Dysregulated heme oxygenase-1^low M2-like macrophages augment lupus nephritis via Bach1 induced by type I interferons

Patients

All of the patients fulfilled the revised 1997 American College of Rheumatology criteria for the classification of systemic lupus erythematosus [21]. Patients enrolled in the study signed a written informed consent form that was approved by ethics committee of Yokohama City University Hospital (B130905030).

Mice

MRL/MpJ JmsSlc^-lpr/lpr (MRL/lpr) mice were obtained from Japan SLC (Hamamatsu, Japan). The Bach1 ^−/− mice (on the C57BL/6J background) used in this study have been described previously [22]. We obtained congenic mice by backcrossing with Bach1^−/− C57/BL6J for 12 generations. Male Bach1^+/− and female Bach1^+/+ MRL/lpr mice were interbred. Mice were genotyped by PCR using primers previously described [23]. Bach1^−/− MRL/lpr female mice and Bach1^+/+ MRL/lpr female mice were used in this study. Animals were maintained under specific pathogen-free conditions within the animal facility at Yokohama City University. Animal treatment protocols were approved by the Yokohama City University animal protocol ethics committee.

Urine was collected for 6 h from individual 24-week-old mice in metabolic cages (Shinano Manufacturing Co., Tokyo, Japan). Urine protein and creatinine concentrations were determined by using DC Protein Assay Reagent (Bio-Rad Laboratories, Hercules, CA, USA) and the Parameter Creatinine assay kit (R&D Systems, Minneapolis, MN, USA). Sera were collected from the tails of 20-week-old mice. Serum anti-double-stranded DNA (anti-dsDNA) antibody (immunoglobulin G [IgG]) and blood urea nitrogen (BUN) were measured using an enzyme-linked immunosorbent assay (Shibayagi, Shibukawa, Japan) [18] and a BUN colorimetric detection kit (Arbor Assays, Ann Arbor, MI, USA).

Mϕ from MRL/lpr mice were collected by peritoneal lavage with ice-cold PBS. These cells underwent positive selection by using a CD11b⁺ MACS antibody (Miltenyi Biotec, Bergisch Gladbach, Germany), followed by incubation at 37 °C for 40 minutes to remove floating cells [24].

In vitro polarization of human M1- and M2-like Mϕ

Human peripheral blood mononuclear cells were obtained from heparinized peripheral blood by gradient density centrifugation using Ficoll-Paque medium. Monocytes were purified by nonmonocyte depletion with antibody-conjugated magnetic-activated cell sorting microbeads (MACS II Monocyte Isolation Kit; Miltenyi Biotec). Cells were cultured in RPMI 1640 medium (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% FBS (2917354; MP Biomedicals, Santa Ana, CA, USA), 50 mg/ml streptomycin, and 50 U/ml penicillin. To differentiate cells into M2- or M1-like Mϕ, 50 ng/ml macrophage colony-stimulating factor (M-CSF) (216-MC; R&D Systems) or 20 ng/ml granulocyte-macrophage colony-stimulating factor (GM-CSF) (215-GM; R&D Systems) was added, respectively [25]. Cells were incubated for 10 consecutive days, and medium change was performed at days 2, 5, and 8. For some experiments, M1- and M2-like Mϕ were stimulated with lipopolysaccharide (LPS) (1 μg/ml, serotype 0111:B4; InvivoGen, San Diego, CA, USA), interferon (IFN)-α2b (1 U/μl; PBL Assay Science, Piscataway, NJ, USA), or IFN-β (1 U/μl; PBL Assay Science).

Immunohistochemical analysis

Paraffinized renal biopsy specimens were preserved at Yokohama City University Hospital. Renal pathological findings were evaluated by the pathologists in accordance with the International Society of Nephrology/Renal Pathology Society (ISN/RPS) 2003 classification of LN [26]. Renal biopsy specimens were sectioned at 3-μm thickness, deparaffinized in xylene, hydrated in ethanol, and pretreated with citrate buffer (10 mM sodium citrate, pH 6.0). Slides were autoclaved for 20 minutes, followed by a 30-minute incubation at room temperature. Endogenous peroxidase activity was disrupted with 0.3% H₂O₂ in methanol for 30 minutes. Immunohistochemistry was performed on consecutive sections using the following antibodies (Abs): CD68 (PGM1) (1:100 dilution, M0876; Dako/Agilent Technologies, Santa Clara, CA, USA), CD163 (1:200 dilution, UKNDL-L-CD163; Novocastra Laboratories, Newcastle, UK), HO-1 (1:500 dilution, ADI-OSA-110; Enzo Biochem, Farmingdale, NY, USA), and pSTAT1 (1:400 dilution, D3B7; Cell Signaling Technology, Danvers, MA, USA), and CMAF (1:50 dilution, M-153, Santa Cruz Biotechnology, Dallas, TX, USA). Abs were applied for 60 minutes at 25 °C. Slides were developed using the Dako EnVision kit (Agilent Technologies). After hematoxylin staining, total numbers of CD68⁺, CD163⁺, pSTAT1⁺, CMAF⁺, and HO-1⁺ cells within the glomeruli and representative ex-glomerular lesions were counted. The number of M1-like Mϕ and M2-like Mϕ were calculated using the following formula: < CD163 × pSTAT1/(pSTAT1 + CMAF) > and < CD163 × CMAF/(pSTAT1 + CMAF) >, where “CD163 × pSTAT1” refers to cells that are double expressors of CD163 and pSTAT1 and “CD163 × CMAF” refers to cells that are double expressors of CD163 and CMAF. For immunocytochemistry, cells incubated on chamber slides were fixed with 4% paraformaldehyde for 10 minutes, followed by application of the Abs and protocols used in the aforementioned immunohistochemical analysis. In immunofluorescence staining, blocking was performed for 1 h by 1% Tris-buffered saline (TBS)-4% goat serum-TBS. Staining was achieved using primary Ab (CD163, pSTAT1, and CMAF) for 45 minutes at room temperature as described above. We used Alexa Fluor 555 antimouse IgG (A21425, dilution 1:500; Thermo Fisher Scientific, Waltham, MA, USA) and Alexa Fluor 488 antirabbit IgG (A11034, dilution 1:500; Thermo Fisher Scientific) as a second antibody.

RT-PCR

Total RNA was extracted using the RNeasy Mini Kit (Qiagen, Hilden, Germany), and complementary DNA was prepared with Invitrogen SuperScript II enzyme (Life Technologies, Carlsbad, CA, USA), according to the manufacturers’ protocols. Primers and probes for human and mouse Hmox1 (Hs00157965_m1, Mm00516004_m1), CD163 (Hs00174705_m1, Mm00474091_m1), Bach1 (Hs00230917_m1), IL-6 (Hs00174131_m1), IL-10 (Hs00961622_m1), and Gapdh (4326317E, Mm99999915_g1) for RT-PCR were purchased from Applied Biosystems (Foster City, CA, USA). Primers for Ifnα and hypoxanthine phosphoribosyltransferase (Hprt) are listed in Additional file 1: Table S1. RT-PCR was performed using TaqMan Fast Advanced Master Mix (Applied Biosystems) or SYBR Green (Fast SYBR Green Master Mix; Applied Biosystems), and the data were analyzed with the StepOnePlus Real-Time PCR System (Applied Biosystems). The data were standardized to the expression of Gapdh or Hprt. The comparative cycle threshold method was used to semiquantify messenger RNA (mRNA) levels.

Western blot analysis

Kidney and spleen samples were lysed in lysis buffer (150 mM NaCl, 50 mM 4-[2-hydroxyethyl]-1-piperazineethanesulfonic acid, 1 mM ethylenediaminetetraacetic acid, 1% Triton X-100) in the presence of a protease inhibitor (Roche, Mannheim, Germany). After 10-minute incubation, cell membranes were disrupted by ultrasonication (Emerson Electric, St. Louis, MO, USA). Supernatants were collected after centrifugation at 15,000 rpm for 30 minutes and adjusted to an appropriate concentration with LDS sample buffer (Life Technologies) and 2-mercaptoethanol (Sigma-Aldrich). Each lysate was resolved by NuPAGE 4–12% Bis-Tris gel electrophoresis (Life Technologies) and transferred onto polyvinylidene fluoride (PVDF) membrane (Merck Millipore, Darmstadt, Germany). After blocking with 5% skim milk PBS, the PVDF membrane was probed with antimouse HO-1 monoclonal antibody (1:500 dilution) or antimouse Bach1 monoclonal antibody (1:100 dilution, F-9; Santa Cruz Biotechnology) overnight at 4 °C, followed by incubation with ECL antimouse IgG horseradish peroxidase-linked whole antibody (1:5000 dilution; GE Healthcare Life Sciences, Little Chalfont, UK) for 60 minutes at room temperature. Blots were developed using ECL Prime Western Blotting Detection Reagent (GE Healthcare Life Sciences) and exposed to the LAS-3000 Mini Imaging System (FUJIFILM, Tokyo, Japan) for 1–5 minutes.

Statistical analysis

Statistical analysis was performed by using Prism software (GraphPad Software, La Jolla, CA, USA). Data are presented as mean and SEM. p < 0.05 was considered statistically significant.

Characteristics of M2-like Mϕ in glomeruli of patients with LN

Expression of CD68, CD163, HO-1, pSTAT1, CMAF, and HO-1 was determined in the renal biopsy specimens using immunohistochemistry. Representative immunohistochemical data from a patient with LN (ISN/RPS class IV-G [A/C]) are shown in Fig. 1a–e. Average cell numbers of CD68, CD163, and HO-1 per glomerulus in the renal tissues from the patients with LN were 9.52, 7.95, and 1.78, respectively (Fig. 1f). We found that numbers of CD163⁺ cells were comparable to numbers of CD68⁺ cells in the glomerulus of LN. We also found that the number of CMAF⁺ M2-like cells was greater than that of pSTAT1⁺ M1-like cells in the glomerulus of LN (Fig. 1g). Average cell numbers of M1-like Mϕ, M2-like Mϕ, and HO-1 per glomerulus of patients with LN were 1.62, 6.33, and 1.78, respectively (Fig. 1h). Double-immunofluorescence staining in a representative case indicated that CD163⁺CMAF⁺ M2-like Mϕ are dominant compared with CD163⁺pSTAT1⁺ cells in the glomeruli of LN (Fig. 1i, j). Numbers of M2-like Mϕ per glomeruli positively correlated with urine protein levels (Fig. 1k), consistent with a previous report [6]. The number of Mϕ in glomerulus were few in LN classes I and II (Additional file 1: Figure S1). In extraglomerular lesions, of LN classes III, IV, and V, the number of M2-like Mϕ was greater than M1-like Mϕ. Also, HO-1⁺ cells were significantly fewer than numbers of M2-like Mϕ (Additional file 1: Figure S2).

Previous reports have shown that HO-1 mediates the anti-inflammatory property of M2 Mϕ [27–29]. Nevertheless, the numbers of HO-1⁺ cells were fewer than those of M2-like Mϕ. These data suggest that HO-1 expression is downregulated in glomerular Mϕ of human LN in spite of the M2-like property.

HO-1 expression is high but is repressed by type I interferons in human M2-like Mϕ generated in vitro

HO-1 is an inducible protein, and the expression is positively and negatively modulated by external environments [27]. We hypothesized that local inflammatory conditions of LN are responsible for the discrepancy between CD163/CMAF positivity and HO-1 expression in glomerular Mϕ. In the present study, we focused on effects of IFN-α on mRNA expression of both molecules in human monocyte-derived M2-like Mϕ generated in vitro, because type I IFN and the related genes are upregulated in patients with LN, including the renal tissues as a so-called IFN signature [1, 3, 30].

Concordant with previous reports [11, 12, 28], our results confirmed that both CD163 and HO-1 mRNA and protein expression was high in M2-like Mϕ compared with M1-like Mϕ in our in vitro culture systems (Fig. 2a, b, g– l ). The in vitro M2-like polarization was associated with increased mRNA of IL-10, another hallmark of M2 Mϕ, whereas that of IL-6 was comparable between M1- and M2-like Mϕ (Fig. 2c, d). The established M2-like Mϕ were further treated with type I IFNs for 4 h. The results showed that HO-1 expression was suppressed by IFN-α in M2-like Mϕ (Fig. 2b, k, and l ) [14, 31], whereas mRNA of Bach1, an HO-1 transcriptional repressor, was upregulated (Fig. 2e). Simultaneously, IL-6 mRNA levels, but not IL-10 levels, were upregulated by IFN-α. The findings were also found in M2-like Mϕ treated with either IFN-β or LPS (Additional file 1: Figure S3A). Moreover, the same results were reproduced in M2-like Mϕ derived from patients with SLE (Fig. 2f). The results suggest that the type I IFN-enriched condition in the glomerular lesions of LN suppresses HO-1 expression through upregulated Bach1 and modulates anti-inflammatory properties in M2-like Mϕ.

HO-1 upregulation by genetic ablation of Bach1 ameliorates LN in mice

We and others have reported that induction of HO-1 expression is beneficial in the treatment of LN [18, 19]. Our in vitro experiments suggest the involvement of Bach1 in IFN-α-dependent reduction of HO-1 expression in LN. To determine the role of Bach1 in the development of LN, we generated LN-prone MRL/lpr mice lacking Bach1 and asked whether recovery of HO-1 expression in M2 Mϕ by Bach1 deficiency leads to amelioration of LN.

Microsatellite analysis (ICLAS Monitoring Center, Kawasaki, Japan) confirmed the genetic purity of Bach1^−/− MRL/MpJ JmsSlc^-lpr/lpr was > 99.4% compared with wild type (Additional file 1: Figure S4). PCR analysis confirmed Bach1 deficiency in these congenic mice (Additional file 1: Figure S5). Using female mice, we observed their phenotypic changes, including renal function, over the time course. As shown in Fig. 3a, the survival rate of Bach1^−/− MRL/lpr mice was better than that of Bach1^+/+ MRL/lpr mice. Urine total protein/creatinine ratio at 24 weeks and serum BUN at 20 weeks were decreased in Bach1^−/− mice compared with Bach1^+/+ mice (Fig. 3b, c), though there was no difference in anti-dsDNA Ab(IgG) between the mice (Fig. 3d). The discrepancy between proteinuria/serum BUN and the serological findings suggest that Bach1 gene targeting modulates the innate immune process after immune complex (IC) deposition, resulting in favorable clinical outcomes of Bach1^−/− MRL/lpr mice. Therefore, we further focused on the analysis of the renal lesions (Fig. 4a–j).

Immunoblot analysis confirmed the deficiency of Bach1 and increased expression of the HO-1 protein in the kidneys and spleens of Bach1^−/− compared with Bach1^+/+ MRL/lpr mice (Fig. 3e). Concordantly, immunohistochemical analysis revealed more HO-1-expressing cells in glomeruli from Bach1^−/− mice than in those from Bach1^+/+ MRL/lpr mice, whereas no differences were found in CD68- or CD163-expressing cells (Fig. 4c, h, and k). However, M2-like polarization was more prominent in glomerular Mϕ of Bach1^−/− MRL/lpr mice than in the wild-type mice, as shown by the increased ratio of CD163⁺ cells per CD68⁺ cells (Fig. 4 l ). CMAF⁺ cells were much more numerous than pSTAT1⁺ cells in both Bach1 ^+/+ and Bach1 ^−/− mice (Fig. 4m, n). The estimated number of M2-like Mϕ was greater than M1 Mϕ in both Bach1^+/+ and Bach1^−/− mice. However, numbers of HO-1⁺ cells in glomeruli were greater in Bach1 ^−/− mice (Fig. 4o). PCR analysis also revealed higher CD163 and HO-1 mRNA expression in the kidneys of Bach1^−/− mice than in wild-type mice, whereas the IFN-α expression level was rather decreased in Bach1^−/− MRL/lpr mice (Fig. 4p–r).

To further compare the characteristics of Mϕ in these mice, Mϕ obtained from peritoneal lavage were examined (Fig. 4s, t). We found that Mϕ from Bach1^−/− MRL/lpr mice expressed higher levels of CD163 and HO-1 mRNA than wild-type mice did, suggesting that M2-like polarization is more evident in Bach1 deficiency, consistent with the results of a previous paper [24]. Collectively, these data suggest that Bach1 negatively regulates HO-1 expression and M2 Mϕ polarization, leading to the inflammation of LN.