Effects of CEACAM1 in oral keratinocytes on HO-1 expression induced by Candida β-glucan particles

Abstract Objective Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) is a member of the carcinoembryonic antigen family. Although its expression has been found in chronic oral inflammatory epithelium, this study aimed to know whether CEACAM1 in oral keratinocytes participates in host immune response against Candida albicans . Methodology We investigated CEACAM1 expression in oral keratinocytes induced by C. albicans as well as by Candida cell wall component β-glucan particles (β-GPs). Furthermore, the effects of CEACAM1 on β-GPs-induced heme oxygenase-1 (HO-1) expression and its related signals were examined. Results Fluorescence staining showed CEACAM1 expression in oral keratinocytes (RT7) cells, whereas quantitative reverse transcription (RT)-PCR indicated that both live and heat-killed C. albicans increased CEACAM1 mRNA expression in RT7 cells. Examinations using quantitative RT-PCR and western blotting indicated that CEACAM1 expression was also increased by β-GPs derived from C. albicans . Specific siRNA for CEACAM1 decreased HO-1 expression induced by β-GPs from C. albicans as well as the budding yeast microorganism Saccharomyces cerevisiae . Moreover, knockdown of CEACAM1 decreased β-GPs-induced ROS activity in the early phase and translocation of Nrf2 into the nucleus. Conclusion CEACAM1 in oral keratinocytes may have a critical role in regulation of HO-1 for host immune defense during Candida infection.


Introduction
Oral candidiasis is an oral mucosal infection caused by Candida species, and C. albicans is the most common associated-pathogen. 1 C. albicans colonizes the surface of oral mucosa, where it adheres, and invades oral keratinocytes, leading to damage during oral infection. 2 On the other hand, oral keratinocytes in the oral mucosal membrane are the first line of defense against C. albicans in innate immune systems. Oral keratinocytes can produce inflammatory cytokines, chemokines, and anti-microbial peptides against live Candida, 3,4,5 and increase the expression of various genes related to inflammatory and stress responses by exposure to heat-killed C. albicans. 6 These reports also indicated that oral keratinocytes can recognize Candida cell wall components, as well as live Candida, and an induce immune response against Candida infection.
Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) is a member of the carcinoembryonic antigen (CEA) family and associates with various signal transduction processes related to cell activation, proliferation, differentiation, and apoptosis. 7 This molecule expression, induced by inflammatory cytokines and specific bacteria, 9,10 takes place on the surface of epithelial and endothelial cells, as well as various immune cell types, such as neutrophils, monocytes, dendritic cells, NK cells, T cells, and B cells. 8 Furthermore, CEACAM1 modulates immune response, which regulates inflammatory signaling and cytokine genes in various cell types. 11,12 In the oral cavity, cases of chronic inflamed oral epithelium present CEACAM 1 expression, such as periodontitis and lichen planus. 13,14 However, if CEACAM1 in oral keratinocytes participates in host defense systems against C. albicans remains unknown.
The β-glucan, a major cell wall component of C.
albicans as well as the budding yeast Saccharomyces cerevisiae, constitutes the inner layer of mannan/ mannoprotein, which is in the outer layer of the Candida cell wall. 15 This component has an important function as an immune modulator, since it can activate immune response in immune cells, such as monocytes and macrophages. 16 Previously, we demonstrated that β-glucan particles (β-GPs) derived from C. albicans enhanced expression of heme oxygenase-1 (HO-1), a stress-inducible gene, in oral keratinocytes, which contributes to host defense against stress caused by Candida infection. 6 However, CEACAM1 functions as a cellular receptor, and interacts with several bacterial pathogens as well as fungi. [17][18][19][20][21] A recent report noted that C. albicans directly interacts with CEACAM1 by the N-terminal IgV-like domain and β-glucan has been found on the surface of human oral epithelium after invasion by Candida. 6 Therefore, β-glucan exposed on the surface of C. albicans may be associated with CEACAM1-mediated immune response in oral keratinocytes.
Based on the hypothesis that CEACAM1 in oral keratinocytes participates in host immune response against C. albicans, we investigated CEACAM1 expression in oral keratinocytes induced by C. albicans and the cell wall component β-GPs. Furthermore, we examined the effects of CEACAM1 on the intercellular signaling pathway involved in β-GP-induced HO-1 expression.

Methodology Reagents
The following antibodies used for immunoblotting buffered saline (PBS) and then heat-killed at 60°C for 30 minutes. 24 β-glucan-containing particles derived from C. albicans C. albicans β-GPs were obtained using a hot alkali and acid method, as previously described. 25 C. albicans IFO1385 was cultured in 1200 mL of Sabouraud's broth medium, then washed with PBS and suspended in 1% (wt/v) NaOH at 100°C for 24 h, and later the supernatant for the alkali-soluble fraction was collected. The insoluble residue was treated with 0.5 M acetic acid at 80°C for 24 h and subjected to repeated washings, then neutralized with PBS (pH 7.4), which the pellet was later lyophilized and stored at 4°C.

Immunocytochemistry
Cells were seeded into two-well chamber slides ability of β-GPs, a C. albicans cell wall component, to affect CEACAM1 expression, and RT-PCR analysis showed that C. albicans β -GPs, as well as heat-killed C. albicans, increased that expression ( Figure 3A).
Composed of a β-glucan layer covalently linked to a variety of cell surface mannoproteins, 27 C. albicans and the budding yeast S. cerevisiae share cell wall structure similarities. S. cerevisiae and C. albicans β-GPs ( Figure 3B) increased CEACAM1 expression, whereas western blotting results indicated that C. albicans β-GPs increased CEACAM1 protein expression, approximately 120 kDa, also noted in a previous report 28 (Figure 4).

Effects of CEACAM1 on β-GPs-induced HO-1 expression
In our previous study, we found that β-GPs from both C. albicans and S. cerevisiae enhance expression of the stress-inducible gene HO-1 in oral keratinocytes. 6 To determine if β-GPs-induced HO-1 expression is associated with CEACAM1 expression, we examined knockdown of CEACAM1, mediated by two different siRNAs ( Figure 5A, B), which showed that knockdown by each siRNA decreased HO-1 expression induced by β-GPs from both C. albicans and S. cerevisiae ( Figure 6A, B). Furthermore, the neutralizing antibody for CEACAM1 also decreased β-GPs-induced HO-1 expression (Figure 7). Therefore, β-GPs-induced HO-1 and CEACAM1expressions are associated.

Effects of CEACAM1 on signaling mediated by β-GPs-induced HO-1 expression
Oxidative stress is caused by elevation of Figure 2-Effects of live and heat-killed C. albicans on CEACAM1 mRNA expression in RT7 cells. Cells were incubated with live (105/mL) or heat-killed (108/mL) C. albicans (CA) for 12 hours. Gene mRNA levels are presented as relative to that of β-actin. Values are shown as fold increase as compared to non-treated cells and presented as the mean ± SD of three independent experiments. *Significantly different from non-treated control (Bonferroni/Dunn's multiple comparison test: p<0.05). reactive oxygen species (ROS). 29 We previously demonstrated that β-GPs increased HO-1 expression by enhancement of intercellular ROS. 6 Another study found ROS-mediated signaling activating nuclear factor erythroid 2-related factor 2 (Nrf2), a recently revealed regulator of cellular resistance to oxidants, which is translocated into the nucleus and increases HO-1 expression. 30 We examined if signaling related to β-GPs-induced HO-1 expression mediates CEACAM1.
In the early phase, the knockdown of CEACAM1 decreased β-GPs-induced intracellular ROS production by its specific siRNA ( Figure 8). Furthermore, Nrf2 expression in transfected cells with CEACAM1 siRNA was high in the nucleus, as compared to control siRNA cells, suggesting a decrease in translocation of Nrf2 into the nucleus by knockdown of CEACAM1 ( Figure   9). Therefore, CEACAM1 is associated with regulation of oxidative stress-mediated signaling related to β-GPs-induced HO-1 expression. Discussion CEACAM1 is found in a variety of human organs, including gastric, colon, lung, breast, endometrium, prostate, and gall bladder tissues. In oral tissues, CEACAM1 was detected in sulcular epithelium sites affected by periodontitis and junctional epithelium in healthy subjects. 13 Some investigators reported that up-regulation of CEACAM1 is associated with development of chronic immune-mediated diseases, Figure 4-Effects of C. albicans β-GPs on CEACAM1 protein expression in RT7 cells Cells were incubated with C. albicans β-glucan-containing particles (CA β-GPs) (200 μg/mL) for 24 hours. Cell extracts were subjected to SDS-PAGE and western blotting with antibodies against CEACAM1 and GAPDH. The experiments were performed at least three times, with representative results shown. with bacterial pathogens such as Neisseria. 9 Klaile, et al. 33 (2017), reported that live C. albicans increase CEACAM1 expression in intestinal epithelial cells.
In this study, we found that heat-killed and live C. albicans cells increase CEACAM1 expression in oral keratinocytes. Therefore, Candida cell wall components can activate immune systems related to CEACAM1 expression in oral keratinocytes.
β-glucan, a cell-wall component of Candida recognized by the innate system, showed a capability Figure 6-Effects of CEACAM1 siRNA on β-GPs-induced HO-1 in RT7 cells Cells were transiently transfected with siRNA for CEACAM1 for 48 hours, then exposed to C. albicans β-glucan-containing particles (CA β-GPs) or S. cerevisiae β-GPs (SC β-GPs) (200 μg/mL) for 12 hours. Gene mRNA levels are presented as relative to that of β-actin. Values are shown as fold increase as compared to control siRNA-transfected cells and presented as the mean ± SD of least three independent experiments. #Significant decrease as compared with the control (Bonferroni/Dunn's multiple comparison test: p<0.05).   -Effects of knockdown of CEACAM1 on β-GPs-induced translocation of Nrf2 into nucleus in RT7 cells Cells were transfected with siRNA for CEACAM1 siRNA for 48 hours, then stimulated with C. albicans β-glucan-containing particles (CA β-GPs) (200 μg/mL) for 4 hours. Whole cell and nuclear extractions were prepared, then subjected to western blot analysis with the Nrf2 antibody. LaminB and GAPDH were used as marker proteins for the nuclear and whole cell fractions, respectively. The experiments were performed at least three times, with representative results shown. 2022;30:e20220158 9/11 latter study showed Dectin-1 functions as a trigger for effective immune response by recognition of β-glucan. We previously found that Dectin-1 was expressed in oral keratinocytes, whereas knockdown of Dectin-1 had no effect on fungal β-GP-induced CEACAM1 expression in the present study (data not shown). Therefore, the regulatory mechanism related to CEACAM1 expression induced by β-GPs in oral keratinocytes is likely different from that induced by Dectin-1.
HO-1 is an enzyme known to degrade heme and generate carbon monoxide and biliverdin, while causing release of iron, which is stored within the iron-binding protein ferritin. 36 Moreover, the enzyme is stress inducible and upregulated in response to cellular stress conditions, such as oxidative stress, caused by reactive oxygen species (ROS). 29 HO-1 has an important role in decreasing oxidantinduced injury during inflammatory processes, whereas the overexpression can promote resistance to apoptosis, induce cell proliferation, and alleviate inflammation. 37  Furthermore, excessive ROS production activates Nrf2/antioxidant response element signaling for defense against intracellular oxidative stress and regulation of increased HO-1 expression, which provide cell protective effects including anti-apoptotic and anti-inflammatory functions. 30,40 Our previous findings showed that β-GPs induced intercellular ROS production in RT7 within 1 h, while translocation of Nrf2 into the nucleus increased HO-1 expression. In the study, a specific siRNA for CEACAM1 inhibited intercellular ROS induction after exposure to β-GPs, suggesting that CEACAM1 acts upstream of ROS and translocation of Nrf2, and regulates HO-1 expression. Based on previous reports and on the present study, we considered that C. albicans binds to CEACAM1 in oral keratinocytes during candida infection of oral epithelium, and that β-glucan exposed on surface of C.

CEACAM1 mediates adhesion and internalization
albicans can activate CEACAM1 expression. Therefore, β-glucan increases ROS-mediated signaling resulting in increased HO-1 expression in oral keratinocytes.
Furthermore, CEACAM1 in oral keratinocytes may participate in host immune defense response against C. albicans infection.

Conclusions
To our knowledge, this is the first study to demonstrate that β-GPs enhance CEACAM1 expression in oral keratinocytes. We found an association between CEACAM1 and β-GPs -induced HO-1 expression via ROS signaling. Therefore, CEACAM1 in oral keratinocytes may have a critical role in regulating HO-1 for host immune defense during Candida infection.