Figure Legends:
Fig 1. H. polygyrusinfection induces an expansion of lung mononuclear phagocytes.
Female BALB/c mice were administered an equal volume of either sterile
dH2O as infection control (i) or 200 H. polygyrus(H. poly) L3 larvae (ii) by oral gavage. Seven, 10, and 14 days post
infection (dpi) lungs were harvested for flow cytometry analysis. (A)
Representative gating strategy for the identification of alveolar
macrophages (CD45+, Ly6G-,
SigF+, CD64+) and mononuclear
phagocytes (CD45+,
Ly6G-, SigF-,
CD11b+, CD64+). (B-C) Absolute
numbers of (B) mononuclear phagocytes and (C) alveolar macrophages at
the indicated time points post H. polygyrus infection. (D)
Representative expression of Ly6C and CD11c by lung
SigF-, CD11b+,
CD64+ cells obtained from H. polygyrus infected
mice at 10 dpi. (E-F) Absolute numbers of (E)
Ly6C+CD11c– and (F)
Ly6C–CD11c+ mononuclear phagocytes
at the indicated time points post H. polygyrus infection. Symbols
represent individual mice with n=6 per group pooled from two independent
experiments. Statistical significance of difference determined with
unpaired two-tailed t-test. *P <0.05,
***P <0.001.
Fig 2. H. polygyrus infection induces circulatory
monocytosis.
Female BALB/c mice were administered an equal volume of either sterile
dH2O as infection control (i) or 200 H. polygyrus(H. poly) L3 larvae (ii) by oral gavage. (A) Flow cytometry to identify
monocytes (CD45+, Lineage-,
CD11b+, Ly6G-,
CD115+), and the monocytic subsets – classical
(Ly6C+, Treml4-) intermediate
(Ly6C+, Treml4+), and non-classical
(Ly6C-, Treml4+) (B) Monocyte
numbers per mL blood at indicated time points post H. polygyrusinfection. (C) Numbers of blood monocyte subtypes 10 dpi with H.
polygyrus . Symbols represent individual mice with n=6 per group pooled
from two independent experiments. Statistical significance of difference
determined with unpaired two-tailed t-test. **P <0.01,
***P <0.001.
Fig 3. H. polygyrus infection induces bone marrow
monopoiesis.
Female BALB/c mice were administered an equal volume of either sterile
dH2O as infection control (i) or 200 H. polygyrus(H. poly) L3 larvae (ii) by oral gavage. (A) Bone marrow colony forming
assays (CFAs) were performed on hind limb bone marrow 4 dpi, assessing
monocytic colony forming units (CFU-M) blast forming unit – erythroid
(BFU-E), colony forming unit granulocytic (CFU-G),
granulocytic/monocytic (CFU-GM), granulocytic/erythroid/monocytic mixed
(GEMM). (B) The increase in CFU-M subsides by 10 dpi. (C) Flow cytometry
analysis of bone marrow characterised bone marrow monocytes
(lineage-, CD16/CD32+,
CD117-, CD115+,
Ly6C+). (D) Numbers of bone marrow monocytes were
assessed throughout H. polygyrus infection. Symbols represent
individual mice n=4 for control and n=6 for H. polygyrus groups
pooled from two independent CFU experiments. Flow cytometry experiments
day 4 and 7 n=6 per group, day 10 n = 9 per group. Statistical
significance of difference determined with unpaired two-tailed t-test.
*P <0.05, **P <0.01.
Fig 4. IFNAR signalling is essential for H. polygyrusinduced blood monocyte and lung mononuclear phagocyte expansion but not
bone marrow monopoiesis.
Female C57BL/6 or Ifnar1-/- mice were administered 200 H.
polygyrus L3 larvae by oral gavage or an equal volume of sterile
dH2O as infection control. 10 dpi lungs were harvested
for flow cytometry analysis. Across C56BL/6 and Ifnar1-/- mice numbers
of (A) lung mononuclear phagocytes (B) blood monocytes and (C) bone
marrow Lyc6+ monocytes were assessed. (D) Bone marrow
colony forming assays (CFAs) were assessed from hind limb bone marrow 10
dpi (other CFA outputs non-significant). Symbols represent individual
mice with n=6 per group pooled from two independent experiments for
lung. One experiment of n=4 per group for blood and bone marrow.
Statistical significance of difference determined with one way ANOVA *P <0.05, ** P <0.01,
***P <0.001, ****P <0.0001.
Fig 5. H. polygyrus infection accelerates accumulation
of mononuclear phagocytes in early RSV infection. (A) Female BALB/c mice
were ntranasally administered 105 pfu RSV then culled
8 hours later and numbers of lung mononuclear phagocytes were assessed
by flow cytometry. (B-E) Female BALB/c mice were administered H.
polygyrus (H. poly) or dH2O infection control by oral
gavage and ten days later infected intranasally with
105 pfu RSV. Eight hours after RSV infection lungs
were analysed by flow cytometry to assess numbers of (B) lung
mononuclear phagocytes, (C) Ly6C+ macrophages and (D)
CD11c+ macrophages. (E) Macrophage subsets were also
assessed 96 hours post RSV administration with and without priorH. polygyrus infection. Symbols represent individual mice with
n=6 per group pooled from two independent experiments. Statistical
significance of difference determined with unpaired two-tailed t-test.
*P <0.05, *P <0.0001.
Fig 6. Circulatory monocytes are necessary and sufficient for
the anti-RSV effects of H. polygyrus infection.
Female BALB/c mice were administered H. polygyrus (H. poly) or
dH2O as a control by oral gavage. At 7 and 9 dpi theH. polygyrus infected animals were administered MC-21 anti-CCR2
antibody via IP injection. (A) Blood monocyte counts at 10 dpi. A subset
of mice were culled at 10 dpi to assess numbers of (B) mononuclear
phagocytes, (C) Ly6C+ macrophages and (D)
CD11C+ macrophages in the lung. All remaining mice
were infected intranasally with 105 pfu RSV. (E) Four
days post RSV infection viral load was assessed by immunoplaque assays.
(F) Bone marrow was collected from female BALB/c mice 10 dpi withH. polygyrus or dH2O as a control. Samples were
enriched for bone marrow monocytes using magnetic bead negative lineage
selection and 2 million monocytes administered intravenously via the
tail vein 30 minutes prior to RSV inoculation. PBS was used as a control
for monocyte administration. Viral load at 4 dpi RSV was assessed by
immunoplaque assay. Symbols represent
individual mice with n=6 per group pooled from two independent
experiments for RSV infection. For dual infection n=11 across two
independent experiments with four samples taken for lung flow cytometry
analysis and remainders for viral load assessment. Eight mice across two
experiments for monocyte transfer experiment. Statistical significance
of difference determined by one-way ANOVA with multiple comparisons.
*P <0.05, ** P <0.01,
****P <0.0001. Counts in (B-D) log transformed prior to
statistical analysis.
Fig S1. Alveolar macrophages in H. polygyrus and
respiratory syncytial virus infections. Female BALB/c mice were administered H. polygyrus (H. poly) or
dH2O infection control by oral gavage and ten days later
infected intranasally with 105 pfu RSV. Alveolar
macrophage numbers assessed at (A) 8 hours and (B) 96 hours post RSV
infection. Female BALB/c mice were administered H. polygyrus (H.
poly) or dH2O as a control by oral gavage. At 7 and 9
dpi the H. polygyrus infected animals were administered MC-21
anti-CCR2 antibody via IP injection. (C) Alveolar macrophage numbers
assessed 10 dpi. Symbols represent individual mice with n=6 per group
pooled from two independent experiments for H. polygyrus RSV
coinfections. For MC21 treatment n=4 per group pooled from two
independent experiments. All groups no significant differences.