Development of Exhausted Memory Monocytes and Underlying Mechanisms.

Pathogenic inflammation and immuno-suppression are cardinal features of exhausted monocytes increasingly recognized in septic patients and murine models of sepsis. However, underlying mechanisms responsible for the generation of exhausted monocytes have not been addressed. In this report, we examined the generation of exhausted primary murine monocytes through prolonged and repetitive challenges with high dose bacterial endotoxin lipopolysaccharide (LPS). We demonstrated that repetitive LPS challenges skew monocytes into the classically exhausted Ly6Chi population, and deplete the homeostatic non-classical Ly6Clo population, reminiscent of monocyte exhaustion in septic patients. scRNAseq analyses confirmed the expansion of Ly6Chi monocyte cluster, with elevation of pathogenic inflammatory genes previously observed in human septic patients. Furthermore, we identified CD38 as an inflammatory mediator of exhausted monocytes, associated with a drastic depletion of cellular NAD+; elevation of ROS; and compromise of mitochondria respiration, representative of septic monocytes. Mechanistically, we revealed that STAT1 is robustly elevated and sustained in LPS-exhausted monocytes, dependent upon the TRAM adaptor of the TLR4 pathway. TRAM deficient monocytes are largely resistant to LPS-mediated exhaustion, and retain the non-classical homeostatic features. Together, our current study addresses an important yet less-examined area of monocyte exhaustion, by providing phenotypic and mechanistic insights regarding the generation of exhausted monocytes.

Dissolvable microneedles based on Panax notoginseng polysaccharide for transdermal drug delivery and skin dendritic cell activation.

This work explored the feasibility of using biological polysaccharide to fabricate dissolvable microneedles (MNs) for the purpose of transdermal drug delivery and skin dendritic cell (DC) activation. Panax notoginseng polysaccharide (PNPS), a naturally derived immunoactive macromolecule, was used to fabricate dissolvable MNs. The prepared PNPS MNs showed a satisfactory mechanical strength and a skin penetration depth. By Franz diffusion cell assay, the PNPS MNs demonstrated a high transdermal delivery amount of model drugs. Furthermore, with the assistance of MNs, PNPS easily penetrated across the stratum corneum and target ear skin DCs, activating the maturation and migration of immunocytes by increasing the expressions of CD40, CD80, CD86, and MHC II of skin DCs. Consequently, the matured DCs migrated to the auricular draining lymph nodes and increased the proportions of CD4+ T and CD8+ T cells. Thus, PNPS might be a promising biomaterial for transdermal drug delivery, with adjuvant potential.

Susceptibility of cyclin-dependent kinase inhibitor 1-deficient mice to rheumatoid arthritis arising from interleukin-1ß-induced inflammation.

We recently reported that cyclin-dependent kinase inhibitor 1 (p21) deficiency induces osteoarthritis susceptibility. Here, we determined the mechanism underlying the effect of p21 in synovial and cartilage tissues in RA. The knee joints of p21-knockout (p21-/-) (n = 16) and wild type C57BL/6 (p21+/+) mice (n = 16) served as in vivo models of collagen antibody-induced arthritis (CAIA). Arthritis severity was evaluated by immunological and histological analyses. The response of p21 small-interfering RNA (siRNA)-treated human RA FLSs (n = 5 per group) to interleukin (IL)-1ß stimulation was determined in vitro. Arthritis scores were higher in p21-/- mice than in p21+/+ mice. More severe synovitis, earlier loss of Safranin-O staining, and cartilage destruction were observed in p21-/- mice compared to p21+/+ mice. p21-/- mice expressed higher levels of IL-1ß, TNF-α, F4/80, CD86, p-IKKα/ß, and matrix metalloproteinases (MMPs) in cartilage and synovial tissues via IL-1ß-induced NF-kB signaling. IL-1ß stimulation significantly increased IL-6, IL-8, and MMP expression, and enhanced IKKα/ß and IκBα phosphorylation in human FLSs. p21-deficient CAIA mice are susceptible to RA phenotype alterations, including joint cartilage destruction and severe synovitis. Therefore, p21 may have a regulatory role in inflammatory cytokine production including IL-1ß, IL-6, and TNF-α.

Thymoquinone ameliorates Pachycondyla sennaarensis venom-induced acute toxic shock in male rats.

BACKGROUND: For many decades, the sting of Samsun ant (Pachycondyla sennaarensis) has been a serious clinical challenge for the people living in some of the major Middle East and Asian countries. In the present study, the therapeutic potential of Nigella sativa derived plant extract component, thymoquinone (TQ) has been tested against the Samsun ant venom (SAV) at the toxic dose in the rats. METHODS: The adult male rats were divided into four groups (n = 10): control, SAV treated, SAV + TQ treated and TQ alone treated. It was found that the sub-lethal dose of SAV alters not only many of the kidney and liver function markers but also induces oxidative stress in the animals. Moreover, the SAV also disturbs various immunological parameters including expression of PMNs, CD-80, CD-86, interleukins and other cytokines compromising the affected organism towards mild to severe allergic reactions including life-risking anaphylaxis. RESULTS: The plant extract, TQ, effectively restores many of the biochemical and oxidative stress parameters comparable to the normal concomitant with improving the immunological aspects that might attributive in relieving from SAV-induced toxicity and allergic reactions in the affected organism to a greater extent. CONCLUSION: Hence, TQ has an excellent antidote property against SAV-induced toxicities in vivo. Although the study is a vivid indication of the potential therapeutic potential of TQ against the SAV induced in vivo toxicity, yet the actual mechanism of interaction translating the toxicity amelioration warrants further investigations.

Rice bran oil ameliorates inflammatory responses by enhancing mitochondrial respiration in murine macrophages.

Previous studies have revealed the anti-inflammatory properties of rice bran oil (RBO), but the detailed mechanisms are poorly understood. Recent studies on the molecular/cellular anti-inflammatory mechanisms of dietary components have demonstrated that mitochondrial respiration plays a key role in macrophage functioning. Since dietary lipids are major substrates for mitochondrial respiration through ß-oxidation, the current study examined whether RBO regulates inflammatory responses by modulating mitochondrial energy metabolism. Palm oil (PO), enriched with palmitic acid which are known to be effectively taken up by cells and used for oxidative phosphorylation, served as a positive control. In the in vitro model of LPS-stimulated RAW 264.7 murine cells, the levels of pro-inflammatory cytokines (IL-6 and TNF-α) in the culture supernatant were significantly reduced by RBO treatment. In contrast, secretion of the anti-inflammatory cytokine IL-10 was upregulated by RBO. Transcription of genes encoding inflammatory mediator molecules (COX-2 and iNOS) and expression of activation markers (CD80, CD86, and MHC-II) in LPS-stimulated RAW 264.7 cells were suppressed by RBO. Mitochondrial respiration (as assessed by an extracellular flux analyzer) increased upon RBO treatment, as the basal respiration, maximal respiration, ATP production, and spare respiratory capacity were upregulated. In an in vivo study, C57BL/6 mice were fed a negative control diet containing corn oil (CO), PO, or RBO for 4 weeks, and bone marrow-derived macrophages (BMDM) were isolated from their tibias and femurs. In pro-inflammatory M1-polarized BMDM (M1-BMDM), the RBO-induced suppression of IL-6 and TNF-α was recapitulated in vivo. Mitochondrial respiration in M1-BMDM also increased following the RBO intervention and the PO control treatment as compared to CO fed negative control. Overall, the current study for the first time demonstrates that RBO regulates inflammatory responses in murine macrophages by upregulating mitochondrial respiration. Further clinical studies are required to validate the animal study.

Gastrodin, a traditional Chinese medicine monomer compound, can be used as adjuvant to enhance the immunogenicity of melanoma vaccines.

Gastrodin (GAS) is a Chinese medicine with wide application for the treatment of nervous system disease. Previous studies reported that GAS exhibited non-specific immunomodulatory activities. To explore the effects of GAS as a vaccine adjuvant, the expression levels of CD80, CD86, MHCI and MHCII activated markers were detected after GAS treatment in vitro and in vivo, and the expression levels of IL-2 and TNF-α in splenocytes were detected after GAS treatment in vivo. Besides, the expression levels of IL-2 and IFN-γ in CD4+T cells and perforin, TNF-α and IFN-γ in CD8+T cells were detected. The effects of GAS on the survival rate and tumor size of tumor-challenged mice and the effect of cytotoxicity on CD8+T cells were also investigated. Our data showed that GAS ameliorated CD8+T cell mediated immune response and significantly improved protection of tumor-challenged animals. The results demonstrated that GAS is a potential adjuvant contributing to anticancer immunomodulation.

The effect of 808 nm and 905 nm wavelength light on recovery after spinal cord injury.

We investigated the effect of a Multiwave Locked System laser (with a simultaneous 808 nm continuous emission and 905 nm pulse emission) on the spinal cord after spinal cord injury (SCI) in rats. The functional recovery was measured by locomotor tests (BBB, Beam walking, MotoRater) and a sensitivity test (Plantar test). The locomotor tests showed a significant improvement of the locomotor functions of the rats after laser treatment from the first week following lesioning, compared to the controls. The laser treatment significantly diminished thermal hyperalgesia after SCI as measured by the Plantar test. The atrophy of the soleus muscle was reduced in the laser treated rats. The histopathological investigation showed a positive effect of the laser therapy on white and gray matter sparing. Our data suggests an upregulation of M2 macrophages in laser treated animals by the increasing number of double labeled CD68+/CD206+ cells in the cranial and central parts of the lesion, compared to the control animals. A shift in microglial/macrophage polarization was confirmed by gene expression analysis by significant mRNA downregulation of Cd86 (marker of inflammatory M1), and non-significant upregulation of Arg1 (marker of M2). These results demonstrated that the combination of 808 nm and 905 nm wavelength light is a promising non-invasive therapy for improving functional recovery and tissue sparing after SCI.

Impact of dibenzocyclooctadiene lignans from Schisandra chinensis on the redox status and activation of human innate immune system cells.

Redox signaling has been established as an essential component of inflammatory responses, and redox active compounds are of interest as potential immunomodulatory agents. Dibenzocyclooctadiene lignans isolated from Schisandra chinensis, a medicinal plant with widespread use in oriental medicine, have been implicated to possess immunomodulatory properties but their effects on the human innate immune system cells have not been described. In this contribution, data are presented on the impact of schisandrin, schisandrin B and schisandrin C on human monocytic cell redox status, as well as their impact on dendritic cell maturation and T cell activation capacity and cytokine production. In THP-1 cells, levels of intracellular reactive oxygen species (ROS) were elevated after 1 h exposure to schisandrin. Schisandrin B and schisandrin C decreased cellular glutathione pools, which is a phenotype previously reported to promote anti-inflammatory functions. Treatment of human primary monocytes with the lignans during their maturation to dendritic cells did not have any effect on the appearance of surface markers HLA-DR and CD86 but schisandrin B and schisandrin C suppressed the secretion of cytokines interleukin (IL)-6, IL-10 and IL-12 by the mature dendritic cells. Dendritic cells maturated in presence of schisandrin C were further cocultured with naïve CD4+ T cells, resulting in reduced IL-12 production. In THP-1 cells, schisandrin B and schisandrin C reduced the IL-6 and IL-12 production triggered by E. coli lipopolysaccharide and IL-12 production induced by an infection with Chlamydia pneumoniae. In conclusion, the studied lignans act as immunomodulatory agents by altering the cytokine secretion, but do not interfere with dendritic cell maturation. And the observed effects may be associated with the ability of the lignans to alter cellular redox status.

Polysaccharide rich extract (PRE) from Tinospora cordifolia inhibits the intracellular survival of drug resistant strains of Mycobacterium tuberculosis in macrophages by nitric oxide induction.

Plethora of clinical and scientific information obtained in recent past has strengthened the idea that targeting critical constituents of host immune system may have beneficial outcomes for the treatment of tuberculosis. Macrophages being the primary host for Mycobacterium tuberculosis, offer an attractive target for modulation. Owing to their negligible toxicity, plant derived polysaccharides with the ability to activate macrophages; are suitable candidates for immunomodulation. In the present study, effects of polysaccharide rich extract (PRE) isolated from Tinospora cordifolia, on the survival of intracellular MTB strains and activation of macrophages were investigated. PRE treatment up regulated the expression of pro-inflammatory cytokines such as IL-ß, TNF-α, IL-6, IL-12, and IFN-γ in RAW 264.7 cell line. Up regulation in the expression of NOS2 was observed along with concomitant enhanced nitric oxide production post PRE treatment. Surface expression of MHC-II and CD-86 was up regulated after PRE treatment. Above results suggested the classical activation of macrophages by PRE treatment. Furthermore, PRE treatment led to the activation of all the three classes of MAPK i.e p38, ERK and JNK MAPKs. Further, PRE up regulated the expression of cytokines, NOS-2, MHC-II and CD-86 in MTB infected macrophages. PRE treatment inhibited the intracellular survival of drug resistant MTB in macrophages which was partially attributed to PRE mediated NO induction. Thus our data demonstrate classical activation of macrophages by PRE treatment and killing of intracellular MTB by NO induction.

Targeted delivery of p-coumaric acid encapsulated mannosylated liposomes to the synovial macrophages inhibits osteoclast formation and bone resorption in the rheumatoid arthritis animal model.

The current study aimed to target the delivery of p-coumaric acid (CA), a dietary polyphenol to the synovial macrophages of AIA rats via mannose incorporated liposomal delivery system (ML) with reference to osteoclastogenesis and bone resorption. In vivo imaging and in vitro drug release study indicated the efficiency of mannosylated liposomes to localize at the site of inflammation and increased sustain drug release respectively. Morphological assessment of isolated synovial macrophages with respect to CD86 (synovial macrophages) and CD51 (pre-/osteoclast) indicated that p-coumaric acid encapsulated mannosylated liposomes (ML-CA) inhibited the osteoclasts differentiation. ML-CA treatment inhibited the TRAP staining, downregulated the expression of MMP-9 and NFATc1 and inflammatory cytokines. The ex-vivo study specified the ability of CA to induce the OPG production in bone marrow stromal cell triggered macrophage-osteoclasts differentiation and to preserve the calcium content. Taken together, our results demonstrated that ML-CA could intervene in the osteoclast formation.

Marina crystal minerals (MCM) activate human dendritic cells to induce CD4+ and CD8+ T cell responses in vitro.

Marina crystal minerals (MCM) are a mixture that contains crystallized minerals along with trace elements extracted from seawater. It is a nutritional supplement that is capable of enhancing natural killer (NK) cell activity and increasing T and B cell proliferation in humans post ingestion. However, its effect on dendritic cells (DCs), the cells that bridge innate and adaptive immunity, is not yet known. In this study, we examine the stimulatory effects of MCM on DCs' maturation and function in vitro. Human monocyte-derived DCs were treated with MCM at two different concentrations (10 and 20 µg/mL) for 24 h. Results showed that MCM treatment activated DCs in a dose-dependent fashion. It caused the upregulation of costimulatory molecules CD80, CD86, and HLA-DR, and prompted the production of DC cytokines, including interleukin (IL)-6, IL-10, tumor necrosis factor (TNF)-α, and IL-1ß, and chemokines (monocyte chemotactic protein-1 (MCP-1)) and interferon-gamma-inducible protein-10 (IP-10). In addition, activated DCs primed CD4+ T cells to secrete significant amounts of interferon gamma (IFN-γ), and they also stimulated CD8+ T cells to express higher amounts of CD107a. These results indicate that MCM is a potentially powerful adjuvant, from natural materials, that activates human DCs in vitro and therefore may suggest its possible use in immune-based therapies against cancer and viral infections.

Effect of isoliquiritigenin for the treatment of atopic dermatitis-like skin lesions in mice.

Atopic dermatitis (AD) is a common chronic inflammatory skin disease characterized with high heterogeneity. Recent studies have suggested that it is driven by both terminal keratinocyte differentiation defects and type 2 immune responses. The mainstay steroid topical therapy has severe side effect and new treatment is in demand. Isoliquiritigenin (ISLG) is a small phenolic bioactive molecule from licorice that has shown multiple pharmacological effects against cancer, inflammatory disorder, and cardiovascular diseases. ISLG was evaluated in AD-like lesion model induced by the repetitive application of 2,4-dinitrochlorobenzene (DNCB) in BALB/c mice. Overall symptom score, serological and molecular changes of the skin lesions were evaluated. ISLG could ameliorate the overall manifestation of AD-like symptoms including scratching behavior incidence and skin lesion severity. At blood level, ISLG significantly suppressed the DNCB-induced IgE and Th2 cytokines up-regulation. At skin lesion site, ISLG also inhibited DNCB-induced pro-inflammatory cytokines like TNF-α, IL-6 as well as IL-4 expressions. In a human monocyte model THP-1, ISLG suppressed the up-regulation of CD86 and CD54 and abolished the DNCB-induced p38-α and ERK activation, suggesting a molecular mechanism for ISLG therapy. This study indicated that ISLG could be a potential therapeutic agent for the treatment of AD.

PeptoSomes for Vaccination: Combining Antigen and Adjuvant in Polypept(o)ide-Based Polymersomes.

In this work, the first vaccine is reported based on a PeptoSome, which contains a model antigen (SIINFEKL) and adjuvant (CpG). PeptoSomes are polypept(o)ide-based polymersomes built of a block-copolymer with polysarcosine (PSar) as the hydrophilic block (X n = 111) and poly(benzyl-glutamic acid) (PGlu(OBn)) as the hydrophobic one (X n = 46). The polypept(o)ide is obtained with low dispersity index of 1.32 by controlled ring-opening polymerization. Vesicle formation by dual centrifugation technique allows for loading of vesicles up to 40 mol%. PeptoSomes are characterized by multiangle dynamic light scattering, static light scattering, and cryogenic transmission electron microscopy (cryoTEM). The PeptoSomes have a hydrodynamic radius of 39.2 nm with a low dispersity (µ 2 = 0.1). The ρ-ratio R g /R h of 0.95 already indicates that vesicles are formed, which can be confirmed by cryoTEM. Loaded PeptoSomes deliver the antigen (SIINFEKL) and an adjuvant (CpG) simultaneously into dendritic cells (DCs). Upon cellular uptake, dendritic cells are stimulated and activated, which leads to expression of cluster of differentiation CD80, CD86, and MHCII, but induces excretion of proinflammatory cytokines (e.g., TNFα). Furthermore, DC-mediated antigen-specific T-cell proliferation is achieved, thus underlining the enormous potential of PeptoSomes as a versatile platform for vaccination.

Vitamin D treatment modulates immune activation in cystic fibrosis.

Persistent inflammatory response in cystic fibrosis (CF) airways is believed to play a central role in the progression of lung damage. Anti-inflammatory treatment may slow lung disease progression, but adverse side effects have limited its use. Vitamin D has immunoregulatory properties. We randomized 16 CF patients to receive vitamin D2, vitamin D3 or to serve as controls, and investigated the effect of vitamin D supplementation on soluble immunological parameters, myeloid dendritic cells (mDCs) and T cell activation. Three months of vitamin D treatment were followed by two washout months. Vitamin D status at baseline was correlated negatively with haptoglobin, erythrocyte sedimentation rate and immunoglobulin A concentration. Total vitamin D dose per kg bodyweight correlated with the down-modulation of the co-stimulatory receptor CD86 on mDCs. Vitamin D treatment was associated with reduced CD279 (PD-1) expression on CD4+ and CD8+ T cells, as well as decreased frequency of CD8+ T cells co-expressing the activation markers CD38 and human leucocyte antigen D-related (HLA-DR) in a dose-dependent manner. There was a trend towards decreased mucosal-associated invariant T cells (MAIT) cell frequency in patients receiving vitamin D and free serum 25-hydroxyvitamin D (free-s25OHD) correlated positively with CD38 expression by these cells. At the end of intervention, the change in free-s25OHD was correlated negatively with the change in CD279 (PD-1) expression on MAIT cells. Collectively, these data indicate that vitamin D has robust pleiotropic immunomodulatory effects in CF. Larger studies are needed to explore the immunomodulatory treatment potential of vitamin D in CF in more detail.

Capparis spinosa Fruit Ethanol Extracts Exert Different Effects on the Maturation of Dendritic Cells.

Capparis spinosa L. (C. spinosa) has been used as food and traditional medicine and shows anti-inflammatory and anti-oxidant activities. Here, we prepared the C. spinosa fruit ethanol extracts (CSEs) using different procedures and investigated the effects of CSE on the maturation of mouse bone marrow-derived dendritic cells (DCs) in the absence or presence of lipopolysaccharide (LPS). DC maturation and cytokine production were detected by flow cytometry and ELISA, respectively. We obtained three different CSEs and dissolved in water or DMSO, named CSE2W, CSEMW, CSE3W, CSE2D, CSEMD, and CSE3D, respectively. These CSEs showed different effects on DC maturation. CSEMW and CSEMD significantly increased the expressions of CD40, CD80, and CD86, in a dose-dependent manner. CSE2W and CSE2D also showed a modest effect on DC maturation, which enhanced the expression of CD40. CSE3W and CSE3D did not change DC maturation but suppressed LPS-induced DC maturation characterized by the decreased levels of CD40 and CD80. CSE3W and CSE3D also significantly inhibited the secretions of IL-12p40, IL-6, IL-1ß, and TNF-α induced by LPS. CSE3W further increased the level of IL-10 induced by LPS. Moreover, CSE3D suppressed LPS-induced DC maturation in vivo, which decreased the expressions of CD40 and CD80. These results suggested that CSE3W and CSE3D might be used to treat inflammatory diseases.

Curcumin ameliorates experimental autoimmune myasthenia gravis by diverse immune cells.

Curcumin is a traditional Asian medicine with diverse immunomodulatory properties used therapeutically in the treatment of many autoimmune diseases. However, the effects of curcumin on myasthenia gravis (MG) remain undefined. Here we investigated the effects and potential mechanisms of curcumin in experimental autoimmune myasthenia gravis (EAMG). Our results demonstrated that curcumin ameliorated the clinical scores of EAMG, suppressed the expression of T cell co-stimulatory molecules (CD80 and CD86) and MHC class II, down-regulated the levels of pro-inflammatory cytokines (IL-17, IFN-γ and TNF-α) and up-regulated the levels of the anti-inflammatory cytokine IL-10, shifted the balance from Th1/Th17 toward Th2/Treg, and increased the numbers of NKR-P1(+) cells (natural killer cell receptor protein 1 positive cells, including NK and NKT cells). Moreover, the administration of curcumin promoted the differentiation of B cells into a subset of B10 cells, increased the anti-R97-166 peptide IgG1 levels and decreased the relative affinity indexes of anti-R97-116 peptide IgG. In summary, curcumin effectively ameliorate EAMG, indicating that curcumin may be a potential candidate therapeutic agent for MG.

Piperlongumine Suppresses Dendritic Cell Maturation by Reducing Production of Reactive Oxygen Species and Has Therapeutic Potential for Rheumatoid Arthritis.

Piperlongumine (PLM) is a natural product from the plant Piper longum that inhibits platelet aggregation, atherosclerosis plaque formation, and tumor cell growth. It has potential value in immunomodulation and the management of autoimmune diseases. In this study, we investigated the role of PLM in regulating the differentiation and maturation of dendritic cells (DCs), a critical regulator of immune tolerance, and evaluated its clinical effects in a rheumatoid arthritis mouse model. We found that PLM treatment reduced LPS-induced murine bone marrow-derived DC maturation, characterized by reduced expression of CD80/86, secretion of MCP-1, IL-12p70, IL-6, TNFα, IFN-γ, and IL-23, and reduced alloproliferation of T cells; however, PLM does not affect cell differentiation. Furthermore, PLM reduced intracellular reactive oxygen species (ROS) production by DCs and inhibited the activation of p38, JNK, NF-κB, and PI3K/Akt signaling pathways. Conversely, PLM increased the expression of GSTP1 and carbonyl reductase 1, two enzymes that counteract ROS effects. ROS inhibition by exogenous N-acetyl-l-cysteine suppressed DC maturation. PLM treatment improved the severity of arthritis and reduced in vivo splenic DC maturation, collagen-specific CD4(+) T cell responses, and ROS production in mice with collagen-induced arthritis. Taken together, these results suggest that PLM inhibits DC maturation by reducing intracellular ROS production and has potential as a therapeutic agent for rheumatoid arthritis.

In vitro inhibitory effects of thymol and carvacrol on dendritic cell activation and function.

CONTEXT: Thyme has been used in traditional medicine for medicinal purposes since ancient times. OBJECTIVE: The objective of this study was to investigate the effects of thymol and carvacrol as two major constituents of thyme on dendritic cells (DCs) maturation and T cell activation. MATERIALS AND METHODS: Splenic DCs were treated with non-cytotoxic concentrations of the components and then analyzed for MHC II, CD86, and CD40 expression by flow cytometry. The effects of compounds on mitogenic, as well as allogenic T cell responses in mixed lymphocyte culture (MLR) and the release of cytokines were investigated. RESULTS: At 0.1 µg/ml, reduced mean fluorescent intensity (MFI) of CD86 for thymol (80.3 ± 0.2% of untreated control) and CD40 for carvacrol (79.5 ± 0.14%) was observed (p < 0.001). Decreased mitogenic T cell proliferation by thymol [proliferation index (PI) from 0.93 ± 0.11 at 1 µg/ml to 0.42 ± 0.16 at 100 µg/ml (p < 0.01)] and carvacrol [PI from 1.08 ± 0.3 at 1 µg/ml to 0.28 ± 0.1 at 100 µg/ml (p < 0.001)] was seen. Ten micrograms/ml thymol (PI, 0.85 ± 0.04) and carvacrol (PI, 0.89 ± 0.03) inhibited allogenic T cell response (p < 0.05). Decreased IFN-γ level in MLR supernatant from 1441 ± 27.7 pg/ml in untreated cells to 944 ± 32.1 at 10 µg/ml of thymol and of carvacrol (886 ± 31.7 pg/ml) (p < 0.01) was found. IL-4 levels were decreased in the presence of both compounds (p < 0.01). CONCLUSION: These data showed the suppressive effects of thymol and carvacrol on DCs maturation and function, as well as T cell responses.

Optimization of the THP-1 activation assay to detect pharmaceuticals with potential to cause immune mediated drug reactions.

Despite important impacts of systemic hypersensitivity induced by pharmaceuticals, for such endpoint no reliable preclinical approaches are available. We previously established an in vitro test to identify contact and respiratory allergens based on interleukin-8 (IL-8) production in THP-1 cells. Here, we challenged it for identification of pharmaceuticals associated with systemic hypersensitivity reactions, with the idea that drug sensitizers share common mechanisms of cell activation. Cells were exposed to drugs associated with systemic hypersensitivity reactions (streptozotocin, sulfamethoxazole, neomycin, probenecid, clonidine, procainamide, ofloxacin, methyl salicylate), while metformin was used as negative drug. Differently to chemicals, drugs tested were well tolerated, except clonidine and probenecid, with no signs of cytotoxicity up to 1-2mg/ml. THP-1 activation assay was adjusted, and conditions, that allow identification of all sensitizing drugs tested, were established. Next, using streptozotocin and selective inhibitors of PKC-ß and p38 MAPK, two pathways involved in chemical allergen-induced cell activation, we tested the hypothesis that similar pathways were also involved in drug-induced IL-8 production and CD86 upregulation. Results indicated that drugs and chemical allergens share similar activation pathways. Finally, we made a structure-activity hypothesis related to hypersensitivity reactions, trying to individuate structural requisite that can be involved in immune mediated adverse reactions.

Modulation of radiochemoimmunotherapy-induced B16 melanoma cell death by the pan-caspase inhibitor zVAD-fmk induces anti-tumor immunity in a HMGB1-, nucleotide- and T-cell-dependent manner.

One prerequisite that radiotherapy (RT) and chemotherapy (CT) result in anti-tumor immune responses is triggering of immunogenic cell death forms such as necroptosis. The latter is inducible by inhibition of apoptosis with the pan-caspase inhibitor zVAD-fmk. The design of multimodal therapies that overcome melanoma's resistance to apoptosis is a big challenge of oncoimmunology. As hints exist that immune stimulation by hyperthermia (HT) augments the efficacy of melanoma therapies and that tumors can be sensitized for RT with zVAD-fmk, we asked whether combinations of RT with dacarbazine (DTIC) and/or HT induce immunogenic melanoma cell death and how this is especially influenced by zVAD-fmk. Necroptosis was inducible in poorly immunogenic B16-F10 melanoma cells and zVAD-fmk generally increased melanoma cell necrosis concomitantly with the release of HMGB1. Supernatants (SNs) of melanoma cells whose cell death was modulated with zVAD-fmk induced an upregulation of the activation markers CD86 and MHCII on macrophages. The same was seen on dendritic cells (DCs), but only when zVAD-fmk was added to multimodal tumor treatments including DTIC. DCs of MyD88 KO mice and DCs incubated with SNs containing apyrase did not increase the expression of these activation markers on their surface. The in vivo experiments revealed that zVAD-fmk decreases the tumor growth significantly and results in a significantly reduced tumor infiltration of Tregs when added to multimodal treatment of the tumor with RT, DTIC and HT. Further, a significantly increased DC and CD8+ T-cell infiltration into the tumor and in the draining lymph nodes was induced, as well as an increased expression of IFNγ by CD8+ T cells. However, zVAD-fmk did not further reduce tumor growth in MyD88 KO mice, mice treated with apyrase or RAG KO mice. We conclude that HMGB1, nucleotides and CD8+ T cells mediate zVAD-fmk induced anti-melanoma immune reactions in multimodal therapy settings.