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Additional file 2 : Supplemental figure 2. Total bacterial 16SrDNA in the stool. Mice (n = 5) were treated by oral vancomycin (VAN) and colistin (CST) or not. Total 16srDNA expressed in relative quantity (fold-decrease) between VAN/CST and untreated controls (reference = 1).

Additional file 5 : Supplemental figure 5. Effects of oral non-absorbable antibiotics on hematopoietic factors and bone marrow monocyte/dendritic cell progenitors. (A) ELISA of hematopoietic factors in serum of mice treated or not (controls) with 7-days oral vancomycin/colistin (VAN/CST) followed by fecal microbiota transplant (VAN/CST + FMT) or not, (B) Flow cytometry of monocyte/DC progenitors in the bone marrow in mice treated or not (controls) with 7-days oral vancomycin/colistin (VAN/CST) followed by fecal microbiota transplant (VAN/CST + FMT) or not. For all experiments, group size 5 mice per group; results are shown as mean ± SD; *: p

Additional file 6 : Supplemental figure 6. Effects of Flt3-Ligand on lung immune cell populations and on outcomes of lung infection. (A) Flow cytometry of monocyte/DC progenitors in the bone marrow of mice treated or not (controls) with 7-days oral vancomycin/colistin (VAN/CST) followed by systemic Flt3-Ligand administration (VAN/CST + Flt3-L) or not, (B) Flow cytometry of immune cells from lungs of mice with 7-days oral vancomycin/colistin (VAN/CST) followed by systemic Flt3-Ligand administration (VAN/CST + Flt3-L) or not. For all experiments, group size 5 mice per group; results are shown as mean ± SD; *: p

Additional file 3 : Supplemental figure 3. Bacterial family as percentages of sequenced 16S rDNA in the lung and gut. Mice were treated or not by oral vancomycin (VAN), and colistin (CST) followed or not by fecal microbiota transplant (FMT) and infected or not by P. aeruginosa PAO1 (n = 2 per group).

Additional file 4 : Supplemental figure 4. Effects of oral non-absorbable antibiotics on lung and spleen immune cell populations. (A) Flow cytometry of immune cells from lung homogenates in mice treated or not (controls) with 7-days oral vancomycin/colistin (VAN/CST) followed by fecal microbiota transplant (VAN/CST + FMT) or not. (B) Flow cytometry of immune cells from spleen homogenates in mice treated or not (controls) with 7-days oral vancomycin/colistin (VAN/CST) followed by fecal microbiota transplant (VAN/CST + FMT) or not. All experiments, group size 5 mice per group, results are shown as mean ± SD; *: p

Additional file 1 : Supplemental figure 1. Gating strategy for neutrophils, antigen-presenting cells, and the significant populations of lymphocytes in the lung. The percentages of natural killer (NK), invariant NKT cells (iNKT), and T Lc with a TCRgd were determined. Among APC, we identified alveolar macrophages (AM), inflammatory monocytes, patrolling monocytes, interstitial macrophages (IM), and conventional dendritic cells (cDC)1 and 2.

Additional file 7.

International audience; Bacterial infections of the respiratory tract constitute a major cause of death worldwide. Given the constant rise in bacterial resistance to antibiotics, treatment failure is increasingly frequent. In this context, innovative therapeutic strategies are urgently needed. Stimulation of innate immune cells in the respiratory tract [via activation of Toll-like receptors (TLRs)] is an attractive approach for rapidly activating the body's immune defenses against a broad spectrum of microorganisms. Previous studies of the TLR5 agonist flagellin in animal models showed that standalone TLR stimulation does not result in the effective treatment of pneumococcal respiratory infection but does significantly improve the therapeutic outcome of concomitant antibiotic treatment. Here, we investigated the antibacterial interaction between antibiotic and intranasal flagellin in a mouse model of pneumococcal respiratory infection. Using various doses of orally administered amoxicillin or systemically administered cotrimoxazole, we found that the intranasal instillation of flagellin (a dose that promotes maximal lung pro-inflammatory responses) induces synergistic rather than additive antibacterial effects against antibiotic-susceptible pneumococcus. We next set up a model of infection with pneumococcus that is resistant to multiple antibiotics in the context of influenza superinfection. Remarkably, the combination of amoxicillin and flagellin effectively treated superinfection with the amoxicillin-resistant pneumococcus since the bacterial clearance was increased by more than 100-fold compared to standalone treatments. Our results also showed that, in response to flagellin, the lung tissue generated an innate immune response even though it had been damaged by the influenza virus and pneumococcal infections. In conclusion, we demonstrated that the selective boosting of lung innate immunity is a conceptually advantageous approach for improving the effectiveness of antibiotic treatment and fighting antibiotic-resistant bacteria.

Secondary bacterial infections contribute to the excess morbidity and mortality of influenza A virus (IAV) infection. Disruption of lung integrity and impaired antibacterial immunity during IAV infection participate in colonization and dissemination of the bacteria out of the lungs. One key feature of IAV infection is the profound alteration of lung myeloid cells, characterized by the recruitment of deleterious inflammatory monocytes. We herein report that IAV infection causes a transient decrease of lung conventional dendritic cells (cDCs) (both cDC1 and cDC2) peaking at day 7 post-infection. While triggering emergency monopoiesis, IAV transiently altered the differentiation of cDCs in the bone marrow, the cDC1-biaised pre-DCs being particularly affected. The impaired cDC differentiation during IAV infection was independent of type I interferons (IFNs), IFN-γ, TNFα and IL-6 and was not due to an intrinsic dysfunction of cDC precursors. The alteration of cDC differentiation was associated with a drop of local and systemic production of Fms-like tyrosine kinase 3 ligand (Flt3-L), a critical cDC differentiation factor. Overexpression of Flt3-L during IAV infection boosted the cDC progenitors’ production in the BM, replenished cDCs in the lungs, decreased inflammatory monocytes’ infiltration and lowered lung damages. This was associated with partial protection against secondary pneumococcal infection, as reflected by reduced bacterial dissemination and prolonged survival. These findings highlight the impact of distal viral infection on cDC genesis in the BM and suggest that Flt3-L may have potential applications in the control of secondary infections., Author summary Secondary bacterial infections constitute a leading cause of mortality during influenza epidemics and pandemics. Acute infections trigger the mobilization of myeloid cells from the bone marrow (BM) to the infected tissues, a process referred to as emergency myelopoiesis. Influenza infection leads to a profound alteration in the pulmonary myeloid cell compartment characterized by the recruitment of inflammatory myeloid cells. The consequences of IAV infection on BM myelopoiesis have yet to be described in detail. Here, we report that influenza infection leads to reduced number of conventional dendritic cells (cDC) in the lungs (with a peak at day 7) and severely impairs DC differentiation in the BM, for the benefit of monopoiesis. The altered cDC differentiation was independent of pro-inflammatory cytokines and was not due to an intrinsic dysfunction of cDC precursors. Defective DC differentiation during influenza was associated with a decrease in the production of the key cDC differentiation factor, Fms-like tyrosine kinase 3 ligand (Flt3-L). Overexpression of Flt3-L during influenza enhanced the number of BM cDC progenitors and restored lung cDC compartment. This phenomenon was associated with a lowered number of lung inflammatory monocytes and reduced lung damages. Importantly, Flt3-L overexpression partially protected against secondary pneumococcal infection characterized by reduced bacterial loads, improved pathological outcomes and prolonged survival. Our results provide new clues as to how influenza infection influences the differentiation of cDCs, and highlight the potential therapeutic value of Flt3-L in a disease in desperate need of better treatments.