Original magnification ×200, scale bar = 50 µm. *P < 0.05, **P < 0.005, and ****P < 0.0001. Data are means ± SE from 38 biopsies in each group analyzed by the Kruskal-Wallis test. AU, arbitrary units.

Intrarenal TLR expression patterns are similar in MPO-AAV and PR3-AAV.

Because MPO-AAV and PR3-AAV have differing genetic associations and may be distinct diseases with syndromic overlap (31), we analyzed TLR staining according to ANCA specificity, finding similar patterns of staining and intensity (Table 5), although numbers in the PR3-AAV group were relatively low.

Table 5.

Comparison of intrarenal TLR expression in MPO-AAV and PR3-AAV patients

	Glomerulus		Tubulointerstitium
	Intensity (AU/GCS)	Area (AU/GCS), %	Intensity (AU/HPF)	Area (AU/HPF), %
MPO-AAV (n = 30)
TLR2a	1.6 ± 1.2	4.8 ± 2.7	3.8 ± 1.5	9.3 ± 2.5
TLR4b	9.3 ± 2.5	26.9 ± 4.4	13.6 ± 2.5	27.4 ± 4.1
TLR9c	3.9 ± 0.8	14.4 ± 2.6	10.5 ± 2.1	18.2 ± 3.0
Significance†	a–b****, b–c****, a–c¶	a–b****, b–c*, a–c****	a–b***, b–c**, a–c**	a–b***, b–c¶, a–c¶
PR3-AAV (n = 8)
TLR2d	2.2 ± 1.1	9.0 ± 2.9	5.6 ± 1.3	11.5 ± 3.0
TLR4e	15.7 ± 2.3	51.0 ± 6.1	26 ± 5.6	41.7 ± 4.6
TLR9f	3.9 ± 1.3	12.9 ± 3.8	3.9 ± 1.3	16.0 ± 4.5
Significance‡	d–e***, e–f***, d–f¶	d–e***, e–f***, d–f¶	d–f*, e–f*, d–f¶	d–e***, e–f*, d–f¶

n, No. of patients; AU, arbitrary units; GCS, glomerular cross section; HPF, high-power field; MPO, myeloperoxidase; AAV, antineutrophil cytoplasmic antibody-associated vasculitis; PR3, proteinase 3; TLR2, Toll-like receptor 2; TLR4, Toll-like receptor 4; TLR9, Toll-like receptor 9. Intensity refers to the intensity of fluorescent staining measured in Image J. %Area refers to the percentage area of the glomerulus with staining.

^†Kruskal-Wallis test, reported as means ± SE of the individual TLRs compared within the MPO-AAV group denoted as TLR2 (a), TLR4 (b), and TLR9 (c).

^‡Kruskal-Wallis test, reported as means ± SE of the individual TLRs compared within the PR3-AAV group denoted as TLR2 (d), TLR4 (e), and TLR9 (f).

^¶Not significant.

^*P < 0.05,

^**P < 0.005,

^***P < 0.001, and

^****P < 0.0001.

Intrarenal TLR expression in AAV compared with minimal change disease and lupus nephritis ISN/RPS Class IV.

Renal biopsies were assessed from subjects with control diseases to determine whether TLR expression differed between different renal diseases (Fig. 6). The control diseases assessed were minimal change disease (n = 8) as a form of nonproliferative GN with preserved renal structure and ISN/RPS Class IV lupus nephritis (LN, n = 8, with the majority classed as active, global). Intrarenal TLR2 expression in AAV was significantly more than in either minimal change disease or Class IV LN (Fig. 6, A, D, G, and J). In both glomerular and tubulointerstitial compartments, TLR4 expression in AAV was more than in minimal change disease but similar to that seen in Class IV LN (Fig. 6, B, E, H, and K). Similar findings were evident for TLR9, although there was a trend in most expression parameters toward increased expression in AAV (Fig. 6, C, F, I, and L).

Fig. 6.

Comparison of Toll-like receptor (TLR) expression in antineutrophil cytoplasmic antibody (ANCA)-associated glomerulonephritis and control disease tissues, minimal change disease, and Class IV International Society of Nephrology/Renal Pathology Society lupus nephritis. TLR2, TLR4, and TLR9 expression both in the intensity of the signal (A–C) and percentage area covered (D–F) is significantly increased within glomeruli of antibody-associated vasculitis (AAV) patients compared with those with minimal change disease. The intensity and extent of TLR2 expression in glomeruli were significantly increased compared with those with lupus nephritis. In the tubulointerstitial compartment, TLR2, TLR4, and TLR9 expression was significantly increased both in intensity of signal (G–I) and the area of the tubulointerstitium expressing the relevant TLR (J–L), in the AAV patient cohort compared with minimal change disease (G–L). *P < 0.05, **P < 0.05, ***P < 0.0005, and ****P < 0.0001. Data are means ± SE from 8 minimal change disease, 10 class IV lupus nephritis, and 38 AAV biopsies, in each group analyzed by the Kruskal-Wallis test. AU, arbitrary units; GCS, glomerular cross section; HPF, high-powered field.

DISCUSSION

These studies demonstrate substantial intrarenal expression of TLR2, TLR4, and TLR9 in patients with AAV. The major findings of the studies are 1) the dominance of TLR4, its inverse correlation with presenting eGFR and correlation with severe glomerular injury, 2) the presence, albeit at a lower level, of TLR2 that was also associated with injury, 3) the clear presence of intrarenal TLR9 without a significant association with injury, 4) the colocalization of the TLR ligands HMGB1 and fibrinogen with TLRs, 5) similar patterns in both MPO-AAV and PR3-AAV, and 6) in class IV LN, the presence of similar degrees of intrarenal TLR4 and TLR9, but a paucity of TLR2 compared with AAV.

Compared with TLR2 and TLR9, TLR4 was more extensively and more strongly expressed in kidneys of AAV patients. TLR4 expression in glomeruli correlated with proportions of glomeruli exhibiting both segmental necrosis and cellular crescent formation, and inversely correlated with eGFR at biopsy. TLR4 was expressed relatively prominently in both endothelial cells and podocytes, consistent with findings in experimental MPO- and ANCA-associated GN, where TLR4 is expressed in glomerular endothelial cells and increases after stimulation with LPS and anti-MPO antibodies (49). TLR4 was also prominent in areas within glomeruli lacking endothelial and podocyte markers: these markers are lost in significant injury (38). Human glomerular endothelial cells and podocytes express the prototypic neutrophil chemoattractant CXCL8 after TLR4 engagement, linking TLR4 expression in AAV to a key mechanism of injury (9, 49). TLR2 was also present in AAV patients’ kidneys, to a lesser degree than TLR4, but significantly more than in MCD or class IV LN. TLR2 expression correlated with the proportion of glomeruli exhibiting acute segmental lesions. TLR2 in other models of disease are important for neutrophil recruitment via CXCL2 expression and Th17 cytokine production (2, 47). Our laboratory has previously shown in murine anti-MPO-GN that the TLR2 ligand pamityol-3-cysteine-serine-lysine-4 can direct autoimmunity by inducing a Th17 CD4 T cell response, inducing accumulation of neutrophils to glomeruli (47). The overexpression of TLR2 observed in AAV biopsies may be indicative of a role for TLR2 in recruiting neutrophils to damaged glomeruli, and perpetuating the vicious cycle of glomerular injury (63). The significant correlations of the percentage of glomeruli affected by segmental necrosis and/or cellular crescents with TLR2 and TLR4 suggest that these TLRs relate to the most active of glomerular lesions. Although TLR9 did not correlate with renal injury, the widespread intrarenal expression of TLR9 does not exclude a local role for TLR in amplifying inflammatory responses. TLR9 plays a role both in murine vasculitis and experimental crescentic glomerulonephritis by directing Th1 immune responses. We have previously demonstrated that TLR9-deficient mice are protected from renal injury, and administration of a TLR9 inhibitor supresses Th1 immune responses, through the reduction of glomerular effector T cell recruitment and subsequent macrophage accumulation (47, 48).

TLRs can influence AAV at multiple stages of disease pathogenesis. There is evidence from human studies and animal models of TLR involvement in loss of tolerance, in influencing the direction of autoimmunity, in the priming of neutrophils (and potentially monocytes), and in triggering disease (17, 18, 47, 50). The presence of TLRs within the kidney in AAV with TLR4 and TLR2 expression being linked to severe acute lesions implies a further role for local TLRs in promoting tissue inflammation in AAV. Although a significant proportion of neutrophils and macrophages expressed TLRs, which is consistent with TLRs contributing to their inflammatory phenotype, the majority of TLR expression was on and within intrinsic renal cells. Mechanistically, intrinsic kidney cells express TLRs and respond to TLR ligands by producing a variety of proinflammatory mediators. Functionally, in experimental AAV, intrinsic cell TLR expression is important in MPO- and ANCA-induced neutrophil localization and injury, and, in models of other renal diseases, intrinsic kidney cell expression of TLR2, TLR4, and TLR9 is also important (4, 6, 35, 56, 61). Thus, when combined with in vitro and in vivo functional studies, the current studies implicate intrarenal expression of TLRs in promoting leukocyte recruitment, local inflammation, and perpetuating local renal tissue injury.

Wang et al. examined TLR expression in AAV kidneys and demonstrated minimal intrarenal TLR9, and an inverse association with histological injury (TLR4) and presenting serum creatinine (TLR2 and TLR4) (58). In the case of TLR2 and TLR4, we found that these proinflammatory molecules did correlate with histological and functional injury. Our current results are concordant with experimental studies and support strategies aimed at limiting TLR’s functions and their consequences. The difference in results between the current study and that of Wang et al. may be influenced by the different methodologies used, such as antigen retrieval methods and the use of immunoperoxidase/chromagen detection for TLR detection as opposed to immunofluorescence. We used intensity of fluorescence since it is directly proportional to the amount of stained antigen. However, analysis of chromagen intensity by Wang et al. may be limited by the difficulty of quantifying the intensity of chromagen staining. In contrast, fluorescent staining has a more dynamic range of intensity to measure. We used the following two methods to assess TLR staining: both intensity and glomerular or tubulointerstitial proportional area positive for the relevant TLR. In addition, the cohorts of patients differed between the two studies. Wang et al. studied only 24 patients with a larger proportion of renal biopsies with glomerular cellular crescents (42% vs. our 27%). Functionally we used eGFR using the Chronic Kidney Disease Epidemiology Collaboration (CDK-EPI) with eGFR being linearly related to renal function as opposed to serum creatinine alone by Wang et al.

In the current studies we have shown that HMGB1 and fibrinogen (DAMPs) are present in glomeruli of patients with AAV and colocalize with TLRs. HMGB1, a known ligand for TLR2, TLR4, and TLR9 (20, 66), is released from both activated and necrotic cells. HMGB1 can mediate glomerular cell proliferation via TLR2 (13), signal through TLR4 to promote inflammation (66), and promote TLR9 activation by enhancing the recognition of CpG-oligodeoxynucleotides (20). Although not all studies are concordant as to whether serum HMGB1 levels reflect disease activity, circulating HMGB1 levels are elevated in AAV and also may prime neutrophils (11, 57, 60). Intraglomerular fibrinogen has long been known to be present in AAV and other forms of rapidly progressive GN (10, 36), as a product of activation of the coagulation system. However, fibrinogen is also a ligand for both TLR2 and TLR4, where it can promote chemokine expression and leukocyte recruitment (4). NETs are present in situ within glomeruli from patients with AAV (22, 38). In addition to other known endogenous TLR ligands, histones released through NETosis or dying intrinsic kidney cells act as DAMPs for TLR2, TLR4, and TLR9 and induce proinflammatory cytokine production (1). In particular, TLR9 senses extracellular DNA from necrotic and NETotic cells, which may amplify neutrophil recruitment and cytokine release (12, 27, 28).

Although there are differences between MPO-AAV and PR3-AAV as diseases (31), the current studies do not demonstrate differences in intrarenal TLR expression between patients who have lost tolerance to either MPO or PR3 (albeit with relatively low numbers of PR3-AAV subjects). TLRs were expressed only at a low level in MCD patients with proteinuria. This could be explained by the preservation of glomerular and tubulointerstitial structure (aside from podocyte foot process fusion) in this cohort of patients compared with that of the AAV patients. The degree of TLR4 and TLR9 expression was similar in AAV and Class IV LN, but TLR2 was significantly increased in AAV. This relatively selective increase in TLR2 expression may imply a pathogenic role for TLR2 in AAV.

Accumulating evidence from human observations and in vitro studies together with animal models of AAV suggest that TLRs play a major role in this disease. The current studies comprehensively demonstrate the pattern and extent of intrarenal TLR2, TLR4, and TLR9 expression in AAV. The key finding was the dominance of TLR4, which not only was the most prominently expressed TLR in this disease but also consistently correlated with important indexes of histological and functional injury. TLR2 expression also correlated with some indexes of severity while TLR9, although definitively present in the kidneys of patients with AAV, did not clearly relate to the degree of renal injury. Based on these findings, TLR4 and potentially TLR2 may be therapeutic targets in AAV.

GRANTS

Support for this study was provided by the National Health and Medical Research Council (NHMRC), Australia.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

K.M.O., A.R.K., and S.R.H. conceived and designed research; K.M.O. and A.L. performed experiments; K.M.O. and S.L.F. analyzed data; K.M.O., A.R.K., and S.R.H. interpreted results of experiments; K.M.O. prepared figures; K.M.O. drafted manuscript; K.M.O., A.R.K., and S.R.H. edited and revised manuscript; K.M.O., S.L.F., A.L., A.R.K., and S.R.H. approved final version of manuscript.