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[Preprint]. 2024 Oct 7:2024.10.07.617039.
doi: 10.1101/2024.10.07.617039.

A chronic Pseudomonas aeruginosa mouse lung infection modeling the pathophysiology and inflammation of human cystic fibrosis

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A chronic Pseudomonas aeruginosa mouse lung infection modeling the pathophysiology and inflammation of human cystic fibrosis

Mylene Vaillancourt et al. bioRxiv. .

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Abstract

Investigation of chronic cystic fibrosis (CF) lung infections has been limited by a lack of murine models that reproduce obstructive lung pathology, chronicity of bacterial infections, and complex inflammation in human CF lung pathology. Three different approaches have been used separately to address these limitations, including using transgenic Scnn1b-Tg mice overexpressing a lung epithelial sodium channel to mimic the mucus-rich and hyperinflammatory CF lung environment, using synthetic CF sputum medium (SCFM) in an acute infection to induce bacterial phenotypes consistent with human CF, or using agar beads to promote chronic infections. Here, we combine these three models to establish a chronic Pseudomonas aeruginosa lung infection model using SCFM agar beads and Scnn1b-Tg mice (SCFM-Tg-mice) to recapitulate nutrients, mucus, and inflammation characteristic of the human CF lung environment. Like people with CF, SCFM-Tg-mice failed to clear bacterial infections. Lung function measurements showed that infected SCFM-Tg-mice had decreased inspiratory capacity and compliance, elevated airway resistance, and significantly reduced FVC and FEV0.1. Using spectral flow cytometry and multiplex cytokine arrays we show that, like people with CF, SCFM-Tg-mice developed inflammation characterized by eosinophil infiltration and Th2 lymphocytic cytokine responses. Chronically infected SCFM-Tg-mice developed an exacerbated mix of innate and Th1, Th2, and Th17-mediated inflammation, causing higher lung cellular damage, and elevated numbers of unusual Siglec F+ neutrophils. Thus, SCFM-Tg-mice represents a powerful tool to investigate bacterial pathogenesis and potential treatments for chronic CF lung infections and reveal a potential role for Siglec F+ neutrophils in CF inflammation.

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Figures

Fig. 1.
Fig. 1.. Bacterial clearance is impaired in Scnn1b-Tg mice and increases airway resistance during chronic infection.
(A) WT C57BL/6 or Scnn1b-Tg mice were intratracheally inoculated with sterile or 1×106 CFU PAO1-laden SCFM2-agar beads for 7 days. (B) Representative microscopic images of sterile and PAO1-laden SCFM2-agar beads. (C) Bacterial load 7 days post-infection. Bacterial load was determined by CFU/mL. (D-F) Lung function measurements obtained using the flexiVent (SCIREQ). (D) Inspiratory capacity using a deep inflation technique. (E) System resistance and elastance parameters acquired by the single frequency forced oscillation maneuver. (F) Airway resistance, tissue resistance (damping) and elastance obtained from the low frequency forced oscillation technique. n=4–5 mice/group. *p<0.05, ** p<0.01. See Table S1 for statistical tests used and exact p-values.
Fig. 2.
Fig. 2.. Scnn1b-Tg mice develop mixed obstructive and restrictive lung disease during chronic infection.
WT C57BL/6 or Scnn1b-Tg mice were intratracheally inoculated with sterile or 1×106 CFU PAO1-laden SCFM2-agar beads for 7 days. (A-D) Lung function measurements obtained using the flexiVent (SCIREQ). (A) Static compliance and hysteresivity obtained by a pressure-volume (PV) loop. (B) Representative image of PV-loop. (C) FVC, FEV0.1, FEV0.1/FVC, PEF and FEF0.1 obtained from the forced expiratory volume perturbation. (D) Representative image of the forced expiratory volume perturbation. n=4–5 mice/group. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. See Table S1 for statistical tests used and exact p-values.
Fig. 3.
Fig. 3.. Chronic infection in WT C57BL/6 or Scnn1b-Tg mice for immune response by flow cytometry.
(A)WT C57BL/6 or Scnn1b-Tg mice were intratracheally inoculated with sterile or 1×106 CFU PAO1-laden SCFM2-agar beads for 7 days. (B) Gating strategy used to identify immune cell response during chronic infection. Cells were isolated from enzymatically digested mouse lungs, and, after the exclusion of doublets and debris, live and immune cells were identified by LIVE/DEAD staining and CD45 staining. Neutrophils (Ly6G+) were isolated and gated for Siglec F marker. Then, Ly6G and Siglec F+ cells were selected to differentiate alveolar macrophages (Siglec-F+, CD11c+) and eosinophils (Siglec-F+, CD11b+, CD11c). T cells (TCRβ+) were then separated from the rest of Sigle F cells. CD4+ and CD8+ were separated from the double-negative (DN) subset. CD4+ and Foxp3+ cells were isolated, while Foxp3 cells were separated by the CD44 and CD62L markers to identify naïve CD4+ T cells (CD44, CD62L+), effector CD4+ T cells (CD44+, CD62L), and central memory CD4+ T cells (CD44+, CD62L+). CD8+ T cells were also separated with the same markers CD44+ and CD62L. Finally, TCR cells were further separated using Ly6C and CD11b markers to identify monocyte-derived macrophages (CD11bHigh, Ly6C+/−, CD64+, FSC-Ahigh), classical monocytes (CD11b+, Ly6C+), and other myeloid-derived cells (CD11b+, Ly6C).
Fig. 4.
Fig. 4.. Scnn1b-Tg mice lung inflammation is characterized by an increase in atypical neutrophils.
(A) Inflammatory cells were increased in both Scnn1b-Tg mice and their WT littermates. (B-E) Different innate cells were upregulated in both genotype during chronic infection. (B) Alveolar macrophages. (C) Monocyte-derived macrophages. (D) Classical monocytes. E. Other myeloid cells. (F) Eosinophils were not upregulated during chronic infection with P. aeruginosa. (G) Neutrophils were upregulated during chronic infection but not modulated by the genotype. (H-I) An atypical Siglec F+ neutrophil subset was upregulated in Scnn1b-Tg mice during chronic infection. n=6 mice/group *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. See Table S1 for statistical tests used and exact p-values.
Fig. 5.
Fig. 5.. Scnn1b-Tg immune response is characterized by effector T cells.
(A) Total T cells were significantly increased during chronic infection and even more in the Scnn1b-Tg mice. (B-C) This increase in T cells was explained by higher numbers of CD4+ (B) and CD8+ (C) T cells. (D) Activation state of CD4+ T cells. No difference was seen in naïve CD4+ T cells. During chronic infection, a significant upregulation of effector T cell was observed in both genotypes and this increase was greater in Scnn1b-Tg mice compared to their WT littermates. A modest but non-significant increase was detected for central memory T cells in infected mice. (E) Activation state of CD8+ T cells. During chronic infection, a significant increase in naïve CD8+ T cell was observed in Scnn1b-Tg mice. Effector T cells were also increased in both genotypes. A modest increase of central memory CD8 was detected for both genotypes. (F) Regulatory T cells were also increased in all infected mice but not modulated by the genotype. G. Double-negative (DN) cells were significantly increased in all infected mice and were significantly higher in Scnn1b-Tg mice compared to their WT littermates. n=6 mice/group *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. See Table S1 for statistical tests used and exact p-values.
Fig. 6.
Fig. 6.. Scnn1b-Tg mice develop exacerbated innate inflammation during chronic infection.
(A) Quantification (pg/μg of proteins) of pro- and anti-inflammatory cytokines and chemokines in whole lung lysates of chronically infected mice. (B) Inflammatory cytokines IL-6, IL-1β, and TNF-α were upregulated in all infected mice. IL-1β and TNF-α levels were higher in Scnn1b-Tg mice compared to their WT littermates. (C) Monocytes/macrophages chemoattractant MIP-1α and CXCL10 were significantly upregulated in infected Scnn1b-Tg mice. (D) Neutrophil chemoattractants were significantly upregulated in infected Scnn1b-Tg mice compared to their WT littermates. (E) Lymphocyte chemoattractant MIP-3α was upregulated in all infected mice but was higher in Scnn1b-Tg mice. IL-15 was increased in infected WT C57BL/6 mice only, while and IL-16 was only upregulated in Scnn1b-Tg mice. n=4–5 mice/group *p<0.05, **p<0.01, ***p<0.001. See Table S1 for statistical tests used and exact p-values.
Fig. 7.
Fig. 7.. Chronic infection leads to dysfunctional lymphoid-mediated inflammation in Scnn1b-Tg mice.
(A) Quantification (pg/μg of proteins) of type 1, 2 and 3 inflammation cytokines and chemokines in whole lung lysates of chronically infected mice. (B) Type 1 inflammation lymphokines IL-2, IL-27p/28/IL-30, and IFN-γ were significantly upregulated in infected Scnn1b-Tg mice. (C) Type 2 inflammation lymphokines IL-4 and IL-5 were upregulated in uninfected Scnn1b-Tg mice. Although IL-4 was downregulated during infection, IL-5 levels were maintained. IL-33 was upregulated in Scnn1b-Tg mice during chronic infection. (D) The type 3 inflammation cytokine IL-17 was upregulated in all mice and further increased in Scnn1b-Tg mice. (E) Z-scores highlight a type 2 and lymphoid inflammation in uninfected Scnn1b-Tg mice compared to their WT littermates. IL-17 is the most differentially upregulated cytokine in these mice during infection. (F) Scnn1b-Tg have higher lung tissue damage at baseline. Chronic infection caused increased lipid peroxidation in all infected mice but was greater in Scnn1b-Tg mice. n=4–5 mice/group *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. See Table S1 for statistical tests used and exact p-values.
Fig. 8.
Fig. 8.. Summary of lung inflammation in C57/BL/6 and Scnn1b-Tg mice during chronic infection with P. aeruginosa.
Healthy lungs from C57BL/6 mice are characterized by alveolar macrophages. Uninfected Scnn1b-Tg mice show underlying inflammation characterized by the presence of alveolar and monocyte-derived macrophages, monocytes, other myeloid cells, and effector T cells. Conventional and Siglec F+ neutrophils are also present in the BAL of uninfected Scnn1b-Tg mice. A type 2 inflammation, demonstrated by the presence of eosinophils and IL-4 and IL-5, is present at baseline in the Scnn1b-Tg lung environment. During chronic infection, both C57BL/6 and Scnn1b-Tg immune responses are characterized by infiltration of innate and T cells and high levels of type 1 and 3 inflammation cytokines and chemokines (IL-1β, IL-2, IL-6, IL-17, TNFα, IFNγ, MIP-1α, MIP-2, MIP-3α and KC/GRO). In addition to being exacerbated, Scnn1b-Tg inflammation is characterized by a sustained type 2 inflammation, a marked IL-17/neutrophil interplay and the recruitment of unconventional Siglec F+ neutrophils. The higher inflammation is associated with higher lung tissue damage in Scnn1b-Tg mice. Cytokines in red font are cytokines expressed in the specific genotypes. Red arrows indicate where the cytokine production is increased relative to the other genotype.

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