2022 Volume 45 Issue 2 Pages 213-219
In the lung alveolar region, the innate immune system serves as an important host defense system. We recently reported that peptide transporter 2 (PEPT2) has an essential role in the uptake of bacterial peptides and induction of innate immune response in alveolar epithelial cells. In this study, we aimed to clarify the effects of corticosteroids on PEPT2 function and PEPT2-dependent innate immune response. NCI-H441 (H441) cells were used as an in vitro model of human alveolar type II epithelial cells, and the effects of dexamethasone (DEX) and budesonide (BUD) on the transport function of PEPT2 and the innate immune response induced by bacterial peptides were examined. PEPT2 function, estimated by measuring β-alanyl-Nε-(7-amino-4-methyl-2-oxo-2H-1-benzopyran-3-acetyl)-L-lysine (β-Ala-Lys-AMCA) uptake in H441 cells, was suppressed by treatment with DEX and BUD in a concentration- and time-dependent manner. The suppression of PEPT2 function was partially recovered by a glucocorticoid receptor antagonist. The expression of PEPT2 and nucleotide-binding oligomerization domain 1 (NOD1) mRNAs was suppressed by treatment with DEX and BUD, while PEPT2 protein level was not changed by these treatment conditions. Additionally, the increased mRNA expression of interleukin (IL)-8 and the increased secretion of IL-8 into the culture medium induced by bacterial peptides were also suppressed by treatment with these corticosteroids. Taken together, these results clearly suggest that corticosteroids suppress PEPT2 function and bacterial peptide-induced innate immune response in alveolar epithelial cells. Therefore, PEPT2- and NOD1-dependent innate immune response induced by bacterial peptides in the lung alveolar region may be suppressed during the inhaled corticosteroid therapy.
The lung is constantly exposed to the external milieu, and therefore has various immune mechanisms as a host defense system against invading pathogens.1,2) Nucleotide-binding oligomerization domain 1 (NOD1) is an intracellular pattern recognition receptor which recognizes bacterial peptidoglycan-derived peptides and activates downstream signaling pathways to initiate the immune response. However, the influx of these peptides into the cells is a prerequisite for the recognition of bacterial peptides by intracellular NOD1. Recently, we reported that bacterial peptidoglycan peptides, including γ-D-glutamyl-meso-diaminopimelic acid (iE-DAP) and L-alanyl-γ-D-glutamyl-meso-diaminopimelic acid (Tri-DAP), induced NOD1-dependent innate immune response after being taken up into the cells by peptide transporter 2 (PEPT2; SLC15A2), using a human alveolar epithelial cell line NCI-H441 (H441).3) Additionally, receptor-interacting serine/threonine-protein kinase 2 and mitogen-activated protein kinase (MAPK) signaling pathways, including p38 MAPK and extracellular signal-regulated kinase, but not c-Jun N-terminal kinase, were found to be involved in the induction of the innate immune response by bacterial peptides. In addition, the nuclear factor-κB (NF-κB) pathway also played a role in the innate immune response.3)
The H441 cell line was originally isolated and established from cells in pericardial fluid of a patient with papillary adenocarcinoma of the lung. Salomon et al.4) reported that H441 cells formed confluent, electrically tight monolayers, with high transepithelial electrical resistance (peak value of 1010 Ω·cm2 after 13 d in culture). They also found that membrane transporter expression, such as organic cation transporters, and their transport activities in H441 cells were similar with those observed in human alveolar epithelial cells in primary culture. Additionally, we found that PEPT2 and NOD1, but not PEPT1, were expressed in H441 cells.3,5) Therefore, H441 cells would be a useful in vitro model to study PEPT2- and NOD1-dependent innate immune responses in alveolar epithelial cells.
Inhaled corticosteroids such as budesonide (BUD) are widely used as the first-line treatment of asthma.6) However, respiratory infections have been indicated as an adverse reaction of inhaled corticosteroids. Inhaled corticosteroid therapy was reported to be associated with a higher risk of pneumonia, especially in patients with chronic obstructive pulmonary disease (COPD).7–9) The higher risk of pneumonia was also reported in asthma patients treated with inhaled corticosteroids.10) However, the role of PEPT2-dependent innate immune response in increased susceptibility to infections under corticosteroid therapy remains unknown. In the present study, we examined the effects of corticosteroids on PEPT2 function and PEPT2- and NOD1-dependent innate immune response induced by bacterial peptidoglycan peptides, using H441 cells.
RPMI-1640 medium, iE-DAP, and Tri-DAP were purchased from Thermo Fisher Scientific, Inc. (Waltham, MA, U.S.A.). Fetal bovine serum (FBS) and insulin-transferrin-sodium selenite (ITS) supplements were purchased from Capricorn Scientific (Ebsdorfergrund, Germany) and Roche (Basel, Switzerland), respectively. β-Alanyl-Nε-(7-amino-4-methyl-2-oxo-2H-1-benzopyran-3-acetyl)-L-lysine (β-Ala-Lys-AMCA), a fluorophore-conjugated dipeptide, was purchased from PEPTIDE INSTITUTE, Inc. (Osaka, Japan). Cefadroxil (CDX) and BUD were purchased from Merck KGaA (Darmstadt, Germany). Dexamethasone (DEX) and RU486 (RU) were purchased from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan) and Cayman Chemical Company (Ann Arbor, MI, U.S.A.), respectively. All other chemicals used were of the highest grade commercially available.
Cell Culture and Corticosteroid TreatmentAfter seeding, H441 cells were cultured in RPMI-1640 medium supplemented with 5% FBS, 100 IU/mL penicillin, 100 µg/mL streptomycin, and 1% sodium pyruvate for 24 h in a humidified atmosphere containing 5% CO2 at 37 °C, followed by culture in RPMI-1640 medium containing 200 nM DEX and ITS supplement until day 8. On day 9, the culture medium was replaced with that containing ITS supplement and various concentrations of DEX or BUD (0–100 nM), and cells were cultured until day 13. Each medium was changed every two days.
Uptake of β-Ala-Lys-AMCA in H441 CellsH441 cells were seeded at a density of 3 × 104 cells/well in 24 well plates. On day 13, H441 cells were preincubated with N-(2-hydroxyethyl)piperazine-N′-2-ethanesulfonic acid (HEPES) buffer (5 mM HEPES, 145 mM NaCl, 3 mM KCl, 1 mM CaCl2, 0.5 mM MgCl2, pH 7.4) for 10 min at 37 °C. Cells were incubated with β-Ala-Lys-AMCA (25 µM) in 2-(N-morpholino)ethanesulfonic acid (MES) buffer (5 mM MES, 145 mM NaCl, 3 mM KCl, 1 mM CaCl2, 0.5 mM MgCl2, and 5 mM D-glucose, pH 6.5) for 60 min. After the cells were washed with ice-cold HEPES buffer, they were solubilized with 0.1% Triton X-100 in phosphate-buffered saline (137 mM NaCl, 3 mM KCl, 8 mM Na2HPO4, 1.5 mM KH2PO4, pH 7.4) for 30 min. Fluorescence of β-Ala-Lys-AMCA in cell lysates was measured using a Hitachi fluorescence spectrophotometer F-2700 (Tokyo, Japan) at Ex 350 nm/Em 455 nm. PEPT2-mediated uptake was calculated by subtracting β-Ala-Lys-AMCA uptake in the presence of CDX (1 mM) from that in the absence of CDX. Cellular protein content was measured by the Lowry method using bovine serum albumin as the standard.
Expression of PEPT2 Protein Detected by Western Blotting and Confocal Laser Scanning MicroscopyThe crude membrane fraction was prepared from H441 cells as described previously.11) The expression of PEPT2 protein in the crude membrane fraction was examined by Western blotting, using rabbit polyclonal anti-PEPT2 antibodies (NBP1-59626; 1 : 1000 dilution) (Novus Biologicals, LLC, Centennial, CO, U.S.A.) as the primary antibody and peroxidase-labelled affinity purified antibodies to rabbit immunoglobulin G (IgG) (H + L) (1 : 2000 dilution) as the secondary antibody, as described previously.5) β-Actin was detected with mouse monoclonal anti-β-actin antibodies (A2228; 1 : 2000 dilution) (Merck KGaA, Darmstadt, Germany) and used as a loading control. The detection was performed using a luminescent image analyzer, LAS 4000plus (GE Healthcare Japan Corporation, Tokyo, Japan).
H441 cells were seeded at a density of 14 × 104 cells/35-mm dish with glass bottom. The cells were used on day 13 for immunostaining of PEPT2 as described previously,5) using rabbit polyclonal anti-PEPT2 antibodies (NBP1-59626; 1 : 1000 dilution) as the primary antibody and fluorescein isothiocyanate-labelled goat anti-rabbit IgG (SA00003-2; 1 : 200 dilution) (Proteintech Group, Inc., IL, U.S.A.) as the secondary antibody. Fluorescence was detected using confocal laser scanning microscopy (CLSM).
Treatment of H441 Cells with Bacterial Peptides, iE-DAP and Tri-DAPH441 cells were seeded at a density of 5 × 104 cells/well in 12 well plates. On day 13, H441 cells were preincubated with serum-free medium for 4 h, and then treated with iE-DAP (10 µg/mL) for 24 h or Tri-DAP (5 µg/mL) for 8 h. Finally, the supernatant was collected for the enzyme-linked immunosorbent assay (ELISA) assay and the cells were lysed for real-time PCR analysis.
Real-Time PCR AnalysisTotal RNA was extracted from cells using a High Pure RNA Isolation Kit (Roche, Basel, Switzerland). Reverse transcription of RNA was performed using ReverTra Ace (TOYOBO, Osaka, Japan) to produce cDNA. mRNA expression was analyzed using a CFX Connect™ Real-Time PCR detection system (Bio-Rad Laboratories, Inc., Hercules, CA, U.S.A.). The primer sequences were as follows: human PEPT2 sense, 5′-AGGAAAATGGCTGTTGGTATGATC-3′ and antisense, 5′-CGCAACTGCAAATGCCAG-3′; human NOD1 sense, 5′-CTCGCAGATGCCTACGTGGAC-3′ and antisense, 5′-GGGCATAGCACAGCACGAAC-3′; human interleukin (IL)-8 sense, 5′-AAGAAACCACCGGAAGGAAC-3′ and antisense, 5′-ATTTGGGGTGGAAAGGTTTG-3′; human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) sense, 5′-ACGGGAAGCTTGTCATCAAT-3′ and antisense, 5′-TGGACTCCACGACGTACTCA-3′. The expression of each mRNA was normalized to that of GAPDH mRNA, a housekeeping gene.
ELISA Assay of IL-8IL-8 protein secretion was quantified by the ELISA kits (Biolegend, San Diego, CA, U.S.A.) according to the protocol supplied in the manufacturer’s manual.
Statistical AnalysesData are expressed as mean ± standard error of the mean (S.E.M.). To evaluate statistical significance, Student's t-test or one-way ANOVA followed by Tukey's test for multiple comparisons were performed. The level of significance was set at ** or †† p < 0.01.
Generally, including our group, DEX (200 nM) is added in the culture medium for H441 cells to obtain stable monolayers and to enhance the expression of alveolar epithelial cell markers such as surfactant proteins.4,5) Therefore, we first examined the effect of DEX in the culture medium on PEPT2 function. PEPT2 function was estimated using β-Ala-Lys-AMCA as a substrate and CDX as an inhibitor. Unexpectedly, the total uptake of β-Ala-Lys-AMCA was higher when the cells were cultured in the absence of DEX than that in the presence of DEX (Fig. 1A), and PEPT2-mediated uptake was approximately 3-fold high in the absence of DEX (Fig. 1B). These results suggest that PEPT2 function is suppressed by the DEX generally added in the culture medium for H441 cells.
H441 cells were treated with (+) or without (−) DEX (200 nM) for 96 h (day 9 to day 13). (A) The total uptake of β-Ala-Lys-AMCA (25 µM) was measured at pH 6.5 for 60 min on day 13 in the absence (open columns) or presence (gray columns) of CDX (1 mM). (B) PEPT2-mediated uptake was calculated by subtracting β-Ala-Lys-AMCA uptake in the presence of CDX from that in the absence of CDX. Each value represents the mean ± S.E.M. (n = 3). ** p < 0.01; significantly different from (A) CDX (−) or (B) DEX (+).
The effect of various concentrations of DEX and BUD (0–100 nM) on PEPT2 function was examined. As shown in Fig. 2, PEPT2-mediated β-Ala-Lys-AMCA uptake in H441 cells was suppressed by both corticosteroids in a concentration-dependent manner. In the following experiments, H441 cells were treated with 50 nM DEX or BUD.
H441 cells were treated without (open circles) or with (closed circles) various concentrations of DEX (A) or BUD (B) for 96 h (day 9 to day 13). The uptake of β-Ala-Lys-AMCA (25 µM) was measured at pH 6.5 for 60 min on day 13. PEPT2-mediated uptake was calculated by subtracting β-Ala-Lys-AMCA uptake in the presence of CDX (1 mM) from that in the absence of CDX. Each value represents the mean ± S.E.M. (n = 3). ** p < 0.01; significantly different from control (0 nM DEX or BUD).
The effect of treatment time with DEX and BUD on PEPT2 function was examined in H441 cells. The suppressive effect of DEX and BUD on PEPT2-mediated β-Ala-Lys-AMCA uptake was dependent on the treatment time, and significant suppression was observed at 24 h and later, after treatment (Fig. 3). Conversely, no suppressive effect was observed when H441 cells were treated with BUD for shorter periods (4–18 h; data not shown). In H441 cells treated with DEX and BUD for 96 h, the suppressive effect on PEPT2 function continued for at least 12 h after removal of these corticosteroids (data not shown).
H441 cells were treated without (open circles; 0 h) or with (closed circles) DEX (50 nM) (A) or BUD (50 nM) (B) for 24–96 h. The uptake of β-Ala-Lys-AMCA (25 µM) was measured at pH 6.5 for 60 min on day 13. PEPT2-mediated uptake was calculated by subtracting β-Ala-Lys-AMCA uptake in the presence of CDX (1 mM) from that in the absence of CDX. Each value represents the mean ± S.E.M. (n = 3). ** p < 0.01; significantly different from the value at 0 h. The dashed line indicates the uptake level at 0 h (100%).
To understand the role of glucocorticoid receptor (GR) in DEX- and BUD-induced suppression of PEPT2 function, the effect of RU (100 nM), a GR antagonist, was examined (Fig. 4). PEPT2-mediated β-Ala-Lys-AMCA uptake suppressed by DEX and BUD was partially recovered by the treatment with RU. Therefore, GR may, partially, be involved in the suppression of PEPT2 function by these corticosteroids.
H441 cells were treated with DEX (50 nM) (A) or BUD (50 nM) (B) for 48 h (day 11 to day 13) in the absence or presence of RU (100 nM). The uptake of β-Ala-Lys-AMCA (25 µM) was measured at pH 6.5 for 60 min on day 13. PEPT2-mediated uptake was calculated by subtracting β-Ala-Lys-AMCA uptake in the presence of CDX (1 mM) from that in the absence of CDX. Each value represents the mean ± S.E.M. (n = 3). ** p < 0.01; significantly different from each control. †† p < 0.01; significantly different from the value with DEX (A) or BUD (B).
The effect of DEX and BUD on mRNA expression of PEPT2 was examined by real-time PCR analysis in H441 cells. Both DEX and BUD significantly suppressed the mRNA expression of PEPT2 (Fig. 5A). The expression of NOD1 mRNA was also suppressed by DEX and BUD (Supplementary Fig. 1). On the other hand, DEX and BUD did not affect PEPT2 protein level in the crude membrane fraction of H441 cells, when estimated by Western blotting (Fig. 5B). To further confirm the effect of DEX and BUD on PEPT2 level, PEPT2 protein expression was examined by CLSM after immunostaining of the cells with PEPT2 antibodies (Fig. 5C). The expression of PEPT2 protein was clearly observed in H441 cells without corticosteroid treatment (control), which was not detected in negative control cells stained without PEPT2 antibodies. Consistent with the result of Western blotting, the expression of PEPT2 protein was not affected by corticosteroid treatment.
H441 cells were treated with DEX (50 nM) or BUD (50 nM) for 96 h (day 9 to day 13). (A) Real-time PCR was performed to analyze the mRNA expression level of PEPT2, which was normalized to GAPDH mRNA expression. The dashed line indicates the control level (100%). (B) Protein expression level of PEPT2 in crude membrane fraction of H441 cells was estimated by Western blotting. (C) Immunostaining of PEPT2 (green) and Hoechst 33342 staining of nuclei (blue) were observed using CLMS. Scale bars: 20 µm. Each value represents the mean ± S.E.M. (n = 3). ** p < 0.01; significantly different from control.
We previously reported that bacterial peptidoglycan peptides, iE-DAP and Tri-DAP, increased mRNA expression and secretion of pro-inflammatory cytokines, including IL-6 and IL-8.3) In this study, the effects of DEX and BUD on the innate immune response induced by iE-DAP and Tri-DAP were examined by measuring mRNA expression and secretion of IL-8. Both iE-DAP and Tri-DAP significantly increased the mRNA expression of IL-8 and secretion of IL-8 protein into the culture medium (Fig. 6). Tri-DAP showed a much more potent induction effect, on both IL-8 mRNA expression and IL-8 secretion, than iE-DAP. DEX and BUD suppressed IL-8 mRNA expression (Figs. 6A, B) and IL-8 secretion (Figs. 6C, D) induced by these bacterial peptides.
H441 cells were treated without (open columns) or with (hatched columns) iE-DAP for 24 h (A, C) and Tri-DAP for 8 h (B, D) following the treatment with DEX (50 nM) or BUD (50 nM) for 96 h (day 9 to day 13). The expression of IL-8 mRNA (A, B) and the secretion of IL-8 protein into the culture medium (C, D) were measured. The dashed line indicates the control level without iE-DAP or Tri-DAP (100%). Each value represents the mean ± S.E.M. (n = 3). ** and †† p < 0.01; significantly different from each control in the absence and presence of iE-DAP or Tri-DAP, respectively.
Alveolar epithelium is composed of two types of cells, type I cells and type II cells. The squamous and extremely thin type I cells are essential for physiological gas exchange, while the cuboidal type II cells have multiple other physiological functions, including surfactant secretion into the alveolar lining fluid. Though both type I and type II cells contribute to host defense, type II cells are assumed to be immunologically more active because they secrete various factors, including cytokines and chemokines, resulting in activation of immune cells, such as alveolar macrophages.2) We and others previously reported that PEPT2, which is essential for the uptake of bacterial peptides such as Tri-DAP and the following induction of innate immune response, was functionally expressed in primary cultured rat type II cells but not in transdifferentiated type I-like cells.12,13) The functional expression of PEPT2 in normal and cystic fibrosis human alveolar type II cells was also shown by immunohistochemistry and ex vivo uptake studies.14)
A549 cells are widely used as an in vitro model of human alveolar type II cells. However, bacterial peptide-induced innate immune response was not observed in A549 cells, even though NOD1, IL-6, and IL-8 mRNAs were expressed in this cell line.3) This is probably due to the lack of functional expression of PEPT2 in A549 cells.3,5) In addition, RLE-6TN, another cell line widely used as an in vitro model of type II cells, also lacked PEPT2 function (data not shown). In contrast, both PEPT2 and NOD1 were expressed in H441 cells, and the innate immune response induced by bacterial peptides was observed in this cell line.3,5) In addition, the innate immune response induced by iE-DAP and Tri-DAP in H441 cells was suppressed by glycylsarcosine, a PEPT2 substrate.3) Taken together, these findings strongly suggest that PEPT2 is essential for the bacterial peptide-induced innate immune response in alveolar epithelial cells. Therefore, as far as we know, H441 cells may be the only cell line which can be used to study the PEPT2- and NOD1-dependent innate immune response in alveolar type II epithelial cells.
After respiratory infection by pathogenic bacteria, bacterial peptides such as iE-DAP and Tri-DAP are generated in the lung by the degradation of peptidoglycan cell wall of most Gram-negative and some Gram-positive bacteria. Lysozyme in the lung is active against these bacteria and induces catalytic degradation of peptidoglycan.15) In addition, PEPT2 was shown to be expressed on the apical membrane of alveolar epithelial cells and H441 cells.5,16) Therefore, in this study, the effects of corticosteroids on PEPT2 function and PEPT2- and NOD1-dependent innate immune response induced by bacterial peptidoglycan peptides were examined using H441 cell monolayers.
In our previous study, D-Ala-Lys-AMCA was used as a substrate of PEPT2,3) while in this study, β-Ala-Lys-AMCA was used, partly because β-Ala-Lys-AMCA uptake in H441 cells was higher than D-Ala-Lys-AMCA uptake in our preliminary experiment. The usefulness of β-Ala-Lys-AMCA as a biosensor to measure the transport activity of PEPT1 and PEPT2 was reported, for example, in renal brush border membrane vesicles.17) However, because no information concerning β-Ala-Lys-AMCA transport in H441 cells was available, we examined general transport characteristics of β-Ala-Lys-AMCA before starting this study. The uptake of β-Ala-Lys-AMCA in H441 cells was time- and temperature-dependent, and uptake at 37 °C, but not at 4 °C, was inhibited by CDX (Supplementary Figs. 2A, B). In contrast, uptake was not affected by cefazolin, which is not a substrate nor an inhibitor of PEPTs (Supplementary Fig. 2C). Additionally, β-Ala-Lys-AMCA uptake in H441 cells was pH-dependent and was maximum at pH 6.5 (Supplementary Fig. 2D). The effect of extracellular pH on PEPT2 activity was previously examined in other cells, and similar bell-shaped uptake profiles against extracellular pH were observed, being maximum at pH 6.0–6.5.18,19) These results suggest that β-Ala-Lys-AMCA uptake in H441 cells is mediated by PEPT2.
There are several reports on the effect of corticosteroids on PEPT1 and PEPT2, though information in alveolar epithelial cells is lacking. DEX treatment upregulated PEPT1 function in mouse intestine,20) downregulated mRNA expression of PEPT1 in rabbit intestine21) and did not affect PEPT1-mediated cephalexin uptake in the human intestinal cell line Caco-2.22) In bovine mammary gland, mRNA expression of PEPT2 was enhanced by hydrocortisone, though the effect of hydrocortisone on the transporter function and protein expression was not examined in that study.23) Thus, the effect of corticosteroids on PEPTs remains controversial or may be different in different cell types and/or animal species. In this study, we found that DEX and BUD potently suppressed PEPT2 function in a human-derived alveolar epithelial cell line H441.
The pharmacological effects of corticosteroids are mediated by several different mechanisms of action, including genomic and non-genomic effects.24,25) The classical genomic effects are mediated by the cytosolic GR, a member of the steroid-hormone-receptor family, while non-genomic effects are caused by the interaction of corticosteroids with the cytosolic GR, membrane-bound GR, or cellular membrane. Generally, non-genomic effects occur very rapidly (e.g., within minutes) after corticosteroid stimulation, while it takes hours or days before induction of genomic effects.24,25) In this study, we observed that the suppression of PEPT2 function induced by the treatment with DEX and BUD was partially recovered by a GR antagonist, RU. Therefore, GR may be partially involved in the suppression of PEPT2 function by these corticosteroids. Additionally, as described in Results, the suppressive effect of BUD on PEPT2 function was observed at 24 h and later after treatment, but not at shorter periods, suggesting that the suppressive effect of DEX and BUD on PEPT2 function may be mediated by their genomic effects. On the other hand, PEPT2 function was also decreased by RU alone. At present, the reason for the inhibition of PEPT2 function by RU is not clear. It may not be related to the antagonistic effect of RU on GR.
The detailed mechanisms underlying the suppression of PEPT2 function by corticosteroids are unclear. Though the expression of PEPT2 mRNA was decreased by corticosteroid treatment, no apparent change in PEPT2 protein expression was observed under the present treatment conditions, as confirmed by Western blotting and by CLSM after immunostaining. Recently, Selo et al.26) examined the effect of inhaled compounds, including BUD and cigarette smoke extract (CSE), on multidrug resistance-associated protein-1 (MRP1; an efflux transporter) using human alveolar type II and type I-like epithelial cells in primary culture and H441 cells. In that study, they first compared the mRNA and protein expression of MRP1 in type II and type I-like epithelial cells and found that MRP1 protein expression was significantly higher in type I-like cells than in type II cells, while the mRNA expression level was similar between these cells. They also demonstrated that BUD and CSE suppressed the transport function of MRP1, while these compounds did not reduce MRP1 protein levels. Thus, as observed in this and their studies, transport function and/or mRNA level does not always correlate with protein abundance, which may be due to the existence of numerous biological mechanisms that decouple protein levels from mRNA levels.27) Therefore, certain factors, directly or indirectly modulating PEPT2 function, may be involved in the suppression of PEPT2 function by corticosteroids. Currently, we hypothesize that the change in Na+/H+ exchanger (NHE) activity, which is known to regulate PEPT2 function,19,28) may be involved in the suppression of PEPT2 function by glucocorticoid treatment. Muto et al.29) reported that NHE activity in vascular smooth muscle cells was inhibited by long-term (24 h) treatment of the cells with glucocorticoid, and intracellular pH was decreased by this treatment (decreased intracellular pH would result in the suppression of PEPT2 activity). Therefore, the change in NHE activity and intracellular pH may be one of possible mechanisms underlying the suppression of PEPT2 function by glucocorticoid, though further studies are needed to conclude the involvement of this mechanism in H441 cells.
In our previous study, the mRNA expression of IL-6 and IL-8 and the secretion of these cytokines into the culture medium were measured as markers of innate immune response.3) In that study, increased level of mRNA expression and protein secretion of IL-8 induced by iE-DAP was much higher than that of IL-6. Therefore, IL-8 was used as a sensitive marker of innate immune response in this study. The innate immune response induced by iE-DAP and Tri-DAP was markedly suppressed by DEX and BUD. The suppression of innate immune response by these corticosteroids would be partially due to the suppression of PEPT2 function, which mediates the influx of iE-DAP and Tri-DAP into H441 cells, and suppression of NOD1 mRNA expression. However, pathways other than the PEPT2- and NOD1-dependent pathway may also be involved because corticosteroids have pleiotropic pharmacological effects.30,31)
Currently, the clinical benefit of corticosteroid treatment for coronavirus disease 2019 (COVID-19) is being extensively studied, though a definitive conclusion on the merit of corticosteroid therapy in the treatment of COVID-19 has yet to be demonstrated.32) The pathways for the induction of immune response are not the same between bacterial and viral infections. However, NOD1 is suggested to be involved in the induction of innate immune response not only by bacterial peptides but also by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).33) In addition, we recently reported that NF-κB pathway played a role in the bacterial peptide-induced innate immune response.3) NF-κB is also one of the key transcription factors involved in the immune response induced by SARS-CoV-2 in lung epithelial cells.33) Therefore, corticosteroids such as DEX may also suppress the immune response induced by SARS-CoV-2, at least in part, by affecting NOD1 and its downstream NF-κB signaling pathway in the lung.
In conclusion, we found that corticosteroids, including DEX and BUD, suppressed PEPT2 function and PEPT2- and NOD1-dependent innate immune response induced by bacterial peptidoglycan-derived peptides in H441 human alveolar epithelial cells. Inhaled corticosteroids are widely used to treat asthma and COPD. These findings may provide novel information to understand the mechanism underlying the increased susceptibility to infections during inhaled corticosteroid therapy.
This study was supported in part by a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (JSPS; 21K066680A). A part of this work was carried out at the Natural Science Center for Basic Research and Development, Hiroshima University.
The authors declare no conflict of interest.
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