Pneumonia causes adverse outcomes including hospitalization, intensive care, and death. Host response is pivotal, with immune activities and cell dysfunctions responsible for pathophysiologies including but not limited to excessive microbial growth, alveolar flooding, and septal destruction. Many diverse biological pathways can lead to adverse outcomes within the infected lungs. A better understanding of the immunological and cellular activities occurring inside severely infected lungs is needed, to distinguish sub-phenotypes of disease and improve the development and application of host-directed therapies. In the proposed studies, we will leverage large sets of post-mortem human pneumonic lung samples collected via rapid autopsy for defining pneumonia sub-phenotypes based on their pulmonary pathobiology. We will integrate semi-quantitative histopathology (scored by board-certified pathologists), quantitative multiplex fluorescent immunohistochemistry (mfIHC; to enumerate and localize immune cells within these lungs), and single nuclei RNA sequencing (snRNA-seq; to broaden and deepen cell and molecular resolution of these lungs) in order to differentiate distinct lung pathobiologies during pneumonia. In addition, we will examine multiple current and emergent experimental models of severe pneumonia to determine which if any recapitulate elements of these pulmonary pathobiologies. We propose to test the central hypothesis that severe pneumonias cluster into distinct lung pathobiology sub-phenotypes, by pursuing the following specific aims: Aim 1) To test whether humans dying with pneumonia segregate into lung pathology sub-phenotypes, using rapid autopsy samples analyzed via histopathologic scoring and quantitative mfIHC. Aim 2) To validate and elucidate pneumonia sub- phenotypes using an independent cohort with microscopy matched to snRNA-seq for defining cell-types and cell-specific gene expression in infected human lungs. Aim 3) To test whether established and emerging experimental models of severe pneumonia recapitulate some and which elements of human pulmonary pathobiology. The proposed studies will be significant for generating discoveries from lung and blood samples of pneumonia cases in elderly subjects, an especially high risk group for pneumonia. Proposed studies will increase resolution into the heterogeneity of pneumonia, a currently pressing research priority. Results will reveal whether sub-phenotypes or select features measured within the pneumonic lungs differ between pneumonias caused by pneumococcus, influenza, and SARS-CoV-2. Defining sub-types of pneumonia with distinct molecular and cellular changes in the lungs will improve the development and use of host-directed approaches for treating or preventing pneumonia.

The proposed studies will help delineate the biological processes occurring in the lungs of pneumonia patients, with cases anticipated to cluster into different types of lung responses. The sub-types of pneumonia will be investigated using innovative experimental systems and defined at both microscopic and molecular levels within the lung. Differentiating pneumonia sub-types will mean that current and emerging treatments can be delivered more precisely, to target those most likely to benefit and spare those who may be harmed instead.