Poly(lactic acid)-poly(ethylene glycol) nanoparticles provide sustained delivery of a Chlamydia trachomatis recombinant MOMP peptide and potentiate systemic adaptive immune responses in mice.

PLA-PEG [poly(lactic acid)-poly (ethylene glycol)], a biodegradable copolymer, is underexploited for vaccine delivery although it exhibits enhanced biocompatibility and slow release immune-potentiating properties. We document here successful encapsulation of M278, a Chlamydia trachomatis MOMP (major outer-membrane protein) peptide, within PLA-PEG nanoparticles by size (~73-100nm), zeta potential (-16 mV), smooth morphology, encapsulation efficiency (~60%), slow release pattern, and non-toxicity to macrophages. Immunization of mice with encapsulated M278 elicited higher M278-specific T-cell cytokines [Th1 (IFN-γ, IL-2), Th17 (IL-17)] and antibodies [Th1 (IgG2a), Th2 (IgG1, IgG2b)] compared to bare M278. Encapsulated-M278 mouse serum inhibited Chlamydia infectivity of macrophages, with a concomitant transcriptional down-regulation of MOMP, its cognate TLR2 and CD80 co-stimulatory molecule. Collectively, encapsulated M278 potentiated crucial adaptive immune responses, which are required by a vaccine candidate for protective immunity against Chlamydia. Our data highlight PLA-PEG's potential for vaccines, which resides in its slow release and potentiating effects to bolster immune responses. FROM THE CLINICAL EDITOR: This study highlights the potential of a PLA-PEG-based nanoparticle formulation containing a major outer membrane protein of chlamydia trachomatis in inducing a sustained enhanced immune response, paving the way to the development of a vaccination strategy against this infection.

PLGA-Chitosan Encapsulated IL-10 Nanoparticles Modulate Chlamydia Inflammation in Mice.

Introduction: Interleukin-10 (IL-10) is a key anti-inflammatory mediator in protecting host from over-exuberant responses to pathogens and play important roles in wound healing, autoimmunity, cancer, and homeostasis. However, its application as a therapeutic agent for biomedical applications has been limited due to its short biological half-life. Therefore, it is important to prolong the half-life of IL-10 to replace the current therapeutic application, which relies on administering large and repeated dosages. Therefore, not a cost-effective approach. Thus, studies that aim to address this type of challenges are always in need. Methods: Recombinant IL-10 was encapsulated in biodegradable nanoparticles (Poly-(Lactic-co-Glycolic Acid) and Chitosan)) by the double emulsion method and then characterized for size, surface charge, thermal stability, cytotoxicity, in vitro release, UV-visible spectroscopy, and Fourier Transform-Infrared Spectroscopy as well as evaluated for its anti-inflammatory effects. Bioactivity of encapsulated IL-10 was evaluated in vitro using J774A.1 macrophage cell-line and in vivo using BALB/c mice. Inflammatory cytokines (IL-6 and TNF-α) were quantified from culture supernatants using specific enzyme-linked immunosorbent assay (ELISA), and significance was analyzed using ANOVA. Results: We obtained a high 96% encapsulation efficiency with smooth encapsulated IL-10 nanoparticles of ~100-150 nm size and release from nanoparticles as measurable to 22 days. Our result demonstrated that encapsulated IL-10 was biocompatible and functional by reducing the inflammatory responses induced by LPS in macrophages. Of significance, we also proved the functionality of encapsulated IL-10 by its capacity to reduce inflammation in BALB/c mice as provoked by Chlamydia trachomatis, an inflammatory sexually transmitted infectious bacterium. Discussion: Collectively, our results show the successful IL-10 encapsulation, slow release to prolong its biological half-life and reduce inflammatory cytokines IL-6 and TNF production in vitro and in mice. Our results serve as proof of concept to further explore the therapeutic prospective of encapsulated IL-10 for biomedical applications, including inflammatory diseases.

Flavonoid naringenin: a potential immunomodulator for Chlamydia trachomatis inflammation.

Chlamydia trachomatis, the agent of bacterial sexually transmitted infections, can manifest itself as either acute cervicitis, pelvic inflammatory disease, or a chronic asymptomatic infection. Inflammation induced by C. trachomatis contributes greatly to the pathogenesis of disease. Here we evaluated the anti-inflammatory capacity of naringenin, a polyphenolic compound, to modulate inflammatory mediators produced by mouse J774 macrophages infected with live C. trachomatis. Infected macrophages produced a broad spectrum of inflammatory cytokines (GM-CSF, TNF, IL-1ß, IL-1α, IL-6, IL-12p70, and IL-10) and chemokines (CCL4, CCL5, CXCL1, CXCL5, and CXCL10) which were downregulated by naringenin in a dose-dependent manner. Enhanced protein and mRNA gene transcript expressions of TLR2 and TLR4 in addition to the CD86 costimulatory molecule on infected macrophages were modulated by naringenin. Pathway-specific inhibition studies disclosed that p38 mitogen-activated-protein kinase (MAPK) is involved in the production of inflammatory mediators by infected macrophages. Notably, naringenin inhibited the ability of C. trachomatis to phosphorylate p38 in macrophages, suggesting a potential mechanism of its attenuation of concomitantly produced inflammatory mediators. Our data demonstrates that naringenin is an immunomodulator of inflammation triggered by C. trachomatis, which possibly may be mediated upstream by modulation of TLR2, TLR4, and CD86 receptors on infected macrophages and downstream via the p38 MAPK pathway.

The anti-inflammatory cytokine, interleukin-10, inhibits inflammatory mediators in human epithelial cells and mouse macrophages exposed to live and UV-inactivated Chlamydia trachomatis.

Chlamydia trachomatis infects macrophages and epithelial cells evoking acute and chronic inflammatory conditions, which, if not controlled, may put patients at risk for major health issues such as pelvic inflammatory disease, chronic abdominal pain, and infertility. Here we hypothesized that IL-10, with anti-inflammatory properties, will inhibit inflammatory mediators that are produced by innate immune cells exposed to C. trachomatis. We used human epithelial (HeLa) cells and mouse J774 macrophages as target cells along with live and UV-inactivated C. trachomatis mouse pneumonitis (MoPn) as stimulants. Confocal microscopy employing an anti-Chlamydia antibody confirmed cells infectivity by day 1, which persisted up to day 3. Kinetics studies revealed that live C. trachomatis induced TNF, IL-6, and IL-8, as a function of time, with day-2 infection inducing the highest cytokine levels. Exogenous IL-10 inhibited TNF, IL-6, and IL-8 as secreted by day-2 infected cells. Similarly, IL-10 diminished cytokine levels as produced by macrophages exposed to UV-inactivated Chlamydia, suggesting the IL-10-mediated inhibition of cytokines is not restricted to live organisms. Our data imply that IL-10 is an important regulator of the initial inflammatory response to C. trachomatis infection and that further investigations be made into IL-10 use to combat inflammation induced by this bacterium.

Anti-inflammatory effects of silver-polyvinyl pyrrolidone (Ag-PVP) nanoparticles in mouse macrophages infected with live Chlamydia trachomatis.

Chlamydia trachomatis is a very common sexually transmissible infection in both developing and developed countries. A hallmark of C. trachomatis infection is the induction of severe inflammatory responses which play critical roles in its pathogenesis. Antibiotics are the only treatment option currently available for controlling C. trachomatis infection; however, they are efficacious only when administered early after an infection. The objectives of this study are to explore alternative strategies in the control and regulation of inflammatory responses triggered by a C. trachomatis infection. We employed silver-polyvinyl pyrrolidone (Ag-PVP) nanoparticles, which have been shown to possess anti-inflammatory properties, as our target and the in vitro mouse J774 macrophage model of C. trachomatis infection. Our hypothesis is that small sizes of Ag-PVP nanoparticles will control inflammatory mediators triggered by a C. trachomatis infection. Cytotoxicity studies using Ag-PVP nanoparticles of 10, 20, and 80 nm sizes revealed >80% macrophage viability up to a concentration of 6.25 µg/mL, with the 10 nm size being the least toxic. All sizes of Ag-PVP nanoparticles, especially the 10 nm size, reduced the levels of the prototypic cytokines, tumor necrosis factor (TNF) and interleukin (IL)-6, as elicited from C. trachomatis infected macrophages. Additionally, Ag-PVP nanoparticles (10 nm) selectively inhibited a broad spectrum of other cytokines and chemokines produced by infected macrophages. Of significance, Ag-PVP nanoparticles (10 nm) caused perturbations in a variety of upstream (toll like receptor 2 [TLR2], nucleotide-binding oligomerization-protein 2 [NOD2], cluster of differentiation [CD]40, CD80, and CD86) and downstream (IL-1 receptor-associated kinase 3 [IRAK3] and matrix metallopeptidase 9 [MMP9]) inflammatory signaling pathways by downregulating their messenger ribonucleic acid (mRNA) gene transcript expressions as induced by C. trachomatis in macrophages. Collectively, our data provides further evidence for the anti-inflammatory properties of Ag-PVP nanoparticles, and opens new possibilities for smaller sizes of Ag-PVP nanoparticles to be employed as regulators of inflammatory responses induced by C. trachomatis.

Chlamydia trachomatis recombinant MOMP encapsulated in PLGA nanoparticles triggers primarily T helper 1 cellular and antibody immune responses in mice: a desirable candidate nanovaccine.

We recently demonstrated by in vitro experiments that PLGA (poly D, L-lactide-co-glycolide) potentiates T helper 1 (Th1) immune responses induced by a peptide derived from the recombinant major outer membrane protein (rMOMP) of Chlamydia trachomatis, and may be a promising vaccine delivery system. Herein we evaluated the immune-potentiating potential of PLGA by encapsulating the full-length rMOMP (PLGA-rMOMP), characterizing it in vitro, and investigating its immunogenicity in vivo. Our hypothesis was that PLGA-rMOMP triggers Th1 immune responses in mice, which are desirable prerequisites for a C. trachomatis candidate nanovaccine. Physical-structural characterizations of PLGA-rMOMP revealed its size (approximately 272 nm), zeta potential (-14.30 mV), apparent spherical smooth morphology, and continuous slow release pattern. PLGA potentiated the ability of encapsulated rMOMP to trigger production of cytokines and chemokines by mouse J774 macrophages. Flow cytometric analyses revealed that spleen cells from BALB/c mice immunized with PLGA-rMOMP had elevated numbers of CD4+ and CD8+ T cell subsets, and secreted more rMOMP-specific interferon-gamma (Th1) and interleukin (IL)-12p40 (Th1/Th17) than IL-4 and IL-10 (Th2) cytokines. PLGA-rMOMP-immunized mice produced higher serum immunoglobulin (Ig)G and IgG2a (Th1) than IgG1 (Th2) rMOMP-specific antibodies. Notably, sera from PLGA-rMOMP-immunized mice had a 64-fold higher Th1 than Th2 antibody titer, whereas mice immunized with rMOMP in Freund's adjuvant had only a four-fold higher Th1 than Th2 antibody titer, suggesting primarily induction of a Th1 antibody response in PLGA-rMOMP-immunized mice. Our data underscore PLGA as an effective delivery system for a C. trachomatis vaccine. The capacity of PLGA-rMOMP to trigger primarily Th1 immune responses in mice promotes it as a highly desirable candidate nanovaccine against C. trachomatis.

Antimicrobial lipids: novel innate defense molecules are elevated in sinus secretions of patients with chronic rhinosinusitis.

BACKGROUND: Airway secretions possess intrinsic antimicrobial properties that contribute to the innate host defense of the respiratory tract. These microbicidal capabilities have largely been attributed to the presence of antibacterial polypeptides. However, recent investigation has indicated that host-derived lipids including cholesteryl esters also exhibit antimicrobial properties. The purpose of this study was to determine whether sinus secretions contain such antimicrobial lipids and to compare the lipid composition in patients with and without chronic rhinosinusitis (CRS). METHODS: Maxillary sinus fluid was obtained via antral lavage from subjects with (seven patients) and without (nine patients) a history of CRS. After specimen collection, total lipid was extracted according to Bligh and Dyer (Bligh EG and Dyer WJ, A rapid method of total lipid extraction and purification, Can J Biochem Physiol 37:911-918, 1959) and lipid profiles were obtained by reverse phase high-performance liquid chromatography on an amide-embedded C18 column. In addition, the neutrophil-specific antimicrobial peptides human neutrophil peptides 1-3 (HNP1-3) were quantified by Western immunoblotting. RESULTS: Lipids, including cholesteryl esters, were identified in the maxillary sinus secretions of patients with and without CRS. However, levels of lipid composition differed between the two groups with CRS patients exhibiting greater amounts of all classes of lipids, reaching over 10-fold higher concentration when compared with non-CRS patients. This increase was independent of HNP1-3 content. CONCLUSION: Sinus secretions of patients with CRS appear to show elevated levels of antimicrobial lipids compared with controls independent from neutrophil influx. This up-regulation suggests that host-derived lipids act as mediators of mucosal immunity in CRS. Further study is necessary to determine if such antimicrobial lipids function alone or synergistically with antibacterial peptides in conferring such inherent microbicidal properties.