Effects of splenectomy on skin inflammation and psoriasis-like phenotype of imiquimod-treated mice

Imiquimod (IMQ) is widely used as animal model of psoriasis, a chronic inflammatory skin disorder. Although topical application of IMQ to back skin causes splenomegaly in mice, how the spleen affects the psoriasis-like phenotype of IMQ-treated mice remains unclear. In this study, we analyzed the cellular composition of spleen and measured metabolites in blood of IMQ-treated mice. We also investigated whether splenectomy influences the degree of skin inflammation and pathology in IMQ-treated mice. Flow cytometry showed that the numbers of CD11b+Ly6c+ neutrophils, Ter119+ proerythroblasts, B220+ B cells, F4/80+ macrophages, and CD11c+ dendritic cells in the spleen were significantly higher in IMQ-treated mice compared to control mice. An untargeted metabolomics analysis of blood identified 14 metabolites, including taurine and 2,6-dihydroxybenzoic acid, whose levels distinguished the two groups. The composition of cells in the spleen and blood metabolites positively correlated with the weight of the spleen. However, splenectomy did not affect IMQ-induced psoriasis-like phenotypes compared with sham-operated mice, although splenectomy increased the expression of interleukin-17A mRNA in the skin of IMQ-treated mice. These data suggest that the spleen does not play a direct role in the development of psoriasis-like phenotype on skin of IMQ-treated mice, though IMQ causes splenomegaly.

performed non-targeted metabolome analysis of blood samples from the two groups and examined correlations between spleen weight and blood metabolites. Finally, we determined whether splenectomy could affect psoriasislike phenotype and skin inflammation in IMQ-treated mice.

Results
Effects of IMQ on weight and cell populations of spleen. Topical application of IMQ caused significantly increased spleen weight in IMQ-treated mice compared to control mice (Fig. 1A,B), consistent with our previous report 22 . The number of total cells of IMQ-treated mice was also significantly higher than those of control mice (Fig. 1C). Spleen cells were analyzed for the percentage and the number of CD11b + Ly6c + neutrophils, Ter119 + proerythroblasts, B220 + B cells, CD3 + T cells, CD4 + T cells, CD8 + T cells, NK1.1 + natural killer (NK) cells, F4/80 + macrophages, and CD11c + dendritic cells (DCs). The number of neutrophils, proerythroblasts, B cells, macrophages, and DCs in the spleen of IMQ-treated mice was significantly higher than those of control mice (Fig. 1C,D). In contrast, there were no differences in the number of T cells (CD3 + , CD4 + , and CD8 + ) and NK cells (Fig. 1C). Data are shown as mean ± SEM (n = 8). One data of Ter119 + proerythroblasts and CD11c + DCs in the control group and one data of CD3 + T cells in the IMQ group were missing because of technical problems. # P (FDR-corrected) < 0.05, ## P (FDR-corrected) < 0.01, ### P (FDR-corrected) < 0.001. NS: not significant. (D): The representative FACS data of CD11b + Ly6c + neutrophils, Ter119 + proerythroblasts, F4/80 + macrophages, and CD11c + DCs in the spleen of control mice and IMQ-treated mice.
The total number of cells in the spleen positively correlated with the cell types (i.e., neutrophils, proerythroblasts, B cells, macrophages, and DCs) in the two groups ( Fig. 2A). Moreover, the weight of spleen positively correlated with the cell types (i.e., neutrophils, proerythroblasts, B cells, macrophages, and DCs) in the two groups (Fig. 2B).
Associations between spleen cells, spleen weight, and plasma metabolites. Spearman correlation analysis was used to quantify the correlations between the spleen cell types, spleen weight, and the fourteen The correlations between the number of total splenic cells and the number of the splenic cell types whose amount significantly increased in IMQ group. (B): The correlations between the weight of spleen and the number of the splenic cell types whose amount significantly increased in IMQ group. The values represent the mean ± SEM (n = 8). www.nature.com/scientificreports/ differential metabolites of plasma. Several cell types and spleen weight were significantly correlated with the plasma metabolites in the two groups (Fig. 4A). The cell types such as total cells, neutrophils, and macrophages were positively associated with plasma metabolites except N,N,N-trimethyl-lysine. The other cell types (B cells, proerythroblasts, and DCs) were positively associated with several metabolites. Spleen weight was also positively associated with plasma metabolites except N,N,N-trimethyl-lysine (Fig. 4A). Spearman correlation was also used to determine if IMQ-related cell types in the spleen and plasma metabolites contribute to splenomegaly. The cell types in the spleen and plasma metabolites differentially abundant in the two groups showed more associations with spleen weight (Fig. 4B). Interestingly, neutrophils in the spleen were positively correlated with spleen weight and plasma metabolites (Fig. 4B). Next, we measured the gene expression levels of IL-17A and IL-23A in the skin. The mRNA levels of IL-17A and IL-23A in the IMQ group were higher than those of the control group (Fig. 5D). Expression of IL-17A mRNA in the splenectomy-IMQ group was significantly higher than that of sham-IMQ group, whereas the expression of IL-23A mRNA was not different between the sham-IMQ group and splenectomy-IMQ group (Fig. 5D). There were also no changes in the body weight between sham group and splenectomy group in both of IMQ group and control group (Fig. 5E).

Discussion
This is the first study to show how the spleen contributes to psoriasis-like phenotypes in IMQ-treated mice. Indeed, topical application of IMQ significantly increased infiltration of immune cells such as neutrophils, DCs, macrophages, and B cells in the spleen, which agrees with previous studies. These cell types correlated with spleen weight, which also correlated with higher levels of 14 metabolites in the plasma of IMQ-treated mice; taurine and 2,6-dihydroxybenzoic acid had especially high VIP values. These metabolites likely contribute to splenomegaly upon IMQ application. Finally, we showed that splenectomy does not affect a psoriasis-like phenotype on the skin of IMQ-treated mice but could potentiate IMQ-induced increases in IL-17A mRNA in the skin.
Neutrophils, proerythroblasts, B cells, macrophages, and DCs significantly enlarged the IMQ-treated spleen; their cell compartments positively correlated with spleen weight. Interestingly, the percentage of B cells in the spleen did not change between the control group and IMQ-treated group. Previously, percentages of macrophages and DCs increased and the percentage of T cells (both CD4 + and CD8 + ) decreased in an IMQ-treated group 9 , consistent with the current data. Topical treatment with IMQ likely induces inflammation, and increased numbers of immune cells cause splenomegaly.
The longitudinal diameter of the spleen and the duration of psoriasis were correlated in a previous study of 79 psoriatic patients 23 , suggesting that increased diameter of the spleen in psoriatic patients with long-term illness is related to chronic inflammation. Future clinical studies are needed to confirm the role of spleen in the pathogenesis of psoriasis 24 .
Non-targeted metabolomics profiling identified 14 metabolites whose levels were significantly different between the IMQ-treated group and the control group. Taurine and 2,6-dihydroxybenzoic acid had the highest VIP values, indicating that they contributed the most to the separation between groups. Taurine plays an important role in inflammation associated with oxidative stress 25,26 and may play a pathological role in psoriasis given its higher levels in blood from patients with psoriasis when compared to healthy controls 27 . Taurine reduced blood levels of IL-6 in patients with traumatic brain injury in a recent randomized double-blind controlled trial 28 . Taurine's anti-inflammatory actions 25,26 in the blood of IMQ-treated mice may compensate for IMQ-induced inflammation in the body. Further studies should explore how taurine affects spleen size in IMQ-treated mice. The function of 2,6-dihydroxybenzoic acid remains unclear but is likely anti-inflammatory 29 . Higher levels of 2,6-dihydroxybenzoic acid in the blood of IMQ-treated mice may also compensate for IMQ-induced inflammation in the body. These metabolites should be targeted in future studies on splenomegaly of IMQ-treated mice.
Spleen enlargement is linked to systemic inflammation 30 . For example, we reported splenomegaly in mice treated with lipopolysaccharide (LPS), and there were positive correlations between spleen weight and blood levels of pro-inflammatory cytokines (i.e., IL-6, tumor necrosis factor-α) [31][32][33] . A chronic social defeat stress (CSDS) model revealed that the spleen weight of susceptible mice with depression-like behaviors was higher than that (B): Volcano plot shows the differential metabolites between the two groups. The X-axis indicates the log2transformed plasma metabolite abundance of fold change, and the Y-axis indicates the -log10-transformed P value using the Wilcoxon rank sum test. Horizontal lines indicate P < 0.05. Increased or decreased metabolites are marked in red and blue, respectively. The size of the dot represents the size of the VIP value. Metabolites with P < 0.05 and VIP > 0.6 are mentioned in text. (C): VIP value of the differential metabolites between the two groups. (D): Log2 transformed data of the abundance of the differential plasma metabolites.  www.nature.com/scientificreports/
Considering the spleen's key role in the immune system 18-21 , we investigated how splenectomy affects psoriasis-like pathology and skin inflammation of IMQ-treated mice. Unexpectedly, splenectomy did not change the psoriasis-like phenotype in IMQ-treated mice. However, we found that splenectomy significantly enhanced IL-17A mRNA in the skin of IMQ-treated mice compared to sham-operated mice. The spleen may not directly impact the psoriasis-like phenotype of IMQ-treated mice, but it does cause splenomegaly.
This study has one limitation. In this study, we used 5% IMQ cream (Beselna cream). The full list of excipients is isostearic acid, benzyl alcohol, cetyl alcohol, stearyl alcohol, white soft paraffin, polysorbate 60, sorbitan stearate, glycerol, methyl hydroxybenzoate, propyl hydroxybenzoate, xanthan gum, and purified water. Walter et al. 37 reported that isostearic acid, a major component, could promote inflammasome activation in cultured keratinocytes, and that it increased the expression of inflammatory cytokines in vivo. These data suggest that isostearic acid may contribute to the observed effects of Beselna cream used in this study 37,38 . In this study, we did not examine the effects of isosteatic acid on spleen function since the company did not disclose the detailed information of excipients including isostearic acid. Further study is needed to investigate the effects of isostearic acid on spleen functions.
In conclusion, this study highlighted the key role of the spleen in chronic inflammation of IMQ-treated mice. The numbers of neutrophils, proerythroblasts, B cells, macrophages, and DCs in the spleen significantly increased, which correlated with higher spleen weight. Metabolomics profiling also revealed metabolites whose roles in psoriasis pathogenesis can be studied further. However, splenectomy did not affect psoriasis-like phenotypes in IMQ-treated mice. Although the spleen may not play a major role in psoriasis-like phenotypes in IMQ-treated mice, topical application of IMQ to back skin causes splenomegaly.

Materials and methods
Animals. Nine-week-old female C57BL/6 mice (weighing 18-21 g, n = 16, Japan SLC Inc., Hamamatsu, Shizuoka, Japan) were used in Experiment 1. Seven-week-old female C57BL/6 mice (weighing 18-21 g, n = 28, Japan SLC Inc., Hamamatsu, Shizuoka, Japan) were used in Experiment 2. Mice were housed (3-4 per cage) under a 12-h/12-h light/dark cycle (lights on between 07:00 and 19:00), with ad libitum access to food (CE-2; CLEA Japan, Inc., Tokyo, Japan) and water. The experimental protocol was approved by the Chiba University Institutional Animal Care and Use Committee (Permission number: 2-433). All procedures were performed in accordance with the relevant guidelines and regulations, and the study complied with ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines. All efforts were made to minimize animal suffering 22 . IMQ treatment. The shaved back skin of mice was treated with 62.5 mg of 5% IMQ cream (Beselna cream; Mochida Pharmaceutical Co., Tokyo, Japan) daily for four consecutive days as previously described 22 . Control mice were treated similarly with 62.5 mg of white petrolatum (Maruishi Pharmaceutical Co., Osaka, Japan). Splenectomy. Splenectomy (or sham) surgery was performed under continuous isoflurane inhalation anesthesia as previously described 39 . Briefly, the mice were anesthetized with 3% isoflurane through an inhalation anesthesia apparatus (KN-1071NARCOBIT-E; Natsume Seisakusho, Tokyo, Japan). In the splenectomy group, each mouse was maintained in a right lateral recumbent position, and an approximately 1-cm incision was made from the abdominal wall under the left costal margin. The skin was dissected, and subcutaneous, muscle, and fascia layers were removed individually until the spleen was exposed. The peripheral ligament of the spleen was separated, associated blood vessels and nerves were ligatured using 6-0 silk sutures, and the spleen was removed by transecting the blood vessels distal to the ligature. Abdominal muscles and the skin incision were closed sequentially using 4-0 silk sutures. The abdominal wall was similarly opened during sham surgery, and the wall was closed immediately after identifying the spleen 39 . In this study, we did not use opioid and/or non-steroidal anti-inflammatory drugs for pain management after surgery.
Sample collection. Experiment 1. After IMQ treatment for four consecutive days, the skin, spleen, and blood samples were collected on day 5. Experiment 2. Splenectomy or sham was carried out on day 1. Mice were treated with IMQ from day 15 to day 18, and skin samples were collected on day 19. The clinical skin score was measured from day 15 to day 19. The degree of skin inflammation was assessed with a cumulative disease severity score, similar to the human Psoriasis Area and Severity Index but without considering the area. Erythema, scaling, and thickening were scored independently from 0 to 4: 0, none; 1, slight; 2, moderate; 3, marked; 4, very marked. The single scores were summed; the highest possible score is 12 9 .  Untargeted metabolomics analysis of plasma samples. Untargeted metabolomics analysis was performed using an ExionLC AD UPLC system (SCIEX, Tokyo, Japan) interfaced with an X500R LC-QToFMS system (SCIEX, Tokyo, Japan) with electrospray ionization (ESI) operating in positive and negative ionization mode, as previous reported 40,41 . First, 100 μL of methanol containing internal standards (100 μM N,N-diethyl-2-phenylacetamide and d-camphor-10-sulfonic acid) was added to the plasma samples (100 μL), and then samples were centrifuged at 14,000 × rpm for 5 min. After centrifugation, the supernatant was transferred to an Amicon® Ultra-0.5 3 kDa filter column (Merck Millipore, Tokyo, Japan) and centrifuged at 14,000 × rpm for 1 h. The filtrate was transferred to glass vials for subsequent analysis.
The metabolomics data was analyzed with Mass Spectrometry-Data Independent AnaLysis (MS-DIAL) software version 4.60 42 and R statistical environment Ver 4.0.5. Only metabolites present in 50% of the samples were measured, and metabolites whose coefficient of variation value was over 30% in pooled QC samples were removed from analysis. Annotation level 2 proposed by Schymanski et al. 43 was used for data analysis.
Histology. Back skin samples from control and IMQ-treated groups were collected and fixed in 10% formalin (FUJIFILM Wako Pure Chemical Corp., Tokyo, Japan). Staining with hematoxylin and eosin (HE) was performed at the Biopathology Institute Co., Ltd (Kunisaki, Oita, Japan) as previously reported 22 . Back skin samples were embedded in paraffin, and 3-μm sections were prepared and stained with HE. Representative images of two groups were obtained using a Keyence BZ-9000 Generation II microscope (Osaka, Japan) as previously reported 22 .
Quantitative real-time polymerase chain reaction. RNA was isolated using TRIzol LS Reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions; cDNA was generated using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Waltham, MA, USA) and TaKaRa polymerase chain reaction (PCR) Thermal Cycler Dice (Takara Bio Inc., Kusatsu, Shiga, Japan), and quantitative real-time PCR was performed using the StepOnePlus Real-Time PCR System (Applied Biosystems, Waltham, MA, USA). The mouse primers for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (4352339E), IL-17A (Mm00439618), and IL-23A (Mm00518984) were obtained from Applied Biosystems. The GAPDH housekeeping gene was used to normalize gene expression.

Statistical analysis.
Data are shown as the mean ± standard error of the mean. Data were analyzed using GraphPad Prism (Tokyo, Japan). Student's t-test was performed to compare spleen weights between the two groups. Spleen cell types and plasma metabolites were compared between the two groups using Mann-Whitney U-test with a false discovery rate (FDR) control. Correlations among spleen weight, spleen cells, and plasma metabolites were evaluated using Spearman's correlation analysis. For multivariate analysis of the metabolome data, orthogonal partial least squares discriminant analysis (OPLS-DA) was performed in Simca-P V.14.0 (Umetrics AB). Metabolites with VIP > 0.6 and p-value < 0.05 (Wilcoxon rank-sum test) were considered differentially abundant. Cumulative skin score, relative mRNA expression of skin, and body weight changes were analyzed with a Kruskal-Wallis test. P < 0.05 (or FDR-corrected P < 0.05) was considered statistically significant.

Data availability
The data that support the findings of this study are available from the corresponding author upon reasonable request. www.nature.com/scientificreports/