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Abstract

Fc gamma receptor IIb (FcgRIIb) is the only inhibitory-FcgR in the FcgR family, and FcgRIIb-deficient (FcgRIIb-/-) mice develop a lupus-like condition with hyper-responsiveness against several stimulations. The activation of aryl hydrocarbon receptor (Ahr), a cellular environmental sensor, might aggravate activity of the lupus-like condition. As such, 1,4-chrysenequinone (1,4-CQ), an Ahr-activator, alone did not induce supernatant cytokines from macrophages, while the 24 h pre-treatment by lipopolysaccharide (LPS), a representative inflammatory activator, prior to 1,4-CQ activation (LPS/1,4-CQ) predominantly induced macrophage pro-inflammatory responses. Additionally, the responses from FcgRIIb-/- macrophages were more prominent than wild-type (WT) cells as determined by (i) supernatant cytokines (TNF-α, IL-6, and IL-10), (ii) expression of the inflammation associated genes (NF-κB, aryl hydrocarbon receptor, iNOS, IL-1β and activating-FcgRIV) and cell-surface CD-86 (a biomarker of M1 macrophage polarization), and (iii) cell apoptosis (Annexin V), with the lower inhibitory-FcgRIIb expression. Moreover, 8-week-administration of 1,4-CQ in 8 week old FcgRIIb-/- mice, a genetic-prone lupus-like model, enhanced lupus characteristics as indicated by anti-dsDNA, serum creatinine, proteinuria, endotoxemia, gut-leakage (FITC-dextran), and glomerular immunoglobulin deposition. In conclusion, an Ahr activation worsened the disease severity in FcgRIIb-/- mice possibly through the enhanced inflammatory responses. The deficiency of inhibitory-FcgRIIb in these mice, at least in part, prominently enhanced the pro-inflammatory responses. Our data suggest that patients with lupus might be more vulnerable to environmental pollutants.

Keywords: FcgRIIb-deficient mice; air pollution; aryl hydrocarbon receptor; systemic lupus erythematosus.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1

Figure 1

Schema of the in vitro…

Figure 1

Schema of the in vitro experiments ( A ) (details in Materials and…

Figure 1 Schema of the in vitro experiments (A) (details in Materials and Methods) and the characteristic responses of RAW246.7, a macrophage cell line, against a single activation by an aryl hydrocarbon receptor activator; 1,4-chrysenequinone (N/1,4-CQ), or lipopolysaccharide (N/LPS) (a positive-control stimulator) or the activation with the pre-treatment protocols (1,4-CQ/LPS and LPS/1,4-CQ) as determined by supernatant cytokines (B–D) and gene expression of inflammatory cytokines (E–G) and the downstream signaling molecules (TLR-4, NF-κB and aryl hydrocarbon receptor) (H–J) are demonstrated. DMEM, Dulbecco’s Modified Eagle Medium (culture media of control group). All experiments were independently performed in triplicate.
Figure 2

Figure 2

The characteristic responses of RAW246.7,…

Figure 2

The characteristic responses of RAW246.7, a macrophage cell line, against a single activation…

Figure 2 The characteristic responses of RAW246.7, a macrophage cell line, against a single activation by an aryl hydrocarbon receptor activator; 1,4-chrysenequinone (N/1,4-CQ), or a positive-control stimulator, lipopolysaccharide (N/LPS), and the activation with the pre-treatment protocols (1,4-CQ/LPS and LPS/1,4-CQ) as demonstrated by gene expression of M1 macrophage polarization (iNOS and IL-1β) (A,B) and Fc gamma receptors (FcgRs) (C–F) are demonstrated. DMEM, Dulbecco’s Modified Eagle Medium (culture media of control group). All experiments were independently performed in triplicate.
Figure 3

Figure 3

Characteristics of the responses in…

Figure 3

Characteristics of the responses in FcgRIIb −/− lupus macrophages (KO) and wild-type cells…

Figure 3 Characteristics of the responses in FcgRIIb−/− lupus macrophages (KO) and wild-type cells (WT) after a single activation by an aryl hydrocarbon receptor activator, 1,4-chrysenequinone (N/1,4-CQ), or lipopolysaccharide (N/LPS) (a positive-control stimulator) (left side of each graph) and the activation after the pre-treatment protocols (1,4-CQ/LPS and LPS/1,4-CQ) (right side of each graph) as determined by supernatant cytokines (A–C) and gene expression of the downstream signaling molecules (TLR-4, NF-κB and aryl hydrocarbon receptor) (D–F) are demonstrated. All experiments were independently performed in triplicate.
Figure 4

Figure 4

Characteristics of the responses in…

Figure 4

Characteristics of the responses in FcgRIIb −/− macrophages (KO) and wild-type cells (WT)…

Figure 4 Characteristics of the responses in FcgRIIb−/− macrophages (KO) and wild-type cells (WT) after a single activation by an aryl hydrocarbon receptor activator, 1,4-chrysenequinone (N/1,4-CQ), or a positive-control stimulator, lipopolysaccharide (N/LPS) and the activation after the pre-treatment protocols (1,4-CQ/LPS and LPS/1,4-CQ) as determined by the expression of macrophage polarization genes for M1 polarization (iNOS and IL-1β), CD-86 (M1 macrophage polarization marker) at 24 h post-stimulation (flow cytometry analysis) (A–C) and expression of Fc gamma receptors (FcgRs) (D–F) are demonstrated. All experiments were independently performed in triplicate.
Figure 5

Figure 5

The quantitative flow-cytometric analysis of…

Figure 5

The quantitative flow-cytometric analysis of FcgRIIb −/− macrophages (KO) and wild-type cells (WT)…

Figure 5 The quantitative flow-cytometric analysis of FcgRIIb−/− macrophages (KO) and wild-type cells (WT) after control media incubation (control), a single activation by an aryl hydrocarbon receptor activator, 1,4-chrysenequinone (N/1,4-CQ), or the 1,4-CQ activation after LPS pre-treatment (LPS/1,4-CQ) as stained by Annexin V and propidium iodide (PI) for the determination of early apoptosis cells (Annexin V positive, PI negative), late apoptosis cells (Annexin V positive, PI positive) and necrotic cells (Annexin V negative, PI positive) (A–C) with the representative flow-cytometric patterns (D) are demonstrated. All experiments were independently performed in triplicate.
Figure 6

Figure 6

Schema of the short-term experiments…

Figure 6

Schema of the short-term experiments (upper part of figure) and the characteristics of…

Figure 6 Schema of the short-term experiments (upper part of figure) and the characteristics of FcgRIIb−/− mice (KO) and wild-type mice (WT) mice after the pre-treatment with ip administration of lipopolysaccharide (LPS) or control phosphate buffer solution (PBS) at 24 h before ip injection with an aryl hydrocarbon receptor activator, 1,4-chrysenequinone (1,4-CQ), or PBS as indicated by serum cytokines (TNF-α, IL-6 and IL-10) (A–C) (n = 5–7/time-point) are demonstrated. Abbreviation of the protocols are PBS/PBS (control), PBS injection prior to PBS; PBS/1,4-CQ (single 1,4-CQ administration), PBS injection prior to 1,4-CQ; LPS/1,4-CQ (LPS pre-treatment before 1,4-CQ), LPS injection prior to 1,4-CQ; LPS/PBS (control of LPS pre-treatment), LPS injection prior to PBS.
Figure 7

Figure 7

Schema of the long-term daily…

Figure 7

Schema of the long-term daily ip administration for 8 weeks with aryl hydrocarbon…

Figure 7 Schema of the long-term daily ip administration for 8 weeks with aryl hydrocarbon receptor activator, 1,4-chrysenequinone (1,4-CQ) or phosphate buffer solution (PBS) control (upper part of the figure) and the characteristics of FcgRIIb−/− mice (KO) and wild-type mice (WT) as determined by lupus characteristics (anti-dsDNA, serum creatinine, and urine protein creatinine index) (A–C), serum cytokines (D–F), gut leakage (FITC-dextran assay and endotoxemia) (G,H), glomerular immunoglobulin deposition score (I), and the representative immunofluorescent glomerular pictures (J) are demonstrated (n = 7–9/time point or group). The original magnification of the glomeruli is 200x; green and blue color demonstrate mouse IgG and glomerular nuclei, respectively. The picture of PBS-administered wild-type control mice (PBS-WT) is not shown due to the similarity to PBS-KO group.
Figure 8

Figure 8

The characteristics of FcgRIIb −/− …

Figure 8

The characteristics of FcgRIIb −/− mice (KO) and wild-type mice (WT) after daily…

Figure 8 The characteristics of FcgRIIb−/− mice (KO) and wild-type mice (WT) after daily 8 week administration of aryl hydrocarbon receptor activator, 1,4-chrysenequinone (1,4-CQ) or phosphate buffer solution (PBS) control as determined by cytokines in renal tissue (A–C) and percentage of glomeruli with glomerular mesangial expansion with the representative H&E-stained histological pictures (original magnification are at 200× and 400×) (D,E) are demonstrated (n = 7–9/group). The pictures of PBS administered WT mice (PBS-WT) are not shown due to the similarity to 1,4-CQ WT group. Arrow head, glomeruli with mesangial expansion; arrow, normal glomeruli.
Figure 9

Figure 9

The proposed hypothesis demonstrates a…

Figure 9

The proposed hypothesis demonstrates a difference between FcgRIIb −/− macrophages and wild-type (WT)…

Figure 9 The proposed hypothesis demonstrates a difference between FcgRIIb−/− macrophages and wild-type (WT) macrophages. In WT, the pre-treatment by lipopolysaccharide (LPS) activates TLR-4, aryl hydrocarbon receptor (Ahr) and Fc gamma receptors (FcgRs), including the activating-FcgRs and an inhibitory-FcgRIIb [66,68,76,79], partly through NF-κB signaling. Then, the enhanced Ahr by LPS accelerates responses against a subsequent Ahr stimulation. Without the inhibitory signaling, inflammatory reaction is increased and inflammation-induced apoptosis is more prominent in FcgRIIb−/− cells than WT [53,54,55]. Both inflammation and apoptosis possibly exacerbate activity of the lupus-like condition [78]: Solid and dot line in black demonstrate direction of the activation by LPS and Ahr activator, respectively. Red- and green-colored dotted lines are an activating and inhibitory signaling, respectively. The symbol +++ and + are the estimated higher and lower intensity of the reaction, respectively.
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