Macrophage Necroptosis Research Articles

Pseudomonas aeruginosa Mediates Host Necroptosis through Rhl-Pqs Quorum Sensing Interaction.

Pseudomonas aeruginosa (P. aeruginosa) is an opportunistic pathogen that can cause serious infections in immunocompromised patients. Quorum sensing (QS), a communication system evolved by P. aeruginosa to survey its density, is well acknowledged to be involved in various activities during bacterial infection. Recent studies have revealed the link between P. aeruginosa QS and host innate immune response. Previous evidence suggests that programmed cell death exists in response to P. aeruginosa infection. However, it remains unclear whether QS plays a role in the host programmed cell death process during the infection. In this study, we found that the deficiency of one of QS subsystems, rhl, markedly increased mouse bone marrow macrophage cell death induced by P. aeruginosa, which was accompanied by elevated phosphorylation of RIPK3 and MLKL. This highly increased necroptosis activation was caused by the upregulation of another QS subsystem, pqs, because the deletion of pqs in rhl-deficient P. aeruginosa abolished macrophage necroptosis invitro and invivo. In sum, our data highlight the cross-talk between P. aeruginosa QS and host necroptosis, which is executed through the rhl-pqs axis.

ASFV infection induces macrophage necroptosis and releases proinflammatory cytokine by ZBP1-RIPK3-MLKL necrosome activation.

African swine fever (ASF) is an infectious disease characterized by hemorrhagic fever, which is highly pathogenic and causes severe mortality in domestic pigs. It is caused by the African swine fever virus (ASFV). ASFV is a large DNA virus and primarily infects porcine monocyte macrophages. The interaction between ASFV and host macrophages is the major reason for gross pathological lesions caused by ASFV. Necroptosis is an inflammatory programmed cell death and plays an important immune role during virus infection. However, whether and how ASFV induces macrophage necroptosis and the effect of necroptosis signaling on host immunity and ASFV infection remains unknown. This study uncovered that ASFV infection activates the necroptosis signaling in vivo and macrophage necroptosis in vitro. Further evidence showed that ASFV infection upregulates the expression of ZBP1 and RIPK3 to consist of the ZBP1-RIPK3-MLKL necrosome and further activates macrophage necroptosis. Subsequently, multiple Z-DNA sequences were predicted to be present in the ASFV genome. The Z-DNA signals were further confirmed to be present and colocalized with ZBP1 in the cytoplasm and nucleus of ASFV-infected cells. Moreover, ZBP1-mediated macrophage necroptosis provoked the extracellular release of proinflammatory cytokines, including TNF-α and IL-1β induced by ASFV infection. Finally, we demonstrated that ZBP1-mediated necroptosis signaling inhibits ASFV replication in host macrophages. Our findings uncovered a novel mechanism by which ASFV induces macrophage necroptosis by facilitating Z-DNA accumulation and ZBP1 necrosome assembly, providing significant insights into the pathogenesis of ASFV infection.

The anti-inflammatory role of zDHHC23 through the promotion of macrophage M2 polarization and macrophage necroptosis in large yellow croaker (Larimichthys crocea).

Zinc finger Asp-His-His-Cys motif-containing (zDHHC) proteins, known for their palmitoyltransferase (PAT) activity, play crucial roles in diverse cellular processes, including immune regulation. However, their non-palmitoyltransferase immunomodulatory functions and involvement in teleost immune responses remain underexplored. In this study, we systematically characterized the zDHHC family in the large yellow croaker (Larimichthys crocea), identifying 22 members. Phylogenetic analysis unveiled that each of the 22 LczDHHCs formed distinct clusters with their orthologues from other teleost species. Furthermore, all LczDHHCs exhibited a highly conserved DHHC domain, as confirmed by tertiary structure prediction. Notably, LczDHHC23 exhibited the most pronounced upregulation following Pseudomonas plecoglossicida (P. plecoglossicida) infection of macrophage/monocyte cells (MO/MΦ). Silencing LczDHHC23 led to heightened pro-inflammatory cytokine expression and diminished anti-inflammatory cytokine levels in MO/MΦ during infection, indicating its anti-inflammatory role. Functionally, LczDHHC23 facilitated M2-type macrophage polarization, as evidenced by a significant skewing of MO/MΦ towards the pro-inflammatory M1 phenotype upon LczDHHC23 knockdown, along with the inhibition of MO/MΦ necroptosis induced by P. plecoglossicida infection. These findings highlight the non-PAT immunomodulatory function of LczDHHC23 in teleost immune regulation, broadening our understanding of zDHHC proteins in host-pathogen interactions, suggesting LczDHHC23 as a potential therapeutic target for immune modulation in aquatic species.

Acute exposure to polystyrene nanoparticles promotes liver injury by inducing mitochondrial ROS-dependent necroptosis and augmenting macrophage-hepatocyte crosstalk

BackgroundThe global use of plastic materials has undergone rapid expansion, resulting in the substantial generation of degraded and synthetic microplastics and nanoplastics (MNPs), which have the potential to impose significant environmental burdens and cause harmful effects on living organisms. Despite this, the detrimental impacts of MNPs exposure towards host cells and tissues have not been thoroughly characterized.ResultsIn the present study, we have elucidated a previously unidentified hepatotoxic effect of 20 nm synthetic polystyrene nanoparticles (PSNPs), rather than larger PS beads, by selectively inducing necroptosis in macrophages. Mechanistically, 20 nm PSNPs were rapidly internalized by macrophages and accumulated in the mitochondria, where they disrupted mitochondrial integrity, leading to heightened production of mitochondrial reactive oxygen species (mtROS). This elevated mtROS generation essentially triggered necroptosis in macrophages, resulting in enhanced crosstalk with hepatocytes, ultimately leading to hepatocyte damage. Additionally, it was demonstrated that PSNPs induced necroptosis and promoted acute liver injury in mice. This harmful effect was significantly mitigated by the administration of a necroptosis inhibitor or systemic depletion of macrophages prior to PSNPs injection.ConclusionCollectively, our study suggests a profound toxicity of environmental PSNP exposure by triggering macrophage necroptosis, which in turn induces hepatotoxicity via intercellular crosstalk between macrophages and hepatocytes in the hepatic microenvironment.

Hydrogen sulfide regulates macrophage polarization and necroptosis to accelerate diabetic skin wound healing

Hydrogen sulfide (H2S), recognized as the third gasotransmitter, plays a pivotal role in the pathophysiological processes of various diseases. Cystathionine γ-lyase (CSE) is the main enzyme for H2S production in the skin. However, effects and mechanisms of H2S in diabetic skin wound healing remain unclear. Our findings revealed a decrease in plasma H2S content in diabetic patients with skin wounds. CSE knockout (KO) diabetic mice resulted in delayed wound healing, reduced blood perfusion, and CD31 expression around the wounds. It also led to increased infiltration of inflammatory cells and M1-type macrophages, decreased collagen levels, α-smooth muscle actin (α-SMA), and proliferating cell nuclear antigen (PCNA) expression. Additionally, there were enhanced expressions of necroptosis related proteins, including receptor interacting protein kinase 1 (RIPK1), RIPK3 and mixed lineage kinase domain like protein (MLKL). In comparison, sodium hydrosulfide (NaHS), H2S donor, accelerated skin wound healing in leptin receptor deficiency (db/db) mice. This acceleration was accompanied by increased blood perfusion and CD31 expression, reduced infiltration of inflammatory cells and M1-type macrophages, elevated collagen levels, α-SMA, and PCNA expressions, and decreased necroptosis-related protein expressions together with nuclear factor-κB (NF-κB) p65 phosphorylation. In conclusion, H2S regulates macrophage polarization and necroptosis, contributing to the acceleration of diabetic skin wound healing. These findings offer a novel strategy for the treatment of diabetic skin wounds.

MiR-223-3p Prevents Necroptotic Macrophage Death by Targeting Ripk3 in a Negative Feedback Loop and Consequently Ameliorates Advanced Atherosclerosis.

The formation of large necrotic cores results in vulnerable atherosclerotic plaques, which can lead to severe cardiovascular diseases. However, the specific regulatory mechanisms underlying the development of necrotic cores remain unclear. To evaluate how the modes of lesional cell death are reprogrammed during the development of atherosclerosis, the expression levels of key proteins that are involved in the necroptotic, apoptotic, and pyroptotic pathways were compared between different stages of plaques in humans and mice. Luciferase assays and loss-of-function studies were performed to identify the microRNA-mediated regulatory mechanism that protects foamy macrophages from necroptotic cell death. The role of this mechanism in atherosclerosis was determined by using a knockout mouse model with perivascular drug administration and tail vein injection of microRNA inhibitors in Apoe-/- mice. Here, we demonstrate that the necroptotic, rather than the apoptotic or pyroptotic, pathway is more activated in advanced unstable plaques compared with stable plaques in both humans and mice, which closely correlates with necrotic core formation. The upregulated expression of Ripk3 (receptor-interacting protein kinase 3) promotes the C/EBPβ (CCAAT/enhancer binding protein beta)-dependent transcription of the microRNA miR-223-3p, which conversely inhibits Ripk3 expression and forms a negative feedback loop to regulate the necroptosis of foamy macrophages. The knockout of the Mir223 gene in bone marrow cells accelerates atherosclerosis in Apoe-/- mice, but this effect can be rescued by Ripk3 deficiency or treatment with the necroptosis inhibitors necrostatin-1 and GSK-872. Like the Mir223 knockout, treating Apoe-/- mice with miR-223-3p inhibitors increases atherosclerosis. Our study suggests that miR-223-3p expression in macrophages protects against atherosclerotic plaque rupture by limiting the formation of necrotic cores, thus providing a potential microRNA therapeutic candidate for atherosclerosis.

Anti-Necroptotic Effects of Itaconate and its Derivatives.

Itaconate is an unsaturated dicarboxylic acid that is derived from the decarboxylation of the Krebs cycle intermediate cis-aconitate and has been shown to exhibit anti-inflammatory and anti-bacterial/viral properties. But the mechanisms underlying itaconate's anti-inflammatory activities are not fully understood. Necroptosis, a lytic form of regulated cell death (RCD), is mediated by receptor-interacting protein kinase 1 (RIPK1), RIPK3, and mixed lineage kinase domain-like protein (MLKL) signaling. It has been involved in the pathogenesis of organ injury in many inflammatory diseases. In this study, we aimed to explore whether itaconate and its derivatives can inhibit necroptosis in murine macrophages, a mouse MPC-5 cell line and a human HT-29 cell line in response to different necroptotic activators. Our results showed that itaconate and its derivatives dose-dependently inhibited necroptosis, among which dimethyl itaconate (DMI) was the most effective one. Mechanistically, itaconate and its derivatives inhibited necroptosis by suppressing the RIPK1/RIPK3/MLKL signaling and the oligomerization of MLKL. Furthermore, DMI promoted the nuclear translocation of Nrf2 that is a critical regulator of intracellular redox homeostasis, and reduced the levels of intracellular reactive oxygen species (ROS) and mitochondrial superoxide (mtROS) that were induced by necroptotic activators. Consistently, DMI prevented the loss of mitochondrial membrane potential induced by the necroptotic activators. In addition, DMI mitigated caerulein-induced acute pancreatitis in mice accompanied by reduced activation of the necroptotic signaling in vivo. Collectively, our study demonstrates that itaconate and its derivatives can inhibit necroptosis by suppressing the RIPK1/RIPK3/MLKL signaling, highlighting their potential applications for treating necroptosis-associated diseases.

PTRF-IL33-ZBP1 signaling mediating macrophage necroptosis contributes to HDM-induced airway inflammation

Polymerase 1 and transcript release factor (PTRF, encoding by Cavin-1) regulates interleukin 33 (IL-33) release, which is implicated in asthma development. Z-DNA binding protein 1 (ZBP1)-sensing Z-RNAs induces necroptosis which causes inflammatory diseases. House dust mite (HDM) is the major source of allergen in house dust and is strongly associated with the development of asthma. Whether PTRF via IL-33 and ZBP1 mediates HDM-induced macrophage necroptosis and airway inflammation remains unclear. Here, we found that deficiency of PTRF could reduce lung IL-33, ZBP1, phosphor-receptor-interacting protein kinase 3 (p-RIPK3), and phosphor-mixed lineage kinase domain-like (p-MLKL) (necroptosis executioner), and airway inflammation in an HDM-induced asthma mouse model. In HDM-treated macrophages, ZBP1, p-RIPK3, and p-MLKL levels were markedly increased, and these changes were reversed by deletion of Cavin-1. Deletion of Il33 also reduced expression of ZBP1, p-RIPK3, and p-MLKL in HDM-challenged lungs. Moreover, IL-33 synergizing with HDM boosted expression of ZBP1, p-RIPK3, and p-MLKL in macrophages. In bronchial epithelial cells rather than macrophages and vascular endothelial cells, PTRF positively regulates IL-33 expression. Therefore, we conclude that PTRF mediates HDM-induced macrophage ZBP1/necroptosis and airway inflammation, and this effect could be boosted by bronchial epithelial cell-derived IL-33. Our findings suggest that PTRF-IL33-ZBP1 signaling pathway might be a promising target for dampening airway inflammation.

Black carbon nanoparticles activate the crosstalk mechanism between necroptosis and macrophage extracellular traps to change macrophages fate

PM2.5 still poses a threat to public health even at very low levels. Black carbon (BC) is a key component of PM2.5. Macrophage extracellular traps (METs) are a means by which macrophages capture and destroy invading pathogens. Necroptosis is an inflammatory programmed cell death. However, there is no research on the crosstalk mechanism between necroptosis and METs after BC exposure. In our study, fluorescence labeling, fluorescent probes, qPCR, and immunofluorescence were applied. Our research found that under normal physiological conditions, when macrophages receive external stimuli (in our experiment, phorbol 12-myristate 13-acetate (PMA)), they will form METs, thus exhibiting innate immune function. However, exposure to BC can cause necroptosis in macrophages accompanied by increased levels of ROS and cytosolic calcium ions as well as altered expression of inflammatory factors and chemokines that prevent the formation of METs, and weakening innate immune function. Notably, inhibition of necroptosis restored the formation of METs, indicating that necroptosis inhibits the formation of METs. Our experiment will enrich the understanding of the mechanism of macrophage injury caused by BC exposure, provide a new direction for studying harmful atmospheric particle toxicity, and propose new therapeutic insights for diseases caused by atmospheric particulate matter. This study is the first to explore the crosstalk mechanism between necroptosis and METs after BC exposure.

Ursolic Acid Promotes Autophagy by Inhibiting Akt/mTOR and TNF-α/TNFR1 Signaling Pathways to Alleviate Pyroptosis and Necroptosis in Mycobacterium tuberculosis-Infected Macrophages.

As a lethal infectious disease, tuberculosis (TB) is caused by Mycobacterium tuberculosis (Mtb). Its complex pathophysiological process limits the effectiveness of many clinical treatments. By regulating host cell death, Mtb manipulates macrophages, the first line of defense against invading pathogens, to evade host immunity and promote the spread of bacteria and intracellular inflammatory substances to neighboring cells, resulting in widespread chronic inflammation and persistent lung damage. Autophagy, a metabolic pathway by which cells protect themselves, has been shown to fight intracellular microorganisms, such as Mtb, and they also play a crucial role in regulating cell survival and death. Therefore, host-directed therapy (HDT) based on antimicrobial and anti-inflammatory interventions is a pivotal adjunct to current TB treatment, enhancing anti-TB efficacy. In the present study, we showed that a secondary plant metabolite, ursolic acid (UA), inhibited Mtb-induced pyroptosis and necroptosis of macrophages. In addition, UA induced macrophage autophagy and enhanced intracellular killing of Mtb. To investigate the underlying molecular mechanisms, we explored the signaling pathways associated with autophagy as well as cell death. The results showed that UA could synergistically inhibit the Akt/mTOR and TNF-α/TNFR1 signaling pathways and promote autophagy, thus achieving its regulatory effects on pyroptosis and necroptosis of macrophages. Collectively, UA could be a potential adjuvant drug for host-targeted anti-TB therapy, as it could effectively inhibit pyroptosis and necroptosis of macrophages and counteract the excessive inflammatory response caused by Mtb-infected macrophages via modulating the host immune response, potentially improving clinical outcomes.

Ozone-oxidized black carbon particles change macrophage fate: Crosstalk between necroptosis and macrophage extracellular traps

The impacts of environmental PM 2.5 on public health have become a major concern all over the world. Many studies have shown that PM 2.5 still poses a threat to public health even at very low levels. Physical or chemical reactions occur between primary particles and other components in the environment, which changes the properties of primary particles. Such newly formed particles with changed properties are called secondary particles. Ozone-oxidized black carbon (oBC) is a key part of PM 2.5 and a representative secondary particle. Macrophages extracellular traps (METs) is a means for macrophages to capture and destroy invading pathogens, thereby exercising innate immunity. Necroptosis is a kind of programmed cell death, which is accompanied by the destruction of membrane integrity, thus inducing inflammatory reaction. However, there is no research on the crosstalk mechanism between necroptosis and MET after oBC exposure. In our study, AO/EB staining, SYTOX Green staining, fluorescent probe, qPCR, Western blot, and immunofluorescence were applied. This experiment found that under normal physiological conditions, when macrophages receive external stimuli (such as pathogens; in our experiment: phorbol 12-myristate 13-acetate (PMA)), they will form METs, capture and kill pathogens, thus exerting innate immune function. However, exposure to oBC can cause necroptosis in macrophages, accompanied by increased levels of reactive oxygen species (ROS) and cytosolic calcium ions, as well as the expression disorder of inflammatory factors and chemokines, and prevent the formation of METs, lose the function of capturing and killing pathogens, and weaken the innate immune function. Notably, inhibition of necroptosis restored the formation of METs, indicating that necroptosis inhibited the formation of METs. This study was the first to explore the crosstalk mechanism between necroptosis and METs after oBC exposure.

1,4-Naphthoquinone-Coated Black Carbon, a Kind of Atmospheric Fine Particulate Matter, Affects Macrophage Fate: New Insights into Crosstalk between Necroptosis and Macrophage Extracellular Traps

1,4-Naphthoquinone-coated BC (1,4 NQ-BC) is an important component of PM2.5 and a representative secondary particle. However, there is no research on the crosstalk mechanism between necroptosis and macrophage extracellular traps (METs) after 1,4 NQ-BC exposure. In this study, we treated RAW264.7 cells with 50, 100, and 200 mg/L 1,4 NQ-BC for 24 h, with 10 μM necrostatin-1 for 24 h, and with 2.5 μM phorbol 12-myristate 13-acetate (PMA) for 3 h. Our experiment revealed that under normal physiological conditions, when macrophages receive external stimuli (such as pathogens; in this experiment, PMA), they will form METs and capture and kill pathogens, thus exerting innate immune function. However, exposure to 1,4 NQ-BC can cause necroptosis in macrophages, accompanied by increased levels of reactive oxygen species (ROS) and cytosolic calcium ions, as well as the expression disorder of inflammatory factors and chemokines, prevent the formation of METs, lead to loss of the function of capturing and killing pathogens, and weaken the innate immune function. Notably, inhibition of necroptosis restored the formation of METs, indicating that necroptosis inhibited the formation of METs. Our study was the first to explore the crosstalk mechanism between necroptosis and METs. This experiment will enrich the mechanism of macrophage injury caused by 1,4 NQ-BC exposure.

TREM-1 triggers necroptosis of macrophages through mTOR-dependent mitochondrial fission during acute lung injury

BackgroundNecroptosis of macrophages is a necessary element in reinforcing intrapulmonary inflammation during acute lung injury (ALI). However, the molecular mechanism that sparks macrophage necroptosis is still unclear. Triggering receptor expressed on myeloid cells-1 (TREM-1) is a pattern recognition receptor expressed broadly on monocytes/macrophages. The influence of TREM-1 on the destiny of macrophages in ALI requires further investigation.MethodsTREM-1 decoy receptor LR12 was used to evaluate whether the TREM-1 activation induced necroptosis of macrophages in lipopolysaccharide (LPS)-induced ALI in mice. Then we used an agonist anti-TREM-1 Ab (Mab1187) to activate TREM-1 in vitro. Macrophages were treated with GSK872 (a RIPK3 inhibitor), Mdivi-1 (a DRP1 inhibitor), or Rapamycin (an mTOR inhibitor) to investigate whether TREM-1 could induce necroptosis in macrophages, and the mechanism of this process.ResultsWe first observed that the blockade of TREM-1 attenuated alveolar macrophage (AlvMs) necroptosis in mice with LPS-induced ALI. In vitro, TREM-1 activation induced necroptosis of macrophages. mTOR has been previously linked to macrophage polarization and migration. We discovered that mTOR had a previously unrecognized function in modulating TREM-1-mediated mitochondrial fission, mitophagy, and necroptosis. Moreover, TREM-1 activation promoted DRP1Ser616 phosphorylation through mTOR signaling, which in turn caused surplus mitochondrial fission-mediated necroptosis of macrophages, consequently exacerbating ALI.ConclusionIn this study, we reported that TREM-1 acted as a necroptotic stimulus of AlvMs, fueling inflammation and aggravating ALI. We also provided compelling evidence suggesting that mTOR-dependent mitochondrial fission is the underpinning of TREM-1-triggered necroptosis and inflammation. Therefore, regulation of necroptosis by targeting TREM-1 may provide a new therapeutic target for ALI in the future.

Necroptosis of macrophage is a key pathological feature in biliary atresia via GDCA/S1PR2/ZBP1/p-MLKL axis

Biliary atresia (BA) is a severe inflammatory and fibrosing neonatal cholangiopathy disease characterized by progressive obstruction of extrahepatic bile ducts, resulting in cholestasis and progressive hepatic failure. Cholestasis may play an important role in the inflammatory and fibrotic pathological processes, but its specific mechanism is still unclear. Necroptosis mediated by Z-DNA-binding protein 1 (ZBP1)/phosphorylated-mixed lineage kinase domain-like pseudokinase (p-MLKL) is a prominent pathogenic factor in inflammatory and fibrotic diseases, but its function in BA remains unclear. Here, we aim to determine the effect of macrophage necroptosis in the BA pathology, and to explore the specific molecular mechanism. We found that necroptosis existed in BA livers, which was occurred in liver macrophages. Furthermore, this process was mediated by ZBP1/p-MLKL, and the upregulated expression of ZBP1 in BA livers was correlated with liver fibrosis and prognosis. Similarly, in the bile duct ligation (BDL) induced mouse cholestatic liver injury model, macrophage necroptosis mediated by ZBP1/p-MLKL was also observed. In vitro, conjugated bile acid-glycodeoxycholate (GDCA) upregulated ZBP1 expression in mouse bone marrow-derived monocyte/macrophages (BMDMs) through sphingosine 1-phosphate receptor 2 (S1PR2), and the induction of ZBP1 was a prerequisite for the enhanced necroptosis. Finally, after selectively knocking down of macrophage S1pr2 in vivo, ZBP1/p-MLKL-mediated necroptosis was decreased, and further collagen deposition was markedly attenuated in BDL mice. Furthermore, macrophage Zbp1 or Mlkl specific knockdown also alleviated BDL-induced liver injury/fibrosis. In conclusion, GDCA/S1PR2/ZBP1/p-MLKL mediated macrophage necroptosis plays vital role in the pathogenesis of BA liver fibrosis, and targeting this process may represent a potential therapeutic strategy for BA.

Severe influenza infection is associated with inflammatory programmed cell death in infected macrophages.

Influenza A virus (IAV) is one of the leading causes of respiratory tract infections in humans, representing a major public health concern. The various types of cell death have a crucial role in IAV pathogenesis because this virus may trigger both apoptosis and necroptosis in airway epithelial cells in parallel. Macrophages play an important role in the clearance of virus particles, priming the adaptive immune response in influenza. However, the contribution of macrophage death to pathogenesis of IAV infection remains unclear. In this work, we investigated IAV-induced macrophage death, along with potential therapeutic intervention. We conducted in vitro and in vivo experiments to evaluate the mechanism and the contribution of macrophages death to the inflammatory response induced by IAV infection. We found that IAV or its surface glycoprotein hemagglutinin (HA) triggers inflammatory programmed cell death in human and murine macrophages in a Toll-like receptor-4 (TLR4)- and TNF-dependent manner. Anti-TNF treatment in vivo with the clinically approved drug etanercept prevented the engagement of the necroptotic loop and mouse mortality. Etanercept impaired the IAV-induced proinflammatory cytokine storm and lung injury. In summary, we demonstrated a positive feedback loop of events that led to necroptosis and exacerbated inflammation in IAV-infected macrophages. Our results highlight an additional mechanism involved in severe influenza that could be attenuated with clinically available therapies.

Low-intensity pulsed ultrasound (LIPUS) switched macrophage into M2 phenotype and mitigated necroptosis and increased HSP 70 in gentamicin-induced nephrotoxicity

Background and aimMany attempts to control acute kidney injury (AKI) have failed due to a lack of understanding of its pathophysiological key components. Macrophages are a crucial determinant of AKI, which can be categorized functionally as M1 pro-inflammatory and M2 anti-inflammatory macrophages. Low-intensity pulsed ultrasound (LIPUS) is currently being investigated as an immune modulator. The present study aimed to explore the potential effects of LIPUS on the polarization of renal macrophages, as well as the possible interplay between macrophage polarization and necroptosis in gentamicin-induced acute kidney injury. MethodAll rats were randomly allocated into one of four groups: control, LIPUS-treated control, gentamicin acute kidney (GM-AKI), and LIPUS-treated GM-AKI. Renal functions, macrophage polarization, necroptosis, and heat shock protein-70 (HSP70) were analyzed using real-time reverse-transcriptase-polymerase chain reaction (rT-PCR), Western Blot, Enzyme-linked immunosorbent assay (ELISA) as well as immunohistological analysis. Resultswe found that LIPUS markedly inhibited the expressions of M1 macrophage-related genes and promoted significantly the expression of M2 macrophages related genes. This was accompanied by an inhibition of necroptosis and a marked reduction of HSP-70, resulting in a reversal of gentamicin-induced renal alteration. ConclusionFunctional switching of macrophage responses from M1 into M2 seems to be a potential approach to ameliorate necroptosis as well as HSP-70 by low pulsed ultrasound waves in GM-AKI.

Interleukin-33 Induces Neutrophil Extracellular Trap (NET) Formation and Macrophage Necroptosis via Enhancing Oxidative Stress and Secretion of Proatherogenic Factors in Advanced Atherosclerosis.

Interleukin-33 (IL-33) acts as an 'alarmin', and its role has been demonstrated in driving immune regulation and inflammation in many human diseases. However, the precise mechanism of action of IL-33 in regulating neutrophil and macrophage functioning is not defined in advanced atherosclerosis (aAT) patients. Further, the role of IL-33 in neutrophil extracellular trap (NET) formation in aAT and its consequent effect on macrophage function is not known. In the present study, we recruited n = 52 aAT patients and n = 52 control subjects. The neutrophils were isolated from both groups via ficoll/percoll-based density gradient centrifugation. The effect of IL-33 on the NET formation ability of the neutrophils was determined in both groups. Monocytes, isolated via a positive selection method, were used to differentiate them into macrophages from each of the study subjects and were challenged by IL-33-primed NETs, followed by the measurement of oxidative stress by calorimetric assay and the expression of the proinflammatory molecules by quantitative PCR (qPCR). Transcript and protein expression was determined by qPCR and immunofluorescence/ELISA, respectively. The increased expression of IL-33R (ST-2) was observed in the neutrophils, along with an increased serum concentration of IL-33 in aAT compared to the controls. IL-33 exacerbates NET formation via specifically upregulating CD16 expression in aAT. IL-33-primed NETs/neutrophils increased the cellular oxidative stress levels in the macrophages, leading to enhanced macrophage necroptosis and the release of atherogenic factors and matrix metalloproteinases (MMPs) in aAT compared to the controls. These findings suggested a pathogenic effect of the IL-33/ST-2 pathway in aAT patients by exacerbating NET formation and macrophage necroptosis, thereby facilitating the release of inflammatory factors and the release of MMPs that may be critical for the destabilization/rupture of atherosclerotic plaques in aAT. Targeting the IL-33/ST-2-NETs axis may be a promising therapeutic target for preventing plaque instability/rupture and its adverse complications in aAT.

Apolipoprotein A1 Protects Against Necrotic Core Development in Atherosclerotic Plaques: PDZK1-Dependent High-Density Lipoprotein Suppression of Necroptosis in Macrophages.

Atherosclerosis is a chronic disease affecting artery wall and a major contributor to cardiovascular diseases. Large necrotic cores increase risk of plaque rupture leading to thrombus formation. Necrotic cores are rich in debris from dead macrophages. Programmed necrosis (necroptosis) contributes to necrotic core formation. HDL (high-density lipoprotein) exerts direct atheroprotective effects on different cells within atherosclerotic plaques. Some of these depend on the SR-B1 (scavenger receptor class B type I) and the adapter protein PDZK1 (postsynaptic density protein/Drosophila disc-large protein/Zonula occludens protein containing 1). However, a role for HDL in protecting against necroptosis and necrotic core formation in atherosclerosis is not completely understood. Low-density lipoprotein receptor-deficient mice engineered to express different amounts of ApoA1 (apolipoprotein A1), or to lack PDZK1 were fed a high fat diet for 10 weeks. Atherosclerotic plaque areas, necrotic cores, and key necroptosis mediators, RIPK3 (receptor interacting protein kinase 3), and MLKL (mixed lineage kinase domain-like protein) were characterized. Cultured macrophages were treated with HDL to determine its effects, as well as the roles of SR-B1, PDZK1, and the PI3K (phosphoinositide 3-kinase) signaling pathway on necroptotic cell death. Genetic overexpression reduced, and ApoA1 knockout increased necrotic core formation and RIPK3 and MLKL within atherosclerotic plaques. Macrophages were protected against necroptosis by HDL and this protection required SR-B1, PDZK1, and PI3K/Akt pathway. PDZK1 knockout increased atherosclerosis in LDLRKO mice, increasing necrotic cores and phospho-MLKL; both of which were reversed by restoring PDZK1 in BM-derived cells. Our findings demonstrate that HDL in vitro and ApoA1, in vivo, protect against necroptosis in macrophages and necrotic core formation in atherosclerosis, suggesting a pathway that could be a target for the treatment of atherosclerosis.

Apigenin suppresses mycoplasma-induced alveolar macrophages necroptosis via enhancing the methylation of TNF-α promoter by PPARγ-Uhrf1 axis

BackgroundMycoplasma-associated pneumonia is characterized by severe lung inflammation and immunological dysfunction. However, current anti-mycoplasma agents used in clinical practice do not prevent dysfunction of alveolar macrophages caused by the high level of the cytokine tumor necrosis factor-α (TNF-α) after mycoplasma infection. Apigenin inhibits the production of TNF-α in variet inflammation associated disease. PurposeThis study aimed to investigate apigenin's effect on mycoplasma-induced alveolar immune cell injury and the mechanism by which it inhibits TNF-α transcription. MethodsIn this study, we performed a mouse model of Mycoplasma hyopneumoniae infection to evaluate the effect of apigenin on reducing mycoplasma-induced alveolar immune cell injury. Furthermore, we carried out transcriptome analysis, RNA interference assay, methylated DNA bisulfite sequencing assay, and chromatin immunoprecipitation assay to explore the mechanism of action for apigenin in reducing TNF-α. ResultsWe discovered that M. hyopneumoniae infection-induced necroptosis in alveolar macrophages MH-S cells and primary mouse alveolar macrophages, which was activated by TNF-α autocrine. Apigenin inhibited M. hyopneumoniae-induced elevation of TNF-α and necroptosis in alveolar macrophages. Apigenin inhibited TNF-a mRNA production via increasing ubiquitin-like with PHD and RING finger domains 1 (Uhrf1)-dependent DNA methylation of the TNF-a promotor. Finally, we demonstrated that apigenin regulated Uhrf1 transcription via peroxisome proliferator activated receptor gamma (PPARγ) activation, which acts as a transcription factor binding to the Uhrf1 promoter and protected infected mice's lungs, and promoted alveolar macrophage survival. ConclutsionThis study identified a novel mechanism of action for apigenin in reducing alveolar macrophage necroptosis via the PPARγ/ Uhrf1/TNF-α pathway, which may have implications for the treatment of Mycoplasma pneumonia.

SIRPα maintains macrophage homeostasis by interacting with PTK2B kinase in Mycobacterium tuberculosis infection and through autophagy and necroptosis.

SummaryBackgroundTo determine whether SIRPα can be a diagnostic marker of pulmonary tuberculosis (PTB) and the molecular mechanism of SIRPα regulating macrophages to kill Mycobacterium tuberculosis (MTB).MethodsMeta-analysis combined with subsequent qRT-PCR, western-blotting and flow cytometry assay were used to detect SIRPα expression in PTB patients. Cell-based assays were used to explore the regulation of macrophage function by SIRPα. SIRPα−/- and wide type macrophages transplanted C57BL/6J mice were used to determine the function of SIRPα on MTB infection in vivo.FindingsSIRPα levels are closely correlated with the treatment outcomes among PTB patients. Cell-based assay demonstrated that MTB significantly induces the expression of SIRPα on macrophages. SIRPα deficiency enhances the killing ability of macrophages against MTB through processes that involve enhanced autophagy and reduced necroptosis of macrophages. Mechanistically, SIRPα forms a direct interaction with PTK2B through its intracellular C-terminal domain, thus inhibiting PTK2B activation in macrophages. Necroptosis inhibition due to SIRPα deficiency requires PTK2B activity. The transfer of SIRPα-deficient bone marrow-derived macrophages (BMDMs) into wild type mice resulted in a drop of bacterial load in the lungs but an enhancement of inflammatory lung damage, and the combination of ulinastatin and SIRPα−/−→WT treatment could decrease the inflammation and maintain the bactericidal capacity.InterpretationOur data define SIRPα a novel biomarker for tuberculosis infection and underlying mechanisms for maintaining macrophage homeostasis.FundingThis work was financially supported by the Chinese National Natural Science Foundation project (No.81401635). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.