Nicole R. Falkowski

Development of Allergic Airway Disease in Mice following Antibiotic Therapy and Fungal Microbiota Increase: Role of Host Genetics, Antigen, and Interleukin-13

ABSTRACT Lending support to the hygiene hypothesis, epidemiological studies have demonstrated that allergic disease correlates with widespread use of antibiotics and alterations in fecal microbiota (“microflora”). Antibiotics also lead to overgrowth of the yeast Candida albicans, which can secrete potent prostaglandin-like immune response modulators, from the microbiota. We have recently developed a mouse model of antibiotic-induced gastrointestinal microbiota disruption that is characterized by stable increases in levels of gastrointestinal enteric bacteria and Candida. Using this model, we have previously demonstrated that microbiota disruption can drive the development of a CD4 T-cell-mediated airway allergic response to mold spore challenge in immunocompetent C57BL/6 mice without previous systemic antigen priming. The studies presented here address important questions concerning the universality of the model. To investigate the role of host genetics, we tested BALB/c mice. As with C57BL/6 mice, microbiota disruption promoted the development of an allergic response in the lungs of BALB/c mice upon subsequent challenge with mold spores. In addition, this allergic response required interleukin-13 (IL-13) (the response was absent in IL-13−/− mice). To investigate the role of antigen, we subjected mice with disrupted microbiota to intranasal challenge with ovalbumin (OVA). In the absence of systemic priming, only mice with altered microbiota developed airway allergic responses to OVA. The studies presented here demonstrate that the effects of microbiota disruption are largely independent of host genetics and the nature of the antigen and that IL-13 is required for the airway allergic response that follows microbiota disruption.

Candida albicans and Bacterial Microbiota Interactions in the Cecum during Recolonization following Broad-Spectrum Antibiotic Therapy

ABSTRACT Candida albicans is a normal member of the gastrointestinal (GI) tract microbiota of healthy humans, but during host immunosuppression or alterations in the bacterial microbiota, C. albicans can disseminate and cause life-threatening illness. The bacterial microbiome of the GI tract, including lactic acid bacteria (LAB), plays a vital role in preventing fungal invasion. However, little is known about the role of C. albicans in shaping the bacterial microbiota during antibiotic recovery. We investigated the fungal burdens in the GI tracts of germfree mice and mice with a disturbed microbiome to demonstrate the role of the microbiota in preventing C. albicans colonization. Histological analysis demonstrated that colonization with C. albicans during antibiotic treatment does not trigger overt inflammation in the murine cecum. Bacterial diversity is reduced long term following cefoperazone treatment, but the presence of C. albicans during antibiotic recovery promoted the recovery of bacterial diversity. Cefoperazone diminishes Bacteroidetes populations long term in the ceca of mice, but the presence of C. albicans during cefoperazone recovery promoted Bacteroidetes population recovery. However, the presence of C. albicans resulted in a long-term reduction in Lactobacillus spp. and promoted Enterococcus faecalis populations. Previous studies have focused on the ability of bacteria to alter C. albicans; this study addresses the ability of C. albicans to alter the bacterial microbiota during nonpathogenic colonization.

Interplay between the Gastric Bacterial Microbiota and Candida albicans during Postantibiotic Recolonization and Gastritis

ABSTRACT The indigenous bacterial microbiome of the stomach, including lactobacilli, is vital in promoting colonization resistance against Candida albicans. However, there are gaps in our understanding about C. albicans gastric colonization versus disease, especially during the postantibiotic recovery phase. This study compared the gastric responses to C. albicans strains CHN1 and SC5314 in microbiome-disturbed and germfree mice to elucidate the contribution of the indigenous microbiota in C. albicans colonization versus disease and yeast-bacterium antagonism during the post-cefoperazone recolonization period. C. albicans can prevent the regrowth of Lactobacillus spp. in the stomach after cefoperazone and promote increased colonization by Enterococcus spp. Using a culture-independent analysis, the effects of oral cefoperazone on the gastric bacterial microbiota were observed to last at least 3 weeks after the cessation of the antibiotic. Disturbance of the gastric bacterial community by cefoperazone alone was not sufficient to cause gastritis, C. albicans colonization was also needed. Gastritis was not evident until after day 7 in cefoperazone-treated infected mice. In contrast, in germfree mice which lack a gastric microbiota, C. albicans induced gastric inflammation within 1 week of inoculation. Therefore, the gastric bacterial community in cefoperazone-treated mice during the first week of postantibiotic recolonization was sufficient to prevent the development of gastritis, despite being ineffective at conferring colonization resistance against C. albicans. Altogether, these data implicate a dichotomy between C. albicans colonization and gastric disease that is bacterial microbiome dependent.

Transient Neutralization of Tumor Necrosis Factor Alpha Can Produce a Chronic Fungal Infection in an Immunocompetent Host: Potential Role of Immature Dendritic Cells

ABSTRACT The mechanisms underlying induction of immune dysregulation and chronic fungal infection by a transient tumor necrosis factor alpha (TNF-α) deficiency remain to be defined. The objective of our studies was to determine the potential contribution of neutropenia and immature dendritic cells to the immune deviation. Administration of an anti-TNF-α monoclonal antibody at day 0 neutralized TNF-α only during the first week of a pulmonary Cryptococcus neoformans infection. Transient neutralization of TNF-α resulted in transient depression of interleukin-12 (IL-12), monocyte chemotactic protein 1 (MCP-1), and gamma interferon (IFN-γ) production but permanently impaired long-term clearance of the infection from the lungs even after the levels of these cytokines increased and a vigorous inflammatory response developed. Early neutrophil recruitment was defective in the absence of TNF-α. However, as demonstrated by neutrophil depletion studies, this did not account for the decrease in IL-12 and IFN-γ levels and did not play a role in establishing chronic pulmonary cryptococcal infection. Transient TNF-α neutralization also produced a deficiency in CD11c+ MHC II+ cells and IL-12 in the lymph nodes, potentially implicating a defect in mature dendritic cell trafficking. Transfer of cryptococcal antigen-pulsed immature dendritic cells into naïve mice prior to intratracheal challenge resulted in the development of a nonprotective immune response to C. neoformans that was similar to that observed in anti-TNF-α-treated mice (increased IL-4, IL-5, and IL-10 levels, pulmonary eosinophilia, and decreased clearance). Thus, stimulation of an antifungal response by immature dendritic cells can result in an immune deviation similar to that produced by transient TNF-α deficiency, identifying a new mechanism by which a chronic fungal infection can occur in an immunocompetent host.

Enrichment of the lung microbiome with gut bacteria in sepsis and the acute respiratory distress syndrome.

Sepsis and the acute respiratory distress syndrome (ARDS) are major causes of mortality without targeted therapies. Although many experimental and clinical observations have implicated gut microbiota in the pathogenesis of these diseases, culture-based studies have failed to demonstrate translocation of bacteria to the lungs in critically ill patients. Here, we report culture-independent evidence that the lung microbiome is enriched with gut bacteria both in a murine model of sepsis and in humans with established ARDS. Following experimental sepsis, lung communities were dominated by viable gut-associated bacteria. Ecological analysis identified the lower gastrointestinal tract, rather than the upper respiratory tract, as the likely source community of post-sepsis lung bacteria. In bronchoalveolar lavage fluid from humans with ARDS, gut-specific bacteria (Bacteroides spp.) were common and abundant, undetected by culture and correlated with the intensity of systemic inflammation. Alveolar TNF-α, a key mediator of alveolar inflammation in ARDS, was significantly correlated with altered lung microbiota. Our results demonstrate that the lung microbiome is enriched with gut-associated bacteria in sepsis and ARDS, potentially representing a shared mechanism of pathogenesis in these common and lethal diseases.

Interleukin-17 Drives Pulmonary Eosinophilia following Repeated Exposure to Aspergillus fumigatus Conidia

ABSTRACT Previous research in our laboratory has demonstrated that repeated intranasal exposure to Aspergillus fumigatus conidia in C57BL/6 mice results in a chronic pulmonary inflammatory response that reaches its maximal level after four challenges. The inflammatory response is characterized by eosinophilia, goblet cell metaplasia, and T helper TH2 cytokine production, which is accompanied by sustained interleukin-17 (IL-17) expression that persists even after the TH2 response has begun to resolve. TH17 cells could develop in mice deficient in gamma interferon (IFN-γ), IL-4, or IL-10. In the lungs of IL-17 knockout mice repeatedly challenged with A. fumigatus conidia, inflammation was attenuated (with the most significant decrease occurring in eosinophils), conidial clearance was enhanced, and the early transient peak of CD4+ CD25+ FoxP3+ cells blunted. IL-17 appeared to play only a minor role in eosinophil differentiation in the bone marrow but a central role in eosinophil extravasation from the blood into the lungs. These observations point to an expanded role for IL-17 in driving TH2-type inflammation to repeated inhalation of fungal conidia.

Bacterial Topography of the Healthy Human Lower Respiratory Tract

ABSTRACT Although culture-independent techniques have refuted lung sterility in health, controversy about contamination during bronchoscope passage through the upper respiratory tract (URT) has impeded research progress. We sought to establish whether bronchoscopic sampling accurately reflects the lung microbiome in health and to distinguish between two proposed routes of authentic microbial immigration, (i) dispersion along contiguous respiratory mucosa and (ii) subclinical microaspiration. During bronchoscopy of eight adult volunteers without lung disease, we performed seven protected specimen brushings (PSB) and bilateral bronchoalveolar lavages (BALs) per subject. We amplified, sequenced, and analyzed the bacterial 16S rRNA gene V4 regions by using the Illumina MiSeq platform. Rigorous attention was paid to eliminate potential sources of error or contamination, including a randomized processing order and the inclusion and analysis of exhaustive procedural and sequencing control specimens. Indices of mouth-lung immigration (mouth-lung community similarity, bacterial burden, and community richness) were all significantly greater in airway and alveolar specimens than in bronchoscope contamination control specimens, indicating minimal evidence of pharyngeal contamination. Ecological indices of mouth-lung immigration peaked at or near the carina, as predicted for a primary immigration route of microaspiration. Bacterial burden, diversity, and mouth-lung similarity were greater in BAL than PSB samples, reflecting differences in the sampled surface areas. (This study has been registered at ClinicalTrials.gov under registration no. NCT02392182.) IMPORTANCE This study defines the bacterial topography of the healthy human respiratory tract and provides ecological evidence that bacteria enter the lungs in health primarily by microaspiration, with potential contribution in some subjects by direct dispersal along contiguous mucosa. By demonstrating that contamination contributes negligibly to microbial communities in bronchoscopically acquired specimens, we validate the use of bronchoscopy to investigate the lung microbiome. This study defines the bacterial topography of the healthy human respiratory tract and provides ecological evidence that bacteria enter the lungs in health primarily by microaspiration, with potential contribution in some subjects by direct dispersal along contiguous mucosa. By demonstrating that contamination contributes negligibly to microbial communities in bronchoscopically acquired specimens, we validate the use of bronchoscopy to investigate the lung microbiome.

Total Parenteral Nutrition–Associated Lamina Propria Inflammation in Mice Is Mediated by a MyD88-Dependent Mechanism

Enteral nutrient deprivation via total parenteral nutrition (TPN) administration leads to local mucosal inflammatory responses, but the underlying mechanisms are unknown. Wild-type (WT) and MyD88−/− mice underwent jugular vein cannulation. One group received TPN without chow, and controls received standard chow. After 7 d, we harvested intestinal mucosally associated bacteria and isolated small-bowel lamina propria (LP) cells. Bacterial populations were analyzed using 454 pyrosequencing. LP cells were analyzed using quantitative PCR and multicolor flow cytometry. WT, control mucosally associated microbiota were Firmicutes-dominant, whereas WT TPN mice were Proteobacteria-domiant. Similar changes were observed in MyD88−/− mice with TPN administration. UniFrac analysis showed divergent small bowel and colonic bacterial communities in controls, merging toward similar microbiota (but distinct from controls) with TPN. The percentage of LP T regulatory cells significantly decreased with TPN in WT mice. F4/80+CD11b+CD11cdull/− macrophage–derived proinflammatory cytokines significantly increased with TPN. These proinflammatory immunologic changes were significantly abrogated in MyD88−/− TPN mice. Thus, TPN administration is associated with significant expansion of Proteobacteria within the intestinal microbiota and increased proinflammatory LP cytokines. Additionally, MyD88 signaling blockade abrogated decline in epithelial cell proliferation and epithelial barrier function loss.

Modulation of Post-Antibiotic Bacterial Community Reassembly and Host Response by Candida albicans

The introduction of Candida albicans into cefoperazone-treated mice results in changes in bacterial community reassembly. Our objective was to use high-throughput sequencing to characterize at much greater depth the specific changes in the bacterial microbiome. The colonization of C. albicans significantly altered bacterial community reassembly that was evident at multiple taxonomic levels of resolution. There were marked changes in the levels of Bacteriodetes and Lactobacillaceae. Lachnospiraceae and Ruminococcaceae, the two most abundant bacterial families, did not change in relative proportions after antibiotics, but there were marked genera-level shifts within these two bacterial families. The microbiome shifts occurred in the absence of overt intestinal inflammation. Overall, these experiments demonstrate that the introduction of a single new microbe in numerically inferior numbers into the bacterial microbiome during a broad community disturbance has the potential to significantly alter the subsequent reassembly of the bacterial community as it recovers from that disturbance.

Repeated exposure to Aspergillus fumigatus conidia results in CD4+ T cell-dependent and -independent pulmonary arterial remodeling in a mixed Th1/Th2/Th17 microenvironment that requires interleukin-4 (IL-4) and IL-10.

ABSTRACT Pulmonary arterial remodeling is a pathological process seen in a number of clinical disease states, driven by inflammatory cells and mediators in the remodeled artery microenvironment. In murine models, Th2 cell-mediated immune responses to inhaled antigens, such as purified Aspergillus allergen, have been reported to induce remodeling of pulmonary arteries. We have previously shown that repeated intranasal exposure of healthy C57BL/6 mice to viable, resting Aspergillus fumigatus conidia leads to the development of chronic pulmonary inflammation and the coevolution of Th1, Th2, and Th17 responses in the lungs. Our objective was to determine whether repeated intranasal exposure to Aspergillus conidia would induce pulmonary arterial remodeling in this mixed Th inflammatory microenvironment. Using weekly intranasal conidial challenges, mice developed robust pulmonary arterial remodeling after eight exposures (but not after two or four). The process was partially mediated by CD4+ T cells and by interleukin-4 (IL-4) production, did not require eosinophils, and was independent of gamma interferon (IFN-γ) and IL-17. Furthermore, remodeling could occur even in the presence of strong Th1 and Th17 responses. Rather than serving an anti-inflammatory function, IL-10 was required for the development of the Th2 response to A. fumigatus conidia. However, in contrast to previous studies of pulmonary arterial remodeling driven by the A. fumigatus allergen, viable conidia also stimulated pulmonary arterial remodeling in the absence of CD4+ T cells. Remodeling was completely abrogated in IL-10−/− mice, suggesting that a second, CD4+ T cell-independent, IL-10-dependent pathway was also driving pulmonary arterial remodeling in response to repeated conidial exposure.