Immunoglobulin-Regulated Expression of Borrelia burgdorferi Outer Surface Protein A In Vivo
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感染与免疫杂志 2005年第6期
Center for Comparative Medicine, Schools of Medicine and Veterinary Medicine, University of California Davis, Davis, California 95616
ABSTRACT
Borrelia burgdorferi, the agent of Lyme disease, down-regulates outer surface protein A (OspA), which is abundantly expressed in ticks, during infection of the mammalian host. In this study we examined the signals that may be responsible for maintaining the OspA-negative state of spirochetes during infection. Transcription of ospA mRNA was found in tissues of C3H-severe combined immunodeficient (C3H-scid) mice, but not immunocompetent C3H mice, inoculated with cultured B. burgdorferi, tick-borne spirochetes, and host-adapted spirochetes. Transcription was more frequent at 4 weeks than at 1 week. Transcription was present at the host-tick interface as early as 24 h after tick attachment but declined at 48 and 72 h. Thus, ospA mRNA transcription in distant tissues and at later times in C3H-scid mice is probably due to up-regulation during infection. Adoptive lymphocyte transfer from nave C3H mice to infected C3H-scid mice resulted in OspA seroconversion, confirming OspA expression in the host. Passive transfer of normal mouse serum, immunoglobulin M (IgM) from normal mouse serum, or IgG from normal mouse serum into infected C3H-scid mice resulted in down-regulation of ospA, but transfer of normal mouse serum depleted of immunoglobulin did not influence ospA mRNA transcription. Collectively, our results indicate that ospA mRNA transcription in the host is regulated by nonspecific immunoglobulin, which may be a natural antibody.
INTRODUCTION
Lyme borreliosis in humans, as well as in domestic and wild animals, is caused by spirochetes belonging to the Borrelia burgdorferi sensu lato complex, including B. burgdorferi sensu stricto in North America. B. burgdorferi is transmitted to and acquired from its hosts by hard tick species belonging to the Ixodes persulcatus complex, which includes Ixodes scapularis and Ixodes pacificus in North America, Ixodes ricinus in Europe, and Ixodes persulcatus in Eastern Europe and Asia (2, 38, 49). In the United States, a wide variety of birds and mammals serve as reservoir hosts, but the principal reservoir host is the white-footed mouse, Peromyscus leucopus, and the white-tailed deer, Odocoileus virginianus, plays a critical role as a host for adult ticks (2). The life cycle of I. scapularis requires at least 2 years, involving larval, nymphal, and adult stages that must feed on hosts while also surviving seasonal weather variations. B. burgdorferi must also persevere through and adapt to these various conditions.
The ability of B. burgdorferi to survive and adapt to these markedly changing conditions is believed to be facilitated by differential expression of various gene products, particularly outer surface proteins (Osps). A notable example is OspA, a major 31-kDa lipoprotein that is abundantly expressed by B. burgdorferi in the midgut of unfed ticks and by spirochetes grown in artificial media but is generally not expressed during infection of mammalian hosts. OspA has been the subject of intensive investigation since its initial discovery (3, 10, 22, 26, 37, 52). Among the factors that have been shown to modulate OspA expression are temperature (34, 48), pH (51, 52), cocultivation with tick cells (34), exposure to tick hemolymph (20), the presence of anti-OspA antibody within feeding ticks (17), the presence of natural antibody (9), and serum starvation (1).
More germane to the role of OspA in the infectious cycle and the reason for investigating its expression under various conditions is the fact that OspA is highly dynamic in the tick and the host. In unfed infected ticks (nymphs and adults) spirochetes are restricted to their midgut and express abundant OspA (16), whereas feeding by the tick stimulates spirochetes to rapidly multiply and migrate to the salivary glands but significantly down-regulates OspA (13, 14, 47, 48). Immunization of hosts against OspA protects against tick-borne infection by killing OspA-expressing spirochetes in the tick midgut during the initial stages of feeding (22). When spirochetes are transmitted to nave hosts, they do not express OspA (24, 30, 40) and are therefore no longer vulnerable to OspA immunity (15). These dynamics are reflected in the sera of most patients and animals following tick-borne infection with B. burgdorferi. Such sera do not contain OspA antibody, whereas sera from experimental animals inoculated by syringe with high doses of cultured B. burgdorferi, which expresses OspA, contain OspA antibodies (5, 6, 8, 19, 23, 36, 41, 42).
In contrast to these findings, OspA antibody has been found in the sera of some human patients late in the course of infection, suggesting OspA expression (19, 27, 28). Furthermore, serial serum samples from Lyme disease patients have revealed seroconversion to OspA coinciding with the severity and onset of arthritis, as well as the duration of arthritis (27, 28). The mechanism for this paradoxical event has not been determined, but one possible explanation is that OspA expression can be increased by spirochetes maintained in an inflammatory environment in vivo (12). In addition to OspA antibody in some patients with chronic infections and disease, low levels of OspA antibody, primarily immunoglobulin M (IgM), have been detected in many patients during early infection, but these antibodies tend to be complexed with antigen, and the response appears to be transient (43-45).
In the immunocompetent mouse model of Lyme disease, seroconversion to OspA and ospA transcription have not been detected during infection following tick-borne infection or infection with host-adapted spirochetes (7, 11, 15, 23-25, 42). We recently published seemingly contrary studies that found low levels of ospA mRNA in the skin, hearts, and tibiotarsal joints of C3H-severe combined immunodeficient (C3H-scid) mice inoculated by syringe with cultured B. burgdorferi. Furthermore, ospA mRNA transcription was most commonly found after 2 or more weeks of infection and at sites distant from the inoculation site, suggesting that ospA mRNA transcription took place by disseminating spirochetes within the host (25).
To further explore this phenomenon, we assessed B. burgdorferi ospA mRNA transcription in C3H-scid mice following syringe inoculation, tick-borne inoculation, or inoculation with host-adapted B. burgdorferi and examined the antibody responses in infected C3H-scid mice that were adoptively reconstituted with lymphocytes from immunocompetent mice. The results led to investigation of the effect of non-OspA (nonspecific) antibody on ospA mRNA transcription. Our studies suggest that host innate immunity, mediated through immunoglobulins, is involved in modulation of OspA expression in vivo.
MATERIALS AND METHODS
Mice. C3H/Smn.CIcrHsd-Prkdcscid (C3H-scid) and C3H/HeN (C3H) specific-pathogen-free mice (3 to 5 weeks old) were purchased from Harlan Sprague-Dawley, Inc. (Indianapolis, Ind.) and Frederick Cancer Research Center (Frederick, MD), respectively. Mice were housed in isolator cages, given food and water ad libitum, and killed by carbon dioxide narcosis, followed by cardiocentesis.
B. burgdorferi. A clonal strain of B. burgdorferi sensu stricto (cN40) was grown in modified Barbour-Stoenner-Kelly (BSK II) medium (2) at 33°C. At the time of necropsy, tissues (blood, inoculation site, and urinary bladder) were cultured in modified BSK II medium, as described previously (6), to confirm the infection status of each mouse.
Mouse inoculation. For syringe inoculation, 104 B. burgdorferi cN40 spirochetes at the mid-log phase in 0.1 ml of BSK II medium were inoculated intradermally at the dorsal thoracic midline into each mouse. For tick-borne inoculation, five nymphal ticks that were infected with B. burgdorferi cN40 were placed on the dorsal thoracic midline and allowed to attach and feed to repletion. For infection with host-adapted spirochetes, 3-mm punches were obtained from ears of infected C3H mice using disposable dermal biopsy punches (Premiere Medical Supply) at 3 weeks after syringe inoculation. The punches were placed in BSK II medium and transplanted beneath the skin of the dorsal thoracic midline through a pocket watch incision, as described previously (4, 7). Recognizing that doses could not be exactly the same with these different types of inocula, we attempted to roughly equilibrate inocula for all of these methods to the 104-spirochete dose inoculated by syringe. For tick inoculation we utilized nymphal ticks with a mean of 5 x 104 spirochetes per tick (unknown numbers were transmitted), and the 3-mm-diameter ear transplants contained a mean of 1.8 x 104 spirochetes, based upon quantitative DNA analysis (see below).
Ticks. I. scapularis ticks were obtained as field-collected adults from southern Connecticut (cordially provided by Durland Fish, Yale University, New Haven, CT), and they produced uninfected larvae for experimental use. All larvae were derived from a single cohort for the experiments described in this study, and a sample of the cohort was confirmed to be uninfected by B. burgdorferi flaB PCR. To generate infected nymphs, larvae were allowed to engorge on C3H mice that had been infected with B. burgdorferi for 2 weeks following syringe inoculation, as described previously (25). A sample of the infected nymphal tick cohort revealed that 97% of the ticks were PCR positive for B. burgdorferi flaB.
Quantitative analysis of B. burgdorferi DNA and mRNA. Tissue samples were weighed, snap-frozen in liquid nitrogen immediately after collection, pulverized, and homogenized, and then they were split equally for use for DNA and RNA extraction. DNA was extracted using DNeasy tissue kits according to the manufacturer's instruction for tissues (QIAGEN, Valencia, CA), and total RNA was purified using RNeasy mini kits according to the manufacturer's instruction for tissues (QIAGEN, Valencia, CA). The copy number of each B. burgdorferi target gene was expressed per mg of tissue. Samples used for RNA analysis were homogenized with a QIAshredder and then treated with RNase-free DNase I prior to elution. The concentration and purity of extracted RNA were determined by measuring the A260 and A280. Extracted total RNA was stored at –80°C until use. For synthesis of cDNA from extracted total RNA, reverse transcription was performed utilizing TaqMan reverse transcription reagents (PE Biosystems, Foster City, CA). cDNA was amplified in a standard heat block instrument after incubation for 10 min at 25°C following transcription at 48°C for 30 min and inactivation at 95°C for 5 min. Real-time PCR was optimized and utilized to quantify B. burgdorferi population dynamics targeting flaB DNA, as well as to evaluate ospA transcriptional activity, targeting ospA cDNA, as described previously (25).
Histology. Hearts and joints (knees, tibiotarsi) were fixed in 10% neutral buffered formalin. Joints were demineralized in decalcifying solution. Formalin-fixed tissues were embedded in paraffin, sectioned at 5 μm, and then stained with hematoxylin using standard histologic techniques.
Serology. An antibody capture enzyme-linked immunosorbent assay (ELISA) was used to determine serum IgM and IgG titers to mouse IgG (heavy and light chains) (Jackson Immuno-Research Laboratories, Inc., West Grove, PA), B. burgdorferi cN40 lysate, and recombinant OspA. Briefly, ELISA plates (Nunc ImmunoMax Maxi-Sorp, Wiesbaden, Germany) were coated with 1 μg/ml of rat anti-mouse IgG (Jackson Immuno-Research Laboratories, Inc.), B. burgdorferi lysate, or recombinant cN40 OspA (21) in carbonate buffer and incubated overnight at 4°C. After the plates were washed and after nonspecific binding was blocked with 1% bovine serum albumin, twofold serial dilutions of sera were titrated in the plates, which were incubated overnight at 4°C. The plates were washed again and then incubated with alkaline phosphatase-conjugated rat-anti mouse IgG(H+L) (Jackson Immuno-Research Laboratories, Inc.). After a final wash, wells were incubated with 1 mg of alkaline phosphatase substrate (Sigma, St. Louis, Mo.) per ml for color development. Absorbance was read with an ELISA reader (Molecular Devices, Sunnyvale, CA) at a test wavelength of 405 nm. Each assay included positive and negative controls for serum. The cutoff point for positive reactions was established at 3 standard deviations above normal mouse serum or control antigen background.
Purification of serum immunoglobulins. Normal mouse serum was obtained from specific-pathogen-free female C3H mice (3 to 5 weeks old). To equilibrate serum to buffer, serum was dialyzed overnight at 4°C against binding buffer (0.1 M sodium phosphate, 0.15 M sodium chloride, pH 7.2). For separation of IgM, the serum was applied to an AminoLink Plus (Pierce Biotechnology, Inc., Rockford, IL) column with immobilized rat anti-mouse IgM antibody. Bound fractions of IgM were eluted with 0.1 M glycine, pH 2.7. For separation of IgG, the remaining serum was applied consecutively to Affinity Pak immobilized protein L (Pierce) and protein G HiTrap affinity (Amersham Biosciences Corporation, Piscataway, NJ) columns. Bound fractions of IgG were eluted with 0.1 M glycine, pH 2.7. The immunoglobulin content of all fractions (normal mouse serum, serum devoid of immunoglobulins, IgG and IgM fractions) was assessed by quantitative ELISA. The concentrations of immunoglobulin in the IgG and IgM fractions were adjusted to the levels detected in the normal mouse serum.
Statistics. Statistical comparisons of mean numbers of spirochetes per mg of tissue for infected C3H-scid and C3H mice were made using StatView (PowerPC version; SAS Institute Inc., Cary, NC). Multiple-comparison analyses were performed with a one-way analysis of variance, followed by a least-difference post hoc test. P values lower than 0.05 were considered significant.
RESULTS
Comparison of ospA mRNA transcription in C3H-scid mice infected by syringe or with tick-borne spirochetes. An experiment was performed not only to confirm previous findings (24) for ospA mRNA transcription in syringe-inoculated C3H-scid mice but also to determine if ospA mRNA transcription was due to the fact that mice were inoculated with cultured spirochetes or if tick-borne spirochetes elicited the same effect. Ten C3H-scid mice were infected with B. burgdorferi by allowing five infected nymphal ticks to feed to repletion. Two days after the ticks were placed on these mice, an additional 10 C3H-scid and five C3H mice were syringe inoculated. The staggered timing of inoculation approximately equilibrated the timing of infection, to reflect the delay of spirochete entry after tick attachment (25, 39). At 1 week after inoculation (9 days after tick attachment), five tick-infected and five syringe-infected C3H-scid mice were necropsied. At 4 weeks after inoculation, five syringe-infected C3H mice, five syringe-infected C3H-scid mice, and five tick-infected C3H-scid mice were necropsied. At necropsy, tissues were cultured to verify infection, and heart base, tibiotarsus, and quadriceps femoris muscle samples were used for PCR analysis (B. burgdorferi flaB DNA, flaB mRNA, and ospA mRNA). Quadriceps muscle was included in order to compare an infected tissue without significant inflammation to the tibiotarsus, which manifested arthritis.
At 1 week, hearts from four of five syringe-infected and four of five tick-infected mice, tibiotarsal tissue from four of five syringe-infected and one of five tick-infected mice, and quadriceps muscle from three of five syringe-inoculated mice had detectable flaB DNA (data not shown). At 4 weeks, all mice, regardless of the inoculum, had detectable flaB DNA in all three tissues. In all flaB DNA-positive samples, transcriptional activity of flaB mRNA was detectable. In contrast, ospA mRNA transcription was detected in only one syringe-infected heart sample at 1 week. At 4 weeks, low levels of ospA mRNA transcription were detected in hearts from three of five syringe-infected and two of five tick-infected mice and in tibiotarsi from two of five syringe-infected and three of five tick-infected mice but not in quadriceps muscle (Fig. 1). Collectively, three of five syringe-infected and three of five tick-inoculated mice had detectable ospA mRNA transcription in one or more tissues at 4 weeks. These results confirm previous studies which demonstrated that ospA mRNA transcription occurred during infection of C3H-scid mice (24). The data also suggest that ospA mRNA transcription, albeit low and not detectable in all samples, is more frequent after 4 weeks of infection than after 1 week and therefore likely to reflect up-regulation during infection of the host. Furthermore, the presence of ospA mRNA transcription in inflamed hearts and joints, but not in uninflamed muscle, supports the observation of other workers (12) that inflammation stimulates ospA mRNA transcription.
Transcription of ospA mRNA at the vector-host interface in C3H-scid mice. The experiment described above suggested that ospA mRNA transcription occurred following tick-borne infection. We next examined the skin of C3H-scid mice at 24, 48, and 72 h after tick attachment to determine if ospA mRNA transcription was present during early transmission (Fig. 2). Fifteen C3H-scid mice were each infested with six nymphal ticks, and two tick attachment sites were collected from each mouse for groups of five mice at 24, 48, and 72 h after tick attachment. Of the samples that were positive for flaB DNA, 4 of 10 samples (from five flaB DNA-positive mice) were positive for ospA mRNA transcription at 24 h; one of nine samples (from four flaB DNA-positive mice) were positive at 48 h; and none of six samples (from three flaB DNA-positive mice) were positive at 72 h. These results confirm previous findings for tick-borne infection in C3H mice (24). They extend the findings to C3H-scid mice and indicate that spirochetes initially transcribe ospA mRNA at 24 h in the tick attachment sites following tick-borne transmission, but spirochetes appear to down-regulate ospA mRNA transcription by 48 to 72 h, despite rising numbers of spirochetes in the skin in an immunodeficient host.
Transcription of ospA mRNA in C3H-scid mice inoculated with host-adapted spirochetes. In the next experiment we sought to determine if ospA mRNA transcription is up-regulated in C3H-scid mice following infection by transplant of ear tissue containing host-adapted spirochetes from immunocompetent C3H mice (in which ospA mRNA transcription does not take place). Ten C3H mice were inoculated by syringe with B. burgdorferi. Three weeks later, these donor mice were necropsied, and the infection status was confirmed by culture. All donor mice were culture positive. Ear tissues from the donor mice were transplanted into C3H-scid mice. At 1 and 4 weeks after inoculation, groups of five and four C3H-scid mice, respectively, were necropsied, tissues were cultured to verify infection, hearts and joints were processed for histology, and the heart base, tibiotarsal joint, and quadriceps muscle were collected for PCR analysis. All mice which received transplants were culture positive.
Carditis and arthritis were present in the four mice examined at 4 weeks but were not evident in the five mice at 1 week. Although flaB mRNA transcription was present in hearts and joints of all five mice at 1 week, no ospA mRNA transcription was detected at this time. In contrast, ospA mRNA transcription was found in three heart samples and one joint sample at 4 weeks (Fig. 3). These results suggest that host-adapted spirochetes (derived from immunocompetent C3H mice), which do not express OspA, appear to up-regulate ospA mRNA transcription during infection of C3H-scid mice.
Transcription of ospA mRNA and seroconversion to OspA in infected C3H-scid mice adoptively reconstituted with lymphocytes from immunocompetent C3H mice. Transcription of ospA mRNA occurred in C3H-scid mice but not in C3H mice, suggesting that ospA mRNA transcription is influenced by innate or acquired host immune factors. Furthermore, although these results indicate that ospA mRNA transcription occurred, they do not necessarily prove that OspA translation and expression occurred. In an effort to prove that OspA protein expression occurred, C3H-scid mice were infected and then immunologically reconstituted and tested for seroconversion to OspA. Twenty C3H-scid mice were syringe inoculated with B. burgdorferi. At 2 weeks after inoculation, 10 C3H-scid mice were reconstituted by intraperitoneal inoculation with 2 x 107 splenocytes and 1 x 107 lymph node cells (total, 3 x 107 cells) from nave C3H mice. At 4 weeks after inoculation, five nonreconstituted C3H-scid mice were necropsied. At 6 weeks after inoculation, five nonreconstituted and five reconstituted (4 weeks after adoptive transfer) mice were necropsied, and at 8 weeks after inoculation, five reconstituted mice were necropsied (6 weeks after reconstitution). The infection status was confirmed by culture. Hearts, joints, and muscles were analyzed by PCR, and serum was tested for antibody. In this experiment, heart base was compared to ventricular muscle (inflamed versus adjacent noninflamed tissue), and the tibiotarsus was compared to quadriceps muscle (inflamed versus adjacent noninflamed tissue).
At necropsy, all mice from both groups (reconstituted and nonreconstituted) were culture and PCR positive. Carditis and arthritis were present in nonreconstituted C3H-scid mice at 4 and 6 weeks, and the arthritis was severe. Inflammatory lesions were present in the heart base but not in the ventricular muscle and were present in the tibiotarsus but not in the quadriceps muscle. Carditis and arthritis were less severe in reconstituted mice at 6 weeks and were regressing at 8 weeks (data not shown). Nonreconstituted C3H-scid mice were examined at 4 weeks to verify transcription of ospA mRNA at a time at which the reconstituted mice would be developing acquired immunity 2 weeks following adoptive transfer. Transcription of ospA mRNA was verified in one or more tissues from four of five mice (two of five heart bases; three of five tibiotarsi) (Fig. 4). Thus, it could be assumed that similarly inoculated but reconstituted C3H-scid mice were likely to immunologically recognize OspA, if it was translated into protein.
Comparing the spirochete loads for different treatment groups of mice at week 6 (Fig. 4) revealed significantly higher spirochete numbers (flaB DNA) in the heart base (P = 0.0007), ventricular muscle (P = 0.00027), and quadriceps muscle (P = 0.00001) of nonreconstituted mice than in the tissues of reconstituted mice. In contrast, reconstitution did not significantly reduce spirochete numbers in tibiotarsal joints (P = 0.37), despite resolving arthritis. Quantitative analysis of flaB mRNA revealed a pattern that followed the quantitative flaB DNA dynamics for both groups of mice and for all tissues. Transcription of ospA mRNA was detected in the heart base in two of five mice and in the tibiotarsus of three of five mice (four of five mice tested had ospA transcription in one or both sites) but not in the ventricular muscle or quadriceps muscle. None of the reconstituted mice had detectable ospA mRNA transcription in any of the tissues. These results confirmed that ospA mRNA transcription occurs in the absence of immunity. Furthermore, they support the observation that ospA mRNA transcription tends to occur in tissues with inflammation (heart base and tibiotarsus), in contrast to adjacent, noninflamed tissues (ventricular muscle and quadriceps muscle).
IgM and IgG were detected in all reconstituted mice at high titers (>1:25,000), confirming that there was successful reconstitution. Antibodies against B. burgdorferi lysates were detected in all reconstituted C3H-scid mice, with the highest titers in mice necropsied at 8 weeks (data not shown). When plates were coated with OspA and probed with sera from reconstituted C3H-scid mice, OspA antibodies were detected at a titer of 1:100 in two of five mice necropsied at 8 weeks (data not shown). Thus, detection of OspA antibodies in reconstituted C3H-scid mice confirmed that the low levels of ospA mRNA transcription in C3H-scid mice are accompanied by OspA translation, based upon seroconversion.
Effect of normal serum on ospA mRNA transcription in infected C3H-scid mice. Other workers (9) have shown that natural antibodies in nave mice interact with spirochetes in feeding ticks and influence OspA expression within the tick. Therefore, the effect of normal mouse serum on ospA mRNA transcription in infected C3H-scid mice was investigated. Twenty C3H-scid mice were syringe inoculated with B. burgdorferi. Ten of the mice were each injected intraperitoneally with 1 ml of normal (uninfected) C3H mouse serum at the time of inoculation and at 2 weeks, and the other 10 mice received no serum. At 4 weeks, mice were necropsied, tissues were cultured to verify infection, and tissues (heart base, ventricular muscle, tibiotarsus, and quadriceps muscle) were processed for B. burgdorferi DNA and RNA analysis.
All mice were culture positive. The numbers of spirochetes, based upon flaB DNA copy numbers, and the levels of flaB mRNA transcription in each tissue were equivalent in the two groups (serum treated and untreated) of C3H-scid mice (Fig. 5). Transcription of ospA mRNA was found in the heart base of 6 of 10 untreated mice, in the ventricular muscle of 2 of 10 untreated mice, and in the tibiotarsus of 4 of 10 untreated mice. Collectively, 7 of 10 untreated mice had ospA mRNA transcription in one or more tissues. No ospA mRNA transcription was detected in the quadriceps muscle from any of the untreated mice. In contrast, no ospA mRNA transcription was detected in the heart base, ventricular muscle, tibiotarsus, or quadriceps muscle of mice treated with normal mouse serum (10 mice had no ospA mRNA transcription in any tissue). Thus, these results suggest that components of serum from nave, immunocompetent mice influence ospA mRNA transcription in the host.
Effect of normal mouse serum and immunoglobulin fractions on ospA mRNA transcription in infected C3H-scid mice. Next, we attempted to localize the effector for down-regulation of ospA mRNA transcription to the immunoglobulin fraction of serum. Twenty C3H-scid mice were syringe inoculated with B. burgdorferi and divided into four equal groups of five mice. Group 1 received 1 ml of normal mouse serum at the time of inoculation and at 2 weeks. Group 2 received 1 ml of normal mouse serum that was depleted of immunoglobulin. Group 3 received 1 ml of the IgG fraction from normal mouse serum. Group 4 received 1 ml of the IgM fraction from normal mouse serum. At 4 weeks, mice were necropsied, tissues were cultured to verify infection, and the heart base, ventricular muscle, tibiotarsus, and quadriceps muscle were processed for B. burgdorferi DNA and RNA analysis.
The various treatments had no effect upon B. burgdorferi flaB DNA levels or flaB mRNA transcription. For the 20 mice and 20 tissue samples from each mouse, ospA mRNA transcription was found only in tissues from three mice (two of three heart base samples, one of three tibiotarsus samples) treated with serum that was depleted of immunoglobulin (Fig. 6). Furthermore, these results confirmed the effect of normal mouse serum and nonspecific immunoglobulins on ospA mRNA transcription.
DISCUSSION
The results of the current study reveal that ospA mRNA transcription takes place during infection of tissues in immunodeficient C3H-scid mice. The level of activity was low, and activity was not detectable in all mice or all samples. Collective analysis of the data, however, suggested that tissues with inflammation (heart base and tibiotarsus) favored ospA mRNA transcription. In contrast, noninflamed tissues (cardiac ventricular muscle and quadriceps muscle), which were consistently infected and contained spirochetes that were actively transcribing flaB, tended not to favor ospA mRNA transcription. These findings support observations of ospA mRNA transcription and OspA expression by spirochetes that were maintained in an inflammatory environment (12). In that study, spirochetes were maintained in dialysis tubing chambers implanted in the peritoneum of mice, which were then were subjected to intraperitoneal treatment with zymosan, a yeast cell extract that induces inflammation. Under such conditions, spirochetes increased transcription of ospA mRNA, as well as expressed increased amounts of OspA. In contrast to the current study, the spirochetes in the peritoneal chambers were culture derived and, even when host adapted, expressed OspA. Nevertheless, these two independent studies clearly demonstrate that OspA can be expressed in vivo and that an inflammatory environment favors ospA mRNA transcription and OspA expression by B. burgdorferi.
In other studies of B. burgdorferi infection of C3H-scid mice workers have not observed ospA mRNA transcription. Liang et al. (31) were unable to detect ospA mRNA transcription in C3H-scid mice using less sensitive microarray approaches. Crother et al. (11) were not able to detect OspA protein in detergent-phase tissue extracts from C3H-scid mice infected for 17 days with B. burgdorferi B31, but other proteins, including VlsE, OspC, and DbpA, were detectable. The inability to detect OspA was possibly due to the very small amounts of OspA in tissues, which the present data suggest, and possibly also due to the early time (17 days). Real-time PCR is a highly sensitive and quantitative means for detecting RNA transcripts in tissues of mice infected with B. burgdorferi, and it has recently been utilized to demonstrate dynamics of gene expression during infection in both immunocompetent and immunodeficient mice (24, 25, 32). Acquired immunity by the host has been shown to significantly reduce, but not eliminate, spirochetes from tissues, with commensurate reduction of transcription of several genes, including flaB, ospC, dbpA, arp (BBF01), vlsE, fbp (BBK32), oppA-2, and p37-42 (BBK47). The only absolute reduction in gene transcription in all tissues under these circumstances was the reduction in ospA transcription (24). These results were confirmed in a recent study that examined dbpA, ospC, BBF01, and vlsE mRNA transcription, but that study involved fewer tissues and ospA mRNA transcription was not examined (32).
It has been generally accepted that B. burgdorferi, which expresses abundant amounts of OspA in unfed ticks, rapidly down-regulates ospA mRNA transcription during tick feeding upon a host (16, 33, 48). The signals for this profound change in surface protein expression are not known, but a recent study has shown that natural antibody in nonimmune mice is a significant factor (9). Ninety percent of spirochetes in the salivary glands of ticks feeding upon B-cell-deficient mice were shown to express OspA, in contrast to only 5% of spirochetes from ticks feeding upon nave, immunocompetent mice. Furthermore, the spirochete numbers in the midguts of ticks were significantly reduced in ticks feeding upon immunocompetent mice compared to B-cell-deficient mice. Passive transfer of normal mouse serum into the B-cell-deficient mice was sufficient to induce these profound shifts in OspA expression. It has been known for some time that natural IgM interacts with OspA on spirochetes grown in vitro (18, 50). Furthermore, sera from several different species of animals, which were not exposed to B. burgdorferi, have been shown to contain natural antibodies that can kill spirochetes in vitro in the presence of complement (29). Although we did not observe reductions in spirochete numbers in infected C3H-scid mice that were passively treated with normal mouse serum, our results complement these other studies by demonstrating the role of natural antibodies as a signal for ospA mRNA transcription modulation in vivo and maintenance of ospA down-regulation during persistent infection of the host.
The down-regulation of ospA mRNA transcription during tick feeding is not absolute, as some spirochetes in the salivary glands of feeding ticks have been shown to express OspA (35, 46-48). In a recent study examining B. burgdorferi population kinetics and gene expression at the host-vector interface, it was shown that spirochetes which initially enter the host at the site of tick feeding transiently transcribe ospA mRNA at 24 h but rapidly down-regulate ospA mRNA transcription by 48 h and beyond in immunocompetent C3H mice (25). The current study confirmed this observation, using C3H-scid mice. Thus, transient OspA expression explains the finding of early antibody responses to OspA, which tend to rapidly decline in both experimental animals and humans (43, 45). Studies of C3H-scid mice infected with cultured (24), host-adapted, or tick-borne spirochetes (current study) indicate that ospA mRNA transcription continues in the host in the absence of immune competence, and the critical signaling factor is natural immunoglobulin.
Because of the relative paucity of spirochetes in tissues and the relatively low level of ospA mRNA transcription in infected mice, we attempted to prove OspA translation and expression indirectly with an adoptive transfer experiment. The results demonstrated that there was seroconversion of infected C3H-scid mice that were reconstituted with normal lymphocytes from nave, immunocompetent C3H mice. However, seroconversion to OspA took place in only two of five mice that were successfully reconstituted. All five mice readily seroconverted to B. burgdorferi. The variable results are likely due to the fact that ospA mRNA transcription was down-regulated during the early phases of reconstitution because of sensitivity to natural immunoglobulin, thereby reducing or eliminating the antigenic stimulus at a time when full immunocompetence was affected. Nevertheless, we established the fact that some of the mice seroconverted specifically to OspA, thereby proving that OspA translation occurred in vivo.
The regulatory effector for ospA transcription in vivo, at least in part, is immunoglobulin. Our study compartmentalized the effector to either IgM or IgG. Nonspecific immunoglobulins, possibly natural antibody, have been shown to significantly influence OspA expression in the tick. In the previous study, as in the current study, the IgM and IgG fractions of normal mouse serum were tested, but only IgM was found to be effective at modifying OspA expression in ticks. It was speculated that the lack of effect of IgG on OspA expression in ticks may have been due to IgG binding proteins in tick saliva (9). It remains to be determined if the effects of immunoglobulins on ospA mRNA transcription and OspA expression are actually due to natural antibodies that are specifically reactive with OspA or due to some other mechanism. If specificity is required, this finding may provide insight into why OspA may be expressed in some Lyme disease patients who seroconvert to OspA during the late stages of infection.
ACKNOWLEDGMENTS
This work was supported by PHS grant R01 AI26815 from the National Institute of Allergy and Infectious Diseases.
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ABSTRACT
Borrelia burgdorferi, the agent of Lyme disease, down-regulates outer surface protein A (OspA), which is abundantly expressed in ticks, during infection of the mammalian host. In this study we examined the signals that may be responsible for maintaining the OspA-negative state of spirochetes during infection. Transcription of ospA mRNA was found in tissues of C3H-severe combined immunodeficient (C3H-scid) mice, but not immunocompetent C3H mice, inoculated with cultured B. burgdorferi, tick-borne spirochetes, and host-adapted spirochetes. Transcription was more frequent at 4 weeks than at 1 week. Transcription was present at the host-tick interface as early as 24 h after tick attachment but declined at 48 and 72 h. Thus, ospA mRNA transcription in distant tissues and at later times in C3H-scid mice is probably due to up-regulation during infection. Adoptive lymphocyte transfer from nave C3H mice to infected C3H-scid mice resulted in OspA seroconversion, confirming OspA expression in the host. Passive transfer of normal mouse serum, immunoglobulin M (IgM) from normal mouse serum, or IgG from normal mouse serum into infected C3H-scid mice resulted in down-regulation of ospA, but transfer of normal mouse serum depleted of immunoglobulin did not influence ospA mRNA transcription. Collectively, our results indicate that ospA mRNA transcription in the host is regulated by nonspecific immunoglobulin, which may be a natural antibody.
INTRODUCTION
Lyme borreliosis in humans, as well as in domestic and wild animals, is caused by spirochetes belonging to the Borrelia burgdorferi sensu lato complex, including B. burgdorferi sensu stricto in North America. B. burgdorferi is transmitted to and acquired from its hosts by hard tick species belonging to the Ixodes persulcatus complex, which includes Ixodes scapularis and Ixodes pacificus in North America, Ixodes ricinus in Europe, and Ixodes persulcatus in Eastern Europe and Asia (2, 38, 49). In the United States, a wide variety of birds and mammals serve as reservoir hosts, but the principal reservoir host is the white-footed mouse, Peromyscus leucopus, and the white-tailed deer, Odocoileus virginianus, plays a critical role as a host for adult ticks (2). The life cycle of I. scapularis requires at least 2 years, involving larval, nymphal, and adult stages that must feed on hosts while also surviving seasonal weather variations. B. burgdorferi must also persevere through and adapt to these various conditions.
The ability of B. burgdorferi to survive and adapt to these markedly changing conditions is believed to be facilitated by differential expression of various gene products, particularly outer surface proteins (Osps). A notable example is OspA, a major 31-kDa lipoprotein that is abundantly expressed by B. burgdorferi in the midgut of unfed ticks and by spirochetes grown in artificial media but is generally not expressed during infection of mammalian hosts. OspA has been the subject of intensive investigation since its initial discovery (3, 10, 22, 26, 37, 52). Among the factors that have been shown to modulate OspA expression are temperature (34, 48), pH (51, 52), cocultivation with tick cells (34), exposure to tick hemolymph (20), the presence of anti-OspA antibody within feeding ticks (17), the presence of natural antibody (9), and serum starvation (1).
More germane to the role of OspA in the infectious cycle and the reason for investigating its expression under various conditions is the fact that OspA is highly dynamic in the tick and the host. In unfed infected ticks (nymphs and adults) spirochetes are restricted to their midgut and express abundant OspA (16), whereas feeding by the tick stimulates spirochetes to rapidly multiply and migrate to the salivary glands but significantly down-regulates OspA (13, 14, 47, 48). Immunization of hosts against OspA protects against tick-borne infection by killing OspA-expressing spirochetes in the tick midgut during the initial stages of feeding (22). When spirochetes are transmitted to nave hosts, they do not express OspA (24, 30, 40) and are therefore no longer vulnerable to OspA immunity (15). These dynamics are reflected in the sera of most patients and animals following tick-borne infection with B. burgdorferi. Such sera do not contain OspA antibody, whereas sera from experimental animals inoculated by syringe with high doses of cultured B. burgdorferi, which expresses OspA, contain OspA antibodies (5, 6, 8, 19, 23, 36, 41, 42).
In contrast to these findings, OspA antibody has been found in the sera of some human patients late in the course of infection, suggesting OspA expression (19, 27, 28). Furthermore, serial serum samples from Lyme disease patients have revealed seroconversion to OspA coinciding with the severity and onset of arthritis, as well as the duration of arthritis (27, 28). The mechanism for this paradoxical event has not been determined, but one possible explanation is that OspA expression can be increased by spirochetes maintained in an inflammatory environment in vivo (12). In addition to OspA antibody in some patients with chronic infections and disease, low levels of OspA antibody, primarily immunoglobulin M (IgM), have been detected in many patients during early infection, but these antibodies tend to be complexed with antigen, and the response appears to be transient (43-45).
In the immunocompetent mouse model of Lyme disease, seroconversion to OspA and ospA transcription have not been detected during infection following tick-borne infection or infection with host-adapted spirochetes (7, 11, 15, 23-25, 42). We recently published seemingly contrary studies that found low levels of ospA mRNA in the skin, hearts, and tibiotarsal joints of C3H-severe combined immunodeficient (C3H-scid) mice inoculated by syringe with cultured B. burgdorferi. Furthermore, ospA mRNA transcription was most commonly found after 2 or more weeks of infection and at sites distant from the inoculation site, suggesting that ospA mRNA transcription took place by disseminating spirochetes within the host (25).
To further explore this phenomenon, we assessed B. burgdorferi ospA mRNA transcription in C3H-scid mice following syringe inoculation, tick-borne inoculation, or inoculation with host-adapted B. burgdorferi and examined the antibody responses in infected C3H-scid mice that were adoptively reconstituted with lymphocytes from immunocompetent mice. The results led to investigation of the effect of non-OspA (nonspecific) antibody on ospA mRNA transcription. Our studies suggest that host innate immunity, mediated through immunoglobulins, is involved in modulation of OspA expression in vivo.
MATERIALS AND METHODS
Mice. C3H/Smn.CIcrHsd-Prkdcscid (C3H-scid) and C3H/HeN (C3H) specific-pathogen-free mice (3 to 5 weeks old) were purchased from Harlan Sprague-Dawley, Inc. (Indianapolis, Ind.) and Frederick Cancer Research Center (Frederick, MD), respectively. Mice were housed in isolator cages, given food and water ad libitum, and killed by carbon dioxide narcosis, followed by cardiocentesis.
B. burgdorferi. A clonal strain of B. burgdorferi sensu stricto (cN40) was grown in modified Barbour-Stoenner-Kelly (BSK II) medium (2) at 33°C. At the time of necropsy, tissues (blood, inoculation site, and urinary bladder) were cultured in modified BSK II medium, as described previously (6), to confirm the infection status of each mouse.
Mouse inoculation. For syringe inoculation, 104 B. burgdorferi cN40 spirochetes at the mid-log phase in 0.1 ml of BSK II medium were inoculated intradermally at the dorsal thoracic midline into each mouse. For tick-borne inoculation, five nymphal ticks that were infected with B. burgdorferi cN40 were placed on the dorsal thoracic midline and allowed to attach and feed to repletion. For infection with host-adapted spirochetes, 3-mm punches were obtained from ears of infected C3H mice using disposable dermal biopsy punches (Premiere Medical Supply) at 3 weeks after syringe inoculation. The punches were placed in BSK II medium and transplanted beneath the skin of the dorsal thoracic midline through a pocket watch incision, as described previously (4, 7). Recognizing that doses could not be exactly the same with these different types of inocula, we attempted to roughly equilibrate inocula for all of these methods to the 104-spirochete dose inoculated by syringe. For tick inoculation we utilized nymphal ticks with a mean of 5 x 104 spirochetes per tick (unknown numbers were transmitted), and the 3-mm-diameter ear transplants contained a mean of 1.8 x 104 spirochetes, based upon quantitative DNA analysis (see below).
Ticks. I. scapularis ticks were obtained as field-collected adults from southern Connecticut (cordially provided by Durland Fish, Yale University, New Haven, CT), and they produced uninfected larvae for experimental use. All larvae were derived from a single cohort for the experiments described in this study, and a sample of the cohort was confirmed to be uninfected by B. burgdorferi flaB PCR. To generate infected nymphs, larvae were allowed to engorge on C3H mice that had been infected with B. burgdorferi for 2 weeks following syringe inoculation, as described previously (25). A sample of the infected nymphal tick cohort revealed that 97% of the ticks were PCR positive for B. burgdorferi flaB.
Quantitative analysis of B. burgdorferi DNA and mRNA. Tissue samples were weighed, snap-frozen in liquid nitrogen immediately after collection, pulverized, and homogenized, and then they were split equally for use for DNA and RNA extraction. DNA was extracted using DNeasy tissue kits according to the manufacturer's instruction for tissues (QIAGEN, Valencia, CA), and total RNA was purified using RNeasy mini kits according to the manufacturer's instruction for tissues (QIAGEN, Valencia, CA). The copy number of each B. burgdorferi target gene was expressed per mg of tissue. Samples used for RNA analysis were homogenized with a QIAshredder and then treated with RNase-free DNase I prior to elution. The concentration and purity of extracted RNA were determined by measuring the A260 and A280. Extracted total RNA was stored at –80°C until use. For synthesis of cDNA from extracted total RNA, reverse transcription was performed utilizing TaqMan reverse transcription reagents (PE Biosystems, Foster City, CA). cDNA was amplified in a standard heat block instrument after incubation for 10 min at 25°C following transcription at 48°C for 30 min and inactivation at 95°C for 5 min. Real-time PCR was optimized and utilized to quantify B. burgdorferi population dynamics targeting flaB DNA, as well as to evaluate ospA transcriptional activity, targeting ospA cDNA, as described previously (25).
Histology. Hearts and joints (knees, tibiotarsi) were fixed in 10% neutral buffered formalin. Joints were demineralized in decalcifying solution. Formalin-fixed tissues were embedded in paraffin, sectioned at 5 μm, and then stained with hematoxylin using standard histologic techniques.
Serology. An antibody capture enzyme-linked immunosorbent assay (ELISA) was used to determine serum IgM and IgG titers to mouse IgG (heavy and light chains) (Jackson Immuno-Research Laboratories, Inc., West Grove, PA), B. burgdorferi cN40 lysate, and recombinant OspA. Briefly, ELISA plates (Nunc ImmunoMax Maxi-Sorp, Wiesbaden, Germany) were coated with 1 μg/ml of rat anti-mouse IgG (Jackson Immuno-Research Laboratories, Inc.), B. burgdorferi lysate, or recombinant cN40 OspA (21) in carbonate buffer and incubated overnight at 4°C. After the plates were washed and after nonspecific binding was blocked with 1% bovine serum albumin, twofold serial dilutions of sera were titrated in the plates, which were incubated overnight at 4°C. The plates were washed again and then incubated with alkaline phosphatase-conjugated rat-anti mouse IgG(H+L) (Jackson Immuno-Research Laboratories, Inc.). After a final wash, wells were incubated with 1 mg of alkaline phosphatase substrate (Sigma, St. Louis, Mo.) per ml for color development. Absorbance was read with an ELISA reader (Molecular Devices, Sunnyvale, CA) at a test wavelength of 405 nm. Each assay included positive and negative controls for serum. The cutoff point for positive reactions was established at 3 standard deviations above normal mouse serum or control antigen background.
Purification of serum immunoglobulins. Normal mouse serum was obtained from specific-pathogen-free female C3H mice (3 to 5 weeks old). To equilibrate serum to buffer, serum was dialyzed overnight at 4°C against binding buffer (0.1 M sodium phosphate, 0.15 M sodium chloride, pH 7.2). For separation of IgM, the serum was applied to an AminoLink Plus (Pierce Biotechnology, Inc., Rockford, IL) column with immobilized rat anti-mouse IgM antibody. Bound fractions of IgM were eluted with 0.1 M glycine, pH 2.7. For separation of IgG, the remaining serum was applied consecutively to Affinity Pak immobilized protein L (Pierce) and protein G HiTrap affinity (Amersham Biosciences Corporation, Piscataway, NJ) columns. Bound fractions of IgG were eluted with 0.1 M glycine, pH 2.7. The immunoglobulin content of all fractions (normal mouse serum, serum devoid of immunoglobulins, IgG and IgM fractions) was assessed by quantitative ELISA. The concentrations of immunoglobulin in the IgG and IgM fractions were adjusted to the levels detected in the normal mouse serum.
Statistics. Statistical comparisons of mean numbers of spirochetes per mg of tissue for infected C3H-scid and C3H mice were made using StatView (PowerPC version; SAS Institute Inc., Cary, NC). Multiple-comparison analyses were performed with a one-way analysis of variance, followed by a least-difference post hoc test. P values lower than 0.05 were considered significant.
RESULTS
Comparison of ospA mRNA transcription in C3H-scid mice infected by syringe or with tick-borne spirochetes. An experiment was performed not only to confirm previous findings (24) for ospA mRNA transcription in syringe-inoculated C3H-scid mice but also to determine if ospA mRNA transcription was due to the fact that mice were inoculated with cultured spirochetes or if tick-borne spirochetes elicited the same effect. Ten C3H-scid mice were infected with B. burgdorferi by allowing five infected nymphal ticks to feed to repletion. Two days after the ticks were placed on these mice, an additional 10 C3H-scid and five C3H mice were syringe inoculated. The staggered timing of inoculation approximately equilibrated the timing of infection, to reflect the delay of spirochete entry after tick attachment (25, 39). At 1 week after inoculation (9 days after tick attachment), five tick-infected and five syringe-infected C3H-scid mice were necropsied. At 4 weeks after inoculation, five syringe-infected C3H mice, five syringe-infected C3H-scid mice, and five tick-infected C3H-scid mice were necropsied. At necropsy, tissues were cultured to verify infection, and heart base, tibiotarsus, and quadriceps femoris muscle samples were used for PCR analysis (B. burgdorferi flaB DNA, flaB mRNA, and ospA mRNA). Quadriceps muscle was included in order to compare an infected tissue without significant inflammation to the tibiotarsus, which manifested arthritis.
At 1 week, hearts from four of five syringe-infected and four of five tick-infected mice, tibiotarsal tissue from four of five syringe-infected and one of five tick-infected mice, and quadriceps muscle from three of five syringe-inoculated mice had detectable flaB DNA (data not shown). At 4 weeks, all mice, regardless of the inoculum, had detectable flaB DNA in all three tissues. In all flaB DNA-positive samples, transcriptional activity of flaB mRNA was detectable. In contrast, ospA mRNA transcription was detected in only one syringe-infected heart sample at 1 week. At 4 weeks, low levels of ospA mRNA transcription were detected in hearts from three of five syringe-infected and two of five tick-infected mice and in tibiotarsi from two of five syringe-infected and three of five tick-infected mice but not in quadriceps muscle (Fig. 1). Collectively, three of five syringe-infected and three of five tick-inoculated mice had detectable ospA mRNA transcription in one or more tissues at 4 weeks. These results confirm previous studies which demonstrated that ospA mRNA transcription occurred during infection of C3H-scid mice (24). The data also suggest that ospA mRNA transcription, albeit low and not detectable in all samples, is more frequent after 4 weeks of infection than after 1 week and therefore likely to reflect up-regulation during infection of the host. Furthermore, the presence of ospA mRNA transcription in inflamed hearts and joints, but not in uninflamed muscle, supports the observation of other workers (12) that inflammation stimulates ospA mRNA transcription.
Transcription of ospA mRNA at the vector-host interface in C3H-scid mice. The experiment described above suggested that ospA mRNA transcription occurred following tick-borne infection. We next examined the skin of C3H-scid mice at 24, 48, and 72 h after tick attachment to determine if ospA mRNA transcription was present during early transmission (Fig. 2). Fifteen C3H-scid mice were each infested with six nymphal ticks, and two tick attachment sites were collected from each mouse for groups of five mice at 24, 48, and 72 h after tick attachment. Of the samples that were positive for flaB DNA, 4 of 10 samples (from five flaB DNA-positive mice) were positive for ospA mRNA transcription at 24 h; one of nine samples (from four flaB DNA-positive mice) were positive at 48 h; and none of six samples (from three flaB DNA-positive mice) were positive at 72 h. These results confirm previous findings for tick-borne infection in C3H mice (24). They extend the findings to C3H-scid mice and indicate that spirochetes initially transcribe ospA mRNA at 24 h in the tick attachment sites following tick-borne transmission, but spirochetes appear to down-regulate ospA mRNA transcription by 48 to 72 h, despite rising numbers of spirochetes in the skin in an immunodeficient host.
Transcription of ospA mRNA in C3H-scid mice inoculated with host-adapted spirochetes. In the next experiment we sought to determine if ospA mRNA transcription is up-regulated in C3H-scid mice following infection by transplant of ear tissue containing host-adapted spirochetes from immunocompetent C3H mice (in which ospA mRNA transcription does not take place). Ten C3H mice were inoculated by syringe with B. burgdorferi. Three weeks later, these donor mice were necropsied, and the infection status was confirmed by culture. All donor mice were culture positive. Ear tissues from the donor mice were transplanted into C3H-scid mice. At 1 and 4 weeks after inoculation, groups of five and four C3H-scid mice, respectively, were necropsied, tissues were cultured to verify infection, hearts and joints were processed for histology, and the heart base, tibiotarsal joint, and quadriceps muscle were collected for PCR analysis. All mice which received transplants were culture positive.
Carditis and arthritis were present in the four mice examined at 4 weeks but were not evident in the five mice at 1 week. Although flaB mRNA transcription was present in hearts and joints of all five mice at 1 week, no ospA mRNA transcription was detected at this time. In contrast, ospA mRNA transcription was found in three heart samples and one joint sample at 4 weeks (Fig. 3). These results suggest that host-adapted spirochetes (derived from immunocompetent C3H mice), which do not express OspA, appear to up-regulate ospA mRNA transcription during infection of C3H-scid mice.
Transcription of ospA mRNA and seroconversion to OspA in infected C3H-scid mice adoptively reconstituted with lymphocytes from immunocompetent C3H mice. Transcription of ospA mRNA occurred in C3H-scid mice but not in C3H mice, suggesting that ospA mRNA transcription is influenced by innate or acquired host immune factors. Furthermore, although these results indicate that ospA mRNA transcription occurred, they do not necessarily prove that OspA translation and expression occurred. In an effort to prove that OspA protein expression occurred, C3H-scid mice were infected and then immunologically reconstituted and tested for seroconversion to OspA. Twenty C3H-scid mice were syringe inoculated with B. burgdorferi. At 2 weeks after inoculation, 10 C3H-scid mice were reconstituted by intraperitoneal inoculation with 2 x 107 splenocytes and 1 x 107 lymph node cells (total, 3 x 107 cells) from nave C3H mice. At 4 weeks after inoculation, five nonreconstituted C3H-scid mice were necropsied. At 6 weeks after inoculation, five nonreconstituted and five reconstituted (4 weeks after adoptive transfer) mice were necropsied, and at 8 weeks after inoculation, five reconstituted mice were necropsied (6 weeks after reconstitution). The infection status was confirmed by culture. Hearts, joints, and muscles were analyzed by PCR, and serum was tested for antibody. In this experiment, heart base was compared to ventricular muscle (inflamed versus adjacent noninflamed tissue), and the tibiotarsus was compared to quadriceps muscle (inflamed versus adjacent noninflamed tissue).
At necropsy, all mice from both groups (reconstituted and nonreconstituted) were culture and PCR positive. Carditis and arthritis were present in nonreconstituted C3H-scid mice at 4 and 6 weeks, and the arthritis was severe. Inflammatory lesions were present in the heart base but not in the ventricular muscle and were present in the tibiotarsus but not in the quadriceps muscle. Carditis and arthritis were less severe in reconstituted mice at 6 weeks and were regressing at 8 weeks (data not shown). Nonreconstituted C3H-scid mice were examined at 4 weeks to verify transcription of ospA mRNA at a time at which the reconstituted mice would be developing acquired immunity 2 weeks following adoptive transfer. Transcription of ospA mRNA was verified in one or more tissues from four of five mice (two of five heart bases; three of five tibiotarsi) (Fig. 4). Thus, it could be assumed that similarly inoculated but reconstituted C3H-scid mice were likely to immunologically recognize OspA, if it was translated into protein.
Comparing the spirochete loads for different treatment groups of mice at week 6 (Fig. 4) revealed significantly higher spirochete numbers (flaB DNA) in the heart base (P = 0.0007), ventricular muscle (P = 0.00027), and quadriceps muscle (P = 0.00001) of nonreconstituted mice than in the tissues of reconstituted mice. In contrast, reconstitution did not significantly reduce spirochete numbers in tibiotarsal joints (P = 0.37), despite resolving arthritis. Quantitative analysis of flaB mRNA revealed a pattern that followed the quantitative flaB DNA dynamics for both groups of mice and for all tissues. Transcription of ospA mRNA was detected in the heart base in two of five mice and in the tibiotarsus of three of five mice (four of five mice tested had ospA transcription in one or both sites) but not in the ventricular muscle or quadriceps muscle. None of the reconstituted mice had detectable ospA mRNA transcription in any of the tissues. These results confirmed that ospA mRNA transcription occurs in the absence of immunity. Furthermore, they support the observation that ospA mRNA transcription tends to occur in tissues with inflammation (heart base and tibiotarsus), in contrast to adjacent, noninflamed tissues (ventricular muscle and quadriceps muscle).
IgM and IgG were detected in all reconstituted mice at high titers (>1:25,000), confirming that there was successful reconstitution. Antibodies against B. burgdorferi lysates were detected in all reconstituted C3H-scid mice, with the highest titers in mice necropsied at 8 weeks (data not shown). When plates were coated with OspA and probed with sera from reconstituted C3H-scid mice, OspA antibodies were detected at a titer of 1:100 in two of five mice necropsied at 8 weeks (data not shown). Thus, detection of OspA antibodies in reconstituted C3H-scid mice confirmed that the low levels of ospA mRNA transcription in C3H-scid mice are accompanied by OspA translation, based upon seroconversion.
Effect of normal serum on ospA mRNA transcription in infected C3H-scid mice. Other workers (9) have shown that natural antibodies in nave mice interact with spirochetes in feeding ticks and influence OspA expression within the tick. Therefore, the effect of normal mouse serum on ospA mRNA transcription in infected C3H-scid mice was investigated. Twenty C3H-scid mice were syringe inoculated with B. burgdorferi. Ten of the mice were each injected intraperitoneally with 1 ml of normal (uninfected) C3H mouse serum at the time of inoculation and at 2 weeks, and the other 10 mice received no serum. At 4 weeks, mice were necropsied, tissues were cultured to verify infection, and tissues (heart base, ventricular muscle, tibiotarsus, and quadriceps muscle) were processed for B. burgdorferi DNA and RNA analysis.
All mice were culture positive. The numbers of spirochetes, based upon flaB DNA copy numbers, and the levels of flaB mRNA transcription in each tissue were equivalent in the two groups (serum treated and untreated) of C3H-scid mice (Fig. 5). Transcription of ospA mRNA was found in the heart base of 6 of 10 untreated mice, in the ventricular muscle of 2 of 10 untreated mice, and in the tibiotarsus of 4 of 10 untreated mice. Collectively, 7 of 10 untreated mice had ospA mRNA transcription in one or more tissues. No ospA mRNA transcription was detected in the quadriceps muscle from any of the untreated mice. In contrast, no ospA mRNA transcription was detected in the heart base, ventricular muscle, tibiotarsus, or quadriceps muscle of mice treated with normal mouse serum (10 mice had no ospA mRNA transcription in any tissue). Thus, these results suggest that components of serum from nave, immunocompetent mice influence ospA mRNA transcription in the host.
Effect of normal mouse serum and immunoglobulin fractions on ospA mRNA transcription in infected C3H-scid mice. Next, we attempted to localize the effector for down-regulation of ospA mRNA transcription to the immunoglobulin fraction of serum. Twenty C3H-scid mice were syringe inoculated with B. burgdorferi and divided into four equal groups of five mice. Group 1 received 1 ml of normal mouse serum at the time of inoculation and at 2 weeks. Group 2 received 1 ml of normal mouse serum that was depleted of immunoglobulin. Group 3 received 1 ml of the IgG fraction from normal mouse serum. Group 4 received 1 ml of the IgM fraction from normal mouse serum. At 4 weeks, mice were necropsied, tissues were cultured to verify infection, and the heart base, ventricular muscle, tibiotarsus, and quadriceps muscle were processed for B. burgdorferi DNA and RNA analysis.
The various treatments had no effect upon B. burgdorferi flaB DNA levels or flaB mRNA transcription. For the 20 mice and 20 tissue samples from each mouse, ospA mRNA transcription was found only in tissues from three mice (two of three heart base samples, one of three tibiotarsus samples) treated with serum that was depleted of immunoglobulin (Fig. 6). Furthermore, these results confirmed the effect of normal mouse serum and nonspecific immunoglobulins on ospA mRNA transcription.
DISCUSSION
The results of the current study reveal that ospA mRNA transcription takes place during infection of tissues in immunodeficient C3H-scid mice. The level of activity was low, and activity was not detectable in all mice or all samples. Collective analysis of the data, however, suggested that tissues with inflammation (heart base and tibiotarsus) favored ospA mRNA transcription. In contrast, noninflamed tissues (cardiac ventricular muscle and quadriceps muscle), which were consistently infected and contained spirochetes that were actively transcribing flaB, tended not to favor ospA mRNA transcription. These findings support observations of ospA mRNA transcription and OspA expression by spirochetes that were maintained in an inflammatory environment (12). In that study, spirochetes were maintained in dialysis tubing chambers implanted in the peritoneum of mice, which were then were subjected to intraperitoneal treatment with zymosan, a yeast cell extract that induces inflammation. Under such conditions, spirochetes increased transcription of ospA mRNA, as well as expressed increased amounts of OspA. In contrast to the current study, the spirochetes in the peritoneal chambers were culture derived and, even when host adapted, expressed OspA. Nevertheless, these two independent studies clearly demonstrate that OspA can be expressed in vivo and that an inflammatory environment favors ospA mRNA transcription and OspA expression by B. burgdorferi.
In other studies of B. burgdorferi infection of C3H-scid mice workers have not observed ospA mRNA transcription. Liang et al. (31) were unable to detect ospA mRNA transcription in C3H-scid mice using less sensitive microarray approaches. Crother et al. (11) were not able to detect OspA protein in detergent-phase tissue extracts from C3H-scid mice infected for 17 days with B. burgdorferi B31, but other proteins, including VlsE, OspC, and DbpA, were detectable. The inability to detect OspA was possibly due to the very small amounts of OspA in tissues, which the present data suggest, and possibly also due to the early time (17 days). Real-time PCR is a highly sensitive and quantitative means for detecting RNA transcripts in tissues of mice infected with B. burgdorferi, and it has recently been utilized to demonstrate dynamics of gene expression during infection in both immunocompetent and immunodeficient mice (24, 25, 32). Acquired immunity by the host has been shown to significantly reduce, but not eliminate, spirochetes from tissues, with commensurate reduction of transcription of several genes, including flaB, ospC, dbpA, arp (BBF01), vlsE, fbp (BBK32), oppA-2, and p37-42 (BBK47). The only absolute reduction in gene transcription in all tissues under these circumstances was the reduction in ospA transcription (24). These results were confirmed in a recent study that examined dbpA, ospC, BBF01, and vlsE mRNA transcription, but that study involved fewer tissues and ospA mRNA transcription was not examined (32).
It has been generally accepted that B. burgdorferi, which expresses abundant amounts of OspA in unfed ticks, rapidly down-regulates ospA mRNA transcription during tick feeding upon a host (16, 33, 48). The signals for this profound change in surface protein expression are not known, but a recent study has shown that natural antibody in nonimmune mice is a significant factor (9). Ninety percent of spirochetes in the salivary glands of ticks feeding upon B-cell-deficient mice were shown to express OspA, in contrast to only 5% of spirochetes from ticks feeding upon nave, immunocompetent mice. Furthermore, the spirochete numbers in the midguts of ticks were significantly reduced in ticks feeding upon immunocompetent mice compared to B-cell-deficient mice. Passive transfer of normal mouse serum into the B-cell-deficient mice was sufficient to induce these profound shifts in OspA expression. It has been known for some time that natural IgM interacts with OspA on spirochetes grown in vitro (18, 50). Furthermore, sera from several different species of animals, which were not exposed to B. burgdorferi, have been shown to contain natural antibodies that can kill spirochetes in vitro in the presence of complement (29). Although we did not observe reductions in spirochete numbers in infected C3H-scid mice that were passively treated with normal mouse serum, our results complement these other studies by demonstrating the role of natural antibodies as a signal for ospA mRNA transcription modulation in vivo and maintenance of ospA down-regulation during persistent infection of the host.
The down-regulation of ospA mRNA transcription during tick feeding is not absolute, as some spirochetes in the salivary glands of feeding ticks have been shown to express OspA (35, 46-48). In a recent study examining B. burgdorferi population kinetics and gene expression at the host-vector interface, it was shown that spirochetes which initially enter the host at the site of tick feeding transiently transcribe ospA mRNA at 24 h but rapidly down-regulate ospA mRNA transcription by 48 h and beyond in immunocompetent C3H mice (25). The current study confirmed this observation, using C3H-scid mice. Thus, transient OspA expression explains the finding of early antibody responses to OspA, which tend to rapidly decline in both experimental animals and humans (43, 45). Studies of C3H-scid mice infected with cultured (24), host-adapted, or tick-borne spirochetes (current study) indicate that ospA mRNA transcription continues in the host in the absence of immune competence, and the critical signaling factor is natural immunoglobulin.
Because of the relative paucity of spirochetes in tissues and the relatively low level of ospA mRNA transcription in infected mice, we attempted to prove OspA translation and expression indirectly with an adoptive transfer experiment. The results demonstrated that there was seroconversion of infected C3H-scid mice that were reconstituted with normal lymphocytes from nave, immunocompetent C3H mice. However, seroconversion to OspA took place in only two of five mice that were successfully reconstituted. All five mice readily seroconverted to B. burgdorferi. The variable results are likely due to the fact that ospA mRNA transcription was down-regulated during the early phases of reconstitution because of sensitivity to natural immunoglobulin, thereby reducing or eliminating the antigenic stimulus at a time when full immunocompetence was affected. Nevertheless, we established the fact that some of the mice seroconverted specifically to OspA, thereby proving that OspA translation occurred in vivo.
The regulatory effector for ospA transcription in vivo, at least in part, is immunoglobulin. Our study compartmentalized the effector to either IgM or IgG. Nonspecific immunoglobulins, possibly natural antibody, have been shown to significantly influence OspA expression in the tick. In the previous study, as in the current study, the IgM and IgG fractions of normal mouse serum were tested, but only IgM was found to be effective at modifying OspA expression in ticks. It was speculated that the lack of effect of IgG on OspA expression in ticks may have been due to IgG binding proteins in tick saliva (9). It remains to be determined if the effects of immunoglobulins on ospA mRNA transcription and OspA expression are actually due to natural antibodies that are specifically reactive with OspA or due to some other mechanism. If specificity is required, this finding may provide insight into why OspA may be expressed in some Lyme disease patients who seroconvert to OspA during the late stages of infection.
ACKNOWLEDGMENTS
This work was supported by PHS grant R01 AI26815 from the National Institute of Allergy and Infectious Diseases.
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