Sergei S. Makarov

NF-κB as a therapeutic target in chronic inflammation: recent advances

Abstract The family of nuclear factor κB (NF-κB) transcription factors is a topic of intense interest in the biomedical community stemming from the role NF-κB plays in almost every aspect of cell regulation: stress responses, immune cell activation, apoptosis, proliferation, differentiation and oncogenic transformation. The objective of this article is to provide an overview of recent developments in the field with an emphasis on the role of NF-κB in chronic inflammation, and to discuss the feasibility of therapeutic approaches based on the specific suppression of the NF-κB pathway.

NF-κB in rheumatoid arthritis: a pivotal regulator of inflammation, hyperplasia, and tissue destruction

The transcription factor NF-κB has been well recognized as a pivotal regulator of inflammation in rheumatoid arthritis (RA), but recent developments revealed a broad involvement of NF-κB in other aspects of RA pathology, including development of T helper 1 responses, activation, abnormal apoptosis and proliferation of RA fibroblast-like synovial cells, and differentiation and activation of bone resorbing activity of osteoclasts. In agreement with this, studies in animal models of RA have demonstrated the high therapeutic efficacy of specific inhibitors of NF-κB pathway, indicating the feasibility of anti-NF-κB therapy for human disease.

Benchmarking Organic Micropollutants in Wastewater, Recycled Water and Drinking Water with In Vitro Bioassays

Thousands of organic micropollutants and their transformation products occur in water. Although often present at low concentrations, individual compounds contribute to mixture effects. Cell-based bioassays that target health-relevant biological endpoints may therefore complement chemical analysis for water quality assessment. The objective of this study was to evaluate cell-based bioassays for their suitability to benchmark water quality and to assess efficacy of water treatment processes. The selected bioassays cover relevant steps in the toxicity pathways including induction of xenobiotic metabolism, specific and reactive modes of toxic action, activation of adaptive stress response pathways and system responses. Twenty laboratories applied 103 unique in vitro bioassays to a common set of 10 water samples collected in Australia, including wastewater treatment plant effluent, two types of recycled water (reverse osmosis and ozonation/activated carbon filtration), stormwater, surface water, and drinking water. Sixty-five bioassays (63%) showed positive results in at least one sample, typically in wastewater treatment plant effluent, and only five (5%) were positive in the control (ultrapure water). Each water type had a characteristic bioanalytical profile with particular groups of toxicity pathways either consistently responsive or not responsive across test systems. The most responsive health-relevant endpoints were related to xenobiotic metabolism (pregnane X and aryl hydrocarbon receptors), hormone-mediated modes of action (mainly related to the estrogen, glucocorticoid, and antiandrogen activities), reactive modes of action (genotoxicity) and adaptive stress response pathway (oxidative stress response). This study has demonstrated that selected cell-based bioassays are suitable to benchmark water quality and it is recommended to use a purpose-tailored panel of bioassays for routine monitoring.

Impact of environmental chemicals on key transcription regulators and correlation to toxicity end points within EPA's ToxCast program.

Exposure to environmental chemicals adds to the burden of disease in humans and wildlife to a degree that is difficult to estimate and, thus, mitigate. The ability to assess the impact of existing chemicals for which little to no toxicity data are available or to foresee such effects during early stages of chemical development and use, and before potential exposure occurs, is a pressing need. However, the capacity of the current toxicity evaluation approaches to meet this demand is limited by low throughput and high costs. In the context of EPAs ToxCast project, we have evaluated a novel cellular biosensor system (Factorial (1) ) that enables rapid, high-content assessment of a compounds impact on gene regulatory networks. The Factorial biosensors combined libraries of cis- and trans-regulated transcription factor reporter constructs with a highly homogeneous method of detection enabling simultaneous evaluation of multiplexed transcription factor activities. Here, we demonstrate the application of the technology toward determining bioactivity profiles by quantitatively evaluating the effects of 309 environmental chemicals on 25 nuclear receptors and 48 transcription factor response elements. We demonstrate coherent transcription factor activity across nuclear receptors and their response elements and that Nrf2 activity, a marker of oxidative stress, is highly correlated to the overall promiscuity of a chemical. Additionally, as part of the ToxCast program, we identify molecular targets that associate with in vivo end points and represent modes of action that can serve as potential toxicity pathway biomarkers and inputs for predictive modeling of in vivo toxicity.

An essential role of NF-κB in the “tumor-like” phenotype of arthritic synoviocytes

A hallmark of rheumatoid arthritis is the formation of an aggressive, tumor-like structure called pannus that erodes the joint. A major cellular component of the pannus is the fibroblast-like synoviocyte (FLS), whose morphology strikingly resembles that of a transformed cell, but underlying mechanisms of this “transformation” are not known. Here, using animal models of rheumatoid arthritis, we show that arthritic FLS contain a substantial (>30%) fraction of bone marrow-derived precursors that can differentiate in vitro into various mesenchymal cell types, but inflammation prevents the multilineage differentiation. We show that the transcription factor NF-κB plays the key role in the repression of osteogenic and adipogenic differentiation of arthritic FLS. Furthermore, we show that specific activation of NF-κB profoundly enhances proliferation, motility, and matrix-degrading activity of FLS. We thus propose that arthritic FLS are mesenchymal stem cells whose differentiation is arrested at early stages of differentiation by activation of NF-κB.

Cytokine biomarkers and chronic pain: association of genes, transcription, and circulating proteins with temporomandibular disorders and widespread palpation tenderness.

Summary Women with widespread vs localized chronic pain exhibit distinctive alterations in molecular‐genetic profiles of the inflammatory mediators MCP‐1, IL‐1ra, IL‐8, and TGFβ1. ABSTRACT For reasons unknown, temporomandibular disorder (TMD) can manifest as localized pain or in conjunction with widespread pain. We evaluated relationships between cytokines and TMD without or with widespread palpation tenderness (TMD−WPT or TMD+WPT, respectively) at protein, transcription factory activity, and gene levels. Additionally, we evaluated the relationship between cytokines and intermediate phenotypes characteristic of TMD and WPT. In a case‐control study of 344 females, blood samples were analyzed for levels of 22 cytokines and activity of 48 transcription factors. Intermediate phenotypes were measured by quantitative sensory testing and questionnaires asking about pain, health, and psychological status. Single nucleotide polymorphisms (SNPs) coding cytokines and transcription factors were genotyped. TMD−WPT cases had elevated protein levels of proinflammatory cytokine monocyte chemotactic protein (MCP‐1) and antiinflammatory cytokine interleukin (IL)‐1ra, whereas TMD+WPT cases had elevated levels of proinflammatory cytokine IL‐8. MCP‐1, IL‐1ra, and IL‐8 were differentially associated with experimental pain, self‐rated pain, self‐rated health, and psychological phenotypes. TMD−WPT and TMD+WPT cases had inhibited transcription activity of the antiinflammatory cytokine transforming growth factor β1 (TGFβ1). Interactions were observed between TGFβ1 and IL‐8 SNPs: an additional copy of the TGFβ1 rs2241719 minor T allele was associated with twice the odds of TMD+WPT among individuals homozygous for the IL‐8 rs4073 major A allele, and half the odds of TMD+WPT among individuals heterozygous for rs4073. These results demonstrate how pro‐ and antiinflammatory cytokines contribute to the pathophysiology of TMD and WPT in genetically susceptible people. Furthermore, they identify MCP‐1, IL‐1ra, IL‐8, and TGFβ1 as potential diagnostic markers and therapeutic targets for pain in patients with TMD.

NF-κB as a target for anti-inflammatory gene therapy: suppression of inflammatory responses in monocytic and stromal cells by stable gene transfer of IκBα cDNA

One of the most challenging issues of anti-inflammatory gene therapy is the complexity of inflammatory pathways. Transcription factor NF-κB plays a pivotal role in activation of multiple inflammatory molecules, and therefore represents the logical target for intervention. We evaluated the feasibility of suppressing the inflammatory responses in different cell lines through specific inhibition of NF-κB by gene transfer of IκBα, the naturally occurring intracellular inhibitor of NF-κB. The IκBα overexpressing cells were established using retroviral gene transfer or stable transfection with the wild-type (wt) IκBα cDNA. In all cell types, overexpression of wt IκBα resulted in a profound (>100-fold) increase of the IκBα message and a moderate (two- to three-fold) increase of the IκBα protein. The effects of the IκBα overexpression on the NF-κB activation and the inflammatory responses varied significantly in different cell lines. In conditionally immortalized human endometrial stromal cells, overexpression of IκBα prevented both interleukin-1 (IL-1)-inducible degradation of endogenous IκBα protein and activation of NF-κB. Accordingly, induction of cytokines interleukin-8 (IL-8) and Groγ was markedly suppressed. In monocytic THP-1 cells, both lipopolysaccharide (LPS)-inducible degradation of IκBα and NF-κB activation were only partially inhibited by overexpression of exogenous IκBα cDNA. None the less, the LPS-induced transcription of IL-1β and secretion of cytokines interleukin-6 (IL-6) and IL-8 were virtually abolished. In epithelial HT-29 cells, no inflammatory responses were inhibited. These results demonstrate the range of responses in various cell lines to gene transfer of IκBα and indicate the feasibility of suppression of inflammatory responses in appropriate target cells and their progeny by suppression of NF-κB.

Sequence motifs associated with hepatotoxicity of locked nucleic acid—modified antisense oligonucleotides

Fully phosphorothioate antisense oligonucleotides (ASOs) with locked nucleic acids (LNAs) improve target affinity, RNase H activation and stability. LNA modified ASOs can cause hepatotoxicity, and this risk is currently not fully understood. In vitro cytotoxicity screens have not been reliable predictors of hepatic toxicity in non-clinical testing; however, mice are considered to be a sensitive test species. To better understand the relationship between nucleotide sequence and hepatotoxicity, a structure–toxicity analysis was performed using results from 2 week repeated-dose-tolerability studies in mice administered LNA-modified ASOs. ASOs targeting human Apolipoprotien C3 (Apoc3), CREB (cAMP Response Element Binding Protein) Regulated Transcription Coactivator 2 (Crtc2) or Glucocorticoid Receptor (GR, NR3C1) were classified based upon the presence or absence of hepatotoxicity in mice. From these data, a random-decision forest-classification model generated from nucleotide sequence descriptors identified two trinucleotide motifs (TCC and TGC) that were present only in hepatotoxic sequences. We found that motif containing sequences were more likely to bind to hepatocellular proteins in vitro and increased P53 and NRF2 stress pathway activity in vivo. These results suggest in silico approaches can be utilized to establish structure–toxicity relationships of LNA-modified ASOs and decrease the likelihood of hepatotoxicity in preclinical testing.

Identification of novel mediators of NF-κB through genome-wide survey of monocyte adherence-induced genes

The transcription factor nuclear factor (NF)‐κB controls the expression of genes involved in inflammation, cell proliferation, apoptosis, and differentiation. Impaired regulation of NF‐κB has been associated with many diseases; thus, there is significant interest in therapeutic approaches based on modulation of this transcription factor. NF‐κB activity is controlled by numerous signaling molecules, many of which are potentially to be identified. Monocytes are principal effectors of the immune system, and monocyte adherence is the first step leading to their activation and differentiation. Adherence induces activation of NF‐κB, resulting in the induction of proinflammatory genes as well as anti‐inflammatory genes, which counterbalance and limit the intensity and duration of NF‐κB activation. Here, to identify novel mediators of NF‐κB signaling, we used the model of monocyte adherence to perform a systematic, genome‐wide survey of adherence‐induced genes. Having isolated mRNAs from nonadherent and adherent primary human monocytes, we constructed suppressive subtraction hybridization libraries containing cDNAs, which were differentially regulated by adherence. Of 366 identified differentially expressed genes, most were found to be up‐regulated by adherence. Having analyzed a subset of these genes, we found that the library was enriched with inhibitors of NF‐κB. Three of those (an orphan nuclear receptor NUR77, a guanosine 5′‐diphosphate/guanosine 5′‐triphosphate exchange factor RABEX5, and a PRK1‐associated protein AWP1) were particularly potent inhibitors of NF‐κB activation. Thus, the collection of monocyte adherence‐regulated genes represents a rich source for the identification of novel components of the machinery that controls NF‐κB activation.

CD14 mediates the innate immune responses to arthritopathogenic peptidoglycan-polysaccharide complexes of Gram-positive bacterial cell walls

Bacterial infections play an important role in the multifactorial etiology of rheumatoid arthritis. The arthropathic properties of Gram-positive bacteria have been associated with peptidoglycan–polysaccharide complexes (PG-PS), which are major structural components of bacterial cell walls. There is little agreement as to the identity of cellular receptors that mediate innate immune responses to PG-PS. A glycosylphosphatidylinositol-linked cell surface protein, CD14, the lipopolysaccharide receptor, has been proposed as a PG-PS receptor, but contradictory data have been reported. Here, we examined the inflammatory and pathogenic responses to PG-PS in CD14 knockout mice in order to examine the role for CD14 in PG-PS-induced signaling. We found that PG-PS-induced responses in vitro, including transient increase in intracellular calcium, activation of nuclear factor-κB, and secretion of the cytokines tumor necrosis factor-α and interleukin-6, were all strongly inhibited in CD14 knockout macrophages. In vivo, the incidence and severity of PG-PS induced acute polyarthritis were significantly reduced in CD14 knockout mice as compared with their wild-type counterparts. Consistent with these findings, CD14 knockout mice had significantly inhibited inflammatory cell infiltration and synovial hyperplasia, and reduced expression of inflammatory cytokines in PG-PS arthritic joints. These results support an essential role for CD14 in the innate immune responses to PG-PS and indicate an important role for CD14 in PG-PS induced arthropathy.