Blockade of CD354 (TREM-1) Ameliorates Anti-GBM-Induced Nephritis

CD354, Triggering Receptor of Myeloid Cells-1 (TREM-1), is a potent amplifier of myeloid immune responses. Our goal was to determine the expression and function of TREM-1 in immune-mediated nephritis. An anti-glomerular basement membrane antibody (anti-GBM)-induced nephritis model was employed, where mice were sensitized with rabbit IgG followed by anti-GBM serum to induce disease. Anti-GBM-treated 129x1/svJ mice developed severe nephritis whereas C57BL/6 (B6) mice were resistant to disease. Anti-GBM disease resulted in elevated renal TREM-1 messenger RNA (mRNA) and protein levels and increased urine TREM-1 levels in 129x1/svJ. TREM-1 blockade with an inhibitory peptide, LP17, inhibited proteinuria and renal disease as measured by glomerulonephritis class, severity of tubulointerstitial disease, crescent formation, and inflammatory cell infiltrates. In sum, TREM-1 is upregulated in renal inflammation and plays a vital role in driving disease. Thus, TREM-1 blockade emerges as a potential therapeutic avenue for immune-mediated renal diseases such as lupus nephritis.


INTRODUCTION
Renal inflammation leading to organ dysfunction and end-stage disease is a common feature of autoimmune diseases such as systemic lupus erythematosus (SLE) [1,2]. Increased knowledge of pathogenic mechanisms could greatly enhance prognosis and treatment of kidney diseases. Murine models suggest that contributing factors to immune-mediated nephritis include autoantibodies such as anti-glomerular antibodies [3,4] and innate immune activation independent of autoantibody production [5,6]. Dendritic cells maintain renal self-tolerance mediated by T regulatory cells [7]. However, resident macrophages and recruited monocytes regulate renal inflammation which can involve renal cells [8,9].
Anti-glomerular basement membrane antibody (anti-GBM)-induced disease or Goodpasture's syndrome is characterized by antibodies with specificities to renal targets, such as collagen type IV in glomerular basement membranes [10]. Murine models mimicking human anti-GBM nephritis require immunization with collagen type IV or heterologous anti-GBM antibodies which initiate an autoimmune antibody-mediated inflammatory response [10]. The murine anti-GBM model shares a number of characteristics with spontaneous lupus murine models in regard to pathogenic mechanisms contributing to nephritis including shared molecular mediators such as complement, FcR, toll-like receptors (TLR), costimulatory receptors, adhesion receptors, cytokines, chemokines, and other mediators [11][12][13]. Thus, the anti-GBM model provides a unique approach for rapidly screening agents with potential therapeutic efficacy for other more protracted varieties of immune-mediated nephritis such as lupus nephritis.
This study was designed to determine whether the novel inhibitory peptide LP17, targeting the CD354 receptor, Triggering Receptor Expressed on Myeloid Cells-1 or TREM-1, could impact anti-GBM nephritis. TREM-1 was first described as a costimulatory receptor on myeloid cells [14,15]. TREM-1 potently amplified the function of TLR and Nod-like receptors (NLR) which resulted in enhanced cytokine production [14,15]. Early studies reported a role for TREM-1 in sepsis models and found that soluble TREM-1 could serve as a biomarker for acute inflammation during infection [16,17]. Protective effects of TREM-1 blockade were also documented in acute infections [14,18]. Subsequently, a role for TREM-1 in the amplification of noninfectious inflammatory responses was reported [14,17,19]. Recently, TREM-1 activation by selfproteins in the absence of TLR/NLRs has been shown to stimulate monocyte cytokine production [20]. Antagonizing TREM-1 with antibodies, fusion proteins, or inhibitory peptides reduces inflammation in experimental arthritis and colitis models [14,21,22]. This report represents a first step toward revealing the contribution of TREM-1 to inflammatory kidney diseases. The current study demonstrates that the LP17 inhibitory peptide targeting TREM-1 profoundly subdues anti-GBM-mediated nephritis.

Mice and Anti-GBM Nephritis Model
Female 129x1/svJ mice and C57BL/6 (B6) controls (Jackson Laboratories) were maintained in a pathogen-free colony. Animal experiments were approved and conducted in accordance with UT Southwestern's Institutional Animal Care and Use Committee guidelines which comply with all applicable international and national guidelines for the care and use of animals. Anti-GBM serum was purchased from Lampire Laboratories [23,24]. Anti-GBM nephritis was induced in 8-12-week-old female mice [4,23]. Briefly, mice were pre-sensitized with rabbit IgG 250 μg/mouse, given intraperitoneally (i.p.) in complete Freund's adjuvant (Sigma). Five days later, the mice received rabbit anti-GBM serum 200 μg IgG/25 g body weight, administered intravenously (i.v.) [4,23]. This dosing regimen was sufficient to induce proteinuria but not mortality. Experiments are representative of three independent experiments.

Sample Preparation and TREM-1 Enzyme-Linked Immunosorbent Assay (ELISA)
All serum and urine samples were collected on the days indicated and centrifuged at 14,000 rpm for 5 min, immediately aliquoted and rapidly frozen. Clear supernatants were assayed for total protein with a Coomassie Plus Kit (Pierce). The serum and urine creatinine levels were measured by capillary electrophoresis diode array detector in the UT Southwestern Metabolomics core. Proteinuria was detected for samples collected over a 24-h period as described [25]. Renal eluates were prepared as described [4,23,24]. Soluble TREM-1 levels were measured by ELISA (R&D Systems).

Illumina Arrays and RT-PCR Analysis
Renal macrophages isolated by Miltenyi CD11b magnetic selection were 95 % purified populations [26]. Total RNA was purified using RNeasy Kits (Qiagen) and the RNA quality was checked with an Agilent Bioanalyzer (Agilent Technologies). Microarray analysis was performed using the Sentrix Mouse-6 v1.1 Illumina Whole Genome Expression Beadchips according to the manufacturer's protocol. Microarray data using background noise subtracted and normalized data (BeadStudio v3.1 software) was assessed as previously reported [27]. Data is shown as the average signal intensity for TREM-1 and TREM-2 messenger RNA (mRNA) levels.
Renal cortex and medullas were isolated under a dissecting microscope (Zeiss) and total cellular RNA was prepared as previously described [25]. Quantitative polymerase chain reaction (Q-PCR) was performed to detect TREM-1 mRNA expression in kidney tissue using the TaqMan reverse transcription reagent (Applied Biosystems). Primers were as follows: mouse TREM-1, forward 5′-GAGCTTGAAGGATGAGGAAGGC-3′ and reverse 5′-CAGAGTCTGTCACTTGAAGGTCAGTC-3′; mouse β-actin, forward 5′-TGGAATCCTGTGG C A T C C A T G A A A C -3 ′ a n d r e v e r s e 5 ′ -TAAAACGCAGCTCAGTAACAGTCCG-3. Expression of TREM-1 was normalized to β-actin expression using the standard ΔCT method.

Statistical Analysis
Data analysis was performed using the Mann-Whitney U test or linear regression as described in the text with either SigmaStat (SPSS, Chicago, IL) or GraphPad Prism software (San Diego, CA).

TREM-1 Blockade Ameliorates Renal Injury
To determine whether TREM-1 played a pathogenic role in anti-GBM-induced nephritis, we treated 129/SvJ mice with an antagonistic TREM-1 peptide, LP17 [18,  peptide. Mice received daily treatment with controls or LP17 peptide. e Serum creatinine levels were elevated on days 14-21 in mice receiving PBS or control peptide, but only slightly elevated in LP17-treated mice. f Proteinuria was observed in control mice, but not in LP17-treated mice through day 21. Statistical analysis was carried out by Mann-Whitney U tests to obtain the P values shown. 31]. Renal function was monitored by assessing serum creatinine levels and urine protein excretion (Fig. 1e, f). Serum creatinine levels were elevated on days 14 and 21 in vehicle and peptide controltreated animals whereas minimal changes were observed in LP17-treated mice (Fig. 1e). Proteinuria was markedly increased in control animals on days 14 and 21, but not in samples from LP17-treated mice (Fig. 1f). These studies suggest that anti-GBM-induced renal impairment was significantly improved by treatment with the LP17 peptide as compared to controls.

Renal Pathology in LP17-Treated Mice
Consistent with reported findings, 129/SvJ kidneys developed severe proliferative glomerulonephritis (GN) including mesangial proliferation with increases in matrices, focal necrosis, destruction of capillary lumens, and crescent formation [12,13,25,28]. Figure 2 shows that renal disease, including GN score, percent crescent formation, and severity of tubulointerstitial (TI) disease, was markedly reduced in LP17 treated mice, commensurate with the dramatic reduction in the renal inflammatory infiltrate and interstitial macrophages in LP17-treated mice.

Correlation of TREM-1 Expression with Renal
Pathology Finally, we correlated the TREM-1 expression in anti-GBM-induced B6 and 129/SvJ kidneys with renal pathology scores as shown in Fig. 3. TREM-1 eluted from the kidneys of anti-GBM-diseased mice with varying disease severity correlated with tubulointerstitial disease (TI) score, glomerulonephritis (GN) score, and serum creatinine levels (Fig. 3a-c).
Similarly, there was a clear correlation between renal pathology and urine soluble TREM-1 levels (Fig. 3d-f). Thus, these studies demonstrate that elevated levels of TREM-1 can be observed in anti-GBM-mediated nephritis and correlate with the degree of renal disease.

DISCUSSION
Initial reports showed that monocytes, macrophages, and neutrophils expressed the TREM-1 receptor and that it was a potent costimulator of proinflammatory cytokines [14][15][16]. More recent studies have found that TREM-1 mRNA and protein were elevated in inflammatory bowel disease, experimental colitis, inflammatory arthritis, and other noninfectious inflammatory diseases [14,21,22]. Moreover, TREM-1 activation in the absence of TLR ligation or other stimuli induced myeloid cell cytokine production including TNF, IL-6, IL-8, and MCP-1 in in vitro cultures and disease models [14,20,21].
The current studies demonstrate that anti-GBM nephritis is associated with increased expression of TREM-1 (CD354) protein and mRNA in renal tissue from the 129x1/SvJ mouse strain in contrast to the nephritis resistant B6 strain. Urine levels of soluble TREM-1 but not serum soluble TREM-1 increased significantly in samples from the disease-susceptible 129x1/SvJ nephritic strain, indicating that the local production of TREM-1 in the inflamed kidney might contribute to disease pathogenesis. Although soluble TREM-1 and TREM-2 have been shown to attenuate or restrain macrophage activation, our data suggest that TREM-1 and TREM-2 expression levels are in part dependent on genetic influences and that increased TREM-1 expression promotes the inflammatory state. Our immunohistochemistry studies in murine and human lupus renal samples indicate that TREM-1 can be expressed by the inflammatory infiltrate and renal epithelial cells in chronic disease (manuscript in preparation). Thus, collectively, our data suggest that genetics could influence the relative expression of TREM-1 by the inflamed target organ.
Our studies are the first to demonstrate the elevated expression of renal TREM-1 in the nephritis-prone 129/ SvJ strain and indicate that TREM-1 plays a critical role in the pathogenesis of inflammatory nephritis. Moreover, these studies demonstrate, for the first time, that TREM-1 is amenable to therapeutic targeting for nephritis. TREM-1 blockade markedly reduced inflammation in immunemediated nephritis. These studies suggest that TREM-1 blockade might represent an effective novel strategy to be incorporated into induction and possibly maintenance regimens for patients with immune-mediated nephritis.

ACKNOWLEDGMENTS
Author Contributions. All authors approved the article for submission and all authors were involved in drafting and/or revising the article. YD oversaw all aspects of the studies including study conception and design and supervised daily tasks for the murine nephritis induction and treatment studies, ELISAs, immunohistochemistry, data interpretation, statistical analysis, and writing of the manuscript; TW carried out array assays, data interpretation, and statistical analysis and reviewed the manuscript; XJZ is an expert in kidney pathobiology and contributed to study design and data interpretation, carried out blinded assessment of kidney scores, and performed histopathological scans and scores; LSD assisted with study supervision, data analysis, data interpretation, statistical analysis, and writing of the manuscript; and CM developed the anti-GBM assay and supervised the study including study conception and study design, data analysis, interpretation of the data and of the statistical analysis, and writing of the manuscript.

COMPLIANCE WITH ETHICAL STANDARDS
Animal experiments were approved and conducted in accordance with UT Southwestern's Institutional Animal Care and Use Committee guidelines which comply with all applicable international and national guidelines for the care and use of animals.
Conflict of Interest. The authors declare that they have no conflict of interest.
Funding. These studies were supported by National Institutes of Health (NIH) Grants R01 AR050812 (PI, Mohan), DK081872 (PI, Mohan), and P50-AR055503 (PI, Mohan). Dr. Davis and Dr. Mohan are also supported by the Alliance for Lupus Research.
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