## 2 Results
### The Composition of the Lung Microbiome and the Levels of SCFAs Are Shaped by the Immune System  
Since the composition and function of the microbiota are tuned by the immune system, we resorted to _RagT\({}^{/}\)_- mice, deficient in B and T lymphocytes, and NSG mice, which show  
Figure 3: The administration of _Klebsiella pneumoniae_ or butyrate has opposite effects in modifying the susceptibility to aspergillosis. Wild\(-\)type mice were administered _Klebsiella pneumoniae_ (**A**,**B**) or pre-treated with piperacillin–tazobactam and administered butyrate or acetate in the drinking water (**C**–**F**) before intranasal infection with _A. fumigatus_ resting conidia. Mice were assessed for fungal growth (Log10 CFU) in the lungs (**A**,**C**), lung histology (periodic acid–Schiff staining, magnification 10\(\times\) and insets 100\(\times\)) (**B**,**D**), mRNA levels of _Ill17a_ and _Ill22_ (**E**), and protein amounts of IL\(-\)1\(\beta\) and TNF\(-\alpha\) (**F**) at 5 (**A**,**B**) and 3 days (**C**–**F**) post infection. In B, the black arrows indicate areas of abnormal histopathology and, in the insets, the infiltration of neutrophils. * \(p<0.05\), **\(\ast\)\(p<0.01\)**, unpaired \(t-\)test. defects in both the innate and adaptive immune responses, to assess the role of the crosstalk between the microbiota and the immune system in response to Aspergillus. Both _Rag1-/-_ and NSG mice were more susceptible to infection compared to wild-type mice at 3 days post infection, as shown by increased fungal burden and more severe histopathology (Figure 4). Conversely, at 7 days post infection, while NSG mice were still more susceptible to infection, in agreement with published data [18], _Rag1-/-_ mice appeared more resistant, as shown by improved histopathology and a trend towards a reduced fungal burden (Figure 4). We evaluated the composition of the lung microbiome in these mice and found significant differences in compositional structure at the phylum level between the different groups, as measured by the Jaccard (\(p\) = 0.007) and Bray-Curtis (\(p\) = 0.016) indexes. We then compared the abundances of the major phyla in _Rag1-/-_, NSG, and wild-type mice at different times post infection. As shown in Figure 5, differences were observed in the composition of lung microbiome at baseline in both _Rag1-/-_ and NSG mice as compared to wild-type mice. Indeed, both strains had lower levels of Firmicutes, increased levels of Bacteroidota (particularly the _Rag1-/-_ strain), and similar levels of Proteobacteria. However, when ANOVA-like differential expression (ALDEx) analysis was applied to the lung microbiome of these mice at baseline to identify the classes that significantly differentiate each group from all the others, only one taxa was identified in _Rag1-/-_ mice, which corresponded to Clostridia, and was found to be expanded as compared to wild-type and NSG mice (Figure 6). Thus, although the baseline levels of Firmicutes were lower, Clostridia were expanded, which would indicate increased levels of strict anaerobes, in _Rag1-/-_ mice. Of interest, in addition to Clostridia, Bacteroidota were also expanded in _Rag1-/-_ mice, particularly late in infection, consistent with the failure of Bacteroidota to thrive in the acidic environment generated by the inflammatory response [19]. Thus, the relative expansion of strictly anaerobes through the course of the infection likely limited the expansion of Proteobacteria in Rag1-/- mice as opposed to NSG mice (Figure 5). Therefore, although the underlying genetic immune deficiencies certainly play a role, it can be speculated that  
Figure 4: The presence of a dysfunctional immune system affects the susceptibility to aspergillilosis. Wild\(-\)type, _Rag1-/-_, and NSG mice were infected i.n. with _A. fumigatus_ resting conidia. Mice were assessed for fungal growth (Log10 CFU) in the lungs (**A**) and lung histology (periodic acid\(-\)Schiff staining, representative sections, magnification 10\(\times\)) (**B**) at 3 and 7 days post infection.