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) Ly6CCD11c+ 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.