Primary cilia as a targetable node between biliary injury, senescence and regeneration in liver transplantation.

BACKGROUND & AIMS: Biliary complications are a major cause of morbidity and mortality in liver transplantation. Up to 25% of patients that develop biliary complications require additional surgical procedures, re-transplantation or die in the absence of a suitable regraft. Here, we investigate the role of the primary cilium, a highly-specialised sensory organelle, in biliary injury leading to post-transplant biliary complications. METHODS: Human biopsies were used to study the structure and function of primary cilia in liver transplant recipients that develop biliary complications (N=7) in comparison with recipients without biliary complications (N=12). To study the biological effects of the primary cilia during transplantation, we generated murine models that recapitulate liver procurement and cold storage, and assessed the elimination of the primary cilia in biliary epithelial cells in the K19CreERTKif3aflox/flox mouse model. To explore the molecular mechanisms responsible for the observed phenotypes we used in vitro models of ischemia, cellular senescence and primary cilia ablation. Finally, we used pharmacological and genetic approaches to target cellular senescence and the primary cilia, both in mouse models and discarded human donor livers. RESULTS: Prolonged ischemic periods before transplantation result in ciliary shortening and cellular senescence, an irreversible cell cycle arrest that blocks regeneration. Our results indicate that primary cilia damage results in biliary injury and a loss of regenerative potential. Senescence negatively impacts primary cilia structure and triggers a negative feedback loop that further impairs regeneration. Finally, we explore how targeted interventions for cellular senescence and/or the stabilisation of the primary cilia improve biliary regeneration following ischemic injury. CONCLUSIONS: Primary cilia play an essential role in biliary regeneration and we demonstrate that senolytics and cilia-stabilising treatments provide a potential therapeutic opportunity to reduce the rate of biliary complications and improve clinical outcomes in liver transplantation. IMPACT AND IMPLICATIONS: Up to 25% of liver transplants result in biliary complications, leading to additional surgery, retransplants, or death. We found that the incidence of biliary complications is increased by damage to the primary cilium, an antenna that protrudes from the cell and is key to regeneration. Here, we show that treatments that preserve the primary cilia during the transplant process provide a potential solution to reduce the rates of biliary complications.

NRF2 Activation Reprograms Defects in Oxidative Metabolism to Restore Macrophage Function in Chronic Obstructive Pulmonary Disease.

Rationale: Chronic obstructive pulmonary disease (COPD) is a disease characterized by persistent airway inflammation and disordered macrophage function. The extent to which alterations in macrophage bioenergetics contribute to impaired antioxidant responses and disease pathogenesis has yet to be fully delineated. Objectives: Through the study of COPD alveolar macrophages (AMs) and peripheral monocyte-derived macrophages (MDMs), we sought to establish if intrinsic defects in core metabolic processes drive macrophage dysfunction and redox imbalance. Methods: AMs and MDMs from donors with COPD and healthy donors underwent functional, metabolic, and transcriptional profiling. Measurements and Main Results: We observed that AMs and MDMs from donors with COPD display a critical depletion in glycolytic- and mitochondrial respiration-derived energy reserves and an overreliance on glycolysis as a source for ATP, resulting in reduced energy status. Defects in oxidative metabolism extend to an impaired redox balance associated with defective expression of the NADPH-generating enzyme, ME1 (malic enzyme 1), a known target of the antioxidant transcription factor NRF2 (nuclear factor erythroid 2-related factor 2). Consequently, selective activation of NRF2 resets the COPD transcriptome, resulting in increased generation of TCA cycle intermediaries, improved energetic status, favorable redox balance, and recovery of macrophage function. Conclusions: In COPD, an inherent loss of metabolic plasticity leads to metabolic exhaustion and reduced redox capacity, which can be rescued by activation of the NRF2 pathway. Targeting these defects, via NRF2 augmentation, may therefore present an attractive therapeutic strategy for the treatment of the aberrant airway inflammation described in COPD.

The emerging role for metabolism in fueling neutrophilic inflammation.

Neutrophils are a critical element of host defense and are rapidly recruited to inflammatory sites. Such sites are frequently limited in oxygen and/or nutrient availability, presenting a metabolic challenge for infiltrating cells. Long believed to be uniquely dependent on glycolysis, it is now clear that neutrophils possess far greater metabolic plasticity than previously thought, with the capacity to generate energy stores and utilize extracellular proteins to fuel central carbon metabolism and biosynthetic activity. Out-with cellular energetics, metabolic programs have also been implicated in the production of neutrophils and their progenitors in the bone marrow compartment, activation of neutrophil antimicrobial responses, inflammatory and cell survival signaling cascades, and training of the innate immune response. Thus, understanding the mechanisms by which metabolic processes sustain changes in neutrophil effector functions and how these are subverted in disease states provides exciting new avenues for the treatment of dysfunctional neutrophilic inflammation which are lacking in clinical practice to date.

Feasibility of Using Detuned Laser as a Placebo In Manual Therapy Research: An Analysis of Participant Perceptions.

OBJECTIVE: The purpose of this study was to determine the feasibility of using detuned laser as a placebo intervention in manual therapy research. METHODS: We performed a secondary data analysis of a randomized controlled trial. In our analysis, 30 participants with chronic ankle instability (manual therapy group: n = 13, age = 33.1 ± 8.1 years, female participants = 50%; detuned laser group: n = 17, age = 31.9 ± 11.8 years, female participants = 72%) were asked to indicate which intervention (manual therapy [active] or detuned laser [placebo]), they thought they had received and to give a confidence rating on their response regarding the received intervention at the conclusion of the course of intervention. Independent t tests were used to compare the groups. Participants in both groups were asked the following open-ended question: "What did you think of the intervention?". RESULTS: There were 52.9% participants in the detuned laser group and 53.8% participants in the manual therapy group who perceived that they had received the active intervention. The confidence ratings about their perceptions (6.7 ± 2.0, detuned laser group; 6.3 ± 2.4, manual therapy group) (P = .66) and the self-reported recovery ratings (1.9 ± 1.5 and 1.8 ± 1.2, respectively) (P = .77) were similar. CONCLUSIONS: Participants in this study confidently perceived that detuned laser was an active intervention. They positively rated their recovery following the course of the placebo intervention and perceived that detuned laser was effective in treating their condition. Therefore, it is feasible for detuned laser to be used as a placebo for manual therapy trials.

Hypoxia shapes the immune landscape in lung injury and promotes the persistence of inflammation.

Hypoxemia is a defining feature of acute respiratory distress syndrome (ARDS), an often-fatal complication of pulmonary or systemic inflammation, yet the resulting tissue hypoxia, and its impact on immune responses, is often neglected. In the present study, we have shown that ARDS patients were hypoxemic and monocytopenic within the first 48 h of ventilation. Monocytopenia was also observed in mouse models of hypoxic acute lung injury, in which hypoxemia drove the suppression of type I interferon signaling in the bone marrow. This impaired monopoiesis resulted in reduced accumulation of monocyte-derived macrophages and enhanced neutrophil-mediated inflammation in the lung. Administration of colony-stimulating factor 1 in mice with hypoxic lung injury rescued the monocytopenia, altered the phenotype of circulating monocytes, increased monocyte-derived macrophages in the lung and limited injury. Thus, tissue hypoxia altered the dynamics of the immune response to the detriment of the host and interventions to address the aberrant response offer new therapeutic strategies for ARDS.

Coagulation factor V is a T-cell inhibitor expressed by leukocytes in COVID-19.

Clotting Factor V (FV) is primarily synthesized in the liver and when cleaved by thrombin forms pro-coagulant Factor Va (FVa). Using whole blood RNAseq and scRNAseq of peripheral blood mononuclear cells, we find that FV mRNA is expressed in leukocytes, and identify neutrophils, monocytes, and T regulatory cells as sources of increased FV in hospitalized patients with COVID-19. Proteomic analysis confirms increased FV in circulating neutrophils in severe COVID-19, and immunofluorescence microscopy identifies FV in lung-infiltrating leukocytes in COVID-19 lung disease. Increased leukocyte FV expression in severe disease correlates with T-cell lymphopenia. Both plasma-derived and a cleavage resistant recombinant FV, but not thrombin cleaved FVa, suppress T-cell proliferation in vitro. Anticoagulants that reduce FV conversion to FVa, including heparin, may have the unintended consequence of suppressing the adaptive immune system.

Neutrophil HIF-1α stabilization is augmented by mitochondrial ROS produced via the glycerol 3-phosphate shuttle.

Neutrophils are predominantly glycolytic cells that derive little ATP from oxidative phosphorylation; however, they possess an extensive mitochondrial network and maintain a mitochondrial membrane potential. Although studies have shown neutrophils need their mitochondria to undergo apoptosis and regulate NETosis, the metabolic role of the respiratory chain in these highly glycolytic cells is still unclear. Recent studies have expanded on the role of reactive oxygen species (ROS) released from the mitochondria as intracellular signaling molecules. Our study shows that neutrophils can use their mitochondria to generate ROS and that mitochondrial ROS release is increased in hypoxic conditions. This is needed for the stabilization of a high level of the critical hypoxic response factor and pro-survival protein HIF-1α in hypoxia. Further, we demonstrate that neutrophils use the glycerol 3-phosphate pathway as a way of directly regulating mitochondrial function through glycolysis, specifically to maintain polarized mitochondria and produce ROS. This illustrates an additional pathway by which neutrophils can regulate HIF-1α stability and will therefore be an important consideration when looking for treatments of inflammatory conditions in which HIF-1α activation and neutrophil persistence at the site of inflammation are linked to disease severity.

The transcription factor EGR2 is indispensable for tissue-specific imprinting of alveolar macrophages in health and tissue repair.

Alveolar macrophages are the most abundant macrophages in the healthy lung where they play key roles in homeostasis and immune surveillance against airborne pathogens. Tissue-specific differentiation and survival of alveolar macrophages rely on niche-derived factors, such as granulocyte-macrophage colony-stimulating factor (GM-CSF) and transforming growth factor­ß (TGF-ß). However, the nature of the downstream molecular pathways that regulate the identity and function of alveolar macrophages and their response to injury remain poorly understood. Here, we identify that the transcription factor EGR2 is an evolutionarily conserved feature of lung alveolar macrophages and show that cell-intrinsic EGR2 is indispensable for the tissue-specific identity of alveolar macrophages. Mechanistically, we show that EGR2 is driven by TGF-ß and GM-CSF in a PPAR-γ­dependent manner to control alveolar macrophage differentiation. Functionally, EGR2 was dispensable for the regulation of lipids in the airways but crucial for the effective handling of the respiratory pathogen Streptococcus pneumoniae. Last, we show that EGR2 is required for repopulation of the alveolar niche after sterile, bleomycin-induced lung injury and demonstrate that EGR2-dependent, monocyte-derived alveolar macrophages are vital for effective tissue repair after injury. Collectively, we demonstrate that EGR2 is an indispensable component of the transcriptional network controlling the identity and function of alveolar macrophages in health and disease.

-------A type I IFN, prothrombotic hyperinflammatory neutrophil signature is distinct for COVID-19 ARDS--.

Background: Acute respiratory distress syndrome (ARDS) is a severe critical condition with a high mortality that is currently in focus given that it is associated with mortality caused by coronavirus disease 2019 (COVID-19). Neutrophils play a key role in the lung injury characteristic of non-COVID-19 ARDS and there is also accumulating evidence of neutrophil mediated lung injury in patients who succumb to infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Methods: We undertook a functional proteomic and metabolomic survey of circulating neutrophil populations, comparing patients with COVID-19 ARDS and non-COVID-19 ARDS to understand the molecular basis of neutrophil dysregulation. Results: Expansion of the circulating neutrophil compartment and the presence of activated low and normal density mature and immature neutrophil populations occurs in ARDS, irrespective of cause. Release of neutrophil granule proteins, neutrophil activation of the clotting cascade and upregulation of the Mac-1 platelet binding complex with formation of neutrophil platelet aggregates is exaggerated in COVID-19 ARDS. Importantly, activation of components of the neutrophil type I interferon responses is seen in ARDS following infection with SARS-CoV-2, with associated rewiring of neutrophil metabolism, and the upregulation of antigen processing and presentation. Whilst dexamethasone treatment constricts the immature low density neutrophil population, it does not impact upon prothrombotic hyperinflammatory neutrophil signatures. Conclusions: Given the crucial role of neutrophils in ARDS and the evidence of a disordered myeloid response observed in COVID-19 patients, this work maps the molecular basis for neutrophil reprogramming in the distinct clinical entities of COVID-19 and non-COVID-19 ARDS.

Macrophage miR-210 induction and metabolic reprogramming in response to pathogen interaction boost life-threatening inflammation.

Unbalanced immune responses to pathogens can be life-threatening although the underlying regulatory mechanisms remain unknown. Here, we show a hypoxia-inducible factor 1α-dependent microRNA (miR)-210 up-regulation in monocytes and macrophages upon pathogen interaction. MiR-210 knockout in the hematopoietic lineage or in monocytes/macrophages mitigated the symptoms of endotoxemia, bacteremia, sepsis, and parasitosis, limiting the cytokine storm, organ damage/dysfunction, pathogen spreading, and lethality. Similarly, pharmacologic miR-210 inhibition improved the survival of septic mice. Mechanistically, miR-210 induction in activated macrophages supported a switch toward a proinflammatory state by lessening mitochondria respiration in favor of glycolysis, partly achieved by downmodulating the iron-sulfur cluster assembly enzyme ISCU. In humans, augmented miR-210 levels in circulating monocytes correlated with the incidence of sepsis, while serum levels of monocyte/macrophage-derived miR-210 were associated with sepsis mortality. Together, our data identify miR-210 as a fine-tuning regulator of macrophage metabolism and inflammatory responses, suggesting miR-210-based therapeutic and diagnostic strategies.

Hypoxia drives murine neutrophil protein scavenging to maintain central carbon metabolism.

Limiting dysfunctional neutrophilic inflammation while preserving effective immunity requires a better understanding of the processes that dictate neutrophil function in the tissues. Quantitative mass-spectrometry identified how inflammatory murine neutrophils regulated expression of cell surface receptors, signal transduction networks, and metabolic machinery to shape neutrophil phenotypes in response to hypoxia. Through the tracing of labeled amino acids into metabolic enzymes, proinflammatory mediators, and granule proteins, we demonstrated that ongoing protein synthesis shapes the neutrophil proteome. To maintain energy supplies in the tissues, neutrophils consumed extracellular proteins to fuel central carbon metabolism. The physiological stresses of hypoxia and hypoglycemia, characteristic of inflamed tissues, promoted this extracellular protein scavenging with activation of the lysosomal compartment, further driving exploitation of the protein-rich inflammatory milieu. This study provides a comprehensive map of neutrophil proteomes, analysis of which has led to the identification of active catabolic and anabolic pathways that enable neutrophils to sustain synthetic and effector functions in the tissues.

Neutrophils Fuel Effective Immune Responses through Gluconeogenesis and Glycogenesis.

Neutrophils can function and survive in injured and infected tissues, where oxygen and metabolic substrates are limited. Using radioactive flux assays and LC-MS tracing with U-13C glucose, glutamine, and pyruvate, we observe that neutrophils require the generation of intracellular glycogen stores by gluconeogenesis and glycogenesis for effective survival and bacterial killing. These metabolic adaptations are dynamic, with net increases in glycogen stores observed following LPS challenge or altitude-induced hypoxia. Neutrophils from patients with chronic obstructive pulmonary disease have reduced glycogen cycling, resulting in impaired function. Metabolic specialization of neutrophils may therefore underpin disease pathology and allow selective therapeutic targeting.

Semaphorin 3F signaling actively retains neutrophils at sites of inflammation.

Neutrophilic inflammation is central to disease pathogenesis, for example, in chronic obstructive pulmonary disease, yet the mechanisms that retain neutrophils within tissues remain poorly understood. With emerging evidence that axon guidance factors can regulate myeloid recruitment and that neutrophils can regulate expression of a class 3 semaphorin, SEMA3F, we investigated the role of SEMA3F in inflammatory cell retention within inflamed tissues. We observed that neutrophils upregulate SEMA3F in response to proinflammatory mediators and following neutrophil recruitment to the inflamed lung. In both zebrafish tail injury and murine acute lung injury models of neutrophilic inflammation, overexpression of SEMA3F delayed inflammation resolution with slower neutrophil migratory speeds and retention of neutrophils within the tissues. Conversely, constitutive loss of sema3f accelerated egress of neutrophils from the tail injury site in fish, whereas neutrophil-specific deletion of Sema3f in mice resulted in more rapid neutrophil transit through the airways, and significantly reduced time to resolution of the neutrophilic response. Study of filamentous-actin (F-actin) subsequently showed that SEMA3F-mediated retention is associated with F-actin disassembly. In conclusion, SEMA3F signaling actively regulates neutrophil retention within the injured tissues with consequences for neutrophil clearance and inflammation resolution.

Hypoxia Modulates Platelet Purinergic Signalling Pathways.

BACKGROUND: Hypoxia resulting from ascent to high-altitude or pathological states at sea level is known to increase platelet reactivity. Previous work from our group has suggested that this may be adenosine diphosphate (ADP)-specific. Given the clinical importance of drugs targeting ADP pathways, research into the impact of hypoxia on platelet ADP pathways is highly important. METHODS: Optimul aggregometry was performed on plasma from 29 lowland residents ascending to 4,700 m, allowing systematic assessment of platelet reactivity in response to several platelet agonists. Aggregometry was also performed in response to ADP in the presence of inhibitors of the two main ADP receptors, P2Y1 and P2Y12 (MRS2500 and cangrelor, respectively). Phosphorylation of vasodilator-stimulated phosphoprotein (VASP), a key determinant of platelet aggregation, was analysed using the VASPFix assay. RESULTS: Hypobaric hypoxia significantly reduced the ability of a fixed concentration of cangrelor to inhibit ADP-induced aggregation and increased basal VASP phosphorylation. However, in the absence of P2Y receptor inhibitors, we did not find evidence of increased platelet sensitivity to any of the agonists tested and found reduced sensitivity to thrombin receptor-activating peptide-6 amide. CONCLUSION: Our results provide evidence of increased P2Y1 receptor activity at high altitude and suggest down-regulation of the P2Y12 pathway through increased VASP phosphorylation. These changes in ADP pathway activity are of potential therapeutic significance to high-altitude sojourners and hypoxic sea level patients prescribed platelet inhibitors and warrant further investigation.

Hypoxia and the regulation of myeloid cell metabolic imprinting: consequences for the inflammatory response.

Inflamed and infected tissue sites are characterised by oxygen and nutrient deprivation. The cellular adaptations to insufficient oxygenation, hypoxia, are mainly regulated by a family of transcription factors known as hypoxia-inducible factors (HIFs). The protein members of the HIF signalling pathway are critical regulators of both the innate and adaptive immune responses, and there is an increasing body of evidence to suggest that the elicited changes occur through cellular metabolic reprogramming. Here, we review the literature on innate immunometabolism to date and discuss the role of hypoxia in innate cell metabolic reprogramming, and how this determines immune responses.

IL4Rα Signaling Abrogates Hypoxic Neutrophil Survival and Limits Acute Lung Injury Responses In Vivo.

Rationale: Acute respiratory distress syndrome is defined by the presence of systemic hypoxia and consequent on disordered neutrophilic inflammation. Local mechanisms limiting the duration and magnitude of this neutrophilic response remain poorly understood. Objectives: To test the hypothesis that during acute lung inflammation tissue production of proresolution type 2 cytokines (IL-4 and IL-13) dampens the proinflammatory effects of hypoxia through suppression of HIF-1α (hypoxia-inducible factor-1α)-mediated neutrophil adaptation, resulting in resolution of lung injury. Methods: Neutrophil activation of IL4Ra (IL-4 receptor α) signaling pathways was explored ex vivo in human acute respiratory distress syndrome patient samples, in vitro after the culture of human peripheral blood neutrophils with recombinant IL-4 under conditions of hypoxia, and in vivo through the study of IL4Ra-deficient neutrophils in competitive chimera models and wild-type mice treated with IL-4. Measurements and Main Results: IL-4 was elevated in human BAL from patients with acute respiratory distress syndrome, and its receptor was identified on patient blood neutrophils. Treatment of human neutrophils with IL-4 suppressed HIF-1α-dependent hypoxic survival and limited proinflammatory transcriptional responses. Increased neutrophil apoptosis in hypoxia, also observed with IL-13, required active STAT signaling, and was dependent on expression of the oxygen-sensing prolyl hydroxylase PHD2. In vivo, IL-4Ra-deficient neutrophils had a survival advantage within a hypoxic inflamed niche; in contrast, inflamed lung treatment with IL-4 accelerated resolution through increased neutrophil apoptosis. Conclusions: We describe an important interaction whereby IL4Rα-dependent type 2 cytokine signaling can directly inhibit hypoxic neutrophil survival in tissues and promote resolution of neutrophil-mediated acute lung injury.