Profiling of cellular immune responses to Mycoplasma pulmonis infection in C57BL/6 and DBA/2 mice.

Mycoplasma infections cause respiratory tract damages and atypical pneumonia, resulting in serious problems in humans and animals worldwide. It is well known that laboratory inbred mouse strains show various susceptibility to Mycoplasma pulmonis (M. pulmonis) infection, which causes murine respiratory mycoplasmosis. In this study, we aimed to demonstrate the difference in cellular immune responses between resistant strain, C57BL/6NCrSlc (B6) and susceptible strain, DBA/2CrSlc (D2) after challenging M. pulmonis infection. D2 mice showed higher amount of bacterial proliferation in lung, higher pulmonary infiltration of immune cells such as neutrophils, macrophages, and lymphocytes, and higher levels of interleukin (IL)-1ß, IL-6, IL-17A, and tumor necrosis factor-α in bronchoalveolar lavage fluid than did B6 mice. The results of this study suggest that D2 mice are more susceptible than B6 mice to M. pulmonis infection due to a hyper-immune inflammatory response.

Interleukin-17A Exacerbates Disease Severity in BALB/c Mice Susceptible to Lung Infection with Mycoplasma pulmonis.

Mycoplasmas are atypical bacteria that disrupt the immune response to promote respiratory tract infections and secondary complications. However, not every immunologic response that protects or damages the host during mycoplasma infection is known. Interleukin-17A (IL-17A) is elevated in individuals infected with mycoplasmas, but how IL-17A and its cellular sources dictate disease outcome remains unclear. Here, IL-17A is hypothesized to worsen disease in individuals susceptible to mycoplasma infection. Thus, monoclonal anti-IL-17A antibodies were given to disease-susceptible BALB/c mice and disease-resistant C57BL/6 mice infected with Mycoplasma pulmonis Neutralizing the function of IL-17A using anti-IL-17A antibodies reduced disease severity during M. pulmonis infection in BALB/c, but not C57BL/6, mice. Neutralizing IL-17A also reduced the incidence of neutrophilic lung lesions during infection in BALB/c mice. Reduced pathology occurred without impacting the bacterial burden, demonstrating that IL-17A is not required for mycoplasma clearance. The main source of IL-17A throughout infection in BALB/c mice was CD4+ T cells, and neutralizing IL-17A after infiltration of the lungs by T cells reduced disease severity, identifying the Th17 response as a herald of late mycoplasma pathology in susceptible mice. Neutralizing IL-17A did not further reduce disease during M. pulmonis infection in BALB/c mice depleted of neutrophils, suggesting that IL-17A requires the presence of pulmonary neutrophils to worsen respiratory pathology. IL-17A is a pathological element of murine respiratory mycoplasma infection. Using monoclonal antibodies to neutralize IL-17A could reduce disease severity during mycoplasma infection in humans and domesticated animals.

Respiratory Pathology and Pathogens in Wild Urban Rats (Rattus norvegicus and Rattus rattus).

Norway (Rattus norvegicus) and black rats (Rattus rattus) are common peridomestic species, yet little is known about wild rat ecology, including their natural diseases. We describe gross and histological lesions in the respiratory tract of a sample of 711 wild urban rats. A subset was examined for 19 distinct categories of histological lesions in the respiratory tract. Testing for known respiratory pathogens included serology and polymerase chain reaction (PCR) of lung samples. Grossly evident lesions were rare (8/711; 1%). Upper respiratory tract inflammation was present in 93 of 107 (87%) rats and included rhinitis, submucosal and periglandular lymphoplasmacytic tracheitis, and/or tracheal intraluminal necrotic debris and was significantly associated (P < .05) with the presence of cilia-associated respiratory bacillus (CARB), Mycoplasma pulmonis, and increased body mass (odds ratio [OR] = 1.09; 95% confidence interval [CI] = 1.05-1.14 per 10 g). Within the lungs, peribronchiolar and/or perivascular lymphoplasmacytic cuffs were present in 152 of 199 rats (76%) and were also significantly associated (P ≤ .02) with CARB, M. pulmonis, and increased body mass (OR = 1.20; 95% CI = 1.14-1.27 per 10 g). Rats were frequently coinfected with M. pulmonis and CARB, and lesions associated with these pathogens were histologically indistinguishable. Pneumocystis sp was detected in 48 of 102 (47%) rats using PCR but was not significantly associated with lesions. This description of pathology in the respiratory system of wild rats demonstrates that respiratory disease is common. Although the impact of these lesions on individual and population health remains to be investigated, respiratory disease may be an important contributor to wild rat morbidity and mortality.

Sero-Prevalence of Rodent Pathogens in India.

Health monitoring is an integral part of laboratory animal quality standards. However, current or past prevalence data as well as regulatory requirements dictate the frequency, type and the expanse of health monitoring. In an effort to understand the prevalence of rodent pathogens in India, a preliminary study was carried out by sero-epidemiology. Sera samples obtained from 26 public and private animal facilities were analyzed for the presence of antibodies against minute virus of mice (MVM), ectromelia virus (ECTV), lymphocytic choriomeningitis virus (LCMV), mouse hepatitis virus (MHV), Sendai virus (SeV), and Mycoplasma pulmonis in mice, and SeV, rat parvo virus (RPV), Kilham's rat virus (KRV) and sialodacryoadenitis virus (SDAV) in rats, by sandwich ELISA. It was observed that MHV was the most prevalent agent followed by Mycoplasma pulmonis and MVM in mice, and SDAV followed by RPV were prevalent in rats. On the other hand, none of the samples were positive for ECTV in mice, or SeV or KRV in rats. Multiple infections were common in both mice and rats. The incidence of MHV and Mycoplasma pulmonis was higher in facilities maintained by public organizations than in vivaria of private organizations, although the difference was not statistically different. On the other hand the prevalence of rodent pathogens was significantly higher in the northern part of India than in the South. These studies form the groundwork for detailed sero-prevalence studies which should further lay the foundations for country-specific guidelines for health monitoring of laboratory animals.

Prior infection exacerbates postoperative cognitive dysfunction in aged rats.

Older patients may experience persisting postoperative cognitive dysfunction (POCD), which is considered to largely depend on surgery-induced (neuro)inflammation. We hypothesize that inflammatory events before surgery could predispose patients to POCD. When part of our aged rats developed Mycoplasma pulmonis, this presented the unique opportunity to investigate whether a pulmonary infection before surgery influences surgery-induced neuroinflammation and POCD. Male 18-mo-old Wistar rats that had recovered from an active mycoplasma infection (infection) and control rats (healthy) were subjected to abdominal surgery and jugular vein catheterization under general anesthesia (surgery) or remained naïve (control). In postoperative week 2, behavioral tests were performed to assess cognitive performance and exploratory behavior. The acute systemic inflammatory response was investigated by measuring plasma IL-6 and IL-12. In the hippocampus, prefrontal cortex and striatum, microglial activity, neurogenesis, and concentrations of IL-6, IL-12, IL1B, and brain-derived neurotropic factor on postoperative day 14 were determined. Rats still showed signs of increased neuroinflammatory activity, as well as cognitive and behavioral changes, 3 wk after the symptoms of infection had subsided. Rats that had experienced infection before surgery exhibited a more generalized and exacerbated postoperative cognitive impairment compared with healthy surgery rats, as well as a prolonged increase in systemic cytokine levels and increased microglial activation in the hippocampus and prefrontal cortex. These findings support the hypothesis that an infection before surgery under general anesthesia exacerbates POCD. Future studies are necessary to determine whether the found effects are aging specific and to investigate the magnitude and time course of this effect in a controlled manner.

Antigen-pulsed bone marrow-derived and pulmonary dendritic cells promote Th2 cell responses and immunopathology in lungs during the pathogenesis of murine Mycoplasma pneumonia.

Mycoplasmas are a common cause of pneumonia in humans and animals, and attempts to create vaccines have not only failed to generate protective host responses, but they have exacerbated the disease. Mycoplasma pulmonis causes a chronic inflammatory lung disease resulting from a persistent infection, similar to other mycoplasma respiratory diseases. Using this model, Th1 subsets promote resistance to mycoplasma disease and infection, whereas Th2 responses contribute to immunopathology. The purpose of the present study was to evaluate the capacity of cytokine-differentiated dendritic cell (DC) populations to influence the generation of protective and/or pathologic immune responses during M. pulmonis respiratory disease in BALB/c mice. We hypothesized that intratracheal inoculation of mycoplasma Ag-pulsed bone marrow-derived DCs could result in the generation of protective T cell responses during mycoplasma infection. However, intratracheal inoculation (priming) of mice with Ag-pulsed DCs resulted in enhanced pathology in the recipient mice when challenged with mycoplasma. Inoculation of immunodeficient SCID mice with Ag-pulsed DCs demonstrated that this effect was dependent on lymphocyte responses. Similar results were observed when mice were primed with Ag-pulsed pulmonary, but not splenic, DCs. Lymphocytes generated in uninfected mice after the transfer of either Ag-pulsed bone marrow-derived DCs or pulmonary DCs were shown to be IL-13(+) Th2 cells, known to be associated with immunopathology. Thus, resident pulmonary DCs most likely promote the development of immunopathology in mycoplasma disease through the generation of mycoplasma-specific Th2 responses. Vaccination strategies that disrupt or bypass this process could potentially result in a more effective vaccination.

EPS-I polysaccharide protects Mycoplasma pulmonis from phagocytosis.

Few mycoplasmal polysaccharides have been described and little is known about their role in pathogenesis. The infection of mice with Mycoplasma pulmonis has been utilized in many in vivo and in vitro studies to gain a better understanding of host-pathogen interactions during chronic respiratory infection. Although alveolar macrophages have a primary role in host defence, M. pulmonis is killed inefficiently in vitro. One antiphagocytic factor produced by the mycoplasma is the family of phase- and size-variable Vsa lipoproteins. However, bacteria generally employ multiple strategies for combating host defences, with capsular polysaccharide often having a key role. We show here that mutants lacking the EPS-I polysaccharide of M. pulmonis exhibit increased susceptibility to binding and subsequent killing by alveolar macrophages. These results give further insight into how mycoplasmas are able to avoid the host immune system and sustain a chronic infection.

Mycoplasma polysaccharide protects against complement.

Although they lack a cell wall, mycoplasmas do possess a glycocalyx. The interactions between the glycocalyx, mycoplasmal surface proteins and host complement were explored using the murine pathogen Mycoplasma pulmonis as a model. It was previously shown that the length of the tandem repeat region of the surface lipoprotein Vsa is associated with susceptibility to complement-mediated killing. Cells producing a long Vsa containing about 40 repeats are resistant to complement, whereas strains that produce a short Vsa of five or fewer repeats are susceptible. We show here that the length of the Vsa protein modulates the affinity of the M. pulmonis EPS-I polysaccharide for the mycoplasma cell surface, with more EPS-I being associated with mycoplasmas producing a short Vsa protein. An examination of mutants that lack EPS-I revealed that planktonic mycoplasmas were highly susceptible to complement killing even when the Vsa protein was long, demonstrating that both EPS-I and Vsa length contribute to resistance. In contrast, the mycoplasmas were resistant to complement even in the absence of EPS-I when the cells were encased in a biofilm.

Effect of experimental genital mycoplasmosis on gene expression in the fetal brain.

Neurodevelopmental disorders may have their origins during intrauterine development. We used a well-defined animal model to test whether hematogenous infection with genital mycoplasma would alter the expression of genes associated with autism spectrum disorders (ASD). In a preliminary experiment, rats were exposed at 14 days gestation (GD14) to Mycoplasma pulmonis or sterile broth and sacrificed at GD18. Infection and inflammation status of the pups was ascertained by culture and cytokine ELISA. Intra-cardiac injection of 10(6)CFU M. pulmonis resulted in amniotic infection of 100% of the pups and was accompanied by higher levels of IL-1ß in amniotic fluids. In a second experiment, animals were infected in a similar manner but dams and their litters were sacrificed at GD18, GD21 or postpartum day 3 (PPD3). Expression of proinflammatory cytokines and neurodevelopmental genes in the fetal brains was evaluated. M. pulmonis infection significantly increased the expression of IL-1ß, TNF-α and COX-2 in fetal and neonatal brains. Expression of GFAP and CD11b, markers for activation on astrocytes and microglial cells, respectively, was also increased for infected animals. M. pulmonis significantly increased SHANK-3 gene expression at GD21 and PPD3 and PCP-2 expression at GD21. No effect of M. pulmonis infection on Reelin, PTEN, BDNF or HGF was detected. These data suggest that M. pulmonis infection at GD14 increases the expression of proinflammatory genes in the perinatal brain. Further studies with earlier time-points of infection and ones that use behavioral outcomes are needed to better understand the potential role of genital mycoplasmosis on pychopathology.

Indirect ELISA and indirect immunofluorescent antibody assay for detecting the antibody against murine norovirus S7 in mice.

To evaluate murine norovirus (MNV) infection in laboratory mice, we attempted to develop an enzyme-linked immunosorbent assay (ELISA) system and an indirect immunofluorescent antibody (IFA) assay for detecting the anti-MNV-S7 antibody in mice. MNV-S7, which was isolated in Japan, was used in both assays. The antigen for ELISA was prepared by ultracentrifugation of culture supernatants of RAW 264 cells infected with MNV-S7. Positive sera were obtained from 6-week-old, female C57BL/6JJcl mice inoculated orally with MNV-S7. IFA against infected RAW 264 cells was able to discriminate positive sera from negative sera. Indirect ELISA was performed using 96-well ELISA plates coated with formalin-treated MNV-S7 antigen. In this ELISA system, mouse sera obtained 2 weeks after infection or later showed significantly high OD values and were judged positive. An equal level of anti-MNV-S7 antibody response was observed in BALB/cAJcl, C57BL/6JJcl, DBA/2JJcl, and Jcl:ICR mice; whereas, C3H/HeJJcl mice demonstrated slightly lower antibody production 4 weeks after infection. We also used this ELISA system to evaluate 77 murine serum samples obtained from 15 conventional mouse rooms in research facilities in Japan and found that approximately half of the serum samples contained antibody to MNV-S7. We found that some serum samples were negative for antibodies to mouse hepatitis virus and Mycoplasma pulmonis but positive for antibody to MNV-S7. The results suggest that the MNV infection is more prevalent than other infections such as mouse hepatitis virus and Mycoplasma pulmonis in conventional mouse colonies in Japan, as is the case in other areas of the world.

NK cells interfere with the generation of resistance against mycoplasma respiratory infection following nasal-pulmonary immunization.

The purpose of the present study was to determine the impact of NK cells on the development of protective adaptive immunity in response to nasal-pulmonary immunization against mycoplasma. Depletion of NK cells before nasal-pulmonary immunization enhanced resistance to mycoplasma respiratory infection. The effect of NK cells on the generation of protective immunity in lungs was dependent on lymphoid cells, as immunization of either SCID mice or immunocompetent mice depleted of CD4(+) T cells did not demonstrate any increased resistance in the presence or absence of NK cells. The presence of NK cells at the time of nasal-pulmonary immunization modulated mycoplasma-specific cytokine responses in lungs and lower respiratory nodes. In particular, NK cells skewed the mycoplasma-specific T cell cytokine responses in the draining lymph nodes to higher IL-4, IL-13, and IL-17 while lowering IFN-gamma responses. Adoptive transfer of total lung lymphocytes isolated from immunized mice into naive mice led to a significant reduction in the mycoplasma numbers in lungs, and the resistance was greater if cells were obtained from immunized mice that were depleted of NK cells. Similar results were obtained if purified B cells, T cells, or CD4(+) T cells were used. Interestingly, this is the first time that a favorable role of functional CD4(+) T cells in mediating protection in mycoplasma respiratory disease was demonstrated. Thus, NK cells can influence the responses of multiple lymphocyte populations capable of mediating resistance to mycoplasma infection.

A novel IL-17-dependent mechanism of cross protection: respiratory infection with mycoplasma protects against a secondary listeria infection.

Immune responses to pathogens occur within the context of current and previous infections. Cross protection refers to the phenomena where infection with a particular pathogen provides enhanced resistance to a subsequent unrelated pathogen in an antigen-independent manner. Proposed mechanisms of antigen-independent cross protection have involved the secretion of IFN-gamma, which activates macrophages, thus providing enhanced innate immunity against the secondary viral or bacterial pathogen. Here we provide evidence that a primary infection with the chronic respiratory pathogen, Mycoplasma pulmonis, provides a novel form of cross protection against a secondary infection with Listeria monocytogenes that is not mediated by IFN-gamma, but instead relies upon IL-17 and mobilization of neutrophils. Mice infected with M. pulmonis have enhanced clearance of L. monocytogenes from the spleen and liver, which is associated with increased numbers of Gr-1(+)CD11b(+) cells and higher levels of IL-17. This enhanced clearance of L. monocytogenes was absent in mice depleted of Gr-1(+) cells or in mice deficient in the IL-17 receptor. Additionally, both the IL-17 receptor and neutrophils were essential for optimal clearance of M. pulmonis. Thus, a natural component of the immune response directed against M. pulmonis was able to enhance clearance of L. monocytogenes.

Risk factors associated with irreversible airflow limitation in asthma.

PURPOSE OF REVIEW: Irreversible airflow limitation develops in some patients with asthma and is related to poorer prognosis. This paper reviews recent literature on natural course, risk factors, and potential mechanisms of persistent airflow limitation in asthma. RECENT FINDINGS: The natural course of persistent airflow limitation in asthma is poorly known, but reduced lung function at disease onset and an increased rate of decline during adult life contribute to its development. Risk factors for progressive irreversible airway obstruction in asthma include adult onset, frequent exacerbations, smoking, occupational exposure, ongoing eosinophilic airway inflammation and airway hyperresponsiveness. Polymorphisms of the ADAM33 gene predict excess decline in lung function, in asthma as well as at population level. It is still not clear how different components of airway remodeling affect lung function in asthma. Airway epithelium and airway smooth muscle seem to be highly important, but the interrelationship between persistent airflow limitation, airway inflammation, remodeling and airway hyperresponsiveness has not been clearly defined. SUMMARY: Whereas several factors have been implicated as being important in the development of fixed airway obstruction in asthma, we are just beginning to explore the different components of airway remodeling and their relevance, deleterious or beneficial, to clinical outcome.

Biofilms protect Mycoplasma pulmonis cells from lytic effects of complement and gramicidin.

The length of the tandem repeat region of the Vsa protein of Mycoplasma pulmonis has previously been shown to modulate the susceptibility of mycoplasmas to killing by complement: cells that produce a short form of the Vsa protein are highly sensitive, and cells producing the long Vsa protein are resistant. In contrast to their differing susceptibilities to complement, the mycoplasmas were highly sensitive to gramicidin irrespective of the length of the Vsa protein produced. We show here that when encased within a biofilm, cells of M. pulmonis producing a short form of the Vsa protein were more resistant to complement and gramicidin than mycoplasmas that were dispersed. The resistance appeared to be localized to those mycoplasmas within tower structures of the biofilms. Biofilm formation may be a mechanism that protects mycoplasmas from host immunity.

T-bet deficiency facilitates airway colonization by Mycoplasma pulmonis in a murine model of asthma.

Epidemiological and clinical evidence suggest a correlation between asthma and infection with atypical bacterial respiratory pathogens. However, the cellular and molecular underpinnings of this correlation remain unclear. Using the T-bet-deficient (T-bet(-/-)) murine model of asthma and the natural murine pathogen Mycoplasma pulmonis, we provide a mechanistic explanation for this correlation. In this study, we demonstrate the capacity of asthmatic airways to facilitate colonization by M. pulmonis and the capacity of M. pulmonis to exacerbate symptoms associated with acute and chronic asthma. This mutual synergism results from an inability of T-bet(-/-) mice to mount an effective immune defense against respiratory infection through release of IFN-gamma and the ability of M. pulmonis to trigger the production of Th2-type cytokines (e.g., IL-4 and IL-5), and Abs (e.g., IgG1, IgE, and IgA), eosinophilia, airway remodeling, and hyperresponsiveness; all pathophysiological hallmarks of asthma. The capacity of respiratory pathogens such as Mycoplasma spp. to dramatically augment the pathological changes associated with asthma likely explains their association with acute asthmatic episodes in juvenile patients and with adult chronic asthmatics, >50% of whom are found to be PCR positive for M. pneumoniae. In conclusion, our study demonstrates that in mice genetically predisposed to asthma, M. pulmonis infection elicits an inflammatory milieu in the lungs that skews the immune response toward the Th2-type, thus exacerbating the pathophysiological changes associated with asthma. For its part, airways exhibiting an asthmatic phenotype provide a fertile environment that promotes colonization by Mycoplasma spp. and one which is ill-equipped to kill and clear respiratory pathogens.

Mast cells protect mice from Mycoplasma pneumonia.

RATIONALE: As the smallest free-living bacteria and a frequent cause of respiratory infections, mycoplasmas are unique pathogens. Mice infected with Mycoplasma pulmonis can develop localized, life-long airway infection accompanied by persistent inflammation and remodeling. OBJECTIVE: Because mast cells protect mice from acute septic peritonitis and gram-negative pneumonia, we hypothesized that they defend against mycoplasma infection. This study tests this hypothesis using mast cell-deficient mice. METHODS: Responses to airway infection with M. pulmonis were compared in wild-type and mast cell-deficient Kit(W-sh)/Kit(W-sh) mice and sham-infected control mice. MEASUREMENTS AND MAIN RESULTS: Endpoints include mortality, body and lymph node weight, mycoplasma antibody titer, and lung mycoplasma burden and histopathology at intervals after infection. The results reveal that infected Kit(W-sh)/Kit(W-sh) mice, compared with other groups, lose more weight and are more likely to die. Live mycoplasma burden is greater in Kit(W-sh)/Kit(W-sh) than in wild-type mice at early time points. Four days after infection, the difference is 162-fold. Titers of mycoplasma-specific IgM and IgA appear earlier and rise higher in Kit(W-sh)/Kit(W-sh) mice, but antibody responses to heat-killed mycoplasma are not different compared with wild-type mice. Infected Kit(W-sh)/Kit(W-sh) mice develop larger bronchial lymph nodes and progressive pneumonia and airway occlusion with neutrophil-rich exudates, accompanied by angiogenesis and lymphangiogenesis. In wild-type mice, pneumonia and exudates are less severe, quicker to resolve, and are not associated with increased angiogenesis. CONCLUSIONS: These findings suggest that mast cells are important for innate immune containment of and recovery from respiratory mycoplasma infection.

Immune complex-dependent remodeling of the airway vasculature in response to a chronic bacterial infection.

Chronic inflammation in the airways is associated with dramatic architectural changes in the walls of the airways and in the vasculature they contain. In this study, we show that the adaptive immune system is essential for airway remodeling that occurs in mice that are chronically infected with the respiratory pathogen Mycoplasma pulmonis. Angiogenesis, lymphangiogenesis, and epithelial remodeling were greatly reduced in mice that lacked B cells. Substantiating a role for Ab and airway immune complexes, we found that the transfer of immune serum to B cell-deficient mice could reconstitute pathogen-induced angiogenesis. Inflammatory cells recruited to the infected airways were activated by the humoral response, and this activation correlated with the induction of genes for remodeling factors such as vascular endothelial growth factor-D. The results reveal a novel pathway whereby T cell-dependent humoral immunity to a persistent airway infection can induce inflammation-dependent angiogenesis, lymphangiogenesis, and chronic airway pathology.

Avoidance of the host immune system through phase variation in Mycoplasma pulmonis.

Phase-variable lipoproteins are commonly found in Mycoplasma species. Mycoplasma pulmonis contains a family of extensively studied phase- and size-variable lipoproteins encoded by the vsa locus. The Vsa surface proteins vary at a high frequency, the in vivo significance of which has yet to be determined. We investigated the role of Vsa phase variation in respect to tissue tropism and avoidance of the immune system in the mouse host. Mycoplasmas were cultured 3, 14, and 21 days postinoculation from the nose, lung, trachea, liver, and spleen of experimentally infected C57BL/6 (wild-type), C57BL/6-RAG-1-/- (RAG-/-), and C57BL/6-inducible nitric oxide synthase (iNOS)-/- (iNOS-/-) mice. In wild-type and iNOS-/- mice, a large number of Vsa variants were seen by 21 days postinoculation. In contrast, little Vsa variation occurred in all tissues of RAG-/- mice. Analysis of isolates from 14 days postinoculation revealed less variation of the Vsa proteins in iNOS-/- mice than in the wild type. Western blot analysis of isolates from each strain of mouse demonstrated that Vsa phase variation occurred independently of size variation, indicating no obvious selection pressure for size variants. Additionally, these experiments provided no evidence that mycoplasmas producing particular Vsa proteins adhered only to specific tissues. The data strongly indicate that Vsa phase variation is a mechanism for avoidance of the immune system with no obvious contribution to tissue tropism.

Experimental genital mycoplasmosis causes increased levels of mRNA for IL-6 and TNF-alpha in the placenta.

PROBLEM: Previous studies with animal models have shown that injection of lipopolysaccharide (LPS) results in fetal loss and increases production of proinflammatory cytokines at the maternal-fetal interface. Most intrauterine infections, however, are associated with Ureaplasma urealyticum, a microorganism that lacks a cell wall and therefore does not contain LPS. Previous work in our laboratory with an animal model for genital infection with a similar organism, Mycoplasma pulmonis, revealed that widespread infection in maternal and fetal tissues can be experimentally induced with minimal manipulation of the animal. For this project, we tested the hypothesis that administration of the organism by a hematogenous route at gestational day (gd) 14 would result in increased tumor necrosis factor (TNF)-alpha and interleukin (IL)-6 production by the placenta. STUDY DESIGN: Timed-pregnant, Sprague-Dawley rats were anesthetized on gd 14 and 10(7) CFU of M. pulmonis strain X1048 or an equivalent volume of sterile medium was injected into the heart. Rats were necropsied on gd 18 or 21, and ex vivo production of TNF-alpha and IL-6 was evaluated from six randomly selected placentas from each litter. The remaining placentas were harvested and either snap-frozen or placed in formalin. Frozen placentas were processed for real-time reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of TNF-alpha and IL-6 mRNA. Formalin-fixed placentas were sectioned and stained with hematoxylin and eosin for lesion analysis. RESULTS: Concentrations of TNF-alpha but not IL-6 were significantly higher in conditioned medium from placentas harvested from infected dams at gd 21. Levels of mRNA for IL-6 and TNF-alpha, however, were increased by M. pulmonis at gd 18 and 21. Analysis of gd 21 placentas by light microscopy revealed that significant histological chorioamnionitis was present in infected animals with accumulations of neutrophils in the capsular decidua. CONCLUSION: These data indicate that experimental infection with M. pulmonis causes histological chorioamnionitis, elevated mRNA levels of TNF-alpha and IL-6 in placental tissues, and the secretion of TNF-alpha by the placenta during late gestation.

Resistance of Mycoplasma pulmonis to complement lysis is dependent on the number of Vsa tandem repeats: shield hypothesis.

The Vsa proteins are associated with the virulence of the murine respiratory pathogen Mycoplasma pulmonis. The antigens consist of a conserved N-terminal region that is combined with one of several different variable C-terminal regions comprised of tandem repeats. M. pulmonis strains that produce VsaA with about 40 tandem repeats do not adhere to polystyrene or erythrocytes and are highly resistant to complement killing. Strains that produce VsaA with three tandem repeats adhere strongly to polystyrene and erythrocytes and are highly susceptible to complement killing. We report here that the resistance to complement lysis was not due to a lack of activation of the complement cascade. Isolation and analysis of M. pulmonis strains that produced Vsa proteins other than VsaA (VsaG and VsaI) with either long or short repeat regions indicated that adherence to polystyrene and resistance to complement were dependent on the length of the repeat region but not on the Vsa type. Furthermore, M. pulmonis Vsa variants were susceptible to the polypeptide pore-forming molecule gramicidin D, independent of the Vsa type and length. Collectively, the data indicate the Vsa proteins nonspecifically mediate M. pulmonis surface interactions and function to sterically hinder access of complement to the mycoplasma cell membrane while permitting access of smaller molecules.