Inhibition of Bruton's Tyrosine Kinase Activity Attenuates Hemorrhagic Shock-Induced Multiple Organ Dysfunction in Rats.

OBJECTIVE: The aim of this study was to investigate (a) the potential of the Bruton's tyrosine kinase (BTK) inhibitors acalabrutinib and fenebrutinib to reduce multiple organ dysfunction syndrome (MODS) in acute (short-term and long-term follow-up) hemorrhagic shock (HS) rat models and (b) whether treatment with either acalabrutinib or fenebrutinib attenuates BTK, NF-κB and NLRP3 activation in HS. BACKGROUND: The MODS caused by an excessive systemic inflammatory response following trauma is associated with a high morbidity and mortality. The protein BTK is known to play a role in the activation of the NLRP3 inflammasome, which is a key component of the innate inflammatory response. However, its role in trauma-hemorrhage is unknown. METHODS: Acute HS rat models were performed to determine the influence of acalabrutinib or fenebrutinib on MODS. The activation of BTK, NF-κB and NLRP3 pathways were analyzed by western blot in the kidney. RESULTS: We demonstrated that (a) HS caused organ injury and/or dysfunction and hypotension (post-resuscitation) in rats, while (b) treatment of HS-rats with either acalabrutinib or fenebrutinib attenuated the organ injury and dysfunction in acute HS models and (c) reduced the activation of BTK, NF- kB and NLRP3 pathways in the kidney. CONCLUSION: Our results point to a role of BTK in the pathophysiology of organ injury and dysfunction caused by trauma/hemorrhage and indicate that BTK inhibitors may be repurposed as a potential therapeutic approach for MODS after trauma and/or hemorrhage.

SUMOylation in peripheral tissues under low perfusion-related pathological states.

SUMOylation is described as a posttranslational protein modification (PTM) that is involved in the pathophysiological processes underlying several conditions related to ischemia- and reperfusion-induced damage. Increasing evidence suggests that, under low oxygen levels, SUMOylation might be part of an endogenous mechanism, which is triggered by injury to protect cells within the central nervous system. However, the role of ischemia-induced SUMOylation in the periphery is still unclear. This article summarizes the results of recent studies regarding SUMOylation profiles in several diseases characterized by impaired blood flow to the cardiorenal, gastrointestinal, and respiratory systems. Our review shows that although ischemic injury per se does not always increase SUMOylation levels, as seen in strokes, it seems that in most cases the positive modulation of protein SUMOylation after peripheral ischemia might be a protective mechanism. This complex relationship warrants further investigation, as the role of SUMOylation during hypoxic conditions differs from organ to organ and is still not fully elucidated.

Resolvin D1 Attenuates the Organ Injury Associated With Experimental Hemorrhagic Shock.

OBJECTIVE: To evaluate the potential changes in the plasma levels of resolvin D1 (RvD1) in patients with trauma and hemorrhage. Having found that trauma results in a profound reduction in plasma RvD1 in patients, we have then investigated the effects of RvD1 on the organ injury and dysfunction associated with hemorrhagic shock (HS) in the rat. BACKGROUND: HS is a common cause of death in trauma due to excessive systemic inflammation and multiple organ failure. RvD1 is a member of the resolvin family of pro-resolution mediators. METHODS: Blood samples were drawn from critically injured patients (n = 27, ACITII-prospective observational cohort study) within 2 hours of injury for targeted liquid chromatography tandem mass spectrometry. HS rats (removal of blood to reduce arterial pressure to 30 ±â€Š2 mm Hg, 90 minutes, followed by resuscitation) were treated with RvD1 (0.3 or 1 µg/kg intravenous (i.v.)) or vehicle (n = 7). Parameters of organ injury and dysfunction were determined. RESULTS: Plasma levels of RvD1 (mg/dL) were reduced in patients with trauma+HS (0.17 ±â€Š0.08) when compared with healthy volunteers (0.76 ±â€Š0.25) and trauma patients (0.62 ±â€Š0.20). In rats with HS, RvD1 attenuated the kidney dysfunction, liver injury, and tissue ischemia. RvD1 also reduced activation of the nuclear factor (NF)-κB pathway and reduced the expression of pro-inflammatory proteins such as inducible nitric oxide synthase, tumor necrosis factor-α, interleukin-1ß, and interleukin-6. CONCLUSION: Plasma RvD1 is reduced in patients with trauma-HS. In rats with HS, administration of synthetic RvD1 on resuscitation attenuated the multiple organ failure associated with HS by a mechanism that involves inhibition of the activation of NF-κB.

Artesunate Protects Against the Organ Injury and Dysfunction Induced by Severe Hemorrhage and Resuscitation.

OBJECTIVE: To evaluate the effects of artesunate on organ injury and dysfunction associated with hemorrhagic shock (HS) in the rat. BACKGROUND: HS is still a common cause of death in severely injured patients and is characterized by impairment of organ perfusion, systemic inflammatory response, and multiple organ failure. There is no specific therapy that reduces organ injury/dysfunction. Artesunate exhibits pharmacological actions beyond its antimalarial activity, such as anticancer, antiviral, and anti-inflammatory effects. METHODS: Rats were submitted to HS. Mean arterial pressure was reduced to 30 mm Hg for 90 minutes, followed by resuscitation. Rats were randomly treated with artesunate (2.4 or 4.8 mg/kg i.v.) or vehicle upon resuscitation. Four hours later, parameters of organ injury and dysfunction were assessed. RESULTS: Artesunate attenuated the multiple organ injury and dysfunction caused by HS. Pathway analysis of RNA sequencing provided good evidence to support an effect of artesunate on the Akt-survival pathway, leading to downregulation of interleukin-1 receptor-associated kinase 1. Using Western blot analysis, we confirmed that treatment of HS rats with artesunate enhanced the phosphorylation (activation) of Protein kinase B (Akt) and endothelial nitric oxide synthase and the phosphorylation (inhibition) of glycogen synthase kinase-3ß (GSK-3ß). Moreover, artesunate attenuated the HS-induced activation of nuclear factor kappa B and reduced the expression of proinflammatory proteins (inducible nitric oxide synthase, tumor necrosis factor-α, and interleukin 6). CONCLUSIONS: Artesunate attenuated the organ injury/dysfunction associated with HS by a mechanism that involves the activation of the Akt-endothelial nitric oxide synthase survival pathway, and the inhibition of glycogen synthase kinase-3ß and nuclear factor kappa B. A phase II clinical trial evaluating the effects of good manufacturing practice-artesunate in patients with trauma and severe hemorrhage is planned.

Inhibition of IκB Kinase Attenuates the Organ Injury and Dysfunction Associated with Hemorrhagic Shock.

Nuclear factor-kappa B (NF-κB) activation is widely implicated in multiple organ failure (MOF); however, a direct inhibitor of IκB kinase (IKK), which plays a pivotal role in the activation of NF-κB, has not been investigated in shock. Thus, the aim of the present work was to investigate the effects of an IKK inhibitor on the MOF associated with hemorrhagic shock (HS). Therefore, rats were subjected to HS and were resuscitated with the shed blood. Rats were treated with the inhibitor of IKK or vehicle at resuscitation. Four hours later, blood and organs were assessed for organ injury and signaling events involved in the activation of NF-κB. Additionally, survival following serum deprivation was assessed in HK-2 cells treated with the inhibitor of IKK. HS resulted in renal dysfunction, lung, liver and muscular injury, and increases in serum inflammatory cytokines. Kidney and liver tissue from HS rats revealed increases in phosphorylation of IKKαß and IκBα, nuclear translocation of NF-κB and expression of inducible isoform of nitric oxide synthase (iNOS). IKK16 treatment upon resuscitation attenuated NF-κB activation and activated the Akt survival pathway, leading to a significant attenuation of all of the above parameters. Furthermore, IKK16 exhibited cytoprotective effects in human kidney cells. In conclusion, the inhibitor of IKK complex attenuated the MOF associated with HS. This effect may be due to the inhibition of the NF-κB pathway and activation of the survival kinase Akt. Thus, the inhibition of the IKK complex might be an effective strategy for the prevention of MOF associated with HS.

Rapid NOS-1-derived nitric oxide and peroxynitrite formation act as signaling agents for inducible NOS-2 expression in vascular smooth muscle cells.

Septic vascular dysfunction is characterized by hypotension and hyporeactivity to vasoconstrictors and nitric oxide (NO), reactive oxygen species and peroxynitrite have a prominent role in this condition. However, the mechanism whereby the vascular dysfunction is initiated is poorly understood. Based on previous studies of our group and the literature,we hypothesize that constitutive nitric oxide synthases (c-NOS) and peroxynitrite may play a role in the development of septic vascular dysfunction. Bacterial lipopolysaccharide (LPS) and interferon-γ (IFN) were used to stimulate rat aorta smooth muscle cells (A7r5) and rat aorta slices. This stimulation led to a rapid (within minutes) production of NO and superoxide anion, which led to peroxynitrite formation. When this rapid initial burst was reduced, through the inhibition of c-NOS and NADPH oxidases (NOX) or the scavenging of NO and superoxide the NF-κB activation, NOS-2 expression and nitrite production were significantly attenuated. Although vascular smooth muscle cells express both c-NOS isoforms, gene knockdown revealed that only NOS-1-dependent NO and peroxynitrite formation are important for the later NOS-2 expression. Similar findings were obtained by knockdown NOX-1 gene, one source of superoxide for peroxynitrite formation. Taking together, we show that smooth muscle cell activation by LPS/IFN leads to a rapid formation of NOS-1-derived NO and NOX-1-derived superoxide, forming peroxynitrite; and that this species act as a trigger for NOS-2 expression through NF-κB activation. Therefore, our findings suggest a critical role for NOS-1 and NOX-1 in the initiation of the vascular dysfunction associated with sepsis and septic shock.

EARLY BLOOD LACTATE AS A BIOMARKER FOR CARDIOVASCULAR COLLAPSE IN EXPERIMENTAL SEPSIS.

ABSTRACT: Cecal ligation and puncture (CLP) is the gold standard model for studying septic shock, which is characterized by hypotension and hyporeactivity to vasoconstrictors. However, approximately 30% of CLP animals do not exhibit cardiovascular changes, requiring more replicates because of the high variability of the model. Therefore, biomarkers enabling the early prediction of cardiovascular collapse in sepsis would greatly benefit sepsis nonclinical studies, refining experimental models and improving clinical translation. Thus, this study aimed to test whether the early increase in lactate levels could predict hypotension and hyporesponsiveness to vasoconstrictors in a rat model of sepsis. Male and female Wistar rats were subjected to CLP or sham procedure. Tail blood lactate was measured 6, 12, and 24 h after surgery. Then, inflammatory, biochemical, and hemodynamic parameters were evaluated. Rats subjected to CLP developed hypotension, hyporesponsiveness to vasoconstrictors, an intense inflammatory process, and increased plasma markers of organ dysfunction. By using receiver operating characteristics curve analysis, we have established that a lactate value of 2.45 mmol/L can accurately discriminate between a rat exhibiting a normal vasoconstrictive response and a vasoplegic rat with 84% accuracy (area under the curve: 0.84; confidence interval [CI]: 0.67-1.00). The sensitivity, which is the ability to identify a diseased rat (true positive), was 75% (CI: 41-95), and the true negative rate was 81% (CI: 57-93). Therefore, early measurement of lactate levels in sepsis could serve as a valuable biomarker for distinguishing vasoplegic rats from those exhibiting normal vasoconstrictive responses.

Cardiovascular effects of Roflumilast during sepsis: Risks or benefits?

BACKGROUND: Phosphodiesterase-4 (PDE4) is responsible for terminating cyclic adenosine monophosphate (cAMP) signalling. PDE4 inhibitors, such as roflumilast (RFM), have anti-inflammatory activity and have been studied in inflammation-induced tissue damage in sepsis. However, the role of RFM on cardiovascular derangements induced by sepsis is still unknown. Thus, we aimed to evaluate the potential effects of RFM on cardiovascular collapse and multiorgan damage caused by sepsis. METHODS: Sepsis was induced by cecal ligation and puncture (CLP) in male rats. Six hours after the CLP or sham procedure, animals were randomly assigned to receive either RFM (0.3 mg/kg) or vehicle subcutaneously, and cardiovascular parameters were assessed 24 h after the surgery and organ/plasma samples were collected for further analyses. RESULTS: Sepsis induced hypotension, tachycardia, reduced renal blood flow (RBF) and hyporeactivity to vasoconstrictors both in vivo and ex vivo. RFM treatment increased systemic cAMP levels and RBF. RFM also attenuated hypoperfusion and liver damage induced by CLP. Furthermore, RFM reduced systemic nitric oxide (NO) levels in septic rats, while there were no changes in hepatic NOS-2 expression. Nevertheless, RFM exacerbated sepsis-induced hypotension and tachycardia without ameliorating vascular hyporeactivity. CONCLUSION: Our data show that PDE-4 inhibition protects septic rats from hepatic injury and improves renal perfusion. However, RFM worsened hemodynamic parameters and showed no protection against sepsis-induced cardiovascular dysfunction and mortality. Thus, despite the anti-inflammatory benefits of RFM, its application in sepsis should be approached cautiously.

Renal protection after hemorrhagic shock in rats: Possible involvement of SUMOylation.

Hemorrhagic shock (HS), a leading cause of preventable death, is characterized by severe blood loss and inadequate tissue perfusion. Reoxygenation of ischemic tissues exacerbates organ damage through ischemia-reperfusion injury. SUMOylation has been shown to protect neurons after stroke and is upregulated in response to cellular stress. However, the role of SUMOylation in organ protection after HS is unknown. This study aimed to investigate SUMOylation-mediated organ protection following HS. Male Wistar rats were subjected to HS (blood pressure of 40 ± 2 mmHg, for 90 min) followed by reperfusion. Blood, kidney, and liver samples were collected at various time points after reperfusion to assess organ damage and investigate the profile of SUMO1 and SUMO2/3 conjugation. In addition, human kidney cells (HK-2), treated with the SUMOylation inhibitor TAK-981 or overexpressing SUMO proteins, were subjected to oxygen and glucose deprivation to investigate the role of SUMOylation in hypoxia/reoxygenation injury. The animals presented progressive multiorgan dysfunction, except for the renal system, which showed improvement over time. Compared to the liver, the kidneys displayed distinct patterns in terms of oxidative stress, apoptosis activation, and tissue damage. The global level of SUMO2/3 in renal tissue was also distinct, suggesting a differential role. Pharmacological inhibition of SUMOylation reduced cell viability after hypoxia-reoxygenation damage, while overexpression of SUMO1 or SUMO2 protected the cells. These findings suggest that SUMOylation might play a critical role in cellular protection during ischemia-reperfusion injury in the kidneys, a role not observed in the liver. This difference potentially explains the renal resilience observed in HS animals when compared to other systems.

Blockade of sympathetic ganglia improves vascular dysfunction in septic shock.

Sepsis/septic shock activates the sympathetic nervous system (SNS) to deal with the infection stress. However, an imbalanced or maladaptive response due to excessive or uncontrolled activation characterizes autonomic dysfunction. Our hypothesis was that reducing this excessive activation of the autonomic nervous system would impact positively in sepsis. Using ganglionic blockers as a pharmacological approach, the main aim of the present report was to assess the role of ganglionic transmission in the vascular dysfunction associated with sepsis.Sepsis was induced in rats by cecal ligation and puncture (CLP). One hour after CLP surgery, rats were treated subcutaneously with hexamethonium (15 mg/kg; ganglionic blocker), pentolinium (5 mg/kg; a blocker with a higher selectivity for sympathetic ganglia compared to hexamethonium), or vehicle (PBS). Basal blood pressure and the response to adrenergic agonists were evaluated at 6 and 24 h after CLP surgery. Reactivity to vasoconstrictors, nitric oxide (NO) synthase 2 (NOS-2) expression, IL-1 and TNF plasma levels, and density of α1 adrenergic receptors were evaluated in the aorta 24 h after CLP.Septic shock resulted in hypotension and hyporesponsiveness to norepinephrine and phenylephrine, increased plasma cytokine levels and NOS-2 expression in the aorta, and decreased α1 receptor density in the same vessel. Pentolinium but not hexamethonium recovered responsiveness and α1 adrenergic receptor density in the aorta. Both blockers normalized the in vivo response to vasoconstrictors, and reduced plasma IL-1 and NOx levels and NOS-2 expression in the aorta.Blockade of ganglionic sympathetic transmission reduced the vascular dysfunction in experimental sepsis. This beneficial effect seems to be, at least in part, due to the preservation of α1 adrenergic receptor density and to reduced NOS-2 expression and may lead to adjuvant ways to treat human sepsis.

Doxycycline reduces liver and kidney injuries in a rat hemorrhagic shock model.

BACKGROUND: Hemorrhagic shock (HS), which causes insufficient tissue perfusion, can result in multiple organ failure (MOF) and death. This study aimed to evaluate whether doxycycline (DOX) protects cardiovascular, kidney, and liver tissue from damage in a rat model of HS. Immediately before the resuscitation, DOX (10 mg/kg; i.v.) was administered, and its protective effects were assessed 24 h later. Mean arterial pressure, renal blood flow, heart rate, vasoactive drug response, and blood markers such as urea, creatinine, AST, ALT, CPK, CPR, and NOx levels were determined. RESULTS: We showed that DOX has a significant effect on renal blood flow and on urea, creatinine, AST, ALT, CPK, and NOx. Morphologically, DOX reduced the inflammatory process in the liver tissue. CONCLUSIONS: We conclude that DOX protects the liver and kidney against injury and dysfunction in a HS model and could be a strategy to reduce organ damage associated with ischemia-and-reperfusion injury.

Lung-brain crosstalk: Behavioral disorders and neuroinflammation in septic survivor mice.

Although studies have suggested an association between lung infections and increased risk of neuronal disorders (e.g., dementia, cognitive impairment, and depressive and anxious behaviors), its mechanisms remain unclear. Thus, an experimental mice model of pulmonary sepsis was developed to investigate the relationship between lung and brain inflammation. Male Swiss mice were randomly assigned to either pneumosepsis or control groups. Pneumosepsis was induced by intratracheal instillation of Klebsiella pneumoniae, while the control group received a buffer solution. The model's validation included assessing systemic markers, as well as tissue vascular permeability. Depression- and anxiety-like behaviors and cognitive function were assessed for 30 days in sepsis survivor mice, inflammatory profiles, including cytokine levels (lungs, hippocampus, and prefrontal cortex) and microglial activation (hippocampus), were examined. Pulmonary sepsis damaged distal organs, caused peripheral inflammation, and increased vascular permeability in the lung and brain, impairing the blood-brain barrier and resulting in bacterial dissemination. After sepsis induction, we observed an increase in myeloperoxidase activity in the lungs (up to seven days) and prefrontal cortex (up to 24 h), proinflammatory cytokines in the hippocampus and prefrontal cortex, and percentage of areas with cells positive for ionized calcium-binding adaptor molecule 1 (IBA-1) in the hippocampus. Also, depression- and anxiety-like behaviors and changes in short-term memory were observed even 30 days after sepsis induction, suggesting a crosstalk between inflammatory responses of lungs and brain.

Pneumonia-induced sepsis in mice: temporal study of inflammatory and cardiovascular parameters.

The aim of the present work is to provide a better comprehension of the pneumonia-induced sepsis model through temporal evaluation of several parameters, and thus identify the main factors that determine mortality in this model. Klebsiella pneumoniae was inoculated intratracheally in anesthetized Swiss male mice. Inflammatory and cardiovascular parameters were evaluated 6, 24 and 48 h after the insult. The results show that severity of infection and the mortality correlated with the amount of bacteria. Six, 24 and 48 h after inoculation, animals presented pathological changes in lungs, increase in cell number in the bronchoalveolar lavage, leukopenia, increase in TNF-α and IL-1ß levels, hypotension and hyporesponsiveness to vasoconstrictors, the two latter characteristics of severe sepsis and septic shock. Significant numbers of bacteria in spleen and heart homogenates indicated infection spreading. Interestingly, NOS-2 expression appeared late after bacteria inoculation, whereas levels of NOS-1 and NOS-3 were unchanged. The high NOS-2 expression coincided with an exacerbated NO production in the infection focus and in plasma, as judging by nitrate + nitrite levels. This study shows that K. pneumoniae inoculation induces a systemic inflammatory response and cardiovascular alterations, which endures at least until 48 h. K. pneumoniae-induced lung infection is a clinically relevant animal model of sepsis and a better understanding of this model may help to increase the knowledge about sepsis pathophysiology.

Cannabinoid CB2 receptor agonist reduces local and systemic inflammation associated with pneumonia-induced sepsis in mice.

Sepsis is a severe condition secondary to dysregulated host response to infection leading to tissue damage and organ dysfunction. Cannabinoid CB2 receptor has modulatory effects on the immune response. Therefore, this study investigated the effects of a cannabinoid CB2 receptor agonist on the local and systemic inflammatory process associated with pneumonia-induced sepsis. Pneumonia-induced sepsis was induced in mice by intratracheal inoculation of Klebsiella pneumoniae. Tissue and bronchoalveolar lavage (BAL) were collected 6, 24, or 48 h after surgery. Mice were treated with CB2 agonist (AM1241, 0.3 and 3 mg/kg, i.p.) and several parameters of inflammation were evaluated 24 h after sepsis induction. Polymorphonuclear cell migration to the infectious focus peaked 24 h after pneumonia-induced sepsis induction in male and female animals. Septic male mice presented a significant reduction of cannabinoid CB2 receptor density in the lung tissue after 24 h, which was not observed in females. CB2 expression in BAL macrophages was also reduced in septic animals. Treatment of septic mice with AM1241 reduced cell migration, local infection, myeloperoxidase activity, protein extravasation, and NOS-2 expression in the lungs. In addition, the treatment reduced plasma IL-1ß, increased IL-10 and reduced the severity and mortality of septic animals. These results suggest that AM1241 promotes an interesting balance in the inflammatory response, maintaining lung function and preventing organ injury. Therefore, cannabinoid CB2 receptors are potential targets to control the excessive inflammatory process that occurs in severe conditions, and agonists of these receptors can be considered promising adjuvants in pneumonia-induced sepsis treatment.

Neuraminidase is a host-directed approach to regulate neutrophil responses in sepsis and COVID-19.

BACKGROUND AND PURPOSE: Neutrophil overstimulation plays a crucial role in tissue damage during severe infections. Because pathogen-derived neuraminidase (NEU) stimulates neutrophils, we investigated whether host NEU can be targeted to regulate the neutrophil dysregulation observed in severe infections. EXPERIMENTAL APPROACH: The effects of NEU inhibitors on lipopolysaccharide (LPS)-stimulated neutrophils from healthy donors or COVID-19 patients were determined by evaluating the shedding of surface sialic acids, cell activation, and reactive oxygen species (ROS) production. Re-analysis of single-cell RNA sequencing of respiratory tract samples from COVID-19 patients also was carried out. The effects of oseltamivir on sepsis and betacoronavirus-induced acute lung injury were evaluated in murine models. KEY RESULTS: Oseltamivir and zanamivir constrained host NEU activity, surface sialic acid release, cell activation, and ROS production by LPS-activated human neutrophils. Mechanistically, LPS increased the interaction of NEU1 with matrix metalloproteinase 9 (MMP-9). Inhibition of MMP-9 prevented LPS-induced NEU activity and neutrophil response. In vivo, treatment with oseltamivir fine-tuned neutrophil migration and improved infection control as well as host survival in peritonitis and pneumonia sepsis. NEU1 also is highly expressed in neutrophils from COVID-19 patients, and treatment of whole-blood samples from these patients with either oseltamivir or zanamivir reduced neutrophil overactivation. Oseltamivir treatment of intranasally infected mice with the mouse hepatitis coronavirus 3 (MHV-3) decreased lung neutrophil infiltration, viral load, and tissue damage. CONCLUSION AND IMPLICATIONS: These findings suggest that interplay of NEU1-MMP-9 induces neutrophil overactivation. In vivo, NEU may serve as a host-directed target to dampen neutrophil dysfunction during severe infections.

The role of nitric oxide in sepsis-associated kidney injury.

Sepsis is one of the leading causes of acute kidney injury (AKI), and several mechanisms including microcirculatory alterations, oxidative stress, and endothelial cell dysfunction are involved. Nitric oxide (NO) is one of the common elements to all these mechanisms. Although all three nitric oxide synthase (NOS) isoforms are constitutively expressed within the kidneys, they contribute in different ways to nitrergic signaling. While the endothelial (eNOS) and neuronal (nNOS) isoforms are likely to be the main sources of NO under basal conditions and participate in the regulation of renal hemodynamics, the inducible isoform (iNOS) is dramatically increased in conditions such as sepsis. The overexpression of iNOS in the renal cortex causes a shunting of blood to this region, with consequent medullary ischemia in sepsis. Differences in the vascular reactivity among different vascular beds may also help to explain renal failure in this condition. While most of the vessels present vasoplegia and do not respond to vasoconstrictors, renal microcirculation behaves differently from nonrenal vascular beds, displaying similar constrictor responses in control and septic conditions. The selective inhibition of iNOS, without affecting other isoforms, has been described as the ideal scenario. However, iNOS is also constitutively expressed in the kidneys and the NO produced by this isoform is important for immune defense. In this sense, instead of a direct iNOS inhibition, targeting the NO effectors such as guanylate cyclase, potassium channels, peroxynitrite, and S-nitrosothiols, may be a more interesting approach in sepsis-AKI and further investigation is warranted.

Pharmacological Inhibition of FAK-Pyk2 Pathway Protects Against Organ Damage and Prolongs the Survival of Septic Mice.

Sepsis and septic shock are associated with high mortality and are considered one of the major public health concerns. The onset of sepsis is known as a hyper-inflammatory state that contributes to organ failure and mortality. Recent findings suggest a potential role of two non-receptor protein tyrosine kinases, namely Focal adhesion kinase (FAK) and Proline-rich tyrosine kinase 2 (Pyk2), in the inflammation associated with endometriosis, cancer, atherosclerosis and asthma. Here we investigate the role of FAK-Pyk2 in the pathogenesis of sepsis and the potential beneficial effects of the pharmacological modulation of this pathway by administering the potent reversible dual inhibitor of FAK and Pyk2, PF562271 (PF271) in a murine model of cecal ligation and puncture (CLP)-induced sepsis. Five-month-old male C57BL/6 mice underwent CLP or Sham surgery and one hour after the surgical procedure, mice were randomly assigned to receive PF271 (25 mg/kg, s.c.) or vehicle. Twenty-four hours after surgery, organs and plasma were collected for analyses. In another group of mice, survival rate was assessed every 12 h over the subsequent 5 days. Experimental sepsis led to a systemic cytokine storm resulting in the formation of excessive amounts of both pro-inflammatory cytokines (TNF-α, IL-1ß, IL-17 and IL-6) and the anti-inflammatory cytokine IL-10. The systemic inflammatory response was accompanied by high plasma levels of ALT, AST (liver injury), creatinine, (renal dysfunction) and lactate, as well as a high, clinical severity score. All parameters were attenuated following PF271 administration. Experimental sepsis induced an overactivation of FAK and Pyk2 in liver and kidney, which was associated to p38 MAPK activation, leading to increased expression/activation of several pro-inflammatory markers, including the NLRP3 inflammasome complex, the adhesion molecules ICAM-1, VCAM-1 and E-selectin and the enzyme NOS-2 and myeloperoxidase. Treatment with PF271 inhibited FAK-Pyk2 activation, thus blunting the inflammatory abnormalities orchestrated by sepsis. Finally, PF271 significantly prolonged the survival of mice subjected to CLP-sepsis. Taken together, our data show for the first time that the FAK-Pyk2 pathway contributes to sepsis-induced inflammation and organ injury/dysfunction and that the pharmacological modulation of this pathway may represents a new strategy for the treatment of sepsis.

Inhibition of Macrophage Migration Inhibitory Factor Activity Attenuates Haemorrhagic Shock-Induced Multiple Organ Dysfunction in Rats.

Objective: The aim of this study was to investigate (a) macrophage migration inhibitory factor (MIF) levels in polytrauma patients and rats after haemorrhagic shock (HS), (b) the potential of the MIF inhibitor ISO-1 to reduce multiple organ dysfunction syndrome (MODS) in acute (short-term and long-term follow-up) HS rat models and (c) whether treatment with ISO-1 attenuates NF-κB and NLRP3 activation in HS. Background: The MODS caused by an excessive systemic inflammatory response following trauma is associated with a high morbidity and mortality. MIF is a pleiotropic cytokine which can modulate the inflammatory response, however, its role in trauma is unknown. Methods: The MIF levels in plasma of polytrauma patients and serum of rats with HS were measured by ELISA. Acute HS rat models were performed to determine the influence of ISO-1 on MODS. The activation of NF-κB and NLRP3 pathways were analysed by western blot in the kidney and liver. Results: We demonstrated that (a) MIF levels are increased in polytrauma patients on arrival to the emergency room and in rats after HS, (b) HS caused organ injury and/or dysfunction and hypotension (post-resuscitation) in rats, while (c) treatment of HS-rats with ISO-1 attenuated the organ injury and dysfunction in acute HS models and (d) reduced the activation of NF-κB and NLRP3 pathways in the kidney and liver. Conclusion: Our results point to a role of MIF in the pathophysiology of trauma-induced organ injury and dysfunction and indicate that MIF inhibitors may be used as a potential therapeutic approach for MODS after trauma and/or haemorrhage.

Neuraminidase inhibitors rewire neutrophil function in vivo in murine sepsis and ex vivo in COVID-19.

Neutrophil overstimulation plays a crucial role in tissue damage during severe infections. Neuraminidase (NEU)-mediated cleavage of surface sialic acid has been demonstrated to regulate leukocyte responses. Here, we report that antiviral NEU inhibitors constrain host NEU activity, surface sialic acid release, ROS production, and NETs released by microbial-activated human neutrophils. In vivo, treatment with Oseltamivir results in infection control and host survival in peritonitis and pneumonia models of sepsis. Single-cell RNA sequencing re-analysis of publicly data sets of respiratory tract samples from critical COVID-19 patients revealed an overexpression of NEU1 in infiltrated neutrophils. Moreover, Oseltamivir or Zanamivir treatment of whole blood cells from severe COVID-19 patients reduces host NEU-mediated shedding of cell surface sialic acid and neutrophil overactivation. These findings suggest that neuraminidase inhibitors can serve as host-directed interventions to dampen neutrophil dysfunction in severe infections.

Angiotensin II receptor type 1 blockade improves hyporesponsiveness to vasopressors in septic shock.

Sepsis activates the renin-angiotensin system and the production of angiotensin II, which has a key role in the regulation of blood pressure through AT1 receptors. However, excessive activation of AT1 receptor is associated with deleterious effects. We investigated the consequences of a differential blockade of AT1 receptor caused by two doses of losartan (0.25 mg/kg or 15 mg/kg, s.c), a selective AT1 receptor antagonist on sepsis outcome. These doses reduced the effect of angiotensin II in normal rats by 30% and >90% 8 h after administration, respectively, but only the higher dose maintained its inhibitory effect (~70%) 24 h after injection. Sepsis was induced by cecal ligation and puncture (CLP). Losartan was injected 2 h after CLP and parameters were evaluated 6 and 24 h after CLP. Septic rats developed hypotension and hyporesponsiveness to vasoconstrictors, an intense inflammatory process and increase in plasma markers of organ dysfunction. The lower dose of losartan improved the vasoconstrictive response to phenylephrine and angiotensin II, reduced lung myeloperoxidase and prevented leukopenia 24 h after CLP, but it did not reduce NOS-2 expression, plasma IL-6 levels or organ injury parameters of septic rats. On the other hand, the higher dose of losartan worsened the response to vasoconstrictors, potentiated the hypotension and increased further levels of creatine, urea and lactate in septic rats. Therefore, an early and partial blockade of AT1 receptor with a low dose of losartan may counteract sepsis-induced refractoriness to vasoconstrictors thus providing an opportunity to improve the outcome of this condition.