Anastasia Nijnik

DNA repair is limiting for haematopoietic stem cells during ageing

Accumulation of DNA damage leading to adult stem cell exhaustion has been proposed to be a principal mechanism of ageing. Here we address this question by taking advantage of the highly specific role of DNA ligase IV in the repair of DNA double-strand breaks by non-homologous end-joining, and by the discovery of a unique mouse strain with a hypomorphic Lig4Y288C mutation. The Lig4Y288C mouse, identified by means of a mutagenesis screening programme, is a mouse model for human LIG4 syndrome, showing immunodeficiency and growth retardation. Diminished DNA double-strand break repair in the Lig4Y288C strain causes a progressive loss of haematopoietic stem cells and bone marrow cellularity during ageing, and severely impairs stem cell function in tissue culture and transplantation. The sensitivity of haematopoietic stem cells to non-homologous end-joining deficiency is therefore a key determinant of their ability to maintain themselves against physiological stress over time and to withstand culture and transplantation.

Modulating immunity as a therapy for bacterial infections

Despite our efforts to halt the increase and spread of antimicrobial resistance, bacteria continue to become less susceptible to antimicrobial drugs over time, and rates of discovery for new antibiotics are declining. Thus, it is essential to explore new paradigms for anti-infective therapy. One promising approach involves host-directed immunomodulatory therapies, whereby natural mechanisms in the host are exploited to enhance therapeutic benefit. The objective is to initiate or enhance protective antimicrobial immunity while limiting inflammation-induced tissue injury. A range of potential immune modulators have been proposed, including innate defence regulator peptides and agonists of innate immune components such as Toll-like receptors and NOD-like receptors.

The roles of cathelicidin LL-37 in immune defences and novel clinical applications.

Purpose of reviewLL-37 is the only member of the cathelicidin family of host defence peptides expressed in humans. It is primarily produced by phagocytic leucocytes and epithelial cells, and mediates a wide range of biological responses: direct killing of microorganisms, chemotaxis and chemokine induction, regulation of inflammatory responses, as well as adjuvant, angiogenic and wound healing effects. In this review we will cover the recent advances in the understanding of LL-37 biology: its activities, the mechanisms of its induction and roles in immune defence. Recent findingsRecent studies advanced our understanding of the mechanisms controlling LL-37 expression, demonstrating the key involvement of the vitamin D3 and the hypoxia response pathways, and the impacts of commensal and pathogenic microorganisms on its production. The synergistic and antagonistic interactions between LL-37 and other immune mediators have been further elucidated. Furthermore, studies in animal models and human patients further characterized the roles of cathelicidins in immunity, with roles in infectious and inflammatory conditions. The underlying properties of LL-37 have been exploited to create innate defence regulator peptides that represent a novel immunomodulatory approach to treating infections. SummaryThe understanding of the biological properties and functions of LL-37 and other host defence peptides advances our knowledge of innate immunity, the interactions of the host with pathogens and the microflora, as well as the pathology of infectious and inflammatory diseases, creating many strategies and opportunities for therapeutic intervention.

Synthetic Cationic Peptide IDR-1002 Provides Protection against Bacterial Infections through Chemokine Induction and Enhanced Leukocyte Recruitment

With the rapid rise in the incidence of multidrug resistant infections, there is substantial interest in host defense peptides as templates for production of new antimicrobial therapeutics. Natural peptides are multifunctional mediators of the innate immune response, with some direct antimicrobial activity and diverse immunomodulatory properties. We have previously developed an innate defense regulator (IDR) 1, with protective activity against bacterial infection mediated entirely through its effects on the immunity of the host, as a novel approach to anti-infective therapy. In this study, an immunomodulatory peptide IDR-1002 was selected from a library of bactenecin derivatives based on its substantially more potent ability to induce chemokines in human PBMCs. The enhanced chemokine induction activity of the peptide in vitro correlated with stronger protective activity in vivo in the Staphylococcus aureus-invasive infection model, with a >5-fold reduction in the protective dose in direct comparison with IDR-1. IDR-1002 also afforded protection against the Gram-negative bacterial pathogen Escherichia coli. Chemokine induction by IDR-1002 was found to be mediated through a Gi-coupled receptor and the PI3K, NF-κB, and MAPK signaling pathways. The protective activity of the peptide was associated with in vivo augmentation of chemokine production and recruitment of neutrophils and monocytes to the site of infection. These results highlight the importance of the chemokine induction activity of host defense peptides and demonstrate that the optimization of the ex vivo chemokine-induction properties of peptides is a promising method for the rational development of immunomodulatory IDR peptides with enhanced anti-infective activity.

Potential of immunomodulatory host defense peptides as novel anti-infectives

A fundamentally new strategy for the treatment of infectious disease is the modulation of host immune responses to enhance clearance of infectious agents and reduce tissue damage due to inflammation. Antimicrobial host defense peptides have been investigated for their potential as a new class of antimicrobial drugs. Recently their immunomodulatory activities have begun to be appreciated. Modulation of innate immunity by synthetic variants of host defense peptides, called innate defense regulators (IDRs), is protective without direct antimicrobial action. We discuss the potential and current limitations in exploiting the immunomodulatory activity of IDRs as a novel anti-infective pathway. IDRs show significant promise and current research is uncovering mechanistic information that will aid in the future development of IDRs for clinical use.

Intracellular Receptor for Human Host Defense Peptide LL-37 in Monocytes

The human cationic host defense peptide LL-37 has a broad range of immunomodulatory, anti-infective functions. A synthetic innate defense regulator peptide, innate defense regulator 1 (IDR-1), based conceptually on LL-37, was recently shown to selectively modulate innate immunity to protect against a wide range of bacterial infections. Using advanced proteomic techniques, ELISA, and Western blotting procedures, GAPDH was identified as a direct binding partner for LL-37 in monocytes. Enzyme kinetics and mobility shift studies also indicated LL-37 and IDR-1 binding to GAPDH. The functional relevance of GAPDH in peptide-induced responses was demonstrated by using gene silencing of GAPDH with small interfering RNA (siRNA). Previous studies have established that the induction of chemokines and the anti-inflammatory cytokine IL-10 are critical immunomodulatory functions in the anti-infective properties of LL-37 and IDR-1, and these functions are modulated by the MAPK p38 pathway. Consistent with that, this study demonstrated the importance of the GAPDH interactions with these peptides since gene silencing of GAPDH resulted in impaired p38 MAPK signaling, downstream chemokine and cytokine transcriptional responses induced by LL-37 and IDR-1, and LL-37-induced cytokine production. Bioinformatic analysis, using InnateDB, of the major interacting partners of GAPDH indicated the likelihood that this protein can impact on innate immune pathways including p38 MAPK. Thus, this study has demonstrated a novel function for GAPDH as a mononuclear cell receptor for human cathelicidin LL-37 and immunomodulatory IDR-1 and conclusively demonstrated its relevance in the functioning of cationic host defense peptides.

Human Cathelicidin Peptide LL-37 Modulates the Effects of IFN-γ on APCs

The human cathelicidin peptide LL-37 is a multifunctional immunomodulatory and antimicrobial host defense peptide of the human immune system. LL-37 modulates host cell responses to microbial stimuli and also affects the action of other endogenous immune mediators such as IL-1β and GM-CSF. This activity of LL-37 is known to be complex, with the functional outcomes being dependent on the cell type and activation status, timing of exposure, and other immune mediators present. It was demonstrated in this study that LL-37 inhibited cellular responses to IFN-γ, the key cytokine of Th1-polarized immunity. The inhibitory activity of LL-37 on IFN-γ responses was characterized in monocytes, macrophages, dendritic cells, and B lymphocytes, showing suppression of cell activation, proliferation, and production of proinflammatory and Th1-polarizing cytokines, and Abs. It was further shown that in monocytes the suppressive effects of LL-37 were mediated through inhibition of STAT1-independent signaling events, involving both the p65 subunit of NF-κB and p38 MAPK. This study suggests that LL-37 modulates IFN-γ responses during both the innate and adaptive phases of immune responses, indicating a new immunomodulatory role for this endogenous peptide. These effects on IFN-γ activity should be taken into consideration in the development of cathelicidin-based peptides for therapeutic applications as immunomodulatory or microbicidal agents.

A novel vaccine adjuvant comprised of a synthetic innate defence regulator peptide and CpG oligonucleotide links innate and adaptive immunity.

There has been an increased demand for the development of novel vaccine adjuvants that lead to enhanced induction of protection from infectious challenges and development of immunological memory. A novel vaccine adjuvant was developed comprising a complex containing CpG oligonucleotide and the synthetic cationic innate defence regulator peptide HH2 that has enhanced immune modulating activities. The complex of HH2 and the CpG oligonucleotide 10101 was a potent inducer of cytokine/chemokine expression ex vivo, retained activity following extended storage, had low associated cytotoxicity, and upregulated surface marker expression in dendritic cells, a critical activity for a vaccine adjuvant. Immunization of mice with a coformulation of the HH2-CpG complex and pertussis toxoid significantly enhanced the induction of toxoid-specific antibody titres when compared to toxoid alone, inducing high titres of IgG1 and IgG2a, typical of a balanced Th1/Th2 response, and also led to high IgA titres. This study demonstrates the potential application of the HH2-CpG complex as a vaccine adjuvant.

The Role of Sphingosine-1-Phosphate Transporter Spns2 in Immune System Function

Sphingosine-1-phosphate (S1P) is lipid messenger involved in the regulation of embryonic development, immune system functions, and many other physiological processes. However, the mechanisms of S1P transport across cellular membranes remain poorly understood, with several ATP-binding cassette family members and the spinster 2 (Spns2) member of the major facilitator superfamily known to mediate S1P transport in cell culture. Spns2 was also shown to control S1P activities in zebrafish in vivo and to play a critical role in zebrafish cardiovascular development. However, the in vivo roles of Spns2 in mammals and its involvement in the different S1P-dependent physiological processes have not been investigated. In this study, we characterized Spns2-null mouse line carrying the Spns2tm1a(KOMP)Wtsi allele (Spns2tm1a). The Spns2tm1a/tm1a animals were viable, indicating a divergence in Spns2 function from its zebrafish ortholog. However, the immunological phenotype of the Spns2tm1a/tm1a mice closely mimicked the phenotypes of partial S1P deficiency and impaired S1P-dependent lymphocyte trafficking, with a depletion of lymphocytes in circulation, an increase in mature single-positive T cells in the thymus, and a selective reduction in mature B cells in the spleen and bone marrow. Spns2 activity in the nonhematopoietic cells was critical for normal lymphocyte development and localization. Overall, Spns2tm1a/tm1a resulted in impaired humoral immune responses to immunization. This study thus demonstrated a physiological role for Spns2 in mammalian immune system functions but not in cardiovascular development. Other components of the S1P signaling network are investigated as drug targets for immunosuppressive therapy, but the selective action of Spns2 may present an advantage in this regard.

The critical role of histone H2A-deubiquitinase Mysm1 in hematopoiesis and lymphocyte differentiation

Stem cell differentiation and lineage specification depend on coordinated programs of gene expression, but our knowledge of the chromatin-modifying factors regulating these events remains incomplete. Ubiquitination of histone H2A (H2A-K119u) is a common chromatin modification associated with gene silencing, and controlled by the ubiquitin-ligase polycomb repressor complex 1 (PRC1) and H2A-deubiquitinating enzymes (H2A-DUBs). The roles of H2A-DUBs in mammalian development, stem cells, and hematopoiesis have not been addressed. Here we characterized an H2A-DUB targeted mouse line Mysm1(tm1a/tm1a) and demonstrated defects in BM hematopoiesis, resulting in lymphopenia, anemia, and thrombocytosis. Development of lymphocytes was impaired from the earliest stages of their differentiation, and there was also a depletion of erythroid cells and a defect in erythroid progenitor function. These phenotypes resulted from a cell-intrinsic requirement for Mysm1 in the BM. Importantly, Mysm1(tm1a/tm1a) HSCs were functionally impaired, and this was associated with elevated levels of reactive oxygen species, γH2AX DNA damage marker, and p53 protein in the hematopoietic progenitors. Overall, these data establish a role for Mysm1 in the maintenance of BM stem cell function, in the control of oxidative stress and genetic stability in hematopoietic progenitors, and in the development of lymphoid and erythroid lineages.