Increased Pathogen Burden Research Articles

RIPK3 and Caspase-8 interpret cytokine signals to regulate ILC3 survival in the gut

Group 3 innate lymphoid cells (ILC3s) are abundant in the developing or healthy intestine to critically support tissue homeostasis in response to microbial colonization. However, intestinal ILC3s are reduced during chronic infections, colorectal cancer, or inflammatory bowel disease (IBD), and the mechanisms driving these alterations remain poorly understood. Here we employed RNA sequencing of ILC3s from IBD patients and observed a significant upregulation of RIPK3, the central regulator of necroptosis, during intestinal inflammation. This was modeled in mice where we found that intestinal ILC3s express RIPK3, with conventional (c)ILC3s exhibiting high RIPK3 and low levels of pro-survival genes relative to lymphoid tissue inducer (LTi)-like ILC3s. ILC3-specific RIPK3 is promoted by gut microbiota, further upregulated following enteric infection, and dependent upon IL-23R and STAT3 signaling. However, lineage-specific deletion of RIPK3 revealed a redundant role in ILC3 survival, due to a blockade of RIPK3-mediated necroptosis by caspase 8, which was also activated in response to enteric infection. In contrast, lineage-specific deletion of caspase 8 resulted in loss of cILC3s from the healthy intestine and all ILC3 subsets during enteric infection, which increased pathogen burdens and gut inflammation. This function of caspase 8 required catalytic activity induced by TNF or TL1A and was dispensable if RIPK3 was simultaneously deleted. Caspase 8 activation and cell death were associated with increased Fas on ILC3s, and the Fas-FasL pathway was upregulated by cILC3s during enteric infection, which could restrain the abundance of intestinal ILC3s. Collectively, these data reveal that interpretation of key cytokine signals controls ILC3 survival following microbial challenge, and that an imbalance of these pathways, such as in IBD or across ILC3 subsets, provokes depletion of tissue-protective ILC3s from the inflamed intestine.

Increased Listeria monocytogenes Dissemination and Altered Population Dynamics in Muc2-Deficient Mice.

The mucin Muc2 is a major constituent of the mucus layer that covers the intestinal epithelium and creates a barrier between epithelial cells and luminal commensal or pathogenic microorganisms. The Gram-positive foodborne pathogen Listeria monocytogenes can cause enteritis and also disseminate from the intestine to give rise to systemic disease. L. monocytogenes can bind to intestinal Muc2, but the influence of the Muc2 mucin barrier on L. monocytogenes intestinal colonization and systemic dissemination has not been explored. Here, we used an orogastric L. monocytogenes infection model to investigate the role of Muc2 in host defense against L. monocytogenes Compared to wild-type mice, we found that Muc2-/- mice exhibited heightened susceptibility to orogastric challenge with L. monocytogenes, with higher mortality, elevated colonic pathology, and increased pathogen burdens in both the intestinal tract and distal organs. In contrast, L. monocytogenes burdens were equivalent in wild-type and Muc2-/- animals when the pathogen was administered intraperitoneally, suggesting that systemic immune defects related to Muc2 deficiency do not explain the heightened pathogen dissemination observed in oral infections. Using a barcoded L. monocytogenes library to measure intrahost pathogen population dynamics, we found that Muc2-/- animals had larger pathogen founding population sizes in the intestine and distal sites than observed in wild-type animals. Comparisons of barcode frequencies suggested that the colon becomes the major source for seeding the internal organs in Muc2-/- animals. Together, our findings reveal that Muc2 mucin plays a key role in controlling L. monocytogenes colonization, dissemination, and population dynamics.

A52 BUTYRATE SUPPLEMENTATION AMELIORATES DAMAGE CAUSED BY ENTERIC CITROBACTER RODENTIUM INFECTION

Abstract Background Patients with inflammatory bowel disease (IBD) often display a dysbiotic microbiome as well as a defective intestinal mucus layer, which appears thinner and more penetrable than the mucus layer of healthy subjects. Tributyrin (TB), a prodrug of butyric acid, has shown beneficial effects in models of IBD due to its anti-inflammatory effects. We previously showed that mice lacking the major intestinal mucin Muc2 (Muc2-/-) or lacking the “Core1” enzyme responsible for glycosylating Muc2 (C1galt1-/-) were highly susceptible to infection by Citrobacter rodentium, a murine model of intestinal inflammation. Aims The study explored the role of gut mucus in providing host defense against C. rodentium, as well as the effects of TB supplementation in the prevention of mucosal damage in this model. Methods Six to ten week old wildtype (WT), Muc2-/-, flox control (C1galt1f/f) and C1galt1-/- mice were infected with C. rodentium (∼2.5 × 108 CFU) by oral gavage. For TB supplementation experiments, mice received 100µL of TB or glycerol as a control by oral gavage every other day starting on day 1 post infection. Mice were monitored daily throughout the experiment and were euthanized at day 6 of infection. Several tissues of interest were collected to verify bacterial colonization in the gut and at systemic sites as well as histological tissue damage. Cecal contents were collected for the analysis of short chain fatty acids, while blood was collected by cardiac puncture after oral gavage with FITC-dextran to measure intestinal permeability. Results While WT and C1galt1f/f mice were only modestly susceptible to C. rodentium infection, Muc2-/- and C1galt1-/- mice displayed dramatically (100 fold) increased pathogen burdens, significantly greater intestinal macroscopic and histopathology scores, and heightened barrier disruption as compared to controls. Moreover, Muc2-/- and C1galt1-/- mice showed significantly lower levels of butyric acid as compared to control mice under baseline conditions. Interestingly, when supplemented with TB, Muc2-/- and C1galt1-/- proved less susceptible to C. rodentium infection, as indicated by reduced weight loss and clinical signs of colitis, while pathogen burdens were greatly reduced as was histological tissue damage, and epithelial barrier dysfunction. The same protection was conferred when TB was administered as a dietary supplementation, thus confirming its beneficial effect in protecting mice against C. rodentium infection. Conclusions These findings demonstrate that intestinal mucus controls host susceptibility to C. rodentium infection via control over butyrate levels, and highlight the need to explore the mechanisms by which gut mucus modulates the resident microbiota and its metabolites. Funding Agencies CCC, CIHR

Development of an Infection Risk Index for Microbiome Targeted Intervention in Children at High-Risk of Multidrug-Resistant Bloodstream Infections

Background Bloodstream infections (BSI) cause significant morbidity and mortality following pediatric hematopoietic stem cell transplantation (HSCT) and are increasingly from multidrug-resistant pathogens. Most of these BSI pathogens arise from the intestines. Increased evidence demonstrates commensal gut anaerobes maintain host resistance from enteric infection, while loss of commensals increases host susceptibility. Antibiotic use reduces gut anaerobes and allows a compensatory increased pathogen burden that precedes BSI; BSI risk is related to colonization burden. Recent reports suggest fecal microbiota transplantation (FMT) can displace an enteric pathogens and broad-spectrum antibiotic resistance genes during restoration of a healthy microbiome. However, the impact of intestinal microbiota on colonization, transmission, and invasive MDR infections is poorly understood in children undergoing HSCT. Methods Using metagenomic shotgun sequencing (MSS) data of longitudinal stool samples from high-risk children, including 63 undergoing HSCT with 166 samples preceding BSI onset (n = 23), we developed a metagenomic-based infection risk index (IRI) from key microbiome features (scaled Brady-Curtis distance relative to healthy controls and relative anaerobe and pathogen abundance). Traditional cart data analysis was compared to advanced machine learning algorithms that consider microbiome distribution, longitudinal sampling, and feature selection. Results A metagenomic-derived IRI from our cohort identified children having up to an 8-fold increased BSI risk. Evaluation of these key microbiome features as a diagnostic screening test by traditional cart analysis accurately identified patients at-risk for BSI with 91% sensitivity using its negative likelihood ratio. However, its specificity of 51% prohibits targeting interventions to those at greatest risk (Figure 1). Therefore, advanced machine learning algorithms were used for metagenomic-derived BSI prediction. While training results suggest overfitting, its performance promises clinical utility with a sensitivity of 92% and specificity of 84% warranting optimization and validation in HSCT patients (Figure 2). Analysis during heterologous FMT interventions in 5 symptomatic high-risk children (including 1 HSCT patient) revealed a concomitant pathogen-dominated gut microbiome harboring a high burden of broad-spectrum antibiotic resistance genes prior to each FMT. Following FMT, we confirmed restoration of a healthy gut microbiome with abundant anaerobes had a low metagenomic-derived IRI in all cases (Figure 3). Conclusions The application of advanced machine learning to shotgun metagenomic data provides the resolution required for accurate BSI prediction to identify HSCT patients at greatest risk preceding an illness providing new opportunity for early FMT intervention to prevent BSI.

Ground flaxseed reverses protection of a reduced-fat diet against Citrobacter rodentium-induced colitis

Flaxseed is high in ω-3 polyunsaturated fatty acids, fiber, and lignans known to lower cholesterol levels. However, its use for prevention or treatment of inflammatory bowel diseases has yielded mixed results, perhaps related to dietary interactions. In this study, we evaluated the impact of ground flaxseed supplementation on the severity of Citrobacter rodentium-induced colitis in the setting of either a high-fat (HF, ~36%kcal) or reduced-fat (RF, ~12%kcal) diet. After weaning, C57BL/6 mice ( n = 8-15/treatment) were fed ground flaxseed (7 g/100 g diet) with either HF (HF Flx) or RF (RF Flx) diets for 4 wk before infection with C. rodentium or sham gavage. Weight changes, mucosal inflammation, pathogen burden, gut microbiota composition, tissue polyunsaturated fatty acids, and cecal short-chain fatty acids were compared over a 14-day infection period. The RF diet protected against C. rodentium-induced colitis, whereas the RF Flx diet increased pathogen burden, exacerbated gut inflammation, and promoted gut dysbiosis. When compared with the RF diet, both HF and HF Flx diets resulted in more severe pathology in response to C. rodentium infection. Our findings demonstrate that although an RF diet protected against C. rodentium-induced colitis and associated gut dysbiosis in mice, beneficial effects were diminished with ground flaxseed supplementation. NEW & NOTEWORTHY Our results demonstrate a strong protective effect of a reduced-fat diet against intestinal inflammation, dysbiosis, and pathogen burden during Citrobacter rodentium-induced colitis. However, ground flaxseed supplementation in the setting of a reduced-fat diet exacerbated colitis despite higher levels of intestinal n-3 polyunsaturated fatty acids and cecal short-chain fatty acids.

Noncanonical inflammasome activation of caspase-4/caspase-11 mediates epithelial defenses against enteric bacterial pathogens.

Inflammasome-mediated host defenses have been extensively studied in innate immune cells. Whether inflammasomes function for innate defense in intestinal epithelial cells, which represent the first line of defense against enteric pathogens, remains unknown. We observed enhanced Salmonella enterica serovar Typhimurium colonization in the intestinal epithelium of caspase-11-deficient mice, but not atsystemic sites. In polarized epithelial monolayers, siRNA-mediated depletion of caspase-4, a human ortholog of caspase-11, also led to increased bacterial colonization. Decreased rates of pyroptotic cell death, a host defense mechanism that extrudes S.Typhimurium-infected cells from the polarized epithelium, accounted for increased pathogen burdens. The caspase-4 inflammasome also governs activation of the proinflammatory cytokine, interleukin (IL)-18, in response to intracellular (S.Typhimurium) and extracellular (enteropathogenic Escherichia coli) enteric pathogens, via intracellular LPS sensing. Therefore, an epithelial cell-intrinsic noncanonical inflammasome plays a critical role inantimicrobial defense at the intestinal mucosal surface.

Gut Microbiota Promote Hematopoiesis to Control Bacterial Infection

The commensal microbiota impacts specific immune cell populations and their functions at peripheral sites, such as gut mucosal tissues. However, it remains unknown whether gut microbiota control immunity through regulation of hematopoiesis at primary immune sites. We reveal that germ-free mice display reduced proportions and differentiation potential of specific myeloid cell progenitors of bothyolk sac and bone marrow origin. Homeostatic innate immune defects may lead to impaired early responses to pathogens. Indeed, following systemic infection with Listeria monocytogenes, germ-free and oral-antibiotic-treated mice display increased pathogen burden and acute death. Recolonization of germ-free mice with a complex microbiota restores defects in myelopoiesis and resistance to Listeria. These findings reveal that gut bacteria direct innate immune cell development via promoting hematopoiesis, contributing to our appreciation of the deep evolutionary connection between mammals and their microbiota.

CD4 + T Cells Drive Goblet Cell Depletion during Citrobacter rodentium Infection

Both idiopathic and infectious forms of colitis disrupt normal intestinal epithelial cell (IEC) proliferation and differentiation, although the mechanisms involved remain unclear. Recently, we demonstrated that infection by the attaching and effacing murine pathogen Citrobacter rodentium leads to a significant reduction in colonic goblet cell numbers (goblet cell depletion). This pathology depends on T and/or B cells, as Rag1(-/-) mice do not suffer this depletion during infection, instead suffering high mortality rates. To address the immune mechanisms involved, we reconstituted Rag(-/-) mice with either CD4(+) or CD8(+) T cells. Both T cell subsets increased Rag1(-/-) mouse survival during infection, with mice that received CD8(+) T cells developing colonic ulcers but not goblet cell depletion. In contrast, mice that received CD4(+) T cells showed goblet cell depletion in concert with exaggerated IEC proliferation. To define the possible involvement of T cell-derived cytokines, we infected gamma interferon receptor gene knockout (IFN-γR(-/-)) mice and wild-type mice given interleukin 17A (IL-17A) neutralizing antibodies and found that IFN-γ signaling was required for both goblet cell depletion and increased IEC proliferation. Immunostaining revealed that C. rodentium cells preferentially localized to nonhyperplastic crypts containing numerous goblet cells, whereas hyperplastic, goblet cell-depleted crypts appeared protected from infection. To address whether goblet cell depletion benefits the C. rodentium-infected host, we increased goblet cell numbers using the γ-secretase inhibitor dibenzazepine (DBZ), which resulted in greatly increased pathogen burdens and mortality rates. These results demonstrate that goblet cell depletion reflects host immunomodulation of IEC homeostasis and reflects a novel host defense mechanism against mucosal-adherent pathogens.

Active vitamin D (1,25-dihydroxyvitamin D3) increases host susceptibility toCitrobacter rodentiumby suppressing mucosal Th17 responses

Vitamin D deficiency affects more that 1 billion people worldwide and is associated with an increased risk of developing a number of inflammatory/autoimmune diseases, including inflammatory bowel disease (IBD). At present, the basis for the impact of vitamin D on IBD and mucosal immune responses is unclear; however, IBD is known to reflect exaggerated immune responses to luminal bacteria, and vitamin D has been shown to play a role in regulating bacteria-host interactions. Therefore, to test the effect of active vitamin D on host responses to enteric bacteria, we gave 1,25(OH)(2)D(3) to mice infected with the bacterial pathogen Citrobacter rodentium, an extracellular microbe that causes acute colitis characterized by a strong Th1/Th17 immune response. 1,25(OH)(2)D(3) treatment of infected mice led to increased pathogen burdens and exaggerated tissue pathology. In association with their increased susceptibility, 1,25(OH)(2)D(3)-treated mice showed substantially reduced numbers of Th17 T cells within their infected colons, whereas only modest differences were noted in Th1 and Treg numbers. In accordance with the impaired Th17 responses, 1,25(OH)(2)D(3)-treated mice showed defects in their production of the antimicrobial peptide REG3γ. Taken together, these studies show that 1,25(OH)(2)D(3) suppresses Th17 T-cell responses in vivo and impairs mucosal host defense against an enteric bacterial pathogen.

Stressor-enhanced colitis is abrogated in mice deficient in CCL2 (61.6)

Abstract Exposing mice to a social stressor called social disruption (SDR) has been reported to increase CD11b+Ly-6ChiCCR2+ inflammatory monocytes in the circulation and spleen. This study was designed to test the hypothesis that stressor-induced exacerbation of pathogen-induced colitis is dependent upon CCL2-mediated recruitment of inflammatory monocytes. Mice deficient in CCL2 (i.e., CCL2-/-), a chemokine involved in the recruitment of CCR2+ monocytes, and wild type C57BL/6 mice were challenged with the colonic pathogen Citrobacter rodentium during exposure to SDR. Exposing mice to SDR, which consists of intermale aggression for 2 hrs a night on 6 consecutive nights, during challenge with C.rodentium significantly increased pathogen-induced colitis. This increase was marked by an enhanced accumulation of F4/80+ colonic monocytes/macrophages, increased pathogen burden and pathogen-induced colonic histopathology, and increased inflammatory cytokine and chemokine gene expression in comparison to C. rodentium-challenged non-stressed control. These stressor effects were dependent upon CCL2, since exposing CCL2-/- mice to SDR did not lead to an increased accumulation of colonic monocytes/macrophages in the colon and did not significantly increase gene expression or pathogen-induced histopathology. Together, these data illustrate the deleterious effects of stressor-enhanced recruitment of inflammatory monocytes during bacterial challenge.

The genetic analysis of tolerance to infections: a review.

Tolerance to infections is defined as the ability of a host to limit the impact of a given pathogen burden on host performance. Uncoupling resistance and tolerance is a challenge, and there is a need to be able to separate them using specific trait recording or statistical methods. We present three statistical methods that can be used to investigate genetics of tolerance-related traits. Firstly, using random regressions, tolerance can be analyzed as a reaction norm slope in which host performance (y-axis) is regressed against an increasing pathogen burden (x-axis). Genetic variance in tolerance slopes is the genetic variance for tolerance. Variation in tolerance can induce genotype re-ranking and changes in genetic and phenotypic variation in host performance along the pathogen burden trajectory, contributing to environment-dependent genetic responses to selection. Such genotype-by-environment interactions can be quantified by combining random regressions and covariance functions. To apply random regressions, pathogen burden of individuals needs to be recorded. Secondly, when pathogen burden is not recorded, the cure model for time-until-death data allows separating two traits, susceptibility and endurance. Susceptibility is whether or not an individual was susceptible to an infection, whereas endurance denotes how long time it took until the infection killed a susceptible animal (influenced by tolerance). Thirdly, the normal mixture model can be used to classify continuously distributed host performance, such as growth rate, into different sub-classes (e.g., non-infected and infected), which allows estimation of host performance reduction specific to infected individuals. Moreover, genetics of host performance can be analyzed separately in healthy and affected animals, even in the absence of pathogen burden and survival data. These methods provide novel tools to increase our understanding on the impact of parasites, pathogens, and production diseases on host traits.

Genetic analysis of tolerance to infections using random regressions: a simulation study

Tolerance to infections is the ability of a host to limit the impact of a given pathogen burden on host performance. This simulation study demonstrated the merit of using random regressions to estimate unbiased genetic variances for tolerance slope and its genetic correlations with other traits, which could not be obtained using the previously implemented statistical methods. Genetic variance in tolerance was estimated as genetic variance in regression slopes of host performance along an increasing pathogen burden level. Random regressions combined with covariance functions allowed genetic variance for host performance to be estimated at any point along the pathogen burden trajectory, providing a novel means to analyse infection-induced changes in genetic variation of host performance. Yet, the results implied that decreasing family size as well as a non-zero environmental or genetic correlation between initial host performance before infection and pathogen burden led to biased estimates for tolerance genetic variance. In both cases, genetic correlation between tolerance slope and host performance in a pathogen-free environment became artificially negative, implying a genetic trade-off when it did not exist. Moreover, recording a normally distributed pathogen burden as a threshold trait is not a realistic way of obtaining unbiased estimates for tolerance genetic variance. The results show that random regressions are suitable for the genetic analysis of tolerance, given suitable data structure collected either under field or experimental conditions.

W48 GENETIC VARIATION WITHIN ADIPONUTRIN IS ASSOCIATED WITH LIPOPROTEIN METABOLISM AND LIVER FUNCTION

We investigated whether associations exist between immune reactions to oxidized low-density lipoproteins (OxLDLs), chronic infections, and carotid atherosclerosis as quantified by ultrasound.Atherosclerosis is a chronic immuno-inflammatory disease wherein both oxidized lipids and infectious agents are incriminated as possible contributors.We measured immunoglobulin (Ig)G and IgM autoantibody titers to copper-oxidized-LDL and malondialdehyde-LDL (OxLDL-AB), IgG and IgM apolipoprotein B-100-immune complexes (ApoB-IC), and titers of antibodies to Escherichia coliand chlamydial lipopolysaccharide (LPS), mycobacterial heat shock protein 65 (mHSP65), Chlamydia pneumoniae, Helicobacter pylori, and cytomegalovirus and evaluated their relationship to cardiovascular risk factors, chronic infections, and incident/progressive carotid atherosclerosis in the Bruneck study.The OxLDL-AB and ApoB-IC levels remained stable over time as indicated by strong correlations between 1995 and 2000 measurements (p < 0.001 each). Significant associations existed between all OxLDL markers and antibody titers to pathogens, especially to E. coli-LPS and mHSP65. Both OxLDL-AB and ApoB-IC levels showed a rise with increasing pathogen burden. Notably, OxLDL-ABs were also elevated in subjects with chronic infection as defined by clinical criteria. Titers of IgG, but not IgM, OxLDL-AB, or ApoB-IC inversely correlated with total cholesterol, LDL cholesterol, and apoB concentrations. The IgG OxLDL markers were positively and IgM markers were inversely associated with incident and progressive carotid atherosclerosis in univariate analyses but were not independent predictors in multivariate analyses.Our study provides evidence for an association between human oxLDL markers and chronic infections. Moreover, in this population-based study, neither IgG nor IgM OxLDL autoantibodies were independently predictive of atherosclerosis progression in the carotid arteries.

Tumor Progression Locus 2 (Map3k8) Is Critical for Host Defense againstListeria monocytogenesand IL-1β Production

Tumor progression locus 2 (Tpl2, also known as Map3k8 and Cot) is a serine-threonine kinase critical in innate immunity, linking toll-like receptors (TLRs) to TNF production through its activation of ERK. Tpl2(-/-) macrophages have abrogated TNF production but overproduce IL-12 in response to TLR ligands. Despite enhanced IL-12 production, Tpl2(-/-) T cells have impaired IFN-gamma production. Therefore, the role of Tpl2 in a bona fide bacterial infection where all of these cytokines are important in host defense is unclear. To address this issue, we infected Tpl2(-/-) mice with the model pathogen Listeria monocytogenes. We found that Tpl2(-/-) mice infected i.v. with L. monocytogenes had increased pathogen burdens compared with wild-type mice and rapidly succumbed to infection. Enhanced susceptibility correlated with impaired signaling through TLR2 and nucleotide-binding oligomerization domain 2, two receptors previously shown to mediate Listeria recognition. Surprisingly, TNF production in response to infection was not significantly impaired, even though Tpl2 has been implicated in the regulation of TNF. We found that the role of Tpl2 has cell-type specific effects in regulating TNF and transduces signals from some, but not all, pattern recognition receptors (PRR). In contrast to the cell-type- and receptor-specific regulation of TNF, we found that Tpl2 is essential for IL-1beta production from both macrophages and dendritic cells. These studies implicate Tpl2 as an important mediator for collaboration of pattern recognition receptors with danger-associated molecular patterns to induce TNF and IL-1beta production and optimal host defense.

Toll-like receptor 2 plays a critical role in maintaining mucosal integrity during Citrobacter rodentium-induced colitis

Inflammatory bowel diseases and infectious gastroenteritis likely occur when the integrity of intestinal barriers is disrupted allowing luminal bacterial products to cross into the intestinal mucosa, stimulating immune cells and triggering inflammation. While specific Toll-like receptors (TLR) are involved in the generation of inflammatory responses against enteric bacteria, their contributions to the maintenance of intestinal mucosal integrity are less clear. These studies investigated the role of TLR2 in a model of murine colitis induced by the bacterial pathogen Citrobacter rodentium. C. rodentium supernatants specifically activated TLR2 in vitro while infected TLR2-/- mice suffered a lethal colitis coincident with colonic mucosal ulcerations, bleeding and increased cell death but not increased pathogen burden. TLR2-/- mice suffered impaired epithelial barrier function mediated via zonula occludens (ZO)-1 in naïve mice and claudin-3 in infected mice, suggesting this could underlie their susceptibility. TLR2 deficiency was also associated with impaired production of IL-6 by bone marrow-derived macrophages and infected colons cultured ex vivo. As IL-6 has antiapoptotic and epithelial repair capabilities, its reduced expression could contribute to the impaired mucosal integrity. These studies report for the first time that TLR2 plays a critical role in maintaining intestinal mucosal integrity during infection by a bacterial pathogen.

Anti-P. gingivalis Response Correlates with Atherosclerosis

Significant associations between atherosclerosis and both Porphyromonas gingivalis, a major periodontopathogen, and the respiratory pathogen, Chlamydia pneumoniae, have been shown. Many individuals with evidence of atherosclerosis demonstrate seropositivity to these pathogens. The aim of the present study was to examine the atherogenic effect of repeated immunizations with either or both of these agents, and to determine if molecular mimicry of bacterial heat-shock protein (HSP), termed GroEL, and host (h) HSP60 was involved. Atherogenesis was examined in apolipoprotein-E-deficient (-/-) mice following intraperitoneal immunizations with P. gingivalis, C. pneumoniae, P. gingivalis, and C. pneumoniae or vehicle. Lesion area in the proximal aorta and levels of serum antibodies to P. gingivalis, C. pneumoniae, and GroEL were measured. The increased pathogen burden of P. gingivalis, but not of C. pneumoniae, enhanced atherosclerosis. hHSP60 was detected in lesions, and in P. gingivalis-immunized mice, lesion development was correlated with anti-GroEL antibody levels, supporting the involvement of molecular mimicry between GroEL and hHSP60.

Oxidized Low-Density Lipoprotein Autoantibodies, Chronic Infections, and Carotid Atherosclerosis in a Population-Based Study

We investigated whether associations exist between immune reactions to oxidized low-density lipoproteins (OxLDLs), chronic infections, and carotid atherosclerosis as quantified by ultrasound. Atherosclerosis is a chronic immuno-inflammatory disease wherein both oxidized lipids and infectious agents are incriminated as possible contributors. We measured immunoglobulin (Ig)G and IgM autoantibody titers to copper-oxidized-LDL and malondialdehyde-LDL (OxLDL-AB), IgG and IgM apolipoprotein B-100-immune complexes (ApoB-IC), and titers of antibodies to Escherichia coli and chlamydial lipopolysaccharide (LPS), mycobacterial heat shock protein 65 (mHSP65), Chlamydia pneumoniae, Helicobacter pylori, and cytomegalovirus and evaluated their relationship to cardiovascular risk factors, chronic infections, and incident/progressive carotid atherosclerosis in the Bruneck study. The OxLDL-AB and ApoB-IC levels remained stable over time as indicated by strong correlations between 1995 and 2000 measurements (p < 0.001 each). Significant associations existed between all OxLDL markers and antibody titers to pathogens, especially to E. coli-LPS and mHSP65. Both OxLDL-AB and ApoB-IC levels showed a rise with increasing pathogen burden. Notably, OxLDL-ABs were also elevated in subjects with chronic infection as defined by clinical criteria. Titers of IgG, but not IgM, OxLDL-AB, or ApoB-IC inversely correlated with total cholesterol, LDL cholesterol, and apoB concentrations. The IgG OxLDL markers were positively and IgM markers were inversely associated with incident and progressive carotid atherosclerosis in univariate analyses but were not independent predictors in multivariate analyses. Our study provides evidence for an association between human oxLDL markers and chronic infections. Moreover, in this population-based study, neither IgG nor IgM OxLDL autoantibodies were independently predictive of atherosclerosis progression in the carotid arteries.

Regulation of Streptococcus pneumoniae distribution by Toll-like receptor 2 in vivo

The phagocyte pattern recognition receptor Toll-like receptor 2 (TLR2) and the multi-receptor adaptor MyD88 contribute to the reduction of bacterial load in infections with intra- and extra-cellular Gram-positive bacteria. Their mechanism of antibacterial action is mostly unresolved but evident in vivo by an increased pathogen burden in infected TLR2 −/− and MyD88 −/− compared to C57BL/6 wild type (wt) mice. We had previously observed higher bacterial numbers in brains of TLR2 −/− than of wt mice with meningitis. Here we study bacteria–phagocyte interaction by comparing S. pneumoniae distribution and localization in wt and TLR2 −/− brain by confocal microscopy using a green fluorescent protein-transformed encapsulated S. pneumoniae (C5017). Colony-forming units were similarly distributed in TLR2 −/− and wt mice and exclusively localized in meninges and ventricles. Bacteria were more abundant in ventricles, in and around TLR2 −/− than wt GLT1v + plexus choroideus epithelial cells. S. pneumoniae were also found in and around Gr-1 + granulocytes, but never in F4/80 + macrophages, Iba1 + microglia, GFAP + astrocytes, Meca-31 + endothelial cells or Neun + neurons of either mouse strain. The results indicate that TLR2 does not change bacterial distribution, but may contribute to antibacterial defense by modulating S. pneumoniae adherence and uptake in plexus epithelia.

Infection-Induced Marginal Zone B Cell Production of Borrelia hermsii-Specific Antibody Is Impaired in the Absence of CD1d

Ab that arise in the absence of T cell help are a critical host defense against infection with the spirochetes Borrelia burgdorferi and Borrelia hermsii. We have previously shown that CD1d-deficient (CD1d(-/-)) mice have impaired resistance to infection with B. burgdorferi. In mice, CD1d expression is highest on marginal zone B (MZB) cells, which produce Ab to blood-borne Ag. In this study we examined MZB cell activation and Ab production in mice infected with B. hermsii, which achieve high levels of bacteremia. We show by flow cytometry that MZB cells associate with B. hermsii and up-regulate the activation markers syndecan I and B7.1 within 16 h of infection. By 24 h, MZB cells secrete B. hermsii-specific IgM, coinciding with the loss of activation marker expression and the reduction in spirochete burden. In contrast, MZB cells from CD1d(-/-) mice remain activated for at least 96 h of infection, but produce only minimal B. hermsii-specific IgM in vivo and ex vivo; pathogen burden in the blood also remains elevated. Wild-type mice depleted of MZB cells using mAb to LFA-1 and alpha(4)beta(1) integrin have reduced serum levels of B. hermsii-specific IgM and increased pathogen burden, similar to B. hermsii-infected CD1d(-/-) mice. Passive transfer of immune mouse serum, but not naive mouse serum, into infected CD1d(-/-) mice leads to down-regulation of activation markers and clearance of B. hermsii from the MZB cells. These results demonstrate that blood-borne spirochetes activate MZB cells to produce pathogen-specific IgM and reveal a role for CD1d in this process.

Impact of pathogen burden in patients with coronary artery disease in relation to systemic inflammation and variation in genes encoding cytokines

The number of infectious pathogens to which an individual has been exposed (pathogen burden) has been linked to the development and the prognosis of coronary artery disease (CAD). The interaction among infection, genetic host susceptibility, and CAD remains unclear. This study was aimed at evaluating the modulation of the association between CAD and pathogen burden, by serum levels of inflammatory markers and polymorphisms of the interleukin (IL)-6 and tumor necrosis factor (TNF)-α genes. Immmunoglobulin (Ig) G or IgA antibodies to 8 pathogens were determined in 991 patients with CAD and 333 control subjects. Serum levels of high-sensitivity C-reactive protein, fibrinogen, IL-6, and TNF-α were also measured. All subjects were genotyped for the IL-6/G-174C, the TNF/C-851T, and the TNF/G-308A polymorphisms. Analysis of single pathogens demonstrated a positive relation to the presence of CAD for some ( Chlamydia pneumoniae, cytomegalovirus, Helicobacter pylori, and herpes virus simplex type 1), but not all pathogens. A strong association between increasing pathogen burden and CAD was confirmed, even after adjustment for risk factors. The prevalence of a high pathogen burden (≥4 pathogens) was 50% in patients and 21% in controls (p <0.0001). A high pathogen burden was associated with decreased high-density lipoprotein cholesterol levels (p <0.001). The association between CAD and pathogen burden was modulated by the IL6/G-174C polymorphism, the odds ratio being higher in heterozygotes than in both types of homozygotes (p <0.05). This interaction appeared to be mediated by variations in serum IL-6 levels. No such interaction was detected with any of the 2 TNF-α polymorphisms.