Establishment of a Patient-Derived, Magnetic Levitation-Based, 3D Spheroid Granuloma Model for Human Tuberculosis.

Tuberculous granulomas that develop in response to Mycobacterium tuberculosis (M.tb) infection are highly dynamic entities shaped by the host immune response and disease kinetics. Within this microenvironment, immune cell recruitment, polarization and activation is driven not only by co-existing cell types and multi-cellular interactions, but also by M.tb-mediated changes involving metabolic heterogeneity, epigenetic reprogramming and rewiring of the transcriptional landscape of host cells. There is an increased appreciation of the in vivo complexity, versatility and heterogeneity of the cellular compartment that constitutes the tuberculosis (TB) granuloma, and the difficulty in translating findings from animal models to human disease. Here we describe a novel biomimetic in vitro 3-dimentional (3D) human lung granuloma model, resembling early innate and adaptive stages of the TB granuloma spectrum, and present results of histological architecture, host transcriptional characterization, mycobacteriological features, cytokine profiles and spatial distribution of key immune cells. A range of manipulations of immune cell populations in these granulomas will allow the study of host/pathogen pathways involved in the outcome of infection, as well as pharmacological interventions.

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Revisiting the role of IRF3 in inflammation and immunity by conditional and specifically targeted gene ablation in mice

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1803936115 ◽

2018 ◽

Vol 115 (20) ◽

pp. 5253-5258 ◽

Cited By ~ 19

Author(s):

Hideyuki Yanai ◽

Shiho Chiba ◽

Sho Hangai ◽

Kohei Kometani ◽

Asuka Inoue ◽

...

Keyword(s):

Immune Cell ◽

Cell Types ◽

Type I ◽

Type I Ifn ◽

Cell Type ◽

Gene Ablation ◽

Cell Type Specific

IFN regulatory factor 3 (IRF3) is a transcription regulator of cellular responses in many cell types that is known to be essential for innate immunity. To confirm IRF3’s broad role in immunity and to more fully discern its role in various cellular subsets, we engineered Irf3-floxed mice to allow for the cell type-specific ablation of Irf3. Analysis of these mice confirmed the general requirement of IRF3 for the evocation of type I IFN responses in vitro and in vivo. Furthermore, immune cell ontogeny and frequencies of immune cell types were unaffected when Irf3 was selectively inactivated in either T cells or B cells in the mice. Interestingly, in a model of lipopolysaccharide-induced septic shock, selective Irf3 deficiency in myeloid cells led to reduced levels of type I IFN in the sera and increased survival of these mice, indicating the myeloid-specific, pathogenic role of the Toll-like receptor 4–IRF3 type I IFN axis in this model of sepsis. Thus, Irf3-floxed mice can serve as useful tool for further exploring the cell type-specific functions of this transcription factor.

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Administration of cardiac mesenchymal cells modulates innate immunity in the acute phase of myocardial infarction in mice

Scientific Reports ◽

10.1038/s41598-020-71580-z ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Yi Kang ◽

Marjan Nasr ◽

Yiru Guo ◽

Shizuka Uchida ◽

Tyler Weirick ◽

...

Keyword(s):

Myocardial Infarction ◽

Innate Immunity ◽

Mechanism Of Action ◽

Cell Activation ◽

Cell Function ◽

Immune Cell ◽

Mesenchymal Cell ◽

Cell Types

Abstract Although cardiac mesenchymal cell (CMC) therapy mitigates post-infarct cardiac dysfunction, the underlying mechanisms remain unidentified. It is acknowledged that donor cells are neither appreciably retained nor meaningfully contribute to tissue regeneration—suggesting a paracrine-mediated mechanism of action. As the immune system is inextricably linked to wound healing/remodeling in the ischemically injured heart, the reparative actions of CMCs may be attributed to their immunoregulatory properties. The current study evaluated the consequences of CMC administration on post myocardial infarction (MI) immune responses in vivo and paracrine-mediated immune cell function in vitro. CMC administration preferentially elicited the recruitment of cell types associated with innate immunity (e.g., monocytes/macrophages and neutrophils). CMC paracrine signaling assays revealed enhancement in innate immune cell chemoattraction, survival, and phagocytosis, and diminished pro-inflammatory immune cell activation; data that identifies and catalogues fundamental immunomodulatory properties of CMCs, which have broad implications regarding the mechanism of action of CMCs in cardiac repair.

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Stimulation of Innate Immunity byIn VivoCyclic di-GMP Synthesis Using Adenovirus

Clinical and Vaccine Immunology ◽

10.1128/cvi.00471-14 ◽

2014 ◽

Vol 21 (11) ◽

pp. 1550-1559 ◽

Cited By ~ 4

Author(s):

Benjamin J. Koestler ◽

Sergey S. Seregin ◽

David P. W. Rastall ◽

Yasser A. Aldhamen ◽

Sarah Godbehere ◽

...

Keyword(s):

Innate Immunity ◽

Mammalian Cells ◽

Immune Cell ◽

Innate Immune ◽

Host Cells ◽

Content Type ◽

Cytokines And Chemokines ◽

Novel Approach

ABSTRACTThe bacterial second messenger cyclic di-GMP (c-di-GMP) stimulates inflammation by initiating innate immune cell recruitment and triggering the release of proinflammatory cytokines and chemokines. These properties make c-di-GMP a promising candidate for use as a vaccine adjuvant, and numerous studies have demonstrated that administration of purified c-di-GMP with different antigens increases protection against infection in animal models. Here, we have developed a novel approach to produce c-di-GMP inside host cells as an adjuvant to exploit a host-pathogen interaction and initiate an innate immune response. We have demonstrated that c-di-GMP can be synthesizedin vivoby transducing a diguanylate cyclase (DGC) gene into mammalian cells using an adenovirus serotype 5 (Ad5) vector. Expression of DGC led to the production of c-di-GMPin vitroandin vivo, and this was able to alter proinflammatory gene expression in murine tissues and increase the secretion of numerous cytokines and chemokines when administered to animals. Furthermore, coexpression of DGC modestly increased T-cell responses to aClostridium difficileantigen expressed from an adenovirus vaccine, although no significant differences in antibody titers were observed. This adenovirus c-di-GMP delivery system offers a novel method to administer c-di-GMP as an adjuvant to stimulate innate immunity during vaccination.

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Upregulation of the Adhesin Gene EPA1 Mediated by PDR1 in Candida glabrata Leads to Enhanced Host Colonization

mSphere ◽

10.1128/msphere.00065-15 ◽

2016 ◽

Vol 1 (2) ◽

Cited By ~ 19

Author(s):

Luis A. Vale-Silva ◽

Beat Moeckli ◽

Riccardo Torelli ◽

Brunella Posteraro ◽

Maurizio Sanguinetti ◽

...

Keyword(s):

Drug Resistance ◽

Epithelial Cells ◽

Candida Glabrata ◽

Resistance Mechanism ◽

Cell Types ◽

Host Cells ◽

Regulation Mechanism ◽

Content Type

ABSTRACT Candida glabrata is an important fungal pathogen in human diseases and is also rapidly acquiring drug resistance. Drug resistance can be mediated by the transcriptional activator PDR1, and this results in the upregulation of multidrug transporters. Intriguingly, this resistance mechanism is associated in C. glabrata with increased virulence in animal models and also with increased adherence to specific host cell types. The C. glabrata adhesin gene EPA1 is a major contributor of virulence and adherence to host cells. Here, we show that EPA1 expression is controlled by PDR1 independently of subtelomeric silencing, a known EPA1 regulation mechanism. Thus, a relationship exists between PDR1, EPA1 expression, and adherence to host cells, which is critical for efficient virulence. Our results demonstrate that acquisition of drug resistance is beneficial for C. glabrata in fungus-host relationships. These findings further highlight the challenges of the therapeutic management of C. glabrata infections in human patients. Candida glabrata is the second most common Candida species causing disseminated infection, after C. albicans. C. glabrata is intrinsically less susceptible to the widely used azole antifungal drugs and quickly develops secondary resistance. Resistance typically relies on drug efflux with transporters regulated by the transcription factor Pdr1. Gain-of-function (GOF) mutations in PDR1 lead to a hyperactive state and thus efflux transporter upregulation. Our laboratory has characterized a collection of C. glabrata clinical isolates in which azole resistance was found to correlate with increased virulence in vivo. Contributing phenotypes were the evasion of adhesion and phagocytosis by macrophages and an increased adhesion to epithelial cells. These phenotypes were found to be dependent on PDR1 GOF mutation and/or C. glabrata strain background. In the search for the molecular effectors, we found that PDR1 hyperactivity leads to overexpression of specific cell wall adhesins of C. glabrata. Further study revealed that EPA1 regulation, in particular, explained the increase in adherence to epithelial cells. Deleting EPA1 eliminates the increase in adherence in an in vitro model of interaction with epithelial cells. In a murine model of urinary tract infection, PDR1 hyperactivity conferred increased ability to colonize the bladder and kidneys in an EPA1-dependent way. In conclusion, this study establishes a relationship between PDR1 and the regulation of cell wall adhesins, an important virulence attribute of C. glabrata. Furthermore, our data show that PDR1 hyperactivity mediates increased adherence to host epithelial tissues both in vitro and in vivo through upregulation of the adhesin gene EPA1. IMPORTANCE Candida glabrata is an important fungal pathogen in human diseases and is also rapidly acquiring drug resistance. Drug resistance can be mediated by the transcriptional activator PDR1, and this results in the upregulation of multidrug transporters. Intriguingly, this resistance mechanism is associated in C. glabrata with increased virulence in animal models and also with increased adherence to specific host cell types. The C. glabrata adhesin gene EPA1 is a major contributor of virulence and adherence to host cells. Here, we show that EPA1 expression is controlled by PDR1 independently of subtelomeric silencing, a known EPA1 regulation mechanism. Thus, a relationship exists between PDR1, EPA1 expression, and adherence to host cells, which is critical for efficient virulence. Our results demonstrate that acquisition of drug resistance is beneficial for C. glabrata in fungus-host relationships. These findings further highlight the challenges of the therapeutic management of C. glabrata infections in human patients.

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Pseudomonas aeruginosa Cytotoxin ExoU Is Injected into Phagocytic Cells during Acute Pneumonia

Infection and Immunity ◽

10.1128/iai.01134-09 ◽

2010 ◽

Vol 78 (4) ◽

pp. 1447-1456 ◽

Cited By ~ 61

Author(s):

Maureen H. Diaz ◽

Alan R. Hauser

Keyword(s):

Pseudomonas Aeruginosa ◽

Immune Cells ◽

Type Iii Secretion ◽

Cell Types ◽

Secretion System ◽

Host Cells ◽

Phagocytic Cells ◽

Acute Pneumonia

ABSTRACT ExoU, a cytotoxin translocated into host cells via the type III secretion system of Pseudomonas aeruginosa, is associated with increased mortality and disease severity. We previously showed that impairment of recruited phagocytic cells allowed survival of ExoU-secreting P. aeruginosa in the lung. Here we analyzed types of cells injected with ExoU in vivo using translational fusions of ExoU with a β-lactamase reporter (ExoU-Bla). Cells injected with ExoU-Bla were detectable in vitro but not in vivo, presumably due to the rapid cytotoxicity induced by the toxin. Therefore, we used a noncytotoxic ExoU variant, designated ExoU(S142A)-Bla, to analyze injection in vivo. We determined that phagocytic cells in the lung were frequently injected with ExoU(S142A). Early during infection, resident macrophages constituted the majority of cells into which ExoU was injected, but neutrophils and monocytes became the predominant types of cells into which ExoU was injected upon recruitment into the lung. We observed a modest preference for injection into neutrophils over injection into other cell types, but in general the repertoire of injected immune cells reflected the relative abundance of these cells in the lung. Our results indicate that phagocytic cells in the lung are injected with ExoU and support the hypothesis that ExoU-mediated impairment of phagocytes has a role in the pathogenesis of pneumonia caused by P. aeruginosa.

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Cell-specific and targeted delivery of RNA moieties

10.1101/2020.01.03.893818 ◽

2020 ◽

Author(s):

Aditi Bhargava ◽

Peter Ohara ◽

Luc Jasmin

Keyword(s):

Nucleic Acids ◽

Targeted Delivery ◽

Immune Cell ◽

Neuronal Cell ◽

Cell Types ◽

Specific Cell ◽

Chemically Modified ◽

Covalently Linked

AbstractDelivery of therapeutic moieties to specific cell types, such as neurons remains a challenge. Genes present in neurons are also expressed in non-neuronal cell types such as glia where they mediate non-targeted related functions. Thus, non-specific targeting of these proteins/channels has numerous unwanted side effects, as is the case with current small molecules or drug therapies. Current methodologies that use nanoparticles, lipid-mediated uptake, or mannitol in conjunction with lipids to deliver double-stranded RNA (dsRNA) have yielded mixed and unreliable results. We used a neuroanatomical tracer (B subunit of Cholera Toxin (CTB)) that binds to the ganglioside receptors (GM1) expressed on cells, including primary sensory neurons to deliver encapsulated dsRNA. This approach greatly improved delivery of dsRNA to the desired cells by enhancing uptake, reducing vehicle-mediated toxicity and protecting nucleotides from degradation by endonucleases. The delivery complex is internalized, and once inside the cell, the dsRNA naturally dissociates itself from the carrier complex and is very effective in knocking down cognate targets, both in vivo and in vitro. Past methods have used CTB-fusion proteins or chemically modified oligos or DNA moieties that have been covalently conjugated to CTB. Furthermore, CTB conjugated to an antigen, protein, or chemically modified nucleic acid is a potent activator of immune cell (T and B cells, macrophages) response, whereas CTB admixed with antigens or unmodified nucleic acids does not evoke this immune response. Importantly, in our method, the nucleic acids are not covalently linked to the carrier molecules. Thus, our method holds strong potential for targeted delivery of therapeutic moieties for cell types expressing GM1 receptors, including neuronal cell types.

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Effects of IL8 and immune cells on the regulation of luteal progesterone secretion

Reproduction ◽

10.1530/rep-13-0602 ◽

2014 ◽

Vol 148 (1) ◽

pp. 21-31 ◽

Cited By ~ 14

Author(s):

Heather Talbott ◽

Abigail Delaney ◽

Pan Zhang ◽

Yangsheng Yu ◽

Robert A Cushman ◽

...

Keyword(s):

Mononuclear Cells ◽

Immune Cell ◽

Neutrophil Migration ◽

Prostaglandin F2α ◽

Cell Types ◽

Luteal Cell ◽

Fibroblast Cells ◽

Progesterone Synthesis

Recent studies have suggested that chemokines may mediate the luteolytic action of prostaglandin F2α (PGF). Our objective was to identify chemokines induced by PGFin vivoand to determine the effects of interleukin 8 (IL8) on specific luteal cell typesin vitro. Mid-cycle cows were injected with saline or PGF, ovaries were removed after 0.5–4 h, and expression of chemokine was analyzed by qPCR.In vitroexpression of IL8 was analyzed after PGF administration and with cell signaling inhibitors to determine the mechanism of PGF-induced chemokine expression. Purified neutrophils were analyzed for migration and activation in response to IL8 and PGF. Purified luteal cell types (steroidogenic, endothelial, and fibroblast cells) were used to identify which cells respond to chemokines. Neutrophils and peripheral blood mononuclear cells (PBMCs) were cocultured with steroidogenic cells to determine their effect on progesterone production.IL8,CXCL2,CCL2, andCCL8transcripts were rapidly increased following PGF treatmentin vivo. The stimulatory action of PGF onIL8mRNA expressionin vitrowas prevented by inhibition of p38 and JNK signaling. IL8, but not PGF, TNF, or TGFB1, stimulated neutrophil migration. IL8 had no apparent action in purified luteal steroidogenic, endothelial, or fibroblast cells, but stimulated ERK phosphorylation in neutrophils. In coculture experiments neither IL8 nor activated neutrophils altered basal or LH-stimulated luteal cell progesterone synthesis. In contrast, activated PBMCs inhibited LH-stimulated progesterone synthesis from cultured luteal cells. These data implicate a complex cascade of events during luteolysis, involving chemokine signaling, neutrophil recruitment, and immune cell action within the corpus luteum.

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Diflunisal targets the HMGB1/CXCL12 heterocomplex and blocks immune cell recruitment

10.1101/563890 ◽

2019 ◽

Cited By ~ 1

Author(s):

Federica De Leo ◽

Giacomo Quilici ◽

Mario Tirone ◽

Valeria Mannella ◽

Francesco De Marchis ◽

...

Keyword(s):

Rational Design ◽

Immune Cell ◽

Inflammatory Responses ◽

Inflammatory Cells ◽

Cell Recruitment ◽

Redox States ◽

Inflammatory Drugs ◽

Anti Inflammatory

AbstractExtracellular HMGB1 triggers inflammation following infection or injury, and supports tumorigenesis in inflammation-related malignancies. HMGB1 has several redox states: reduced HMGB1 recruits inflammatory cells to injured tissues forming a heterocomplex with CXCL12 and signaling via its receptor CXCR4; disulfide-containing HMGB1 binds to TLR4 and promotes inflammatory responses. Here we show that Diflunisal, an aspirin-like nonsteroidal anti-inflammatory drug (NSAID) that has been in clinical use for decades, specifically inhibits in vitro and in vivo the chemotactic activity of HMGB1 at nanomolar concentrations, at least in part by binding directly to both HMGB1 and CXCL12 and disrupting their heterocomplex. Importantly, Diflunisal does not inhibit TLR4-dependent responses. Our findings clarify the mode of action of Diflunisal, and open the way to the rational design of functionally specific anti-inflammatory drugs.

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Fibroblast membrane-camouflaged nanoparticles for inflammation treatment in the early stage

International Journal of Oral Science ◽

10.1038/s41368-021-00144-2 ◽

2021 ◽

Vol 13 (1) ◽

Author(s):

Lizhong Sun ◽

Libang He ◽

Wei Wu ◽

Li Luo ◽

Mingyue Han ◽

...

Keyword(s):

Cell Membrane ◽

Immune Cell ◽

Early Stage ◽

Specific Treatment ◽

Cell Types ◽

Loss Of Function ◽

Tissue Repair And Regeneration ◽

Inflammatory Conditions

AbstractUnrestrained inflammation is harmful to tissue repair and regeneration. Immune cell membrane-camouflaged nanoparticles have been proven to show promise as inflammation targets and multitargeted inflammation controls in the treatment of severe inflammation. Prevention and early intervention of inflammation can reduce the risk of irreversible tissue damage and loss of function, but no cell membrane-camouflaged nanotechnology has been reported to achieve stage-specific treatment in these conditions. In this study, we investigated the prophylactic and therapeutic efficacy of fibroblast membrane-camouflaged nanoparticles for topical treatment of early inflammation (early pulpitis as the model) with the help of in-depth bioinformatics and molecular biology investigations in vitro and in vivo. Nanoparticles have been proven to act as sentinels to detect and competitively neutralize invasive Escherichia coli lipopolysaccharide (E. coli LPS) with resident fibroblasts to effectively inhibit the activation of intricate signaling pathways. Moreover, nanoparticles can alleviate the secretion of multiple inflammatory cytokines to achieve multitargeted anti-inflammatory effects, attenuating inflammatory conditions in the early stage. Our work verified the feasibility of fibroblast membrane-camouflaged nanoparticles for inflammation treatment in the early stage, which widens the potential cell types for inflammation regulation.

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PARG Suppresses Tumorigenesis and Downregulates Genes Controlling Angiogenesis, Inflammatory Response, and Immune Cell Recruitment

10.21203/rs.3.rs-1149954/v1 ◽

2022 ◽

Author(s):

Sarah Johnson ◽

Yaroslava Karpova ◽

Danping Guo ◽

Atreyi Ghatak ◽

Dmitriy A. Markov ◽

...

Keyword(s):

Immune Cell ◽

Critical Role ◽

Tumor Growth Inhibition ◽

Rna Seq ◽

Cell Recruitment ◽

Tumor Onset ◽

Tumor Tissues ◽

Immune Cell Recruitment

Abstract Chemokines are highly expressed in tumor microenvironment and play a critical role in all aspects of tumorigenesis, including the recruitment of tumor-promoting immune cells, activation of cancer-associated fibroblasts, angiogenesis, metastasis, and growth. Poly(ADP-ribose) polymerase (PARP) is a multi-target transcription regulator with high levels of poly(ADP-ribose) (pADPr) being reported in a variety of cancers. Furthermore, poly(ADP-ribose) glycohydrolase (PARG), an enzyme that degrades pADPr, has been reported to be downregulated in tumor tissues with abnormally high levels of pADPr. In conjunction to this, we have recently reported that the reduction of pADPr, by either pharmacological inhibition of PARP or PARG’s overexpression, disrupts renal carcinoma cell malignancy in vitro. Here, we use 3T3 mouse embryonic fibroblasts, a universal model for malignant transformation, to follow the effect of PARG upregulation on cells’ tumorigenicity in vivo. We found that the overexpression of PARG in mouse allografts produces significantly smaller tumors with a delay in tumor onset. As downregulation of PARG has also been implicated in promoting the activation of pro-inflammatory genes, we also followed the gene expression profile of PARG-overexpressing 3T3 cells using RNA-seq approach and observed that chemokine transcripts are significantly reduced in those cells. Our data suggest that the upregulation of PARG may be potentially useful for the tumor growth inhibition in cancer treatment and as anti-inflammatory intervention.

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