Borrelia burgdorferi, the Lyme disease agent, causes joint inflammation in an experimental murine model. Inflammation occurs, in part, due to the ability of B. burgdorferi to induce the production of proinflammatory cytokines and a strong CD4+ T helper type 1 response. The mechanisms by which spirochetes induce these responses are not completely known, although transcription factors, such as NF-κB in phagocytic cells, initiate the proinflammatory cytokine burst. We show here that the mitogen-activated protein (MAP) kinase of 38 kDa (p38 MAP kinase) is involved in the proinflammatory cytokine production elicited by B. burgdorferi Ags in phagocytic cells and the development of murine Lyme arthritis. B. burgdorferi Ags activated p38 MAP kinase in vitro, and the use of a specific inhibitor repressed the spirochete-induced production of TNF-α. The infection of mice that are deficient for a specific upstream activator of the kinase, MAP kinase kinase 3, resulted in diminished proinflammatory cytokine production and the development of arthritis, without compromising the ability of CD4+ T cells to respond to borrelial Ags or the production of specific Abs. Overall, these data indicated that the p38 MAP kinase pathway plays an important role in B. burgdorferi-elicited inflammation and point to potential new therapeutic approaches to the treatment of inflammation induced by the spirochete.
Spirochete adaptation in vivo is associated with preferential Borrelia burgdorferi gene expression. In this paper, we show that the administration of B. burgdorferi-immune sera to IFN-γR-deficient mice that have been infected with B. burgdorferi N40 for 4 days causes spirochete clearance. In contrast, immune sera-mediated clearance of B. burgdorferi N40 is not apparent in immunocompetent mice, suggesting a role for IFN-γ-mediated responses in B. burgdorferi N40 host adaptation. B. burgdorferi-immune sera also induces clearance of B. burgdorferi N40 that have been passaged in vitro 75 times (B. burgdorferi N40-75), a derivative of B. burgdorferi N40 that does not rapidly adapt in vivo in immunocompetent mice. B. burgdorferi N40-75 produce lower levels of IFN-γ and IL-12 in mice than does B. burgdorferi N40, and the administration of these cytokines to B. burgdorferi N40-75-infected mice results in an increased spirochetal burden, further indicating that IFN-γ-mediated events promote B. burgdorferi survival. Differential immunoscreening and RT-PCR demonstrate that IFN-γ-mediated signals facilitate spirochete recombination at the variable major protein like sequence locus, a site for early antigenic variation in vivo, and that recombination rates by B. burgdorferi N40 are lower in IFN-γR-deficient mice than in control animals. These results suggest that the murine immune response can promote the in vivo adaptation of B. burgdorferi.
Cyclooxygenase (Cox) is a key enzyme in the biosynthetic metabolism of prostaglandins. The inducible isoform of Cox-2 has been implicated in inflammation and its specific inhibition can be used to treat noninfectious inflammatory diseases, such as rheumatoid arthritis. Borrelia burgdorferi, the agent of Lyme disease, can induce joint inflammation. Here we show that B. burgdorferi induced the upregulation of cox-2 gene expression in murine joints at the onset of arthritis in infected mice. The level of mRNA expression correlated with the degree of inflammation. The specific inhibition of Cox-2 diminished the degree of joint inflammation, without affecting B. burgdorferi-specific antibody or cytokine responses. Cox-2 activity is therefore associated with the genesis of infectious arthritis caused by B. burgdorferi.
Lyme arthritis; Inflammation; Cyclooxygenase-2; Borrelia burgdorferi
The Lyme disease agent, Borrelia burgdorferi, is maintained in a tick–mouse cycle1,2.Here we show that B. burgdorferi usurps a tick salivary protein, Salp15 (ref. 3), to facilitate the infection of mice. The level of salp15 expression was selectively enhanced by the presence of B. burgdorferi in Ixodes scapularis, first indicating that spirochaetes might use Salp15 during transmission. Salp15 was then shown to adhere to the spirochaete, both in vitro and in vivo, and specifically interacted with B. burgdorferi outer surface protein C. The binding of Salp15 protected B. burgdorferi from antibody-mediated killing in vitro and provided spirochaetes with a marked advantage when they were inoculated into naive mice or animals previously infected with B. burgdorferi. Moreover, RNA interference-mediated repression of salp15 in I. scapularis drastically reduced the capacity of tick-borne spirochaetes to infect mice. These results show the capacity of a pathogen to use a secreted arthropod protein to help it colonize the mammalian host.
Salp15 is an Ixodes scapularis salivary protein that inhibits CD4+ T cell activation through the repression of TCR ligation-triggered calcium fluxes and IL-2 production. We show in this study that Salp15 binds specifically to the CD4 coreceptor on mammalian host T cells. Salp15 specifically associates through its C-terminal residues with the outermost two extracellular domains of CD4. Upon binding to CD4, Salp15 inhibits the subsequent TCR ligation-induced T cell signaling at the earliest steps including tyrosine phosphorylation of the Src kinase Lck, downstream effector proteins, and lipid raft reorganization. These results provide a molecular basis to understanding the immunosuppressive activity of Salp15 and its specificity for CD4+ T cells.
Arthopods, such as Ixodes ticks, serve as vectors for many human pathogens. The arthropod gut presents a pivotal microbial entry point and determines pathogen colonization and survival. We show that the gut microbiota of Ixodes scapularis, a major vector of the Lyme disease spirochete Borrelia burgdorferi, influence spirochete colonization of ticks. Perturbing the gut microbiota of larval ticks reduced Borrelia colonization, with dysbiosed larvae displaying decreased expression of the transcription factor STAT. Diminished STAT expression corresponded to lower expression of peritrophin, a key glycoprotein scaffold of the glycan-rich mucus-like peritrophic matrix (PM) that separates the gut lumen from the epithelium. The integrity of the I. scapularis PM was essential for B. burgdorferi to efficiently colonize the gut epithelium. These data elucidate a functional link between the gut microbiota, STAT-signaling, and pathogen colonization in the context of the gut epithelial barrier of an arthropod vector.
Ixodes scapularis; gut microbiota; epithelial barrier; Borrelia burgdorferi
As microbial drug-resistance increases, there is a critical need for new classes of compounds to combat infectious diseases. The Ixodes scapularis tick antifreeze glycoprotein, IAFGP, functions as an anti-virulence agent against diverse bacteria including methicillin-resistant Staphylococcus aureus. Recombinant IAFGP and a peptide, P1, derived from this protein bind to microbes and alter biofilm formation. Transgenic iafgp-expressing flies and mice challenged with bacteria, as well as wild-type animals administered P1, were resistant to infection, septic shock, or biofilm development on implanted catheter tubing. These data show that an antifreeze protein facilitates host control of bacterial infections and suggest new therapeutic strategies to counter pathogens.
Malaria, a mosquito-borne disease caused by Plasmodium species, causes substantial morbidity and mortality throughout the world. Plasmodium sporozoites mature in oocysts formed in the mosquito gut wall and then invade the salivary glands, where they remain until transmitted to the vertebrate host during a mosquito bite. The Plasmodium circumsporozoite protein (CSP) binds to salivary glands and plays a role in the invasion of this organ by sporozoites. We identified an Anopheles salivary gland protein, named CSP-binding protein (CSPBP), that interacts with CSP. Downregulation of CSPBP in mosquito salivary glands inhibited invasion by Plasmodium organisms. In vivo bioassays showed that mosquitoes that were fed blood with CSPBP antibody displayed a 25% and 90% reduction in the parasite load in infected salivary glands 14 and 18 days after the blood meal, respectively. These results suggest that CSPBP is important for the infection of the mosquito salivary gland by Plasmodium organisms and that blocking CSPBP can interfere with the Plasmodium life cycle.
Anopheles gambiae; circumsporozoite protein; invasion; Sporozoite
La7, an immunogenic outer membrane lipoprotein of Borrelia burgdorferi, produced during infection, has been shown to play a redundant role in mammalian infectivity. Here we show that La7 facilitates pathogen survival in all tested phases of the vector-specific spirochete life cycle, including tick-to-host transmission. Unlike wild type or la7-complemented isolates, isogenic La7-deficient spirochetes are severely impaired in their ability to persist within feeding ticks during acquisition from mice, in quiescent ticks during larval-nymphal inter-molt, and in subsequent pathogen transmission from ticks to naïve hosts. Analysis of gene expression during the major stages of the tick-rodent infection cycle showed increased expression of la7 in the vector and a swift downregulation in the mammalian hosts. Co-immunoprecipitation studies coupled with liquid chromatography-mass spectrometry analysis further suggested that La7, a highly conserved and abundant inner membrane protein, is involved in protein-protein interaction with a discrete set of borrelial ligands although biological significance of such interactions remains unclear. Further characterization of vector-induced membrane antigens like La7 and its interacting partners will likely aid in our understanding of the molecular details of B. burgdorferi persistence and transmission through a complex enzootic cycle.
Borrelia burgdorferi; Lyme disease; La7 protein; transmission; tick-borne
Borrelia burgdorferi transmission to the vertebrate host commences with growth of the spirochete in the tick gut and migration from the gut to the salivary glands. This complex process, involving intimate interactions of the spirochete with the gut epithelium, is pivotal to transmission. We utilized a yeast surface display library of tick gut proteins to perform a global screen for tick gut proteins that might interact with Borrelia membrane proteins. A putative fibronectin type III domain-containing tick gut protein (Ixofin3D) was most frequently identified from this screen and prioritized for further analysis. Immunization against Ixofin3D and RNA interference-mediated reduction in expression of Ixofin3D resulted in decreased spirochete burden in tick salivary glands and in the murine host. Microscopic examination showed decreased aggregation of spirochetes on the gut epithelium concomitant with reduced expression of Ixofin3D. Our observations suggest that the interaction between Borrelia and Ixofin3D facilitates spirochete congregation to the gut during transmission, and provides a “molecular exit” direction for spirochete egress from the gut.
Lyme borreliosis, the most common vector-borne illness in Northeastern parts of USA, is caused by Borrelia burgdorferi sensu lato spirochetes, and transmitted by the Ixodes scapularis ticks. Currently there is no vaccine available to prevent Lyme borreliosis. A better understanding of tick proteins that interact with Borrelia to facilitate spirochete transmission could identify new targets for the development of a tick-based vaccine to prevent Lyme borreliosis. Spirochete growth and exit from the gut is central to transmission, and might involve intimate interactions between the spirochete and the tick gut. We therefore performed a global screen to identify Borrelia-interacting tick gut proteins. One of the four Borrelia-interacting tick proteins, referred to as Ixofin3D, was further characterized. RNA-interference-mediated down-regulation of Ixofin3D resulted in decreased spirochete numbers in the salivary glands and consequently decreased transmission to the host during tick feeding. We demonstrate that Ixofin3D aids spirochete congregation to the gut epithelium, a critical first step that might direct spirochete exit from the gut.
Generation of active procoagulant cofactor FVa and its subsequent association with the enzyme FXa to form the prothrombinase complex is a pivotal initial event in blood coagulation and has been the subject of investigative effort, speculation and controversy. The current paradigm assumes that FV activation is initiated by limited proteolysis by traces of (meizo) thrombin.
Methods and Results
Recombinant tick salivary protein TIX-5 was produced and anticoagulant properties were studied using plasma, whole blood and purified systems. Here we report that TIX-5 specifically inhibits FXa-mediated FV activation involving the B-domain of FV and show that FXa activation of FV is pivotal for plasma and blood clotting. In line, tick feeding is impaired on TIX-5 immune rabbits displaying the in vivo importance of TIX-5.
Our data elucidate a unique molecular mechanism by which ticks inhibit the host's coagulation system. Based on our data we propose a revised blood coagulation scheme wherein direct FXa-mediated FV activation occurs in the initiation phase during which thrombin-mediated FV activation is restrained by fibrinogen and inhibitors.
anticoagulants; coagulation; tick salivary protein; initiation phase
Dengue virus (DENV), a flavivirus of global importance, is transmitted to humans by mosquitoes. In this study, we developed in vitro and in vivo models of saliva-mediated enhancement of DENV infectivity. Serine protease activity in Aedes aegypti saliva augmented virus infectivity in vitro by proteolyzing extracellular matrix proteins, thereby increasing viral attachment to heparan sulfate proteoglycans and inducing cell migration. A serine protease inhibitor reduced saliva-mediated enhancement of DENV in vitro and in vivo, marked by a 100-fold reduction in DENV load in murine lymph nodes. A saliva-mediated infectivity enhancement screen of fractionated salivary gland extracts identified serine protease CLIPA3 as a putative cofactor, and short interfering RNA knockdown of CLIPA3 in mosquitoes demonstrated its role in influencing DENV infectivity. Molecules in mosquito saliva that facilitate viral infectivity in the vertebrate host provide novel targets that may aid in the prevention of disease.
Serum from �%^4% of residents was positive for infection, compared with �%^9% for B. burgdorferi.
Borrelia miyamotoi sensu lato, a relapsing fever Borrelia sp., is transmitted by the same ticks that transmit B. burgdorferi (the Lyme disease pathogen) and occurs in all Lyme disease�?"endemic areas of the United States. To determine the seroprevalence of IgG against B. miyamotoi sensu lato in the northeastern United States and assess whether serum from B. miyamotoi sensu lato�?"infected persons is reactive to B. burgdorferi antigens, we tested archived serum samples from area residents during 1991�?"2012. Of 639 samples from healthy persons, 25 were positive for B. miyamotoi sensu lato and 60 for B. burgdorferi. Samples from �%^10% of B. miyamotoi sensu lato�?"seropositive persons without a recent history of Lyme disease were seropositive for B. burgdorferi. Our resultsA suggest thatA human B. miyamotoiA sensu latoA infection may be common in southern New England and that B. burgdorferi antibody testing is not an effective surrogate for detecting B. miyamotoi sensu lato infection.
Borrelia miyamotoi infection; tick-borne disease; ticks; relapsing fever; seroprevalence; seroreactivity; Borrelia miyamotoi sensu lato; United States; northeastern United States; spirochete; bacteria; Borrelia burgdorferi; New York State; New England; Lyme disease
The past decade has seen an explosion in research focusing on innate
immunity. Through a wide range of mechanisms including phagocytosis,
intracellular killing, and activation of pro-inflammatory or antiviral cytokine
production via pattern recognition receptors, the cells of the innate immune
system initiate and support adaptive immunity. The effects of aging on innate
immune responses remain incompletely understood, particularly in humans. Here,
we review advances in the study of human immunosenescence in the diverse cells
of the innate immune system, including neutrophils, monocytes, macrophages, NK
and NKT cells, and dendritic cells—with a focus on consequences for the
response to infection or vaccination in old age.
A mark-release-recapture study was conducted during 2007 through 2010 in six, tick-infested sites in Connecticut, United States to measure changes in antibody titers for Borrelia burgdorferi sensu stricto, Anaplasma phagocytophilum, and Babesia microti in Peromyscus leucopus (white-footed mice). There was an overall recapture rate of 40%, but only four tagged mice were caught in ≥2 yr. Sera from 561 mice were analyzed for total antibodies to B. burgdorferi and A. phagocytophilum by using whole-cell or recombinant (VlsE or protein 44) antigens in a solid-phase enzyme-linked immunosorbent assay or to whole-cell B. microti by indirect fluorescent antibody staining. Antibody prevalences were highly variable for B. burgdorferi (from 56% to 98%), A. phagocytophilum (from 11% to 85%), and B. microti (from 11% to 84%) depending on the site and time of sampling. Of 463 mice with antibodies, 206 (45%) had antibodies to all three pathogens. Changes in antibody status for some mice from negative to positive (117 seroconversions) or from positive to negative (55 reversions) were observed. Seroconversions were observed in 10.1% of 417 mice for B. burgdorferi, 18.0% of 306 mice for A. phagocytophilum, and 6.6% of 304 mice for B. microti; reversion rates were 5.3, 5.9, and 4.9%, respectively. Antibodies to all pathogens persisted in some mice over several weeks while, in others, there were marked declines in titration end points to negative status. The latter may indicate elimination of a certain pathogen, such as A. phagocytophilum, or that mouse immune systems ceased to produce antibodies despite an existing patent infection.
Anaplasma phagocytophilum; antibodies; Babesia microti; Borrelia burgdorferi; Peromyscus leucopus
Induction of type I interferon is a central event of innate immunity,
essential for host defense. Here we report that the transcription factor ELF4 is
induced by type I interferon and upregulates interferon expression in a
feed-forward loop. ELF4 deficiency leads to reduced interferon production,
resulting in enhanced susceptibility to West Nile virus encephalitis in mice.
After viral infection, ELF4 is recruited by STING, interacts with and is
activated by the MAVS-TBK1 complex, and translocates into the nucleus to bind
interferon promoters. Cooperative binding with ELF4 increases the binding
affinity of interferon regulatory factors IRF3 and IRF7, which is mediated by
EICE elements. Thus, in addition to identifying a regulator of innate immune
signaling, we uncovered a role for EICE elements in interferon
We evaluated Toll-like receptor (TLR) function in primary human dendritic cells from 104 young (age 21–30) and older (≥ 65 years) individuals. We used multicolor flow cytometry and intracellular cytokine staining of myeloid (mDC) and plasmacytoid (pDC) DCs and found substantial decreases in older, compared to young individuals in TNF-α, IL-6 and/or IL-12 (p40) production in mDCs and in TNF-α and IFN-α production in pDCs in response to TLR1/2, TLR2/6, TLR3, TLR5, and TLR8 engagement in mDCs and TLR7 and TLR9 in pDCs. These differences were highly significant after adjustment for heterogeneity between young and older groups (e.g. gender, race, body mass index [BMI], number of comorbid medical conditions) using mixed effect statistical modeling. Studies of surface and intracellular expression of TLR proteins, and of TLR gene expression in purified mDCs and pDCs revealed potential contributions for both transcriptional and post-transcriptional mechanisms in these age-associated effects. Moreover, intracellular cytokine production in the absence of TLR ligand stimulation was elevated in cells from older, compared to young individuals, suggesting a dysregulation of cytokine production that may limit further activation by TLR engagement. Our results provide evidence for immunosenescence in dendritic cells; notably, defects in cytokine production were strongly associated with poor antibody response to influenza immunization, a functional consequence of impaired TLR function in the aging innate immune response.
The circadian system ensures the generation and maintenance of self-sustained ~24 h rhythms in physiology that are linked to internal and environmental changes. In mammals, daily variations in light intensity and other cues are integrated by a hypothalamic master clock that conveys circadian information to peripheral molecular clocks that orchestrate physiology. Multiple immune parameters also vary throughout the day and disruption of circadian homeostasis is associated with immune-related disease. Here we discuss the molecular links between the circadian and immune systems and examine their outputs and disease implications. Understanding the mechanisms that underlie circadian-immune crosstalk may prove valuable for devising novel prophylactic and therapeutic interventions.
circadian; immunity; daily rhythms
The gram-negative obligate intracellular bacterium Anaplasma phagocytophilum is the causative agent of human granulocytic anaplasmosis (HGA), an emerging tick-borne infectious disease occuring worldwide. HGA is generally self-limiting, however, the underlying mechanisms, particularly the innate immune pathways that mediate the immune clearance of A. phagocytophilum, are less understood. We herein report an unexpected role for Receptor interacting protein-2 (Rip2), the adaptor protein for the Nod-Like Receptors (NLRs), Nod1/Nod2, in the host immune response against A. phagocytophilum infection. Although A. phagocytophilum genome is reported to lack the genes encoding the known ligands of Nod1 and Nod2, its infection up-regulated the transcription of Rip2 in human primary neutrophils. Our results revealed that Rip2 deficient mice had significantly higher bacterial load than wild type controls throughout the infection period. In addition, the Rip2 deficient mice took strikingly longer duration to clear A. phagocytophilum infection. Detailed analysis identified that interferon gamma (IFNγ) and interleukin -18 (IL-18) but not interleukin -12 (IL-12), macrophage inflammatory protein-2 (MIP-2), and KC response were diminished in A. phagocytophilum-challenged Rip2 deficient mice. Together, these results revealed that Rip2 play important roles in the immune control of A. phagocytophilum, and may contribute to our understanding of the host response to Rickettsiales.
Rip2; Anaplasma phagocytophilum; NLR; IFNγ
Ixodes scapularis transmits the agent of human granulocytic anaplasmosis, among other pathogens. The mechanisms used by the tick to control Anaplasma phagocytophilum are not known. We demonstrate that the I. scapularis Janus kinase (JAK)–signaling transducer activator of transcription (STAT) pathway plays a critical role in A. phagocytophilum infection of ticks. The A. phagocytophilum burden increases in salivary glands and hemolymph when the JAK-STAT pathway is suppressed by RNA interference. The JAK-STAT pathway exerts its anti-Anaplasma activity presumably through STAT-regulated effectors. A salivary gland gene family encoding 5.3-kDa antimicrobial peptides is highly induced upon A. phagocytophilum infection of tick salivary glands. Gene expression and electrophoretic mobility shift assays showed that the 5.3-kDa antimicrobial peptide–encoding genes are regulated by tick STAT. Silencing of these genes increased A. phagocytophilum infection of tick salivary glands and transmission to mammalian host. These data suggest that the JAK-STAT signaling pathway plays a key role in controlling A. phagocytophilum infection in ticks by regulating the expression of antimicrobial peptides.
Summary: West Nile Virus was introduced into the Western Hemisphere during the late summer of 1999 and has been causing significant and sometimes severe human diseases since that time. This article briefly touches upon the biology of the virus and provides a comprehensive review regarding recent discoveries about virus transmission, virus acquisition, and human infection and disease.
Vaccines that activate humoral and cell-mediated immune responses are urgently needed for many infectious agents, including the flaviviruses dengue and West Nile (WN) virus. Vaccine development would be greatly facilitated by a new approach, in which nanoscale modules (Ag, adjuvant, and carrier) are assembled into units that are optimized for stimulating immune responses to a specific pathogen. Toward that goal, we formulated biodegradable nanoparticles loaded with Ag and surface modified with the pathogen-associated molecular pattern CpG oligodeoxynucleotides. We chose to evaluate our construct using a recombinant envelope protein Ag from the WN virus and tested the efficiency of this system in eliciting humoral and cellular responses and providing protection against the live virus. Animals immunized with this system showed robust humoral responses polarized toward Th1 immune responses compared with predominately Th2-biased responses with the adjuvant aluminum hydroxide. Immunization with CpG oligodeoxynucleotide-modified nanoparticles resulted in a greater number of circulating effector T cells and greater activity of Ag-specific lymphocytes than unmodified nanoparticles or aluminum hydroxide. Ultimately, compared with alum, this system offered superior protection in a mouse model of WN virus encephalitis.
Research on syphilis, a sexually transmitted infection caused by the non-cultivatable spirochete Treponema pallidum, has been hampered by the lack of an inbred animal model. We hypothesized that Toll-like receptor (TLR)-dependent responses are essential for clearance of T. pallidum and, consequently, compared infection in wild-type (WT) mice and animals lacking MyD88, the adaptor molecule required for signaling by most TLRs. MyD88-deficient mice had significantly higher pathogen burdens and more extensive inflammation than control animals. Whereas tissue infiltrates in WT mice consisted of mixed mononuclear and plasma cells, infiltrates in MyD88-deficient animals were predominantly neutrophilic. Although both WT and MyD88-deficient mice produced antibodies that promoted uptake of treponemes by WT macrophages, MyD88-deficient macrophages were deficient in opsonophagocytosis of treponemes. Our results demonstrate that TLR-mediated responses are major contributors to the resistance of mice to syphilitic disease and that MyD88 signaling and FcR-mediated opsonophagocytosis are linked to the macrophage-mediated clearance of treponemes.