Immune Responses In Human Cells Research Articles

Diverse modulatory effects of bibenzyls from Dendrobium species on human immune cell responses under inflammatory conditions.

Dendrobium plants are widely used in traditional Chinese medicine. Their secondary metabolites such as bibenzyls and phenanthrenes show various pharmacological benefits such as immunomodulation and inhibitory effects on cancer cell growth. However, our previous study also showed that some of these promising compounds (i.e., gigantol and cypripedin) also induced the expression of inflammatory cytokines including TNF in human monocytes, and thus raising concerns about the use of these compounds in clinical application. Furthermore, the effects of these compounds on other immune cell populations, apart from monocytes, remain to be investigated. In this study, we evaluated immunomodulatory effects of seven known bibenzyl compounds purified from Dendrobium species in human peripheral blood mononuclear cells (PBMCs) that were stimulated with lipopolysaccharide (LPS). Firstly, using flow cytometry, moscatilin (3) and crepidatin (4) showed the most promising dose-dependent immunomodulatory effects among all seven bibenzyls, determined by significant reduction of TNF expression in LPS-stimulated CD14+ monocytes. Only crepidatin at the concentration of 20 μM showed a significant cytotoxicity, i.e., an increased cell death in late apoptotic state. In addition, deep immune profiling using high-dimensional single-cell mass cytometry (CyTOF) revealed broad effects of Dendrobium compounds on diverse immune cell types. Our findings suggest that to precisely evaluate therapeutic as well as adverse effects of active natural compounds, a multi-parameter immune profiling targeting diverse immune cell population is required.

Insights into the molecular basis and mechanism of heme-triggered TLR4 signalling: The role of heme-binding motifs in TLR4 and MD2.

Haemolytic disorders, such as sickle cell disease, are accompanied by the release of high amounts of labile heme into the intravascular compartment resulting in the induction of proinflammatory and prothrombotic complications in affected patients. In addition to the relevance of heme-regulated proteins from the complement and blood coagulation systems, activation of the TLR4 signalling pathway by heme was ascribed a crucial role in the progression of these pathological processes. Heme binding to the TLR4-MD2 complex has been proposed recently, however, essential mechanistic information of the processes at the molecular level, such as heme-binding kinetics, the heme-binding capacity and the respective heme-binding sites (HBMs) is still missing. We report the interaction of TLR4, MD2 and the TLR4-MD2 complex with heme and the consequences thereof by employing biochemical, spectroscopic, bioinformatic and physiologically relevant approaches. Heme binding occurs transiently through interaction with up to four HBMs in TLR4, two HBMs in MD2 and at least four HBMs in their complex. Functional studies highlight that mutations of individual HBMs in TLR4 preserve full receptor activation by heme, suggesting that heme interacts with TLR4 through different binding sites independently of MD2. Furthermore, we confirm and extend the major role of TLR4 for heme-mediated cytokine responses in human immune cells.

Characterization of endotoxin free protein production of brain-derived neurotrophic factor (BDNF) for the study of Parkinson model in SH-SY5Y differentiated cells

Human neuronal cells are a more appropriate cell model for neurological disease studies such as Alzheimer and Parkinson's disease. SH-SY5Y neuroblastoma cells have been widely used for differentiation into a mature neuronal cell phenotype. The cellular differentiation process begins with retinoic acid incubation, followed by incubation with brain-derived neurotrophic factor (BDNF), a recombinant protein produced in E. coli cells. Endotoxin or lipopolysaccharide (LPS) is the major component of the outer membrane of bacterial cells that triggers the activation of pro-inflammatory cytokines and ultimately cell death. Consequently, any endotoxin contamination of the recombinant BDNF used for cell culture experiments would impact on data interpretation. Therefore, in this study, we expressed the BDNF recombinant protein in bacterial endotoxin-free cells that were engineered to modify the oligosaccharide chain of LPS rendering the LPS unable to trigger the immune response of human cells. The expression of DCX and MAP-2 in differentiated cells indicate that in-house and commercial BDNF are equally effective in inducing differentiation. This suggests that our in-house BDNF protein can be used to differentiate SH-SY5Y neuroblastoma cells without the need for an endotoxin removal step.

RAGE antagonism with azeliragon improves xenograft rejection by T cells in humanized mice.

The receptor for advanced glycation endproducts (RAGE) is involved in multiple inflammatory processes. RAGE participates in adaptive and innate immune responses but its role in human immune cell responses has not been directly tested in vivo. We treated humanized mice (NSG) with the small molecule antagonist of RAGE, azeliragon, (AZ), and measured effects on xenogeneic (B6) skin graft rejection. AZ delayed the median time to xenograft rejection (22 vs 56 days, P = 0.0001). PD-1 expression on CD4+ and CD8+ T cells was lower following AZ therapy. Transcriptome studies showed inhibition of pathways in splenocytes with AZ including IL-23, IL-17A and IL-1β signaling. The serum levels of IL-1β and IL-17A in AZ treated mice were reduced in mice that did not reject skin grafts. The RAGE antagonist prevented xenograft rejection by human immune cells in a murine model. A RAGE antagonist may be a useful inhibitor of adaptive human immune responses.

Effect of Sublethal Concentration of Malathion Insecticide on Innate Immune System, Immune Function and Hemocytes of Adult Drosophila melanogaster

Invertebrate organisms mostly have innate immune system. Hemocytes are the immune cells of the innate immunity in invertebrates. Our study was designed to observe the effect of malathion insecticide on the innate immune system, immune function and hemocytes of Drosophila melanogaster. A hemocytometer was used for total count of hemocytes which showed a reduced percentage of live cells (from 91.63% to 44.57%) due to the treatment. For differential count, the hemolymph was spread as a smear on a clean grease free slide. It was observed that the plasmatocyte count considerably decreased under the effect of malathion (from 78% to 44.66%). Lamellocytes also showed a decline in their number (from 18.33% to 12.61%). But, a trivial increase (from 3.67% to 3.98%) in crystal cell count was recorded. Our result suggests that malathion has mostly negative effect on hemocytes. This study could help in understanding of insect defense mechanism against such hazardous chemicals. Additionally, the function of human macrophages is analogous to the function of plasmatocytes. Hence, this work could possibly aid us in studying the response of human immune cells against organophosphates like malathion.

Activation of Anti-SARS-CoV-2 Human CTLs by Extracellular Vesicles Engineered with the N Viral Protein.

We propose an innovative anti-SARS-CoV-2 immune strategy based on extracellular vesicles (EVs) inducing an anti-SARS-CoV-2 N CD8+ T cytotoxic lymphocyte (CTL) immune response. We previously reported that the SARS-CoV-2 N protein can be uploaded at high levels in EVs upon fusion with Nefmut, i.e., a biologically inactive HIV-1 Nef mutant incorporating into EVs at quite high levels. Here, we analyze the immunogenic properties in human cells of EVs engineered with SARS-CoV-2 N fused at the C-terminus of either Nefmut or a deletion mutant of Nefmut referred to as NefmutPL. The analysis of in vitro-produced EVs has supported the uploading of N protein when fused with truncated Nefmut. Mice injected with DNA vectors expressed each fusion protein developed robust SARS-CoV-2 N-specific CD8+ T cell immune responses. When ex vivo human dendritic cells were challenged with EVs engineered with either fusion products, the induction of a robust N-specific CTL activity, as evaluated by both CD107a and trogocytosis assays, was observed. Through these data we achieved the proof-of-principle that engineered EVs can be instrumental to elicit anti-SARS-CoV-2 CTL immune response in human cells. This achievement represents a mandatory step towards the upcoming experimentations in pre-clinical models focused on intranasal administration of N-engineered EVs.

Mass Cytometry Exploration of Immunomodulatory Responses of Human Immune Cells Exposed to Silver Nanoparticles.

Increasing production and application of silver nanoparticles (Ag NPs) have raised concerns on their possible adverse effects on human health. However, a comprehensive understanding of their effects on biological systems, especially immunomodulatory responses involving various immune cell types and biomolecules (e.g., cytokines and chemokines), is still incomplete. In this study, a single-cell-based, high-dimensional mass cytometry approach is used to investigate the immunomodulatory responses of Ag NPs using human peripheral blood mononuclear cells (hPBMCs) exposed to poly-vinyl-pyrrolidone (PVP)-coated Ag NPs of different core sizes (i.e., 10-, 20-, and 40-nm). Although there were no severe cytotoxic effects observed, PVPAg10 and PVPAg20 were excessively found in monocytes and dendritic cells, while PVPAg40 displayed more affinity with B cells and natural killer cells, thereby triggering the release of proinflammatory cytokines such as IL-2, IL-17A, IL-17F, MIP1β, TNFα, and IFNγ. Our findings indicate that under the exposure conditions tested in this study, Ag NPs only triggered the inflammatory responses in a size-dependent manner rather than induce cytotoxicity in hPBMCs. Our study provides an appropriate ex vivo model to better understand the human immune responses against Ag NP at a single-cell level, which can contribute to the development of targeted drug delivery, vaccine developments, and cancer radiotherapy treatments.

SELECTIVELY IMPROVEMENT OF THE RESPONSE OF IMMUNE CELLS TO OPPORTUNISTIC MICROORGANISMS: AN INNOVATIVE PROPOSAL FOR IBD FROM AN ANCIENT GRAIN

Abstract BACKGROUND Inflammatory Bowel Disease (IBD) results from an unbalanced immune response to the gut microbiome. There is a lack of proposals that integrate and may balance the multiple factors (Immunity, microbiome and diet) that together condition the onset and evolution of the disease. D-fagomine is a small molecule, naturally present in buckwheat grain. It Is a monosaccharide mimetic showing glycosidase enzyme inhibition properties. Clinical studies have shown its efficacy to control postprandial glycemic response. Subsequent research “in vitro” had also shown D-fagomine to inhibit the fimbriae adhesion and biofilm formation of some proteobacteria, including ie E. coli or Salmonella. ORIGINAL AIM The aim of the study was to confirm the effects of D-fagomine on the adhesion to human gut mucosa using adherent-invasive E coli (AIEC) that is a microorganism usually linked to IBD. METHOD Ex vivo human resected colonic mucosa was cultured (4 h) with AIEC inoculum and D-fagomine at different range of doses (0-5000 ppm). The amount of E. coli in tissue and supernatant was quantified, measuring also TNF-alfa and LDH. RESULTS It was confirmed the inhibition of AIEC adhesion to the colonic mucosa in a direct dose-response relationship. Totally unexpected, D-fagomine was found to modulate TNF-alfa in an apparent double sigmoid curve with the max activity in the range of 50 ppm (Figure 1). This curve corresponds to an activator of the innate immune cells with mechanism related to complement activation, (ie mannose, betaglucan). Finally D-fagomine was helping the mucosa to detect and overcome the evasion mechanism of AIEC at the range of 50 ppm. Additional studies were considered necessary to confirm these results with different models. ADDED AIM The novel aim was to assess the human immune cell response (other than colonic mucosa) in the dose range of 50 ppm D-fagomine in front of different microorganisms able to evade the human immune system. ADDED METHOD Ex vivo human blood was cultured with D-fagomine (50 ppm) and the correspondent inoculum of different models: bacteria = E. coli (AIEC) and fungus = Blastomyces sp. TNF-alfa was quantified. ADDED RESULTS D-fagomine, at 50 ppm, stimulates TNF-alfa response of human immune cells against AIEC and also Blastomyces (Figure 2). Both models give coherent results, and confirm that D-fagomine at the range of 50 ppm improves the capacity of the innate immune cells to detect human opportunistic microorganisms. CONCLUSIONS Our results have shown for the first time D-fagomine helping human immune cells to develop a more efficient immune response to better control the presence of opportunistic microorganisms. This is of great interest in IBD and also synergic with D-fagomine activity of delaying the release of sugars from diet to the gut, reducing then the negative effects of a Western Diet to humans and also to its microbiome.

P715 Selective enhancement of immune cell response to opportunistic microorganisms: An innovative proposal for Inflammatory Bowel Disease from an ancient grain

Abstract Background Inflammatory Bowel Disease (IBD) results from an unbalanced immune response to the gut microbiome. There is a lack of proposals that integrate and may balance the multiple factors that together condition the onset and evolution of the disease: immunity, microbiome and diet. D-fagomine is a small molecule, naturally present in buckwheat grain. It Is a monosaccharide mimetic showing glycosidase enzyme inhibition properties. Clinical studies have shown its efficacy to control postprandial glycemic response. Subsequent research “in vitro” had also shown D-fagomine to inhibit the fimbriae adhesion and biofilm formation of some proteobacteria, including ie E. coli or Salmonella. The original aim of the study was to confirm the effects of D-fagomine on the adhesion to human gut mucosa using adherent-invasive E coli (AIEC) that is a microorganism usually linked to IBD. Methods A) Ex vivo human resected colonic mucosa was cultured (4 h) with AIEC inoculum and D-fagomine at different range of doses (0-5000 ppm). The amount of E. coli in tissue and supernatant was quantified, measuring also TNF-alfa and LDH. B) Ex vivo human blood was cultured with D-fagomine (50 ppm) and the correspondent inoculum of different microorganism models able to evade the human immune system: bacteria = E. coli (AIEC) and fungus = Blastomyces sp. TNF-alfa was quantified. Results It was confirmed the inhibition of AIEC adhesion to the colonic mucosa in a direct dose-response relationship. Totally unexpected, D-fagomine was found to modulate TNF-alfa in an apparent double sigmoid curve with the max activity in the range of 50 ppm (Fig 1). This curve corresponds to an activator of the innate immune cells with mechanism related to complement activation, (ie mannose, betaglucan). Finally D-fagomine was helping the mucosa to detect and overcome the evasion mechanism of AIEC at the range of 50 ppm. Additional studies (method B) were carried out with the result that D-fagomine, at 50 ppm, stimulates TNF-alfa response of human immune cells against AIEC and also Blastomyces (Fig 2). Both models give coherent results, and confirm that D-fagomine at the range of 50 ppm improves the capacity of the innate immune cells to detect human opportunistic microorganisms. Conclusion Our results have shown for the first time D-fagomine helping human immune cells to develop a more efficient immune response to better control the presence of opportunistic microorganisms. This is of great interest in IBD and is also synergistic with the other known properties of D-fagomine, which overall could finally help reduce some of the negative effects of the Western diet on humans and also their microbiome.

Chemo-Enzymatic Modification of the 5' Cap Maintains Translation and Increases Immunogenic Properties of mRNA.

Eukaryotic mRNAs are emerging modalities for protein replacement therapy and vaccination. Their 5′ cap is important for mRNA translation and immune response and can be naturally methylated at different positions by S‐adenosyl‐l‐methionine (AdoMet)‐dependent methyltransferases (MTases). We report on the cosubstrate scope of the MTase CAPAM responsible for methylation at the N 6‐position of adenosine start nucleotides using synthetic AdoMet analogs. The chemo‐enzymatic propargylation enabled production of site‐specifically modified reporter‐mRNAs. These cap‐propargylated mRNAs were efficiently translated and showed ≈3‐fold increased immune response in human cells. The same effects were observed when the receptor binding domain (RBD) of SARS‐CoV‐2—a currently tested epitope for mRNA vaccination—was used. Site‐specific chemo‐enzymatic modification of eukaryotic mRNA may thus be a suitable strategy to modulate translation and immune response of mRNAs for future therapeutic applications.

Humanized Mice Exhibit Exacerbated Abscess Formation and Osteolysis During the Establishment of Implant-Associated Staphylococcus aureus Osteomyelitis.

Staphylococcus aureus is the predominant pathogen causing osteomyelitis. Unfortunately, no immunotherapy exists to treat these very challenging and costly infections despite decades of research, and numerous vaccine failures in clinical trials. This lack of success can partially be attributed to an overreliance on murine models where the immune correlates of protection often diverge from that of humans. Moreover, S. aureus secretes numerous immunotoxins with unique tropism to human leukocytes, which compromises the targeting of immune cells in murine models. To study the response of human immune cells during chronic S. aureus bone infections, we engrafted non-obese diabetic (NOD)–scid IL2Rγnull (NSG) mice with human hematopoietic stem cells (huNSG) and analyzed protection in an established model of implant-associated osteomyelitis. The results showed that huNSG mice have increases in weight loss, osteolysis, bacterial dissemination to internal organs, and numbers of Staphylococcal abscess communities (SACs), during the establishment of implant-associated MRSA osteomyelitis compared to NSG controls (p < 0.05). Flow cytometry and immunohistochemistry demonstrated greater human T cell numbers in infected versus uninfected huNSG mice (p < 0.05), and that T-bet+ human T cells clustered around the SACs, suggesting S. aureus-mediated activation and proliferation of human T cells in the infected bone. Collectively, these proof-of-concept studies underscore the utility of huNSG mice for studying an aggressive form of S. aureus osteomyelitis, which is more akin to that seen in humans. We have also established an experimental system to investigate the contribution of specific human T cells in controlling S. aureus infection and dissemination.

The long non-coding RNA LUCAT1 is a negative feedback regulator of interferon responses in humans

Long non-coding RNAs are important regulators of biological processes including immune responses. The immunoregulatory functions of lncRNAs have been revealed primarily in murine models with limited understanding of lncRNAs in human immune responses. Here, we identify lncRNA LUCAT1 which is upregulated in human myeloid cells stimulated with lipopolysaccharide and other innate immune stimuli. Targeted deletion of LUCAT1 in myeloid cells increases expression of type I interferon stimulated genes in response to LPS. By contrast, increased LUCAT1 expression results in a reduction of the inducible ISG response. In activated cells, LUCAT1 is enriched in the nucleus where it associates with chromatin. Further, LUCAT1 limits transcription of interferon stimulated genes by interacting with STAT1 in the nucleus. Together, our study highlights the role of the lncRNA LUCAT1 as a post-induction feedback regulator which functions to restrain the immune response in human cells.

SMARCB1 Acts as a Quiescent Gatekeeper for Cell Cycle and Immune Response in Human Cells.

Switch/sucrose non-fermentable (SWI/SNF)-related matrix-associated actin-dependent regulator of chromatin (SMARC) subfamily B member 1 (SMARCB1) is a core subunit of the switch/sucrose non-fermentable (SWI/SNF) complex, one of the adenosine triphosphate (ATP)-dependent chromatin remodeler complexes. The unique role of SMARCB1 has been reported in various cellular contexts. Here, we focused on the general role of the ubiquitous expression of SMARCB1 in a normal cell state. We selected ARPE19 (human primary retinal pigment epithelium) and IMR90 (from human fetal lung fibroblasts) cell lines as they have completely different contexts. Furthermore, although these cell lines have been immortalized, they are relatively close to normal human cells. The loss of SMARCB1 in ARPE19 and IMR90 cells reduced cell cycle progression via the upregulation of P21. Transcriptome analysis followed by SMARCB1 knockdown in both cell lines revealed that SMARCB1 was not only involved in cell maintenance but also conferred immunomodulation. Of note, SMARCB1 bound to interleukin (IL) 6 promoter in a steady state and dissociated in an active immune response state, suggesting that SMARCB1 was a direct repressor of IL6, which was further confirmed via loss- and gain-of-function studies. Taken together, we demonstrated that SMARCB1 is a critical gatekeeper molecule of the cell cycle and immune response.

Dendritic Cells (DCs) as "Fire Accelerants" of Hantaviral Pathogenesis.

Hantaviruses are widespread zoonotic pathogens found around the globe. Depending on their geographical location, hantaviruses can cause two human syndromes, haemorrhagic fever with renal syndrome (HFRS) or hantavirus pulmonary syndrome (HPS). HPS and HFRS have many commonalities amongst which excessive activation of immune cells is a prominent feature. Hantaviruses replicate in endothelial cells (ECs), the major battlefield of hantavirus-induced pathogenesis, without causing cytopathic effects. This indicates that a misdirected response of human immune cells to hantaviruses is causing damage. As dendritic cells (DCs) orchestrate antiviral immune responses, they are in the focus of research analysing hantavirus-induced immunopathogenesis. In this review, we discuss the interplay between hantaviruses and DCs and the immunological consequences thereof.

Intratumoral immunotherapy with TLR7/8 agonist MEDI9197 modulates the tumor microenvironment leading to enhanced activity when combined with other immunotherapies

BackgroundImmune checkpoint blockade (ICB) promotes adaptive immunity and tumor regression in some cancer patients. However, in patients with immunologically “cold” tumors, tumor-resident innate immune cell activation may be required to prime an adaptive immune response and so exploit the full potential of ICB. Whilst Toll-like receptor (TLR) agonists have been used topically to successfully treat some superficial skin tumors, systemic TLR agonists have not been well-tolerated.MethodsThe response of human immune cells to TLR7 and 8 agonism was measured in primary human immune cell assays. MEDI9197 (3M-052) was designed as a novel lipophilic TLR7/8 agonist that is retained at the injection site, limiting systemic exposure. Retention of the TLR7/8 agonist at the site of injection was demonstrated using quantitative whole-body autoradiography, HPLC-UV, and MALDI mass spectrometry imaging. Pharmacodynamic changes on T cells from TLR7/8 agonist treated B16-OVA tumors was assessed by histology, quantitative real time PCR, and flow cytometry. Combination activity of TLR7/8 agonism with immunotherapies was assessed in vitro by human DC-T cell MLR assay, and in vivo using multiple syngeneic mouse tumor models.ResultsTargeting both TLR7 and 8 triggers an innate and adaptive immune response in primary human immune cells, exemplified by secretion of IFNα, IL-12 and IFNγ. In contrast, a STING or a TLR9 agonist primarily induces release of IFNα. We demonstrate that the TLR7/8 agonist, MEDI9197, is retained at the sight of injection with limited systemic exposure. This localized TLR7/8 agonism leads to Th1 polarization, enrichment and activation of natural killer (NK) and CD8+ T cells, and inhibition of tumor growth in multiple syngeneic models. The anti-tumor activity of this TLR7/8 agonist is enhanced when combined with T cell-targeted immunotherapies in pre-clinical models.ConclusionLocalized TLR7/8 agonism can enhance recruitment and activation of immune cells in tumors and polarize anti-tumor immunity towards a Th1 response. Moreover, we demonstrate that the anti-tumor effects of this TLR7/8 agonist can be enhanced through combination with checkpoint inhibitors and co-stimulatory agonists.

Posttranslational Regulation of IL-23 Production Distinguishes the Innate Immune Responses to Live Toxigenic versus Heat-Inactivated Vibrio cholerae.

Vibrio cholerae infection provides long-lasting protective immunity, while oral, inactivated cholera vaccines (OCV) result in more-limited protection. To identify characteristics of the innate immune response that may distinguish natural V. cholerae infection from OCV, we stimulated differentiated, macrophage-like THP-1 cells with live versus heat-inactivated V. cholerae with and without endogenous or exogenous cholera holotoxin (CT). Interleukin 23A gene (IL23A) expression was higher in cells exposed to live V. cholerae than in cells exposed to inactivated organisms (mean change, 38-fold; 95% confidence interval [95% CI], 4.0 to 42; P < 0.01). IL-23 secretion was also higher in cells exposed to live V. cholerae than in cells exposed to inactivated V. cholerae (mean change, 5.6-fold; 95% CI, 4.4 to 11; P < 0.001). This increase in IL-23 secretion was more marked than for other key innate immune cytokines (e.g., IL-1β and IL-6) and dependent on exposure to the combination of both live V. cholerae and CT. While IL-23 secretion was reduced following stimulation with either heat-inactivated wild-type V. cholerae or a live isogenic ctxAB mutant of V. cholerae, the addition of exogenous CT restored IL-23 secretion in combination with the live isogenic ctxAB mutant V. cholerae, but not when it was paired with stimulation by heat-inactivated V. cholerae The posttranslational regulation of IL-23 under these conditions was dependent on the activity of the cysteine protease cathepsin B. In humans, IL-23 promotes the differentiation of Th17 cells to T follicular helper cells, which maintain and support long-term memory B cell generation after infection. Based on these findings, the stimulation of IL-23 production may be a determinant of protective immunity following V. cholerae infection.IMPORTANCE An episode of cholera provides better protection against reinfection than oral cholera vaccines, and the reasons for this are still under study. To better understand this, we compared the immune responses of human cells exposed to live Vibrio cholerae with those of cells exposed to heat-killed V. cholerae (similar to the contents of oral cholera vaccines). We also compared the effects of active cholera toxin and the inactive cholera toxin B subunit (which is included in some cholera vaccines). One key immune signaling molecule, IL-23, was uniquely produced in response to the combination of live bacteria and active cholera holotoxin. Stimulation with V. cholerae that did not produce the active toxin or was killed did not produce an IL-23 response. The stimulation of IL-23 production by cholera toxin-producing V. cholerae may be important in conferring long-term immunity after cholera.

Bacteriophage GH15: Developing A Novel Weapon Against MRSA

In recent years, numerous bacterial species have developed antibiotic resistance due to the overuse of antibiotics in the home, health care setting, and in agriculture. Alternative methods of treatment, including phage therapy (PT), have been proposed as solutions to this problem. PT is showing promise as an alternative method of treatment against the bacteria methicillin-resistant Staphylococcus aureus (MRSA). MRSA is a virulent and antibiotic resistant bacterium capable of causing infections of the skin, respiratory system, and various other body systems. In this research proposal, we propose investigating the use of the Staphylococcal bacteriophage (phage) GH15 as a therapeutic agent against MRSA infections due to its broad host range, its lack of bacterial virulence genes, and its strong ability to lyse various strains of MRSA. Specifically, we propose to evaluate the tail fibre genes of GH15 contributing to the phage’s host range, in addition to the ability of the phage to induce antiphage humoral immune responses in human cells, in the interest of exploring GH15 as a therapeutic agent for use in PT, specifically against MRSA.

Structural variants of Salmonella Typhimurium lipopolysaccharide induce less dimerization of TLR4/MD-2 and reduced pro-inflammatory cytokine production in human monocytes

Salmonella enterica serovar Typhimurium (S. Typhimurium) changes the structure of its lipopolysaccharide (LPS) in response to the environment. The two main LPS variants found in S. Typhimurium correspond to LPS with a hepta-acylated lipid A (LPS 430) and LPS with modified phosphate groups on its lipid A (LPS 435). We have previously shown that these modified LPS have a lower capacity than wild type (WT) LPS to induce the production of pro-inflammatory cytokines in mice. Nevertheless, it is not know if LPS 430 and LPS 435 could also subvert the innate immune responses in human cells. In this study, we found that LPS 430 and LPS 435 were less efficient than WT LPS to induce the production of pro-inflammatory cytokines by human monocytes, in addition we found a decreased dimerization of the TLR4/MD-2 complex in response to LPS 430, suggesting that structurally modified LPS are sensed differently than WT LPS by this receptor; however, LPS 430 and 435 induced similar activation of the transcription factors NF-κB p65, IRF3, p38 and ERK1/2 than WT LPS. Microarray analysis of LPS 430- and LPS 435-activated monocytes revealed a gene transcription profile with differences only in the expression levels of microRNA genes compared to the profile induced by WT LPS, suggesting that the lipid A modifications present in LPS 430 and LPS 435 have a moderate effect on the activation of the human TLR4/MD-2 complex. Our results are relevant to understand LPS modulation of immune responses and this knowledge could be useful for the development of novel adjuvants and immunomodulators.

Specificity and functional interplay between influenza virus PA-X and NS1 shutoff activity.

Influenza A viruses modulate host antiviral responses to promote viral growth and pathogenicity. Through viral PA-X and NS1 proteins, the virus is capable of suppressing host protein synthesis, termed “host shutoff.” Although both proteins are known to induce general shutoff, specificity of target genes and their functional interplay in mediating host shutoff are not fully elucidated. In this study, we generated four recombinant influenza A/California/04/2009 (pH1N1) viruses containing mutations affecting the expression of active PA-X and NS1. We analyzed viral growth, general shutoff activity, specificity of mRNA targets, and viral gene expressions. Our results showed that PA-X was the major contributor in reducing general host protein expression in the virus-infected cells. Intriguingly, our transcriptomic analysis from infected human airway A549 cells indicate that shutoff-active NS1 specifically targeted host mRNAs related to interferon (IFN) signaling pathways and cytokine release. Specificity of target mRNAs was less evident in PA-X, although it preferentially degraded genes associated with cellular protein metabolism and protein repair. Interestingly, in the presence of shutoff-active NS1, PA-X also degraded viral mRNAs, especially NS segments. The virus expressing shutoff-active NS1 with reduced amount of PA-X expression most efficiently suppressed antiviral and innate immune responses in human cells, indicating that influenza virus needs to optimize the contribution of these two shutoff proteins to circumvent host responses for its optimum growth.

Inducible Nitric Oxide Synthase Is a Key Host Factor for Toxoplasma GRA15-Dependent Disruption of the Gamma Interferon-Induced Antiparasitic Human Response.

Although Toxoplasma virulence mechanisms targeting gamma interferon (IFN-γ)-induced cell-autonomous antiparasitic immunity have been extensively characterized in mice, the virulence mechanisms in humans remain uncertain, partly because cell-autonomous immune responses against Toxoplasma differ markedly between mice and humans. Despite the identification of inducible nitric oxide synthase (iNOS) as an anti-Toxoplasma host factor in mice, here we show that iNOS in humans is a pro-Toxoplasma host factor that promotes the growth of the parasite. The GRA15 Toxoplasma effector-dependent disarmament of IFN-γ-induced parasite growth inhibition was evident when parasite-infected monocytes were cocultured with hepatocytes. Interleukin-1β (IL-1β), produced from monocytes in a manner dependent on GRA15 and the host's NLRP3 inflammasome, combined with IFN-γ to strongly stimulate iNOS expression in hepatocytes; this dramatically reduced the levels of indole 2,3-dioxygenase 1 (IDO1), a critically important IFN-γ-inducible anti-Toxoplasma protein in humans, thus allowing parasite growth. Taking the data together, Toxoplasma utilizes human iNOS to antagonize IFN-γ-induced IDO1-mediated cell-autonomous immunity via its GRA15 virulence factor.IMPORTANCEToxoplasma, an important intracellular parasite of humans and animals, causes life-threatening toxoplasmosis in immunocompromised individuals. Gamma interferon (IFN-γ) is produced in the host to inhibit the proliferation of this parasite and eventually cause its death. Unlike mouse disease models, which involve well-characterized virulence strategies that are used by Toxoplasma to suppress IFN-γ-dependent immunity, the strategies used by Toxoplasma in humans remain unclear. Here, we show that GRA15, a Toxoplasma effector protein, suppresses the IFN-γ-induced indole-2,3-dioxygenase 1-dependent antiparasite immune response in human cells. Because NLRP3-dependent production of IL-1β and nitric oxide (NO) in Toxoplasma-infected human cells is involved in the GRA15-dependent virulence mechanism, blocking NO or IL-1β production in the host could represent a novel therapeutic approach for treating human toxoplasmosis.