Isabelle Douchet

Platelet CD154 Potentiates Interferon-α Secretion by Plasmacytoid Dendritic Cells in Systemic Lupus Erythematosus

In the autoimmune disease lupus, platelets activated by self-antigens contribute to pathology by triggering the secretion of interferon from immune cells. Taming the Big Bad Wolf Systemic lupus erythematosus (SLE)—a name some attribute to this disorder’s wolf-like ability to “devour” the affected organs—is an autoimmune inflammatory disease. It can affect virtually any part of the body, but often targets skin, kidney, and joints. A variety of immunological factors have been proposed to contribute to SLE, in particular the type I interferon (IFN) system, which is normally activated in response to viruses. Here, Duffau et al. point to platelets as the culprits in causing aberrant activation of IFN-α (a member of the type I IFN group) in lupus patients and suggest that a drug that blocks platelet activation could be a promising new treatment. A protein called CD154 (CD40 ligand) is found on T cells, where it helps to defend the body by activating cytotoxic immune cells during viral infections. It is also found on the surface of platelets that are activated for clotting and may contribute to the pathogenesis of inflammatory states such as atherosclerosis and autoimmune disorders, including SLE. Here, the authors collected platelets from patients experiencing SLE flare-ups of varying severity, as well as healthy controls, and demonstrated that CD154 abundance and shedding from platelets correlated with disease severity. Moreover, exposure of platelets from healthy donors to serum from patients with active SLE or to immune complexes similar to those in SLE patients triggered an increase in activation and CD154 production. These activated platelets, in turn, signaled to antigen-presenting cells to produce IFN-α, thus propagating an inflammatory cycle, both in vitro and in a murine model of lupus. To further test these ideas, Duffau et al. depleted the platelets in lupus-prone mice, which decreased inflammation in the animals’ kidneys, a commonly affected organ in lupus. They achieved a similar outcome by treating the mice with clopidogrel, an inhibitor of platelet activation already commonly used in patients with heart disease and stroke. In addition to experiencing less kidney damage, the clopidogrel-treated mice with lupus lived for an extra 3 months. The current mainstay of treatment for SLE is immunosuppressive therapy, achieved with steroids and chemotherapy-like medications. These drugs have numerous toxic effects, not the least of which is the immunosuppression itself, which predisposes patients to infections. Being able to treat lupus with an antiplatelet medication such as clopidogrel, which has few side effects, would markedly improve these patients’ safety and quality of life. A similar approach may prove useful in other autoimmune diseases such as rheumatoid arthritis, where it would also provide a badly needed alternative to immunosuppression. Systemic lupus erythematosus (SLE) is a systemic inflammatory autoimmune disease characterized by the involvement of multiple organs and an immune response against nuclear components. Although its pathogenesis remains poorly understood, type I interferon (IFN) and CD40 ligand (CD154) are known to contribute. Because platelets are involved in inflammatory processes and represent a major reservoir of CD154, we hypothesized that they participate in SLE pathogenesis. Here, we have shown that in SLE patients, platelets were activated by circulating immune complexes composed of autoantibodies bound to self-antigens through an Fc-γ receptor IIa (CD32)–dependent mechanism. Further, platelet activation correlated with severity of the disease and activated platelets formed aggregates with antigen-presenting cells, including monocytes and plasmacytoid dendritic cells. In vitro, activated platelets enhanced IFN-α secretion by immune complex–stimulated plasmacytoid dendritic cells through a CD154-CD40 interaction. Finally, in lupus-prone mice, depletion of platelets or administration of the P2Y(12) receptor antagonist (clopidogrel) improved all measures of disease and overall survival; transfusion of activated platelets worsened the disease course. Together, these data identify platelet activation as an important contributor to SLE pathogenesis and suggest that this process and its sequelae may provide a new therapeutic target.

Characterization of an exported monoglyceride lipase from Mycobacterium tuberculosis possibly involved in the metabolism of host cell membrane lipids

The Rv0183 gene of the Mycobacterium tuberculosis H37Rv strain, which has been implicated as a lysophospholipase, was cloned and expressed in Escherichia coli. The purified Rv0183 protein did not show any activity when lysophospholipid substrates were used, but preferentially hydrolysed monoacylglycerol substrates with a specific activity of 290 units x mg(-1) at 37 degrees C. Rv0183 hydrolyses both long chain di- and triacylglycerols, as determined using the monomolecular film technique, although the turnover was lower than with MAG (monoacyl-glycerol). The enzyme shows an optimum activity at pH values ranging from 7.5 to 9.0 using mono-olein as substrate and is inactivated by serine esterase inhibitors such as E600, PMSF and tetrahydrolipstatin. The catalytic triad is composed of Ser110, Asp226 and His256 residues, as confirmed by the results of site-directed mutagenesis. Rv0183 shows 35% sequence identity with the human and mouse monoglyceride lipases and well below 15% with the other bacterial lipases characterized so far. Homologues of Rv0183 can be identified in other mycobacterial genomes such as Mycobacterium bovis, Mycobacterium smegmatis, and even Mycobacterium leprae, which is known to contain a low number of genes involved in the replication process within the host cells. The results of immunolocalization studies performed with polyclonal antibodies raised against the purified recombinant Rv0183 suggested that the enzyme was present only in the cell wall and culture medium of M. tuberculosis. Our results identify Rv0183 as the first exported lipolytic enzyme to be characterized in M. tuberculosis and suggest that Rv0183 may be involved in the degradation of the host cell lipids.

Expression of specific inflammasome gene modules stratifies older individuals into two extreme clinical and immunological states

Low-grade, chronic inflammation has been associated with many diseases of aging, but the mechanisms responsible for producing this inflammation remain unclear. Inflammasomes can drive chronic inflammation in the context of an infectious disease or cellular stress, and they trigger the maturation of interleukin-1β (IL-1β). Here we find that the expression of specific inflammasome gene modules stratifies older individuals into two extremes: those with constitutive expression of IL-1β, nucleotide metabolism dysfunction, elevated oxidative stress, high rates of hypertension and arterial stiffness; and those without constitutive expression of IL-1β, who lack these characteristics. Adenine and N4-acetylcytidine, nucleotide-derived metabolites that are detectable in the blood of the former group, prime and activate the NLRC4 inflammasome, induce the production of IL-1β, activate platelets and neutrophils and elevate blood pressure in mice. In individuals over 85 years of age, the elevated expression of inflammasome gene modules was associated with all-cause mortality. Thus, targeting inflammasome components may ameliorate chronic inflammation and various other age-associated conditions.

Lipolytic enzymes in Mycobacterium tuberculosis

Mycobacterium tuberculosis is a bacterial pathogen that can persist for decades in an infected patient without causing a disease. In vivo, the tubercle bacillus present in the lungs store triacylglycerols in inclusion bodies. The same process can be observed in vitro when the bacteria infect adipose tissues. Indeed, before entering in the dormant state, bacteria accumulate lipids originating from the host cell membrane degradation and from de novo synthesis. During the reactivation phase, these lipids are hydrolysed and the infection process occurs. The degradation of both extra and intracellular lipids can be directly related to the presence of lipolytic enzymes in mycobacteria, which have been ignored during a long period particularly due to the difficulties to obtain a high expression level of these enzymes in M. tuberculosis. The completion of the M. tuberculosis genome offered new opportunity to this kind of study. The aim of this review is to focus on the recent results obtained in the field of mycobacterium lipolytic enzymes and although no experimental proof has been shown in vivo, it is tempting to speculate that these enzymes could be involved in the virulence and pathogenicity processes.

Biochemical properties and three-dimensional structures of two extracellular lipolytic enzymes from Bacillus subtilis

This article reviews our present knowledge on the extracellular lipolytic enzymes LipA and LipB from Bacillus subtilis. Growth of B. subtilis to the late logarithmic growth phase results in a total lipolytic activity of 12–18 units per liter of culture supernatant. Immunodetection with LipA- and LipB-specific antibodies indicated a differential expression of both lipolytic enzymes depending on the composition of the growth medium. LipA was produced in rich and in minimal medium, whereas LipB was present only in rich medium. The lipA and lipB genes were cloned and overexpressed in B. subtilis and Escherichia coli, the corresponding proteins purified to electrophoretic homogeneity and their substrate specificities, pH- and temperature stabilities were determined. The active site residue Ser78 of LipB is located in the consensus sequence Ala–X–Ser–X–Gly where the alanine replaces a glycine found in most of the bacterial lipases. The role of this Ala-residue was investigated by constructing LipB variant A76G thereby restoring the canonical lipase consensus motif. When compared with wild-type LipB this variant showed a markedly reduced thermostability at pH 11 but an increased stability at pH 5–7. These findings were rationalized by building a three-dimensional structural model of LipB using the atomic coordinates of the LipA crystal structure, which was solved recently. The LipB model structure revealed that 43 out of 45 residues, which are different from LipA, were located on the surface of LipB. The surface-exposed amino acids including those located at the rim of the active site cleft may cause the differences in specific activities between LipA and LipB.

Kinetic behaviour of pancreatic lipase in five species using emulsions and monomolecular films of synthetic glycerides

In the absence of colipase and bile salts, using tributyrin emulsions or monomolecular films of dicaprin at low surface pressure, we observed that no significant lipase activity can be measured with Human Pancreatic Lipase (HuPL), Horse Pancreatic Lipase (HoPL) or Dog Pancreatic Lipase (DPL). Only Porcine Pancreatic Lipase (PPL) and recombinant Guinea Pig Pancreatic Lipase Related Protein of type 2 (r-GPL) hydrolyse pure tributyrin in the absence of any additive, as well as dicaprin films at low surface pressures. The former lipases may lack enzyme activity because of irreversible interfacial denaturation due to the high energy existing at the tributyrin/water interface and at the dicaprin film surface at low surface pressures. The enzyme denaturation cannot be reflected in the number of disulfide bridges, since all the pancreatic lipases tested here contain six disulfide bridges, but behaved very differently at interfaces. We propose to use the surface pressure threshold, as determined using the monomolecular technique, as a criterion for classifying lipases in terms of their sensitivity to interfacial denaturation.

Purification of human gastric lipase by immunoaffinity and quantification of this enzyme in the duodenal contents using a new ELISA procedure

Human gastric lipase (HGL) is the first lipolytic enzyme involved in the digestion of dietary lipids along the gastrointestinal tract. We describe an improved procedure for isolating the enzyme using immunoaffinity chromatography in combination with ion-exchange chromatography. The purified enzyme, showing a single band on SDS-PAGE, expressed a specific activity of 1000 U/mg using tributyrin as the substrate. We also describe a specific enzyme-linked immunosorbent assay (ELISA) procedure for measuring duodenal HGL levels. The ELISA was performed using an anti-HGL polyclonal antibody (pAb) as the captor antibody and a biotinylated monoclonal antibody (mAb) as the detector antibody. With the double sandwich ELISA technique, HGL in the range of 1-60 ng/ml was measured in less than 5 h. Identical HGL concentrations were obtained using the above ELISA procedure when compared to those based on the enzymatic activity using the potentiometric method (correlation coefficient: r = 0.95). No significant interference from other duodenal components was observed, as proved by the quantitative HGL determinations performed on intestinal samples.

Potential Role of Mycoplasma hominis in Interleukin (IL)–17–Producing CD4+ T-Cell Generation Via Induction of IL-23 Secretion by Human Dendritic Cells

BACKGROUND Mycoplasma hominis, a human urogenital pathogen, is involved in genital and extragenital infections and arthritis, particularly in immunocompromised patients. The interleukin (IL) 23/T helper (Th) 17 axis is associated with inflammatory and autoimmune diseases. The aim of this study was to assess the IL-23 response to M. hominis in human dendritic cells (DCs) and the CD4(+) T-cell differentiation in response to M. hominis-infected DCs. METHODS Human monocyte-derived DCs were cultured with phosphate-buffered saline, lipopolysaccharide, or M. hominis PG21. Cocultures with heterologous T cells were performed. Extracts from M. hominis were separated and incubated with DCs. Isolates from different clinical syndromes were tested. RESULTS M. hominis induced the maturation of human DCs with predominant IL-23 secretion in a Toll-like receptor 2-dependent manner. The in vitro immunomodulatory capacity of M. hominis was contained in a lipoprotein-enriched fraction from the mycoplasma. M. hominis-activated DCs induced IL-17-producing CD4(+) T cells. Interestingly, clinical isolates differed in their ability to promote IL-23 secretion by DCs. CONCLUSIONS Taken together, our findings demonstrate a major role for the IL-23/Th17 axis in the defense against M. hominis and indicate a potential role for these bacteria in inflammatory and autoimmune diseases.

Interfacial binding and activity of lipases at the lipid–water interface: effects of Gum Arabic and surface pressure

We investigated the effects of Gum Arabic (GA) on the interfacial binding and activity of human pancreatic lipase (HPL) on trioctanoylglycerol (TC8) emulsion. Using the pH-stat technique, in the presence of sodium taurodeoxycholate (NaTDC) at concentration of 0.5 mM or higher, GA had no effect on the HPL activity measured in the presence of colipase. However, in the absence of bile salts or at low bile salt concentration, GA inhibited the HPL activity when TC8 emulsion was used as substrate. At 3% (w/v, final concentration), GA strongly desorbed pure HPL from the TC8 interface and the classical anchoring effect of colipase was clearly observed. An excellent correlation was therefore observed between the interfacial binding and the lipase activity of HPL as measured on a TC8 emulsion. In conclusion, GA was found to be surface active and to have similar effects to those of bile salts on the interfacial binding and activity of HPL. Using the monomolecular film technique, coupled to an ELISA, the penetration of Humicola lanuginosa lipase (HLL) into monomolecular films of non-hydrolysable enantiomeric substrate analogues (1,2-didecanoyl-3-deoxyamino-3-O-methyl glycerol (R3) or 2,3-didecanoyl-3-deoxyamino-1-O-methyl glycerol (S3)) spread at the air–water interface was monitored at constant surface pressure. By combining the surface area variations and the surface excess of HLL, the cross-sectional area of HLL molecule was estimated and a model for its penetration/orientation into the monomolecular films of R3 or S3 was proposed. No significant difference was found in term of HLL molecular area when using these films, indicating that the chirality of the lipid has no significant effect on HLL penetration. Nevertheless, This penetration was found to be deeper at low surface pressures.

Interfacial and/or molecular recognition by lipases of mixed monomolecular films of 1,2-dicaprin and chiral organophosphorus glyceride analogues?

Abstract Enantiomerically pure sn-1,2- and sn-2,3-O-didecanoylphosphonoglycerides, were synthesised and investigated as inhibitors of human pancreatic lipase (HPL) and human gastric lipase (HGL). The inhibition studies were performed using the monomolecular film technique coupled with ELISA tests. All the four stereoisomers investigated reduced the hydrolysis of 1,2-dicaprin by both lipases. With HPL, they exhibited a rather weak inhibitory power and no significant differences were observed among them. With HGL, the inhibition depended much more strongly on the chirality at the sn-2 carbon of the glycerol backbone, while the chirality at the phosphorus had no influence. Moreover, a clear correlation was observed between the HGL surface concentration and the inhibitor surface molar fraction (α50) leading to half inhibition. The greatest enzymatic inhibition was observed with films containing the enantiomeric inhibitor to which the HGL was best adsorbed, assuming thus that the interfacial lipase binding was controlled by a supramolecular chiral recognition process.