Treatment Of Bone Marrow-derived Macrophages Research Articles

A novel NLRP3 inhibitor as a therapeutic agent against monosodium urate-induced gout.

Since NEK7 is critical for NLRP3 inflammasome activation, NEK7 inhibitors could be employed as therapeutic agents against gout, a representative disease caused by NLRP3 inflammasome. We designed NEK7 inhibitors based on biochemical kinome profiling of 2,7-substituted thieno[3,2-d]pyrimidine derivatives (SLC3031~3035 and SLC3037). Inflammasome activation was assessed by ELISA of IL-1b and immunoblotting of IL-1b maturation after treatment of bone marrow-derived macrophages with LPS+monosodium urate (MSU). NLPR3 binding to NEK7 and oligomerization were examined using immunoprecipitation and Blue Native gel electrophoresis, respectively. In vivo effect was investigated by studying gross and histopathological changes of food pad tissue of MSU-injected mice, together with assays of maturation of IL-1b and ASC speck in the tissue. SLC3037 inhibited inflammasome by MSU and other inflammasome activators through blockade of NLRP3 binding to NEK7 or oligomerization, and subsequent ASC oligomerization/phosphorylation. SLC3037 significantly reduced foot pad thickness and inflammation by MSU, which was superior to the effects of colchicine. SLC3037 significantly reduced content or maturation of IL-1b and ASC speck in the food pad. The number and height of intestinal villi were decreased by colchicine but not by SLC3037. SLC3037, a NLRP3 inhibitor blocking NEK7 binding to NLRP3, could be a novel agent against diseases associated with NLRP3 inflammasome activation such as gout, cardiovascular diseases, metabolic syndrome or neurodegenerative diseases.

CTRP6 promotes the macrophage inflammatory response, and its deficiency attenuates LPS-induced inflammation

Macrophages play critical roles in inflammation and tissue homeostasis, and their functions are regulated by various autocrine, paracrine, and endocrine factors. We have previously shown that CTRP6, a secreted protein of the C1q family, targets both adipocytes and macrophages to promote obesity-linked inflammation. However, the gene programs and signaling pathways directly regulated by CTRP6 in macrophages remain unknown. Here, we combine transcriptomic and phosphoproteomic analyses to show that CTRP6 activates inflammatory gene programs and signaling pathways in mouse bone marrow-derived macrophages (BMDMs). Treatment of BMDMs with CTRP6 upregulated proinflammatory, and suppressed the antiinflammatory, gene expression. We also showed that CTRP6 activates p44/42-MAPK, p38-MAPK, and NF-κB signaling pathways to promote inflammatory cytokine secretion from BMDMs, and that pharmacologic inhibition of these signaling pathways markedly attenuated the effects of CTRP6. Pretreatment of BMDMs with CTRP6 also sensitized and potentiated the BMDMs response to lipopolysaccharide (LPS)-induced inflammatory signaling and cytokine secretion. Consistent with the metabolic phenotype of proinflammatory macrophages, CTRP6 treatment induced a shift toward aerobic glycolysis and lactate production, reduced oxidative metabolism, and elevated mitochondrial reactive oxygen species production in BMDMs. Importantly, in accordance with our invitro findings, BMDMs from CTRP6-deficient mice were less inflammatory at baseline and showed a marked suppression of LPS-induced inflammatory gene expression and cytokine secretion. Finally, loss of CTRP6 in mice also dampened LPS-induced inflammation and hypothermia. Collectively, our findings suggest that CTRP6 regulates and primes the macrophage response to inflammatory stimuli and thus may have a role in modulating tissue inflammatory tone in different physiological and disease contexts.

Macrophage AMPK β1 activation by PF-06409577 reduces the inflammatory response, cholesterol synthesis, and atherosclerosis in mice

Atherosclerotic cardiovascular disease is characterized by both chronic low-grade inflammation and dyslipidemia. The AMP-activated protein kinase (AMPK) inhibits cholesterol synthesis and dampens inflammation but whether pharmacological activation reduces atherosclerosis is equivocal. In the current study, we found that the orally bioavailable and highly selective activator of AMPKβ1 complexes, PF-06409577, reduced atherosclerosis in two mouse models in a myeloid-derived AMPKβ1 dependent manner, suggesting a critical role for macrophages. In bone marrow-derived macrophages (BMDMs), PF-06409577 dose dependently activated AMPK as indicated by increased phosphorylation of downstream substrates ULK1 and acetyl-CoA carboxylase (ACC), which are important for autophagy and fatty acid oxidation/de novo lipogenesis, respectively. Treatment of BMDMs with PF-06409577 suppressed fatty acid and cholesterol synthesis and transcripts related to the inflammatory response while increasing transcripts important for autophagy through AMPKβ1. These data indicate that pharmacologically targeting macrophage AMPKβ1 may be a promising strategy for reducing atherosclerosis.

MyD88 and not TRIF knockout is sufficient to abolish LPS-induced inflammatory responses in bone-derived macrophages.

Macrophages play an important role in the response to infection and/or repair of injury in tissues. To examine the NF-κB pathway in response to an inflammatory stimulus, we used wild-type bone-marrow-derived macrophages (BMDMs) or BMDMs with knockout (KO) of myeloid differentiation primary response 88 (MyD88) and/or Toll/interleukin-1 receptor domain-containing adapter-inducing interferon-β (TRIF) via CRISPR/Cas9. Following treatment of BMDMs with lipopolysaccharide (LPS) to induce an inflammatory response, translational signalling of NF-κB was quantified via immunoblot and cytokines were measured. Our findings reveal that MyD88 KO, but not TRIF KO, decreased LPS-induced NF-κB signalling, and 10% expression of basal MyD88 expression was sufficient to partially rescue the abolished inflammatory cytokine secretion observed upon MyD88 KO.

Lipoxin A4, Maresin 1, and Resolvin D1 As Key Factors for the Actions of Plasminogen/Plasmin on Inflammation Resolution

The plasminogen/plasmin (Plg/Pla) system plays a fundamental role in modulating fibrinolysis and many other biological processes, including extracellular matrix degradation, cell migration, and tissue repair. Recent studies highlight the role of the Plg/Pla system in key steps of inflammation resolution, which is mediated, at least in part, by the glucocorticoid-induced protein annexin A1. However, it is unclear whether Plg/Pla might also modulate specialized proresolving mediators (SPMs) derived from omega-3 and omega-6 fatty acids, including lipoxin A4 (LXA4), maresin 1 (MaR1), and resolvin D1 (RvD1). Therefore, we investigated the ability of Plg/Pla to modulate the synthesis of LXA4, MaR1, and RvD1, as well as the involvement of these SPMs in the proresolving effects elicited by Plg or Pla using both in vitro and in vivo strategies. Treatment of bone marrow-derived macrophages (BMDMs) with Plg/Pla (4 μg/mL) induced the release of LXA4, MaR1, and RvD1 as well as increased mRNA levels of their lipoxygenase (LO) synthesizing enzymes 5-LO and 15-LO. Plg treatment also increased the levels of the anti-inflammatory cytokine IL-10 and reduced the levels of the pro-inflammatory cytokine TNF-α in BMDMs. Moreover, Plg decreased the levels of TNF-α and IL-1β induced by lipopolysaccharide (LPS) stimulus of the human monocytic cell line THP-1. Interestingly, LXA4 was able to induce Plg release in THP-1 cells. Notably, Pla (4 μg, i.p.) was able to increase the efferocytosis of apoptotic neutrophils in wild-type (SV129) mice, but not in 5-LO deficient mice (5-LO-/-). Taken together, these findings suggest that Plg and Pla increase the levels of SPMs and IL-10, and modulate the levels of LPS-elicited pro-inflammatory cytokines. Mechanistically, the action of Plg/Pla on efferocytosis was 5-LO dependent, suggesting that SPMs mediate this proresolving effect of Plg/Pla. Moreover, the reciprocal modulation between the Plg/Pla system and SPMs pathways elucidated should be explored in future studies on candidate targets for therapies for inflammatory diseases. Keywords: Inflammation resolution; plasminogen/plasmin system; pro-resolving lipid mediators. Financial support: FAPEMIG, CNPq, CAPES.

OxLDL induces membrane structure rearrangement leading to biomechanics alteration and migration deficiency in macrophage

Cholesterol sequestration from plasma membrane has been shown to induce lipid packing disruption, causing actin cytoskeleton reorganization and polymerization, increasing cell stiffness and inducing lysosomal exocytosis in non-professional phagocytes. Similarly, oxidized form of low-density lipoprotein (oxLDL) has also been shown to disrupt lipid organization and packing in endothelial cells, leading to biomechanics alterations that interfere with membrane injury and repair. For macrophages, much is known about oxLDL effects in cell activation, cytokine production and foam cell formation. However, little is known about its impact in the organization of macrophage membrane structured domains and cellular mechanics, the focus of the present study. Treatment of bone marrow-derived macrophages (BMDM) with oxLDL not only altered membrane structure, and potentially the distribution of raft domains, but also induced actin rearrangement, diffuse integrin distribution and cell shrinkage, similarly to observed upon treatment of these cells with MβCD. Those alterations led to decreased migration efficiency. For both treatments, higher co-localization of actin cytoskeleton and GM1 was observed, indicating a similar mechanism of action involving raft-like domain dynamics. Lastly, like MβCD treatment, oxLDL also induced lysosomal spreading in BMDM. We propose that OxLDL induced re-organization of membrane/cytoskeleton complex in macrophages can be attributed to the insertion of oxysterols into the membrane, which lead to changes in lipid organization and disruption of membrane structure, similar to the effect of cholesterol depletion by MβCD treatment. These results indicate that oxLDL can induce physical alterations in the complex membrane/cytoskeleton of macrophages, leading to significant biomechanical changes that compromise cell behavior.

Intestinal Metabolites Influence Macrophage Phagocytosis and Clearance of Bacterial Infection

The metabolite-rich environment that is the intestinal lumen contains metabolic by-products deriving from microbial fermentation and host cell metabolism, with resident macrophages being constantly exposed to this metabolic flux. Succinate, lactate and itaconate are three metabolites secreted by primed macrophages due to a fragmented tri-carboxylic acid (TCA) cycle. Additionally, succinate and lactate are known by-products of microbial fermentation. How these metabolites impact biological functioning of resident macrophages particularly in response to bacterial infection remains poorly understood. We have investigated the potential influence of these metabolites on macrophage phagocytosis and clearance of Escherichia coli (E. coli) infection. Treatment of murine bone-marrow-derived macrophages (BMDMs) with succinate reduced numbers of intracellular E. coli early during infection, while lactate-treated BMDMs displayed no difference throughout the course of infection. Treatment of BMDMs with itaconate lead to higher levels of intracellular E. coli early in the infection with bacterial burden subsequently reduced at later time-points compared to untreated macrophages, indicative of enhanced engulfment and killing capabilities of macrophages in response to itaconate. Expression of engulfment mediators MARCKS, RhoB, and CDC42 were reduced or unchanged following succinate or lactate treatment and increased in itaconate-treated macrophages following E. coli infection. Nitric oxide (NO) levels varied while pro- and anti-inflammatory cytokines differed in secretory levels in all metabolite-treated macrophages post-infection with E. coli or in response to lipopolysaccharide (LPS) stimulation. Finally, the basal phenotypic profile of metabolite-treated macrophages was altered according to marker gene expression, describing how fluid macrophage phenotype can be in response to the microenvironment. Collectively, our data suggests that microbe- and host-derived metabolites can drive distinct macrophage functional phenotypes in response to infection, whereby succinate and itaconate regulate phagocytosis and bactericidal mechanisms, limiting the intracellular bacterial niche and impeding the pathogenesis of infection.

Apoptosis inhibitor of macrophage (AIM) contributes to IL-10-induced anti-inflammatory response through inhibition of inflammasome activation

Apoptosis inhibitor of macrophage (AIM) modulates the signaling in inflammatory responses, including infection, cancer, or other immune diseases. Recent studies suggest that like interleukin-10 (IL-10), AIM is involved in alternatively activated (M2) macrophage polarization. We aimed to understand whether and how AIM is involved in IL-10-induced inhibition of inflammasome activation and resolution of inflammation. First, we demonstrated that IL-10 induced increases in mRNA and protein expression of AIM in murine bone marrow-derived macrophages (BMDM). In addition, genetic and pharmacologic inhibition of STAT3 (signal transducer and activator of transcription 3) reduced IL-10-induced AIM expression. We also found that IL-10-induced STAT3 activity enhanced the AIM promoter activity by directly binding the promoter of the AIM gene. Additionally, reduction of LPS/adenosine triphosphate (ATP)-induced IL-1β production and caspase-1 activation by IL-10 was reversed in BMDM from AIM−/− mice. Treatment of BMDM from both wild type (WT) and IL-10−/− mice with recombinant AIM showed the inhibitory effects on IL-1β and IL-18 production and caspase-1 activation. Endogenous and exogenous AIM inhibited apoptosis-associated speck-like protein containing a caspase activation and recruitment domain (ASC) speck formation. In LPS-induced acute peritonitis, inhibition of IL-1β and IL-18 production in peritoneal lavage fluid (PLF) and serum, reduction of caspase-1 activation in peritoneal macrophages, and reduction of numbers of neutrophils and peritoneal macrophages in PLF by administration of IL-10 were not evident in AIM−/− mice. Our in vitro and in vivo data reveal a novel role of AIM in the inhibition of inflammasome-mediated caspase-1 activation and IL-1β and IL-18 production.

Three Scrophularia Species (Scrophularia buergeriana, S. koraiensis, and S. takesimensis) Inhibit RANKL-Induced Osteoclast Differentiation in Bone Marrow-Derived Macrophages.

Scrophulariae Radix, derived from the dried roots of Scrophularia ningpoensis Hemsl. or S. buergeriana Miq, is a traditional herbal medicine used in Asia to treat rheumatism, arthritis, and pharyngalgia. However, the effects of Scrophularia buergeriana, S. koraeinsis, and S. takesimensis on osteoclast formation and bone resorption remain unclear. In this study, we investigated the morphological characteristics and harpagoside content of S. buergeriana, S. koraiensis, and S. takesimensis, and compared the effects of ethanol extracts of these species using nuclear factor (NF)-κB ligand (RANKL)-mediated osteoclast differentiation. The harpagoside content of the three Scrophularia species was analyzed by high-performance liquid chromatography–mass spectrometry (HPLC/MS). Their therapeutic effects were evaluated by tartrate-resistant acid phosphatase (TRAP)-positive cell formation and bone resorption in bone marrow-derived macrophages (BMMs) harvested from ICR mice. We confirmed the presence of harpagoside in the Scrophularia species. The harpagoside content of S. buergeriana, S. koraiensis, and S. takesimensis was 1.94 ± 0.24 mg/g, 6.47 ± 0.02 mg/g, and 5.50 ± 0.02 mg/g, respectively. Treatment of BMMs with extracts of the three Scrophularia species inhibited TRAP-positive cell formation in a dose-dependent manner. The area of hydroxyapatite-absorbed osteoclasts was markedly decreased after treatment with the three Scrophularia species extracts. Our results indicated that the three species of the genus Scrophularia might exert preventive effects on bone disorders by inhibiting osteoclast differentiation and bone resorption, suggesting that these species may have medicinal and functional value.

Enhanced Expression of Catalase in Mitochondria Modulates NF-κB-Dependent Lung Inflammation through Alteration of Metabolic Activity in Macrophages.

NF-κB is a reduction-oxidation-sensitive transcription factor that plays a key role in regulating the immune response. In these studies, we intended to investigate the role of mitochondrial-derived reactive oxygen species in regulating NF-κB activation by studying transgenic mice that overexpress mitochondrial-targeted human catalase (mCAT). We treated wild-type (WT) and mCAT mice with intratracheal instillation of Escherichia coli LPS and found that mCAT mice had exaggerated NF-κB activation in the lungs, increased neutrophilic alveolitis, and greater lung inflammation/injury compared with WT mice. Additional studies using bone marrow chimeras revealed that this hyperinflammatory phenotype was mediated by immune/inflammatory cells. Mechanistic studies using bone marrow-derived macrophages (BMDMs) showed that LPS treatment induced a sustained increase in NF-κB activation and expression of NF-κB-dependent inflammatory mediators in mCAT BMDMs compared with WT BMDMs. Further investigations showed that cytoplasmic, but not mitochondrial, hydrogen peroxide levels were reduced in LPS-treated mCAT BMDMs. However, mCAT macrophages exhibited increased glycolytic and oxidative metabolism, coupled with increased ATP production and an increased intracellular NADH/NAD+ ratio compared with BMDMs from WT mice. Treatment of BMDMs with lactate increased the intracellular NADH/NAD+ ratio and upregulated NF-κB activation after LPS treatment, whereas treatment with a potent inhibitor of the mitochondrial pyruvate carrier (UK5099) decreased the NADH/NAD+ ratio and reduced NF-κB activation. Taken together, these findings point to an increased availability of reducing equivalents in the form of NADH as an important mechanism by which metabolic activity modulates inflammatory signaling through the NF-κB pathway.

Maresin 1 resolves aged-associated macrophage inflammation to improve bone regeneration.

Inflammaging is associated with poor tissue regeneration observed in advanced age. Specifically, protracted inflammation after acute injury has been associated with decreased bone fracture healing and increased rates of nonunion in elderly patients. Here, we investigated the efficacy of using Maresin 1 (MaR1), an omega-3 fatty acid-derived pro-resolving agent, to resolve inflammation after tibial fracture injury and subsequently improving aged bone healing. Aged (24-month-old mice) underwent tibial fracture surgery and were either treated with vehicle or MaR1 3 days after injury. Fracture calluses were harvested 7 days, 14 days, 21 days, and 28 days after injury to investigate inflammatory response, cartilage development, bone deposition, and mechanical integrity, respectively. Healing bones from MaR1-treated mice displayed decreased cartilage formation and increased bone deposition which resulted in increased structural stiffness and increased force to fracture in the later stages of repair. In the early stages, MaR1 treatment decreased the number of pro-inflammatory macrophages within the fracture callus and decreased the level of inflammatory biomarkers in circulation. In tissue culture models, MaR1 treatment of bone marrow-derived macrophages from aged mice protected cells form a pro-inflammatory phenotype and induced an anti-inflammatory fate. Furthermore, the secretome of MaR1-treated bone marrow-derived macrophages was identified as osteoinductive, enhancing osteoblast differentiation of bone marrow stromal cells. Our findings here identify resolution of inflammation, and MaR1 itself, to be a point of intervention to improve aged bone healing.

Mitochondrial DNA from hepatocytes induces upregulation of interleukin-33 expression of macrophages in nonalcoholic steatohepatitis

ObjectiveIn the present study, we propose that lipotoxicity induces the release of mitochondrial DNA (mtDNA) from hepatocytes, which in turn upregulates IL-33 expression in macrophages. MethodsThe mtDNA levels of plasma were determined in methionine- and mholine-deficient diet (MCD)-fed mice and NASH patients. Cultured hepatocytes were pre-incubated with Mito-TEMPO or rapamycin and were then stimulated with palmitic acid. The mtDNA levels in the cytosol were measured. The mtDNA from hepatocytes of mice was added to bone marrow-derived macrophages (BMDMs) in the presence of IRS (TLR9 antagonist). The expression of IL-33 in BMDMs was measured. ResultsLevels of mtDNA were higher in NASH patients and MCD-fed mice. Treatment of hepatocytes with palmitic acid in vitro induced mtDNA release into cytosol, which was attenuated by mito-TEMPO or rapamycin, and aggravated by inhibition of autophagy. Treatment of BMDMs with mtDNA enhanced IL-33 expression, which was attenuated by knockdown of TLR9. Treatment of BMDMs with mtDNA enhanced lipopolysaccharide (LPS)-induced production of IL-1β and TNF-α, which was attenuated by pretreatment with soluble ST2. ConclusionmtDNA released from injured hepatocytes under lipid overload induced the upregulation of IL-33 expression in macrophages via TLR9, and enhanced LPS-induced inflammatory cytokine production.

4-Hydroxybenzaldehyde Restricts the Intracellular Growth of Toxoplasma gondii by Inducing SIRT1-Mediated Autophagy in Macrophages.

Toxoplasma gondii is an intracellular protozoan parasite that infects approximately one third of the human population worldwide. Considering the toxicity and side effects of anti-toxoplasma medications, it is important to develop effective drug alternatives with fewer and less severe off-target effects. In this study, we found that 4-hydroxybenzaldehyde (4-HBA) induced autophagy and the expression of NAD-dependent protein deacetylase sirtuin-1 (SIRT1) in primary murine bone marrow-derived macrophages (BMDMs). Interestingly, treatment of BMDMs with 4-HBA significantly reduced the number of macrophages infected with T. gondii and the proliferation of T. gondii in infected cells. This effect was impaired by pretreating the macrophages with 3-methyladenine or wortmannin (selective autophagy inhibitors) or with sirtinol or EX527 (SIRT1 inhibitors). Moreover, we found that pharmacological inhibition of SIRT1 prevented 4-HBA-mediated expression of LC3-phosphatidylethanolamine conjugate (LC3-II) and the colocalization of T. gondii parasitophorous vacuoles with autophagosomes in BMDMs. These data suggest that 4-HBA promotes antiparasitic host responses by activating SIRT1-mediated autophagy, and 4-HBA might be a promising therapeutic alternative for the treatment of toxoplasmosis.

Role of iron metabolism in the immunosuppression mediated by myeloid cells in glioblastoma patients

Abstract Background Glioblastoma multiforme (GBM) is one of the most malignant primary brain tumor and is known to create an immunosuppressive microenvironment that hampers the efficacy of therapies. We demonstrated that GBM microenvironment is infiltrated with a large proportion of bone marrow-derived macrophages (BMDMs) that possess a strong immune suppressive activity. Following 5-aminolevulinic acid (5-ALA) administration to patients before surgery, BMDMs show the highest protoporphyrin IX (PPIX) fluorescence emission, compared to immune and non-immune cells. Since PPIX is the precursor of heme we wondered if the presence of this metabolite could be the consequence of an altered iron metabolism in BMDMs, and if this pathway could be linked to their immunosuppressive activity. Methods GBM tissues were dissociated and subjected to immunomagnetic or FACS sorting for the separation of BMDMs. The expression of HMOX1, the gene coding for heme oxygenase-1 (HO-1) which is a central enzyme in the iron pathway, was analyzed by RT-PCR. BMDM immune suppressive activity was evaluated as the ability to suppress the proliferation of T lymphocytes also in the presence of HO-1 inhibitors. In order to optimize the experimental conditions, immunosuppressive macrophages derived from M-CSF treated monocytes from healthy donors were set up. Another in vitro model of immunosuppressive cells, bone marrow derived-myeloid derived suppressor cells (BM-MDSCs), was tested. Results HMOX1 expression was found to be upregulated in BMDMs compared to other cells. The treatment of BMDMs with a HO-1 inhibitor was able to restore T cells proliferation in immune suppressive assays. The recovery of immunosuppression following treatment with the inhibitor has also been demonstrated in macrophages and in BM-MDSCs. Conclusion Our results show that the connection between iron metabolism and immune response could be exploited from a therapeutic point of view. In particular, HO-1 plays a role in BMDM-induced immunosuppression and could represent a new target to relieve the immunosuppressive microenvironment present in GBM patients. Legal entity responsible for the study University of Padua. Funding AIRC. Disclosure All authors have declared no conflicts of interest.

MDM2 inhibitor APG-115 synergizes with PD-1 blockade through enhancing antitumor immunity in the tumor microenvironment

BackgroundProgrammed death-1 (PD-1) immune checkpoint blockade has achieved clinical successes in cancer therapy. However, the response rate of anti-PD-1 agents remains low. Additionally, a subpopulation of patients developed hyperprogressive disease upon PD-1 blockade therapy. Combination therapy with targeted agents may improve immunotherapy. Recent studies show that p53 activation in the myeloid linage suppresses alternative (M2) macrophage polarization, and attenuates tumor development and invasion, leading to the hypothesis that p53 activation may augment antitumor immunity elicited by anti-PD-1 therapy.MethodUsing APG-115 that is a MDM2 antagonist in clinical development as a pharmacological p53 activator, we investigated the role of p53 in immune modulation and combination therapy with PD-1 blockade.ResultsIn vitro treatment of bone marrow-derived macrophages with APG-115 resulted in activation of p53 and p21, and a decrease in immunosuppressive M2 macrophage population through downregulation of c-Myc and c-Maf. Increased proinflammatory M1 macrophage polarization was observed in the spleen from mice treated with APG-115. Additionally, APG-115 has co-stimulatory activity in T cells and increases PD-L1 expression in tumor cells. In vivo, APG-115 plus anti-PD-1 combination therapy resulted in enhanced antitumor activity in Trp53wt, Trp53mut, and Trp53-deficient (Trp53−/−) syngeneic tumor models. Importantly, such enhanced activity was abolished in a syngeneic tumor model established in Trp53 knockout mice. Despite differential changes in tumor-infiltrating leukocytes (TILs), including the increases in infiltrated cytotoxic CD8+ T cells in Trp53wt tumors and M1 macrophages in Trp53mut tumors, a decrease in the proportion of M2 macrophages consistently occurred in both Trp53wt and Trp53mut tumors upon combination treatment.ConclusionOur results demonstrate that p53 activation mediated by APG-115 promotes antitumor immunity in the tumor microenvironment (TME) regardless of the Trp53 status of tumors per se. Instead, such an effect depends on p53 activation in Trp53 wild-type immune cells in the TME. Based on the data, a phase 1b clinical trial has been launched for the evaluation of APG-115 in combination with pembrolizumab in solid tumor patients including those with TP53mut tumors.

5946The immune protein MD-2 is associated with early death in dilated cardiomyopathy and increases M1 macrophage polarization and recruitment in vitro

Abstract Objective Dilated cardiomyopathy (DCM) is characterized by systolic dysfunction and dilatation of ventricles. Myocardial inflammation and leukocyte activation and recruitment play a major role in the development and progression of disease. Myeloid differentiation factor-2 (MD-2) is the TLR (toll like receptor)-4 co-receptor and has been shown to be an important risk predictor for mortality of DCM patients. It is expressed in various cell types and mediates TLR-4 dependent inflammation/activation processes. Purpose We examined the impact of MD-2 on mortality of DCM patients and on polarization and recruitment of monocytes in vitro. Methods In 77 DCM patients, divided by median time point of death after first hospital admission into early and late death and alive group, MD-2 was quantified by means of ELISA. In THP-1 monocytes, cytokine secretion was quantified by ELISA after 72h treatment with MD-2 (5μg/mL). Bone marrow derived macrophages (BMDM) were generated from MD-2 KO and WT mice. NFkB phosphorylation (10min) and changes in gene expression (4h) of different adhesion molecules was quantified after treatment with 1 or 10ng/mL LPS. In human umbilical vein endothelial cells (HUVEC), protein kinase B (PKB) phosphorylation was quantified after 15min of treatment with 10ng/mL LPS or 5μg/mL MD-2. CCL2 gene expression in lysed cells (4h) and CCL2 secretion in supernatants (48h) were quantified to. Adhesion of monocytes on treated HUVEC was determined by FACS (Fig.1a). Initial HUVEC treatment with MD-2 (5μg/mL) or LPS (10 or 100ng/mL) took place for 48h. Results We found significant increased MD-2 in early (591.3ng/mL; N=18) vs late death (p=0.015) (369.2ng/mL; N=17) and alive (p≤0.0001) (303.2ng/mL; N=42) patients. Treatment of THP-1 cells (N=5) with MD-2 lead to a significantly increased secretion of inflammatory cytokines IL-8 (p=0.012), IP-10 (p=0.029), and MCP-1 (p=0.032) but not of anti-inflammatory cytokines IL-4, IL-10 and IL-13. Treatment of BMDM obtained from MD-2 KO and WT mice with 10ng/mL LPS lead to a increased phosphorylation of NFkB (N=4; p=0.022) and increased gene expression (N=6) of adhesion molecules VLA-4 (p=0.006) and ICAM-1 (p=0.049) in WT mice but not in KO mice. In HUVEC, LPS (p=0.008) and MD-2 induced a comparable increased phosphorylation of PKB (p=0.008) as well as an increase of CCL2 gene expression (p=0.029) and protein amount (p=0.039). Furthermore, treatment of HUVEC with both MD-2 (p=0.015) and LPS (p=0.0001) lead to a significant increase in monocyte adhesion (Fig.1). Conclusion The impact of MD-2 on cardiac inflammation and macrophage recruitment has not been described yet. In this study, we showed that, in DCM, elevated levels of sMD-2 are associated with early death. Furthermore, we could demonstrate that MD-2 enhances the process of HUVEC based monocyte recruitment. Finally, we could show that MD-2 induces inflammatory monocyte activity and triggers polarization of macrophages towards an inflammatory phenotype.

Abstract 3192: Activation of p53 in the tumor microenvironment by MDM2 inhibitor APG-115 synergizes with PD-1 blockade independently of p53 status of tumor cells

Abstract Blockade of the checkpoint inhibitor programmed death 1 (PD-1) has gained big success in cancer therapy. However, the response rate of anti-PD1 agents remains low. Molecularly targeted agents offer selectivity and high tumor response rates, but patients develop resistance to these drugs inevitably. Combinations of targeted agents and immunotherapy provide new opportunities to improve cancer treatments. Recent studies found that p53 activation in the myeloid linage suppressed alternative (M2) macrophage polarization and attenuates tumor development and invasion, leading to the hypothesis that p53 activation may further augment antitumor immunity elicited by anti-PD-1 therapy. APG-115 is an orally active, selective, small molecule inhibitor of the MDM2-p53 protein-protein interaction. APG-115 acts as an antitumor agent by activating the p53 tumor suppressor in p53 wild-type tumors. However, its role in regulating immune responses remained unknown. In this study, we investigated the role of APG-115 in immune modulation both in vitro and in vivo. Enhanced antitumor activity was first demonstrated in p53 wild-type MH-22A, p53 mutant MC38, and p53 knockout (p53-/-) MH-22A syngeneic tumor models after the combination treatment of APG-115 and anti-PD-1 antibody. Despite differential changes in tumor-infiltrating leukocytes, including an increase in cytotoxic CD8+ T cells in the p53 wild-type tumors and an increase in proinflammatory M1 macrophages in the p53 mutant tumors, the combination treatment consistently reduced immunosuppressive M2 macrophages in the tumor microenvironment regardless of p53 status of tumor cells. In addition, in vitro, the treatment of bone marrow-derived macrophages with APG-115 resulted in activation of p53 and p21 gene expression, as well as a decrease in M2 macrophages population and reduction of c-MYC and M2-related gene expression. Moreover, enhanced M1 macrophage polarization in the spleen was also observed in naïve mice treated with APG-115. Furthermore, APG-115 increased production of multiple proinflammatory cytokines, including IFN-γ, TNF-α, IL-2 and IL-6, in stimulated T cells. Collectively, for the first time, our findings suggest that p53 activation by a pharmacological MDM2 inhibitor enables reversal of immunosuppressive tumor microenvironment and enhance antitumor immunity independently of p53 status of tumors. Specifically, in complementary to PD-1 blockade that predominantly activates cytotoxic CD8+ T cell populations, APG-115 primarily targets tumor-associated macrophages. Collectively, our data provide a rationale for applying the combination of APG-115 plus PD-1 blockade to a broader patient population with p53 mutant tumors. Accordingly, a clinical trial of APG-115 in combination with pembrolizumab in metastatic melanoma patients regardless of p53 status has been initiated in the USA. Citation Format: Douglas D. Fang, Qiuqiong Tang, Yanhui Kong, Qixin Wang, Jiaxing Gu, Xu Fang, Peng Zou, Tao Rong, Jingwen Wang, Dajun Yang, Yifan Zhai. Activation of p53 in the tumor microenvironment by MDM2 inhibitor APG-115 synergizes with PD-1 blockade independently of p53 status of tumor cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3192.

Inhibiting Extracellular Cathepsin D Reduces Hepatic Steatosis in Sprague⁻Dawley Rats †.

Dietary and lifestyle changes are leading to an increased occurrence of non-alcoholic fatty liver disease (NAFLD). Using a hyperlipidemic murine model for non-alcoholic steatohepatitis (NASH), we have previously demonstrated that the lysosomal protease cathepsin D (CTSD) is involved with lipid dysregulation and inflammation. However, despite identifying CTSD as a major player in NAFLD pathogenesis, the specific role of extracellular CTSD in NAFLD has not yet been investigated. Given that inhibition of intracellular CTSD is highly unfavorable due to its fundamental physiological function, we here investigated the impact of a highly specific and potent small-molecule inhibitor of extracellular CTSD (CTD-002) in the context of NAFLD. Treatment of bone marrow-derived macrophages with CTD-002, and incubation of hepatic HepG2 cells with a conditioned medium derived from CTD-002-treated macrophages, resulted in reduced levels of inflammation and improved cholesterol metabolism. Treatment with CTD-002 improved hepatic steatosis in high fat diet-fed rats. Additionally, plasma levels of insulin and hepatic transaminases were significantly reduced upon CTD-002 administration. Collectively, our findings demonstrate for the first time that modulation of extracellular CTSD can serve as a novel therapeutic modality for NAFLD.

Anti-inflammatory activity of small-molecule antagonists of Toll-like receptor 2 (TLR2) in mice

Toll-like receptor 2 (TLR2) is currently investigated as a potential therapeutic target in diseases with underlying inflammation like sepsis and arthritis. We reported the discovery, by virtual screening and biological testing, of eight TLR2 antagonists (AT1-AT8) which showed TLR2-inhibitory activity in human cells (Murgueitio et al., 2014). In this study, we have deepened in the mechanism of action and selectivity (TLR2/1 or TLR2/6) of those compounds in mouse primary cells and in vivo. The antagonists reduced, in a dose-dependent way the TNFα production (e.g. AT5 IC50 7.4 μM) and also reduced the nitric oxide (NO) formation in mouse bone marrow-derived macrophages (BMDM). Treatment of BMDM with the antagonists showed that downstream of TLR2, MAPKs phosphorylation and IkBα degradation was reduced. Notably, in a mouse model of tri-acylated lipopeptide (Pam3CSK4)-induced inflammation, AT5 attenuated the TNFα and IL-6 inflammatory response. Further, the effect of AT5 in the stimulation of BMDM by the endogenous alarmin HMGB1 was investigated. Our results indicate that AT4-AT7 and, particularly AT5 appear as good starting points for the development of inhibitors targeting TLR2 in inflammatory disorders.

Mycobacterium tuberculosis acyl carrier protein inhibits macrophage apoptotic death by modulating the reactive oxygen species/c-Jun N-terminal kinase pathway

Mycobacterial acyl carrier protein (AcpM; Rv2244) is a meromycolate extension acyl carrier protein of Mycobacterium tuberculosis (Mtb), which participates in multistep mycolic acid biosynthesis. However, the function of AcpM in host–mycobacterium interactions during infection remains largely uncharacterized. Here we show that AcpM inhibits host cell apoptosis during mycobacterial infection. To examine the function of AcpM during infection, we generated a recombinant Mycobacterium smegmatis (M. smegmatis) strain overexpressing AcpM (Ms_AcpM) and a strain transformed with an empty vector (Ms_Vec). Ms_AcpM promoted intracellular survival of M. smegmatis and led to a significant decrease in the death rate of primary bone marrow-derived macrophages (BMDMs). Importantly, Ms_AcpM showed significantly decreased reactive oxygen species (ROS) generation and activation of c-Jun N-terminal kinase (JNK) signaling compared with Ms_Vec. In addition, treatment of BMDMs with recombinant AcpM significantly inhibited the apoptosis and ROS/JNK signaling induced by M. smegmatis. Moreover, recombinant AcpM enhanced intracellular survival of Mtb H37Rv. Taken together, these results indicate that AcpM plays a role as a virulence factor by modulating host cell apoptosis during mycobacterial infection.