RESUMO
Neutrophil-myeloperoxidase (MPO) is a heme-containing peroxidase which produces excess amounts of hypochlorous acid during inflammation. While pharmacological MPO inhibition mitigates all indices of experimental colitis, no studies have corroborated the role of MPO using knockout (KO) models. Therefore, we investigated MPO deficient mice in a murine model of colitis. Wild type (Wt) and MPO-deficient mice were treated with dextran sodium sulphate (DSS) in a chronic model of experimental colitis with three acute cycles of DSS-induced colitis over 63 days, emulating IBD relapse and remission cycles. Mice were immunologically profiled at the gut muscoa and the faecal microbiome was assessed via 16S rRNA amplicon sequencing. Contrary to previous pharmacological antagonist studies targeting MPO, MPO-deficient mice showed no protection from experimental colitis during cyclical DSS-challenge. We are the first to report drastic faecal microbiota shifts in MPO-deficient mice, showing a significantly different microbiome profile on Day 1 of treatment, with a similar shift and distinction on Day 29 (half-way point), via qualitative and quantitative descriptions of phylogenetic distances. Herein, we provide the first evidence of substantial microbiome shifts in MPO-deficiency, which may influence disease progression. Our findings have significant implications for the utility of MPO-KO mice in investigating disease models.
Assuntos
Colite , Sulfato de Dextrana , Modelos Animais de Doenças , Microbioma Gastrointestinal , Camundongos Knockout , Peroxidase , Animais , Peroxidase/metabolismo , Peroxidase/genética , Camundongos , Colite/microbiologia , Colite/induzido quimicamente , Colite/genética , Fezes/microbiologia , Deleção de Genes , RNA Ribossômico 16S/genética , Camundongos Endogâmicos C57BLRESUMO
Despite numerous screening tools for colorectal cancer (CRC), 25% of patients are diagnosed with advanced disease. Novel diagnostic technologies that are early, accurate, and rapid are imperative to assess the therapeutic efficacy of clinical drugs and identify new biomarkers of treatment response. Here Raman spectroscopy (RS) was used to track metabolic reprogramming in KRAS-mutant HCT116 and SW837 cells, and KRAS wild-type CC cells. RS combined with multivariate analysis methods distinguished nonresponsive, partially responsive, and responsive cells treated with cetuximab, a monoclonal antibody for EGFR inhibition, sotorasib, a clinically approved KRAS inhibitor, and various doses of trametinib, an inhibitor of the MAPK pathway. Cells treated with a combination of subtoxic doses of trametinib and BKM120, an inhibitor of the PI3K pathway, showed a synergistic response between the two pathways. Using a supervised machine learning regression model, we established a scoring methodology trained to a priori predict therapeutic response to new treatment combinations. RS metabolites were verified with mass spectrometry, and enrichment pathways were identified, including amino acid, purine, and nicotinate and nicotinamide metabolism that differentiated monotherapy from combination therapy. Our approach may ultimately be applicable to patient-derived primary cells and cultures of patient tumors to predict effective drugs for individualized care.
RESUMO
Ulcerative colitis is a condition characterised by the infiltration of leukocytes into the gastrointestinal wall. Leukocyte-MPO catalyses hypochlorous acid (HOCl) and hypothiocyanous acid (HOSCN) formation from chloride (Cl-) and thiocyanous (SCN-) anions, respectively. While HOCl indiscriminately oxidises biomolecules, HOSCN primarily targets low-molecular weight protein thiols. Oxidative damage mediated by HOSCN may be reversible, potentially decreasing MPO-associated host tissue destruction. This study investigated the effect of SCN- supplementation in a model of acute colitis. Female mice were supplemented dextran sodium sulphate (DSS, 3% w/v) in the presence of 10 mM Cl- or SCN- in drinking water ad libitum, or with salts (NaCl and NaSCN only) or water only (controls). Behavioural studies showed mice tolerated NaSCN and NaCl-treated water with water-seeking frequency. Ion-exchange chromatography showed increased fecal and plasma SCN- levels in thiocyanate supplemented mice; plasma SCN- reached similar fold-increase for smokers. Overall there was no difference in weight loss and clinical score, mucin levels, crypt integrity and extent of cellular infiltration between DSS/SCN- and DSS/Cl- groups. Neutrophil recruitment remained unchanged in DSS-treated mice, as assessed by fecal calprotectin levels. Total thiol and tyrosine phosphatase activity remained unchanged between DSS/Cl- and DSS/SCN- groups, however, colonic tissue showed a trend in decreased 3-chlorotyrosine (1.5-fold reduction, p < 0.051) and marked increase in colonic GCLC, the rate-limiting enzyme in glutathione synthesis. These data suggest that SCN- administration can modulate MPO activity towards a HOSCN-specific pathway, however, this does not alter the development of colitis within a DSS murine model.
Assuntos
Colite , Colo , Sulfato de Dextrana/toxicidade , Peroxidase/metabolismo , Tiocianatos/farmacologia , Animais , Colite/induzido quimicamente , Colite/tratamento farmacológico , Colite/enzimologia , Colite/patologia , Colo/enzimologia , Colo/patologia , Modelos Animais de Doenças , Feminino , CamundongosRESUMO
Thiocyanate (SCN-) is a pseudohalide anion omnipresent across mammals and is particularly concentrated in secretions within the oral cavity, digestive tract and airway. Thiocyanate can outcompete chlorine anions and other halides (F-, Br-, I-) as substrates for myeloperoxidase by undergoing two-electron oxidation with hydrogen peroxide. This forms their respective hypohalous acids (HOX where X- = halides) and in the case of thiocyanate, hypothiocyanous acid (HOSCN), which is also a bactericidal oxidative species involved in the regulation of commensal and pathogenic microflora. Disease may dysregulate redox processes and cause imbalances in the oxidative profile, where typically favoured oxidative species, such as hypochlorous acid (HOCl), result in an overabundance of chlorinated protein residues. As such, the pharmacological capacity of thiocyanate has been recently investigated for its ability to modulate myeloperoxidase activity for HOSCN, a less potent species relative to HOCl, although outcomes vary significantly across different disease models. To date, most studies have focused on therapeutic effects in respiratory and cardiovascular animal models. However, we note other conditions such as rheumatic arthritis where SCN- administration may worsen patient outcomes. Here, we discuss the pathophysiological role of SCN- in diseases where MPO is implicated.
Assuntos
Peroxidase/metabolismo , Febre Reumática/patologia , Tiocianatos/farmacologia , Animais , Humanos , Febre Reumática/tratamento farmacológico , Febre Reumática/enzimologiaRESUMO
Several diseases have been reported as affecting endangered wild sea turtle population worldwide, including spirorchiidiasis. This parasitic infection results in serious circulatory disorders in sea turtles, as well as tissue damage due to the presence of spirorchiids eggs. However, few reports of organs severely affected by tissue replacement caused by granulomatous inflammatory processes due to spirorchiidiasis in sea turtles are available. In this regard, this study describes massive lesions in 16 juvenile green turtles from southeastern Brazil presenting no other detectable diseases or injuries, associated to parasitic compression of air spaces, parasitic thyroid atrophy, parasitic encephalic compression and parasitic splenic lymphoid depletion. These rare injuries were categorized as extremely severe, affecting most spirorchiidiasis-infected organs. Spirorchiidiasis was, thus, noted herein as capable of causing a variety of lethal injuries to vital or extremely important organs in sea turtles. Spirorchiidiasis should, therefore, also be considered a potential cause of death in stranded green sea turtle monitoring efforts.
RESUMO
Inflammatory bowel disease (IBD) is a chronic condition characterised by leukocyte recruitment to the gut mucosa. Leukocyte myeloperoxidase (MPO) produces the two-electron oxidant hypochlorous acid (HOCl), damaging tissue and playing a role in cellular recruitment, thereby exacerbating gut injury. We tested whether the MPO-inhibitor, 4-Methoxy-TEMPO (MetT), ameliorates experimental IBD. Colitis was induced in C57BL/6 mice by 3% w/v dextran-sodium-sulfate (DSS) in drinking water ad libitum over 9-days with MetT (15â¯mg/kg; via i. p. injection) or vehicle control (10% v/v DMSO+90% v/v phosphate buffered saline) administered twice daily during DSS challenge. MetT attenuated body-weight loss (50%, pâ¯<â¯0.05, nâ¯=â¯6), improved clinical score (53%, pâ¯<â¯0.05, nâ¯=â¯6) and inhibited serum lipid peroxidation. Histopathological damage decreased markedly in MetT-treated mice, as judged by maintenance of crypt integrity, goblet cell density and decreased cellular infiltrate. Colonic Ly6C+, MPO-labelled cells and 3-chlorotyrosine (3-Cl-Tyr) decreased in MetT-treated mice, although biomarkers for nitrosative stress (3-nitro-tyrosine-tyrosine; 3-NO2-Tyr) and low-molecular weight thiol damage (assessed as glutathione sulfonamide; GSA) were unchanged. Interestingly, MetT did not significantly impact colonic IL-10 and IL-6 levels, suggesting a non-immunomodulatory pathway. Overall, MetT ameliorated the severity of experimental IBD, likely via a mechanism involving the modulation of MPO-mediated damage.
Assuntos
Colite/etiologia , Colite/patologia , Óxidos N-Cíclicos/farmacologia , Sulfato de Dextrana/efeitos adversos , Suscetibilidade a Doenças , Substâncias Protetoras/farmacologia , Animais , Biópsia , Colite/diagnóstico por imagem , Colite/tratamento farmacológico , Modelos Animais de Doenças , Imuno-Histoquímica , Doenças Inflamatórias Intestinais/etiologia , Doenças Inflamatórias Intestinais/metabolismo , Doenças Inflamatórias Intestinais/patologia , Camundongos , Imagem Óptica , Oxirredução , Estresse Oxidativo , FenótipoRESUMO
Chronic inflammatory bowel disease (IBD) is a condition with multifactorial pathophysiology. To date, there is no permanent cure and the disease is primarily managed by immunosuppressive drugs; long-term use promotes serious side effects including increased risk malignancies. The current study aimed to target neutrophil-myeloperoxidase, a key contributor to the pathogenesis of IBD, through the use of AZD3241that inhibits extracellular myeloperoxidase. Experimental colitis was induced in C57BL/6 male mice by 2% dextran sodium sulfate in drinking water ad libitum over 9 days. Mice received either normal drinking water and peanut butter (control), 2% DSS in drinking water and peanut butter or 2% DSS in drinking water and AZD3241 (30 mg/kg) dispersed in peanut butter daily for 9 days. Administered AZD3241 attenuated body weight loss (10% p<0.05) and improved clinical score (9 fold p<0.05; a score comprising the time-dependent assessment of stool consistency and extent of rectal bleeding), loss of colonic crypts (p<0.001), preserved surface epithelium (p<0.001) and enhanced expression of the transcription factor Nrf-2 (regulator of antioxidants) and enhanced expression of the downstream antioxidant response element haeoxygenase-1 (HO-1) in the colon tissue. Also, the concentration of fecal hemoglobin and the myeloperoxidase specific oxidative damage biomarker 3-chlorotyrosine in the colon were significantly decreased in the presence of AZD3241. This latter result was consistent with AZD3241 inhibiting MPO activity in vitro. Overall, AZD3241 ameliorated the course and severity of experimental colitis through ameliorating MPO derived tissue damage and could be considered a potential therapeutic option, subject to further validation in chronic IBD models.