Nicotinamide mononucleotide and nicotinamide riboside attenuate cytokine production in human keratinocytes via suppression of p38 Pathway
Chen Xie, Maria Alejandra Molina Velandia, Samantha Marecek, Miriam Khalil, Jijun Hao
TL;DR
NMN and NR reduce inflammation in skin cells by blocking the p38 pathway, suggesting they could be new treatments for atopic dermatitis.
Contribution
NMN and NR are identified as potential AD treatments via p38 inhibition in keratinocytes.
Findings
NMN and NR reduce inflammatory gene expression in cytokine-stimulated keratinocytes.
Both compounds inhibit p38 MAPK phosphorylation without affecting cell viability.
NMN shows broader anti-inflammatory effects than NR.
Abstract
Atopic dermatitis (AD) is a chronic inflammatory skin disorder characterized by impaired epidermal barrier function and immune dysregulation. Current therapies are largely symptomatic and frequently associated with adverse effects, underscoring the need for safer and more effective treatment strategies. Nicotinamide mononucleotide (NMN) and nicotinamide riboside (NR) are precursors of nicotinamide adenine dinucleotide (NAD⁺) and have been reported to regulate inflammatory signaling and cellular metabolism. This study investigated the anti-inflammatory effects of NMN and NR in an in vitro model of AD. Human keratinocyte HaCaT cells were stimulated with TNF-α and IFN-γ to mimic AD-associated inflammation and pretreated with NMN or NR. Quantitative PCR analysis demonstrated that NMN significantly and dose-dependently reduced mRNA expression of IL-1β, macrophage-derived chemokine…
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Taxonomy
TopicsDermatology and Skin Diseases · Sirtuins and Resveratrol in Medicine · PARP inhibition in cancer therapy
Introduction
Atopic dermatitis is a chronic, pruritic, inflammatory skin disease that affects both humans and pets. It is characterized by a complex interplay of genetic, immunological and environmental factors that lead to disrupted skin barriers and heightened immune responses. In humans, it is among the most common dermatologic conditions, affecting up to 20% of children and 10% of adults worldwide [1]. Dogs are also frequently affected, where it is termed canine atopic dermatitis [2, 3]. While clinical manifestations may vary across species, the underlying pathophysiological mechanisms are largely conserved including disrupted epidermal barrier integrity, an overactive T-helper 2 (Th2)-mediated immune response and alterations in skin microbiota. Current treatments are primarily aimed at managing symptoms rather than addressing underlying disease mechanisms, offering only temporary relief and often causing side effects [4–6].
Given these limitations, interest is growing in therapies that target the drivers of inflammation and barrier dysfunction in atopic dermatitis. NAD⁺ is a critical coenzyme involved in energy metabolism, mitochondrial function and immune regulation [7, 8]. NAD⁺ depletion is linked to oxidative stress, impaired DNA repair and heightened inflammation, whereas boosting NAD⁺ has shown benefits for skin rejuvenation and psoriasis-like dermatitis in mice [9–11]. Despite its therapeutic potential, NAD⁺ is biologically unstable. In contrast, its precursors NMN and NR are stable, naturally occurring, and safe in humans and dogs [12–14]. NMN is directly converted to NAD⁺ by NMNAT, while NR is first converted to NMN before entering the biosynthetic pathway. However, their roles in atopic dermatitis remain poorly explored, and no studies have investigated NR in this disease, and only two have assessed NMN in murine models [15, 16].
Keratinocytes are central to atopic dermatitis pathogenesis, contributing to barrier maintenance and inflammatory signaling. HaCaT cells, a human keratinocyte line stimulated with TNF-α and IFN-γ, are widely used to investigate potential anti-inflammatory therapies [17]. In atopic dermatitis, TNF-α and IFN-γ synergistically activate keratinocytes, driving excessive production of pro-inflammatory mediators such as IL-1β, IL6, IL8, TSLP, TARC, MDC, and CCL5/RANTES [18, 19]. These cytokines promote immune cell recruitment and chronic inflammation. In this study, we examined the anti-inflammatory effects of NMN and NR in HaCaT cells stimulated with TNF-α/IFN-γ.
Materials and methods
Cell culture and reagents
The human keratinocyte HaCaT cell line was kindly provided by Professor Ying Huang (College of Pharmacy, Western University of Health Sciences, Pomona, CA, USA). It was cultured in high glucose Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (GibcoTM, Thermo Fisher Scientific, Inc.) and 100 U/ml penicillin and 100 µg/ml streptomycin at 37˚C in a humidified 5% CO2 incubator.
Cell viability assay
Cell viability was measured using the CellTiter 96^®^ AQueous One Solution Cell Proliferation Assay (Promega, Madison, WI, USA), based on a colorimetric MTS assay. HaCaT cells were plated in 96-well plates and incubated overnight at 37 °C in a 5% CO₂ incubator, followed by 24 h culture with varying concentrations of NMN or NR. Then, 20 µL of reagent was added to each well containing 100 µL of medium for 1 h. Absorbance was measured at 490 nm on a POLARstar spectrophotometer (BMG Labtech, Cary, NC, USA). Results were expressed as the percentage of treated versus untreated controls using the equation: Viable% = Absorbance(test)/Absorbance(control) × 100, with control cell viability set as 100%. All experiments were performed in triplicate.
Reverse transcription quantitative PCR (RT-qPCR)
HaCaT cells were seeded in 6-well plates and cultured for 24 h. Cells were pretreated with NMN (0.5, 1, 5 mM) or NR (0.5, 1, 2 mM) for 1 h, followed by TNF-α/IFN-γ (10 ng/mL) stimulation for 24 h. RNA was extracted using the RNeasy^®^ Mini Kit (Qiagen, Hilden, Germany). First-strand cDNAs were synthesized with the High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific, Waltham, MA) according to the manufacturer’s instructions. Quantitative real-time PCR was performed using 2× Fast SYBR Green Master Mix (Thermo Fisher Scientific, Waltham, MA) in triplicate on a Bio-Rad CFX Real-Time PCR system. GAPDH served as internal control. The primer sets used in this study are: Human GAPDH (5’-GGTGTGAACCATGAGAAGTATGA-3’ forward, 5’-GTCCTTCCACGATACCAAAG-3’ reverse), Human IL-1β (5’-ATGGACAAGCTGAGGA AGATG − 3’ forward, 5’-CCCATGTGTCGAAGAAGATAGG-3’ reverse), Human MDC (5’-CGCGTG GTGAAACACTTCTA-3’ forward, 5’-GATCGGCACAGATCTC CTTATC-3’ reverse), Human CCL17 (5’-CTTAGAAAGCTGAAGACGTGGTA-3’ forward, 5’-TCTTCACTCTCTTGTTGTTGGG-3’ reverse), Human IL8 (5’-ACTGAGAGTGATTGAGA GTGGAC-3’ forward), 5’-AACCCTCTGCACCCAGTTTTC − 3’ reverse), Human TSLP (5’-TATGAGTGGGACCAAA AGTACCG-3’ forward, 5’-GGGATTGAAGGTTAGGCTCTGG-3’ reverse), Human RANTES (5’-TGCCCACATCAAGGAGTATTT-3’ forward, 5’-GATGTACTCCC GAACCCATTT-3’ reverse).
Western blot analysis
HaCaT cells were seeded in 6-well plates and cultured for 24 h. Cells were pretreated with NMN (0.5, 1, 5 mM) or NR (0.5, 1, 2 mM) for 1 h, then stimulated with TNF-α/IFN-γ (10 ng/mL) for 1 h. To examine the involvement of the p38 MAPK pathway, HaCaT cells were pretreated with 10 µM p38 MAPK inhibitor SB203580 (MedChemExpress, NJ, USA) for 2 h, followed by NMN or NR treatment for 1 h and subsequent stimulation with TNF-α/IFN-γ (10 ng/mL) for 1 h. After washing with cold PBS, cells were lysed in RIPA buffer (Sigma) containing protease and phosphatase inhibitors (Roche). Samples were denatured at 95 °C for 5 min, separated by SDS-PAGE, and transferred to PVDF membranes (Millipore). Membranes were blocked with Odyssey Blocking Buffer (Li-Cor) for 1 h at room temperature and incubated with primary antibodies (Cell Signaling Technology): AKT, p-AKT, ERK, p-ERK, JNK, p-JNK, p38, p-p38, NF-κB p65, p-p65, IκBα, p-IκBα, and β-actin. Secondary antibodies included IRDye 680 goat anti-rabbit IgG and IRDye 800CW goat anti-mouse IgG (Li-Cor). Protein bands were visualized, and densitometry was performed using Image Studio analyzer.
Cellular NAD+ level assessment
Cellular NAD⁺ levels were measured using an NAD/NADH cell-based assay kit (Cayman Chemical, MI, USA) according to the manufacturer’s protocol. HaCaT cells were seeded at a density of 1 × 10⁴ cells per well in a 96-well plate and cultured overnight at 37 °C in a humidified incubator with 5% CO₂. Cells were then pretreated with NMN (0.5, 1, or 5 mM) or NR (0.5, 1, or 2 mM) for 1 h, followed by stimulation with TNF-α/IFN-γ (10 ng/mL) for 24 h. After treatment, the culture medium was removed, and cells were washed with assay buffer and centrifuged at 500 × g for 5 min. Following aspiration of the assay buffer, cells were incubated with permeabilization buffer under gentle shaking for 30 min at room temperature, then centrifuged at 1,000 × g for 10 min at room temperature. A total of 100 µL of standards or supernatant from each well was transferred to a new clear-bottom 96-well plate, followed by the addition of 100 µL of reaction buffer. Plates were incubated for 90 min on a shaker, and absorbance was measured at 450 nm using a POLARstar spectrophotometer (BMG Labtech, Cary, NC, USA).
Statistical analysis
The data are presented as the mean ± SEM. Differences were evaluated by One-Way ANOVA. When significance was detected, the post hoc comparisons between the different groups were made using Bonferroni’s test for multiple comparisons. The probability value of p < 0.05 was considered statistically significant.
Results
NMN and NR exhibit no cytotoxicity in HaCaT cells
To assess the cytotoxicity of NMN and NR, HaCaT cells were treated with increasing concentrations of NMN (0.5, 1, and 5 mM) or NR (0.5, 1, and 2 mM) for 24 h. Cell viability was determined using the MTS assay. Both NMN and NR showed no significant cytotoxic effects, with cell viability maintained above 95% across all concentrations tested (Supplementary Fig. 1). These results demonstrate that NMN and NR are well tolerated by HaCaT cells at the concentrations used in this study.
NMN and NR suppress TNF-α/IFN-γ-induced proinflammatory gene expression
To confirm that NMN and NR indeed enhance cellular NAD^+^ levels, we used the NAD/NADH cell-based assay kit to measure cellular NAD⁺ levels. The result indicated both NMN and NR statistically significantly up-regulated cellular NAD⁺ levels in the TNF-α/IFN-γ-treated HaCaT cells (Supplementary Fig. 2).
We next evaluated the anti-inflammatory effects of NMN and NR by quantifying mRNA levels of key cytokines and chemokines implicated in atopic dermatitis, respectively. HaCaT cells were pretreated with NMN at various concentrations (0.5, 1, and 5 mM) and then stimulated with TNF-α and IFN-γ, followed by measurement of the expression levels of several inflammatory mediators by RT-qPCR (Fig. 1). As expected, stimulation with TNF-α and IFN-γ markedly increased the expression of IL-1β, MDC and CCL5, in comparison to the controls in the HaCaT cells. Treatments with NMN significantly attenuated their expression, particularly at 5 mM concentration higher doses. NMN also suppressed the expression of CCL17, a chemokine involved in T-cell recruitment in a dose-dependent fashion. Moreover, IL8, a potent neutrophil chemoattractant, showed the most dramatic reduction across all NMN concentrations, indicating a strong and consistent anti-inflammatory response. Lastly, NMN treatment led to a significant decrease in TSLP levels, a cytokine associated with epithelial cell–mediated inflammation and allergic responses. Additionally, we examined anti-inflammatory effects of NR in HaCaT cells. Upon stimulation with TNF-α and IFN-γ, all the pro-inflammatory cytokines and chemokines including IL-1β, MDC, CCL5, CCL17, IL8, and TSLP, were markedly upregulated. NR treatments significantly attenuated these responses except IL-1β (Fig. 2).
Fig. 1NMN suppress inflammatory gene expression in TNF-α/IFN-γ-stimulated HaCaT cells. HaCaT cells were pretreated with NMN (0.5, 1, and 5 mM) for 1 h and then stimulated with TNF-α and IFN-γ (10 ng/mL each) for 24 h. mRNA levels of IL-1β, MDC, CCL5, CCL17 (TARC), IL8, and TSLP were quantified by RT-qPCR. Gene expression levels were normalized to GAPDH and expressed as fold change relative to untreated control cells. Data represent mean ± SEM from three independent biological experiments (n = 3). Statistical significance was determined by one-way ANOVA followed by Bonferroni post hoc test. **p < 0.01, ***p < 0.001, ****p < 0.0001 versus TNF-α/IFN-γ–treated group
Fig. 2NR suppress inflammatory gene expression in TNF-α/IFN-γ-stimulated HaCaT cells. HaCaT cells were pretreated with NR (0.5, 1, and 2 mM) for 1 h and then stimulated with TNF-α and IFN-γ (10 ng/mL each) for 24 h. mRNA levels of IL-1β, MDC, CCL5, CCL17 (TARC), IL8, and TSLP were measured by RT-qPCR. Target gene expression was normalized to GAPDH and expressed as fold change relative to untreated control cells. Data represent mean ± SEM from three independent biological experiments (n = 3). Statistical analysis was performed using one-way ANOVA with Bonferroni post hoc test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 versus TNF-α/IFN-γ–treated group
Collectively, these results demonstrate that NMN and NR suppress proinflammatory cytokine and chemokine expression induced by TNF-α/IFN-γ in HaCaT cells, highlighting their anti-inflammatory potential.
NMN and NR inhibit p38 MAPK activation
To identify the signaling mechanism underlying the anti-inflammatory effects, we examined phosphorylation of p38 MAPK in HaCaT cells stimulated with TNF-α and IFN-γ. As shown in Fig. 3, treatment with pro-inflammatory cytokines led to a marked increase in phosphorylated p38 (p-p38), indicating activation of this signaling pathway.
Fig. 3NMN and NR suppress TNF-α/IFN-γ-induced p38 phosphorylation in HaCaT cells. (A) Representative Western blots showing phosphorylated p38 (p-p38) and total p38 in HaCaT cells pretreated with NMN (0.5, 1, and 5 mM) or NR (0.5, 1, and 2 mM) for 1 h followed by TNF-α/IFN-γ (10 ng/mL) stimulation for 1 h. β-actin served as loading control. (B) Densitometric quantification of p-p38 normalized to total p38. Statistical significance was determined by one-way ANOVA with Bonferroni post hoc test. ***p < 0.001, ****p < 0.0001 versus TNF-α/IFN-γ–treated group
As shown in Fig. 3A, Western blot analysis demonstrated that TNF-α/IFN-γ stimulation led to a marked increase in p-p38 levels, indicating activation of the MAPK signaling pathway. Treatment with increasing concentrations of NMN (0.5, 1, and 5 mM) or NR (0.5, 1, and 2 mM) significantly reduced the levels of p-p38, while total p38 expression remained unchanged. The quantitative analysis of Fig. 3A confirmed significant reductions in p-p38 levels with both compounds with statistical significance observed at all doses tested (p < 0.001, p < 0.0001). (Fig. 3B). These findings demonstrate that NMN and NR inhibit the TNF-α/IFN-γ-induced activation of the p38 MAPK pathway, supporting their potential as modulators of inflammatory signaling pathways.
To further confirm these findings, HaCaT cells were pretreated with the p38 MAPK inhibitor SB203580, with or without NMN or NR, followed by TNF-α/IFN-γ stimulation. Western blot analysis showed that SB203580 markedly attenuated p38 phosphorylation, and that NMN and NR further reduced p38 phosphorylation beyond SB203580 treatment alone (Supplementary Fig. 3).
NMN and NR do not alter AKT, ERK, JNK, or NF-κB signaling pathways
To further investigate the impacts of NMN and NR on other inflammatory signaling, we evaluated the phosphorylation status of key signaling molecules involved in the AKT, ERK, JNK, and NF-κB pathways, all of which are critically implicated in keratinocyte function and the pathogenesis of atopic dermatitis. As shown in Fig. 4, TNF-α/IFN-γ stimulation markedly increased phosphorylation of NF-κB p65 and its upstream regulator IκBα, confirming effective activation of the NF-κB pathway. In contrast, phosphorylation levels of AKT, ERK, and JNK were not altered by TNF-α/IFN-γ treatment under the experimental conditions. Importantly, co-treatment with NMN or NR did not produce detectable changes in the phosphorylation of AKT, ERK, JNK, NF-κB p65, or IκBα compared with TNF-α/IFN-γ treatment alone. Collectively, these results indicate that NMN and NR do not regulate AKT, ERK, JNK, or NF-κB signaling in TNF-α/IFN-γ–stimulated HaCaT cells.
Fig. 4NMN and NR do not regulate AKT, ERK, JNK, or NF-κB signaling in TNF-α/IFN-γ–stimulated HaCaT cells. Western blot analysis was performed to assess phosphorylated protein levels of AKT, ERK, JNK, NF-κB p65, and IκBα in HaCaT cells pretreated with NMN or NR, followed by stimulation with TNF-α/IFN-γ. The result indicates that neither NMN nor NR inhibited the phosphorylation of these signaling molecules, indicating that their anti-inflammatory effects are not mediated through the AKT, ERK, JNK, or NF-κB pathways
Discussions
This study demonstrates that both NMN and NR possess potent anti-inflammatory properties in human keratinocyte HaCaT cells stimulated with TNF-α and IFN-γ, a widely used model of atopic dermatitis (AD) inflammation. Both NAD⁺ precursors significantly reduced expression of key cytokines and chemokines including MDC, CCL5, CCL17/TARC, IL8 and TSLP, molecules upregulated in human and canine AD and associated with Th2 responses. Interestingly, NMN, but not NR, suppressed IL-1β expression, likely reflecting differences in sensitivity to NAD⁺ levels or activation of distinct pathways. Many suppressed chemokines are regulated via p38 MAPK, which our data show is inhibited by both compounds. In contrast, IL-1β may be more tightly controlled by SIRT1-mediated NF-κB inhibition, requiring stronger NAD⁺ elevation that NMN may achieve more efficiently by bypassing NR kinase.
Mechanistically, our data highlights p38 MAPK as a central target of NMN and NR. Western blot analysis showed both markedly suppressed TNF-α/IFN-γ-induced p38 phosphorylation, consistent with downregulation of inflammatory genes. Given p38 MAPK’s pivotal role in keratinocyte cytokine production, this inhibition likely explains much of the anti-inflammatory effect. Importantly, neither compound exhibited cytotoxicity at tested concentrations, supporting safety in vitro. These findings align with prior in vivo studies demonstrating NMN’s efficacy in DNFB-induced mouse AD [15]. To our knowledge, this is the first report identifying NR as an anti-inflammatory agent in keratinocytes, expanding the therapeutic scope of NAD⁺ precursors. Furthermore, NMN and NR did not affect other signaling pathways examined, including AKT, ERK, JNK, NF-κB p65, or IκBα, suggesting that their modulatory effects are preferentially mediated through the p38 MAPK pathway.
Nevertheless, several limitations of this study should be acknowledged. First, all experiments were performed using a single immortalized keratinocyte cell line (HaCaT), which may not fully recapitulate the complexity and heterogeneity of primary human keratinocytes or intact skin tissue. Second, the findings are based exclusively on in-vitro assays and have not yet been validated in three-dimensional skin models or in-vivo systems. Therefore, caution is warranted when extrapolating these results to physiological or clinical settings. Additional studies using primary human keratinocytes, organotypic skin equivalents and animal models of AD will be necessary to confirm the translational relevance of NMN and NR and to better define their mechanisms of action.
In summary, NMN and NR hold promise as therapeutic agents for AD and other inflammatory skin diseases. Their ability to suppress proinflammatory signaling in keratinocytes, key effector cells in skin immunity, underscores their relevance to disease pathogenesis. Future studies using 3D human epidermis and animal models are warranted to validate these findings, assess pharmacokinetics, and evaluate long-term efficacy and safety. These investigations may ultimately support development of NAD⁺-based topical or systemic treatments for AD in both human and veterinary contexts.
Supplementary Information
Below is the link to the electronic supplementary material.
Supplementary Material 1
The reference list from the paper itself. Each links out to its DOI / PubMed record.
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