Engineering DNA nanoparticles as immunomodulatory reagents that activate regulatory T cells

Nanoparticles containing DNA complexed with the cationic polymer polyethylenimine are efficient vehicles to transduce DNA into cells and organisms. DNA/polyethylenimine nanoparticles (DNPs) also elicit rapid and systemic release of proinflammatory cytokines that promote antitumor immunity. In this study, we report that DNPs possess previously unrecognized immunomodulatory attributes due to rapid upregulation of IDO enzyme activity in lymphoid tissues of mice. IDO induction in response to DNP treatment caused dendritic cells and regulatory T cells (Tregs) to acquire potent regulatory phenotypes. As expected, DNP treatment stimulated rapid increase in serum levels of IFN type I (IFN-αβ) and II (IFN-γ), which are both potent IDO inducers. IDO-mediated Treg activation was dependent on IFN type I receptor signaling, whereas IFN-γ receptor signaling was not essential for this response. Moreover, systemic IFN-γ release was caused by TLR9-dependent activation of NK cells, whereas TLR9 signaling was not required for IFN-αβ release. Accordingly, DNPs lacking immunostimulatory TLR9 ligands in DNA stimulated IFN-αβ production, induced IDO, and promoted regulatory outcomes, but did not stimulate potentially toxic, systemic release of IFN-γ. DNP treatment to induce IDO and activate Tregs blocked Ag-specific T cell responses elicited in vivo following immunization and suppressed joint pathology in a model of immune-mediated arthritis. Thus, DNPs lacking TLR9 ligands may be safe and effective reagents to protect healthy tissues from immune-mediated destruction in clinical hyperimmune syndromes.     

Homology modelling of CB1 receptor and selection of potential inhibitor against Obesity

Obesity and patient morbidity has become a health concern worldwide. Obesity is associated with over activity of the endocannabinoid system, which is involved in the regulation of appetite, lipogenesis and insulin resistance. Hypothalamic cannabinoid-1 receptor (CB1R) inverse agonists reduce body weight and improve cardiometabolic abnormalities in experimental and human obesity but displayed neuropsychiatric side effects. Hence, there is a need to develop therapeutics which employs blocking peripheral CB1 receptors and still achieve substantial weight loss. In view of the same, adipose tissue CB1 receptors are employed for this study since it is more specific in reducing visceral fat. Computer aided structure based virtual screening finds application to screen novel inhibitors and develop highly selective and potential drug. The rational drug design requires crystal structure for the CB1 receptor. However, the structure for the CB1 receptor is not available in its native form. Thus, we modelled the crystal structure using a lipid G-Protein coupled receptor (PDB: 3V2W, chain A) as template. Furthermore, we have screened a herbal ligand Quercetin [- 2- (3, 4-dihydroxyphenyl) - 3, 5, 7-trihydroxychromen-4-one] a flavonol present in Mimosa pudica based on its better pharmacokinetics and bioavailability profile. This ligand was selected as an ideal lead molecule. The docking of quercetin with CB1 receptor showed a binding energy of -6.56 Kcal/mol with 4 hydrogen bonds, in comparison to the known drug Rimonabant. This data finds application in proposing antagonism of CB1 receptor with Quercetin, for controlling obesity.     

Enrichment of regulatory CD4(+)CD25(+) T cells by inhibition of phospholipase D signaling

Antigen stimulation of lymphocytes induces upregulation of phospholipase D (PLD) activity, but the biological significance of PLD-mediated signaling in T cells has not been well established. Here we demonstrate that PLD signaling is essential for proliferation of mouse CD8(+) T cells and CD4(+)CD25(-) T cells, but is not required for proliferation of CD4(+)CD25(+) regulatory T cells. We exploited this observation to develop an efficient method to enrich for regulatory T cells starting from preparations of total CD4(+) T lymphocytes. Inhibition of PLD signaling blocked effector T-cell proliferation after T cell-antigen receptor (TCR) engagement, but had no significant effect on the proliferation of CD4(+)CD25(+) T cells with regulatory functions. Consequently, cells expanded in vitro for one week by antigen receptor stimulation with PLD signal inhibition were markedly enriched for regulatory T cells.     

Cutting edge: induced indoleamine 2,3 dioxygenase expression in dendritic cell subsets suppresses T cell clonal expansion

In mice, immunoregulatory APCs express the dendritic cell (DC) marker CD11c, and one or more distinctive markers (CD8alpha, B220, DX5). In this study, we show that expression of the tryptophan-degrading enzyme indoleamine 2,3 dioxygenase (IDO) is selectively induced in specific splenic DC subsets when mice were exposed to the synthetic immunomodulatory reagent CTLA4-Ig. CTLA4-Ig did not induce IDO expression in macrophages or lymphoid cells. Induction of IDO completely blocked clonal expansion of T cells from TCR transgenic mice following adoptive transfer, whereas CTLA4-Ig treatment did not block T cell clonal expansion in IDO-deficient recipients. Thus, IDO expression is an inducible feature of specific subsets of DCs, and provides a potential mechanistic explanation for their T cell regulatory properties.     

Hoxa9 transforms primary bone marrow cells through specific collaboration with Meis1a but not Pbx1b.

Hoxa9, Meis1 and Pbx1 encode homeodomaincontaining proteins implicated in leukemic transformation in both mice and humans. Hoxa9, Meis1 and Pbx1 proteins have been shown to physically interact with each other, as Hoxa9 cooperatively binds consensus DNA sequences with Meis1 and with Pbx1, while Meis1 and Pbx1 form heterodimers in both the presence and absence of DNA. In this study, we sought to determine if Hoxa9 could transform hemopoietic cells in collaboration with either Pbx1 or Meis1. Primary bone marrow cells, retrovirally engineered to overexpress Hoxa9 and Meis1a simultaneously, induced growth factor-dependent oligoclonal acute myeloid leukemia in <3 months when transplanted into syngenic mice. In contrast, overexpression of Hoxa9, Meis1a or Pbx1b alone, or the combination of Hoxa9 and Pbx1b failed to transform these cells acutely within 6 months post-transplantation. Similar results were obtained when FDC-P1 cells, engineered to overexpress these genes, were transplanted to syngenic recipients. Thus, these studies demonstrate a selective collaboration between a member of the Hox family and one of its DNA-binding partners in transformation of hemopoietic cells.     

Immunopotentiating effects of Azadirachta indica (Neem) dry leaves powder in broilers, naturally infected with IBD virus

Effects of feeding of powdered dry leaves of A. indica (AI) were investigated on humoral and cell mediated immune responses, in a flock of broilers which had survived an outbreak of infectious bursal disease (IBD). AI (2 g/kg) treatment significantly enhanced the antibody titres against new castle disease virus (NCDV) antigen and also potentiated the inflammatory reactions to 1. Chloro-2,4-di- nitro benzene (DNCB) inskin contact test. The results indicate that AI could be beneficial in immunosuppressed condition like IBD, in poultry.     

Mannich reaction: an approach for the synthesis of water soluble mulundocandin analogues

Semisynthetic modifications at Hydroxy tyrosine (Htyr) unit of mulundocandin (1) were carried out to improve its aqueous solubility. A single step introduction of substituted aminomethyl groups at the ortho position(s) of phenolic hydroxyl of HTyr unit of mulundocandin has been achieved in 7-85% yield. The in vitro screening of Mannich products against Candida albicans and Aspergillus fumigatus, retained the in vivo activity of parent by oral and intraperitoneal route. Compound 20, showed significant improvement in activity over mulundocandin (1) and activity compares well with that of fluconazole.     

Upregulation of Programmed Death-1 and Its Ligand in Cardiac Injury Models: Interaction with GADD153

Purpose

Programmed Death-1 (PD-1) and its ligand, PD-L1, are regulators of immune/ inflammatory mechanisms. We explored the potential involvement of PD-1/PD-L1 pathway in the inflammatory response and tissue damage in cardiac injury models.

Experimental Design

Ischemic-reperfused and cryoinjured hearts were processed for flow cytometry and immunohistochemical studies for determination of cardiac PD-1 and PD-L1 in the context of assessment of the growth arrest- and DNA damage-inducible protein 153 (GADD153) which regulates both inflammation and cell death. Further, we explored the potential ability of injured cardiac cells to influence proliferation of T lymphocytes.

Results

The isolated ischemic-reperfused hearts displayed marked increases in expression of PD-1 and PD-L1 in cardiomyocytes; however, immunofluorescent studies indicate that PD-1 and PD-L1 are not primarily co-expressed on the same cardiomyocytes. Upregulation of PD-1/PD-L1 was associated with a) marked increases in GADD153 and interleukin (IL)-17 but a mild increase in IL-10 and b) disruption of mitochondrial membrane potential (ψ_m) as well as apoptotic and necrotic cell death. Importantly, while isotype matching treatment did not affect the aforementioned changes, treatment with the PD-L1 blocking antibody reversed those effects in association with marked cardioprotection. Further, ischemic-reperfused cardiac cells reduced proliferation of T lymphocytes, an effect partially reversed by PD-L1 antibody. Subsequent studies using the cryoinjury model of myocardial infarction revealed significant increases in PD-1, PD-L1, GADD153 and IL-17 positive cells in association with significant apoptosis/necrosis.

Conclusions

The data suggest that upregulation of PD-1/PD-L1 pathway in cardiac injury models mediates tissue damage likely through a paracrine mechanism. Importantly, inhibition of T cell proliferation by ischemic-reperfused cardiac cells is consistent with the negative immunoregulatory role of PD-1/PD-L1 pathway, likely reflecting an endogenous cardiac mechanism to curtail the deleterious impact of infiltrating immune cells to the damaged myocardium. The balance of these countervailing effects determines the extent of cardiac injury.     

Towards the characterization of the hidden world of small proteins in Staphylococcus aureus,a proteogenomics approach

Small proteins play essential roles in bacterial physiology and virulence, however, automated algorithms for genome annotation are often not yet able to accurately predict the corresponding genes. The accuracy and reliability of genome annotations, particularly for small open reading frames (sORFs), can be significantly improved by integrating protein evidence from experimental approaches. Here we present a highly optimized and flexible bioinformatics workflow for bacterial proteogenomics covering all steps from (i) generation of protein databases, (ii) database searches and (iii) peptide-to-genome mapping to (iv) visualization of results. We used the workflow to identify high quality peptide spectrum matches (PSMs) for small proteins (≤ 100 aa, SP100) in Staphylococcus aureus Newman. Protein extracts from S. aureus were subjected to different experimental workflows for protein digestion and prefractionation and measured with highly sensitive mass spectrometers. In total, 175 proteins with up to 100 aa (SP100) were identified. Out of these 24 (ranging from 9 to 99 aa) were novel and not contained in the used genome annotation.144 SP100 are highly conserved and were found in at least 50% of the publicly available S. aureus genomes, while 127 are additionally conserved in other staphylococci. Almost half of the identified SP100 were basic, suggesting a role in binding to more acidic molecules such as nucleic acids or phospholipids.