Divergent Macrophage Responses to Influenza A Virus and Streptococcus pneumoniae: Co-infection Drives Bacterial Dominance whereas Superinfection Favors Viral Priming

Estanislao Nistal-Villan^1,2*Javier Arranz-Herrero^1,2,3,4,5*Jana Baranda^1,2,3,4,5Sergio Rius-Rocabert^1,2,4,5Ivan Gonzalez-Ruiz^1,2,4,5Alberto Miranda-Bedate^6,7Elena Pinelli⁶Vicent Tur-Planells^1,2,4,5,8Ines Inchausti-Moya^1,2,4,5Sara Izpura Luis^1,2,4,5María Paloma Reche^1,2,4,5Paloma Fernández Martínez^1,4,5Gustavo Del Real⁹Adolfo García-Sastre^{10,11,12,13,8}César B Gutiérrez Martín¹⁴Jordi Ochando^15,3

• ¹CEU San Pablo University, Madrid, Spain
• ²Universidad CEU San Pablo Facultad de Farmacia, Madrid, Spain
• ³Instituto de Salud Carlos III, Madrid, Spain
• ⁴CEU Universities, Madrid, Spain
• ⁵Institute of Applied Molecular Medicine (IMMA), Department of Basic Medical Sciences, Facultad de Medicina, Instituto de Medicina Molecular Aplicada-Nemesio Díez (IMMA-ND), Universidad San Pablo-CEU, Madrid, Spain
• ⁶Rijksinstituut voor Volksgezondheid en Milieu, Bilthoven, Netherlands
• ⁷Amsterdam UMC Locatie AMC, Amsterdam, Netherlands
• ⁸Icahn School of Medicine at Mount Sinai Department of Microbiology, New York, United States
• ⁹Instituto Nacional de Investigacion y Tecnologia Agraria y Alimentaria, Madrid, Spain
• ¹⁰Global Health and Emerging Pathogens Institute, Icahn School of Medicine., New York, United States
• ¹¹Icahn School of Medicine at Mount Sinai Lillian and Henry M Stratton-Hans Popper Department of Pathology Molecular and Cell-Based Medicine, New York, United States
• ¹²The Icahn Genomics Institute, Icahn School of Medicine at Mount Sinai, New York, United States
• ¹³The Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, United States
• ¹⁴Department of Animal Health, Faculty of Veterinary, Universidad de León, León, Spain
• ¹⁵Department of Oncological Sciences, Icahn School of Medicine at Mount Sinai, New Yorl, United States

Summary with Context (200 words): Respiratory coinfections involving Influenza viruses, including Influenza A viruses (IAV) and bacteria, significantly worsen disease severity and remain a major public health concern, particularly during seasonal and pandemic flu outbreaks. Among bacterial pathogens, Streptococcus pneumoniae (Spn) and Streptococcus suis cause secondary infections in humans and swine respectively following influenza. The immunological mechanisms driving coinfection severity, especially the differences between simultaneous and sequential infections, are incompletely defined. We developed an in vitro differentiated bone marrow-derived macrophages (BMDMs) model to examine transcriptional and protein-level responses during IAV-Spn coinfection or sequential infection. BMDMs were infected with IAV and Spn either simultaneously or with a 48-hour delay. RNA-Seq and OLINK proteomic analyses revealed that simultaneous coinfection elicits a synergistic inflammatory response similar to that caused by Spn alone, with strong activation of NF-κB-dependent genes. In sequential superinfection, responses were shaped by viral priming, with bacterial challenge further amplifying genes linked to inflammation and fibrin clot formation, potentially contributing to disease severity. These effects were consistent across different IAV subtypes when tested in combination with porcine Streptococcus suis serotypes that impose a comparable burden in pigs during influenza coinfection. Additionally, age is a determinant of BMDM responses. This model offers an advantageous tool for studying coinfection dynamics in human and veterinary medicine.