Macrophage Killing Research Articles

Hemolymph of triatomines presents fungistatic activity against Cryptococcus neoformans and improves macrophage function through MCP-I/TNF-α increase.

Background: Triatomines are blood-feeding arthropods belonging to the subfamily Triatominae (Hemiptera; Reduviidae), capable of producing immunomodulatory and water-soluble molecules in their hemolymph, such as antimicrobial peptides (AMPs). In this work, we evaluated the antifungal and immunomodulatory activity of the hemolymph of Meccus pallidipennis (MPH) and Rhodnius prolixus (RPH) against Cryptococcus neoformans. Methods:We assessed the activity of the hemolymph of both insects on fungal growth by a minimum inhibitory concentration (MIC) assay. Further, RAW 264.7 macrophages were cultivated with hemolymph and challenged with C. neoformans. Then, their phagocytic and killing activities were assessed. The cytokines MCP-1, IFN-γ, TNF-α, IL-10, IL-12, and IL-6 were measured in culture supernatants 4- and 48-hours post-infection. Results:Both hemolymph samples directly affected the growth rate of the fungus in a dose-dependent manner. Either MPH or RPH was capable of inhibiting fungal growth by at least 70%, using the lowest dilution (1:20). Treatment of RAW 264.7 macrophages with hemolymph of both insects was capable of increasing the production of MCP-I and TNF-α. In addition, when these cells were stimulated with hemolymph in the presence of C. neoformans, a 2- and a 4-fold increase in phagocytic rate was observed with MPH and RPH, respectively, when compared to untreated cells. For the macrophage killing activity, MPH decreased in approximately 30% the number of viable yeasts inside the cells compared to untreated control; however, treatment with RPH could not reduce the total number of viable yeasts. MPH was also capable of increasing MHC-II expression on macrophages. Regarding the cytokine production, MCP-I and TNF-α, were increased in the supernatant of macrophages treated with both hemolymphs, 4 and 48 hours after stimulation.Conclusion:These results suggested that hemolymph of triatomines may represent a source of molecules capable of presenting antifungal and immunomodulatory activity in macrophages during fungal infection.

Deficiency of GPI Glycan Modification by Ethanolamine Phosphate Results in Increased Adhesion and Immune Resistance of Aspergillus fumigatus.

Glycosylphosphatidylinositol (GPI)-anchored proteins play important roles in maintaining the function of the cell wall and participating in pathogenic processes. The addition and removal of phosphoethanolamine (EtN-P) on the second mannose residue in the GPI anchor are vital for maturation and sorting of GPI-anchored proteins. Previously, we have shown that deletion of the gpi7, the gene that encodes an EtN-P transferase responsible for the addition of EtN-P to the second mannose residue of the GPI anchor, leads to the mislocalization of GPI-anchored proteins, abnormal polarity, reduced conidiation, and fast germination in Aspergillus fumigatus. In this report, the adherence and virulence of the A. fumigatus gpi7 deletion mutant were further investigated. The germinating conidia of the mutant exhibited an increased adhesion and a higher exposure of cell wall polysaccharides. Although the virulence was not affected, an increased adherence and a stronger inflammation response of the mutant were documented in an immunocompromised mouse model. An in vitro assay confirmed that the Δgpi7 mutant induced a stronger immune response and was more resistant to killing. Our findings, for the first time, demonstrate that in A. fumigatus, GPI anchoring is required for proper organization of the conidial cell wall. The lack of Gpi7 leads to fast germination, stronger immune response, and resistance to macrophage killing.

Role of RIPK1 in SMAC mimetics-induced apoptosis in primary human HIV-infected macrophages

Macrophages serve as viral reservoirs due to their resistance to apoptosis and HIV-cytopathic effects. We have previously shown that inhibitor of apoptosis proteins (IAPs) confer resistance to HIV-Vpr-induced apoptosis in normal macrophages. Herein, we show that second mitochondrial activator of caspases (SMAC) mimetics (SM) induce apoptosis of monocyte-derived macrophages (MDMs) infected in vitro with a R5-tropic laboratory strain expressing heat stable antigen, chronically infected U1 cells, and ex-vivo derived MDMs from HIV-infected individuals. To understand the mechanism governing SM-induced cell death, we show that SM-induced cell death of primary HIV-infected macrophages was independent of the acquisition of M1 phenotype following HIV infection of macrophages. Instead, SM-induced cell death was found to be mediated by IAPs as downregulation of IAPs by siRNAs induced cell death of HIV-infected macrophages. Moreover, HIV infection caused receptor interacting protein kinase-1 (RIPK1) degradation which in concert with IAP1/2 downregulation following SM treatment may result in apoptosis of macrophages. Altogether, our results show that SM selectively induce apoptosis in primary human macrophages infected in vitro with HIV possibly through RIPK1. Moreover, modulation of the IAP pathways may be a potential strategy for selective killing of HIV-infected macrophages in vivo.

Anatomical Site-Specific Carbohydrate Availability Impacts Streptococcus pneumoniae Virulence and Fitness during Colonization and Disease.

Streptococcus pneumoniae colonizes the nasopharynx asymptomatically but can also cause severe life-threatening disease. Importantly, stark differences in carbohydrate availability exist between the nasopharynx and invasive disease sites, such as the bloodstream, which most likely impact S. pneumoniae's behavior. Herein, using chemically defined medium (CDM) supplemented with physiological levels of carbohydrates, we examined how anatomical site-specific carbohydrate availability impacted S. pneumoniae physiology and virulence. S. pneumoniae cells grown in CDM modeling the nasopharynx (CDM-N) had reduced metabolic activity and a lower growth rate, demonstrated mixed acid fermentation with marked H2O2 production, and were in a carbon-catabolite repression (CCR)-derepressed state versus S. pneumoniae cells grown in CDM modeling blood (CDM-B). Using transcriptome sequencing (RNA-seq), we determined the transcriptome for the S. pneumoniae wild-type (WT) strain and its isogenic CCR-deficient mutant in CDM-N and CDM-B. Genes with altered expression as a result of changes in carbohydrate availability or catabolite control protein deficiency, respectively, were primarily involved in carbohydrate metabolism, but also encoded established virulence determinants, such as polysaccharide capsule and surface adhesins. We confirmed that anatomical site-specific carbohydrate availability directly influenced established S. pneumoniae virulence traits. S. pneumoniae cells grown in CDM-B formed shorter chains, produced more capsule, were less adhesive, and were more resistant to macrophage killing in an opsonophagocytosis assay. Moreover, growth of S. pneumoniae in CDM-N or CDM-B prior to the challenge of mice impacted relative fitness in a colonization model and invasive disease model, respectively. Thus, anatomical site-specific carbohydrate availability alters S. pneumoniae physiology and virulence, in turn promoting anatomical site-specific fitness.

Engineered Hypoimmune Allogeneic CAR T Cells Exhibit Innate and Adaptive Immune Evasion Even after Sensitization in Humanized Mice and Retain Potent Anti-Tumor Activity

Engineered Hypoimmune Allogeneic CAR T Cells Exhibit Innate and Adaptive Immune Evasion Even after Sensitization in Humanized Mice and Retain Potent Anti-Tumor Activity

Evaluation of Berberine as an Adjunct to TB Treatment.

Tuberculosis (TB) is the global health problem with the second highest number of deaths from a communicable disease after COVID-19. Although TB is curable, poor health infrastructure, long and grueling TB treatments have led to the spread of TB pandemic with alarmingly increasing multidrug-resistant (MDR)-TB prevalence. Alternative host modulating therapies can be employed to improve TB drug efficacies or dampen the exaggerated inflammatory responses to improve lung function. Here, we investigated the adjunct therapy of natural immune-modulatory compound berberine in C57BL/6 mouse model of pulmonary TB. Berberine treatment did not affect Mtb growth in axenic cultures; however, it showed increased bacterial killing in primary murine bone marrow-derived macrophages and human monocyte-derived macrophages. Ad libitum berberine administration was beneficial to the host in combination with rifampicin and isoniazid. Berberine adjunctive treatment resulted in decreased lung pathology with no additive or synergistic effects on bacterial burdens in mice. Lung immune cell flow cytometry analysis showed that adjunctive berberine treatment decreased neutrophil, CD11b+ dendritic cell and recruited interstitial macrophage numbers. Late onset of adjunctive berberine treatment resulted in a similar phenotype with consistently reduced numbers of neutrophils both in lungs and the spleen. Together, our results suggest that berberine can be supplemented as an immunomodulatory agent depending on the disease stage and inflammatory status of the host.

Growth medium and nitric oxide alter Mycobacterium abscessus morphotype and virulence

Mycobacterium abscessus complex (MABC) infections cause significant morbidity and mortality among patients with chronic lung disease, like cystic fibrosis. MABC exists in smooth (S) and rough (R) morphotypes, but triggers of morphotype switching and associated pathogenicity or antimicrobial susceptibility are poorly understood. We demonstrate that M. abscessus subspecies abscessus (Mab), massiliense (Mms), and bolletii (Mbl) cultured in Middlebrook (MB) broth exhibit S morphotype, whereas the bacteria grown in Luria Bertani (LB) broth adopt the R morphotype, characterized by low glycopeptidolipid (GPL) expression. The components of broth that mediate this selection are complex, with albumin supplementation promoting growth of S morphotype, but not sufficient for complete selection. Consistent with the findings of other groups, R forms of Mab, Mms and Mbl selected by LB broth were internalized in RAW 264.7 macrophages with higher efficiency than S. Intracellular survival of broth-selected organisms was variable and was higher for S Mab, but lower for S Mms and Mbl. It is proposed that growth in R morphotype is induced during stress conditions, such as nutrient poor environments or during inflammation. One key component of inflammation is release of nitric oxide. We demonstrated that a nitric oxide donor (DETA-NONOate) appears to induce growth in an R morphotype, as indicated by reduced GPL expression of Mab. Mab treated with DETA-NONOate also enhanced susceptibility to azithromycin at sub-MIC concentrations. In conclusion, morphotype and macrophage intracellular bacterial load of MABC subspecies can be manipulated by growing the bacteria in different culture conditions. Nitric oxide may also drive morphotype selection and enhanced azithromycin activity against Mab and macrophage killing.

Role of acuK in Control of Iron Acquisition and Gluconeogenesis in Talaromyces marneffei.

Talaromyces marneffei is a dimorphic pathogenic fungus causing opportunistic infection in immunocompromised patients. It is a facultative intracellular pathogen and is usually found inside the host macrophages during infection. Alternative carbons and iron are the important nutrients associated with intracellular survival and pathogenesis of T. marneffei. This study reported the importance of the transcription factor AcuK in control of gluconeogenesis and iron acquisition in T. marneffei. Deletion of acuK gene in T. marneffei resulted in retardation of growth and germination in both mold and yeast phases. Microscopically, ΔacuK showed double nuclei hyphae. However, the yeast cells showed normal morphology. The ΔacuK failed to grow in iron-limiting conditions. Additionally, it could not grow in a medium containing gluconeogenic carbon sources. Moreover, ΔacuK showed higher susceptibility to macrophage killing than the wild type. These results demonstrated that AcuK controlled both iron acquisition and gluconeogenesis, and it could contribute to the pathogenicity of this fungus.

Neutrophil extracellular traps enhance macrophage killing of bacterial pathogens.

Neutrophils and macrophages are critical to the innate immune response, but cooperative mechanisms used by these cells to combat extracellular pathogens are not well understood. This study reveals that S100A9-deficient neutrophils produce higher levels of mitochondrial superoxide in response to Staphylococcus aureus and, as a result, form neutrophil extracellular traps (suicidal NETosis). Increased suicidal NETosis does not improve neutrophil killing of S. aureus in isolation but augments macrophage killing. NET formation enhances antibacterial activity by increasing phagocytosis by macrophages and by transferring neutrophil-specific antimicrobial peptides to them. Similar results were observed in response to other phylogenetically distinct bacterial pathogens including Streptococcus pneumoniae and Pseudomonas aeruginosa, implicating this as an immune defense mechanism that broadly enhances antibacterial activity. These results demonstrate that achieving maximal bactericidal activity through NET formation requires macrophages and that accelerated and more robust suicidal NETosis makes neutrophils adept at increasing antibacterial activity, especially when A9 deficient.

Identification of novel genes involved in apoptosis of HIV-infected macrophages using unbiased genome-wide screening

BackgroundMacrophages, besides resting latently infected CD4+ T cells, constitute the predominant stable, major non-T cell HIV reservoirs. Therefore, it is essential to eliminate both latently infected CD4+ T cells and tissue macrophages to completely eradicate HIV in patients. Until now, most of the research focus is directed towards eliminating latently infected CD4+ T cells. However, few approaches have been directed at killing of HIV-infected macrophages either in vitro or in vivo. HIV infection dysregulates the expression of many host genes essential for the survival of infected cells. We postulated that exploiting this alteration may yield novel targets for the selective killing of infected macrophages.MethodsWe applied a pooled shRNA-based genome-wide approach by employing a lentivirus-based library of shRNAs to screen novel gene targets whose inhibition should selectively induce apoptosis in HIV-infected macrophages. Primary human MDMs were infected with HIV-eGFP and HIV-HSA viruses. Infected MDMs were transfected with siRNAs specific for the promising genes followed by analysis of apoptosis by flow cytometry using labelled Annexin-V in HIV-infected, HIV-exposed but uninfected bystander MDMs and uninfected MDMs. The results were analyzed using student’s t-test from at least four independent experiments.ResultsWe validated 28 top hits in two independent HIV infection models. This culminated in the identification of four target genes, Cox7a2, Znf484, Cstf2t, and Cdk2, whose loss-of-function induced apoptosis preferentially in HIV-infected macrophages. Silencing these single genes killed significantly higher number of HIV-HSA-infected MDMs compared to the HIV-HSA-exposed, uninfected bystander macrophages, indicating the specificity in the killing of HIV-infected macrophages. The mechanism governing Cox7a2-mediated apoptosis of HIV-infected macrophages revealed that targeting respiratory chain complex II and IV genes also selectively induced apoptosis of HIV-infected macrophages possibly through enhanced ROS production.ConclusionsWe have identified above-mentioned novel genes and specifically the respiratory chain complex II and IV genes whose silencing may cause selective elimination of HIV-infected macrophages and eventually the HIV-macrophage reservoirs. The results highlight the potential of the identified genes as targets for eliminating HIV-infected macrophages in physiological environment as part of an HIV cure strategy.

Abstract LB144: Overexpression of CD47 protects hypoimmune CAR T cells from innate immune cell killing

Abstract Off-the-shelf CAR T cells could offer advantages over autologous strategies, including ease of manufacturing, quality control and avoidance of malignant contamination and T cell dysfunction. TCR editing can effectively prevent graft-versus-host reactions. However, the vigorous host-versus-graft immune response against histoincompatible T cells prevents expansion and persistence of allogeneic CAR T cells and mitigates the efficacy of this approach. A major challenge is that, while HLA deletion can result in adaptive immune evasion, innate reactivity is enhanced. CD47 overexpression can block both NK cell and macrophage killing (J Exp Med (2021) 218 (3): e20200839), and we hypothesized that T cells would lose their immunogenicity when human leukocyte antigen (HLA) class I and II genes are inactivated and CD47 is over-expressed. We describe here the engineering of human immune evasive CAR T cells based on our previously described hypoimmune technology. Human T cells from healthy donors were obtained by leukapheresis. CRISPR/Cas9 technology was used to delete b2m, CIITA, and TCR and lentiviral transduction to overexpress CD47 and CD19CAR. Control T cells were unmanipulated except for overexpression of CD19CAR containing a 41BB costimulatory domain. When transplanted into allogeneic humanized mice, hypoimmunogenic HLA-I/II- TCR- CD47+ CD19CAR+ T cells evade immune recognition by T and B cells compared to CD19CAR+ T cells generated from the same human donor using ELISPOT and flow cytometry analysis. Innate immune cell assays show that CD47 overexpression protects HLA-I/II deficient CAR T cells from NK cell and macrophage killing in vitro and in vivo. Relative CD47 expression levels were analyzed to understand the relevance of CD47 for protection from macrophage and NK cell killing. A blocking antibody against CD47 made the hypoimmunogenic CAR T cells susceptible to macrophage and NK cell killing, confirming the importance of CD47 overexpression to evade innate immune clearance. The use of CD47 blocking could additionally be envisioned as a safety strategy for our hypoimmunogenic CAR T cells. Neither isolated CD47 overexpression nor all three hypoimmune modifications or knockout of the TCR showed any effect on the cytotoxic potential of CAR+ T cells. Hypoimmune CAR+ T cells retain their antitumor activity in the Nalm-6 B cell leukemia model in vitro and clear leukemic cells in NSG mice across a range of tumor cell: CAR T cell ratios comparable to unmodified CAR T cells. These findings show that hypoimmunogenic CAR T cells are functionally immune evasive in allogeneic recipients with cytotoxic anti-tumor capacity and suggest they could provide universal CAR T cells that is able to persist without immunosuppression. Blocking CD47 could additionally serve as safety strategy for our hypoimmunogenic CAR T cells. Citation Format: Xiaomeng Hu, Mo Dao, Kathy White, Ryan Clarke, Sam Landry, Ron Basco, Corie Gattis, Eleonore Tham, Emily Luo, Andrew Tucker, Christopher Bandoro, Elaine Chu, Junmo Kim, Chi Young, William E. Dowdle, Edward J. Rebar, Terry J. Fry, Sonja Schrepfer. Overexpression of CD47 protects hypoimmune CAR T cells from innate immune cell killing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB144.

Bortezomib-Activated Macrophages Contribute to Multiple Myeloma Progression Via Cancer Stem Cell Enrichment

Introduction: Multiple myeloma (MM) is a progressive malignancy of plasma cells. Bortezomib, a novel proteosome inhibitor, was approved for MM treatment, demonstrating revolutionary clinical results, including a significant improvement in patient outcome. However, mechanisms involved in bortezomib resistance in case of treatment failure require investigation. We previously showed that similar to chemotherapeutic agents, bortezomib induced host effects which could contribute to MM aggressive course and relapse. Mice primed with bortezomib and subsequently implanted with MM cells exhibited increased mortality rate compared to mice injected with the vehicle control. We found that bortezomib promoted the activation of pro-inflammatory macrophages, that in turn, affected MM cell aggressiveness. The current study has explored the mechanisms through which bortezomib-activated macrophages could contribute to MM aggressiveness, with the focus on MM cancer stem cells (CSC), known to be the subset of MM cells with therapy resistance properties.Methods: CAG and RPMI human cell lines were cultured with plasma and macrophage-conditioned medium obtained from mice, 24 hours after bortezomib administration. MM CSCs were phenotypically defined as CD138-/CD20+ cells and functionally defined by increased aldehyde dehydrogenase activity and presence of side population.Results: A 5-day exposure of MM cells to plasma from bortezomib-treated mice resulted in enriched CSC population, as compared to the cells cultured with plasma from control mice. Similarly, the medium conditioned with bortezomib-activated macrophages promoted enrichment of the CSCs in MM cell lines tested. Furthermore, this medium exhibited significantly increased IL-1 β levels compared to those observed in the medium conditioned with control macrophages, suggesting a role of this pro-inflammatory cytokine in MM CSC-mediated effects. To test this ability, RPMI cells were cultured with IL-1β at escalating concentrations. The resultant distinct upsurge in the CSC population could indicate the impact of IL-1β on MM CSC enrichment. As CD47 expressed by various hematopoietic cells is known to contribute to phagocytosis and macrophage killing evasion, the present study assessed CD47 expression on MM CSCs cultured with IL-1β. A significant expression of CD47 was demonstrated in MM-IL1β-enriched CSCs when compared to control MM CSCs.Conclusion: Overall, the results of the current study demonstrate that along with beneficial therapeutic effects in MM, bortezomib, interacting with host, is capable of promoting resistance to therapy by enriching the MM CSC population. This may be partially attributed to secretion of IL-1β from bortezomib-activated pro-inflammatory macrophages and enhanced CD47 expression by CSCs. Blocking such bortezomib pro-tumorigenic effects could improve therapy outcome in MM patients. DisclosuresNo relevant conflicts of interest to declare.

Complement Receptor 3, but not Fcγ Receptor III, abolishes the intracellular survival advantage of virulent Rhodococcus equi in murine macrophages

Abstract Complement receptor 3 (CR3) and Fc gamma receptor III (FcγRIII) are proposed to be essential for mediating Rhodococcus equi entry and killing in macrophages, respectively. Our objective was to investigate the role of each of these receptors in phagocytosis and intracellular replication of R. equi by macrophages. Wild-type (WT), CR3−/−, and FcγRIII−/− J774A.1 macrophages were infected with either virulent or avirulent opsonized R. equi. Quantitative culture was performed immediately post-infection (T0) and after 48 h (T48). Data were analyzed using mixed-effects modeling with the following outcomes: 1) ratio of inoculum:T0 (phagocytosis); and, 2) ratio of T48:T0 (intracellular survival). Additionally, WT, CR3−/−, and FcγRIII−/− macrophages were infected with virulent or avirulent GFP+ R. equi to confirm intracellular infection by confocal microscopy. Our results demonstrate that phagocytosis of either virulent or avirulent R. equi was unaffected by knocking out CR3 or FcγRIII, and imaging revealed that CR3−/− and FcγRIII−/− internalized both R. equi strains similarly to WT macrophages. As expected, intracellular survival in WT was significantly higher for virulent than avirulent R. equi. In CR3−/−, however, no significant difference in survival between virulent and avirulent strains was observed. Knocking out FcγRIII did not alter R. equi intracellular replication. Our findings demonstrate that CR3 is not necessary for R. equi infection and show that CR3 deletion eliminated the intracellular survival advantage of virulent strains. Last, FcγRIII-mediated phagocytosis does not improve intracellular killing as previously suggested.

S100A9 modulates neutrophil extracellular trap formation thereby enhancing macrophage killing of bacterial pathogens.

Abstract Professional phagocytes form the backbone of the innate immune response and are equipped with an arsenal of antimicrobial processes. While many of these processes have been explored in isolation, how immune cells cooperatively combat bacterial pathogens at the site of infection is unclear. The proteins forming calprotectin, S100A8 and S100A9, are the most abundant cytosolic proteins in neutrophils and important components of the innate immune response. Surprisingly, S100A9-deficient (A9−/−) mice are protected from Staphylococcus aureus infection with lower bacterial burdens in the heart. We have uncovered an intracellular function for S100A9, where A9−/− neutrophils produce more mitochondrial superoxide, thereby promoting the terminal formation of neutrophil extracellular traps (suicidal NETosis). Increased suicidal NETosis does not heighten neutrophil killing of S. aureus in isolation, but rather augments macrophage (Mϕ) killing. More specifically, NET formation enhances Mϕ antibacterial activity coinciding with Mϕ phagocytosis of S. aureus and transferring neutrophil-specific antimicrobial peptides to Mϕs. Finally, similar results are observed in response to other phylogenetically distinct bacterial pathogens implicating this as an immune defense mechanism that broadly enhances antibacterial activity. Based on these results, we propose that intracellular S100A9 is a critical rheostat for neutrophil function during infection. Further, NET formation acts as a conduit for neutrophils to enhance the antibacterial activity of Mϕs, where accelerated suicidal NETosis by A9−/− neutrophils makes them especially adept at increasing the antibacterial activity of Mϕs against multiple bacterial pathogens.

Candida glabrata produces a melanin-like pigment that protects against stress conditions encountered during parasitism.

Aim: Melanin has been linked to pathogenesis in several fungi. They often produce melanin-like pigments in the presence of L-dihydroxyphenylalanine (L-DOPA), but this is poorly studied in Candida glabrata. Methods & materials:C. glabrata was grown in minimal medium with or without L-DOPA supplementation and submitted to a chemical treatment with denaturant and hot acid. Results:C. glabrata turned black when grown in the presence of L-DOPA, whereas cells grown without L-DOPA supplementation remained white. Biophysical properties demonstrated that the pigment was melanin. Melanized C. glabrata cells were effectively protected from azoles and amphotericin B, incubation at 42°Cand macrophage killing. Conclusion: In the presence of L-DOPA, C. glabrata produces melanin, increases antifungal resistance and enhances host survival.

Fingolimod Potentiates the Antifungal Activity of Amphotericin B.

Candida albicans (C. albicans) is an opportunistic human fungal pathogen that can cause severe infection in clinic. Its incidence and mortality rate has been increasing rapidly. Amphotericin B (AMB), the clinical golden standard antifungal agent, has severe side effects that limit its clinical application. Thus, lowering the concentration and increasing the efficacy of AMB in a combinatorial antifungal therapy have been pursued by both industry and academia. Here we identify that fingolimod (FTY720), an immunomodulatory drug used for oral treatment of relapsing-remitting multiple sclerosis, can potentiate the efficacy of AMB against C. albicans growth synergistically. Furthermore, we observe an antifungal efficacy of FTY720 in combination with AMB against diverse fungal pathogens. Intriguingly, cells treated with both drugs are hypersensitive to endothelial endocytosis and macrophage killing. This is later found to be due to the hyperaccumulation of reactive oxygen species and the corresponding increase in activities of superoxide dismutase and catalase in the cells that received combinatorial treatment. Therefore, the combination of AMB and FTY720 provides a promising antifungal strategy.

TPL‐2 kinase induces phagosome acidification to promote macrophage killing of bacteria

Tumour progression locus 2 (TPL‐2) kinase mediates Toll‐like receptor (TLR) activation of ERK1/2 and p38α MAP kinases in myeloid cells to modulate expression of key cytokines in innate immunity. This study identified a novel MAP kinase‐independent regulatory function for TPL‐2 in phagosome maturation, an essential process for killing of phagocytosed microbes. TPL‐2 catalytic activity was demonstrated to induce phagosome acidification and proteolysis in primary mouse and human macrophages following uptake of latex beads. Quantitative proteomics revealed that blocking TPL‐2 catalytic activity significantly altered the protein composition of phagosomes, particularly reducing the abundance of V‐ATPase proton pump subunits. Furthermore, TPL‐2 stimulated the phosphorylation of DMXL1, a regulator of V‐ATPases, to induce V‐ATPase assembly and phagosome acidification. Consistent with these results, TPL‐2 catalytic activity was required for phagosome acidification and the efficient killing of Staphylococcus aureus and Citrobacter rodentium following phagocytic uptake by macrophages. TPL‐2 therefore controls innate immune responses of macrophages to bacteria via V‐ATPase induction of phagosome maturation.

Embryonic-Derived Myb- Macrophages Enhance Bacterial Clearance and Improve Survival in Rat Sepsis.

Peritoneal resident macrophages play a key role in combating sepsis in the peritoneal cavity. We sought to determine if peritoneal transplantation of embryonic Myb− “peritoneal-like” macrophages attenuate abdominal fecal sepsis. Directed differentiation of rodent pluripotent stem cells (PSCs) was used in factor-defined media to produce embryonic-derived large “peritoneal-like” macrophages (Ed-LPM) that expressed peritoneal macrophage markers and demonstrated phagocytic capacity. Preclinical in vivo studies determined Ed-LPM efficacy in rodent abdominal fecal sepsis with or without Meropenem. Ex vivo studies explored the mechanism and effects of Ed-LPM on host immune cell number and function, including phagocytosis, reactive oxygen species (ROS) production, efferocytosis and apoptosis. Ed-LPM reduced sepsis severity by decreasing bacterial load in the liver, spleen and lungs. Ed-LPM therapy significantly improved animal survival by ~30% and reduced systemic bacterial burden to levels comparable to Meropenem therapy. Ed-LPM therapy decreased peritoneal TNFα while increasing IL-10 concentrations. Ed-LPMs enhanced peritoneal macrophage phagocytosis of bacteria, increased macrophage production of ROS and restored homeostasis via apoptosis and efferocytosis-induced clearance of neutrophils. In conclusion, Ed-LPM reduced systemic sepsis severity, improved survival and reduced bacterial load by enhancing peritoneal macrophage bacterial phagocytosis and killing and clearance of intra-peritoneal neutrophils. Macrophage therapy may be a potential strategy to address sepsis.

Genome-wide association analysis identified both RNA-seq and DNA variants associated to paratuberculosis in Canadian Holstein cattle \u2018in vitro\u2019 experimentally infected macrophages

BackgroundMycobacterium avium ssp. paratuberculosis (MAP) is the causative agent of paratuberculosis, or Johne’s disease (JD), an incurable bovine disease. The evidence for susceptibility to MAP disease points to multiple interacting factors, including the genetic predisposition to a dysregulation of the immune system. The endemic situation in cattle populations can be in part explained by a genetic susceptibility to MAP infection. In order to identify the best genetic improvement strategy that will lead to a significant reduction of JD in the population, we need to understand the link between genetic variability and the biological systems that MAP targets in its assault to dominate macrophages. MAP survives in macrophages where it disseminates. We used next-generation RNA (RNA-Seq) sequencing to study of the transcriptome in response to MAP infection of the macrophages from cows that have been naturally infected and identified as positive for JD (JD (+); n = 22) or negative for JD (healthy/resistant, JD (−); n = 28). In addition to identifying genetic variants from RNA-seq data, SNP variants were also identified using the Bovine SNP50 DNA chip.ResultsThe complementary strategy allowed the identification of 1,356,248 genetic variants, including 814,168 RNA-seq and 591,220 DNA chip variants. Annotation using SnpEff predicted that the 2435 RNA-seq genetic variants would produce high functional effect on known genes in comparison to the 33 DNA chip variants. Significant variants from JD(+/−) macrophages were identified by genome-wide association study and revealed two quantitative traits loci: BTA4 and 11 at (P < 5 × 10− 7). Using BovineMine, gene expression levels together with significant genomic variants revealed pathways that potentially influence JD susceptibility, notably the energy-dependent regulation of mTOR by LKB1-AMPK and the metabolism of lipids.ConclusionIn the present study, we succeeded in identifying genetic variants in regulatory pathways of the macrophages that may affect the susceptibility of cows that are healthy/resistant to MAP infection. RNA-seq provides an unprecedented opportunity to investigate gene expression and to link the genetic variations to biological pathways that MAP normally manipulate during the process of killing macrophages. A strategy incorporating functional markers into genetic selection may have a considerable impact in improving resistance to an incurable disease. Integrating the findings of this research into the conventional genetic selection program may allow faster and more lasting improvement in resistance to bovine paratuberculosis in dairy cattle.

A Novel Aza-Derivative Inhibits agr Quorum Sensing Signaling and Synergizes Methicillin-Resistant Staphylococcus aureus to Clindamycin.

Increasing antibiotic resistance and diminishing pharmaceutical industry investments have increased the need for molecules that can treat infections caused by dangerous pathogens such as methicillin-resistant Staphylococcus aureus (MRSA). Quorum Sensing (QS) is a signaling mechanism that regulates bacterial virulence in pathogens. A report demonstrating that the anti-inflammatory drug Diflunisal reduces MRSA virulence factors’ expression prompted us to design, synthesize and test 16 aza-analogs as inhibitors of S. aureus virulence factors controlled by the accessory gene regulator (agr) QS system. At first, we evaluated by qRT-PCR the activity of compounds on rnaIII expression, a QS related gene. Azan-7 was the most active molecule tested and it did not show cytotoxic activity in human cell lines. Moreover, we demonstrated that it did not affect bacterial proliferation. Regulation of MRSA virulence genes by Azan-7 was investigated using qRT-PCR and RNAseq. Azan-7 significantly reduced hla, psmα, hysA, agrA, cap1A, and cap1C gene expression. In silico docking demonstrated that Azan-7 binds the response regulator AgrA. This data was confirmed by electrophoretic mobility shift assay (EMSA) reporting that Azan-7 binding to AgrA protein strongly reduced the AgrA-DNA complex formation at the P3 promoter region involved in the regulation of rnaIII transcription. Azan-7 inhibited MRSA-mediated haemolysis, reduced survival of the pathogen at low pH levels, and increased macrophage killing. In addition, Azan-7 enhanced MRSA susceptibility to clindamycin both in planktonic growth and biofilm. Azan-7 did not induce resistance over 10 days in culture. It was equally active against all the AgrA MRSA subtypes encountered among clinical isolates, but it was not active against Staphylococcus epidermidis, although the AgrA proteins show an approximate 80% homology. These results demonstrate that Azan-7 inhibits the expression of MRSA virulence factors by interfering in the QS and synergizes MRSA biofilm with clindamycin, indicating the compound as a promising candidate for the treatment of MRSA infections.