Neeloffer Mookherjee

Cationic Host Defence Peptides: Multifaceted Role in Immune Modulation and Inflammation

Host defence peptides (HDPs) are innate immune effector molecules found in diverse species. HDPs exhibit a wide range of functions ranging from direct antimicrobial properties to immunomodulatory effects. Research in the last decade has demonstrated that HDPs are critical effectors of both innate and adaptive immunity. Various studies have hypothesized that the antimicrobial property of certain HDPs may be largely due to their immunomodulatory functions. Mechanistic studies revealed that the role of HDPs in immunity is very complex and involves various receptors, signalling pathways and transcription factors. This review will focus on the multiple functions of HDPs in immunity and inflammation, with special reference to cathelicidins, e.g. LL-37, certain defensins and novel synthetic innate defence regulator peptides. We also discuss emerging concepts of specific HDPs in immune-mediated inflammatory diseases, including the potential use of cationic peptides as therapeutics for immune-mediated inflammatory disorders.

Inflammatory Cytokines IL-32 and IL-17 Have Common Signaling Intermediates despite Differential Dependence on TNF-Receptor 1

Cytokines IL-32 and IL-17 are emerging as critical players in the pathophysiology of immune-mediated chronic inflammatory diseases. It has been speculated that the molecular mechanisms governing IL-32– and IL-17–mediated cellular responses are differentially dependent on the TNF pathway. In this study, kinome analysis demonstrated that following stimulation with cytokine IL-32, but not IL-17, there was increased phosphorylation of a peptide target corresponding to TNF-R1. Consistent with this observation, blocking TNF-R1 resulted in a suppression of IL-32–induced downstream responses, indicating that IL-32–mediated activity may be dependent on TNF-R1. In contrast, blocking TNF-R1 did not affect IL-17–induced downstream responses. Kinome analysis also implicated p300 (transcriptional coactivator) and death-associated protein kinase-1 (DAPK-1) as signaling intermediates for both IL-32 and IL-17. Phosphorylation of p300 and DAPK-1 upon stimulation with either IL-32 or IL-17 was confirmed by immunoblots. The presence of common targets was supported by results demonstrating similar downstream responses induced in the presence of IL-32 and IL-17, such as transcriptional responses and the direct activation of NF-κB. Furthermore, knockdown of p300 and DAPK-1 altered downstream responses induced by IL-32 and IL-17, and impacted certain cellular responses induced by TNF-α and IL-1β. We hypothesize that p300 and DAPK-1 represent nodes where the inflammatory networks of IL-32 and IL-17 overlap, and that these proteins would affect both TNF-R1–dependent and –independent pathways. Therefore, p300 and DAPK-1 are viable potential therapeutic targets for chronic inflammatory diseases.

Modulation of interleukin-1β-induced inflammatory responses by a synthetic cationic innate defence regulator peptide, IDR-1002, in synovial fibroblasts

IntroductionInnate defence regulator (IDR) peptides are synthetic cationic peptides, variants of naturally occurring innate immune effector molecules known as host defence peptides. IDR peptides were recently demonstrated to limit infection-associated inflammation selectively without compromising host innate immune functions. This study examined the impact of a 12-amino acid IDR peptide, IDR-1002, in pro-inflammatory cytokine interleukin (IL)-1β-induced responses in synovial fibroblasts, a critical cell type in the pathogenesis of inflammatory arthritis.MethodsHuman fibroblast-like synoviocytes (FLS) were stimulated with IL-1β in the presence and absence of IDR-1002. Production of enzyme matrix metalloproteinase-3 (MMP-3) and IL-1-receptor antagonist (IL-1RA) was monitored by enzyme-linked immunosorbent assay (ELISA), and various chemokines were evaluated by using multiplex cytometric bead array. Transcriptional responses were analyzed by quantitative real-time PCR. The impact on IL-1β-induced proteome was investigated by quantitative proteomics by using isobaric tags. IL-1β-induced pathways altered by IDR-1002 implicated by the proteomics analyses were further investigated by using various immunochemical assays. Cellular uptake of the peptide was monitored by using a biotinylated IDR-1002 peptide followed by microscopy probing with streptavidin-Alexa Fluor.ResultsThis study demonstrated that IDR-1002 suppressed the production of IL-1β-induced MMP-3 and monocyte chemotactic protein-1 (MCP-1); in contrast, IDR-1002 enhanced the production of IL-1RA, without neutralizing all chemokine responses. IDR-1002 altered the IL-1β-induced proteome primarily by altering the expression of members of nuclear factor kappa-B (NF-κB) and c-Jun N-terminal kinase (JNK) pathways. The proteomics data also suggested that IDR-1002 was altering the transcription factor HNF-4α-mediated responses, known to be critical in metabolic regulation. With various immunochemical assays, it was further demonstrated that IL-1β-induced NF-κB, JNK, and p38 mitogen-activated protein kinase (MAPK) activations were significantly suppressed by IDR-1002.ConclusionsThis study demonstrates the ability of an innate immune-modulatory IDR-peptide to influence the IL-1β-induced regulatory pathways and selectively to suppress inflammatory responses in synovial fibroblasts. The results of this study provide a rationale for examining the use of IDR-peptides as potential therapeutic candidates for chronic inflammatory diseases such as inflammatory arthritis.

Multiple immune-modulatory functions of cathelicidin host defense peptides.

An essential function of innate immunity is initiating inflammatory responses to limit the spread of invading pathogens, followed by regulatory mechanisms aimed at resolving inflammation and returning the immune system to homeostasis. These meticulously regulated processes are triggered by various pathogen-associated molecular patterns or endogenous damage-associated molecular patterns. A group of natural peptides that have gained notoriety in recent years as immune-modulatory molecules contributing to both resolution of infections and inflammation, thus playing a role in maintaining homeostasis are host defense peptides (HDPs). These are gene-encoded cationic short peptides, less than 50 amino acids with an overall positive charge of +2 to +7, and ≥30% of hydrophobic residues. The two most well characterized families of HDPs in mammals are cathelicidins and defensins. We will focus on cathelicidin HDPs in this review. Genes encoding cathelicidin HDPs have a highly conserved N-terminal cathelin domain in their precursor protein (Tomasinsig and Zanetti, 2005). This conserved cathelin domain has helped in the discovery of new cathelicidins from diverse species including non-mammalian species such as chicken, trout, and hagfish. The biological active mature mammalian cathelicidin peptides are processed from their precursor protein by proteolytic cleavage by serine proteases, e.g., proteinase 3, elastase, and kallikrein (Shinnar et al., 2003; Morizane et al., 2010). The biologically active mature cathelicidins are diverse in their sequence and structure, and can be classified into four structural groups; amphipathic α-helices (e.g., murine CRAMP), β-hairpin molecules (e.g., porcine protegrin 1), those with extended structures (e.g., bovine indolicidin), and cyclic peptides (e.g., bactenecin). Mammalian cathelicidins are found in granules of neutrophils as well as other cell types such as myeloid precursors, epithelial cells, mast cells, lymphocytes, and keratinocytes. These peptides are expressed in a wide variety of tissues (oral cavity, skin, intestine, lungs, cervix, etc.) and found in body fluids such as plasma, breast milk, saliva, gastric juice, semen, sweat, and bronchoalveolar fluid (Nijnik and Hancock, 2009). The biologically active mature cathelicidin peptides have been demonstrated to mediate a wide range of activity from antimicrobial to immune-modulatory (discussed below). Some of the most well studied cathelicidins are human LL-37, murine CRAMP, bovine BMAP-28, and porcine PR-39.

Amphiphilic Tobramycins with Immunomodulatory Properties

Amphiphilic aminoglycosides (AAGs) are an emerging source of antibacterials to combat infections caused by antibiotic-resistant bacteria. Mode-of-action studies indicate that AAGs predominately target bacterial membranes, thereby leading to depolarization and increased permeability. To assess whether AAGs also induce host-directed immunomodulatory responses, we determined the AAG-dependent induction of cytokines in macrophages in the absence or presence of lipopolysaccharide (LPS). Our results show for the first time that AAGs can boost the innate immune response, specifically the recruitment of immune cells such as neutrophils required for the resolution of infections. Moreover, AAGs can selectively control inflammatory responses induced in the presence of endotoxins to prevent septic shock. In conclusion, our study demonstrates that AAGs possess multifunctional properties that combine direct antibacterial activity with host-directed clearance effects reminiscent of those of host-defense peptides.

Vitamin D in a Northern Canadian First Nation Population: Dietary Intake, Serum Concentrations and Functional Gene Polymorphisms

The wide spectrum of vitamin D activity has focused attention on its potential role in the elevated burden of disease in a northern Canadian First Nations (Dené) cohort. Vitamin D insufficiency, and gene polymorphisms in the vitamin D receptor (VDR) and vitamin D binding protein (VDBP) have been implicated in susceptibility to infectious and chronic diseases. The objectives of this study were to determine the contribution of vitamin D from food, and measure the serum concentrations of 25-hydroxyvitamin D3 (25-OHD3) and VDBP in Dené participants. Single nucleotide polymorphisms (SNPs) associated with the dysregulation of the innate immune response were typed and counted. Potential correlations between the SNPs and serum concentrations of 25-OHD3 and VDBP were evaluated. Venous blood was collected in summer and winter over a one-year period and analyzed for 25-OHD3 and VDBP concentrations (N = 46). A questionnaire was administered to determine the amount of dietary vitamin D consumed. Sixty-one percent and 30% of the participants had 25-OHD3 serum concentrations <75 nmol/L in the winter and summer respectively. Mean vitamin D binding protein concentrations were within the normal range in the winter but below normal in the summer. VDBP and VDR gene polymorphisms affect the bioavailability and regulation of 25-OHD3. The Dené had a high frequency of the VDBP D432E-G allele (71%) and the Gc1 genotype (90%), associated with high concentrations of VDBP and a high binding affinity to 25-OHD3. The Dené had a high frequency of VDR Fok1-f allele (82%), which has been associated with a down-regulated Th1 immune response. VDBP and VDR polymorphisms, and low winter 25-OHD3 serum concentrations may be risk factors for infectious diseases and chronic conditions related to the dysregulation of the vitamin D pathway.

Effect of vitamin D supplementation on Mycobacterium tuberculosis-induced innate immune responses in a Canadian Dené First Nations cohort.

Canadian First Nations (FN) population experiences a high burden of tuberculosis. Vitamin D is known to enhance the expression of innate immune effectors, including cathelicidin LL-37, for protection against infections. In this study we performed longitudinal analyses to investigate the impact of vitamin D supplementation on macrophage responses to Mycobacterium tuberculosis (Mtb) lipoprotein (TLR2/1L), in Canadian Dené FN participants compared to Caucasian participants. Serum 25(OH)D and LL-37 levels were evaluated by ELISA. Transcriptional responses and protein expression of TLR2/1L-induced LL-37 and other innate immune cytokines were monitored in monocyte-derived macrophages (MDMs) before and after 8 months of vitamin D supplementation. In this study we showed that serum levels of LL-37 decreased after vitamin D supplementation in both Dené and Caucasian participants. There was no difference in TLR2/1L-induced LL-37 expression in MDMs in the two groups, either pre- or post-vitamin D supplementation. However, vitamin D supplementation markedly enhanced TLR2/1L-induced responses in MDMs e.g. IL-6, IL-12 and IL-23 among Caucasians but not in the Dené participants. In contrast, after vitamin D supplementation TLR2/1L-induced responses e.g. IL-1β, IL-8 and IL-12 were significantly reduced in the Dené MDMs. These results indicate that vitamin D supplementation enhanced TLR2/1L-induced innate immune macrophage responses in the Caucasian but not in the Dené participants. We hypothesize that cytokines may be differentially regulated in Canadian FN compared to Caucasians, in particular those that influence Th-1 and Th-17 responses required for the control of Mtb.

Functions of Cationic Host Defense Peptides in Immunity

Cationic host defense peptides are a widely distributed family of immunomodulatory molecules with antimicrobial properties. The biological functions of these peptides include the ability to influence innate and adaptive immunity for efficient resolution of infections and simultaneous modulation of inflammatory responses. This unique dual bioactivity of controlling infections and inflammation has gained substantial attention in the last three decades and consequent interest in the development of these peptide mimics as immunomodulatory therapeutic candidates. In this review, we summarize the current literature on the wide range of functions of cationic host defense peptides in the context of the mammalian immune system.

Human cathelicidin LL-37 and its derivative IG-19 regulate interleukin-32-induced inflammation

Human cathelicidin LL‐37 protects against infections and endotoxin‐induced inflammation. In a recent study we have shown that IG‐19, an LL‐37‐derived peptide, protects in a murine model of arthritis. Cytokine interleukin‐32 (IL‐32) is elevated and directly associated with the disease severity of inflammatory arthritis. Therefore, in this study we examined the effects of LL‐37 and IG‐19 on IL‐32‐induced responses in human peripheral blood‐derived mononuclear cells (PBMC) and macrophages. We showed that CD14+ monocytes are the primary cells that produce pro‐inflammatory tumour necrosis factor‐α (TNF‐α) following stimulation of PBMC with IL‐32. We demonstrated that LL‐37 and IG‐19 significantly suppress IL‐32‐induced production of pro‐inflammatory cytokines, e.g. TNF‐α and IL‐1β, without altering chemokine production. In contrast, LL‐37 and IG‐19 enhance the production of the anti‐inflammatory cytokine IL‐1RA. Further mechanistic studies revealed that LL‐37 and IG‐19 suppress IL‐32‐mediated phosphorylation of Fyn (Y420) Src kinase. In contrast, IL‐32‐mediated phosphorylation of AKT‐1 (T308) and MKP‐1 (S359) is not suppressed by the peptides. LL‐37 and IG‐19 alone induce the phosphorylation of MKP‐1 (S359), which is a known negative regulator of inflammation. Furthermore, the peptides induce the activity of p44/42 mitogen‐activated protein kinase, which is known to phosphorylate MKP‐1 (S359). This is the first study to demonstrate the regulation of IL‐32‐induced inflammation by LL‐37 and its derivative peptide IG‐19. The mechanistic results from this study suggest that regulation of immune‐mediated inflammation by these peptides may be controlled by the dual phosphatase MKP‐1. We speculate that LL‐37 and its derivatives may contribute to the control of immune‐mediated inflammatory diseases.

High degree of correlation between whole blood and PBMC expression levels of miR-155 and miR-146a in healthy controls and rheumatoid arthritis patients.

Elevated expression of specific microRNAs (miRNA) in peripheral blood-derived mononuclear cells (PBMC), particularly miR-146a and miR-155, is associated with rheumatoid arthritis (RA). Whole blood has not been explored as a potential clinical material for monitoring the expression of miRNAs in RA. We sought to determine whether miRNA levels detected in whole blood samples correlated with those detected in simultaneously isolated peripheral blood-derived mononuclear cells (PBMC) from the same individuals, thus establishing the feasibility of using whole blood as a viable clinical material for monitoring miRNA expression in RA and other disorders. We demonstrated a highly significant linear correlation between miR-146a and miR-155 expression in PBMC and whole blood, from both healthy individuals and RA patients. Whole blood samples accurately reflect miRNA levels in PBMC and would be useful in monitoring the expression of miRNAs as biomarkers. The detection of miRNA levels in samples that are readily obtained in routine clinical practice, such as whole blood, enhances their potential utility in detecting changes in the immunological mechanisms underlying autoimmune diseases such as RA.