## 2 Results  
### Aspergillus Infection Is Associated with the Production of SCFAs  
We have previously shown that strictly anaerobic bacteria, such as _Prevotella or_ and _Peptostreptococcus anaerbius_, representative members of Bacteriodota and Firmicutes (order Clostridiales), respectively, protected against infection in a murine model of aspergillosis [17]. Since anaerobic bacteria produce SCFAs, we evaluated the presence of butyric acid and isobutyric acid in in vitro cultures and found that _Prevotella or_ and _Peptostreptococcus anaerobius_ produced the two SCFAs both in the presence and in the absence of _Aspergillus_ (Figure 1).  
Figure 1: Strictly anaerobic bacteria release SCFAs in culture with _A. fumigatus_. _A. fumigatus_ conidia were co\(-\)cultured in the absence or presence of either _Prevotella or_ or _Peptostreptococcus anaerobius_ for 24 and 72 h, and the levels of butyric acid and isobutyric acid were determined in the medium by mass spectrometry. A representative experiment is shown.  
We then evaluated whether SCFAs were produced in vivo by resorting to a murine model of aspergillillosis. Mice were infected intranasally (i.n.) with _A. fumigatus_ conidiaia, and a panel of SCFAs was quantified in the lung at different times post infection. As shown in Figure 2A, butyric acid and its isomer isobutyric acid were increased at 7 days post infection along with acetic acid, while propanoic, isovaleric and valeric acids were not increased. These results indicate that _Aspergillus_ infection is associated with the production of specific SCFAs.  
Figure 2: SCFAs accumulate in the lung during infection in a strain-dependent manner. Wild\(-\)type (**A**), _RagT\({}^{-}\),_ NSG (**B**), and \(p47^{\textit{thmax-}/-}\) (**C**) mice were infected with _A. fumigatus_, and the levels of SCFAs were measured in the lung homogenates at different times post infection by mass spectrometry. * \(p<0.05\); ** \(p<0.01\); *** \(p<0.001\); 3 or 7 vs. 0 dpi. Unpaired \(t-\)test or one-way ANOVA.  
Manipulating the Composition of the Microbiome or Their Products Alters the Susceptibility to Aspergillus Infection  
Since strictly anaerobic bacteria protected against aspergillosis by preventing the expansion of pathogenic Proteobacteria [17], we evaluated whether tipping the balance between anaerobic bacteria and Proteobacteria could affect the susceptibility to infection. For this purpose, upon administration of the Proteobacteria _Klebsiella pneumoniae_ in mice with aspergillosis, we found a higher fungal burden (Figure 3A) and a more severe histopathology (Figure 3B) in _Klebsiella_-exposed mice as compared to wild-type mice, confirming that Proteobacteria may negatively affect the course of the infection.  
We then administered butyrate acid, as a metabolic product of anaerobic bacteria, in the drinking water of mice before infection with _A. fumigatus_ conidia. Mice were pre-treated with piperacillin-tazobactam to lower the basal levels of SCFAs. As a result, we found that butyric acid, while not significantly reducing the fungal burden (Figure 3C), greatly ameliorated lung histopathology (Figure 3D) and reduced the expression level of inflammatory _Ill17a_ (Figure 3E). In contrast, administration of acetic acid, for which levels were also increased upon infection with _A. fumigatus_, did not play a protective role (Figure 3C,D) and increased the levels of the inflammatory cytokines IL-1\(\beta\) and TNF-\(\alpha\) (Figure 3F).  
All in all, these results indicate that the composition of the microbiome and the levels of their metabolites play a critical role in the susceptibility to _Aspergillus_ infection and are amenable to intervention.