Prior oral non-absorbable antibiotic-induced gut dysbiosis worsens outcomes of P. aeruginosa lung infection
First, we assessed if oral non-absorbable antibiotics induced gut dysbiosis. The gut microbiota from antibiotic treated-mice before P. aeruginosa intra-nasal challenge was altered compared to control (figure 2A). We also observed an eradication of several bacterial families like Muribaculaceae, Prevotellaceae, and Lachnospiraceae and an expansion of Burkholderiaceae, Clostridiales, and Lactobacillaceae. Eradication of several bacterial families is associated with a decrease of gut microbiota diversity (figure 2B). These modifications are observed with a decrease of only one log of 16srDNA compared to untreated mice (supplemental figure 2). As expected, we did not observe any modification of the effect of prior oral non-absorbable on the composition and the diversity of the lung microbiota before P. aeruginosa infection (Figure 2 C-D).
Then, we assessed if prior oral non-absorbable antibiotic modified outcomes of P. aeruginosa acute pneumonia. Antibiotics administered in drinking water seven days before P. aeruginosa intra-nasal challenge were associated with worse infection outcomes (figure 3). P. aeruginosa load increases of 103 CFU and 102 in the lung and in the spleen respectively in antibiotics condition (figure 3A, B). Lung injury is increased in antibiotic pre-treated mice when compared to untreated controls (3.5±1.4% vs. 1.8±1.1%, p < 0.05) (figure 3C). Finally, the survival of antibiotic-treated mice survival decreased to 20% from 100% in untreated controls (p<0.05) (figure 3D). Overall, acute sublethal P. aeruginosa pneumonia became lethal in antibiotic-treated mice compared to untreated controls.
To demonstrate the causality of gut dysbiosis in worse outcomes of P. aeruginosa infected mice, we treated dysbiosis using fecal microbiota transplant (FMT). Following dysbiosis-inducing antibiotics, FMT also restored the composition and the diversity of the gut microbiota equivalent to controls (figure 2A-B) and did not modify the composition and the diversity of the lung microbiota (figure 2C-D). Of note, P. aeruginosa intra-nasal challenge did not further modify the gut microbiota shift observed in antibiotic-treated mice (supplemental figure 3). As expected, P. aeruginosa intra-nasal challenge resulted in a lung microbiota dominated by Pseudomonadaceae (supplemental figure 3). Finally, FMT before P. aeruginosa inoculation restored pneumonia outcomes to those of control mice (figure 3A-B-C-D).
Oral non-absorbable antibiotic-induced gut dysbiosis results in widespread lung and spleen immune depression associated with altered myelopoiesis
Next, we assessed whether antibiotic-induced gut dysbiosis had any effects on baseline lung and circulating cellular immune profiles as a potential mechanism underlying worse outcomes following P. aeruginosa lung infection.
Cell population analysis in lung tissue showed that antibiotics resulted in widespread depression of lung cellular immunity with a significant decrease in macrophages, cDC2, inflammatory monocytes, neutrophils, γδ-T cells, NKs, and iNKT cells (figure 4A). FMT restored most of these alterations in macrophages, cDC2, inflammatory monocytes, neutrophils, and iNKT cells (figure 4A), establishing a significant role of antibiotic-induced gut dysbiosis in this widespread depression of the lung immune response. Likewise, antibiotics resulted in widespread depression of most studied spleen immune cell populations (p< 0.05 except for a trend in NK cells), also partially restored by FMT (figure 4B).
Because the spleen is a hematopoietic organ in mice, we sought to determine the effects of antibiotics on immune cell hematopoietic factors, specifically on circulating serum levels of GM-CSF, M-CSF, and Flt3-Ligand (figure 5A). While circulating levels of GM-CSF were at the detection threshold (1pg/mL) and remained unaltered, antibiotics induced a significant decrease in Flt3-Ligand, and only a trend in M-CSF decrease. Given the known role of Flt3-Ligand as a major hematopoietic stimulating factor, mainly for monocyte and DC progenitors , we studied the effects of antibiotics and FMT on bone marrow monocyte and DC progenitors (figure 5B and supplemental figure 5). We found that antibiotics were associated with a significant decrease in bone marrow progenitors specific to resident monocytes (MonoLy6C-) and several specific to DCs (total pre-DCs, pre-DCs 1 and 2 and cCD2 biased pre-DCs). Among these, FMT following antibiotics restored levels of pre-DCs 1. FMT also stimulated the expansion of bone marrow progenitors common to monocytes/macrophages/DCs (MDP), common dendritic cell progenitors (CDP), and specific to monocytes and macrophages (cMoPs). Our results suggest that the effects of antibiotic-induced dysbiosis on monocyte/macrophage and DC progenitors may be involved in the widespread antibiotic-induced lung immune depression associated with worse P. aeruginosa lung infection outcomes.
Hematopoietic cytokine Flt3-L stimulates bone marrow progenitor and lung immune cell expansion and restores outcomes of P. aeruginosa lung infection following oral non-absorbable antibiotics
Systemic Flt3-L following antibiotics stimulated the expansion of several progenitors (MDPs, CDPs, and cDC-1 biased pre-DCs) (figure 6A and supplemental figure 6). Flt3-Ligand administration partially restored or overstimulated the expansion of depressed alveolar macrophages, cDC2, inflammatory monocytes, neutrophils, NK, and iNKT (figure 6B and supplemental figure 6).
Finally, the effects of Flt3-Ligand (Flt3-L) administration following antibiotics on the outcomes of sublethal P. aeruginosa lung infection were similar to those of FMT. Thus, Flt3-L administration leads to a decrease permeability index in VAN/CST mice group compared to VAN/CST + FLT-3 mice group (Figure 6C). Additionally, Flt3-L administration leads lead to a decrease of P. aeruginosa load in both lung and spleen in VAN/CST mice group compared to VAN/CST + Flt-3L mice group to a level similar to the controls (Figure 6D-E). Finally, Flt3-Ligand was also associated with decreased mortality in a lethal P. aeruginosa lung infection model, from 100% to 40% (p<0.05) similar to the effects of FMT (figure 6F).