Prenatal infection with Mycoplasma pulmonis in rats exaggerates the angiotensin II pressor response in adult offspring.

Exposure to different stressors in utero is linked to adult diseases such as obesity and hypertension. In this study, the impact of prenatal infection (PNI) on adult body weight and cardiovascular function was evaluated using a naturally occurring rodent pathogen, Mycoplasma pulmonis (MP). Pregnant Sprague-Dawley rats were infected with MP on gestationalday 14 and gave birth naturally. Adult PNI offspring weighed more than controls, but resting mean arterial pressure (MAP) was unchanged. Subcutaneous injection of angiotensin II (10 µg/kg) elicited a rise in MAP that was greater in both male and female PNI offspring compared with controls (P < 0.03). The accompanying reflex bradycardia was similar to the controls, suggesting that PNI induced baroreflex dysfunction. Subcutaneous nicotine administration, a potent cardiorespiratory stimulus, also elicited a transient rise in MAP that was generally greater in the PNI group, but the change in MAP from baseline was only significant in the PNI females compared with controls (P < 0.03). Elevated body weight and cardiovascular reactivity in the PNI offspring was associated with an increase in the ratio of hypothalamic corticotrophin-releasing hormone receptors type 1 to type 2 gene expression in both sexes compared with controls. These findings support previous studies demonstrating that PNI induces alterations in cardiovascular function and body weight. Yet, unlike previous studies utilizing other models of PNI (e.g., endotoxin), MP PNI did not induce resting hypertension. Thus, our study provides a foundation for future studies evaluating the cardiovascular risks of offspring exposed to microbial challenges in utero.

Decreased Expression of Vascular Endothelial Growth Factor Receptor 1 Contributes to the Pathogenesis of Hereditary Hemorrhagic Telangiectasia Type 2.

BACKGROUND: Hereditary Hemorrhagic Telangiectasia type 2 (HHT2) is an inherited genetic disorder characterized by vascular malformations and hemorrhage. HHT2 results from ACVRL1 haploinsufficiency, the remaining wild-type allele being unable to contribute sufficient protein to sustain endothelial cell function. Blood vessels function normally but are prone to respond to angiogenic stimuli, leading to the development of telangiectasic lesions that can bleed. How ACVRL1 haploinsufficiency leads to pathological angiogenesis is unknown. METHODS: We took advantage of Acvrl1+/- mutant mice that exhibit HHT2 vascular lesions and focused on the neonatal retina and the airway system after Mycoplasma pulmonis infection, as physiological and pathological models of angiogenesis, respectively. We elucidated underlying disease mechanisms in vitro by generating Acvrl1+/- mouse embryonic stem cell lines that underwent sprouting angiogenesis and performed genetic complementation experiments. Finally, HHT2 plasma samples and skin biopsies were analyzed to determine whether the mechanisms evident in mice are conserved in humans. RESULTS: Acvrl1+/- retinas at postnatal day 7 showed excessive angiogenesis and numerous endothelial "tip cells" at the vascular front that displayed migratory defects. Vascular endothelial growth factor receptor 1 (VEGFR1; Flt-1) levels were reduced in Acvrl1+/- mice and HHT2 patients, suggesting similar mechanisms in humans. In sprouting angiogenesis, VEGFR1 is expressed in stalk cells to inhibit VEGFR2 (Flk-1, KDR) signaling and thus limit tip cell formation. Soluble VEGFR1 (sVEGFR1) is also secreted, creating a VEGF gradient that promotes orientated sprout migration. Acvrl1+/- embryonic stem cell lines recapitulated the vascular anomalies in Acvrl1+/- (HHT2) mice. Genetic insertion of either the membrane or soluble form of VEGFR1 into the ROSA26 locus of Acvrl1+/- embryonic stem cell lines prevented the vascular anomalies, suggesting that high VEGFR2 activity in Acvrl1+/- endothelial cells induces HHT2 vascular anomalies. To confirm our hypothesis, Acvrl1+/- mice were infected by Mycoplasma pulmonis to induce sustained airway inflammation. Infected Acvrl1+/- tracheas showed excessive angiogenesis with the formation of multiple telangiectases, vascular defects that were prevented by VEGFR2 blocking antibodies. CONCLUSIONS: Our findings demonstrate a key role of VEGFR1 in HHT2 pathogenesis and provide mechanisms explaining why HHT2 blood vessels respond abnormally to angiogenic signals. This supports the case for using anti-VEGF therapy in HHT2.

Autoimmune-Disease-Prone NOD Mice Help To Reveal a New Genetic Locus for Reducing Pulmonary Disease Caused by Mycoplasma pulmonis.

Mycoplasmas are bacterial pathogens of a range of animals, including humans, and are a common cause of respiratory disease. However, the host genetic factors that affect resistance to infection or regulate the resulting pulmonary inflammation are not well defined. We and others have previously demonstrated that nonobese diabetic (NOD) mice can be used to investigate disease loci that affect bacterial infection and autoimmune diabetes. Here we show that NOD mice are more susceptible than C57BL/6 (B6) mice to infection with Mycoplasma pulmonis, a natural model of pulmonary mycoplasmosis. The lungs of infected NOD mice had higher loads of M. pulmonis and more severe inflammatory lesions. Moreover, congenic NOD mice that harbored different B6-derived chromosomal intervals enabled identification and localization of a new mycoplasmosis locus, termed Mpr2, on chromosome 13. These congenic NOD mice demonstrated that the B6 allele for Mpr2 reduced the severity of pulmonary inflammation caused by infection with M. pulmonis and that this was associated with altered cytokine and chemokine concentrations in the infected lungs. Mpr2 also colocalizes to the same genomic interval as Listr2 and Idd14, genetic loci linked to listeriosis resistance and autoimmune diabetes susceptibility, respectively, suggesting that allelic variation within these loci may affect the development of both infectious and autoimmune disease.

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.

Impact of gestational nicotine exposure on intrauterine and fetal infection in a rodent model.

We investigated the interaction between prenatal nicotine exposure and intrauterine infection using established rat models. Beginning at gestation day (GD) 6, dams were continuously infused with either saline or 6 mg/kg/day nicotine (Nic). At GD 14, dams received either sterile broth or 105 colony-forming units Mycoplasma pulmonis (MP), resulting in four treatment groups: control (4 dams, 33 fetal units); MP only (5 dams, 55 fetal units); Nic only (5 dams, 61 fetal units), and Nic + MP (7 dams, 82 fetal units). At GD 18, nicotine exposure significantly increased (P ≤ 0.02) the percentage of amniotic fluids and fetuses infected by MP but did not impact colonization rates of maternal sites. Nicotine exposure significantly reduced the numbers of MP in the placenta required for high microbial loads (≥104 color-changing units) in the amniotic fluid (P < 0.01). Fetal inflammatory response lesions were most extensive in the Nic only and Nic + MP groups (P < 0.0001). Control and MP only placentas were interleukin (IL)10-dominant, consistent with an M2/Th2 environment. Placentas exposed to nicotine shifted to a neutral environment, with equivalent levels of interferon gamma (IFNG) and IL10. Both IL6 and tumor necrosis factor (TNF) levels in amniotic fluid were highly elevated when both nicotine and infection were present. Our study suggests that prenatal exposure to nicotine increases the risk for intrauterine infection, lowers the infectious dose required to breach the placental barrier and infect the amniotic fluid and fetus, and alters the pathology and inflammatory profile associated with maternal and fetal sites.

Synergistic actions of blocking angiopoietin-2 and tumor necrosis factor-α in suppressing remodeling of blood vessels and lymphatics in airway inflammation.

Remodeling of blood vessels and lymphatics are prominent features of sustained inflammation. Angiopoietin-2 (Ang2)/Tie2 receptor signaling and tumor necrosis factor-α (TNF)/TNF receptor signaling are known to contribute to these changes in airway inflammation after Mycoplasma pulmonis infection in mice. We determined whether Ang2 and TNF are both essential for the remodeling on blood vessels and lymphatics, and thereby influence the actions of one another. Their respective contributions to the initial stage of vascular remodeling and sprouting lymphangiogenesis were examined by comparing the effects of function-blocking antibodies to Ang2 or TNF, given individually or together during the first week after infection. As indices of efficacy, vascular enlargement, endothelial leakiness, venular marker expression, pericyte changes, and lymphatic vessel sprouting were assessed. Inhibition of Ang2 or TNF alone reduced the remodeling of blood vessels and lymphatics, but inhibition of both together completely prevented these changes. Genome-wide analysis of changes in gene expression revealed synergistic actions of the antibody combination over a broad range of genes and signaling pathways involved in inflammatory responses. These findings demonstrate that Ang2 and TNF are essential and synergistic drivers of remodeling of blood vessels and lymphatics during the initial stage of inflammation after infection. Inhibition of Ang2 and TNF together results in widespread suppression of the inflammatory response.

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.

Preferential lymphatic growth in bronchus-associated lymphoid tissue in sustained lung inflammation.

Lymphatics proliferate, become enlarged, or regress in multiple inflammatory lung diseases in humans. Lymphatic growth and remodeling is known to occur in the mouse trachea in sustained inflammation, but whether intrapulmonary lymphatics exhibit similar plasticity is unknown. We examined the time course, distribution, and dependence on vascular endothelial growth factor receptor (VEGFR)-2/VEGFR-3 signaling of lung lymphatics in sustained inflammation. Lymphatics in mouse lungs were examined under baseline conditions and 3 to 28 days after Mycoplasma pulmonis infection, using prospero heomeobox 1-enhanced green fluorescence protein and VEGFR-3 as markers. Sprouting lymphangiogenesis was evident at 7 days. Lymphatic growth was restricted to regions of bronchus-associated lymphoid tissue (BALT), where VEGF-C-producing cells were scattered in T-cell zones. Expansion of lung lymphatics after infection was reduced 68% by blocking VEGFR-2, 83% by blocking VEGFR-3, and 99% by blocking both receptors. Inhibition of VEGFR-2/VEGFR-3 did not prevent the formation of BALT. Treatment of established infection with oxytetracycline caused BALT, but not the lymphatics, to regress. We conclude that robust lymphangiogenesis occurs in mouse lungs after M. pulmonis infection through a mechanism involving signaling of both VEGFR-2 and VEGFR-3. Expansion of the lymphatic network is restricted to regions of BALT, but lymphatics do not regress when BALT regresses after antibiotic treatment. The lung lymphatic network can thus expand in sustained inflammation, but the expansion is not as reversible as the accompanying inflammation.

Neutrophil dependence of vascular remodeling after Mycoplasma infection of mouse airways.

Vascular remodeling is a feature of sustained inflammation in which capillaries enlarge and acquire the phenotype of venules specialized for plasma leakage and leukocyte recruitment. We sought to determine whether neutrophils are required for vascular remodeling in the respiratory tract by using Mycoplasma pulmonis infection as a model of sustained inflammation in mice. The time course of vascular remodeling coincided with the influx of neutrophils during the first few days after infection and peaked at day 5. Depletion of neutrophils with antibody RB6-8C5 or 1A8 reduced neutrophil influx and vascular remodeling after infection by about 90%. Similarly, vascular remodeling after infection was suppressed in Cxcr2(-/-) mice, in which neutrophils adhered to the endothelium of venules but did not extravasate into the tissue. Expression of the venular adhesion molecule P-selectin increased in endothelial cells from day 1 to day 3 after infection, as did expression of the Cxcr2-receptor ligands Cxcl1 and Cxcl2. Tumor necrosis factor α (TNFα) expression increased more than sixfold in the trachea of wild-type and Cxcr2(-/-) mice, but intratracheal administration of TNFα did not induce vascular remodeling similar to that seen in infection. We conclude that neutrophil influx is required for remodeling of capillaries into venules in the airways of mice with Mycoplasma infection and that TNFα signaling is necessary but not sufficient for vascular remodeling.

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.

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.

Rhamnose biosynthesis in mycoplasmas requires precursor glycans larger than monosaccharide.

Despite the apparent absence of genes coding for the known pathways for biosynthesis, the monosaccharide rhamnose was detected in the d configuration in Mycoplasma pneumoniae and Mycoplasma pulmonis, and in both the d and l configurations in Mycoplasma arthritidis. Surprisingly, the monosaccharide glucose was not a precursor for rhamnose biosynthesis and was not incorporated at detectable levels in glucose-containing polysaccharides or glycoconjugates. In contrast, carbon atoms from starch, a polymer of glucose, were incorporated into rhamnose in each of the three species examined. When grown in a serum-free medium supplemented with starch, M. arthritidis synthesized higher levels of rhamnose, with a shift in the relative amounts of the d and l configurations. Our findings suggest the presence of a novel pathway for rhamnose synthesis that is widespread in the genus Mycoplasma.

Cathepsin L protects mice from mycoplasmal infection and is essential for airway lymphangiogenesis.

Cathepsin L (Ctsl) is a proposed therapeutic target to control inflammatory responses in a number of disease states. However, Ctsl is thought to support host defense via its involvement in antigen presentation pathways. Hypothesizing that Ctsl helps combat bacterial infection, we investigated its role in Mycoplasma pulmonis-infected mice as a model of acute and chronic infectious airway inflammation. Responses to the airway inoculation of mycoplasma were compared in Ctsl(-/-) and Ctsl(+/+) mice. After infection, Ctsl(-/-) mice demonstrated more body weight loss, greater mortality (22% versus 0%, respectively), and heavier lungs than Ctsl(+/+) mice, but had smaller bronchial lymph nodes. The burden of live mycoplasma in lungs was 247-fold greater in Ctsl(-/-) mice than in Ctsl(+/+) mice after infection for 3 days. Ctsl(-/-) mice exhibited more severe pneumonia and neutrophil-rich, airway-occlusive exudates, which developed more rapidly than in Ctsl(+/+) mice. Compared with the conspicuous remodeling of lymphatics after infection in Ctsl(+/+) mice, little lymphangiogenesis occurred in Ctsl(-/-) mice, but blood vessel remodeling and tissue inflammation were similarly severe. Titers of mycoplasma-reactive IgM, IgA, and IgG in blood in response to live and heat-killed organisms were similar to those in Ctsl(+/+) mice. However, enzyme-linked immunosorbent spot assays revealed profound reductions in the cellular IFN-γ response to mycoplasma antigen. These findings suggest that Ctsl helps contain mycoplasma infection by supporting lymphangiogenesis and cellular immune responses to infection, and our findings predict that the therapeutic inhibition of Ctsl could increase the severity of mycoplasmal infections.

Plasticity of button-like junctions in the endothelium of airway lymphatics in development and inflammation.

Endothelial cells of initial lymphatics have discontinuous button-like junctions (buttons), unlike continuous zipper-like junctions (zippers) of collecting lymphatics and blood vessels. Buttons are thought to act as primary valves for fluid and cell entry into lymphatics. To learn when and how buttons form during development and whether they change in disease, we examined the appearance of buttons in mouse embryos and their plasticity in sustained inflammation. We found that endothelial cells of lymph sacs at embryonic day (E)12.5 and tracheal lymphatics at E16.5 were joined by zippers, not buttons. However, zippers in initial lymphatics decreased rapidly just before birth, as buttons appeared. The proportion of buttons increased from only 6% at E17.5 and 12% at E18.5 to 35% at birth, 50% at postnatal day (P)7, 90% at P28, and 100% at P70. In inflammation, zippers replaced buttons in airway lymphatics at 14 and 28 days after Mycoplasma pulmonis infection of the respiratory tract. The change in lymphatic junctions was reversed by dexamethasone but not by inhibition of vascular endothelial growth factor receptor-3 signaling by antibody mF4-31C1. Dexamethasone also promoted button formation during early postnatal development through a direct effect involving glucocorticoid receptor phosphorylation in lymphatic endothelial cells. These findings demonstrate the plasticity of intercellular junctions in lymphatics during development and inflammation and show that button formation can be promoted by glucocorticoid receptor signaling in lymphatic endothelial cells.

The Vsa shield of Mycoplasma pulmonis is antiphagocytic.

The infection of mice with Mycoplasma pulmonis is a model for studying chronic mycoplasmal respiratory disease. Many in vivo and in vitro studies have used the organism to gain a better understanding of host-pathogen interactions in chronic respiratory infection. The organism's Vsa proteins contain an extensive tandem repeat region. The length of the tandem repeat unit varies from as few as 11 amino acids to as many as 19. The number of tandem repeats can be as high as 60. The number of repeats varies at a high frequency due to slipped-strand mispairing events that occur during DNA replication. When the number of repeats is high, e.g., 40, the mycoplasma is resistant to lysis by complement but does not form a robust biofilm. When the number of repeats is low, e.g., 5, the mycoplasma is killed by complement when the cells are dispersed but has the capacity to form a biofilm that resists complement. Here, we examine the role of the Vsa proteins in the avoidance of phagocytosis and find that cells producing a protein with many tandem repeats are relatively resistant to killing by macrophages. These results may be pertinent to understanding the functions of similar proteins that have extensive repeat regions in other microbes.

Neutrophil histamine contributes to inflammation in mycoplasma pneumonia.

Mycoplasmas cause chronic inflammation and are implicated in asthma. Mast cells defend against mycoplasma infection and worsen allergic inflammation, which is mediated partly by histamine. To address the hypothesis that mycoplasma provokes histamine release, we exposed mice to Mycoplasma pulmonis, comparing responses in wild-type and mast cell-deficient KitW-sh/KitW-sh (W-sh) mice. Low histamine levels in uninfected W-sh mice confirmed the conventional wisdom that mast cells are principal sources of airway and serum histamine. Although mycoplasma did not release histamine acutely in wild-type airways, levels rose up to 50-fold above baseline 1 week after infection in mice heavily burdened with neutrophils. Surprisingly, histamine levels also rose profoundly in infected W-sh lungs, increasing in parallel with neutrophils and declining with neutrophil depletion. Furthermore, neutrophils from infected airway were highly enriched in histamine compared with naive neutrophils. In vitro, mycoplasma directly stimulated histamine production by naive neutrophils and strongly upregulated mRNA encoding histidine decarboxylase, the rate-limiting enzyme in histamine synthesis. In vivo, treatment with antihistamines pyrilamine or cimetidine decreased lung weight and severity of pneumonia and tracheobronchitis in infected W-sh mice. These findings suggest that neutrophils, provoked by mycoplasma, greatly expand their capacity to synthesize histamine, thereby contributing to lung and airway inflammation.

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.

Pericyte requirement for anti-leak action of angiopoietin-1 and vascular remodeling in sustained inflammation.

Blood vessel leakiness is an early, transient event in acute inflammation but can also persist as vessels undergo remodeling in sustained inflammation. Angiopoietin/Tie2 signaling can reduce the leakiness through changes in endothelial cells. The role of pericytes in this action has been unknown. We used the selective PDGF-B-blocking oligonucleotide aptamer AX102 to determine whether disruption of pericyte-endothelial crosstalk alters vascular leakiness or remodeling in the airways of mice under four different conditions: i) baseline, ii) acute inflammation induced by bradykinin, iii) sustained inflammation after 7-day infection by the respiratory pathogen Mycoplasma pulmonis, or iv) leakage after bradykinin challenge in the presence of vascular stabilization by the angiopoietin-1 (Ang1) mimic COMP-Ang1 for 7 days. AX102 reduced pericyte coverage but did not alter the leakage of microspheres from tracheal blood vessels at baseline or after bradykinin; however, AX102 exaggerated leakage at 7 days after M. pulmonis infection and increased vascular remodeling and disease severity at 14 days. AX102 also abolished the antileakage effect of COMP-Ang1 at 7 days. Together, these findings show that pericyte contributions to endothelial stability have greater dependence on PDGF-B during the development of sustained inflammation, when pericyte dynamics accompany vascular remodeling, than under baseline conditions or in acute inflammation. The findings also show that the antileakage action of Ang1 requires PDGF-dependent actions of pericytes in maintaining endothelial stability.

Lymphotoxin-alpha contributes to lymphangiogenesis.

Lymphotoxin-α (LTα), lymphotoxin-ß (LTß), and tumor necrosis factor-α (TNFα) are inflammatory mediators that play crucial roles in lymphoid organ development. We demonstrate here that LTα also contributes to the function of lymphatic vessels and to lymphangiogenesis during inflammation. LTα(-/-) mice exhibited reduced lymph flow velocities and increased interstitial fluid pressure. Airways of LTß(-/-) mice infected with Mycoplasma pulmonis had significantly more lymphangiogenesis than wild type (WT) or LTα(-/-) mice, as did the skin draining immunization sites of LTß(-/-) mice. Macrophages, B cells, and T cells, known sources of LT and TNFα, were apparent in the skin surrounding the immunization sites as were LTα, LTß, and TNFα mRNAs. Ectopic expression of LTα led to the development of LYVE-1 and Prox1-positive lymphatic vessels within tertiary lymphoid organs (TLOs). Quantification of pancreatic lymphatic vessel density in RIPLTαLTß(-/-) and WT mice revealed that LTα was sufficient for inducing lymphangiogenesis and that LTß was not required for this process. Kidneys of inducible LTα transgenic mice developed lymphatic vessels before the appearance of obvious TLOs. These data indicate that LTα plays a significant role in lymphatic vessel function and in inflammation-associated lymphangiogenesis.

Angiopoietin/Tie2 signaling transforms capillaries into venules primed for leukocyte trafficking in airway inflammation.

Vascular endothelial growth factor (VEGF) is a key angiogenic factor in tumors, but less is known about what drives vascular remodeling in inflammation, where plasma leakage and leukocyte influx are prominent features. In chronic airway inflammation in mice infected by the bacterium Mycoplasma pulmonis (M. pulmonis), the segment of the microvasculature that supports leukocyte adhesion and migration expands through remodeling of capillaries into vessels with features of venules. Here, we report that the angiopoietin/Tie2 pathway is an essential driving force for capillary remodeling into venules in M. pulmonis-infected mouse airways. Similar to M. pulmonis infection, systemic overexpression of angiopoietin-1 (Ang1) resulted in remodeling of airway capillaries into venular-like vessels that expressed venous markers like P-selectin, ICAM-1, and EphB4 and were sites of leukocyte adhesion during lipopolysaccharide-induced acute inflammation. Ang1 and Ang2 protein increased in M. pulmonis-infected mouse airways but came from different cellular sources: Ang1 was expressed in infiltrating neutrophils and Ang2 in endothelial cells. Indeed, systemic administration of soluble Tie2 inhibited capillary remodeling, induction of venous markers, and leukocyte influx in M. pulmonis-infected mouse airways. Together, these findings suggest that blockade of the Ang/Tie2 pathway may represent a therapeutic approach in airway inflammation.