Activation of murine dendritic cells and macrophages induced by Salmonella enterica serovar Typhimurium

Macrophages and dendritic cells (DCs) are antigen-presenting cells (APCs), and the direct involvement of both cell types in the immune response to Salmonella has been identified. In this study we analysed the phenotypic and functional changes that take place in murine macrophages and DCs in response to live and heat-killed Salmonella enterica serovar Typhimurium. Both types of cell secreted proinflammatory cytokines and nitric oxide (NO) in response to live and heat-killed salmonellae. Bacterial stimulation also resulted in up-regulation of costimulatory molecules on macrophages and DCs. The expression of major histocompatibility complex (MHC) class II molecules by macrophages and DCs was differentially regulated by interferon (IFN)-gamma and salmonellae. Live and heat-killed salmonellae as well as lipopolysaccharide (LPS) inhibited the up-regulation of MHC class II expression induced by IFN-gamma on macrophages but not on DCs. Macrophages as well as DCs presented Salmonella-derived antigen to CD4 T cells, although DCs were much more efficient than macrophages at stimulating CD4 T-cell cytokine release. Macrophages are effective in the uptake and killing of bacteria whilst DCs specialize in antigen presentation. This study showed that the viability of salmonellae was not essential for activation of APCs but, unlike live bacteria, prolonged contact with heat-killed bacteria was necessary to obtain maximal expression of the activation markers studied.     

The inositol phosphatase SHIP controls Salmonella enterica serovar Typhimurium infection in vivo

The SH2 domain-containing inositol 5'-phosphatase, SHIP, negatively regulates various hematopoietic cell functions and is critical for maintaining immune homeostasis. However, whether SHIP plays a role in controlling bacterial infections in vivo remains unknown. Salmonella enterica causes human salmonellosis, a disease that ranges in severity from mild gastroenteritis to severe systemic illness, resulting in significant morbidity and mortality worldwide. The susceptibility of ship(+/+) and ship(-/-) mice and bone marrow-derived macrophages to S. enterica serovar Typhimurium infection was compared. ship(-/-) mice displayed an increased susceptibility to both oral and intraperitoneal serovar Typhimurium infection and had significantly higher bacterial loads in intestinal and systemic sites than ship(+/+) mice, indicating a role for SHIP in the gut-associated and systemic pathogenesis of serovar Typhimurium in vivo. Cytokine analysis of serum from orally infected mice showed that ship(-/-) mice produce lower levels of Th1 cytokines than do ship(+/+) animals at 2 days postinfection, and in vitro analysis of supernatants taken from infected bone marrow-derived macrophages derived to mimic the in vivo ship(-/-) alternatively activated (M2) macrophage phenotype correlated with these data. M2 macrophages were the predominant population in vivo in both oral and intraperitoneal infections, since tissue macrophages within the small intestine and peritoneal macrophages from ship(-/-) mice showed elevated levels of the M2 macrophage markers Ym1 and Arginase 1 compared to ship(+/+) cells. Based on these data, we propose that M2 macrophage skewing in ship(-/-) mice contributes to ineffective clearance of Salmonella in vivo.     

In vitro and in vivo assessment of Salmonella enterica serovar Typhimurium DT104 virulence

Multidrug-resistant Salmonella enterica serovar Typhimurium phage type DT104 has become a widespread cause of human and other animal infection worldwide. The severity of clinical illness in S. enterica serovar Typhimurium DT104 outbreaks has led to the suggestion that this strain possesses enhanced virulence. In the present study, in vitro and in vivo virulence-associated phenotypes of several clinical isolates of S. enterica serovar Typhimurium DT104 were examined and compared to S. enterica serovar Typhimurium ATCC 14028s. The ability of these DT104 isolates to survive within murine peritoneal macrophages, invade cultured epithelial cells, resist antimicrobial actions of reactive oxygen and nitrogen compounds, and cause lethal infection in mice were assessed. Our results failed to demonstrate that S. enterica serovar Typhimurium DT104 isolates are more virulent than S. enterica serovar Typhimurium ATCC 14028s.     

Adaptive acid tolerance response in Salmonella enterica serovar Typhimurium and Salmonella enterica serovar Typhi

The survival of bacteria in various environments depends on a number of protective responses including acid tolerance response (ATR). In this study, ATR phenomenon was compared in Salmonella enterica serovar Typhi 6 and Salmonella enterica serovar Typhimurium 98 under different culture conditions. Survival of the adapted culture (pre-acid shocked to pH 5.5) was significantly better (p < 0.05) as compared to control, unadapted culture after acid shock at pH 3.3. However, the ATR varied with the serovar, incubation temperature and the growth medium used (all p-values < 0.05). S. Typhi 6 failed to grow in pH 3.3 at 45 degrees C. The addition of tetracycline or chloramphenicol (1.0 microg ml(-1)) to adapted cultures during or after acid shock (pH 3.3) had no effect on ATR expression. In S. Typhimurium 98, growth was increased by 10% or greater in adapted culture (when grown at pH 3.3) as compared to growth observed with an unadapted culture (when grown at pH 7.3) on transfer to fresh growth medium at pH 7.3. A poor ATR observed in non-growing S. Typhimurium 98 suspensions clearly showed that ATR is an energy-consuming process. Storage of S. Typhimurium 98 cultures in pH 4.5 nutrient broth at 4 degrees C demonstrated that prolonged exposure to acidic conditions is more detrimental in comparison to the cultures stored at pH 7.3 at this temperature.     

H2-M3 major histocompatibility complex class Ib-restricted CD8 T cells induced by Salmonella enterica serovar Typhimurium infection recognize proteins released by Salmonella serovar Typhimurium

Salmonella enterica serovar Typhimurium causes a typhoid-like disease in mice which has been studied extensively as a model for typhoid fever in humans. CD8 T cells contribute to protection against S. enterica serovar Typhimurium in mice, but little is known about the specificity and major histocompatibility complex (MHC) restriction of the response. We report here that CD8 T-cell lines derived from S. enterica serovar Typhimurium-infected BALB/c mice lysed bone marrow macrophages infected with S. enterica serovar Typhimurium or pulsed with proteins from S. enterica serovar Typhimurium culture supernatants. Cytoxicity was beta-2-microglobulin dependent and largely TAP dependent, although not MHC class Ia restricted, as target cells of several different MHC haplotypes were lysed. The data suggested the participation of class Ib MHC molecules although no evidence for the presence of Qa1-restricted T cells could be found, unlike in previous reports. Instead, the T-cell lines lysed H2-M3-transfected fibroblasts infected with S. enterica serovar Typhimurium SL3261 or treated with Salmonella culture supernatants. Thus, this report increases the number of MHC class Ib antigen-presenting molecules known for Salmonella antigens to three: Qa-1, HLA-E, and now H2-M3. It also expands the range of pathogens that induce H2-M3-restricted CD8 T cells to include an example of gram-negative bacteria.     

Caspase-3-dependent phagocyte death during systemic Salmonella enterica serovar Typhimurium infection of mice

Growth of Salmonella enterica in mammalian tissues results from continuous spread of bacteria to new host cells. Our previous work indicated that infective S. enterica are liberated from host cells via stochastic necrotic burst independently of intracellular bacterial numbers. Here we report that liver phagocytes can undergo apoptotic caspase-3-mediated cell death in vivo, with apoptosis being a rare event, more prevalent in heavily infected cells. The density-dependent apoptotic cell death is likely to constitute an alternative mechanism of bacterial spread as part of a bet-hedging strategy, ensuring an ongoing protective intracellular environment in which some bacteria can grow and persist.     

Salmonella enterica serovar Typhimurium expressing mutant lipid A with decreased endotoxicity causes maturation of murine dendritic cells

A major Salmonella component involved in cellular activation is the lipopolysaccharide (LPS) molecule which can act as a dendritic cell (DC) stimulator. The structure of the lipid A domain of the LPS molecule dictates its immunostimulatory capacity with various cell types. In this study, the role of lipid A as an integral component of Salmonella in stimulating murine DCs was studied by using a Salmonella enterica serovar Typhimurium lpxM mutant with defective lipid A. This study revealed that a mutation in lpxM did not significantly affect the ability of bacteria to activate DCs. Although the lpxM mutant less tumor necrosis factor alpha, interleukin-1beta, and inducible nitric oxide synthase than the parental strain, this was only seen at lower multiplicities of infection (MOIs). Both strains upregulated surface molecule expression on DCs and augmented the T-cell-stimulating capacity of these cells in an MOI-independent manner. Thus, the lpxM mutation did not appear to affect the stimulatory capacity of the Salmonella mutant.     

Salmonella enterica serovar typhimurium-induced maturation of bone marrow-derived dendritic cells

Murine bone marrow-derived dendritic cells (DC) can phagocytose and process Salmonella enterica serovar Typhimurium for peptide presentation on major histocompatibility complex class I (MHC-I) and MHC-II molecules. To investigate if a serovar Typhimurium encounter with DC induces maturation and downregulates their ability to present antigens from subsequently encountered bacteria, DC were pulsed with serovar Typhimurium 24 h prior to coincubating with Escherichia coli expressing the model antigen Crl-OVA. Quantitating presentation of OVA epitopes contained within Crl-OVA showed that Salmonella-pulsed DC had a reduced capacity to process Crl-OVA-expressing E. coli for OVA(257-264)/K(b) and OVA(265-277)/I-A(b) presentation. In addition, time course studies of DC pulsed with Crl-OVA-expressing serovar Typhimurium showed that OVA(257-264)/K(b) complexes could stimulate CD8OVA T-hybridoma cells for <24 h following a bacterial pulse, while OVA(265-277)/I-A(b) complexes could stimulate OT4H T-hybridoma cells for >24 but <48 h. The phoP-phoQ virulence locus of serovar Typhimurium also influenced the ability of DC to process Crl-OVA-expressing serovar Typhimurium for OVA(265-277)/I-A(b) presentation but not for OVA(257-264)/K(b) presentation. Furthermore, pulsing of DC with serovar Typhimurium followed by incubation for 24 or 48 h altered surface expression of MHC-I, MHC-II, CD40, CD54, CD80, and CD86, generating a DC population with a uniform, high expression level of these molecules. Finally, neither the serovar Typhimurium phoP-phoQ locus nor lipopolysaccharides (LPS) containing lipid A modifications purified from phoP mutant strains had a different effect on DC maturation from that of wild-type serovar Typhimurium or purified wild-type LPS. Thus, these data show that Salmonella or Salmonella LPS induces maturation of DC and that this process is not altered by the Salmonella phoP virulence locus. However, phoP did influence OVA(265-277)/I-A(b) presentation by DC infected with Crl-OVA-expressing serovar Typhimurium when quantitated after 2 h of bacterial infection.     

Induction of a novel chicken Toll-like receptor following Salmonella enterica serovar Typhimurium infection

Toll-like receptors (TLRs) are a group of highly conserved molecules that initiate the innate immune response to pathogens by recognizing structural motifs expressed by microbes. We have identified a novel TLR, TLR15, by bioinformatic analysis of the chicken genome, which is distinct from any known vertebrate TLR and thus appears to be avian specific. The gene for TLR15 was sequenced and is found on chromosome 3, and it has archetypal TIR and transmembrane domains and a distinctive arrangement of extracellular leucine-rich regions. mRNA for TLR15 was detected in the spleen, bursa, and bone marrow of healthy chickens, suggesting a role for this novel receptor in constitutive host defense. Following in vivo Salmonella enterica serovar Typhimurium infection, quantitative real-time PCR demonstrated significant upregulation of TLR15 in the cecum of infected chickens. Interestingly, similar induction of TLR2 expression following infection was also observed. In vitro studies revealed TLR15 upregulation in chicken embryonic fibroblasts stimulated with heat-killed S. enterica serovar Typhimurium. Collectively, these results suggest a role for the TLR in avian defense against bacterial infection. We hypothesize that TLR15 may represent an avian-specific TLR that has been either retained in chicken and lost in other taxa or gained in the chicken.     

Abelson tyrosine kinase facilitates Salmonella enterica serovar Typhimurium entry into epithelial cells

The intracellular gram-negative bacterial pathogen Salmonella enterica serovar Typhimurium gains entry into nonphagocytic cells by manipulating the assembly of the host actin cytoskeleton. S. enterica serovar Typhimurium entry requires a functional type III secretion system, a conduit through which bacterial effector proteins are directly translocated into the host cytosol. We and others have previously reported the enhancement of tyrosine kinase activities during Salmonella serovar Typhimurium infection; however, neither specific kinases nor their targets have been well characterized. In this study, we investigated the roles of the cellular Abelson tyrosine kinase (c-Abl) and the related protein Arg in the context of serovar Typhimurium infection. We found that bacterial internalization was inhibited by more than 70% in cells lacking both c-Abl and Arg and that treatment of wild-type cells with a pharmaceutical inhibitor of the c-Abl kinase, STI571 (imatinib), reduced serovar Typhimurium invasion efficiency to a similar extent. Bacterial infection led to enhanced phosphorylation of two previously identified c-Abl substrates, the adaptor protein CT10 regulator of kinase (CrkII) and the Abelson-interacting protein Abi1, a component of the WAVE2 complex. Furthermore, overexpression of the nonphosphorylatable form of CrkII resulted in decreased invasion. Taken together, these findings indicate that c-Abl is activated during S. enterica serovar Typhimurium infection and that its phosphorylation of multiple downstream targets is functionally important in bacterial internalization.     

Virulent Salmonella enterica serovar typhimurium evades adaptive immunity by preventing dendritic cells from activating T cells

Dendritic cells (DCs) constitute the link between innate and adaptive immunity by directly recognizing pathogen-associated molecular patterns (PAMPs) in bacteria and by presenting bacterial antigens to T cells. Recognition of PAMPs renders DCs as professional antigen-presenting cells able to prime na?ve T cells and initiate adaptive immunity against bacteria. Therefore, interfering with DC function would promote bacterial survival and dissemination. Understanding the molecular mechanisms that have evolved in virulent bacteria to evade activation of adaptive immunity requires the characterization of virulence factors that interfere with DC function. Salmonella enterica serovar Typhimurium, the causative agent of typhoid-like disease in the mouse, can prevent antigen presentation to T cells by avoiding lysosomal degradation in DCs. Here, we show that this feature of virulent Salmonella applies in vivo to prevent activation of adaptive immunity. In addition, this attribute of virulent Salmonella requires functional expression of a type three secretion system (TTSS) and effector proteins encoded within the Salmonella pathogenicity island 2 (SPI-2). In contrast to wild-type virulent Salmonella, mutant strains carrying specific deletions of SPI-2 genes encoding TTSS components or effectors proteins are targeted to lysosomes and are no longer able to prevent DCs from activating T cells in vitro or in vivo. SPI-2 mutant strains are attenuated in vivo, showing reduced tissue colonization and enhanced T-cell activation, which confers protection against a challenge with wild-type virulent Salmonella. Our data suggest that impairment of DC function by the activity of SPI-2 gene products is crucial for Salmonella pathogenesis.     

Interplay between MgtC and PagC in Salmonella enterica serovar Typhimurium

In Salmonella enterica serovar Typhimurium, MgtC and PagC are positively regulated by the PhoP-PhoQ two-component system, which is activated under magnesium deprivation. Both MgtC and PagC are of unknown function but have been involved in intramacrophage survival. We have found that the amount of PagC is lowered in a DeltamgtC mutant strain grown in magnesium depleted medium. However, the effect of MgtC on PagC does not account for the growth defect of a DeltamgtC mutant in macrophages since, in contrast to previous reports, our results indicate that PagC does not contribute to intramacrophage survival. In addition, a pagC null mutant is only poorly attenuated in Nramp1-negative or Nramp1-positive mice. On the other hand, a mgtC null mutant is significantly more attenuated with Nramp1-positive than Nramp1-negative mice, suggesting that a functional Nramp1 (Slc11a1) further limits the multiplication of this mutant within the host.     

Increased susceptibility of C1q-deficient mice to Salmonella enterica serovar Typhimurium infection.

The role of the complement system in host defense against Salmonella infection is poorly defined. Bacterial cell wall O-antigen polysaccharide can activate the alternative pathway in vitro. No studies, however, have elucidated the role of the classical pathway in immunity to Salmonella spp. in vivo. C1q-deficient mice (C1qa(-/-)) on a 129/Sv genetic background and strain-matched controls were infected intraperitoneally and intravenously with Salmonella enterica serovar Typhimurium and monitored over a 14-day period. After inoculation by either route, the C1qa(-/-) mice were found to be significantly more susceptible to Salmonella infection. Hepatic and splenic bacterial counts, performed at various time points, showed increased numbers of colonies in complement-deficient mice compared to controls. Analysis of blood clearance showed no difference between the two experimental groups during the first 15 min. However, after 20 min and until 6 h postinfection, numbers of circulating bacteria were significantly higher in complement-deficient mice. In vitro experiments using either resident or thioglycolate-elicited peritoneal macrophages showed a significant increase in the number of bacteria inside C1q-deficient macrophages compared to controls irrespective of the serum used for opsonizing the bacteria. These findings could not be explained either by an increased bacterial uptake, analyzed in vitro and in vivo using green fluorescent protein-tagged salmonellae, or by a defect in the respiratory burst or in NO production. The data presented here suggest the possibility of novel pathways by which C1q may modulate the pathogenesis of infectious diseases caused by intracellular pathogens.     

Role for inducible costimulator in control of Salmonella enterica serovar Typhimurium infection in mice

Inducible costimulator (ICOS) is expressed on activated T cells and plays a key role in sustaining and enhancing the effector function of CD4 T cells. Given the function of this molecule in sustaining T-cell responses, we reasoned that ICOS might play an important role in a prolonged infection model, such as Salmonella infection of mice. To test this hypothesis, wild-type (WT) and ICOS-deficient (ICOS-/-) mice were infected systemically with a Salmonella enterica serovar Typhimurium strain expressing the chicken ovalbumin gene (Salmonella-OVA). ICOS-/- mice exhibited greater splenomegaly than WT mice and showed delayed bacterial clearance. The acquired immune response in this model was slow to develop. Maximal T-cell responses to Salmonella-OVA were detected at 3 weeks postinfection in both WT and ICOS-/- mice. CD4 T-cell-dependent gamma interferon production and a class switch to immunoglobulin G2a were severely reduced in ICOS-/- mice. ICOS-/- mice also exhibited a substantial defect in antigen-specific CD8 T-cell responses. In vitro, the effect of anti-ICOS on CD8 T-cell division was greater when CD8 T cells rather than CD4 T cells expressed ICOS, suggesting that the in vivo effects of ICOS on CD8 T cells could be direct. Taken together, these studies show that ICOS plays a critical role in control of Salmonella infection in mice, with effects on antibody, Th1, and CD8 T-cell responses.     

Expansion of Paneth cell population in response to enteric Salmonella enterica serovar Typhimurium infection

Paneth cells residing at the base of the small intestinal crypts contribute to the mucosal intestinal first line defense by secreting granules filled with antimicrobial polypeptides including lysozyme. These cells derive from the columnar intestinal stem cell located at position 0 and the transit amplifying cell located at position +4 in the crypts. We have previously shown that Salmonella enterica serovar Typhimurium (ST), a leading cause of gastrointestinal infections in humans, effects an overall reduction of lysozyme in the small intestine. To extend this work, we examined small-intestinal tissue sections at various time points after ST infection to quantify and localize expression of lysozyme and assess Paneth cell abundance, apoptosis, and the expression of Paneth cell differentiation markers. In response to infection with ST, the intestinal Paneth cell-specific lysozyme content, the number of lysozyme-positive Paneth cells, and the number of granules per Paneth cell decreased. However, this was accompanied by increases in the total number of Paneth cells and the frequency of mitotic events in crypts, by increased staining for the proliferation marker PCNA, primarily at the crypt side walls where the transit amplifying cell resides and not at the crypt base, and by apoptotic events in villi. Furthermore, we found a time-dependent upregulation of first β-catenin, followed by EphB3, and lastly Sox9 in response to ST, which was not observed after infection with a Salmonella pathogenicity island 1 mutant deficient in type III secretion. Our data strongly suggest that, in response to ST infection, a Paneth cell differentiation program is initiated that leads to an expansion of the Paneth cell population and that the transit amplifying cell is likely the main progenitor responder. Infection-induced expansion of the Paneth cell population may represent an acute intestinal inflammatory response similar to neutrophilia in systemic infection.