Interleukin 17A is an immune marker for chlamydial disease severity and pathogenesis in the koala (Phascolarctos cinereus)

The koala (Phascolarctos cinereus) is an iconic Australian marsupial species that is facing many threats to its survival. Chlamydia pecorum infections are a significant contributor to this ongoing decline. A major limiting factor in our ability to manage and control chlamydial disease in koalas is a limited understanding of the koala’s cell-mediated immune response to infections by this bacterial pathogen. To identify immunological markers associated with chlamydial infection and disease in koalas, we used koala-specific Quantitative Real Time PCR (qrtPCR) assays to profile the cytokine responses of Peripheral Blood Mononuclear Cells (PBMCs) collected from 41 koalas with different stages of chlamydial disease. Target cytokines included the principal Th1 (Interferon gamma; IFNγ), Th2 (Interleukin 10; IL10), and pro-inflammatory cytokines (Tumor Necrosis Factor alpha; TNFα). A novel koala-specific IL17A qrtPCR assay was also developed as part of this study to quantitate the gene expression of this Th17 cytokine in koalas. A statistically significant higher IL17A gene expression was observed in animals with current chlamydial disease compared to animals with asymptomatic chlamydial infection. A modest up-regulation of pro-inflammatory cytokines, such as TNFα and IFNγ, was also observed in these animals with signs of current chlamydial disease. IL10 gene expression was not evident in the majority of animals from both groups. Future longitudinal studies are now required to confirm the role played by cytokines in pathology and/or protection against C. pecorum infection in the koala.

Proinflammatory cytokines regulate cementogenic differentiation of periodontal ligament cells by Wnt/Ca2+ signaling pathway

Periodontal inflammation can inhibit cell differentiation of periodontal ligament cells (PDLCs), resulting in decreased bone/cementum regeneration ability. The Wnt signaling pathway, including canonical Wnt/β-catenin signaling and noncanonical Wnt/Ca2+ signaling, plays essential roles in cell proliferation and differentiation during tooth development. However, little is still known whether noncanonical Wnt/Ca2+ signaling cascade could regulate cementogenic/osteogenic differentiation capability of PDLCs within an inflammatory environment. Therefore, in this study, human PDLCs (hPDLCs) and their cementogenic differentiation potential were investigated in the presence of cytokines. The data demonstrated that both cytokines interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-α) inhibited cell proliferation, relative alkaline phosphatase activity, bone/cementum-related gene/protein expression, and canonical Wnt pathway-related gene/protein expression in hPDLCs. Interestingly, both cytokines upregulated the noncanonical Wnt/Ca2+ signaling-related gene and protein expression in hPDLCs. When the Wnt/Ca2+ pathway was blocked by Ca2+/calmodulin-dependent protein kinase II inhibitor KN93, even in the presence of IL-6 and TNF-α, cementogenesis could be stimulated in hPDLCs. Our data indicate that the Wnt/Ca2+ pathway plays an inhibitory role on PDLC cementogenic differentiation in inflammatory microenvironments. Therefore, targeting the Wnt/Ca2+ pathway may provide a novel therapeutic approach to improve periodontal regeneration for periodontal diseases.

Sonic hedgehog-Dependent Induction of MicroRNA 31 and MicroRNA 150 Regulates Mycobacterium bovis BCG-Driven Toll-Like Receptor 2 Signaling

Hedgehog (HH) signaling is a significant regulator of cell fate decisions during embryogenesis, development, and perpetuation of various disease conditions. Testing whether pathogen-specific HH signaling promotes unique innate recognition of intracellular bacteria, we demonstrate that among diverse Gram-positive or Gram-negative microbes, Mycobacterium bovis BCG, a vaccine strain, elicits a robust activation of Sonic HH (SHH) signaling in macrophages. Interestingly, sustained tumor necrosis factor alpha (TNF-alpha) secretion by macrophages was essential for robust SHH activation, as TNF-alpha(-/-) macrophages exhibited compromised ability to activate SHH signaling. Neutralization of TNF-alpha or blockade of TNF-alpha receptor signaling significantly reduced the infection-induced SHH signaling activation both in vitro and in vivo. Intriguingly, activated SHH signaling downregulated M. bovis BCG-mediated Toll-like receptor 2 (TLR2) signaling events to regulate a battery of genes associated with divergent functions of M1/M2 macrophages. Genome-wide expression profiling as well as conventional gain-of-function or loss-of-function analysis showed that SHH signaling-responsive microRNA 31 (miR-31) and miR-150 target MyD88, an adaptor protein of TLR2 signaling, thus leading to suppression of TLR2 responses. SHH signaling signatures could be detected in vivo in tuberculosis patients and M. bovis BCG-challenged mice. Collectively, these investigations identify SHH signaling to be what we believe is one of the significant regulators of host-pathogen interactions.

The WNT Signaling Pathway Contributes to Dectin-1-Dependent Inhibition of Toll-Like Receptor-Induced Inflammatory Signature

Macrophages regulate cell fate decisions during microbial challenges by carefully titrating signaling events activated by innate receptors such as dectin-1 or Toll-like receptors (TLRs). Here, we demonstrate that dectin-1 activation robustly dampens TLR-induced proinflammatory signature in macrophages. Dectin-1 induced the stabilization of beta-catenin via spleen tyrosine kinase (Syk)-reactive oxygen species (ROS) signals, contributing to the expression of WNT5A. Subsequently, WNT5A-responsive protein inhibitors of activated STAT (PIAS-1) and suppressor of cytokine signaling 1 (SOCS-1) mediate the downregulation of IRAK-1, IRAK-4, and MyD88, resulting in decreased expression of interleukin 12 (IL-12), IL-1 beta, and tumor necrosis factor alpha (TNF-alpha). In vivo activation of dectin-1 with pathogenic fungi or ligand resulted in an increased bacterial burden of Mycobacteria, Klebsiella, Staphylococcus, or Escherichia, with a concomitant decrease in TLR-triggered proinflammatory cytokines. All together, our study establishes a new role for dectin-1-responsive inhibitory mechanisms employed by virulent fungi to limit the proinflammatory environment of the host.

Sonic hedgehog-Dependent Induction of MicroRNA 31 and MicroRNA 150 Regulates Mycobacterium bovis BCG-Driven Toll-Like Receptor 2 Signaling

Hedgehog (HH) signaling is a significant regulator of cell fate decisions during embryogenesis, development, and perpetuation of various disease conditions. Testing whether pathogen-specific HH signaling promotes unique innate recognition of intracellular bacteria, we demonstrate that among diverse Gram-positive or Gram-negative microbes, Mycobacterium bovis BCG, a vaccine strain, elicits a robust activation of Sonic HH (SHH) signaling in macrophages. Interestingly, sustained tumor necrosis factor alpha (TNF-alpha) secretion by macrophages was essential for robust SHH activation, as TNF-alpha(-/-) macrophages exhibited compromised ability to activate SHH signaling. Neutralization of TNF-alpha or blockade of TNF-alpha receptor signaling significantly reduced the infection-induced SHH signaling activation both in vitro and in vivo. Intriguingly, activated SHH signaling downregulated M. bovis BCG-mediated Toll-like receptor 2 (TLR2) signaling events to regulate a battery of genes associated with divergent functions of M1/M2 macrophages. Genome-wide expression profiling as well as conventional gain-of-function or loss-of-function analysis showed that SHH signaling-responsive microRNA 31 (miR-31) and miR-150 target MyD88, an adaptor protein of TLR2 signaling, thus leading to suppression of TLR2 responses. SHH signaling signatures could be detected in vivo in tuberculosis patients and M. bovis BCG-challenged mice. Collectively, these investigations identify SHH signaling to be what we believe is one of the significant regulators of host-pathogen interactions.

The Secreted Protein Rv1860 of Mycobacterium tuberculosis Stimulates Human Polyfunctional CD8(+) T Cells

We previously reported that Rv1860 protein from Mycobacterium tuberculosis stimulated CD4(+) and CD8(+) T cells secreting gamma interferon (IFN-gamma) in healthy purified protein derivative (PPD)-positive individuals and protected guinea pigs immunized with a DNA vaccine and a recombinant poxvirus expressing Rv1860 from a challenge with virulent M. tuberculosis. We now show Rv1860-specific polyfunctional T (PFT) cell responses in the blood of healthy latently M. tuberculosis-infected individuals dominated by CD8(+) T cells, using a panel of 32 overlapping peptides spanning the length of Rv1860. Multiple subsets of CD8(+) PFT cells were significantly more numerous in healthy latently infected volunteers (HV) than in tuberculosis (TB) patients (PAT). The responses of peripheral blood mononuclear cells (PBMC) from PAT to the peptides of Rv1860 were dominated by tumor necrosis factor alpha (TNF-alpha) and interleukin-10 (IL-10) secretions, the former coming predominantly from non-T cell sources. Notably, the pattern of the T cell response to Rv1860 was distinctly different from those of the widely studied M. tuberculosis antigens ESAT-6, CFP-10, Ag85A, and Ag85B, which elicited CD4(+) T cell-dominated responses as previously reported in other cohorts. We further identified a peptide spanning amino acids 21 to 39 of the Rv1860 protein with the potential to distinguish latent TB infection from disease due to its ability to stimulate differential cytokine signatures in HV and PAT. We suggest that a TB vaccine carrying these and other CD8(+) T-cell-stimulating antigens has the potential to prevent progression of latent M. tuberculosis infection to TB disease.

Cytokine pathway disruption in a mouse model of schizophrenia induced by Munc18-1a overexpression in the brain

Background: An accumulating body of evidence points to the significance of neuroinflammation and immunogenetics in schizophrenia, and an imbalance of cytokines in the central nervous system (CNS) has been suggested to be associated with the disorder. Munc18-overexpressing mice (Munc18-OE) have provided a model for the study of the alterations that may underlie the symptoms of subjects with schizophrenia. The aim of the present study was to elucidate the involvement of neuroinflammation and cytokine imbalance in this model. Methods: Cytokines were evaluated in the cortex and the striatum of Munc18-OE and wild-type (WT) mice by enzyme-linked immunosorbent assay (ELISA). Protein levels of specific microglia and macrophage, astrocytic and neuroinflammation markers were quantified by western blot in the cortex and the striatum of Munc18-OE and WT mice. Results: Each cytokine evaluated (Interferon-gamma (IFN-gamma), Tumor Necrosis Factor-alpha (TNF-alpha), Interleukin-2 (IL-2) and CCL2 chemokine) was present at higher levels in the striatum of Munc18-OE mice than WT. Cortical TNF-alpha and IL-2 levels were significantly lower in Munc18-OE mice than WT mice. The microglia and macrophage marker CD11b was lower in the cortexes of Munc18-OE mice than WT, but no differences were observed in the striatum. Glial Fibrillary Acidic Protein (GFAP) and Nuclear Factor-kappaB (NF-kappa B)p65 levels were not different between the groups. Interleukin-1beta (IL-1 beta) and IL-6 levels were beneath detection limits. Conclusions: The disrupted levels of cytokines detected in the brain of Munc18-OE mice was found to be similar to clinical reports and endorses study of this type for analysis of this aspect of the disorder. The lower CD11b expression in the cortex but not in the striatum of the Munc18-OE mice may reflect differences in physiological activity. The cytokine expression pattern observed in Munc18-OE mice is similar to a previously published model of schizophrenia caused by maternal immune activation. Together, these data suggest a possible role for an immune imbalance in this disorder.

Short-term in vitro responses of human peripheral blood monocytes to ferritic stainless steel fiber networks.

Beneficial effects on bone-implant bonding may accrue from ferromagnetic fiber networks on implants which can deform in vivo inducing controlled levels of mechanical strain directly in growing bone. This approach requires ferromagnetic fibers that can be implanted in vivo without stimulating undue inflammatory cell responses or cytotoxicity. This study examines the short-term in vitro responses, including attachment, viability, and inflammatory stimulation, of human peripheral blood monocytes to 444 ferritic stainless steel fiber networks. Two types of 444 networks, differing in fiber cross section and thus surface area, were considered alongside austenitic stainless steel fiber networks, made of 316L, a widely established implant material. Similar high percent seeding efficiencies were measured by CyQuant® on all fiber networks after 48 h of cell culture. Extensive cell attachment was confirmed by fluorescence and scanning electron microscopy, which showed round monocytes attached at various depths into the fiber networks. Medium concentrations of lactate dehydrogenase (LDH) and tumor necrosis factor alpha (TNF-α) were determined as indicators of viability and inflammatory responses, respectively. Percent LDH concentrations were similar for both 444 fiber networks at all time points, whereas significantly lower than those of 316L control networks at 24 h. All networks elicited low-level secretions of TNF-α, which were significantly lower than that of the positive control wells containing zymosan. Collectively, the results indicate that 444 networks produce comparable responses to medical implant grade 316L networks and are able to support human peripheral blood monocytes in short-term in vitro cultures without inducing significant inflammatory or cytotoxic effects.

Effects of cyanobacterial toxin microcystin-LR on the transcription levels of immune-related genes in grass carp Ctenopharyngodon idella

Recent studies in mammals have revealed that the cyanobacterial toxin MC-LR suppresses immune functions. Nevertheless, immunotoxic effects of microcystins have been little studied in fish. In this paper, we present the profiles of the immune modulation of MC-LR in grass carp, and quantitative real-time PCR methodology was developed for the measurement of relative transcription changes of six immune-related genes in the spleen and head kidney of the grass carp Ctenopharyngodon idella, which were intraperitoneally injected with 50 mu g MC-LR center dot kg(-1) body weight in a three-week period. This study was focused exclusively on gene transcription level changes at different time points after MC-LR exposure, so, only one dose was given. The investigated genes were interleukin-1 beta (IL-1 beta), tumor necrosis factor-alpha (TNF-alpha), type I interferon (Type I IFN), peptidoglycan recognition protein-L (PGRP-L), immunoglobulin M (IgM) and major histocompatibility complex class I (MHC-I) genes. The results demonstrated that the transcription levels of the TNF-alpha, type I IFN, and PGRP-L genes in the spleen and head kidney were significantly low at all time points, and those of IL-1 beta were significantly low in the head kidney at different time points. In addition, IgM and MHC-I transcription levels were only significantly low in the spleen and head kidney at 21 d postinjection. The changes in the transcription levels of immune-related genes induced by MC-LR confirmed its effect on inhibiting immune function at the transcription level.

Immunomodulation and antitumor activity of kappa-carrageenan oligosaccharides

The modulation of carrageenan oligosaccharides from Kappaphycus striatum on the immune system in S 180-bearing mice was investigated. The mice inoculated with S180 cell suspension were treated p.o. with carrageenan oligosaccharides (50, 100 and 200 mu g/g) for 14 days. The effects of carrageenan oligosaccharides on transplantable tumors and macrophage phagocytosis, quantitative hemolysis of sheep red blood cells (QHS),. lymphocyte proliferation, the activity of natural killer cells (NK), production of interleukin-2 (IL-2) and tumor necrosis factor-alpha (TNF-alpha) were studied. Carrageenan oligosaccharides could significantly inhibit the growth of transplantable sarcoma S180 and increase macrophage phagocytosis, the form of antibody secreted by spleen cells, spleen lymphocyte proliferation, NK cells activity, serumal IL-2 and TNF-alpha level in S 180-bearing mice. Considering all these results, it is suggested that carrageenan oligosaccharides exert their antitumor effect by promoting the immune system. (c) 2005 Elsevier Ireland Ltd. All rights reserved.

Proteasome and NF-kappa B inhibiting phaeophytins from the green alga Cladophora fascicularis

Chemical examination of the green alga Cladophora fascicularis resulted in the isolation and characterization of a new porphyrin derivative, porphyrinolactone (1), along with five known phaeophytins 2-6 and fourteen sterols and cycloartanes. The structure of 1 was determined on the basis of spectroscopic analyses and by comparison of its NMR data with those of known phaeophytins. Compounds 1-6 displayed moderate inhibition of tumor necrosis factor alpha (TNF-alpha) induced nuclear factor-kappa B (NF-kappa B) activation, while 2 and 4 displayed potential inhibitory activity toward proteasome chymotripsin-like activation. The primary structure-activity relationship was also discussed.

Repeated concanavalin A challenge in mice induces an interleukin 10-producing phenotype and liver fibrosis.

Weekly injections of Concanavalin A (Con A) were performed in BALB/c mice to evaluate the pattern of cytokine production and liver injury. High serum levels of tumor necrosis factor alpha (TNF-alpha), interleukin 2 (IL-2), IL-4, and interferon gamma (IFN-gamma) were found in the serum after the first 2 injections of Con A but rapidly decreased from the third injection. Conversely, IL-10 serum levels after repeated Con A challenge increased by 7 times from week 1 to 20. In vivo depletion studies indicated that CD4(+) T cells are essential in IL-10 production. Hepatocyte necrosis was only observed after the first injections of Con A whereas centrilobular inflammatory infiltrates persisted up to 20 weeks. Perisinusoidal liver fibrosis was also increasingly detected in BALB/c mice, whereas no fibrous change was observed in nude mice after 6 weeks of Con A challenge. The number of stellate cells, detected by immunostaining, increased after 20 weeks of Con A injections. Liver cytokine messenger RNA (mRNA) expression after 20 weeks showed expression of transforming growth factor beta1 (TGF-beta1), IL-10, and IL-4 whereas IL-2 was no more expressed. The present study shows that mice repeatedly injected with Con A develop liver fibrosis. The cytokine-release pattern observed after 1 injection of Con A is rapidly shifted towards an immunomodulatory phenotype characterized by the systemic production of large amounts of IL-10.

A genetically engineered thermally responsive sustained release curcumin depot to treat neuroinflammation.

Radiculopathy, a painful neuroinflammation that can accompany intervertebral disc herniation, is associated with locally increased levels of the pro-inflammatory cytokine tumor necrosis factor alpha (TNFα). Systemic administration of TNF antagonists for radiculopathy in the clinic has shown mixed results, and there is growing interest in the local delivery of anti-inflammatory drugs to treat this pathology as well as similar inflammatory events of peripheral nerve injury. Curcumin, a known antagonist of TNFα in multiple cell types and tissues, was chemically modified and conjugated to a thermally responsive elastin-like polypeptide (ELP) to create an injectable depot for sustained, local delivery of curcumin to treat neuroinflammation. ELPs are biopolymers capable of thermally-triggered in situ depot formation that have been successfully employed as drug carriers and biomaterials in several applications. ELP-curcumin conjugates were shown to display high drug loading, rapidly release curcumin in vitro via degradable carbamate bonds, and retain in vitro bioactivity against TNFα-induced cytotoxicity and monocyte activation with IC50 only two-fold higher than curcumin. When injected proximal to the sciatic nerve in mice via intramuscular (i.m.) injection, ELP-curcumin conjugates underwent a thermally triggered soluble-insoluble phase transition, leading to in situ formation of a depot that released curcumin over 4days post-injection and decreased plasma AUC 7-fold.

Rapid complete response of metastatic melanoma in a patient undergoing ipilimumab immunotherapy in the setting of active ulcerative colitis.

While blockade of the cytotoxic T-lymphocyte antigen-4 (CTLA-4) T cell regulatory receptor has become a commonly utilized strategy in the management of advanced melanoma, many questions remain regarding the use of this agent in patient populations with autoimmune disease. We present a case involving the treatment of a patient with stage IV melanoma and ulcerative colitis (UC) with anti-CTLA-4 antibody immunotherapy. Upon initial treatment, the patient developed grade III colitis requiring tumor necrosis factor-alpha (TNF-α) blocking antibody therapy, however re-treatment with anti-CTLA-4 antibody following a total colectomy resulted in a rapid complete response accompanied by the development of a tracheobronchitis, a previously described extra-intestinal manifestation of UC. This case contributes to the evolving literature on the use of checkpoint inhibitors in patients also suffering from autoimmune disease, supports future clinical trials investigating the use of these agents in patients with autoimmune diseases, and suggests that an understanding of the specific molecular pathways involved in a patient's autoimmune pathology may provide insight into the development of more effective novel combinatorial immunotherapeutic strategies.

Drug Delivery through the Blood-Brain Barrier through Use of Vascular Cell Adhesion Molecule 1- Targeted Nanoparticles

The blood brain barrier (BBB) is a semi-permeable membrane separating the brain from the bloodstream, preventing many drugs that treat neurological diseases, such as Alzheimer’s and Parkinson’s, from reaching the brain. Our project aimed to create a novel drug delivery system targeting the brain during neural inflammation. We developed a cationic solid lipid nanoparticle (CSLN) complex composed of cationic nanoparticles, biotin, streptavidin, and anti-vascular cell adhesion molecule-1 (anti- VCAM-1) antibodies. The anti-VCAM-1 antibody is used to target VCAM-1, a cell adhesion protein found on the BBB endothelium. VCAM-1 expression is elevated in the presence of inflammatory molecules, such as tumor necrosis factor-alpha (TNF- α). Through the use of a simple BBB model, results showed that our novel drug delivery system experienced some level of success in targeting the brain inflammation due to increasing TNF-α concentrations. This is promising for drug delivery research and provides support for VCAM-1 targeting using more robust and complex BBB models.