Murine Lung Microbiome Webinar Recap

On June 8, Dr. Kenneth Klingenberg Barfod — of the National Research Centre for the Working Environment, Denmark — presented a webinar on the importance of the murine lung microbiome, how the lung microbiome in mice can be manipulated experimentally, and its applications in disease modeling.

Compared to the extensively studied gut microbiome, the lung microbiome is a niche research area with relatively few published reports. A common — mistaken — assumption has been that healthy lungs are sterile, though no modern studies to date have shown absence of microorganisms in the lungs¹. While challenges exist in sampling from the lungs, the presence of a lung microbiome in healthy humans, and mice, is now considered a fact.

Dr. Barfod discussed how the presence of microbes in healthy lungs is a balance between immigration and elimination.

The Lung Microbiome in Health and Disease

The composition of the lung microbiome has been correlated to various human diseases: asthma, acute respiratory distress syndrome, chronic obstructive pulmonary disease, HIV, and cystic fibrosis.

In the webinar, Dr. Barfod explained how the lung microbiome is expected to be under the influence of many of the same factors known to impact the gut microbiome: Mode of delivery, antibiotics, breast vs. formula feeding, diet, infection and vaccination history, genetics, environmental impact such as pollution, vitamin D levels due to sun exposure, and more. All these factors may shape the immune system of the lungs, either directly or through alteration of the lung microbiome.

The gut microbiota is the largest and most diverse community of the human microbiome, and therefore perceived as the larger influence on human health relative to the lung microbiome. However, in relation to the risk of lung diseases specifically, Dr. Barfod theorized that the lung microbiome in early life may be a more important determiner than the gut microbiome (Figure 1), but that this may be opposite later in life.

Mice are relevant animal models for investigating causality of the lung microbiome in disease. Dr. Barfod emphasized the need for establishing actual, casual relationships for moving beyond the observed correlations between human disease and the lung microbiome.

Figure 1: Theoretical Importance of Microbiota. Courtesy of K.K. Barfod.

In this regard, animal models were highlighted as crucial. The in vivo approach can be used to gain mechanistic insight through manipulations of the lung microbiome with antibiotics, probiotics, pulmonary medicines, or by creating genetically modified animals to mimic human diseases and conditions. Additionally, animal models allow for sampling approaches not possible in humans.

The research contributions of Dr. Barfod and his colleagues have shown that there is an actual microbial community in the lungs of mice, not just contamination by or a reflection of the gut microbiota^3,4, and that the murine lung microbiome may be manipulated by intranasal antibiotic treatment³. This vindicates mice as relevant models in lung microbiome research.

Microbiome Study Design Factors

Dr. Barfod highlighted the myriad of factors that may affect and potentially bias animal studies. As a scientist, it is essential to know if your study subjects are independent of each other for choosing the proper analytical and statistical approaches. Some of the things you would want to know are:

Dr. Barfod included a useful example of a sound experimental design, taking these factors into account for optimal utilization of animal models.

As differences in the lung microbiota has been observed between male and female mice^3,4,5, he also put forward the relevance of including both sexes in the experiment.

Sampling and Studying the Murine Lung Microbiome

Dr. Barfod went on to discuss common technical challenges when studying the murine lung microbiome. As the bacterial DNA yield of the lungs is very low relative to the gut, it is particularly challenging to perform DNA extraction and account for contamination when sequencing the lung microbiome.

Several helpful tips were given during the webinar, such as performing analysis on sequencing data with and without subtraction of OTUs found in blank control samples. Dr. Barfod shared with the audience the observation that contamination could drive clustering of the samples, and that the clustering disappeared after removing the OTUs from the blank controls.

Also, technical advice for sampling of sterile bronchoalveolar lavage (BAL) fluid was given at the webinar, such as bleeding mice before BAL to obtain more fluid. Ligation of one lung lobe was mentioned as a means to obtain more experimental endpoints; i.e. one lobe can used for BAL and the other for other purposes, such as looking for tissue-dwelling microbes.

Dr. Barfod highlighted that the BAL procedure can easily be implemented as an add-on in gut microbiome studies to enable more experimental endpoints and increase discovery in the research field. Using both "ends" of the animals — looking at the lung as well as the gut microbiome — is meaningful from a harms reduction perspective, as more questions can then be answered using the same number of animals.

Germ-free mice were mentioned as a feasible way to manipulate the microbiota and dissect importance of specific organism or communities; e.g. through inoculation with probiotic strains or human microbiotas.

Murine Lung Microbiome Q&A

The webinar ended with questions from the audience. There wasn't time to address all questions during the webinar, so we present them here with Dr. Barfod's answers.

• Q: Are there any publications looking at the effects of lung microbiota on lung immune system?

Kenneth Klingenberg Barfod (KKB): Yes, there are a couple of good ones. I can recommend these two: Gollwitzer et al. 2014⁶ and Richmond et al. 2016⁷.

• Q: Can microbiota transfer from gut to lung? And how?

KKB: I guess you can inhale them, other routes are highly speculatory. During birth, I find it rather likely from mother to offspring.

• Q: Can you separate the host DNA from the bacterial DNA?

KKB: We spun the BAL fluids to separate mouse cells from the supernatant.

• Q: Is there a difference in tissue interface for microbiota (mucosa layer), as has been seen in cecal mucosa in gut?

KKB: I don't know, but it sounds interesting. I would suggest a dual sequencing approach where you sequence 16S and host factors from the same tissue sample grid.

• Q: How do you account for cell or mucus associated bacteria using BAL?

KKB: You don't. Or you sequence all three components as I did in my 2013 paper⁴ to show the difference between the locations.

• Q: How would you study biofilms in your model?

KKB: I don't think our lab mice have biofilms, but in the paper by Yun et al. 2014⁸ they could FISH stain bacteria in wild caught mice.

• Q: If there is little overlap from the gut microbiome to the lung, can the lung microbiome transfer to gut?

KKB: Yes, we cough and swallow a lot of mucus containing bugs. Whether they stay viable through the stomach is unknown, but they still might have an influence on immune parameters.

• Q: In terms of a gut-lung axis, is there a microbiome in the blood in the naive murines?

KKB: That is a good question. I would speculate that you in the healthy state have some parts of broken down bacteria being moved around in the blood. There are some studies in rats that show fecal bacteria being move to the breastmilk — I guess as a way of letting the offspring sample certain bacteria that have been broken partially down so they can't be dangerous?

• Q: Is there a way to specifically deplete microbiota from the lung without touching the gut microbiome? What about germ free mice?

KKB: Yes, Vancomycin is used as a red label inhalation antibiotic. I have experimentally used it in one of my papers³. Germ-free mice really are the way to go if you want to see what bugs do what and to show causality.

• Q: How do you isolate a lung lobe prior to lavage and do so in a sterile manner?

KKB: If you use the BAL technique, I think you will not contaminate the inside of the lung by binding off one lung lobe with string from the outside? I guess it is less easy if you want the lung tissue as a whole to be sterile.

References:

1. Dickson, R. P.; Huffnagle, G. B. The Lung Microbiome: New Principles for Respiratory Bacteriology in Health and Disease. PLoS Pathog. 2015, 11 (7), e1004923 DOI: 10.1371/journal.ppat.1004923.

2. Dickson, R. P.; Erb-Downward, J. R.; Freeman, C. M.; McCloskey, L.; Beck, J. M.; Huffnagle, G. B.; Curtis, J. L. Spatial Variation in the Healthy Human Lung Microbiome and the Adapted Island Model of Lung Biogeography. Ann. Am. Thorac. Soc. 2015, 12 (6), 821-830 DOI: 10.1513/AnnalsATS.201501-029OC.

3. Barfod, K. K.; Vrankx, K.; Mirsepasi-Lauridsen, H. C.; Hansen, J. S.; Hougaard, K. S.; Larsen, S. T.; Ouwenhand, A. C.; Krogfelt, K. A. The Murine Lung Microbiome Changes During Lung Inflammation and Intranasal Vancomycin Treatment. Open Microbiol. J. 2015, 9 (1), 167-179 DOI: 10.2174/1874285801509010167.

4. Barfod, K. K.; Roggenbuck, M.; Hansen, L. H.; Schjørring, S.; Larsen, S. T.; Sørensen, S. J.; Krogfelt, K. A. The murine lung microbiome in relation to the intestinal and vaginal bacterial communities. BMC Microbiol. 2013, 13, 303 DOI: 10.1186/1471-2180-13-303.

5. Roggenbuck, M.; Anderson, D.; Barfod, K. K.; Feelisch, M.; Geldenhuys, S.; Sørensen, S. J.; Weeden, C. E.; Hart, P. H.; Gorman, S. Vitamin D and allergic airway disease shape the murine lung microbiome in a sex-specific manner. Respir. Res. 2016, 17 (1), 116 DOI: 10.1186/s12931-016-0435-3.

6. Gollwitzer, E. S.; Saglani, S.; Trompette, A.; Yadava, K.; Sherburn, R.; McCoy, K. D.; Nicod, L. P.; Lloyd, C. M.; Marsland, B. J. Lung microbiota promotes tolerance to allergens in neonates via PD-L1. Nat. Med. 2014, 20 (6), 642-647 DOI: 10.1038/nm.3568.

7. Richmond, B. W.; Brucker, R. M.; Han, W.; Du, R.-H.; Zhang, Y.; Cheng, D.-S.; Gleaves, L.; Abdolrasulnia, R.; Polosukhina, D.; Clark, P. E.; et al. Airway bacteria drive a progressive COPD-like phenotype in mice with polymeric immunoglobulin receptor deficiency. Nat. Commun. 2016, 7, 11240 DOI: 10.1038/ncomms11240.

8. Yun, Y.; Srinivas, G.; Kuenzel, S.; Linnenbrink, M.; Alnahas, S.; Bruce, K. D.; Steinhoff, U.; Baines, J. F.; Schaible, U. E. Environmentally determined differences in the murine lung microbiota and their relation to alveolar architecture. PLoS One 2014, 9 (12), e113466 DOI: 10.1371/journal.pone.0113466.