Macrophage Survival Research Articles

Mycobacterium tuberculosis Acetyltransferase Suppresses Oxidative Stress by Inducing Peroxisome Formation in Macrophages.

Mycobacterium tuberculosis (Mtb) inhibits host oxidative stress responses facilitating its survival in macrophages; however, the underlying molecular mechanisms are poorly understood. Here, we identified a Mtb acetyltransferase (Rv3034c) as a novel counter actor of macrophage oxidative stress responses by inducing peroxisome formation. An inducible Rv3034c deletion mutant of Mtb failed to induce peroxisome biogenesis, expression of the peroxisomal β-oxidation pathway intermediates (ACOX1, ACAA1, MFP2) in macrophages, resulting in reduced intracellular survival compared to the parental strain. This reduced virulence phenotype was rescued by repletion of Rv3034c. Peroxisome induction depended on the interaction between Rv3034c and the macrophage mannose receptor (MR). Interaction between Rv3034c and MR induced expression of the peroxisomal biogenesis proteins PEX5p, PEX13p, PEX14p, PEX11β, PEX19p, the peroxisomal membrane lipid transporter ABCD3, and catalase. Expression of PEX14p and ABCD3 was also enhanced in lungs from Mtb aerosol-infected mice. This is the first report that peroxisome-mediated control of ROS balance is essential for innate immune responses to Mtb but can be counteracted by the mycobacterial acetyltransferase Rv3034c. Thus, peroxisomes represent interesting targets for host-directed therapeutics to tuberculosis.

Mycobacterium tuberculosis Rv0309 Dampens the Inflammatory Response and Enhances Mycobacterial Survival.

To reveal functions of novel Mycobacterium tuberculosis (M. tb) proteins responsible for modulating host innate immunity is essential to elucidation of mycobacterial pathogenesis. In this study, we aimed to identify the role of a putative protein Rv0309 encoded within RD8 of M. tb genome in inhibiting the host inflammatory response and the underlying mechanism, using in-vitro and in-vivo experiments. A recombinant M. smegmatis strain Ms_rv0309 expressing Rv0309 and a mutant Bacillus Calmette-Guérin (BCG)ΔRS01790 strain with deletion of BCG_RS01790, 100% homologue of Rv0309 in BCG, were constructed. Rv0309 was found to localize in the cell wall and be able to decrease cell wall permeability. Purified recombinant rRv0309 protein inhibited lipopolysaccharide-induced IL-6 release in RAW264.7 cells. BCG_RS01790 in BCG or Rv0309 in Ms_rv0309 strain greatly inhibited production of IL-6, IL-1β, and TNF-α in RAW264.7 cells. Similarly, BCGΔRS01790 strongly induced expression of these cytokines compared with wild-type BCG and complement strain, cBCGΔRS01790::RS01790. Further BCG_RS01790 or Rv0309 suppressed cytokine production through NF-κB p65/IκBα and MAPK ERK/JNK signaling. Importantly, BCG_RS01790 in BCG and Rv0309 in Ms_rv0309 strain enhanced mycobacterial survival in macrophages. Mice infected with BCGΔRS01790 exhibited high levels of IFN-γ, TNF-α and IL-1β, and large numbers of neutrophils and lymphocytes in the early stage, and minimal lung bacterial load and inflammatory damage in late stage of the experiment. In conclusion, the cell wall protein Rv0309 or BCG_RS01790 enhanced mycobacterial intracellular survival after infection likely through inhibition of the pro-inflammatory response and decrease of bacterial cell wall permeability, thereby contributing to mycobacterial pathogenesis.

Structural investigation on SPI-6-associated Salmonella typhimurium VirG-like stress protein that promotes pathogen survival in macrophages.

Enteric microbial pathogenesis, remarkably a complex process, is achieved by virulence factors encoded by genes located within regions of the bacterial genome termed pathogenicity islands. Salmonella pathogenicity islands (SPI) encodes proteins, that are essential virulence determinants for pathogen colonization and virulence. In addition to the well-characterized SPI-1 and SPI-2 proteins, which are required for bacterial invasion and intracellular replication, respectively, SPI-6 (formerly known as Salmonella enterica centisome 7 island [SCI]) encoding proteins are also known to play pivotal role in Salmonella pathogenesis. However, the underlying molecular mechanism of these proteins remained elusive. To gain molecular insights into SPI-6-associated proteins, in this study, a SPI-6 Salmonella typhimurium VirG-like protein (STV) is characterized using interdisciplinary experimental approaches including X-ray crystallography, nuclear magnetic resonance (NMR) spectroscopy and infection assays. The high-resolution crystal structure, determined by the single-wavelength anomalous dispersion (SAD) method, reveals that STV belongs to the LTxxQ motif family. Solution-state NMR spectroscopy studies reveal that STV form a dimer involving interconnected helices. Interestingly, functional studies show that STV influence pathogen persistence inside macrophages in vitro at later stages of infection. Altogether, our findings suggest that STV, a member of the LTxxQ stress protein family, modulates bacterial survival mechanism in macrophages through SPI-1 and SPI-2 genes, respectively.

System Analysis of Adaptor-Related Protein Complex 1 Subunit Mu 2 (AP1M2) on Malignant Tumors: A Pan-Cancer Analysis.

Objective To identify new tumor marker genes available for early tumor screening, differentially expressed gene profiles of multiple tumors were compared using Genotype-Tissue Expression (GTEx), Cancer Cell Line Encyclopedia (CCLE), and The Cancer Genome Atlas (TCGA) databases. As AP1M2 was highly and differentially expressed in invasive breast carcinoma, the purpose of this study was to explore the association of AP1M2 gene with the survival, immune invasion, and tumor neoantigens of patients on a pan-cancer basis. Methods The expression and distribution of AP1M2 gene in tumor tissues and the corresponding normal control tissues were analyzed using the pan-cancer databases GTEx, CCLE, and TCGA. Kaplan-Meyer survival plots and proportional hazards model (COX) were employed to evaluate actions of AP1M2 on the clinical prognosis of tumor patients. Subsequently, the association of AP1M2 expression with immune invasion in different tumor types was explored. Simultaneously, the investigation of the interrelationship of AP1M2 and tumor neoantigens of the immune system, unstable microsatellite, DNA repair genes, and DNA methyltransferases were explored, and the mutation frequency of AP1M2 gene in diverse tumors was studied. Several tumor types were analyzed using gene-set enrichment analysis (GSEA). Results AP1M2 was abundantly expressed in a wide range of cancers, and its expression level was positively correlated with the outcome of tumor victims. Through a study on AP1M2 action on clinical prognosis and immune infiltration in tumor patients, AP1M2 expression in breast-infiltrating carcinoma was found to be highly associated with patients' overall survival and infiltration levels of macrophages, dendritic cells, T cells (CD4+ and CD8+), and B cells. Also, AP1M2 expression was positively correlated with tumor immune neoantigens and microsatellite instability in breast invasive carcinoma. The effect of AP1M2 on tumors was analyzed by GSEA, and findings demonstrated that AP1M2 expression levels in most tumors influenced the activation of tumor-associated pathways and immune-associated pathways. Conclusions These findings suggest that AP1M2 expression levels are significantly correlated to patients' outcomes and levels of immune infiltration in most cancer types, including T cells (CD8+ and CD4+), macrophages, neutrophils, and dendritic cells (DCs), particularly in breast cancer. The results indicate that AP1M2 may influence the tumor environment of invasive breast cancer patients and it may be a target contributing to early screening and treatment for breast cancer, helping improve the efficiency of early screening and overall survival rate in invasive breast cancer patients.

Nanoparticle Internalization Promotes the Survival of Primary Macrophages.

Macrophages, a class of tissue resident innate immune cells, are responsible for sequestering foreign objects through the process of phagocytosis, making them a promising target for immune-modulation via particulate engineering. Here, we report that nanoparticle (NP) dosing and cellular internalization via phagocytosis significantly enhances survival of ex vivo cultures of primary bone marrow-derived, alveolar, and peritoneal macrophages over particle-free controls. The enhanced survival is attributed to suppression of caspase-dependent apoptosis and is linked to phagocytosis and lysosomal signaling. Uniquely, poly(ethylene glycol)-based NP treatment extended cell viability in the absence of macrophage polarization and enhanced expression of pro-survival B cell lymphoma-2 (Bcl-2) protein in macrophages following multiple routes of in vivo administration. The enhanced survival phenomenon is also applicable to NPs of alternative chemistries, indicating the potential universality of this phenomenon with relevant drug delivery particles. These findings provide a framework for extending the lifespan of primary macrophages ex vivo for drug screening, vaccine studies, and cell therapies and has implications for any in vivo particulate immune-engineering applications.

The Dual Role of CCR5 in the Course of Influenza Infection: Exploring Treatment Opportunities.

Influenza is one of the most relevant respiratory viruses to human health causing annual epidemics, and recurrent pandemics. Influenza disease is principally associated with inappropriate activation of the immune response. Chemokine receptor 5 (CCR5) and its cognate chemokines CCL3, CCL4 and CCL5 are rapidly induced upon influenza infection, contributing to leukocyte recruitment into the airways and a consequent effective antiviral response. Here we discuss the existing evidence for CCR5 role in the host immune responses to influenza virus. Complete absence of CCR5 in mice revealed the receptor’s role in coping with influenza via the recruitment of early memory CD8+ T cells, B cell activation and later recruitment of activated CD4+ T cells. Moreover, CCR5 contributes to inflammatory resolution by enhancing alveolar macrophages survival and reprogramming macrophages to pro-resolving phenotypes. In contrast, CCR5 activation is associated with excessive recruitment of neutrophils, inflammatory monocytes, and NK cells in models of severe influenza pneumonia. The available data suggests that, while CCL5 can play a protective role in influenza infection, CCL3 may contribute to an overwhelming inflammatory process that can harm the lung tissue. In humans, the gene encoding CCR5 might contain a 32-base pair deletion, resulting in a truncated protein. While discordant data in literature regarding this CCR5 mutation and influenza severity, the association of CCR5delta32 and HIV resistance fostered the development of different CCR5 inhibitors, now being tested in lung inflammation therapy. The potential use of CCR5 inhibitors to modulate the inflammatory response in severe human influenza infections is to be addressed.

The role of antibiotic resistance mobile genetic element MCR-1 in enhancing bacterial survival in macrophages

Background: Antimicrobial resistance (AMR) determinants such as mobile colistin resistance (MCR-1) that encodes colistin resistance are increasingly spreading in healthcare-associated and community-acquired infections.1 Colistin, a cationic peptide antibiotic, resistance is encoded by the MCR-1 gene that functions as phosphoethanolamine (PEA) transferase which adds a PEA moiety to lipid A head group rendering it resistant to host antimicrobial cationic peptides (AMPs).2,3 The given hypothesis is that MCR-1 harboring bacteria survive longer in macrophages by evading AMPs. This study aims to investigate the role of MCR-1 in enhancing bacterial survival in macrophages. Methods: Eight E. coli strains were used in the study in which 4 strains were MCR-1 positive and 4 strains were negative. MCR-1 was confirmed by Polymerase Chain Reaction (PCR), and colistin and polymyxin minimal inhibitory concentrations (MICs) were determined using the microdilution method. Macrophage bactericidal assay was employed to examine bacterial survival using adherent murine RAW264 macrophages in an in-vitro bacterial infection model. Briefly, Macrophages were infected with E. coli strains at a multiplicity of infection (MOI) of 50 for 1 hour. The survival of bacteria associated with macrophages was quantified by agar plating method to calculate colony forming units (CFU/ml). Cytokines released from infected macrophages were quantified using ELISA method respectively. Results: Colistin MICs for MCR-1 positive E. coli strains were >25 μg/ml, whereas MCR-1 negative E. coli MICs < 6.2 μg/ml. E. coli strains encoding MCR-1 survived significantly more in association with macrophages (p = 0.024) compared to MCR-1 negative E. coli strains. Further, E.coli strains encoding MCR-1 induced slightly less IL-1β release from infected macrophages compared to E. coli strains without MCR-1 (p = 0.05). Taken together, the data suggest that MCR-1 enhanced bacterial survival in association with macrophages and modulated innate immune responses which may lead to treatment failure. Conclusion: MCR-1 encoding E. coli strains conferred resistance to colistin and survived more in association with macrophages.

Novel genetic polymorphisms identified in the clinical isolates of Mycobacterium tuberculosis PE_PGRS33 gene modulate cytokines expression and promotes survival in macrophages

ObjectivePE_PGRS33 is a member of multi-gene family restricted to pathogenic Mycobacteria and their functions remain elusive. PE_PGRS33 is highly polymorphic in nature to evade host’s immune response. Therefore, we investigated PE_PGRS33 gene polymorphisms in clinical isolates and functional characterization using in vitro experiments. MethodsA total of 103 clinical isolates were recruited. Genomic DNA was extracted, PE_PGRS33 gene amplification, sequencing. Afterward, we have cloned, expressed PE_PGRS33 wild type and three polymorphic alleles in M. smegmatis. Further, performed in vitro stresses assays, THP-1 differentiated macrophage infection assays followed by quantification of cytokine expression. ResultsWe have identified nine novel polymorphisms and also demonstrated that in comparison to M. smegmatis expressing wild type/Mut_alleles displayed altered in growth kinetics and colony morphology. M. smegmatis harbouring Mut_allele3 survived better under oxidative, acidic stress and were resistant to lysozyme treatment. qRT-PCR of cytokines TNF-α, IL-12 and IL-10 after infection with recombinant M. smegmatis showed two Mut_allele (Mut_allele1 and Mut_allele3) induced higher expression of TNF-α, IL-12, while IL-10 expression was decreased in both mutant alleles as compared to wild type PE_PGRS33 at each experimental time point. ConclusionResults of our functional study suggest that PE_PGRS33 gene polymorphisms aid in the survival or persistence of M. tuberculosis and differentially modulate the expression of various cytokines. Overall this study suggests that Mtb clinical strains harbouring different PE_PGRS33 alleles could act as a virulence determinant by differentially regulating pathways essential for the pathogen’s ability to adapt inside the host.

Endoplasmic Reticulum Stress in Osteoarthritis: A Novel Perspective on the Pathogenesis and Treatment.

Osteoarthritis (OA), the most common degenerative joint disease, causes an enormous socioeconomic burden due to its disabling properties and high prevalence. Increasing evidence suggests that OA is a whole-joint disease involving cartilage degradation, synovitis, meniscal lesions, and subchondral bone remodeling. Endoplasmic reticulum (ER) stress is the accumulation of misfolded/unfolded proteins in the ER. Recent studies have found that ER stress is involved in the OA pathological changes by influencing the physiological function and survival of chondrocytes, fibroblast-like synoviocytes, synovial macrophages, meniscus cells, osteoblasts, osteoclasts, osteocytes, and bone marrow mesenchymal stem cells. Therefore, ER stress is an attractive and promising target for OA. However, although targeting ER stress has been proven to alleviate OA progression in vitro and in vivo, the treatments for OA remain in preclinical stage and require further investigation.

AKT Isoforms in Macrophage Activation, Polarization, and Survival.

Macrophages display an array of activation phenotypes depending on the activation signal and the cellular microenvironment. The type and magnitude of the response depend on signaling molecules as well as on the epigenetic and metabolic status of the cells at the time of activation. The AKT family of kinases consists of three isoforms encoded by independent genes possessing similar functions and structures. Generation of research tools such as isoform-specific gene deletion mutant mice and cells and isoform-specific antibodies has allowed us to understand the role of each kinase isoform in macrophage activation and homeostasis. This chapter discusses the current evidence on the role of AKT kinases in macrophage activation, polarization, and homeostasis, highlighting the gaps in knowledge and future challenges in the field.

Abstract IA01: Unconventional ER stress response pro-tumorigenic polarization and survival in TAMs

Abstract Tumor-associated macrophages (TAMs) display pro-tumorigenic phenotypes for supporting tumor progression in response to microenvironmental cues imposed by tumor and stromal cells. However, the underlying mechanisms by which tumor cells instruct TAM behavior remain elusive. Here we uncover that tumor cell-derived glucosylceramide stimulated unconventional endoplasmic reticulum (ER) stress responses by inducing reshuffling of lipid composition and saturation on the ER membrane in macrophages, which induced IRE1-mediated spliced XBP1 production and STAT3 activation. The cooperation of spliced XBP1 and STAT3 reinforced the pro-tumorigenic phenotype and expression of immunosuppressive genes. Ablation of XBP-1 expression with genetic manipulation or ameliorating ER stress responses by facilitating LPCAT3-mediated incorporation of unsaturated lipids to the phosphatidylcholine hampered pro-tumorigenic phenotype and survival in TAMs. Together, our findings reveal the unexpected roles of tumor cell-produced lipids that simultaneously orchestrate macrophage polarization and survival in tumors via induction of ER stress responses and therapeutic targets for sustaining host anti-tumor immunity. Citation Format: Ping-Chih Ho. Unconventional ER stress response pro-tumorigenic polarization and survival in TAMs [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2021 Oct 5-6. Philadelphia (PA): AACR; Cancer Immunol Res 2022;10(1 Suppl):Abstract nr IA01.

A novel leishmanial copper P-type ATPase plays a vital role in parasite infection and intracellular survival

Copper (Cu) is essential for all life forms; however, in excess, it becomes toxic. Toxic properties of Cu are known to be utilized by host species against various pathogenic invasions. Leishmania, in both free-living and intracellular forms, exhibits appreciable tolerance toward Cu stress. While determining the mechanism of Cu-stress evasion employed by Leishmania, we identified and characterized a hitherto unknown Cu-ATPase in Leishmania major and established its role in parasite survival in host macrophages. This novel L. major Cu-ATPase, LmATP7, exhibits homology with its orthologs at multiple motifs. In promastigotes, LmATP7 primarily localized at the plasma membrane. We also show that LmATP7 exhibits Cu-dependent expression patterns and complements Cu transport in a Cu-ATPase-deficient yeast strain. Promastigotes overexpressing LmATP7 exhibited higher survival upon Cu stress, indicating efficacious Cu export compared with Wt and heterozygous LmATP7 knockout parasites. We further explored macrophage–Leishmania interactions with respect to Cu stress. We found that Leishmania infection triggers upregulation of major mammalian Cu exporter, ATP7A, in macrophages, and trafficking of ATP7A from the trans-Golgi network to endolysosomes in macrophages harboring amastigotes. Simultaneously, in Leishmania, we observed a multifold increase in LmATP7 transcripts as the promastigote becomes established in macrophages and morphs to the amastigote form. Finally, overexpressing LmATP7 in parasites increases amastigote survivability within macrophages, whereas knocking it down reduces survivability drastically. Mice injected in their footpads with an LmATP7-overexpressing strain showed significantly larger lesions and higher amastigote loads as compared with controls and knockouts. These data establish the role of LmATP7 in parasite infectivity and intramacrophagic survivability.

TCM Regulates PI3K/Akt Signal Pathway to Intervene Atherosclerotic Cardiovascular Disease.

Vascular endothelial injury is the initial stage of atherosclerosis (AS). Stimulating and activating the phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) signaling pathway can regulate the expression of vascular endothelial cytokines, thus affecting the occurrence and development of AS. In addition, the PI3K/Akt signaling pathway can regulate the polarization and survival of macrophages and the expression of inflammatory factors and platelet function, thus influencing the progression of AS. In recent years, traditional Chinese medicine (TCM) has been widely recognized for its advantages of fewer side effects, multiple pathways, and multiple targets. Also, the research of TCM regulation of AS via the PI3K/Akt signaling pathway has achieved certain results. This study aimed to analyze the characteristics of the PI3K/Akt signaling pathway and its role in the pathogenesis of AS, as well as the role of Chinese medicine in regulating the PI3K/Akt signaling pathway. The findings are expected to provide a theoretical basis for the clinical treatment and pathological mechanism research of AS.

Glutamate dehydrogenase enables Salmonella to survive under oxidative stress and escape from clearance in macrophages.

Oxidative stress and the production of reactive oxygen species (ROS) are a biological threat to bacteria, which induce the synthesis of proteins and production of antioxidants to combat it. Herein, we report that glutamate dehydrogenase (GDH) of Salmonella can assimilate ammonium into glutamate and promote the generation of glutathione (GSH) to combat oxidative damage. Oxidation induces the transcription of gdhA, which encodes GDH, and activates the enzymatic activity of GDH. The ΔgdhA mutant Salmonella strain showed decreased levels of GSH and reduced survival in macrophages, and this growth deficiency could be partially restored by overexpression of GDH and complementation with its downstream metabolites. Therefore, GDH plays a critical role in the growth of Salmonella in oxidative environments, especially under low energy supply.

Mycobacterium abscessus Genetic Determinants Associated with the Intrinsic Resistance to Antibiotics.

Mycobacterium abscessus subsp. abscessus (MAB) is a fast-growing nontuberculous mycobacterium causing pulmonary infections in immunocompromised and immunocompetent individuals. The treatment of MAB infections in clinics is extremely challenging, as this organism is naturally resistant to most available antibiotics. There is limited knowledge on the mechanisms of MAB intrinsic resistance and on the genes that are involved in the tolerance to antimicrobials. To identify the MAB genetic factors, including the components of the cell surface transport systems related to the efflux pumps, major known elements contributing to antibiotic resistance, we screened the MAB transposon library of 2000 gene knockout mutants. The library was exposed at either minimal inhibitory (MIC) or bactericidal concentrations (BC) of amikacin, clarithromycin, or cefoxitin, and MAB susceptibility was determined through the optical density. The 98 susceptible and 36 resistant mutants that exhibited sensitivity below the MIC and resistance to BC, respectively, to all three drugs were sequenced, and 16 mutants were found to belong to surface transport systems, such as the efflux pumps, porins, and carrier membrane enzymes associated with different types of molecule transport. To establish the relevance of the identified transport systems to antibiotic tolerance, the gene expression levels of the export related genes were evaluated in nine MAB clinical isolates in the presence or absence of antibiotics. The selected mutants were also evaluated for their ability to form biofilms and for their intracellular survival in human macrophages. In this study, we identified numerous MAB genes that play an important role in the intrinsic mechanisms to antimicrobials and further demonstrated that, by targeting components of the drug efflux system, we can significantly increase the efficacy of the current antibiotics.

Lipid droplet accumulation occurs early following Salmonella infection and contributes to intracellular bacterial survival and replication.

Salmonellosis is a public health problem caused by Salmonella sp., a highly adapted facultative intracellular pathogen. After internalization, Salmonella sp. Manipulates several host processes, mainly through the activation of the type III secretion system (T3SS), including modification of host lipid metabolism and lipid droplet (LD) accumulation. LDs are dynamic and complex lipid-rich organelles involved in several cellular processes. The present study investigated the mechanism involved in LD biogenesis in Salmonella-infected macrophages and its role in bacterial pathogenicity. Here, we reported that S. Typhimurium induced a rapid time-dependent increase of LD formation in macrophages. The LD biogenesis was demonstrated to depend on Salmonella's viability and SPI1-related T3SS activity, with the participation of Toll-Like Receptor (TLR) signaling. We also observed that LD accumulation occurs through TLR2-dependent signaling and is counter-regulated by TLR4. Last, the pharmacologic modulation of LD formation by inhibiting diacylglycerol O-acyltransferase 1 (DGAT1) and cytosolic phospholipase A2 (cPLA2) significantly reduced the intracellular bacterial proliferation and impaired the prostaglandin E2 (PGE2 ) synthesis. Collectively, our data suggest the role of LDs on S. typhimurium intracellular survival and replication in macrophages. This data set provides new perspectives for future investigations about LDs in host-pathogen interaction.

MicroRNA hsa-let-7a facilitates staphylococcal small colony variants survival in the THP-1 macrophages by reshaping inflammatory responses

Recent studies have provided emerging evidence of the critical involvement of microRNAs in host immune defence against bacterial infection and that likewise the expression of the miRNAs is profoundly impacted by a variety of pathogens to subvert the immune response. Here, we report the role of hsa-let-7a miRNA in response to Staphylococcus epidermidis Small Colony Variants infection. We also assessed whether the expression levels of inflammatory cytokines associated with the hsa-let-7a are manipulated by the pathogen and the effect of the IFN-γ priming on the expression of hsa-let-7a and the fate of SCVs/WTs in infected macrophages. A striking observation was the downregulation of the let-7a miRNA upon challenge of the THP-1 activated cells with the SCV isolates while no significant changes in expression were noticed after the infection of macrophages with their WT counterparts. Staphylococcus epidermidis WT and SCV strains were found to invade and survive in macrophages. A significant reduction in bacterial load for both phenotypes was observed in macrophages treated with let-7a mimic compared to untreated ones. Survival of WTs was augmented in cells treated with the inhibitor in 4 out of 5 strains as compared to the number of bacteria recovered from non-transfected cells. At the same time, let-7a inhibitor did not influence on the survival of SCVs in macrophages as their number was comparable to number recovered from non-transfected cells. When the ratio of both let-7a cytokine targets was compared, anti-inflammatory IL-10 cytokine was induced by SCVs predominantly, while the macrophage challenge with WTs was characterized by the inflammatory cytokine profile with high IL-6 and low IL-10 production. Moreover, the balance between pro-inflammatory and anti-inflammatory cytokines has been expectedly retrieved when macrophages were transfected with let-7a mimic before infection with WT or SCV strains. The results also show that IFN-γ likely regulates the macrophage environment contributing to the inflammatory response and elimination of bacteria from intracellular milieu by augmenting the synthesis of pro-inflammatory cytokines and supressing the anti-inflammatory IL-10. Our work has shown that SCVs have the potential to regulate the let-7a miRNA to balance the pro-inflammatory IL-6 with anti-inflammatory IL-10 and this mechanism is one of the ways in a complex regulatory network adopted by SCVs to promote their survival.

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

Alveolar macrophages are the most abundant macrophages in the healthy lung where they play key roles in homeostasis and immune surveillance against air-borne pathogens. Tissue-specific differentiation and survival of alveolar macrophages relies on niche-derived factors, such as granulocyte-macrophage colony stimulating factor 2 (GM-CSF) and transforming growth factor beta (TGF-β). However, the nature of the downstream molecular pathways that regulate the identity and function of alveolar macrophages and their response to injury remains poorly understood. Here, we identify that the transcription factor EGR2 is an evolutionarily conserved feature of lung alveolar macrophages and show that cell-intrinsic EGR2 is indispensable for the tissue-specific identity of alveolar macrophages. Mechanistically, we show that EGR2 is driven by TGF-β and GM-CSF in a PPAR-γ-dependent manner to control alveolar macrophage differentiation. Functionally, EGR2 was dispensable for regulation of lipids in the airways, but crucial for the effective handling of the respiratory pathogen Streptococcus pneumoniae. Finally, we show that EGR2 is required for repopulation of the alveolar niche following sterile, bleomycin-induced lung injury and demonstrate that EGR2-dependent, monocyte-derived alveolar macrophages are vital for effective tissue repair following injury. Collectively, we demonstrate that EGR2 is an indispensable component of the transcriptional network controlling the identity and function of alveolar macrophages in health and disease.

Exposure to Acyl Homoserine Lactone Enhances Survival of Streptococcus pyogenes in Murine Macrophages.

Streptococcus pyogenes is an opportunistic pathogen causing infections of the skin and upper respiratory tract of the human host. Due to the polymicrobial community present in the human host, S. pyogenes comes across several interspecies signalling molecules. Among these molecules, N-(3-oxododecanoyl)-L-homoserine lactone (Oxo-C12) modulates the morphology, thereby enhancing virulence characteristics of S. pyogenes. After the initial attachment of the bacteria to the host cell, the pathogen needs to invade the host immune system for a successful infection to occur. The host immune system is activated upon infection, where macrophages engulf the pathogen, thereby killing the bacteria. However, S. pyogenes have evolved various strategies to evade the host immune response. In this study, we investigate the role of Oxo-C12 in enhancing the survival of S. pyogenes M3 in murine macrophages. The observed Oxo-C12-mediated increased survival in murine macrophages was through increased lysozyme and acid stress resistance. Moreover, Oxo-C12 increased the survival of S. pyogenes in normal human serum. Thus, understanding the role of interspecies signalling in enhancing the survival strategies of S. pyogenes in the host will further help fill the gap for therapeutics development.

TAMI-34. TARGETING CSF1R AND PD1 IN EXPERIMENTAL GLIOMA

Abstract The treatment of glioblastoma remains a challenge. Novel therapeutic strategies are urgently needed. Targeting the immunosuppressive glioblastoma-associated microenvironment is an interesting approach in this regard. Tumor-associated macrophages represent a distinct population of tumor-infiltrating immune cells with tumor-promoting features. The colony stimulating factor-1/ colony stimulating factor-1 receptor (CSF-1/CSF1R) axis plays an important role for macrophage differentiation and survival inside the tumor microenvironment. We thus aimed at investigating the antiglioma activity of CSF1R inhibition alone or in combination with blockade of programmed death (PD) 1. We detected CSF1R expression in paired tissue samples of primary and corresponding progressive glioblastoma. We investigated anti-CSF1R treatment alone or in combination with anti-PD1 antibodies in the orthotopic syngeneic VM/Dk SMA560 glioma mouse model, evaluated post-treatment effects and assessed treatment-induced cytotoxicity in a coculture model of patient-derived microtumors (PDM) and autologous tumor-infiltrating lymphocytes (TILs) ex vivo. Anti-CSF1R monotherapy increased the latency until the onset of neurological symptoms. Combinations of anti-CSF1R and anti-PD1 antibodies prolonged the latency until the onset of neurological symptoms and led to long-term survivors in vivo. Immunohistochemical analysis of post-treatment SMA-560 glioma revealed a modulation of the microenvironment, including increased T cell infiltration and reduced numbers of tumor-associated macrophages. Furthermore, we observed treatment-induced cytotoxicity of combined anti-CSF1R and anti-PD1 treatment in the PDM/TILs cocultures ex vivo. Taken together, our data indicates that CSF1R is a promising therapeutic target for glioblastoma, potentially in combination with PD1 inhibition.