Priming of Salmonella enterica Serovar Typhi-Specific CD8+ T Cells by Suicide Dendritic Cell Cross-Presentation in Humans

Background

The emergence of antibiotic-resistant strains of Salmonella enterica serovar Typhi (S. Typhi), the etiologic agent of typhoid fever, has aggravated an already important public health problem and added new urgency to the development of more effective typhoid vaccines. To this end it is critical to better understand the induction of immunity to S. Typhi. CD8⁺ T cells are likely to play an important role in host defense against S. Typhi by several effector mechanisms, including killing of infected cells and IFN-γ secretion. However, how S. Typhi regulates the development of specific CD8⁺ responses in humans remains unclear. Recent studies in mice have shown that dendritic cells (DC) can either directly (upon uptake and processing of Salmonella) or indirectly (by bystander mechanisms) elicit Salmonella-specific CD8⁺ T cells.

Methodology/Principal Findings

We report here that upon infection with live S. Typhi, human DC produced high levels of pro-inflammatory cytokines IL-6, IL-8 and TNF-α, but low levels of IL-12 p70 and IFN-γ. In contrast, DC co-cultured with S. Typhi-infected cells, through suicide cross-presentation, uptake S. Typhi-infected human cells and release high levels of IFN-γ and IL-12p70, leading to the subsequent presentation of bacterial antigens and triggering the induction of memory T cells, mostly CD3⁺CD8⁺CD45RA⁻CD62L⁻ effector/memory T cells.

Conclusions/Significance

This study is the first to demonstrate the effect of S. Typhi on human DC maturation and on their ability to prime CD8⁺ cells and highlights the significance of these phenomena in eliciting adaptive immunity to S. Typhi.     

Telomere-Mediated Chromosomal Instability Triggers TLR4 Induced Inflammation and Death in Mice

Background

Telomeres are essential to maintain chromosomal stability. Cells derived from mice lacking telomerase RNA component (mTERC^−/− mice) display elevated telomere-mediated chromosome instability. Age-dependent telomere shortening and associated chromosome instability reduce the capacity to respond to cellular stress occurring during inflammation and cancer. Inflammation is one of the important risk factors in cancer progression. Controlled innate immune responses mediated by Toll-like receptors (TLR) are required for host defense against infection. Our aim was to understand the role of chromosome/genome instability in the initiation and maintenance of inflammation.

Methodology/Principal Findings

We examined the function of TLR4 in telomerase deficient mTERC^−/− mice harbouring chromosome instability which did not develop any overt immunological disorder in pathogen-free condition or any form of cancers at this stage. Chromosome instability was measured in metaphase spreads prepared from wildtype (mTERC^+/+), mTERC^+/− and mTERC^−/− mouse splenocytes. Peritoneal and/or bone marrow-derived macrophages were used to examine the responses of TLR4 by their ability to produce inflammatory mediators TNFα and IL6. Our results demonstrate that TLR4 is highly up-regulated in the immune cells derived from telomerase-null (mTERC^−/−) mice and lipopolysaccharide, a natural ligand for TLR4 stabilises NF-κB binding to its promoter by down-regulating ATF-3 in mTERC^−/− macrophages.

Conclusions/Significance

Our findings implied that background chromosome instability in the cellular level stabilises the action of TLR4-induced NF-κB action and sensitises cells to produce excess pro-inflammatory mediators. Chromosome/genomic instability data raises optimism for controlling inflammation by non-toxic TLR antagonists among high-risk groups.     

CD8+ DC,but Not CD8?DC,Isolated from BCG-Infected Mice Reduces Pathological Reactions Induced by Mycobacterial Challenge Infection

Background

Tuberculosis is a mycobacterial infection causing worldwide public health problems but the available vaccine is far from ideal. Type-1 T cell immunity has been shown to be critical for host defence against tuberculosis infection, but the role of dendritic cell (DC) subsets in pathogenesis of mycobacterial infection remains unclear.

Methodology/Principal Findings

We examined the effectiveness of dendritic cell (DC) subsets in BCG-infected mice in generating immune responses beneficial for pathogen clearance and reduction of pathological reactions in the tissues following challenge infection. Our data showed that only the adoptive transfer of the subset of CD8α⁺ DC isolated from infected mice (iCD8⁺ DC) generated significant protection, demonstrated by less mycobacterial growth and pathological changes in the lung and liver tissues in iCD8⁺ DC recipients than sham-treated control mice. The adoptive transfer of the CD8α⁻DC from the infected mice (iCD8⁻ DC) not only failed to reduce bacterial growth, but enhanced inflammation characterized by diffuse heavy cellular infiltration. Notably, iCD8⁻ DC produced significantly higher levels of IL-10 than iCD8⁺ DC and promoted more Th2 cytokine responses in in vitro DC-T cell co-culture and in vivo adoptive transfer experiments.

Conclusions/Significance

The data indicate that in vivo BCG-primed CD8⁺ DC is the dominant DC subset in inducing protective immunity especially for reducing pathological reactions in infected tissues. The finding has implications for the rational improvement of the prophylactic and therapeutic approaches for controlling tuberculosis infection and related diseases.     

Jak3 Is Involved in Dendritic Cell Maturation and CCR7-Dependent Migration

Background

CCR7-mediated signalling is important for dendritic cell maturation and homing to the lymph nodes. We have previously demonstrated that Jak3 participates in the signalling pathway of CCR7 in T lymphocytes.

Methodology and Principal Findings

Here, we used Jak3^−/− mice to analyze the role of Jak3 in CCR7-mediated dendritic cells migration and function. First, we found no differences in the generation of DCs from Jak3^−/− bone marrow progenitors, when compared to wild type cells. However, phenotypic analysis of the bone marrow derived DCs obtained from Jak3^−/− mice showed reduced expression of co-stimulatory molecules compared to wild type (Jak3^+/+). In addition, when we analyzed the migration of Jak3^−/− and Jak3^+/+ mature DCs in response to CCL19 and CCL21 chemokines, we found that the absence of Jak3 results in impaired chemotactic responses both in vitro and in vivo. Moreover, lymphocyte proliferation and contact hypersensitivity experiments showed that DC-mediated T lymphocyte activation is reduced in the absence of Jak3.

Conclusion/Significance

Altogether, our data provide strong evidence that Jak3 is important for DC maturation, migration and function, through a CCR7-mediated signalling pathway.     

Variable Nav1.5 Protein Expression from the Wild-Type Allele Correlates with the Penetrance of Cardiac Conduction Disease in the Scn5a +/? Mouse Model

Background

Loss-of-function mutations in SCN5A, the gene encoding Na_v1.5 Na⁺ channel, are associated with inherited cardiac conduction defects and Brugada syndrome, which both exhibit variable phenotypic penetrance of conduction defects. We investigated the mechanisms of this heterogeneity in a mouse model with heterozygous targeted disruption of Scn5a (Scn5a ^+/− mice) and compared our results to those obtained in patients with loss-of-function mutations in SCN5A.

Methodology/Principal Findings

Based on ECG, 10-week-old Scn5a ^+/− mice were divided into 2 subgroups, one displaying severe ventricular conduction defects (QRS interval>18 ms) and one a mild phenotype (QRS≤18 ms; QRS in wild-type littermates: 10–18 ms). Phenotypic difference persisted with aging. At 10 weeks, the Na⁺ channel blocker ajmaline prolonged QRS interval similarly in both groups of Scn5a ^+/− mice. In contrast, in old mice (>53 weeks), ajmaline effect was larger in the severely affected subgroup. These data matched the clinical observations on patients with SCN5A loss-of-function mutations with either severe or mild conduction defects. Ventricular tachycardia developed in 5/10 old severely affected Scn5a ^+/− mice but not in mildly affected ones. Correspondingly, symptomatic SCN5A–mutated Brugada patients had more severe conduction defects than asymptomatic patients. Old severely affected Scn5a ^+/− mice but not mildly affected ones showed extensive cardiac fibrosis. Mildly affected Scn5a ^+/− mice had similar Na_v1.5 mRNA but higher Na_v1.5 protein expression, and moderately larger I_Na current than severely affected Scn5a ^+/− mice. As a consequence, action potential upstroke velocity was more decreased in severely affected Scn5a ^+/− mice than in mildly affected ones.

Conclusions

Scn5a ^+/− mice show similar phenotypic heterogeneity as SCN5A-mutated patients. In Scn5a ^+/− mice, phenotype severity correlates with wild-type Na_v1.5 protein expression.     

CD4+ and CD8+ T Cells Can Act Separately in Tumour Rejection after Immunization with Murine Pneumotropic Virus Chimeric Her2/neu Virus-Like Particles

Background

Immunization with murine pneumotropic virus virus-like particles carrying Her2/neu (Her2MPtVLPs) prevents tumour outgrowth in mice when given prophylactically, and therapeutically if combined with the adjuvant CpG. We investigated which components of the immune system are involved in tumour rejection, and whether long-term immunological memory can be obtained.

Methodology and Results

During the effector phase in BALB/c mice, only depletion of CD4⁺ and CD8⁺ in combination, with or without NK cells, completely abrogated tumour protection. Depletion of single CD4⁺, CD8⁺ or NK cell populations only had minor effects. During the immunization/induction phase, combined depletion of CD4⁺ and CD8⁺ cells abolished protection, while depletion of each individual subset had no or negligible effect. When tumour rejection was studied in knock-out mice with a C57Bl/6 background, protection was lost in CD4^−/−CD8^−/− and CD4^−/−, but not in CD8^−/− mice. In contrast, when normal C57Bl/6 mice were depleted of different cell types, protection was lost irrespective of whether only CD4⁺, only CD8⁺, or CD4⁺ and CD8⁺ cells in combination were eradicated. No anti-Her2/neu antibodies were detected but a Her2/neu-specific IFNγ response was seen. Studies of long-term memory showed that BALB/c mice could be protected against tumour development when immunized together with CpG as long as ten weeks before challenge.

Conclusion

Her2MPtVLP immunization is efficient in stimulating several compartments of the immune system, and induces an efficient immune response including long-term memory. In addition, when depleting mice of isolated cellular compartments, tumour protection is not as efficiently abolished as when depleting several immune compartments together.     

Maternal Infection with Trypanosoma cruzi and Congenital Chagas Disease Induce a Trend to a Type 1 Polarization of Infant Immune Responses to Vaccines

Background

We previously showed that newborns congenitally infected with Trypanosoma cruzi (M+B+) display a strong type 1 parasite-specific T cell immune response, whereas uninfected newborns from T. cruzi-infected mothers (M+B−) are prone to produce higher levels of proinflammatory cytokines than control neonates (M−B−). The purpose of the present study was to determine if such fetal/neonatal immunological environments could alter the response to standard vaccines administered in early life.

Methodology

Infants (6–7 months old) living in Bolivia, an area highly endemic for T. cruzi infection, and having received Bacillus Calmette Guerin (BCG), hepatitis B virus (HBV), diphtheria and tetanus vaccines, were enrolled into the M+B+, M+B−, M−B− groups mentioned above. The production of IFN-γ and IL-13, as markers of Th1 and Th2 responses respectively, by peripherical blood mononuclear cells stimulated with tuberculin purified protein derivative of Mycobacterium tuberculosis (PPD) or the vaccinal antigens HBs, diphtheria toxoid (DT) or tetanus toxoid (TT), as well as circulating levels of IgG antibodies against HBsAg, DT and TT were analyzed in infants. Cellular responses to the superantigen SEB were also monitored in M+B+, M+B−, M−B−infants and newborns.

Principal Findings

M+B+ infants developed a stronger IFN-γ response to hepatitis B, diphtheria and tetanus vaccines than did M+B− and M−B− groups. They also displayed an enhanced antibody production to HBsAg. This was associated with a type 1-biased immune environment at birth, since cells of M+B+ newborns produced higher IFN-γ levels in response to SEB. M+B− infants produced more IFN-γ in response to PPD than the other groups. IL-13 production remained low and similar in all the three groups, whatever the subject''s ages or vaccine status.

Conclusion

These results show that: i) both maternal infection with T. cruzi and congenital Chagas disease do not interfere with responses to BCG, hepatitis B, diphtheria and tetanus vaccines in the neonatal period, and ii) the overcoming of immunological immaturity by T. cruzi infection in early life is not limited to the development of parasite-specific immune responses, but also tends to favour type 1 immune responses to vaccinal antigens.     

PPARα Is Essential for Microparticle-Induced Differentiation of Mouse Bone Marrow-Derived Endothelial Progenitor Cells and Angiogenesis

Background

Bone marrow-derived endothelial progenitor cells (EPCs) are critical for neovascularization. We hypothesized that microparticles (MPs), small fragments generated from the plasma membrane, can activate angiogenic programming of EPCs.

Methodology/Principal Findings

We studied the effects of MPs obtained from wild type (MPs^PPARα+/+) and knock-out (MPs^{PPARα−/−}) mice on EPC differentiation and angiogenesis. Bone marrow-derived cells were isolated from WT or KO mice and were cultured in the presence of MPs^PPARα+/+ or MPs^{PPARα−/−} obtained from blood of mice. Only MPs^PPARα+/+ harboring PPAR^α significantly increased EPC, but not monocytic, differentiation. Bone marrow-derived cells treated with MPs^PPARα+/+ displayed increased expression of pro-angiogenic genes and increased in vivo angiogenesis. MPs^PPARα+/+ increased capillary-like tube formation of endothelial cells that was associated with enhanced expressions of endothelial cell-specific markers. Finally, the effects of MPs^PPARα+/+ were mediated by NF-κB-dependent mechanisms.

Conclusions/Significance

Our results underscore the obligatory role of PPARα carried by MPs for EPC differentiation and angiogenesis. PPARα-NF-κB-Akt pathways may play a pivotal stimulatory role for neovascularization, which may, at least in part, be mediated by bone marrow-derived EPCs. Improvement of EPC differentiation may represent a useful strategy during reparative neovascularization.     

Double Negative (CD3+4?8?) TCRαβ Splenic Cells from Young NOD Mice Provide Long-Lasting Protection against Type 1 Diabetes

Background

Double negative CD3⁺4⁻8⁻ TCRαβ splenic cells (DNCD3) can suppress the immune responses to allo and xenografts, infectious agents, tumors, and some autoimmune disorders. However, little is known about their role in autoimmune diabetes, a disease characterized by the reduction of insulin production subsequent to destruction of pancreatic β-cells by a polyclonal population of self-reactive T-cells. Herein, we analyzed the function and phenotype of DNCD3 splenic cells in young NOD mice predisposed to several autoimmune disorders among which, the human-like autoimmune diabetes.

Methodology/Principal Findings

DNCD3 splenic cells from young NOD mice (1) provided long-lasting protection against diabetes transfer in NOD/Scid immunodeficient mice, (2) proliferated and differentiated in the spleen and pancreas of NOD/Scid mice and pre-diabetic NOD mice into IL-10-secreting T_R-1 like cells in a Th2-like environment, and (3) their anti-diabetogenic phenotype is CD3⁺(CD4⁻CD8⁻)CD28⁺CD69⁺CD25^low Foxp3⁻ iCTLA-4⁻TCRαβ⁺ with a predominant Vβ13 gene usage.

Conclusions/Significance

These findings delineate a new T regulatory component in autoimmune diabetes apart from that of NKT and CD4⁺CD25^high Foxp3⁺T-regulatory cells. DNCD3 splenic cells could be potentially manipulated towards the development of autologous cell therapies in autoimmune diabetes.     

Thymic Alterations in GM2 Gangliosidoses Model Mice

Background

Sandhoff disease is a lysosomal storage disorder characterized by the absence of β-hexosaminidase and storage of GM2 ganglioside and related glycolipids. We have previously found that the progressive neurologic disease induced in Hexb ^−/− mice, an animal model for Sandhoff disease, is associated with the production of pathogenic anti-glycolipid autoantibodies.

Methodology/Principal Findings

In our current study, we report on the alterations in the thymus during the development of mild to severe progressive neurologic disease. The thymus from Hexb ^−/− mice of greater than 15 weeks of age showed a marked decrease in the percentage of immature CD4⁺/CD8⁺ T cells and a significantly increased number of CD4⁺/CD8⁻ T cells. During involution, the levels of both apoptotic thymic cells and IgG deposits to T cells were found to have increased, whilst swollen macrophages were prominently observed, particularly in the cortex. We employed cDNA microarray analysis to monitor gene expression during the involution process and found that genes associated with the immune responses were upregulated, particularly those expressed in macrophages. CXCL13 was one of these upregulated genes and is expressed specifically in the thymus. B1 cells were also found to have increased in the thy mus. It is significant that these alterations in the thymus were reduced in FcRγ additionally disrupted Hexb ^−/− mice.

Conclusions/Significance

These results suggest that the FcRγ chain may render the usually poorly immunogenic thymus into an organ prone to autoimmune responses, including the chemotaxis of B1 cells toward CXCL13.     

Attenuation of Reactive Gliosis Does Not Affect Infarct Volume in Neonatal Hypoxic-Ischemic Brain Injury in Mice

Background

Astroglial cells are activated following injury and up-regulate the expression of the intermediate filament proteins glial fibrillary acidic protein (GFAP) and vimentin. Adult mice lacking the intermediate filament proteins GFAP and vimentin (GFAP^−/−Vim^−/−) show attenuated reactive gliosis, reduced glial scar formation and improved regeneration of neuronal synapses after neurotrauma. GFAP^−/−Vim^−/− mice exhibit larger brain infarcts after middle cerebral artery occlusion suggesting protective role of reactive gliosis after adult focal brain ischemia. However, the role of astrocyte activation and reactive gliosis in the injured developing brain is unknown.

Methodology/Principal Findings

We subjected GFAP^−/−Vim^−/− and wild-type mice to unilateral hypoxia-ischemia (HI) at postnatal day 9 (P9). Bromodeoxyuridine (BrdU; 25 mg/kg) was injected intraperitoneally twice daily from P9 to P12. On P12 and P31, the animals were perfused intracardially. Immunohistochemistry with MAP-2, BrdU, NeuN, and S100 antibodies was performed on coronal sections. We found no difference in the hemisphere or infarct volume between GFAP^−/−Vim^−/− and wild-type mice at P12 and P31, i.e. 3 and 22 days after HI. At P31, the number of NeuN⁺ neurons in the ischemic and contralateral hemisphere was comparable between GFAP^−/−Vim^−/− and wild-type mice. In wild-type mice, the number of S100⁺ astrocytes was lower in the ipsilateral compared to contralateral hemisphere (65.0±50.1 vs. 85.6±34.0, p<0.05). In the GFAP^−/−Vim^−/− mice, the number of S100⁺ astrocytes did not differ between the ischemic and contralateral hemisphere at P31. At P31, GFAP^−/−Vim^−/− mice showed an increase in NeuN⁺BrdU⁺ (surviving newly born) neurons in the ischemic cortex compared to wild-type mice (6.7±7.7; n = 29 versus 2.9±3.6; n = 28, respectively, p<0.05), but a comparable number of S100⁺BrdU⁺ (surviving newly born) astrocytes.

Conclusions/Significance

Our results suggest that attenuation of reactive gliosis in the developing brain does not affect the hemisphere or infarct volume after HI, but increases the number of surviving newborn neurons.     

In Situ Patrolling of Regulatory T Cells Is Essential for Protecting Autoimmune Exocrinopathy

Background

Migration of T cells, including regulatory T (Treg) cells, into the secondary lymph organs is critically controlled by chemokines and adhesion molecules. However, the mechanisms by which Treg cells regulate organ-specific autoimmunity via these molecules remain unclear. Although we previously reported autoimmune exocrinopathy resembling Sjögren''s syndrome (SS) in the lacrimal and salivary glands from C-C chemokine receptor 7 (CCR7)-deficient mice, it is still unclear whether CCR7 signaling might specifically affect the dynamics and functions of Treg cells in vivo. We therefore investigated the cellular mechanism for suppressive function of Treg cells via CCR7 in autoimmunity using mouse models and human samples.

Methods and Findings

Patrolling Treg cells were detected in the exocrine organs such as lacrimal and salivary glands from normal mice that tend to be targets for autoimmunity while the Treg cells were almost undetectable in the exocrine glands of CCR7 ^−/− mice. In addition, we found the significantly increased retention of CD4⁺CD25⁺Foxp3⁺ Treg cells in the lymph nodes of CCR7 ^−/− mice with aging. Although Treg cell egress requires sphingosine 1-phosphate (S1P), chemotactic function to S1P of CCR7−/− Treg cells was impaired compared with that of WT Treg cells. Moreover, the in vivo suppression activity was remarkably diminished in CCR7 ^−/− Treg cells in the model where Treg cells were co-transferred with CCR7 ^−/− CD25^-CD4⁺ T cells into Rag2 ^−/− mice. Finally, confocal analysis showed that CCR7⁺Treg cells were detectable in normal salivary glands while the number of CCR7⁺Treg cells was extremely decreased in the tissues from patients with Sjögren''s syndrome.

Conclusions

These results indicate that CCR7 essentially governs the patrolling functions of Treg cells by controlling the traffic to the exocrine organs for protecting autoimmunity. Characterization of this cellular mechanism could have clinical implications by supporting development of new diagnosis or treatments for the organ-specific autoimmune diseases such as Sjögren''s syndrome and clarifying how the local immune system regulates autoimmunity.     

Interaction between Age and Obesity on Cardiomyocyte Contractile Function: Role of Leptin and Stress Signaling

Objectives

This study was designed to evaluate the interaction between aging and obesity on cardiac contractile and intracellular Ca²⁺ properties.

Methods

Cardiomyocytes from young (4-mo) and aging (12- and 18-mo) male lean and the leptin deficient ob/ob obese mice were treated with leptin (0.5, 1.0 and 50 nM) for 4 hrs in vitro. High fat diet (45% calorie from fat) and the leptin receptor mutant db/db obesity models at young and older age were used for comparison. Cardiomyocyte contractile and intracellular Ca²⁺ properties were evaluated including peak shortening (PS), maximal velocity of shortening/relengthening (± dL/dt), time-to-PS (TPS), time-to-90% relengthening (TR₉₀), intracellular Ca²⁺ levels and decay. O₂ ⁻ levels were measured by dihydroethidium fluorescence.

Results

Our results revealed reduced survival in ob/ob mice. Aging and obesity reduced PS, ± dL/dt, intracellular Ca²⁺ rise, prolonged TR₉₀ and intracellular Ca²⁺ decay, enhanced O₂ ⁻ production and p ^47phox expression without an additive effect of the two, with the exception of intracellular Ca²⁺ rise. Western blot analysis exhibited reduced Ob-R expression and STAT-3 phosphorylation in both young and aging ob/ob mice, which was restored by leptin. Aging and obesity reduced phosphorylation of Akt, eNOS and p38 while promoting pJNK and pIκB. Low levels of leptin reconciled contractile, intracellular Ca²⁺ and cell signaling defects as well as O₂ ⁻ production and p ^47phox upregulation in young but not aging ob/ob mice. High level of leptin (50 nM) compromised contractile and intracellular Ca²⁺ response as well as O₂ ⁻ production and stress signaling in all groups. High fat diet-induced and db/db obesity displayed somewhat comparable aging-induced mechanical but not leptin response.

Conclusions

Taken together, our data suggest that aging and obesity compromise cardiac contractile function possibly via phosphorylation of Akt, eNOS and stress signaling-associated O₂ ⁻ release.     

Abnormal Placental Development and Early Embryonic Lethality in EpCAM-Null Mice

Background

EpCAM (CD326) is encoded by the tacstd1 gene and expressed by a variety of normal and malignant epithelial cells and some leukocytes. Results of previous in vitro experiments suggested that EpCAM is an intercellular adhesion molecule. EpCAM has been extensively studied as a potential tumor marker and immunotherapy target, and more recent studies suggest that EpCAM expression may be characteristic of cancer stem cells.

Methodology/Principal Findings

To gain insights into EpCAM function in vivo, we generated EpCAM −/− mice utilizing an embryonic stem cell line with a tacstd1 allele that had been disrupted. Gene trapping resulted in a protein comprised of the N-terminus of EpCAM encoded by 2 exons of the tacstd1 gene fused in frame to βgeo. EpCAM +/− mice were viable and fertile and exhibited no obvious abnormalities. Examination of EpCAM +/− embryos revealed that βgeo was expressed in several epithelial structures including developing ears (otocysts), eyes, branchial arches, gut, apical ectodermal ridges, lungs, pancreas, hair follicles and others. All EpCAM −/− mice died in utero by E12.5, and were small, developmentally delayed, and displayed prominent placental abnormalities. In developing placentas, EpCAM was expressed throughout the labyrinthine layer and by spongiotrophoblasts as well. Placentas of EpCAM −/− embryos were compact, with thin labyrinthine layers lacking prominent vascularity. Parietal trophoblast giant cells were also dramatically reduced in EpCAM −/− placentas.

Conclusion

EpCAM was required for differentiation or survival of parietal trophoblast giant cells, normal development of the placental labyrinth and establishment of a competent maternal-fetal circulation. The findings in EpCAM-reporter mice suggest involvement of this molecule in development of vital organs including the gut, kidneys, pancreas, lungs, eyes, and limbs.     

Antigen-specific B cells reactivate an effective cytotoxic T cell response against phagocytosed Salmonella through cross-presentation

Background

The eradication of facultative intracellular bacterial pathogens, like Salmonella typhi, requires the concerted action of both the humoral immune response and the cytotoxic CD8⁺ T cell response. Dendritic cells (DCs) are considered to orchestrate the cytotoxic CD8⁺ T cell response via cross-presentation of bacterial antigens onto MHC class I molecules. Cross-presentation of Salmonella by DCs however, is accompanied by the induction of apoptosis in the DCs. Besides antibody production, B cells are required to clear Salmonella infection for other unknown reasons.

Methodology/Principal Findings

Here we show that Salmonella-specific B cells that phagocytose Salmonella upon BCR-ligation reactivate human memory CD8⁺ T cells via cross-presentation yielding a Salmonella-specific cytotoxic T cell response. The reactivation of CD8⁺ T cells is dependent on CD4⁺ T cell help. Unlike the DCs, B cell-mediated cross-presentation of Salmonella does not coincide with apoptosis.

Conclusions/Significance

B cells form a new player in the activation of the cytotoxic effector arm of the immune response and the generation of effective adaptive immunity in Salmonella infection.     

CXCL14 Deficiency in Mice Attenuates Obesity and Inhibits Feeding Behavior in a Novel Environment

Background

CXCL14 is a chemoattractant for macrophages and immature dendritic cells. We recently reported that CXCL14-deficient (CXCL14 ^−/−) female mice in the mixed background are protected from obesity-induced hyperglycemia and insulin resistance. The decreased macrophage infiltration into visceral adipose tissues and the increased insulin sensitivity of skeletal muscle contributed to these phenotypes.

Methodology/Principal Findings

In this study, we performed a comprehensive study for the body weight control of CXCL14 ^−/− mice in the C57BL/6 background. We show that both male and female CXCL14 ^−/− mice have a 7–11% lower body weight compared to CXCL14 ^+/− and CXCL14 ^+/+ mice in adulthood. This is mainly caused by decreased food intake, and not by increased energy expenditure or locomotor activity. Reduced body weight resulting from the CXCL14 deficiency was more pronounced in double mutant CXCL14^−/− ob/ob and CXCL14 ^−/−A^y mice. In the case of CXCL14 ^−/−A^y mice, oxygen consumption was increased compared to CXCL14 ^+/−A^y mice, in addition to the reduced food intake. In CXCL14 ^−/− mice, fasting-induced up-regulation of Npy and Agrp mRNAs in the hypothalamus was blunted. As intracerebroventricular injection of recombinant CXCL14 did not change the food intake of CXCL14 ^−/− mice, CXCL14 could indirectly regulate appetite. Intriguingly, the food intake of CXCL14 ^−/− mice was significantly repressed when mice were transferred to a novel environment.

Conclusions/Significance

We demonstrated that CXCL14 is involved in the body weight control leading to the fully obese phenotype in leptin-deficient or A^y mutant mice. In addition, we obtained evidence indicating that CXCL14 may play an important role in central nervous system regulation of feeding behavior.     

The Mutyh Base Excision Repair Gene Influences the Inflammatory Response in a Mouse Model of Ulcerative Colitis

Background

The Mutyh DNA glycosylase is involved in the repair of oxidized DNA bases. Mutations in the human MUTYH gene are responsible for colorectal cancer in familial adenomatous polyposis. Since defective DNA repair genes might contribute to the increased cancer risk associated with inflammatory bowel diseases, we compared the inflammatory response of wild-type and Mutyh^−/− mice to oxidative stress.

Methodology/Principal Findings

The severity of colitis, changes in expression of genes involved in DNA repair and inflammation, DNA 8-oxoguanine levels and microsatellite instability were analysed in colon of mice treated with dextran sulfate sodium (DSS). The Mutyh^−/− phenotpe was associated with a significant accumulation of 8-oxoguanine in colon DNA of treated mice. A single DSS cycle induced severe acute ulcerative colitis in wild-type mice, whereas lesions were modest in Mutyh^−/− mice, and this was associated with moderate variations in the expression of several cytokines. Eight DSS cycles caused chronic colitis in both wild-type and Mutyh^−/− mice. Lymphoid hyperplasia and a significant reduction in Foxp3⁺ regulatory T cells were observed only in Mutyh^−/− mice.

Conclusions

The findings indicate that, in this model of ulcerative colitis, Mutyh plays a major role in maintaining intestinal integrity by affecting the inflammatory response.     

DNA Polymerase δ Is Required for Early Mammalian Embryogenesis

Background

In eukaryotic cells, DNA polymerase δ (Polδ), whose catalytic subunit p125 is encoded in the Pold1 gene, plays a central role in chromosomal DNA replication, repair, and recombination. However, the physiological role of the Polδ in mammalian development has not been thoroughly investigated.

Methodology/Principal Findings

To examine this role, we used a gene targeting strategy to generate two kinds of Pold1 mutant mice: Polδ-null (Pold1 ^−/−) mice and D400A exchanged Polδ (Pold1 ^exo/exo) mice. The D400A exchange caused deficient 3′–5′ exonuclease activity in the Polδ protein. In Polδ-null mice, heterozygous mice developed normally despite a reduction in Pold1 protein quantity. In contrast, homozygous Pold1 ^−/− mice suffered from peri-implantation lethality. Although Pold1 ^−/− blastocysts appeared normal, their in vitro culture showed defects in outgrowth proliferation and DNA synthesis and frequent spontaneous apoptosis, indicating Polδ participates in DNA replication during mouse embryogenesis. In Pold1 ^exo/exo mice, although heterozygous Pold1 ^exo/+ mice were normal and healthy, Pold1 ^exo/exo and Pold1 ^exo/− mice suffered from tumorigenesis.

Conclusions

These results clearly demonstrate that DNA polymerase δ is essential for mammalian early embryogenesis and that the 3′–5′ exonuclease activity of DNA polymerase δ is dispensable for normal development but necessary to suppress tumorigenesis.