CexE Is a Coat Protein and Virulence Factor of Diarrheagenic Pathogens.

CexE is a 12 kDa protein that was originally reported to be present in just three strains of enterotoxigenic Escherichia coli (ETEC); a frequent cause of diarrheal illnesses worldwide. However, an examination of sequenced genomes has revealed that CexE is actually present in a majority of ETEC strains. In addition, homologs of CexE are present in enteroaggregative E. coli (EAEC), Yersinia enterocolitica, Providencia alcalifaciens, and Citrobacter rodentium. Although it has been hypothesized that CexE and its homologs are virulence factors, this has yet to be tested. Thus the primary aim of this study was to determine if these proteins contribute to pathogenicity. Our secondary aim was determine if they are secreted coat proteins. Here we report that all neonatal mice infected with a wild-type strain of C. rodentium perished. In contrast a cexE mutant was significantly attenuated with 45% neonate survival. In adult mice the wild-type strain reached significantly higher loads in the large intestines and were shed in greater numbers than cexE mutants. Secretion of the CexE homolog in EAEC is dependent upon an atypical Type I secretion system that accepts its client from the periplasm rather than the cytoplasm. Insertion mutants of cexC, the putative ATPase of the CexE secretion system, were attenuated in our murine model. In vitro we found that CexC is required for the secretion of CexE to the outer membranes of both ETEC and C. rodentium. Secretion is not constitutive because CexE accumulates in the periplasm when the two pathogens are cultured under noninducing conditions. Although secretion conditions differ between ETEC and C. rodentium, secreted CexE remains predominantly associated with the outer membranes of both species. In aggregate these findings demonstrate that CexE is a secreted coat protein and virulence factor that promotes colonization of host intestinal tissues by enteric pathogens.

The eIF2α Kinase Heme-Regulated Inhibitor Protects the Host from Infection by Regulating Intracellular Pathogen Trafficking.

The host employs both cell-autonomous and system-level responses to limit pathogen replication in the initial stages of infection. Previously, we reported that the eukaryotic initiation factor 2α (eIF2α) kinases heme-regulated inhibitor (HRI) and protein kinase R (PKR) control distinct cellular and immune-related activities in response to diverse bacterial pathogens. Specifically for Listeria monocytogenes, there was reduced translocation of the pathogen to the cytosolic compartment in HRI-deficient cells and consequently reduced loading of pathogen-derived antigens on major histocompatibility complex class I (MHC-I) complexes. Here we show that Hri-/- mice, as well as wild-type mice treated with an HRI inhibitor, are more susceptible to listeriosis. In the first few hours of L. monocytogenes infection, there was much greater pathogen proliferation in the liver of Hri-/- mice than in the liver of Hri+/+ mice. Further, there was a rapid increase of serum interleukin-6 (IL-6) levels in Hri+/+ mice in the first few hours of infection whereas the increase in IL-6 levels in Hri-/- mice was notably delayed. Consistent with these in vivo findings, the rate of listeriolysin O (LLO)-dependent pathogen efflux from infected Hri-/- macrophages and fibroblasts was significantly higher than the rate seen with infected Hri+/+ cells. Treatment of cells with an eIF2α kinase activator enhanced both the HRI-dependent and PKR-dependent infection phenotypes, further indicating the pharmacologically malleability of this signaling pathway. Collectively, these results suggest that HRI mediates the cellular confinement and killing of virulent L. monocytogenes in addition to promoting a system-level cytokine response and that both are required to limit pathogen replication during the first few hours of infection.

Specialized Proresolving Mediators Rescue Infant Mice from Lethal Citrobacter rodentium Infection and Promote Immunity against Reinfection.

Infants are generally highly susceptible to oral pathogens. Intestinal infection and the associated diarrhea are significant global causes of morbidity and mortality in infants. Among the enteric pathogens, enteropathogenic Escherichia coli (EPEC) stands out as showing the highest risk for infection-induced death in infants ≤12 months old. We have developed an experimental model of infant infection with EPEC, using the mouse-specific pathogen Citrobacter rodentium Our murine infant model is similar to EPEC infection in human infants since infant mice are much more susceptible to C. rodentium infection than adult mice; infants infected with 50-fold fewer bacteria than the standard adult dose uniformly succumbed to the infection. Infant infection is characterized by high early and sustained bacterial titers and profound intestinal inflammation associated with extensive necrosis and systemic dissemination of the bacteria. Therefore, it seems likely that infant deaths result from sepsis secondary to intestinal damage. Recently, specialized proresolving mediators (SPM) have been found to exert profound beneficial effects in adult models of infection. Thus, we investigated the actions of two proresolving lipid mediators, resolvin D1 (RvD1) and resolvin D5 (RvD5), on the course of infection in infants. Strikingly, postinfection treatment with RvD1 and RvD5 reduced bacterial loads, mitigated inflammation, and rescued the infants from death. Furthermore, postinfection treatment with RvD1 and RvD5 led to protection from reinfection associated with C. rodentium-specific IgG responses comparable to those in adults. These results indicate that SPM may provide novel therapeutic tools for the treatment of pathological intestinal infections in infants.

Rapid CD8+ Function Is Critical for Protection of Neonatal Mice from an Extracellular Bacterial Enteropathogen.

Both human and murine neonates are characteristically highly susceptible to bacterial infections. However, we recently discovered that neonatal mice are surprisingly highly resistant to oral infection with Yersinia enterocolitica. This resistance was linked with activation of both innate and adaptive responses, involving innate phagocytes, CD4+ cells, and B cells. We have now extended these studies and found that CD8+ cells also contribute importantly to neonatal protection from Y. enterocolitica. Strikingly, neonatal CD8+ cells in the mesenteric lymph nodes (MLN) are rapidly mobilized, increasing in proportion, number, and IFNγ production as early as 48 h post infection. This early activation appears to be critical for protection since B2m-/- neonates are significantly more susceptible than wt neonates to primary Y. enterocolitica infection. In the absence of CD8+ cells, Y. enterocolitica rapidly disseminated to peripheral tissues. Within 48 h of infection, both the spleens and livers of B2m-/-, but not wt, neonates became heavily colonized, likely leading to their deaths from sepsis. In contrast to primary infection, CD8+ cells were dispensable for the generation of immunological memory protective against secondary infection. These results indicate that CD8+ cells in the neonatal MLN contribute importantly to protection against an extracellular bacterial enteropathogen but, notably, they appear to act during the early innate phase of the immune response.

Murine neonates infected with Yersinia enterocolitica develop rapid and robust proinflammatory responses in intestinal lymphoid tissues.

Neonatal animals are generally very susceptible to infection with bacterial pathogens. However, we recently reported that neonatal mice are highly resistant to orogastric infection with Yersinia enterocolitica. Here, we show that proinflammatory responses greatly exceeding those in adults arise very rapidly in the mesenteric lymph nodes (MLN) of neonates. High-level induction of proinflammatory gene expression occurred in the neonatal MLN as early as 18 h postinfection. Marked innate phagocyte recruitment was subsequently detected at 24 h postinfection. Enzyme-linked immunosorbent spot assay (ELISPOT) analyses indicated that enhanced inflammation in neonatal MLN is contributed to, in part, by an increased frequency of proinflammatory cytokine-secreting cells. Moreover, both CD11b(+) and CD11b(-) cell populations appeared to play a role in proinflammatory gene expression. The level of inflammation in neonatal MLN was also dependent on key bacterial components. Y. enterocolitica lacking the virulence plasmid failed to induce innate phagocyte recruitment. In contrast, tumor necrosis factor alpha (TNF-α) protein expression and neutrophil recruitment were strikingly higher in neonatal MLN after infection with a yopP-deficient strain than with wild-type Y. enterocolitica, whereas only modest increases occurred in adults. This hyperinflammatory response was associated with greater colonization of the spleen and higher mortality in neonates, while there was no difference in mortality among adults. This model highlights the dynamic levels of inflammation in the intestinal lymphoid tissues and reveals the protective (wild-type strain) versus harmful (yopP-deficient strain) consequences of inflammation in neonates. Moreover, these results reveal that the neonatal intestinal lymphoid tissues have great potential to rapidly mobilize innate components in response to infection with bacterial enteropathogens.

Neonatal immunology: responses to pathogenic microorganisms and epigenetics reveal an "immunodiverse" developmental state.

Neonatal animals have heightened susceptibility to infectious agents and are at increased risk for the development of allergic diseases, such as asthma. Experimental studies using animal models have been quite useful for beginning to identify the cellular and molecular mechanisms underlying these sensitivities. In particular, results from murine neonatal models indicate that developmental regulation of multiple immune cell types contributes to the typically poor responses of neonates to pathogenic microorganisms. Surprisingly, however, animal studies have also revealed that responses at mucosal surfaces in early life may be protective against primary or secondary disease. Our understanding of the molecular events underlying these processes is less well developed. Emerging evidence indicates that the functional properties of neonatal immune cells and the subsequent maturation of the immune system in ontogeny may be regulated by epigenetic phenomena. Here, we review recent findings from our group and others describing cellular responses to infection and developmentally regulated epigenetic processes in the newborn.

The murine Th2 locus undergoes epigenetic modification in the thymus during fetal and postnatal ontogeny.

Epigenetic modifications play a central role in the differentiation and function of immune cells in adult animals. Developmentally regulated epigenetic patterns also appear to contribute to the ontogeny of the immune system. We show here that the epigenetic profile of the T-helper (Th) 2 locus undergoes changes in T lineage cells beginning in mid-gestation and extending throughout the first week of life. In particular, regulatory regions of the Th2 locus are largely methylated at CpG residues among fetal liver common lymphoid progenitor cells. The locus subsequently becomes highly hypomethylated among the downstream progeny of these cells within the fetal thymus. This hypomethylated state is preserved until birth when the locus becomes rapidly re-methylated, achieving adult-like status by 3-6 days post birth. Notably, the capacity for rapid, high level Th2 cytokine production is lost in parallel with this re-methylation. In vitro organ culture and in vivo transplantation experiments indicate that signals from the adult environment are required to achieve the postnatal methylated state. Together, these findings indicate that the Th2 bias of neonates may be conferred, in part, by an epigenetic profile inherited from fetal life. However, the fetal program is rapidly terminated post birth by the development of signals leading to the acquisition of adult-like epigenetic patterns.

Host innate recognition of an intestinal bacterial pathogen induces TRIF-dependent protective immunity.

Toll-like receptor 4 (TLR4), which signals through the adapter molecules myeloid differentiation factor 88 (MyD88) and toll/interleukin 1 receptor domain-containing adapter inducing IFN-ß (TRIF), is required for protection against Gram-negative bacteria. TRIF is known to be important in TLR3-mediated antiviral signaling, but the role of TRIF signaling against Gram-negative enteropathogens is currently unknown. We show that TRIF signaling is indispensable for establishing innate protective immunity against Gram-negative Yersinia enterocolitica. Infection of wild-type mice rapidly induced both IFN-ß and IFN-γ in the mesenteric lymph nodes. In contrast, TRIF-deficient mice were defective in these IFN responses and showed impaired phagocytosis in regional macrophages, resulting in greater bacterial dissemination and mortality. TRIF signaling may be universally important for protection against Gram-negative pathogens, as TRIF-deficient macrophages were also impaired in killing both Salmonella and Escherichia coli in vitro. The mechanism of TRIF-mediated protective immunity appears to be orchestrated by macrophage-induced IFN-ß and NK cell production of IFN-γ. Sequential induction of IFN-ß and IFN-γ leads to amplification of macrophage bactericidal activity sufficient to eliminate the invading pathogens at the intestinal interface. Our results demonstrate a previously unknown role of TRIF in host resistance to Gram-negative enteropathogens, which may lead to effective strategies for combating enteric infections.

Immune responses of female BALB/c and C57BL/6 neonatal mice to vaccination or intestinal infection are unaltered by exposure to breast milk lycopene.

Lycopene, a carotenoid produced by some commonly consumed plants such as tomatoes, is not synthesized by animals. Thus, the levels of lycopene found in the breast milk of lactating females reflect the dietary lycopene supply. Lycopene has potent antioxidant activity but has also been implicated in modulating immune function. Therefore, lycopene in breast milk has the potential to affect the development and/or function of the immune system in the suckling pups. Here, we have investigated the impact of breast milk lycopene on systemic and mucosal immunity in mouse neonates. Diets containing 0.3 g/kg lycopene (Lyc) or control (Con) diets were fed to mouse dams beginning at late gestation and continuing throughout lactation. Seven-day-old female BALB/c pups were parenterally immunized with a model vaccine antigen dinitrophenyl-keyhole limpet hemocyanin (DNP-KLH) and then reimmunized as adults. The levels of DNP-KLH-specific IgG in the sera as well as keyhole limpet hemocyanin-specific IFNγ and IL-4 production by splenic CD4(+) cells were similar in the Lyc and Con pups. In addition, female neonatal (d7) C57BL/6 Lyc and Con pups were infected orally with the enteropathogen Yersinia enterocolitica. Breast milk lycopene had no effect on the recruitment of neutrophils to intestinal lymphoid tissues or on bacterial tissue colonization of the intestines, spleens, and livers. Thus, suckling pups exposed to lycopene in breast milk appear to develop normal innate and adaptive responses both systemically and at intestinal mucosal surfaces.

Yersinia enterocolitica promotes robust mucosal inflammatory T-cell immunity in murine neonates.

Mucosal immunity to gastrointestinal pathogens in early life has been studied only slightly. Recently, we developed an infection model in murine neonates using the gastroenteric pathogen Yersinia enterocolitica. Here, we report that oral infection of neonatal mice with low doses of virulent Y. enterocolitica leads to vigorous intestinal and systemic adaptive immunity. Y. enterocolitica infection promoted the development of anti-LcrV memory serum IgG1 and IgG2a responses of comparable affinity and magnitude to adult responses. Strikingly, neonatal mesenteric lymph node CD4(+) T cells produced Yersinia-specific gamma interferon (IFN-gamma) and interleukin-17A (IL-17A), exceeding adult levels. The robust T- and B-cell responses elicited in neonates exposed to Y. enterocolitica were associated with long-term protection against mucosal challenge with this pathogen. Using genetically deficient mice, we found that IFN-gamma and CD4(+) cells, but not B cells, are critical for protection of neonates during primary Y. enterocolitica infection. In contrast, adults infected with low bacterial doses did not require either cell population for protection. CD4-deficient neonatal mice adoptively transferred with CD4(+) cells from wild-type, IFN-gamma-deficient, or IL-17AF-deficient mice were equally protected from infection. These data demonstrate that inflammatory CD4(+) T cells are required for protection of neonatal mice and that this protection may not require CD4-derived IFN-gamma, IL-17A, or IL-17F. Overall, these studies support the idea that Y. enterocolitica promotes the development of highly inflammatory mucosal responses in neonates and that intestinal T-cell function may be a key immune component in protection from gastrointestinal pathogens in early life.

The pendulum swings: Tolerance versus priming to NIMA.

Fetal and/or perinatal exposure to noninherited maternal antigens (NIMA) has been reported to induce NIMA-specific tolerance. This tolerant state is highly beneficial in transplantation settings; enhanced graft acceptance has been observed when transplanted tissues express NIMA. Reduction in severe graft-vs-host disease has also been noted when bone marrow grafts originate from donors exposed to NIMA in early life. However, there is emerging evidence that exposure to NIMA can alternatively lead to specific priming. The processes regulating tolerance versus priming to NIMA are poorly understood and probably multifactorial. Based on studies in both humans and mice, we propose that both the quality and the quantity of NIMA exposure will be found to be key determinants of these opposing outcomes.

Neonatal immunity: faulty T-helpers and the shortcomings of dendritic cells.

Immunity in the newborn is characterized by minimal T helper (Th)1 function but an excess of Th2 activity. Since Th1 lymphocytes are important to counter microbes and Th2 cells favor allergies, the newborn faces susceptibility to microbial infections and allergic reactions. Delayed maturation of certain dendritic cells leads to limited interleukin (IL)-12 production during the neonatal period. The Th2 cytokine locus of neonatal CD4(+) T cells is poised epigenetically for rapid and robust production of IL-4 and IL-13. Together, these circumstances lead to efficient differentiation of Th2 cells and the expression of an IL-4Ralpha/IL-13Ralpha1 heteroreceptor on Th1 cells. Upon re-challenge, Th2 cells rapidly produce IL-4 which utilizes the heteroreceptor to drive apoptosis of Th1 cells, thus yielding the Th2 bias of neonatal immunity.

Murine neonatal recent thymic emigrants are phenotypically and functionally distinct from adult recent thymic emigrants.

In contrast to adults, the murine neonatal CD4+ compartment contains a high frequency of recent thymic emigrants (RTEs). However, the functional capabilities of these cells in neonates are relatively unknown. Moreover, it has not been determined whether RTEs from neonates and adults are comparable. Here we have directly compared neonatal and adult CD4+ RTEs for the first time, using a transgenic mouse strain that allows for the identification and purification of RTEs. Our data demonstrate that RTEs from murine neonates and adults are phenotypically and functionally distinct. In particular, although the magnitude of RTEs cytokine responses from both age groups is dependent on the conditions of activation, neonatal RTEs always exhibited higher levels of effector Th1/Th2 cytokine production than adult RTEs. In addition, neonatal, but not adult, RTEs showed early proliferation in response to stimulation with interleukin-7 alone. This was associated with faster kinetics of interleukin-7Ralpha down-regulation and higher levels of pSTAT5 in neonatal RTEs. These quantitative and qualitative differences in the neonatal and adult RTEs populations may at least partially explain the diverse responses that are elicited in vivo in neonates in response to different conditions of antigen exposure.

Murine neonates develop vigorous in vivo cytotoxic and Th1/Th2 responses upon exposure to low doses of NIMA-like alloantigens.

Early life exposure to noninherited maternal antigens (NIMAs) may occur via transplacental transfer and/or breast milk. There are indications that early life exposure to NIMAs may lead to lifelong tolerance. However, there is mounting evidence that exposure to NIMAs may also lead to immunologic priming. Understanding how these different responses arise could be critical in transplantation with donor cells expressing NIMAs. We recently reported that murine neonates that received a transplant of low doses of NIMA-like alloantigens develop vigorous memory cytotoxic responses, as assessed by in vitro assays. Here, we demonstrate that robust allospecific cytotoxicity is also manifest in vivo. Importantly, at low doses, NIMA-expressing cells induced the development of in vivo cytotoxicity during the neonatal period. NIMA-exposed neonates also developed vigorous primary and memory allospecific Th1/Th2 responses that exceeded the responses of adults. Overall, we conclude that exposure to low doses of NIMA-like alloantigens induces robust in vivo cytotoxic and Th1/Th2 responses in neonates. These findings suggest that early exposure to low levels of NIMA may lead to long-term immunologic priming of all arms of T-cell adaptive immunity, rather than tolerance.

Essential role of TNF receptor superfamily 25 (TNFRSF25) in the development of allergic lung inflammation.

We identify the tumor necrosis factor receptor superfamily 25 (TNFRSF25)/TNFSF15 pair as critical trigger for allergic lung inflammation, which is a cardinal feature of asthma. TNFRSF25 (TNFR25) signals are required to exert T helper cell 2 (Th2) effector function in Th2-polarized CD4 cells and co-stimulate interleukin (IL)-13 production by glycosphingolipid-activated NKT cells. In vivo, antibody blockade of TNFSF15 (TL1A), which is the ligand for TNFR25, inhibits lung inflammation and production of Th2 cytokines such as IL-13, even when administered days after airway antigen exposure. Similarly, blockade of TNFR25 by a dominant-negative (DN) transgene, DN TNFR25, confers resistance to lung inflammation in mice. Allergic lung inflammation-resistant, NKT-deficient mice become susceptible upon adoptive transfer of wild-type NKT cells, but not after transfer of DN TNFR25 transgenic NKT cells. The TNFR25/TL1A pair appears to provide an early signal for Th2 cytokine production in the lung, and therefore may be a drug target in attempts to attenuate lung inflammation in asthmatics.

Polynucleotide phosphorylase independently controls virulence factor expression levels and export in Yersinia spp.

Previously, it was shown that optimal functioning of the Yersinia type III secretion system (T3SS) in cell culture infection assays requires the exoribonuclease polynucleotide phosphorylase (PNPase) and that normal T3SS activity could be restored in the Deltapnp strains by expressing just the approximately 70-aa S1 RNA-binding domain of PNPase. Here, it is shown that the Yersinia Deltapnp strain is less virulent in the mouse compared with the isogenic wild-type strain. To begin to understand what could be limiting T3SS activity in the absence of PNPase, T3SS-encoding transcripts and proteins in the YersiniaDeltapnp strains were analyzed. Surprisingly, it was found that the Deltapnp Yersinia strains possessed enhanced levels of T3SS-encoding transcripts and proteins compared with the wild-type strains. We then found that an S1 variant containing a disruption in its RNA-binding subdomain was inactive in terms of restoring normal T3SS activity. However, T3SS expression levels did not differ between Deltapnp strains expressing active and inactive S1 proteins, further showing that T3SS activity and expression levels, at least as related to PNPase and its S1 domain, are not linked. The results suggest that PNPase affects the expression and activity of the T3SS by distinct mechanisms and that the S1-dependent effect on T3SS activity involves an RNA intermediate.