Mouse Model Of Respiratory Infection Research Articles

Peptide-mimetic treatment of Pseudomonas aeruginosa in a mouse model of respiratory infection

The rise of drug resistance has become a global crisis, with >1 million deaths due to resistant bacterial infections each year. Pseudomonas aeruginosa, in particular, remains a serious problem with limited solutions due to complex resistance mechanisms that now lead to more than 32,000 multidrug-resistant (MDR) infections and over 2000 deaths in the U.S. annually. While the emergence of resistant bacteria has become ominously common, identification of useful new drug classes has been limited over the past over 40 years. We found that a potential novel therapeutic, the peptide-mimetic TM5, is effective at killing P. aeruginosa and displays sufficiently low toxicity in mammalian cells to allow for use in treatment of infections. Interestingly, TM5 kills P. aeruginosa more rapidly than traditional antibiotics, within 30–60 min in vitro, and is effective against a range of clinical isolates, including extensively drug resistant strains. In vivo, TM5 significantly reduced bacterial load in the lungs within 24 h compared to untreated mice and demonstrated few adverse effects. Taken together, these observations suggest that TM5 shows promise as an alternative therapy for MDR P. aeruginosa respiratory infections.

B-007 Measurement of Cytokines in Bronchoalveolar Lavage (BAL) Fluid and CSF Supports Model Development for Discovery Programs

Abstract In our laboratory, increased demand for cytokine measurements to support both safety and efficacy endpoints across discovery programs has also resulted in cytokine analyses in biological fluids other than routine serum or plasma samples. Recently we have evaluated cytokines in bronchoalveolar lavage (BAL) fluid and CSF to support a mouse respiratory infection and a rat neuroinflammatory model, respectively. Fit-for-purpose validation of these sample types is necessary to confirm cytokine methods are suitable for use, including selection of optimal sample dilutions to reduce or eliminate any matrix effects or interferences. Meso Scale Discovery® (MSD Gaithersburg, MD) methodology was used for measuring cytokines and validation parameters included precision and accuracy of calibration curves (CV% and %Relative Error <20%) to confirm LLOQ and ULOQ for each cytokine method, precision of results for quality control and study samples (CV ≤ 20%) and linearity of response in samples. In development of the mouse respiratory infection model, cytokines IL-1β, IL-6, KC/GRO and MCP-1 were increased in BAL fluid (up to 2892-fold) 20 h post-infection compared to sham and vehicle control groups. Once the model was established, treatment of mice with Compound A demonstrated statistical decreases in these cytokines in BAL fluid compared to an infected control group. Dilutional linearity demonstrated that a minimal required dilution of 1:2 was required for mouse BAL fluids. In support of development of the rat neuroinflammatory model, cytokines IL-1β, IL-6, KC/GRO, MCP-1 and TNF-α were increased (325X, 13X, 28X, 23X and 7.7X, respectively) in CSF compared to plasma at 24 h post treatment. Dilutional linearity evaluations demonstrated that CSF samples can be run neat but a 1:4 dilution was required for plasma samples. In conclusion, fit-for-purpose validation confirmed MSD methods were acceptable for use in supporting these drug discovery models.

WS07.05 Unraveling the pathogenicity of Mycobacterium abscessus clinical isolates in cystic fibrosis pulmonary epithelial cell and mouse models of respiratory infection

Mycobacterium abscessus (Mabs) infections can be caused by strains belonging to different dominant circulating clones (DCCs) or displaying bacterial morphotypes in people with cystic fibrosis (CF).

Adaptive immune protection of the middle ears differs from that of the respiratory tract.

The efficacy of the adaptive immune system in the middle ear (ME) is well established, but the mechanisms are not as well defined as those of gastrointestinal or respiratory tracts. While cellular elements of the adaptive response have been detected in the MEs following infections (or intranasal immunizations), their specific contributions to protecting the organ against reinfections are unknown. How immune protection mechanisms of the MEs compares with those in the adjacent and attached upper and lower respiratory airways remains unclear. To address these knowledge gaps, we used an established mouse respiratory infection model that we recently showed also involves ME infections. Bordetella bronchiseptica delivered to the external nares of mice in tiny numbers very efficiently infects the respiratory tract and ascends the Eustachian tube to colonize and infect the MEs, where it causes severe but acute inflammation resembling human acute otitis media (AOM). Since this AOM naturally resolves, we here examine the immunological mechanisms that clear infection and protect against subsequent infection, to guide efforts to induce protective immunity in the ME. Our results show that once the MEs are cleared of a primary B. bronchiseptica infection, the convalescent organ is strongly protected from reinfection by the pathogen despite its persistence in the upper respiratory tract, suggesting important immunological differences in these adjacent and connected organs. CD4+ and CD8+ T cells trafficked to the MEs following infection and were necessary to robustly protect against secondary challenge. Intranasal vaccination with heat killed B. bronchiseptica conferred robust protection against infection to the MEs, even though the nasopharynx itself was only partially protected. These data establish the MEs as discrete effector sites of adaptive immunity and shows that effective protection in the MEs and the respiratory tract is significantly different. This model system allows the dissection of immunological mechanisms that can prevent bacteria in the nasopharynx from ascending the ET to colonize the ME.

513 Unravelling the pathogenicity of Mycobacterium abscessus clinical isolates in cystic fibrosis pulmonary epithelial cell and mouse models of respiratory infection

513 Unravelling the pathogenicity of Mycobacterium abscessus clinical isolates in cystic fibrosis pulmonary epithelial cell and mouse models of respiratory infection

A Mouse Model of Pseudomonas aeruginosa Pneumonia.

The rapid innate immune response to respiratory infection is essential to prevent the systemic dissemination of pathogens. This chapter outlines an experimental mouse model of respiratory infection by gram-negative Pseudomonas aeruginosa and analyses of leukocyte trafficking in the lungs. The reader will learn two methods to induce respiratory infection in mice that differ in whether the initial bolus is targeted within a specific lobe of the lung. We then describe a technique based on tissue digestion and flow cytometry that allows the investigator to distinguish leukocytes within different compartments of the lung, and discuss the advantages and limitations to such an approach.

Cyclooxygenase inhibitors impair CD4 T cell immunity and exacerbate Mycobacterium tuberculosis infection in aerosol-challenged mice

Tuberculosis, caused by infection with Mycobacterium tuberculosis (Mtb), kills over 1.6 million people each year despite availability of antibiotics. The increase in drug resistant Mtb strains is a major public health emergency and host-directed therapy as adjunct to antibiotic treatment has gained increased interest. Cyclooxygenase inhibitors (COXi) are frequently used drugs to alleviate tuberculosis related symptoms. Mouse studies of acute intravenous Mtb infection have suggested a potential benefit of COXi for host-directed therapy. Here we show that COXi treatment (ibuprofen and celecoxib) is detrimental to Mtb control in different mouse models of respiratory infection. This effect links to impairments of the Type-1 helper (Th1) T-cell response as CD4 T-cells in COXi-treated animals have significantly decreased Th1 differentiation, reduced IFNγ expression and decreased protective capacity upon adoptive transfer. If confirmed in clinical trials, these findings could have major impact on global health and question the use of COXi for host-directed therapy.

P133 Set of preclinical mouse models of respiratory infection to evaluate antibiotic efficacy

P133 Set of preclinical mouse models of respiratory infection to evaluate antibiotic efficacy

Impaired induction of Slc26a4 promotes respiratory acidosis and severe, steroid-resistant asthma

Abstract CO2 produced by systemic cellular respiration is hydrated into carbonic acid (H2CO3) that dissociates into H+ and HCO3−. These are transported in the plasma to the lungs where HCO3− is converted back into H2CO3 and CO2 that are expelled through breathing. Evidence suggests that dysfunction of the mechanisms that govern these processes may result in the development of respiratory acidosis (RA) and the cardinal features of severe, steroid-resistant (SSR) asthma. Reduced lung function, which occurs in SSR asthma, impairs removal of volatile H2CO3 and CO2, resulting in acid accumulation and increased arterial PaCO2. Patients with severe asthma often develop complications from increased PaCO2, which skews the PaCO2/HCO3− ratio resulting in increased H+ concentration and reduced pH. We developed three mouse models of respiratory infection and ovalbumin-induced SSR allergic airways disease (SSRAAD) that are highly representative of SSR asthma in humans. We used these models to show a role for impaired homeostatic acid-base balance in SSR asthma. All three infections suppress the induction of the expression of the chloride (Cl−)/HCO− pump, Slc26a4, in the airway mucosa in AAD. Importantly, SSRAAD is associated with increased levels of free H+ ions in bronchoalveolar lavage fluid. Administration of Slc26a4-specific siRNA in steroid-sensitive AAD, which mimics the effect of decreased Slc26a4 responses in SSRAAD, induced RA and steroid-resistant airway inflammation and AHR. Importantly, treatment of RA with NaHCO3 during infection-induced SSRAAD suppressed steroid-resistant AHR. Thus, we have identified a previously unrecognised role for deficient Slc26a4 responses that result in the development of RA and the pathogenesis of SSRAAD.

Resistance to Acute Macrophage Killing Promotes Airway Fitness of Prevalent Community-Acquired Staphylococcus aureus Strains.

The incidence of methicillin-resistant Staphylococcus aureus (MRSA) pneumonia in otherwise healthy individuals is increasing. To investigate the mechanism underlying the epidemiological success of predominant community-associated (CA)-MRSA strains, we examined their fitness traits during the initial interaction between bacteria and the host occurring in the lower airway. Using a mouse respiratory infection model, we show that clinical isolates often responsible for CA infections are highly resistant to clearance from healthy airways, whereas S. aureus strains not as prevalent or traditionally associated with hospital-associated infections are relatively susceptible. Mechanistically, the competitive fitness of S. aureus is a result of both agr-dependent and -independent resistance to innate bacterial killing. Furthermore, we show that rather than evasion from neutrophil-dependent bactericidal process, the observed S. aureus fitness in the lower airways is due to its intrinsic resistance to resident alveolar macrophage-mediated intracellular killing. Importantly, we demonstrate that the virulence determinants responsible for bacterial persistence in immune-competent mice are dispensable in mice with predisposing conditions such as influenza infection. Taken together, these novel findings of the improved competence of predominant CA-MRSA strains to survive innate killing in healthy hosts, particularly at the very beginning stage of infection, provide a unique insight into their epidemiological success.

Characterization of a large, proteolytically processed cowpox virus membrane glycoprotein conserved in most chordopoxviruses

Most poxvirus proteins are either highly conserved and essential for basic steps in replication or less conserved and involved in host interactions. Homologs of the CPXV219 protein, encoded by cowpox virus, are present in nearly all chordopoxvirus genera and some species have multiple copies. The CPXV219 homologs have estimated masses of greater than 200kDa, making them the largest known poxvirus proteins. We showed that CPXV219 was expressed early in infection and cleaved into N- and C-terminal fragments that remained associated. The protein has a signal peptide and transited the secretory pathway where extensive glycosylation and proteolytic cleavage occurred. CPXV219 was located by immunofluorescence microscopy in association with the endoplasmic reticulum, Golgi apparatus and plasma membrane. In non-permeabilized cells, CPXV219 was accessible to external antibody and biotinylation. Mutants that did not express CPXV219 replicated normally in cell culture and retained virulence in a mouse respiratory infection model.

MicroRNA-21 drives severe, steroid-insensitive experimental asthma by amplifying PI3K-mediated suppression of HDAC2 (HYP7P.262)

Abstract BACKGROUND: Severe, steroid-insensitive (SSI) asthma is a substantial clinical problem. Effective treatments are urgently required, however, their development is hampered by a lack of understanding of the mechanisms that promote disease. SSI asthma is associated with respiratory infections and non-eosinophilic endotypes of disease, including neutrophilic asthma. OBJECTIVES: To develop and use mouse models of SSI neutrophilic asthma to investigate pathogenic mechanisms involving microRNA (miR)-21, phosphoinositide-3-kinase (PI3K) and histone deacetylase (HDAC)2 in order to identify new therapeutic approaches. METHODS: Novel mouse models of respiratory infection and ovalbumin-induced, SSI neutrophilic allergic airway disease (SSIAAD) in BALB/c mice were developed. The roles of infection-induced miR-21 expression and PI3K-dependent signalling in the lung were examined using a specific miR-21 inhibitor (antagomir-21) and the pan-PI3K inhibitor LY294002. RESULTS: Infection induced a miR-21-dependent, PI3K-mediated signalling pathway that decreased nuclear HDAC2 levels and promoted steroid-insensitive neutrophilic inflammation and airway hyper-responsiveness (AHR) in AAD. Inhibition of miR-21 or PI3K suppressed nuclear pAkt levels and restored HDAC2 levels and steroid sensitivity. CONCLUSIONS: We have identified a novel role for a miR-21/PI3K/HDAC2 signalling axis in SSIAAD. Our data highlights miR-21 as a novel target for treating this form of asthma.

Antibody-mediated inhibition of Bordetella pertussis adenylate cyclase–haemolysin-induced macrophage cytotoxicity is influenced by variations in the bacterial population

Whooping cough is a vaccine-preventable disease presenting with epidemic cycles linked to natural and/or vaccine-driven evolution of the aetiological agent of the disease, Bordetella pertussis. Adenylate cyclase-haemolysin (AC-Hly) is a major toxin produced by this pathogen, which mediates macrophage apoptosis in vitro and in vivo. While current acellular pertussis vaccine (APV) formulations do not include AC-Hly, they all contain pertussis toxin and can comprise filamentous haemagglutinin (FHA), which interacts with AC-Hly, and pertactin (PRN), which has been hypothesized also to interact with AC-Hly. We aimed to study the capacity of specific antibodies to inhibit the in vitro B. pertussis AC-Hly-mediated cytotoxicity of J774A.1 murine macrophages in a background of a changing bacterial population. We demonstrate that: (i) clinical isolates of different types or PRN phenotype are all cytotoxic and lethal in the mouse model of respiratory infection; (ii) lack of PRN production does not impact AC-Hly-related phenotypes; (iii) anti-AC-Hly antibodies inhibit cell lysis whatever the phenotype of the isolate, while anti-PRN antibodies significantly inhibit cell lysis provided the isolate produces this antigen, which might be relevant in vivo for APV-induced immunity; and (iv) anti-FHA antibodies only inhibit lysis induced by isolates collected in 2012, maybe indicating specific characteristics of epidemic lineages of B. pertussis.

Differential expression of Bordetella pertussis iron transport system genes during infection

Temporal expression patterns of the Bordetella pertussis alcaligin, enterobactin and haem iron acquisition systems were examined using alcA-, bfeA- and bhuR-tnpR recombinase fusion strains in a mouse respiratory infection model. The iron systems were differentially expressed in vivo, showing early induction of the alcaligin and enterobactin siderophore systems, and delayed induction of the haem system in a manner consistent with predicted changes in host iron source availability during infection. Previous mixed infection competition studies established the importance of alcaligin and haem utilization for B. pertussis in vivo growth and survival. In this study, the contribution of the enterobactin system to the fitness of B. pertussis was confirmed using wild-type and enterobactin receptor mutant strains in similar competition infection experiments. As a correlate to the in vivo expression studies of B. pertussis iron systems in mice, sera from uninfected and B. pertussis-infected human donors were screened for antibody reactivity with Bordetella iron-repressible cell envelope proteins. Pertussis patient sera recognized multiple iron-repressible proteins including the known outer membrane receptors for alcaligin, enterobactin and haem, supporting the hypothesis that B. pertussis is iron-starved and responds to the presence of diverse iron sources during natural infection.

Phg, a novel member of the autotransporter family present in Bordetella species

Several proteins encoded in the genomes of Bordetella species show significant sequence similarity to the autotransporter domains of surface exposed or secreted virulence factors of bordetellae such as pertactin, tracheal colonization factor or Vag8. One of these putative autotransporters, provisionally termed Phg, is encoded by the pertactin homologous gene (phg), which is highly conserved in Bordetella pertussis, B. bronchiseptica and B. parapertussis, but absent in B. avium and B. petrii. In contrast to homologues with documented functions in host interaction and virulence, several key amino acids probably involved in proteolytic processing of the autotransporter domain are not conserved in Phg. The transcription start site of phg was identified by primer extension analysis, but differential transcription of phg could not be detected in B. bronchiseptica strains under conditions that lead to enhanced expression of other known Bordetella autotransporter proteins. A mutant of B. pertussis was constructed in which major parts of phg are substituted by a kanamycin resistance cassette. Virulence testing of this mutant in a mouse respiratory infection model showed the same colonization properties as the wild-type strain.

Reduced Susceptibility to Respiratory Infection Following Moderate Exercise

0908 Moderate exercise training has been associated with a decreased risk for URTI. It has been hypothesized that lung macrophages may play a role in the increased resistance to respiratory infection following moderate exercise. PURPOSE: To determine the effects of lung macrophage depletion on the benefits of moderate exercise on susceptibility to infection using our mouse model of respiratory infection (Davis et.al. J. Appl. Physiol Physiol. 83 (5): 1461–6 1997). METHODS: Male CD-1 mice were randomly assigned to one of four groups (n = 17–21 per group). Mice were administered 100μl of clodronate (EX-CL2MDP and CON-CL2MDP) or phosphate buffered saline (EX-PBS and CON-PBS) encapsulated in liposomes, via an intra-nasal route following exercise or rest on the 4th day of training and again on the 6th day following infection. Macrophages ingest the liposomes and are killed by the clodronate through apoptosis. This dose has previously been found to deplete approximately 75% of lung macrophages. Exercise mice (EX-PBS and EX-CL2MDP) ran for 1h on a treadmill for 6 consecutive days at 36m/min, 8% grade. CON mice (CON-PBS and CONCL 2MDP) remained in their cages. Following rest or EX on the last day of training, mice were intra-nasally inoculated with a standardized dose (1.6 × 105 PFUs) of Herpes Simplex Virus-1. They were monitored twice daily for morbidity and mortality as well as symptom severity for 21 days. RESULTS: EX decreased morbidity by 36% (P = 0.0017) and mortality by 61% (P < 0.0001) as well as the symptom severity score on days 5 through 9 (P < 0.0001). Depletion of macrophages in the lungs negated the beneficial effects of moderate exercise; there were no differences between the EX- CL2MDP group and the CON-PBS group in morbidity (89% versus 95%), mortality (79% versus 95%) or symptom severity score. CONCLUSION: Results indicate that macrophages play an important role in mediating the beneficial effects of moderate exercise on susceptibility to respiratory infection in mice. Previously we have demonstrated an association between increased macrophage anti-viral activity and decreased susceptibility to infection. This study however, has specifically tested the causal role of lung macrophages in improved resistance to infection in this model.

Effects of Moderate Exercise on Susceptibility to Respiratory Infection in Aged Mice

1751 Ageing has been associated with a decline in several aspects of the immune system. Moderate exercise may restore immune function in aged mice and reduce susceptibility to respiratory infection. PURPOSE: We studied the effects of moderate exercise training on susceptibility to infection in aged mice using our mouse model of respiratory infection (Davis et.al. J. Appl. Physiol Physiol. 83 (5): 1461–6 1997). Viral titers and inflammatory cytokine responses in the lung were also determined. METHODS: Female CD-1 mice were randomly assigned to one of three groups (n = 14–18/group). Exercise aged mice (EX-aged) (18 months old) ran on a treadmill for 45 min/day, 6 days a week for 6 weeks at 18m/min, 5% grade. Control aged mice (CON-aged) (18 months old) and control adult mice (CON-adult) (5 months old) remained in their cages during this time. Following the last day of training or rest, mice were intranasally inoculated with Herpes Simplex Virus-1 (HSV-1). They were monitored twice daily for morbidity and mortality as well as symptom severity for 21 days. Additional mice (n = 10/per group) were inoculated with HSV-1 and sacrificed on day 4 following infection. Lungs were analyzed for viral titers and inflammatory cytokine responses. RESULTS: Ageing was associated with a significant increase in morbidity (47%, P = 0.0015), mortality (45%, P = 0.007) and symptom severity score on days 5 through 11 post-infection (P<0.05). Exercise reduced the increase in morbidity (23%, P = 0.009) and symptom severity score on days 5, 7 and 8 post-infection (P<0.05). There was no difference in mortality. Ageing resulted in a trend toward an increase in lung viral titers (P = 0.1) with an associated increase in IL-1β (P<0.001). Exercise reduced this increase in lung viral titers (P = 0.07) and IL-1β (P = 0.003). CONCLUSION: These data suggest that moderate exercise training can help restore some of the negative effects of ageing on immune function and therefore reduce susceptibility to infection in mice. Funded by a grant from the American College of Sports Medicine

EFFECTS OF OAT BETA-GLUCAN ON SUSCEPTIBILITY TO INFECTION AND MACROPHAGE ANTI-VIRAL RESISTANCE FOLLOWING STRENUOUS EXERCISE

Intense exercise is associated with increased risk for URTI. Consumption of oat beta-glucan (Obg), a soluble fiber, is a mild immune system enhancer that may offset immune suppression with intense training. PURPOSE: We studied the effects of Obg on susceptibility to infection following short-term exhaustive exercise training using our mouse model of respiratory infection (Davis et.al. J. Appl. Physiol. 83 (5): 1461-6 1997). Macrophage (Mo) anti-viral resistance was also determined as a potential mechanism of this effect. METHODS: Male CD-1 mice (n = 96) were randomly assigned to one of four groups. Exercise mice (EX-obg and EX-h2o) ran to volitional fatigue on a treadmill for 3 consecutive days at 36m/min, 8% grade. Control mice (C-obg and C-h2o) were exposed to the same environment, but remained in their cages throughout the exercise period. Obg was consumed in the drinking water (3.6mg/day) (EX-obg and C-obg) for 10 consecutive days prior to virus inoculation. Following rest or exercise on the last day of training, mice were intra-nasally inoculated with herpes simplex virus-1 (HSV-1). They were monitored twice daily for morbidity and mortality for 21 days. Additional mice (n = 72) were sacrificed following exercise; peritoneal macrophages were obtained via i.p. lavage and assayed for anti-viral resistance to HSV-1. RESULTS: EX increased morbidity by 25% and mortality by 17% (P < 0.05), and ingestion of Obg prevented this increase in morbidity and mortality (P < 0.05). EX also decreased Mo anti-viral resistance (P < 0.05) and Obg prevented this decrease (P < 0.05). CONCLUSION: These data suggest that consumption of Obg may offset the increased risk of infection during heavy training, and that this maybe mediated, at least in part, by an increase in Mo anti-viral function.

Role of the polymorphic region 1 of the Bordetella pertussis protein pertactin in immunity.

In several countries pertussis is re-emerging, despite a high vaccination coverage. It is suggested that antigenic divergence between Bordetella pertussis vaccine strains and circulating strains, in particular with respect to pertactin, has contributed to pertussis re-emergence. Polymorphism in pertactin is essentially limited to region 1, which is composed of repeats and is located adjacent to an Arg-Gly-Asp motif implicated in adherence. Evidence is provided for the immunological relevance of polymorphism in region 1. Region 1 was found to contain a B-cell epitope recognized in both humans and mice. Furthermore, variation in region 1 affected antibody binding and, in a mouse respiratory infection model, the efficacy of a whole-cell vaccine. Moreover, passive and active immunization indicated that region 1 confers protective immunity. An mAb directed against a linear conserved epitope conferred cross-immunity against isolates with distinct pertactin variants. The results indicate an important role of region 1 of pertactin in immunity.

Bordetella pertussis TonB, a Bvg-independent virulence determinant.

In gram-negative bacteria, high-affinity iron uptake requires the TonB/ExbB/ExbD envelope complex to release iron chelates from their specific outer membrane receptors into the periplasm. Based on sequence similarities, the Bordetella pertussis tonB exbB exbD locus was identified on a cloned DNA fragment. The tight organization of the three genes suggests that they are cotranscribed. A putative Fur-binding sequence located upstream from tonB was detected in a Fur titration assay, indicating that the tonB exbB exbD operon may be Fur-repressed in high-iron growth conditions. Putative structural genes of the beta-subunit of the histone-like protein HU and of a new two-component regulatory system were identified upstream from tonB and downstream from exbD, respectively. A B. pertussis DeltatonB exbB::Km(r) mutant was constructed by allelic exchange and characterized. The mutant was impaired for growth in low-iron medium in vitro and could not use ferrichrome, desferal, or hemin as iron sources. Levels of production of the major bacterial toxins and adhesins were similar in the TonB(+)/TonB(-) pair. The DeltatonB exbB mutant was still responsive to chemical modulators of virulence; thus, the BvgA/BvgS two-component system is not TonB dependent. Nevertheless, in vivo in the mouse respiratory infection model, the colonization ability of the mutant was reduced compared to the parental strain.