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CD22 and CD72 contribute to the development of scleroderma in a murine model
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CD22 and CD72 contribute to the development of scleroderma in a murine model Chunyan Zhaoa , Takashi Matsushitaa , Vinh Thi Ha Nguyena , Momoko Tennichia , Manabu Fujimotob , Kazuhiko Takeharaa , Yasuhito Hamaguchia,* a b
Department of Dermatology, Faculty of Medicine, Institute of Medical, Pharmaceutical and Health Sciences, Kanazawa University, Ishikawa, Japan Department of Dermatology, Course of Integrated Medicine, Graduate School of Medicine, Osaka University, Osaka, Japan
A R T I C L E I N F O
A B S T R A C T
Article history: Received 13 September 2019 Received in revised form 1 December 2019 Accepted 10 December 2019
Background: Systemic sclerosis (SSc) is a systemic autoimmune disease that is characterized by excessive fibrosis. CD22 and CD72 are B cell-specific cell surface molecules that negatively regulate B cell function. Objective: The aim of the present study was to investigate the roles of CD22 and CD72 in a murine scleroderma model. Methods: The experimental fibrosis model was generated by subcutaneous injection of bleomycin or hypochlorous acid (HOCL) into wild-type (WT), CD22-deficient (CD22 / ), CD72-deficient (CD72 / ) and CD22 and CD72 double-deficient (CD22 / /CD72 / ) mice. We histologically assessed skin fibrosis and inflammatory cell infiltration. Cytokine and chemokine expression levels were measured by real-time polymerase chain reaction. Results: The severity of fibrosis in the skin and lung was significantly less in CD22 / , CD72 / , and CD22 / /CD72 / mice than in WT mice in the bleomycin-induced model. In the skin of bleomycintreated mice, the numbers of CD3+ T cells, CD8+ T cells, and F4/80+ macrophages were significantly lower in CD22 / , CD72 / , and CD22 / /CD72 / mice than in WT mice. The expression levels of mRNAs for IL-6, TNF-α, TGF-β, CTGF, IL-1β, IL-13, CXCL2, and ICAM-1 were significantly lower in CD22 / , CD72 / , and CD22 / /CD72 / mice than in WT mice. In the HOCL-induced model, both skin and lung fibrosis were ameliorated in CD22 / , CD72 / and CD22 / /CD72 / mice compared to WT mice. Conclusion: These results indicate that CD22 and CD72 likely play crucial roles in skin and lung fibrosis. Moreover, the inhibition of CD22 and CD72 function has potential as a therapeutic approach to SSc. © 2019 Japanese Society for Investigative Dermatology. Published by Elsevier B.V. All rights reserved.
Keywords: CD22 CD72 B cell Bleomycin Fibrosis
1. Introduction Systemic sclerosis (SSc) is characterized by vascular alterations and extensive fibrosis of the skin, lungs and other internal organs. Although the pathogenesis of SSc has not been elucidated, B cell abnormalities likely play an important role [1]. CD19, a B-cellspecific cell surface molecule that defines signal thresholds critical for humoral immune responses and autoimmunity, is overexpressed on B cells from patients with SSc [2]. In the tight-skin (TSK/+) mouse, a genetic model for human SSc, TSK/+ mice deficient in CD19 expression demonstrated significantly decreased skin fibrosis [3]. B-cell depletion with anti-CD20 monoclonal antibody ameliorated the development of skin sclerosis and autoantibody production in a
* Corresponding author at: Department of Dermatology, Faculty of Medicine, Institute of Medical, Pharmaceutical and Health Sciences, Kanazawa University, 13-1 Takaramachi, Kanazawa, Ishikawa 920-8641, Japan. E-mail address: [email protected] (Y. Hamaguchi).
murine model [4]. Thus, B cells appear to play important roles in the pathogenesis of SSc and might represent therapeutic targets. CD22 is a B cell-specific transmembrane protein [5]. The cytoplasmic domain of CD22 contains 6 tyrosine residues, including immune receptor tyrosine-based inhibition motifs (ITIMs) [5,6]. CD22-deficient (CD22 / ) B cells show strongly increased Ca2+ mobilization after BCR cross-linking [7]. The overall function of CD22 on BCR signaling is inhibitory. CD72 is a type II membrane protein and a member of the C-type lectin superfamily. CD72 contains an ITIM and an ITIM-like sequence in its cytoplasmic tail. Upon recruitment of SHP-1 to phosphorylated ITIM, CD72 negatively regulates BCR signaling. Thus far, collaboration between CD22 and CD72 in the development of fibrosis has not been examined in detail. Deficiency of inhibitory molecules on the B cell surface enhances the immune response. For instance, mice deficient in FcgRIIB, the inhibitory Fc receptors for IgG-containing IC, exhibited augmented IgG-mediated passive cutaneous anaphylaxis [8] as well as reverse passive alveolitis [9]. Contrary to this hypothesis,
https://doi.org/10.1016/j.jdermsci.2019.12.007 0923-1811/ © 2019 Japanese Society for Investigative Dermatology. Published by Elsevier B.V. All rights reserved.
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we have previously reported that the combined loss of CD22 and CD72 resulted in reduced edema and hemorrhage in the reverse Arthus reaction model [10]. In the present study, we show that the loss of either CD22 or CD72 resulted in reduced fibrosis in both skin and lung in a murine fibrosis model. Given their proinflammatory roles, CD22 and CD72 might be potential therapeutic targets for the treatment of fibrotic disease, such as SSc. 2. Materials and methods 2.1. Mice C57BL/6 wild-type (WT) mice were purchased from CLEA Japan, Inc. (Shizuoka, Japan). CD22-deficient (CD22 / ) (C57BL/6 129) mice were generated as described [7] and backcrossed to a C57BL/6 strain 12 times. CD72-deficient (CD72 / ) mice were purchased from The Jackson Laboratory. CD22 / /CD72 / mice were generated, as previously described [10]. All mice used in experiments were 8–12 weeks of age and were housed in a specific pathogen-free barrier facility and screened regularly for pathogens. The Committee on Animal Experimentation of Kanazawa University Graduate School of Medical Science approved all studies and procedures. 2.2. Flow cytometric analysis of immune cells Single cell suspensions of bone marrow (bilateral femurs), spleens and peripheral lymph nodes (paired axillary and inguinal) were generated by gentle dissection [11]. Single cell leukocyte suspensions (0.5–1.0 106) were stained, as described [11]. Cells were analyzed using a FACSCanto II flow cytometer (BD Biosciences, San Jose, CA). Data were analyzed using FlowJo software (Tree Star, Ashland, OR). Antibodies used in this study included the following: anti-B220 (RA3-6B2), anti-CD21 (7G6), anti-CD24 (M1/69), anti-CD5 (53-7.3), anti-IA/IB (M5/114.15.2), anti-CD19 (1D3) (BD Bioscience), anti-IgM (MA-69) and CD11b (M1/70) (BioLegend, San Diego, CA). 2.3. B cell turnover measurement B cell turnover in mice was measured by bromodeoxyuridine (BrdU) uptake. Mice were fed BrdU (B23151, Invitrogen, Carlsbad, CA) for 7 days and killed. The percentage of BrdU+ B cells in each tissue (bone marrow, blood, spleen) was determined by immunofluorescence staining, as described [12]. 2.4. Intradermal treatment with bleomycin Bleomycin was dissolved in sterile saline at 1 mg/mL. The mice received every other day intradermal injections of 300 mL of bleomycin into their shaved backs (the para-midline, lower back region) with a 27-gauge needle for 4 weeks. 2.5. Intradermal treatment with hypochlorous acid (HOCL) Skin and lung fibrosis was also induced by intradermal injection of hypochlorous acid (HOCL), as previously described [13] with minor modifications. Briefly, HOCL was produced by adding 166 mL of NaClO solution (9.6% as active chlorine) to 11.1 mL of KH2PO4 solution (100 mM, pH 7.2) and 400 mL (2 injections of 200 mL each) of HOCL solution were intradermally injected into the shaved backs of the mice, using a 27-gauge needle, daily for 6 weeks. 2.6. Histologic examination of lung and skin fibrosis All skin sections were taken from the bleomycin- or HOCLinjected region of the lower back and were obtained as fullthickness sections extending down to the body wall musculature
on day 28 (bleomycin model) or day 42 (HOCL model), as previously described [14,15]. Sections (6 mm in thickness) were stained with hematoxylin and eosin (H&E), Masson trichrome, or van Gieson reagents to identify collagen deposition. Expression of α-smooth muscle actin (α-SMA) was analyzed by using anti-α-SMA antibodies (Abs) conjugated to alkaline phosphatase (SigmaAldrich, St. Louis, MO). Dermal thickness, which was defined as the thickness of skin from the top of the granular layer to the junction between the dermis and intradermal fat, was evaluated independently by 2 investigators (ZC and YH) in a blinded manner. Whole lungs were harvested from the bleomycin- or HOCLtreated mice and inflated, as previously described [14]. The severity of lung inflammation was determined by a semiquantitative scoring system as previously described [16]. All sections were scored independently by 2 investigators (ZC and YH) in a blinded manner. Using the free-hand tool in the Photoshop Elements 3.0 software package (Adobe Systems), specific staining with Masson’s trichrome, van Gieson reagents, and α-SMA was quantified in the whole lung and skin sections. 2.7. Measurement of collagen contents in tissue samples The content of collagen in the skin and lung tissues was measured on day 28 (bleomycin model) or day 42 (HOCL model), as previously described [14,15]. Tissue samples were embedded in paraffin and sections approximately 15 mm thick were obtained. The method is based on the selective binding of Sirius red F3BA and Fast green FCF to collagens and non-collagenous proteins, respectively [17]. 2.8. Immunohistochemical staining Skin and lung tissues were harvested prior to and at 7 and 14 days of bleomycin treatment. The numbers of infiltrating CD3+ T cells, CD4+ T cells, CD8+ T cells, myeloperoxidase (MPO)-positive neutrophils, F4/80+ macrophages, NK1.1+ NK cells, and B220 + B cells were assessed by immunostaining, as described previously [14,15]. Sections were incubated with rat mAbs specific for CD3 (Serotec, Oxford, UK), CD4, CD8, CD45R/B220 (BD Biosciences), F4/80 (Abcam, Cambridge, UK) or NK1.1+ (BioLegend) or with rabbit mAb specific for myeloperoxidase (MPO, Neomarkers). Rat IgG (Southern Biotechnology Associates, Birmingham, AL) was used as a control for nonspecific staining. Stained cells were counted in five random grids under high-magnification (400) on a light microscope. Each section was examined independently by 2 investigators (ZC and YH), in a blinded manner, and the mean was used for analysis. 2.9. Reverse transcriptase-polymerase chain reaction (RT-PCR) Skin and lung tissues were harvested 7 or 14 days after bleomycin administration. The expression levels of mRNAs for Tnf, Il1b, Il6, Il10, Il13, Ifng, Tgfb1, connective tissue growth factor (CTGF/Ccn2), Icam1, Cxcl2, Ccl2 and Ccl3 were analyzed using a real-time polymerase chain reaction (RT-PCR) quantification method. Glyceraldehyde-3phosphate dehydrogenase mRNA (Gapdh) was used to normalize the mRNAs levels. The relative expression of RT-PCR products was determined using the DDCT method to compare target genes with housekeeping Gapdh gene mRNA expression [18]. 2.10. Regulatory T cell depletion in the bleomycin-induced fibrosis model To deplete regulatory T cells (Tregs), mice were given a single intraperitoneal (i.p.) injection of 500 mL of anti-CD25 Abs (PC61) (BioLegend) on day 0 [19]. Isotype-matched control Abs were given to WT mice. Bleomycin-induced fibrosis was induced 7 days after i.p. injection of anti-CD25 Abs or control Abs.
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2.11. Adoptive transfer experiment
2.12. Statistical analysis
Splenic B cells from WT mice were enriched by using a pan-B cell isolation kit (Miltenyi Biotec, Bergisch Gladbach, Germany). One day before induction with BLM, a total of 20 106 purified B cells was intravenously transferred into CD22 / , CD72 / , or CD22 / /CD72-/mice.
Student’s t-test was used for determining the level of significance of differences in sample means, and Bonferroni’s test was used for multiple comparisons. A P value < 0.05 was considered significant. All data are shown as means SEM. Statistical analyses were performed using JMP version 10 statistical software (Cary, NC).
Fig. 1. Skin fibrosis induced by intradermal treatment with bleomycin. CD22 / , CD72 / , CD22 / /CD72 / , and WT mice were treated every other day by intradermal injection of bleomycin for 28 days. A. Representative histological sections of skin tissue obtained from each bleomycin-treated mouse strain. Sections were stained with hematoxylin and eosin (H&E), Masson’s trichrome, or α-SMA. B–C. Analysis of the skin for determination of dermal thickness (B) and collagen content (C). D–E. Histologic evaluation of skin fibrosis. Size of fibrotic areas, as evaluated by van Gieson’s staining (D) and percentage of staining with α-SMA (E). All values represent the means SEM of results obtained from 5 to 10 mice in each group. *, p < 0.05; **, p < 0.01.
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3. Results 3.1. B cell phenotypes in the mutant mice B cell phenotypes CD22 / , CD72 / , and CD22 / /CD72 / mice are shown in Supplemental Fig. 1 and Supplemental
Table 1. The immunophenotyping results for CD22 / and CD72 / mice were consistent with those previously reported [6,7,20,21] and CD22 / /CD72 / mice did not show any phenotypes beyond those found in single CD22 / or CD72 / mice. BrdU incorporation studies showed an increase in B cell turnover both in the periphery (blood and spleen) and bone
Fig. 2. Lung fibrosis induced by intradermal treatment with bleomycin. CD22 / , CD72 / , CD22 / /CD72 / , and WT mice were treated every other day by intradermal injection of bleomycin for 28 days. A. Representative histological sections of lung tissue obtained from each bleomycin-treated mouse strain. Sections were stained with hematoxylin and eosin (H&E), Masson’s trichrome, or α-SMA. B–C. Analysis of the lungs for determination of lung fibrosis score (B) and collagen content (C). D–E. Histologic evaluation of lung fibrosis. Size of fibrotic areas, as evaluated by van Gieson’s staining (D) and percentage of staining with α-SMA (E). All values represent the mean SEM of results obtained from 5 to 10 mice in each group. *, p < 0.05; **, p < 0.01.
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marrow of CD22 / /CD72 / mice compared to WT mice, but a decrease compared to CD22 / mice. These data suggested that CD22 and CD72 play important roles in the efficiency of B cell developmental transition. In addition, B cell phenotypes of the mutant mice showed that CD22 and CD72 have redundant functions in some aspects of cell signaling.
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3.2. Amelioration of bleomycin-induced skin fibrosis by loss of CD22 and CD72 We assessed the results of intradermal administration of bleomycin to CD22 / , CD72 / , CD22 / /CD72 / and WT mice. CD22 or CD72 deficiency caused a significant reduction in the
Fig. 3. Inflammatory cell infiltration in the skin of CD22 / , CD72 / , CD22 / /CD72 / , and WT mice during intradermal bleomycin treatment. Numbers of cells per high powered field: CD3+ T cells (A), CD4+ T cells (B), CD8+ T cells (C), F4/80+ macrophages (D), myeloperoxidase (MPO)-positive neutrophils (E) and NK1.1+ NK cells (F) on days 7 and 14. Bars show the means SEM of results obtained from 5 to 10 mice in each group. *, p < 0.05; **, p < 0.01. Representative images showing CD3 (G) and F4/80 (H) staining in tissue sections from CD22 / , CD72 / , CD22 / /CD72 / , and WT mice. Original magnification, x 400.
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mean dermal thickness compared to WT mice (p < 0.05 for each), as evaluated by H&E staining or Masson’s trichrome staining (Fig. 1A and B). The loss of both CD22 and CD72 significantly reduced the mean dermal thickness compared to WT mice (p < 0.05). The degree of reduction observed when both CD22 and CD72 were absent was similar to that observed with single molecule deficiency. The local skin collagen content and the size of fibrotic areas, as evaluated by van Gieson’s staining, were consistent with the findings for dermal thickness (Fig. 1C and 1D). Likewise, the frequencies of α-SMA– positive myofibroblasts were significantly reduced in CD22 / , CD72 / and CD22 / /CD72-/- mice compared to WT mice
(Fig. 1A and E). In contrast, the degrees of skin fibrosis were comparable among mutant mice. Thus, loss of CD22 or CD72, or both ameliorated skin fibrosis induced by daily intradermal bleomycin treatment. 3.3. Bleomycin-induced lung fibrosis after loss of CD22 and CD72 We assessed lung fibrosis in mutant and WT mice (Fig. 2A). The lung fibrosis scores were significantly lower in CD22 / , CD72 / , and CD22 / /CD72-/- mice compared to WT mice (p < 0.05 for each; Fig. 2B). The local lung collagen content and the size of fibrotic areas, as evaluated by van Gieson’s staining, were
Fig. 4. mRNA expression levels of cytokines and chemokines in the skin of CD22 / , CD72 / , CD22 / /CD72 / , and WT mice during intradermal bleomycin treatment. The mRNA amounts were measured by real-time PCR and normalized to Gapdh mRNA. Bars show the means SEM of results obtained with 5 mice in each group. *, p < 0.05; **, p < 0.01.
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consistent with the findings for lung fibrosis score (Fig. 2C and D). Likewise, the frequencies of α-SMA–positive myofibroblasts were significantly reduced in CD22 / , CD72 / and CD22 / /CD72-/mice compared to WT mice (p < 0.01 for each; Fig. 2A and E). In contrast, the severities of lung fibrosis were similar among mutant mice. Thus, in the same manner as observed in skin, loss of CD22 or CD72, or both ameliorated lung fibrosis induced by intradermal bleomycin treatment.
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3.4. Leukocyte infiltration in bleomycin-induced skin fibrosis Leukocyte infiltration of the skin was assessed in tissue sections after 7 or 14 days of bleomycin treatment (Fig. 3). Infiltrating CD3+ T cell numbers after 7 or 14 days of bleomycin treatment were significantly reduced in CD22 / , CD72 / , and CD22 / /CD72 / mice compared with WT mice (p < 0.01 for each; Fig. 3A and G). CD8+ T cells were also significantly decreased
Fig. 5. Skin and lung fibrosis induced by intradermal treatment with hypochlorous acid (HOCL). CD22 / , CD72 / , CD22 / /CD72 / and WT mice were treated with daily intradermal injections of HOCL for 42 days. A. Representative histological sections of skin tissues obtained from each HOCL-treated mouse strain. Sections were stained with hematoxylin and eosin (H&E) or Masson’s trichrome. B. Analysis of the skin tissues for determination of dermal thickness (left panel) and collagen content (right panel). C. Representative histological sections of lung tissue obtained from each HOCL-treated mouse strain. Sections were stained with H&E or Masson’s trichrome. D. Analysis of the lungs for determination of lung fibrosis score (left panel) and collagen content (right panel). All values represent the means SEM of results obtained from 5 to 10 mice in each group. *, p < 0.05; **, p < 0.01.
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in CD22 / , CD72 / and CD22 / /CD72 / mice compared with WT mice (p < 0.01 for each; Fig. 3C) at 7 days. However, a significant reduction was observed only between CD72 / and WT mice at 14 days (p < 0.01; Fig. 3C). F4/80+ macrophage numbers were significantly decreased in CD22 / , CD72 / , and CD22 / /CD72 / mice compared with WT mice after 7 days (p < 0.01 for each) and 14 days (p < 0.05,
p < 0.01, and p < 0.01, respectively) after bleomycin treatment (Fig. 3D and H). MPO+ neutrophil numbers were significantly reduced in CD72 / and CD22 / /CD72 / mice compared with WT mice after 7 days (p < 0.01 for each) and 14 days (p < 0.05 and p < 0.01, respectively) of bleomycin treatment (Fig. 3E). CD22 / mice showed a decrease in the number of infiltrating neutrophils after 7
Fig. 6. Adoptive transfer experiment to CD22 / , CD72 / , and CD22 / /CD72 / mice. Isolated B cells from WT mice were intravenously transferred into CD22 / , CD72 / , and CD22 / /CD72 / mice after which skin and lung fibrosis were induced by intradermal treatment with bleomycin. A. Representative histological sections of skin tissues obtained from each bleomycin-treated mouse strain. Sections were stained with hematoxylin and eosin (H&E) or Masson’s trichrome. B. Analysis of skin tissues for determination of dermal thickness (left panel) and collagen content (right panel). C. Representative histological sections of lung tissue obtained from each bleomycin-treated mouse strain. Sections were stained with H&E or Masson’s trichrome. D. Analysis of the lungs for determination of lung fibrosis score (left panel) and collagen content (right panel). All values represent the means SEM of results obtained from 3 to 5 mice in each group.
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or 14 days of bleomycin treatment, but the differences did not reach significance. Although there were several significant differences in infiltrating CD4+ T and NK1.1+ NK cell numbers between knockout and WT mice, the degree of infiltration was not associated with the severity of fibrosis. We detected almost no or very few B220+ B cells in the skin of normal and fibrotic mice (data not shown). These results suggest that CD22 and CD72 cooperatively play important roles in the migration of T cells, neutrophils and macrophages into inflamed skin tissue in a bleomycin fibrosis model. 3.5. Cytokine and chemokine mRNA expression in bleomycin-induced skin fibrosis We investigated whether CD22 and CD72 deficiencies affected the expression of fibrogenic and anti-fibrogenic factors in the skin of mice receiving bleomycin (Fig. 4). Regarding fibrogenic cytokines, Tnf and Il13 mRNA expression levels in CD22 / , CD72 / , and CD22 / /CD72 / mice were significantly decreased 7 days after bleomycin treatment. Il1b and Il6 mRNA expression levels in CD22 / , CD72 / , and CD22 / /CD72 / mice were significantly decreased 14 days after bleomycin treatment. Tgfb1 and Ccn2 mRNA expression levels in CD22 / , CD72 / , and CD22 / /CD72 / mice were significantly decreased both 7 and 14 days after bleomycin treatment. Regarding anti-fibrogenic cytokines, Il10 mRNA expression levels in CD22 / , CD72 / , and CD22 / /CD72 / mice were significantly increased both 7 and 14 days after bleomycin treatment, except for the comparison between CD22 / and WT mice at day 14. Although IFN-g is considered an anti-fibrogenic cytokine, Ifng mRNA expression levels in CD22 / , CD72 / and CD22 / /CD72 / mice were significantly decreased 7 days after bleomycin treatment. Thus, the reduced skin inflammatory responses caused by the loss of CD22 and/or CD72 molecules were associated with a reduced release of profibrogenic and an increased release of anti-fibrogenic cytokines. Regarding chemokine expression, Icam1 and Cxcl2 mRNA expression levels in CD22 / , CD72 / , and CD22 / /CD72 / mice were significantly decreased on day 7. Although several significant differences were observed between knockout and WT mice for Ccl2 and Ccl3 mRNAs expression levels, there was no clear association between the severity of fibrosis and the mRNA expression levels of these chemokines. Therefore, CD22 and CD72 can affect Icam1 and Cxcl2 expression, leading to the regulation of inflammatory cell infiltration in bleomycin-induced fibrosis. 3.6. Skin and lung fibrosis in mice treated with HOCL We asked whether the roles of CD22 and CD72 observed in the bleomycin model were a general phenomenon. In the HOCL-induced fibrosis model, both skin and lung fibrosis were significantly ameliorated in CD22 / , CD72 / and CD22 / /CD72 / mice compared to WT mice (Fig. 5A-D). Thus, the role of CD22 and CD72 in the development of fibrosis was confirmed in a different fibrosis model. 3.7. Adoptive transfer experiment in mutant mice Further adoptive transfer experiments were conducted to confirm the role of CD22 and CD72 in the development of fibrosis. The degree of skin and lung fibrosis in CD22 / , CD72 / and CD22 / /CD72 / mice that received isolated B cells from WT mice was comparable to that in WT mice (Fig. 6A-D). Therefore, CD22 and CD72 have pivotal roles in the development of fibrosis.
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3.8. Treg cell depletion in mice treated with bleomycin The reverse Arthus reaction model using CD22 / and CD72 / mice showed that Treg cells had a crucial role in the development of vasculitis [10]. Therefore, we conducted Treg cell depletion therapy in bleomycin-induced fibrosis. The significant differences regarding the degree of both skin and lung fibrosis were unchanged between WT mice treated with control Abs and knockout mice treated with anti-CD25 Abs (data not shown). Therefore, Treg cells did not have a substantial role in CD22 / and CD72 / mice in bleomycin-induced fibrosis. 4. Discussion This study is the first to elucidate the contributions of CD22 and CD72 (alone and together) on the development of bleomycininduced fibrosis. In the present study, bleomycin-induced skin and lung fibrosis were significantly attenuated in CD22 / /CD72 / mice compared with WT mice (Fig. 1 and 2). Moreover, inflammatory leukocyte infiltration, which was composed primarily of T cells and macrophages in the skin lesions, was significantly decreased in knockout mice compared with WT mice during bleomycin-induced skin fibrosis (Fig. 3). Reduced infiltration of inflammatory leukocytes was correlated with a decrease in the pro-fibrotic cytokines TNF-α, IL-1β, IL-6, IL-13, TGF-β and CTGF and the chemokines ICAM-1 and CXCL2 (Fig. 4). The roles of CD22 and CD72 in skin and lung fibrosis were confirmed in another model in which fibrosis was induced by HOCL (Fig. 5). Adoptive transfer of B cells from WT mice into knockout mice exacerbated the fibrosis to the same degree as WT mice (Fig. 6). Taken together, these results demonstrate that CD22 and CD72 contribute to the development of fibrosis by regulating the migration of leukocytes via control of the production of cytokines and chemokines. Mice lacking CD22 or CD72 exhibited augmented B cell activation, a reasonable result considering that CD22 and CD72 are inhibitory co-receptors that control BCR signaling strength and determine the fate of B cells, leading to the induction of various autoimmune conditions [6,7,20,22]. In contrast, in the murine fibrosis model, the degree of fibrosis was diminished in CD22 / , CD72 / , and CD22 / /CD72 / mice in the present study. Sato et al. reported that CD22 negatively regulated antigen receptor signaling in the absence of antigen; however, activation of CD22deficient B cells by prolonged IgM cross-linking resulted in modest B cell proliferation, demonstrating that CD22 positively regulates antigen receptor signaling in the presence of antigen [7]. In addition, in the reverse Arthus reaction model, deficiencies of CD22 and CD72 resulted in the amelioration of vasculitis [10]. Therefore, CD22 and CD72 may have pro-inflammatory roles in the development of fibrosis. Further experiments using other murine disease models are needed in order to confirm the contribution of CD22 and CD72 in the development of various immune-mediated diseases. Neutrophils and macrophage have substantial roles in the bleomycin-mediated fibrosis model [23]. In contrast, the contribution of CD8+ T cells to the development of fibrosis has not been predominant in previous studies. In the present study, neutrophil infiltration was significantly decreased in CD72 / and CD22 / /CD72 / mice, but not in CD22 / mice and infiltration of CD8+ T cells and F4/80+ macrophages was most associated with the degree of skin fibrosis. Therefore, a crucial leukocyte subset may be different in the experimental systems. In the reverse Arthus reaction model using CD22 / and CD72 / mice, CD3+ T cells and F4/80+ macrophages were significantly decreased in CD22 / /CD72 / mice compared with WT mice [10]. It is likely that CD22 and CD72 are required for the recruitment of T cells and macrophages to the inflamed
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lesions in immune responses. Additional experiments using other murine disease models are needed to confirm this hypothesis. Cytokines and chemokines play central roles in the pathophysiology of fibrosis in both SSc patients and murine fibrosis models [24–26]. Skin and lung fibrosis were attenuated in B cell-specific, IL6-deficient mice, whereas B cell-specific IL-10-deficient mice showed more severe fibrosis [27]. TGF-β-induced subcutaneous fibrosis and subsequent CTGF application caused persistent fibrosis in a murine model [28,29]. ICAM-1 can be a potential serum biomarker for fibrosis and vascular involvement of SSc [30]. The present study suggests that in a bleomycin-induced fibrosis model, CD22 and CD72 might regulate lymphocyte recruitment through ICAM-1 and CXCL2 chemokine-related pathways resulting in the reduced production of pro-fibrotic cytokines, including IL-6, IL-1β, TNF-α, IL-13, TGF-β, and CTGF and the increased production of anti-fibrotic cytokines such as IL-10. Alternatively, altered cell infiltration of CD8 + T cells, macrophages, and neutrophils may affect the production of cytokines and chemokines. Further study is required to identify the mechanisms underlying the regulation of cytokine and chemokine production. A multitude of regulatory cell subsets modulate immune responses. The impairment of these populations can cause various immune-mediated diseases. CD22 / /CD72 / mice have significantly increased Treg cells in the steady state and small alterations of the frequency and absolute number of Treg cells in the spleen (but not in the skin) influence the severity of vasculitis [10]. However, increased severity of skin and lung fibrosis in knockout mice with Treg cell depletion was not observed in the present study. Therefore, a mechanism independent of Treg cells regulates the immune response of bleomycin-induced fibrosis in CD22 / and CD72 / mice. Targeting specific B cell surface molecules represents a promising approach for the treatment of autoimmune diseases. The efficacy of Epratuzumab (a humanized anti-CD22 Ab) has been reported in patients with systemic lupus erythematosus and Sjogren syndrome [31–33]. Our findings indicate that both CD22 and CD72 play important roles in the development of fibrosis in a murine model, and that they could be potential targets for the treatment of fibrotic diseases. However, the effect of simultaneous inhibition of both CD22 and CD72 may be limited, because no additive effect of inhibiting CD72 in addition to the blockade of CD22 was observed. Thus, in skin and lung fibrosis, single deficiency of either CD22 or CD72 was enough to reduce skin and lung fibrosis. Financial support None. Declaration of Competing Interest The authors have declared no conflicts of interest. Acknowledgements We thank Ms. M. Matsubara and Y. Yamada for technical assistance. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.jdermsci.2019.12.007. References [1] A. Forestier, T. Guerrier, M. Jouvray, J. Giovannelli, G. Lefevre, V. Sobanski, et al., Altered B lymphocyte homeostasis and functions in systemic sclerosis, Autoimmun. Rev. 17 (2018) 244–255.
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CD22 and CD72 contribute to the development of scleroderma in a murine model Chunyan Zhaoa , Takashi Matsushitaa , Vinh Thi Ha Nguyena , Momoko Tennichia , Manabu Fujimotob , Kazuhiko Takeharaa , Yasuhito Hamaguchia,* a b
Department of Dermatology, Faculty of Medicine, Institute of Medical, Pharmaceutical and Health Sciences, Kanazawa University, Ishikawa, Japan Department of Dermatology, Course of Integrated Medicine, Graduate School of Medicine, Osaka University, Osaka, Japan
A R T I C L E I N F O
A B S T R A C T
Article history: Received 13 September 2019 Received in revised form 1 December 2019 Accepted 10 December 2019
Background: Systemic sclerosis (SSc) is a systemic autoimmune disease that is characterized by excessive fibrosis. CD22 and CD72 are B cell-specific cell surface molecules that negatively regulate B cell function. Objective: The aim of the present study was to investigate the roles of CD22 and CD72 in a murine scleroderma model. Methods: The experimental fibrosis model was generated by subcutaneous injection of bleomycin or hypochlorous acid (HOCL) into wild-type (WT), CD22-deficient (CD22 / ), CD72-deficient (CD72 / ) and CD22 and CD72 double-deficient (CD22 / /CD72 / ) mice. We histologically assessed skin fibrosis and inflammatory cell infiltration. Cytokine and chemokine expression levels were measured by real-time polymerase chain reaction. Results: The severity of fibrosis in the skin and lung was significantly less in CD22 / , CD72 / , and CD22 / /CD72 / mice than in WT mice in the bleomycin-induced model. In the skin of bleomycintreated mice, the numbers of CD3+ T cells, CD8+ T cells, and F4/80+ macrophages were significantly lower in CD22 / , CD72 / , and CD22 / /CD72 / mice than in WT mice. The expression levels of mRNAs for IL-6, TNF-α, TGF-β, CTGF, IL-1β, IL-13, CXCL2, and ICAM-1 were significantly lower in CD22 / , CD72 / , and CD22 / /CD72 / mice than in WT mice. In the HOCL-induced model, both skin and lung fibrosis were ameliorated in CD22 / , CD72 / and CD22 / /CD72 / mice compared to WT mice. Conclusion: These results indicate that CD22 and CD72 likely play crucial roles in skin and lung fibrosis. Moreover, the inhibition of CD22 and CD72 function has potential as a therapeutic approach to SSc. © 2019 Japanese Society for Investigative Dermatology. Published by Elsevier B.V. All rights reserved.
Keywords: CD22 CD72 B cell Bleomycin Fibrosis
1. Introduction Systemic sclerosis (SSc) is characterized by vascular alterations and extensive fibrosis of the skin, lungs and other internal organs. Although the pathogenesis of SSc has not been elucidated, B cell abnormalities likely play an important role [1]. CD19, a B-cellspecific cell surface molecule that defines signal thresholds critical for humoral immune responses and autoimmunity, is overexpressed on B cells from patients with SSc [2]. In the tight-skin (TSK/+) mouse, a genetic model for human SSc, TSK/+ mice deficient in CD19 expression demonstrated significantly decreased skin fibrosis [3]. B-cell depletion with anti-CD20 monoclonal antibody ameliorated the development of skin sclerosis and autoantibody production in a
* Corresponding author at: Department of Dermatology, Faculty of Medicine, Institute of Medical, Pharmaceutical and Health Sciences, Kanazawa University, 13-1 Takaramachi, Kanazawa, Ishikawa 920-8641, Japan. E-mail address: [email protected] (Y. Hamaguchi).
murine model [4]. Thus, B cells appear to play important roles in the pathogenesis of SSc and might represent therapeutic targets. CD22 is a B cell-specific transmembrane protein [5]. The cytoplasmic domain of CD22 contains 6 tyrosine residues, including immune receptor tyrosine-based inhibition motifs (ITIMs) [5,6]. CD22-deficient (CD22 / ) B cells show strongly increased Ca2+ mobilization after BCR cross-linking [7]. The overall function of CD22 on BCR signaling is inhibitory. CD72 is a type II membrane protein and a member of the C-type lectin superfamily. CD72 contains an ITIM and an ITIM-like sequence in its cytoplasmic tail. Upon recruitment of SHP-1 to phosphorylated ITIM, CD72 negatively regulates BCR signaling. Thus far, collaboration between CD22 and CD72 in the development of fibrosis has not been examined in detail. Deficiency of inhibitory molecules on the B cell surface enhances the immune response. For instance, mice deficient in FcgRIIB, the inhibitory Fc receptors for IgG-containing IC, exhibited augmented IgG-mediated passive cutaneous anaphylaxis [8] as well as reverse passive alveolitis [9]. Contrary to this hypothesis,
https://doi.org/10.1016/j.jdermsci.2019.12.007 0923-1811/ © 2019 Japanese Society for Investigative Dermatology. Published by Elsevier B.V. All rights reserved.
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we have previously reported that the combined loss of CD22 and CD72 resulted in reduced edema and hemorrhage in the reverse Arthus reaction model [10]. In the present study, we show that the loss of either CD22 or CD72 resulted in reduced fibrosis in both skin and lung in a murine fibrosis model. Given their proinflammatory roles, CD22 and CD72 might be potential therapeutic targets for the treatment of fibrotic disease, such as SSc. 2. Materials and methods 2.1. Mice C57BL/6 wild-type (WT) mice were purchased from CLEA Japan, Inc. (Shizuoka, Japan). CD22-deficient (CD22 / ) (C57BL/6 129) mice were generated as described [7] and backcrossed to a C57BL/6 strain 12 times. CD72-deficient (CD72 / ) mice were purchased from The Jackson Laboratory. CD22 / /CD72 / mice were generated, as previously described [10]. All mice used in experiments were 8–12 weeks of age and were housed in a specific pathogen-free barrier facility and screened regularly for pathogens. The Committee on Animal Experimentation of Kanazawa University Graduate School of Medical Science approved all studies and procedures. 2.2. Flow cytometric analysis of immune cells Single cell suspensions of bone marrow (bilateral femurs), spleens and peripheral lymph nodes (paired axillary and inguinal) were generated by gentle dissection [11]. Single cell leukocyte suspensions (0.5–1.0 106) were stained, as described [11]. Cells were analyzed using a FACSCanto II flow cytometer (BD Biosciences, San Jose, CA). Data were analyzed using FlowJo software (Tree Star, Ashland, OR). Antibodies used in this study included the following: anti-B220 (RA3-6B2), anti-CD21 (7G6), anti-CD24 (M1/69), anti-CD5 (53-7.3), anti-IA/IB (M5/114.15.2), anti-CD19 (1D3) (BD Bioscience), anti-IgM (MA-69) and CD11b (M1/70) (BioLegend, San Diego, CA). 2.3. B cell turnover measurement B cell turnover in mice was measured by bromodeoxyuridine (BrdU) uptake. Mice were fed BrdU (B23151, Invitrogen, Carlsbad, CA) for 7 days and killed. The percentage of BrdU+ B cells in each tissue (bone marrow, blood, spleen) was determined by immunofluorescence staining, as described [12]. 2.4. Intradermal treatment with bleomycin Bleomycin was dissolved in sterile saline at 1 mg/mL. The mice received every other day intradermal injections of 300 mL of bleomycin into their shaved backs (the para-midline, lower back region) with a 27-gauge needle for 4 weeks. 2.5. Intradermal treatment with hypochlorous acid (HOCL) Skin and lung fibrosis was also induced by intradermal injection of hypochlorous acid (HOCL), as previously described [13] with minor modifications. Briefly, HOCL was produced by adding 166 mL of NaClO solution (9.6% as active chlorine) to 11.1 mL of KH2PO4 solution (100 mM, pH 7.2) and 400 mL (2 injections of 200 mL each) of HOCL solution were intradermally injected into the shaved backs of the mice, using a 27-gauge needle, daily for 6 weeks. 2.6. Histologic examination of lung and skin fibrosis All skin sections were taken from the bleomycin- or HOCLinjected region of the lower back and were obtained as fullthickness sections extending down to the body wall musculature
on day 28 (bleomycin model) or day 42 (HOCL model), as previously described [14,15]. Sections (6 mm in thickness) were stained with hematoxylin and eosin (H&E), Masson trichrome, or van Gieson reagents to identify collagen deposition. Expression of α-smooth muscle actin (α-SMA) was analyzed by using anti-α-SMA antibodies (Abs) conjugated to alkaline phosphatase (SigmaAldrich, St. Louis, MO). Dermal thickness, which was defined as the thickness of skin from the top of the granular layer to the junction between the dermis and intradermal fat, was evaluated independently by 2 investigators (ZC and YH) in a blinded manner. Whole lungs were harvested from the bleomycin- or HOCLtreated mice and inflated, as previously described [14]. The severity of lung inflammation was determined by a semiquantitative scoring system as previously described [16]. All sections were scored independently by 2 investigators (ZC and YH) in a blinded manner. Using the free-hand tool in the Photoshop Elements 3.0 software package (Adobe Systems), specific staining with Masson’s trichrome, van Gieson reagents, and α-SMA was quantified in the whole lung and skin sections. 2.7. Measurement of collagen contents in tissue samples The content of collagen in the skin and lung tissues was measured on day 28 (bleomycin model) or day 42 (HOCL model), as previously described [14,15]. Tissue samples were embedded in paraffin and sections approximately 15 mm thick were obtained. The method is based on the selective binding of Sirius red F3BA and Fast green FCF to collagens and non-collagenous proteins, respectively [17]. 2.8. Immunohistochemical staining Skin and lung tissues were harvested prior to and at 7 and 14 days of bleomycin treatment. The numbers of infiltrating CD3+ T cells, CD4+ T cells, CD8+ T cells, myeloperoxidase (MPO)-positive neutrophils, F4/80+ macrophages, NK1.1+ NK cells, and B220 + B cells were assessed by immunostaining, as described previously [14,15]. Sections were incubated with rat mAbs specific for CD3 (Serotec, Oxford, UK), CD4, CD8, CD45R/B220 (BD Biosciences), F4/80 (Abcam, Cambridge, UK) or NK1.1+ (BioLegend) or with rabbit mAb specific for myeloperoxidase (MPO, Neomarkers). Rat IgG (Southern Biotechnology Associates, Birmingham, AL) was used as a control for nonspecific staining. Stained cells were counted in five random grids under high-magnification (400) on a light microscope. Each section was examined independently by 2 investigators (ZC and YH), in a blinded manner, and the mean was used for analysis. 2.9. Reverse transcriptase-polymerase chain reaction (RT-PCR) Skin and lung tissues were harvested 7 or 14 days after bleomycin administration. The expression levels of mRNAs for Tnf, Il1b, Il6, Il10, Il13, Ifng, Tgfb1, connective tissue growth factor (CTGF/Ccn2), Icam1, Cxcl2, Ccl2 and Ccl3 were analyzed using a real-time polymerase chain reaction (RT-PCR) quantification method. Glyceraldehyde-3phosphate dehydrogenase mRNA (Gapdh) was used to normalize the mRNAs levels. The relative expression of RT-PCR products was determined using the DDCT method to compare target genes with housekeeping Gapdh gene mRNA expression [18]. 2.10. Regulatory T cell depletion in the bleomycin-induced fibrosis model To deplete regulatory T cells (Tregs), mice were given a single intraperitoneal (i.p.) injection of 500 mL of anti-CD25 Abs (PC61) (BioLegend) on day 0 [19]. Isotype-matched control Abs were given to WT mice. Bleomycin-induced fibrosis was induced 7 days after i.p. injection of anti-CD25 Abs or control Abs.
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2.11. Adoptive transfer experiment
2.12. Statistical analysis
Splenic B cells from WT mice were enriched by using a pan-B cell isolation kit (Miltenyi Biotec, Bergisch Gladbach, Germany). One day before induction with BLM, a total of 20 106 purified B cells was intravenously transferred into CD22 / , CD72 / , or CD22 / /CD72-/mice.
Student’s t-test was used for determining the level of significance of differences in sample means, and Bonferroni’s test was used for multiple comparisons. A P value < 0.05 was considered significant. All data are shown as means SEM. Statistical analyses were performed using JMP version 10 statistical software (Cary, NC).
Fig. 1. Skin fibrosis induced by intradermal treatment with bleomycin. CD22 / , CD72 / , CD22 / /CD72 / , and WT mice were treated every other day by intradermal injection of bleomycin for 28 days. A. Representative histological sections of skin tissue obtained from each bleomycin-treated mouse strain. Sections were stained with hematoxylin and eosin (H&E), Masson’s trichrome, or α-SMA. B–C. Analysis of the skin for determination of dermal thickness (B) and collagen content (C). D–E. Histologic evaluation of skin fibrosis. Size of fibrotic areas, as evaluated by van Gieson’s staining (D) and percentage of staining with α-SMA (E). All values represent the means SEM of results obtained from 5 to 10 mice in each group. *, p < 0.05; **, p < 0.01.
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3. Results 3.1. B cell phenotypes in the mutant mice B cell phenotypes CD22 / , CD72 / , and CD22 / /CD72 / mice are shown in Supplemental Fig. 1 and Supplemental
Table 1. The immunophenotyping results for CD22 / and CD72 / mice were consistent with those previously reported [6,7,20,21] and CD22 / /CD72 / mice did not show any phenotypes beyond those found in single CD22 / or CD72 / mice. BrdU incorporation studies showed an increase in B cell turnover both in the periphery (blood and spleen) and bone
Fig. 2. Lung fibrosis induced by intradermal treatment with bleomycin. CD22 / , CD72 / , CD22 / /CD72 / , and WT mice were treated every other day by intradermal injection of bleomycin for 28 days. A. Representative histological sections of lung tissue obtained from each bleomycin-treated mouse strain. Sections were stained with hematoxylin and eosin (H&E), Masson’s trichrome, or α-SMA. B–C. Analysis of the lungs for determination of lung fibrosis score (B) and collagen content (C). D–E. Histologic evaluation of lung fibrosis. Size of fibrotic areas, as evaluated by van Gieson’s staining (D) and percentage of staining with α-SMA (E). All values represent the mean SEM of results obtained from 5 to 10 mice in each group. *, p < 0.05; **, p < 0.01.
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marrow of CD22 / /CD72 / mice compared to WT mice, but a decrease compared to CD22 / mice. These data suggested that CD22 and CD72 play important roles in the efficiency of B cell developmental transition. In addition, B cell phenotypes of the mutant mice showed that CD22 and CD72 have redundant functions in some aspects of cell signaling.
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3.2. Amelioration of bleomycin-induced skin fibrosis by loss of CD22 and CD72 We assessed the results of intradermal administration of bleomycin to CD22 / , CD72 / , CD22 / /CD72 / and WT mice. CD22 or CD72 deficiency caused a significant reduction in the
Fig. 3. Inflammatory cell infiltration in the skin of CD22 / , CD72 / , CD22 / /CD72 / , and WT mice during intradermal bleomycin treatment. Numbers of cells per high powered field: CD3+ T cells (A), CD4+ T cells (B), CD8+ T cells (C), F4/80+ macrophages (D), myeloperoxidase (MPO)-positive neutrophils (E) and NK1.1+ NK cells (F) on days 7 and 14. Bars show the means SEM of results obtained from 5 to 10 mice in each group. *, p < 0.05; **, p < 0.01. Representative images showing CD3 (G) and F4/80 (H) staining in tissue sections from CD22 / , CD72 / , CD22 / /CD72 / , and WT mice. Original magnification, x 400.
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mean dermal thickness compared to WT mice (p < 0.05 for each), as evaluated by H&E staining or Masson’s trichrome staining (Fig. 1A and B). The loss of both CD22 and CD72 significantly reduced the mean dermal thickness compared to WT mice (p < 0.05). The degree of reduction observed when both CD22 and CD72 were absent was similar to that observed with single molecule deficiency. The local skin collagen content and the size of fibrotic areas, as evaluated by van Gieson’s staining, were consistent with the findings for dermal thickness (Fig. 1C and 1D). Likewise, the frequencies of α-SMA– positive myofibroblasts were significantly reduced in CD22 / , CD72 / and CD22 / /CD72-/- mice compared to WT mice
(Fig. 1A and E). In contrast, the degrees of skin fibrosis were comparable among mutant mice. Thus, loss of CD22 or CD72, or both ameliorated skin fibrosis induced by daily intradermal bleomycin treatment. 3.3. Bleomycin-induced lung fibrosis after loss of CD22 and CD72 We assessed lung fibrosis in mutant and WT mice (Fig. 2A). The lung fibrosis scores were significantly lower in CD22 / , CD72 / , and CD22 / /CD72-/- mice compared to WT mice (p < 0.05 for each; Fig. 2B). The local lung collagen content and the size of fibrotic areas, as evaluated by van Gieson’s staining, were
Fig. 4. mRNA expression levels of cytokines and chemokines in the skin of CD22 / , CD72 / , CD22 / /CD72 / , and WT mice during intradermal bleomycin treatment. The mRNA amounts were measured by real-time PCR and normalized to Gapdh mRNA. Bars show the means SEM of results obtained with 5 mice in each group. *, p < 0.05; **, p < 0.01.
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consistent with the findings for lung fibrosis score (Fig. 2C and D). Likewise, the frequencies of α-SMA–positive myofibroblasts were significantly reduced in CD22 / , CD72 / and CD22 / /CD72-/mice compared to WT mice (p < 0.01 for each; Fig. 2A and E). In contrast, the severities of lung fibrosis were similar among mutant mice. Thus, in the same manner as observed in skin, loss of CD22 or CD72, or both ameliorated lung fibrosis induced by intradermal bleomycin treatment.
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3.4. Leukocyte infiltration in bleomycin-induced skin fibrosis Leukocyte infiltration of the skin was assessed in tissue sections after 7 or 14 days of bleomycin treatment (Fig. 3). Infiltrating CD3+ T cell numbers after 7 or 14 days of bleomycin treatment were significantly reduced in CD22 / , CD72 / , and CD22 / /CD72 / mice compared with WT mice (p < 0.01 for each; Fig. 3A and G). CD8+ T cells were also significantly decreased
Fig. 5. Skin and lung fibrosis induced by intradermal treatment with hypochlorous acid (HOCL). CD22 / , CD72 / , CD22 / /CD72 / and WT mice were treated with daily intradermal injections of HOCL for 42 days. A. Representative histological sections of skin tissues obtained from each HOCL-treated mouse strain. Sections were stained with hematoxylin and eosin (H&E) or Masson’s trichrome. B. Analysis of the skin tissues for determination of dermal thickness (left panel) and collagen content (right panel). C. Representative histological sections of lung tissue obtained from each HOCL-treated mouse strain. Sections were stained with H&E or Masson’s trichrome. D. Analysis of the lungs for determination of lung fibrosis score (left panel) and collagen content (right panel). All values represent the means SEM of results obtained from 5 to 10 mice in each group. *, p < 0.05; **, p < 0.01.
Please cite this article in press as: C. Zhao, et al., CD22 and CD72 contribute to the development of scleroderma in a murine model, J Dermatol Sci (2019), https://doi.org/10.1016/j.jdermsci.2019.12.007
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in CD22 / , CD72 / and CD22 / /CD72 / mice compared with WT mice (p < 0.01 for each; Fig. 3C) at 7 days. However, a significant reduction was observed only between CD72 / and WT mice at 14 days (p < 0.01; Fig. 3C). F4/80+ macrophage numbers were significantly decreased in CD22 / , CD72 / , and CD22 / /CD72 / mice compared with WT mice after 7 days (p < 0.01 for each) and 14 days (p < 0.05,
p < 0.01, and p < 0.01, respectively) after bleomycin treatment (Fig. 3D and H). MPO+ neutrophil numbers were significantly reduced in CD72 / and CD22 / /CD72 / mice compared with WT mice after 7 days (p < 0.01 for each) and 14 days (p < 0.05 and p < 0.01, respectively) of bleomycin treatment (Fig. 3E). CD22 / mice showed a decrease in the number of infiltrating neutrophils after 7
Fig. 6. Adoptive transfer experiment to CD22 / , CD72 / , and CD22 / /CD72 / mice. Isolated B cells from WT mice were intravenously transferred into CD22 / , CD72 / , and CD22 / /CD72 / mice after which skin and lung fibrosis were induced by intradermal treatment with bleomycin. A. Representative histological sections of skin tissues obtained from each bleomycin-treated mouse strain. Sections were stained with hematoxylin and eosin (H&E) or Masson’s trichrome. B. Analysis of skin tissues for determination of dermal thickness (left panel) and collagen content (right panel). C. Representative histological sections of lung tissue obtained from each bleomycin-treated mouse strain. Sections were stained with H&E or Masson’s trichrome. D. Analysis of the lungs for determination of lung fibrosis score (left panel) and collagen content (right panel). All values represent the means SEM of results obtained from 3 to 5 mice in each group.
Please cite this article in press as: C. Zhao, et al., CD22 and CD72 contribute to the development of scleroderma in a murine model, J Dermatol Sci (2019), https://doi.org/10.1016/j.jdermsci.2019.12.007
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or 14 days of bleomycin treatment, but the differences did not reach significance. Although there were several significant differences in infiltrating CD4+ T and NK1.1+ NK cell numbers between knockout and WT mice, the degree of infiltration was not associated with the severity of fibrosis. We detected almost no or very few B220+ B cells in the skin of normal and fibrotic mice (data not shown). These results suggest that CD22 and CD72 cooperatively play important roles in the migration of T cells, neutrophils and macrophages into inflamed skin tissue in a bleomycin fibrosis model. 3.5. Cytokine and chemokine mRNA expression in bleomycin-induced skin fibrosis We investigated whether CD22 and CD72 deficiencies affected the expression of fibrogenic and anti-fibrogenic factors in the skin of mice receiving bleomycin (Fig. 4). Regarding fibrogenic cytokines, Tnf and Il13 mRNA expression levels in CD22 / , CD72 / , and CD22 / /CD72 / mice were significantly decreased 7 days after bleomycin treatment. Il1b and Il6 mRNA expression levels in CD22 / , CD72 / , and CD22 / /CD72 / mice were significantly decreased 14 days after bleomycin treatment. Tgfb1 and Ccn2 mRNA expression levels in CD22 / , CD72 / , and CD22 / /CD72 / mice were significantly decreased both 7 and 14 days after bleomycin treatment. Regarding anti-fibrogenic cytokines, Il10 mRNA expression levels in CD22 / , CD72 / , and CD22 / /CD72 / mice were significantly increased both 7 and 14 days after bleomycin treatment, except for the comparison between CD22 / and WT mice at day 14. Although IFN-g is considered an anti-fibrogenic cytokine, Ifng mRNA expression levels in CD22 / , CD72 / and CD22 / /CD72 / mice were significantly decreased 7 days after bleomycin treatment. Thus, the reduced skin inflammatory responses caused by the loss of CD22 and/or CD72 molecules were associated with a reduced release of profibrogenic and an increased release of anti-fibrogenic cytokines. Regarding chemokine expression, Icam1 and Cxcl2 mRNA expression levels in CD22 / , CD72 / , and CD22 / /CD72 / mice were significantly decreased on day 7. Although several significant differences were observed between knockout and WT mice for Ccl2 and Ccl3 mRNAs expression levels, there was no clear association between the severity of fibrosis and the mRNA expression levels of these chemokines. Therefore, CD22 and CD72 can affect Icam1 and Cxcl2 expression, leading to the regulation of inflammatory cell infiltration in bleomycin-induced fibrosis. 3.6. Skin and lung fibrosis in mice treated with HOCL We asked whether the roles of CD22 and CD72 observed in the bleomycin model were a general phenomenon. In the HOCL-induced fibrosis model, both skin and lung fibrosis were significantly ameliorated in CD22 / , CD72 / and CD22 / /CD72 / mice compared to WT mice (Fig. 5A-D). Thus, the role of CD22 and CD72 in the development of fibrosis was confirmed in a different fibrosis model. 3.7. Adoptive transfer experiment in mutant mice Further adoptive transfer experiments were conducted to confirm the role of CD22 and CD72 in the development of fibrosis. The degree of skin and lung fibrosis in CD22 / , CD72 / and CD22 / /CD72 / mice that received isolated B cells from WT mice was comparable to that in WT mice (Fig. 6A-D). Therefore, CD22 and CD72 have pivotal roles in the development of fibrosis.
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3.8. Treg cell depletion in mice treated with bleomycin The reverse Arthus reaction model using CD22 / and CD72 / mice showed that Treg cells had a crucial role in the development of vasculitis [10]. Therefore, we conducted Treg cell depletion therapy in bleomycin-induced fibrosis. The significant differences regarding the degree of both skin and lung fibrosis were unchanged between WT mice treated with control Abs and knockout mice treated with anti-CD25 Abs (data not shown). Therefore, Treg cells did not have a substantial role in CD22 / and CD72 / mice in bleomycin-induced fibrosis. 4. Discussion This study is the first to elucidate the contributions of CD22 and CD72 (alone and together) on the development of bleomycininduced fibrosis. In the present study, bleomycin-induced skin and lung fibrosis were significantly attenuated in CD22 / /CD72 / mice compared with WT mice (Fig. 1 and 2). Moreover, inflammatory leukocyte infiltration, which was composed primarily of T cells and macrophages in the skin lesions, was significantly decreased in knockout mice compared with WT mice during bleomycin-induced skin fibrosis (Fig. 3). Reduced infiltration of inflammatory leukocytes was correlated with a decrease in the pro-fibrotic cytokines TNF-α, IL-1β, IL-6, IL-13, TGF-β and CTGF and the chemokines ICAM-1 and CXCL2 (Fig. 4). The roles of CD22 and CD72 in skin and lung fibrosis were confirmed in another model in which fibrosis was induced by HOCL (Fig. 5). Adoptive transfer of B cells from WT mice into knockout mice exacerbated the fibrosis to the same degree as WT mice (Fig. 6). Taken together, these results demonstrate that CD22 and CD72 contribute to the development of fibrosis by regulating the migration of leukocytes via control of the production of cytokines and chemokines. Mice lacking CD22 or CD72 exhibited augmented B cell activation, a reasonable result considering that CD22 and CD72 are inhibitory co-receptors that control BCR signaling strength and determine the fate of B cells, leading to the induction of various autoimmune conditions [6,7,20,22]. In contrast, in the murine fibrosis model, the degree of fibrosis was diminished in CD22 / , CD72 / , and CD22 / /CD72 / mice in the present study. Sato et al. reported that CD22 negatively regulated antigen receptor signaling in the absence of antigen; however, activation of CD22deficient B cells by prolonged IgM cross-linking resulted in modest B cell proliferation, demonstrating that CD22 positively regulates antigen receptor signaling in the presence of antigen [7]. In addition, in the reverse Arthus reaction model, deficiencies of CD22 and CD72 resulted in the amelioration of vasculitis [10]. Therefore, CD22 and CD72 may have pro-inflammatory roles in the development of fibrosis. Further experiments using other murine disease models are needed in order to confirm the contribution of CD22 and CD72 in the development of various immune-mediated diseases. Neutrophils and macrophage have substantial roles in the bleomycin-mediated fibrosis model [23]. In contrast, the contribution of CD8+ T cells to the development of fibrosis has not been predominant in previous studies. In the present study, neutrophil infiltration was significantly decreased in CD72 / and CD22 / /CD72 / mice, but not in CD22 / mice and infiltration of CD8+ T cells and F4/80+ macrophages was most associated with the degree of skin fibrosis. Therefore, a crucial leukocyte subset may be different in the experimental systems. In the reverse Arthus reaction model using CD22 / and CD72 / mice, CD3+ T cells and F4/80+ macrophages were significantly decreased in CD22 / /CD72 / mice compared with WT mice [10]. It is likely that CD22 and CD72 are required for the recruitment of T cells and macrophages to the inflamed
Please cite this article in press as: C. Zhao, et al., CD22 and CD72 contribute to the development of scleroderma in a murine model, J Dermatol Sci (2019), https://doi.org/10.1016/j.jdermsci.2019.12.007
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lesions in immune responses. Additional experiments using other murine disease models are needed to confirm this hypothesis. Cytokines and chemokines play central roles in the pathophysiology of fibrosis in both SSc patients and murine fibrosis models [24–26]. Skin and lung fibrosis were attenuated in B cell-specific, IL6-deficient mice, whereas B cell-specific IL-10-deficient mice showed more severe fibrosis [27]. TGF-β-induced subcutaneous fibrosis and subsequent CTGF application caused persistent fibrosis in a murine model [28,29]. ICAM-1 can be a potential serum biomarker for fibrosis and vascular involvement of SSc [30]. The present study suggests that in a bleomycin-induced fibrosis model, CD22 and CD72 might regulate lymphocyte recruitment through ICAM-1 and CXCL2 chemokine-related pathways resulting in the reduced production of pro-fibrotic cytokines, including IL-6, IL-1β, TNF-α, IL-13, TGF-β, and CTGF and the increased production of anti-fibrotic cytokines such as IL-10. Alternatively, altered cell infiltration of CD8 + T cells, macrophages, and neutrophils may affect the production of cytokines and chemokines. Further study is required to identify the mechanisms underlying the regulation of cytokine and chemokine production. A multitude of regulatory cell subsets modulate immune responses. The impairment of these populations can cause various immune-mediated diseases. CD22 / /CD72 / mice have significantly increased Treg cells in the steady state and small alterations of the frequency and absolute number of Treg cells in the spleen (but not in the skin) influence the severity of vasculitis [10]. However, increased severity of skin and lung fibrosis in knockout mice with Treg cell depletion was not observed in the present study. Therefore, a mechanism independent of Treg cells regulates the immune response of bleomycin-induced fibrosis in CD22 / and CD72 / mice. Targeting specific B cell surface molecules represents a promising approach for the treatment of autoimmune diseases. The efficacy of Epratuzumab (a humanized anti-CD22 Ab) has been reported in patients with systemic lupus erythematosus and Sjogren syndrome [31–33]. Our findings indicate that both CD22 and CD72 play important roles in the development of fibrosis in a murine model, and that they could be potential targets for the treatment of fibrotic diseases. However, the effect of simultaneous inhibition of both CD22 and CD72 may be limited, because no additive effect of inhibiting CD72 in addition to the blockade of CD22 was observed. Thus, in skin and lung fibrosis, single deficiency of either CD22 or CD72 was enough to reduce skin and lung fibrosis. Financial support None. Declaration of Competing Interest The authors have declared no conflicts of interest. Acknowledgements We thank Ms. M. Matsubara and Y. Yamada for technical assistance. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.jdermsci.2019.12.007. References [1] A. Forestier, T. Guerrier, M. Jouvray, J. Giovannelli, G. Lefevre, V. Sobanski, et al., Altered B lymphocyte homeostasis and functions in systemic sclerosis, Autoimmun. Rev. 17 (2018) 244–255.
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Please cite this article in press as: C. Zhao, et al., CD22 and CD72 contribute to the development of scleroderma in a murine model, J Dermatol Sci (2019), https://doi.org/10.1016/j.jdermsci.2019.12.007