Antimicrobial activities of phenoloxidasegenerated reactive compounds and regulation of immune response by a serpin from Manduca sexta

Scope and Method of Study: Phenoloxidase (PO) and its activation system are implicated in several defense responses of insects. Upon wounding or infection, inactive prophenoloxidase (proPO) is converted to active PO through a network of serine proteases and their homologs. PO generates reactive compounds such as 5,6- dihydroxyindole (DHI), which have a broad-spectrum antimicrobial activity. The regulation of proteolytic activation cascade of immune response involves serine protease inhibitor (serpin) family molecules. Many serpins are found to play key roles in negatively regulating immune defense system through irreversibly inhibiting target serine proteases, not only to humoral but also to the cellular responses. A new gene, serpin-10, was found in Manduca sexta and expressed in insect cell via infection of recombinant baculovirus contain serpin-10 cDNA fragment. The serpin10 antiserum was prepared and used for determining protein level in hemolymph and affinity chromatography to analysis immune complex formation with serpin-10.Findings and Conclusions: Here we demonstrate that DHI and its spontaneous oxidation products are active against bacteria, fungi, viruses, and parasitic wasp eggs. These results established that proPO activation is an integral component of the insect defense system. A possible mechanism of DHI toxicity is the cross-linking of proteins and nucleic acid via its oxidization products. The transcript level of M. sexta serpin-10 is higher in nerve system, trachea, and fat body than in other tissues. The transcriptional level does not change a lot during 4th and 5th instar larval stages, but decreases remarkably in middle and late pupa stage, and then recovers in adults. This gene is suppressed in fat body, which indicates it may have a relationship with immune response to pathogen invasion. The protein level of serpin-10 is quite stable in hemolymph. M. sexta serpin10 forms a covalent complex with an unknown serine protease. HP1 is present in the immune complex via non-covalent association

Comparative genomic analysis of the Tribolium immune system

The annotation, and comparison with homologous genes in other species, of immunity-related genes in the Tribolium castaneum genome allowed the identification of around 300 candidate defense proteins, and revealed a framework of information on Tribolium immunity

Rapamycin-Loaded Leukosomes Reverse Vascular Inflammation

Rationale: Through localized delivery of rapamycin via a biomimetic drug delivery system, it is possible to reduce vascular inflammation and thus the progression of vascular disease. Objective: Use..

Presenilin1 regulates Th1 and Th17 effector responses but is not required for experimental autoimmune encephalomyelitis.

Multiple Sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system (CNS) where pathology is thought to be regulated by autoreactive T cells of the Th1 and Th17 phenotype. In this study we sought to understand the functions of Presenilin 1 (PSEN1) in regulating T cell effector responses in the experimental autoimmune encephalomyelitis (EAE) murine model of MS. PSEN1 is the catalytic subunit of γ-secretase a multimolecular protease that mediates intramembranous proteolysis. γ-secretase is known to regulate several pathways of immune importance. Here we examine the effects of disrupting PSEN1 functions on EAE and T effector differentiation using small molecule inhibitors of γ-secretase (GSI) and T cell-specific conditional knockout mice (PSEN1 cKO). Surprisingly, blocking PSEN1 function by GSI treatment or PSEN1 cKO had little effect on the development or course of MOG35-55-induced EAE. In vivo GSI administration reduced the number of myelin antigen-specific T cells and suppressed Th1 and Th17 differentiation following immunization. In vitro, GSI treatment inhibited Th1 differentiation in neutral but not IL-12 polarizing conditions. Th17 differentiation was also suppressed by the presence of GSI in all conditions and GSI-treated Th17 T cells failed to induce EAE following adoptive transfer. PSEN cKO T cells showed reduced Th1 and Th17 differentiation. We conclude that γ-secretase and PSEN1-dependent signals are involved in T effector responses in vivo and potently regulate T effector differentiation in vitro, however, they are dispensable for EAE

Increased surfactant protein D fails to improve bacterial clearance and inflammation in serpinB1-/- mice

Previously, we described the protective role of the neutrophil serine protease inhibitor serpinB1 in preventing early mortality of Pseudomonas aeruginosa lung infection by fostering bacterial clearance and limiting inflammatory cytokines and proteolytic damage. Surfactant protein D (SP-D), which maintains the antiinflammatory pulmonary environment and mediates bacterial removal, was degraded in infected serpinB1-deficient mice. Based on the hypothesis that increased SP-D would rescue or mitigate the pathological effects of serpinB1 deletion, we generated two serpinB1(-/-) lines overexpressing lung-specific rat SP-D and inoculated the mice with P. aeruginosa. Contrary to predictions, bacterial counts in the lungs of SP-D(low)serpinB1(-/-) and SP-D(high) serpinB1(-/-) mice were 4 logs higher than wild-type and not different from serpinB1(-/-) mice. SP-D overexpression also failed to mitigate inflammation (TNF-α), lung injury (free protein, albumin), or excess neutrophil death (free myeloperoxidase, elastase). These pathological markers were higher for infected SP-D(high)serpinB1(-/-) mice than for serpinB1(-/-) mice, although the differences were not significant after controlling for multiple comparisons. The failure of transgenic SP-D to rescue antibacterial defense of serpinB1-deficient mice occurred despite 5-fold or 20-fold increased expression levels, largely normal structure, and dose-dependent bacteria-aggregating activity. SP-D of infected wild-type mice was intact in 43-kD monomers by reducing SDS-PAGE. By contrast, proteolytic fragments of 35, 17, and 8 kD were found in infected SP-D(low)serpinB1(-/-), SP-D(high) serpinB1(-/-) mice, and serpinB1(-/-) mice. Thus, although therapies to increase lung concentration of SP-D may have beneficial applications, the findings suggest that therapy with SP-D may not be beneficial for lung inflammation or infection if the underlying clinical condition includes excess proteolysis

Presenilin1 regulates Th1 and Th17 effector responses but is not required for experimental autoimmune encephalomyelitis

<div><p>Multiple Sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system (CNS) where pathology is thought to be regulated by autoreactive T cells of the Th1 and Th17 phenotype. In this study we sought to understand the functions of Presenilin 1 (PSEN1) in regulating T cell effector responses in the experimental autoimmune encephalomyelitis (EAE) murine model of MS. PSEN1 is the catalytic subunit of γ-secretase a multimolecular protease that mediates intramembranous proteolysis. γ-secretase is known to regulate several pathways of immune importance. Here we examine the effects of disrupting PSEN1 functions on EAE and T effector differentiation using small molecule inhibitors of γ-secretase (GSI) and T cell-specific conditional knockout mice (PSEN1 cKO). Surprisingly, blocking PSEN1 function by GSI treatment or PSEN1 cKO had little effect on the development or course of MOG35-55-induced EAE. <i>In vivo</i> GSI administration reduced the number of myelin antigen-specific T cells and suppressed Th1 and Th17 differentiation following immunization. <i>In vitro</i>, GSI treatment inhibited Th1 differentiation in neutral but not IL-12 polarizing conditions. Th17 differentiation was also suppressed by the presence of GSI in all conditions and GSI-treated Th17 T cells failed to induce EAE following adoptive transfer. PSEN cKO T cells showed reduced Th1 and Th17 differentiation. We conclude that γ-secretase and PSEN1-dependent signals are involved in T effector responses <i>in vivo</i> and potently regulate T effector differentiation <i>in vitro</i>, however, they are dispensable for EAE.</p></div

GSI treatment inhibits effector differentiation, activation and proliferation <i>in vitro</i>.

<p>A-E. Effects of GSI on Th1 differentiation. Splenocytes were stimulated <i>in vitro</i> with anti-CD3 and anti-CD28 for 72 hours in the presence of anti-IL-4 and either DMSO or GSI. A-C. IFNγ and Tbet expression were determined by intracellular staining and flow cytometry. A. Representative flow cytometry plots showing IFNγ and Tbet expression. B. IFNγ expression was measured by intracellular flow cytometry. C. Tbet expression was measured by intracellular flow cytometry. D and E. Expression analysis of IL12Rβ1 and IL12Rβ2. Total cellular RNA was isolated from each culture and target gene expression determined by real time PCR. D. Expression of IL12Rβ1. E. Expression of IL12Rβ2. F-J. Effects of GSI on T cell activation. Splenocytes from 2D2 TCR transgenic mice were activated <i>in vitro</i> with MOG35-55 peptide in the presence of DMSO or GSI. At 72 hours of stimulation, flow cytometry was used to measure expression of the activation markers CD25, CD44 and CD69. F. Representative flow cytometry plots showing CD25 and CD44 expression. G. The percentage of T cells expressing CD25. H. Expression of CD44 by T cells. I. The percentages of T cells expressing CD69. J. Quantitation of CD25 expression on activated T cells (CD4 and CD44 gated events). K-N Effects of GSI on T cell proliferation. Splenocytes were labeled with Cell-trace and stimulated for 96 hours with antibodies to CD3 and CD28 in the presence of DMSO or DBZ. Th1 differentiation was promoted by the addition of anti-IL-4 alone (neutral), or in combination with IL-2. K. Proliferation and intracellular IFNγ staining were detected by flow cytometry. L and M. Division index and Proliferation index were calculated using gates to measure the percentages of T cells within each cell division. O. Expression of IFNγ by T cells that had undergone 4 or more cell divisions. P. Expression of CD25 by T cells cultured in the absence or presence of IL-2. Flow cytometry plots are gated on live CD4+ T cells. Results shown are representative of at least two experiments. The numbers in FACS plots indicate cell percentages within each quadrant. Individual symbols indicate results from replicate wells. Open circles indicate treatment with DMSO, filled squares indicate GSI treatment. Error bars indicate SEM. Asterisks indicate significant differences (* p<0.05, ** p<0.01 and ***p <0.001).</p