Cytokine biomarkers of exacerbations in sputum from Chronic Obstructive Pulmonary Disease patients: a prospective cohort study.

BACKGROUND: We determined the relationships between cytokine expression in sputum and clinical data to characterise and understand Chronic Obstructive Pulmonary Disease (COPD) exacerbations in COPD patients. METHODS: We measured 30 cytokines in 936 sputum samples, collected at stable state (ST) and exacerbation (EX) visits from 99 participants in the Acute Exacerbation and Respiratory InfectionS in COPD (AERIS) study (NCT01360398, www.clinicaltrials.gov). We determined their longitudinal expression and examined differential expression based on disease status or exacerbation type. RESULTS: Of the cytokines, 17 were suitable for analysis. As for disease states, in EX sputum samples, IL-17A, TNF-α, IL-1ß, and IL-10 were significantly increased compared to ST sputum samples, but a logistic mixed model could not predict disease state. As for exacerbation types, bacteria-associated exacerbations showed higher expression of IL-17A, TNF-α, IL-1ß, and IL-1α. IL-1α, IL-1ß, and TNF-α were identified as suitable biomarkers for bacteria-associated exacerbation. Bacteria-associated exacerbations also formed a cluster separate from other exacerbation types in principal component analysis. CONCLUSIONS: Measurement of cytokines in sputum from COPD patients could help identify bacteria-associated exacerbations based on increased concentrations of IL-1α, IL-1ß, or TNF-α. This finding may provide a point-of-care assessment to distinguish a bacterial exacerbation of COPD from other exacerbation types.

The Human Pathogen Streptococcus pyogenes Releases Lipoproteins as Lipoprotein-rich Membrane Vesicles.

Bacterial lipoproteins are attractive vaccine candidates because they represent a major class of cell surface-exposed proteins in many bacteria and are considered as potential pathogen-associated molecular patterns sensed by Toll-like receptors with built-in adjuvanticity. Although Gram-negative lipoproteins have been extensively characterized, little is known about Gram-positive lipoproteins. We isolated from Streptococcus pyogenes a large amount of lipoproteins organized in vesicles. These vesicles were obtained by weakening the bacterial cell wall with a sublethal concentration of penicillin. Lipid and proteomic analysis of the vesicles revealed that they were enriched in phosphatidylglycerol and almost exclusively composed of lipoproteins. In association with lipoproteins, a few hypothetical proteins, penicillin-binding proteins, and several members of the ExPortal, a membrane microdomain responsible for the maturation of secreted proteins, were identified. The typical lipidic moiety was apparently not necessary for lipoprotein insertion in the vesicle bilayer because they were also recovered from the isogenic diacylglyceryl transferase deletion mutant. The vesicles were not able to activate specific Toll-like receptor 2, indicating that lipoproteins organized in these vesicular structures do not act as pathogen-associated molecular patterns. In light of these findings, we propose to name these new structures Lipoprotein-rich Membrane Vesicles.

Modulation of endotoxicity of Shigella generalized modules for membrane antigens (GMMA) by genetic lipid A modifications: relative activation of TLR4 and TLR2 pathways in different mutants.

Outer membrane particles from Gram-negative bacteria are attractive vaccine candidates as they present surface antigens in their natural context. We previously developed a high yield production process for genetically derived particles, called generalized modules for membrane antigens (GMMA), from Shigella. As GMMA are derived from the outer membrane, they contain immunostimulatory components, especially lipopolysaccharide (LPS). We examined ways of reducing their reactogenicity by modifying lipid A, the endotoxic part of LPS, through deletion of late acyltransferase genes, msbB or htrB, in GMMA-producing Shigella sonnei and Shigella flexneri strains. GMMA with resulting penta-acylated lipid A from the msbB mutants showed a 600-fold reduced ability, and GMMA from the S. sonnei ΔhtrB mutant showed a 60,000-fold reduced ability compared with GMMA with wild-type lipid A to stimulate human Toll-like receptor 4 (TLR4) in a reporter cell line. In human peripheral blood mononuclear cells, GMMA with penta-acylated lipid A showed a marked reduction in induction of inflammatory cytokines (S. sonnei ΔhtrB, 800-fold; ΔmsbB mutants, 300-fold). We found that the residual activity of these GMMA is largely due to non-lipid A-related TLR2 activation. In contrast, in the S. flexneri ΔhtrB mutant, a compensatory lipid A palmitoleoylation resulted in GMMA with hexa-acylated lipid A with ∼10-fold higher activity to stimulate peripheral blood mononuclear cells than GMMA with penta-acylated lipid A, mostly due to retained TLR4 activity. Thus, for use as vaccines, GMMA will likely require lipid A penta-acylation. The results identify the relative contributions of TLR4 and TLR2 activation by GMMA, which need to be taken into consideration for GMMA vaccine development.

Development and Application of a Novel SPE-Method for Bioassay-Guided Fractionation of Marine Extracts.

The biological diversity of marine habitats is a unique source of chemical compounds with potential use as pharmaceuticals, cosmetics and dietary supplements. However, biological screening and chemical analysis of marine extracts pose specific technical constraints and require adequate sample preparation. Here we report an improved method on Solid Phase Extraction (SPE) to fractionate organic extracts containing high concentration of salt that hampers the recovery of secondary metabolites. The procedure uses a water suspension to load the extracts on a poly(styrene-divynylbenzene)-based support and a stepwise organic solvent elution to effectively desalt and fractionate the organic components. The novel protocol has been tested on MeOH-soluble material from three model organisms (Reniera sarai, Dendrilla membranosa and Amphidinium carterae) and was validated on a small panel of 47 marine samples, including sponges and protists, within discovery programs for identification of immuno-stimulatory and anti-infective natural products.

Adjuvanticity of the oil-in-water emulsion MF59 is independent of Nlrp3 inflammasome but requires the adaptor protein MyD88.

Oil-in-water emulsions have been successfully used to increase the efficacy, immunogenicity, and cross-protection of human vaccines; however, their mechanism of action is still largely unknown. Nlrp3 inflammasome has been previously associated to the activity of alum, another adjuvant broadly used in human vaccines, and MyD88 adaptor protein is required for the adjuvanticity of most Toll-like receptor agonists. We compared the contribution of Nlrp3 and MyD88 to the adjuvanticity of alum, the oil-in-water emulsion MF59, and complete Freund's adjuvant in mice using a three-component vaccine against serogroup B Neisseria meningitidis (rMenB). Although the basal antibody responses to the nonadjuvanted rMenB vaccine were largely dependent on Nlrp3, the high-level antibody responses induced by alum, MF59, or complete Freund's adjuvant did not require Nlrp3. Surprisingly, we found that MF59 requires MyD88 to enhance bactericidal antibody responses to the rMenB vaccine. Because MF59 did not activate any of the Toll-like receptors in vitro, we propose that MF59 requires MyD88 for a Toll-like receptor-independent signaling pathway.

Lung mucosal immunity to NTHi vaccine antigens: Antibodies in sputum of chronic obstructive pulmonary disease patients.

Chronic obstructive pulmonary disease (COPD) is a common chronic respiratory illness in older adults. A major cause of COPD-related morbidity and mortality is acute exacerbation of COPD (AECOPD). Bacteria in the lungs play a role in exacerbation development, and the most common pathogen is non-typeable Haemophilus influenzae (NTHi). A vaccine to prevent AECOPD containing NTHi surface antigens was tested in a clinical trial. This study measured IgG and IgA against NTHi vaccine antigens in sputum. Sputum samples from 40 COPD patients vaccinated with the NTHi vaccine were collected at baseline and 30 days after the second dose. IgG and IgA antibodies against the target antigens and albumin were analyzed in the sputum. We compared antibody signals before and after vaccination, analyzed correlation with disease severity and between sputum and serum samples, and assessed transudation. Antigen-specific IgG were absent before vaccination and present with high titers after vaccination. Antigen-specific IgA before and after vaccination were low but significantly different for two antigens. IgG correlated between sputum and serum, and between sputum and disease severity. Sputum albumin was higher in patients with severe COPD than in those with moderate COPD, suggesting changes in transudation played a role. We demonstrated that immunization with the NTHi vaccine induces antigen-specific antibodies in sputum. The correlation between IgG from sputum and serum and the presence of albumin in the sputum of severe COPD patients suggested transudation of antibodies from the serum to the lungs, although local IgG production could not be excluded.Clinical Trial Registration: NCT02075541.

What is the context? Chronic obstructive pulmonary disease (COPD) is the most common chronic respiratory illness in older adults and the third leading cause of death worldwide.One bacterium in the lungs, non-typeable Haemophilus influenzae (NTHi), is responsible for acute exacerbation of the disease, characterized by an increase in airway wall inflammation and symptoms, leading to high morbidity and mortality.A vaccine targeting NTHi was previously developed but did not show efficacy in reducing exacerbations in COPD patients, probably because the vaccine did not elicit an immune response in the lung mucosae, where the bacteria are located.What is the impact? Parenteral immunization with new vaccines targeting NTHi is able to elicit immune defense at the level of lung mucosae.Now that antibodies can be measured in sputum, new vaccines against COPD exacerbations or other lung infections can be tested for efficacy in the actual target tissue.Also, lung immunity against specific pathogens can now be tested.What is new? We determined that antigen-specific antibodies were present in the lungs after vaccination; these were assessed in sputum after vaccination with NTHi surface antigens.NTHi-specific IgG were present in the lungs and appeared to have arrived there primarily by transudation, a type of leakage from the serum to the lung mucosae.Transudation appeared to be stronger in severe than in moderate COPD patients.

Systems analysis of human responses to an aluminium hydroxide-adsorbed TLR7 agonist (AS37) adjuvanted vaccine reveals a dose-dependent and specific activation of the interferon-mediated antiviral response.

The candidate Adjuvant System AS37 contains a synthetic toll-like receptor agonist (TLR7a) adsorbed to alum. In a phase I study (NCT02639351), healthy adults were randomised to receive one dose of licensed alum-adjuvanted meningococcal serogroup C (MenC-CRM197) conjugate vaccine (control) or MenC-CRM197 conjugate vaccine adjuvanted with AS37 (TLR7a dose 12.5, 25, 50 or 100 µg). A subset of 66 participants consented to characterisation of peripheral whole blood transcriptomic responses, systemic cytokine/chemokine responses and multiple myeloid and lymphoid cell responses as exploratory study endpoints. Blood samples were collected pre-vaccination, 6 and 24 h post-vaccination, and 3, 7, 28 and 180 days post-vaccination. The gene expression profile in whole blood showed an early, AS37-specific transcriptome response that peaked at 24 h, increased with TLR7a dose up to 50 µg and generally resolved within one week. Five clusters of differentially expressed genes were identified, including those involved in the interferon-mediated antiviral response. Evaluation of 30 cytokines/chemokines by multiplex assay showed an increased level of interferon-induced chemokine CXCL10 (IP-10) at 24 h and 3 days post-vaccination in the AS37-adjuvanted vaccine groups. Increases in activated plasmacytoid dendritic cells (pDC) and intermediate monocytes were detected 3 days post-vaccination in the AS37-adjuvanted vaccine groups. T follicular helper (Tfh) cells increased 7 days post-vaccination and were maintained at 28 days post-vaccination, particularly in the AS37-adjuvanted vaccine groups. Moreover, most of the subjects that received vaccine containing 25, 50 and 100 µg TLR7a showed an increased MenC-specific memory B cell responses versus baseline. These data show that the adsorption of TLR7a to alum promotes an immune signature consistent with TLR7 engagement, with up-regulation of interferon-inducible genes, cytokines and frequency of activated pDC, intermediate monocytes, MenC-specific memory B cells and Tfh cells. TLR7a 25-50 µg can be considered the optimal dose for AS37, particularly for the adjuvanted MenC-CRM197 conjugate vaccine.

HIV-1 Tat binds to SH3 domains: cellular and viral outcome of Tat/Grb2 interaction.

The Src-homology 3 (SH3) domain is one of the most frequent protein recognition modules (PRMs), being represented in signal transduction pathways and in several pathologies such as cancer and AIDS. Grb2 (growth factor receptor-bound protein 2) is an adaptor protein that contains two SH3 domains and is involved in receptor tyrosine kinase (RTK) signal transduction pathways. The HIV-1 transactivator factor Tat is required for viral replication and it has been shown to bind directly or indirectly to several host proteins, deregulating their functions. In this study, we show interaction between the cellular factor Grb2 and the HIV-1 trans-activating protein Tat. The binding is mediated by the proline-rich sequence of Tat and the SH3 domain of Grb2. As the adaptor protein Grb2 participates in a wide variety of signaling pathways, we characterized at least one of the possible downstream effects of the Tat/Grb2 interaction on the well-known IGF-1R/Raf/MAPK cascade. We show that the binding of Tat to Grb2 impairs activation of the Raf/MAPK pathway, while potentiating the PKA/Raf inhibitory pathway. The Tat/Grb2 interaction affects also viral function by inhibiting the Tat-mediated transactivation of HIV-1 LTR and viral replication in infected primary microglia.

Human plasmacytoid dendritic cells are unresponsive to bacterial stimulation and require a novel type of cooperation with myeloid dendritic cells for maturation.

Dendritic cell (DC) populations play unique and essential roles in the detection of pathogens, but information on how different DC types work together is limited. In this study, 2 major DC populations of human blood, myeloid (mDCs) and plasmacytoid (pDCs), were cultured alone or together in the presence of pathogens or their products. We show that pDCs do not respond to whole bacteria when cultured alone, but mature in the presence of mDCs. Using purified stimuli, we dissect this cross-talk and demonstrate that mDCs and pDCs activate each other in response to specific induction of only one of the cell types. When stimuli for one or both populations are limited, they synergize to reach optimal activation. The cross-talk is limited to enhanced antigen presentation by the nonresponsive population with no detectable changes in the quantity and range of cytokines produced. We propose that each population can be a follower or leader in immune responses against pathogen infections, depending on their ability to respond to infectious agents. In addition, our results indicate that pDCs play a secondary role to induce immunity against human bacterial infections, which has implications for more efficient targeting of DC populations with improved vaccines and therapeutics.

Tubulin-mediated binding of human immunodeficiency virus-1 Tat to the cytoskeleton causes proteasomal-dependent degradation of microtubule-associated protein 2 and neuronal damage.

One of the hallmarks of human immunodeficiency virus (HIV)-1 associated pathology in the CNS is deterioration of neuronal processes. Although there is mounting evidence of neuronal toxicity and cell death induced by the HIV-1 transactivating factor Tat, the molecular events linked directly to its detrimental effect on neuronal cells remain unclear. In this study, we used rat embryonic cortical neurons and demonstrated that Tat causes rapid degradation of microtubule-associated protein 2 (MAP2) and the collapse of cytoskeletal filaments. The mechanism of Tat action on MAP2 stability involved Tat-mediated translocation of the proteasome to the site of microtubule filaments. Immunohistochemical analysis of clinical samples from patients with HIV encephalopathy further revealed a significant decrease in MAP2 with predominant cytoplasmic 20S in cortical neurons near microglial nodules. These findings indicate a novel mechanism for the action of Tat on neuronal cells. It involves proteasome-mediated MAP2 degradation and may account for the loss of MAP2 and neuronal damage observed in the brain of AIDS patients with neurological dysfunctions.

A new TLR2 agonist promotes cross-presentation by mouse and human antigen presenting cells.

Cross-presentation is the process by which professional APCs load peptides from an extracellularly derived protein onto class I MHC molecules to trigger a CD8(+) T cell response. The ability to enhance this process is therefore relevant for the development of antitumor and antiviral vaccines. We investigated a new TLR2-based adjuvant, Small Molecule Immune Potentiator (SMIP) 2.1, for its ability to stimulate cross-presentation. Using OVA as model antigen, we demonstrated that a SMIP2.1-adjuvanted vaccine formulation induced a greater CD8(+) T cell response, in terms of proliferation, cytokine production and cytolytic activity, than a non-adjuvanted vaccine. Moreover, using an OVA-expressing tumor model, we showed that the CTLs induced by the SMIP2.1 formulated vaccine inhibits tumor growth in vivo. Using a BCR transgenic mouse model we found that B cells could cross-present the OVA antigen when stimulated with SMIP2.1. We also used a flow cytometry assay to detect activation of human CD8(+) T cells isolated from human PBMCs of cytomegalovirus-seropositive donors. Stimulation with SMIP2.1 increased the capacity of human APCs, pulsed in vitro with the pp65 CMV protein, to activate CMV-specific CD8(+) T cells. Therefore, vaccination with an exogenous antigen formulated with SMIP2.1 is a successful strategy for the induction of a cytotoxic T cell response along with antibody production.

Rational design of small molecules as vaccine adjuvants.

Adjuvants increase vaccine potency largely by activating innate immunity and promoting inflammation. Limiting the side effects of this inflammation is a major hurdle for adjuvant use in vaccines for humans. It has been difficult to improve on adjuvant safety because of a poor understanding of adjuvant mechanism and the empirical nature of adjuvant discovery and development historically. We describe new principles for the rational optimization of small-molecule immune potentiators (SMIPs) targeting Toll-like receptor 7 as adjuvants with a predicted increase in their therapeutic indices. Unlike traditional drugs, SMIP-based adjuvants need to have limited bioavailability and remain localized for optimal efficacy. These features also lead to temporally and spatially restricted inflammation that should decrease side effects. Through medicinal and formulation chemistry and extensive immunopharmacology, we show that in vivo potency can be increased with little to no systemic exposure, localized innate immune activation and short in vivo residence times of SMIP-based adjuvants. This work provides a systematic and generalizable approach to engineering small molecules for use as vaccine adjuvants.

Src kinases are required for a balanced production of IL-12/IL-23 in human dendritic cells activated by Toll-like receptor agonists.

BACKGROUND: Pathogen recognition by dendritic cells (DC) is crucial for the initiation of both innate and adaptive immune responses. Activation of Toll-like Receptors (TLRs) by microbial molecular patterns leads to the maturation of DC, which present the antigen and activate T cells in secondary lymphoid tissues. Cytokine production by DC is critical for shaping the adaptive immune response by regulating T helper cell differentiation. It was previously shown by our group that Src kinases play a key role in cytokines production during TLR4 activation in human DC. PRINCIPAL FINDINGS: In this work we investigated the role of Src kinases during different TLRs triggering in human monocyte-derived DC (MoDC). We found that Src family kinases are important for a balanced production of inflammatory cytokines by human MoDC upon stimulation of TLR3 and 8 with their respective agonists. Disruption of this equilibrium through pharmacological inhibition of Src kinases alters the DC maturation pattern. In particular, while expression of IL-12 and other inflammatory cytokines depend on Src kinases, the induction of IL-23 and co-stimulatory molecules do not. Accordingly, DC treated with Src inhibitors are not compromised in their ability to induce CD4 T cell proliferation and to promote the Th17 subset survival but are less efficient in inducing Th1 differentiation. CONCLUSIONS: We suggest that the pharmacological modulation of DC maturation has the potential to shape the quality of the adaptive immune response and could be exploited for the treatment of inflammation-related diseases.

Introduction of zwitterionic motifs into bacterial polysaccharides generates TLR2 agonists able to activate APCs.

It was shown previously that bacterial polysaccharides (PS), which naturally contain both positive and negative charges, are able to activate T cells and APCs. However, the vast majority of bacterial PS are anionic and do not have these properties. In this study, we show that chemical introduction of positive charges into naturally anionic bacterial PS confers to the resulting zwitterionic PS (ZPS) the ability to activate pure human monocytes, monocyte-derived dendritic cells, and mouse bone marrow-derived dendritic cells, as do natural bacterial ZPS. Cells are induced to up-regulate MHC class II and costimulatory molecules and to produce cytokines. In mixed monocyte-T cell cocultures, ZPS induce MHC II-dependent T cell proliferation and up-regulation of activation markers. These stimulatory qualities of ZPS disappear when the positive charge is chemically removed from the molecules and thus the zwitterionic motif is destroyed. The ability of natural and chemically derived ZPS to activate APCs can be blocked by anti-TLR2 mAbs, and TLR2 transfectants show reporter gene transcription upon incubation with ZPS. In conclusion, the generation of a zwitterionic motif in bacterial PS confers the ability to activate both APCs and T cells. This finding has important implications for the design of novel polysaccharide vaccines.

Cross talk between growth factors and viral and cellular factors alters neuronal signaling pathways: implication for HIV-associated dementia.

HIV-associated dementia (HAD) is a serious neurological disorder affecting about 7% of people with AIDS. In the brain, HIV-1 infects a restricted number of cell types, being primarily present in macrophages and microglial cells, less abundant in astrocytes, and rarely seen in oligodendrocytes and neurons. Lack of a productive HIV-1 infection of neuronal cells suggests the presence of an indirect pathway by which the virus may determine the brain pathology and neuronal dysfunction seen in AIDS patients. Among the participants in this event, viral proteins including gp120 and Tat, along with host factors including cytokines, chemokines, and several signaling pathways have received considerable attention. In this article, we discuss the most recent concepts pertaining to the mechanisms of HIV-1-induced neuronal dysfunction by highlighting the interplay between signal transduction pathways activated by viral and host factors and their consequences in neuronal cell function.