Interactions between naïve and infected macrophages reduce Mycobacterium tuberculosis viability

A high intracellular bacillary load of Mycobacterium tuberculosis in macrophages induces an atypical lysosomal cell death with early features of apoptosis that progress to necrosis within hours. Unlike classical apoptosis, this cell death mode does not appear to diminish M. tuberculosis viability. We previously reported that culturing heavily infected macrophages with na?ve macrophages produced an antimicrobial effect, but only if na?ve macrophages were added during the pre-necrotic phase of M. tuberculosis-induced cell death. In the present study we investigated the mechanism of antimicrobial activity in co-cultures, anticipating that efferocytosis of bacilli in apoptotic bodies would be required. Confocal microscopy revealed frustrated phagocytosis of M. tuberculosis-infected macrophages with no evidence that significant numbers of bacilli were transferred to the na?ve macrophages. The antimicrobial effect of na?ve macrophages was retained when they were separated from infected macrophages in transwells, and conditioned co-culture supernatants transferred antimicrobial activity to cultures of infected macrophages alone. Antimicrobial activity in macrophage co-cultures was abrogated when the na?ve population was deficient in IL-1 receptor or when the infected population was deficient in inducible nitric oxide synthase. The participation of nitric oxide suggested a conventional antimicrobial mechanism requiring delivery of bacilli to a late endosomal compartment. Using macrophages expressing GFP-LC3 we observed the induction of autophagy specifically by a high intracellular load of M. tuberculosis. Bacilli were identified in LC3-positive compartments and LC3-positive compartments were confirmed to be acidified and LAMP1 positive. Thus, the antimicrobial effect of na?ve macrophages acting on M. tuberculosis in heavily-infected macrophages is contact-independent. Interleukin-1 provides an afferent signal that induces an as yet unidentified small molecule which promotes nitric oxide-dependent antimicrobial activity against bacilli in autolysosomes of heavily infected macrophages. This cooperative, innate antimicrobial interaction may limit the maximal growth rate of M. tuberculosis prior to the expression of adaptive immunity in pulmonary tuberculosis.     

Calcineurin inhibitors suppress cytokine production from memory T cells and differentiation of naïve T cells into cytokine-producing mature T cells

T cells have been classified as belonging to the Th1 or Th2 subsets according to the production of defining cytokines such as IFN-γ and IL-4. The discovery of the Th17 lineage and regulatory T cells shifted the simple concept of the Th1/Th2 balance into a 4-way mechanistic pathway of local and systemic immunological activity. Clinically, the blockage of cytokine signals or non-specific suppression of cytokine predominance by immunosuppressants is the first-line treatment for inflammatory T cell-mediated disorders. Cyclosporine A (CsA) and Tacrolimus (Tac) are commonly used immunosuppressants for the treatment of autoimmune disease, psoriasis, and atopic disorders. Many studies have shown that these compounds suppress the activation of the calcium-dependent phosphatase calcineurin, thereby inhibiting T-cell activation. Although CsA and Tac are frequently utilized, their pharmacological mechanisms have not yet been fully elucidated.In the present study, we focused on the effects of CsA and Tac on cytokine secretion from purified human memory CD4(+)T cells and the differentiation of na?ve T cells into cytokine-producing memory T cells. CsA or Tac significantly inhibited IFN-γ, IL-4, and IL-17 production from memory T cells. These compounds also inhibited T cell differentiation into the Th1, Th2, and Th17 subsets, even when used at a low concentration. This study provided critical information regarding the clinical efficacies of CsA and Tac as immunosuppressants.     

IL-6 inhibits IFN-γ induced autophagy in Mycobacterium tuberculosis H37Rv infected macrophages

The significance of IL-6 production in tuberculosis is yet to be fully elucidated, although it is known for quite some time that IL-6 interferes with IFN-γ induced signal. In order to know which cellular process induced by IFN-γ is actually counteracted by IL-6, we studied the role of IL-6 on IFN-γ induced autophagy formation in virulent Mycobacterium tuberculosis infection in THP-1 cells, since it is well characterized that induction of autophagy by IFN-γ eliminates intracellular mycobacterium by overcoming the phagosome maturation block imposed by bacilli. We report here that IL-6 inhibits both IFN-γ and starvation induced autophagy in M. tuberculosis H37Rv infected cells. M. tuberculosis H37Rv infection results in time dependent production of IL-6 in THP-1 cells and neutralization of this endogenous IL-6 by anti-IL-6 antibody significantly enhances the IFN-γ mediated killing of the intracellular bacteria. IL-6 time dependently lowers Atg12-Atg5 complex and therefore inhibits autophagosome biogenesis rather than autophagolysosome formation. IL-6 also affects IFN-γ mediated stimulation of mTOR, p-38 and JNK pathways. These results clearly indicate that virulent mycobacteria strategically upregulate IL-6 production to combat innate immunity.     

Cells previously desensitized to type 1 interferons display different mechanisms of activation of stat-dependent gene expression from naïve cells

Over the past decade, a wealth of knowledge has been obtained concerning the mechanisms by which interferons (IFNs) and other cytokines activate or down-regulate immediate early genes via the Jak/Stat pathway. In contrast, little information is available on interferon-activated gene expression in na?ve cells compared with cells that have been desensitized and subsequently resensitized to the actions of these cytokines. In na?ve cells, the ISG54 gene is activated via IFN beta-stimulated formation of ISGF3, a heterotrimeric DNA binding complex consisting of p48 (IRF9) and tyrosine-phosphorylated Stat1 and Stat2. In contrast, in previously desensitized cells IFN beta weakly stimulates the assembly of an ISGF3-like complex that lacks Stat1, even though ISG54 mRNA induction is the same as in na?ve cells. The lack of Stat1 tyrosine phosphorylation and DNA binding is due to increased activity of a protein-tyrosine phosphatase. In cells that do not express the tyrosine phosphatase Tc-PTP, the rate of Stat1 dephosphorylation is the same in na?ve and previously desensitized cells. These results implicate Tc-PTP in a novel role in the regulation of type 1 interferon-stimulated gene expression.     

Inability to demonstrate fish-to-fish transmission of Ichthyophonus from laboratory infected Pacific herring Clupea pallasii to naïve conspecifics

The parasite Ichthyophonus is enzootic in many marine fish populations of the northern Atlantic and Pacific Oceans. Forage fishes are a likely source of infection for higher trophic level predators; however, the processes that maintain Ichthyophonus in forage fish populations (primarily clupeids) are not well understood. Lack of an identified intermediate host has led to the convenient hypothesis that the parasite can be maintained within populations of schooling fishes by waterborne fish-to-fish transmission. To test this hypothesis we established Ichthyophonus infections in Age-1 and young-of-the-year (YOY) Pacific herring Clupea pallasii (Valenciennes) via intraperitoneal (IP) injection and cohabitated these donors with na?ve conspecifics (sentinels) in the laboratory. IP injections established infection in 75 to 84% of donor herring, and this exposure led to clinical disease and mortality in the YOY cohort. However, after cohabitation for 113 d no infections were detected in na?ve sentinels. These data do not preclude the possibility of fish-to-fish transmission, but they do suggest that other transmission processes are necessary to maintain Ichthyophonus in wild Pacific herring populations.     

Glycans define the stemness of naïve and primed pluripotent stem cells

Cell surface glycans are tissue-specific and developmentally regulated. They function as essential modulators in cell-cell interactions, cell-extracellular matrix interactions, and ligand-receptor interactions, binding to various ligands, including Wnt, fibroblast growth factors, and bone morphogenetic proteins. Embryonic stem (ES) cells, originally derived from the inner cell mass of blastocysts, have the essential characteristics of pluripotency and self-renewal. Recently, it has been proposed that mouse and human conventional ES cells are present in different developmental stages, namely pre-implantation blastocyst and post-implantation blastocyst stages, also called the naïve state and the primed state, respectively. They therefore require different extrinsic signals for the maintenance of self-renewal and pluripotency, and also appear to require different surface glycans. Understanding of molecular mechanisms involving glycans in self-renewal and pluripotency of ES cells is increasingly important for potential clinical applications, as well as for basic research. This review focuses on the roles of glycans in the two different states of pluripotent stem cells, namely the naïve state and the primed state, and the transition between these two states.     

Differential expression of FCRLA in naïve and activated mouse B cells

FCRLA is an intracellular B cell protein that belongs to the FcR-like family. Using newly generated FCRLA-specific antibodies, we studied the constitutive expression pattern of mouse FCRLA and monitored changes during an immune response and following in vitro B cell activation. All B cell subpopulations examined expressed FCRLA. However, the level of FCRLA expression is determined by the stage of B cell differentiation. Low expression of FCRLA is characteristic of naïve follicular and marginal zone B cells. High expression was detected in a small fraction of activated B cells scattered along migratory pathways in the lymphoid tissues. FCRLA-bright cells could be subdivided into two subpopulations, with high and low/undetectable level of intracellular immunoglobulins, which phenotypically resemble either plasma or memory B cells. High expression of FCRLA in subset(s) of terminally differentiated B-cells suggests that, being an ER protein, FCRLA may participate in the regulation of immunoglobulin assembly and secretion.     

Long-term self-renewal of naïve neural stem cells in a defined condition

During embryonic development, neural stem cells (NSCs) emerge as early as the neural plate stage and give rise to the nervous system. Early-stage NSCs express Sry-related-HMG box-1 (Sox1) and are biased towards neuronal differentiation. However, long-term maintenance of early-stage NSCs in vitro remains a challenge. Here, we report development of a defined culture condition for the long-term maintenance of Sox1-positive early-stage mouse NSCs. The proliferative ability of these Sox1-positive NSCs was confirmed by clonal propagation. Compared to the NSCs cultured using the traditional culture condition, the long-term self-renewing Sox1-positive NSCs efficiently differentiate into neurons and exhibit an identity representative of the anterior and midbrain regions. These early-stage Sox1-positive NSCs could also be switched to late-stage NSCs by being cultured with bFGF/EGF, which can then differentiate into astrocytes and oligodendrocytes. The long-term self-renewing Sox1-positive NSCs were defined as naïve NSCs, based on their high neuronal differentiation capacity and anterior regional identity. This culture condition provides a robust platform for further dissection of the NSC self-renewal mechanism and promotes potential applications of NSCs for cell-based therapy on nervous system disorders.     

Non-random tissue distribution of human naïve umbilical cord matrix stem cells

AIM: To determine the tissue and temporal distribution of human umbilical cord matrix stem (hUCMS) cells in severe combined immunodeficiency (SCID) mice. METHODS: For studying the localization of hUCMS cells, tritiated thymidine-labeled hUCMS cells were injected in SCID mice and tissue distribution was quantitatively determined using a liquid scintillation counter at days 1, 3, 7 and 14. Furthermore, an immunofluorescence detection technique was employed in which anti-human mitochondrial antibody was used to identify hUCMS cells in mouse tissues. In order to visualize the distribution of transplanted hUCMS cells in H&E stained tissue sections, India Black ink 4415 was used to label the hUCMS cells. RESULTS: When tritiated thymidine-labeled hUCMS cells were injected systemically (iv) in female SCID mice, the lung was the major site of accumulation at 24 h after transplantation. With time, the cells migrated to other tissues, and on day three, the spleen, stomach, and small and large intestines were the major accumulation sites. On day seven, a relatively large amount of radioactivity was detected in the adrenal gland, uterus, spleen, lung, and digestive tract. In addition, labeled cells had crossed the blood brain barrier by day 1. CONCLUSION: These results indicate that peripherally injected hUCMS cells distribute quantitatively in a tissue-specific manner throughout the body.     

1,25-dihydroxyvitamin D3 impairs NF-κB activation in human naïve B cells

1α,25-dihydroxyvitamin D(3) (calcitriol), the bioactive metabolite of vitamin D, modulates the activation and inhibits IgE production of anti-CD40 and IL-4 stimulated human peripheral B cells. Engagement of CD40 results in NF-κB p50 activation, which is essential for the class switch to IgE. Herein, we investigated by which mechanism calcitriol modulates NF-κB mediated activation of human na?ve B cells. Na?ve B cells were predominantly targeted by calcitriol in comparison with memory B cells as shown by pronounced induction of the VDR target gene cyp24a1. Vitamin D receptor activation resulted in a strongly reduced p105/p50 protein and mRNA expression in human na?ve B cells. This effect is mediated by impaired nuclear translocation of p65 and consequently reduced binding of p65 to its binding site in the p105 promoter. Our data indicate that the vitamin D receptor reduces NF-κB activation by interference with NF-κB p65 and p105. Thus, the vitamin D receptor inhibits costimulatory signal transduction in na?ve B cells, namely by reducing CD40 signaling.     

The activation, by antigen, of naïve TCR transgenic CD4 T cells cultured at physiological, rather than artificially high, frequencies more accurately reflects the in vivo activation of normal numbers of naïve CD4(+) T cells

The majority of in vitro studies investigating the activation of na?ve TCR transgenic T cells routinely employ an artificially high frequency of such cells. To assess whether employing high frequencies of TCR transgenic cells in vitro accurately reflects the in vivo activation of a normal number of T cells, we cultured between 300 and 3×10(6) Rag2(-/-) DO11.10 T cells per well under otherwise identical conditions. We find that those T cells cultured at low frequencies proliferate more and are more potently activated, as assessed by the expression of CD44 and CD62L, each giving rise to a much larger number of cytokine producing cells, comparable to the number generated in vivo when a normal number of CD4(+) T cells are activated. The effect of T cell frequency on the level of their activation was not due to differences in MHCII or CD80/86 expression by B cells, the major APC population present, nor to increased death of B cells in high frequency cultures. Taken together, our observations illustrate the necessity of culturing na?ve TCR transgenic CD4(+) T cells at a physiological frequency if one is to more accurately recapitulate the in vivo activation of na?ve CD4(+) T cells.     

miRNA profiling of naïve, effector and memory CD8 T cells

microRNAs have recently emerged as master regulators of gene expression during development and cell differentiation. Although profound changes in gene expression also occur during antigen-induced T cell differentiation, the role of miRNAs in the process is not known. We compared the miRNA expression profiles between antigen-specific na?ve, effector and memory CD8+ T cells using 3 different methods--small RNA cloning, miRNA microarray analysis and real-time PCR. Although many miRNAs were expressed in all the T cell subsets, the frequency of 7 miRNAs (miR-16, miR-21, miR-142-3p, miR-142-5p, miR-150, miR-15b and let-7f) alone accounted for approximately 60% of all miRNAs, and their expression was several fold higher than the other expressed miRNAs. Global downregulation of miRNAs (including 6/7 dominantly expressed miRNAs) was observed in effector T cells compared to na?ve cells and the miRNA expression levels tended to come back up in memory T cells. However, a few miRNAs, notably miR-21 were higher in effector and memory T cells compared to na?ve T cells. These results suggest that concomitant with profound changes in gene expression, miRNA profile also changes dynamically during T cell differentiation. Sequence analysis of the cloned mature miRNAs revealed an extensive degree of end polymorphism. While 3'end polymorphisms dominated, heterogeneity at both ends, resembling drosha/dicer processing shift was also seen in miR-142, suggesting a possible novel mechanism to generate new miRNA and/or to diversify miRNA target selection. Overall, our results suggest that dynamic changes in the expression of miRNAs may be important for the regulation of gene expression during antigen-induced T cell differentiation. Our study also suggests possible novel mechanisms for miRNA biogenesis and function.     

Human papilloma virus–specific T cells can be generated from naïve T cells for use as an immunotherapeutic strategy for immunocompromised patients

Human papilloma virus (HPV) is a known cause of cervical cancer, squamous cell carcinoma and laryngeal cancer. Although treatments exist for HPV-associated malignancies, patients unresponsive to these therapies have a poor prognosis. Recent findings from vaccine studies suggest that T-cell immunity is essential for disease control. Because Epstein-Barr Virus (EBV)-specific T cells have been highly successful in treating or preventing EBV-associated tumors, we hypothesized that the development of a manufacturing platform for HPV-specific T cells from healthy donors could be used in a third-party setting to treat patients with high-risk/relapsed HPV-associated cancers. Most protocols for generating virus-specific T cells require prior exposure of the donor to the targeted virus and, because the seroprevalence of high-risk HPV types varies greatly by age and ethnicity, manufacturing of donor-derived HPV-specific T cells has proven challenging. We, therefore, made systematic changes to our current Good Manufacturing Practice (GMP)-compliant protocols to improve antigen presentation, priming and expansion for the manufacture of high-efficacy HPV-specific T cells. Like others, we found that current methodologies fail to expand HPV-specific T cells from most healthy donors. By optimizing dendritic cell maturation and function with lipopolysaccharide (LPS) and interferon (IFN)γ, adding interleukin (IL)-21 during priming and depleting memory T cells, we achieved reliable expansion of T cells specific for oncoproteins E6 and E7 to clinically relevant amounts (mean, 578-fold expansion; n?=?10), which were polyfunctional based on cytokine multiplex analysis. In the third-party setting, such HPV-specific T-cell products might serve as a potent salvage therapy for patients with HPV-associated diseases.     

Naïve blood monocytes suppress T-cell function. A possible mechanism for protection from autoimmunity

In certain disease context, cells of the monocyte/macrophage lineage are known to exhibit T-cell suppressor function. However, whether na?ve monocytes are also able to suppress T-cell responses has not been previously investigated. In this study, we have discovered that CD11b(+)Ly6G(-) mononuclear cells in the blood of na?ve mice are potent suppressors of T-cell proliferation in vitro. The suppression of T-cell proliferation requires cell-cell contact and is partially dependent on nitric oxide production. Following the induction of experimental autoimmune encephalomyelitis in mice, the suppressor function of this blood CD11b(+)Ly6G(-) cell population is impaired. Therefore, blood CD11b(+)Ly6G(-) cells appear to be intrinsically suppressive and may have a key role in maintaining immune homoeostasis. Loss of this suppressive function may contribute to development of autoimmunity.     

Porcine induced pluripotent stem cells analogous to naïve and primed embryonic stem cells of the mouse

Authentic or na?ve embryonic stem cells (ESC) have probably never been derived from the inner cell mass (ICM) of pig blastocysts, despite over 25 years of effort. Recently, several groups, including ours, have reported induced pluripotent stem cells (iPSC) from swine by reprogramming somatic cells with a combination of four factors, OCT4 (POU5F1)/SOX2/KLF4/c-MYC delivered by retroviral transduction. The porcine (p) iPSC resembled human (h) ESC and the mouse "Epiblast stem cells" (EpiSC) in their colony morphology and expression of pluripotent genes, and are likely dependent on FGF2/ACTIVIN/NODAL signaling, therefore representing a primed ESC state. These cells are likely to advance swine as a model in biomedical research, since grafts could potentially be matched to the animal that donated the cells for re-programming. The objective of the present work has been to develop na?ve piPSC. Employing a combination of seven reprogramming factors assembled on episomal vectors, we successfully reprogrammed porcine embryonic fibroblasts on a modified LIF-medium supplemented with two kinase inhibitors; CHIR99021, which inhibits GSK-3beta, and PD0325901, a MEK inhibitor. The derived piPSC bear a striking resemblance to na?ve mESC in colony morphology, are dependent on LIF to maintain an undifferentiated phenotype, and express markers consistent with pluripotency. They exhibit high telomerase activity, a short cell cycle interval, and a normal karyotype, and are able to generate teratomas. Currently, the competence of these lines for contributing to germ-line chimeras is being tested.     

Study of naïve and memory cells in a cohort of Egyptian chronic granulomatous disease patients

Abstract

Context: Chronic granulomatous disease (CGD) is a primary immunodeficiency disorder caused by inherited defects in the NADPH oxidase complex which may be involved in important pathways that connect innate and adaptive immunity. Objectives: Characterize the naive and memory compartment of B and T lymphocytes in patients with CGD. Methods: Twenty CGD patients and twenty healthy controls matched for age and sex were enrolled in this study. Flow cytometric assessment of the naïve and memory compartments of peripheral blood lymphocytes was done using cell surface markers CD45RO, CD45RA, CD27, CD3 and CD19. Results: There were 15 (79%) autosomal recessive CGD patients (8 females (53%) and 7 males (47%), 100% positive parental consanguinity) and four (21%) X-linked CGD patients. On comparing the 3 groups; AR CGD, X-linked CGD and controls, there was a positive statistical significant difference for the percentage and absolute count of CD19?+?CD27+ memory B cell (p?=?0.028 and p?=?0.047 respectively), CD45RA cells (with p values of p?=?0.000 and 0.033, respectively), the naïve compartment CD3?+?CD45RA+ cells percentage and absolute counts (p?=?0.005, 0.01respectively), CD3?+?CD27?+?cells percentage and absolute counts (p?=?0.001, 0.012 respectively), CD3?+?CD45RA?+?CD27+ cells percentage and absolute counts (p?=?0.015, 0.005, respectively). The significance was mainly attributed to the decrease in the X-linked group than control group. Conclusion: There was an altered naïve and memory B profile in CGD patients, this may increase susceptibility of the patients to opportunistic infections and autoimmune disorders. T-cell alterations have to be interpreted cautiously especially in the presence of infections.