Triple-negative breast cancer cells must evade immune surveillance to metastasize to distant sites, yet this process is not well understood. The Eyes absent (EYA) family of proteins, which are crucial for embryonic development, become dysregulated in cancer, where they have been shown to mediate proliferation, migration, and invasion. Our study reveals an unusual mechanism by which EYA3 reduces the presence of cytotoxic natural killer (NK) cells in the premetastatic niche (PMN) to enhance metastasis, independent of its effects on the primary tumor. We find that EYA3 up-regulates nuclear factor κB signaling to enhance CCL2 expression, which, in contrast to previous findings, suppresses cytotoxic NK cell activation in vitro and their infiltration into the PMN in vivo. These findings uncover an unexpected role for CCL2 in inhibiting NK cell responses at the PMN and suggest that targeting EYA3 could be an effective strategy to reactivate antitumor immune responses to inhibit metastasis.

Multiple mechanisms were proposed to mediate the nuclear import of TAZ/YAP, transcriptional co-activators regulating organ growth and regeneration. Our earlier observations showed that TAZ/YAP harbor a C-terminal, unconventional nuclear localization signal (NLS). Here, we show that this sequence, necessary and sufficient for basal, ATP-independent nuclear import, contains an indispensable central methionine flanked by negatively charged residues. Based on these features, we define the M-motif and propose that it is a new class of NLS, also present and import-competent in other cellular (STAT1 and cyclin B1) and viral (ORF6 of SARS-CoV2, VSV-M) proteins. Accordingly, ORF6 SARS-Cov2 competitively inhibits TAZ/YAP uptake, while TAZ abrogates STAT1 import. Similar to viral M-motif proteins, TAZ binds RAE1 and inhibits classic nuclear protein import, including that of antiviral factors (IRF3 and NF-κB). However, RAE1 is dispensable for TAZ import itself. Thus, the TAZ/YAP NLS has a dual function: it mediates unconventional nuclear import and inhibits classic import, contributing to the suppression of antiviral responses.

Trials have shown promising clinical activity of the selective SYK inhibitor entospletinib in patients with high expressing HOXA9/MEIS1 acute leukemias. As the development of resistance mechanisms is a common problem in the use of targeted drugs, we performed a chemical library screen to identify drug sensitivities in SYK inhibitor resistant AML cells. We identified that SYK inhibitor resistant cells displayed an increased sensitivity to glucocorticoids. Glucocorticoids are potent immunosuppressants which work in part by inhibiting the transcription of cytokine genes. RNA sequencing of entospletinib resistant cells revealed a strong enrichment of inflammatory response and TNFα signaling via NF-κB gene sets in comparison to naive cells. Naive AML cells treated with entospletinib showed a strong downregulation of the same gene sets which were upregulated in the resistant state. Our data suggest that inflammatory signaling pathways play a role in entospletinib resistant AML cells.

Thyroid cancer is rapidly increasing worldwide, with some patients facing poor prognosis and recurrence despite current treatments. Chitosan-based nanoparticles have exhibited exciting antitumor efficacy both in vitro and in vivo, which indicates that there is vast scope of clinical application. This study develops a anhydride-modified chitosan and anhydride-modified chitosan‑silver nanoparticles, encapsulated in γ-cyclodextrin to help drug delivery by safe way and enhance thyroid cancer therapy.

5H pyrolo(3,4-b)pyrazin-5,7-(6H)-dione-6-(N-chitosanimide nanoparticle(composite constructed with nano silver (B1) was prepared and the optimized formula was further investigated regarding FT-IR, X-RD, SEM and TEM. Furthermore, it was encapsulated in γ-CD, and an in vivo study was conducted to investigate its anticancer activity. The binding affinities of 2,3-Pyrazinedicarboxylic anhydride to inhibitor of kappa B kinase beta (IKK-β) was demonstrated by molecular docking.

SEM and TEM revealed that Ag NPs were mostly uniformly incorporated into the 5H pyrolo(3,4-b)pyrazin-5,7-(6H)-dione 6-(N-chitosanimide nanoparticle, while FT-IR and X-RD findings verified the formation of 5H pyrolo(3,4-b)pyrazin-5,7-(6H)-dione-6-(N-chitosanimide nanoparticle)/composite constructed with nano silver and encapsulated in γ-CD (B2). γ-CD encapsulation induced a significant enhancement in pyrazine thyroid antitumor activity in xenografic model.

B2 could be considered a promising formula for suppression of thyroid cancer by modulating NF-κB signaling pathway, and hence, future studies could be planned to transfer our formula to the clinical field.

TANK-binding kinase 1 (TBK1) is a key signaling kinase involved in innate immune and inflammatory responses. TBK1 drives immune cells to participate in the inflammatory response by activating the NF-κB and interferon regulatory factor signaling pathways in immune cells, promoting the expression of pro-inflammatory genes, and regulating immune cell function. Thus, it plays a crucial role in initiating a signaling cascade that establishes an inflammatory environment. In inflammation-related diseases, TBK1 acts as a bridge linking inflammation to immunity, metabolism, or tumorigenesis, playing an important role in the pathogenesis of immune-mediated inflammatory diseases, metabolic, inflammatory syndromes, and inflammation-associated cancers by regulating the activation of inflammatory pathways and the production of inflammatory cytokines in cells. In this review, we focused on the mechanisms of TBK1 in immune cells and inflammatory-related diseases, providing new insights for further studies targeting TBK1 as a potential treatment for inflammation-related diseases. Thus, optimizing and investigating highly selective cell-specific TBK1 inhibitors is important for their application in these diseases.

The IκB kinase (IKK) complex plays a central role in many signaling pathways that activate NF-κB, which turns on a battery of genes important for immune response, inflammation, and cancer development. Ubiquitination is one of the most prevalent post-translational modifications of proteins and is best known for targeting substrates for proteasomal degradation. The investigations of NF-κB signaling pathway primed the unveiling of the non-degradative roles of protein ubiquitination. The NF-κB-essential modulator (NEMO) is the IKK regulatory subunit that is essential for IKK activation by diverse intrinsic and extrinsic stimuli. The studies centered on NEMO as a polyubiquitin-binding protein have remarkably advanced understandings of how NEMO transmits signals to NF-κB activation and have laid a foundation for determining the molecular events demonstrating non-degradative ubiquitination as a major driving element in IKK activation. Furthermore, these studies have largely solved the enigma that IKK can be activated by diverse pathways that employ distinct sets of intermediaries in transmitting signals. NEMO and NEMO-related proteins that include optineurin, ABIN1, ABIN2, ABIN3, and CEP55, as non-degradative ubiquitin chain receptors, play a key role in sensing and transmitting ubiquitin signals embodied in different topologies of polyubiquitin chains for a variety of cellular processes and body responses. Studies of these multifaceted proteins in ubiquitin sensing have promoted understanding about the functions of non-degradative ubiquitination in intracellular signaling, protein trafficking, proteostasis, immune response, DNA damage response, and cell cycle control. In this review, I will also discuss how dysfunction in the NEMO family of protein-mediated non-degradative ubiquitin signaling is associated with various diseases, including immune disorders, neurodegenerative diseases, and cancer, and how microbial virulence factors target NEMO to induce pathogenesis or manipulate host response. A profound understanding of the molecular bases for non-degradative ubiquitin signaling will be valuable for developing tailored approaches for therapeutic purposes.

IKKα, encoded by CHUK, is crucial in the non-canonical NF-κB pathway and part of the IKK complex activating the canonical pathway alongside IKKβ. The absence of IKKα causes fetal encasement syndrome in humans, fatal in utero, while an impaired IKKα-NIK interaction was reported in a single patient and causes combined immunodeficiency. Here, we describe compound heterozygous variants in the kinase domain of IKKα in a female patient with hypogammaglobulinemia, recurrent lung infections, and Hay-Wells syndrome-like features. We showed that both variants were loss-of-function. Non-canonical NF-κB activation was profoundly diminished in stromal and immune cells while the canonical pathway was unexpectedly partially impaired. Reintroducing wt CHUK restored non-canonical NF-κB activation. The patient had neutralizing autoantibodies against type I IFN, akin to non-canonical NF-κB pathway deficiencies. Thus, this is the first case of biallelic CHUK mutations disrupting IKKα kinase function, broadening non-canonical NF-κB defect understanding, and suggesting IKKα's role in canonical NF-κB target gene expression in humans.

The cytosolic nucleic acid sensors RIG-I and cGAS induce type-I interferon (IFN)-mediated immune responses to RNA and DNA viruses, respectively. So far no connection between the two cytosolic pathways upstream of IKK-like kinase activation has been investigated. Here, we identify heterogeneous nuclear ribonucleoprotein M (hnRNPM) as a positive regulator of IRF3 phosphorylation and type-I IFN induction downstream of both cGAS and RIG-I. Combining interactome analysis with genome editing, we further uncover the RNA-binding protein ELAV-like protein 1 (ELAVL1; also known as human antigen R, HuR) as an hnRNPM interactor. Depletion of hnRNPM or ELAVL1 impairs type-I IFN induction by herpes simplex virus 1 or Sendai virus. In addition, we show that hnRNPM and ELAVL1 interact with TANK-binding kinase 1, IκB kinase ε, IκB kinase β, and NF-κB p65. Our confocal microscopy experiments demonstrate cytosolic and perinuclear interactions between hnRNPM, ELAVL1, and TBK1. Furthermore, pharmacological inhibition of ELAVL1 strongly reduces cytokine release from type-I interferonopathy patient fibroblasts. The RNA-binding proteins hnRNPM and ELAVL1 are the first non-redundant regulators to bridge the cGAS/STING and RIG-I/MAVS pathways. Overall, our study characterizes the hnRNPM-ELAVL1 complex as a novel system promoting antiviral defense, pointing to a potential therapeutic target to reduce auto-inflammation in patients with type-I interferonopathies.

NLRP3 inflammasome activation is a pivotal area of research in innate immunity, yet the precise priming and activation signal remain unclear. In this study, we demonstrate that glycolysis inhibitor 2-Deoxy-D-glucose (2DG) triggers NLRP3-driven pyroptosis in human leukemia monocyte THP-1 cells by interfering glycosylation rather than glycolysis, which occurs independent of potassium efflux but requires the involvement of glycolysis rate-limiting enzyme PFKP. Using a CRISPR-Cas9 mediated large-scale screen, with 2DG as a new tool for probing NLRP3 activation, we identified that TLR2, rather than TLR4, initiates a rapid and robust priming signal for NLRP3 inflammasome activation. Importantly, both TLR2 and TLR4 depend entirely on MyD88, but not TRIF, for signal transduction. Furthermore, we discovered that TAK1, IKKβ and NEMO, but not IKKα, are essential for the priming signal. Additionally, we observed that deficiency in the linear ubiquitin assembly complex (LUBAC) subunits HOIP and HOIL-1, but not SHARPIN, is sufficient to inhibit 2DG-induced pyroptotic cell death. Collectively, our study reveals some common mechanism in the NLRP3 priming signals, as well as specific mechanisms upstream of NLRP3 triggered by 2DG, and underscores the potential of 2DG as a trigger to facilitate further detailed analysis of the underlying mechanisms of NLRP3 inflammasome activation. One Sentence Summary: Priming signal by IKKβ is essential for NLRP3 activation.

African swine fever virus (ASFV) is the causative agent of a lethal disease in pigs. Highly virulent strains of ASFV are known to suppress the induction of type I interferons (IFNs), while naturally attenuated strains do not exhibit this capability. Thus, the ability to suppress IFN is assumed to be associated with viral virulence. ASFV genome encodes many proteins capable of disrupting crucial components of host immune response pathways. Notably, these viral proteins interfere with the induction of type I IFNs by targeting various steps of the cGAS-STING signaling pathway. Additionally, certain viral proteins impede the expression of interferon-stimulated genes by interfering with the JAK-STAT pathway. Consequently, ASFV proteins hamper both IFN production and the induction of antiviral responses by IFNs. This review article summarizes the viral proteins responsible for suppressing various steps of the cGAS-STING and JAK-STAT signaling pathways and discusses the potential application of this knowledge to the rational design of a live-attenuated ASFV vaccine.

Background and Objectives: Age-related macular degeneration (AMD) is the leading cause of blindness, affecting millions worldwide. Its pathogenesis involves the death of the retinal pigment epithelium (RPE), followed by photoreceptor degeneration. Although AMD is multifactorial, various genetic markers are strongly associated with the disease and may serve as biomarkers for evaluating treatment efficacy. This study investigates TAB2 rs237025, IKBKB rs13278372, and IKBKG rs2472395 variants and their respective serum protein concentrations in relation to AMD occurrence and exudative AMD treatment response to anti-VEGF treatment. Materials and Methods: The case-control study involved 961 individuals, and they were divided into three groups: control, early AMD, and exudative AM patients. Genotyping of selected SNPs were conducted using a real-time polymerase chain reaction method (RT-PCR). Based on the clinical OCT and BCVA data, patients with exudative AMD were categorized into one of two groups: responders and non-responders. The data obtained were analyzed using the "IBM SPSS Statistics 29.0" software program. Results: Our study revealed that TAB2 rs237025 allele A was identified as a risk factor for early and exudative AMD development. The same associations remained only in females with exudative AMD but not in males, suggesting gender-specific pathogenetic pathways in exudative AMD. Analysis of IKBKB rs13278372 or serum IKBKB protein associations with early or exudative AMD occurrence in the Lithuanian population revealed no significant associations. On the other hand, we found that each A allele of IKBKB rs13278372 was associated with a worse response to anti-VEGF treatment (OR = 0.347; 95% CI: 0.145-0.961; p = 0.041). These results suggest a potential marker for future studies evaluating anti-VEGF treatment for exudative AMD patients. IKBKG rs2472395 was a protective variant for early AMD in males and for exudative AMD in females only. Also, IKBKG protein concentration was lower in exudative AMD relative to the control group (median (IQR): 0.442 (0.152) vs. 0.538 (0.337), p = 0.015). Moreover, exudative AMD patients who carry the GG genotype of IKBKG rs2472394 exhibited significantly reduced serum IKBKG concentrations compared to the controls (median (IQR): 0.434 (0.199) vs. 0.603 (0.335), p = 0.012), leading to the hypothesis that the IKBKG rs2472394 variant might play a role in protein concentration differences and exudative AMD development. Conclusions: Our study identified the TAB2 rs237025 allele A as a significant risk factor for both early and exudative AMD, with gender-specific associations observed in females with exudative AMD, suggesting distinct pathogenetic pathways. While IKBKB rs13278372 and serum IKBKB protein levels showed no significant association with AMD development, the A allele of IKBKB rs13278372 was associated with a worse response to anti-VEGF treatment, indicating its potential as a marker for treatment outcomes. Additionally, the IKBKG rs2472395 variant was found to be protective for early AMD in males and exudative AMD in females, and lower IKBKG protein levels were associated with exudative AMD, particularly in patients with the GG genotype of IKBKG rs2472394, suggesting its role in protein concentration and disease progression. These findings highlight genetic markers that may contribute to AMD pathogenesis and treatment response.

Ovarian cancer is the leading cause of death among gynecologic malignancies. Despite recent advancements in targeted therapies such as PARP inhibitors, recurrence is common and frequently resistant to existing therapies. A powerful diagnostic tool, coupled with a comprehensive understanding of its implications, is crucial. HE4, a clinical serum biomarker for ovarian cancer, has shown efficacy in monitoring malignant phenotypes, yet little is known about its biological role and regulatory mechanisms. Our research demonstrates that HE4 expression in ovarian cancer can be regulated by the NF-κB signaling pathway. We found that the activation of NF-κB signaling by tumor necrosis factor (TNF)-α, a cytokine found in ovarian cancer tumors and ascites, enhanced the secretion of HE4 while its inhibition suppressed HE4 levels. Nuclear translocation of the NF-κB component p65 was found to be critical for HE4 expression; induced NF-κB activation through p65 expression or constitutive IKK2 activity elevated HE4 expression, while p65 knockdown had the opposite effect. Furthermore, we observed that NF-κB mediated HE4 expression at the transcriptional level. Our data also suggests that there is a regulatory role for HE4 in the expression of α5-Integrin, a crucial adhesion molecule in ovarian cancer metastasis; HE4 knockdown corresponded with reduced α5-Integrin expression, cell migration and cell adhesion to fibronectin.

Viruses elicit long-term adaptive responses in the tissues they infect. Understanding viral adaptions in humans is difficult in organs such as the heart, where primary infected material is not routinely collected. In search of asymptomatic infections with accompanying host adaptions, we mined for cardio-pathogenic viruses in the unaligned reads of nearly 1000 human hearts profiled by RNA sequencing. Among virus-positive cases (~20%), we identified three robust adaptions in the host transcriptome related to inflammatory nuclear factor κB (NF-κB) signaling and posttranscriptional regulation by the p38-MK2 pathway. The adaptions are not determined by the infecting virus, and they recur in infections of human or animal hearts and cultured cardiomyocytes. Adaptions switch states when NF-κB or p38-MK2 is perturbed in cells engineered for chronic infection by the cardio-pathogenic virus, coxsackievirus B3. Stratifying viral responses into reversible adaptions adds a targetable systems-level simplification for infections of the heart and perhaps other organs.

Cleavage of the innate immune receptor NLRP1B by various microbial proteases causes the proteasomal degradation of its N-terminal fragment and the subsequent release of a C-terminal fragment that forms an inflammasome. We reported previously that metabolic stress caused by intracellular bacteria triggers NLRP1B activation, but the mechanism by which this occurs was not elucidated. Here we demonstrate that TLR4 signaling in metabolically stressed macrophages promotes the formation of a TRIF/RIPK1/caspase-8 complex. Caspase-8 activity, induced downstream of this TLR4 pathway or through a distinct TNF receptor pathway, causes cleavage and activation of NLRP1B, which facilitates the maturation of both pro-caspase-1 and pro-caspase-8. Thus, our findings indicate that caspase-8 and NLRP1B generate a positive feedback loop that amplifies cell death processes and promotes a pro-inflammatory response through caspase-1. The ability of NLRP1B to detect caspase-8 activity suggests that this pattern recognition receptor may play a role in the defense against a variety of pathogens that induce apoptosis.

Dairy cattle with high (HM) versus low muscle (LM) reserves as determined by longissimus dorsi muscle depth (LDD) in late gestation exhibit differential muscle mobilization related to subsequent milk production. Moreover, branched-chain volatile fatty acid (BCVFA) supplementation increased blood glucose levels. We hypothesized that differences in HM and LM reflect distinct muscle metabolism and that BCVFA supplementation altered metabolic pathways. At 42 days before expected calving (BEC), Holstein dairy cows were enrolled in a 2 × 2 factorial study of diet and muscle reserves, by assignment to control (CON)- or BCVFA-supplemented diets and LDD of HM (>4.6 cm) or LM (≤4.6 cm) groups: HM-CON (n = 13), HM-BCVFA (n = 10), LM-CON (n = 9), and LM-BCVFA (n = 9). Longisumus dorsi muscle was biopsied at 21 days BEC, total RNA was isolated, and protein-coding gene expression was measured with RNA sequencing. Between HM and LM, 713 genes were differentially expressed and 481 between BCVFA and CON (P < 0.05). Transcriptional signatures indicated differential distribution of type II fibers between groups, with MYH1 greater in LM cattle and MYH2 greater in HM cattle (P < 0.05). Signatures of LM cattle relative to HM cattle indicated greater activation of autophagy, ubiquitin-proteasome, and Ca2+-calpain pathways. HM cattle displayed greater expression of genes that encode extracellular matrix proteins and factors that regulate their proteolysis and turnover. BCVFA modified transcriptomes by increasing expression of genes that regulate fatty acid degradation and flux of carbons into the tricarboxylic acid cycle as acetyl CoA. Molecular signatures support distinct metabolic strategies between LM and HM cattle and that BCVFA supplementation increased substrates for energy generation.NEW & NOTEWORTHY Muscle biopsies of the longissimus dorsi of prepartum dairy cattle indicate that molecular signatures support distinct metabolic strategies between low- and high-muscle cattle and that branched-chain volatile fatty acid supplementation increased substrates for energy generation.

IkappaB kinase beta (IKKβ) is a key member of IκB kinases and functions importantly in interferon (IFN) signaling. Phosphorylation and ubiquitination are involved in the activation of IKKβ. A20 is a de-ubiquitin enzyme and functions as a suppressor in inflammation signaling, which has been reported to be phosphorylated and activated by IKKβ. However, the role and relationship of IKKβ and A20 in teleost remains unclear. In this study, IKKβ (bcIKKβ) and A20 (bcA20) of black carp (Mylopharyngodon piceus) have been cloned and characterized. Overexpressed bcIKKβ in EPC cells showed strong anti-viral ability by activating both NF-κB and IFN signaling. EPC cells stable expressing bcIKKβ presented improved anti-viral activity as well. The interaction between bcA20 and bcIKKβ was identified, and overexpression of bcA20 was able to suppress bcIKKβ-mediated activation of NF-κB and IFN signaling. Meanwhile, knock-down of A20 increased host the antiviral ability of host cells. Importantly, it has been identified that bcA20 was able to remove K27-linked ubiquitination and decrease the phosphorylation of bcIKKβ. Thus, our data conclude that bcA20 suppresses the anti-viral activity of bcIKKβ and removes its K27-linked ubiquitination, which presents a new mechanism of IKKβ regulation.

Patients with Triple Negative Breast Cancer (TNBC) exhibit high rates of metastases and poor prognoses. The Eyes absent (EYA) family of proteins are developmental transcriptional cofactors/phosphatases that are re-expressed and/or upregulated in numerous cancers. Herein, we demonstrate that EYA3 correlates with decreased survival in breast cancer, and that it strongly, and specifically, regulates metastasis via a novel mechanism that involves NF-kB signaling and an altered innate immune profile at the pre-metastatic niche (PMN). Remarkably, restoration of NF-kB signaling downstream of Eya3 knockdown (KD) restores metastasis without restoring primary tumor growth, isolating EYA3/NF-kB effects to the metastatic site. We show that secreted CCL2, regulated downstream of EYA3/NF-kB, specifically decreases cytotoxic NK cells in the PMN and that re-expression of Ccl2 in Eya3 -KD cells is sufficient to rescue activation/levels of cytotoxic NK cells in vitro and at the PMN, where EYA3-mediated decreases in cytotoxic NK cells are required for metastatic outgrowth. Importantly, analysis of public breast cancer datasets uncovers a significant correlation of EYA3 with NF-kB/CCL2, underscoring the relevance of EYA3/NF-kB/CCL2 to human disease. Our findings suggest that inhibition of EYA3 could be a powerful means to re-activate the innate immune response at the PMN, inhibiting TNBC metastasis.

EYA3 promotes metastasis of TNBC cells by promoting NF-kB-mediated CCL2 expression and inhibiting cytotoxic NK cells at the pre-metastatic niche, highlighting a potential therapeutic target in this subset of breast cancer.

Exposure to excess ammonia-N (NH3/NH4+) in aquaculture can disrupt physiological function in shrimp leading to enhanced oxidative stress and apoptosis, but little is known concerning the post-transcriptional regulation mechanism. In this study, the first miR-200 family member in crustacean was identified and characterized from Litopenaeus vannamei (designed as Lva-miR-8-3p). Lva-miR-8-3p was highly expressed in eyestalks, brainganglion, and gills. The expression of Lva-miR-8-3p in gills significantly decreased after ammonia-N stress, and Lva-miR-8-3p was confirmed to target IKKβ 3'UTR for negatively regulating IKKβ/NF-κB pathway. Overexpression of miR-8-3p promoted the hemolymph ammonia-N accumulation, total hemocyte count (THC) decrease, and gills tissue damage, thus resulting in a decreased survival rate of ammonia-exposed shrimp. Besides, Lva-miR-8-3p silencing could enhance the antioxidant enzymes activities and reduce the oxidative damage, whereas overexpression of Lva-miR-8-3p exerted the opposite effects. Furthermore, Lva-miR-8-3p overexpression was found to aggravate ammonia-N induced apoptosis in gills. In primarily cultured hemocytes, the cell viability decreased, the ROS content and caspase-3 activity increased after agomiR-8-3p transfection, while antagomiR-8-3p transfection caused the opposite change except the cell viability. These findings indicate that Lva-miR-8-3p acts as a post-transcriptional regulator in ammonia-N induced antioxidant response and apoptosis by negatively regulating IKKβ/NF-κB pathway.

The aim of this study was to determine associations of TRAF2 (rs867186), TAB2 (rs237025), IKBKB (rs13278372) gene polymorphisms and TRAF2, TAB2, IKBKB protein levels with clinical and morphological features of pituitary adenomas (PAs).

This case-control study included 459 individuals divided into two groups: a control group (n = 320) and a group of individuals with PAs (n = 139). DNA from peripheral blood leukocytes was isolated using salt precipitation and column method. Real-time PCR was used for TRAF2 (rs867186), TAB2 (rs237025), and IKBKB (rs13278372) SNP genotyping, and TRAF2, TAB2, IKBKB protein concentration measurements were performed by immunoenzymatic analysis tests using a commercial ELISA kit according to the manufacturer's recommendations. The labeling index Ki-67 was determined by immunohistochemical analysis using a monoclonal antibody (clone SP6; Spring Bioscience Corporation). Statistical data analysis was performed using the programs "IMB SPSS Statistics 29.0".

We found significant differences in TRAF2 (rs867186) genotypes (AA, AG, GG) between groups: 79.1%, 17.3%, 3.6% vs. 55.3%, 20.9%, 23.8% (p < 0.001). The G allele was less frequent in the PA group than in controls (12.2% vs. 34.2%, p < 0.001). The AG and GG genotypes reduced PA occurrence by 1.74-fold and 9.43-fold, respectively, compared to AA (p < 0.001). In the dominant model, GG and AG genotypes reduced PA odds by 3.07-fold, while in the recessive model, the GG genotype reduced PA odds by 8.33-fold (p < 0.001). Each G allele decreased PA odds by 2.49-fold in the additive model (p < 0.001). Microadenomas had significant genotype differences compared to controls: 81.3%, 18.8%, 0.0% vs. 55.3%, 20.9%, 23.8% (p < 0.001), with the G allele being less frequent (9.4% vs. 34.2%, p < 0.001). In macroadenomas, genotype differences were 78%, 16.5%, 5.5% vs. 55.3%, 20.9%, 23.8% (p < 0.001), and the G allele was less common (13.7% vs. 34.2%, p < 0.001). The dominant model showed that GG and AG genotypes reduced microadenoma odds by 3.5-fold (p = 0.001), and each G allele reduced microadenoma odds by 3.1-fold (p < 0.001). For macroadenomas, the GG genotype reduced odds by 6.1-fold in the codominant model (p < 0.001) and by 2.9-fold in GG and AG genotypes combined compared to AA (p < 0.001). The recessive model indicated the GG genotype reduced macroadenoma odds by 5.3-fold (p < 0.001), and each G allele reduced odds by 2.2-fold in the additive model (p < 0.001).

The TRAF2 (rs867186) G allele and GG genotype are significantly associated with reduced odds of pituitary adenomas, including both microadenomas and macroadenomas, compared to the AA genotype. These findings suggest a protective role of the G allele against the occurrence of these tumors.

Receptor-interacting protein kinase 1 (RIPK1) plays a crucial role in controlling inflammation and cell death. Its function is tightly controlled through post-translational modifications, enabling its dynamic switch between promoting cell survival and triggering cell death. Phosphorylation of RIPK1 at various sites serves as a critical mechanism for regulating its activity, exerting either activating or inhibitory effects. Perturbations in RIPK1 phosphorylation status have profound implications for the development of severe inflammatory diseases in humans. This review explores the intricate regulation of RIPK1 phosphorylation and dephosphorylation and highlights the potential of targeting RIPK1 phosphorylation as a promising therapeutic strategy for mitigating human diseases.

Tumor heterogeneity poses a significant challenge in osteosarcoma (OS) treatment. In this regard, the "omics" era has constantly expanded our understanding of biomarkers and altered signaling pathways (i.e., PI3K/AKT/mTOR, WNT/β-catenin, NOTCH, SHH/GLI, among others) involved in OS pathophysiology. Despite different players and complexities, many commonalities have been described, among which the nuclear factor kappa B (NF-κB) stands out. Its altered activation is pervasive in cancer, with pleiotropic action on many disease-relevant traits. Thus, in the scope of this article, we highlight the evidence of NF-κB dysregulation in OS and its integration with other cancer-related pathways while we summarize the repertoire of compounds that have been described to interfere with its action. In silico strategies were used to demonstrate that NF-κB is closely coordinated with other commonly dysregulated signaling pathways not only by functionally interacting with several of their members but also by actively participating in the regulation of their transcription. While existing inhibitors lack selectivity or act indirectly, the therapeutic potential of targeting NF-κB is indisputable, first for its multifunctionality on most cancer hallmarks, and secondly, because, as a common downstream effector of the many dysregulated pathways influencing OS aggressiveness, it turns complex regulatory networks into a simpler picture underneath molecular heterogeneity.

IKKα, encoded by CHUK , is crucial in the non-canonical NF-κB pathway and part of the IKK complex activating the canonical pathway alongside IKKβ. Absence of IKKα cause fetal encasement syndrome in human, fatal in utero, while an impaired IKKα-NIK interaction was reported in a single patient and cause combined immunodeficiency. Here, we describe compound heterozygous variants in the kinase domain of IKKα in a female patient with hypogammaglobulinemia, recurrent lung infections, and Hay-Wells syndrome-like features. We showed that both variants were loss-of-function. Non-canonical NF-κB activation was profoundly diminished in stromal and immune cells while the canonical pathway was partially impaired. Reintroducing wild-type CHUK restored non-canonical NF-κB activation. The patient had neutralizing autoantibodies against type I IFN, akin to non-canonical NF-κB pathway deficiencies. Thus, this is the first case of bi-allelic CHUK mutations disrupting IKKα kinase function, broadening non-canonical NF-κB defect understanding and suggesting IKKα's role in canonical NF-κB target gene expression in human.

Epitranscriptomic RNA modifications are crucial for the maintenance of glioma stem cells (GSCs), the most malignant cells in glioblastoma (GBM). 3-methylcytosine (m3C) is a new epitranscriptomic mark on RNAs and METTL8 represents an m3C writer that is dysregulated in cancer. Although METTL8 has an established function in mitochondrial tRNA (mt-tRNA) m3C modification, alternative splicing of METTL8 can also generate isoforms that localize to the nucleolus where they may regulate R-loop formation. The molecular basis for METTL8 dysregulation in GBM, and which METTL8 isoform(s) may influence GBM cell fate and malignancy remain elusive. Here, we investigated the role of METTL8 in regulating GBM stemness and tumorigenicity. In GSC, METTL8 is exclusively localized to the mitochondrial matrix where it installs m3C on mt-tRNAThr/Ser(UCN) for mitochondrial translation and respiration. High expression of METTL8 in GBM is attributed to histone variant H2AZ-mediated chromatin accessibility of HIF1α and portends inferior glioma patient outcome. METTL8 depletion impairs the ability of GSC to self-renew and differentiate, thus retarding tumor growth in an intracranial GBM xenograft model. Interestingly, METTL8 depletion decreases protein levels of HIF1α, which serves as a transcription factor for several receptor tyrosine kinase (RTK) genes, in GSC. Accordingly, METTL8 loss inactivates the RTK/Akt axis leading to heightened sensitivity to Akt inhibitor treatment. These mechanistic findings, along with the intimate link between METTL8 levels and the HIF1α/RTK/Akt axis in glioma patients, guided us to propose a HIF1α/Akt inhibitor combination which potently compromises GSC proliferation/self-renewal in vitro. Thus, METTL8 represents a new GBM dependency that is therapeutically targetable.

Trauma is statistically a significant cause of mortality among patients across countries. Nevertheless, the precise correlation between genetic diagnostic markers and the intricate mechanism of trauma remains indistinct.

Our study exclusively centered on trauma patients and selected three trauma-related datasets from the Gene Expression Omnibus (GEO) database, all of which had blood samples collected within post-traumatic 12 h. Differential gene screening, the WGCNA and Cytoscape software were employed to analyze the two datasets, with a particular emphasis on the top 100 genes selected based on MCC algorithm scores. A logistic diagnostic model was constructed by analyzing the intersection genes in the third dataset, leading to the identification of diagnostic biomarkers with high efficiency. The global immune landscape of these patients was extensively investigated using a multidimensional approach. Meanwhile, the underlying pathological and physiological mechanisms associated with early trauma status are summarized by integrating existing literature.

Out of these two GEO datasets, 21 overlapping genes were identified and incorporated into in the logistic diagnostic model constructed in the GSE36809 dataset. A panel of 9 genes was uncovered as a diagnostic biomarker, and their expression and correlation were subsequently verified. Additionally, by virtue of various algorithms, the findings revealed an upregulation of neutrophil expression and a downregulation of CD8+ T cell expression, indicating characteristic early trauma-induced inflammation activation and immune suppression. The correlation observed between the feature genes and immune cells serves to validate the exceptional diagnostic capability of these 9 genes in identifying trauma status and their promising potential for patients who could benefit from targeted immune interventions. Drawing from these findings, the discussion section offers insights into the underlying pathological and physiological mechanisms at play.

Our research has discovered a novel diagnostic biomarker and unveiled its association with post-traumatic immune alterations. This breakthrough enables accurate and timely diagnosis of early trauma, facilitating the implementation of appropriate healthcare interventions.

Viruses elicit long-term adaptive responses in the tissues they infect. Understanding viral adaptions in humans is difficult in organs such as the heart, where primary infected material is not routinely collected. In search of asymptomatic infections with accompanying host adaptions, we mined for cardio-pathogenic viruses in the unaligned reads of nearly one thousand human hearts profiled by RNA sequencing. Among virus-positive cases (~20%), we identified three robust adaptions in the host transcriptome related to inflammatory NFκB signaling and post-transcriptional regulation by the p38-MK2 pathway. The adaptions are not determined by the infecting virus, and they recur in infections of human or animal hearts and cultured cardiomyocytes. Adaptions switch states when NFκB or p38-MK2 are perturbed in cells engineered for chronic infection by the cardio-pathogenic virus, coxsackievirus B3. Stratifying viral responses into reversible adaptions adds a targetable systems-level simplification for infections of the heart and perhaps other organs.

Maternal decidual cells are crucial for the maintenance of canine pregnancy as they are the only cells expressing the nuclear progesterone (P4) receptor (PGR) in the placenta. Interfering with P4/PGR signaling adversely affects decidual cells and terminates pregnancy. Although immortalized dog uterine stromal (DUS) cells can be decidualized in vitro using cAMP, the involvement of cAMP-dependent kinases in canine decidualization had not been investigated. Therefore, the present project investigated changes in the kinome of DUS cells following in vitro decidualization, using the serine/threonine kinase (STK) PamChip assay (PamGene). Decidualization led to a predicted activation of 85 STKs in DUS cells, including protein kinase (PK) A, PKC, extracellular signal-regulated kinase (ERK)1/2 and other mitogen-activated protein kinases (MAPKs), calcium/calmodulin-dependent protein kinases (CAMKs), and Akt1/2. In addition, blocking PGR with type 2 antigestagens (aglepristone or mifepristone) decreased the activity of virtually all kinases modulated by decidualization. The underlying transcriptional effects were inferred from comparison with available transcriptomic data on antigestagen-mediated effects in DUS cells. In targeted studies, interfering with PKA or MAPK kinase (MEK)1/2 resulted in downregulation of important decidualization markers (e.g., insulin-like growth factor 1 (IGF1), prostaglandin E2 synthase (PTGES), prolactin receptor (PRLR), PGR, and prostaglandin-endoperoxide synthase 2 (PTGS2/COX2)). Conversely, blocking of PKC decreased the mRNA availability of IGF1, PGR, and PTGS2, but not of PTGES and PRLR. Moreover, suppressing PKA decreased the phosphorylation of the transcription factors cJUN and CREB, whereas blocking of PKC affected only cJUN. This first kinomics analysis to target decidualization showed an increased activity of a wide range of STKs, which could be hindered by disrupting P4/PGR signaling. Decidualization appears to be regulated in a kinase-dependent manner, with PKA and PKC evoking different effects.

The growing demands for gene therapy have encouraged development of safe and efficient lentiviral vector (LV) preparation. While much progress has been made in this field, it remains to be explored how to boost its production from producer cells. This paper reports that transient co-expression of RelA or several mitogen-activated protein kinase kinase kinases (MAP3Ks) with packaging constructs can potently enhance LV production in HEK293T producer cells. Adding in transfection a small amount of effector plasmid is sufficient to achieve 3- to 4-fold enhancement, which can further be augmented by co-expression of IκB kinase 2 or HIV Tat. It is also shown that expression of RelA or MAP3K1 can increase LV production in HEK293T/17SF cells grown in suspension. These results indicate that stimulation of intracellular signaling pathways in producer cells represents a powerful means for enhancing LV production.

Toll-like receptor 5 (TLR5), serving as a sensor of bacterial flagellin, mediates the innate immune response to actively engage in the host's immune processes against pathogen invasion. However, the mechanism underlying TLR5-mediated immune response in fish remains unclear. Despite the presumed cell surface expression of TLR5 member form (TLR5M), its trafficking dynamics remain elusive. Here, we have identified Epinephelus coioides TLR5M as a crucial mediator of Vibrio flagellin-induced cytokine expression in grouper cells. EcTLR5M facilitated the activation of NF-κB signaling pathway in response to flagellin stimulation and exerted a modest influence on the mitogen-activated protein kinase (MAPK)-extracellular regulated kinase (ERK) signaling. The trafficking chaperone Unc-93 homolog B1 (EcUNC93B1) participated in EcTLR5M-mediated NF-κB signaling activation and downstream cytokine expression. In addition, EcUNC93B1 combined with EcTLR5M to mediate its exit from the endoplasmic reticulum, and also affected its post-translational maturation. Collectively, these findings first discovered that EcTLR5M mediated the flagellin-induced cytokine expression primarily by regulating the NF-κB signaling pathway, and EcUNC93B1 mediated EcTLR5M function through regulating its trafficking and post-translational maturation. This research expanded the understanding of fish innate immunity and provided a novel concept for the advancement of anti-vibrio immunity technology.

Marek's disease virus (MDV) strain GX0101 was the first reported field strain of recombinant gallid herpesvirus type 2 (GaHV-2). However, the splenic proteome of MDV-infected chickens remains unclear. In this study, a total of 28 1-day-old SPF chickens were intraperitoneally injected with chicken embryo fibroblast (CEF) containing 2000 PFU GX0101. Additionally, a control group, consisting of four one-day-old SPF chickens, received intraperitoneal equal doses of CEF. Blood and various tissue samples were collected at different intervals (7, 14, 21, 30, 45, 60, and 90 days post-infection; dpi) for histopathological, real-time PCR, and label-free quantitative analyses. The results showed that the serum expressions of MDV-related genes, meq and gB, peaked at 45 dpi. The heart, liver, and spleen were dissected at 30 and 45 dpi, and their hematoxylin-eosin staining indicated that virus infection compromised the normal organizational structure at 45 dpi. Particularly, the spleen structure was severely damaged, and the lymphocytes in the white medulla were significantly reduced. Furthermore, liquid chromatography-mass spectrometry (LC-MS) and label-free techniques were used to analyze the difference in splenic proteome profiles of the experimental and control groups at 30 and 45 dpi. Proteomic analysis identified 1660 and 1244 differentially expressed proteins (DEPs) at 30 and 40 dpi, respectively, compared with the uninfected spleen tissues. According to GO analysis, these DEPs were involved in processes such as organelle organization, cellular component biogenesis, cellular component assembly, anion binding, small molecule binding, metal ion binding, cation binding, cytosol, nuclear part, etc. Additionally, KEGG analysis indicated that the following pathways were linked to MDV-induced inflammation, apoptosis, and tumor: Wnt, Hippo, AMPK, cAMP, Notch, TGF-β, PI3K-Akt, Rap1, Ras, Calcium, NF-κB, PPAR, cGMP-PKG, Apoptosis, VEGF, mTOR, FoxO, TNF, JAK-STAT, MAPK, Prion disease, T cell receptor, and B cell receptor. We finally screened 674 DEPs that were linked to MDV infection in spleen tissue. This study improves our understanding of the MDV response mechanism in the spleen.

Cognate interaction between CD4+ effector memory T (TEM) cells and dendritic cells (DCs) induces innate inflammatory cytokine production, resulting in detrimental autoimmune pathology and cytokine storms. While TEM cells use tumor necrosis factor (TNF) superfamily ligands to activate DCs, whether TEM cells prompt other DC-intrinsic changes that influence the innate inflammatory response has never been investigated. We report the surprising discovery that TEM cells trigger double-strand DNA breaks via mitochondrial reactive oxygen species (ROS) production in interacting DCs. Initiation of the DNA damage response in DCs induces activation of a cyclic guanosine monophosphate (GMP)-AMP synthase (cGAS)-independent, non-canonical stimulator of interferon genes (STING)-TNF receptor-associated factor 6 (TRAF6)-nuclear factor κB (NF-κB) signaling axis. Consequently, STING-deficient DCs display reduced NF-κB activation and subsequent defects in transcriptional induction and functional production of interleukin-1β (IL-1β) and IL-6 following their interaction with TEM cells. The discovery of TEM cell-induced innate inflammation through DNA damage and a non-canonical STING-NF-κB pathway presents this pathway as a potential target to alleviate T cell-driven inflammation in autoimmunity and cytokine storms.

N-terminal phosphorylation at residues T3 and S13 is believed to have important beneficial implications for the biological and pathological properties of mutant huntingtin, where inhibitor of nuclear factor kappa B kinase subunit beta (IKBKB) was identified as a candidate regulator of huntingtin N-terminal phosphorylation. The paucity of mechanistic information on IKK pathways, together with the lack of sensitive methods to quantify endogenous huntingtin phosphorylation, prevented detailed study of the role of IKBKB in Huntington's disease. Using novel ultrasensitive assays, we demonstrate that IKBKB can regulate endogenous S13 huntingtin phosphorylation in a manner, dependent on its kinase activity and known regulators. We found that the ability of IKBKB to phosphorylate endogenous huntingtin S13 is mediated through a non-canonical interferon regulatory factor3-mediated IKK pathway, distinct from the established involvement of IKBKB in mutant huntingtin's pathological mechanisms mediated via the canonical pathway. Furthermore, increased huntingtin S13 phosphorylation by IKBKB resulted in decreased aggregation of mutant huntingtin in cells, again dependent on its kinase activity. These findings point to a non-canonical IKK pathway linking S13 huntingtin phosphorylation to the pathological properties of mutant huntingtin aggregation, thought to be significant to Huntington's disease.

Radiation therapy (RT) provides therapeutic benefits for patients with glioblastoma (GBM), but inevitably induces poorly understood global changes in GBM and its microenvironment (TME) that promote radio-resistance and recurrence. Through a cell surface marker screen, we identified that CD142 (tissue factor or F3) is robustly induced in the senescence-associated β-galactosidase (SA-βGal)-positive GBM cells after irradiation. F3 promotes clonal expansion of irradiated SA-βGal+ GBM cells and orchestrates oncogenic TME remodeling by activating both tumor-autonomous signaling and extrinsic coagulation pathways. Intratumoral F3 signaling induces a mesenchymal-like cell state transition and elevated chemokine secretion. Simultaneously, F3-mediated focal hypercoagulation states lead to activation of tumor-associated macrophages (TAMs) and extracellular matrix (ECM) remodeling. A newly developed F3-targeting agent potently inhibits the aforementioned oncogenic events and impedes tumor relapse in vivo. These findings support F3 as a critical regulator for therapeutic resistance and oncogenic senescence in GBM, opening potential therapeutic avenues.

Protein interaction networks underlie countless biological mechanisms. However, most protein interaction predictions are based on biological evidence that are biased to well-known protein interaction or physical evidence that exhibits low accuracy for weak interactions and requires high computational power. In this study, a novel method has been suggested to predict protein interaction partners by investigating narrow funnel-like interaction energy distribution. In this study, it was demonstrated that various protein interactions including kinases and E3 ubiquitin ligases have narrow funnel-like interaction energy distribution. To analyze protein interaction distribution, modified scores of iRMS and TM-score are introduced. Then, using these scores, algorithm and deep learning model for prediction of protein interaction partner and substrate of kinase and E3 ubiquitin ligase were developed. The prediction accuracy was similar to or even better than that of yeast two-hybrid screening. Ultimately, this knowledge-free protein interaction prediction method will broaden our understanding of protein interaction networks.

The nuclear factor kappa B (NF-κB) is a family of transcription factors that, beyond their numberless functions in various cell processes, play a pivotal role in regulating immune cell activation. Two main pathways-canonical and non-canonical-are responsible for NF-κB activation and heterodimer translocation into the nucleus. A complex crosstalk between NF-κB signaling and metabolism is emerging in innate immunity. Metabolic enzymes and metabolites regulate NF-κB activity in many cases through post-translational modifications such as acetylation and phosphorylation. On the other hand, NF-κB affects immunometabolic pathways, including the citrate pathway, thereby building an intricate network. In this review, the emerging findings about NF-κB function in innate immunity and the interplay between NF-κB and immunometabolism have been discussed. These outcomes allow for a deeper comprehension of the molecular mechanisms underlying NF-κB function in innate immune cells. Moreover, the new insights are important in order to perceive NF-κB signaling as a potential therapeutic target for inflammatory/immune chronic diseases.

Coronavirus disease 2019 (COVID-19) is a global pandemic caused by SARS-CoV-2 infection. Patients with severe COVID-19 exhibit robust induction of proinflammatory cytokines, which are closely associated with the development of acute respiratory distress syndrome. However, the underlying mechanisms of the NF-κB activation mediated by SARS-CoV-2 infection remain poorly understood. Here, we screened SARS-CoV-2 genes and found that ORF3a induces proinflammatory cytokines by activating the NF-κB pathway. Moreover, we found that ORF3a interacts with IKKβ and NEMO and enhances the interaction of IKKβ-NEMO, thereby positively regulating NF-κB activity. Together, these results suggest ORF3a may play pivotal roles in the pathogenesis of SARS-CoV-2 and provide novel insights into the interaction between host immune responses and SARS-CoV-2 infection.

Epithelial-mesenchymal transition (EMT), which is well known for its role in embryonic development, malignant transformation, and tumor progression, has also been implicated in a variety of retinal diseases, including proliferative vitreoretinopathy (PVR), age-related macular degeneration (AMD), and diabetic retinopathy. EMT of the retinal pigment epithelium (RPE), although important in the pathogenesis of these retinal conditions, is not well understood at the molecular level. We and others have shown that a variety of molecules, including the co-treatment of human stem cell-derived RPE monolayer cultures with transforming growth factor beta (TGF-β) and the inflammatory cytokine tumor necrosis factor alpha (TNF-α), can induce RPE-EMT; however, small molecule inhibitors of RPE-EMT have been less well studied. Here, we demonstrate that BAY651942, a small molecule inhibitor of nuclear factor kapa-B kinase subunit beta (IKKβ) that selectively targets NF-κB signaling, can modulate TGF-β/TNF-α-induced RPE-EMT. Next, we performed RNA-seq studies on BAY651942 treated hRPE monolayers to dissect altered biological pathways and signaling events. Further, we validated the effect of IKKβ inhibition on RPE-EMT-associated factors using a second IKKβ inhibitor, BMS345541, with RPE monolayers derived from an independent stem cell line. Our data highlights the fact that pharmacological inhibition of RPE-EMT restores RPE identity and may provide a promising approach for treating retinal diseases that involve RPE dedifferentiation and EMT.

Non-small cell lung cancer (NSCLC) accounts for 80-85% cases of lung cancer cases. Diagnosis at advanced stages is common, after which therapy-refractory disease progression frequently occurs. Therefore, a better understanding of the molecular mechanisms that control NSCLC progression is necessary to develop new therapies. Overexpression of IκB kinase α (IKKα) in NSCLC correlates with poor patient survival. IKKα is an NF-κB-activating kinase that is important in cell survival and differentiation, but its regulation of oncogenic signaling is not well understood. We recently demonstrated that IKKα promotes NSCLC cell migration by physically interacting with dopamine- and cyclic AMP-regulated phosphoprotein, Mr 32000 (DARPP-32), and its truncated splice variant, t-DARPP. Here, we show that IKKα phosphorylates DARPP-32 at threonine 34, resulting in DARPP-32-mediated inhibition of protein phosphatase 1 (PP1), subsequent inhibition of PP1-mediated dephosphorylation of ERK, and activation of ERK signaling to promote lung oncogenesis. Correspondingly, IKKα ablation in human lung adenocarcinoma cells reduced their anchorage-independent growth in soft agar. Mice challenged with IKKα-ablated HCC827 cells exhibited less lung tumor growth than mice orthotopically administered control HCC827 cells. Our findings suggest that IKKα drives NSCLC growth through the activation of ERK signaling via DARPP-32-mediated inhibition of PP1 activity.

Increasing amounts of nanoplastics (NPs) in the environment are a great threat to human health, causing intestinal inflammation when consumed through seafood and water. There is, however, still a lack of understanding of the immunomodulatory role of NPs in immune cells, especially the early signal events behind inflammation resulting from NPs ingestion. In this study, we explored the dynamic internalization of polystyrene NPs and their carboxy and amino-functionalized products (PS, PS-COOH and PS-NH₂) followed by activation of ROS-MAPK/NF-κB signaling pathways in macrophage RAW 264.7. The inflammatory and cytotoxic potentials of NPs were evaluated by ELISA and apoptosis assays. Results showed that PS-COOH accumulated most in cells and induced more pronounced ROS and apoptosis than PS, PS-NH₂ and PS-μm. PS-COOH and PS-NH₂ showed stronger MAPK/NF-κB activation potential to at a low concentration of 10 μg/mL than unmodified PS, followed by production of IL-6 and TNF-α cytokines. Furthermore, PS-COOH induced MAPK/NF-κB activation and cytokine secretion could be inhibited by NAC, indicating that ROS was responsible for signal dysregulation and immunogenicity of PS-COOH, but not for PS-NH₂. The results suggested that the MAPK and NF-κB pathways were involved in NPs-induced macrophage inflammation, which was influenced by surface functionalization of NPs, with carboxylated PS NPs exhibiting a greater pro-inflammatory and cytotoxic potential.