Alumina Ceramic Exacerbates the Inflammatory Disease by Activation of Macrophages and T Cells

(1) Background: Aluminum oxide (Al2O3) ceramic is one of the materials used for artificial joints, and it has been known that their fine particles (FPs) are provided by the wear of the ceramic. Al2O3 FPs have been shown to induce macrophage activation in vitro; however, the inflammatory effect in vivo has not been studied. (2) Methods: We examined the in vivo effect of Al2O3 FPs on the innate and adaptive immune cells in the mice. (3) Results: Al2O3 FPs promoted the activation of spleen macrophages; however, conventional dendritic cells (cDCs), plasmacytoid DCs (pDCs), and natural killer (NK) cells were not activated. In addition, increases in the CD4 and CD8 T cells was induced in the spleens of the mice treated with Al2O3 FPs, which differentiated into interferon-gamma (IFN-γ)-producing helper T1 (Th1) and cytotoxic T1 (Tc1) cells. Finally, the injection of Al2O3 FPs exacerbated dextran sulfate sodium (DSS)-induced inflammation in the colon, mediated by activated and increased number of CD4 and CD8 T cells. (4) Conclusions: These data demonstrate that FPs of Al2O3 ceramic may contribute to the exacerbation of inflammatory diseases in the patients.


Introduction
Alumina ceramic articulation, made of aluminum oxide (Al 2 O 3 ), is one of the bearing surfaces of hip arthroplasty, which is well known to have high resistance to wear and high chemical stability [1][2][3]. For these reasons, alumina ceramic articulation has been used for several decades.
The hip joint is the biggest joint of the body, which endures three times the body weight during walking. Joints perform tens of thousands of frictional movements in their lifetime [4]. Although the wear rate of alumina ceramic has been shown to be less than 1 µm/year, the fine particles (FPs) of Al 2 O 3 are generated by continuous frictional movement, which may promote inflammation in the adjacent tissue of implant [2,3,5,6]. Previous studies have shown that the treatment of Al 2 O 3 FPs induced activation of human macrophages, which produced higher levels of tumor necrosis factor-α (TNF-α) [7][8][9][10]. These inflammatory responses are capable of causing disastrous situation such as aseptic loosening [11]. However, the effects of Al 2 O 3 FPs on the other immune cells, and in vivo effects in mouse models, have not been studied.
Inflammation is a reaction of the body's defense system to tackle the pathogens [12,13]. Although the inflammation contributes in protecting the body by immune activation, it also induces damage to healthy tissues [12]. Various stimuli including pathogens, radiation, antigen, and damaged cells can promote inflammation, which is harmful to the healthy tissue [14][15][16]. Inflammatory responses are classified as acute and chronic reactions [17,18]. The acute inflammatory response is an initial response to the stimulus, which progresses within a short period of time, and is mainly involved in the innate immune cells such as macrophages, dendritic cells (DCs), and natural killer (NK) cells [19][20][21]. The DCs contain two main subsets, conventional DCs (cDCs) and plasmacytoid DCs (pDCs) [22]. The pDCs are the main cells that protect body from viral infection and produce interferon-alpha (IFN-α) in responses to the virus [23]. On the other hand, cDCs and macrophages are representative antigen-presenting cells (APCs) against microbes, which further induce the activation of T and B cells [24][25][26][27]. The activated DCs and macrophages upregulate co-stimulator, major histocompatibility complex, and pro-inflammatory cytokine production, which promote the differentiation and activation of T cells [28][29][30].
Although it has been shown that the Al 2 O 3 FPs induced the macrophage activation in vitro, the effects of Al 2 O 3 FPs in the induction of inflammation in peripheral tissue in vivo in the mouse have not been studied. In addition, there was no report of the effect of Al 2 O 3 FPs-induced inflammation in autoimmune disease. So, this study confirmed whether Al 2 O 3 FPs exacerbates the inflammatory response by inducing IBD, the most representative autoimmune disease, into a mouse model. Therefore, we conducted this study to confirm our hypothesis that the Al 2 O 3 FPs may induce the activation of innate and adaptive immune cells in vivo in the mice and exacerbate the tissue inflammation.   Then, we evaluated the inflammatory properties of Al 2 O 3 FPs in the mouse in vivo, especially on innate immune cells. The C57BL/6 mice were intraperitoneally (i.p.) injected with 10 mg/kg of Al 2 O 3 FPs. The lipopolysaccharide (LPS) was also injected into mice as a positive control. Six hours post-injection, the spleen was harvested, revealing that the total number of splenocytes was not changed by Al 2 O 3 FPs (Figure 2a). We further analyzed the activation of innate immune cells by Al 2 O 3 FPs. As shown in Figure 2b, the pDCs (CD317 + B220 + cells), cDCs (CD11c + lineagecells), macrophages (CD11b + CD11ccells), and NK cells (NK1.1 + CD3cells) were defined in the spleen. We found that the number of macrophages was significantly increased by the treatment with Al 2 O 3 FPs, whereas the number of NK cells tended to decrease (Figure 2c). The numbers of pDCs and cDCs were unchanged in the spleen compared to those in the phosphate buffered saline (PBS)-treated controls ( Figure 2c). Thus, these data indicate that the Al 2 O 3 FPs promote an increase in the number of macrophages in the spleen.  The absolute numbers of pDCs, cDCs, macrophages and NK cells analyzed in the spleen. All data are representatives and averages of six independent samples (2 mice for three experiments, two-way ANOVA, mean ± SEM). * and ** represent p < 0.05 and p < 0.01, respectively. (c) The absolute numbers of pDCs, cDCs, macrophages and NK cells analyzed in the spleen. All data are representatives and averages of six independent samples (2 mice for three experiments, two-way ANOVA, mean ± SEM). * and ** represent p < 0.05 and p < 0.01, respectively.

Al 2 O 3 FPs Promoted the Activation of Spleen Macrophages
We further examined whether the Al 2 O 3 FPs can induce the activation of innate immune cells. The treatment with 10 mg/kg of Al 2 O 3 FPs did not upregulate the expression levels of co-stimulators and major histocompatibility complex (MHC) class I and II in pDCs and cDCs, although the CD86 expression levels were considerably increased in cDCs (Figure 3a,b and Figure S1). In contrast with DCs, the co-stimulator and MHC class I and II levels in the macrophages were significantly increased by the treatment with 10 mg/kg of Al 2 O 3 FPs (Figure 3c and Figure S1). The expression of co-stimulators and MHC class were not considerably changed in pDCs, cDCs and macrophages at 1 and 5 mg/kg of Al 2 O 3 FPs ( Figure S1). Moreover, the co-stimulators were rapidly reduced 18 h after treatment of 10 mg/kg of Al 2 O 3 FPs, whereas the MHC class I and II levels were significantly upregulated in the macrophages ( Figure S2). On the other hand, the treatment with Al 2 O 3 FPs did not promote the activation of NK cells in the spleen, although the CD69 expression levels were slightly increased ( Figure S3). expression levels were considerably increased in cDCs (Figures 3a,b and S1). In contrast with DCs, the co-stimulator and MHC class I and II levels in the macrophages were significantly increased by the treatment with 10 mg/kg of Al2O3 FPs (Figures 3c and S1). The expression of co-stimulators and MHC class were not considerably changed in pDCs, cDCs and macrophages at 1 and 5 mg/kg of Al2O3 FPs ( Figure S1). Moreover, the co-stimulators were rapidly reduced 18 h after treatment of 10 mg/kg of Al2O3 FPs, whereas the MHC class I and II levels were significantly upregulated in the macrophages ( Figure S2). On the other hand, the treatment with Al2O3 FPs did not promote the activation of NK cells in the spleen, although the CD69 expression levels were slightly increased ( Figure S3).
We then examined the production of pro-inflammatory cytokines in the serum in response to Al2O3 FPs. The concentrations of tumor necrosis factor-alpha (TNF-α), interleukin (IL)-6, and IL-12p70 in the serum were substantially increased by Al2O3 FPs (Figure 3d). Thus, these data suggested that the Al2O3 FPs induce the activation of spleen macrophages, which contributes to the production of inflammatory cytokines in the serum. . Al2O3 FPs promoted the activation of macrophages, but not pDCs and cDCs. The C57BL/6 mice were i.p. injected with 10 mg/kg of Al2O3 FPs. Spleens were harvested six hours post-injection, followed by flow cytometric analysis of the pDCs, cDCs and macrophages. The expression levels of co-stimulator and major histocompatibility complex (MHC) class I and II were shown in (a) pDCs, (b) cDCs and (c) macrophages (n = 6 mice, two-way ANOVA, mean ± SEM). (d) Concentration of tumor necrosis factor-alpha (TNF-α), interleukin (IL)-6, and IL-12p70 were measured by Enzyme Linked Immunosorbent Assay (ELISA). The data are averages of six independent samples (2 mice for three experiments, two-way ANOVA, mean ± SEM). * and ** represent p < 0.05 and p < 0.01, respectively. FPs promoted the activation of macrophages, but not pDCs and cDCs. The C57BL/6 mice were i.p. injected with 10 mg/kg of Al 2 O 3 FPs. Spleens were harvested six hours post-injection, followed by flow cytometric analysis of the pDCs, cDCs and macrophages. The expression levels of co-stimulator and major histocompatibility complex (MHC) class I and II were shown in (a) pDCs, (b) cDCs and (c) macrophages (n = 6 mice, two-way ANOVA, mean ± SEM). (d) Concentration of tumor necrosis factor-alpha (TNF-α), interleukin (IL)-6, and IL-12p70 were measured by Enzyme Linked Immunosorbent Assay (ELISA). The data are averages of six independent samples (2 mice for three experiments, two-way ANOVA, mean ± SEM). * and ** represent p < 0.05 and p < 0.01, respectively.
We then examined the production of pro-inflammatory cytokines in the serum in response to Al 2 O 3 FPs. The concentrations of tumor necrosis factor-alpha (TNF-α), interleukin (IL)-6, and IL-12p70 in the serum were substantially increased by Al 2 O 3 FPs (Figure 3d). Thus, these data suggested that the Al 2 O 3 FPs induce the activation of spleen macrophages, which contributes to the production of inflammatory cytokines in the serum.

Al 2 O 3 FPs Elicited Helper T 1 (Th1) and Cytotoxic T 1 (Tc1) Cell Responses
Since the treatment with Al 2 O 3 FPs promoted the activation of macrophages and production of pro-inflammatory cytokines, we examined whether the Al 2 O 3 FPs induce activation and differentiation of T cells in adaptive immunity. C57BL/6 mice were i.p. injected with PBS, Al 2 O 3 FPs, and LPS daily for 3 days, and their spleens were harvested on the day 4 as shown in the schematic diagram in Figure 4a. NK1.1 -CD3 + cells in the live leukocytes were defined as T cells, which were further divided into CD4 and CD8 cells (Figure 4b). The numbers of total CD4 and CD8 T cells were significantly increased by the treatment with Al 2 O 3 FPs (Figure 4c). Moreover, interferon-gamma (IFN-γ)-producing CD4 and CD8 T cells in the spleen were markedly increased by Al 2 O 3 FPs, indicating that the Al 2 O 3 FPs promote differentiation of Th1 and Tc1 cells (Figure 4d,e). In contrast with the interferon-gamma (IFN-γ) producing T cells, IL-4-and IL-17-producing CD4 T cells and IL-4-producing CD8 T cells were not changed considerably (Figure 4d,e). In addition, the concentration of IFN-γ in the serum was also elevated substantially by the treatment with Al 2 O 3 FPs (Figure 4f). Thus, these data suggested that Al 2 O 3 FPs elicit the Th1 and Tc1 responses in the mouse spleen.

Al2O3 FPs Elicited Helper T 1 (Th1) and Cytotoxic T 1 (Tc1) Cell Responses.
Since the treatment with Al2O3 FPs promoted the activation of macrophages and production of pro-inflammatory cytokines, we examined whether the Al2O3 FPs induce activation and differentiation of T cells in adaptive immunity. C57BL/6 mice were i.p. injected with PBS, Al2O3 FPs, and LPS daily for 3 days, and their spleens were harvested on the day 4 as shown in the schematic diagram in Figure 4a. NK1.1 -CD3 + cells in the live leukocytes were defined as T cells, which were further divided into CD4 and CD8 cells (Figure 4b). The numbers of total CD4 and CD8 T cells were significantly increased by the treatment with Al2O3 FPs (Figure 4c). Moreover, interferon-gamma (IFN-γ)-producing CD4 and CD8 T cells in the spleen were markedly increased by Al2O3 FPs, indicating that the Al2O3 FPs promote differentiation of Th1 and Tc1 cells (Figure 4d,e). In contrast with the interferon-gamma (IFN-γ) producing T cells, IL-4-and IL-17-producing CD4 T cells and IL-4-producing CD8 T cells were not changed considerably (Figure 4d,e). In addition, the concentration of IFN-γ in the serum was also elevated substantially by the treatment with Al2O3 FPs (Figure 4f). Thus, these data suggested that Al2O3 FPs elicit the Th1 and Tc1 responses in the mouse spleen.

DSS-Induced Colitis Was Exacerbated by Al 2 O 3 FPs
Our data on the Al 2 O 3 FPs-mediated induction of Th1 and T1c immune responses prompted us to examine whether the Al 2 O 3 FPs exacerbate the progression of Th1-mediated inflammatory disease. The effects of Al 2 O 3 FPs were examined in the DSS-induced colitis in the mice, where Th1 cells are the main contributors. C57BL/6 mice were randomly divided into four groups (five mice for each group) as shown in the schematic diagram (Figure 5a). The administration of DSS did not reduce the length of colon, whereas the additional treatment with Al 2 O 3 FPs induced significant decrease in the length of colon compared to the controls (Figure 5b,c). Histological analysis showed that the DSS administration induced mild infiltration of leukocytes in the colon, which was substantially increased by the additional treatment with Al 2 O 3 FPs (Figure 5d). Consistent with the histology data, the colon infiltrated number of leukocytes and the number of leukocytes in the mesenteric lymph node (mLN) was significantly increased by the treatment with DSS and Al 2 O 3 FPs compared to those in the controls (Figure 5e). Therefore, these data suggested that the DSS-induced colitis was exacerbated by Al 2 O 3 FPs in the mice.

DSS-Induced Colitis was Exacerbated by Al2O3 FPs
Our data on the Al2O3 FPs-mediated induction of Th1 and T1c immune responses prompted us to examine whether the Al2O3 FPs exacerbate the progression of Th1-mediated inflammatory disease. The effects of Al2O3 FPs were examined in the DSS-induced colitis in the mice, where Th1 cells are the main contributors. C57BL/6 mice were randomly divided into four groups (five mice for each group) as shown in the schematic diagram (Figure 5a). The administration of DSS did not reduce the length of colon, whereas the additional treatment with Al2O3 FPs induced significant decrease in the length of colon compared to the controls (Figure 5b,c). Histological analysis showed that the DSS administration induced mild infiltration of leukocytes in the colon, which was substantially increased by the additional treatment with Al2O3 FPs (Figure 5d). Consistent with the histology data, the colon infiltrated number of leukocytes and the number of leukocytes in the mesenteric lymph node (mLN) was significantly increased by the treatment with DSS and Al2O3 FPs compared to those in the controls (Figure 5e). Therefore, these data suggested that the DSS-induced colitis was exacerbated by Al2O3 FPs in the mice.   (Figure 6a,b). Moreover, the TNF-α-, IFN-γ-, and IL-17A-producing CD4 T cells in the colon and mLN were significantly increased by Al 2 O 3 FPs compared to those in controls (Figure 6c,d). Furthermore, IFN-γ-producing CD8 cells were also increased dramatically, whereas TNF-α-producing CD8 T cells were not increased compared to those in controls (Figure 6c,d). The serum concentration of IFN-γ was also elevated significantly upon treatment with DSS and Al 2 O 3 FPs in the mice, whereas that of TNF-α was not increased upon treatment with DSS and Al 2 O 3 FPs compared to treatment with the Al 2 O 3 FPs alone and DSS alone ( Figure S4). Thus, these data suggested that the Th1 and Tc1 immune responses induced by Al 2 O 3 FPs contributed in the exacerbation of the DSS-induced colitis.

Al2O3 FPs-Induced Th1 and Tc1 Cells Contributed in the Exacerbation of Colitis
We further examined whether the Th1 and Tc1 responses induced by Al2O3 FPs contributed in the exacerbation of DSS-induced colitis. Treatment with DSS and Al2O3 FPs elicited great increases in the number of total T cells in the colon and mLN as well as CD4 and CD8 T cells compared to those in controls (Figure 6a,b). Moreover, the TNF-α-, IFN-γ-, and IL-17A-producing CD4 T cells in the colon and mLN were significantly increased by Al2O3 FPs compared to those in controls (Figure 6c,d). Furthermore, IFN-γ-producing CD8 cells were also increased dramatically, whereas TNF-αproducing CD8 T cells were not increased compared to those in controls (Figure 6c,d). The serum concentration of IFN-γ was also elevated significantly upon treatment with DSS and Al2O3 FPs in the mice, whereas that of TNF-α was not increased upon treatment with DSS and Al2O3 FPs compared to treatment with the Al2O3 FPs alone and DSS alone ( Figure S4). Thus, these data suggested that the Th1 and Tc1 immune responses induced by Al2O3 FPs contributed in the exacerbation of the DSSinduced colitis.  Figure 5.

(a) Total numbers of colon infiltrated T cells, CD4 T cells and CD8 T cells. (b) The number of total T cells, CD4 T cells and CD8 T cells in mLNs. (c) The numbers of cytokine producing CD4 and CD8 T cells in colon. (d) Intracellular
cytokine-producing CD4 and CD8 T cells in the mLNs (n = 5, two-way ANOVA, mean ± SEM). * and ** represent p < 0.05 and p < 0.01, respectively.

Discussion
Total hip arthroplasty has been proven to be an effective procedure to restore patients' damaged joints by replacing them with the artificial joints [39]. Bearing surface is the most important part for the longevity of the arthroplasty. Although the bearing options have evolved in recent decades, the artificial joint is a place where frictional motion continues to occur and is permanently unavailable due to the generation of the wear particles and dissociation of the insert [4]. It can also lead to complications by biological reactions such as inflammation caused by FPs [40]. There are various  Figure 5.

(a) Total numbers of colon infiltrated T cells, CD4 T cells and CD8 T cells. (b) The number of total T cells, CD4 T cells and CD8 T cells in mLNs. (c) The numbers of cytokine producing CD4 and CD8 T cells in colon. (d) Intracellular
cytokine-producing CD4 and CD8 T cells in the mLNs (n = 5, two-way ANOVA, mean ± SEM). * and ** represent p < 0.05 and p < 0.01, respectively.

Discussion
Total hip arthroplasty has been proven to be an effective procedure to restore patients' damaged joints by replacing them with the artificial joints [39]. Bearing surface is the most important part for the longevity of the arthroplasty. Although the bearing options have evolved in recent decades, the artificial joint is a place where frictional motion continues to occur and is permanently unavailable due to the generation of the wear particles and dissociation of the insert [4]. It can also lead to complications by biological reactions such as inflammation caused by FPs [40]. There are various materials for artificial joints, including metal and polyethylene. Alumina ceramic has attracted the attention due to its high chemical, physical and biological stability. However, inflammation has been often seen around the implant tissue despite being an artificial joint using Al 2 O 3 [3]. Moreover, tissue fibrosis at the periprosthetic joint capsule collected during a revision surgery was significantly increased than in the metal and polyethylene articulation due to inflammatory response [5].
It has been shown that the Al 2 O 3 induces the activation of human macrophages [2,3,40]. In addition, we further found that the systemic injection of Al 2 O 3 FPs promoted the activation of macrophages, Th1, and Tc1 cells, leading to exacerbation of the inflammatory diseases such as colitis. Therefore, these data are the first report demonstrating the possibility of inflammation exacerbation by Al 2 O 3 FPs in the human.
Macrophages contain two phenotypes: M1 and M2 [41,42]. M1 macrophages contribute in the clearance of microbes as inflammatory immune responses, especially the production of pro-inflammatory cytokines and upregulation of co-stimulators and MHC molecules [43]. In contrast to the M1 phenotype, the M2 macrophages contribute to the repairing of the inflamed tissue by suppressing immune activation [44]. Therefore, M1 and M2 macrophages are competitive in immune responses. In this study, we found that the treatment with the Al 2 O 3 FPs promoted upregulation of co-stimulators and production of pro-inflammatory cytokines in the macrophages, indicating the activation of the M1 macrophages. Thus, Al 2 O 3 FPs contribute to inflammation of the peripheral tissue instead of M2-mediated tissue-repair.
DSS is one of the materials that induces colitis in the mouse model [45,46]. It causes the loss of the colon's epithelial barrier function and induces infiltration of the immune cells in the colon, which induces the inflammation of the tissues [36,45]. The DSS-induced colitis is mediated by the Th1 and Th17 cells [47,48]. It is well known that the continuous administration of the DSS, such as 7 days administration, promotes serious inflammation of the colon [49]. We administrated the DSS for 5 days and followed by the treatment with the distilled water or Al 2 O 3 FPs for the determination of the effect of Al 2 O 3 FPs on the colitis. The 5-day administration of DSS did not promote serious inflammation of the colon, and the exacerbation effect of Al 2 O 3 FPs in the mice was evaluated. Additional treatment with the Al 2 O 3 FPs in the DSS-induced colitis in the mice further upregulated the infiltration of T cells and pro-inflammatory cytokines. Thus, these data demonstrated the exacerbation of inflammatory diseases by the Al 2 O 3 FPs.
Treatment with the Al 2 O 3 FPs induced the Th1 and Tc1 responses in the mice. In the DSS-colitis models, the additional treatment with the Al 2 O 3 FPs further increased the Th1 and Tc1 cells in the colon. Interestingly, IL-17-producing CD4 T cells were also increased in the DSS-induced colitis mice by Al 2 O 3 FPs; however, Al 2 O 3 FPs treatment alone did not induce Th17 responses in the mice in vivo. Such different effects on the induction of the Th17 response may be due to the synergistic effect of DSS and Al 2 O 3 FPs. Although the Al 2 O 3 FPs did not promote significant difference in the induction of Th17 responses, treatment with Al 2 O 3 FPs after DSS treatment might increase the Th17 response synergistically. In our next study, we will evaluate whether the DSS-induced increases in the levels of RORγδ, a transcription factor of Th17 cells [50], are further increased by the Al 2 O 3 FPs.

Mice
C57BL/6 mice were purchased from Hyochang Science (Daegu, Korea) and used at the age of 5 to 8 weeks. The mice were kept under pathogen-free standard conditions in a room with sterilized chow and water, 20-25 • C, and 40-60% humidity. The study was approved by the Yeungnam University Institutional Animal Care and Use Committee (IACUC) (21 July 2020) of Yeungnam University, Korea, and it is recorded recorded by the animal protocol number 2020-011, in compliance with the Animal Protection Act, the Law on Laboratory Animals, and the IACUC regulation of Yeungnam University.
All the experiments in this study were performed with the efforts to minimize the pain to the animals, and CO 2 gas was used for euthanasia in accordance with the humanitarian end-point standards in terms of ethics.

Transmission Electron Microscopy (TEM)
The size and shape of Al 2 O 3 FPs were determined by TEM (H-7600 transmission electron microscope, Hitachi, Japan). Sample was prepared by making suspension of Al 2 O 3 FPs in distilled water (DW), followed by deposition onto the TEM grid.

Preparation of Single Cell Suspension from Tissues
The spleens, colon, and mLNs were harvested and digested with digestion buffer containing DNase I (Sigma-Aldrich, MO, USA, #DN25) and collagenase D (Sigma-Aldrich, MO, USA, #11088858001) for 20 min at room temperature (RT). After washing with PBS, the pellets were re-suspended in 5 mL of histopaque 1.077 (Sigma-Aldrich, MO, USA, #10771), forming a layer on the fresh 5 mL histopaque, followed by fresh media on the top. The cells were centrifuged for 10 min at 1836× g. The leukocytes-containing fraction, with a density less than 1.077 g/mL, was collected [51].

Stimulation and Intracellular Staining of T cells
The single cells were incubated with ionomycin (1 Mm, #I9657) and phorbol 12-

Mouse Model of DSS-Induced Colitis
The mice were randomly divided into 4 groups (5 mice in each group). The first group was a control group provided with normal water daily. In the second group, in addition to providing normal water daily, Al 2 O 3 FP (10 mg/kg) was i.p. injected from day 6 to 8. The 3rd and 4th groups were provided with 5% DSS in the water for 5 days. From day 6, the 3rd group was provided with normal water and daily injection of PBS whereas the 4th group was provided with normal water and injection of Al 2 O 3 FPs for 3 days.

Histological Analysis
The small fractions of colon in each group were fixed in 4% formaldehyde. The colons were dehydrated overnight with 50%, 70%, 95%, and 100% ethanol. The colons were then embedded in paraffin. The paraffin blocks were cut into 6-mm-thick sections, placed on glass slides, and dried at 50-55 • C on a hot plate. The sections were deparaffinized with xylene, rehydrated, and then stained with H&E (Sigma-Aldrich, MO, USA, #H3136 and #E4009). These sections were dehydrated, covered with a cover glass with Canada balsam (Sigma-Aldrich, MO, USA, #C1795), and observed under a microscope.

Statistical Analyses
All the data are shown as mean ± standard error of the mean (SEM). Data sets were analyzed using a two-way ANOVA (Tukey multiple comparison test) and Mann-Whitney t-test. p values < 0.05 were considered to have a statistically significant difference. p < 0.05 was expressed as "*".and p < 0.01 was expresses as "**".

Conclusions
In this study, we demonstrated that Al 2 O 3 FPs induces the activation of macrophages, which elicits differentiation and activation of CD4 T and CD8 T cells. In addition, Al 2 O 3 FPs promoted study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.