Dysregulated heme oxygenase-1low M2-like macrophages augment lupus nephritis via Bach1 induced by type I interferons
Innate immunity including macrophages (Mϕ) in lupus nephritis (LN) has been gaining attention, but roles of Mϕ in LN remain uncertain.
Immunohistochemical staining was performed to determine CD68, CD163, heme oxygenase (HO)-1 (a stress-inducible heme-degrading enzyme with anti-inflammatory property), pSTAT1, and CMAF-expressing Mϕ in the glomeruli of patients with LN. Effects of type I interferons on the expression levels of CD163, HO-1, BTB and CNC homology 1 (Bach1; a transcriptional HO-1 repressor), interleukin (IL)-6, and IL-10 by human M2-like Mϕ, which were differentiated in vitro from peripheral monocytes with macrophage colony-stimulating factor, were assessed by RT-PCR and immunocytostaining. Clinical manifestations, anti-double-stranded DNA (anti-dsDNA), and local HO-1 expression were compared in Bach1-deficient and wild-type MRL/lpr mice.
The number of glomerular M2-like Mϕ correlated with the amounts of proteinuria in patients with LN. Unlike monocyte-derived M2-like Mϕ, HO-1 expression was defective in the majority of glomerular M2-like Mϕ of patients with LN. Stimulation of human M2-like Mϕ with type I interferons led to reduced HO-1 expression and increased Bach1 and IL-6 expression. Bach1-deficient MRL/lpr mice exhibited increased HO-1 expression in kidneys, prolonged survival, reduced urine proteins, and serum blood urea nitrogen levels, but serum anti-dsDNA antibody levels were comparable. Increased expression of CD163 and HO-1 was found in peritoneal Mϕ from Bach1-deficient MRL/lpr mice.
Our data suggest that dysregulated M2-like Mϕ play a proinflammatory role in LN. Bach1 is a potential therapeutic target that could restore the anti-inflammatory property of M2 Mϕ.
KeywordsLupus nephritis Heme oxygenase 1 Bach1 Type I interferons Macrophage polarization
BTB and CNC homology 1
Blood urea nitrogen
Complement component C3
Complement component C4
Glyceraldehyde 3-phosphate dehydrogenase
Granulocyte-macrophage colony-stimulating factor
Genome-wide association studies
Heme oxygenase 1
International Society of Nephrology/Renal Pathology Society
Macrophage colony-stimulating factor
Nuclear factor erythroid 2-related factor 2
Phosphorylated signal transducer and activator of transcription 1
Systemic lupus erythematosus
Systemic Lupus Erythematosus Disease Activity Index
Systemic lupus erythematosus (SLE) is an autoimmune disease with a broad spectrum of clinical presentations . Lupus nephritis (LN) occurs in approximately 25–50% of patients with SLE and remains one of the leading causes of morbidity . New immunosuppressive therapies such as mycophenolate mofetil have been improving disease outcomes in patients with SLE, but some patients are refractory to standard treatments [3, 4]. Unlike rheumatoid arthritis, development of biologics to treat LN has been challenging, partially owing to its disease heterogeneity . Thus, unmet needs remain for patients with LN who are refractory to conventional remission-induction therapy.
Although abnormalities in acquired immunity, such as the presence of autoreactive T and B cells and autoantibodies, are considered a hallmark of SLE, recent studies have also highlighted the critical roles of innate immunity, including macrophages (Mϕ), in SLE. It has been shown that Mϕ are abundantly present in LN glomerulus and that the number of glomerular Mϕ positively correlates with proteinuria level . Moreover, depletion of Mϕ ameliorated antibody-induced LN in a nephritis model . Recently, the novel concept of Mϕ subsets emerged, comprising proinflammatory, classically activated M1 Mϕ and anti-inflammatory, alternatively activated M2 Mϕ . The balance between M1 and M2 Mϕ has been implicated in the pathogenesis of nephritis . In an adriamycin-induced nephritis model, IL-10/transforming growth factor-β-modified M2 Mϕ adequately protected against renal injury . In renal biopsy specimens from patients with LN, M2c Mϕ (CD68+/CD163+) predominates over M1 Mϕ (CD68+/inducible nitric oxide synthase-positive) . Collectively, these data suggest potential roles of M2 Mϕ in LN.
M2 Mϕ highly express CD163, a scavenger receptor of hemoglobin-derived heme that is widely accepted as one of the surface markers for M2 Mϕ [11, 12]. Also, phosphorylated signal transducer and activator of transcription 1 (pSTAT1) and CMAF have been proposed as M1 and M2 Mϕ markers . Haptoglobin-bound heme captured by CD163 is engulfed into Mϕ lysosomes and degraded into biliverdin and Fe2+ by inducible enzyme heme oxygenase (HO)-1. Among the leukocytes, HO-1 is expressed mainly in monocytes/Mϕ lineage cells . Expression of HO-1 is tightly controlled by the transcriptional balance between activator nuclear factor erythroid 2-related factor 2 (Nrf2) and repressor BTB and CNC homology 1 (Bach1) [15, 16]. We and others have previously shown that HO-1 has anti-inflammatory effects and that its induction is beneficial for the treatment of various inflammatory animal models . We previously reported that peritoneal injection of hemin, a chemical inducer of HO-1, into lupus-prone MRL/lpr mice suppressed proteinuria and kidney injury . In line with our findings, Nrf2-deficient mice developed lupus-like autoimmune nephritis , whereas treatment with Nrf2 activator dimethyl fumarate ameliorated pristane-induced LN . These results reinforce the notion that induction of HO-1 could be beneficial for the treatment of LN. However, it is still unclear whether M2 Mϕ play a pathological role in human LN or whether induction of HO-1 is useful for the treatment of patients with LN.
In the present study, we demonstrate that M2-like Mϕ lacking HO-1 expression are found in LN kidneys. Supplementation of HO-1 by targeting Bach1 genes ameliorated LN in mice, suggesting that dysregulated HO-1low M2 Mϕ contribute to augmenting the inflammation of LN.
All of the patients fulfilled the revised 1997 American College of Rheumatology criteria for the classification of systemic lupus erythematosus . Patients enrolled in the study signed a written informed consent form that was approved by ethics committee of Yokohama City University Hospital (B130905030).
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 . 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 . 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)  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 .
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 . 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).
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 . 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.
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 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
Clinical features of patients with lupus nephritis at renal biopsy
Characteristics of all patients with SLE (n = 19)
Female sex, n (%)
31.0 ± 10.9
Duration of SLE, years
6.1 ± 7.6
Duration of LN, years
2.8 ± 5.8
Initial diagnosis of LN at renal biopsy, n (%)
ISN/RPS classification, n
16.0 ± 8.9
PSL, n (%)
Dosage of PSL, mg/day
15.0 ± 12.4
Concomitant immunosuppressantsb, n (%)
Urine protein, g/24 h
1.88 ± 1.93
Serum C3, mg/dl
60.1 ± 22.7 (normal range 70–129)
Serum C4, mg/dl
10.6 ± 6.9 (normal range 12–36)
Serum creatinine, mg/dl
0.65 ± 0.18 (normal range 0.48–0.82)
Anti-DNA- or anti-dsDNA antibody-positivec, n (%)
Previous reports have shown that HO-1 mediates the anti-inflammatory property of M2 Mϕ [27, 28, 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 . 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].
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.
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 . Collectively, these data suggest that Bach1 negatively regulates HO-1 expression and M2 Mϕ polarization, leading to the inflammation of LN.
In the present study, we showed high numbers of M2-like Mϕ than M1-like Mϕ within the glomerulus of patients with LN, suggesting an M2 shift in LN. However, HO-1 expression was reduced in these abundantly present M2-like Mϕ (Fig. 1). Moreover, human monocyte-derived M2-like Mϕ treated with type I IFNs showed reduced HO-1 and increased Bach1 expression. Mϕ from Bach1-deficient mice showed an M2 shift along with high HO-1 expression, consistent with a previous report . These data suggest that HO-1-expressing M2 Mϕ are necessary to regulate LN.
Over the past few decades, authors of hundreds of papers have reported the anti-inflammatory properties of HO-1 in various inflammation settings. Indeed, HO-1 deficiency in humans and mice exhibits marked inflammation caused by oxidative stress [32, 33]. Moreover, we previously reported that HO-1 knockdown by small interfering RNA resulted in an enhanced inflammatory response in human monocytes . Therefore, it is likely that these “HO-1-deficient M2-like Mϕ” in LN are proinflammatory rather than anti-inflammatory as in ordinary M2 Mϕ. Indeed, the results of the present study show low HO-1 and high IL-6 production in human M2 Mϕ stimulated with IFN-α (Fig. 2b and d). Besides, a previous study demonstrated that chemical induction of HO-1 was beneficial in the LN murine model . Along the same line, the present study provides hope that the cell population could be reversed into anti-inflammatory M2 Mϕ through induction of HO-1 via inhibition of Bach1.
In the present study, we demonstrate that Bach1 is induced by type I IFNs in human M2-like Mϕ (Fig. 2e). Elucidating the mechanisms of how type I IFNs regulate Bach1 expression is essential. Bach1 was initially identified as a Maf-binding protein, and it now is well established that Bach1 forms a heterodimer with small Maf . A recent study showed that MafB is involved in the regulation of IRF3-dependent type I IFN-inducible genes . Interestingly, genome-wide association studies (GWAS) identified Bach2, which belongs to the same family as Bach1, as being associated with SLE . These findings and our data suggest that Bach1 is critically involved in type I IFN-mediated inflammation, including SLE.
Bach2 regulates antibody class switch in B cells , thus contributing to acquired immunity because it is identified as a susceptible gene in GWAS of various autoimmune diseases . In contrast, a primary source of Bach1 is monocytes (BioGPS GeneAtlas U133A, gcrma), and Bach1 was identified as one of the core Mϕ-associated genes in mice , suggesting that Bach1 plays a strong role in innate immunity. In this context, Bach1-deficient mice showed a milder LN phenotype than wild-type mice without affecting anti-dsDNA antibody production (Fig. 3c). This fact suggests that innate immune responses after IC deposition on glomerulus are also significant in this model. Although the mainstay of current treatment strategies for LN is to suppress acquired immunity, including pathogenic autoantibody production, our data support an alternative treatment strategy directed against innate immunity via Bach1. The approach is expected to have an advantage over conventional therapies regarding safety issues, especially for complications with infection because systemic immunosuppressive effects are marginal.
There are several possible strategies to treat LN by targeting the Bach1, HO-1, and M2 Mϕ. First, inhibition of Bach1 transcription is promising. Recently, the novel Bach1 inhibitor HPP971, which increases HO-1 expression, was developed . Nrf2, a competitor to Bach1, is an alternative because Nrf2-deficient mice showed a phenotype resembling LN . Treatment with novel Nrf2 inducer TFM-735 ameliorated experimental autoimmune encephalomyelitis . Finally, supplementation or generation of normal HO-1-expressing M2 Mϕ could also be beneficial for LN. It has been shown that adoptive transplant of M2 Mϕ, but not of M1 Mϕ, reduced SLE severity in clodronate- and activated lymphocyte-derived DNA-treated mice .
Our data suggest that functional alteration of M2 Mϕ plays an important role in LN and that Bach1 is a therapeutic target for LN.
We express our gratitude to Yukihiro Toyota, Kento Ichikawa, Takayuki Akagi, and Masato Kawashima for their assistance. We thank Tom Kiper for his critical review in preparing the manuscript.
YKi is supported by grants from the Japanese Society for the Promotion of Science Grants-in-Aid for Scientific Research (grants 26713036, 15 K15374), the Yokohama Foundation for Advancement of Medical Science, the Naito Foundation, the Uehara Memorial Foundation, the Japan Intractable Diseases Research Foundation, and the Japan Rheumatism Foundation. RY is supported by grants from the Japan Society for the Promotion of Science Grant-in-Aid for Scientific Research (grant 26461468), Takeda Science Foundation, Mochida Memorial Foundation for Medical and Pharmaceutical Research, and Yokohama Foundation for Advancement of Medical Science. MTak is supported by grants from the Japan Society for the Promotion of Science Grant-in-Aid for Scientific Research (grant 26461469). MTam is supported by grants from the Japan Society for the Promotion of Science Grant-in-Aid for Scientific Research (grant 26860756). KN is supported by grants from the Japan Society for the Promotion of Science Grant-in-Aid for Scientific Research (grant 16 K08698). This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sector.
Availability of data and materials
The datasets used and analyzed during the present study are available from the corresponding author on reasonable request.
DK performed experiments, analyzed data, and wrote the manuscript. MTam, KTM, YKu, H.Nakano, and IK performed some experiments. MTam, MTak, KN, RY, KI, IA, and H.Nakajima coordinated research work and critically revised the manuscript. YKi conceived of the experimental design, analyzed data, and wrote the manuscript. All authors read and approved the final manuscript.
Ethics approval and consent to participate
Each patients enrolled in this study signed a written informed consent form, which was approved by the ethics committee of Yokohama City University Hospital (B130905030). 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.
Consent for publication
The authors declare that they have no competing interests.
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