RESUMEN

The innate immune system is the first line of host defense against infection by pathogenic microorganisms, among which macrophages are important innate immune cells. Macrophages are widely distributed throughout the body and recognize and eliminate viruses through pattern recognition receptors (PRRs) to sense pathogen-associated molecular patterns (PAMPs). In the present chapter, we provide detailed protocols for vesicular stomatitis virus (VSV) amplification, VSV titer detection, isolation of mouse primary peritoneal macrophages, in vitro and in vivo VSV infection, detection of interferon-beta (IFN-ß) expression, and lung injury. These protocols provide efficient and typical methods to evaluate virus-induced innate immunity in vitro and in vivo.

Asunto(s)

Inmunidad Innata , Interferón beta , Macrófagos Peritoneales , Vesiculovirus , Animales , Ratones , Macrófagos Peritoneales/inmunología , Macrófagos Peritoneales/virología , Macrófagos Peritoneales/metabolismo , Interferón beta/inmunología , Interferón beta/metabolismo , Interferón beta/genética , Vesiculovirus/inmunología , Vesiculovirus/genética , Estomatitis Vesicular/inmunología , Estomatitis Vesicular/virología , Virus de la Estomatitis Vesicular Indiana/inmunología , Receptores de Reconocimiento de Patrones/metabolismo , Receptores de Reconocimiento de Patrones/inmunología

RESUMEN

Endometriosis is a chronic inflammatory disease that causes debilitating pelvic pain in women. Macrophages are considered to be key players in promoting disease progression, as abundant macrophages are present in ectopic lesions and elevated in the peritoneum. In the present study, we examined the role of GATA6+ peritoneal macrophages on endometriosis-associated hyperalgesia using mice with a specific myeloid deficiency of GATA6. Lesion induction induced the disappearance of TIM4hi MHCIIlo residential macrophages and the influx of increased Ly6C+ monocytes and TIM4lo MHCIIhi macrophages. The recruitment of MHCIIhi inflammatory macrophages was extensive in Mac Gata6 KO mice due to the severe disappearance of TIM4hi MHCIIlo residential macrophages. Ki67 expression confirmed GATA6-dependent proliferative ability, showing different proliferative phenotypes of TIM4+ residential macrophages in Gata6f/f and Mac Gata6 KO mice. Peritoneal proinflammatory cytokines were elevated after lesion induction. When cytokine levels were compared between Gata6f/f and Mac Gata6 KO mice, TNFα at day 21 in Gata6f/f mice was higher than in Mac Gata6 KO mice. Lesion induction increased both abdominal and hind paw sensitivities. Gata6f/f mice tended to show higher sensitivity in the abdomen after day 21. Elevated expression of TRPV1 and CGRP was observed in the dorsal root ganglia after ELL induction in Gata6f/f mice until days 21 and 42, respectively. These results support that peritoneal GATA6+ macrophages are involved in the recruitment and reprogramming of monocyte-derived macrophages. The extensive recruitment of monocyte-derived macrophages in Mac Gata6 KO mice might protect against inflammatory stimuli during the resolution phase, whereas GATA6 deficiency did not affect lesion initiation and establishment at the acute phase of inflammation. GATA6+ residential macrophages act to sustain local inflammation in the peritoneum and sensitivities in the neurons, reflecting endometriosis-associated hyperalgesia.

Asunto(s)

Endometriosis , Factor de Transcripción GATA6 , Macrófagos Peritoneales , Animales , Femenino , Ratones , Citocinas/metabolismo , Modelos Animales de Enfermedad , Endometriosis/inmunología , Endometriosis/patología , Endometriosis/metabolismo , Ganglios Espinales/metabolismo , Ganglios Espinales/inmunología , Factor de Transcripción GATA6/metabolismo , Factor de Transcripción GATA6/genética , Hiperalgesia/etiología , Hiperalgesia/metabolismo , Hiperalgesia/inmunología , Macrófagos Peritoneales/inmunología , Macrófagos Peritoneales/metabolismo , Ratones Endogámicos C57BL , Ratones Noqueados , Peritoneo/patología , Peritoneo/inmunología , Canales Catiónicos TRPV/metabolismo , Canales Catiónicos TRPV/genética

RESUMEN

Macrophages polarize into alternatively activated M2 macrophages through interleukin (IL)-4, and they express high levels of arginase-1, which promotes anti-inflammatory responses. Several studies have confirmed the anti-inflammatory effects of cyclin-dependent kinase (CDK) 8/19 inhibition, and hence, numerous CDK8/19 inhibitors, such as BRD6989, have been developed. However, the effects of CDK8/19 inhibitors on arginase-1 expression in macrophages have not yet been elucidated. This study investigated the effects of CDK8/19 inhibitor on arginase-1 expression in IL-4-activated macrophages. The results showed that BRD6989 increased arginase-1 expression transcriptionally in murine peritoneal macrophages and the murine macrophage cell line RAW264.7 in an IL-4-dependent manner. In addition, the results indicated that BRD6989 enhances signal transducer and activator of transcription (STAT) 6 phosphorylation. Meanwhile, BRD6989 exhibited the capability to activate p38 mitogen-activated protein kinase (MAPK） even in the absence of IL-4 stimulation. Moreover, we observed that a p38 MAPK inhibitor suppressed the BRD6989-induced increase in arginase-1 expression. Besides, BRD6989 increased the surface expression of CD206, an M2 macrophage marker. Thus, this study demonstrated for the first time that CDK8/19 inhibition increases arginase-1 expression, suggesting that this mechanism involves the activation of STAT6 and p38 MAPK. This finding implies that CDK8/19 inhibition may facilitate the production of anti-inflammatory M2 macrophages.

Asunto(s)

Arginasa , Quinasa 8 Dependiente de Ciclina , Quinasas Ciclina-Dependientes , Interleucina-4 , Factor de Transcripción STAT6 , Proteínas Quinasas p38 Activadas por Mitógenos , Animales , Arginasa/metabolismo , Arginasa/antagonistas & inhibidores , Factor de Transcripción STAT6/metabolismo , Ratones , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidores , Células RAW 264.7 , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Quinasas Ciclina-Dependientes/metabolismo , Interleucina-4/metabolismo , Quinasa 8 Dependiente de Ciclina/antagonistas & inhibidores , Quinasa 8 Dependiente de Ciclina/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Fosforilación/efectos de los fármacos , Macrófagos Peritoneales/efectos de los fármacos , Macrófagos Peritoneales/metabolismo , Activación Enzimática/efectos de los fármacos , Flavonoides , Piperidinas , Quinasa 9 Dependiente de la Ciclina

RESUMEN

Substances of silver nanoparticles dialyzed through a 13 kDa membrane, synthesized in a medium of humic ligands modified with hydroquinone and 2-hydroxynaphthoquinone from PowHumus brown coal, specifically enhance the M2 properties of peritoneal macrophages due to inhibition of NO synthase and significant activation of arginase, thus enhancing anti-inflammatory properties of cells. In small, but effective concentrations, they do not have cytotoxic properties and do not contain pyrogenic impurities. The studied humates are able to influence the mechanisms of immune response formation and are an effective means for correcting inflammation and regeneration.

Asunto(s)

Arginasa , Arginina , Sustancias Húmicas , Macrófagos Peritoneales , Plata , Animales , Macrófagos Peritoneales/efectos de los fármacos , Macrófagos Peritoneales/metabolismo , Ratones , Arginina/farmacología , Arginina/química , Arginasa/metabolismo , Plata/química , Plata/farmacología , Nanopartículas del Metal/química , Hidroquinonas/farmacología , Hidroquinonas/química , Masculino , Óxido Nítrico/metabolismo , Óxido Nítrico Sintasa/metabolismo , Naftoquinonas/farmacología , Naftoquinonas/química

RESUMEN

Obesity has reached global epidemic proportions, and even though its effects are well-documented, studying the interactions among all influencing factors is crucial for a better understanding of its physiopathology. In a high-fat-diet-induced obesity animal model using C57BL/6J mice, behavioural responses were assessed through a battery of tests, while stress biomarkers and systemic inflammatory cytokines were measured using an Enzyme-Linked ImmunoSorbent Assay and a Bio-Plex Multiplex System. The peritoneal macrophage microbicide capacity was analysed via flow cytometry, and crown-like structures (CLSs) in white adipose tissue (WAT) were evaluated through staining techniques. Results indicated that obese mice exhibited increased body weight, hyperglycaemia, and hyperlipidaemia after 18 weeks on a high-fat diet, as well as worse physical conditions, poorer coordination and balance, and anxiety-like behaviour. Differences in corticosterone and noradrenaline concentrations were also found in obese animals, revealing a stress response and noradrenergic dysregulation, along with a weakened innate immune response characterized by a lower microbicide capacity, and the presence of an underlying inflammation evidenced by more CLSs in WAT. Altogether, these findings indicate that obesity deteriorates the entire stress, inflammatory, metabolic, sensorimotor and anxiety-like behavioural axis. This demonstrates that jointly evaluating all these aspects allows for a deeper and better exploration of this disease and its associated comorbidities, emphasizing the need for individualized and context-specific strategies for its management.

Asunto(s)

Conducta Animal , Corticosterona , Dieta Alta en Grasa , Ratones Endogámicos C57BL , Obesidad , Animales , Dieta Alta en Grasa/efectos adversos , Obesidad/metabolismo , Obesidad/psicología , Masculino , Corticosterona/sangre , Ratones , Citocinas/metabolismo , Citocinas/sangre , Tejido Adiposo Blanco/metabolismo , Inflamación , Estrés Fisiológico , Macrófagos Peritoneales/metabolismo , Macrófagos Peritoneales/inmunología , Modelos Animales de Enfermedad , Norepinefrina/sangre , Norepinefrina/metabolismo , Ansiedad/etiología , Hiperglucemia

RESUMEN

Trilobolide and its analogues belong to the guaianolide type of sesquiterpene lactones, which are characteristic and widely distributed within the families Asteraceae and Apiaceae. Certain guaianolides are receiving continuously increasing attention for their promising sarco-endoplasmic reticulum Ca2+-ATPase (SERCA)-inhibitory activity. However, because of their alkylation capabilities, they are generally toxic. Therefore, the search for compounds with significant immunobiological properties but with decreased cytotoxicities suitable for use in immune-based pharmacotherapy is ongoing. Therefore, we extended our previous investigation of the immunobiological effects of trilobolide to a series of structurally related guaianolides and germacranolides. To evaluate the relationship, we tested a series of selected derivatives containing α-methyl lactone or exomethylene lactone ring. For a wider comparison, we also included some of their glycosidic derivatives. We assessed the in vitro immunobiological effects of the tested compounds on nitric oxide (NO) production, cytokine secretion, and prostaglandin E2 (PGE2) release by mouse peritoneal cells, activated primarily by lipopolysaccharide (LPS), and evaluated their viability. The inhibitory effects of the apparently most active substance, 8-deoxylactucin, seem to be the most promising.

Asunto(s)

Lactonas , Óxido Nítrico , Sesquiterpenos de Germacrano , Sesquiterpenos de Guayano , Animales , Óxido Nítrico/metabolismo , Ratones , Sesquiterpenos de Germacrano/farmacología , Sesquiterpenos de Germacrano/química , Sesquiterpenos de Guayano/farmacología , Sesquiterpenos de Guayano/química , Lactonas/farmacología , Lactonas/química , Lipopolisacáridos/farmacología , Sesquiterpenos/farmacología , Sesquiterpenos/química , Citocinas/metabolismo , Dinoprostona/metabolismo , Dinoprostona/biosíntesis , Macrófagos Peritoneales/efectos de los fármacos , Macrófagos Peritoneales/metabolismo , Butiratos , Furanos

RESUMEN

The environmental factor aryl hydrocarbon receptor (AhR), a key protein connecting the external environmental signals (e.g., environmental endocrine disruptor TCDD) to internal cellular processes, is involved in the activation of peripheral macrophages and inflammatory response in human body. Thus, there is widespread interest in finding compounds to anti-inflammatory response in macrophages by targeting human AhR. Here, ensemble docking based-virtual screening was first used to screen a library (~200,000 compounds) against human AhR ligand binding domain (LBD) and 25 compounds were identified as potential inhibitors. Then, 9 out of the 25 ligands were found to down-regulate the mRNA expression of CYP1A1 (a downstream gene of AhR signaling) in AhR overexpressing macrophages. The most potent compound AE-411/41415610 was selected for further study and found to reduce both mRNA and protein expressions level of CYP1A1 in mouse peritoneal macrophage. Moreover, protein chip signal pathway analysis indicated that AE-411/41415610 play a role in regulating JAK-STAT and AKT-mTOR pathways. In sum, the discovered hits with novel scaffolds provided a starting point for future design of more effective AhR-targeted lead compounds to regulate CYP1A1 expression of inflammatory peritoneal macrophages.

Asunto(s)

Citocromo P-450 CYP1A1 , Simulación del Acoplamiento Molecular , Receptores de Hidrocarburo de Aril , Transducción de Señal , Receptores de Hidrocarburo de Aril/metabolismo , Citocromo P-450 CYP1A1/metabolismo , Citocromo P-450 CYP1A1/genética , Animales , Ligandos , Ratones , Humanos , Transducción de Señal/efectos de los fármacos , Macrófagos/metabolismo , Macrófagos/efectos de los fármacos , Macrófagos Peritoneales/metabolismo , Macrófagos Peritoneales/efectos de los fármacos , Inflamación/metabolismo , Inflamación/tratamiento farmacológico , Proteínas Proto-Oncogénicas c-akt/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Sitios de Unión

RESUMEN

AIM: To study the activity of antitumor immunity effectors and to analyze possible mechanisms of peritoneal Mph M1/M2 repolarization of Balb/c mice under the influence of lectin from B. subtilis IMV B-7724 in the dynamics of the model tumor growth. MATERIALS AND METHODS: Studies were performed on Balb/c mice; Ehrlich adenocarcinoma (АСЕ) was used as an experimental tumor. Lectin from B. subtilis IMV B-7724 was administered to ACE-bearing mice at a dose of 1 mg/kg of body weight, 10 times. Immunological testing was performed on days 21 and 28 after tumor grafting. The functional activity of peritoneal macrophages (Mph), natural killer (NK) cells, cytotoxic lymphocytes (CTL), and cytokine levels (IFN-γ, IL-4) were studied by the standard methods. mRNA expression levels of transcription factors STAT-1, STAT-6, IRF5, and IRF4 in Mph were evaluated. RESULTS: The administration of lectin from B. subtilis IMV B-7724 to mice with solid ACE led to the preservation of the initial functional state of peritoneal Mph M1 during the experiment. The bacterial lectin ensured the preservation of the cytotoxic activity of CD8+ T-lymphocytes and a significant (p < 0.05) increase in the NK activity (by 2.7 times compared to the intact animals and by 12.9 times compared to the untreated mice). A strong positive correlation was noted between the levels of the functional activity of Mph and CD8+ T-lymphocytes of animals with tumors and the indices of the antitumor effectiveness of bacterial lectin. The indirect polarization of Mph was evidenced by a strong positive correlation between the level of the NO/Arg ratio (which characterizes the direction of Mph polarization) and the cytotoxic activity of CD8+ T-lymphocytes, NK cells, and the expression of STAT1/STAT6 (the 21st day) and IRF5/IRF4 (the 28th day). CONCLUSION: In ACE-bearing mice, repolarization of the peritoneal Mph toward M1 can occur not only due to the direct action of bacterial lectin on the cellular receptors but also with the involvement of other effectors of antitumor immunity (NK cells, T-lymphocytes). The transcription factors of the STAT and IRF signaling pathways are involved in the polarization process.

Asunto(s)

Células Asesinas Naturales , Macrófagos Peritoneales , Ratones Endogámicos BALB C , Animales , Ratones , Macrófagos Peritoneales/inmunología , Macrófagos Peritoneales/metabolismo , Macrófagos Peritoneales/efectos de los fármacos , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/metabolismo , Carcinoma de Ehrlich/inmunología , Carcinoma de Ehrlich/patología , Carcinoma de Ehrlich/metabolismo , Bacillus subtilis , Citocinas/metabolismo , Linfocitos T Citotóxicos/inmunología , Linfocitos T Citotóxicos/metabolismo

RESUMEN

The non-neural cholinergic system plays a critical role in regulating immune equilibrium and tissue homeostasis. While the expression of choline acetyltransferase (ChAT), the enzyme catalyzing acetylcholine biosynthesis, has been well documented in lymphocytes, its role in the myeloid compartment is less understood. Here, we identify a significant population of macrophages (Mϕs) expressing ChAT and synthesizing acetylcholine in the resolution phase of acute peritonitis. Using Chat-GFP reporter mice, we observed marked upregulation of ChAT in monocyte-derived small peritoneal Mϕs (SmPMs) in response to Toll-like receptor agonists and bacterial infections. These SmPMs, phenotypically and transcriptionally distinct from tissue-resident large peritoneal macrophages, up-regulated ChAT expression through a MyD88-dependent pathway involving MAPK signaling. Notably, this process was attenuated by the TRIF-dependent TLR signaling pathway, and our tests with a range of neurotransmitters and cytokines failed to induce a similar response. Functionally, Chat deficiency in Mϕs led to significantly decreased peritoneal acetylcholine levels, reduced efferocytosis of apoptotic neutrophils, and a delayed resolution of peritonitis, which were reversible with exogenous ACh supplementation. Intriguingly, despite B lymphocytes being a notable ChAT-expressing population within the peritoneal cavity, Chat deletion in B cells did not significantly alter the resolution process. Collectively, these findings underscore the crucial role of Mϕ-derived acetylcholine in the resolution of inflammation and highlight the importance of the non-neuronal cholinergic system in immune regulation.

Asunto(s)

Acetilcolina , Colina O-Acetiltransferasa , Macrófagos Peritoneales , Peritonitis , Animales , Colina O-Acetiltransferasa/metabolismo , Colina O-Acetiltransferasa/genética , Peritonitis/inmunología , Peritonitis/metabolismo , Ratones , Macrófagos Peritoneales/metabolismo , Macrófagos Peritoneales/inmunología , Acetilcolina/metabolismo , Factor 88 de Diferenciación Mieloide/metabolismo , Factor 88 de Diferenciación Mieloide/genética , Ratones Endogámicos C57BL , Transducción de Señal , Inflamación/metabolismo , Inflamación/patología , Linfocitos B/inmunología , Linfocitos B/metabolismo , Receptores Toll-Like/metabolismo , Fagocitosis , Macrófagos/metabolismo , Macrófagos/inmunología , Ratones Noqueados

RESUMEN

Eleven compounds including caffeic acid (CA), 4 kinds of caffeoylquinic acid (CQA) and 6 kinds of dicaffeoylquinic acid (DCQA), were selected to evaluate the anti-inflammatory effectiveness using mouse primary peritoneal macrophages in the absence or presence of lipopolysaccharide (LPS). The optimal non-cytotoxic doses of each individual compound were determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Pro-inflammatory (TNF-α, IL-1ß, IL-6) and anti-inflammatory (IL-10) cytokines secreted by treated macrophages were analyzed using the enzyme-linked immunosorbent assay. Cytokine secretion profiles of each individual test sample at optimal non-cytotoxic doses were further analyzed using Principal Component Analysis (PCA). The results showed that CA and all selected CQAs exhibited lower cytotoxicity (IC50: >50 µmol/l). Both CA and 5-CQA were found to have the most significant contributions for inhibiting pro-inflammatory cytokines, but increasing anti-inflammatory cytokine secretions, evidencing that CA at 10 µmol/l and 5-CQA at 25 µmol/l can be qualified as potent anti-inflammatory agents for treating inflammation-related diseases.

Asunto(s)

Antiinflamatorios , Ácidos Cafeicos , Citocinas , Análisis de Componente Principal , Animales , Ácidos Cafeicos/farmacología , Ácidos Cafeicos/química , Ratones , Citocinas/metabolismo , Citocinas/inmunología , Antiinflamatorios/farmacología , Antiinflamatorios/química , Macrófagos Peritoneales/efectos de los fármacos , Macrófagos Peritoneales/metabolismo , Macrófagos Peritoneales/inmunología , Células Cultivadas , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Macrófagos/metabolismo

RESUMEN

Corynebacterium pseudotuberculosis (C. p), a facultative intracellular bacterium, is an important zoonotic pathogen that causes abscesses and pyogenic granulomas. The relationship between gut microbiota and host health or diseases has received increasing attention. However, the role of gut microbiota in the process of C. p infection is still unclear. In this study, we established a C. p infection model in C57BL/6 mice and examined the impact of preemptive oral administration Lactobacillus acidophilus (L. acidophilus) on infection. Our findings revealed that C. p infection led to pronounced pathological alterations in the liver and kidneys, characterized by abscess formation, intense inflammatory responses, and bacterial overload. Remarkably, these deleterious effects were greatly relieved by oral administration of L. acidophilus before infection with C. p. Additionally, we further found that during C. p infection, peritoneal macrophages (PMs) of mice orally administered with L. acidophilus accumulated more rapidly at sites of infection. Furthermore, our results showed that PMs from mice with oral L. acidophilus administration showed a stronger C. p clearance effect, and this was mediated by high expression of LC3-II protein. Meanwhile, oral administration of L. acidophilus protected the gut microbiota disorder in C57BL/6 mice caused by C. p infection. In summary, our study demonstrates that oral administration of L. acidophilus confers effective protection against C. p infection in C57BL/6 mice by modulating macrophage autophagy, thereby augmenting bacterial clearance and preserving gut microbiota and function stability. These findings position L. acidophilus as a viable probiotic candidate for the clinical prevention of C. p infection. IMPORTANCE: Corynebacterium pseudotuberculosis (C. p) is known to induce a range of chronic diseases in both animals and humans. Currently, clinical treatment for C. p infection mainly relies on antibiotic therapy or surgical intervention. However, excessive use of antibiotics may increase the risk of drug-resistant strains, and the effectiveness of treatment remains unsatisfactory. Furthermore, surgical procedures do not completely eradicate pathogens and can easily cause environmental pollution. Probiotic interventions are receiving increasing attention for improving the body's immune system and maintaining health. In this study, we established a C. p infection model in C57BL/6 mice to explore the impact of Lactobacillus acidophilus during C. p infection. Our results showed that L. acidophilus effectively protected against C. p infection by regulating the autophagy of macrophages and maintaining intestinal microbiota homeostasis. This study may provide a new strategy for the prevention of C. p infection.

Asunto(s)

Autofagia , Infecciones por Corynebacterium , Corynebacterium pseudotuberculosis , Microbioma Gastrointestinal , Lactobacillus acidophilus , Ratones Endogámicos C57BL , Animales , Autofagia/efectos de los fármacos , Microbioma Gastrointestinal/efectos de los fármacos , Microbioma Gastrointestinal/fisiología , Lactobacillus acidophilus/fisiología , Ratones , Infecciones por Corynebacterium/prevención & control , Infecciones por Corynebacterium/microbiología , Homeostasis/efectos de los fármacos , Probióticos/administración & dosificación , Probióticos/farmacología , Macrófagos Peritoneales/inmunología , Macrófagos Peritoneales/microbiología , Macrófagos Peritoneales/efectos de los fármacos , Macrófagos Peritoneales/metabolismo , Macrófagos/inmunología , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Modelos Animales de Enfermedad

RESUMEN

BACKGROUND: The surplus cytokines remaining after use in the early stages of the inflammatory response stimulate immune cells even after the response is over, causing a secondary inflammatory response and ultimately damaging the host, which is called a cytokine storm. Inhibiting heat shock protein 90 (Hsp90), which has recently been shown to play an important role in regulating inflammation in various cell types, may help control excessive inflammatory responses and cytokine storms. METHODS: We discovered an anti-inflammatory compound by measuring the inhibitory effect of CD86 expression on spleen DCs (sDCs) using the chemical compounds library of Hsp90 inhibitors. Subsequently, to select the hit compound, the production of cytokines and expression of surface molecules were measured on the bone marrow-derived DCs (BMDCs) and peritoneal macrophages. Then, we analyzed the response of antigen-specific Th1 cells. Finally, we confirmed the effect of the compound using acute lung injury (ALI) and delayed-type hypersensitivity (DTH) models. RESULTS: We identified Be01 as the hit compound, which reduced CD86 expression the most in sDCs. Treatment with Be01 decreased the production of pro-inflammatory cytokines (IL-6, TNF-α, and IL-1ß) in BMDC and peritoneal macrophages stimulated by LPS. Under the DTH model, Be01 treatment reduced ear swelling and pro-inflammatory cytokines in the spleen. Similarly, Be01 treatment in the ALI model decreased neutrophil infiltration and lower levels of secreted cytokines (IL-6, TNF-α). CONCLUSIONS: Reduction of CD80 and CD86 expression on DCs by Be01 indicates reduced secondary inflammatory response by Th1 cells, and reduced release of pro-inflammatory cytokines by peritoneal macrophages may initially control the cytokine storm.

Asunto(s)

Antiinflamatorios , Citocinas , Células Dendríticas , Proteínas HSP90 de Choque Térmico , Macrófagos Peritoneales , Ratones Endogámicos C57BL , Animales , Células Dendríticas/efectos de los fármacos , Células Dendríticas/inmunología , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Proteínas HSP90 de Choque Térmico/metabolismo , Citocinas/metabolismo , Antiinflamatorios/farmacología , Antiinflamatorios/uso terapéutico , Ratones , Macrófagos Peritoneales/efectos de los fármacos , Macrófagos Peritoneales/inmunología , Macrófagos Peritoneales/metabolismo , Hipersensibilidad Tardía/tratamiento farmacológico , Hipersensibilidad Tardía/inmunología , Antígeno B7-2/metabolismo , Lesión Pulmonar Aguda/tratamiento farmacológico , Lesión Pulmonar Aguda/inmunología , Células Cultivadas , Síndrome de Liberación de Citoquinas/tratamiento farmacológico , Síndrome de Liberación de Citoquinas/inmunología , Células TH1/inmunología , Células TH1/efectos de los fármacos , Inflamación/tratamiento farmacológico , Inflamación/inmunología , Femenino , Modelos Animales de Enfermedad , Bazo/inmunología , Bazo/efectos de los fármacos

RESUMEN

INTRODUCTION: A dysregulated immune system is a major driver of the mortality and long-term morbidity from sepsis. With respect to macrophages, it has been shown that phenotypic changes are critical to effector function in response to acute infections, including intra-abdominal sepsis. Invariant natural killer T cells (iNKT cells) have emerged as potential central regulators of the immune response to a variety of infectious insults. Specifically, various iNKT cell:macrophage interactions have been noted across a spectrum of diseases, including acute events such as sepsis. However, the potential for iNKT cells to affect peritoneal macrophages during an abdominal septic event is as yet unknown. METHODS: Cecal ligation and puncture (CLP) was performed in both wild type (WT) and invariant natural killer T cell knockout (iNKT-/-) mice. 24 h following CLP or sham operation, peritoneal macrophages were collected for analysis. Analysis of macrophage phenotype and function was undertaken to include analysis of bactericidal activity and cytokine or superoxide production. RESULTS: Within iNKT-/- mice, a greater degree of intraperitoneal macrophages in response to the sepsis was noted. Compared to WT mice, within iNKT-/- mice, CLP did induce an increase in CD86+ and CD206+, but no difference in CD11b+. Unlike WT mice, intra-abdominal sepsis within iNKT-/- mice induced an increase in Ly6C-int (5.2% versus 14.9%; P < 0.05) and a decrease in Ly6C-high on peritoneal macrophages. Unlike phagocytosis, iNKT cells did not affect macrophage bactericidal activity. Although iNKT cells did not affect interleukin-6 production, iNKT cells did affect IL-10 production and both nitrite and superoxide production from peritoneal macrophages. CONCLUSIONS: The observations indicate that iNKT cells affect specific phenotypic and functional aspects of peritoneal macrophages during polymicrobial sepsis. Given that pharmacologic agents that affect iNKT cell functioning are currently in clinical trial, these findings may have the potential for translation to critically ill surgical patients with abdominal sepsis.

Asunto(s)

Macrófagos Peritoneales , Ratones Endogámicos C57BL , Ratones Noqueados , Células T Asesinas Naturales , Sepsis , Animales , Macrófagos Peritoneales/inmunología , Macrófagos Peritoneales/metabolismo , Sepsis/inmunología , Sepsis/microbiología , Células T Asesinas Naturales/inmunología , Ratones , Masculino , Superóxidos/metabolismo , Citocinas/metabolismo , Modelos Animales de Enfermedad

RESUMEN

Background: The disruption of the circadian clock is associated with inflammatory and immunological disorders. BMAL2, a critical circadian protein, forms a dimer with CLOCK, activating transcription. Extracellular cold-inducible RNA-binding protein (eCIRP), released during sepsis, can induce macrophage endotoxin tolerance. We hypothesized that eCIRP induces BMAL2 expression and promotes macrophage endotoxin tolerance through triggering receptor expressed on myeloid cells-1 (TREM-1). Methods: C57BL/6 wild-type (WT) male mice were subjected to sepsis by cecal ligation and puncture (CLP). Serum levels of eCIRP 20 h post-CLP were assessed by ELISA. Peritoneal macrophages (PerM) were treated with recombinant mouse (rm) CIRP (eCIRP) at various doses for 24 h. The cells were then stimulated with LPS for 5 h. The levels of TNF-α and IL-6 in the culture supernatants were assessed by ELISA. PerM were treated with eCIRP for 24 h, and the expression of PD-L1, IL-10, STAT3, TREM-1 and circadian genes such as BMAL2, CRY1, and PER2 was assessed by qPCR. Effect of TREM-1 on eCIRP-induced PerM endotoxin tolerance and PD-L1, IL-10, and STAT3 expression was determined by qPCR using PerM from TREM-1-/- mice. Circadian gene expression profiles in eCIRP-treated macrophages were determined by PCR array and confirmed by qPCR. Induction of BMAL2 activation in bone marrow-derived macrophages was performed by transfection of BMAL2 CRISPR activation plasmid. The interaction of BMAL2 in the PD-L1 promoter was determined by computational modeling and confirmed by the BIAcore assay. Results: Serum levels of eCIRP were increased in septic mice compared to sham mice. Macrophages pre-treated with eCIRP exhibited reduced TNFα and IL-6 release upon LPS challenge, indicating macrophage endotoxin tolerance. Additionally, eCIRP increased the expression of PD-L1, IL-10, and STAT3, markers of immune tolerance. Interestingly, TREM-1 deficiency reversed eCIRP-induced macrophage endotoxin tolerance and significantly decreased PD-L1, IL-10, and STAT3 expression. PCR array screening of circadian clock genes in peritoneal macrophages treated with eCIRP revealed the elevated expression of BMAL2, CRY1, and PER2. In eCIRP-treated macrophages, TREM-1 deficiency prevented the upregulation of these circadian genes. In macrophages, inducible BMAL2 expression correlated with increased PD-L1 expression. In septic human patients, blood monocytes exhibited increased expression of BMAL2 and PD-L1 in comparison to healthy subjects. Computational modeling and BIAcore assay identified a putative binding region of BMAL2 in the PD-L1 promoter, suggesting BMAL2 positively regulates PD-L1 expression in macrophages. Conclusion: eCIRP upregulates BMAL2 expression via TREM-1, leading to macrophage endotoxin tolerance in sepsis. Targeting eCIRP to maintain circadian rhythm may correct endotoxin tolerance and enhance host resistance to bacterial infection.

Asunto(s)

Proteínas de Unión al ARN , Sepsis , Animales , Humanos , Masculino , Ratones , Factores de Transcripción ARNTL/genética , Modelos Animales de Enfermedad , Endotoxinas/inmunología , Tolerancia Inmunológica , Lipopolisacáridos/inmunología , Macrófagos/inmunología , Macrófagos/metabolismo , Macrófagos Peritoneales/inmunología , Macrófagos Peritoneales/metabolismo , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Sepsis/inmunología , Sepsis/metabolismo , Receptor Activador Expresado en Células Mieloides 1/inmunología , Receptor Activador Expresado en Células Mieloides 1/genética , Receptor Activador Expresado en Células Mieloides 1/metabolismo

RESUMEN

BACKGROUND: Endometria are one of the important components of the uterus, which is located in the peritoneal cavity. Endometrial injury usually leads to intrauterine adhesions (IUA), accompanied by inflammation and cell death. We previously reported that both the endometrial ferroptosis was increased and monocytes/macrophages were involved in endometrial injury of IUA. Large peritoneal macrophages (LPMs) are recently reported to migrate into the injured tissues and phagocytose dead cells to repair the tissues. We previously demonstrated that mesenchymal stromal cells (MSCs) had made excellent progress in the repair of endometrial injury. However, it is unclear whether MSCs regulate the LPM efferocytosis against ferroptotic monocytes/macrophages in the injured endometria. METHODS: Here, endometrial injury in IUA mouse model was conducted by uterine curettage and LPS injection surgery and the samples were collected at different times to detect the changes of LPMs and ferroptotic monocytes/macrophages. We conducted LPMs depletion assay in vivo and LPMs and Erastin-induced ferroptotic THP-1 cells coculture systems in vitro to detect the LPM efferocytosis against ferroptotic monocytes/macrophages. The IUA model was treated with MSCs, and their effects on LPMs and endometrial repair were analyzed. Flow cytometry, western blotting, quantitative real-time PCR, immunohistochemical analysis, ELISA, and RNA-sequencing were performed. RESULTS: We found that LPMs migrated to the injured uteri in response to the damage in early phase (3 h), and sustained to a later stage (7 days). Astonishingly, we found that ferroptotic monocytes/macrophages were significantly increased in the injured uteri since 12 h after injury. Moreover, LPMs cocultured with Erastin-induced ferroptotic THP-1 cells in vitro, efferocytosis of LPMs against ferroptotic monocytes/macrophages was emerged. The mRNA expression profiles revealed that LPM efferocytosis against ferroptotic monocytes/macrophages was an induction of glycolysis program and depended on the PPARγ-HK2 pathway. Importantly, we validated that MSCs promoted the efferocytic capability and migration of LPMs to the injured uteri via secreting stanniocalcin-1 (STC-1). CONCLUSION: The data collectively demonstrated first the roles of LPMs via removal of ferroptotic monocytes/macrophages and provided a novel mechanism of MSCs in repairing the endometrial injury.

Asunto(s)

Macrófagos Peritoneales , Células Madre Mesenquimatosas , Monocitos , Femenino , Animales , Ratones , Células Madre Mesenquimatosas/metabolismo , Células Madre Mesenquimatosas/citología , Monocitos/metabolismo , Monocitos/citología , Humanos , Macrófagos Peritoneales/metabolismo , Endometrio/lesiones , Endometrio/metabolismo , Endometrio/citología , Endometrio/patología , Fagocitosis , Ratones Endogámicos C57BL , Modelos Animales de Enfermedad , Eferocitosis

RESUMEN

N-acetyl glucosamine (NAG) is a natural amino sugar found in various human tissues with previously described anti-inflammatory effects. Various chemical modifications of NAG have been made to promote its biomedical applications. In this study, we synthesized two bi-deoxygenated NAG, BNAG1 and BNAG2 and investigated their anti-inflammatory properties, using an in vivo and in vitro inflammation mouse model induced by lipopolysaccharide (LPS). Among the parent molecule NAG, BNAG1 and BNAG2, BNAG1 showed the highest inhibition against serum levels of IL-6 and TNF α and the leukocyte migration to lungs and peritoneal cavity in LPS challenged mice, as well as IL-6 and TNF α production in LPS-stimulated primary peritoneal macrophages. BNAG2 displayed an anti-inflammatory effect which was comparable to NAG. These findings implied potential application of these novel NAG derivatives, especially BNAG1, in treatment of certain inflammation-related diseases.

Asunto(s)

Acetilglucosamina , Antiinflamatorios , Lipopolisacáridos , Macrófagos Peritoneales , Factor de Necrosis Tumoral alfa , Animales , Acetilglucosamina/farmacología , Ratones , Antiinflamatorios/farmacología , Antiinflamatorios/síntesis química , Macrófagos Peritoneales/efectos de los fármacos , Macrófagos Peritoneales/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Interleucina-6/metabolismo , Interleucina-6/sangre , Inflamación/tratamiento farmacológico , Masculino , Modelos Animales de Enfermedad

RESUMEN

Acute lung injury (ALI) remains a significant clinical challenge due to the absence of effective treatment alternatives. This study presents a new method that employs a screening platform focusing on MyD88 affinity, anti-inflammatory properties, and toxicity. This platform was used to evaluate a 300-compound library known for its anti-inflammatory potential. Among the screened compounds, Bicyclol emerged as a standout, exhibiting MyD88 binding and a significant reduction in LPS-stimulated pro-inflammatory factors production in mouse primary peritoneal macrophages. By targeting MyD88, Bicyclol disrupts the MyD88/TLR4 complex and MyD88 polymer formation, thereby mitigating the MAPKs and NF-κB signaling pathways. In vivo experiments further confirmed Bicyclol's efficacy, demonstrating alleviated ALI symptoms, decreased inflammatory cytokines level, and reduced inflammatory cells presence in lung tissues. These findings were associated with a decrease in mortality in LPS-challenged mice. Overall, Bicyclol represents a promising treatment option for ALI by specifically targeting MyD88 and limiting inflammatory responses.

Asunto(s)

Lesión Pulmonar Aguda , Compuestos de Bifenilo , Lipopolisacáridos , Ratones Endogámicos C57BL , Factor 88 de Diferenciación Mieloide , Animales , Lesión Pulmonar Aguda/inducido químicamente , Lesión Pulmonar Aguda/prevención & control , Lesión Pulmonar Aguda/metabolismo , Lesión Pulmonar Aguda/patología , Lipopolisacáridos/toxicidad , Factor 88 de Diferenciación Mieloide/metabolismo , Ratones , Masculino , Compuestos de Bifenilo/farmacología , Antiinflamatorios/farmacología , Macrófagos Peritoneales/efectos de los fármacos , Macrófagos Peritoneales/metabolismo , FN-kappa B/metabolismo , Transducción de Señal/efectos de los fármacos , Receptor Toll-Like 4/metabolismo , Citocinas/metabolismo , Pulmón/efectos de los fármacos , Pulmón/patología , Pulmón/metabolismo

RESUMEN

The NOD-like receptor family protein 3 (NLRP3) inflammasome is a crucial complex for the host to establish inflammatory immune responses and plays vital roles in a series of disorders, including Alzheimer's disease and acute peritonitis. However, its regulatory mechanism remains largely unclear. Zinc finger antiviral protein (ZAP), also known as zinc finger CCCH-type antiviral protein 1 (ZC3HAV1), promotes viral RNA degradation and plays vital roles in host antiviral immune responses. However, the role of ZAP in inflammation, especially in NLRP3 activation, is unclear. Here, we show that ZAP interacts with NLRP3 and promotes NLRP3 oligomerization, thus facilitating NLRP3 inflammasome activation in peritoneal macrophages of C57BL/6 mice. The shorter isoform of ZAP (ZAPS) appears to play a greater role than the full-length isoform (ZAPL) in HEK293T cells. Congruously, Zap-deficient C57BL/6 mice may be less susceptible to alum-induced peritonitis and lipopolysaccharide-induced sepsis in vivo. Therefore, we propose that ZAP is a positive regulator of NLRP3 activation and a potential therapeutic target for NLRP3-related inflammatory disorders.

Asunto(s)

Inflamasomas , Proteína con Dominio Pirina 3 de la Familia NLR , Peritonitis , Animales , Humanos , Masculino , Ratones , Células HEK293 , Inflamasomas/metabolismo , Inflamasomas/inmunología , Lipopolisacáridos/inmunología , Macrófagos Peritoneales/inmunología , Macrófagos Peritoneales/metabolismo , Ratones Endogámicos C57BL , Ratones Noqueados , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/genética , Peritonitis/inmunología , Peritonitis/inducido químicamente , Multimerización de Proteína , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/genética , Sepsis/inmunología , Sepsis/metabolismo

RESUMEN

LPS induced sepsis is a complex process involving various immune cells and signaling molecules. Dysregulation of macrophage polarization and ROS production contributed to the pathogenesis of sepsis. PGP is a transmembrane transporter responsible for the efflux of a number of drugs and also expressed in murine macrophages. Natural products have been shown to decrease inflammation and expression of efflux transporters. However, no treatment is currently available to treat LPS induced sepsis. Verapamil and Tangeretin also reported to attenuate lipopolysaccharide-induced inflammation. However, the effects of verapamil or tangeretin on lipopolysaccharide (LPS)-induced sepsis and its detailed anti-inflammatory mechanism have not been reported. Here, we have determined that verapamil and tangeretin protects against LPS-induced sepsis by suppressing M1 macrophages populations and also through the inhibition of P-glycoprotein expression via downregulating STAT1/STAT3 and upregulating SOCS3 expression in macrophages. An hour before LPS (10 mg/kg) was administered; mice were given intraperitoneal injections of either verapamil (5 mg/kg) or tangeretin (5 mg/kg). The peritoneal macrophages from different experimental groups of mice were isolated. Hepatic, pulmonary and splenic morphometric analyses revealed that verapamil and tangeretin decreased the infiltration of neutrophils into the tissues. Verapamil and tangeritin also enhanced the activity of SOD, CAT, GRX and GSH level in all the tissues tested. verapamil or tangeretin pre-treated mice shifted M1 macrophages to M2 type possibly through the inhibition of P-glycoprotein expression via downregulating STAT1/STAT3 and upregulating SOCS3 expression. Hence, both these drugs have shown protective effects in sepsis via suppressing iNOS, COX-2, oxidative stress and NF-κB signaling in macrophages. Therefore, in our study we can summarize that mice were treated with either Vera or Tan before LPS administration cause an elevated IL-10 by the macrophages which enhances the SOCS3 expression, and thereby able to limits STAT1/STAT3 inter-conversion in the macrophages. As a result, NF-κB activity is also getting down regulated and ultimately mitigating the adverse effect of inflammation caused by LPS in resident macrophages. Whether verapamil or tangeretin offers such protection possibly through the inhibition of P-glycoprotein expression in macrophages needs clarification with the bio availability of these drugs under PGP inhibited conditions is a limitation of this study.

Asunto(s)

Flavonas , Lipopolisacáridos , Factor de Transcripción STAT1 , Factor de Transcripción STAT3 , Proteína 3 Supresora de la Señalización de Citocinas , Verapamilo , Animales , Verapamilo/farmacología , Factor de Transcripción STAT1/metabolismo , Proteína 3 Supresora de la Señalización de Citocinas/metabolismo , Proteína 3 Supresora de la Señalización de Citocinas/genética , Flavonas/farmacología , Flavonas/uso terapéutico , Ratones , Factor de Transcripción STAT3/metabolismo , Masculino , Sepsis/tratamiento farmacológico , Sepsis/inmunología , Sepsis/metabolismo , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Antiinflamatorios/farmacología , Antiinflamatorios/uso terapéutico , Regulación hacia Abajo/efectos de los fármacos , Macrófagos Peritoneales/efectos de los fármacos , Macrófagos Peritoneales/metabolismo , Macrófagos Peritoneales/inmunología , Células Cultivadas , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Macrófagos/inmunología , Transducción de Señal/efectos de los fármacos , Regulación hacia Arriba/efectos de los fármacos

RESUMEN

Sepsis is a systemic inflammatory response syndrome caused by trauma or infection, which can lead to multiple organ dysfunction. In severe cases, sepsis can also progress to septic shock and even death. Effective treatments for sepsis are still under development. This study aimed to determine if targeting the PI3K/Akt signaling with CAL-101, a PI3K p110δ inhibitor, could alleviate lipopolysaccharide (LPS)-induced sepsis and contribute to immune tolerance. Our findings indicated that CAL-101 treatment improved survival rates and alleviated the progression of LPS-induced sepsis. Compared to antibiotics, CAL-101 not only restored the Th17/regulatory T cells (Treg) balance but also enhanced Treg cell function. Additionally, CAL-101 promoted type 2 macrophage (M2) polarization, inhibited TNF-α secretion, and increased IL-10 secretion. Moreover, CAL-101 treatment reduced pyroptosis in peritoneal macrophages by inhibiting caspase-1/gasdermin D (GSDMD) activation. This study provides a mechanistic basis for future clinical exploration of targeted therapeutics and immunomodulatory strategies in the treatment of sepsis.

Asunto(s)

Fosfatidilinositol 3-Quinasa Clase I , Ratones Endogámicos C57BL , Piroptosis , Sepsis , Linfocitos T Reguladores , Células Th17 , Animales , Ratones , Fosfatidilinositol 3-Quinasa Clase I/antagonistas & inhibidores , Fosfatidilinositol 3-Quinasa Clase I/metabolismo , Macrófagos/inmunología , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Macrófagos Peritoneales/inmunología , Macrófagos Peritoneales/efectos de los fármacos , Macrófagos Peritoneales/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3/farmacología , Piroptosis/efectos de los fármacos , Sepsis/inmunología , Sepsis/tratamiento farmacológico , Transducción de Señal/efectos de los fármacos , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/efectos de los fármacos , Células Th17/inmunología , Células Th17/efectos de los fármacos