Macrophage Survival Research Articles

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.

Significance of macrophage infiltration in the prognosis of lung adenocarcinoma patients evaluated by scRNA and bulkRNA analysis.

PurposeTo investigate the significance of macrophage infiltration to the prognosis of lung adenocarcinoma.MethodsR language bioinformatics analysis technology, was used to obtain macrophage infiltration-related module genes through WGCNA (Weighted Gene Co-Expression Network Analysis). Marker genes of macrophage subtypes were identified using single-cell sequencing of lung adenocarcinoma tissue. Risk score models were constructed and validated using external data cohorts and clinical samples.ResultsAnalysis of cohorts TCGA-LUAD, GSE11969, GSE31210, GSE50081, GSE72094 and GSE8894, revealed a negative correlation between macrophage infiltration and survival. Immunohistochemical analyses of clinical samples were consistent with these data. Based on cell-cluster-markers and TAMs-related-genes, TOP8 genes were obtained (C1QTNF6, CCNB1, FSCN1, HMMR, KPNA2, PRC1, RRM2, and TK1) with a significant association to prognosis. Risk score models including 9 factors (C1QTNF6, FSCN1, KPNA2, GLI2, TYMS, BIRC3, RBBP7, KRT8, GPR65) for prognosis were constructed. The efficacy, stability and generalizability of the risk score models were validated using multiple data cohorts (GSE19188, GSE26939, GSE31210, GSE50081, GSE42127, and GSE72094).ConclusionsMacrophage infiltration negatively correlates with prognosis in patients with lung adenocarcinoma. Based on cell-cluster-markers and TAMs-related-genes, both TOP8 genes (C1QTNF6, CCNB1, FSCN1, HMMR, KPNA2, PRC1, RRM2, TK1) and risk score models using C1QTNF6, FSCN1, KPNA2, GLI2, TYMS, BIRC3, RBBP7, KRT8, GPR65 could predict disease prognosis.

512 A phoRP-regulated pilin in Mycobacterium abscessus is required for association with and survival in macrophages and cystic fibrosis mice

512 A phoRP-regulated pilin in Mycobacterium abscessus is required for association with and survival in macrophages and cystic fibrosis mice

343 (PB123) - HX301, a potent CSF1R inhibitor, suppresses tumor associated M2 macrophage (TAM), enhancing tumor immunity and causing transit tumor inhibition in syngeneic EMT-6 tumors

Background. Tumor associated macrophage (TAM) plays important immunosuppression role within the tumor microenvironment (TME). CSF1R is a receptor tyrosine kinase (RTK) responsible for the growth, survival and polarization (toward M2) of macrophages, thus implicated in pathogenesis of immunological disorders, as well as immuno-oncology (IO). High CSF1R expression is reported to be correlated to poorer prognosis of cancers CSF1R inhibitors (CSF1Ri), both TKIs or antibodies, have been proposed as potential cancer treatment strategy, although yet to be proven in the clinics.

Mycobacterium tuberculosis PPE7 Enhances Intracellular Survival of Mycobacterium smegmatis and Manipulates Host Cell Cytokine Secretion Through Nuclear Factor Kappa B and Mitogen-Activated Protein Kinase Signaling.

The PE/PPE family proteins of Mycobacterium tuberculosis have been associated with its virulence and interaction with the host immune system. The highly virulent modern lineage of M. tuberculosis possesses a lineage-specific PPE gene (PPE7), which arises from an ancestral mutation and is rarely studied. Here we examined the role of PPE7 in mycobacterial pathogenicity and survival by expressing M. tuberculosis PPE7 in Mycobacterium smegmatis. We show that, PPE7 activates host inflammation by increasing expression of pro-inflammatory cytokines including tumor necrosis factor-alpha (TNF-α), interleukin (IL)-1β, and IL-6, while suppressing the expression of anti-inflammatory cytokines such as IL-10, possibly through the nuclear factor kappa B, ERK1/2, and p38 mitogen-activated protein kinase pathways. Overexpressing PPE7 in M. smegmatis could enhance bacterial intracellular survival of infected macrophages. Furthermore, higher level of bacterial persistence, higher levels of TNF-α, IL-1β, and IL-6 cytokines, and more injury in the lung, liver, and spleen tissues of infected mice has been discovered. In conclusion, PPE7 could manipulate host immune response and increase bacterial persistence.

S5.3d How mitochondrial complex I proteins in Candida albicans moderate phagocytosis and the production of pro-inflammatory cytokines in murine macrophages and dendritic cells

S5.3 Cellular pleomorphism and fungal virulence, September 22, 2022, 3:00 PM - 4:30 PM ObjectivesInhibition of respiration in Candida albicans impairs its colonization in the host tissues and causes avirulence in a murine vascular candidiasis model. Accordingly, blockage of the mitochondrial electron transport chain (ETC) of C. albicans by respiratory inhibitors promotes phagocytosis by increasing exposure of glucan which could be due to the mannan reduction. In our model, we have reported that 85% mannan reduction in goa1Δ, a deletion mutant of an ETC Complex I (CI) regulator, oppositely decreased phagocytosis. To understand such a difference, we broaden our investigation with three CI respiratory subunit mutants, which are either fungal-specific (nuo1Δ and nuo2Δ) or broadly conserved subunits (ndh51Δ) for cell wall analysis and innate immune responses.MethodsWe characterized mutant cell wall defects in these mutants, then analyzed their respective survival in macrophages. Fungal internalization into macrophages was visualized under fluorescent microscopy and live-cell imaging and analyzed through flow cytometry analysis. Cytokine production in dendritic cells (DCs) infected by fungal cells was measured by xMAP technology and the transcriptional profiles of murine macrophages-infected by different mutants were compared.ResultsWe find that phosphopeptidomannan (PPM) reduction in goa1Δ and nuo1Δ and phospholipomannan (PLM) reduction in nuo2Δ correlate with massive inhibition of cytokine. PPM loss in nuo1Δ or goa1Δ fails to promote phagocytosis but promotes opsonized neutrophil killing. The cause of PPM insufficiency results from reduced phosphorylation of the Cek1 MAPK in goa1Δ and nuo1Δ. In contrast other three mutants, phagocytosis and cytokine production of ndh51Δ more resemble WT cells, which have shown an ∼ 30% glucan reduction due to a defective Mek1 MAPK response. The divergent immune responses to these CI mutants are shown at the transcriptional level in infected macrophages. We noted that those well-characterized host receptors such as dectins and TLR2/4 for PPM, PLM, and glucan ligands are not significantly affected at 1 h post-infection. However, the scavenger receptor CD36, integrin ICAM, and growth factor receptors are downregulated along with a generally downregulated endocytosis and antigen processing/presentation. In addition, the host metabolic processes, oxidative stress-induced senescence, apoptosis, and signaling pathways such as Ras1/Erk5, the cAMP/CREB, and TLR9 pathway, are each individually affected in the host cells.ConclusionWe speculate that mitochondrial signals of fungal origin may also be sensed by the host immune cells to coordinate the immune responses together with cell replication and metabolism during the early stage of infection.

S4.3b Candida glabrata-host interactions

S4.3 Emergent species of the Candida genus, September 22, 2022, 10:30 AM - 12:00 PM Candida glabrata is among the most common pathogenic yeasts of humans. Yet, this fungus is phylogenetically, genetically, and phenotypically very different from other clinically relevant Candida species. This includes an unusual high level of intrinsic resistance to antifungals such as azoles. Furthermore, in contrast to the rather aggressive pathogenicity mechanisms of the dominant Candida species, C. albicans, C. glabrata seems to have evolved strategies that are based on stealth, evasion and persistence, without causing severe host cell damage during infection. For example, C. glabrata is unable to invade epithelial cells in vitro and causes only low levels of damage in standard in vitro infection models. However, the addition of a single common host protein, albumin, strongly enhances the damage potential to vaginal epithelial cells. Similar to other Candida species, the potential of C. glabrata to survive in human blood is low. However, C. glabrata can survive within macrophages for long periods without causing host cell damage and responses. This intracellular stage requires the acquisition of nutrients, such as biotin, facilitated by defined biotin up-take systems. Survival in macrophages is further supported by transient mitochondria dysfunction: C. glabrata is able to switch into a petite phenotype upon interaction with human phagocytes. The petite phenotype significantly differs from non-petite cells in its growth behavior, its interactions with the immune system, and even its resistance to antifungal drugs. Effectively, the interaction with phagocytes can render C. glabrata mostly resistant to commonly used azole drugs, and vice versa (‘cross resistance’), providing a principle mechanism by which intrinsic antifungal resistance may have evolved.SourcesSeider et al. (2011) The facultative intracellular pathogen Candida glabrata subverts macrophage cytokine production and phagolysosome maturation. J Immunol. 187(6):3072-86.Brunke and Hube. (2013) Two unlike cousins: Candida albicans and C. glabrata infection strategies. Cell Microbiol. 15(5):701-8.Kämmer et al. (2020) Survival strategies of pathogenic Candida species in human blood show independent and specific adaptations. mBio 11(5):e02435-20.Sprenger et al. (2020) Fungal biotin homeostasis is essential for immune evasion after macrophage phagocytosis and virulence. Cell Microbiol. 22(7):e13197.Pekmezovic et al. (2021) Candida pathogens induce protective mitochondria-associated type I interferon signalling and a damage-driven response in vaginal epithelial cells. Nat Microbiol. 6(5):643-657.Pekmezovic et al. (2021) Human albumin enhances the pathogenic potential of Candida glabrata on vaginal epithelial cells. PLoS Pathog. 17(10):e1010037.Siscar-Lewin et al. (2021) Transient mitochondria dysfunction confers fungal cross-resistance against phagocytic killing and fluconazole. mBio 12(3):e0112821.

P343 Novel hydrophobic binding surface proteins are instrumental for phagocytosis of Lichtheimia corymbifera by macrophages

Poster session 3, September 23, 2022, 12:30 PM - 1:30 PMObjectivesPerform proteomic analysis of the spore surface alongside secretome analysis of two strains of Lichtheimia corymbifera.Evaluate interaction, recognition, and phagocytosis of Lichtheimia spores by murine alveolar macrophages.MethodsTwo strains of L. corymbifera (JMRC: FSU: 09682 and JMRC: FSU: 10164) were used in this study. For phagocytosis assay, the spores were labeled with 0.1 mg/ml Fluorescein isothiocyanate (FITC) (Sigma Aldrich Chemie) in 0.1 M Na2CO3 for 30 min 146 at 30°C. Murine alveolar macrophages MH-S (ATCC:CRL-2019) were cultivated in RPMI-1640 (Sigma, 30-2001) supplemented.Identification of the surface proteins of L. corymbifera was carried out as described before with minor modification.1 The supernatants were stored at −80°C for liquid chromatography-mass spectrometry (LC-MS/MS) analysis. Secreted proteins of L. corymbifera were determined as described in a previous study with minor modification2.The raw files generated by the LC-MS/MS were further processed by the software Proteome Discoverer v1.4.0.288 (Thermo). Tandem mass spectra were searched against the NCBI L. corymbifera protein database.3 Approximately 10 000 MH-S cells were cultivated onto 96-well microplates (NUNC 163320) in 100 μL RPMI-1640 medium (Sigma, 30-2001). Images were acquired by the Zeiss Axio Observer 7 Spinning Disk Confocal Microscope (ZEISS, Jena, Germany) and processed with ZEN 2.1 Software (ZEISS) by 63x objective lens.ResultsAbundant surface proteins were found which serve as Candidates for secretome analysis. A total of 113 proteins were identified. Thirty proteins were confirmed to be on the spore surface based on the presence of signal peptides3. The following proteins were predominantly identified: Spore coat protein (CotH), hydrophobic surface binding protein A (HsbA), aconitase, ricin-like lectin, two transition elongation factors, multi-copper oxidase, heat shock protein 70 family (Hsp70), malate synthase, putative allergen Candidates, etc.These proteins were heterologsly overexpressed in yeast. Successful overexpression was confirmed by LC-MS/MS. The yeast mutants were subjected to phagocytosis assays (Fig. 1). The role of surface proteins in macrophages is discussed.ConclusionThe surface proteins are instrumental for recognition of L. corymbifera by macrophages and for intracellular survival in macrophages. The role of surface proteins is discussed in the light of evolution from environmental to human pathogenic fungus.Sources:Voltersen V, Blango MG, Herrmann S, et al. Proteome Analysis Reveals the Conidial Surface Protein CcpA Essential for Virulence of the Pathogenic Fungus Aspergillus fumigatus. mBio. 2018;9(5). doi:10.1128/MBIO.01557-18Thürich J, Meichsner D, Furch ACU, et al. Arabidopsis thaliana responds to colonisation of Piriformospora indica by secretion of symbiosis-specific proteins. PLOS ONE. 2018;13(12):e0209658. doi:10.1371/JOURNAL. PONE.0209658.Schwartze VU, Winter S, Shelest E, et al. Gene Expansion Shapes Genome Architecture in the Human Pathogen Lichtheimia corymbifera: An Evolutionary Genomics Analysis in the Ancient Terrestrial Mucorales (Mucoromycotina). PLOS Genetics. 2014;10(8):e1004496. doi:10.1371/JOURNAL.PGEN.1004496.

Streptococcus agalactiae cadD alleviates metal stress and promotes intracellular survival in macrophages and ascending infection during pregnancy

Perinatal infection with Streptococcus agalactiae, or Group B Streptococcus (GBS), is associated with preterm birth, neonatal sepsis, and stillbirth. Here, we study the interactions of GBS with macrophages, essential sentinel immune cells that defend the gravid reproductive tract. Transcriptional analyses of GBS-macrophage co-cultures reveal enhanced expression of a gene encoding a putative metal resistance determinant, cadD. Deletion of cadD reduces GBS survival in macrophages, metal efflux, and resistance to metal toxicity. In a mouse model of ascending infection during pregnancy, the ΔcadD strain displays attenuated bacterial burden, inflammation, and cytokine production in gestational tissues. Furthermore, depletion of host macrophages alters cytokine expression and decreases GBS invasion in a cadD-dependent fashion. Our results indicate that GBS cadD plays an important role in metal detoxification, which promotes immune evasion and bacterial proliferation in the pregnant host.

Effect of Mycobacterium tuberculosis higBA on Bacterial Stress Response and Intracellular Infection and Immunity

To investigate the effect of Mycobacterium tuberculosis ( Mtb) higBA on bacterial stress response and intracellular infection and immunity. The target gene amplified from Mtb H37Rv genome was cloned to the vector and then transferred to Mycobacterium smegmatis ( Ms) to construct a recombinant strain. Stress response experiment and Raw264.7 mouse macrophage infection was carried out with Ms_higBA, the recombinant strain, and Ms_ vec, the vector strain. Tests were conducted to measure bacterial colony forming unit (CFU) and transcriptional levels of cytokines, including interleukin ( IL)-1β, IL-6, IL-10, IL-12 p40, interferon ( IFN)- γ, tumor necrosis factor ( TNF)- α, and inducible nitric oxide synthase ( iNOS). The recombinant strain, Ms_higBA, was constructed successfully. According to the findings of the stress response experiment, higBA could indeed enhance bacterial survival under certain conditions of in vitro culture. Intracellular infection experiment demonstrated that higBA enhanced bacterial survival in macrophages and influenced the transcriptional level of cytokines. The higBA genes from Mtb play a role in bacterial stress response and intracellular infection and immunity.

Macrophage Polarization Mediated by Mitochondrial Dysfunction Induces Adipose Tissue Inflammation in Obesity.

Obesity is one of the prominent global health issues, contributing to the growing prevalence of insulin resistance and type 2 diabetes. Chronic inflammation in adipose tissue is considered as a key risk factor for the development of insulin resistance and type 2 diabetes in obese individuals. Macrophages are the most abundant immune cells in adipose tissue and play an important role in adipose tissue inflammation. Mitochondria are critical for regulating macrophage polarization, differentiation, and survival. Changes to mitochondrial metabolism and physiology induced by extracellular signals may underlie the corresponding state of macrophage activation. Macrophage mitochondrial dysfunction is a key mediator of obesity-induced macrophage inflammatory response and subsequent systemic insulin resistance. Mitochondrial dysfunction drives the activation of the NLRP3 inflammasome, which induces the release of IL-1β. IL-1β leads to decreased insulin sensitivity of insulin target cells via paracrine signaling or infiltration into the systemic circulation. In this review, we discuss the new findings on how obesity induces macrophage mitochondrial dysfunction and how mitochondrial dysfunction induces NLRP3 inflammasome activation. We also summarize therapeutic approaches targeting mitochondria for the treatment of diabetes.

Primary Murine Macrophages as a Tool for Virulence Factor Discovery in Coxiella burnetii.

ABSTRACTCoxiella burnetii requires a type IVB secretion system (T4SS) to promote intracellular replication and virulence. We hypothesized that Coxiella employs its T4SS to secrete effectors that enable stealthy colonization of immune cells. To address this, we used RNA sequencing to compare the transcriptional response of murine bone marrow-derived macrophages (BMDM) infected with those of wild-type Coxiella and a T4SS-null mutant at 8 and 24 h postinfection. We found a T4SS-independent upregulation of proinflammatory transcripts which was consistent with a proinflammatory polarization phenotype. Despite this, infected BMDM failed to completely polarize, as evidenced by modest surface expression of CD38 and CD11c, nitrate production, and reduced proinflammatory cytokine and chemokine secretion compared to positive controls. As these BMDM permitted replication of C. burnetii, we employed them to identify T4SS effectors that are essential in the specific cellular context of a primary macrophage. We found five Himar1 transposon mutants in T4SS effectors that had a replication defect in BMDM but not J774A.1 cells. The mutants were also attenuated in a SCID mouse model of infection. Among these candidate virulence factors, we found that CBU1639 contributed to the inhibition of macrophage proinflammatory responses to Coxiella infection. These data demonstrate that while T4SS is dispensable for the stealthy invasion of primary macrophages, Coxiella has evolved multiple T4SS effectors that specifically target macrophage function to proliferate within that specific cellular context.IMPORTANCE Coxiella burnetii, the causative agent of Q fever, preferentially infects macrophages of the respiratory tract when causing human disease. This work describes how primary macrophages respond to C. burnetii at the earliest stages of infection, before bacterial replication. We found that while infected macrophages increase expression of proinflammatory genes after bacterial entry, they fail to activate the accompanying antibacterial functions that might ultimately control the infection. This disconnect between initial response and downstream function was not mediated by the bacterium’s type IVB secretion system, suggesting that Coxiella has other virulence factors that dampen host responses early in the infection process. Nevertheless, we were able to identify several type IVB secreted effectors that were specifically required for survival in macrophages and mice. This work is the first to identify type IVB secretion effectors that are specifically required for infection and replication within primary macrophages.

Regulatory cross-talk supports resistance to Zn intoxication in Streptococcus.

Metals such as copper (Cu) and zinc (Zn) are important trace elements that can affect bacterial cell physiology but can also intoxicate bacteria at high concentrations. Discrete genetic systems for management of Cu and Zn efflux have been described in several bacterial pathogens, including streptococci. However, insight into molecular cross-talk between systems for Cu and Zn management in bacteria that drive metal detoxification, is limited. Here, we describe a biologically consequential cross-system effect of metal management in group B Streptococcus (GBS) governed by the Cu-responsive copY regulator in response to Zn. RNAseq analysis of wild-type (WT) and copY-deficient GBS subjected to metal stress revealed unique transcriptional links between the systems for Cu and Zn detoxification. We show that the Cu-sensing role of CopY extends beyond Cu and enables CopY to regulate Cu and Zn stress responses that effect changes in gene function for central cellular processes, including riboflavin synthesis. CopY also supported GBS intracellular survival in human macrophages and virulence during disseminated infection in mice. In addition, we show a novel role for CovR in modulating GBS resistance to Zn intoxication. Identification of the Zn resistome of GBS using TraDIS revealed a suite of genes essential for GBS growth in metal stress. Several of the genes identified are novel to systems that support bacterial survival in metal stress and represent a diverse set of mechanisms that underpin microbial metal homeostasis during cell stress. Overall, this study reveals a new and important mechanism of cross-system complexity driven by CopY in bacteria to regulate cellular management of metal stress and survival.

097 Epidermal integrin α3β1 is a regulator of cytokine, CSF-1, and of crosstalk from keratinocytes to dermal macrophages

As the major cell surface receptors for the extracellular matrix, integrins regulate adhesion and migration, and have been shown to drive tumor growth and progression. Our earlier findings have identified integrin α3β1 as a key regulator of the keratinocyte secretome and of the skin tumor microenvironment, indicating its potential efficacy as a therapeutic target. Previous mass spectrometric analysis of conditioned medium from immortalized keratinocytes indicated that α3β1 positively regulates colony-stimulating factor 1 (CSF-1), a secreted cytokine that is a primary regulator of macrophage differentiation, proliferation, and survival.

Human iNKT Cells Modulate Macrophage Survival and Phenotype.

CD1d-restricted invariant Natural Killer T (iNKT) cells are unconventional innate-like T cells whose functions highly depend on the interactions they establish with other immune cells. Although extensive studies have been reported on the communication between iNKT cells and macrophages in mice, less data is available regarding the relevance of this crosstalk in humans. Here, we dove into the human macrophage-iNKT cell axis by exploring how iNKT cells impact the survival and polarization of pro-inflammatory M1-like and anti-inflammatory M2-like monocyte-derived macrophages. By performing in vitro iNKT cell-macrophage co-cultures followed by flow cytometry analysis, we demonstrated that antigen-stimulated iNKT cells induce a generalized activated state on all macrophage subsets, leading to upregulation of CD40 and CD86 expression. CD40L blocking with a specific monoclonal antibody prior to co-cultures abrogated CD40 and CD86 upregulation, thus indicating that iNKT cells required CD40-CD40L co-stimulation to trigger macrophage activation. In addition, activated iNKT cells were cytotoxic towards macrophages in a CD1d-dependent manner, killing M1-like macrophages more efficiently than their naïve M0 or anti-inflammatory M2-like counterparts. Hence, this work highlighted the role of human iNKT cells as modulators of macrophage survival and phenotype, untangling key features of the human macrophage-iNKT cell axis and opening perspectives for future therapeutic modulation.

Hypoxia-induced polypoid giant cancer cells in glioma promote the transformation of tumor-associated macrophages to a tumor-supportive phenotype.

AimsPolypoid giant cancer cells (PGCCs) represent a unique subgroup of stem‐like cells, acting as a critical factor in promoting the recurrence of various solid tumors. The effect of PGCCs on the tumor malignancy of glioma and its immune microenvironment remains unclear.MethodsBioinformatic analysis was performed to investigate the relationship between M2 tumor‐associated macrophages (TAMs) infiltration and survival of glioblastoma (GBM) patients. The spatial location of M2 TAMs in GBM was also investigated using the Ivy Glioblastoma Atlas Project (Ivy GAP) database. PGCCs were quantified in glioma of different grades. CoCl2 was used to induce PGCCs in cultures of A172 cells. PGCCs, and their progeny cells in cultures were further evaluated for morphological features, tumorsphere formation, and TAMs activation.ResultsThe magnitude of M2 TAMs infiltration is significantly correlated with poor survival in GBM patients. M2 TAMs were enriched in the perinecrotic zone (PNZ) of GBM and positively correlated with hypoxic levels. Increased PGCCs were detected in glioma specimens of higher grades. CoCl2 induced hypoxia and the transformation of A172 cultures into PGCCs, producing the progeny cells, PGCCs‐Dau, through asymmetric division. PGCCs and PGCCs‐Dau possessed tumor stem cell‐like features, while PGCCs‐Dau enhanced the polarization of TAMs into an M2 phenotype with relevance to immunosuppression and malignancy in GBM.ConclusionsPGCCs promote malignancy and immune‐suppressive microenvironment in GBM. PGCCs or their progeny cells may be a potential therapeutic target for GBM.

The host micro-RNA cfa-miR-346 is induced in canine leishmaniasis

BackgroundLeishmaniases are a group of anthropo-zoonotic parasitic diseases caused by a protozoan of the Leishmania genus, affecting both humans and other vertebrates, including dogs. L. infantum is responsible for the visceral and occasionally cutaneous form of the disease in humans and canine leishmaniasis. Previously, we have shown that L. infantum induces a mild but significant increase in endoplasmic reticulum (ER) stress expression markers to promote parasites survival in human and murine infected macrophages. Moreover, we demonstrated that the miRNA hsa-miR-346, induced by the UPR-activated transcription factor sXBP1, was significantly upregulated in human macrophages infected with different L. infantum strains. However, the ER stress response in infected dogs, which represent an important reservoir for Leishmania parasite, was described once recently, whereas the miR-346 expression was not reported before. Therefore, this study aimed to investigate these pathways in the canine macrophage-like cell line DH82 infected by Leishmania spp. and to evaluate the presence of cfa-miR-346 in plasma of non-infected and infected dogs. The DH82 cells were infected with L. infantum and L. braziliensis parasites and the expression of cfa-mir-346 and several ER stress markers was evaluated by quantitative PCR (qPCR) at different time points. Furthermore, the cfa-miR-346 was monitored in plasma collected from non-infected dogs (n = 11) and dogs naturally infected by L. infantum (n = 18).ResultsThe results in DH82 cells showed that cfa-mir-346 was induced at both 24 h and 48 h post-infection with all Leishmania strains but not with tunicamycin, accounting for a mechanism of induction independent from sXBP1, unlike what was previously observed in human cell lines. Moreover, the cfa-miR-346 expression analysis on plasma revealed a significant increase in infected dogs compared to non-infected dogs.ConclusionsHere for the first time, we report the upregulation of cfa-miR-346 induced by Leishmania infection in canine macrophage-like cells and plasma samples of naturally infected dogs. According to our results, the cfa-miR-346 appears to be linked to infection, and understanding its role and identifying its target genes could contribute to elucidate the mechanisms underlying the host–pathogen interaction in leishmaniasis.

NAD kinase promotes Staphylococcus aureus pathogenesis by supporting production of virulence factors and protective enzymes.

Nicotinamide adenine dinucleotide phosphate (NADPH) is the primary electron donor for reductive reactions that are essential for the biosynthesis of major cell components in all organisms. Nicotinamide adenine dinucleotide kinase (NADK) is the only enzyme that catalyzes the synthesis of NADP(H) from NAD(H). While the enzymatic properties and physiological functions of NADK have been thoroughly studied, the role of NADK in bacterial pathogenesis remains unknown. Here, we used CRISPR interference to knock down NADK gene expression to address the role of this enzyme in Staphylococcus aureus pathogenic potential. We find that NADK inhibition drastically decreases mortality of zebrafish infected with S. aureus. Furthermore, we show that NADK promotes S. aureus survival in infected macrophages by protecting bacteria from antimicrobial defense mechanisms. Proteome-wide data analysis revealed that production of major virulence-associated factors is sustained by NADK. We demonstrate that NADK is required for expression of the quorum-sensing response regulator AgrA, which controls critical S. aureus virulence determinants. These findings support a key role for NADK in bacteria survival within innate immune cells and the host during infection.

Abstract 3133: Glucose is important for macrophage metabolism and response to immunotherapy in a pancreatic cancer model

Abstract Introduction: Pancreatic cancer (PC) is resistant to systemic therapies. This is at least in part due to the characteristically harsh tumor microenvironment which limits the delivery of systemic therapies and is not conducive to anti-tumor immune cell function. Unfortunately, development of novel therapeutics is arduous. Thus, identification of methods to augment the efficacy of existing therapeutics, such as immunotherapies, represents an attractive option. Herein, we attempt to sensitize PC to a colony stimulating factor 1 receptor (CSF1R) inhibitor by augmenting peripheral glucose levels. Methods Murine PC cells (KPC) and bone marrow derived macrophages were cultured in hypoglycemic (≤2.5mM) or hyperglycemic (25mM) conditions. Phenotypic (western blot, flow cytometry), metabolic (seahorse, LC-MS metabolomics), and cell survival (clonogenic assay) assays were performed. KPC cells were orthotopically injected into the pancreas of immunocompetent mice for survival studies. Mice were randomized to pexidartinib (a CSF1R inhibitor) or vehicle and received either normal water of 30% dextrose (D30). Results: In a co-culture experiment, tumor-supporting M2 macrophages increased KPC cell growth by 30%, whereas tumor-fighting M1 macrophages decreased KPC growth by 95%. M1 macrophages were poorly suited to survive in hypoglycemic conditions as compared to hyperglycemic conditions (relative survival: 38% vs 100%, p<0.01). M2 macrophage survival was equivalent (93% vs 100%, p=0.39). When cultured in hypoglycemic conditions, M1 macrophages adopted an M2-like metabolic profile (i.e., increased TCA cycle metabolites and decreased glycolytic metabolites) assessed by both seahorse and LC-MS metabolomics. As glucose concentrations decreased in culture media over time, protein levels of an M1 marker (inducible nitric oxide, iNOS) decreased and an M2 marker (arginase) appeared, suggesting a phenotypic switch. Pexidartinib augmented M1 iNOS protein levels and simultaneously decreased M2 arginase levels. On an in vitro flow cytometric analysis, the percentage of M2 macrophages (CD206+, CD301+, F480+, CD11b+) decreased as glucose concentrations were increased (33.7% vs 38.0%), and decreased further when pexidartinib was combined with hyperglycemia (20.5%). Mice treated with pexidartinib and D30 had improved median survival relative to mice receiving pexidartinib alone (median survival: 42 (IQR: 38, 52) vs 34 (IQR: 32, 38) days, p<0.05) in two independent experiments. Of note, pexidartinib alone did not improve survival as compared to vehicle or D30. Conclusion: Higher glucose conditions promote M1 macrophage survival and function, while preventing the switch to an M2 phenotype. Also, higher peripheral glucose levels appear to sensitize PC to a CSF1R inhibitor. We plan to validate these findings using other immunotherapeutics (e.g., checkpoint inhibitors). Citation Format: Jonathan J. Hue, Mehrdad Zarei, Hallie J. Graor, Erryk S. Katayama, Omid Hajihassani, Alexander W. Loftus, Luke D. Rothermel, Ali Vaziri-Gohar, Jordan M. Winter. Glucose is important for macrophage metabolism and response to immunotherapy in a pancreatic cancer model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3133.

Overexpression of VPS11 antagonizes the promoting effect of miR-542-3p on Mycobacterium tuberculosis survival in macrophages by regulating autophagy

Impaired autophagy is an important cause of Mycobacterium tuberculosis survival in macrophages. VPS11 is an important regulator of autophagy; decreased VPS11 expression has been observed in macrophages after tuberculosis (TB) infection. Gene ontology data revealed that various miRNAs (for example, miR-542-3p) were upregulated in macrophages upon TB infection; thus, these miRNAs were likely to reduce VPS11 expression. In this study, both TB patients and healthy subjects were enrolled, and the levels of VPS11 and some miRNAs in their blood macrophages were measured. Moreover, various macrophages were cultured and infected with M. tuberculosis. Luciferase reporter, RNA pulldown, and RNA immunoprecipitation assays were performed to determine the regulatory effect of miR-542-3p on VPS11 expression. Results showed that VPS11 expression was downregulated, whereas miR-542-3p expression was upregulated in blood macrophages after TB infection. TB infection reduced VPS11 levels in two human macrophages in vitro, but not in mouse macrophages. This might be because the seed sequence exists in the VPS11 3’ untranslated region in humans, but is absent in mice and rats. miR-542-3p promoted M. tuberculosis survival in human macrophages, but VPS11 overexpression antagonized the promoting effect of miR-542-3p. Further, VPS11 was confirmed as a target of miR-542-3p. Overexpression of VPS11 or depletion of miR-542-3p promoted autophagy, which was suppressed upon TB infection. In summary, VPS11 overexpression antagonized the promoting effect of miR-542-3p on M. tuberculosis survival in macrophages by regulating autophagy.