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).