Rapamycin Restores Different Patterns of Cytokine Expression to Dexamethasone Treatment on CD14++CD16+ Monocytes from Steroid-Resistant Asthma Patients

Abstract

Objective: We aimed to investigate the differences in interleukin (IL)-10, IL-1β, IL-6, and tumor necrosis factor (TNF)-α expression in lipopolysaccharide (LPS)-stimulated CD14++CD16+ monocytes obtained from asthmatics after dexamethasone or dexamethasone plus rapamycin treatments between clinical steroid responders (R) and non-responders (NR). Methods: Cytokine expressions in LPS-stimulated CD14++CD16+ p-mammalian target of rapamycin (mTOR) monocytes from R and NR were determined using flow cytometry. Results: IL-10^high CD14++CD16+ p-mTOR population following LPS stimulation increased in the R group although decreased in the NR group with dexamethasone treatment. IL-1β^high population decreased in the R group although increased in the NR group. Rapamycin treatment after LPS and dexamethasone resulted in a significant increase in the IL-10^high population and a significant decrease in the IL-1β^high population in the NR group. Conclusion: Dexamethasone treatment resulted in different patterns of change in cytokine expressions in LPS-stimulated CD14++CD16+ p-mTOR monocytes between the R and NR. mTOR inhibition can restore steroid responsiveness involving IL-10 and IL-1β in CD14++CD16+ p-mTOR monocytes.

INTRODUCTION

Glucocorticoids are used to reduce inflammatory conditions in chronic inflammatory diseases such as allergic diseases, rheumatoid diseases. Steroids effectively control airway inflammation in asthma.^1,2) However, the treatment responses to glucocorticoids and their side effects vary between individuals,^3,4) and some patients with asthma show poor responses to steroids (so-called steroid-resistant patients). Individual diversities have been demonstrated in glucocorticoid receptor, glucocorticoid receptor binding, chaperone protein, glucocorticoid metabolic enzymes, or corticosteroid-binding globulin transporters in asthma.⁵⁾ Asthma patients with poor responses to steroids pose a real burden in clinical practice and many investigators have constantly been searching for the mechanisms of steroid resistance.⁶⁾ According to previous studies, signaling pathways related to phosphoinositide 3-kinases (PI3K) and mitogen-activated protein kinase (MAPK) are important for the production of inflammatory substances in immune cells.^7,8) In addition, a possible association between PI3K/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) and steroid resistance in immune cells has been proposed.^9,10)

mTOR, a conserved serine/threonine kinase, is a member of the PI3K related kinase family,¹¹⁾ and a crucial regulator of protein synthesis, cell growth, metabolism, and aging.¹²⁾ It is activated and plays a key role in various diseases such as cancer, neurodegenration, inflammatory diseases.^13,14) Previous studies have shown that mTOR silencing attenuates lipopolysaccharide (LPS)-induced inflammation^15,16) and mTOR directly affects the inflammatory response. In addition, it showed beneficial effects involving in the glucocorticoid metabolic pathway.^17,18) There has been evidence that the mTOR pathway increases glucocorticoid sensitivity in malignancy,¹⁹⁾ and that the function of methylprednisolone in autoimmune hepatitis is mediated via the mTOR pathway.²⁰⁾

Monocytes also contribute to steroid resistance by amplifying inflammation and cytokine production and facilitating dendritic cell and inflammatory macrophage differentiation.²¹⁾ The activation of mTOR seen in monocytes, when stimulated by LPS, results in the change of pro-inflammatory and anti-inflammatory cytokines.²²⁾ Dexamethasone treatment altered mTOR activation during this process²³⁾ and it could be suggested that the activity of mTOR in monocytes is related to steroid resistance. Human monocytes are commonly classified according to cd14 and cd16 expression on the surface, and it is believed that their functions and phenotypes are related to the expression intensities of these molecules.^24,25) Classical monocytes are a subset characterized by CD14++CD16−, intermediate monocytes with CD14++CD16+, and non-classical monocytes with CD14+CD16++. A recent study reported that intermediate monocytes, also known as inflammatory monocytes in asthma patients and other inflammatory diseases, are important for the development of inflammation and steroid resistance.²⁶⁾ However, the exact mechanism by which monocytes contribute to steroid resistance remains unknown.

Based on this evidence, glucocorticoids can control the transcription of pro- and anti-inflammatory cytokine genes through the mTOR pathway. The authors evaluated the role of CD14++CD16+ monocytes in steroid resistance to demonstrate that the responsiveness to glucocorticoids could be enhanced through the regulation of the mTOR pathway in individuals with genetic variation in the responsiveness to glucocorticoids. We treated peripheral blood mononuclear cells (PBMCs) of asthma patients with LPS, dexamethasone, and rapamycin, gated monocyte (CD14++CD16+ cells) population, and measured mTOR activation and inflammatory cytokine expression.

MATERIALS AND METHODS

Study Population

The clinical characteristics are presented in Table 1. We prospectively enrolled patients with asthma at Seoul National University Hospital, Seoul, Republic of Korea. Patients with a history of variable respiratory symptoms such as wheezing, shortness of breath, chest tightness, and cough were diagnosed with asthma by physicians’ decision. Variable expiratory airflow limitation or positive methacholine provocation test (PC₂₀ ≤ 16 mg/mL) confirmed the diagnosis. After diagnosis, all patients were treated with medium-dose ICSs²⁷⁾ and regularly followed up; pulmonary function measurements were performed every 12 weeks. Current smokers and former smokers over 10 pack-year were excluded from the study. We evaluated patients’ responsiveness to treatment by lung function changes between at baseline and at 3 months after asthma treatment (Fig. 1). Steroid responders (R) were defined as patients with 12% or more than 12% improvement in FEV₁ compared to baseline values within 3 months after initiation of treatment, and non-responders (NR) was defined as patients with less than 12% improvement.²⁸⁾ Control complained of no respiratory symptoms and showed negative results in the methacholine bronchial provocation test. This study was approved by the Institutional Review Board of the Seoul National University Hospital (H-1408-051-601), and informed consent was obtained from all study participants.

Table 1. Clinical Characteristics of Study Population

	Responders (n = 15)	Non-responders (n = 15)
Age (year)	48.3 ± 3.8	50.3 ± 4.9
Female (%)	66.7	60.0
BMI (kg/m2)	23.8 ± 0.8	24.9 ± 0.9
Baseline FEV1 (%)	63.9 ± 4.1	83.6 ± 5.1
Baseline FEV1 (mL)	1812.0 ± 122.7	2298.0 ± 197.3
Baseline FEV1/FVC	69.2 ± 2.7	75.4 ± 2.7
FeNO (ppb)	62.0 ± 13.4	52.5 ± 10.5
Peripheral eosinophil (106/L)	418.7 ± 82.1	289.4 ± 92.2
Total IgE (IU/mL)	615.9 ± 229.3	570.4 ± 289.9
Atopy (%)	71.4	55.6
Smoking
Non-smoker (%)	92.9	76.9
Smoker (<10PY) (%)	7.1	23.1
Asthma medication (%)
ICS, ICS/LABA	100.0	100.0
LAMA	0.0	0.0
Methylxanthine	13.3	20.0
Leukotriene modifier	70.0	40.0
SABA	0.0	0.0
Oral steroid	13.3	33.3
Comorbid conditions (%)
Hypertension	13.3	33.3
Diabetes	0.0	20.0
Cardiovascular	6.7	20.0
Rheumatis	0.0	0.0
Malignancy	0.0	6.7
Thyroid disease	6.7	20.0

BMI, body mass index; FEV₁, forced expiratory volume in 1 s; FeNO, fractional exhaled nitric oxide; ICS, inhaled corticosteroid; LABA, long-acting β₂-agonist; SABA, short-acting β₂-agonist.

Fig. 1. Lung Function (FEV₁) of Patients in Responders and Non-responders before and after Steroid Treatment

A, B. The absolute value of FEV₁ (A) and predicted value of FEV₁ (B) of patients at the baseline and 3 months after steroid treatment. The blue dots represent patients in responders and the red dots represents patients in non-responders. C, D. Box plots presenting absolute (C) and percent (D) values of changes in lung function between baseline and 3 months of steroid treatment in responders and non-responders.

Peripheral Blood Sample Collection

PBMCs were prepared from buffy coats by density gradient centrifugation using Ficoll-Paque Plus (GE Healthcare, Chicago, IL, U.S.A.). The cells were stored in freezing solution at −196 °C before the experiment.

PBMC Culture

PBMCs (5 × 10⁵ cells/well) were seeded in RPMI 1640 (Thermo Fisher Scientific, Waltham, MA, U.S.A.) containing 10% charcoal stripped fetal bovine serum (Thermo Fisher Scientific) and 1% penicillin-streptomycin (Thermo Fisher Scientific) and were stimulated with 50 ng/mL lipopolysaccharide²⁹⁾ (LPS, Escherichia coli 0111:B4, Sigma-Aldrich, U.S.A.) with/without 10⁻⁷ M dexamethasone²⁸⁾ (Calbiochem, U.S.A.) or 10 nM Rapamycin²²⁾ (Santa Cruz Biotechnology, U.S.A.) overnight. Cells were also treated with Golgi stop (BD Bioscience, U.S.A.).

Measurement of Cytokine Expression Using Flow Cytometry

Cells were incubated for 30 min at 4 °C in the dark with the surface markers APC-conjugated anti-human CD14 antibody (17-0149-42; 1 : 50, isotype control 17-4714-81; 1 : 1000, Thermo Fisher Scientific) and Alexa 700-conjugated anti-human CD16 antibody (302026; 1 : 50, isotype control 400143; 1 : 1000, Biolegend, San Diego, CA, U.S.A.). After incubation, the cells were washed with 1 mL FACS buffer followed by centrifugation. After fix/perm staining, cells were stained with phenylephrine (PE)-conjugated anti-phospho-mTOR antibody (12-9718-42; 1 : 100, isotype control 12-4724-81; 1 : 1000, Thermo Fisher Scientific), PE/texas red-conjugated anti-interleukin (IL)-6 (501121; 1 : 100, isotype control 400445;1 : 1000, Biolegend), PE/cy7-conjugated anti-IL-10 (501419; 1 : 100, isotype control 400415; 1 : 1000, Biolegend), fluorescein isothiocyanate (FITC)-conjugated anti-IL-1β (511705; 1 : 100, isotype control 400309; 1 : 1000, Biolegend), or BV421-conjugated anti-tumor necrosis factor (TNF)-α (502932; 1 : 100, isotype control 400157; 1 : 1000, Biolegend) antibodies. For flow cytometry analysis, at least 5 × 10⁴ cells were acquired using an LSR Fortessa (BD Biosciences, San Jose, CA, U.S.A.). Flow cytometry data were analyzed using FlowJo 10.6.1 (Tree Star Inc., Ashland, OR, U.S.A.).

Statistics

The results are expressed as means ± standard deviation (S.D.), and statistical differences among groups were assessed using the unpaired two-tailed t-test and nonparametric Mann–Whitney test. Statistical analyses were performed using GraphPad Prism (GraphPad Software, La Jolla, CA, U.S.A.). Statistical significance was set at p < 0.05.

RESULTS

Changes in CD14++CD16+ Monocytes after LPS Stimulation

Figure 2A shows gating plots with anti-CD14 and anti-CD16 antibodies of PBMCs from the NR, R, and C groups after medium or LPS stimulation. The population of CD14++CD16+ monocytes significantly decreased only in the R group after LPS stimulation than the medium. No significant difference in CD14++CD16+ populations and phospho-mTOR population of CD14++CD16+ monocyte from the NR or C groups was observed between medium and LPS stimulation (Figs. 2B, C).

Fig. 2. The Population of CD14++CD16+ Monocyte and Phospho-mTOR of CD14++CD16+ Monocyte in PBMC upon LPS Stimulation

A. Gating strategy of CD14 and CD16 on peripheral blood monocyte cell. CD14++CD16+ population was separated by anti-CD14 and anti-CD16 antibodies. B. The frequency of CD14++CD16+ monocyte in 3 groups. C. The frequency of phospho-mTOR of CD14++CD16+ monocyte in 3 groups (Responder, Non-Responder, and Control) was represented as Medium or LPS stimulation. Each data represent mean ± S.D. of 15 (NR and R) and 10 (control) samples. * p < 0.05.

Changes in Inflammatory and Anti-inflammatory Cytokine Expressions in CD14++CD16+ p-mTOR Monocytes after LPS Stimulation

We evaluated phospho-mTOR (p-mTOR; an activated form of mTOR) expression in CD14++CD16+ monocytes according to the expression of inflammatory cytokines, such as IL-1β, IL-6, and TNF-α, and the anti-inflammatory cytokine IL-10 after LPS stimulation. The co-expression levels of p-mTOR and each inflammatory cytokine after LPS stimulation were evaluated as shifting patterns based on the intensity of cytokine expressing p-mTOR. The CD14++ CD16+ monocytes were divided into intermediate expression and high expression populations. As shown in Figs. 3A–D, among CD14++CD16+ p-mTOR monocytes, the population with high expression of IL-10, IL-1β, IL-6, and TNF-α increased after LPS stimulation compared to the medium. After LPS stimulation, cytokine-positive total populations (both intermediate and high expression) in CD14++CD16+ p-mTOR monocytes increased in all the three groups (R, NR, and C) (Figs. 4A–L), and no significant differences in the level of increase were observed among the groups. Moreover, cytokine-negative populations in CD14++CD16+ p-mTOR monocytes decreased in all three groups after LPS stimulation (Figs. 4M–P).

Fig. 3. The Plot of Cytokines with Phospho-mTOR in CD14++CD16+ Population upon LPS Stimulation

A–D. The expression change of IL-10 (A), IL-1β (B), IL-6 (C) and TNF-α (D) with phospho-mTOR (p-mTOR) in CD14++CD16+ population upon LPS stimulation. Upper and low panels display Medium or LPS stimulation.int; intermediate, hi; high.

Fig. 4. The Difference of Cytokines with Phospho-mTOR in R, NR and Control Groups

A–D. The expression of p-mTOR + IL10+ (A), p-mTOR + IL1β+ (B), p-mTOR + IL6+ (C) and p-mTOR + TNFα+ (D) in CD14++CD16+ population from R, NR, and control groups. E–H. The expression of p-mTOR + IL10 hi (E), p-mTOR + IL1β hi (F), p-mTOR + IL6 hi (G) and p-mTOR + TNFα hi (H) in CD14++CD16+ population from R, NR and control group. I–L. The expression of p-mTOR + IL10 int (I), p-mTOR + IL1β int (J), p-mTOR + IL6 int (K) and p-mTOR + TNFα int (L) in CD14++CD16+ population from R, NR and control group. M-P. The expression of p-mTOR + IL10-(M), p-mTOR + IL1β-(N), p-mTOR + IL6-(O) and p-mTOR + TNFα-(P) in CD14++CD16+ population from R, NR, and control groups. Y axis represents percent (%) of increment on LPS stimulation compared to Medium. Each data represent mean ± S.D. of 15 (NR and R) and 10 (control) samples. hi; high, int; intermediate, R; Responder, NR; Non-responder, C; Control.

Differences in Pro-inflammatory and Anti-inflammatory Cytokine Expressions in LPS-Stimulated CD14++CD16+ p-mTOR Monocytes after Dexamethasone Treatment between R and NR Groups

We evaluated the effect of dexamethasone on LPS-induced changes in CD14++CD16+ monocytes. There were no significant differences in the frequencies of CD14++CD16+ monocytes between LPS and LPS plus dexamethasone in all groups (R, NR, and C groups) (Fig. 5A). Figure 5B shows changes in cytokine expression (negative, intermediate, and high expression) in LPS-stimulated CD14++CD16+ p-mTOR monocytes after dexamethasone treatment. IL-10+ (Fig. 6A), IL-6+ (Fig. 6C), and TNF-α+ (Fig. 6D) populations in CD14++CD16+ p-mTOR monocytes decreased after dexamethasone treatment in both the R and NR groups; however, no significant differences were observed between the two groups. Furthermore, the IL-1β+ population (Fig. 6B) increased in both the R and NR groups, although there was no difference between the two groups. However, after dexamethasone treatment, the inflammatory cytokine high expression populations showed different patterns between the N and NR groups. The IL-10^high expression population in LPS-stimulated CD14++CD16+ p-mTOR monocytes increased in the R group following dexamethasone treatment, whereas it decreased in the NR group (Fig. 6E). In contrast, the IL-1β^high expression population decreased in the R group but increased in the NR group upon dexamethasone treatment (Fig. 6F). Both IL-6^high (Fig. 6G) and TNF-α^high (Fig. 6H) expression populations decreased in the R and NR groups after dexamethasone treatment. In the TNF-α^high expression population, a significantly greater decrease was observed in the NR group than in the R group following dexamethasone treatment.

Fig. 5. The Population of CD14++CD16+ and Expression of Cytokines with Phospho-mTOR upon LPS Stimulation or LPS + Dexa

A. The frequency of CD14++CD16+ monocyte was separated by anti-CD14 and anti-CD16 antibodies. The population of 3 groups (R, NR, and Control) was represented by LPS stimulation or LPS + dexa. B. The expression change of IL-10, IL-1β, IL-6, and TNF-α with phospho-mTOR (p-mTOR) in CD14++CD16+ population upon LPS stimulation or LPS + dexa. Upper and Low panels display LPS stimulation or LPS + dexa. Each data represent mean ± S.D. of 15 (NR and R) and 10 (control) samples. int; intermediate, hi; high, dexa; dexamethasone, R; Responder, NR; Non-responder, C; Control

Fig. 6. The Change of Expression of Cytokines with Phospho-mTOR upon LPS + Dexa in R and NR Groups

A–D. The expression of p-mTOR + IL10+ (A), p-mTOR + IL1β+ (B), p-mTOR + IL6+ (C) and p-mTOR + TNFα+ (D) in CD14++CD16+ population upon LPS + dexa from R and NR groups. E–H. The expression of p-mTOR + IL10 hi (E), p-mTOR + IL1β hi (F), p-mTOR + IL6 hi (G) and p-mTOR + TNFα hi (H) in CD14++CD16+ population from R and NR groups. Y axis represents percent (%) of decrement upon LPS + dexa compared to LPS stimulation. Each data represent mean ± S.D. of 15 (NR and R) samples. hi; high, R; Responder, NR; Non-responder. * p < 0.05.

The results of changes in cytokine intermediate expression populations after dexamethasone treatment in LPS-stimulated CD14++CD16+ p-mTOR monocytes are presented in supplementary figures. IL-10 (Supplementary Fig. 1A), IL-6 (Supplementary Fig. 1A), and TNF-α from the NR group (Supplementary Fig. 1D) decreased after dexamethasone treatment, but IL-1β (Supplementary Fig. 1B) and TNF-α from the R group (Supplementary Fig. 1D) increased. However, there was no difference between the R and NR groups. In contrast to the cytokine-positive populations, cytokine-negative populations in LPS-stimulated CD14++CD16+ p-mTOR monocytes increased in cases of IL-10, IL-6, and TNF-α following dexamethasone treatment (Supplementary Figs. 1E, G, H). IL-1β negative population (Supplementary Fig. 1F) decreased in the R group but increased in the NR group. In the treatment with dexamethasone alone, there was no change in each cytokine compared to that in the medium. (data not shown).

The Effect of Rapamycin on the IL-10 and IL-1β High Expression Populations in LPS-Stimulated CD14++CD16+ p-mTOR Monocytes in R and NR Groups

We evaluated the effect of rapamycin on the IL-10^high and IL-1β^high expression populations in LPS-stimulated CD14++CD16+ p-mTOR monocytes, which showed different aspects of changes after dexamethasone treatment in the NR group contrary to the R group. Rapamycin treatment after LPS plus dexamethasone resulted in a significant increase in the IL-10^high expression population (Fig. 7A) but a significant decrease of IL-1β^high expression in the NR group (Fig. 7B). The same tendency was observed with rapamycin treatment after LPS treatment. In the R group, rapamycin treatment after LPS plus dexamethasone showed no remarkable change in the IL-10^high and IL-1β^high expression populations (Figs. 7C, D). Rapamycin treatment alone resulted in no changes in inflammatory cytokine expression in populations compared to the medium (data not shown).

Fig. 7. The Effect of Rapamycin on IL-10^hi and IL1β^hi Expressions with Phospho-mTOR upon LPS + Dexa in R and NR Groups

A, B. The expression of p-mTOR + IL10^hi (A), p-mTOR + IL1β^hi (B) in CD14++CD16+ population on L + D, L + D + R and L + R from NR group. C, D. The expression of p-mTOR + IL10^hi (C), p-mTOR + IL1β^hi (D) in CD14++CD16+ population on L + D, L + D + R and L + R from R group. Y axis represent percent (%) of decrement on LPS + dexa compared to LPS stimulation. Each data represent mean ± S.D. of 15 (NR and R) samples. L + D; LPS + Dexamethasone, L + D + R; LPS + Dexamethasone + Rapamycin, L + R; LPS + Rapamycin. * p < 0.05.

DISCUSSION

This study determined IL-1β, IL-6, TNF-α, and IL-10 expression in CD14++CD16+ p-mTOR monocytes obtained from asthmatics after LPS or LPS plus dexamethasone treatment and evaluated whether there were differences in cytokine expression according to the clinical response to steroids in patients.

Dexamethasone treatment resulted in different patterns of changes in populations with high cytokine expression in LPS-stimulated CD14++CD16+ p-mTOR monocytes between the R and NR groups. The IL-10^high expression population increased in the R group although decreased in the NR group with dexamethasone treatment. Furthermore, the IL-1β^high expression population decreased in the R group although increased in the NR group. Thereafter, we assessed the additional effects of rapamycin on IL-10^high and IL-1β ^high expression populations in LPS-stimulated CD14++CD16+ p-mTOR monocytes. It was found that rapamycin treatment after dexamethasone resulted in a significant increase in the IL-10^high expression population and a significant decrease in the IL-1β^high expression population in the NR group. However, rapamycin treatment, in addition to dexamethasone, showed no significant changes in either IL-10^high or IL-1β^high expression populations in the R group. To the best of our knowledge, this is the first study analyzing p-mTOR and cytokine co-expressed CD14++CD16+ monocytes using flow cytometry in predicting steroid responses in humans.

Previous studies have demonstrated the difference between steroid-sensitive and steroid-resistant patients at the molecular level.^28,30) Patients were grouped according to the degree of improvement in steroid treatment. The secretion of cytokines involved in airway inflammation was closely related to steroid resistance. Proinflammatory cytokines such as IL-2, IL-4, IL-13, and IL-8 affect glucocorticoid binding affinity,^30–32) whereas TNF-α and IL-10 influence the sensitivity to dexamethasone in monocytes.³⁾ One study showed that the response to dexamethasone could be controlled by manipulating specific cytokines.³³⁾ Among several immune cells that produce anti- and pro-inflammatory cytokines, monocytes play a central role in the innate immune system.³⁴⁾ They play a pivotal role in the generation and resolution of inflammation, and in the removal of pathogens or apoptotic cell debris. Intermediate monocytes, a subpopulation of monocytes characterized by CD14++CD16+, are thought to be more associated with steroid resistance in asthma.³⁵⁾ It has been reported that the number of CD14++CD16+ cells was higher in steroid-resistant patients than in steroid-sensitive patients, and this subpopulation cell also increased during dexamethasone treatment. However, information regarding the changes in cytokines originating from this cell subpopulation is inadequate, and further investigation is necessary.

mTOR, a serine/threonine kinase, is a crucial regulator of protein synthesis, cell growth, metabolism, and aging¹²⁾ and plays a key role in various diseases such as cancer, aging, inflammatory diseases, and asthma.¹³⁾ Previous studies have shown that mTOR silencing attenuates LPS-induced inflammation,^15,16) and mTOR directly affects the inflammatory response. In addition, it is involved in the glucocorticoid metabolic pathway and has beneficial effects on glucocorticoids.^17,18) There has been evidence that the mTOR pathway increases glucocorticoid sensitivity in malignancy,¹⁹⁾ and that the function of methylprednisolone in autoimmune hepatitis is mediated via the mTOR pathway.²⁰⁾ mTOR controls NFκB via IKK.³⁶⁾ Based on this evidence, glucocorticoids can control the transcription of pro- and anti-inflammatory cytokine genes through the mTOR pathway. The authors hypothesized that the responsiveness to glucocorticoids could be enhanced through the regulation of the mTOR pathway in individuals with genetic variation in the glucocorticoid pathway and aimed to investigate the co-expression of phospho-mTOR and cytokines as targets of the CD14++CD16+ population.

In this study, the co-expression levels of p-mTOR and cytokines were presented as shifting patterns upon LPS stimulation. Based on the intensity of cytokines with p-mTOR, we conducted the analysis by dividing them into negative, intermediate-positive, and high-positive populations. The results showed differences between the R and NR groups in the expression of IL-10 and IL-1β after dexamethasone treatment. In contrast, LPS stimulation itself did not lead to differences in cytokine expression between the two groups. The IL-10 high population with p-mTOR decreased, and the IL-1β high population increased in the NR group, contrary to the R group. These two cytokines are important in the mechanism of steroid-resistant asthma. The IL-1β-dependent response accompanying excessive NLRP3 (nucleotide-binding oligomerization domain-like receptor family, pyrin domain-containing 3) inflammasome plays a key role in severe, steroid-resistant asthma.³⁷⁾ IL-10 is dependent on mTOR signaling in LPS-stimulated monocytes, and some studies have suggested that CD14++CD16+ is the main source.³⁸⁾ It has also been reported that IL-10 expression is decreased in steroid resistance.³⁹⁾ In conclusion, this study confirmed that the different patterns of cytokine expression in the NR group were reversed with rapamycin co-treatment. Therefore, our results suggest that individual differences in responsiveness to mTOR could be a mechanism for steroid resistance. It also solidifies earlier studies that mTOR inhibition may ameliorate the response to glucocorticoids.^17–20)

To verify our results, it is necessary to reveal the amount of IL10^high and IL-1β^high populations with p-mTOR+, which were presented differently in the R and NR groups, affect the actual protein levels. Therefore, these monocytes should be isolated and tested in the future. In the final experiment, we attempted to confirm whether the p-mTOR+IL10^highand p-mTOR+IL-1β^high populations, which showed different responses to dexamethasone in the two groups, could be reversed by rapamycin treatment. When rapamycin was treated with dexamethasone, the opposite reactions observed in the NR group were reversed. Since some studies have suggested the synergistic effect of dexamethasone and rapamycin,⁴⁰⁾ this study also shows the possibility that rapamycin can modulate the reactivity toward dexamethasone. According to the studies presented on their synergistic effect, it was reported that the simultaneous treatment of dexamethasone and rapamycin increased c-Jun inhibition and Akt activation.^41,42) The c-Jun transcription factor has been reported to be essential for the induction of IL-1β gene expression.⁴³⁾ There is also a study that an increase in Akt activation causes an increase in IL-10.⁴⁴⁾ Therefore, rapamycin can be assumed to modulate the effect of dexamethasone by the above mentioned mechanism. It is known that LPS-stimulated monocyte secretion of cytokines increases nuclear factor-kappaB (NF-κB) through PI3K/AKT/mTOR and other signaling pathways such as p38/extracellular signal-regulated kinase (ERK)/c-Jun N-terminal kinase (JNK).⁴⁵⁾ Therefore, it is thought that CD14++CD16+ cells can also be involved in various signaling pathway-related factors other than mTOR in cytokine expression. However, it was confirmed that the major inflammatory factors IL-1β, IL-6, and TNF-α, known as pro-inflammatory factors and IL-10, are known to be directly related to mTOR activation in the known steroid resistance-related potential population (CD14++CD16+). It can be concluded that this is significant because their changes were analyzed.

CONCLUSION

In conclusion, our results demonstrated that dexamethasone treatment resulted in different patterns of changes in IL-10^high and IL-1β^high expression populations upon LPS-stimulated CD14++CD16+ p-mTOR monocytes between the R and NR groups, which were restored by mTOR inhibition.

Acknowledgments

This study was supported by a Grant from the SNUH Research Fund (Grant No. 04-2018-0060).

Author Contributions

Hyun Seung Lee contributed to the study in conception, data collection and analysis, literature review, manuscript writing, and critical review.

Heung-Woo Park contributed to the study in materials, data collection, manuscript writing, and critical review.

Suh-Young Lee contributed to the study in conception, design, supervision, materials, data collection, literature review, manuscript writing, and critical review.

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

This article contains supplementary materials.

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