Oxidized DNA fragments exit mitochondria via mPTP- and VDAC-dependent channels to activate NLRP3 inflammasome and interferon signaling.

Mitochondrial DNA (mtDNA) escaping stressed mitochondria provokes inflammation via cGAS-STING pathway activation and, when oxidized (Ox-mtDNA), it binds cytosolic NLRP3, thereby triggering inflammasome activation. However, it is unknown how and in which form Ox-mtDNA exits stressed mitochondria in non-apoptotic macrophages. We found that diverse NLRP3 inflammasome activators rapidly stimulated uniporter-mediated calcium uptake to open mitochondrial permeability transition pores (mPTP) and trigger VDAC oligomerization. This occurred independently of mtDNA or reactive oxygen species, which induce Ox-mtDNA generation. Within mitochondria, Ox-mtDNA was either repaired by DNA glycosylase OGG1 or cleaved by the endonuclease FEN1 to 500-650 bp fragments that exited mitochondria via mPTP- and VDAC-dependent channels to initiate cytosolic NLRP3 inflammasome activation. Ox-mtDNA fragments also activated cGAS-STING signaling and gave rise to pro-inflammatory extracellular DNA. Understanding this process will advance the development of potential treatments for chronic inflammatory diseases, exemplified by FEN1 inhibitors that suppressed interleukin-1ß (IL-1ß) production and mtDNA release in mice.

Autophagy, Inflammation, and Immunity: A Troika Governing Cancer and Its Treatment.

Autophagy, a cellular waste disposal process, has well-established tumor-suppressive properties. New studies indicate that, in addition to its cell-autonomous anti-tumorigenic functions, autophagy inhibits cancer development by orchestrating inflammation and immunity. While attenuating tumor-promoting inflammation, autophagy enhances the processing and presentation of tumor antigens and thereby stimulates anti-tumor immunity. Although cancer cells can escape immunosurveillance by tuning down autophagy, certain chemotherapeutic agents with immunogenic properties may enhance anti-tumor immunity by inducing autophagic cell death. Understanding the intricate and complex relationships within this troika and how they are affected by autophagy enhancing drugs should improve the efficacy of cancer immunotherapy.

NF-κB Restricts Inflammasome Activation via Elimination of Damaged Mitochondria.

Nuclear factor κB (NF-κB), a key activator of inflammation, primes the NLRP3-inflammasome for activation by inducing pro-IL-1ß and NLRP3 expression. NF-κB, however, also prevents excessive inflammation and restrains NLRP3-inflammasome activation through a poorly defined mechanism. We now show that NF-κB exerts its anti-inflammatory activity by inducing delayed accumulation of the autophagy receptor p62/SQSTM1. External NLRP3-activating stimuli trigger a form of mitochondrial (mt) damage that is caspase-1- and NLRP3-independent and causes release of direct NLRP3-inflammasome activators, including mtDNA and mtROS. Damaged mitochondria undergo Parkin-dependent ubiquitin conjugation and are specifically recognized by p62, which induces their mitophagic clearance. Macrophage-specific p62 ablation causes pronounced accumulation of damaged mitochondria and excessive IL-1ß-dependent inflammation, enhancing macrophage death. Therefore, the "NF-κB-p62-mitophagy" pathway is a macrophage-intrinsic regulatory loop through which NF-κB restrains its own inflammation-promoting activity and orchestrates a self-limiting host response that maintains homeostasis and favors tissue repair.

Metformin inhibition of mitochondrial ATP and DNA synthesis abrogates NLRP3 inflammasome activation and pulmonary inflammation.

Acute respiratory distress syndrome (ARDS), an inflammatory condition with high mortality rates, is common in severe COVID-19, whose risk is reduced by metformin rather than other anti-diabetic medications. Detecting of inflammasome assembly in post-mortem COVID-19 lungs, we asked whether and how metformin inhibits inflammasome activation while exerting its anti-inflammatory effect. We show that metformin inhibited NLRP3 inflammasome activation and interleukin (IL)-1ß production in cultured and alveolar macrophages along with inflammasome-independent IL-6 secretion, thus attenuating lipopolysaccharide (LPS)- and SARS-CoV-2-induced ARDS. By targeting electron transport chain complex 1 and independently of AMP-activated protein kinase (AMPK) or NF-κB, metformin blocked LPS-induced and ATP-dependent mitochondrial (mt) DNA synthesis and generation of oxidized mtDNA, an NLRP3 ligand. Myeloid-specific ablation of LPS-induced cytidine monophosphate kinase 2 (CMPK2), which is rate limiting for mtDNA synthesis, reduced ARDS severity without a direct effect on IL-6. Thus, inhibition of ATP and mtDNA synthesis is sufficient for ARDS amelioration.

New mitochondrial DNA synthesis enables NLRP3 inflammasome activation.

Dysregulated NLRP3 inflammasome activity results in uncontrolled inflammation, which underlies many chronic diseases. Although mitochondrial damage is needed for the assembly and activation of the NLRP3 inflammasome, it is unclear how macrophages are able to respond to structurally diverse inflammasome-activating stimuli. Here we show that the synthesis of mitochondrial DNA (mtDNA), induced after the engagement of Toll-like receptors, is crucial for NLRP3 signalling. Toll-like receptors signal via the MyD88 and TRIF adaptors to trigger IRF1-dependent transcription of CMPK2, a rate-limiting enzyme that supplies deoxyribonucleotides for mtDNA synthesis. CMPK2-dependent mtDNA synthesis is necessary for the production of oxidized mtDNA fragments after exposure to NLRP3 activators. Cytosolic oxidized mtDNA associates with the NLRP3 inflammasome complex and is required for its activation. The dependence on CMPK2 catalytic activity provides opportunities for more effective control of NLRP3 inflammasome-associated diseases.

Interleukin-17 receptor a signaling in transformed enterocytes promotes early colorectal tumorigenesis.

Interleukin-17A (IL-17A) is a pro-inflammatory cytokine linked to rapid malignant progression of colorectal cancer (CRC) and therapy resistance. IL-17A exerts its pro-tumorigenic activity through its type A receptor (IL-17RA). However, IL-17RA is expressed in many cell types, including hematopoietic, fibroblastoid, and epithelial cells, in the tumor microenvironment, and how IL-17RA engagement promotes colonic tumorigenesis is unknown. Here we show that IL-17RA signals directly within transformed colonic epithelial cells (enterocytes) to promote early tumor development. IL-17RA engagement activates ERK, p38 MAPK, and NF-κB signaling and promotes the proliferation of tumorigenic enterocytes that just lost expression of the APC tumor suppressor. Although IL-17RA signaling also controls the production of IL-6, this mechanism makes only a partial contribution to colonic tumorigenesis. Combined treatment with chemotherapy, which induces IL-17A expression, and an IL-17A neutralizing antibody enhanced the therapeutic responsiveness of established colon tumors. These findings establish IL-17A and IL-17RA as therapeutic targets in colorectal cancer.

Cirmtuzumab blocks Wnt5a/ROR1 stimulation of NF-κB to repress autocrine STAT3 activation in chronic lymphocytic leukemia.

Coculture of nurse-like cells (NLCs) with chronic lymphocytic leukemia (CLL) cells induced leukemia cell phosphorylation of STAT3 (pSTAT3), which could be blocked by anti-Wnt5a antibodies or the anti-ROR1 monoclonal antibody, cirmtuzumab. Time-course studies revealed Wnt5a could induce activation of NF-κB within 30 minutes, but required more than 3 hours to induce pSTAT3. Culture of isolated CLL cells for 24 hours revealed Wnt5a-induced expression of interleukin 6 (IL-6), IL-8, CCL2, CCL3, CCL4, and CXCL1, which in turn could induce pSTAT3 in unstimulated CLL cells within 30 minutes. We found that Wnt5a could induce CLL cell expression of NF-κB target genes, including IL-6, and that this effect could be blocked by cirmtuzumab or drugs that inhibit NF-κB. Examination of CLL cells and plasma collected from patients treated with cirmtuzumab revealed reduced levels of phosphorylated p65 and diminished expression of NF-κB and STAT3 target genes in CLL cells, as well as lower plasma levels of IL-6, in the samples after therapy. Collectively, these studies indicate that Wnt5a/ROR1-dependent signaling contributes to CLL cell activation of NF-κB, which in turn causes autocrine IL-6-induced activation of pSTAT3. As such, this study demonstrates that cirmtuzumab can inhibit leukemia cell activation of both NF-κB and STAT3 in patients with CLL.

Immunosuppressive plasma cells impede T-cell-dependent immunogenic chemotherapy.

Cancer-associated genetic alterations induce expression of tumour antigens that can activate CD8(+) cytotoxic T cells (CTLs), but the microenvironment of established tumours promotes immune tolerance through poorly understood mechanisms. Recently developed therapeutics that overcome tolerogenic mechanisms activate tumour-directed CTLs and are effective in some human cancers. Immune mechanisms also affect treatment outcome, and certain chemotherapeutic drugs stimulate cancer-specific immune responses by inducing immunogenic cell death and other effector mechanisms. Our previous studies revealed that B cells recruited by the chemokine CXCL13 into prostate cancer tumours promote the progression of castrate-resistant prostate cancer by producing lymphotoxin, which activates an IκB kinase α (IKKα)-BMI1 module in prostate cancer stem cells. Because castrate-resistant prostate cancer is refractory to most therapies, we examined B cell involvement in the acquisition of chemotherapy resistance. Here we focus on oxaliplatin, an immunogenic chemotherapeutic agent that is effective in aggressive prostate cancer. We show that mouse B cells modulate the response to low-dose oxaliplatin, which promotes tumour-directed CTL activation by inducing immunogenic cell death. Three different mouse prostate cancer models were refractory to oxaliplatin unless genetically or pharmacologically depleted of B cells. The crucial immunosuppressive B cells are plasmocytes that express IgA, interleukin (IL)-10 and programmed death ligand 1 (PD-L1), the appearance of which depends on TGFß receptor signalling. Elimination of these cells, which also infiltrate human-therapy-resistant prostate cancer, allows CTL-dependent eradication of oxaliplatin-treated tumours.

Hexokinase 2 as a novel selective metabolic target for rheumatoid arthritis.

OBJECTIVES: Recent studies indicate that glucose metabolism is altered in rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS). Hexokinases (HKs) catalyse the first step in glucose metabolism, and HK2 constitutes the principal HK inducible isoform. We hypothesise that HK2 contributes to the synovial lining hypertrophy and plays a critical role in bone and cartilage damage. METHODS: HK1 and HK2 expression were determined in RA and osteoarthritis (OA) synovial tissue by immunohistochemistry. RA FLS were transfected with either HK1 or HK2 siRNA, or infected with either adenovirus (ad)-GFP, ad-HK1 or ad-HK2. FLS migration and invasion were assessed. To study the role of HK2 in vivo, 108 particles of ad-HK2 or ad-GFP were injected into the knee of wild-type mice. K/BxN serum transfer arthritis was induced in HK2F/F mice harbouring Col1a1-Cre (HK2Col1), to delete HK2 in non-haematopoietic cells. RESULTS: HK2 is particular of RA histopathology (9/9 RA; 1/8 OA) and colocalises with FLS markers. Silencing HK2 in RA FLS resulted in a less invasive and migratory phenotype. Consistently, overexpression of HK2 resulted in an increased ability to migrate and invade. It also increased extracellular lactate production. Intra-articular injection of ad-HK2 in normal knees dramatically increased synovial lining thickness, FLS activation and proliferation. HK2 was highly expressed in the synovial lining after K/BxN serum transfer arthritis. HK2Col1 mice significantly showed decreased arthritis severity, bone and cartilage damage. CONCLUSION: HK2 is specifically expressed in RA synovial lining and regulates FLS aggressive functions. HK2 might be an attractive selective metabolic target safer than global glycolysis for RA treatment.

Autophagy, NLRP3 inflammasome and auto-inflammatory/immune diseases.

Loss of homeostasis, as a result of pathogen invasion or self imbalance, causes tissue damage and inflammation. In addition to its well-established role in promoting clearance of pathogens or cell corpses, inflammation is also key to drive tissue repair and regeneration. Conserved from flies to humans, a transient, well-balanced inflammatory response is critical for restoration of tissue homeostasis after damage. The absence of such a response can result in failure of tissue repair, leading to the development of devastating immunopathologies and degenerative diseases. Studies in the past decade collectively suggest that a malfunction of NLRP3 inflammasome, a key tissue damage sensor, is a dominant driver of various autoinflammatory and autoimmune diseases, including gout, rheumatoid arthritis, and lupus. It is therefore crucial to understand the biology and regulation of NLRP3 inflammasome and determine its affect in the context of various diseases. Of note, various studies suggest that autophagy, a cellular waste removal and rejuvenation process, serves an important role as a macrophage-intrinsic negative regulator of NLRP3 inflammasome. Here, we review recent advances in understanding how autophagy regulates NLRP3 inflammasome activity and discuss the implications of this regulation on the pathogenesis of autoinflammatory and autoimmune diseases.

Integrin-linked kinase plays a key role in the regulation of angiotensin II-induced renal inflammation.

ILK (integrin-linked kinase) is an intracellular serine/threonine kinase involved in cell-matrix interactions. ILK dysregulation has been described in chronic renal disease and modulates podocyte function and fibrosis, whereas data about its role in inflammation are scarce. AngII (angiotensin II) is a pro-inflammatory cytokine that promotes renal inflammation. AngII blockers are renoprotective and down-regulate ILK in experimental kidney disease, but the involvement of ILK in the actions of AngII in the kidney has not been addressed. Therefore we have investigated whether ILK signalling modulates the kidney response to systemic AngII infusion in wild-type and ILK-conditional knockout mice. In wild-type mice, AngII induced an inflammatory response, characterized by infiltration of monocytes/macrophages and lymphocytes, and up-regulation of pro-inflammatory factors (chemokines, adhesion molecules and cytokines). AngII activated several intracellular signalling mechanisms, such as the NF-κB (nuclear factor κB) transcription factor, Akt and production of ROS (reactive oxygen species). All these responses were prevented in AngII-infused ILK-deficient mice. In vitro studies characterized further the mechanisms regulating the inflammatory response modulated by ILK. In cultured tubular epithelial cells ILK blockade, by siRNA, inhibited AngII-induced NF-κB subunit p65 phosphorylation and its nuclear translocation. Moreover, ILK gene silencing prevented NF-κB-related pro-inflammatory gene up-regulation. The results of the present study demonstrate that ILK plays a key role in the regulation of renal inflammation by modulating the canonical NF-κB pathway, and suggest a potential therapeutic target for inflammatory renal diseases.

The C-terminal module IV of connective tissue growth factor is a novel immune modulator of the Th17 response.

Connective tissue growth factor (CTGF/CCN2) is a matricellular protein susceptible to proteolytic degradation. CCN2 levels have been suggested as a potential risk biomarker in several chronic diseases. In body fluids, CCN2 full-length and its degradation fragments can be found; however, their in vivo effects are far from being elucidated. CCN2 was described as a profibrotic mediator, but this concept is changing to a proinflammatory cytokine. In vitro, CCN2 full-length and its C-terminal module IV (CCN2(IV)) exert proinflammatory properties. Emerging evidence suggest that Th17 cells, and its effector cytokine IL-17A, participate in chronic inflammatory diseases. Our aim was to explore whether CCN2(IV) could regulate the Th17 response. In vitro, stimulation of human naive CD4+ T lymphocytes with CCN2(IV) resulted in differentiation to Th17 phenotype. The in vivo effects of CCN2(IV) were studied in C57BL/6 mice. Intraperitoneal administration of recombinant CCN2(IV) did not change serum IL-17A levels, but caused an activation of the Th17 response in the kidney, characterized by interstitial infiltration of Th17 (IL17A+/CD4+) cells and upregulation of proinflammatory mediators. In CCN2(IV)-injected mice, elevated renal levels of Th17-related factors (IL-17A, IL-6, STAT3 and RORγt) were found, whereas Th1/Th2 cytokines or Treg-related factors (TGF-ß and Foxp-3) were not modified. Treatment with an anti-IL-17A neutralizing antibody diminished CCN2(IV)-induced renal inflammation. Our findings unveil that the C-terminal module of CCN2 induces the Th17 differentiation of human Th17 cells and causes a renal Th17 inflammatory response. Furthermore, these data bear out that IL-17A targeting is a promising tool for chronic inflammatory diseases, including renal pathologies.

Role of mitochondria-bound HK2 in rheumatoid arthritis fibroblast-like synoviocytes.

Background: Glucose metabolism, specifically, hexokinase 2 (HK2), has a critical role in rheumatoid arthritis (RA) fibroblast-like synoviocyte (FLS) phenotype. HK2 localizes not only in the cytosol but also in the mitochondria, where it protects mitochondria against stress. We hypothesize that mitochondria-bound HK2 is a key regulator of RA FLS phenotype. Methods: HK2 localization was evaluated by confocal microscopy after FLS stimulation. RA FLSs were infected with Green fluorescent protein (GFP), full-length (FL)-HK2, or HK2 lacking its mitochondrial binding motif (HK2ΔN) expressing adenovirus (Ad). RA FLS was also incubated with methyl jasmonate (MJ; 2.5 mM), tofacitinib (1 µM), or methotrexate (1 µM). RA FLS was tested for migration and invasion and gene expression. Gene associations with HK2 expression were identified by examining single-cell RNA sequencing (scRNA-seq) data from murine models of arthritis. Mice were injected with K/BxN serum and given MJ. Ad-FLHK2 or Ad-HK2ΔN was injected into the knee of wild-type mice. Results: Cobalt chloride (CoCl2) and platelet-derived growth factor (PDGF) stimulation induced HK2 mitochondrial translocation. Overexpression of the HK2 mutant and MJ incubation reversed the invasive and migrative phenotype induced by FL-HK2 after PDGF stimulation, and MJ also decreased the expression of C-X-C Motif Chemokine Ligand 1 (CXCL1) and Collagen Type I Alpha 1 Chain (COL1A1). Of interest, tofacitinib but not methotrexate had an effect on HK2 dissociation from the mitochondria. In murine models, MJ treatment significantly decreased arthritis severity, whereas HK2FL was able to induce synovial hypertrophy as opposed to HK2ΔN. Conclusion: Our results suggest that mitochondrial HK2 regulates the aggressive phenotype of RA FLS. New therapeutic approaches to dissociate HK2 from mitochondria offer a safer approach than global glycolysis inhibition.

HSP27/HSPB1 as an adaptive podocyte antiapoptotic protein activated by high glucose and angiotensin II.

Apoptosis is a driving force of diabetic end-organ damage, including diabetic nephropathy (DN). However, the mechanisms that modulate diabetes-induced cell death are not fully understood. Heat shock protein 27 (HSP27/HSPB1) is a cell stress protein that regulates apoptosis in extrarenal cells and is expressed by podocytes exposed to toxins causing nephrotic syndrome. We investigated the regulation of HSPB1 expression and its function in podocytes exposed to factors contributing to DN, such as high glucose and angiotensin (Ang) II. HSPB1 expression was assessed in renal biopsies from patients with DN, minimal change disease or focal segmental glomerulosclerosis (FSGS), in a rat model of diabetes induced by streptozotocin (STZ) and in Ang II-infused rats. The regulation of HSPB1 was studied in cultured human podocytes and the function of HSPB1 expressed in response to pathophysiologically relevant stimuli was explored by short interfering RNA knockdown. Total kidney HSPB1 mRNA and protein expression was increased in rats with STZ-induced diabetes and in rats infused with Ang II. Upregulation of HSPB1 protein was confirmed in isolated diabetic glomeruli. Immunohistochemistry showed increased glomerular expression of HSPB1 in both models and localized glomerular HSPB1 to podocytes. HSPB1 protein was increased in glomerular podocytes from patients with DN or FSGS. In cultured human podocytes HSPB1 mRNA and protein expression was upregulated by high glucose concentrations and Ang II. High glucose, but not Ang II, promoted podocyte apoptosis. HSPB1 short interfering RNA (siRNA) targeting increased apoptosis in a high-glucose milieu and sensitized to Ang II or TGFß1-induced apoptosis by promoting caspase activation. In conclusion, both high glucose and Ang II contribute to HSPB1 upregulation. HSPB1 upregulation allows podocytes to better withstand an adverse high-glucose or Ang II-rich environment, such as can be found in DN.

Synovial inflammation in osteoarthritis progression.

Osteoarthritis (OA) is a progressive degenerative disease resulting in joint deterioration. Synovial inflammation is present in the OA joint and has been associated with radiographic and pain progression. Several OA risk factors, including ageing, obesity, trauma and mechanical loading, play a role in OA pathogenesis, likely by modifying synovial biology. In addition, other factors, such as mitochondrial dysfunction, damage-associated molecular patterns, cytokines, metabolites and crystals in the synovium, activate synovial cells and mediate synovial inflammation. An understanding of the activated pathways that are involved in OA-related synovial inflammation could form the basis for the stratification of patients and the development of novel therapeutics. This Review focuses on the biology of the OA synovium, how the cells residing in or recruited to the synovium interact with each other, how they become activated, how they contribute to OA progression and their interplay with other joint structures.

Monosodium urate crystals regulate a unique JNK-dependent macrophage metabolic and inflammatory response.

Monosodium urate crystals (MSUc) induce inflammation in vivo without prior priming, raising the possibility of an initial cell-autonomous phase. Here, using genome-wide transcriptomic analysis and biochemical assays, we demonstrate that MSUc alone induce a metabolic-inflammatory transcriptional program in non-primed human and murine macrophages that is markedly distinct to that induced by LPS. Genes uniquely upregulated in response to MSUc belong to lipid and amino acid metabolism, glycolysis, and SLC transporters. This upregulation leads to a metabolic rewiring in sera from individuals and mice with acute gouty arthritis. Mechanistically, the initiating inflammatory-metabolic changes in acute gout flares are regulated through a persistent expression and increased binding of JUN to the promoter of target genes through JNK signaling-but not P38-in a process that is different than after LPS stimulation and independent of inflammasome activation. Finally, pharmacological JNK inhibition limits MSUc-induced inflammation in animal models of acute gouty inflammation.

Epigenetic Regulation of Nutrient Transporters in Rheumatoid Arthritis Fibroblast-like Synoviocytes.

OBJECTIVE: Since previous studies indicate that metabolism is altered in rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS), we undertook this study to determine if changes in the genome-wide chromatin and DNA states in genes associated with nutrient transporters could help to identify activated metabolic pathways in RA FLS. METHODS: Data from a previous comprehensive epigenomic study in FLS were analyzed to identify differences in genome-wide states and gene transcription between RA and osteoarthritis. We utilized the single nearest genes to regions of interest for pathway analyses. Homer promoter analysis was used to identify enriched motifs for transcription factors. The role of solute carrier transporters and glutamine metabolism dependence in RA FLS was determined by small interfacing RNA knockdown, functional assays, and incubation with CB-839, a glutaminase inhibitor. We performed 1 H nuclear magnetic resonance to quantify metabolites. RESULTS: The unbiased pathway analysis demonstrated that solute carrier-mediated transmembrane transport was one pathway associated with differences in at least 4 genome-wide states or gene transcription. Thirty-four transporters of amino acids and other nutrients were associated with a change in at least 4 epigenetic marks. Functional assays revealed that solute carrier family 4 member 4 (SLC4A4) was critical for invasion, and glutamine was sufficient as an alternate source of energy to glucose. Experiments with CB-839 demonstrated decreased RA FLS invasion and proliferation. Finally, we found enrichment of motifs for c-Myc in several nutrient transporters. CONCLUSION: Our findings demonstrate that changes in the epigenetic landscape of genes are related to nutrient transporters, and metabolic pathways can be used to identify RA-specific targets, including critical solute carrier transporters, enzymes, and transcription factors, to develop novel therapeutic agents.

Lights and shadows of proteomic technologies for the study of protein species including isoforms, splicing variants and protein post-translational modifications.

Recent reviews pinpointed the enormous diversity of proteins found in living organisms, especially in higher eukaryotes. Protein diversity is driven through three main processes: first, at deoxyribonucleic acid (DNA) level (i.e. gene polymorphisms), second, at precursor messenger ribonucleic acid (pre-mRNA) or messenger ribonucleic acid (mRNA) level (i.e. alternative splicing, also termed as differential splicing) and, finally, at the protein level (i.e. PTM). Current proteomic technologies allow the identification, characterization and quantitation of up to several thousands of proteins in a single experiment. Nevertheless, the identification and characterization of protein species using these technologies are still hampered. Here, we review the use of the terms "protein species" and "protein isoform." We evidence that the appropriate selection of the database used for searches can impede or facilitate the identification of protein species. We also describe examples where protein identification search engines systematically fail in the attribution of protein species. We briefly review the characterization of protein species using proteomic technologies including gel-based, gel-free, bottom-up and top-down analysis and discuss their limitations. As an example, we discuss the theoretical characterization of the two human choline kinase species, α-1 and α-2, sharing the same catalytic activity but generated by alternative splicing on CHKA gene.

Autophagy-mitophagy induction attenuates cardiovascular inflammation in a murine model of Kawasaki disease vasculitis.

Kawasaki disease (KD) is the leading cause of acquired heart disease among children. Murine and human data suggest that the NLRP3-IL-1ß pathway is the main driver of KD pathophysiology. NLRP3 can be activated during defective autophagy/mitophagy. We used the Lactobacillus casei cell wall extract (LCWE) murine model of KD vasculitis to examine the role of autophagy/mitophagy on cardiovascular lesion development. LCWE-injected mice had impaired autophagy/mitophagy and increased levels of ROS in cardiovascular lesions, together with increased systemic 8-OHdG release. Enhanced autophagic flux significantly reduced cardiovascular lesions in LCWE-injected mice, whereas autophagy blockade increased inflammation. Vascular smooth muscle cell-specific deletion of Atg16l1 and global Parkin-/- significantly increased disease formation, supporting the importance of autophagy/mitophagy in this model. Ogg1-/- mice had significantly increased lesions with increased NLRP3 activity, whereas treatment with MitoQ reduced vascular tissue inflammation, ROS production, and systemic 8-OHdG release. Treatment with MN58b or Metformin (increasing AMPK and reducing ROS) resulted in decreased cardiovascular lesions. Our results demonstrate that impaired autophagy/mitophagy and ROS-dependent damage exacerbate the development of murine KD vasculitis. This pathway can be efficiently targeted to reduce disease severity. These findings enhance our understanding of KD pathogenesis and identify potentially novel therapeutic avenues for KD treatment.

CTGF promotes inflammatory cell infiltration of the renal interstitium by activating NF-kappaB.

Connective tissue growth factor (CTGF) is an important profibrotic factor in kidney diseases. Blockade of endogenous CTGF ameliorates experimental renal damage and inhibits synthesis of extracellular matrix in cultured renal cells. CTGF regulates several cellular responses, including adhesion, migration, proliferation, and synthesis of proinflammatory factors. Here, we investigated whether CTGF participates in the inflammatory process in the kidney by evaluating the nuclear factor-kappa B (NF-kappaB) pathway, a key signaling system that controls inflammation and immune responses. Systemic administration of CTGF to mice for 24 h induced marked infiltration of inflammatory cells in the renal interstitium (T lymphocytes and monocytes/macrophages) and led to elevated renal NF-kappaB activity. Administration of CTGF increased renal expression of chemokines (MCP-1 and RANTES) and cytokines (INF-gamma, IL-6, and IL-4) that recruit immune cells and promote inflammation. Treatment with a NF-kappaB inhibitor, parthenolide, inhibited CTGF-induced renal inflammatory responses, including the up-regulation of chemokines and cytokines. In cultured murine tubuloepithelial cells, CTGF rapidly activated the NF-kappaB pathway and the cascade of mitogen-activated protein kinases, demonstrating crosstalk between these signaling pathways. CTGF, via mitogen-activated protein kinase and NF-kappaB activation, increased proinflammatory gene expression. These data show that in addition to its profibrotic properties, CTGF contributes to the recruitment of inflammatory cells in the kidney by activating the NF-kappaB pathway.