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1.  TLR2 Signaling Decreases Transmission of Streptococcus pneumoniae by Limiting Bacterial Shedding in an Infant Mouse Influenza A Co-infection Model 
PLoS Pathogens  2014;10(8):e1004339.
While the importance of transmission of pathogens is widely accepted, there is currently little mechanistic understanding of this process. Nasal carriage of Streptococcus pneumoniae (the pneumococcus) is common in humans, especially in early childhood, and is a prerequisite for the development of disease and transmission among hosts. In this study, we adapted an infant mouse model to elucidate host determinants of transmission of S. pneumoniae from inoculated index mice to uninfected contact mice. In the context of co-infection with influenza A virus, the pneumococcus was transmitted among wildtype littermates, with approximately half of the contact mice acquiring colonization. Mice deficient for TLR2 were colonized to a similar density but transmitted S. pneumoniae more efficiently (100% transmission) than wildtype animals and showed decreased expression of interferon α and higher viral titers. The greater viral burden in tlr2−/− mice correlated with heightened inflammation, and was responsible for an increase in bacterial shedding from the mouse nose. The role of TLR2 signaling was confirmed by intranasal treatment of wildtype mice with the agonist Pam3Cys, which decreased inflammation and reduced bacterial shedding and transmission. Taken together, these results suggest that the innate immune response to influenza virus promotes bacterial shedding, allowing the bacteria to transit from host to host. These findings provide insight into the role of host factors in the increased pneumococcal carriage rates seen during flu season and contribute to our overall understanding of pathogen transmission.
Author Summary
In this study, we sought to identify factors contributing to the transmission of the bacterial pathogen Streptococcus pneumoniae (the pneumococcus), a major cause of otitis media, pneumonia, and septicemia. Often found as a co-infection with other bacterial and viral pathogens, the pneumococcus is commonly carried by young children and is spread by close human contact, most likely through large droplet respiratory secretions. The specific determinants of bacterial transmission, however, have not been identified. This report details our use of an infant mouse model of transmission, which includes influenza A co-infection, to elucidate the mechanism of host-to-host transmission. We found that the inflammatory response to influenza, which is aggravated in the context of weakened host defense, promotes transmission by inducing bacterial shedding from the mouse nose. These results show how a bacterial pathogen exploits the host immune response to spread from one host to the next.
PMCID: PMC4148449  PMID: 25166617
2.  The transcription factor Foxp1 is a critical negative regulator of the differentiation of follicular helper T cells 
Nature immunology  2014;15(7):667-675.
CD4+ follicular helper T cells (TFH cells) are essential for germinal center (GC) responses and long-lived antibody responses. Here we report that naive CD4+ T cells deficient in the transcription factor Foxp1 ‘preferentially’ differentiated into TFH cells, which resulted in substantially enhanced GC and antibody responses. We found that Foxp1 used both constitutive Foxp1A and Foxp1D induced by stimulation of the T cell antigen receptor (TCR) to inhibit the generation of TFH cells. Mechanistically, Foxp1 directly and negatively regulated interleukin 21 (IL-21); Foxp1 also dampened expression of the costimulatory molecule ICOS and its downstream signaling at early stages of T cell activation, which rendered Foxp1-deficient CD4+ T cells partially resistant to blockade of the ICOS ligand (ICOSL) during TFH cell development. Our findings demonstrate that Foxp1 is a critical negative regulator of TFH cell differentiation.
PMCID: PMC4142638  PMID: 24859450
3.  Viral antigen induces differentiation of Foxp3+ natural regulatory T cells in influenza virus-infected mice 
We have examined the formation, participation and functional specialization of virus-reactive Foxp3+ regulatory T cells (Tregs) in a mouse model of influenza virus infection. “Natural” Tregs generated intra-thymically based on interactions with a self-peptide proliferated in response to a homologous viral antigen in the lungs, and to a lesser extent in the lung-draining mediastinal LN (medLN), of virus-infected mice. By contrast, conventional CD4+ T cells with identical TCR specificity underwent little or no conversion to become “adaptive” Tregs. The virus-reactive Tregs in the medLN and the lungs of infected mice upregulated a variety of molecules associated with Treg activation, and also acquired expression of molecules (T-bet, Blimp-1 and IL-10) that confer functional specialization to Tregs. Notably, however, the phenotypes of the T-bet+ Tregs obtained from these sites were distinct, since Tregs isolated from the lungs expressed significantly higher levels of T-bet, Blimp-1 and IL-10 than did Tregs from the medLN. Adoptive transfer of antigen-reactive Tregs led to decreased proliferation of anti-viral CD4+ and CD8+ effector T cells in the lungs of infected hosts, while depletion of Tregs had a reciprocal effect. These studies demonstrate that thymically-generated Tregs can become activated by a pathogen-derived peptide and acquire discrete T-bet+ Treg phenotypes while participating in and modulating an antiviral immune response.
PMCID: PMC3703618  PMID: 23667113
4.  Commensal Bacteria Calibrate the Activation Threshold of Innate Antiviral Immunity 
Immunity  2012;37(1):158-170.
Signals from commensal bacteria can influence immune cell development and susceptibility to infectious or inflammatory diseases. However, the mechanisms by which commensal bacteria regulate protective immunity after exposure to systemic pathogens remain poorly understood. Here, we demonstrate that antibiotic-treated (ABX) mice exhibit impaired innate and adaptive antiviral immune responses and substantially delayed viral clearance after exposure to systemic LCMV or mucosal influenza virus. Furthermore, ABX mice exhibited severe bronchiole epithelial degeneration and increased host mortality after influenza virus infection. Genome-wide transcriptional profiling of macrophages isolated from ABX mice revealed decreased expression of genes associated with antiviral immunity. Moreover, macrophages from ABX mice exhibited defective responses to type I and type II IFNs and impaired capacity to limit viral replication. Collectively, these data indicate that commensal-derived signals provide tonic immune stimulation that establishes the activation threshold of the innate immune system required for optimal antiviral immunity.
PMCID: PMC3679670  PMID: 22705104
5.  Cooperativity Between CD8+ T Cells, Non-Neutralizing Antibodies, and Alveolar Macrophages Is Important for Heterosubtypic Influenza Virus Immunity 
PLoS Pathogens  2013;9(3):e1003207.
Seasonal epidemics of influenza virus result in ∼36,000 deaths annually in the United States. Current vaccines against influenza virus elicit an antibody response specific for the envelope glycoproteins. However, high mutation rates result in the emergence of new viral serotypes, which elude neutralization by preexisting antibodies. T lymphocytes have been reported to be capable of mediating heterosubtypic protection through recognition of internal, more conserved, influenza virus proteins. Here, we demonstrate using a recombinant influenza virus expressing the LCMV GP33-41 epitope that influenza virus-specific CD8+ T cells and virus-specific non-neutralizing antibodies each are relatively ineffective at conferring heterosubtypic protective immunity alone. However, when combined virus-specific CD8 T cells and non-neutralizing antibodies cooperatively elicit robust protective immunity. This synergistic improvement in protective immunity is dependent, at least in part, on alveolar macrophages and/or other lung phagocytes. Overall, our studies suggest that an influenza vaccine capable of eliciting both CD8+ T cells and antibodies specific for highly conserved influenza proteins may be able to provide heterosubtypic protection in humans, and act as the basis for a potential “universal” vaccine.
Author Summary
Influenza virus continues to pose a significant risk to global health and is responsible for thousands of deaths each year in the United States. This threat is largely due to the ability of the influenza virus to undergo rapid changes, allowing it to escape from immune responses elicited by previous infections or vaccinations. Certain internal determinants of the influenza virus are largely conserved across different viral strains and represent attractive targets for potential “universal” influenza vaccines. Here, we demonstrated that cross-subtype protection against the influenza virus could be obtained through simultaneous priming of multiple arms of the immune response against conserved elements of the influenza virus. These results suggest a novel strategy that could potentially form a primary component of a universal influenza vaccine capable of providing long-lasting protection.
PMCID: PMC3597515  PMID: 23516357
6.  Pharmacologic Activation of the Innate Immune System to Prevent Respiratory Viral Infections 
Drugs that can rapidly inhibit respiratory infection from influenza or other respiratory pathogens are needed. One approach is to engage primary innate immune defenses against viral infection, such as activating the IFN pathway. In this study, we report that a small, cell-permeable compound called 5,6-di-methylxanthenone-4-acetic acid (DMXAA) can induce protection against vesicular stomatitis virus in vitro and H1N1 influenza A virus in vitro and in vivo through innate immune activation. Using the mouse C10 bronchial epithelial cell line and primary cultures of nasal epithelial cells, we demonstrate DMXAA activates the IFN regulatory factor-3 pathway leading to production of IFN-β and subsequent high-level induction of IFN-β–dependent proteins, such as myxovirus resistance 1 (Mx1) and 2′,5′-oligoadenylate synthetase 1 (OAS1). Mice treated with DMXAA intranasally elevate mRNA/protein expression of Mx1 and OAS1 in the nasal mucosa, trachea, and lung. When challenged intranasally with a lethal dose of H1N1 influenza A virus, DMXAA reduced viral titers in the lungs and protected 80% of mice from death, even when given at 24 hours before infection. These data show that agents, like DMXAA, that can directly activate innate immune pathways, such as the IFN regulatory factor-3/IFN-β system, in respiratory epithelial cells can be used to protect from influenza pneumonia and potentially in other respiratory viral infections. Development of this approach in humans could be valuable for protecting health care professionals and “first responders” in the early stages of viral pandemics or bioterror attacks.
PMCID: PMC3265219  PMID: 21148741
innate immunity; interferon; influenza; pneumonia; bronchial epithelium
7.  Vaccination with M2e-Based Multiple Antigenic Peptides: Characterization of the B Cell Response and Protection Efficacy in Inbred and Outbred Mice 
PLoS ONE  2011;6(12):e28445.
The extracellular domain of the influenza A virus protein matrix protein 2 (M2e) is remarkably conserved between various human isolates and thus is a viable target antigen for a universal influenza vaccine. With the goal of inducing protection in multiple mouse haplotypes, M2e-based multiple antigenic peptides (M2e-MAP) were synthesized to contain promiscuous T helper determinants from the Plasmodium falciparum circumsporozoite protein, the hepatitis B virus antigen and the influenza virus hemagglutinin. Here, we investigated the nature of the M2e-MAP-induced B cell response in terms of the distribution of antibody (Ab) secreting cells (ASCs) and Ab isotypes, and tested the protective efficacy in various mouse strains.
Methodology/Principal Findings
Immunization of BALB/c mice with M2e-MAPs together with potent adjuvants, CpG 1826 oligonucleotides (ODN) and cholera toxin (CT) elicited high M2e-specific serum Ab titers that protected mice against viral challenge. Subcutaneous (s.c.) and intranasal (i.n.) delivery of M2e-MAPs resulted in the induction of IgG in serum and airway secretions, however only i.n. immunization induced anti-M2e IgA ASCs locally in the lungs, correlating with M2-specific IgA in the bronchio-alveolar lavage (BAL). Interestingly, both routes of vaccination resulted in equal protection against viral challenge. Moreover, M2e-MAPs induced cross-reactive and protective responses to diverse M2e peptides and variant influenza viruses. However, in contrast to BALB/c mice, immunization of other inbred and outbred mouse strains did not induce protective Abs. This correlated with a defect in T cell but not B cell responsiveness to the M2e-MAPs.
Anti-M2e Abs induced by M2e-MAPs are highly cross-reactive and can mediate protection to variant viruses. Although synthetic MAPs are promising designs for vaccines, future constructs will need to be optimized for use in the genetically heterogeneous human population.
PMCID: PMC3236751  PMID: 22180783
8.  Protective antiviral antibody responses in a mouse model of influenza virus infection require TACI 
The Journal of Clinical Investigation  2011;121(10):3954-3964.
Antiviral Abs, for example those produced in response to influenza virus infection, are critical for virus neutralization and defense against secondary infection. While the half-life of Abs is short, Ab titers can last a lifetime due to a subset of the Ab-secreting cells (ASCs) that is long lived. However, the mechanisms governing ASC longevity are poorly understood. Here, we have identified a critical role for extrinsic cytokine signals in the survival of respiratory tract ASCs in a mouse model of influenza infection. Irradiation of mice at various time points after influenza virus infection markedly diminished numbers of lung ASCs, suggesting that they are short-lived and require extrinsic factors in order to persist. Neutralization of the TNF superfamily cytokines B lymphocyte stimulator (BLyS; also known as BAFF) and a proliferation-inducing ligand (APRIL) reduced numbers of antiviral ASCs in the lungs and bone marrow, whereas ASCs in the spleen and lung-draining lymph node were surprisingly unaffected. Mice deficient in transmembrane activator and calcium-modulator and cyclophilin ligand interactor (TACI), a receptor for BLyS and APRIL, mounted an initial antiviral B cell response similar to that generated in WT mice but failed to sustain protective Ab titers in the airways and serum, leading to increased susceptibility to secondary viral challenge. These studies highlight the importance of TACI signaling for the maintenance of ASCs and protection against influenza virus infection.
PMCID: PMC3195469  PMID: 21881204
9.  ICOS expression by effector T cells influences the ability of regulatory T cells to inhibit anti-chromatin B cell responses in recipient mice 
Journal of autoimmunity  2009;34(4):460-468.
T regulatory cells are critical for the prevention of autoimmunity. Specifically, Treg cells can control anti-chromatin antibody production in vivo, and this correlates with decreased ICOS expression on CD4+ T helper cells. Here we test the significance of high ICOS expression by T effector cells, firstly in terms of the anti-chromatin B cell response, and secondly on the ability of Treg cells to suppress T cell help. We bred CD4+ T cell receptor transgenic mice with mice that carry the Roquinsan/san mutation. The Roquin gene functions to limit ICOS mRNA such that CD4 T cells from mutant mice express elevated ICOS. Using an in vivo model, TS1.Roquinsan/san Th cells were compared with wild-type TS1 Th cells with regard to their ability to help anti-chromatin B cells in the presence or absence of Treg cells. Both TS1 and TS1.Roquinsan/san Th cells induced anti-chromatin IgMa antibodies, but the TS1.Roquinsan/san Th cells resulted in the recovery of more class-switched and germinal center B cells. Neither source of Th cells were capable of inducing long-lived autoantibodies. Treg cells completely suppressed anti-chromatin IgMa antibody production and reduced anti-chromatin B cell recovery induced by TS1 Th cells. Importantly, this suppression was less effective when TS1.Roquinsan/san Th cells were used. Thus, high ICOS levels on effector T cells results in autoimmunity by augmenting the autoreactive B cell response and by dampening the effect of Treg cell suppression.
PMCID: PMC2859982  PMID: 20022728
regulatory T cells; autoreactive B cells; ICOS expression
10.  Efficient help for autoreactive B cell activation requires CD4+ T cell recognition of an agonist peptide at the effector stage 
European journal of immunology  2009;39(9):2377-2382.
T cell recognition of peptide/MHC complexes is flexible and can lead to differential activation, but how interactions with agonist (full activation) or partial agonist (suboptimal activation) peptides can shape immune responses in vivo is not well characterized. We investigated the effect of stimulation by agonist or partial agonist ligands during initial CD4+ T cell priming, and subsequent T-B cell cognate interactions, on antibody production by anti-chromatin B cells. We found that autoantibody production required TCR recognition of an agonist peptide at the effector stage of B cell activation. However, interaction with a weak agonist ligand at this effector stage failed to promote efficient autoantibody production, even if the CD4+ T cells were fully primed by an agonist peptide. These studies suggest that the reactivity of the TCR for a target self-peptide during CD4+ T-B cell interaction can be a critical determinant in restraining anti-chromatin autoantibody production.
PMCID: PMC3015147  PMID: 19662636
T cells; B cells; antibodies; autoimmunity
11.  Role of B cells in Systemic Lupus Erythematosus and Rheumatoid Arthritis 
Current opinion in immunology  2008;20(6):639-645.
B cell tolerance to many self-proteins is actively maintained by either purging self-reactive B receptors through clonal deletion and receptor editing, or by functional silencing known as anergy. However, these processes are clearly incomplete as B cell-driven autoimmune diseases still occur. The significance of B cells in two such diseases, rheumatoid arthritis and systemic lupus erythematosus, is highlighted by the ameliorative effects of B cell depletion. It remains to be determined, however, whether the key role of the B cell in autoimmune disease is autoantibody production or another antibody-independent function.
PMCID: PMC2646198  PMID: 18775493
12.  Autoantibody production in lpr/lpr gld/gld mice reflects accumulation of CD4+ effector cells that are resistant to regulatory T cell activity1 
Journal of autoimmunity  2008;31(2):98-109.
In Fas/FasL deficient mice anti-chromatin Ab production is T cell dependent and is not apparent until after 10 weeks of age. Early control of anti-chromatin antibodies may be due to the counterbalancing influence of Treg cells. Here we show that Treg cells block lpr/lpr gld/gld Th cells from providing help to anti-chromatin B cells in an in vivo transfer system. Interestingly, the percentage and absolute numbers of Foxp3+ Treg cells is elevated in BALB/c-lpr/lpr gld/gld mice and increases with age compared to BALB/c mice. The majority of Foxp3 expression is found in the B220− CD4+ T cell population, and Foxp3-expressing cells are localized in the splenic PALS (periarteriolar lymphocyte sheath). Strikingly, although the lack of functional Fas/FasL does not affect the ability of Treg cells to block Th cell proliferation, Treg cells can block the IFN-γ differentiation of Th cells from BALB/c or young BALB-lpr/lpr gld/gld mice but not of pre-existing Th1 cells from older BALB/c-lpr/lpr gld/gld mice. Thus, we suggest autoantibody production is not caused by the lack of Treg cells but by a defect in activation-induced cell death that leads to the accumulation of T effector cells that are resistant to regulatory T cell activity.
PMCID: PMC2585757  PMID: 18539433
autoantibody; Fas/FasL; IFN-γ; T regulatory
13.  B cell activator PAX5 promotes lymphomagenesis through stimulation of B cell receptor signaling 
The Journal of Clinical Investigation  2007;117(9):2602-2610.
The presumed involvement of paired box gene 5 (PAX5) in B-lymphomagenesis is based largely on the discovery of Pax5-specific translocations and somatic hypermutations in non-Hodgkin lymphomas. Yet mechanistically, the contribution of Pax5 to neoplastic growth remains undeciphered. Here we used 2 Myc-induced mouse B lymphoma cell lines, Myc5-M5 and Myc5-M12, which spontaneously silence Pax5. Reconstitution of these cells with Pax5–tamoxifen receptor fusion protein (Pax5ER TAM) increased neoplastic growth in a hormone-dependent manner. Conversely, expression of dominant-negative Pax5 in murine lymphomas and Pax5 knockdown in human lymphomas negatively affected cell expansion. Expression profiling revealed that Pax5 was required to maintain mRNA levels of several crucial components of B cell receptor (BCR) signaling, including CD79a, a protein with the immunoreceptor tyrosine-based activation motif (ITAM). In contrast, expression of 2 known ITAM antagonists, CD22 and PIR-B, was suppressed. The key role of BCR/ITAM signaling in Pax5-dependent lymphomagenesis was corroborated in Syk, an ITAM-associated tyrosine kinase. Moreover, we observed consistent expression of phosphorylated BLNK, an activated BCR adaptor protein, in human B cell lymphomas. Thus, stimulation of neoplastic growth by Pax5 occurs through BCR and is sensitive to genetic and pharmacological inhibitors of this pathway.
PMCID: PMC1950455  PMID: 17717600
14.  Characterization of marginal zone B cell precursors 
The Journal of Experimental Medicine  2005;202(9):1225-1234.
Selection of recently formed B cells into the follicular or marginal zone (MZ) compartments is proposed to occur by way of proliferative intermediates expressing high levels of CD21/35 and CD23. However, we show that CD21/35high CD23+ splenocytes are not enriched for proliferative cells, and do not contribute substantially to the generation of follicular B cells. Instead, ontogenic relationships, steady-state labeling kinetics, and adoptive transfer experiments suggest that CD21/35high CD23+ splenocytes serve primarily as precursors for MZ B cells, although their developmental potential seems to be broader and is influenced by environmental cues that are associated with lymphopenia. Furthermore, CD21/35high CD23+ splenocytes share several key functional characteristics with MZ B cells, including their capacity to trap T-independent antigen and a heightened proliferative response to LPS. These observations challenge previous models of peripheral B cell maturation, and suggest that MZ B cells develop by way of CD21/35high CD23+ intermediates.
PMCID: PMC2213231  PMID: 16260487
15.  Activation of diverse repertoires of autoreactive T cells enhances the loss of anti-dsDNA B cell tolerance 
Journal of Clinical Investigation  2003;112(9):1361-1371.
CD4+ helper T cells play a critical role in the production of the antinuclear autoantibodies that characterize systemic lupus erythematosus in mice and humans. A key issue is whether this help is derived from a diverse repertoire of autoreactive CD4+ T cells or from a select number of T cells of limited specificity. We used the chronic graft-versus-host disease model to define the diversity of the CD4+ T cell repertoire required to induce the autoantibody response. By transferring clonally restricted versus clonally diverse populations of MHC class II–reactive CD4+ T cells, we show that the loss of B cell tolerance to nuclear antigens has two distinct components with different CD4+ cell requirements. Activation of limited repertoires of CD4+ T cells was sufficient for the expansion of anergized anti–double-stranded DNA B cells and production of IgM autoantibodies. Unexpectedly, we found that CD4+ T cell diversity was necessary for CD4+ T cell trafficking into the follicle and for the generation of isotype-switched IgG autoantibodies. Importantly, combining two limited repertoires of T cells provides sufficient CD4+ T cell diversity to drive antinuclear Ab production. These data demonstrate that a diverse CD4+ T cell repertoire is required to generate a sustained effector B cell response capable of mediating systemic autoimmunity.
PMCID: PMC233020  PMID: 14597762
16.  MRL-lpr/lpr Mice Exhibit a Defect in Maintaining Developmental Arrest and Follicular Exclusion of Anti–double-stranded DNA B Cells  
The Journal of Experimental Medicine  1999;189(11):1799-1814.
A hallmark of systemic lupus erythematosus and the MRL murine model for lupus is the presence of anti–double-stranded (ds)DNA antibodies (Abs). To identify the steps leading to the production of these Abs in autoimmune mice, we have compared the phenotype and localization of anti-dsDNA B cells in autoimmune (MRL+/+ and lpr/lpr) mice with that in nonautoimmune (BALB/c) mice. Anti-dsDNA B cells are actively regulated in BALB/c mice as indicated by their developmental arrest and accumulation at the T–B interface of the splenic follicle. In the MRL genetic background, anti-dsDNA B cells are no longer developmentally arrested, suggesting an intrinsic B cell defect conferred by MRL background genes. With intact Fas, they continue to exhibit follicular exclusion; however, in the presence of the lpr/lpr mutation, anti-dsDNA B cells are now present in the follicle. Coincident with the altered localization of anti-dsDNA B cells is a follicular infiltration of CD4 T cells. Together, these data suggest that MRL mice are defective in maintaining the developmental arrest of autoreactive B cells and indicate a role for Fas in restricting entry into the follicle.
PMCID: PMC2193088  PMID: 10359584
tolerance; Fas; autoimmunity; antinuclear antibody; splenic architecture
17.  Regulation of Anti–double-stranded DNA B Cells in Nonautoimmune Mice: Localization to the T–B Interface of the Splenic Follicle  
The Journal of Experimental Medicine  1997;186(8):1257-1267.
Systemic lupus erythematosus (SLE) and the MRL-lpr/lpr murine model for SLE are characterized by the presence of serum anti–double-stranded (ds)DNA antibodies (Abs), whereas nonautoimmune individuals have negligible levels of these Abs. To increase the frequency of anti-DNA B cells and identify the mechanisms involved in their regulation in nonautoimmune mice, we have used Ig transgenes (tgs). In the present study, we used the VH3H9 heavy (H) chain tg which expresses an H chain that was repeatedly isolated from anti-dsDNA Abs from MRL-lpr/lpr mice. Because the VH3H9 H chain can pair with endogenous L chains to generate anti–single-stranded DNA, anti-dsDNA, and non-DNA B cells, this allowed us to study the regulation of anti-dsDNA B cells in the context of a diverse B cell repertoire. We have identified anti-dsDNA B cells that are located at the T–B interface in the splenic follicle where they have an increased in vivo turnover rate. These anti-dsDNA B cells exhibit a unique surface phenotype suggesting developmental arrest due to antigen exposure.
PMCID: PMC2199093  PMID: 9334365

Results 1-17 (17)