# Adalimumab inflammatory signaling pathways ## Adalimumab modulates NF-κB, JAK/STAT, TNFα-TGFβ, Notch-1, IL-17, and IL-12/23 signaling pathways through direct TNF-α blockade, with pathway-specific effects varying by cell type, disease context, and treatment duration, mediated by coordinated changes in gene expression, protein levels, and microRNA regulation. # Abstract Adalimumab modulates multiple interconnected inflammatory signaling pathways through direct TNF-α blockade and subsequent downstream effects. In keratinocytes, adalimumab primarily targets NF-κB signaling by downregulating IKBKB, IRAK1, TRAF2, MAP3K7, and TNFAIP3, with IKBKB functioning as a central hub in protein-protein interaction networks. In T cells, the dominant mechanism involves JAK/STAT pathway modulation, with adalimumab increasing STAT6 phosphorylation and restoring STAT4 activation to healthy levels, while disrupting Th1, Th17, and Th22 pathways in psoriasis patients. Adalimumab also affects the TNFα-TGFβ signaling axis differentially depending on disease context, effectively decreasing downstream inflammation in extended-to-be juvenile idiopathic arthritis synoviocytes but showing paradoxical increases in persistent disease. A novel mechanism was identified in inflammatory bowel disease involving Notch-1, which binds directly to TNFα and mediates adalimumab’s effects on T cell cycling but not apoptosis. MicroRNAs including miR-1297, miR-30a, miR-95-5p, miR-125b, and miR-4329 emerged as important regulators that fine-tune pathway responses, with temporal dynamics showing initial broad transcriptional changes followed by progressive miRNA restriction. These pathway modulations translate into reduced inflammatory mediator production (CRP, TNF, IL-6, GlycA) and clinical improvements in psoriasis and rheumatoid arthritis, though adverse effects on HDL metabolism occurred at 52 weeks. The heterogeneous findings across studies reflect cell type-specific inflammatory network architecture rather than contradictory results: keratinocytes rely on NF-κB signaling, T cells use JAK/STAT for differentiation, and synoviocytes employ the TNFα-TGFβ axis. Context-dependent responses suggest adalimumab is most effective when TNFα drives downstream inflammatory cascades, with limited efficacy in contexts where alternative pathways predominate or disease has progressed to TNFα-independent inflammation. ## Results ### Characteristics of Included Studies The included studies investigated adalimumab effects on inflammatory signaling pathways across diverse experimental contexts, ranging from in vitro cell culture systems to clinical trials in patients with inflammatory diseases. | Study | Full text retrieved? | Experimental context | Cell type/tissue | Disease model | Inflammatory stimulus | |--------------------------------------|---------------------|-------------------------------|------------------------------------|----------------------------------|-------------------------------| | N. Mehta et al., 2018 | Yes | Randomized controlled trial | Patient samples | Psoriasis (moderate-to-severe) | Adalimumab (blocks TNF-α) | | B. Grabarek et al., 2019 | No | Cell culture | Normal human dermal fibroblasts | Psoriasis | None mentioned | | B. Grabarek et al., 2018 | No | Cell culture | Normal human dermal fibroblasts | None (healthy cells) | Adalimumab (8 µg/ml) | | Paulina Buda et al., 2023 | Yes | Cell culture | Human keratinocytes (HaCaT) | Psoriasis | Lipopolysaccharide A (LPS) | | Aleksandra Plata-Babula et al., 2025| Yes | Cell culture | Immortalized keratinocytes (HaCaT) | Psoriasis | Lipopolysaccharide (LPS) | | Megan M. Simonds et al., 2023 | Yes | Ex vivo cell culture | Fibroblast-like synoviocytes | Juvenile idiopathic arthritis | Adalimumab (TNFα inhibitor) | | Iwona Adwent et al., 2020 | No | Cell culture | Normal human fibroblasts | General fibroblast response | Lipopolysaccharide (LPS) | | N. Aerts et al., 2010 | No | Patient samples | CD4+ T cells (PBMCs) | Rheumatoid arthritis | Anti-CD3/anti-CD28, IL-4, IL-12 | | L. Werner et al., 2011 | No | Patient samples or ex vivo tissue| T cells | Inflammatory bowel disease | TNFα | | Li Luan et al., 2015 | No | Patient samples | Th1, Th17, Th22 cells (blood) | Psoriasis (moderate-to-severe) | None mentioned | Treatment parameters varied across studies. In vitro studies typically used adalimumab concentrations of 8 µg/ml or 10 µg/ml for durations of 2, 8, and 24 hours. Clinical trials administered adalimumab subcutaneously with an initial dose of 80 mg followed by 40 mg every other week for 12 weeks, with one study extending treatment to 52 weeks. Several in vitro studies employed LPS pretreatment at 1 µg/ml for 8 hours before adalimumab addition. ## Inflammatory Pathways Modulated by Adalimumab ### NF-κB Signaling Pathway The NF-κB pathway emerged as a central target of adalimumab modulation in keratinocytes. Adalimumab reversed LPS-induced upregulation of multiple NF-κB-associated genes, including IKBKB, IRAK1, TRAF2, MAP3K7, and TNFAIP3. STRING analysis identified IKBKB as a central hub in the protein-protein interaction network, coordinating immune and apoptotic signals. This downregulation occurred consistently across multiple timepoints (2, 8, and 24 hours), with early suppression of certain genes and broader inhibition at later timepoints. The NF-κB pathway also showed crosstalk with MAPK and JAK/STAT pathways, and participates in the TNF-α/IL-23/IL-17 axis that is central to psoriatic inflammation. Gene ontology enrichment highlighted IκB kinase/NF-κB, TNF-mediated, and Toll-like receptor signaling pathways as primary targets. Adalimumab’s effects on NF-κB signaling were validated at both mRNA and protein levels, with marked reductions in protein concentrations following treatment. The IL-17 signaling pathway, which activates NF-κB and MAPK cascades, also showed significant modulation, with adalimumab affecting chemokine and cytokine signaling pathways most strongly. ### JAK/STAT Signaling Pathway JAK/STAT pathway modulation occurred across multiple experimental contexts. In normal human dermal fibroblasts, adalimumab affected 18 of 37 JAK/STAT-associated mRNAs, with time-dependent changes in regulatory microRNAs (20 miRNAs at 2 hours, 9 at 8 hours, and 3 at 24 hours). Specific pathway components showed distinct responses: in rheumatoid arthritis patients, adalimumab increased Th2-associated STAT6 phosphorylation in both freshly isolated and stimulated CD4+ T cells, while restoring Th1-associated STAT4 phosphorylation to levels observed in healthy individuals. In LPS-stimulated keratinocytes, adalimumab’s effects on the IL-12/23-dependent JAK/STAT pathway were particularly notable. The treatment silenced SOCS3 and IL-6 expression while causing overexpression of STAT1, STAT3, STAT5, JAK3, and IL-6R. Strong regulatory connections were identified between JAK3 and hsa-miR-373-5p (target score 96), and between SOCS3/STAT5 and hsa-miR-1827 (target score 96). These changes were consistent regardless of exposure time, indicating sustained pathway modulation. In fibroblasts exposed to LPS, adalimumab caused silencing of TNF-α, IFN-γ, IL-17, IL12A, IL12B, and IL23A expression, affecting genes dependent on IL-12 receptor interactions. The effects on TNF-α and IFN-γ were most pronounced, with lesser modulation of IL-17. ### TNFα-TGFβ Signaling Axis In juvenile idiopathic arthritis synoviocytes, adalimumab demonstrated differential effects on persistent versus extended-to-be (ETB) fibroblast-like synoviocytes. After 24 hours of treatment, persistent FLS showed significant elevation of TNFα (FC=1.2, p=0.001), TGFβ (FC=1.5, p=0.001), lymphotoxin alpha (FC=4.3, p=0.015), sTNFRI (FC=5.1, p=0.008), and sTNFRII (FC=3.8, p=0.025). In contrast, ETB FLS exhibited a different pattern, with TNFα slightly elevated (FC=1.04, p=0.023) but TGFβ significantly decreased (FC=1.03, p=0.037). This suggests that adalimumab effectively decreases inflammation downstream of initial TNFα signaling in ETB cells, as TNFα signaling is upstream of TGFβ, with TGFβ acting as an intermediary for MAPK and NF-κB activation. ### Notch-1 Signaling Pathway In inflammatory bowel disease, adalimumab revealed a novel mechanism involving Notch-1 signaling. Notch-1 mucosal expression increased in response to anti-TNFα treatment, and Notch-1 was found to bind directly to TNFα. Functionally, Notch-1 mediated the inhibitory effects of adalimumab on T cell cycling but not apoptosis, indicating pathway-specific regulation. Notch-1 inhibition prevented anti-TNFα-induced T cell cycle arrest while apoptosis induction remained intact. This demonstrates crosstalk between Notch-1 and TNFα pathways, with Notch-1 function being regulated by TNFα inhibitors. ### Th1/Th17/Th22 Pathways Clinical studies in psoriasis patients demonstrated that adalimumab disrupts multiple T helper cell pathways simultaneously. At baseline, psoriasis patients showed elevated frequencies of Th1, Th17, and Th22 cells compared to healthy controls, along with higher expression of associated transcription factors (T-bet, RORγt, AHR) and cytokines (IFN-γ, IL-17, IL-22, IL-6, TNF-α). After 12 weeks of adalimumab therapy, significant declines occurred in cell frequencies, transcription factor expression, and cytokine levels, indicating broad immunomodulation across these pathways. ## Molecular Mechanisms of Pathway Modulation ### Direct and Indirect Effects Adalimumab’s primary direct mechanism involves blocking TNF-α through antibody binding, preventing TNF-α from interacting with its receptors. This direct inhibition triggers cascading indirect effects on downstream pathways. In keratinocytes, adalimumab directly affected NF-κB pathway gene expression, while in fibroblasts, it directly influenced JAK/STAT pathway genes. The IL-17 signaling pathway showed both direct effects on gene expression and indirect effects through modulation of six signaling pathways and 19 biological processes. ### Gene Expression and Protein Level Changes Adalimumab induced coordinated changes at transcriptional and translational levels. In LPS-stimulated keratinocytes, adalimumab downregulated NF-κB-associated genes (IKBKB, IRAK1, TRAF2, MAP3K7, TNFAIP3) with concordant protein-level reductions. Gene ontology analysis revealed enrichment in immune regulation, apoptosis, and NF-κB signaling pathways. However, discrepancies between mRNA and protein levels occurred in some cases, suggesting post-translational regulation. For example, in keratinocytes exposed to LPS and adalimumab, JAK3, SOCS3, and STAT5 showed overexpression at the mRNA level but decreased protein concentrations. ### MicroRNA Involvement MicroRNAs emerged as important mediators of adalimumab’s effects. In NF-κB pathway modulation, anti-inflammatory miRNAs including miR-1297, miR-30a, miR-95-5p, and miR-125b showed reciprocal expression changes consistent with anti-inflammatory activity, while pro-survival miRNAs like miR-4329 and miR-20b-3p were downregulated. In the JAK/STAT pathway, adalimumab altered miRNA expression in a time-dependent manner (20 miRNAs at 2 hours, 9 at 8 hours, 3 at 24 hours), suggesting dynamic regulation. Specific miRNA-mRNA interactions were identified, including hsa-miR-373-5p regulating JAK3 and hsa-miR-1827 regulating SOCS3 and STAT5. ### Protein-Protein Interactions STRING analysis and experimental data revealed critical protein-protein interactions affected by adalimumab. IKBKB functioned as a central hub in the NF-κB pathway protein-protein interaction network. In IL-12/23 signaling, JAK3, SOCS3, and STAT5 showed altered expression affecting JAK/STAT signaling dynamics. Direct binding between Notch-1 and TNFα was demonstrated, providing a mechanism for crosstalk between these pathways. ## Functional Consequences of Pathway Modulation ### Inflammatory Mediator Production Adalimumab reduced key inflammatory markers across multiple studies. In psoriasis patients, treatment decreased serum CRP, IL-6, TNF, and glycoprotein acetylation (GlycA) at both 12 and 52 weeks. The reduction in these systemic inflammatory markers was statistically significant compared to placebo. At the cellular level, adalimumab caused silencing of mRNA and protein expression for TNF-α, IFN-γ, IL-17, IL12A, IL12B, and IL23A in LPS-exposed fibroblasts. In keratinocytes, adalimumab silenced SOCS3 and IL-6 expression. However, context-dependent responses occurred. In juvenile idiopathic arthritis synoviocytes, persistent FLS showed increased TNFα, TGFβ, lymphotoxin alpha, sTNFRI, and sTNFRII after adalimumab treatment, while extended-to-be FLS exhibited decreased TGFβ alongside reduced G-CSF, IFNγ, IL-10, M-CSF, MIP-1-alpha, and MIP-1-delta. In psoriasis patients, while TNF and GlycA decreased, IL-6 levels paradoxically increased at the end of the 52-week extension period. ### Effects on Cell Function Adalimumab influenced T cell function through multiple mechanisms. In inflammatory bowel disease, treatment induced T cell apoptosis, inhibited activation, reduced cytokine secretion, and restricted cell cycling. The cell cycle restriction was mediated by Notch-1, while apoptosis occurred through a Notch-1-independent mechanism. In rheumatoid arthritis, IFN-γ production increased in adalimumab-treated patients compared to untreated individuals, while IL-4, IL-6, and IL-12 production remained unchanged. In psoriasis, adalimumab treatment resulted in significant declines in Th1, Th17, and Th22 cell frequencies, indicating effects on T cell differentiation or proliferation. Transcription factor levels (T-bet, RORγt, AHR) decreased concordantly. ### Clinical and Phenotypic Improvements Clinical improvements correlated with pathway modulation. In psoriasis, adalimumab improved skin disease and systemic inflammation markers, with GlycA improvement potentially explaining beneficial effects observed in observational studies. Treatment led to clinical improvement concurrent with disruption of Th1, Th17, and Th22 pathways. In keratinocytes, adalimumab promoted resolution of inflammation and restoration of epidermal homeostasis, with potential restoration of apoptotic balance and normalization of epidermal architecture. ### Biomarker Changes Several biomarkers emerged as indicators of pathway activity. Modulation of IL-6 levels served as a marker of response to TNF-α inhibitor treatment. Changes in miRNA expression profiles (including hsa-miR-373-5p and hsa-miR-1827, as well as miR-1297, miR-30a, miR-95-5p, miR-125b, and miR-4329) showed potential as biomarkers for treatment monitoring and patient stratification. Increases in Notch-1 mucosal expression reflected anti-TNFα treatment response. ### Adverse Effects Adalimumab demonstrated potential adverse effects on lipid metabolism. At 52 weeks, cholesterol efflux and high-density lipoprotein particle count (HDL-p) were reduced, with no beneficial impact on metabolic markers including insulin, adiponectin, and leptin. Phototherapy showed more favorable effects on HDL-p at 12 weeks compared to adalimumab. These findings suggest that while adalimumab reduces systemic inflammatory markers, it may have unfavorable effects on HDL function and lipid metabolism. ## Synthesis The heterogeneous findings across studies can be reconciled by considering experimental context, disease model, and pathway-specific effects. **Context-Dependent Pathway Responses** The divergent effects of adalimumab on inflammatory mediators in different cell types reflect fundamental differences in cellular inflammatory programs. In juvenile idiopathic arthritis synoviocytes, persistent FLS showed paradoxical increases in TNFα, TGFβ, and soluble TNF receptors after adalimumab treatment, while extended-to-be FLS demonstrated the expected anti-inflammatory response with decreased TGFβ. This differential response occurs because persistent and extended-to-be JIA represent distinct disease phenotypes at the molecular level. Patients were classified before disease progression, and the synoviocytes from extended-to-be patients were already primed for more aggressive inflammation even before clinical extension occurred. The fact that adalimumab effectively suppresses inflammation in extended-to-be FLS but not persistent FLS suggests it is most beneficial in contexts where TNFα signaling drives downstream inflammatory cascades. In persistent disease, alternative pathways may predominate, rendering TNFα blockade less effective or even triggering compensatory inflammatory responses. **Cell Type-Specific Mechanisms** The molecular mechanisms by which adalimumab modulates pathways vary substantially by cell type. In keratinocytes, adalimumab primarily targets NF-κB signaling, downregulating IKBKB, IRAK1, TRAF2, MAP3K7, and TNFAIP3. In T cells, the dominant mechanism involves JAK/STAT pathway modulation, with increased STAT6 phosphorylation and restored STAT4 activation. In synoviocytes, the TNFα-TGFβ axis predominates. This heterogeneity reflects the cell type-specific wiring of inflammatory networks: keratinocytes, as barrier cells, rely heavily on NF-κB for antimicrobial and inflammatory responses; T cells use JAK/STAT for differentiation and effector function; and mesenchymal cells like synoviocytes employ TGFβ for tissue remodeling. These are not contradictory findings but rather demonstrate that adalimumab’s anti-TNFα activity has downstream consequences tailored to each cell type’s unique signaling architecture. **Temporal Dynamics of Response** Time-dependent effects reveal that pathway modulation by adalimumab follows distinct kinetics for different regulatory mechanisms. Gene expression changes occurred within 2 hours, but microRNA profiles showed progressive restriction over time (20 differentially expressed miRNAs at 2 hours, 9 at 8 hours, 3 at 24 hours). This temporal pattern suggests an initial broad transcriptional response followed by selective stabilization of specific regulatory programs. Protein-level changes lagged behind mRNA changes, consistent with the time required for protein turnover. Clinical studies demonstrated sustained effects at 12 weeks and 52 weeks, but with emergence of secondary changes such as increased IL-6 at later timepoints. This late IL-6 increase likely represents a compensatory response to chronic TNFα blockade, as cells attempt to restore inflammatory capacity through alternative pathways. **Disease Model Considerations** The use of LPS to model inflammation in several in vitro studies provides mechanistic insights but has limitations for extrapolating to human disease. LPS activates Toll-like receptor 4, triggering acute inflammatory responses that may not fully recapitulate chronic inflammatory conditions like psoriasis or rheumatoid arthritis. Studies using primary cells from patients showed different response patterns than LPS-stimulated normal cells, suggesting that chronic disease primes cells for distinct inflammatory responses. For example, in rheumatoid arthritis patients, STAT4 phosphorylation was absent in untreated patients but restored by adalimumab, whereas in LPS-stimulated fibroblasts, adalimumab simply suppressed LPS-induced gene expression. This indicates that adalimumab may correct disease-specific signaling defects in patient cells beyond simple anti-inflammatory effects. **Pathway Crosstalk and Network Effects** The observation that adalimumab affects multiple pathways simultaneously (NF-κB, JAK/STAT, IL-17, IL-12/23, Notch-1) reflects extensive crosstalk in inflammatory networks rather than off-target effects. TNFα sits at a central node in inflammatory signaling, and its blockade reverberates through interconnected pathways. For instance, NF-κB interacts with MAPK and JAK/STAT pathways, and the TNF-α/IL-23/IL-17 axis involves NF-κB signaling. TGFβ acts as an intermediary for MAPK and NF-κB activation downstream of TNFα. Notch-1 binds directly to TNFα and mediates specific downstream effects on cell cycling. These interactions mean that inhibiting TNFα does not simply shut down one linear pathway but rather reconfigures a network of interconnected signaling nodes. The specific consequences depend on which alternative pathways are available in each cell type and disease context. **MicroRNA-Mediated Fine-Tuning** The involvement of microRNAs in adalimumab’s effects provides an additional layer of regulation that reconciles some apparent contradictions. For example, JAK3, SOCS3, and STAT5 showed overexpression at the mRNA level but decreased protein concentrations in keratinocytes. The identification of miR-373-5p and miR-1827 as regulators of these genes explains this discrepancy: adalimumab induces compensatory transcriptional upregulation, but microRNAs suppress translation, resulting in net protein reduction. This post-transcriptional regulation allows cells to modulate pathway activity without proportional gene expression changes and may represent a mechanism for fine-tuning inflammatory responses. The observation that miRNA changes show distinct temporal dynamics compared to mRNA changes suggests they serve as a secondary regulatory layer that refines initial transcriptional responses. **Implications for Clinical Use** The context-dependent effects have implications for predicting clinical response. Adalimumab appears most effective when TNFα drives downstream inflammatory cascades through pathways like TGFβ, NF-κB, and JAK/STAT. In conditions where alternative cytokine pathways predominate or where disease has progressed to TNFα-independent inflammation, response may be limited or paradoxical. The differential response in persistent versus extended-to-be JIA synoviocytes exemplifies this principle: early intervention in patients whose disease is still TNFα-dependent may be more effective than treatment after alternative pathways are established. The adverse effects on HDL metabolism suggest that pathway modulation has trade-offs, with anti-inflammatory benefits potentially offset by metabolic complications in some patients. Biomarkers including IL-6 levels, miRNA profiles, and Notch-1 expression may help identify patients most likely to benefit from adalimumab and monitor pathway-specific responses during treatment.