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The very common condition of sinusitis is characterized by persistent inflammation of the nasal cavity, which contributes to chronic rhinosinusitis and morbidity of cystic fibrosis patients. Colonization by opportunistic pathogens such as Staphylococcus aureus and Pseudomonas aeruginosa triggers inflammation that is exacerbated by defects in the innate immune response. Pathophysiological mechanisms underlying initial colonization of the sinuses are not well established. Despite their extensive use, current murine models of acute bacterial rhinosinusitis have not improved the understanding of early disease stages due to analytical limitations. In this study, a model is described that is technically simple, allows non-invasive tracking of bacterial infection, and screening of host-responses to infection and therapies. The model was modified to investigate longer-term infection and disease progression by using a less virulent, epidemic P. aeruginosa cystic fibrosis clinical isolate LESB65. Tracking of luminescent bacteria was possible after intranasal infections, which were sustained for up to 120 h post-infection, without compromising the overall welfare of the host. Production of reactive oxidative species was associated with neutrophil localization to the site of infection in this model. Further, host-defense peptides administered by Respimat® inhaler or intranasal instillation reduced bacterial burden and impacted disease progression as well as cytokine responses associated with rhinosinusitis. Thus, future studies using this model will improve our understanding of rhinosinusitis etiology and early stage pathogenesis, and can be used to screen for the efficacy of emerging therapies pre-clinically.

Host defense peptides (HDPs), also known as antimicrobial peptides, are naturally occurring polypeptides (~12–50 residues) composed of cationic and hydrophobic amino acids that adopt an amphipathic conformation upon folding usually after contact with membranes. HDPs have a variety of biological activities including immunomodulatory, anti-inflammatory, anti-bacterial, and anti-biofilm functions. Although HDPs have the potential to address the global threat of antibiotic resistance and to treat immune and inflammatory disorders, they have yet to achieve this promise. Indeed, there are several challenges associated with bringing peptide-based drug candidates from the lab bench to clinical practice, including identifying appropriate indications, stability, toxicity, and cost. These challenges can be addressed in part by the development of innate defense regulator (IDR) peptides and peptidomimetics, which are synthetic derivatives of HDPs with similar or better efficacy, increased stability, and reduced toxicity and cost of the original HDP. However, one of the largest gaps between basic research and clinical application is the validity and translatability of conventional model systems, such as cell lines and animal models, for screening HDPs and their derivatives as potential drug therapies. Indeed, such translation has often relied on animal models, which have only limited validity. Here we discuss the recent development of human organoids for disease modeling and drug screening, assisted by the use of omics analyses. Organoids, developed from primary cells, cell lines, or human pluripotent stem cells, are three-dimensional, self-organizing structures that closely resemble their corresponding in vivo organs with regards to immune responses, tissue organization, and physiological properties; thus, organoids represent a reliable method for studying efficacy, formulation, toxicity and to some extent drug stability and pharmacodynamics. The use of patient-derived organoids enables the study of patient-specific efficacy, toxicogenomics and drug response predictions. We outline how organoids and omics data analysis can be leveraged to aid in the clinical translation of IDR peptides.

Canadian Indigenous peoples (First Nations and Inuit) exhibit a high burden of infectious diseases including tuberculosis influenced by societal factors, and biological determinants. Toll-like receptor (TLR)-mediated innate immune responses are the first line of defence against infections. We examined the production of a panel of 30 cytokines in peripheral blood-derived mononuclear cells (PBMC) isolated from Indigenous and non-Indigenous participants, following stimulation with five different TLR ligands. The levels of TLR-induced pro-inflammatory cytokines such as IL-12/23p40, IL-16, and IFN-γ, and chemokines (MCP-4, MDC and eotaxin) were different between Indigenous compared to non-Indigenous participants. Antimicrobial cationic host defence peptides (CHDP) induced by TLR activation are critical for resolution of infections and modulate the TLR-to-NFκB pathway to alter downstream cytokine responses. Therefore, we examined the expression of human CHDP defensins and cathelicidin in PBMC. mRNA expression of genes encoding for def-A1 and def-B1 were significantly higher following stimulation with TLR ligands in Indigenous compared to non-Indigenous participants. The purinergic receptor P2X7 known to be activated by ATP released following TLR stimulation, is a receptor for CHDP. Therefore, we further examined single nucleotide polymorphisms (SNP) in P2X7. Indigenous participants had a significantly higher percentage of a P2X7 SNP which is associated with reduced function and lower ability to clear infections. These results suggest that a higher frequency of non-functional P2X7 receptors may influence the activity of downstream immune mediators required for resolution of infections such as pro-inflammatory cytokines and CHDP defensins, thus contributing to higher burden of infections in Indigenous population.

The anti-endotoxin activity of the cationic peptide LL-37 and its derivative IG-19 is attributed to electrostatic interaction of the peptides&rsquo, positive charge with negatively charged bacterial lipopolysaccharides (LPS), and in part to the alteration of intracellular mechanisms independent of peptide binding to LPS. We examined the immunomodulatory responses induced by IG-19 and four IG-19-derived scrambled peptides (IG-19a&ndash, d), in the presence and absence of LPS, in macrophages and peripheral blood-derived mononuclear cells. All peptides had identical net charge (+5) and amino acid composition, but different hydrophobicity and &alpha, helical propensity. Peptide IG-19 suppressed LPS-induced cytokine/chemokine production by &gt, 90%, IG-19a and IG-19b suppressed it by 40&ndash, 50%, and IG-19c and IG-19d did not suppress cytokine/chemokine production at all. In silico prediction algorithms and the peptide retention time (RT) on a C18 RP HPLC column indicated a linear association between &alpha, helical propensity and hydrophobicity with the ability of the peptides to inhibit LPS-induced responses. Peptide RT exhibited a significant correlation (&gt, 70%) between the suppression of LPS-induced cytokine/chemokine production and peptide-induced production of the anti-inflammatory cytokine IL-1RA. These results indicate that RT on a C18 column can be used as a predictor for the immunomodulatory functions of cationic peptides. Overall, we demonstrated that the immunomodulatory functions of LL-37-derived peptides with identical positive charge and amino acid composition are directly associated with the predicted &alpha, helical propensity and hydrophobicity of the peptides.

An impediment in the development of new therapeutic strategies for chronic inflammatory diseases is the limited understanding of underlying molecular mechanisms. The objective of this study was to identify newly synthesized (nascent) proteins induced by critical inflammatory cytokines TNF-α and IL-1β in human monocytic THP-1 cells. We optimized methods to combine two different approaches, bio‐orthogonal non-canonical amino acid tagging (BONCAT) along with proteomics using isobaric tags (iTRAQ). BONCAT employed the incorporation of l -azidohomoalanine (AHA), an analog of methionine, into TNF-α or IL-1β induced nascent proteins. The AHA-containing nascent proteins were tagged with alkyne-biotin to allow enrichment using avidin affinity purification. The differential expressions of the enriched proteins were further determined using iTRAQ reagents and mass spectrometry (MS). The combination of BONCAT and proteomics represents a unique approach that has uncovered the nascent proteome induced by inflammatory cytokines TNF-α and IL-1β.

Summary 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.

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.