FK506 impairs neutrophil migration that results in increased polymicrobial sepsis susceptibility.

OBJECTIVE: This study aimed to investigate the effects of FK506 on experimental sepsis immunopathology. It investigated the effect of FK506 on leukocyte recruitment to the site of infection, systemic cytokine production, and organ injury in mice with sepsis. METHODS: Using a murine cecal ligation and puncture (CLP) peritonitis model, the experiments were performed with wild-type (WT) mice and mice deficient in the gene Nfat1 (Nfat1-/-) in the C57BL/6 background. Animals were treated with 2.0 mg/kg of FK506, subcutaneously, 1 h before the sepsis model, twice a day (12 h/12 h). The number of bacteria colony forming units (CFU) was manually counted. The number of neutrophils in the lungs was estimated by the myeloperoxidase (MPO) assay. The expression of CXCR2 in neutrophils was determined using flow cytometry analysis. The expression of inflammatory cytokines in macrophage was determined using ELISA. The direct effect of FK506 on CXCR2 internalization was evaluated using HEK-293T cells after CXCL2 stimulation by the BRET method. RESULTS: FK506 treatment potentiated the failure of neutrophil migration into the peritoneal cavity, resulting in bacteremia and an exacerbated systemic inflammatory response, which led to higher organ damage and mortality rates. Failed neutrophil migration was associated with elevated CXCL2 chemokine plasma levels and lower expression of the CXCR2 receptor on circulating neutrophils compared with non-treated CLP-induced septic mice. FK506 did not directly affect CXCL2-induced CXCR2 internalization by transfected HEK-293 cells or mice neutrophils, despite increasing CXCL2 release by LPS-treated macrophages. Finally, the CLP-induced response of Nfat1-/- mice was similar to those observed in the Nfat1+/+ genotype, suggesting that the FK506 effect is not dependent on the NFAT1 pathway. CONCLUSION: Our data indicate that the increased susceptibility to infection of FK506-treated mice is associated with failed neutrophil migration due to the reduced membrane availability of CXCR2 receptors in response to exacerbated levels of circulating CXCL2.

The metabolic function of pyruvate kinase M2 regulates reactive oxygen species production and microbial killing by neutrophils.

Neutrophils rely predominantly on glycolytic metabolism for their biological functions, including reactive oxygen species (ROS) production. Although pyruvate kinase M2 (PKM2) is a glycolytic enzyme known to be involved in metabolic reprogramming and gene transcription in many immune cell types, its role in neutrophils remains poorly understood. Here, we report that PKM2 regulates ROS production and microbial killing by neutrophils. Zymosan-activated neutrophils showed increased cytoplasmic expression of PKM2. Pharmacological inhibition or genetic deficiency of PKM2 in neutrophils reduced ROS production and Staphylococcus aureus killing in vitro. In addition, this also resulted in phosphoenolpyruvate (PEP) accumulation and decreased dihydroxyacetone phosphate (DHAP) production, which is required for de novo synthesis of diacylglycerol (DAG) from glycolysis. In vivo, PKM2 deficiency in myeloid cells impaired the control of infection with Staphylococcus aureus. Our results fill the gap in the current knowledge of the importance of lower glycolysis for ROS production in neutrophils, highlighting the role of PKM2 in regulating the DHAP and DAG synthesis to promote ROS production in neutrophils.

Platelets Fuel the Inflammasome Activation of Innate Immune Cells.

The inflammasomes control the bioactivity of pro-inflammatory cytokines of the interleukin (IL)-1 family. The inflammasome assembled by NLRP3 has been predominantly studied in homogeneous cell populations in vitro, neglecting the influence of cellular interactions that occur in vivo. Here, we show that platelets boost the inflammasome capacity of human macrophages and neutrophils and are critical for IL-1 production by monocytes. Platelets license NLRP3 transcription, thereby enhancing ASC oligomerization, caspase-1 activity, and IL-1ß secretion. Platelets influence IL-1ß production in vivo, and blood platelet counts correlate with plasmatic IL-1ß levels in malaria. Furthermore, we reveal an enriched platelet gene signature among the highest-expressed transcripts in IL-1ß-driven autoinflammatory diseases. The platelet effect is independent of cell-to-cell contact, platelet-derived lipid mediators, purines, nucleic acids, and a host of platelet cytokines, and it involves the triggering of calcium-sensing receptors on macrophages. Hence, platelets provide an additional layer of regulation of inflammasomes and IL-1-driven inflammation.

Subtotal nephrectomy inhibits the gastric emptying of liquid in awake rats.

Homeostasis of blood volume (BV) is attained through a functional interaction between the cardiovascular and renal systems. The gastrointestinal tract also adjusts its permeability and motor behavior after acute BV imbalances. We evaluated the effect of progressive nephron loss on gut motility. Male Wistar rats were subjected or not (sham) to 5/6 partial nephrectomy (PNX) in two steps (0 and 7th day). After further 3, 7, or 14 days, PNX and sham operation (control) rats were instrumented to monitor mean arterial pressure (MAP), central venous pressure (CVP), heart rate (HR), and blood collection for biochemical analysis. The next day, they were gavage fed with a liquid test meal (phenol red in glucose solution), and fractional dye recovery determined 10, 20, or 30 min later. The effect of nonhypotensive hypovolemia and the role of neuroautonomic pathways on PNX-induced gastric emptying (GE) delay were also evaluated. Compared with the sham-operated group, PNX rats exhibited higher (P < 0.05) MAP and CVP values as well as increased values of gastric dye recovery, phenomenon proportional to the BV values. Gastric retention was prevented by prior hypovolemia, bilateral subdiaphragmatic vagotomy, coelic ganglionectomy + splanchnicectomy, guanethidine, or atropine pretreatment. PNX also inhibited (P < 0.05) the marker's progression through the small intestine. In anesthetized rats, PNX increased (P < 0.05) gastric volume, measured by a balloon catheter in a barostat system. In conclusion, the progressive loss of kidney function delayed the GE rate, which may contribute to gut dysmotility complaints associated with severe renal failure.

Stable experimental model of carotid artery saccular aneurysm in swine using the internal jugular vein.

OBJECTIVE: To develop an experimental model of stable saccular aneurysm in carotid of pigs using the internal jugular vein. METHODS: In 12 healthy pigs, weighing between 25 and 5 0kg, five males and seven females, we made a right common carotid artery aneurysm. After elliptical arteriotomy, we carried out a terminolateral anastomosis with the distal stump of the internal jugular vein. Aneurysm volume was calculated so that the value did not exceed 27 times the area of the arteriotomy. After six days angiography and microscopic examination were performed to assess patency of the aneurysm and the presence of total or partial thrombosis. RESULTS: There was a significant weight gain of pigs in the time interval between the manufacture of the aneurysm and angiography (p = 0.04). Aneurysmal patency was observed in ten pigs (83%). Operative wound infections occurred in two animals (16.6%), both with early onset, three days after the making of the aneurysm. Histological analysis showed aneurysm thrombus partially occluding the light in nine pigs (75%). In these animals, it was observed that on average 9% of the aneurysmal diameter was filled with thrombi. CONCLUSION: It was possible to develop a stable experimental model of saccular aneurysms in pig carotid artery by use of the internal jugular vein.

Modelo experimental estável de aneurisma sacular em artéria carótida de suínos utilizando veia jugular interna / Stable experimental model of carotid artery saccular aneurysm in swine using the internal jugular vein

OBJETIVO: Desenvolver um modelo experimental estável de aneurisma sacular em carótida de suínos utilizando veia jugular interna. MÉTODOS: Em 12 suínos sadios, com peso variando entre 25 e 50kg, cinco machos e sete fêmeas, foi confeccionado aneurisma na artéria carótida comum direita. Após arteriotomia elíptica, foi realizada anastomose terminolateral com coto distal de veia jugular interna. O volume do aneurisma era calculado de maneira que o valor não excedesse em 27 vezes o valor da área da arteriotomia. Após seis dias, era realizada angiografia e análise microscópica do aneurisma para avaliar perviedade e trombose parcial ou total. RESULTADOS: Houve ganho de peso significante dos suínos no intervalo de tempo entre a confecção do aneurisma e a angiografia (p = 0,04). Foi observada perviedade aneurismática em dez suínos (83%). Ocorreram infecções de feridas operatórias em dois animais (16,6%), ambas com início de aparecimento em três dias após a confecção do aneurisma. Análise histológica dos aneurismas mostrou trombos ocluindo parcialmente a luz em nove suínos (75%). Nesses animais, observou-se que, em média, 9% da luz aneurismática estava preenchida por trombos. CONCLUSÃO: Pôde ser desenvolvido um modelo experimental estável de aneurisma sacular em carótida de suínos utilizando veia jugular interna.

OBJECTIVE: To develop an experimental model of stable saccular aneurysm in carotid of pigs using the internal jugular vein. METHODS: In 12 healthy pigs, weighing between 25 and 50kg, five males and seven females, we made a right common carotid artery aneurysm. After elliptical arteriotomy, we carried out a terminolateral anastomosis with the distal stump of the internal jugular vein. Aneurysm volume was calculated so that the value did not exceed 27 times the area of the arteriotomy. After six days angiography and microscopic examination were performed to assess patency of the aneurysm and the presence of total or partial thrombosis. RESULTS: There was a significant weight gain of pigs in the time interval between the manufacture of the aneurysm and angiography (p = 0.04). Aneurysmal patency was observed in ten pigs (83%). Operative wound infections occurred in two animals (16.6%), both with early onset, three days after the making of the aneurysm. Histological analysis showed aneurysm thrombus partially occluding the light in nine pigs (75%). In these animals, it was observed that on average 9% of the aneurysmal diameter was filled with thrombi. CONCLUSION: It was possible to develop a stable experimental model of saccular aneurysms in pig carotid artery by use of the internal jugular vein.