Exogenous M-CSF Protects from Lethal a. Fumigatus pulmonary Infection By Tissue-Resident Alveolar Macrophages Early after Hematopoietic Cell Transplantation

In the time window of bone marrow engraftment and immune reconstitution, patients undergoing hematopoietic cell transplantation (HCT) are vulnerable to life-threatening opportunistic infections, such as invasive fungal infections of the lung. Innate immune mechanisms, importantly tissue-resident alveolar macrophages (AMs), have been described as a first line of defense against fungal pathogens Therefore, we addressed how defined cytokines orchestrate AM responses to A. fumigatus in vivo after allogeneic HCT (allo-HCT). We employed super-resolution and dynamic confocal microscopy to study the impact of cytokines, mainly M-CSF and IL-34, on primary AMs phenotype and migration behavior in vitro. Additionally, we performed functional in vitro assays to study AMs phagocytic behavior. To study the outcome of exogenous cytokine stimulation on the pulmonary immune response in vivo we employed mouse models of allo-HCT (8 Gy TBI, C57Bl/6àBALB/c) and invasive A. fumigatus infection. To this end, we monitored the survival and clinical performance of the mice and investigated local host-pathogen interaction using flow cytometry and 3D light sheet fluorescence microscopy. HCT recipients survived A. fumigatus infection when infected 6 days but not 4 days after HCT. AMs showed the highest frequency, proliferation and phagocytic activity in the lung, when compared to neutrophils and monocytes, suggesting that tissue-resident AMs were responsible for protecting mice from A. fumigatus infection. This observation was confirmed by selective AM depletion, which rendered mice vulnerable to uncontainable infection. Subsequently, we investigated whether cytokines could boost AMs to achieve earlier protection. In vitro, M-CSF but not IL-34, increased AMs migration speed (0.52 vs. 0.3 µm/min, respectively) and versatility (diffusion co-efficient of 0.78 vs. 0.6 µm2/min, respectively). Next, we treated HCT-recipients with M-CSF and subsequently infected them intratracheally with A. fumigatus 4 days after HCT. M-CSF boosted myelopoiesis by 2-fold and, importantly, locally expanded tissue-resident AMs by 1.5-fold. Additionally, M-CSF improved AM killing capacity and protected 90% of HCT recipients from lethal A. fumigatus infection on day 4 after HCT. Additionally, M-CSF fostered lung tissue integrity and reduced serum levels of pro-inflammatory cytokines. Finally, M-CSF treatment did not protect from lethal aspergillosis upon local depletion of AMs in HCT recipients supporting the importance of a functional pool of tissue-resident AMs to protect from pulmonary infections maintaining lung function. Conclusively, these data support that M-CSF holds great potential for clinical application by fostering tissue-resident AMs responsiveness to preserve lung function and protect from early A. fumigatus infections in HCT recipients.