Regulatory T cells (Tregs) and their therapeutic potential against autoimmune disorders - Advances and challenges.

Autoimmune diseases are caused when immune cells act against self-protein. This biological self-non-self-discrimination phenomenon is controlled by a distinct group of lymphocytes known as regulatory T cells (Tregs), which are key inflammatory response regulators and play a pivotal role in immune tolerance and homeostasis. Treg-mediated robust immunosuppression provides self-tolerance and protection against autoimmune diseases. However, once this system fails to operate or poorly operate, it leads to an extreme situation where immune system reacts against self-antigens and destroys host organs, thus causing autoimmune diseases. Tregs can target both innate and adaptive immunity via modulating multiple immune cells such as neutrophils, monocytes, antigen-presenting cells, B cells, and T cells. This review highlights the Treg-mediated immunosuppression, role of several markers and their interplay during Treg development and differentiation, and advances in therapeutic aspects of Treg cells to reduce severity of autoimmunity-related conditions along with emphasizing limitations and challenges of their usages.

Differential responses of chicken monocyte-derived dendritic cells infected with Salmonella Gallinarum and Salmonella Typhimurium.

Salmonella enterica serovar Gallinarum is a host-restricted bacterial pathogen that causes a serious systemic disease exclusively in birds of all ages. Salmonella enterica serovar Typhimurium is a host-generalist serovar. Dendritic cells (DCs) are key antigen-presenting cells that play an important part in Salmonella host-restriction. We evaluated the differential response of chicken blood monocyte-derived dendritic cells (chMoDCs) exposed to S. Gallinarum or S. Typhimurium. S. Typhimurium was found to be more invasive while S. Gallinarum was more cytotoxic at the early phase of infection and later showed higher resistance against chMoDCs killing. S. Typhimurium promoted relatively higher upregulation of costimulatory and other immune function genes on chMoDCs in comparison to S. Gallinarum during early phase of infection (6 h) as analyzed by real-time PCR. Both Salmonella serovars strongly upregulated the proinflammatory transcripts, however, quantum was relatively narrower with S. Gallinarum. S. Typhimurium-infected chMoDCs promoted relatively higher proliferation of naïve T-cells in comparison to S. Gallinarum as assessed by mixed lymphocyte reaction. Our findings indicated that host restriction of S. Gallinarum to chicken is linked with its profound ability to interfere the DCs function. Present findings provide a valuable roadmap for future work aimed at improved vaccine strategies against this pathogen.

Brucella abortus S19 rfbD mutant is highly attenuated, DIVA enable and confers protection against virulent challenge in mice.

Brucella abortus S19 is an important tool for controlling bovine brucellosis across the globe. However, vaccination with S19 suffers critical shortcomings such as, presence of residual virulence, induction of abortion and sero-diagnostic interference. In this study, rfbD gene deleted mutant S19 was developed. The mutant strain designated S19ΔR displayed rough LPS phenotype, which was confirmed by acriflavine dye-agglutination and LPS-SDS-PAGE analysis. The virulence was amply reduced as suggested by increased sensitivity to complement killing; reduction in splenic-bacterial load and the recovery time RT50 as validated in mice model. Anti-brucella humoral response was significantly lower as compared to S19 immunization. The minimal induction of Brucella specific IgG1, IgG2a & IgG2b, and IgG3 resulted in no apparent reactivity to RBPT antigen. S19ΔR showed protective index of 1.90 against virulent challenge. S19ΔR being highly attenuated and DIVA compatible may facilitate a platform for developing a safer bovine adulthood vaccine.

Combination of TLR2 and TLR3 agonists derepress infectious bursal disease virus vaccine-induced immunosuppression in the chicken.

Live intermediate plus infectious bursal disease virus (IBDV) vaccines (hot vaccines) are used for protection against the virulent IBDV strains in young chickens. We evaluated the potential of Toll-like receptor (TLR) agonists to alleviate hot vaccine-induced immunosuppression. The combination of Pam3CSK4 and poly I:C synergistically upregulated IFN-ß, IFN-γ, IL-12, IL-4, and IL-13 transcripts and cross-inhibited IL-1ß, IL-10, and iNOS transcripts in the chicken peripheral blood mononuclear cells (PBMCs) as analyzed by quantitative real-time PCR. Further, four-week old specific pathogen free White Leghorn chickens (n = 60) were randomly divided into six groups and either immunized with hot IBDV vaccine with or without Pam3CSK4 and/or poly I:C or not vaccinated to serve as controls. The results indicated that poly I:C alone and in combination with Pam3CSK4 alleviated vaccine-induced immunosuppression, as evidenced by greater weight gain, increased overall antibody responses to both sheep erythrocytes and live infectious bronchitis virus vaccine, upregulated IFN-γ transcripts and nitric oxide production by PBMCs (P < 0.05), and lower bursal lesion score in the experimental birds. In conclusion, poly I:C alone and its combination with Pam3CSK4 reduced the destruction of B cells as well as bursal damage with restoration of function of T cells and macrophages when used with a hot IBDV vaccine.

Development and evaluation of a gold nanoparticle-based immunochromatographic strip test for the detection of canine parvovirus.

Canine parvovirus (CPV) is the leading viral cause of enteritis in dogs and occurs mainly in 6- to 8-week-old pups. Rapid diagnosis of CPV under field conditions is now possible due to commercially available immunochromatographic (IC) assays. However, these commercial kits are somewhat expensive because they utilize a minimum of two monoclonal antibodies (mAbs) targeting different epitopes as capture and detector antibodies. Using only a single mAb for both capture and detection purpose may reduce the sensitivity of the assay. In the present study, efforts were made to develop an economical assay that can be utilized for diagnosis of CPV under Indian conditions with a high level of confidence. Rabbit polyclonal antibodies (pAbs) generated against recombinant truncated VP2 proteins of CPV were used as capture antibodies because they can be produced economically, while a commercial anti-CPV mAb was used as the detector antibody. The detection limit of the test strip was 6.6×105 TCID50/ml, and it specifically detected CPV-2, CPV-2a and CPV-2b while displaying no cross-reactivity with other common canine enteric pathogens. The relative sensitivity/specificity of pAb based strip test was 71%/92% and 71%/100% in relation to the hemagglutination test and a commercial IC kit, respectively, with substantial agreement. In addition, two commercially available mAbs targeting different epitopes were also used for development of another IC assay, which showed sensitivity, and specificity of 82%/87% and 90%/98% in relation to the hemagglutination test and commercial kit. Hence, the present strip test based on a combination of mAb and pAb provides an acceptable alternative for onsite and cost-effective diagnosis of CPV infection.

Comparative roles of clpA and clpB in the survival of S. Typhimurium under stress and virulence in poultry.

By assisting in the proteolysis, disaggregation and refolding of the aggregated proteins, Caseinolytic proteases (Clps) enhance the cellular survival under stress conditions. In the current study, comparative roles of two such Clps, ClpA (involved in proteolysis) and ClpB (involved in protein disaggregation and refolding) in the survival of Salmonella Typhimurium (S. Typhimurium) under different stresses and in virulence have been investigated. clpA and clpB gene deletion mutant strains (∆clpA and ∆clpB) of S. Typhimurium have been hypersensitive to 42 °C, HOCl and paraquat. However, the ∆clpB strain was comparatively much more susceptible (p < 0.001) to the above stresses than ∆clpA strain. ∆clpB strain also showed reduced survival (p < 0.001) in poultry macrophages. The hypersusceptibilities of ∆clpB strain to oxidants and macrophages were restored in plasmid based complemented (∆clpB + pclpB) strain. Further, the ∆clpB strain was defective for colonization in the poultry caecum and showed decreased dissemination to the spleen and liver. Our findings suggest that the role of ClpB is more important than the role of ClpA for the survival of S. Typhimurium under stress and colonization in chickens.

Role of different receptors and actin filaments on Salmonella Typhimurium invasion in chicken macrophages.

Bacterial attachment to host cell is the first event for pathogen entry. The attachment is mediated through membrane expressed adhesins present on the organism and receptors on the cell surface of host. The objective of this study was to investigate the significance of Fc receptors (FcRs), actin filament polymerization, mannose receptors (MRs), carbohydrate moieties like N-linked glycans and sialic acid on chicken macrophages for invasion of S. Typhimurium. Opsonisation of S. Typhimurium resulted in three folds more invasion in chicken monocyte derived macrophages. Cytochalasin D, an inhibitor of actin filament polymerization prevented uptake of S. Typhimurium. Pre-incubation of macrophages with cytochalasin D, showed severe decrease (28 folds) in S. Typhimurium invasion. Next we attempted to analyse the role of carbohydrate receptors of macrophages in S. Typhimurium invasion. Treatment of macrophages with methyl α-d-mannopyranoside, PNGase F and neuraminidase, showed 2.5, 5 and 2.5 folds decrease in invasion respectively. Our data suggest that deglycosylation of N-linked glycans including sialic acid by PNGase F is more effective in inhibition of S. Typhimurium invasion than neuraminidase which removes only sialic acid. These findings suggested FcRs, actin filament polymerization, MRs, N-linked glycans and sialic acid may act as gateway for entry of S. Typhimurium.

Salmonella Typhimurium methionine sulfoxide reductase A (MsrA) prefers TrxA in repairing methionine sulfoxide.

Intraphagocytic survival of Salmonella Typhimurium (ST) depends (at least in part) upon its ability to repair oxidant-damaged macromolecules. Met residues either free or in protein bound form are highly susceptible to phagocyte-generated oxidants. Oxidation of Mets leads to Met-SO formation, consequently loss of protein functions that results in cell death. Methionine sulfoxide reductase (Msr) reductively repairs Met-SO to Met in the presence of thioredoxin (trx) and thioredoxin reductase (trxR). Earlier we reported that methionine sulfoxide reductase A (msrA) gene deletion strain of ST suffered oxidative stress.[1] Thioredoxin system of ST comprises of two thioredoxins (trxA and trxC) and one thioredoxin reductase (trxB). Preferred trx utilized in MsrA-mediated repair of Met-SO is not known. In current study, we cloned, expressed, and purified ST TrxA, TrxB, TrxC, and MsrA in recombinant forms. The migration of TrxA, TrxB, TrxC, and MsrA proteins was approximately 10, 36, 16, and 26 kDa on SDS-gels. The nicotinamide adenine dinucleotide phosphate hydrogen (NADPH)-linked reductase assays interpreted that MsrA utilized two times more NADPH for the reduction of S-methyl p-tolyl sulfoxide when TrxA was included in the assays as compared to TrxC.

Contribution of protein isoaspartate methyl transferase (PIMT) in the survival of Salmonella Typhimurium under oxidative stress and virulence.

The enteric pathogen Salmonella Typhimurium (ST) survives inside the oxidative environment of phagocytic cells. Phagocyte generated oxidants primarily target proteins and modify amino acids in them. These modifications render the targeted proteins functionally inactive. Conversion of Asp to iso-Asp is one of the several known oxidant mediated amino acids modifications. By repairing iso-Asp to Asp, protein-isoaspartyl methyltransferase (PIMT) maintains the activities of proteins and thus helps in cellular survival under oxidative stress. To elucidate the role of PIMT in ST survival under oxidative stress, we have constructed a pimt gene deletion strain (Δpimt strain) of ST. The Δpimt strain grows normally in various culture media in vitro. However, in comparison to wild type ST, the Δpimt strain is found significantly (p<0.001) more susceptible to H2O2 and hypochlorite (HOCl). Further, the Δpimt mutant strain shows hypersusceptibility (p<0.001) to INF-γ stimulated macrophages. This susceptibility is reversed by pharmacological inhibition of reactive oxygen species (ROS) but not reactive nitrogen species (RNS) production. Further, plasmid based complementation enhances the survival of Δpimt mutant strain against oxidants in vitro and also inside the macrophages. In mice model, the LD50 for wild type ST and mutant Δpimt has been 1.73×10(4) and 1.38×10(5), respectively. Further, the mutant strain shows reduced dissemination to spleen and liver in mice. Following infection with a mixture of wild type ST and the Δpimt mutant (co-infection experiment), we recover significantly (p<0.001) less numbers of mutant bacteria from the spleen and liver of mice.

Methionine sulfoxide reductase A (MsrA) contributes to Salmonella Typhimurium survival against oxidative attack of neutrophils.

Salmonella Typhimurium (ST) must evade neutrophil assault for infection establishment in the host. Myeloperoxidase generated HOCl is the key antimicrobial agent produced by the neutrophils; and methionine (Met) residues are the primary targets of this oxidant. Oxidation of Mets leads to methionine sulfoxide (Met-SO) formation and consequently compromises the protein function(s). Methionine sulfoxide reductase A (MsrA) reductively repairs Met-SO to Mets. In this manner, MsrA maintains the function(s) of key proteins which are important for virulence of ST and enhance the survival of this bacterium under oxidative stress. We constructed msrA gene deletion strain (ΔmsrA). The primers located in the flanking regions to ΔmsrA gene amplified 850 and 300 bp amplicons in ST and ΔmsrA strains, respectively. The ΔmsrA strain grew normally in in vitro broth culture. However, ΔmsrA strain showed high susceptibility (p<0.001) to very low concentrations of HOCl which was restored (at least in part) by plasmid based complementation. ΔmsrA strain was hypersensitive (than ST) to the granules isolated from neutrophils. Further, the ΔmsrA strain was significantly (p<0.05) more susceptible to neutrophil mediated killing.

Experimental iron-inactivated Pasteurella multocida A: 1 vaccine adjuvanted with bacterial DNA is safe and protects chickens from fowl cholera.

Fowl cholera is a serious problem in large and small scale poultry production. The present study describes the development and testing of an inactivated whole-cell, low-cost, safe, and effective vaccine for fowl cholera based on a previous work (Vaccine 23:5590-5598). Pasteurella multocida A: 1 grown in the presence of low FeCl(3) concentrations, inactivated with higher concentrations of FeCl(3), and adjuvanted with bacterial DNA from P. multocida B: 2 containing immunostimulatory CpG motifs protect chickens with a lethal P. multocida A: 1 challenge. Chickens were immunized with two whole-cell inactivated vaccine doses at 4 weeks apart and challenged 4 weeks after booster immunization. Experimental vaccines were pure, easy injectable, and caused very little distress in chickens due to their aqueous consistency. Vaccines and bacterial DNA (bDNA) posed no safety problems when chickens were injected subcutaneously (s.c.) with a single, double, and overdose of these preparations. Immunized chickens produced systemic IgY antibodies (Ab) responses and vaccine adjuvanted with bDNA protected 100% chickens from lethal intrapertoneal (i.p.) P. multocida A: 1 challenge. This work suggests that use of bDNA as an adjuvant can improve the cost-effectiveness of inactivated veterinary vaccines for their use in developing countries. Our future studies will focus on safety and potency evaluation of experimental and current vaccines using bDNA as an adjuvant.