Immunization with a whole cell vaccine reduces pneumococcal nasopharyngeal density and shedding, and middle ear infection in mice.

Pneumococcal Conjugate Vaccines (PCVs) have substantially reduced the burden of disease caused by Streptococcus pneumoniae (the pneumococcus). However, protection is limited to vaccine serotypes, and when administered to children who are colonized with pneumococci at the time of vaccination, immune responses to the vaccine are blunted. Here, we investigate the potential of a killed whole cell pneumococcal vaccine (WCV) to reduce existing pneumococcal carriage and mucosal disease when given therapeutically to infant mice colonized with pneumococci. We show that a single dose of WCV reduced pneumococcal carriage density in an antibody-dependent manner. Therapeutic vaccination induced robust immune responses to pneumococcal surface antigens CbpA, PspA (family 1) and PiaA. In a co-infection model of otitis media, a single dose of WCV reduced pneumococcal middle ear infection. Lastly, in a two-dose model, therapeutic administration of WCV reduced nasal shedding of pneumococci. Taken together, our data demonstrate that WCV administered in colonized mice reduced pneumococcal density in the nasopharynx and the middle ear, and decreased shedding. WCVs would be beneficial in low and middle-income settings where pneumococcal carriage in children is high.

Synergism and Antagonism of Bacterial-Viral Coinfection in the Upper Respiratory Tract.

Streptococcus pneumoniae (the pneumococcus) is a leading cause of pneumonia in children under 5 years of age. Coinfection by pneumococci and respiratory viruses enhances disease severity. Little is known about pneumococcal coinfections with respiratory syncytial virus (RSV). Here, we developed a novel infant mouse model of coinfection using pneumonia virus of mice (PVM), a murine analogue of RSV, to examine the dynamics of coinfection in the upper respiratory tract, an anatomical niche that is essential for host-to-host transmission and progression to disease. Coinfection increased damage to the nasal tissue and increased production of the chemokine CCL3. Nasopharyngeal pneumococcal density and shedding in nasal secretions were increased by coinfection. In contrast, coinfection reduced PVM loads in the nasopharynx, an effect that was independent of pneumococcal strain and the order of infection. We showed that this "antagonistic" effect was absent using either ethanol-killed pneumococci or a pneumococcal mutant deficient in capsule production and incapable of nasopharyngeal carriage. Colonization with a pneumococcal strain naturally unable to produce capsule also reduced viral loads. The pneumococcus-mediated reduction in PVM loads was caused by accelerated viral clearance from the nasopharynx. Although these synergistic and antagonistic effects occurred with both wild-type pneumococcal strains used in this study, the magnitude of the effects was strain dependent. Lastly, we showed that pneumococci can also antagonize influenza virus. Taken together, our study has uncovered multiple novel facets of bacterial-viral coinfection. Our findings have important public health implications, including for bacterial and viral vaccination strategies in young children. IMPORTANCE Respiratory bacterial-viral coinfections (such as pneumococci and influenza virus) are often synergistic, resulting in enhanced disease severity. Although colonization of the nasopharynx is the precursor to disease and transmission, little is known about bacterial-viral interactions that occur within this niche. In this study, we developed a novel mouse model to examine pneumococcal-viral interactions in the nasopharynx with pneumonia virus of mice (PVM) and influenza. We found that PVM infection benefits pneumococci by increasing their numbers in the nasopharynx and shedding of these bacteria in respiratory secretions. In contrast, we discovered that pneumococci decrease PVM numbers by accelerating viral clearance. We also report a similar effect of pneumococci on influenza. By showing that coinfections lead to both synergistic and antagonistic outcomes, our findings challenge the existing dogma in the field. Our work has important applications and implications for bacterial and viral vaccines that target these microbes.

Excessive Reactive Oxygen Species Inhibit IL-17A+ γδ T Cells and Innate Cellular Responses to Bacterial Lung Infection.

Aims: Excessive reactive oxygen species (ROS) are detrimental to immune cellular functions that control pathogenic microbes; however, the mechanisms are poorly understood. Our aim was to determine the immunological consequences of increased ROS levels during acute bacterial infection. Results: We used a model of Streptococcus pneumoniae (Spn) lung infection and superoxide dismutase 3-deficient (SOD3-/-) mice, as SOD3 is a major antioxidant enzyme that catalyses the dismutation of superoxide radicals. First, we observed that in vitro, macrophages from SOD3-/- mice generated excessive phagosomal ROS during acute bacterial infection. In vivo, there was a significant reduction in infiltrating neutrophils in the bronchoalveolar lavage fluid and reduced peribronchial and alveoli inflammation in SOD3-/- mice 2 days after Spn infection. Annexin V/propidium iodide staining revealed enhanced apoptosis in neutrophils from Spn-infected SOD3-/- mice. In addition, SOD3-/- mice showed an altered macrophage phenotypic profile, with markedly diminished recruitment of monocytes (CD11clo, CD11bhi) in the airways. Further investigation revealed significantly lower levels of the monocyte chemokine CCL-2, and cytokines IL-23, IL-1ß, and IL-17A in Spn-infected SOD3-/- mice. There were also significantly fewer IL-17A-expressing gamma-delta T cells (γδ T cells) in the lungs of Spn-infected SOD3-/- mice. Innovation: Our data demonstrate that SOD3 deficiency leads to an accumulation of phagosomal ROS levels that initiate early neutrophil apoptosis during pneumococcal infection. Consequent to these events, there was a failure to initiate innate γδ T cell responses. Conclusion: These studies offer new cellular and mechanistic insights into how excessive ROS can regulate innate immune responses to bacterial infection.

Effect of a pneumococcal whole cell vaccine on influenza A-induced pneumococcal otitis media in infant mice.

The pneumococcus remains a common cause of otitis media (OM) despite the widespread introduction of pneumococcal conjugate vaccines. In mice, a pneumococcal whole cell vaccine (WCV) induces serotype-independent protection against pneumococcal colonisation and invasive disease via TH17- and antibody-mediated immunity, respectively. We investigated the effect of WCV on influenza A-induced pneumococcal OM in an infant mouse model. C57BL/6 mice were immunised subcutaneously with a single dose of WCV or adjuvant at 6 days of age, infected with pneumococci (EF3030 [serotype 19F] or PMP1106 [16F]) at 12 days of age, and given influenza A virus (A/Udorn/72/307 [H3N2], IAV) at 18 days of age to induce pneumococcal OM. Pneumococcal density in middle ear and nasopharyngeal tissues was determined 6 and 12 days post-virus. Experiments were repeated in antibody (B6.µMT-/-)- and CD4+ T-cell-deficient mice to investigate the immune responses involved. A single dose of WCV did not prevent the development of pneumococcal OM, nor accelerate pneumococcal clearance compared with mice receiving adjuvant alone. However, WCV reduced the density of EF3030 in the middle ear at 6 days post-viral infection (p = 0.022), and the density of both isolates in the nasopharynx at 12 days post-viral infection (EF3030, p = 0.035; PMP1106, p = 0.011), compared with adjuvant alone. The reduction in density in the middle ear required antibodies and CD4+ T cells: WCV did not reduce EF3030 middle ear density in B6.µMT-/- mice (p = 0.35) nor in wild-type mice given anti-CD4 monoclonal antibody before and after IAV inoculation (p = 0.91); and WCV-immunised CD4+ T cell-deficient GK1.5 mice had higher levels of EF3030 in the middle ear than their adjuvant-immunised counterparts (p = 0.044). A single subcutaneous dose of WCV reduced pneumococcal density in the middle ears of co-infected mice in one of two strains tested, but did not prevent OM from occurring in this animal model.

Mus musculus deficient for secretory antibodies show delayed growth with an altered urinary metabolome.

BACKGROUND: The polymeric immunoglobulin receptor (pIgR) maintains the integrity of epithelial barriers by transporting polymeric antibodies and antigens through the epithelial mucosa into the lumen. In this study, we examined the role of pIgR in maintaining gut barrier integrity, which is important for the normal development in mice. METHODS: Cohorts of pIgR-/- mice and their wildtype controls were housed under Specific Pathogen Free (SPF) conditions and monitored for weight gain as an indicator of development over time. The general physiology of the gastrointestinal tract was analysed using immunohistochemistry in young (8-12 weeks of age) and aged mice (up to 18 months of age), and the observed immunopathology in pIgR-/- mice was further characterised using flow cytometry. Urinary metabolites were analysed using gas chromatography-mass spectrometry (GC-MS), which revealed changes in metabolites that correlated with age-related increase in gut permeability in pIgR-/- mice. RESULTS: We observed that pIgR-/- mice exhibited delayed growth, and this phenomenon is associated with low-grade gut inflammation that increased with ageing. The gross intraepithelial lymphocytic (IEL) infiltration characteristic of pIgR-/- mice was redefined as CD8α+αß+ T cells, the majority of which expressed high levels of CD103 and CD69 consistent with tissue resident memory T cells (TRM). Comparison of the urinary metabolome between pIgR-/- and wild-type mice revealed key changes in urinary biomarkers fucose, glycine and Vitamin B5, suggestive of altered mucosal permeability. A significant increase in gut permeability was confirmed by analysing the site-specific uptake of sugar probes in different parts of the intestine. CONCLUSION: Our data show that loss of the secretory antibody system in mice results in enhanced accumulation of inflammatory IELs in the gut, which likely reflects ongoing inflammation in reaction to gut microbiota or food antigens, leading to delayed growth in pIgR-/- mice. We demonstrate that this leads to the presence of a unique urinary metabolome profile, which may provide a biomarker for altered gut permeability.

Unique Transcriptional Architecture in Airway Epithelial Cells and Macrophages Shapes Distinct Responses following Influenza Virus Infection Ex Vivo.

Airway epithelial cells and macrophages differ markedly in their responses to influenza A virus (IAV) infection. To investigate transcriptional responses underlying these differences, purified subsets of type II airway epithelial cells (ATII) and alveolar macrophages (AM) recovered from the lungs of mock- or IAV-infected mice at 9 h postinfection were subjected to RNA sequencing. This time point was chosen to allow for characterization of cell types first infected with the virus inoculum, prior to multicycle virus replication and the infiltration of inflammatory cells into the airways. In the absence of infection, AM predominantly expressed genes related to immunity, whereas ATII expressed genes consistent with their physiological roles in the lung. Following IAV infection, AM almost exclusively activated cell-intrinsic antiviral pathways that were dependent on interferon (IFN) regulatory factor 3/7 (IRF3/7) and/or type I IFN signaling. In contrast, IAV-infected ATII activated a broader range of physiological responses, including cell-intrinsic antiviral pathways, which were both independent of and dependent on IRF3/7 and/or type I IFN. These data suggest that transcriptional profiles hardwired during development are a major determinant underlying the different responses of ATII and AM to IAV infection.IMPORTANCE Airway epithelial cells (AEC) and airway macrophages (AM) represent major targets of influenza A virus (IAV) infection in the lung, yet the two cell types respond very differently to IAV infection. We have used RNA sequencing to define the host transcriptional responses in each cell type under steady-state conditions as well as following IAV infection. To do this, different cell subsets isolated from the lungs of mock- and IAV-infected mice were subjected to RNA sequencing. Under steady-state conditions, AM and AEC express distinct transcriptional activities, consistent with distinct physiological roles in the airways. Not surprisingly, these cells also exhibited major differences in transcriptional responses following IAV infection. These studies shed light on how the different transcriptional architectures of airway cells from two different lineages drive transcriptional responses to IAV infection.

Aspirin-triggered resolvin D1 reduces pneumococcal lung infection and inflammation in a viral and bacterial coinfection pneumonia model.

Formyl peptide receptor 2/lipoxin A4 (LXA4) receptor (Fpr2/ALX) co-ordinates the transition from inflammation to resolution during acute infection by binding to distinct ligands including serum amyloid A (SAA) and Resolvin D1 (RvD1). Here, we evaluated the proresolving actions of aspirin-triggered RvD1 (AT-RvD1) in an acute coinfection pneumonia model. Coinfection with Streptococcus pneumoniae and influenza A virus (IAV) markedly increased pneumococcal lung load and neutrophilic inflammation during the resolution phase. Fpr2/ALX transcript levels were increased in the lungs of coinfected mice, and immunohistochemistry identified prominent Fpr2/ALX immunoreactivity in bronchial epithelial cells and macrophages. Levels of circulating and lung SAA were also highly increased in coinfected mice. Therapeutic treatment with exogenous AT-RvD1 during the acute phase of infection (day 4-6 post-pneumococcal inoculation) significantly reduced the pneumococcal load. AT-RvD1 also significantly reduced neutrophil elastase (NE) activity and restored total antimicrobial activity in bronchoalveolar lavage (BAL) fluid (BALF) of coinfected mice. Pneumonia severity, as measured by quantitating parenchymal inflammation or alveolitis was significantly reduced with AT-RvD1 treatment, which also reduced the number of infiltrating lung neutrophils and monocytes/macrophages as assessed by flow cytometry. The reduction in distal lung inflammation in AT-RvD1-treated mice was not associated with a significant reduction in inflammatory and chemokine mediators. In summary, we demonstrate that in the coinfection setting, SAA levels were persistently increased and exogenous AT-RvD1 facilitated more rapid clearance of pneumococci in the lungs, while concurrently reducing the severity of pneumonia by limiting excessive leukocyte chemotaxis from the infected bronchioles to distal areas of the lungs.

Reduced PU.1 expression underlies aberrant neutrophil maturation and function in ß-thalassemia mice and patients.

ß-Thalassemia is associated with several abnormalities of the innate immune system. Neutrophils in particular are defective, predisposing patients to life-threatening bacterial infections. The molecular and cellular mechanisms involved in impaired neutrophil function remain incompletely defined. We used the Hbbth3/+ ß-thalassemia mouse and hemoglobin E (HbE)/ß-thalassemia patients to investigate dysregulated neutrophil activity. Mature neutrophils from Hbbth3/+ mice displayed a significant reduction in chemotaxis, opsonophagocytosis, and production of reactive oxygen species, closely mimicking the defective immune functions observed in ß-thalassemia patients. In Hbbth3/+ mice, the expression of neutrophil CXCR2, CD11b, and reduced NAD phosphate oxidase components (p22phox, p67phox, and gp91phox) were significantly reduced. Morphological analysis of Hbbth3/+ neutrophils showed that a large percentage of mature phenotype neutrophils (Ly6GhiLy6Clow) appeared as band form cells, and a striking expansion of immature (Ly6GlowLy6Clow) hyposegmented neutrophils, consisting mainly of myelocytes and metamyelocytes, was noted. Intriguingly, expression of an essential mediator of neutrophil terminal differentiation, the ets transcription factor PU.1, was significantly decreased in Hbbth3/+ neutrophils. In addition, in vivo infection with Streptococcus pneumoniae failed to induce PU.1 expression or upregulate neutrophil effector functions in Hbbth3/+ mice. Similar changes to neutrophil morphology and PU.1 expression were observed in splenectomized and nonsplenectomized HbE/ß-thalassemia patients. This study provides a mechanistic insight into defective neutrophil maturation in ß-thalassemia patients, which contributes to deficiencies in neutrophil effector functions.

Neonatal pneumococcal colonisation caused by Influenza A infection alters lung function in adult mice.

There is emerging epidemiological data to suggest that upper respiratory tract bacterial colonisation in infancy may increase the risk of developing respiratory dysfunction later in life, and respiratory viruses are known to precipitate persistent colonisation. This study utilized a neonatal mouse model of Streptococcus pneumonia (SP) and influenza A virus (IAV) co-infection, where bronchoalveolar leukocyte infiltration had resolved by adulthood. Only co-infection resulted in persistent nasopharyngeal colonisation over 40 days and a significant increase in airway resistance in response to in vivo methacholine challenge. A significant increase in hysteresivity was also observed in IAV and co-infected mice, consistent with ventilatory heterogeneity and structural changes in the adult lung. Airway hyper-responsiveness was not associated with a detectable increase in goblet cell transdifferentiation, peribronchial smooth muscle bulk or collagen deposition in regions surrounding the airways. Increased reactivity was not observed in precision cut lung slices challenged with methacholine in vitro. Histologically, the airway epithelium appeared normal and expression of epithelial integrity markers (ZO-1, occludin-1 and E-cadherin) were not altered. In summary, neonatal co-infection led to persistent nasopharyngeal colonisation and increased airway responsiveness that was not associated with detectable smooth muscle or mucosal epithelial abnormalities, however increased hysteresivity may reflect ventilation heterogeneity.

Increased autoimmune diabetes in pIgR-deficient NOD mice is due to a "Hitchhiking" interval that refines the genetic effect of Idd5.4.

Selective breeding to introduce a gene mutation from one mouse strain onto the genetic background of another strain invariably produces "hitchhiking" (i.e. flanking) genomic intervals, which may independently affect a disease trait of interest. To investigate a role for the polymeric Ig receptor in autoimmune diabetes, a congenic nonobese diabetic (NOD) mouse strain was generated that harbors a Pigr null allele derived from C57BL/6 (B6) mice. These pIgR-deficient NOD mice exhibited increased serum IgA along with an increased diabetes incidence. However, the Pigr null allele was encompassed by a relatively large "hitchhiking" genomic interval that was derived from B6 mice and overlaps Idd5.4, a susceptibility locus for autoimmune diabetes. Additional congenic NOD mouse strains, harboring smaller B6-derived intervals, confirmed Idd5.4 independently of the other three known susceptibility loci on chromosome 1, and further localized Idd5.4 to an interval proximal to Pigr. Moreover, these congenic NOD mice showed that B6 mice harbor a more diabetogenic allele than NOD mice for this locus. The smallest B6-derived interval encompassing the Pigr null allele may, however, confer a small degree of protection against diabetes, but this protection appears to be dependent on the absence of the diabetogenic B6 allele for Idd5.4. This study provides another example of the potential hidden effects of "hitchhiking" genomic intervals and how such intervals can be used to localize disease susceptibility loci.

Analysis of Salmonella enterica serovar Typhimurium variable-number tandem-repeat data for public health investigation based on measured mutation rates and whole-genome sequence comparisons.

Variable-number tandem repeats (VNTRs) mutate rapidly and can be useful markers for genotyping. While multilocus VNTR analysis (MLVA) is increasingly used in the detection and investigation of food-borne outbreaks caused by Salmonella enterica serovar Typhimurium (S. Typhimurium) and other bacterial pathogens, MLVA data analysis usually relies on simple clustering approaches that may lead to incorrect interpretations. Here, we estimated the rates of copy number change at each of the five loci commonly used for S. Typhimurium MLVA, during in vitro and in vivo passage. We found that loci STTR5, STTR6, and STTR10 changed during passage but STTR3 and STTR9 did not. Relative rates of change were consistent across in vitro and in vivo growth and could be accurately estimated from diversity measures of natural variation observed during large outbreaks. Using a set of 203 isolates from a series of linked outbreaks and whole-genome sequencing of 12 representative isolates, we assessed the accuracy and utility of several alternative methods for analyzing and interpreting S. Typhimurium MLVA data. We show that eBURST analysis was accurate and informative. For construction of MLVA-based trees, a novel distance metric, based on the geometric model of VNTR evolution coupled with locus-specific weights, performed better than the commonly used simple or categorical distance metrics. The data suggest that, for the purpose of identifying potential transmission clusters for further investigation, isolates whose profiles differ at one of the rapidly changing STTR5, STTR6, and STTR10 loci should be collapsed into the same cluster.

Congenic mice reveal genetic epistasis and overlapping disease loci for autoimmune diabetes and listeriosis.

The nonobese diabetic (NOD) mouse strain serves as a genomic standard for assessing how allelic variation for insulin-dependent diabetes (Idd) loci affects the development of autoimmune diabetes. We previously demonstrated that C57BL/6 (B6) mice harbor a more diabetogenic allele than NOD mice for the Idd14 locus when introduced onto the NOD genetic background. New congenic NOD mouse strains, harboring smaller B6-derived intervals on chromosome 13, now localize Idd14 to an ~18-Mb interval and reveal a new locus, Idd31. Notably, the B6 allele for Idd31 confers protection against diabetes, but only in the absence of the diabetogenic B6 allele for Idd14, indicating genetic epistasis between these two loci. Moreover, congenic mice that are more susceptible to diabetes are more resistant to Listeria monocytogenes infection. This result co-localizes Idd14 and Listr2, a resistance locus for listeriosis, to the same genomic interval and indicates that congenic NOD mice may also be useful for localizing resistance loci for infectious disease.

Evaluation of intra- and extra-epithelial secretory IgA in chlamydial infections.

Immunoglobulin A is an important mucosal antibody that can neutralize mucosal pathogens by either preventing attachment to epithelia (immune exclusion) or alternatively inhibit intra-epithelial replication following transcytosis by the polymeric immunoglobulin receptor (pIgR). Chlamydia trachomatis is a major human pathogen that initially targets the endocervical or urethral epithelium in women and men, respectively. As both tissues contain abundant secretory IgA (SIgA) we assessed the protection afforded by IgA targeting different chlamydial antigens expressed during the extra- and intra-epithelial stages of infection. We developed an in vitro model using polarizing cells expressing the murine pIgR together with antigen-specific mouse IgA, and an in vivo model using pIgR(-/-) mice. Secretory IgA targeting the extra-epithelial chlamydial antigen, the major outer membrane protein, significantly reduced infection in vitro by 24% and in vivo by 44%. Conversely, pIgR-mediated delivery of IgA targeting the intra-epithelial inclusion membrane protein A bound to the inclusion but did not reduce infection in vitro or in vivo. Similarly, intra-epithelial IgA targeting the secreted protease Chlamydia protease-like activity factor also failed to reduce infection. Together, these data suggest the importance of pIgR-mediated delivery of IgA targeting extra-epithelial, but not intra-epithelial, chlamydial antigens for protection against a genital tract infection.

Antibodies mediate formation of neutrophil extracellular traps in the middle ear and facilitate secondary pneumococcal otitis media.

Otitis media (OM) (a middle ear infection) is a common childhood illness that can leave some children with permanent hearing loss. OM can arise following infection with a variety of different pathogens, including a coinfection with influenza A virus (IAV) and Streptococcus pneumoniae (the pneumococcus). We and others have demonstrated that coinfection with IAV facilitates the replication of pneumococci in the middle ear. Specifically, we used a mouse model of OM to show that IAV facilitates the outgrowth of S. pneumoniae in the middle ear by inducing middle ear inflammation. Here, we seek to understand how the host inflammatory response facilitates bacterial outgrowth in the middle ear. Using B cell-deficient infant mice, we show that antibodies play a crucial role in facilitating pneumococcal replication. We subsequently show that this is due to antibody-dependent neutrophil extracellular trap (NET) formation in the middle ear, which, instead of clearing the infection, allows the bacteria to replicate. We further demonstrate the importance of these NETs as a potential therapeutic target through the transtympanic administration of a DNase, which effectively reduces the bacterial load in the middle ear. Taken together, these data provide novel insight into how pneumococci are able to replicate in the middle ear cavity and induce disease.

Contribution of secretory antibodies to intestinal mucosal immunity against Helicobacter pylori.

The natural immune response to Helicobacter pylori neither clears infection nor prevents reinfection. However, the ability of secretory antibodies to influence the course of H. pylori infection has not been determined. We compared the natural progression of H. pylori infection in wild-type C57BL/6 mice with that in mice lacking the polymeric immunoglobulin receptor (pIgR) that is essential for the secretion of polymeric antibody across mucosal surfaces. H. pylori SS1-infected wild-type and pIgR knockout (KO) mice were sampled longitudinally for gastrointestinal bacterial load, antibody response, and histological changes. The gastric bacterial loads of wild-type and pIgR KO mice remained constant and comparable at up to 3 months postinfection (mpi) despite SS1-reactive secretory IgA in the intestinal contents of wild-type mice at that time. Conversely, abundant duodenal colonization of pIgR KO animals contrasted with the near-total eradication of H. pylori from the intestine of wild-type animals by 3 mpi. H. pylori was cultured only from the duodenum of those animals in which colonization in the distal gastric antrum was of sufficient density for immunohistological detection. By 6 mpi, the gastric load of H. pylori in wild-type mice was significantly lower than in pIgR KO animals. While there was no corresponding difference between the two mouse strains in gastric pathology results at 6 mpi, reductions in gastric bacterial load correlated with increased gastric inflammation together with an intestinal secretory antibody response in wild-type mice. Together, these results suggest that naturally produced secretory antibodies can modulate the progress of H. pylori infection, particularly in the duodenum.

Systemic infection of Mice with Listeria monocytogenes to characterize host immune responses.

Listeria monocytogenes is a Gram-positive facultative intracellular bacterium that is widely used to characterize bacterial pathogenesis and host immunity. Here, we describe a set of basic methods and techniques to infect mice with L. monocytogenes, measure bacterial load in tissues, and analyze immune cell subsets responding to infection in the spleen and liver. In addition, a specialized method for immune cell depletion is incorporated within the overall protocol, along with suggestions at various points in the protocol for minimizing experimental variability in mouse infection studies using L. monocytogenes. Finally, we highlight a number of experimental strategies for which L. monocytogenes has facilitated research into host immune responses and bacterial pathogenesis.

Increased antigen specific T cell numbers in the absence of altered migration or division rates as a result of mucosal cholera toxin administration.

Cholera toxin (CT) is a mucosal adjuvant capable of inducing strong immune responses to co-administered antigens following oral or intranasal immunization of mice. To date, the direct effect of CT on antigen-specific CD4(+) T cell migration and proliferation profiles in vivo is not well characterized. In this study, the effect of CT on the migration pattern and proliferative responses of adoptively transferred, CD4(+) TCR transgenic T cells in orally or intranasally vaccinated mice, was analyzed by flow cytometry. GFP-expressing or CFSE-labeled OT-II lymphocytes were adoptively transferred to naïve C57BL/6 mice, and mice were subsequently vaccinated with OVA with or without CT via the oral or intranasal route. CT did not alter the migration pattern of antigen-specific T cells, regardless of the route of immunization, but increased the number of transgenic CD4(+) T cells in draining lymphoid tissue. This increase in the number of transgenic CD4(+) T cells was not due to cells undergoing more rounds of cellular division in vivo, suggesting that CT may exert an indirect adjuvant effect on CD4(+) T cells. The findings reported here suggest that CT functions as a mucosal adjuvant by increasing the number of antigen specific CD4(+) T cells independent of their migration pattern or kinetics of cellular division.

Influenza A virus induced bacterial otitis media is independent of virus tropism for α2,6-linked sialic acid.

BACKGROUND: Otitis media (OM) affects ≥80% of children before the age of three. OM can arise following co-infection with influenza A virus (IAV) and the bacterium Streptococcus pneumoniae. We have previously shown that H3 IAV strains (such as Udorn/72) induced a higher rate of bacterial OM than H1 strains (such as PR8/34). This was associated with more efficient replication of H3 strains in the middle ear. FINDINGS: Here, we assess if the increased replication of IAV strains such as Udorn/72 in the middle ear is dependent upon the binding of the viral HA to α2,6-linked sialic acid. Using murine and in vitro models, the present study shows that recognition of α2,6-linked sialic acid was not required to facilitate bacterial OM. CONCLUSIONS: Taken together, these data suggest that other features of the HA mediate bacterial OM.

Absence of platelet endothelial cell adhesion molecule 1, PECAM-1/CD31, in vivo increases resistance to Salmonella enterica serovar Typhimurium in mice.

PECAM-1/CD31 is known to regulate inflammatory responses and exhibit pro- and anti-inflammatory functions. This study was designed to determine the functional role of PECAM-1 in susceptibility to murine primary in vivo infection with Salmonella enterica serovar Typhimurium and in in vitro inflammatory responses of peritoneal macrophages. Lectin profiling showed that cellular PECAM-1 and recombinant human PECAM-1-Ig chimera contain high levels of mannose sugars and N-acetylglucosamine. Consistent with this carbohydrate pattern, both recombinant human and murine PECAM-1-Ig chimeras were shown to bind S. Typhimurium in a dose-dependent manner in vitro. Using oral and fecal-oral transmission models of S. Typhimurium SL1344 infection, PECAM-1(-/-) mice were found to be more resistant to S. Typhimurium infection than wild-type (WT) C57BL/6 mice. While fecal shedding of S. Typhimurium was comparable in wild-type and PECAM-1(-/-) mice, the PECAM-1-deficient mice had lower bacterial loads in systemic organs such as liver, spleen, and mesenteric lymph nodes than WT mice, suggesting that extraintestinal dissemination was reduced in the absence of PECAM-1. This reduced bacterial load correlated with reduced tumor necrosis factor (TNF), interleukin-6 (IL-6), and monocyte chemoattractant protein (MCP) levels in sera of PECAM-1(-/-) mice. Following in vitro stimulation of macrophages with either whole S. Typhimurium, lipopolysaccharide (LPS) (Toll-like receptor 4 [TLR4] ligand), or poly(I·C) (TLR3 ligand), production of TNF and IL-6 by PECAM-1(-/-) macrophages was reduced. Together, these results suggest that PECAM-1 may have multiple functions in resistance to infection with S. Typhimurium, including binding to host cells, extraintestinal spread to deeper tissues, and regulation of inflammatory cytokine production by infected macrophages.

Influenza-induced inflammation drives pneumococcal otitis media.

Influenza A virus (IAV) predisposes individuals to secondary infections with the bacterium Streptococcus pneumoniae (the pneumococcus). Infections may manifest as pneumonia, sepsis, meningitis, or otitis media (OM). It remains controversial as to whether secondary pneumococcal disease is due to the induction of an aberrant immune response or IAV-induced immunosuppression. Moreover, as the majority of studies have been performed in the context of pneumococcal pneumonia, it remains unclear how far these findings can be extrapolated to other pneumococcal disease phenotypes such as OM. Here, we used an infant mouse model, human middle ear epithelial cells, and a series of reverse-engineered influenza viruses to investigate how IAV promotes bacterial OM. Our data suggest that the influenza virus HA facilitates disease by inducing a proinflammatory response in the middle ear cavity in a replication-dependent manner. Importantly, our findings suggest that it is the inflammatory response to IAV infection that mediates pneumococcal replication. This study thus provides the first evidence that inflammation drives pneumococcal replication in the middle ear cavity, which may have important implications for the treatment of pneumococcal OM.