Expanded circulatory monocytes are necessary and sufficient for enhanced immunity against RSV infection.
To assess if the H. polygyrus- induced increase in lung mononuclear phagocytes contributes to the previously reported H. polygyrus induced antiviral effect against RSV, monocytes were depleted through administration of anti-CCR2 (MC-21) antibodies seven and nine days after H. polygyrus infection. Flow cytometry analysis of tail vein blood 10 dpi confirmed that the antibody treatment reversed the expansion of circulatory monocytes, returning their numbers to steady state levels (Fig. 6A). Anti-CCR2 treatment also prevented the increases in mononuclear phagocytes (Fig. 6B), Ly6C+macrophages (Fig. 6C) and CD11c+ macrophages (Fig. 6D) in the lung. Alveolar macrophage numbers appeared to be unaffected by anti-CCR2 treatment, as expected for this tissue resident population without overt inflammatory stimuli (Fig. S1C). Mice were then intranasally infected with 105 pfu RSV and their lungs were assessed for viral load by immunoplaque assay four days post RSV infection (14 days after H. polygyrus infection). The RSV load was significantly reduced following H. polygyrus infection, as expected. However, anti-CCR2 treatment prevented this decrease in RSV load (Fig. 6E). This demonstrates that increases in circulatory monocytes and lung mononuclear phagocytes are required for the H. polygyrus induced protective effect against RSV infection.
Finally, to complement these depletion experiments we used adoptive monocyte transfer to test if elevated numbers of blood monocytes are sufficient to replicate the H. polygyrus effect and limit RSV infection. Female BALB/c mice were infected with H. polygyrus and ten days later bone marrow was isolated and enriched for monocytes to 94% using negative lineage selection beads to minimise monocyte activation. Two million monocytes from H. polygyrus infected or control mice were intravenously transferred to naïve mice to raise their number in the circulation immediately prior to RSV infection. Analysis of the peak viral load four days after RSV infection showed significant decreases in RSV titres after adoptive monocyte transfer from both control and H. polygyrus infected animals. Taken together, these data demonstrate that increasing the numbers of circulatory monocytes is sufficient to increase anti-RSV immunity irrespective of the infection state of the donors (Fig. 6F).