Designed protein receptors hold diagnostic and therapeutic promise. We now report the design of five consensus leucine‐rich repeat proteins (CLRR4–8) based on the LRR domain of nucleotide‐binding ...oligomerization domain (NOD)‐like receptors involved in the innate immune system. The CLRRs bind muramyl dipeptide (MDP), a bacterial cell wall component, with micromolar affinity. The overall Kd app values ranged from 1.0 to 57 μM as measured by fluorescence quenching experiments. Biphasic fluorescence quenching curves were observed in all CLRRs, with higher affinity Kd1 values ranging from 0.04 to 4.5 μM, and lower affinity Kd2 values ranging from 3.1 to 227 μM. These biphasic binding curves, along with the docking studies of MDP binding to CLRR4, suggest that at least two MDPs bind to each protein. Previously, only single MDP binding was reported. This high‐capacity binding of MDP promises small, soluble, stable CLRR scaffolds as candidates for the future design of pathogen biosensors.
The Crosstalk between Nrf2 and Inflammasomes Hennig, Paulina; Garstkiewicz, Martha; Grossi, Serena ...
International journal of molecular sciences,
02/2018, Letnik:
19, Številka:
2
Journal Article
Recenzirano
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The Nrf2 (nuclear factor E2-related factor or nuclear factor (erythroid-derived 2)-like 2) transcription factor is a key player in cytoprotection and activated in stress conditions caused by reactive ...oxygen species (ROS) or electrophiles. Inflammasomes represent central regulators of inflammation. Upon detection of various stress factors, assembly of the inflamasome protein complex results in activation and secretion of proinflammatory cytokines. In addition, inflammasome activation causes pyroptosis, a lytic form of cell death, which supports inflammation. There is growing evidence of a crosstalk between the Nrf2 and inflammasome pathways at different levels. For example, Nrf2 activating compounds inhibit inflammasomes and consequently inflammation. This review summarizes what is known about the complex and predominantly antagonistic relationship of both stress-activated pathways.
In mammals, white adipose tissue (WAT) stores and releases lipids, whereas brown adipose tissue (BAT) oxidizes lipids to fuel thermogenesis. In obese individuals, WAT undergoes profound changes; it ...expands, becomes dysfunctional, and develops a low-grade inflammatory state. Importantly, BAT content and activity decline in obese subjects, mainly as a result of the conversion of brown adipocytes to white-like unilocular cells. Here, we show that BAT “whitening” is induced by multiple factors, including high ambient temperature, leptin receptor deficiency, β-adrenergic signaling impairment, and lipase deficiency, each of which is capable of inducing macrophage infiltration, brown adipocyte death, and crown-like structure (CLS) formation. Brown-to-white conversion and increased CLS formation were most marked in BAT from adipose triglyceride lipase (Atgl)-deficient mice, where, according to transmission electron microscopy, whitened brown adipocytes contained enlarged endoplasmic reticulum, cholesterol crystals, and some degenerating mitochondria, and were surrounded by an increased number of collagen fibrils. Gene expression analysis showed that BAT whitening in Atgl-deficient mice was associated to a strong inflammatory response and NLRP3 inflammasome activation. Altogether, the present findings suggest that converted enlarged brown adipocytes are highly prone to death, which, by promoting inflammation in whitened BAT, may contribute to the typical inflammatory state seen in obesity.
Non-alcoholic steatohepatitis (NASH) is a key step in the progression of non-alcoholic fatty liver disease (NAFLD), which causes serious health problems worldwide. The nucleotide-binding ...oligomerization domain, leucine-rich repeat-containing receptor-containing pyrin domain 3 (NLRP3) inflammasome and pyroptosis play crucial roles in the progression of NASH. Our team has provided clinical evidence of the effects of glucagon-like peptide-1 (GLP-1) on the improvement in liver function and histological resolution of NAFLD. Preliminary work has demonstrated that GLP-1 inhibited NLRP3 inflammasome activation in a mouse model of NAFLD. We further explored the potential molecular mechanisms underlying the anti-inflammatory effect of liraglutide, a long-acting GLP-1 analog, in the treatment of NASH. We established a HepG2 cell model of NASH using double stimulation with palmitic acid and lipopolysaccharide to assess NLRP3 inflammasome and pyroptotic cell activity and to evaluate mitochondrial function and mitophagy. Liraglutide reduced lipid accumulation, inhibited NLRP3 inflammasome and pyroptosis activation, attenuated mitochondrial dysfunction and reactive oxygen species generation, augmented mitophagy in hepatocytes. Mitophagy inhibition with 3-methyladenine/PINK1-directed siRNA weakened the liraglutide-mediated suppression of inflammatory injury. We propose that liraglutide suppresses NLRP3 inflammasome-induced hepatocyte pyroptosis via mitophagy to slow the progression of NASH.
The liver is the primary site of inflammation caused by bacterial endotoxins in sepsis, and septic acute liver injury (SALI) is usually associated with poor outcomes in sepsis. Forsythiaside A (FTA), ...an active constituent of Forsythia suspensa, has been reported to have anti-inflammatory properties, antioxidant properties, and protective properties against neuroinflammation, sepsis, and edema.Therefore, the purpose of the present study was to examine FTA's potential effects on lipopolysaccharide (LPS)-induced SALI in mice.Our results indicated that pretreatment with FTA significantly attenuated aspartate aminotransferase (AST) and aminoleucine transferase (ALT) levels in plasma, ameliorated histopathological damage, inhibited hepatocyte apoptosis, diminished the expression of tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6 in the liver from mice exposed to LPS. Furthermore, our data showed that the administration of LPS resulted in robust endoplasmic reticulum (ER) stress response, as evidenced by GRP78 upregulation, p-PERK activation, elF2α phosphorylation, and ATF4 and CHOP overexpression in the liver. This, in turn, led to nucleotide-binding oligomerization domain-like receptor pyrin domain containing 3 (NLRP3) inflammasome activation, including the cleavage of caspase-1, secretion of IL-1β, and pyroptotic cell death in the liver specimens. Importantly, the ER stress response induced by the LPS challenge was blocked by FTA administration. Correspondingly, NLRP3 inflammasome activation was significantly ameliorated by the pretreatment with FTA. Thus, we demonstrated that FTA pretreatment could protect mice from LPS-induced SALI, and its protective effects were possibly mediated by inhibiting ER stress response and subsequent NLRP3 inflammasome activation.
Background and Objective
Diabetes influences the frequency and development of periodontitis. Inflammation of human periodontal ligament cells (HPDLCs) participates in this pathologic process. Hence, ...this study aims to explore whether advanced glycation end products (AGEs), by‐products of diabetes, could exaggerate inflammation induced by muramyl dipeptide (MDP) in HPDLCs, and whether nucleotide‐binding oligomerization domain‐like receptors (NLRs) signaling pathway was involved.
Material and Methods
Human periodontal ligament cells were pre‐treated with 100 μg/mL AGEs for 24 hours and stimulated with 10 μg/mL MDP for 24 hours. IL‐6, IL‐1β, and RAGE were detected, and the activation of NF‐κB signaling pathway was observed. The expression of NLRs was evaluated with or without silencing RAGE or blocking NF‐κB pathway under AGEs stimulation. Statistical analyses were performed by using independent sample t test.
Results
Advanced glycation end products induced significant increase of inflammatory cytokines in HPDLCs (P < 0.05). Results of western blot (WB) showed that after 45 minutes stimulation of AGEs, p‐p65/p65 ratio peaked; AGEs promoted the expression of NLRP1, NLRP3, and apoptosis‐associated speck‐like protein containing a CARD (ASC). After silencing RAGE or blocking NF‐κB pathway, the up‐regulation of NLRs protein caused by AGEs was attenuated. Additionally, AGEs pre‐treatment could enhance the inflammatory response of MDP and the expression of NLRs, which were demonstrated by more expression of IL‐6, IL‐1β, NOD2, NLRP1, NLRP3, and ASC.
Conclusion
Advanced glycation end products induced inflammatory response in HPDLCs via NLRP1‐inflammasome and NLRP3‐inflammasome activation in which NF‐κB signal pathway was involved. Besides, AGEs promoted the inflammatory response of MDP via NOD2, NLRP1‐inflammasome, and NLRP3‐inflammasome.
Mitochondria in innate immune signaling Banoth, Balaji; Cassel, Suzanne L
Translational research : the journal of laboratory and clinical medicine,
12/2018, Letnik:
202
Journal Article
Recenzirano
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Mitochondria are functionally versatile organelles. In addition to their conventional role of meeting the cell's energy requirements, mitochondria also actively regulate innate immune responses ...against infectious and sterile insults. Components of mitochondria, when released or exposed in response to dysfunction or damage, can be directly recognized by receptors of the innate immune system and trigger an immune response. In addition, despite initiation that may be independent from mitochondria, numerous innate immune responses are still subject to mitochondrial regulation as discrete steps of their signaling cascades occur on mitochondria or require mitochondrial components. Finally, mitochondrial metabolites and the metabolic state of the mitochondria within an innate immune cell modulate the precise immune response and shape the direction and character of that cell's response to stimuli. Together, these pathways result in a nuanced and very specific regulation of innate immune responses by mitochondria.
The NOD-, LRR-, and pyrin domain-containing protein 3 (NLRP3) inflammasome is the platform for IL-1β maturation, aimed at mediating a rapid immune response against danger signals which must be ...tightly regulated. Insulin is well known as the critical hormone in the maintenance of glucose in physiologic response. Previous studies have proved insulin has the anti-inflammatory effect but the molecular mechanism of immunomodulation provided by insulin is not clear so far. Here we investigated whether insulin reduces inflammation by regulating the NLRP3 inflammasome. In the present study, we used LPS and ATP to induce the intracellular formation of the NLRP3 inflammasome. Insulin inhibited the secretion of IL-1β by preventing the assembly of the ASC in THP-1 cells and human CD14
monocyte-derived macrophages. The phosphorylation status of Syk, p38 mitogen-activated protein kinase (MAPK) and ASC were altered by insulin. These effects were attenuated in THP-1 cells transfected with small interfering RNA targeting insulin receptors.
, administration of glucose-insulin-potassium reduced serum IL-1β level, intestinal ASC speck formation, local macrophage infiltration and alleviated intestinal injury in mice exposed to LPS. Insulin may play an immunomodulatory role in anti-inflammation by regulating the NLRP3 inflammasome.
Type 2 diabetes mellitus (T2DM) may affect the oral microbial community, exacerbating periodontal inflammation; however, its pathogenic mechanisms remain unclear. As nucleotide-binding ...oligomerization domain 2 (NOD2) plays a crucial role in the activation during periodontitis (PD), it is hypothesized that changes in the oral microbial community due to diabetes enhance periodontal inflammation through the activation of NOD2.
We collected subgingival plaque from 180 subjects who were categorized into two groups based on the presence or absence of T2DM. The composition of oral microbiota was detected by 16S rRNA high-throughput sequencing. In animal models of PD with or without T2DM, we assessed alveolar bone resorption by micro-computerized tomography and used immunohistochemistry to detect NOD2 expression in alveolar bone. Primary osteoblasts were cultured in osteogenic induction medium with high or normal glucose and treated with inactivated bacteria. After 24 h of inactivated bacteria intervention, the osteogenic differentiation ability was detected by alkaline phosphatase (ALP) staining, and the expressions of NOD2 and interleukin-12 (IL-6) were detected by western blot.
The relative abundance of Parvimonas and Filifactor in the T2DM group was increased compared to the group without T2DM. In animal models, alveolar bone mass was decreased in PD, particularly in T2DM with PD (DMPD) group, compared to controls. Immunohistochemistry revealed NOD2 in osteoblasts from the alveolar bone in both the PD group and DMPD group, especially in the DMPD group. In vitro, intervention with inactivated Parvimonas significantly reduced ALP secretion of primary osteoblasts in high glucose medium, accompanied by increased expression of NOD2 and IL-6.
The results suggest that T2DM leading to PD may be associated with the activation of NOD2 by Parvimonas.
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