Gary B. Huffnagle

Analysis of the Lung Microbiome in the “Healthy” Smoker and in COPD

Although culture-independent techniques have shown that the lungs are not sterile, little is known about the lung microbiome in chronic obstructive pulmonary disease (COPD). We used pyrosequencing of 16S amplicons to analyze the lung microbiome in two ways: first, using bronchoalveolar lavage (BAL) to sample the distal bronchi and air-spaces; and second, by examining multiple discrete tissue sites in the lungs of six subjects removed at the time of transplantation. We performed BAL on three never-smokers (NS) with normal spirometry, seven smokers with normal spirometry (“heathy smokers”, HS), and four subjects with COPD (CS). Bacterial 16 s sequences were found in all subjects, without significant quantitative differences between groups. Both taxonomy-based and taxonomy-independent approaches disclosed heterogeneity in the bacterial communities between HS subjects that was similar to that seen in healthy NS and two mild COPD patients. The moderate and severe COPD patients had very limited community diversity, which was also noted in 28% of the healthy subjects. Both approaches revealed extensive membership overlap between the bacterial communities of the three study groups. No genera were common within a group but unique across groups. Our data suggests the existence of a core pulmonary bacterial microbiome that includes Pseudomonas, Streptococcus, Prevotella, Fusobacterium, Haemophilus, Veillonella, and Porphyromonas. Most strikingly, there were significant micro-anatomic differences in bacterial communities within the same lung of subjects with advanced COPD. These studies are further demonstration of the pulmonary microbiome and highlight global and micro-anatomic changes in these bacterial communities in severe COPD patients.

Antibiotic-induced shifts in the mouse gut microbiome and metabolome increase susceptibility to Clostridium difficile infection

Antibiotics can have significant and long lasting effects on the gastrointestinal tract microbiota, reducing colonization resistance against pathogens including Clostridium difficile. Here we show that antibiotic treatment induces substantial changes in the gut microbial community and in the metabolome of mice susceptible to C. difficile infection. Levels of secondary bile acids, glucose, free fatty acids, and dipeptides decrease, whereas those of primary bile acids and sugar alcohols increase, reflecting the modified metabolic activity of the altered gut microbiome. In vitro and ex vivo analyses demonstrate that C. difficile can exploit specific metabolites that become more abundant in the mouse gut after antibiotics, including primary bile acid taurocholate for germination, and carbon sources mannitol, fructose, sorbitol, raffinose and stachyose for growth. Our results indicate that antibiotic-mediated alteration of the gut microbiome converts the global metabolic profile to one that favors C. difficile germination and growth.

The ‘microflora hypothesis’ of allergic diseases

Increasingly, epidemiologic and clinical data support the hypothesis that perturbations in the gastrointestinal (GI) microbiota because of antibiotic use and dietary differences in ‘industrialized’ countries have disrupted the normal microbiota‐mediated mechanisms of immunological tolerance in the mucosa, leading to an increase in the incidence of allergic airway disease. The data supporting this ‘microflora hypothesis’ includes correlations between allergic airway disease and (1) antibiotic use early in life, (2) altered fecal microbiota and (3) dietary changes over the past two decades. Our laboratory has recently demonstrated that mice can develop allergic airway responses to allergens if their endogenous microbiota is altered at the time of first allergen exposure. These experimental and clinical observations are consistent with other studies demonstrating that the endogenous microbiota plays a significant role in shaping the development of the immune system. Data are beginning to accumulate that a ‘balanced’ microbiota plays a positive role in maintaining mucosal immunologic tolerance long after post‐natal development. Other studies have demonstrated that even small volumes delivered to the nasopharynx largely end up in the GI tract, suggesting that airway tolerance and oral tolerance may operate simultaneously. The mechanism of microbiota modulation of host immunity is not known; however, host and microbial oxylipins are one potential set of immunomodulatory molecules that may control mucosal tolerance. The cumulative data are beginning to support the notion that probiotic and prebiotic strategies be considered for patients coming off of antibiotic therapy.

Leptin-Deficient Mice Exhibit Impaired Host Defense in Gram-Negative Pneumonia

Leptin is an adipocyte-derived hormone that is secreted in correlation with total body lipid stores. Serum leptin levels are lowered by the loss of body fat mass that would accompany starvation and malnutrition. Recently, leptin has been shown to modulate innate immune responses such as macrophage phagocytosis and cytokine synthesis in vitro. To determine whether leptin plays a role in the innate host response against Gram-negative pneumonia in vivo, we compared the responses of leptin-deficient and wild-type mice following an intratracheal challenge of Klebsiella pneumoniae. Following K. pneumoniae administration, we observed increased leptin levels in serum, bronchoalveolar lavage fluid, and whole lung homogenates. In a survival study, leptin-deficient mice, as compared with wild-type mice, exhibited increased mortality following K. pneumoniae administration. The increased susceptibility to K. pneumoniae in the leptin-deficient mice was associated with reduced bacterial clearance and defective alveolar macrophage phagocytosis in vitro. The exogenous addition of very high levels of leptin (500 ng/ml) restored the defect in alveolar macrophage phagocytosis of K. pneumoniae in vitro. While there were no differences between wild-type and leptin-deficient mice in lung homogenate cytokines TNF-α, IL-12, or macrophage-inflammatory protein-2 after K. pneumoniae administration, leukotriene synthesis in lung macrophages from leptin-deficient mice was reduced. Leukotriene production was restored by the addition of exogenous leptin (500 ng/ml) to macrophages in vitro. This study demonstrates for the first time that leptin-deficient mice display impaired host defense in bacterial pneumonia that may be due to a defect in alveolar macrophage phagocytosis and leukotriene synthesis.

CCR2 expression determines T1 versus T2 polarization during pulmonary Cryptococcus neoformans infection.

Pulmonary clearance of the encapsulated yeast Cryptococcus neoformans requires the development of T1-type immunity. The objective of this study was to determine the role of CCR2 in leukocyte recruitment and development of T1-type cell-mediated immunity during pulmonary C. neoformans infection. Intratracheal inoculation of C. neoformans into CCR2 knockout (CCR2−/−) mice produced a prolonged pulmonary infection (5000-fold CFU at 6 wk compared with CCR2+/+ mice) and significant dissemination to the spleen and brain (160- and 800-fold greater). In addition, CCR2 deficiency resulted in significantly reduced recruitment of macrophages (weeks 1–3) and CD8+ T cells (weeks 1–2) into the lungs. The immune response in CCR2−/− mice was characterized by chronic pulmonary eosinophilia, crystal deposition in the lungs, pulmonary leukocyte production of IL-4 and IL-5 but not IFN-γ, lack of anticryptococcal delayed-type hypersensitivity, and high levels of serum IgE. These results demonstrate that expression of CCR2 is required for the development of a T1-type response to C. neoformans infection and lack of CCR2 results in a switch to a T2-type response. Thus, CCR2 plays a critical role in promoting the development of T1- over T2-type immune responses in the lung following cryptococcus infection.

Role of Antibiotics and Fungal Microbiota in Driving Pulmonary Allergic Responses

ABSTRACT Over the past four decades, there has been a significant increase in allergy and asthma in westernized countries, which correlates with alterations in fecal microbiota (microflora) and widespread use of antibiotics (the “hygiene hypothesis”). Antibiotics also lead to overgrowth of the yeast Candida albicans, which can secrete potent prostaglandin-like immune response modulators. We have developed a mouse model of antibiotic-induced microbiota disruption that includes stable increases in gastrointestinal (GI) enteric bacteria and GI Candida levels with no introduction of microbes into the lungs. Mice are treated for 5 days with cefoperazone in the drinking water, followed by a single oral gavage of C. albicans. This results in alterations of GI bacterial populations and increased yeast numbers in the GI microbiota for at least 2 to 3 weeks and can drive the development of a CD4 T-cell-mediated allergic airway response to subsequent mold spore (Aspergillus fumigatus) exposure in immunocompetent mice without previous systemic antigen priming. The allergic response in the lungs is characterized by increased levels of eosinophils, mast cells, interleukin-5 (IL-5), IL-13, gamma interferon, immunoglobulin E, and mucus-secreting cells. In the absence of antibiotics, mice exposed to Aspergillus spores do not develop an allergic response in the airways. This study provides the first experimental evidence to support a role for antibiotics and fungal microbiota in promoting the development of allergic airway disease. In addition, these studies also highlight the concept that events in distal mucosal sites such as the GI tract can play an important role in regulating immune responses in the lungs.

Analysis of the Upper Respiratory Tract Microbiotas as the Source of the Lung and Gastric Microbiotas in Healthy Individuals

ABSTRACT No studies have examined the relationships between bacterial communities along sites of the upper aerodigestive tract of an individual subject. Our objective was to perform an intrasubject and intersite analysis to determine the contributions of two upper mucosal sites (mouth and nose) as source communities for the bacterial microbiome of lower sites (lungs and stomach). Oral wash, bronchoalveolar lavage (BAL) fluid, nasal swab, and gastric aspirate samples were collected from 28 healthy subjects. Extensive analysis of controls and serial intrasubject BAL fluid samples demonstrated that sampling of the lungs by bronchoscopy was not confounded by oral microbiome contamination. By quantitative PCR, the oral cavity and stomach contained the highest bacterial signal levels and the nasal cavity and lungs contained much lower levels. Pyrosequencing of 16S rRNA gene amplicon libraries generated from these samples showed that the oral and gastric compartments had the greatest species richness, which was significantly greater in both than the richness measured in the lungs and nasal cavity. The bacterial communities of the lungs were significantly different from those of the mouth, nose, and stomach, while the greatest similarity was between the oral and gastric communities. However, the bacterial communities of healthy lungs shared significant membership with the mouth, but not the nose, and marked subject-subject variation was noted. In summary, microbial immigration from the oral cavity appears to be the significant source of the lung microbiome during health, but unlike the stomach, the lungs exhibit evidence of selective elimination of Prevotella bacteria derived from the upper airways. IMPORTANCE We have demonstrated that the bacterial communities of the healthy lung overlapped those found in the mouth but were found at lower concentrations, with lower membership and a different community composition. The nasal microbiome, which was distinct from the oral microbiome, appeared to contribute little to the composition of the lung microbiome in healthy subjects. Our studies of the nasal, oral, lung, and stomach microbiomes within an individual illustrate the microbiological continuity of the aerodigestive tract in healthy adults and provide culture-independent microbiological support for the concept that microaspiration is common in healthy individuals. We have demonstrated that the bacterial communities of the healthy lung overlapped those found in the mouth but were found at lower concentrations, with lower membership and a different community composition. The nasal microbiome, which was distinct from the oral microbiome, appeared to contribute little to the composition of the lung microbiome in healthy subjects. Our studies of the nasal, oral, lung, and stomach microbiomes within an individual illustrate the microbiological continuity of the aerodigestive tract in healthy adults and provide culture-independent microbiological support for the concept that microaspiration is common in healthy individuals.

Urokinase is required for the pulmonary inflammatory response to Cryptococcus neoformans. A murine transgenic model.

Urokinase (uPA) is hypothesized to provide proteolytic activity enabling inflammatory cells to traverse tissues during recruitment, and it is implicated as a cytokine modulator. Definitive evaluation of these hypotheses in vivo has previously been impossible because uPA could not completely and irreversibly be eliminated. This limitation has been overcome through the development of uPA-deficient transgenic mice (uPA-/-). Using these mice, we evaluated the importance of uPA in the pulmonary inflammatory response to Cryptococcus neoformans (strain 52D). C. neoformans was inoculated into uPA-/- and control mice (uPA+/+), and cell recruitment to the lungs was quantitated. The number of CFU in lung, spleen and brain was determined to assess clearance, and survival curves were generated. By day 21 after inoculation, uPA-/- mice had markedly fewer pulmonary inflammatory (CD45+), CD4+, and CD11b/CD18+ cells compared with uPA+/+ controls (P<0.0007); pulmonary CFUs in the uPA-/- mice continued to increase, whereas CFUs diminished in uPA+/+ mice(P<0.005). In survival studies, only 3/19 uPA+/+ mice died, whereas 15/19 uPA-/- mice died (p<0.001). We have demonstrated that uPA is required for a pulmonary inflammatory response to C. neoformans. Lack of uPA results in inadequate cellular recruitment, uncontrolled infection, and death.

The role of CD4+ and CD8+ T cells in the protective inflammatory response to a pulmonary cryptococcal infection

Moderately virulent strains of Cryptococcusneoformans, inoculated via the trachea, cause a pulmonary infection in BALB/c mice that was gradually resolved by T lymphocyte‐dependent mechanisms. The current studies, using monoclonal antibodies to deplete T cell subsets, demonstrated that CD4+ and CD8+ T cells combined to mediate a prominent pulmonary inflammatory infiltrate that included lymphocytes, macrophages, neutrophils, and eosinophils. The inflammatory response peaked 2 weeks after infection and coincided with the beginning of gradual pulmonary clearance of the infection. CD4/CD8 double, deficiency (4‐8‐) markedly reduced the influx of all cells into the lungs. A CD4 deficiency had a more profound effect on the total number of inflammatory cells recruited to the lungs than a CD8 deficiency. Depletion of either CD8+ or CD4+ T cells significantly decreased pulmonary macrophages and neutrophils, but only a CD4 deficiency prevented the influx of eosinophils. Recruitment of CD8+ T cells occurred independently of CD4+ T cells, but CD4+ T cell recruitment to the lungs was significantly reduced in CD8‐deficient mice. Mitogen‐stimulated infiltrating lung lymphocytes from infected 4+8+ mice secreted both T helper cell type 1 (Th1) [interferon‐γ (IFN‐γ) and interleukin‐2 (IL‐2)] and Th2 (IL‐4, IL‐5, and IL‐10) cytokines. CD4 deficiency resulted in loss of T cells secreting IL‐4, IL‐5, and IL‐10. However, residual CD8+ T cells still secreted IL‐2 and IFN‐γ. Lung T cells from CD8‐deficient mice secreted similar levels of IL‐4, IL‐5, and IL‐10 on a per lung basis compared with 4+8+ mice despite decreased numbers of CD4+ T cells, but secreted reduced levels of IFN‐γ. These experiments indicate that (1) CD4+ T cells play a dominant role in recruiting macrophages and granulocytes to the lung and (2) CD8+ T cells also mediate cellular recruitment, increase the magnitude of CD4+ T cell numbers in the infiltrate, and contribute to the local secretion of IFN‐γ. Thus, these studies demonstrate that CD8+ T cells can independently mediate an inflammatory response to a large, particulate, extracellular antigen, a role heretofore attributed almost solely to CD4+ T cells. J. Leukoc. Biol. 55: 35–42; 1994.

Pathogenic Yeasts Cryptococcus neoformans and Candida albicans Produce Immunomodulatory Prostaglandins

ABSTRACT Enhanced prostaglandin production during fungal infection could be an important factor in promoting fungal colonization and chronic infection. Host cells are one source of prostaglandins; however, another potential source of prostaglandins is the fungal pathogen itself. Our objective was to determine if the pathogenic yeastsCryptococcus neoformans and Candida albicansproduce prostaglandins and, if so, to begin to define the role of these bioactive lipids in yeast biology and disease pathogenesis. C. neoformans and C. albicans both secreted prostaglandins de novo or via conversion of exogenous arachidonic acid. Treatment with cyclooxygenase inhibitors dramatically reduced the viability of the yeast and the production of prostaglandins, suggesting that an essential cyclooxygenase like enzyme may be responsible for fungal prostaglandin production. A PGE series lipid was purified from both C. albicans and C. neoformans and was biologically active on both fungal and mammalian cells. Fungal PGEx and synthetic PGE2 enhanced the yeast-to-hypha transition in C. albicans. Furthermore, in mammalian cells, fungal PGEx down-modulated chemokine production, tumor necrosis factor alpha production, and splenocyte proliferation while up-regulating interleukin 10 production. These are all activities previously documented for mammalian PGE2. Thus, eicosanoids are produced by pathogenic fungi, are critical for growth of the fungi, and can modulate host immune functions. The discovery that pathogenic fungi produce and respond to immunomodulatory eicosanoids reveals a virulence mechanism that has potentially great implications for understanding the mechanisms of chronic fungal infection, immune deviation, and fungi as disease cofactors.