Contribution of Puma to Inflammatory Resolution During Early Pneumococcal Pneumonia

Abstract

Apoptosis of cells at the site of infection is a requirement for shutdown of inflammatory signaling, avoiding tissue damage, and preventing progression of sepsis. Puma^+/+ and Puma^-/- mice were challenged with TIGR4 strain pneumococcus and cytokines were quantitated from lungs and blood using a magnetic bead panel analysis. Puma^-/- mice exhibited higher lung and blood cytokine levels of several major inflammatory cytokines, including IL-6, G-CSF, RANTES, IL-12, IFN-ϒ, and IP-10. Puma^-/- mice were more susceptible to bacterial dissemination and exhibited more weight loss than their wild-type counterparts. RNA sequencing analysis of whole pulmonary tissue revealed Puma-dependent regulation of Nrxn2, Adam19, and Eln. Enrichment of gene ontology groups differentially expressed in Puma^-/- tissues were strongly correlated to IFN-β and -ϒ signaling. Here, we demonstrate for the first time the role of Puma in prohibition of the cytokine storm during bacterial pneumonia. These findings further suggest a role for targeting immunomodulation of IFN signaling during pulmonary inflammation. Additionally, our findings suggest previously undemonstrated roles for genes encoding regulatory and binding proteins during the early phase of the innate immune response of pneumococcal pneumonia.

Keywords: PUMA (p53 upregulated modulator of apoptosis); apoptosis; inflammation; innate immunity; streptococcus pneumoniae (pneumococcus).

Figure 1

Puma attenuates sepsis and morbidity. (A) Mice were intranasally challenged with 2 x 10⁵ total CFU. Pneumonia was allowed to develop for 24 h, at which time mice were euthanized and tissues were sampled. For each group, WT (n = 10), KO (n = 7). Horizontal lines represent means. A Mann-Whitney U test was performed, α = 0.05. (B) Mice were weighed immediately before challenge and before sacrifice. A two-tailed, unpaired T-test was performed, α = 0.05. *p = 0.0003.

Figure 2

Puma protects against tissue inflammation. (A, B) Puma^+/+ mice exhibited mild peribronchioloar inflammation with edema and neutrophils and macrophages. (C–E) Puma^-/- mice exhibited moderate peribronchiolar/perivascular inflammation, abundant neutrophils, macrophages, vasculitis (eosinophilic material, vascular thickening). Scale bars: A–C, E, 50 μm; D, 20 μm. Images shown are representative of the pathology described in the results from 3 sections each from 5 mice.

Figure 3

Puma-dependent host responses during early pneumococcal pneumonia suppress production of cytokines in the lung. Cytokines expressed more than 3-fold in Puma^-/- mouse lungs (A) and sera (B) are shown here. Cytokines were detected via magnetic bead panel using flow cytometry analysis. Values are reported as pg/mL. Mann-Whitney U tests were performed to determine significance, α = 0.05.

Figure 4

Gene ontology analysis reveals Puma limits IL-12 and Type I and II IFN signaling. Genes significantly expressed (red dots) in their respective GO groups are plotted against all other genes (blue dots). Significance was determined using the Kolmogorov-Smirnov test and p-values were adjusted for multiple-testing. The area above the black line indicates higher expression in Puma^-/- mice, while the area under the black line indicates higher expression in Puma^+/+ mice.