SINAPs: A Software Tool for Analysis and Visualization of Interaction Networks of Molecular Dynamics Simulations.

As long as the structural study of molecular mechanisms requires multiple molecular dynamics reflecting contrasted bioactive states, the subsequent analysis of molecular interaction networks remains a bottleneck to be fairly treated and requires a user-friendly 3D view of key interactions. Structural Interaction Network Analysis Protocols (SINAPs) is a proprietary python tool developed to (i) quickly solve key interactions able to distinguish two protein states, either from two sets of molecular dynamics simulations or from two crystallographic structures, and (ii) render a user-friendly 3D view of these key interactions through a plugin of UCSF Chimera, one of the most popular open-source viewing software for biomolecular systems. Through two case studies, glucose transporter-1 (GLUT-1) and A2A adenosine receptor (A2AR), SINAPs easily pinpointed key interactions observed experimentally and relevant for their bioactivities. This very effective tool was thus applied to identify the amino acids involved in the molecular enzymatic mechanisms ruling the activation of an immunomodulator drug candidate, P28 glutathione-S-transferase (P28GST). SINAPs is freely available at https://github.com/ParImmune/SINAPs.

Safety of P28GST, a Protein Derived from a Schistosome Helminth Parasite, in Patients with Crohn's Disease: A Pilot Study (ACROHNEM).

Despite the development of novel therapies, inflammatory bowel diseases remain an innovative treatment challenge. Helminth therapy is a new promising approach, and a key issue is the identification of helminth-derived anti-inflammatory mediators. P28 glutathione-S-transferase (P28GST), a protein derived from schistosomes, a trematode parasitic helminth, was shown to reduce intestinal inflammation in experimental colitis by down-regulating the Th1/Th17 response. In this multicenter, open-label, pilot Phase 2a study, we evaluated the safety of P28GST administered to patients with mild Crohn's disease (CD). We enrolled 10 patients with a baseline Crohn's disease activity index (CDAI) value <220. Eight patients received two to three subcutaneous injections of recombinant P28GST with adjuvant. This three-month treatment was followed by a nine-month monitoring period. The primary endpoints were the monthly rate and seriousness of adverse events (AEs). Secondary endpoints were clinical recurrence, assessed with the CDAI as well as the levels of immunologic and inflammatory blood and tissue markers. The most common AEs were local or regional events at the injection site and gastrointestinal disorders. At three months after the first injection, CDAI scores and blood calprotectin levels decreased in parallel. These results indicate that P28GST showed promise as a safe and new therapeutic option for treating CD.

Escherichia coli mastitis strains: In vitro phenotypes and severity of infection in vivo.

Mastitis remains a major infection of dairy cows and an important issue for dairy farmers and the dairy industry, in particular infections due to Escherichia coli strains. So far, properties specific to E. coli causing mastitis remain ill defined. In an attempt to better understand the properties required for E. coli to trigger mastitis, we used a range of in vitro assays to phenotypically characterize four E. coli strains, including the prototypical E. coli mastitis strain P4, possessing different relative abilities to cause mastitis in a mouse model. Our results indicate that a certain level of serum resistance might be required for colonization of the mammary gland. Resistance to neutrophil killing is also likely to contribute to a slower clearance of bacteria and higher chances to colonize the udder. In addition, we show that the four different strains do induce a pro-inflammatory response by mammary epithelial cells but with different intensities. Interestingly, the prototypical mastitis strain P4 actually induces the less intense response while it is responsible for the most severe infections in vivo. Altogether, our results suggest that different strategies can be used by E. coli strains to colonize the mammary gland and cause mastitis.

IL-17A Is an Important Effector of the Immune Response of the Mammary Gland to Escherichia coli Infection.

The cytokine IL-17A has been shown to play critical roles in host defense against bacterial and fungal infections at different epithelial sites, but its role in the defense of the mammary gland (MG) has seldom been investigated, although infections of the MG constitute the main pathology afflicting dairy cows. In this study, we showed that IL-17A contributes to the defense of the MG against Escherichia coli infection by using a mouse mastitis model. After inoculation of the MG with a mastitis-causing E. coli strain, the bacterial load increased rapidly, triggering an intense influx of leukocytes into mammary tissue and increased concentrations of IL-6, IL-22, TNF-α, and IL-10. Neutrophils were the first cells that migrated intensely to the mammary tissue, in line with an early production of CXCL2. Depletion of neutrophils induced an increased mammary bacterial load. There was a significant increase of IL-17-containing CD4(+) αß T lymphocyte numbers in infected glands. Depletion of IL-17A correlated with an increased bacterial colonization and IL-10 production. Intramammary infusion of IL-17A at the onset of infection was associated with markedly decreased bacterial numbers, decreased IL-10 production, and increased neutrophil recruitment. Depletion of CD25(+) regulatory T cells correlated with a decreased production of IL-10 and a reduced bacterial load. These results indicate that IL-17A is an important effector of MG immunity to E. coli and suggest that an early increased local production of IL-17A would improve the outcome of infection. These findings point to a new lead to the development of vaccines against mastitis.

Investigating the contribution of IL-17A and IL-17F to the host response during Escherichia coli mastitis.

Mastitis remains a major disease of cattle with a strong impact on the dairy industry. There is a growing interest in understanding how cell mediated immunity contributes to the defence of the mammary gland against invading mastitis causing bacteria. Cytokines belonging to the IL-17 family, and the cells that produce them, have been described as important modulators of the innate immunity, in particular that of epithelial cells. We report here that expression of IL-17A and IL-17F genes, encoding two members of the IL-17 family, are induced in udder tissues of cows experimentally infected with Escherichia coli. The impact of IL-17A on the innate response of bovine mammary epithelial cells was investigated using a newly isolated cell line, the PS cell line. We first showed that PS cells, similar to primary bovine mammary epithelial cells, were able to respond to agonists of TLR2 and to LPS, provided CD14 was added to the culture medium. We then showed that secretion of CXCL8 and transcription of innate immunity related-genes by PS cells were increased by IL-17A, in particular when these cells were stimulated with live E. coli bacteria. Together with data from the literature, these results support the hypothesis that IL-17A and IL-17 F could play an important role in mediating of host-pathogen interactions during mastitis.

Plasmodium berghei histamine-releasing factor favours liver-stage development via inhibition of IL-6 production and associates with a severe outcome of disease.

Plasmodium spp., which causes malaria, produces a histamine-releasing factor (HRF), an orthologue of mammalian HRF. Histamine-releasing factor produced by erythrocytic stages of the parasite is thought to play a role in the pathogenesis of severe malaria. Here, we show in a rodent model that HRF is not important during the erythrocytic but pre-erythrocytic phase of infection, which mainly consists in the transformation in the liver of the mosquito-injected parasite form into the erythrocyte-infecting form. Development of P. berghei ANKA cl15cy1 liver stages lacking HRF is impaired and associated with an early rise in systemic IL-6, a cytokine that strongly suppresses development of Plasmodium liver stages. The defect is rescued by injection of anti-IL-6 antibodies or infection in IL-6-deficient mice and parasite HRF is sufficient to decrease IL-6 synthesis, indicating a direct role of parasite HRF in reducing host IL-6. The target cells modulated by HRF for IL-6 production at early time points during liver infection are neutrophils. Parasite HRF is thus used to down-regulate a cytokine with anti-parasite activity. Our data also highlight the link between a prolonged transition from liver to blood-stage infection and reduced incidence of experimental cerebral malaria.

Repertoire of Escherichia coli agonists sensed by innate immunity receptors of the bovine udder and mammary epithelial cells.

Escherichia coli is a frequent cause of clinical mastitis in dairy cows. It has been shown that a prompt response of the mammary gland after E. coli entry into the lumen of the gland is required to control the infection, which means that the early detection of bacteria is of prime importance. Yet, apart from lipopolysaccharide (LPS), little is known of the bacterial components which are detected by the mammary innate immune system. We investigated the repertoire of potential bacterial agonists sensed by the udder and bovine mammary epithelial cells (bMEC) during E. coli mastitis by using purified or synthetic molecular surrogates of bacterial agonists of identified pattern-recognition receptors (PRRs). The production of CXCL8 and the influx of leucocytes in milk were the readouts of reactivity of stimulated cultured bMEC and challenged udders, respectively. Quantitative PCR revealed that bMEC in culture expressed the nucleotide oligomerization domain receptors NOD1 and NOD2, along with the Toll-like receptors TLR1, TLR2, TLR4, and TLR6, but hardly TLR5. In line with expression data, bMEC proved to react to the cognate agonists C12-iE-DAP (NOD1), Pam3CSK4 (TLR1/2), Pam2CSK4 (TLR2/6), pure LPS (TLR4), but not to flagellin (TLR5). As the udder reactivity to NOD1 and TLR5 agonists has never been reported, we tested whether the mammary gland reacted to intramammary infusion of C12-iE-DAP or flagellin. The udder reacted to C12-iE-DAP, but not to flagellin, in line with the reactivity of bMEC. These results extend our knowledge of the reactivity of the bovine mammary gland to bacterial agonists of the innate immune system, and suggest that E. coli can be recognized by several PRRs including NOD1, but unexpectedly not by TLR5. The way the mammary gland senses E. coli is likely to shape the innate immune response and finally the outcome of E. coli mastitis.

Critical role of the neutrophil-associated high-affinity receptor for IgE in the pathogenesis of experimental cerebral malaria.

The role of the IgE-FcεRI complex in malaria severity in Plasmodium falciparum-hosting patients is unknown. We demonstrate that mice genetically deficient for the high-affinity receptor for IgE (FcεRIα-KO) or for IgE (IgE-KO) are less susceptible to experimental cerebral malaria (ECM) after infection with Plasmodium berghei (PbANKA). Mast cells and basophils, which are the classical IgE-expressing effector cells, are not involved in disease as mast cell-deficient and basophil-depleted mice developed a disease similar to wild-type mice. However, we show the emergence of an FcεRI(+) neutrophil population, which is not observed in mice hosting a non-ECM-inducing PbNK65 parasite strain. Depletion of this FcεRI(+) neutrophil population prevents ECM, whereas transfer of this population into FcεRIα-KO mice restores ECM susceptibility. FcεRI(+) neutrophils preferentially home to the brain and induce elevated levels of proinflammatory cytokines. These data define a new pathogenic mechanism of ECM and implicate an FcεRI-expressing neutrophil subpopulation in malaria disease severity.

Histamine H(3) receptor-mediated signaling protects mice from cerebral malaria.

BACKGROUND: Histamine is a biogenic amine that has been shown to contribute to several pathological conditions, such as allergic conditions, experimental encephalomyelitis, and malaria. In humans, as well as in murine models of malaria, increased plasma levels of histamine are associated with severity of infection. We reported recently that histamine plays a critical role in the pathogenesis of experimental cerebral malaria (CM) in mice infected with Plasmodium berghei ANKA. Histamine exerts its biological effects through four different receptors designated H1R, H2R, H3R, and H4R. PRINCIPAL FINDINGS: In the present work, we explored the role of histamine signaling via the histamine H3 receptor (H3R) in the pathogenesis of murine CM. We observed that the lack of H3R expression (H3R(-/-) mice) accelerates the onset of CM and this was correlated with enhanced brain pathology and earlier and more pronounced loss of blood brain barrier integrity than in wild type mice. Additionally tele-methylhistamine, the major histamine metabolite in the brain, that was initially present at a higher level in the brain of H3R(-/-) mice was depleted more quickly post-infection in H3R(-/-) mice as compared to wild-type counterparts. CONCLUSIONS: Our data suggest that histamine regulation through the H3R in the brain suppresses the development of CM. Thus modulating histamine signaling in the central nervous system, in combination with standard therapies, may represent a novel strategy to reduce the risk of progression to cerebral malaria.

[Role of histamine and histamine receptors in the pathogenesis of malaria]. / Rôle de l'histamine et des récepteurs histaminiques dans la pathogenèse du paludisme.

A hallmark of the host response to Plasmodium parasite is an inflammatory reaction characterized by elevated histaminemia levels. Since histamine, which acts through four different receptors and which synthesis is under the control of the histidine decarboxylase (HDC), is endowed with pro-inflammatory and immunosuppressive activities, we hypothesized that this vaso-active amine may participe to malaria pathogenesis. Combining genetic and pharmacologic approaches by using H1R(-/-), H2R(-/-), H3R(-/-), HDC(-/-) mice and H1R, H2R-, and H3R-antagonists, respectively, we found that cerebral malaria-associated pathogenetic processes such as blood brain barrier disruption, and T lymphocyte sequestration to cerebral vascular endothelium in mice were associated with histamine production. The identification of this novel inflammatory pathway and its implication in Plasmodium infection may lead to novel strategies to manipulate the anti-Plasmodium immune response and may provide new therapeutic tools to alleviate malaria disease.

Inhibition of histamine-mediated signaling confers significant protection against severe malaria in mouse models of disease.

From the inoculation of Plasmodium sporozoites via Anopheles mosquito bites to the development of blood-stage parasites, a hallmark of the host response is an inflammatory reaction characterized by elevated histamine levels in the serum and tissues. Given the proinflammatory and immunosuppressive activities associated with histamine, we postulated that this vasoactive amine participates in malaria pathogenesis. Combined genetic and pharmacologic approaches demonstrated that histamine binding to H1R and H2R but not H3R and H4R increases the susceptibility of mice to infection with Plasmodium. To further understand the role of histamine in malaria pathogenesis, we used histidine decarboxylase-deficient (HDC(-/-)) mice, which are free of histamine. HDC(-/-) mice were highly resistant to severe malaria whether infected by mosquito bites or via injection of infected erythrocytes. HDC(-/-) mice displayed resistance to two lethal strains: Plasmodium berghei (Pb) ANKA, which triggers cerebral malaria (CM), and Pb NK65, which causes death without neurological symptoms. The resistance of HDC(-/-) mice to CM was associated with preserved blood-brain barrier integrity, the absence of infected erythrocyte aggregation in the brain vessels, and a lack of sequestration of CD4 and CD8 T cells. We demonstrate that histamine-mediated signaling contributes to malaria pathogenesis. Understanding the basis for these biological effects of histamine during infection may lead to novel therapeutic strategies to alleviate the severity of malaria.

Immunocompetence does not correlate with resistance to helminth parasites in house mouse subspecies and their hybrids.

A central point not yet elucidated in immuno-ecological studies is whether the different ways to assess the immune status of individuals are good indicators of parasite resistance of the host. We investigate the relationship between immunocompetence and the resistance/susceptibility phenotype of five strains of two European subspecies of the house mouse and their hybrids derived from natural populations. The assessment of immunocompetence was measured indirectly by the mass of the spleen and directly by the phytohaemagglutinin (PHA) test. The resistance of each mouse strain to common helminths was previously determined by the distribution of parasite burdens obtained after experimental infection. We found no relationship between the resistance/susceptibility phenotype of strains and the level of PHA response or the size of the spleen. A significant negative correlation was found between residuals of log spleen mass (controlled for body mass) and PHA response, suggesting that these two components of immune function are not independent measures of defence against parasitism. We discuss the validity of these two techniques of measuring immunocompetence and emphasize the importance of taking into account the complexity of the immune system at structural and functional levels in understanding the component of selection attributable to parasitism in the evolution of immunocompetence.

Recombination does not generate pinworm susceptibility during experimental crosses between two mouse subspecies.

The susceptibility to Aspiculuris tetraptera of European Mus musculus hybrids is thought to reflect the disruption of genomic co-adaptation through recombination of the parental genomes. Here, we compared the susceptibility to this parasite between parents and experimental hybrids (intersubspecific until F4, intrasubspecific F1, F2) to clarify the contributions of heterosis and subspecies incompatibility. F1 showed hybrid vigor. Unlike intrasubspecific F2, intersubspecific F2 were less resistant than F1, but revealed no increased susceptibility relative to the parents. Intersubspecific F3 and F4 showed the same hybrid vigor as F1. Heterosis contributed most to the resistance, but the differences between intra- and intersubspecific F2 suggested genomic incompatibilities between subspecies. However, the susceptibility did not increase through the recombination process, showing that disruption of co-adaptation does not directly affect resistance. Even if previous studies still support the selective role of parasites in the current hybrid zone, an alternative hypothesis on the origin of hybrid susceptibility is warranted.