Mycoplasma Pulmonis Research Articles

Critical Role of VEGF-C/VEGFR-3 Signaling in Innate and Adaptive Immune Responses in Experimental Obliterative Bronchiolitis

Chronic inflammation, a hallmark of obliterative bronchiolitis, is known to induce lymphangiogenesis. We therefore studied the role of lymphangiogenic vascular endothelial growth factor C (VEGF-C), its receptor VEGFR-3, and lymphangiogenesis during development of experimental obliterative bronchiolitis [ie, obliterative airway disease (OAD)] in rat tracheal allografts. The functional importance of VEGF-C was investigated by adenovirus-mediated overexpression of VEGF-C (AdVEGF-C), and by inhibition of VEGF-C activity with VEGFR-3-Ig (AdVEGFR-3-Ig). Analyses included histology, immunohistochemistry, and real-time RT-PCR 10 and 30 days after transplantation. In the course of OAD development, lymphangiogenesis was induced in the airway wall during the alloimmune response, which was reversed by cyclosporine A in a dose-dependent fashion. VEGF-C overexpression in tracheal allografts induced epithelial activation, neutrophil chemotaxis, and a shift toward a Th17 adaptive immune response, followed by enhanced lymphangiogenesis and the development of OAD. In contrast, inhibition of VEGF-C activity with VEGFR-3-Ig inhibited lymphangiogenesis and angiogenesis and reduced infiltration of CD4(+) T cells and the development of OAD. Lymphangiogenesis was linked to T-cell responses during the development of OAD, and VEGF-C/VEGFR-3 signaling modulated innate and adaptive immune responses in the development of OAD in rat tracheal allografts. Our results thus suggest VEGFR-3-signaling as a novel strategy to regulate T-cell responses in the development of obliterative bronchiolitis after lung transplantation.

Respiratory diseases in rats

ABSTRACT:Respiratory disease due to infectious disease is one of the most common presentations in rats but the cause is in many cases multifactorial. Predisposing factors are generally related to poor husbandry and diet. Respiratory disease is caused primarily by infectious agents such as Mycoplasma pulmonis, Streptococcus pneumoniae, Corynebacterium kutscheri, cilia associated respiratory (CAR) bacillus, Sendai virus and coronavirus. Other causes are cardiovascular diseases, neoplastic diseases and trauma of the upper and lower respiratory tract. A presumptive diagnosis can be made by good history and physical examination. However, many diagnostic tests are best performed under general anaesthesia. Medical treatment generally includes oxygen therapy, fluid therapy, analgesia and administration of antibiotics, the use of mucolytic agents and bronchodilators and supportive care. Medications can be administrated orally, parentally or via nebulisation.

Common Bacterium Induces Histamine Production in Neutrophils

Airway inflammation and constriction associated with the release of histamine is a hallmark of asthma and allergies. Mast cells are considered the main producers of histamine.1 But new research indicates that the common environmental bacterium Pseudomonas aeruginosa can induce neutrophils, a type of white blood cell, to produce and release very high levels of histamine as well.2 Acute bacterial and viral infections often trigger serious asthma attacks that result in hospitalization, and many of these patients have elevated levels of neutrophils in their airways.3 “We suspected that neutrophils may [produce] mediators that worsen airway constriction,” says George Caughey, chief of the Pulmonary and Critical Care Medicine Section at the Veterans Affairs Medical Center and a professor at the University of California, San Francisco, who led the study. A few years ago, Caughey’s laboratory studied mice infected with the bacterium Mycoplasma pulmonis and found that their neutrophils released histamine.2 The researchers speculated that the more common and more virulent P. aeruginosa—which, like M. pulmonis, is associated with chronic airway infections—also may trigger histamine production by neutrophils. To explore the connection, the researchers infected mouse neutrophils with two clinical laboratory strains of P. aeruginosa. Strain PA103 quickly killed the neutrophils, and no extra histamine was produced. The other strain, PAO1, provoked neutrophils to increase histamine production by as much as six times. However, mast cells treated with PAO1 produced no extra histamine. When mice were infected with PAO1, they developed bronchitis and pneumonia, and lung histamine levels rose by about three times. The histamine surge was tracked to histidine decarboxylase, a key enzyme for histamine synthesis—neutrophils made more of the enzyme when infected with P. aeruginosa.2 Pseudomonas aeruginosa The underlying causes of asthma and allergies are not well understood, but the findings suggest that P. aeruginosa may trigger inflammation via neutrophils. Patients with chronic airway diseases often experience cycles of bacterial infections followed by inflammation.4 Excess histamine generated by neutrophils with chronic P. aeruginosa infection could contribute to the narrowing of the airways observed in asthma, allergies, chronic bronchitis, cystic fibrosis, and other lung diseases, according to Caughey. The production of histamine by neutrophils “is a significant finding because histamine is a major player in the respiratory tract. This opens the door for new ways to look at bacterial-associated allergic inflammation,” says John Wherry, an associate professor of microbiology at the University of Pennsylvania, Philadelphia. Understanding why only certain strains induce neutrophil histamine production “could reveal insights into how pathogens evolve to evade host immunity,” Wherry says. Caughey plans to test whether more strains of P. aeruginosa, other bacteria, and viruses also cause neutrophils to produce histamine. “The most common causes of severe asthma attacks are viruses,” he says. The next step would be to demonstrate that neutrophils make histamine in response to microbial infections in people. If this proves to be the case, Caughey says, “our research could improve the understanding of inflammation in bacterial infections and help us craft therapies for relief of inflammation and its consequences.”

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.

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.

Mycoplasma pulmonisVsa proteins and polysaccharide modulate adherence to pulmonary epithelial cells

The Mycoplasma pulmonis Vsa proteins are a family of size- and phase-variable lipoproteins that shield the mycoplasmas from complement and modulate attachment to abiotic surfaces. Mycoplasmas producing a long Vsa protein hemadsorb poorly and yet are proficient at colonizing rats and mice. The effect of the length of the Vsa protein on the attachment of mycoplasmas to epithelial cells has not been previously explored. We find that independent of Vsa isotype, mycoplasmas producing a long Vsa protein with many tandem repeats adhere poorly to murine MLE-12 cells compared with mycoplasmas producing a short Vsa. We also find that mutants lacking the EPS-I polysaccharide of M.pulmonis exhibited decreased adherence to MLE-12 cells, even though it has been shown previously that such mutants have an enhanced ability to form a biofilm.

Pathology in Practice

Pathology in Practice

Accommodation of profound sequence differences at the interfaces of eubacterial RNA polymerase multi-protein assembly.

Evolutionarily divergent proteins have been shown to change their interacting partners. RNA polymerase assembly is one of the rare cases which retain its component proteins in the course of evolution. This ubiquitous molecular assembly, involved in transcription, consists of four core subunits (alpha, beta, betaprime, and omega), which assemble to form the core enzyme. Remarkably, the orientation of the four subunits in the complex is conserved from prokaryotes to eukaryotes although their sequence similarity is low. We have studied how the sequence divergence of the core subunits of RNA polymerase is accommodated in the formation of the multi-molecular assembly, with special reference to eubacterial species. Analysis of domain composition and order of the core subunits in >85 eubacterial species indicates complete conservation. However, sequence analysis indicates that interface residues of alpha and omega subunits are more divergent than those of beta, betaprime, and sigma70 subunits. Although beta and betaprime are generally well-conserved, residues involved in interaction with divergent subunits are not conserved. Insertions/deletions are also observed near interacting regions even in case of the most conserved subunits, beta and betaprime. Homology modelling of three divergent RNA polymerase complexes, from Helicobacter pylori, Mycoplasma pulmonis and Onion yellows phytoplasma, indicates that insertions/deletions can be accommodated near the interface as they generally occur at the periphery. Evaluation of the modeled interfaces indicates that they are physico-chemically similar to that of the template interfaces in Thermus thermophilus, indicating that nature has evolved to retain the obligate complex in spite of substantial substitutions and insertions/deletions.

INFECÇÃO POR Mycoplasma pulmonis EM RATOS WISTAR PROVENIENTES DE BIOTÉRIO

Mycoplasma pulmonis, the agent of the Murine Respiratory Mycoplasmosis (MRM), is one of the most pathogenic mycoplasma species for laboratory rodents. Mortality and morbidity in infected animals may be elevated, especially in long term studies or in animals with subclinical infection exacerbated by experimental procedures. Therefore, its most significant impact may be due to the interference in the interpretation of experimental results. The aim of this work was to evaluate the rate of M. pulmonis infection in rats from the animal house of the Regional University of Blumenau, by culture and PCR, bred specifically for scientific experimentation purposes. Among the 20 animals studied, 15 (75%) had bronchoalveolar samples with positive result for M. pulmonis by PCR, and two presented growth in culture with SP4 medium. Our results demonstrate a high rate of mycoplasma infection in laboratory rats from the animal house of the university, in spite of the knowledge about this issue since the 1990’s.

Rapid Identification of <i>Mycoplasma pulmonis</i> Isolated from Laboratory Mice and Rats Using Matrix-Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry

Mycoplasma species identification is based on biochemical, immunological, and molecular methods that require several days for accurate identification. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a novel method for identification of bacteria and has recently been introduced into the clinical microbiology laboratory as a rapid and accurate technique. This method allows a characteristic mass spectral fingerprint to be obtained from whole inactivated mycoplasmal cells. In this study, we evaluated the performance of the MALDI-TOF MS for the identification of Mycoplasma by comparison with standard sequence analysis of 16S rRNA. We developed the first database of MALDI-TOF MS profiles of Mycoplasma species, containing Mycoplasma pulmonis, M. arthritidis, and M. neurolyticum, which are the most common pathogens in mice and/or rats, and species-specific spectra were recorded. Using the database, 6 clinical isolates were identified. Six tracheal swabs from 4 mice and 2 rats were cultured on PPLO agar for 4 to 7 days, and the colonies were directly applied to analyze the protein profiles. Five strains were identified as M. pulmonis, and 1 strain from a mouse was identified as M. neurolyticum (spectral scores were >2.00); the results were consistent with the results of the 16S rRNA gene sequence analysis (homologies>97.0%). These data indicate that MALDI-TOF MS can be used as a clearly rapid, accurate, and cost-effective method for the identification of M. pulmonis isolates, and this system may represent a serious alternative for clinical laboratories to identify Mycoplasma species.

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-1β 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-1β, 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.

Strain-dependent induction of neutrophil histamine production and cell death byPseudomonas aeruginosa

Airway diseases often feature persistent neutrophilic inflammation and infection. In cystic fibrosis bronchitis, for example, Pseudomonas aeruginosa is isolated frequently. Previously, this laboratory revealed that neutrophils become major sources of histamine in mice with tracheobronchitis caused by the wall-less bacterium Mycoplasma pulmonis. To test the hypothesis that more-broadly pathogenic P. aeruginosa (which expresses cell wall-associated LPS and novel toxins) has similar effects, we incubated naïve mouse neutrophils with two strains of P. aeruginosa. Strain PAO1 greatly increased neutrophil histamine content and secretion, whereas strain PA103 depressed histamine production by killing neutrophils. The histamine-stimulating capacity of PAO1, but not PA103-mediated toxicity, persisted in heat-killed organisms. In PAO1-infected mice, lung and neutrophil histamine content increased. However, PAO1 did not alter production by mast cells (classical histamine reservoirs), which also resisted PA103 toxicity. To explore mechanisms of neutrophil-selective induction, we measured changes in mRNA encoding histidine decarboxylase (rate-limiting for histamine synthesis), probed involvement of endotoxin-TLR pathways in Myd88-deficient neutrophils, and examined contributions of pyocyanin and exotoxins. Results revealed that PAO1 increased histamine production by up-regulating histidine decarboxylase mRNA via pathways largely independent of TLR, pyocyanin, and type III secretion system exotoxins. PAO1 also increased histidine decarboxylase mRNA in neutrophils purified from infected lung. Stimulation required direct contact with neutrophils and was blocked by phagocytosis inhibitor cytochalasin D. In summary, Pseudomonas-augmented histamine production by neutrophils is strain-dependent in vitro and likely mediated by up-regulation of histidine decarboxylase. These findings raise the possibility that Pseudomonas-stimulated neutrophils can enhance airway inflammation by producing histamine.

SPARC Oppositely Regulates Inflammation and Fibrosis in Bleomycin-Induced Lung Damage

Fibrosis results from inflammatory tissue damage and impaired regeneration. In the context of bleomycin-induced pulmonary fibrosis, we demonstrated that the matricellular protein termed secreted protein acidic and rich in cysteine (SPARC) distinctly regulates inflammation and collagen deposition, depending on its cellular origin. Reciprocal Sparc(-/-) and wild-type (WT) bone marrow chimeras revealed that SPARC expression in host fibroblasts is required and sufficient to induce collagen fibrosis in a proper inflammatory environment. Accordingly, Sparc(-/-) >WT chimeras showed exacerbated inflammation and fibrosis due to the inability of Sparc(-/-) macrophages to down-regulate tumor necrosis factor production because of impaired responses to tumor growth factor-β. Hence, the use of bone marrow cells expressing a dominant-negative form of tumor growth factor-β receptor type II under the monocyte-specific CD68 promoter, as a decoy, phenocopied Sparc(-/-) donor chimeras. Our results point to an unexpected dual role of SPARC in oppositely influencing the outcome of fibrosis.

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.

Diagnóstico histopatológico e molecular da infecção por Mycoplasma sp. em ratos mantidos em biotério convencional

The chronic infection by Mycoplasma pulmonis is frequently found in rats kept in conventional premises and may influence the results of the experiments conducted using these animals. During the clinical presentation of the infection animals develop among other manifestations clinical signs of respiratory system impairment. On the present study, we report the diagnosis the histopathological and molecular diagnosis of Mycoplasma sp. infection in Wistar rats both presenting clinical signs of infection and health animals kept in the same colony. The efficacy of therapeutic and prophylactic actions is discussed.

Angiopoietin-2-Driven Vascular Remodeling in Airway Inflammation

Vascular remodeling is a feature of chronic inflammation during which capillaries transform into venules that expand the region of the vasculature in which leakage and leukocyte emigration both occur. Recently, we found that angiopoietin/Tie2 receptor signaling drives the transformation of capillaries into venules at an early stage of the sustained inflammatory response in the airways of mice infected with Mycoplasma pulmonis. However, the precise contributions of both angiopoietin-1 (Ang1) and angiopoietin-2 (Ang2) are not clear. In this study, we sought to determine the contribution of Ang2 to this vascular remodeling. Ang2 mRNA expression levels increased and phosphorylated Tie2 immunoreactivity in mucosal blood vessels decreased, indicative of diminished receptor signaling after infection. Selective inhibition of Ang2 throughout the infection by administration of either of two distinct function-blocking antibodies reduced the suppression of Tie2 phosphorylation and decreased the remodeling of mucosal capillaries into venules, the amount of leukocyte influx, and disease severity. These findings are consistent with Ang2 acting as an antagonist of Tie2 receptors and the reduction of Tie2 phosphorylation in endothelial cells rendering the vasculature more responsive to cytokines that promote both vascular remodeling and the consequences of inflammation after M. pulmonis infection. By blocking such changes, Ang2 inhibitors may prove beneficial in the treatment of sustained inflammation in which vascular remodeling, leakage, and leukocyte influx contribute to its pathophysiology.

Lymphotoxin-alpha contributes to lymphangiogenesis

AbstractLymphotoxin-α (LTα), lymphotoxin-β (LTβ), 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 LTβ−/− mice infected with Mycoplasma pulmonis had significantly more lymphangiogenesis than wild type (WT) or LTα−/− mice, as did the skin draining immunization sites of LTβ−/− 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, LTβ, 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 RIPLTαLTβ−/− and WT mice revealed that LTα was sufficient for inducing lymphangiogenesis and that LTβ 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.

Commentary

The authors recently assessed the likelihood that lifetime cancer bioassays of aspartame, methanol, and methyl tertiary butyl ether conducted with conventional (not specific pathogen free) Sprague-Dawley rats were compromised by Mycoplasma pulmonis disease. From the tumor data and other information, the authors concluded that the rats used in these bioassays likely had M pulmonis disease and that lesions of the disease were plausibly interpreted as lymphoma. Subsequently, they analyzed the nonneoplastic lesion data from these bioassays for occurrence of inflammatory lesions and found that 2,267 of 2,960 rats (76.6%) were reported to have bronchitis, the signature lesion of M pulmonis disease, and that 633 rats (21.4%) were reported to have otitis, another common lesion of the disease. Also, documentation is now available containing serologic evidence of mycoplasma infection in the rats. In contrast, the reports of 6 National Toxicology Program bioassays based on specific pathogen-free Sprague-Dawley rats listed no instances of bronchitis or otitis. These findings provide substantial additional evidence that the bioassays in question were compromised by M pulmonis disease. Therefore, the reported induction of lymphoma in these studies should not be considered in cancer risk assessments. The authors also found that inflammatory lesions were prevalent in lymph nodes, thymus, pleura, and brain. Finally, they found that of all 328 cases of lymphoimmunoblastic lymphoma affecting the lung (the primary form of lymphoma reported), 218 (66.5%) occurred within the first 104 weeks of the studies, showing that occurrence of such lesions was not due to appearance in rats surviving beyond that interval.

Fewer essential genes in mycoplasmas than previous studies suggest

Here, we describe mutants of Mycoplasma pulmonis that were obtained using a minitransposon, Tn4001TF1, which actively transposes but is then unable to undergo subsequent excision events. Using Tn4001TF1, we disrupted 39 genes previously thought to be essential for growth. Thus, the number of genes required for growth has been overestimated. This study also revealed evidence of gene duplications in M. pulmonis and identified chromosome segregation proteins that are dispensable in mycoplasmas but essential in Bacillus subtilis.

Toll-like receptor 2 (TLR2) plays a major role in innate resistance in the lung against murine Mycoplasma.

Mycoplasma lipoproteins are recognized by Toll-like receptors (TLR), but TLRs' role in responses to infection are unknown. Mycoplasma pulmonis is a naturally occurring respiratory pathogen in mice. In the current study, we used TLR-transfected HEK cells and TLR2−/− bone marrow-derived dendritic cells to demonstrate TLR2-mediated events are important in the initial host-mycoplasma interactions promoting cytokine responses. As we found alveolar macrophages expressed TLR1, TLR2 and TLR6 mRNAs, a role for TLR2 in innate immune clearance in lungs was examined. Three days post-infection, TLR2−/− mice had higher M. pulmonis numbers in lungs, but not in nasal passages. However, TLR2−/− mice had higher lung cytokine levels, indicating TLR2-independent mechanisms are also involved in host responses. Thus, TLR2 plays a critical role in the ability of innate immunity to determine M. pulmonis numbers in the lung, and it is likely that early after respiratory infection that TLR2 recognition of M. pulmonis triggers initial cytokine responses of host cells.