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Andre M Vale,
Pratibha Kapoor,
Greg A Skibinski,
Ada Elgavish,
Tamer I Mahmoud,
Cosima Zemlin,
Michael Zemlin,
Peter D Burrows,
Alberto Nobrega, John F Kearney,
David E Briles,
Harry W Schroeder
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ABSTRACT: Selection and physiological production of protective natural antibodies (NAbs) have been associated with exposure to endogenous antigens. The extent to which this association depends on germline NAb sequence is uncertain. Here we show that alterations in germline DH sequence can sever the association between the production of self-reactive NAbs and NAbs that afford protection against a pathogen. In unmanipulated hosts, the availability of the evolutionarily conserved DFL16.1 gene segment sequence profoundly affected the serum levels of NAbs against bacterial phosphorylcholine but not oxidized low-density lipoprotein. Mice with partially altered DFL16.1 sequence could use N nucleotides to recreate the amino acid sequence associated with the classical protective T15 idiotype-positive NAbs, whereas those without DFL16.1 could not. DFL16.1 gene-deficient mice proved more susceptible to challenge with live Streptococcus pneumoniae. Our findings indicate that although production of self-reactive NAbs can be independent of germline DH sequence, their capacity to provide protection against pathogens cannot. The potential relevance of these findings for the rational design of vaccines is discussed.
Journal of Experimental Medicine 04/2013; · 13.85 Impact Factor
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ABSTRACT: There has been a sharp rise in allergic asthma and asthma-related deaths in the developed world, in contrast to many childhood illnesses that have been reduced or eliminated. The hygiene hypothesis proposes that excessively sanitary conditions early in life result in autoimmune and allergic phenomena because of a failure of the immune system to receive proper microbial stimulation during development. We demonstrate that Abs generated against conserved bacterial polysaccharides are reactive with and dampen the immune response against chitin and Aspergillus fumigatus. A reduction in Ag uptake, cell influx, cell activation, and cytokine production occurred in the presence of anti-polysaccharide Abs, resulting in a striking decrease in the severity of allergic airway disease in mice. Overall, our results suggest that Ag exposure--derived from environmental sources, self-antigens, or vaccination--during the neonatal period has dramatic effects on the adult Ab response and modifies the development of allergic airway disease.
The Journal of Immunology 07/2012; 189(5):2246-56. · 5.79 Impact Factor
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ABSTRACT: Many bacteria-associated polysaccharides induce long-lived Ab responses that protect against pathogenic microorganisms. The maintenance of polysaccharide-specific Ab titers may be due to long-lived plasma cells or ongoing Ag-driven B cell activation due to polysaccharide persistence. BALB/c and V(H)J558.3 transgenic mice respond to α1→3-dextran (DEX) by generating a peak anti-DEX response at 7 d, followed by maintenance of serum Ab levels for up to 150 d. Analysis of the cellular response to DEX identified a population of short-lived, cyclophosphamide-sensitive DEX-specific plasmablasts in the spleen, and a quiescent, cyclophosphamide-resistant DEX-specific Ab-secreting population in the bone marrow. BrdU pulse-chase experiments demonstrated the longevity of the DEX-specific Ab-secreting population in the bone marrow. Splenic DEX-specific plasmablasts were located in the red pulp with persisting DEX-associated CD11c(+) dendritic cells 90 d after immunization, whereas DEX was not detected in the bone marrow after 28 d. Selective depletion of short-lived DEX-specific plasmablasts and memory B1b B cells using cyclophosphamide and anti-CD20 treatment had a minimal impact on the maintenance of serum anti-DEX Abs. Collectively, these findings demonstrate that the maintenance of serum polysaccharide-specific Abs is the result of continuous Ag-driven formation of short-lived plasmablasts in the spleen and a quiescent population of Ab-secreting cells maintained in the bone marrow for a long duration.
The Journal of Immunology 11/2011; 188(1):57-67. · 5.79 Impact Factor
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ABSTRACT: Mammalian antimicrobial peptides (AMPs) play an important role in host defense via direct antimicrobial activity as well as immune regulation. The mouse cathelin-related antimicrobial peptide (mCRAMP), produced from the mouse gene Camp, is the only mouse cathelicidin identified and the ortholog of the human gene encoding the peptide LL-37. This study tested the hypothesis that mouse B and T cells produce and respond to mCRAMP. We show that all mature mouse B-cell subsets, including follicular (FO), marginal zone (MZ), B1a, and B1b cells, as well as CD4(+) and CD8(+) T cells produce Camp mRNA and mCRAMP protein. Camp(-/-) B cells produced equivalent levels of IgM, IgG3, and IgG2c but less IgG1 and IgE, while Camp(-/-) CD4(+) T cells cultured in Th2-inducing conditions produced more IL-4-expressing cells when compared with WT cells, effects that were reversed upon addition of mCRAMP. In vivo, Camp(-/-) mice immunized with TNP-OVA absorbed in alum produced an enhanced TNP-specific IgG1 response when compared with WT mice. ELISpot analysis revealed increased numbers of TNP-specific IgG1-secreting splenic B cells and FACS analysis revealed increased CD4(+) T-cell IL-4 expression. Our results suggest that mCRAMP differentially regulates B- and T-cell function and implicate mCRAMP in the regulation of adaptive immune responses.
European Journal of Immunology 07/2011; 41(10):3006-16. · 5.10 Impact Factor
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ABSTRACT: Anti-polysaccharide Ab responses in mice are often oligoclonal, and the mechanisms involved in Ag-specific clone production and selection remain poorly understood. We evaluated the relative contribution of D(H) germline content versus N nucleotide addition in a classic oligoclonal, T-independent Ab response (α 1→3 dextran [DEX]) by challenging adult TdT-sufficient (TdT(+/+)) and TdT-deficient (TdT(-/-)) gene-targeted mice, limited to the use of a single D(H) gene segment (D-limited mice), with Enterobacter cloacae. D-limited mice achieved anti-DEX-specific levels of Abs that were broadly comparable to those of wild-type (WT) BALB/c mice. Sequence analysis of the third CDR of the H chain intervals obtained by PCR amplification of V(H) domain DNA from DEX-specific plasmablasts revealed the near universal presence of an aspartic acid residue (D99) at the V-D junction, irrespective of the composition of the D(H) locus. Although WT mice were able to use germline D(H) (DQ52, DSP, or DST) gene segment sequence, TdT activity, or both to produce D99, all three D-limited mouse strains relied exclusively on N addition. Additionally, in the absence of TdT, D-limited mice failed to produce a DEX response. Coupled with previous studies demonstrating a reduced response to DEX in TdT(-/-) mice with a WT D(H) locus, we concluded that in the case of the anti-DEX repertoire, which uses a short third CDR of the H chain, the anti-DEX response relies more intensely on sequences created by postnatal N nucleotide addition than on the germline sequence of the D(H).
The Journal of Immunology 06/2011; 187(2):879-86. · 5.79 Impact Factor
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ABSTRACT: The marginal zone (MZ) of the mouse spleen contains macrophages that express receptors that trap pathogens, including the scavenger receptor macrophage receptor with a collagenous structure and the C-type lectin specific intracellular adhesion molecule-grabbing nonintegrin receptor 1 (SIGN-R1). We previously reported that expression of SIGN-R1 was decreased in CD19-deficient mice. In this study, we demonstrate that SIGN-R1 is expressed on a subset of macrophage receptor with a collagenous structure (MARCO)(+) macrophages. This subset is diminished when MZ B cells are absent due to either genetic developmental defects or following transient migration of B cells out of the MZ. When B cells return to the MZ, there is a delay in recovery of SIGN-R1-expressing macrophages. During this period, capture of Ficoll, which for the macrophages requires SIGN-R1, remains defective not only by the macrophages, but also by the B cells. Thus, MZ B cells regulate expression of molecules on macrophages that are important for trapping Ag, which, in turn, is required for Ag capture by the B cells.
The Journal of Immunology 02/2011; 186(4):2172-81. · 5.79 Impact Factor
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ABSTRACT: The outermost layer of the Bacillus anthracis spore, the exosporium, is composed of a paracrystalline basal layer and an external hair-like nap. The nap is formed from a single collagen-like glycoprotein, while the basal layer contains many different proteins, including a 186-amino acid protein called ExsB. In this study, we discovered that ExsB is unusually highly phosphorylated, with at least 14 of its 19 threonine residues modified. The phosphorylated threonines are included in seven contiguous approximately 12-residue imperfect repeats, which presumably contain kinase recognition sequences. We demonstrated that a B. anthracis DeltaexsB mutant unable to synthesize ExsB produced spores with an exosporium that was readily sloughed, indicating that ExsB was required for stable exosporium attachment. This unstable exosporium also lacked the enzyme alanine racemase, which is normally tightly associated with the exosporium. Additionally, purified DeltaexsB spores lacking a visible exosporium were devoid of most exosporium proteins but, surprisingly, retained the putative exosporium proteins BxpC and CotB-1. Finally, we showed that transcription of the exsB gene occurred only during the late stages of sporulation, and we used an active and phosphorylated ExsB-EGFP fusion protein to monitor ExsB localization to wild-type and DeltabxpB mutant exosporia.
Molecular Microbiology 04/2010; 76(6):1527-38. · 5.01 Impact Factor
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ABSTRACT: An understanding of Ab responses to polysaccharides associated with pathogenic microorganisms is of importance for improving vaccine design, especially in neonates that respond poorly to these types of Ags. In this study, we have investigated the role of the lymphoid-specific enzyme TdT in generating B cell clones responsive to alpha-1,3 dextran (DEX). TdT is a DNA polymerase that plays a major role in generating diversity of lymphocyte AgRs during V(D)J recombination. In this study, we show that the DEX-specific Ab response is lower, and the dominant DEX-specific J558 idiotype (Id) is not detected in TdT(-/-) mice when compared with wild-type (WT) BALB/c mice. Nucleotide sequencing of H chain CDR3s of DEX-specific plasmablasts, sorted postimmunization, showed that TdT(-/-) mice generate a lower frequency of the predominant adult molecularly determined clone J558. Complementation of TdT expression in TdT(-/-) mice by early forced expression of the short splice variant of TdT-restored WT proportions of J558 Id+ clones and also abrogated the development of the minor M104E Id+ clones. J558 Id V(D)J rearrangements are detected as early as 7 d after birth in IgM-negative B cell precursors in the liver and spleen of WT and TdT-transgenic mice but not in TdT(-/-) mice. These data show that TdT is essential for the generation of the predominant higher-affinity DEX-responsive J558 clone.
The Journal of Immunology 12/2009; 184(2):851-8. · 5.79 Impact Factor
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ABSTRACT: B1b B cells generate a novel form of memory and provide Ab mediated-protection to persisting bacterial pathogens. To understand how B1b B cells establish memory to polysaccharide Ags, we studied an oligoclonal B cell response to alpha-1,3 dextran (DEX) expressed on Enterobacter cloacae. B cells specific for DEX enrich in the marginal zone (MZ) and B1b B cell populations. After E. cloacae immunization, MZ B cells were responsible for the generation of initial peak DEX-specific Ab titers, whereas, DEX-specific B1b B cells expanded and played an important role in boosted production of DEX-specific Ab titers upon E. cloacae rechallenge. Cell transfer experiments demonstrate that B1b B cells possess the capacity for both robust proliferation and plasma cell differentiation, thus distinguishing themselves from MZ B cells, which uniformly commit to plasma cell differentiation. These results define B1b B cells as the principal reservoir for memory to bacterial-associated polysaccharide Ags.
The Journal of Immunology 11/2009; 183(10):6359-68. · 5.79 Impact Factor
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ABSTRACT: In mice, the absence of terminal deoxynucleotidyl transferase (Tdt) expression during fetal and neonatal life provides a window in development where clones of lymphocytes are generated that provide protective immunity. Introducing premature Tdt activity interferes with the development of these clones and results in an impaired ability to make protective antibodies. Conversely, gene-targeted disruption of Tdt prevents N additions at all stages of T and B-lymphocyte development and promotes the development of fetal-like T and B-cell clones into adulthood, with accompanying alterations in repertoire. The alternative splice forms of Tdt may be necessary to provide regulatory mechanisms to restrict N addition to appropriate stages of the developmental pathways, the details of which are being revealed. The evidence continues to build that Tdt is a key player in influencing the outcome of V(D)J recombination during lymphocyte and repertoire development.
Immunological Reviews 09/2009; 175(1):150 - 157. · 11.15 Impact Factor
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ABSTRACT: Anthrax, a potentially lethal disease of animals and humans, is caused by the Gram-positive spore-forming bacterium Bacillus anthracis. The outermost exosporium layer of B. anthracis spores contains an external hair-like nap formed by the glycoprotein BclA. Recognition of BclA by the integrin Mac-1 promotes spore uptake by professional phagocytes, resulting in the carriage of spores to sites of spore germination and bacterial growth in distant lymphoid organs. We show that CD14 binds to rhamnose residues of BclA and acts as a coreceptor for spore binding by Mac-1. In this process, CD14 induces signals involving TLR2 and PI3k that promote inside-out activation of Mac-1, thereby enhancing spore internalization by macrophages. As observed with mice lacking Mac-1, CD14(-/-) mice are also more resistant than wild-type mice to infection by B. anthracis spores. Additionally, after B. anthracis spore challenge of CD14(-/-) mice, interference with the CD14-mediated signaling pathways results in increased mortality. Our results show that the binding and uptake of B. anthracis spores by phagocytic cells is a dynamic process and involves multiple receptors and signaling pathways.
Proceedings of the National Academy of Sciences 08/2009; 106(33):13957-62. · 9.68 Impact Factor
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ABSTRACT: Cathelicidins are a family of cationic peptides expressed in mammals that possess numerous bactericidal and immunomodulatory properties. In vitro analyses showed that human, mouse, and pig cathelicidins inhibited Bacillus anthracis bacterial growth at micromolar concentrations in the presence or absence of capsule. Combined in vitro analyses of the effects of each peptide on spore germination and vegetative outgrowth by time lapse phase contrast microscopy, transmission electron microscopy, and flow cytometric analysis showed that only the pig cathelicidin was capable of directly arresting vegetative outgrowth and killing the developing bacilli within the confines of the exosporium. C57BL/6 mice were protected from spore-induced death by each cathelicidin in a time- and dose-dependent manner. Protection afforded by the porcine cathelicidin was due to its bactericidal effects, whereas the human and mouse cathelicidins appeared to mediate protection through increased recruitment of neutrophils to the site of infection. These findings suggest that cathelicidins might be utilized to augment the initial innate immune response to B. anthracis spore exposure and prevent the development of anthrax.
The Journal of Immunology 11/2008; 181(7):4989-5000. · 5.79 Impact Factor
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ABSTRACT: Paired Ig-like receptors of activating (PIR-A) and inhibitory (PIR-B) isoforms are expressed by many hematopoietic cells, including B lymphocytes and myeloid cells. To determine the functional roles of PIR-A and PIR-B in primary bacterial infection, PIR-B-deficient (PIR-B(-/-)) and wild-type (WT) control mice were injected i.v. with an attenuated strain of Salmonella enterica Typhimurium (WB335). PIR-B(-/-) mice were found to be more susceptible to Salmonella infection than WT mice, as evidenced by high mortality rate, high bacterial loads in the liver and spleen, and a failure to clear bacteria from the circulation. Although blood levels of major cytokines and Salmonella-specific Abs were mostly comparable in the two groups of mice, distinct patterns of inflammatory lesions were found in their livers at 7-14 days postinfection: diffuse spreading along the sinusoids in PIR-B(-/-) mice vs nodular restricted localization in WT mice. PIR-B(-/-) mice have more inflammatory cells in the liver but fewer B cells and CD8(+) T cells in the spleen than WT mice at 14 days postinfection. PIR-B(-/-) bone marrow-derived macrophages (BMMphi) failed to control intracellular replication of Salmonella in vitro, in part due to inefficient phagosomal oxidant production, when compared with WT BMMphi. PIR-B(-/-) BMMphi also produced more nitrite and TNF-alpha upon exposure to Salmonella than WT BMMphi did. These findings suggest that the disruption of PIR-A and PIR-B balance affects their regulatory roles in host defense to bacterial infection.
The Journal of Immunology 10/2008; 181(6):4229-39. · 5.79 Impact Factor
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Annals of the New York Academy of Sciences 06/2008; 764(1):207 - 221. · 3.15 Impact Factor
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ABSTRACT: Marginal zone (MZ) B cells play an important role in the clearance of blood-borne bacterial infections via rapid T-independent IgM responses. We have previously demonstrated that MZ B cells respond rapidly and robustly to bacterial particulates. To determine the MZ-specific genes that are expressed to allow for this response, MZ and follicular (FO) B cells were sort purified and analyzed via DNA microarray analysis. We identified 181 genes that were significantly different between the two B cell populations. Ninety-nine genes were more highly expressed in MZ B cells while 82 genes were more highly expressed in FO B cells. To further understand the molecular mechanisms by which MZ B cells respond so rapidly to bacterial challenge, Id-positive and -negative MZ B cells were sort purified before (0 h) or after (1 h) i.v. immunization with heat-killed Streptococcus pneumoniae, R36A, and analyzed via DNA microarray analysis. We identified genes specifically up-regulated or down-regulated at 1 h following immunization in the Id-positive MZ B cells. These results give insight into the gene expression pattern in resting MZ vs FO B cells and the specific regulation of gene expression in Ag-specific MZ B cells following interaction with Ag.
The Journal of Immunology 06/2008; 180(10):6663-74. · 5.79 Impact Factor
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ABSTRACT: Of a number of mAbs made by immunization with sort-purified marginal zone (MZ) B cells, one was shown to recognize the mouse scavenger receptor CD36. Although CD36 is expressed by most resting MZ B cells and not by follicular and B1 B cells, it is rapidly induced on follicular B cells in vitro following TLR and CD40 stimulation. In response to T-independent and T-dependent Ag challenge, we found that CD36 was expressed on IgM+ plasma cells, but down-regulated on isotype-switched plasma cells in vivo. Although development, localization, and phenotype of MZ B cells in CD36-/- mice appeared normal, there was a minor block in the transitional stages of mature B cell development. In both primary and secondary Ab responses to heat-killed Streptococcus pneumoniae (R36A strain), both phosphoryl choline (PC)-specific IgM and IgG levels in CD36-/- mice were slightly reduced compared with wild-type mice. In addition, mice deficient in both TLR2 and CD36 produced significantly reduced levels of anti-PC IgG titers than those of single gene-deficient mice, suggesting that they may cooperate in an anti-PC Ab response. Collectively, these results show that CD36 does not affect the development of B cells, but modulates both primary and secondary anti-PC Ab responses during S. pneumoniae infection similarly to TLR2.
The Journal of Immunology 02/2008; 180(1):230-7. · 5.79 Impact Factor
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ABSTRACT: Anthrax, a disease caused by Bacillus anthracis, affects animals and humans. Because the inert spore is the infectious form of the organism that first contacts the potential host, the interaction between the host and spore exosporium is vital to the initiation of disease. Here, we demonstrate that the integrin Mac-1 is essential for the recognition of the major exosporium protein BclA by phagocytic cells. Expression of Mac-1, but not p150/95, in CHO cells markedly enhanced infection with Sterne strain of B. anthracis spores (WT spores). Conversely, CD11b(-/-) macrophages demonstrated a significant decrease in spore uptake when compared with macrophages from normal C57BL/6 mice. However, when CD11b(-/-) macrophages were infected with DeltabclA spores, spore ingestion was no different from their C57BL/6 counterparts. DeltabclA spores were also efficiently internalized by all CHO cell lines tested, independently of Mac-1 expression. Taken together, these results show that there is an alternative Mac-1-independent pathway involved in spore uptake that is unmasked only in the absence of BclA. Survival studies, using C57BL/6 and CD11b(-/-) mice, revealed that CD11b(-/-) mice are more resistant to infection with WT but not DeltabclA spores. Our experiments also show that DeltabclA spores are more virulent than WT spores in C57BL/6 and A/J mice. Overall, our data indicate that the Mac-1/BclA interaction may play a major role in B. anthracis pathogenesis by promoting spore uptake by professional phagocytes and subsequent access to a favorable niche for transport, germination, and outgrowth in lymphoid tissues.
Proceedings of the National Academy of Sciences 02/2008; 105(4):1261-6. · 9.68 Impact Factor
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ABSTRACT: Spores of Bacillus anthracis are enclosed by an exosporium composed of a basal layer and an external hair-like nap. The nap is formed by a collagen-like glycoprotein called BclA, while the basal layer contains many different proteins, one of which is a spore-specific alanine racemase (Alr). In this study, we employed fluorescence microscopy and a fluorescently labelled anti-Alr monoclonal antibody (mAb) to examine the distribution of Alr within the exosporium. Binding of the mAb occurred over approximately three-quarters of the exosporium but not in a cap-like region at one end of the spore, indicating the absence or inaccessibility of Alr in this region. We also determined that the cap-like region, or cap, corresponds to the first part of the exosporium assembled within the mother cell during sporulation and the only part of the exosporium assembled in a DeltaexsY mutant strain of B. anthracis. Our results provide the first direct evidence that exosporium assembly is a non-uniform process and suggest that exosporium formation is discontinuous. Finally, we demonstrated that during spore germination and outgrowth, the outgrowing cell always escapes from its exosporium shell by popping through the cap, suggesting that the cap is designed to facilitate the emergence of the outgrowing cell.
Molecular Microbiology 05/2007; 64(2):359-67. · 5.01 Impact Factor
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Annals of the New York Academy of Sciences 12/2006; 651(1):10 - 20. · 3.15 Impact Factor
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ABSTRACT: Two members of the recently identified FcR homolog (FcRH) family in mice demonstrate preferential B cell expression. One of these, FcRH3, encodes a type I transmembrane protein with five extracellular Ig domains and a cytoplasmic tail with a consensus ITIM and a noncanonical ITAM. Analysis of full-length cDNAs from five different mouse strains defines two FcRH3 alleles. A panel of FcRH3-specific mAbs was generated to define its expression pattern and functional potential on B lineage cells. Although poorly detected on the majority of bone marrow or peripheral blood cells, FcRH3 was readily identified on splenic marginal zone (MZ) and MZ precursor B cells, but not on the bulk of newly formed B cells, follicular B cells, germinal center B cells, and plasma cells. In the peritoneal cavity, FcRH3 was found on B1 cells, and not on the majority of B2 cells. Consistent with its possession of an ITIM and ITAM-like sequence, FcRH3 was tyrosine phosphorylated following pervanadate treatment, and its coligation with the BCR inhibited calcium mobilization. These results suggest FcRH3 is a novel immunoregulatory marker of MZ and B1 B lineage cells.
The Journal of Immunology 12/2006; 177(10):6815-23. · 5.79 Impact Factor
