Attenuation of relapsing fever neuroborreliosis in mice by IL-17A blockade.

Relapsing fever due to Borrelia hermsii is characterized by recurrent bacteremia episodes. However, infection of B. hermsii, if not treated early, can spread to various organs including the central nervous system (CNS). CNS disease manifestations are commonly referred to as relapsing fever neuroborreliosis (RFNB). In the mouse model of B. hermsii infection, we have previously shown that the development of RFNB requires innate immune cells as well as T cells. Here, we found that prior to the onset of RFNB, an increase in the systemic proinflammatory cytokine response followed by sustained levels of IP-10 concurrent with the CNS disease phase. RNA sequencing analysis of the spinal cord tissue during the disease phase revealed an association of the interleukin (IL)-17 signaling pathway in RFNB. To test a possible role for IL-17 in RFNB, we compared B. hermsii infection in wild-type and IL-17A-/- mice. Although the onset of bacteremia and protective anti-B. hermsii antibody responses occurred similarly, the blood-brain barrier permeability, proinflammatory cytokine levels, immune cell infiltration in the spinal cord, and RFNB manifestations were significantly diminished in IL-17A-/- mice compared to wild-type mice. Treatment of B. hermsii-infected wild-type mice with anti-IL-17A antibody ameliorated the severity of spinal cord inflammation, microglial cell activation, and RFNB. These data suggest that the IL-17 signaling pathway plays a major role in the pathogenesis of RFNB, and IL-17A blockade may be a therapeutic modality for controlling neuroborreliosis.

The Immunoglobulin M Response to Pneumococcal Polysaccharide Vaccine Is Sufficient for Conferring Immunity.

In mice, pneumococcal polysaccharide (PPS) vaccines generate antigen-specific immunoglobulin M (IgM) and immunoglobulins G1, G2, and G3. Antibody and complement-dependent opsonophagocytosis correlates with the protection induced by PPS vaccines in vivo. Since IgM is a very efficient immunoglobulin isotype in activating the complement system, we evaluated whether anti-PPS IgM alone is sufficient to confer protective immunity to Streptococcus pneumoniae. We found that immunization of wild-type and activation-induced cytidine deaminase-deficient mice capable of producing only IgM with Pneumovax 23 generated comparable anti-PPS IgM and resistance to lethal systemic challenge with S pneumoniae. These data suggest that an IgM response to PPS vaccines is sufficient for conferring immunity.

IL-7 Enables Antibody Responses to Bacterial Polysaccharides by Promoting B Cell Receptor Diversity.

Polysaccharide vaccines such as the Vi polysaccharide (ViPS) of Salmonella enterica serovar Typhi induce efficient Ab responses in adults but not in young children. The reasons for this difference are not understood. IL-7 dependency in B cell development increases progressively with age. IL-7Rα-mediated signals are required for the expression of many VH gene segments that are distal to DH-JH in the IgH locus and for the complete diversification of the BCR repertoire. Therefore, we hypothesized that B cells generated in the absence of IL-7 do not recognize a wide range of Ags because of a restricted BCR repertoire. Compared with adult wildtype mice, young wildtype mice and IL-7-deficient adult mice generated a significantly reduced Ab response to ViPS. Additionally, ViPS-binding B cells in adult wildtype mice predominantly used distal VH gene segments. Transgenic expression of either IL-7 or a BCR encoded by a distal VH gene segment permitted young mice to respond efficiently to bacterial polysaccharides. These results indicate that restricted VH gene usage early in life results in a paucity of Ag-specific B cell precursors, thus limiting antipolysaccharide responses.

An Unmutated IgM Response to the Vi Polysaccharide of Salmonella Typhi Contributes to Protective Immunity in a Murine Model of Typhoid.

T cell-dependent B cell responses typically develop in germinal centers. Abs generated during such responses are isotype switched and have a high affinity to the Ag because of somatic hypermutation of Ab genes. B cell responses to purified polysaccharides are T cell independent and do not result in the formation of bona fide germinal centers, and the dominant Ab isotype produced during such responses is IgM with very few or no somatic mutations. Activation-induced cytidine deaminase (AID) is required for both somatic hypermutation and Ig isotype switching in humans and mice. To test the extent to which unmutated polysaccharide-specific IgM confers protective immunity, we immunized wildtype and AID-/- mice with either heat-killed Salmonella enterica serovar Typhi (S. Typhi) or purified Vi polysaccharide (ViPS). We found that wildtype and AID-/- mice immunized with heat-killed S. Typhi generated similar anti-ViPS IgM responses. As expected, wildtype, but not AID-/- mice generated ViPS-specific IgG. However, the differences in the Ab-dependent killing of S. Typhi mediated by the classical pathway of complement activation were not statistically significant. In ViPS-immunized wildtype and AID-/- mice, the ViPS-specific IgM levels and S. Typhi bactericidal Ab titers at 7 but not at 28 d postimmunization were also comparable. To test the protective immunity conferred by these immunizations, mice were challenged with a chimeric S. Typhimurium strain expressing ViPS. Compared with their naive counterparts, immunized wildtype and AID-/- mice exhibited significantly reduced bacterial burden regardless of the route of infection. These data indicate that an unmutated IgM response to ViPS contributes to protective immunity to S. Typhi.

Terminal Deoxynucleotidyl Transferase Is Not Required for Antibody Response to Polysaccharide Vaccines against Streptococcus pneumoniae and Salmonella enterica Serovar Typhi.

B cell antigen receptor (BCR) diversity increases by several orders of magnitude due to the action of terminal deoxynucleotidyl transferase (TdT) during V(D)J recombination. Unlike adults, infants have limited BCR diversity, in part due to reduced expression of TdT. Since human infants and young mice respond poorly to polysaccharide vaccines, such as the pneumococcal polysaccharide vaccine Pneumovax23 and Vi polysaccharide (ViPS) of Salmonella enterica serovar Typhi, we tested the contribution of TdT-mediated BCR diversity in response to these vaccines. We found that TdT+/- and TdT-/- mice generated comparable antibody responses to Pneumovax23 and survived Streptococcus pneumoniae challenge. Moreover, passive immunization of B cell-deficient mice with serum from Pneumovax23-immunized TdT+/- or TdT-/- mice conferred protection. TdT+/- and TdT-/- mice generated comparable levels of anti-ViPS antibodies and antibody-dependent, complement-mediated bactericidal activity against S Typhi in vitro To test the protective immunity conferred by ViPS immunization in vivo, TdT+/- and TdT-/- mice were challenged with a chimeric Salmonella enterica serovar Typhimurium strain expressing ViPS, since mice are nonpermissive hosts for S Typhi infection. Compared to their unimmunized counterparts, immunized TdT+/- and TdT-/- mice challenged with ViPS-expressing S Typhimurium exhibited a significant reduction in the bacterial burden and liver pathology. These data suggest that the impaired antibody response to the Pneumovax23 and ViPS vaccines in the young is not due to limited TdT-mediated BCR diversification.

B cell multitasking is required to control nematode infection.

In this issue of Immunity, Wojciechowski et al. (2009) demonstrate that in addition to producing antibodies, B cells play pivotal roles in specific-antigen presentation and cytokine production for optimal T helper 2 responses required for protection against Heligmosomoides polygyrus.

Impaired B cell receptor signaling is responsible for reduced TACI expression and function in X-linked immunodeficient mice.

Immune response to T cell independent type 2 (TI-2) Ags, such as bacterial polysaccharides, is severely impaired in X-linked immunodeficient (XID) mice. In this study, we investigated the involvement of a proliferation-inducing ligand (APRIL) or BAFF and their receptors in the unresponsiveness of XID mouse to TI-2 Ags. We discovered that whereas serum BAFF levels were increased, the expression of the APRIL and BAFF receptor transmembrane activator and calcium-modulator and cyclophilin ligand interactor (TACI) was severely reduced in XID B cells. Moreover, B cells from XID mouse were unable to secrete Igs in response to APRIL or BAFF. In correlation with reduced TACI expression and impaired TACI function, APRIL or BAFF did not activate the classical NF-κB pathway in XID cells. Also correlating with the unaltered expression of BAFF receptor, BAFF stimulation induced the activation of the alternative NF-κB pathway in XID cells. Moreover, activation of MAPK pathway was ablated in APRIL-stimulated XID cells. Prestimulation of XID B cells with the TLR9 agonist, CpG led to a significant increase in TACI expression and restored TACI-mediated functions. CpG prestimulation also restored TACI-mediated signaling in APRIL- or BAFF-stimulated XID B cells. Finally, immunization of XID mouse with the prototype TI-2 Ag NP-Ficoll induced IgG and IgM Abs when CpG was given with NP-Ficoll. Collectively, these results suggest that reduced TACI expression is responsible for the unresponsiveness of XID mouse to TI-2 Ags and BCR activation controls TACI expression.

Efficient B cell responses to Borrelia hermsii infection depend on BAFF and BAFFR but not TACI.

T cell-independent antibody responses develop rapidly, within 3 to 4 days, and are critical for preventing blood-borne pathogens from evolving into life-threatening infections. The interaction of BAFF, also known as BLyS, with its receptors BAFFR and TACI on B cells is critical for B cell homeostasis and function. Using a synthetic polysaccharide antigen, it has previously been shown that TACI is critical for T cell-independent antibody responses. To examine the role of BAFFR and TACI in T cell-independent antibody responses to an active infection, we utilized the Borrelia hermsii infection system. In this infection system, T cell-independent responses mediated by the B1b cell subset are critical for controlling bacteremia. We found that B1b cells express BAFFR and TACI and that the surface expression of both receptors is upregulated on B1b cells following exposure to whole B. hermsii cells. Surprisingly, we found that TACI(-/-) mice are not impaired either in specific antibody responses to B. hermsii or in controlling B. hermsii bacteremia. In contrast, TACI-deficient mice immunized with heat-killed type 3 serotype pneumococcus cells are impaired in generating pneumococcal polysaccharide-specific responses and succumb to challenge with live type 3 serotype pneumococcus, indicating that TACI is required for T cell-independent antibody responses to bacterial-associated polysaccharides. Although we have found that TACI is dispensable for controlling B. hermsii infection, mice deficient in BAFFR or BAFF exhibit impairment in B. hermsii-specific IgM responses and clearance of bacteremia. Collectively, these data indicate a disparity in the roles for TACI and BAFFR in primary T cell-independent antibody responses to bacterial pathogens.

Characteristics of Borrelia hermsii infection in human hematopoietic stem cell-engrafted mice mirror those of human relapsing fever.

Rodents are natural reservoirs for a variety of species of Borrelia that cause relapsing fever (RF) in humans. The murine model of this disease recapitulates many of the clinical manifestations of the human disease and has revealed that T cell-independent antibody responses are required to resolve the bacteremic episodes. However, it is not clear whether such protective humoral responses are mounted in humans. We examined Borrelia hermsii infection in human hematopoietic stem cell-engrafted nonobese diabetic/SCID/IL-2Rγ(null) mice: "human immune system mice" (HISmice). Infection of these mice, which are severely deficient in lymphoid and myeloid compartments, with B. hermsii resulted in persistent bacteremia. In contrast, this infection in HISmice resulted in recurrent episodes of bacteremia, the hallmark of RF. The resolution of the primary episode of bacteremia was concurrent with the generation of B. hermsii-specific human IgM. Remarkably, HISmice generated antibody responses to the B. hermsii outer-membrane protein Factor H binding protein A. Sera from humans infected by B. hermsii have a similar reactivity, and studies in mice have shown that this response is generated by the B1b cell subset. HISmice contain several B-cell subsets, including those with the phenotype CD20(+)CD27(+)CD43(+)CD70(-), a proposed human equivalent of mouse B1 cells. Reduction of B cells by administration of anti-human CD20 antibody resulted in diminished anti-B. hermsii responses and persistent bacteremia in HISmice. These data indicate that analysis of B. hermsii infection in HISmice will serve as a model in which to study the cellular and molecular mechanisms involved in controlling human RF.

TLR4 Ligands in Typhoid Vi Polysaccharide Subunit Vaccines Contribute to Immunogenicity.

Activation of B cells and T cells requires the engagement of costimulatory signaling pathways in addition to Ag receptor signaling for efficient immune responses. None of the typhoid Vi polysaccharide (ViPS) subunit vaccines contains adjuvants that could activate costimulatory signaling pathways, yet these vaccines are very immunogenic. I hypothesized that residual TLR ligands present in the ViPS preparation used for making typhoid subunit vaccines account for the robust immune response generated by these vaccines. I show the presence of endotoxin, a potent agonist of TLR4, in ViPS preparations and ViPS vaccines. Furthermore, I found that ViPS obtained from various sources induces the production of proinflammatory cytokines such as IL-6 from mouse peritoneal exudate cells. Unconjugated and tetanus toxoid-conjugated ViPS vaccines activate human and mouse TLR4. Mice deficient in TLR4 or the signaling adaptors MyD88 and Trif (Toll/IL-1R domain-containing adapter inducing IFN-ß) are severely impaired in generating anti-ViPS responses to these vaccines. Elimination of the TLR4 agonist in ViPS preparation resulted in the loss of immunogenicity, and addition of lipid A, a known TLR4 agonist, restored the immunogenicity. These data highlight the importance of associated TLR ligands in the immunogenicity of ViPS subunit vaccines.

A TLR4 ligand-based adjuvant for promoting the immunogenicity of typhoid subunit vaccines.

None of the typhoid Vi Polysaccharide (ViPS) subunit vaccines incorporate adjuvants, and the immunogenicity of ViPS vaccines (e.g. Typbar TCV® and Typhim Vi®) is in part due to associated TLR4 ligands such as endotoxin present in these vaccines. Since endotoxin content in vaccines is variable and kept very low due to inherent toxicity, it was hypothesized that incorporating a defined amount of a non-toxic TLR4-ligand such as monophosphoryl lipid A in ViPS vaccines would improve their immunogenicity. To test this hypothesis, a monophosphoryl lipid A-based adjuvant formulation named Turbo was developed. Admixing Turbo with Typbar TCV® (ViPS-conjugated to tetanus toxoid) increased the levels of anti-ViPS IgM, IgG1, IgG2b, IgG2a/c, and IgG3 in inbred and outbred mice. In infant mice, a single immunization with Turbo adjuvanted Typbar TCV® resulted in a significantly increased and durable IgG response and improved the control of bacterial burden compared to mice immunized without Turbo. Similarly, when adjuvanted with Turbo, the antibody response and control of bacteremia were also improved in mice immunized with Typhim Vi®, an unconjugated vaccine. The immunogenicity of unconjugated ViPS is inefficient in young mice and is lost in adult mice when immunostimulatory ligands in ViPS are removed. Nevertheless, when adjuvanted with Turbo, poorly immunogenic ViPS induced a robust IgG response in young and adult mice, and this was observed even under antigen-limiting conditions. These data suggest that incorporation of Turbo as an adjuvant will make typhoid vaccines more immunogenic regardless of their intrinsic immunogenicity or conjugation status and maximize the efficacy across all ages.

Monophosphoryl Lipid A-based Adjuvant to Promote the Immunogenicity of Multivalent Meningococcal Polysaccharide Conjugate Vaccines.

Activation of the adaptive immune system requires the engagement of costimulatory pathways in addition to B and T cell Ag receptor signaling, and adjuvants play a central role in this process. Many Gram-negative bacterial polysaccharide vaccines, including the tetravalent meningococcal conjugate vaccines (MCV4) and typhoid Vi polysaccharide vaccines, do not incorporate adjuvants. The immunogenicity of typhoid vaccines is due to the presence of associated TLR4 ligands in these vaccines. Because the immunogenicity of MCV4 is poor and requires boosters, I hypothesized that TLR4 ligands are absent in MCV4 and that incorporation of a TLR4 ligand-based adjuvant would improve their immunogenicity. Consistent with this hypothesis, two Food and Drug Administration-approved MCV4 vaccines, MENVEO and MenQuadfi, lack TLR4 ligands. Admixing monophosphoryl lipid A, a TLR4 ligand-based adjuvant formulation named "Turbo" with MCV4 induced significantly improved IgM and IgG responses to all four meningococcal serogroup polysaccharides in adult and aged mice after a single immunization. Furthermore, in infant mice, a single booster was sufficient to promote a robust IgG response and 100% seroconversion when MCV4 was adjuvanted with Turbo. Turbo upregulated the expression of the costimulatory molecules CD40 and CD86 on B cells, and Turbo-driven adjuvanticity is lost in mice deficient in CD40 and CD86. These data suggest that Turbo induces the required costimulatory molecules for its adjuvant activity and that incorporation of Turbo could make bacterial polysaccharide vaccines more immunogenic, minimize booster requirements, and be cost-effective, particularly for those individuals in low- and middle-income and disease-endemic countries.

Identification of collaborative cross mouse strains permissive to Salmonella enterica serovar Typhi infection.

Salmonella enterica serovar Typhi is the causative agent of typhoid fever restricted to humans and does not replicate in commonly used inbred mice. Genetic variation in humans is far greater and more complex than that in a single inbred strain of mice. The Collaborative Cross (CC) is a large panel of recombinant inbred strains which has a wider range of genetic diversity than laboratory inbred mouse strains. We found that the CC003/Unc and CC053/Unc strains are permissive to intraperitoneal but not oral route of S. Typhi infection and show histopathological changes characteristic of human typhoid. These CC strains are immunocompetent, and immunization induces antigen-specific responses that can kill S. Typhi in vitro and control S. Typhi in vivo. Our results indicate that CC003/Unc and CC053/Unc strains can help identify the genetic basis for typhoid susceptibility, S. Typhi virulence mechanism(s) in vivo, and serve as a preclinical mammalian model system to identify effective vaccines and therapeutics strategies.

Induction of distinct neurologic disease manifestations during relapsing fever requires T lymphocytes.

Relapsing fever borreliosis is a multisystemic infection characterized primarily by bacteremia but can extend to the CNS. The incidence of CNS disease manifestations in humans depends on the infecting relapsing fever Borrelia species. In the murine model of Borrelia hermsii infection we found high incidence of distinct signs of CNS disease that ranged from a flaccid tail to complete paralysis of hind limbs. Infiltration of large number of T cells into the spinal cord of B. hermsii-infected mice and the upregulation of MHC class II and CD80 on infiltrating macrophages and on microglial cells suggested a role for T cell and Ag-presenting cell interactions in this pathogenesis. Indeed, B. hermsii infection did not induce CNS disease manifestations in T cell-deficient mice (TCR-beta x delta(-/-)), although it resulted in bacteremia comparable to wild-type (Wt) level. Moreover, the infiltration of immune cells into the spinal cord of TCR-beta x delta(-/-) mice was reduced and the resident microglial cells were not activated. Histopathological analysis of lumbar sections of the spinal cord confirmed severe inflammation in Wt but not in TCR-beta x delta(-/-) mice. Induction of CNS disease was dependent on the B. hermsii strain as well as on the ability of the host to control bacteremia. Mice that are impaired in controlling B. hermsii, such as CD14(-/-) mice, exhibited more severe CNS disease than Wt mice. This study demonstrates that distinct neurologic disease manifestations develop during relapsing fever and that T cells play a critical role in the induction of neuropathogenesis.

IL-7-dependent B lymphocytes are essential for the anti-polysaccharide response and protective immunity to Streptococcus pneumoniae.

Young children are impaired in their response to T cell-independent (TI) Ags, such as pneumococcal polysaccharide (PPS). B lymphopoeisis early in life is IL-7 independent, whereas in adults it is IL-7 dependent. Therefore, we hypothesized that IL-7-driven B lymphopoiesis plays a critical role in promoting Ab responses to TI Ags. Young but not adult mice are impaired in responses to PPS vaccination and to 4-hydroxy-3-nitrophenyl-acetyl-Ficoll, a widely studied model TI Ag, and B1b cells generate Ab responses to these Ags. In this paper, we show that, despite having B1b, B1a, and MZ B cells-all of which are involved in TI responses-young wild-type or adult mice deficient either in IL-7 or in IL-7Ralpha are severely impaired in anti-PPS responses and do not survive Streptococcus pneumoniae challenge, indicating IL-7-dependent B cells are required for TI immunity. Consistent with this, PPS immunization induced a robust TI response in young IL-7 transgenic mice that was comparable to adult wild-type responses. Moreover, immunized young or adult IL-7 transgenic mice were completely resistant to S. pneumoniae challenge. Our data indicate that activating the IL-7 signaling pathway could restore impaired TI responses in the young.

Effective preexposure and postexposure prophylaxis of rabies with a highly attenuated recombinant rabies virus.

Rabies remains an important public health problem with more than 95% of all human rabies cases caused by exposure to rabid dogs in areas where effective, inexpensive vaccines are unavailable. Because of their ability to induce strong innate and adaptive immune responses capable of clearing the infection from the CNS after a single immunization, live-attenuated rabies virus (RV) vaccines could be particularly useful not only for the global eradication of canine rabies but also for late-stage rabies postexposure prophylaxis of humans. To overcome concerns regarding the safety of live-attenuated RV vaccines, we developed the highly attenuated triple RV G variant, SPBAANGAS-GAS-GAS. In contrast to most attenuated recombinant RVs generated thus far, SPBAANGAS-GAS-GAS is completely nonpathogenic after intracranial infection of mice that are either developmentally immunocompromised (e.g., 5-day-old mice) or have inherited deficits in immune function (e.g., antibody production or type I IFN signaling), as well as normal adult animals. In addition, SPBAANGAS-GAS-GAS induces immune mechanisms capable of containing a CNS infection with pathogenic RV, thereby preventing lethal rabies encephalopathy. The lack of pathogenicity together with excellent immunogenicity and the capacity to deliver immune effectors to CNS tissues makes SPBAANGAS-GAS-GAS a promising vaccine candidate for both the preexposure and postexposure prophylaxis of rabies.

The Lack of Natural IgM Increases Susceptibility and Impairs Anti-Vi Polysaccharide IgG Responses in a Mouse Model of Typhoid.

Circulating IgM present in the body prior to any apparent Ag exposure is referred to as natural IgM. Natural IgM provides protective immunity against a variety of pathogens. Salmonella enterica serovar Typhi (S. Typhi) is the causative agent of typhoid fever in humans. Because mice are not permissive to S. Typhi infection, we employed a murine model of typhoid using S. enterica serovar Typhimurium expressing the Vi polysaccharide (ViPS) of S. Typhi (S. Typhimurium strain RC60) to evaluate the role of natural IgM in pathogenesis. We found that natural mouse IgM binds to S. Typhi and S. Typhimurium. The severity of S. Typhimurium infection in mice is dependent on presence of the natural resistance-associated macrophage protein 1 (Nramp1) allele; therefore, we infected mice deficient in secreted form of IgM (sIgM) on either a Nramp1-resistant (129S) or -susceptible (C57BL/6J) background. We found that the lack of natural IgM results in a significantly increased susceptibility and an exaggerated liver pathology regardless of the route of infection or the Nramp1 allele. Reconstitution of sIgM-/- mice with normal mouse serum or purified polyclonal IgM restored the resistance to that of sIgM+/+ mice. Furthermore, immunization of sIgM-/- mice with heat-killed S. Typhi induced a significantly reduced anti-ViPS IgG and complement-dependent bactericidal activity against S. Typhi in vitro, compared with that of sIgM+/+ mice. These findings indicate that natural IgM is an important factor in reducing the typhoid severity and inducing an optimal anti-ViPS IgG response to vaccination.

T-cell-independent immune responses do not require CXC ligand 13-mediated B1 cell migration.

The dynamic movement of B cells increases the probability of encountering specific antigen and facilitates cell-cell interactions required for mounting a rapid antibody response. B1a and B1b cells are enriched in the coelomic cavity, contribute to T-cell-independent (TI) antibody responses, and increase in number upon antigen exposure. B1 cell movement is largely governed by Cxc ligand 13 (Cxcl13), and mice deficient in this chemokine have a severe reduction in peritoneal B1 cells. In this study, we examined the role of Cxcl13-dependent B cell migration using Borrelia hermsii infection or intraperitoneal immunization with pneumococcal polysaccharide or 4-hydroxy-3-nitrophenyl-acetyl (NP)-Ficoll, all of which induce robust antibody responses from B1b cells. Surprisingly, we found that antibody responses to B. hermsii or to FhbA, an antigenic target of B1b cells, and the resolution of bacteremia were indistinguishable between wild-type and Cxcl13-/- mice. Importantly, we did not observe an expansion of peritoneal B1b cell numbers in Cxcl13-/- mice. Nonetheless, mice that had resolved infection were resistant to reinfection, indicating that the peritoneal B1b cell reservoir is not required for controlling B. hermsii. Furthermore, despite a reduced peritoneal B1b compartment, immunization with pneumococcal polysaccharide vaccine yielded comparable antigen-specific antibody responses in wild-type and Cxcl13-/- mice and conferred protection against Streptococcus pneumoniae. Likewise, immunization with NP-Ficoll elicited similar antibody responses in wild-type and Cxcl13-/- mice. These data demonstrate that homing of B1 cells into the coelomic cavity is not a requirement for generating protective TI antibody responses, even when antigen is initially localized to this anatomical compartment.

Toll-like receptor 2 deficiency results in impaired antibody responses and septic shock during Borrelia hermsii infection.

Overwhelming bacteremia is a leading cause of death. To understand the mechanisms involved in protective antibody and pathological inflammatory responses during bacteremia, we have been studying the murine model of Borrelia hermsii infection. Toll-like receptor (TLR) signaling plays an important role in generating the rapid anti-B. hermsii antibody responses required for the resolution of bacteremia. Using NF-κB reporter assays, we found that B. hermsii activates TLR2 and TLR9. However, TLR2(-/-) TLR9(-/-) mice exhibited an impairment in anti-B. hermsii antibody responses similar to that of TLR2(-/-) mice. Moreover, the impairment in the antibody responses of TLR2(-/-) mice or TLR2(-/-) TLR9(-/-) mice coincides with an order-of-magnitude-higher bacteremia, and death results from septic shock, as evidenced by a dysregulated systemic cytokine response and characteristic organ pathology. Since TLR2 appears to be the major extracellular sensor stimulated by B. hermsii, we hypothesized that during elevated bacteremia the activation of intracellular sensors of bacteria triggers dysregulated inflammation in TLR2(-/-) mice. Indeed, blocking the internalization of B. hermsii prevented the induction of inflammatory cytokine responses in TLR2-deficient cells. Furthermore, we found that B. hermsii activates the cytoplasmic sensor nucleotide-binding oligomerization domain 2 (NOD2). Macrophages deficient in both TLR2 and NOD2 have impaired cytokine responses to B. hermsii compared to cells lacking TLR2 alone, and B. hermsii-infected TLR2(-/-) NOD2(-/-) mice exhibited improved survival compared to TLR2(-/-) mice. These data demonstrate that TLR2 is critical for protective immunity and suggest that, during heightened bacteremia, recognition of bacterial components by intracellular sensors can lead to pathological inflammatory responses.

Genetic control of the innate immune response to Borrelia hermsii influences the course of relapsing fever in inbred strains of mice.

Host susceptibility to infection is controlled in large measure by the genetic makeup of the host. Spirochetes of the genus Borrelia include nearly 40 species of vector-borne spirochetes that are capable of infecting a wide range of mammalian hosts, causing Lyme disease and relapsing fever. Relapsing fever is associated with high-level bacteremia, as well as hematologic manifestations, such as thrombocytopenia (i.e., low platelet numbers) and anemia. To facilitate studies of genetic control of susceptibility to Borrelia hermsii infection, we performed a systematic analysis of the course of infection using immunocompetent and immunocompromised inbred strains of mice. Our analysis revealed that sensitivity to B. hermsii infections is genetically controlled. In addition, whereas the role of adaptive immunity to relapsing fever-causing spirochetes is well documented, we found that innate immunity contributes significantly to the reduction of bacterial burden. Similar to human infection, the progression of the disease in mice was associated with thrombocytopenia and anemia. Histological and fluorescence in situ hybridization (FISH) analysis of infected tissues indicated that red blood cells (RBCs) were removed by tissue-resident macrophages, a process that could lead to anemia. Spirochetes in the spleen and liver were often visualized associated with RBCs, lending support to the hypothesis that direct interaction of B. hermsii spirochetes with RBCs leads to clearance of bacteria from the bloodstream by tissue phagocytes.