IL-21 signaling is essential for optimal host resistance against Mycobacterium tuberculosis infection

IL-21 is produced predominantly by activated CD4(+) T cells and has pleiotropic effects on immunity via the IL-21 receptor (IL-21R), a member of the common gamma chain (gamma(c)) cytokine receptor family. We show that IL-21 signaling plays a crucial role in T cell responses during Mycobacterium tuberculosis infection by augmenting CD8(+) T cell priming, promoting T cell accumulation in the lungs, and enhancing T cell cytokine production. In the absence of IL-21 signaling, more CD4(+) and CD8(+) T cells in chronically infected mice express the T cell inhibitory molecules PD-1 and TIM-3. We correlate these immune alterations with increased susceptibility of IL-21R(-/-) mice, which have increased lung bacterial burden and earlier mortality compared to WT mice. Finally, to causally link the immune defects with host susceptibility, we use an adoptive transfer model to show that IL-21R(-/-) T cells transfer less protection than WT T cells. These results prove that IL-21 signaling has an intrinsic role in promoting the protective capacity of T cells. Thus, the net effect of IL-21 signaling is to enhance host resistance to M. tuberculosis. These data position IL-21 as a candidate biomarker of resistance to tuberculosis.This work was supported by National Institutes of Health Grants R21 AI100766, R01 AI106725, and P01 AI073748

Plant virus particles carrying tumour antigen activate TLR7 and induce high levels of protective antibody

Induction of potent antibody is the goal of many vaccines targeted against infections or cancer. Modern vaccine designs that use virus-like particles (VLP) have shown efficacy for prophylactic vaccination against virus-associated cancer in the clinic. Here we used plant viral particles (PVP), which are structurally analogous to VLP, coupled to a weak idiotypic (Id) tumour antigen, as a conjugate vaccine to induce antibody against a murine B-cell malignancy. The Id-PVP vaccine incorporates a natural adjuvant, the viral ssRNA, which acts via TLR7. It induced potent protective anti-Id antibody responses in an in vivo mouse model, superior to the "gold standard" Id vaccine, with prevalence of the IgG2a isotype. Combination with alum further increased antibody levels and maintained the IgG2a bias. Engagement of TLR7 in vivo was followed by secretion of IFN-? by plasmacytoid dendritic cells and by activation of splenic CD11chi conventional dendritic cells. The latter was apparent from up-regulation of co-stimulatory molecules and from secretion of a wide range of inflammatory cytokines and chemokines including the Th1-governing cytokine IL-12, in keeping with the IgG2a antibody isotype distribution. PVP conjugates are a novel cancer vaccine design, offering an attractive molecular form, similar to VLP, and providing T-cell help. In contrast to VLP, they also incorporate a safe "in-built" ssRNA adjuvant

Animal welfare in studies on murine tuberculosis : assessing progress over a 12-year period and the need for further improvement

There is growing concern over the welfare of animals used in research, in particular when these animals develop pathology. The present study aims to identify the main sources of animal distress and to assess the possible implementation of refinement measures in experimental infection research, using mouse models of tuberculosis (TB) as a case study. This choice is based on the historical relevance of mouse studies in understanding the disease and the present and long-standing impact of TB on a global scale. Literature published between 1997 and 2009 was analysed, focusing on the welfare impact on the animals used and the implementation of refinement measures to reduce this impact. In this 12-year period, we observed a rise in reports of ethical approval of experiments. The proportion of studies classified into the most severe category did however not change significantly over the studied period. Information on important research parameters, such as method for euthanasia or sex of the animals, were absent in a substantial number of papers. Overall, this study shows that progress has been made in the application of humane endpoints in TB research, but that a considerable potential for improvement remains.Nuno H. Franco is funded by Fundação para a Ciência e Tecnologia (SFRH/BD/38337/2007). This work is funded by FEDER funds through the Operational Competitiveness Programme - COMPETE and by national funds through FCT - Fundação para a Ciência e Tecnologia under the project FCOMP-01-0124-FEDER-022718 (PEst-C/SAU/LA0002/2011

Human and murine clonal CD8+ T cell expansions arise during tuberculosis because of TCR selection

The immune system can recognize virtually any antigen, yet T cell responses against several pathogens, including Mycobacterium tuberculosis, are restricted to a limited number of immunodominant epitopes. The host factors that affect immunodominance are incompletely understood. Whether immunodominant epitopes elicit protective CD8+ T cell responses or instead act as decoys to subvert immunity and allow pathogens to establish chronic infection is unknown. Here we show that anatomically distinct human granulomas contain clonally expanded CD8+ T cells with overlapping T cell receptor (TCR) repertoires. Similarly, the murine CD8+ T cell response against M. tuberculosis is dominated by TB10.44-11-specific T cells with extreme TCRß bias. Using a retro genic model of TB10.44-11-specific CD8+ Tcells, we show that TCR dominance can arise because of competition between clonotypes driven by differences in affinity. Finally, we demonstrate that TB10.4-specific CD8+ T cells mediate protection against tuberculosis, which requires interferon-? production and TAP1-dependent antigen presentation in vivo. Our study of how immunodominance, biased TCR repertoires, and protection are inter-related, provides a new way to measure the quality of T cell immunity, which if applied to vaccine evaluation, could enhance our understanding of how to elicit protective T cell immunity.This work was supported by the Portuguese Foundation for Science and Technology individual fellowship (CNA) www.fct.pt, a National Institutes of Health Grant R01 AI106725 (SMB) www.nih.gov, and a Center for AIDS Research Grant P30 AI 060354 (SMB) www.nih.gov. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Early severe inflammatory responses to uropathogenic E. coli predispose to chronic and recurrent urinary tract infection

Chronic infections are an increasing problem due to the aging population and the increase in antibiotic resistant organisms. Therefore, understanding the host-pathogen interactions that result in chronic infection is of great importance. Here, we investigate the molecular basis of chronic bacterial cystitis. We establish that introduction of uropathogenic E. coli (UPEC) into the bladders of C3H mice results in two distinct disease outcomes: resolution of acute infection or development of chronic cystitis lasting months. The incidence of chronic cystitis is both host strain and infectious dose-dependent. Further, development of chronic cystitis is preceded by biomarkers of local and systemic acute inflammation at 24 hours post-infection, including severe pyuria and bladder inflammation with mucosal injury, and a distinct serum cytokine signature consisting of elevated IL-5, IL-6, G-CSF, and the IL-8 analog KC. Mice deficient in TLR4 signaling or lymphocytes lack these innate responses and are resistant, to varying degrees, to developing chronic cystitis. Treatment of C3H mice with the glucocorticoid anti-inflammatory drug dexamethasone prior to UPEC infection also suppresses the development of chronic cystitis. Finally, individuals with a history of chronic cystitis, lasting at least 14 days, are significantly more susceptible to redeveloping severe, chronic cystitis upon bacterial challenge. Thus, we have discovered that the development of chronic cystitis in C3H mice by UPEC is facilitated by severe acute inflammatory responses early in infection, which subsequently are predisposing to recurrent cystitis, an insidious problem in women. Overall, these results have significant implications for our understanding of how early host-pathogen interactions at the mucosal surface determines the fate of disease

Exosomes Derived from M. Bovis BCG Infected Macrophages Activate Antigen-Specific CD4+ and CD8+ T Cells In Vitro and In Vivo

Activation of both CD4+ and CD8+ T cells is required for an effective immune response to an M. tuberculosis infection. However, infected macrophages are poor antigen presenting cells and may be spatially separated from recruited T cells, thus limiting antigen presentation within a granuloma. Our previous studies showed that infected macrophages release from cells small membrane-bound vesicles called exosomes which contain mycobacterial lipid components and showed that these exosomes could stimulate a pro-inflammatory response in naïve macrophages. In the present study we demonstrate that exosomes stimulate both CD4+ and CD8+ splenic T cells isolated from mycobacteria-sensitized mice. Although the exosomes contain MHC I and II as well as costimulatory molecules, maximum stimulation of T cells required prior incubation of exosomes with antigen presenting cells. Exosomes isolated from M. bovis and M. tuberculosis infected macrophages also stimulated activation and maturation of mouse bone marrow-derived dendritic cells. Interestingly, intranasal administration of mice with exosomes isolated from M. bovis BCG infected macrophages induce the generation of memory CD4+ and CD8+ T cells. The isolated T cells also produced IFN-γ upon restimulation with BCG antigens. The release of exosomes from infected macrophages may overcome some of the defects in antigen presentation associated with mycobacterial infections and we suggest that exosomes may be a promising M. tuberculosis vaccine candidate

How “Humane” Is Your Endpoint?—Refining the Science-Driven Approach for Termination of Animal Studies of Chronic Infection

Public concern on issues such as animal welfare or the scientific validity and clinical value of animal research is growing, resulting in increasing regulatory demands for animal research. Abiding to the most stringent animal welfare standards, while having scientific objectives as the main priority, is often challenging. To do so, endpoints of studies involving severe, progressive diseases need to be established considering how early in the disease process the scientific objectives can be achieved. We present here experimental studies of tuberculosis (TB) in mice as a case study for an analysis of present practice and a discussion of how more refined science-based endpoints can be developed. A considerable proportion of studies in this field involve lethal stages, and the establishment of earlier, reliable indicators of disease severity will have a significant impact on animal welfare. While there is an increasing interest from scientists and industry in moving research in this direction, this is still far from being reflected in actual practice. We argue that a major limiting factor is the absence of data on biomarkers that can be used as indicators of disease severity. We discuss the possibility of complementing the widely used weight loss with other relevant biomarkers and the need for validation of these parameters as endpoints. Promotion of ethical guidelines needs to be coupled with systematic research in order to develop humane endpoints beyond the present euthanasia of moribund animals. Such research, as we propose here for chronic infection, can show the way for the development and promotion of welfare policies in other fields of research. Research on chronic infection relies heavily on the use of animals, as only the integral animal body can model the full aspect of an infection. That animals are generally made to develop a disease in infection studies exacerbates the tension between human benefit and animal well-being, which characterizes all biomedical research with animals. Scientists typically justify animal research with reference to potential human benefits, but if accepting the assumption that human benefits can offset animal suffering, it still needs to be argued that the same benefits could not be achieved with less negative effects on animal welfare. Reducing the animal welfare problems associated with research (“refinement” [1]) is therefore crucial in order to render animal-based research less of an ethical problem and to assure public trust in research. Studies that are designed to measure time of death or survival percentages present a particularly challenging situation in which at least some of the animals are made to die from the disease. These studies are frequent in experimental research on severe infections. The scientific community, industry, and regulatory authorities have responded to the ethical concerns over studies in which animals die from severe disease by developing new policies and guidelines for the implementation of humane endpoints as a key refinement measure (e.g., [2]–[4]). The most widely used definition considers a humane endpoint to be the earliest indicator in an animal experiment of severe pain, severe distress, suffering, or impending death [5], underlining that ideally such indicators should be identified before the onset of the most severe effects. Euthanizing animals, rather than awaiting their “spontaneous” death, is important to avoid unnecessary suffering in studies in which data on survival is thought to be required for scientific or legal reasons. However, several questions remain open regarding how humane endpoints are to be applied to address real animal welfare problems. We used TB experiments in mice as a case study to highlight the potential to establish biomarkers of disease progress that can replace survival time as a measure of disease severity.Fundação para a Ciência e Tecnologia (SFRH/BD/38337/2007)

The IL-1-Like Cytokine IL-33 Is Constitutively Expressed in the Nucleus of Endothelial Cells and Epithelial Cells In Vivo: A Novel ‘Alarmin’?

BACKGROUND: Interleukin-33 (IL-33) is an IL-1-like cytokine ligand for the IL-1 receptor-related protein ST2, that activates mast cells and Th2 lymphocytes, and induces production of Th2-associated cytokines in vivo. We initially discovered IL-33 as a nuclear factor (NF-HEV) abundantly expressed in high endothelial venules from lymphoid organs, that associates with chromatin and exhibits transcriptional regulatory properties. This suggested that, similarly to IL-1alpha and chromatin-associated cytokine HMGB1, IL-33 may act as both a cytokine and a nuclear factor. Although the activity of recombinant IL-33 has been well characterized, little is known yet about the expression pattern of endogenous IL-33 in vivo. METHODOLOGY/PRINCIPAL FINDINGS: Here, we show that IL-33 is constitutively and abundantly expressed in normal human tissues. Using a combination of human tissue microarrays and IL-33 monoclonal and polyclonal antibodies, we found that IL-33 is a novel nuclear marker of the endothelium widely expressed along the vascular tree. We observed abundant nuclear expression of IL-33 in endothelial cells from both large and small blood vessels in most normal human tissues, as well as in human tumors. In addition to endothelium, we also found constitutive nuclear expression of IL-33 in fibroblastic reticular cells of lymphoid tissues, and epithelial cells of tissues exposed to the environment, including skin keratinocytes and epithelial cells of the stomach, tonsillar crypts and salivary glands. CONCLUSIONS/SIGNIFICANCE: Together, our results indicate that, unlike inducible cytokines, IL-33 is constitutively expressed in normal human tissues. In addition, they reveal that endothelial cells and epithelial cells constitute major sources of IL-33 in vivo. Based on these findings, we speculate that IL-33 may function, similarly to the prototype 'alarmin' HMGB1, as an endogenous 'danger' signal to alert the immune system after endothelial or epithelial cell damage during trauma or infection

IL-33 Is Produced by Mast Cells and Regulates IgE-Dependent Inflammation

Background: IL-33 is a recently characterized IL-1 family cytokine and found to be expressed in inflammatory diseases, including severe asthma and inflammatory bowl disease. Recombinant IL-33 has been shown to enhance Th2-associated immune responses and potently increase mast cell proliferation and cytokine production. While IL-33 is constitutively expressed in endothelial and epithelial cells, where it may function as a transcriptional regulator, cellular sources of IL-33 and its role in inflammation remain unclear. Methodology/Principal Findings: Here, we identify mast cells as IL-33 producing cells. IgE/antigen activation of bone marrow-derived mast cells or a murine mast cell line (MC/9) significantly enhanced IL-33. Conversely, recombinant IL-33 directly activated mast cells to produce several cytokines including IL-4, IL-5 and IL-6 but not IL-33. We show that expression of IL-33 in response to IgE-activation required calcium and that ionomycin was sufficient to induce IL-33. In vivo, peritoneal mast cells expressed IL-33 and IL-33 levels were significantly lower within the skin of mast cell deficient mice, compared to littermate controls. Local activation of mast cells promotes edema, followed by the recruitment of inflammatory cells. We demonstrate using passive cutaneous anaphylaxis, a mast cell-dependent model, that deficiency in ST2 or antibody blockage of ST2 or IL-33 ablated the late phase inflammatory response but that the immediate phase response was unaffected. IL-33 levels in the skin were significantly elevated only during the late phase

DING Proteins from Phylogenetically Different Species Share High Degrees of Sequence and Structure Homology and Block Transcription of HIV-1 LTR Promoter

Independent research groups reported that DING protein homologues isolated from bacterial, plant and human cells demonstrate the anti-HIV-1 activity. This might indicate that diverse organisms utilize a DING-mediated broad-range protective innate immunity response to pathogen invasion, and that this mechanism is effective also against HIV-1. We performed structural analyses and evaluated the anti-HIV-1 activity for four DING protein homologues isolated from different species. Our data show that bacterial PfluDING, plant p38SJ (pDING), human phosphate binding protein (HPBP) and human extracellular DING from CD4 T cells (X-DING-CD4) share high degrees of structure and sequence homology. According to earlier reports on the anti-HIV-1 activity of pDING and X-DING-CD4, other members of this protein family from bacteria and humans were able to block transcription of HIV-1 and replication of virus in cell based assays. The efficacy studies for DING-mediated HIV-1 LTR and HIV-1 replication blocking activity showed that the LTR transcription inhibitory concentration 50 (IC50) values ranged from 0.052–0.449 ng/ml; and the HIV-1 replication IC50 values ranged from 0.075–0.311 ng/ml. Treatment of cells with DING protein alters the interaction between p65-NF-κB and HIV-1 LTR. Our data suggest that DING proteins may be part of an innate immunity defense against pathogen invasion; the conserved structure and activity makes them appealing candidates for development of a novel therapeutics targeting HIV-1 transcription