Dynamic changes in thymic sub-populations during acute and long-term infections with virulent and virulence-attenuated Salmonella Typhimurium strains in C57BL/6 and autoimmune-prone lpr mice.

SALMONELLA: Typhimurium infection in mice results in drastic loss of immature CD4- CD8- double negative (DN) and CD4+ CD8+ double positive (DP) thymic subsets compared to mature single positive (SP) subsets. We investigated changes in thymocyte sub-populations post infection with a wild type (WT) virulent strain and ΔrpoS, a virulence-attenuated strain, of Salmonella Typhimurium in C57BL/6 (B6) and Fas-deficient autoimmune-prone lpr mice. The WT strain caused acute thymic atrophy with greater loss of thymocytes in lpr mice compared to B6 mice. Infection with ΔrpoS caused progressive thymic atrophy in B6 and lpr mice. Analysis of thymocyte subsets revealed that immature thymocytes including the DN, immature single positive (ISP), and DP thymocytes underwent extensive loss. SP thymocytes were more resistant to loss in WT-infected B6 mice, whereas WT-infected lpr and ΔrpoS-infected mice exhibited depletion of SP thymocytes. Overall, thymocyte sub-populations exhibited differential susceptibilities depending on bacterial virulence and the host background.

Particle uptake driven phagocytosis in macrophages and neutrophils enhances bacterial clearance.

Humans are exposed to numerous synthetic foreign particles in the form of drug delivery systems and diagnostic agents. Specialized immune cells (phagocytes) clear these particles by phagocytosing and attempting to degrade them. The process of recognition and internalization of the particles may trigger changes in the function of phagocytes. Some of these changes, especially the ability of a particle-loaded phagocyte to take up and neutralize pathogens, remains poorly studied. Herein, we demonstrate that the uptake of non-stimulatory cargo-free particles enhances the phagocytic ability of monocytes, macrophages and neutrophils. The enhancement in phagocytic ability was independent of particle properties, such as size or the base material constituting the particle. Additionally, we show that the increased phagocytosis was not a result of cellular activation or cellular heterogeneity but was driven by changes in cell membrane fluidity and cellular compliance. A consequence of the enhanced phagocytic activity was that particulate-laden immune cells neutralize Escherichia coli (E. coli) faster in culture. Moreover, when administered in mice as a prophylactic, particulates enable faster clearance of E. coli and Staphylococcus epidermidis. Together, we demonstrate that the process of uptake induces cellular changes that favor additional phagocytic events. This study provides insights into using non-stimulatory cargo-free particles to engineer immune cell functions for applications involving faster clearance of phagocytosable abiotic and biotic material.

Autoimmune-prone lpr mice exhibit a prolonged but lethal infection with an attenuated Salmonella Typhimurium strain.

Autoimmunity can potentially pre-dispose to, exacerbate or ameliorate pathogenic infections. The current study was designed to compare and understand the infection outcomes with Salmonella enterica serovar Typhimurium ATCC 14028s (S. Typhimurium) wild type (WT) and attenuated ΔrpoS strains, in autoimmune-prone lpr mice. C57BL/6 (B6) and B6/lpr (lpr) 6-8 weeks old mice were orally infected with S. Typhimurium WT and ΔrpoS strains. Disease outcomes were assessed with respect to survival, organ bacterial load, tissue damage and inflammation in infected mice. The acute infection stage (day 4) was examined and compared to the later stages (up to day 12) post ΔrpoS infection. S. Typhimurium WT exhibited an acute and lethal infection in both B6 and lpr mice. However, the ΔrpoS strain exhibited prolonged infection with reduced mortality in B6 mice but complete mortality in lpr mice. During late infection, bacterial load and serum IFNγ levels were higher in the ΔrpoS strain infected lpr mice compared to B6 mice. The ΔrpoS strain infected lpr mice also exhibited greater bacterial faecal shedding and greater tissue histopathological changes. Interestingly, ΔrpoS-infected B6 mice displayed minimal microbial load in the brain; however, sustained brain bacterial load was observed in ΔrpoS-infected lpr mice, corresponding to abnormal gait. Overall, S. Typhimurium ΔrpoS is competent in establishing infection but compromised in sustaining it. Nonetheless, lpr mice are less efficient in controlling this attenuated infection. The findings from the study demonstrate that genetic pre-disposition to autoimmunity is sufficient for greater host susceptibility to infection by attenuated S. Typhimurium strains.

Comparative evaluation of structure and characteristic of peptidyl-prolyl cis-trans isomerase proteins and their function in Salmonella Typhimurium stress responses and virulence.

Peptidyl-prolyl cis-trans isomerases (PPIase) exhibit chaperone activity and assist in protein folding by increasing the rate of cis-trans transition on proline-peptide bonds. The current study aimed to identify and characterize three genes, ppiA, ppiB, and ppiC, which encode proteins of the PPIase family in the bacterium Salmonella enterica serovar Typhimurium. Salmonella Typhimurium is a facultative intracellular zoonotic pathogen that causes food- and water-borne gastroenteritis in humans (leading to bacteremia in immune-compromised subjects). Recombinant clones for the three genes were constructed and sequenced and the sequences submitted to NCBI GenBank. Three-dimensional structures for the corresponding proteins were predicted by comparative modeling. A maximum-likelihood phylogenetic gene tree constructed for the three genes showed a low evolutionary mean diversity, indicating strong evolutionary conservation. Further, single-gene deletion mutant strains, generated for the respective genes, were observed to be more susceptible to the stationary phase of growth and heat stress conditions and showed reduced survival within macrophage cells line. The present study thus indicates that ppiA, ppiB, and ppiC genes are conserved among Salmonella genome, are critical for the growth of Salmonella Typhimurium in the examined stress conditions, and may play a role in its responses and virulence.

Comparative analysis of thymic subpopulations during different modes of atrophy identifies the reactive oxygen species scavenger, N-acetyl cysteine, to increase the survival of thymocytes during infection-induced and lipopolysaccharide-induced thymic atrophy.

The development of immunocompetent T cells entails a complex pathway of differentiation in the thymus. Thymic atrophy occurs with ageing and during conditions such as malnutrition, infections and cancer chemotherapy. The comparative changes in thymic subsets under different modes of thymic atrophy and the mechanisms involved are not well characterized. These aspects were investigated, using mice infected with Salmonella Typhimurium, injection with lipopolysaccharide (LPS), an inflammatory but non-infectious stimulus, etoposide (Eto), a drug used to treat some cancers, and dexamethasone (Dex), a steroid used in some inflammatory diseases. The effects on the major subpopulations of thymocytes based on multicolour flow cytometry studies were, first, the CD4-  CD8- double-negative (DN) cells, mainly DN2-4, were reduced with infection, LPS and Eto treatment, but not with Dex. Second, the CD8+  CD3lo immature single-positive cells (ISPs) were highly sensitive to infection, LPS and Eto, but not Dex. Third, treatment with LPS, Eto and Dex reduced all three subpopulations of CD4+  CD8+ double-positive (DP) thymocytes, i.e. DP1, DP2 and DP3, but the DP3 subset was relatively more resistant during infection. Fourth, both CD4+ and CD8+ single-positive (SP) thymocytes were lowered by Eto and Dex, but not during infection. Notably, LPS lowered CD4+ SP subsets, whereas the CD8+ SP subsets were relatively more resistant. Interestingly, the reactive oxygen species quencher, N-acetyl cysteine, greatly improved the survival of thymocytes, especially DNs, ISPs and DPs, during infection and LPS treatment. The implications of these observations for the development of potential thymopoietic drugs are discussed.

Differential susceptibility and maturation of thymocyte subsets during Salmonella Typhimurium infection: insights on the roles of glucocorticoids and Interferon-gamma.

The thymus is known to atrophy during infections; however, a systematic study of changes in thymocyte subpopulations has not been performed. This aspect was investigated, using multi-color flow cytometry, during oral infection of mice with Salmonella Typhimurium (S. Typhimurium). The major highlights are: First, a block in the developmental pathway of CD4-CD8- double negative (DN) thymocytes is observed. Second, CD4+CD8+ double positive (DP) thymocytes, mainly in the DP1 (CD5loCD3lo) and DP2 (CD5hiCD3int), but not DP3 (CD5intCD3hi), subsets are reduced. Third, single positive (SP) thymocytes are more resistant to depletion but their maturation is delayed, leading to accumulation of CD24hiCD3hi SP. Kinetic studies during infection demonstrated differences in sensitivity of thymic subpopulations: Immature single positive (ISP) > DP1, DP2 > DN3, DN4 > DN2 > CD4+ > CD8+. Upon infection, glucocorticoids (GC), inflammatory cytokines, e.g. Ifnγ, etc are induced, which enhance thymocyte death. Treatment with RU486, the GC receptor antagonist, increases the survival of most thymic subsets during infection. Studies with Ifnγ-/- mice demonstrated that endogenous Ifnγ produced during infection enhances the depletion of DN2-DN4 subsets, promotes the accumulation of DP3 and delays the maturation of SP thymocytes. The implications of these observations on host cellular responses during infections are discussed.