Gut microbial dysbiosis occurring during pulmonary fungal infection in rats is linked to inflammation and depends on healthy microbiota composition

While the effect of gut microbiota and/or inflammation on a distant body site, including the lungs (gut–lung axis), has been well characterized, data about the influence of lung microbiota and lung inflammation on gut homeostasis (lung–gut axis) are scarce. Using a well-characterized model of pulmonary infection with the fungus Aspergillus fumigatus, we investigated alterations in the lung and gut microbiota by next-generation sequencing of the V3–V4 regions of total bacterial DNA. Pulmo- nary inflammation due to the fungus A. fumigatus caused bacterial dysbiosis in both lungs and gut, but with different characteristics. While increased alpha diversity and unchanged bacterial composition were noted in the lungs, dysbiosis in the gut was characterized by decreased alpha diversity indices and modified bacterial composition. The altered homeostasis in the lungs allows the immigration of new bacterial species of which 41.8% were found in the feces, indicating that some degree of bacterial migration from the gut to the lungs occurs. On the contrary, the dysbiosis occurring in the gut during pulmonary infection was a consequence of the local activity of the immune system. In addition, the alteration of gut microbiota in response to pulmonary infection depends on the bacterial composition before infection, as no changes in gut bacterial microbiota were detected in a rat strain with diverse gut bacteria. The data presented support the existence of the lung–gut axis and provide additional insight into this mechanism. IMPORTANCE Data regarding the impact of lung inflammation and lung microbiota on GIT are scarce, and the mechanisms of this interaction are still unknown. Using a well-characterized model of pulmonary infection caused by the opportunistic fungus Aspergillus fumigatus, we observed bacterial dysbiosis in both the lungs and gut that supports the existence of the lung–gut axis. KEYWORDS fungal lung infection, gastrointestinal microbiota, lung microbiota, lung-gut axis, rats B acteria inhabit every part of the human body, but most of them are found in the gut. Gut microbiota are responsible for many functions, including nutrient metabolism, immunomodulation, maintenance of host physiology, and protection against pathogen overgrowth (1). To date, numerous scientific studies confirm the important role of gut bacteria in health and disease. This microbial community impacts not only local immunity but also a distant body site, such as the lungs. Disturbances in gut bacterial composition have been linked to asthma (2), chronic obstructive pulmonary disease (3), cystic fibrosis (4), and lung cancer (5). Furthermore, pulmonary involvement was noted in inflammatory gastrointestinal disease characterized by microbial dysbiosis (6), Month XXXX Volume 0 Issue 0 10.1128/spectrum.01990-23 1 Editor Agostinho Carvalho, University of Minho, Braga, Portugal Address correspondence to Maja Tolinacki, [email protected]. The authors declare no conflict of interest. See the funding table on p. 15. Received 11 May 2023 Accepted 25 July 2023 Published 25 August 2023 Copyright © 2023 Popovic et al. This is an open- access article distributed under the terms of the Creative Commons Attribution 4.0 International license. Downloaded from https://journals.asm.org/journal/spectrum on 09 October 2023 by 147.91.199.205. supporting the existence of a gut–lung axis. The gut bacterial microbiota or some of their constituents impact the immune response in the lungs against viruses (7–9), bacteria (10–13), fungi (14), and allergic airway inflammation (15) mainly through the effect of the gut microbiota (or their metabolites) on the immune cell activity. While the gut–lung axis is well characterized, the influence of the lung microbiota as well as lung inflammation on gut homeostasis has attracted much more attention in recent years. The first indication of the lung–gut axis was a higher prevalence (compared to healthy subjects) of gastrointestinal symptoms in patients with asthma (16) and chronic obstructive pulmonary disease (17). The existence of gastrointestinal symptoms in patients with pulmonary virus infection has also been documented (18). Gastrointesti- nal symptoms (abdominal pain, nausea, vomiting, and diarrhea) were noted in 11.6% of children with influenza infection (18), and a later study showed a decrease in alpha diversity in the feces of influenza-infected patients compared to healthy controls (19). Fecal bacterial samples from patients with COVID-19 infection were shown to cluster separately from those in healthy controls as well, but in the majority of these patients, SARS-Cov-2 could be detected in the feces (20). Experimental studies in mice confirmed the occurrence of gut dysbiosis following respiratory influenza virus infection (21–25) and respiratory syncytial virus infection (24), despite the fact that the virus has not been detected in the gut (21, 22, 24, 25). It has been shown that the alteration of gut microbiota is a consequence of infection with live virus particles, as administration of an attenuated influenza vaccine had no effect on the microbiota (24). Bacterial dysbiosis in the gut also occurs following pulmonary bacterial infection. A decrease in alpha diversity indices and differential relative abundance of fecal microbiota were noted in patients with pulmonary tuberculosis (26, 27) and in mice infected with Mycobacterium tuberculosis (28) and Klebsiella pneumoniae (29). Even administration of the major component of the outer membrane of Gram-negative bacteria, lipopolysac- charide, to the lungs caused gut bacteria dysbiosis (30). In addition to pulmonary infections caused by viruses or bacteria, alteration of the gut microbiota was noted in mice exposed to hyperoxia (31) and in patients with lung cancer (compared to healthy individuals) (32) indicating that pulmonary inflammation/injury affects the gut microbiota regardless of its origin. Despite a growing body of evidence for interaction between the lungs and gut, there is still a lot of work to be done to understand this crosstalk. There are virtually no data regarding gut microbiota changes during pulmonary infection caused by fungi. Our previous study showed an alteration in immune-mediated homeostasis of the gut in a rat model of sublethal pulmonary infection with A. fumigatus (33). Using the same experimental model of infection in Dark Agouti (DA) rat strain, we aimed to investigate changes in the lung and gut microbiota by next-generation sequencing of the V3–V4 regions of total bacterial DNA in these two organs. Possible mechanisms of lung–gut communication were also investigated. In addition, to examine whether gut dysbiosis is a general characteristic during pulmonary fungal infection, we analyzed feces from infected Albino Oxford (AO) rats, a strain that develop quantitatively different immune response to fungus A. fumigatus (34) and whose gut microbiota was previously shown to respond differently to oral cadmium administra- tion (35) compared to DA rats

Oral warfarin affects some aspects of systemic immunomodulation with topical dinitrochlorobenzene (DNCB) in rats

Purpose: The efficacy of topical dinitrochlorobenzene (DNCB) in the treatment of some skin dermatoses is based both on local and systemic effects. It is not known, however, whether it can be applied to patients receiving some other therapy associated with systemic immunomodulation. The aim of the present paper using a rat model was to examine whether oral warfarin (WF) intake, as shown by others and by us, had an immunomodulatory potential to interfere with effects of topical DNCB as systemic immunotherapy. Materials and methods: Rats received 3.5 mg/l of WF sodium in drinking water for 30 days and were thereafter skin-sensitized with 0.4% DNCB. Changes in the oxidative activity (myeloperoxidase/MPO, reduction of nitroblue tetrazolium/NBT and nitric oxide/NO production) as well as tumor necrosis factor (TNF) production by peripheral blood polymorphonuclear cells (PMN) were measured and compared with PMN from sensitized unexposed to WF rats. Results: WF intake enhanced some aspects of PMN activity (intracellular MPO activity and unstimulated NO production) as well as their responsiveness to exogenous stimulation (NBT reduction and TNF production from sensitized animals). However, WF also decreased PMN responsiveness of NO production to stimulation. WF affected NO and TNF production solely by PMN, as no effect on these activities of peripheral blood mononuclear cells was seen. Conclusion: Having in mind that polymorphonuclear leukocytes are the most abundant cell type in peripheral blood in humans, increase of basic aspects of PMN activity described in the present paper might be relevant for consideration of using WF as therapeutic modality in patients topically treated with DNCB

Pulmonary Aspergillus fumigatus infection in rats affects gastrointestinal homeostasis

Microbiota inhabiting mucosal tissues is involved in maintenance of their immune homeostasis. Growing body of evidence indicate that dysbiosis in gut influence immune responses at distal sites including lungs. There are also reports concerning gut involvement with pulmonary injury/inflammation in settings of respiratory viral and bacterial infections. The impact of infections with other microorganisms on gut homeostasis is not explored. In this study, the rat model of sublethal pulmonary infection with Aspergillus fumigants was used to investigate the effect of fungal respiratory infection on gut immune-mediated homeostasis. Signs of intestinal damage, intestinal and gut-draining lymphoid tissue cytokine responses and gut bacterial microbiota diversity were examined. Intestinal injury, inflammatory cell infiltration, as well as increased levels of intestinal interferon-gamma (IFN-gamma) and interleukin-17 (IL-17) (as opposed to unchanged levels of anti-inflammatory cytokine IL-10) during the two-week period depict intestinal inflammation in rats with pulmonary A. fumigates infection. It could not be ascribed to the fungus as it was not detected in the intestine of infected rats. Increased production of pro-inflammatory cytokines by major gut-draining mesenteric lymph nodes point to these lymphoid organs as places of generation of cytokine-producing cells. No changes in spleen or systemic cytokine responses was observed, showing lack of the effects of pulmonary A. fumigatus infection outside mucosal immune system. Drop of intestinal bacterial microbiota diversity (disappearance of several bacterial bands) was noted early in infection with normalization starting from day seven. From day three, appearance of new bacterial bands (unique to infected individuals, not present in controls) was seen, and some of them are pathogens. Alterations in intestinal bacterial community might have affected intestinal immune tolerance contributing to inflammation. Disruption of gut homeostasis during pulmonary infection might render gastrointestinal tract more susceptible to variety of physiological and pathological stimuli. Data which showed for the first time gut involvement with pulmonary infection with A. fumigatus provide the baseline for future studies of the impact of fungal lung infections to gut homeostasis, particularly in individuals susceptible to these infections

Lung microbiota changes during pulmonary Aspergillus fumigatus infection in rats

Since the realization that the lungs are not sterile but are normally inhabited by various bacterial species, studies have been conducted to define healthy lung microbiota and to investigate whether it changes during lung diseases, infections, and inflammation. Using next-generation sequencing, we investigated bacterial microbiota from whole lungs in two rat strains (previously shown to differ in gut microbiota composition) in a healthy state and during pulmonary infection caused by the opportunistic fungus Aspergillus fumigatus. No differences in alpha diversity indices and microbial composition between DA and AO rats before infection were noted. Fungal infection caused dysbiosis in both rat strains, characterized by increased alpha diversity indices and unchanged beta diversity. The relative abundance of genera and species was increased in DA but decreased in AO rats during infection. Changes in lung microbiota coincided with inflammation (in both rat strains) and oxidative stress (in DA rats). Disparate response of lung microbiota in DA and AO rats to pulmonary fungal infection might render these two rat strains differentially susceptible to a subsequent inflammatory insult