Inactivating a cellular intrinsic immune defense mediated by Daxx is the mechanism through which the human cytomegalovirus pp71 protein stimulates viral immediate-early gene expression

Human cytomegalovirus (HCMV) masterfully evades adaptive and innate immune responses, allowing infection to be maintained and periodically reactivated for the life of the host. Here we show that cells also possess an intrinsic immune defense against HCMV that is disarmed by the virus. In HCMV-infected cells, the promyelocytic leukemia nuclear body (PML-NB) protein Daxx silences viral immediate-early gene expression through the action of a histone deacetylase. However, this antiviral tactic is efficiently neutralized by the viral pp71 protein, which is incorporated into virions, delivered to cells upon infection, and mediates the proteasomal degradation of Daxx. This work demonstrates the mechanism through which pp71 activates viral immediate-early gene expression in HCMV-infected cells. Furthermore, it provides insight into how a PML-NB protein institutes an intrinsic immune defense against a DNA virus and how HCMV pp71 inactivates this defense.     

Profiles of gene expression in human autoimmune disease

Human autoimmune diseases arise from complex interactions between genetic and environmental factors, result from immune attack upon target tissues, and affect 3–5% of the population. We compared gene expression profiles (>4000 genes) in the peripheral blood mononuclear cells of normal individuals after immunization to individuals with four different autoimmune diseases (rheumatoid arthritis, systemic lupus erythematosus, insulin-dependent diabetes mellitus, and multiple sclerosis). All autoimmune individuals, including unaffected first-degree relatives, share a common gene expression profile that is completely distinct from the immune profile. Therefore, this expression pattern is not simply a recapitulation of the immune response to nonself, is not a result of the disease process, and results, as least in part, from genetic factors. Surprisingly, these genes are clustered in chromosomal domains suggesting there is some genomewide logic to this unique expression pattern. These data argue that that there is a constant pattern of gene expression in autoimmunity that is independent of the specific autoimmune disease and clinical parameters associated with any individual autoimmune disease.     

Identification of novel IL-4/Stat6-regulated genes in T lymphocytes

IL-4, primarily produced by T cells, mast cells, and basophiles, is a cytokine which has pleiotropic effects on the immune system. IL-4 induces T cells to differentiate to Th2 cells and activated B lymphocytes to proliferate and to synthesize IgE and IgG1. IL-4 is particularly important for the development and perpetuation of asthma and allergy. Stat6 is the protein activated by signal transduction through the IL-4R, and studies with knockout mice demonstrate that Stat6 is critical for a number of IL-4-mediated functions including Th2 development and production of IgE. In the present study, novel IL-4- and Stat6-regulated genes were discovered by using Stat6(-/-) mice and Affymetrix oligonucleotide arrays. Genes regulated by IL-4 were identified by comparing the gene expression profile of the wild-type T cells induced to polarize to the Th2 direction (CD3/CD28 activation + IL-4) to gene expression profile of the cells induced to proliferate (CD3/CD28 activation alone). Stat6-regulated genes were identified by comparing the cells isolated from the wild-type and Stat6(-/-) mice; in this experiment the cells were induced to differentiate to the Th2 direction (CD3/CD28 activation + IL-4). Our study demonstrates that a number a novel genes are regulated by IL-4 through Stat6-dependent and -independent pathways. Moreover, elucidation of kinetics of gene expression at early stages of cell differentiation reveals several genes regulated rapidly during the process, suggesting their importance for the differentiation process.     

RNA silencing: small RNAs as ubiquitous regulators of gene expression

'RNA silencing' is the suppression of gene expression through nucleotide sequence-specific interactions that are mediated by RNA. Initially identified as an immune system that is targeted against transposons and viruses, RNA silencing is emerging as a fundamental regulatory process that is likely to affect many layers of endogenous gene expression in most, if not all, eukaryotes.     

1,25-Dihyroxyvitamin D3 promotes FOXP3 expression via binding to vitamin D response elements in its conserved noncoding sequence region

FOXP3-positive regulatory T (Treg) cells are a unique subset of T cells with immune regulatory properties. Treg cells can be induced from non-Treg CD4(+) T cells (induced Treg [iTreg] cells) by TCR triggering, IL-2, and TGF-β or retinoic acid. 1,25-Dihyroxyvitamin D(3) [1,25(OH)(2)VD(3)] affects the functions of immune cells including T cells. 1,25(OH)(2)VD(3) binds the nuclear VD receptor (VDR) that binds target DNA sequences known as the VD response element (VDRE). Although 1,25(OH)(2)VD(3) can promote FOXP3 expression in CD4(+) T cells with TCR triggering and IL-2, it is unknown whether this effect of 1,25(OH)(2)VD(3) is mediated through direct binding of VDR to the FOXP3 gene without involving other molecules. Also, it is unclear whether FOXP3 expression in 1,25(OH)(2)VD(3)-induced Treg (VD-iTreg) cells is critical for the inhibitory function of these cells. In this study, we demonstrated the presence of VDREs in the intronic conserved noncoding sequence region +1714 to +2554 of the human FOXP3 gene and the enhancement of the FOXP3 promoter activity by such VDREs in response to 1,25(OH)(2)VD(3). Additionally, VD-iTreg cells suppressed the proliferation of target CD4(+) T cells and this activity was dependent on FOXP3 expression. These findings suggest that 1,25(OH)(2)VD(3) can affect human immune responses by regulating FOXP3 expression in CD4(+) T cells through direct VDR binding to the FOXP3 gene, which is essential for inhibitory function of VD-iTreg cells.     

Dynamic changes in the localization of MAPK cascade components controlling pathogenesis-related (PR) gene expression during innate immunity in parsley

The activation of mitogen-activated protein kinase (MAPK) cascades is an important mechanism for stress adaptation through the control of gene expression in mammals, yeast, and plants. MAPK activation has emerged as a common mechanism by which plants trigger pathogen defense responses following innate immune recognition of potential microbial pathogens. We are studying the non-host plant defense response of parsley to attempted infection by Phytophthora species using an experimental system of cultured parsley cells and the Phytophthora-derived Pep-13 peptide elicitor. Following receptor-mediated recognition of this peptide, parsley cells trigger a multifaceted innate immune response, involving the activation of three MAPKs that have been shown to function in the oxidative burst-independent activation of defense gene expression. Using this same experimental model we now report the identification of a MAPK kinase (MAPKK) that functions upstream in this pathway. This kinase, referred to as PcMKK5 based on sequence similarity to Arabidopsis thaliana AtMKK5, is activated in parsley cells following Pep-13 treatment and functions as an in vivo activator of all three MAPKs previously shown to be involved in this response. Gain- and loss-of-function mutant versions of PcMKK5, when used in protoplast co-transfection assays, demonstrated that kinase activity of PcMKK5 is required for PR gene promoter activation following Pep-13 treatment. Furthermore, using specific antibodies and immunofluorescent labeling, we demonstrate that activation of MAPKs in parsley cells correlates with an increase in their nuclear localization, which is not detectable for activated PcMKK5. These results suggest that activation of gene expression through MAPK cascades during innate immune responses in plants involves dynamic changes in the localization of the proteins involved, which may reflect the distribution of key protein substrates for the activated MAPKs.     

A novel non-viral delivery method that enables efficient engineering of primary human T cells for ex vivo cell therapy applications

Background aimsNext-generation immune cell therapy products will require complex modifications using engineering technologies that can maintain high levels of cell functionality. Non-viral engineering methods have the potential to address limitations associated with viral vectors. However, while electroporation is the most widely used non-viral modality, concerns about its effects on cell functionality have led to the exploration of alternative approaches. Here the authors have examined the suitability of the Solupore non-viral delivery system for engineering primary human T cells for cell therapy applications.MethodsThe Solupore system was used to deliver messenger RNA (mRNA) and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) guide RNA ribonucleoprotein (RNP) cargos to T cells, and efficiency was measured by flow cytometry. Cell perturbation was assessed by immune gene expression profiling, including an electroporation comparator. In vitro and in vivo cytotoxicity of chimeric antigen receptor (CAR) T cells generated using the Solupore system was evaluated using a real-time cellular impedance assay and a Raji-luciferase mouse tumor model, respectively.ResultsEfficient transfection was demonstrated through delivery of mRNA and CRISPR CAS9 RNP cargos individually, simultaneously and sequentially using the Solupore system while consistently maintaining high levels of cell viability. Gene expression profiling revealed minimal alteration in immune gene expression, demonstrating the low level of perturbation experienced by the cells during this transfection process. By contrast, electroporation resulted in substantial changes in immune gene expression in T cells. CAR T cells generated using the Solupore system exhibited efficient cytotoxicity against target cancer cells in vitro and in vivo.ConclusionsThe Solupore system is a non-viral means of simply, rapidly and efficiently delivering cargos to primary human immune cells with retention of high cell viability and functionality.     

Low molecular weight hyaluronan mediated CD44 dependent induction of IL-6 and chemokines in human dermal fibroblasts potentiates innate immune response

Complex regulation of the wound healing process involves multiple interactions among stromal tissue cells, inflammatory cells, and the extracellular matrix. Low molecular weight hyaluronan (LMW HA) derived from the degradation of high molecular weight hyaluronan (HMW HA) is suggested to activate cells involved in wound healing through interaction with HA receptors. In particular, receptor CD44 is suggested to mediate cell response to HA of different MW, being the main cell surface HA receptor in stromal tissue and immune cells. However, the response of dermal fibroblasts, the key players in granulation tissue formation within the wound healing process, to LMW HA and their importance for the activation of immune cells is unclear. In this study we show that LMW HA (4.3 kDa) induced pro-inflammatory cytokine IL-6 and chemokines IL-8, CXCL1, CXCL2, CXCL6 and CCL8 gene expression in normal human dermal fibroblasts (NHDF) that was further confirmed by increased levels of IL-6 and IL-8 in cell culture supernatants. Conversely, NHDF treated by HMW HA revealed a tendency to decrease the gene expression of these cytokine and chemokines when compared to untreated control. The blockage of CD44 expression by siRNA resulted in the attenuation of IL-6 and chemokines expression in LMW HA treated NHDF suggesting the involvement of CD44 in LMW HA mediated NHDF activation. The importance of pro-inflammatory mediators produced by LMW HA triggered NHDF was evaluated by significant activation of blood leukocytes exhibited as increased production of IL-6 and TNF-α. Conclusively, we demonstrated a pro-inflammatory response of dermal fibroblasts to LMW HA that was transferred to leukocytes indicating the significance of LMW HA in the inflammatory process development during the wound healing process.     

Mesenchymal stem cells as carrier of the therapeutic agent in the gene therapy of blood disorders

Nonhematopoietic stem cells as a delivery platform of therapeutic useful genes have attracted widespread attention in recent years, owing to gained a long lifespan, easy separation, high proliferation, and high transfection capacity. Mesenchymal stem/stromal cells (MSCs) are the choice of the cells for gene and cell therapy due to high self-renewal capacity, high migration rate to the site of the tumor, and with immune suppressive and anti-inflammatory properties. Hence, it has a high potential of safety genetic modification of MSCs for antitumor gene expression and has paved the way for the clinical application of these cells to target the therapy of cancers and other diseases. The aim of gene therapy is targeted treatment of cancers and diseases through recovery, change, or enhancement cell performance to the sustained secretion of useful therapeutic proteins and induction expression of the functional gene in intended tissue. Recent developments in the vectors designing leading to the increase and durability of expression and improvement of the safety of the vectors that overcome a lot of problems, such as durability of expression and the host immune response. Nowadays, gene therapy approach is used by MSCs as a delivery vehicle in the preclinical and the clinical trials for the secretion of erythropoietin, recombinant antibodies, coagulation factors, cytokines, as well as angiogenic inhibitors in many blood disorders like anemia, hemophilia, and malignancies. In this study, we critically discuss the status of gene therapy by MSCs as a delivery vehicle for the treatment of blood disorders. Finally, the results of clinical trial studies are assessed, highlighting promising advantages of this emerging technology in the clinical setting.     

Fungal proteases induce Th2 polarization through limited dendritic cell maturation and reduced production of IL-12

Allergens are capable of polarizing the T cell immune response toward a Th2 cytokine profile in a process that is mediated by dendritic cells (DCs). Proteases derived from Aspergillus species (Aspergillus proteases; AP) have been shown to induce a Th2-like immune response when administered directly to the airway and without adjuvant or prior priming immunizations at sites remote from the lung in models of allergic airway disease. To explore mechanisms that underlie the Th2 immune response, we have investigated the effect of AP on DC function. We found that human DCs derived from CD14(+) monocytes from healthy donors underwent partial maturation when incubated with AP. Naive allogeneic T cells primed with AP-activated DCs proliferated and displayed enhanced production of IL-4 and reduced expression of IFN-gamma as compared with naive T cells primed with LPS-activated DCs. Global gene expression analysis of DCs revealed relatively low expression of IL-12p40 in AP-activated DCs as compared with those activated by LPS, and this was confirmed at the protein level by ELISA. Exogenous IL-12p70 added to cocultures of DCs and T cells resulted in reduced IL-4 and increased IFN-gamma expression when DCs were activated with AP. When the proteolytic activity of AP was neutralized by chemical inactivation it failed to up-regulate costimulatory molecules on DCs, and these DCs did not prime a Th2 response in naive T cells. These findings provide a mechanism for explaining how proteolytically active allergens could preferentially induce Th2 responses through limited maturation of DCs with reduced production of IL-12.