Mevalonate Biosynthesis Intermediates Are Key Regulators of Innate Immunity in Bovine Endometritis.

Metabolic changes can influence inflammatory responses to bacteria. To examine whether localized manipulation of the mevalonate pathway impacts innate immunity, we exploited a unique mucosal disease model, endometritis, where inflammation is a consequence of innate immunity. IL responses to pathogenic bacteria and LPS were modulated in bovine endometrial cell and organ cultures by small molecules that target the mevalonate pathway. Treatment with multiple statins, bisphosphonates, squalene synthase inhibitors, and small interfering RNA showed that inhibition of farnesyl-diphosphate farnesyl transferase (squalene synthase), but not 3-hydroxy-3-methylglutaryl-CoA reductase or farnesyl diphosphate synthase, reduced endometrial organ and cellular inflammatory responses to pathogenic bacteria and LPS. Although manipulation of the mevalonate pathway reduced cellular cholesterol, impacts on inflammation were independent of cholesterol concentration as cholesterol depletion using cyclodextrins did not alter inflammatory responses. Treatment with the isoprenoid mevalonate pathway-intermediates, farnesyl diphosphate and geranylgeranyl diphosphate, also reduced endometrial cellular inflammatory responses to LPS. These data imply that manipulating the mevalonate pathway regulates innate immunity within the endometrium, and that isoprenoids are regulatory molecules in this process, knowledge that could be exploited for novel therapeutic strategies.

Alendronate inhalation ameliorates elastase-induced pulmonary emphysema in mice by induction of apoptosis of alveolar macrophages.

Alveolar macrophages play a crucial role in the pathogenesis of emphysema, for which there is currently no effective treatment. Bisphosphonates are widely used to treat osteoclast-mediated bone diseases. Here we show that delivery of the nitrogen-containing bisphosphonate alendronate via aerosol inhalation ameliorates elastase-induced emphysema in mice. Inhaled, but not orally ingested, alendronate inhibits airspace enlargement after elastase instillation, and induces apoptosis of macrophages in bronchoalveolar fluid via caspase-3- and mevalonate-dependent pathways. Cytometric analysis indicates that the F4/80(+)CD11b(high)CD11c(mild) population characterizing inflammatory macrophages, and the F4/80(+)CD11b(mild)CD11c(high) population defining resident alveolar macrophages take up substantial amounts of the bisphosphonate imaging agent OsteoSense680 after aerosol inhalation. We further show that alendronate inhibits macrophage migratory and phagocytotic activities and blunts the inflammatory response of alveolar macrophages by inhibiting nuclear factor-κB signalling. Given that the alendronate inhalation effectively induces apoptosis in both recruited and resident alveolar macrophages, we suggest this strategy may have therapeutic potential for the treatment of emphysema.

Immune modulation by zoledronic acid in human myeloma: an advantageous cross-talk between Vγ9Vδ2 T cells, αß CD8+ T cells, regulatory T cells, and dendritic cells.

Vγ9Vδ2 T cells play a major role as effector cells of innate immune responses against microbes, stressed cells, and tumor cells. They constitute <5% of PBLs but can be expanded by zoledronic acid (ZA)-treated monocytes or dendritic cells (DC). Much less is known about their ability to act as cellular adjuvants bridging innate and adaptive immunity, especially in patients with cancer. We have addressed this issue in multiple myeloma (MM), a prototypic disease with several immune dysfunctions that also affect γδ T cells and DC. ZA-treated MM DC were highly effective in activating autologous γδ T cells, even in patients refractory to stimulation with ZA-treated monocytes. ZA inhibited the mevalonate pathway of MM DC and induced the intracellular accumulation and release into the supernatant of isopentenyl pyrophosphate, a selective γδ T cell activator, in sufficient amounts to induce the proliferation of γδ T cells. Immune responses against the tumor-associated Ag survivin (SRV) by MHC-restricted, SRV-specific CD8(+) αß T cells were amplified by the concurrent activation of γδ T cells driven by autologous DC copulsed with ZA and SRV-derived peptides. Ancillary to the isopentenyl pyrophosphate-induced γδ T cell proliferation was the mevalonate-independent ZA ability to directly antagonize regulatory T cells and downregulate PD-L2 expression on the DC cell surface. In conclusion, ZA has multiple immune modulatory activities that allow MM DC to effectively handle the concurrent activation of γδ T cells and MHC-restricted CD8(+) αß antitumor effector T cells.

Simvastatin inhibits airway hyperreactivity: implications for the mevalonate pathway and beyond.

RATIONALE: Statin use has been linked to improved lung health in asthma and chronic obstructive pulmonary disease. We hypothesize that statins inhibit allergic airway inflammation and reduce airway hyperreactivity via a mevalonate-dependent mechanism. OBJECTIVES: To determine whether simvastatin attenuates airway inflammation and improves lung physiology by mevalonate pathway inhibition. METHODS: BALB/c mice were sensitized to ovalbumin over 4 weeks and exposed to 1% ovalbumin aerosol over 2 weeks. Simvastatin (40 mg/kg) or simvastatin plus mevalonate (20 mg/kg) was injected intraperitoneally before each ovalbumin exposure. MEASUREMENTS AND MAIN RESULTS: Simvastatin reduced total lung lavage leukocytes, eosinophils, and macrophages (P < 0.05) in the ovalbumin-exposed mice. Cotreatment with mevalonate, in addition to simvastatin, reversed the antiinflammatory effects seen with simvastatin alone (P < 0.05). Lung lavage IL-4, IL-13, and tumor necrosis factor-alpha levels were all reduced by treatment with simvastatin (P < 0.05). Simvastatin treatment before methacholine bronchial challenge increased lung compliance and reduced airway hyperreactivity (P = 0.0001). CONCLUSIONS: Simvastatin attenuates allergic airway inflammation, inhibits key helper T cell type 1 and 2 chemokines, and improves lung physiology in a mouse model of asthma. The mevalonate pathway appears to modulate allergic airway inflammation, while the beneficial effects of simvastatin on lung compliance and airway hyperreactivity may be independent of the mevalonate pathway. Simvastatin and similar agents that modulate the mevalonate pathway may prove to be treatments for inflammatory airway diseases, such as asthma.

Inhibitory effects of fluvastatin on cytokine and chemokine production by peripheral blood mononuclear cells in patients with allergic asthma.

BACKGROUND: Statins have anti-inflammatory effects on immune cells. OBJECTIVE: To investigate the immunomodulatory effects of fluvastatin on peripheral blood mononuclear cells (PBMCs) after allergen-specific and non-allergen-specific stimulation in patients with asthma and in healthy subjects. METHODS: PBMCs from seven patients with asthma who showed elevated immunoglobulin (Ig)E to house dust mite were isolated and stimulated with Dermatofagoides farinae, purified protein derivative, and phytohaemagglutinin (PHA) in the presence or absence of fluvastatin. PBMCs from seven healthy subjects were stimulated with PHA. The effects of fluvastatin on cell proliferation and production of cytokines (interferon [IFN]-gamma and interleukin [IL]-5) and chemokines (chemokine CXC motif, ligand [CXCL10], and CC chemokine ligand [CCL17]) were measured. Migration of T helper (Th)1 and Th2 cell lines was also investigated. The expression of CXCR3 and CCR4 was analysed with flow cytometry. Steroid-insensitive PBMCs induced by preculture with IL-2 and IL-4 were also evaluated. Some experiments were performed in the presence of mevalonic acid. RESULTS: Fluvastatin inhibited the proliferation of PBMCs and decreased the production of IL-5, IFN-gamma, CCL17, and CXCL10 after allergen-specific and non-allergen-specific stimulation; all these effects, except for decreased CXCL10 production, were partially reversed by mevalonic acid. Culture supernatants obtained in the presence of fluvastatin prevented the migration of Th1 and Th2 cell lines in a dose-dependent manner. In addition, CCR4 and CXCR3 expression on CD4(+) T cells was not affected by the presence of fluvastatin. Fluvastatin inhibited the proliferative response of steroid-insensitive PBMCs to phytohaemagglutinin. CONCLUSION: Fluvastatin has inhibitory effects on cytokine and chemokine production, and thus might be used as a potential therapeutic agent in severe asthma.

MICA expressed by multiple myeloma and monoclonal gammopathy of undetermined significance plasma cells Costimulates pamidronate-activated gammadelta lymphocytes.

Amino-biphosphonates (like pamidronate) activate human Vgamma9/Vdelta2 T lymphocytes and promote their cytotoxicity against multiple myeloma cells. T-cell receptor (TCR)-mediated effector functions of gammadelta cells are enhanced upon triggering of the activating receptor NKG2D by MICA, a stress-inducible antigen expressed by epithelial and some hematopoietic tumors, including multiple myeloma. Here we show that MICA was expressed not only by myeloma cell lines and by 6 of 10 primary multiple myeloma cells from patients but also by bone marrow plasma cells from all (six of six) patients with preneoplastic gammopathy (monoclonal gammopathy of undetermined significance, MGUS), a direct precursor of multiple myeloma. Moreover, compared with multiple myeloma plasma cells, MICA was expressed by MGUS plasma cells at significantly (P < 0.05) higher levels. MICA expressed by myeloma cell lines contributed to killing and IFN-gamma production by Vgamma9/Vdelta2 cells only upon pamidronate treatment, suggesting a dual interaction between Vgamma9/Vdelta2 lymphocytes and multiple myeloma plasma cells involving both TCR triggering and NKG2D-mediated signals. Finally, MICA enhanced killing of freshly derived, pamidronate-treated multiple myeloma cells from patients by gammadelta cells, as indicated by the significantly (P < 0.05) higher gammadelta cytotoxicity against MICA-positive rather than MICA-negative multiple myeloma cells. Our results indicate that MICA expressed by monoclonal plasma cells is functional and correlates with disease stages, suggesting a role for the molecule in the immune surveillance against multiple myeloma. Moreover, pamidronate-activated Vgamma9/Vdelta2 lymphocytes can be exploited in the immune therapy of early stages multiple myeloma and possibly of premalignant disease.

The bisphosphonate acute phase response: rapid and copious production of proinflammatory cytokines by peripheral blood gd T cells in response to aminobisphosphonates is inhibited by statins.

The bisphosphonates are a novel class of drug that have been registered for various clinical applications worldwide. Bisphosphonates, and in particular the aminobisphosphonates (nBPs), are known to have a number of side-effects including a rise in body temperature and accompanying flu-like symptoms that resemble a typical acute phase response. The mechanism for this response has been partially elucidated and appears to be associated with the release of tumour necrosis factor (TNF)alpha and interleukin (IL)6, although the effector cells that release these cytokines and the mechanism of action remain enigmatic. Here, we show that the nBP-induced acute phase response differs from the typical acute phase response in that CD14+ cells such as monocytes and macrophages are not the primary cytokine producing cells. We show that by inhibiting the mevalonate pathway, nBPs induce rapid and copious production of TNFalpha and IL6 by peripheral blood gammadelta T cells. Prior treatment with statins, which inhibit 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase, blocks nBP-induced production of these proinflammatory cytokines by gammadelta T cells and may offer a means of avoiding the associated acute phase response. In addition, our findings provide a further mechanism for the anti-inflammatory effects attributed to inhibitors of HMG CoA reductase.

Human T cell receptor gammadelta cells recognize endogenous mevalonate metabolites in tumor cells.

T lymphocytes expressing the T cell receptor (TCR)-gammadelta recognize unknown antigens on tumor cells. Here we identify metabolites of the mevalonate pathway as the tumor ligands that activate TCR-gammadelta cells. In tumor cells, blockade of hydroxy-methylglutaryl-CoA reductase (HMGR), the rate limiting enzyme of the mevalonate pathway, prevents both accumulation of mevalonate metabolites and recognition by TCR-gammadelta cells. When metabolite accumulation is induced by overexpressing HMGR or by treatment with nitrogen-containing bisphosphonate drugs, tumor cells derived from many tissues acquire the capacity to stimulate the same TCR-gammadelta population. Accumulation of mevalonate metabolites in tumor cells is a powerful danger signal that activates the immune response and may represent a novel target of tumor immunotherapy.

Enzyme immunoassay of urinary mevalonic acid and its clinical application.

We have developed an enzyme immunoassay for mevalonic acid (MVA), using a specific monoclonal antibody. The intra- and interassay coefficients of variation calculated on two urine samples were 3.3% and 3.4%, respectively, in the intraassay precision test and 3.5% and 6.9% in the interassay evaluation. Pravastatin, a 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitor, was administered to nine healthy men, and in all cases, their MVA excretion rates then decreased. The more MVA that was excreted in the urine before the pravastatin administration, the greater a reduction of MVA excretion was observed. The daily MVA excretions in healthy men (n = 120) and women (n = 105) were 2.32 micromol/day (SD, 0.82 micromol/day) and 1.85 micromol/day (SD, 0.47 micromol/day), respectively. In streptozotocin-induced diabetic rats (n = 14), the plasma cholesterol concentrations and MVA excretion rates were increased, and a positive correlation was observed between the plasma cholesterol and the urinary MVA concentrations.

Feasibility of an immunoassay for mevalonolactone.

Mevalonic acid is a key intermediate in a broad spectrum of cellular biological processes and their regulation. Availability of a rapid, sensitive and accurate method for its assay would be highly useful. Therefore, the feasibility of developing an immunoassay for mevalonic acid in biological samples was explored. The strategy employed was to synthesize several racemic haptens structurally resembling R-mevalonolactone, the cyclic form of mevalonic acid present at lower pH and presumed to be more antigenic. Two of these haptens were coupled to keyhole limpet hemocyanin, and the resulting conjugates were used successfully to generate antibodies in rabbits. The first antiserum bound to R,S-mevalonolactone much more effectively at pH 4.0 than at pH 6.0, consistent with the structural resemblance of the haptens to the lactone form. This antiserum also bound the free hapten from which it was generated and two others of different structure with comparable effectiveness; and slightly better than it bound R,S-mevalonolactone at pH 4.0. Similar results were obtained with the antiserum to the second hapten. The binding of either antiserum to the natural enantiomer, R-mevalonolactone, was 20 times weaker than to R,S-mevalonolactone, suggesting that the nonbiological enantiomer was more antigenic. Nevertheless, the results demonstrate that an immunochemical approach to accurate quantitation of mevalonic acid in biological samples is feasible.