Macrophage Response Research Articles

Obesity alters the macrophages' response to Leishmania major in C57BL/6 mice.

Obesity is a global pandemic associated with several comorbidities, such as cardiovascular diseases and type 2 diabetes. It is also a predisposing factor for infectious diseases, increasing mortality rates. Moreover, diet-induced obesity can cause metabolic fluctuations that affect macrophage differentiation in various organs. In this sense, we investigated how bone marrow-derived macrophages and tissue-resident macrophages in the skin, which have been differentiated in a host with metabolic syndrome and with previous inflammatory burden, respond to Leishmania major infection. Our findings suggest that bone marrow-derived macrophages from obese C57BL/6 mice, even when cultivated in vitro with inflammatory stimuli, are more susceptible to L. major. These macrophages produce less tumor necrosing factor (TNF) and nitric oxide (NO) and show higher arginase activity. Furthermore, obese mice infected with an intermediate dose of L. major in the skin had more severe lesions when analyzed for ulceration, diameter, thickness, and parasite burden. The increase in lesion severity in obese mice was associated with a higher frequency of tissue-resident macrophages, which are less efficient in killing parasites. We also used CCR2-/- mice, which predominantly have tissue-resident macrophages, and found that lesion resolution was delayed in association with CCR2 deficiency. Additionally, obesity potentiated tissue damage, resulting in higher frequency of tissue-resident macrophages. Our results demonstrate that obesity can alter macrophage responses to infection, leading to increased susceptibility to L. major and more severe cutaneous leishmaniasis. These findings may have important implications for managing obesity-related infections and the development of new therapies for cutaneous leishmaniasis.

Macrophage energy metabolism in cardiometabolic disease.

In a rapidly expanding body of literature, the major role of energy metabolism in determining the response and polarization status of macrophages has been examined, and it is currently a very active area of research. The metabolic flux through different metabolic pathways in the macrophage is interconnected and complex and could influence the polarization of macrophages. Earlier studies suggested glucose flux through cytosolic glycolysis is a prerequisite to trigger the pro-inflammatory phenotypes of macrophages while proposing that fatty acid oxidation is essential to support anti-inflammatory responses by macrophages. However, recent studies have shown that this understanding is oversimplified and that the metabolic control of macrophage polarization is highly complex and not fully defined yet. In this review, we systematically reviewed and summarized the literature regarding the role of energy metabolism in controlling macrophage activity and how that might be altered in cardiometabolic diseases, namely heart failure, obesity, and diabetes. We critically appraised the experimental studies and methodologies in the published studies. We also highlighted the challenging concepts in macrophage metabolism and identified several research questions yet to be addressed in future investigations.

GAPDH-Silence Microsphere via Reprogramming Macrophage Metabolism and eradicating Bacteria for Diabetic infection bone regeneration

Macrophage metabolism dysregulation, which is exacerbated by persistent stimulation in infectious and inflammatory diseases, such as diabetic infectious bone defects (DIBD), eventually leads to the failure of bone repair. Here, we have developed an injectable, macrophage-modulated GAPDH-Silence drug delivery system. This microsphere comprises chondroitin sulfate methacrylate (CM) and methacrylated gelatin (GM), while the dimethyl fumarate (DMF)-loaded liposome (D-lip) is encapsulated within the microsphere (CM@GM), named D-lip/CM@GM. Triggered by the over-expressed collagenase in DIBD, the microspheres degrade and release the encapsulated D-lip. D-lip could modulate metabolism by inhibiting GAPDH, which suppresses the over-activation of glycolysis, thus preventing the inflammatory response of macrophages in vitro. While beneficial for macrophages, D-lip/CM@GM is harmful to bacteria. GAPDH, while crucial for glycolysis of staphylococcal species (S. aureus), can be effectively countered by D-lip/CM@GM. We are utilizing existing drugs in innovative ways to target central metabolism for effective eradication of bacteria. In the DIBD model, our results confirmed that the D-lip/CM@GM enhanced bacteria clearance and reprogrammed dysregulated metabolism, thereby significantly improving bone regeneration. In conclusion, this GAPDH-Silence microsphere system may provide a viable strategy to promote diabetic infection bone regeneration.

B10 cells regulate macrophage polarization to alleviate inflammation and bone loss in periodontitis.

The polarization of macrophages into an anti-inflammatory phenotype is crucial for resolving periodontal inflammation. It has been reported that B10 cells can regulate the immune response of macrophages during inflammation and are also able to regulate inflammation in periodontitis. However, whether B10 cells' regulation function in periodontitis is related to macrophage polarization remains unclear. This study aims to investigate whether B10 cells can regulate macrophage polarization in periodontitis. Macrophages were cocultured with B10 cells in vitro for 5days. After coculture, macrophages were obtained for analysis directly or followed by stimulation with Pg-LPS/IFN-γ or IL-4/IL-13. Flow cytometry and/or reverse transcriptase-polymerase chain reaction (RT-PCR) were employed to detect the expression of IL-1β, iNOS, TNF-α, CD206, and ARG-1 in macrophages. B10 cells were transferred on the 5th day after ligation in wild or macrophage-depletion mice. Toluidine blue and TRAP staining were used to evaluate alveolar bone resorption and osteoclast activation. Immunohistochemistry was employed to detect the expression of CD68, IL-1β, TNF-α, iNOS, ARG-1, and IL-10. Immunofluorescence was used to detect the expression of CD68+CD86+M1 macrophages and CD68+CD206+M2 macrophages. In vitro, B10 cells inhibit the expression of IL-1β, iNOS, and TNF-α in macrophages while increasing the expression of CD206 and ARG-1. In experimental periodontitis, B10 cells inhibit the polarization of CD68+CD86+M1 macrophages and iNOS expression but enhance the polarization of CD68+CD206+M2 macrophages and ARG-1 expression. Importantly, the depletion of macrophages partially weakened the regulation function of B10 cells in periodontitis. B10 cells promote M2 macrophage polarization, inhibit M1 macrophage polarization in periodontitis, and alleviate periodontitis partially by regulating macrophage polarization.

Molecular and functional in vivo characterisation of murine Dectin-1 isoforms.

Dectin-1 is a C-type lectin-receptor involved in sensing fungi by innate immune cells. Encoded by the Clec7a gene, Dectin-1 exists in two major splice isoforms, Dectin-1a and 1b, which differ in the presence of a membrane-proximal stalk domain. As reported previously, this domain determines degradative routes for Dectin-1a and 1b leading to the generation of a stable N-terminal fragment exclusively from Dectin-1a. Here, we narrow down the responsible part of the stalk and demonstrate the stabilisation of the Dectin-1a N-terminal fragment in tetraspanin-enriched microdomains. C57BL/6 and BALB/c mice show divergent Dectin-1 isoform expression patterns, which are caused by a single nucleotide polymorphism in exon 3 of the Clec7a gene, leading to a non-sense Dectin-1a mRNA in C57BL/6 mice. Using backcrossing, we generated mice with the C57BL/6 Clec7a allele on a BALB/c background and compared these to the parental strains. Expression of the C57BL/6 allele leads to the exclusive presence of the Dectin-1b protein. Furthermore, it was associated with higher Dectin-1 mRNA expression, but less Dectin-1 at the cell surface according to flow cytometry. In neutrophils, this altered ROS production induced by Dectin-1 model ligands, while cellular responses in macrophages and dendritic cells were not significantly influenced by the Dectin-1 isoform pattern.

Bioinspired micro-nano hierarchical delivery system for sequential targeted therapy of inflammatory bowel disease

Inflammatory bowel disease (IBD) constitutes an immense health and economic burden worldwide, highlighting the need for improved therapy. Up to now, various delivery systems have been proposed to enhance IBD treatment. Herein, inspired by cellular communication in nature, we present a novel micro-nano hierarchical microsphere system with the enhanced targeting capability for IBD treatment. The hierarchical microspheres are designed for oral delivery, with astaxanthin nanoparticles in the core and Zein protein shell. Similar to a stepwise process of cellular communication, microscale spheres can escape from the gastric acid due to Zein coating. Once reach into the intestine, mucoadhesive Zein coating and the core could be enzymatically digested, following the targeted release of astaxanthin nanoparticles. Particularly, these astaxanthin nanoparticles are endowed with the mitochondria targeting capability, thereby greatly attenuating pro-inflammatory response of macrophages. Based on these features, practical performances of the hierarchical delivery system have been investigated in both acute and chronic colitis model. Animal experiments have revealed the desired therapeutic effects of the hierarchical microspheres by inhibiting inflammatory responses and modulating gut microbiota. These results suggested the present hierarchical delivery system offers a stepwise strategy for nanodrug delivery to the intestine as well as the effective treatment of IBD.

LRG1 promotes atherosclerosis by inducing macrophage M1-like polarization

Atherosclerosis is a chronic inflammatory disease of the arterial wall characterized by the accumulation of cholesterol-rich lipoproteins in macrophages. How macrophages commit to proinflammatory polarization under atherosclerosis conditions is not clear. Report here that the level of a circulating protein, leucine-rich alpha-2 glycoprotein 1 (LRG1), is elevated in the atherosclerotic tissue and serum samples from patients with coronary artery disease (CAD). LRG1 stimulated macrophages to proinflammatory M1-like polarization through the activation of extracellular signal-regulated kinase 1/2 (ERK1/2) and c-Jun N-terminal kinase (JNK) pathways. The LRG1 knockout mice showed significantly delayed atherogenesis progression and reduced levels of macrophage-related proinflammatory cytokines in a high-fat diet-induced Apoe-/- mouse atherosclerosis model. An anti-LRG1 neutralizing antibody also effectively blocked LRG1-induced macrophage M1-like polarization in vitro and conferred therapeutic benefits to animals with ApoE deficiency-induced atherosclerosis. LRG1 may therefore serve as an additional biomarker for CAD and targeting LRG1 could offer a potential therapeutic strategy for CAD patients by mitigating the proinflammatory response of macrophages.

TARGETED TEMPERATURE MANAGEMENT AT 36°C IMPROVES SURVIVAL AND PROTECTS TISSUES BY MITIGATING THE DELETERIOUS INFLAMMATORY RESPONSE FOLLOWING HEMORRHAGIC SHOCK.

Hemorrhagic shock (HS) is a life-threatening condition with high mortality rates despite current treatments. This study investigated whether targeted temperature management (TTM) could improve outcomes by modulating inflammation and protecting organs following HS. Using a rat model of HS, TTM was applied at 33°C and 36°C after fluid resuscitation. Surprisingly, TTM at 33°C increased mortality, while TTM at 36°C significantly improved survival rates. It also reduced histological damage in lung and kidney tissues, lowered serum lactate levels, and protected against apoptosis and excessive reactive oxygen species production. TTM at 36°C inhibited the release of high mobility group box 1 protein (HMGB1), a key mediator of inflammation, and decreased proinflammatory cytokine levels in the kidneys and lungs. Moreover, it influenced macrophage behavior, suppressing the harmful M1 phenotype while promoting the beneficial M2 polarization. Cytokine array analysis confirmed reduced levels of proinflammatory cytokines with TTM at 36°C. These results collectively highlight the potential of TTM at 36°C as a therapeutic approach to improve outcomes in HS. By addressing multiple aspects of injury and inflammation, including modulation of macrophage responses and cytokine profiles, TTM at 36°C offers promising implications for critical care management after HS, potentially reducing mortality and improving patient recovery.

Low pH modulates lipopolysaccharide-induced tumor necrosis factor-alpha expression and macropinocytotic activity in RAW264.7 cells

Inflammation triggers various types of diseases that need to be addressed. Macrophages play important roles in the inflammatory responses. As atherosclerosis progresses, macrophages transform into foam cells. Extracellular acidification is observed at and around bacterial infection and atherosclerotic sites. However, the effects of acidification on the inflammatory response of macrophages and the progression of atherosclerosis have not been fully understood. This study investigates the impact of extracellular acidification on lipopolysaccharide-induced tumor necrosis factor-alpha (TNF-α) expression and macropinocytotic activity in RAW264.7 cells. TNF-α expression is measured by real-time polymerase chain reaction (relative value to glyceraldehyde-3-phosphate dehydrogenase expression). Macropinocytotic activity is measured by neutral red uptake (absorbance at 540 nm). Results show that TNF-α expression increased with decreasing extracellular pH in both un-foamed and foamed cells. Macropinocytotic activity was upregulated at pH 6.8 in un-foamed cells, but downregulated in foamed cells stimulated at low pH. Proton-sensing G protein-coupled receptors (GPCRs) were involved in the expression of TNF-α and in the macropinocytotic activity of foamed cells. In conclusion, this study reveals that extracellular acidification differently affect various inflammatory responses such as LPS-induced TNF-α expression and macropinocytotic activity of RAW264.7 cells and different proton-sensing GPCRs are involved in the different inflammatory responses.

The potential therapeutic role of itaconate and mesaconate on the detrimental effects of LPS-induced neuroinflammation in the brain

Despite advances in antimicrobial and anti-inflammatory treatment, inflammation and its consequences remain a major challenge in the field of medicine. Inflammatory reactions can lead to life-threatening conditions such as septic shock, while chronic inflammation has the potential to worsen the condition of body tissues and ultimately lead to significant impairment of their functionality. Although the central nervous system has long been considered immune privileged to peripheral immune responses, recent research has shown that strong immune responses in the periphery also affect the brain, leading to reactive microglia, which belong to the innate immune system and reside in the brain, and neuroinflammation. The inflammatory response is primarily a protective mechanism to defend against pathogens and tissue damage. However, excessive and chronic inflammation can have negative effects on neuronal structure and function. Neuroinflammation underlies the pathogenesis of many neurological and neurodegenerative diseases and can accelerate their progression. Consequently, targeting inflammatory signaling pathways offers potential therapeutic strategies for various neuropathological conditions, particularly Parkinson’s and Alzheimer’s disease, by curbing inflammation. Here the blood–brain barrier is a major hurdle for potential therapeutic strategies, therefore it would be highly advantageous to foster and utilize brain innate anti-inflammatory mechanisms. The tricarboxylic acid cycle-derived metabolite itaconate is highly upregulated in activated macrophages and has been shown to act as an immunomodulator with anti-inflammatory and antimicrobial functions. Mesaconate, an isomer of itaconate, similarly reduces the inflammatory response in macrophages. Nevertheless, most studies have focused on its esterified forms and its peripheral effects, while its influence on the CNS remained largely unexplored. Therefore, this study investigated the immunomodulatory and therapeutic potential of endogenously synthesized itaconate and its isomer mesaconate in lipopolysaccharide (LPS)-induced neuroinflammatory processes. Our results show that both itaconate and mesaconate reduce LPS-induced neuroinflammation, as evidenced by lower levels of inflammatory mediators, reduced microglial reactivity and a rescue of synaptic plasticity, the cellular correlate of learning and memory processes in the brain. Overall, this study emphasizes that both itaconate and mesaconate have therapeutic potential for neuroinflammatory processes in the brain and are of remarkable importance due to their endogenous origin and production, which usually leads to high tolerance.

Effects of Fasting on THP1 Macrophage Metabolism and Inflammatory Profile.

Fasting can affect the body's inflammatory response, and this has been linked to potential health benefits, including improvements for people with rheumatic diseases. In this work, we evaluated, in vitro, how changes in nutrient availability alter the inflammatory response of macrophages. Macrophage-differentiated THP1 cells were cultured, deprived of FCS or subjected to cycles of FCS deprivation and restoration to mimic intermittent fasting. Changes in the macrophage phenotype, the cells' response to inflammatory stimuli and the level of mitochondrial alteration were assessed. The results indicate that while periods of serum starvation are associated with a decrease in IL1β and TNFα expression, consistent with an anti-inflammatory response, intermittent serum starvation cycles promote a pro-inflammatory phenotype. Rapid changes in reducing capacity and mitochondrial response were also observed. Of note, while some changes, such as the production of oxygen free radicals, were reversed with refeeding, others, such as a decrease in reducing capacity, were maintained and even increased. This study shows that different fasting protocols can have diverging effects and highlights that time-limited nutrient changes can significantly affect macrophage functions in cell cultures. These findings help elucidate some of the mechanisms by which specific fasting dietary interventions could help control inflammatory diseases.

Photoaging of polystyrene-based microplastics amplifies inflammatory response in macrophages

The continuous release of municipal and industrial products into the environment poses a growing concern for public health. Among environmental pollutants, polystyrene (PS) stands out as a primary constituent of environmental plastic waste, given its widespread use and high production rates owing to its durability and user-friendly properties. The detection of polystyrene microparticles (PS-MPs) in various living organisms has been well-documented, posing a serious threat due to their potential passage into the human ecosystem. In this manuscript, we aimed to study the toxicological effects of low concentrations of pristine and photoaged PS-MPs in a murine macrophage cell line. To this purpose, PS-MPs were photoaged by indoor exposure to visible light to simulate environmental weathering due to solar irradiation (PS-MPs3h). Physical characterization revealed that the irradiation treatment results in particle degradation and the possible release of nanoparticles. Monocultures of the RAW264.7 cell line were then exposed to PS-MPs and PS-MPs3h at concentrations comparable to experimental measurements from biological samples, to assess cytotoxicity, intracellular oxidative stress, primary genotoxicity, and inflammatory effects. Significant toxicity-related outcomes were observed in cells treated with both pristine PS-MPs and PS-MPs3h even at low concentrations (0,10 μg/ml and 1 μg/ml). PS-MPs3h exhibited greater adverse effects compared to PS-MPs, including reduced cell viability, increased ROS production, elevated DNA damage, and upregulation of IL-6 and NOS2 gene expression. Therefore, we can conclude that changes induced by environmental aging in the physicochemical composition of PS microplastics play a crucial role in the adverse health outcomes associated with microplastic exposure.

Phosphatidylserine liposomes induce a phagosome acidification-dependent and ROS-mediated intracellular killing of Mycobacterium abscessus in human macrophages.

Mycobacterium abscessus (Mab) is an opportunistic nontuberculous mycobacterium responsible of difficult-to-treat pulmonary infections in vulnerable patients, such as those suffering from Cystic Fibrosis (CF), where it represents a major cause of morbidity and mortality. Additionally, due to the intrinsic extensive antimicrobial resistance spectrum displayed by this species and the side effects reported for some available antibiotics, the therapeutic management of such infections remains extremely difficult. In the present study, we show that phosphatidylserine liposomes (PS-L) enhance intracellular mycobacterial killing of Mab infected human macrophages with functional or pharmacologically inhibited cystic fibrosis conductance regulator (CFTR), by a mechanism involving phagosome acidification and reactive oxygen species (ROS) production. Additionally, PS-L significantly reduce proinflammatory response of Mab infected macrophages in terms of NF-kB activation and TNF-α production, irrespective of CFTR inhibition. Altogether, these results represent the proof of concept for a possible future development of PS-L as a therapeutic strategy against difficult-to-treat Mab infection.

Macrophage WDFY3, a protector against autoimmunity.

Efficient efferocytosis is essential for maintaining homeostasis. Excessive apoptotic cell (AC) death and impaired macrophage efferocytosis lead to autoantigen release and autoantibody production, immune activation, and organ damage. It remains unclear whether these immunogenic autoantigens are the sole cause of increased autoimmunity or if efferocytosis of ACs directly influences macrophage function, impacting their ability to activate T cells and potentially amplifying autoimmune responses. Additionally, it has not been established if enhancing macrophage efferocytosis or modulating macrophage responses to AC engulfment can be protective in autoimmune-like disorders. Our previous work showed WDFY3 is crucial for efficient macrophage efferocytosis. This study reveals that myeloid knockout of Wdfy3 exacerbates autoimmunity in young mice with increased AC burden by systemic injections of ACs and in middle-aged mice developing spontaneous autoimmunity, whereas ectopic overexpression of WDFY3 suppresses autoimmunity in these models. Macrophages, as efferocytes, can activate T cells and the inflammasome upon engulfing ACs, which are suppressed by overexpressing WDFY3. This work uncovered the role of WDFY3 as a protector against autoimmunity by promoting macrophage efferocytosis thus limiting autoantigen production, as well as mitigating T cell activation and inflammasome activation.

Lipid nanoparticle encapsulation of a Delta spike-CD40L DNA vaccine improves effectiveness against Omicron challenge in Syrian hamsters

The effectiveness of mRNA vaccines largely depends on their lipid nanoparticle (LNP) component. Herein, we investigate the effectiveness of DLin-KC2-DMA (KC2) and SM-102-based LNPs for the intramuscular delivery of a plasmid encoding B.1.617.2 (Delta) spike fused with CD40 ligand. LNP encapsulation of this CD40L-adjuvanted DNA vaccine with either LNP formulation drastically enhanced antibody responses, enabling neutralization of heterologous Omicron variants. The DNA-LNP formulations provided excellent protection from homologous challenge, reducing viral replication, and preventing histopathological changes in the pulmonary tissues. Moreover, theDNA-LNP vaccines maintained a high level of protection against heterologous Omicron BA.5 challenge despite a reduced neutralizing response. In addition, we observed that DNA-LNP vaccination led to the pulmonary downregulation of interferon signaling, interleukin-12 signaling, and macrophage response pathways following SARS-CoV-2 challenge, shedding some light on the mechanisms underlying the prevention of pulmonary injury. These results highlight the potential combination of molecular adjuvants with LNP-based vaccine delivery to induce greater and broader immune responses capable of preventing inflammatory damage and protecting against emerging variants. These findings could be informative for the future design of both DNA and mRNA vaccines.

The miR-26a/SIRT6/HIF-1α axis regulates glycolysis and inflammatory responses in host macrophages during Mycobacterium tuberculosis infection.

Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis. Here, a macrophage infection model was used to unravel the role of the histone deacetylase sirtuin 6 (SIRT6) in Mtb-triggered regulation of the innate immune response. Mtb infection downregulated microRNA-26a and upregulated its target SIRT6. SIRT6 suppressed glycolysis and expression of HIF-1α-dependent glycolytic genes during infection. In addition, SIRT6 regulated the levels of intracellular succinate which controls stabilization of HIF-1α, as well as the release of interleukin (IL)-1β. Furthermore, SIRT6 inhibited inducible nitric oxide synthase (iNOS) and proinflammatory IL-6 but augmented anti-inflammatory arginase expression. The miR-26a/SIRT6/HIF-1α axis therefore regulates glycolysis and macrophage immune responses during Mtb infection. Our findings link SIRT6 to rewiring of macrophage signaling pathways facilitating dampening of the antibacterial immune response.

ALDH2 deficiency augments atherosclerosis through the USP14-cGAS-dependent polarization of proinflammatory macrophages

The aldehyde dehydrogenase 2 (ALDH2) rs671 polymorphism commonly exists in the East Asian populations and is associated with high risks of cardiovascular disease (CVD). However, the cellular and molecular mechanisms that underlie the ALDH2 rs671 mutant-linked high CVD remain elusive. Here, we show that macrophages derived from human ALDH2 rs671 carriers and ALDH2 knockout mice exhibited an enhanced pro-inflammatory macrophage phenotype and an impaired anti-inflammatory macrophage phenotype. Transplanting bone marrow from ALDH2−/−ApoE−/− to ApoE−/− mice significantly increased atherosclerotic plaque growth and pro-inflammatory macrophage polarization in vivo. Mechanistically, ALDH2 inhibited activation of the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway in macrophages. Pharmacological inhibition of cGAS by RU.521 completely neutralized ALDH2-deficiency-induced macrophage polarization. In-depth mechanistic investigation showed that ALDH2 accelerated cGAS K48-linked polyubiquitination degradation at lysine 282 in macrophages by reducing the interaction between ubiquitin-specific protease 14 (USP14) and cGAS, mainly through its enzymatic role in mitigating 4-hydroxy-2-nonenal (4-HNE) accumulation. Consistently, USP14 knockdown in bone marrow cells alleviated proinflammatory responses in macrophages and protected against atherosclerosis. Our findings provide new mechanistic insights of ALDH2 deficiency-associated proinflammation and atherosclerosis and new therapeutic and preventive paradigms for treatment of atherosclerosis-associated CVD.

Signalling Pathways of Inflammation and Cancer in Human Mononuclear Cells: Effect of Nanoparticle Air Pollutants.

Fine inhalable particulate matter (PM) triggers an inflammatory response in the airways and activates mononuclear cells, mediators of tissue homeostasis, and tumour-promoting inflammation. We have assessed ex vivo responses of human monocytes and monocyte-derived macrophages to standardised air pollutants: carbon black, urban dust, and nanoparticulate carbon black, focusing on their pro-inflammatory and DNA-damaging properties. None of the PM (100 μg/mL/24 h) was significantly toxic to the cells, aside from inducing oxidative stress, fractional DNA damage, and inhibiting phagocytosis. TNFα was only slightly increased. PM nanoparticles increase the expression and activate DNA-damage-related histone H2A.X as well as pro-inflammatory NF-κB. We have shown that the urban dust stimulates the pathway of DNA damage/repair via the selective post-translational phosphorylation of H2A.X while nanoparticulate carbon black increases inflammation via activation of NF-κB. Moreover, the inflammatory response to lipopolysaccharide was significantly stronger in macrophages pre-exposed to urban dust or nanoparticulate carbon black. Our data show that airborne nanoparticles induce PM-specific, epigenetic alterations in the subsets of cultured mononuclear cells, which may be quantified using binary fluorescence scatterplots. Such changes intercede with inflammatory signalling and highlight important molecular and cell-specific epigenetic mechanisms of tumour-promoting inflammation.

Origin recognition complex subunit 6 (ORC6) is a key mediator of LPS-induced NFκB activation and the pro-inflammatory response

Lipopolysaccharide (LPS)-activated pro-inflammatory responses play a critical role in sepsis, a life-threatening condition. This study investigates the role of origin recognition complex subunit 6 (ORC6) in LPS responses in macrophages and monocytes. Silencing ORC6 using targeted shRNA significantly reduced LPS-induced expression and production of IL-1β (interleukin-1 beta), TNF-α (tumor necrosis factor alpha), and IL-6 (interleukin-6) in THP-1 human macrophages, peripheral blood mononuclear cells (PBMCs), and bone marrow-derived macrophages (BMDMs). Additionally, ORC6 knockout (KO) via the CRISPR/Cas9 method in THP-1 macrophages inhibited LPS-induced pro-inflammatory responses, while ectopic overexpression of ORC6 enhanced LPS-induced expression and production of pro-inflammatory cytokines. ORC6 is crucial for the activation of the nuclear factor kappa B (NFκB) signaling cascade in macrophages and monocytes. LPS-induced NFκB activation was largely inhibited by ORC6 silencing or KO, but potentiated following ORC6 overexpression. Mechanistically, ORC6 associated with nuclear p65 after LPS stimulation, an interaction necessary for NFκB activation. Overexpression of ORC6 did not recover the reduced pro-inflammatory response to LPS in THP-1 macrophages with silenced p65. Furthermore, the NFκB inhibitor BMS-345,541 nearly eliminated the pro-inflammatory response enhanced by ORC6 overexpression in response to LPS. Further studies revealed that ORC6 depletion inhibited NFκB activation induced by double-stranded RNA (dsRNA) and high mobility group box 1 (HMGB1) in THP-1 macrophages. In vivo experiments demonstrated that macrophage-specific knockdown of ORC6 protected mice from LPS-induced septic shock and inhibited LPS-stimulated production of IL-1β, TNF-α, and IL-6 in mouse serum. ORC6 silencing also inhibited LPS-induced NFκB activation in ex vivo cultured PBMCs following macrophage-specific knockdown of ORC6. These findings highlight ORC6 as a pivotal mediator in LPS-induced NFκB activation and the pro-inflammatory response in sepsis, suggesting that targeting ORC6 could be a novel therapeutic strategy for managing sepsis and related inflammatory conditions.

Transcriptional analysis of efferocytosis in mouse skin wounds.

Defects in apoptotic cell clearance, or efferocytosis, can cause inflammatory diseases and prevent tissue repair due in part to inducing a pro-repair transcriptional program in phagocytic cells like macrophages. While the cellular machinery and metabolic pathways involved in efferocytosis have been characterized, the precise efferocytic response of macrophages is dependent on the identity and macromolecular cues of apoptotic cells, and the complex tissue microenvironment in which efferocytosis occurs. Here, we find that macrophages undergoing active efferocytosis in mid-stage mouse skin wounds in vivo display a pro-repair gene program, while efferocytosis of apoptotic skin fibroblasts in vitro also induces an inflammatory transcription response. These data provide a resource for understanding how the skin wound environment influences macrophage efferocytosis and will be useful for future investigations that define the role of efferocytosis during tissue repair.