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.

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.

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.

BAFF receptor and TACI in B-1b cell maintenance and antibacterial responses.

Although evidence of the protective immunity conferred by B-1b cells (CD19(+) B220(+) IgM(hi) Mac1(+) CD5(-)) has been established, the mechanisms governing the maintenance and activation of B-1b cells following pathogen encounter remain unclear. B cell-activating factor (BAFF) and a proliferation-inducing ligand (APRIL) mediate their function in mature B cells through the BAFF receptor (BAFFR) and transmembrane activator and CAML interactor (TACI). BAFFR-deficient mice have lower numbers of B-1b cells, and this reduction is directly proportional to BAFFR levels. The generation of B-1b cells is also dependent on the strength of B cell receptor (BCR) signaling. Mice with impaired BCR signaling, such as X-linked immunodeficient (xid) mice, have B-1b cell deficiency, indicating that both BCR- and BAFFR-mediated signaling are critical for B-1b cell homeostasis. Borrelia hermsii induces expansion and persistence of B-1b cells in xid mice, and these B-1b cells provide a heightened protective response. Toll-like receptor (TLR)-mediated stimulation of xid B cells results in a significant increase in TACI expression and restoration of TACI-mediated functions. The activation of TLR signaling by B. hermsii and BCR/TLR costimulation-mediated upregulation of BAFFR and TACI on B-1b cells suggests that B-1b cell maintenance and function following bacterial exposure may depend on BAFFR- and TACI-mediated signaling. In fact, the loss of both BAFFR and TACI results in a greater impairment in anti-B. hermsii responses compared to deficiency of BAFFR or TACI alone.

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.

Deciphering the role of Toll-like receptors in humoral responses to Borreliae.

The bacteria of the genus Borrelia are arthropod-borne spirochetes that cause relapsing fever and Lyme disease in humans. Like most arthropod-borne pathogens, Borreliae must survive in the periphery of their vertebrate hosts to allow for transmission to another arthropod vector. These spatial and temporal restrictions require that Borreliae evade the adaptive immune response. Borreliae have evolved genetic mechanisms that alter their surface protein expression, thereby altering the antigenic target presented to the host. To control Borreliae infection, the host relies on a rapid humoral response. While it is clear that B cell antigen receptor signaling is a critical requirement for the specific antibody responses, growing evidence suggests that additional signaling by innate immune receptors such as Toll-like receptors is necessary for optimal T cell-dependent and T cell-independent antibody responses. This review is focused on the role of Toll-like receptors in B cell responses to relapsing fever and Lyme disease Borreliae.

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.