Toll-like receptor activation regulates the paracrine effect of adipose-derived mesenchymal stem cells on reversing osteoarthritic phenotype of chondrocytes.

BACKGROUND: The therapeutic efficacy of intra-articular mesenchymal stem cells (MSCs) injection for patients with osteoarthritis (OA) currently exhibits inconsistency, and the underlying mechanism remains elusive. It has been postulated that the immunomodulatory properties and paracrine activity of MSCs might be influenced by the inflammatory micro-environment within osteoarthritic joints, potentially contributing to this observed inconsistency. METHODS: Adipose-derived MSCs (ADSCs) were isolated from SD rats and pre-treated with Toll-like receptor 3 (TLR3) agonist Poly I:C or Toll-like receptor 4 (TLR4) agonist LPS. The pre-treated ADSCs were then co-cultured with IL-1ß-induced osteoarthritic chondrocytes using a Transwell system to analyze the paracrine effect of ADSCs on reversing the osteoarthritic phenotype of chondrocytes. RESULTS: RT-PCR and Western blot analysis revealed that Poly I:C and LPS pre-treatments up-regulated the expression of IL-10 and IL-6 in ADSCs, respectively. Furthermore, only Poly I:C-preconditioned ADSCs significantly promoted proliferation while inhibiting apoptosis in IL-1ß-treated chondrocytes. Additionally, Poly I:C-preconditioned ADSCs downregulated MMP13 expression while upregulating aggrecan and collagen II expression levels in IL-1ß-treated chondrocytes. CONCLUSIONS: TLR3 activation polarizes ADSCs into an immunomodulatory phenotype distinct from TLR4 activation, exerting differential effects on reversing the osteoarthritic phenotype of chondrocytes; thus indicating that MSCs' paracrine effect regulated by TLRs signaling impacts the efficacy of intra-articular MSCs injection.

Synergistic Inhibitory Effect of Lactobacillus Cell Lysates and Butyrate on Poly I:C-Induced IL-8 Production in Human Intestinal Epithelial Cells.

Postbiotics include cell lysates (CLs), enzymes, cell wall fragments, and heat-killed bacteria derived from probiotics. Although postbiotics are increasingly being considered for their potential health-promoting properties, the effects of postbiotics on virus-mediated inflammatory responses in the intestine have not been elucidated. Hence, the present study aimed to examine whether CLs of Lactipantibacillus plantarum (LP CL) and Lacticaseibacillus rhamnosus GG (LR CL) could inhibit virus-mediated inflammatory responses in the human intestinal epithelial cell line HT-29 in vitro. Pretreatment with LP CL and LR CL significantly inhibited interleukin (IL)-8 production, which was induced by poly I:C, a synthetic analog of double-stranded RNA (dsRNA) viruses, at the mRNA and protein levels in HT-29 cells. However, peptidoglycans and heat-killed L. plantarum and L. rhamnosus GG did not effectively inhibit IL-8 production. LP CL and LR CL attenuated the poly I:C-induced phosphorylation of ERK and JNK and the activation of NF-κB, suggesting that these CLs could inhibit poly I:C-induced IL-8 production by regulating intracellular signaling pathways in HT-29 cells. Furthermore, among the short-chain fatty acids, butyrate enhanced the inhibitory effect of CLs on poly I:C-induced IL-8 production at the mRNA and protein levels in HT-29 cells, while acetate and propionate did not. Taken together, these results suggest that both LP CL and LR CL could act as potent effector molecules that can inhibit virus-mediated inflammatory responses and confer synergistic inhibitory effects with butyrate in human intestinal epithelial cells.

Paternal immune activation by Poly I:C modulates sperm noncoding RNA profiles and causes transgenerational changes in offspring behavior.

Paternal pre-conceptual environmental experiences, such as stress and diet, can affect offspring brain and behavioral phenotypes via epigenetic modifications in sperm. Furthermore, maternal immune activation due to infection during gestation can reprogram offspring behavior and brain functioning in adulthood. However, the effects of paternal pre-conceptual exposure to immune activation on the behavior and physiology of offspring (F1) and grand-offspring (F2) are not currently known. We explored effects of paternal pre-conceptual exposure to viral-like immune activation on F1 and F2 behavioral and physiological phenotypes using a C57BL/6J mouse model. Males were treated with a single injection (intraperitoneal) of the viral mimetic polyinosinic:polycytidylic acid (Poly I:C: 12 mg/kg) then bred with naïve female mice four weeks after the Poly I:C (or 0.9% saline control) injection. The F1 offspring of Poly I:C treated fathers displayed increased depression-like behavior in the Porsolt swim test, an altered stress response in the novelty-suppressed feeding test, and significant transcriptomic changes in their hippocampus. Additionally, the F1 male offspring of Poly I:C treated F0 males showed significantly increased immune responsivity after a Poly I:C immune challenge (12 mg/kg). Furthermore, the F2 male grand-offspring took longer to enter and travelled significantly shorter distances in the light zone of the light/dark box. An analysis of the small noncoding RNA profiles in sperm from Poly I:C treated males and their male offspring revealed significant effects of Poly I:C on the sperm microRNA content at the time of conception and on the sperm PIWI-interacting RNA content of the male offspring. Notably, eight miRNAs with an FDR < 0.05 (miR-141-3p, miR-126b-5p, miR-669o-5p, miR-10b-3p, miR-471-5p, miR-463-5p, miR-148b-3p, and miR-181c-5p) were found to be significantly downregulated in the sperm of Poly I:C treated males. Collectively, we demonstrate that paternal pre-conceptual exposure to a viral immune challenge results in both intergenerational and transgenerational effects on brain and behavior that may be mediated by alterations in the sperm small noncoding RNA content.

Targeting TLR4/3 using chlorogenic acid ameliorates LPS+POLY I:C-induced acute respiratory distress syndrome via alleviating oxidative stress-mediated NLRP3/NF-κB axis.

Acute lung injury (ALI) or acute respiratory distress syndrome (ARDS) is a life-threatening condition caused due to significant pulmonary and systemic inflammation. Chlorogenic acid (CGA) has been shown to possess potent antioxidant, anti-inflammatory, and immunoprotective properties. However, the protective effect of CGA on viral and bacterial-induced ALI/ARDS is not yet explored. Hence, the current study is aimed to evaluate the preclinical efficacy of CGA in lipopolysaccharide (LPS) and polyinosinic:polycytidylic acid (POLY I:C)-induced ALI/ARDS models in vitro and in vivo. Human airway epithelial (BEAS-2B) cells exposed to LPS+POLY I:C significantly elevated oxidative stress and inflammatory signaling. Co-treatment with CGA (10 and 50 µM) prevented inflammation and oxidative stress mediated by TLR4/TLR3 and NLRP3 inflammasome axis. BALB/c mice, when chronically challenged with LPS+POLY I:C showed a significant influx of immune cells, up-regulation of pro-inflammatory cytokines, namely: IL-6, IL-1ß, and TNF-α, and treatment with intranasal CGA (1 and 5 mg/kg) normalized the elevated levels of immune cell infiltration as well as pro-inflammatory cytokines. D-Dimer, the serum marker for intravascular coagulation, was significantly increased in LPS+ POLY I:C challenged animals which was reduced with CGA treatment. Further, CGA treatment also has a beneficial effect on the lung and heart, as shown by improving lung physiological and cardiac functional parameters accompanied by the elevated antioxidant response and simultaneous reduction in tissue damage caused by LPS+POLY I:C co-infection. In summary, these comprehensive, in vitro and in vivo studies suggest that CGA may be a viable therapeutic option for bacterial and viral-induced ALI-ARDS-like pathology.

Ibrutinib Prevents Acute Lung Injury via Multi-Targeting BTK, FLT3 and EGFR in Mice.

Ibrutinib has potential therapeutic or protective effects against viral- and bacterial-induced acute lung injury (ALI), likely by modulating the Bruton tyrosine kinase (BTK) signaling pathway. However, ibrutinib has multi-target effects. Moreover, immunity and inflammation targets in ALI treatment are poorly defined. We investigated whether the BTK-, FLT3-, and EGFR-related signaling pathways mediated the protective effects of ibrutinib on ALI. The intratracheal administration of poly I:C or LPS after ibrutinib administration in mice was performed by gavage. The pathological conditions of the lungs were assessed by micro-CT and HE staining. The levels of neutrophils, lymphocytes, and related inflammatory factors in the lungs were evaluated by ELISA, flow cytometry, immunohistochemistry, and immunofluorescence. Finally, the expression of proteins associated with the BTK-, FLT3-, and EGFR-related signaling pathways were evaluated by Western blotting. Ibrutinib (10 mg/kg) protected against poly I:C-induced (5 mg/kg) and LPS-induced (5 mg/kg) lung inflammation. The wet/dry weight ratio (W/D) and total proteins in the bronchoalveolar lavage fluid (BALF) were markedly reduced after ibrutinib (10 mg/kg) treatment, relative to the poly I:C- and LPS-treated groups. The levels of ALI indicators (NFκB, IL-1ß, IL-6, TNF-α, IFN-γ, neutrophils, and lymphocytes) were significantly reduced after treatment. Accordingly, ibrutinib inhibited the poly I:C- and LPS-induced BTK-, FLT3-, and EGFR-related pathway activations. Ibrutinib inhibited poly I:C- and LPS-induced acute lung injury, and this may be due to its ability to suppress the BTK-, FLT3-, and EGFR-related signaling pathways. Therefore, ibrutinib is a potential protective agent for regulating immunity and inflammation in poly I:C- and LPS-induced ALI.

Poly I:C Exacerbates Airway Inflammation and Remodeling in Cigarette Smoke-Exposed Mice.

BACKGROUND: Chronic obstructive pulmonary disease (COPD) is a chronic respiratory disorder characterized by chronic inflammation and airway remodeling. Cigarette smoke (CS) and respiratory viruses are major causes of COPD development and exacerbation, but the mechanisms of these compounding factors on inflammation and pathological changes in airway structure still need further investigation. PURPOSE: This work aimed to investigate the effects and mechanisms of Poly I:C on pathological changes in CS-induced COPD mice, such as airway inflammation and remodeling. METHODS: From 1 to 8 weeks, the mice were exposed to CS, Poly I:C, or a combination of both. To compare the pathological changes among different groups over time, the mice were sacrificed at week 4, 8, 16, and 24, then the lungs were harvested to measure pulmonary pathology, inflammatory cytokines, and airway remodeling. RESULTS: Our data revealed that the fundamental characteristics of COPD, such as pulmonary pathological damage, the release of inflammatory mediators, and the remodeling of airway walls, were observed at week 8 in CS-exposed mice and these pathological changes persisted to week 16. Compared with the CS group, the pathological changes, including decreased lung function, inflammatory cell infiltration, alveolar destruction, and airway wall thickening, were weaker in the Poly I:C group. These pathological changes were observed at week 8 and persisted to week 16 in Poly I:C-induced mice. Furthermore, Poly I:C exacerbated lung tissue damage in CS-induced COPD mice. The decreased lung function, airway inflammation and remodeling were observed in the combined group at week 4, and these pathological changes persisted to week 24. Our research indicated that Poly I:C enhanced the expression of p-P38, p-JNK and p-NF-κB in CS-exposed mice. CONCLUSION: Poly I:C could promote airway inflammation and remodeling in CS-induced COPD mice probably by NF-κB and MAPK signaling.

Preweaning environmental enrichment alters neonatal ultrasonic vocalisations in a rat model for prenatal infections.

OBJECTIVE: Maternal infections are a well-known risk factor for neurodevelopmental defects. Such defects are associated with a range of symptoms, and environmental enrichment (EE) could be a promising approach to rehabilitate these. We used the well-established prenatal poly I:C (polyinosinic-polycytidylic acid) model in rats to examine the effects of preweaning EE on rat pups' ultrasonic vocalisations (USVs) when separated from their mothers. USVs are one of the earliest indicators of a pup's functional level and, thus, well-suited as a marker of neurodevelopmental abnormalities. METHODS: We used a two-by-two factorial design in which pregnant Sprague-Dawley rats received either saline or the viral mimic poly I:C, and one group of pups was exposed to preweaning enrichment. We measured maternal separation-induced USVs both before postnatal day (PND) 7 and after preweaning enrichment on PND 14. RESULTS: Poly I:C significantly reduced the number of USVs on PND 7. EE interacted with the poly I:C treatment in that poly I:C pups in the enrichment group called more, whereas saline pups in the enriched environment called less on PND 14 than the respective controls. CONCLUSION: We showed that the effects of maternal poly I:C on the offspring's USVs could be reduced by early EE. If replicated, it could open novel and safe avenues for treating children of mothers who were exposed to infections during pregnancy.

Docosahexaenoic Acid Ameliorates the Toll-Like Receptor 22-Triggered Inflammation in Fish by Disrupting Lipid Raft Formation.

BACKGROUND: Although dietary DHA alleviates Toll-like receptor (TLR)-associated chronic inflammation in fish, the underlying mechanism is not well understood. OBJECTIVES: This study aimed to explore the role of Tlr22 in the innate immunity of large yellow croaker and investigate the anti-inflammatory effects of DHA on Tlr22-triggered inflammation. METHODS: Head kidney-derived macrophages of croaker and HEK293T cells were or were not pretreated with 100 µM DHA for 10 h prior to polyinosinic-polycytidylic acid (poly I:C) stimulation. We executed qRT-PCR, immunoblotting, and lipidomic analysis to examine the impact of DHA on Tlr22-triggered inflammation and membrane lipid composition. In vivo, croakers (12.03 ± 0.05 g) were fed diets containing 0.2% [control (Ctrl)], 0.8%, and 1.6% DHA for 8 wk before injection with poly I:C. Inflammatory genes expression and rafts-related lipids and protein expression were measured in the head kidney. Data were analyzed by ANOVA or Student t test. RESULTS: The activation of Tlr22 by poly I:C induced inflammation, and DHA diminished Tlr22-targeted inflammatory gene expression by 56-73% (P ≤ 0.05). DHA reduced membrane sphingomyelin (SM) and SFA-containing phosphatidylcholine (SFA-PC) contents, as well as lipid raft marker caveolin 1 amounts. Furthermore, lipid raft disruption suppressed Tlr22-induced Nf-κb and interferon h activation and p65 nuclear translocation. In vivo, expression of Tlr22 target inflammatory genes was 32-64% lower in the 1.6% DHA group than in the Ctrl group upon poly I:C injection (P ≤ 0.05). Also, the 1.6% DHA group showed a reduction in membrane SM and SFA-PC contents, accompanied by a decrease in caveolin 1 amounts, compared with the Ctrl group. CONCLUSIONS: The activation of Tlr22 signaling depends on lipid rafts, and DHA ameliorates the Tlr22-triggered inflammation in both head kidney and head kidney-derived macrophages of croaker partially by altering membrane SMs and SFA-PCs that are required for lipid raft organization.

In vitro 2D and 3D cancer models to evaluate compounds that modulate macrophage polarization.

In vitro cancer models that can identify novel immunomodulating compounds are essential. Using a 3D multicellular tumor spheroid (MCTS) model comprising cancer cells, fibroblasts, and macrophages, we tested tumor-associated macrophage (TAM)-inhibiting compounds (CCL2 Ab, CSF1R inhibitor, CSF1R Ab) and TAM-reprograming compounds (poly I:C, CD40 Ab, CD40 ligand) for their effects on monocyte infiltration and polarization in tumor spheroids. For characterization of macrophage polarization, we measured the expression of CD206, CD163, CD86, MHC II, CD40, and CD14 and measured 43 soluble factors in the 3D MCTS cultures. 2D macrophage models were evaluated for comparison. A CSF1R inhibitor prevented infiltration of monocytes into pancreatic cancer spheroids, and macrophages treated with the inhibitor showed decreased expression of M2 markers. Treatment with a CD40 ligand and poly I:C induced M1 macrophage polarization in our models. We propose that these models can be used to improve the drug screening process of anti-cancer immunotherapies targeting macrophages.

Metformin Protects Against Inflammation, Oxidative Stress to Delay Poly I:C-Induced Aging-Like Phenomena in the Gut of an Annual Fish.

Metformin, a clinical agent of type 2 diabetes, is reported as a potential geroprotector. Viral infection induces phenotypes of senescence in human T cells, and polyinosinic:polycytidylic acid (poly I:C), a viral mimic, induces upregulation of senescence-associated beta-galactosidase (SA-ß-gal) activity in the ovary of the annual fish Nothobranchius guentheri. However, the effects and mechanisms of metformin on poly I:C-induced aging-like phenomena are poorly understood in vertebrates. In this study, the activity of SA-ß-gal increased in the gut of 12-month-old fish and poly I:C-injected 6-month-old fish, compared to 6-month-old control fish, indicating that poly I:C induces aging-like phenomena in the gut of the fish. Metformin supplementation retarded accumulation of SA-ß-gal in the gut of old fish and poly I:C-treated young fish. The results of qPCR analysis showed that metformin reduced NF-κB-mediated inflammatory response including the decreased level of proinflammatory cytokine IL-8 and increased expression of anti-inflammatory cytokine IL-10 in the gut of the fish with natural aging and poly I:C-injected 6-month-old fish. Metformin also exhibited antioxidant effects, as it reduced reactive oxygen species production that is associated with the upregulation of FoxO3a and PGC-1α in the gut of 6-month-old fish with poly I:C injection. Expression of AMPK and SIRT1 was reduced in the gut of 6-month-old fish with poly I:C treatment, and feeding metformin reversed these declines. Taken together, the present study suggested that poly I:C injection led to aging-like phenomena in the gut and metformin activated AMPK and SIRT1 to reduce NF-κB-mediated inflammation and resist oxidative stress via enhanced expression of FoxO3a and PGC-1α and finally delayed gut aging in vertebrates.

SARS-CoV-2 Nucleocapsid Protein Targets RIG-I-Like Receptor Pathways to Inhibit the Induction of Interferon Response.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the coronavirus disease 2019 (COVID-19) that has resulted in the current pandemic. The lack of highly efficacious antiviral drugs that can manage this ongoing global emergency gives urgency to establishing a comprehensive understanding of the molecular pathogenesis of SARS-CoV-2. We characterized the role of the nucleocapsid protein (N) of SARS-CoV-2 in modulating antiviral immunity. Overexpression of SARS-CoV-2 N resulted in the attenuation of retinoic acid inducible gene-I (RIG-I)-like receptor-mediated interferon (IFN) production and IFN-induced gene expression. Similar to the SARS-CoV-1 N protein, SARS-CoV-2 N suppressed the interaction between tripartate motif protein 25 (TRIM25) and RIG-I. Furthermore, SARS-CoV-2 N inhibited polyinosinic: polycytidylic acid [poly(I:C)]-mediated IFN signaling at the level of Tank-binding kinase 1 (TBK1) and interfered with the association between TBK1 and interferon regulatory factor 3 (IRF3), subsequently preventing the nuclear translocation of IRF3. We further found that both type I and III IFN production induced by either the influenza virus lacking the nonstructural protein 1 or the Zika virus were suppressed by the SARS-CoV-2 N protein. Our findings provide insights into the molecular function of the SARS-CoV-2 N protein with respect to counteracting the host antiviral immune response.

Application of a Scavenger Receptor A1-Targeted Polymeric Prodrug Platform for Lymphatic Drug Delivery in HIV.

We have developed a macromolecular prodrug platform based on poly(l-lysine succinylated) (PLS) that targets scavenger receptor A1 (SR-A1), a receptor expressed by myeloid and endothelial cells. We demonstrate the selective uptake of PLS by murine macrophage, RAW 264.7 cells, which was eliminated upon cotreatment with the SR-A inhibitor polyinosinic acid (poly I). Further, we observed no uptake of PLS in an SR-A1-deficient RAW 264.7 cell line, even after 24 h incubation. In mice, PLS distributed to lymphatic organs following i.v. injection, as observed by ex vivo fluorescent imaging, and accumulated in lymph nodes following both i.v. and i.d. administrations, based on immunohistochemical analysis with high-resolution microscopy. As a proof-of-concept, the HIV antiviral emtricitabine (FTC) was conjugated to the polymer's succinyl groups via ester bonds, with a drug loading of 14.2% (wt/wt). The prodrug (PLS-FTC) demonstrated controlled release properties in vitro with a release half-life of 15 h in human plasma and 29 h in esterase-inhibited plasma, indicating that drug release occurs through both enzymatic and nonenzymatic mechanisms. Upon incubation of PLS-FTC with human peripheral blood mononuclear cells (PBMCs), the released drug was converted to the active metabolite FTC triphosphate. In a pharmacokinetic study in rats, the prodrug achieved ∼7-19-fold higher concentrations in lymphatic tissues compared to those in FTC control, supporting lymphatic-targeted drug delivery. We believe that the SR-A1-targeted macromolecular PLS prodrug platform has extraordinary potential for the treatment of infectious diseases.

A Novel Subset of Anti-Inflammatory CD138+ Macrophages Is Deficient in Mice with Experimental Lupus.

Dead cells accumulating in the tissues may contribute to chronic inflammation. We examined the cause of impaired apoptotic cell clearance in human and murine lupus. Dead cells accumulated in bone marrow from lupus patients but not from nonautoimmune patients undergoing myeloablation, where they were efficiently removed by macrophages (MΦ). Impaired apoptotic cell uptake by MΦ also was seen in mice treated i.p. with pristane (develop lupus) but not mineral oil (MO) (do not develop lupus). The inflammatory response to both pristane and MO rapidly depleted resident (Tim4+) large peritoneal MΦ. The peritoneal exudate of pristane-treated mice contained mainly Ly6Chi inflammatory monocytes; whereas in MO-treated mice, it consisted predominantly of a novel subset of highly phagocytic MΦ resembling small peritoneal MΦ (SPM) that expressed CD138+ and the scavenger receptor Marco. Treatment with anti-Marco-neutralizing Abs and the class A scavenger receptor antagonist polyinosinic acid inhibited phagocytosis of apoptotic cells by CD138+ MΦ. CD138+ MΦ expressed IL-10R, CD206, and CCR2 but little TNF-α or CX3CR1. They also expressed high levels of activated CREB, a transcription factor implicated in generating alternatively activated MΦ. Similar cells were identified in the spleen and lung of MO-treated mice and also were induced by LPS. We conclude that highly phagocytic, CD138+ SPM-like cells with an anti-inflammatory phenotype may promote the resolution of inflammation in lupus and infectious diseases. These SPM-like cells are not restricted to the peritoneum and may help clear apoptotic cells from tissues such as the lung, helping to prevent chronic inflammation.

Combinatorial contextualization of peptidic epitopes for enhanced cellular immunity.

Invocation of cellular immunity by epitopic peptides remains largely dependent on empirically developed protocols, such as interfusion of aluminum salts or emulsification using terpenoids and surfactants. To explore novel vaccine formulation, epitopic peptide motifs were co-programmed with structural motifs to produce artificial antigens using our "motif-programming" approach. As a proof of concept, we used an ovalbumin (OVA) system and prepared an artificial protein library by combinatorially polymerizing MHC class I and II sequences from OVA along with a sequence that tends to form secondary structures. The purified endotoxin-free proteins were then examined for their ability to activate OVA-specific T-cell hybridoma cells after being processed within dendritic cells. One clone, F37A (containing three MHC I and two MHC II OVA epitopes), possessed a greater ability to evoke cellular immunity than the native OVA or the other artificial antigens. The sensitivity profiles of drugs that interfered with the F37A uptake differed from those of the other artificial proteins and OVA, suggesting that alteration of the cross-presentation pathway is responsible for the enhanced immunogenicity. Moreover, F37A, but not an epitopic peptide, invoked cellular immunity when injected together with monophosphoryl lipid A (MPL), and retarded tumor growth in mice. Thus, an artificially synthesized protein antigen induced cellular immunity in vivo in the absence of incomplete Freund's adjuvant or aluminum salts. The method described here could be potentially used for developing vaccines for such intractable ailments as AIDS, malaria and cancer, ailments in which cellular immunity likely play a crucial role in prevention and treatment.

Macrophage- and neutrophil-derived TNF-α instructs skin langerhans cells to prime antiviral immune responses.

Dendritic cells are major APCs that can efficiently prime immune responses. However, the roles of skin-resident Langerhans cells (LCs) in eliciting immune responses have not been fully understood. In this study, we demonstrate for the first time, to our knowledge, that LCs in cynomolgus macaque skin are capable of inducing antiviral-specific immune responses in vivo. Targeting HIV-Gag or influenza hemagglutinin Ags to skin LCs using recombinant fusion proteins of anti-Langerin Ab and Ags resulted in the induction of the viral Ag-specific responses. We further demonstrated that such Ag-specific immune responses elicited by skin LCs were greatly enhanced by TLR ligands, polyriboinosinic polyribocytidylic acid, and R848. These enhancements were not due to the direct actions of TLR ligands on LCs, but mainly dependent on TNF-α secreted from macrophages and neutrophils recruited to local tissues. Skin LC activation and migration out of the epidermis are associated with macrophage and neutrophil infiltration into the tissues. More importantly, blocking TNF-α abrogated the activation and migration of skin LCs. This study highlights that the cross-talk between innate immune cells in local tissues is an important component for the establishment of adaptive immunity. Understanding the importance of local immune networks will help us to design new and effective vaccines against microbial pathogens.

Signaling through Toll-like receptor 3 and Dectin-1 potentiates the capability of human monocyte-derived dendritic cells to promote T-helper 1 and T-helper 17 immune responses.

BACKGROUND AIMS: Recent studies have shown that the ligation of Toll-like receptor 3 (TLR3) or Dectin-1 on human monocyte-derived dendritic cells (MoDC) elicits their maturation, but with a different outcome on immunomodulation. Therefore the aim of this work was to study the response of MoDC to the combined effect of polyinosinic:polycytydilic acid [Poly (I:C)] and curdlan, selective TLR3 and Dectin-1 agonists, respectively. METHODS: Immature MoDC, generated from human monocytes, were treated with Poly (I:C), curdlan or their combination for 2 days. Phenotypic characteristics of MoDC were determined by flow cytometry, and cytokine production was measured by enzyme-linked immunosorbent assay (ELISA) and FlowCytomix, while the stimulatory capability of MoDC was tested using a mixed leukocyte reaction assay. RESULTS: The combination of Poly (I:C) and curdlan induced phenotypic maturation of MoDC with the capability to stimulate an alloreactive response. Such treated MoDC up-regulated the production of interleukin (IL)-12, IL-23 and IL-10, compared with the effect of Poly (I:C) alone. Curdlan-treated MoDC stimulated the production of IL-17 by alloreactive CD4 (+) T cells more strongly than Poly (I:C)-treated MoDC. The opposite effect was observed for interferon(IFN)-γ production. When combined, these agonists primed MoDC to increase further the production of IFN-γ by CD4 (+) T cells in co-culture, especially those of naive (CD45RA (+)) phenotype, and IL-17 by memory (CD45RO (+)) CD4 (+) T cells. CONCLUSIONS: Ligation of TLR3 and Dectin-1 receptor up-regulates T-helper (Th) 1 and Th17 immune responses compared with single agonists. These findings may have therapeutic implications for the use of MoDC in immunotherapy.

Selective targeting of microglia by quantum dots.

BACKGROUND: Microglia, the resident immune cells of the brain, have been implicated in brain injury and various neurological disorders. However, their precise roles in different pathophysiological situations remain enigmatic and may range from detrimental to protective. Targeting the delivery of biologically active compounds to microglia could help elucidate these roles and facilitate the therapeutic modulation of microglial functions in neurological diseases. METHODS: Here we employ primary cell cultures and stereotaxic injections into mouse brain to investigate the cell type specific localization of semiconductor quantum dots (QDs) in vitro and in vivo. Two potential receptors for QDs are identified using pharmacological inhibitors and neutralizing antibodies. RESULTS: In mixed primary cortical cultures, QDs were selectively taken up by microglia; this uptake was decreased by inhibitors of clathrin-dependent endocytosis, implicating the endosomal pathway as the major route of entry for QDs into microglia. Furthermore, inhibiting mannose receptors and macrophage scavenger receptors blocked the uptake of QDs by microglia, indicating that QD uptake occurs through microglia-specific receptor endocytosis. When injected into the brain, QDs were taken up primarily by microglia and with high efficiency. In primary cortical cultures, QDs conjugated to the toxin saporin depleted microglia in mixed primary cortical cultures, protecting neurons in these cultures against amyloid beta-induced neurotoxicity. CONCLUSIONS: These findings demonstrate that QDs can be used to specifically label and modulate microglia in primary cortical cultures and in brain and may allow for the selective delivery of therapeutic agents to these cells.

Aß potentiates inflammatory activation of glial cells induced by scavenger receptor ligands and inflammatory mediators in culture.

Alzheimer disease (AD) is a neurodegenerative disorder characterized by the accumulation of ß amyloid (Aß) aggregates. Aß induces the inflammatory activation of glia, inducing secretion of Interleukin 1ß (IL1ß), nitric oxide (NO) and superoxide radicals. The specific receptor responsible for the induction of inflammatory activation by Aß, is still an open question. We propose that scavenger receptors (SR) participate in the activation of glia by Aß. We assessed production of NO, synthesis of IL1ß and activation of ERK, JNK and NF-κB signaling pathways by Western blot, in primary rat glial cultures exposed to SR ligands (fucoidan and Poly I), LPS + IFNγ (LI), and Aß. Poly I but not fucoidan nor fibrillar Aß increased threefold NO production by astrocytes in a time-dependent manner. Fucoidan and Poly I increased 5.5- and 3.5-fold NO production by microglia, and co-stimulation with Aß increased an additional 60% NO induced by SR ligands. Potentiation by Aß was observed later for astrocytes than for microglia. In astrocytes, co-stimulation with Aß potentiated ERK and JNK activation in response to Fucoidan and Poly I, whereas it reduced induction of JNK activation by LI and left unaffected NF-κB activation induced by LI. Levels of pro-IL1ß in astrocytes increased with Aß, SR ligands and LI, and were potentiated by co-stimulation with Aß. Our results suggest that SRs play a role on inflammatory activation, inducing production of NO and IL1ß, and show potentiation by Aß. Potentiation of the inflammatory response of Aß could be meaningful for the activation of glia observed in AD.

Polyinosinic acid decreases sequestration and improves systemic therapy of measles virus.

Off-target binding or vector sequestration can significantly limit the efficiency of systemic virotherapy. We report here that systemically administered oncolytic measles virus (MV) was rapidly sequestered by the mononuclear phagocytic system (MPS) of the liver and spleen in measles receptor CD46-positive and CD46-negative mice. Since scavenger receptors on Kupffer cells are responsible for the elimination of blood-borne pathogens, we investigated here if MV uptake was mediated by scavenger receptors on Kupffer cells. Pretreatment of cells with poly(I), a scavenger receptor ligand, reduced MV expression by 99% in murine (J774A.1) macrophages and by 50% in human (THP-1) macrophages. Pre-dosing of mice with poly(I) reduced MPS sequestration of MV and increased circulating levels of MV by 4 to 15-folds at 2 min post virus administration. Circulating virus was still detectable 30 min post infusion in mice pre-dosed with poly(I) whereas no detectable MV was found at 5-10 min post infusion if mice did not receive poly(I). MPS blockade by poly(I) enhanced virus delivery to human ovarian SKOV3ip.1 and myeloma KAS6/1 xenografts in mice. Higher gene expression and improved control of tumor growth was noted early post therapy. Based on these results, incorporation of MPS blockade into MV treatment regimens is warranted.

Influence of preparation method on polynucleotide conformation and pharmacological activity of lipoplex.

Conformations of polyinosinic acid [poly(I)] and polycytidylic acid [poly(C)] in liposomes (lipoplex) were investigated by both circular dichroism (CD) spectroscopy and fluorescence resonance energy transfer (FRET) measurements, and compared with those in aqueous solution. The results indicate that poly(I) and poly(C) take double-stranded structure in aqueous solution at pH 6.5-7.5 in the presence of NaCl at higher concentration than 50mM. Although lipoplex was prepared without NaCl to avoid aggregation of lipoplex particles, poly(I) and poly(C) were double-stranded in pre-mixed poly(I)/poly(C) lipoplex (pre-mixed LIC), prepared by adding a mixed solution of poly(I) and poly(C) to the cationic liposomes. However, poly(I) and poly(C) did not take double-stranded structure in separately mixed LIC, prepared by separate addition of poly(I) solution and poly(C) solution to the cationic liposomes. The physicochemical properties (particle diameter and zeta potential) of pre-mixed LIC and separately mixed LIC were not different, but the anti-proliferative effect of pre-mixed LIC on human epidermoid carcinoma A431 cells was about eight times greater than that of separately mixed LIC. Our results indicate that polynucleotide conformation in lipoplex is markedly influenced by the preparation method, and the polynucleotide conformation in lipoplex has a substantial effect on pharmacological activity.