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\begin{document}

\title{Helminth induced monocytosis conveys protection from respiratory
syncytial virus infection in mice.}



\author[1]{Matthew Burgess O}%
\author[1]{Piotr Janas}%
\author[1]{Karla Berry}%
\author[2]{Hannah Mayr}%
\author[3]{Matthias Mack}%
\author[1]{Stephen Jenkins J}%
\author[1]{Calum Bain C}%
\author[4]{Henry J. McSorley}%
\author[1]{Jürgen Schwarze}%
\affil[1]{The University of Edinburgh Centre for Inflammation Research}%
\affil[2]{Medizinische Universitat Wien}%
\affil[3]{Universitat Regensburg}%
\affil[4]{University of Dundee Division of Cell Signalling and Immunology}%


\vspace{-1em}



  
  \date{May 24, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
\textbf{Background} Respiratory syncytial virus (RSV) infection in
infants is a major cause of viral bronchiolitis and hospitalisation. We
have previously shown in a murine model that ongoing infection with the
gut helminth \emph{Heligmosomoides polygyrus} ( \emph{H. polygyrus})
protects against RSV infection through type I interferon (IFN-I)
dependent reduction of viral load. Yet, the cellular basis for this
protection has remained elusive. Given that recruitment of mononuclear
phagocytes to the lung is critical for early RSV infection control, we
assessed their role in this coinfection model. \textbf{Methods} Mice
were infected by oral gavage with \emph{H. polygyrus}. Myeloid immune
cell populations were assessed by flow cytometry in lung, blood and bone
marrow throughout infection and after secondary infection with RSV.
Monocyte numbers were depleted by anti-CCR2 antibody or increased by
intravenous transfer of enriched monocytes. \textbf{Results} \emph{H.
polygyrus} infection induces bone marrow monopoiesis, increasing
circulatory monocytes and lung mononuclear phagocytes in a IFN-I
signalling dependent manner. This expansion causes enhanced lung
mononuclear phagocyte counts early in RSV infection that may contribute
to the reduction of RSV load. Depletion or supplementation of
circulatory monocytes prior to RSV infection confirms that these are
both necessary and sufficient for helminth induced antiviral protection.
\textbf{Conclusions} \emph{H. polygyrus} infection induces systemic
monocytosis contributing to elevated mononuclear phagocyte numbers in
the lung. These cells are central to an anti-viral effect that reduces
the peak viral load in RSV infection. Treatments to promote or modulate
these cells may provide novel paths to control RSV infection in high
risk individuals.%
\end{abstract}%



\sloppy


\emph{Title:}

Helminth induced monocytosis conveys protection from respiratory
syncytial virus infection in mice.

\emph{Short title:}

Helminth induced monocytes reduce RSV load.

\emph{Authors:}

Matthew O Burgess\textsubscript{1} Piotr Janas\textsubscript{1} Karla
Berry\textsubscript{1} Hannah Mayr\textsubscript{1} Matthias
Mack\textsubscript{2} Stephen J Jenkins\textsubscript{1} Calum C
Bain\textsubscript{1} Henry J McSorley\textsubscript{3} Jurgen
Schwarze\textsubscript{1, 4}

\emph{Author's affiliations:}

1 Centre for Inflammation Research, University of Edinburgh, United
Kingdom. 2 Department of Nephrology, University Hospital Regensburg,
Germany. 3 Cell signalling and Immunology, School of Life Sciences,
University of Dundee, United Kingdom, 4 Child Life and Health,
University of Edinburgh, United Kingdom.

Correspondence address for Hannah Mayr is now Institute of Medical
Genetics, Medical University of Vienna, Austria.

\emph{Acknowledgments:}

Study design by MOB, SJJ, CBB, HJMcS and JS. Experiments performed by
MOB with PJ, KB for flow cytometry and with HM for Methocult assays.
MOB, CCB and HJMcS analysed and discussed the data. MOB and JS drafted
the manuscript and MOB, HM, SJJ, CCB, HJMcS and JS critically reviewed
and revised the manuscript. JS obtained the funding. MM developed and
provided the MC-21 antibody. All authors gave final approval to the
version submitted for publication.

Flow cytometry data was generated with support from the QMRI Flow
Cytometry and cell sorting facility, University of Edinburgh. This work
was funded by UKRI-MRC grant MR/T029668/1. This UK funded award is part
of the EDCTP2 programme supported by the European Union. CCB is
supported by a Sir Henry Dale Fellowship jointly funded by the Wellcome
Trust and the Royal Society (grant number 206234/Z/17/Z). We thank Amy
Buck (University of Edinburgh, United Kingdom) for providing \emph{H.
polygyrus} L3 larvae and Samanta Mariani (University of Edinburgh,
United Kingdom) for advice on bone marrow Methocult assays.

\emph{Abstract:}

\textbf{Background} Respiratory syncytial virus (RSV) infection in
infants is a major cause of viral bronchiolitis and hospitalisation. We
have previously shown in a murine model that ongoing infection with the
gut helminth \emph{Heligmosomoides polygyrus} (\emph{H. polygyrus} )
protects against RSV infection through type I interferon (IFN-I)
dependent reduction of viral load. Yet, the cellular basis for this
protection has remained elusive. Given that recruitment of mononuclear
phagocytes to the lung is critical for early RSV infection control, we
assessed their role in this coinfection model.

\textbf{Methods} Mice were infected by oral gavage with \emph{H.
polygyrus} . Myeloid immune cell populations were assessed by flow
cytometry in lung, blood and bone marrow throughout infection and after
secondary infection with RSV. Monocyte numbers were depleted by
anti-CCR2 antibody or increased by intravenous transfer of enriched
monocytes.

\textbf{Results} \emph{H. polygyrus} infection induces bone marrow
monopoiesis, increasing circulatory monocytes and lung mononuclear
phagocytes in a IFN-I signalling dependent manner. This expansion causes
enhanced lung mononuclear phagocyte counts early in RSV infection that
may contribute to the reduction of RSV load. Depletion or
supplementation of circulatory monocytes prior to RSV infection confirms
that these are both necessary and sufficient for helminth induced
antiviral protection.

\textbf{Conclusions} \emph{H. polygyrus} infection induces systemic
monocytosis contributing to elevated mononuclear phagocyte numbers in
the lung. These cells are central to an anti-viral effect that reduces
the peak viral load in RSV infection. Treatments to promote or modulate
these cells may provide novel paths to control RSV infection in high
risk individuals.

\emph{Keywords:}

Helminth, Innate immunity, Mononuclear phagocyte, RSV, Virus

\emph{Main Text:}

\textbf{Introduction}

Respiratory syncytial virus (RSV) induced viral bronchiolitis is a major
cause of infant hospitalisation worldwide\textsuperscript{1}. Adult
infection is frequently associated with mild respiratory illness but can
also cause significant morbidity and mortality in the elderly and
immunocompromised individuals\textsuperscript{2,3}. There is as yet no
active vaccination towards or specific therapy for RSV infection
although many promising candidates are currently in clinical trials.
Treatment is limited to supportive measures and prophylaxis for high
risk infants with the anti-RSV F-protein antibody palivizumab, a high
cost option with limited effectiveness\textsuperscript{4,5}.

Novel approaches to managing RSV, and greater understanding of the
immune response to infection, are still required. We have previously
reported that an ongoing infection with the murine enteric
helminth\emph{Heligmosomoides polygyrus} (\emph{H. polygyrus} ) is able
to improve RSV infection outcomes by suppressing the peak viral load
early in infection\textsuperscript{6}. This work found that the adaptive
immune response to \emph{H. polygyrus} infection, the type 2 immune
response, characteristic of helminthic infection and typified by robust
IL-33, IL-4 and IL-13 signalling\textsuperscript{7}, as well as the
anti-microbial peptide LL-37 are not required for the \emph{H.
polygyrus} induced anti-viral immunity in the lung. Rather, type I
interferon (IFN-I) signalling, induction of interferon beta (IFN\selectlanguage{greek}β\selectlanguage{english}) and
interferon stimulated genes (ISGs), including \emph{Rsad2} (encoding
viperin) and \emph{Oas1a} are central to the \emph{H. polygyrus} induced
protective effect against RSV infection. Many ISGs have antiviral
functions and both viperin and OAS can limit RSV
infection\textsuperscript{8,9}. Almost all cells of the lung have the
capacity to both produce one or more of the family of IFN-Is and to
respond to IFN-I signalling through the dedicated receptor IFNalpha
receptor (IFNAR) \textsuperscript{10-12}. This broad potential space for
IFN-I signalling led us to question which cells may be contributing
to\emph{H. polygyrus} induced anti-viral effects. Mononuclear
phagocytes, including monocytes and macrophages, are known responders to
IFN-Is and have been linked to early control of RSV
infection\textsuperscript{13,14}. IFN-Is can regulate monocyte
recruitment during inflammation, skew hematopoietic output, and through
interferon regulatory factors promote differentiation to, and
polarisation of, macrophages\textsuperscript{15-18}. Monocytes can also
be potent produces of IFN-Is, especially IFN\selectlanguage{greek}β, \selectlanguage{english}in response to stimuli
including RSV\textsuperscript{19}. We therefore hypothesised that lung
mononuclear phagocytes following \emph{H. polygyrus} infection mediate
the helminth induced protective effect against RSV infection.

\textbf{Materials and Methods}

\emph{Animals}

BALB/c mice were purchased from Charles River (Margate, Kent, UK).
C57BL/6 and \emph{Ifnar1\textsuperscript{--/--}} (C57BL/6
background)\textsuperscript{20} mice were bred in-house at the
University of Edinburgh. Eight -- 12 week old mice were infected by oral
gavage with 200 stage 3 \emph{H. polygyrus} larvae. Some animals were
administered 20 \selectlanguage{greek}µ\selectlanguage{english}g of anti-CCR2 antibody (MC-21, gift M. Mack) by
intra-peritoneal injection seven and nine days later. Other animals were
given 2x10\textsuperscript{6} enriched bone marrow monocytes by
intravenous injection. Some animals were infected with RSV
(10\textsuperscript{5}plaque forming units) intranasally ten days after
\emph{H. polygyrus}infection. After monocyte transfer RSV was
administered 30 minutes later. All procedures were approved by local
ethical review committee and the UK Home Office.

\emph{Parasites and viral stocks}

The parasite life cycle was maintained as previously
described\textsuperscript{21}. Plaque-purified human RSV (Strain A2;
ATCC, VA, USA) was grown in HEp-2 cells as previously
described\textsuperscript{22}.

\emph{RSV immunoplaque assay}

RSV titres were assessed by immunoplaque assay. Lung homogenates were
titrated onto HEp-2 cell monolayers in 96-well plates. Twenty-four hours
later monolayers were fixed and permeabilised with 2\%
H\textsubscript{2}O\textsubscript{2} in methanol then bound with
biotin-conjugate goat anti-RSV antibody (BioRad; Watford, Hertfordshire,
UK). Plaques were visualised with extravadin peroxidase and AEC red
(SigmaAldrich; Glasgow, Scotland, UK) and infection units observed by
light microscopy.

\emph{L-gene real time PCR}

Lungs were homogenized in 1 mL of TRIzol (ThermoFisher Scientific;
Waltham, MA, USA). cDNA was made from the two \selectlanguage{greek}μ\selectlanguage{english}g extracted RNA using HC
cDNA RT kit (ThermoFisher Scientific; Waltham, MA, USA) according to
manufacturer's instructions. Custom primers and probe for RSV L gene
were used as previously described\textsuperscript{6}.

\emph{Colony forming assays}

Bone marrow colony forming assays were performed with MethoCult GF M3434
(StemCell Technologies; Cambridge, Cambridgeshire, UK) per
manufacturer's instructions. Bone marrow from hind limb tibia and femur
flushes was washed, 70 \selectlanguage{greek}µ\selectlanguage{english}M filtered and then resuspended in MethoCult GF
M3434 at 1x10\textsuperscript{4} cells per 35mm culture dish in
duplicate. Plates were incubated at 37\textsuperscript{o}C 5\%
CO\textsubscript{2} in humidified chambers. After 10-12 days colonies
were characterised to lineage and counted.

\emph{Flow cytometry}

Following digestion with Liberase (385\selectlanguage{greek}µ\selectlanguage{english}g/mL PBS; SigmaAldrich; Glasgow,
Scotland, UK) and DNaseI (0.5mg/mL; SigmaAldrich; Glasgow, Scotland, UK)
and red blood cell lysis (Hybrimax buffer; SigmaAldrich; Glasgow,
Scotland, UK) 2x10\textsuperscript{6} lung cells per panel were stained
with LIVE/DEAD fixable near-IR dead cell kit (ThermoFisher Scientific;
Waltham, MA, USA). The following anti-mouse antibodies were used for
immune cell characterisation; Brilliant Violet 510 CD11c (N418;
Biolegend; London, UK), Brilliant Violet 570 CD11b (M1/70; Biolegend;
London, UK), Brilliant Violet 605 CD45 (30-F11; Biolegend; London, UK),
AlexaFluor 647 CD64 (X54-5/7.1; Biolegend; London, UK), AlexaFluor 700
Ly6C (HK1.4; Biolegend; London, UK), phycoerythrin Siglec-F (E50-2440;
BD Biosciences; Wokingham, Berkshire, UK), eFluor 450 Ly6G (1A8;
ThermoFisher Scientific; Waltham, MA, USA). Fc-receptor binding was
blocked using anti-mouse CD16/CD32 antibody (2.4G2) (BD Biosciences;
Wokingham, Berkshire, UK).

Red blood cell were lysed (RBC lysis buffer; Biolegend; London, UK) and
50 \selectlanguage{greek}µ\selectlanguage{english}l per panel was stained with dead cell kit as above and the
following antibodies used; Lineage dump (Brilliant Violet 421 Siglec F
(E50-2440; BD Biosciences), Pacific Blue CD3 (17A2; Biolegend; London,
UK), CD19 (6D5; Biolegend; London, UK), and CD49b (PK136; Biolegend;
London, UK)), Brilliant Violet 510 Ly6G (1A8; Biolegend; London, UK),
Brilliant Violet 605 CD45 (30-F11; Biolegend; London, UK), Brilliant
Violet 650 CD11b (M1/70; Biolegend; London, UK), phycoerythrin Treml4
(16E5; Biolegend; London, UK), allophycocyanin CD115 (AFS98; Biolegend;
London, UK), AlexaFluor 700 Ly6C (HK1.4; Biolegend; London, UK).
Fc-receptor binding was blocked as above.

2x10\textsuperscript{6} bone marrow cells were stained with dead cell
kit as above, lineage dump (as above plus Pacific Blue Ly6G (1A8;
Biolegend; London, UK), TER-119 (TER-119; Biolegend; London, UK), CD11c
(N418; Biolegend; London, UK)), BV711 CD16/CD32 (93; Biolegend; London,
UK), PE-Cy7 CD117 (2B8; Biolegend; London, UK), allophycocyanin CD115
(AFS98; Biolegend; London, UK) and AlexaFluor 700 Ly6C (HK1.4;
Biolegend; London, UK). Fc-receptor binding was blocked using purified
mouse serum (M5905; SigmaAldrich; Glasgow, Scotland, UK).

Flow cytometry samples were analysed with an Aurora cytometer (Cytek;
Amsterdam, The Netherlands) and analysis performed with FCS Express 7
(De Novo Software; Pasadena, CA, USA).

\emph{Monocyte isolation}

To enrich bone marrow monocytes for cell transfer experiments mouse
monocyte isolation Kit (130-100-629; Miltenyl Biotec; Bisley, Surrey,
UK) was used.

\emph{Statistical analysis}

All data were analysed using Prism 9 software (GraphPad Software;
Boston, MA, USA). Data from two groups were analysed by unpaired\emph{t}
test, while data from more than two groups were analysed by one-way
ANOVA with Tukey's multiple comparisons test with a single pooled
variance. Unless otherwise stated, differences are statistically
non-significant. \emph{**** p} \textless{}0.0001, \emph{***
p}\textless{}0.001; \emph{** p} \textless{}0.01; \emph{*
p}\textless{}0.05.

\textbf{Results}

\emph{H. polygyrus infection induces blood monocytosis and increases
lung mononuclear phagocytes} .

The early response to RSV infection in mice drives the recruitment of
monocytes to the lung causing an accumulation of mononuclear
phagocytes\textsuperscript{13}. In the initial hours of infection these
cells are predominantly Ly6C\textsuperscript{hi}, later downregulating
Ly6C and becoming predominantly CD11c\textsuperscript{+}. Flow
cytometric analysis was used to assess if these populations were also
affected by \emph{H. polygyrus} infection. Female Balb/c mice were
infected with \emph{H. polygyrus} and lungs assessed for innate immune
cell composition through the later stages of infection. Cells of the
monocyte-macrophage lineage were identified by their expression of the
high affinity Fc receptor, CD64 (also known as Fc\selectlanguage{greek}γ\selectlanguage{english}R1), and subdivided on
the basis of SiglecF expression. SiglecF expressing
CD64\textsuperscript{+} cells represent alveolar macrophages, whereas
the SiglecF\textsuperscript{--} compartment will contain interstitial
macrophages and some monocytes and their progeny. Compared with sham
infection controls there was an increase in the numbers of mononuclear
phagocytes (CD45\textsuperscript{+}, Ly6G\textsuperscript{-},
SiglecF\textsuperscript{-}, CD11b\textsuperscript{+},
CD64\textsuperscript{+}, Fig. 1A) in the lungs of \emph{H.
polygyrus}infected mice from ten days post infection, subsiding yet
still elevated at 14 days. (Fig. 1B). In comparison, the resident
alveolar macrophage compartment (CD45\textsuperscript{+},
Ly6G\textsuperscript{-}, SiglecF\textsuperscript{+},
CD11b\textsuperscript{-}, CD64\textsuperscript{+}, Fig. 1A) remained
unchanged (Fig. 1C). The expansion was predominantly due to
Ly6C\textsuperscript{+} macrophages with a variable increase in
CD11c\textsuperscript{+} only at day ten (Fig. 1D-F).

A large proportion of the recruited macrophages seen in response to lung
inflammatory stimuli are derived from circulating
monocytes\textsuperscript{23,24}. It is therefore likely that the
vascular monocyte reservoir would reflect the observed influx of
mononuclear phagocytes to the lung. Peripheral blood was sampled from
mice throughout \emph{H. polygyrus} infection to assess circulatory
monocyte counts by flow cytometry (CD45\textsuperscript{+},
Lin\textsuperscript{-}, CD11b\textsuperscript{+},
Ly6G\textsuperscript{-}, CD115\textsuperscript{+}, Fig. 2A). Elevated
total monocyte numbers were observed from seven to ten days post
infection (Fig. 2B) and their numbers returned to baseline by 14 days
post infection. Circulatory monocytes in the mouse can be distinguished
into three phenotypic subtypes; classical, intermediate and
non-classical monocytes\textsuperscript{25,26}. Using the markers Ly6C
and Treml4 to separate these three populations (Fig. 2A), the \emph{H.
polygyrus} induced expansion of monocytes was found to be predominantly
in Ly6C\textsuperscript{+}Treml4\textsuperscript{-} classical monocytes
(Fig. 2C).

\emph{H. polygyrus infection induces bone marrow monopoiesis.}

Under normal physiological conditions, murine classical monocytes have a
half-life of just over one day in the circulation, with constant
replenishment from the bone marrow\textsuperscript{27,28}. To understand
if the monocytosis in blood during \emph{H. polygyrus} infection
reflected changes at the level of bone marrow haematopoiesis, we used
colony forming assays to assess the progenitor lineage potential
during\emph{H. polygyrus} infection. Four days post infection there was
an increase in the number of monocyte-committed progenitors (Colony
Forming Unit -- Monocytic; CFU-M) compared to uninfected controls (Fig.
3A). Although this elevation was short lived and lost by 10 dpi (Fig.
3B), enumeration of classical monocytes (Lin\textsuperscript{-},
CD16/CD32\textsuperscript{+}, CD117\textsuperscript{-},
CD115\textsuperscript{+}, Ly6C\textsuperscript{+}, Fig. 3C) in the bone
marrow confirmed that their numbers increase in the context of \emph{H.
polygyrus} infection (Fig. 3D).

\emph{H. polygyrus infection induced increase in blood monocytes and
lung mononuclear phagocytes is IFN-I signalling dependent.}

IFN-I signalling through IFNAR is essential to the \emph{H.
polygyrus}induced anti-viral effect\textsuperscript{6}. To test if the
expansion in mononuclear phagocytes was dependent on this signalling
axis, cell counts were assessed 10 dpi with H. polygyrus
in\emph{Ifnar1\textsuperscript{--/--}} mice, which are maintained on a
C57BL/6 background. Importantly, the \emph{H. polygyrus} induced
expansion of lung mononuclear phagocytes (Fig. 4A) and of circulatory
monocytes (Fig. 4B) were evident in control C57BL/6 mice but lost in the
absence of IFN-I signalling. In contrast, IFNAR-deficient mice still had
increases in committed monocyte progenitors in the bone marrow
after\emph{H. polygyrus} , albeit to a lower level than in wild type
controls. Ly6C\textsuperscript{+} monocyte numbers in the bone marrow
also seemed to increase after \emph{H. polygyrus} , but without reaching
statistical significance (Fig. 4C,D).

\emph{H. polygyrus monocytosis results in higher numbers of lung
mononuclear phagocytes early after RSV infection.}

RSV induced monocyte influx and subsequent accumulation of lung
mononuclear phagocytes is crucial for the production of cytokines
associated with the antiviral response\textsuperscript{13}. To ascertain
if this early influx was altered following \emph{H. polygyrus}infection,
we used flow cytometry to assess the monocyte-macrophage compartment
eight hours post RSV infection. RSV infection alone increased the
numbers of mononuclear phagocytes in the lung (Fig. 5A), as others have
observed, but after prior infection with \emph{H. polygyrus} their
counts were significantly higher than after RSV infection alone (Fig.
5B). This expansion was seen in both Ly6C\textsuperscript{+} and
CD11c\textsuperscript{+} macrophages (Fig. 5C,D). Four days after RSV
infection, numbers of Ly6C\textsuperscript{+}macrophages remained higher
in mice with prior \emph{H. polygyrus}infection, while the increase in
numbers of CD11c\textsuperscript{+}macrophages did not persist (Fig.
5E). Alveolar macrophage numbers were unaffected by prior \emph{H.
polygyrus} infection at both 8 and 96 hours post RSV infection (Fig.
S1A,B). These findings demonstrate that\emph{H. polygyrus} infection
leads to numbers of lung mononuclear phagocytes that exceed those found
early after RSV infection alone, raising the possibility that high
numbers of these cells confers an early protective effect against RSV
infection.

\emph{Expanded circulatory monocytes are necessary and sufficient for
enhanced immunity against RSV infection.}

To assess if the \emph{H. polygyrus-} induced increase in lung
mononuclear phagocytes contributes to the previously reported \emph{H.
polygyrus} induced antiviral effect against RSV, monocytes were depleted
through administration of anti-CCR2 (MC-21) antibodies seven and nine
days after \emph{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\textsuperscript{+}macrophages (Fig. 6C) and
CD11c\textsuperscript{+} 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
10\textsuperscript{5} pfu RSV and their lungs were assessed for viral
load by immunoplaque assay four days post RSV infection (14 days after
\emph{H. polygyrus} infection). The RSV load was significantly reduced
following \emph{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 \emph{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 \emph{H. polygyrus} effect and limit RSV
infection. Female BALB/c mice were infected with \emph{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 \emph{H. polygyrus} infected or
control mice were intravenously transferred to na\selectlanguage{ngerman}ïve 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 \emph{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).

\textbf{Discussion}

\emph{H. polygyrus} infection reduces RSV infection peak viral load,
secondary immuno-pathology, and associated lung function
impairment\textsuperscript{6}. We now describe how this helminth
infection induces enhanced monopoiesis in the bone marrow, elevated
circulatory monocytes and increased recruitment of monocyte derived
mononuclear phagocytes to the lung. Using monocyte ablation and transfer
we show the functional importance of circulatory monocyte derived cells
in the \emph{H. polygyrus} -induced anti-viral effect against RSV.
Elevated numbers of circulatory leukocytes over the first two weeks
of\emph{H. polygyrus} infection, including lymphocytes, neutrophils and
monocytes have previously been described but only in C57BL/6
mice\textsuperscript{29,30}. The correlative observations of bone marrow
myelopoiesis to drive this expansion in circulatory monocytes, and the
subsequent elevation of lung mononuclear phagocytes are novel to our
study. We expect that the mononuclear phagocyte population in the lung
is directly derived from circulatory monocytes. This is supported by the
circulatory increase being observed from 7 dpi, whereas lung mononuclear
phagocytes remain unchanged until 10 dpi. The absence of the increase in
lung mononuclear phagocytes when circulatory monocytes are ablated
supports this interpretation. However, due to the potential for
persistence of CCR2 on tissue mononuclear
phagocytes\textsuperscript{31}we cannot discount the possibility that we
directly ablated cells in the lung as well.

A growing body of work has assessed the interplay between parasitic and
viral infections\textsuperscript{32}. \emph{H. polygyrus} infection has
been previously described to suppress influenza A (IAV) infections
although the mechanism was
undetermined\textsuperscript{33}.\emph{Trichinella spiralis} infection
improves disease outcome to IAV in a gut damage dependent mechanism, but
this was through suppression of secondary inflammation, rather than
reduction in viral titres and no alteration in the mononuclear phagocyte
compartment was observed\textsuperscript{34}. \emph{Schistosoma mansoni}
(\emph{S. mansoni} ) infection is protective against secondary infection
with IAV and in pneumonia virus of mice (PVM) infection it gave a small
reduction in peak PVM load\textsuperscript{35}. \emph{S. mansoni} larvae
pass through the lung in mice so their effect on respiratory viral
infection is likely due to local inflammatory responses rather than to
the systemic response observed with the strictly enteric \emph{H.
polygyrus} . A mononuclear phagocyte response was not reported but
others have described \emph{S. mansoni} to drive recruitment of
alternatively activated macrophages\textsuperscript{36}. Their local
immunosuppressive effect is often detrimental to viral infection
control, with both\emph{H. polygyrus} and \emph{S. mansoni} infection
reactivating latent murine \selectlanguage{greek}γ-\selectlanguage{english}herpesvirus infections by suppressing the
antiviral IFN response\textsuperscript{37}.

The promotion of alternative activated macrophages in helminth infection
has also been described in tissues peripheral to the gut. \emph{H.
polygyrus} induces heart macrophages with a strong type 2 polarisation
which appear at 28 dpi\textsuperscript{38}. The absence of elevated
blood monocytes at this late time point, and the very small increase in
the number of monocytes and mononuclear phagocytes expressing the M2
marker CD301 (predominantly regulatory macrophages, data not shown) at
10 dpi in our study, further indicate that different mechanisms are
responsible for the early induction of lung mononuclear phagocytes
after\emph{H. polygyrus} infection and a later type-2 immunity driven
expansion of alternatively activated macrophages.

An active factor driving the early \emph{H. polygyrus} effects observed
in our study has not yet been identified but the fact that both the
antiviral effect and the expansion of both blood monocytes and lung
mononuclear phagocytes depend on IFNAR signalling likely implies a role
for IFN-Is either as direct effectors or stimuli for a secondary signal.
The monocytic response to IFN-I signalling is key to myeloid responses
in many other infections. IFNAR-deficient mice have been used widely in
SARS-CoV-2 models where this disruption impairs recruitment of
Ly6C\textsuperscript{+} monocytes to the lung\textsuperscript{39,40}. In
other inflammatory conditions this may be due to decreased turnover to
Ly6C\textsuperscript{-} monocytes, but the overall increase in total
monocyte and mononuclear phagocyte numbers in \emph{H.
polygyrus}infection suggests other additional
effects\textsuperscript{14}. IFNAR signalling has also been linked to
mononuclear phagocyte function in IAV infection\textsuperscript{41,42}
and to inflammatory monocyte accumulation in mucosal herpes simplex
virus infection\textsuperscript{43}. IFN-I signalling is also key to the
accumulation of inflammatory Ly6C\textsuperscript{+} mononuclear
phagocytes in the lung associated with older age, with a phenotype
similar to that seen in viral infections such as
IAV\textsuperscript{44}. In PVM infection IFNAR signalling has no effect
on monocytic cells but is required for the induction of inflammatory
conventional dendritic cells, indicating a likely infection specificity
of myeloid IFN-I signalling requirements\textsuperscript{45}. However,
dendritic cells express CD11c and may contribute to the
CD11c\textsuperscript{+} macrophages we have found to expand
after\emph{H. polygyrus} infection. Indeed the ongoing difficulty in
characterising the mononuclear phagocyte sub-compartments in murine
pulmonary inflammation (recently reviewed\textsuperscript{46,47}) due to
shared expression of the key markers used for steady state
characterisation is also a limitation in this study. It is important to
note that similar to the work in our study the use of global IFNAR
knockouts in the studies above means that despite the usually high
expression of IFNAR on monocytes, their response may be dependent on
secondary cells.

Effects of IFN-Is on blood monocytes are less well studied including
in\emph{Ifnar1} deficiency. The historical characterisations of
IFNAR-deficient mice showed elevated circulatory monocytes, counter to
our model, but the increased risk of infection in IFNAR-deficient mice,
at a time before modern infection control measures were available, may
have contributed to this effect\textsuperscript{20}. In human peripheral
blood mononuclear cells (PBMCs) classical monocytes are more responsive
to IFN-Is than non-classical monocytes due to differential abundance of
IFNAR\textsuperscript{48} and culture of PBMCs with IFN-Is will alter
their phenotype\textsuperscript{49-51}. An expansion of circulatory
monocytes and lung CD64\textsuperscript{+} cells, similar to our
findings, was observed in idiopathic pulmonary fibrosis patients and
associated with increased circulatory IFN-Is\textsuperscript{52}.
Exogenous treatment with IFN\selectlanguage{greek}α \selectlanguage{english}for two to four weeks in asthma patients
also increased the numbers of circulating monocytes and increased their
antigen presenting phenotype\textsuperscript{53}.

Clearly, from the array of responses described above the context of
IFN-I signalling is crucial. The outcome of IFN-I stimulation is
modulated by concomitant signalling/environmental factors that certainly
also play a role in \emph{H. polygyrus} responses\textsuperscript{54}.
One such environmental factor is the microbiome and it's metabolic
products, the presence of which is essential for the \emph{H.
polygyrus}antiviral effect\textsuperscript{6}. While we were unable to
test the impact of the microbiome in the present study, interactions
between IFN-I production and microbiome components have been well
described\textsuperscript{55,56} and the microbiome is known to be
modulated in helminth infections including \emph{H.
polygyrus}infection\textsuperscript{57,58}.

The apparent lack of dependency on IFNAR signalling for bone marrow
monopoiesis in this model suggests alternative signalling requirements.
Interferon treatments have been described to affect
lymphopoiesis\textsuperscript{59}, IFN-I suppresses neutrophil
differentiation\textsuperscript{60,61}, and IFN\selectlanguage{greek}γ \selectlanguage{english}promotes
myelopoiesis\textsuperscript{62,63}. Parasitic modulation of the bone
marrow has also been described, with \emph{Trichuris muris}
and\emph{Trichuris gondii} infections modulating haematopoiesis in an
IFN\selectlanguage{greek}γ \selectlanguage{english}dependent manner\textsuperscript{64,65}. There is also substantial
evidence that interferon regulatory factor 5 (IRF5) is a key regulator
of hematopoietic development, particularly in myeloid
lineages\textsuperscript{66,67}. \emph{H. polygyrus} has been recently
described to induce IFN-\selectlanguage{greek}γ \selectlanguage{english}(IFN-II) early in
infection\textsuperscript{68} which may be responsible for the initial
induction of monopoiesis. The deeply interlinked feedback loops between
IFN-I and IFN-II signalling may explain why we see a reduced effect on
monocytosis in the bone marrow\textsuperscript{69}.

The mechanism of the anti-viral effect of \emph{H. polygyrus} -induced
mononuclear phagocytes is as yet unclear. In RSV infection mononuclear
phagocytes are known to produce TNF\selectlanguage{greek}α \selectlanguage{english}which is important for control of
viral expansion, providing a possible mechanism\textsuperscript{13} but
their direct effects on viral load were not assessed. Our findings
directly demonstrate, for the first time, that elevating mononuclear
phagocyte numbers in the lung prior to RSV infection can reduce viral
load. Monocytes/macrophages are also known to produce IFN\selectlanguage{greek}β, \selectlanguage{english}viperin and
OAS in response to viral exposure, thus the expansion of mononuclear
phagocytes in the lung may explain the previously observed increase in
these antiviral factors in whole lung samples during \emph{H.
polygyrus}infection\textsuperscript{19,70-73}.

In conclusion, we show that intestinal helminth infection can induce
transient systemic monocytosis and increased numbers of mononuclear
phagocytes within the lung that are essential to reducing the burden of
viral respiratory infection. Due to the IFNAR dependency of this
expansion in the blood and lung we hypothesise that helminth induced
IFN-Is or secondary signalling products play a key role in these
effects. Future work will be required to identify the specific factors
that act upon monocytes to drives these effects and to further
characterise the specific anti-viral mechanisms of these cells in RSV
infection.

\emph{References:}

1. Estimates of the global, regional, and national morbidity, mortality,
and aetiologies of lower respiratory infections in 195 countries,
1990-2016: a systematic analysis for the Global Burden of Disease Study
2016. \emph{Lancet Infect Dis.}2018;18(11):1191-1210.

2. Falsey AR, Hennessey PA, Formica MA, Cox C, Walsh EE. Respiratory
syncytial virus infection in elderly and high-risk adults. \emph{N Engl
J Med.} 2005;352(17):1749-1759.

3. Nguyen-Van-Tam JS, O'Leary M, Martin ET, et al. Burden of respiratory
syncytial virus infection in older and high-risk adults: a systematic
review and meta-analysis of the evidence from developed countries.
\emph{Eur Respir Rev.} 2022;31(166).

4. Binns E, Tuckerman J, Licciardi PV, Wurzel D. Respiratory syncytial
virus, recurrent wheeze and asthma: A narrative review of
pathophysiology, prevention and future directions.\emph{J Paediatr Child
Health.} 2022;58(10):1741-1746.

5. Soto JA, Stephens LM, Waldstein KA, Canedo-Marroquin G, Varga SM,
Kalergis AM. Current Insights in the Development of Efficacious Vaccines
Against RSV. \emph{Front Immunol.}2020;11:1507.

6. McFarlane AJ, McSorley HJ, Davidson DJ, et al. Enteric
helminth-induced type I interferon signaling protects against pulmonary
virus infection through interaction with the microbiota. \emph{J Allergy
Clin Immunol.} 2017(140):1068-1078.

7. Maizels RM. Regulation of immunity and allergy by helminth parasites.
\emph{Allergy.} 2020;75(3):524-534.

8. Schoggins JW, Wilson SJ, Panis M, et al. A diverse range of gene
products are effectors of the type I interferon antiviral response.
\emph{Nature.} 2011;472(7344):481-485.

9. Schoggins JW. Interferon-Stimulated Genes: What Do They All Do?
\emph{Annu Rev Virol.} 2019;6(1):567-584.

10. Sposito B, Broggi A, Pandolfi L, et al. The interferon landscape
along the respiratory tract impacts the severity of COVID-19.
\emph{Cell.} 2021;184(19):4953-4968 e4916.

11. McNab F, Mayer-Barber K, Sher A, Wack A, O'Garra A. Type I
interferons in infectious disease. \emph{Nat Rev Immunol.}
2015;15(2):87-103.

12. Khaitov MR, Laza-Stanca V, Edwards MR, et al. Respiratory virus
induction of alpha-, beta- and lambda-interferons in bronchial
epithelial cells and peripheral blood mononuclear cells. \emph{Allergy.}
2009;64(3):375-386.

13. Goritzka M, Makris S, Kausar F, et al. Alveolar macrophage-derived
type I interferons orchestrate innate immunity to RSV through
recruitment of antiviral monocytes. \emph{J Exp Med.}
2015;212(5):699-714.

14. Lee PY, Li Y, Kumagai Y, et al. Type I interferon modulates monocyte
recruitment and maturation in chronic inflammation. \emph{Am J Pathol.}
2009;175(5):2023-2033.

15. Krausgruber T, Blazek K, Smallie T, et al. IRF5 promotes
inflammatory macrophage polarization and TH1-TH17 responses. \emph{Nat
Immunol.} 2011;12(3):231-238.

16. Askenasy N. Interferon and tumor necrosis factor as humoral
mechanisms coupling hematopoietic activity to inflammation and injury.
\emph{Blood Rev.} 2015;29(1):11-15.

17. Essers MA, Offner S, Blanco-Bose WE, et al. IFNalpha activates
dormant haematopoietic stem cells in vivo.\emph{Nature.}
2009;458(7240):904-908.

18. Dalmas E, Toubal A, Alzaid F, et al. Irf5 deficiency in macrophages
promotes beneficial adipose tissue expansion and insulin sensitivity
during obesity. \emph{Nat Med.}2015;21(6):610-618.

19. Kim TH, Kim CW, Oh DS, Jung HE, Lee HK. Monocytes Contribute to
IFN-beta Production via the MyD88-Dependent Pathway and Cytotoxic T-Cell
Responses against Mucosal Respiratory Syncytial Virus Infection.
\emph{Immune Netw.}2021;21(4):e27.

20. Muller U, Steinhoff U, Reis LF, et al. Functional role of type I and
type II interferons in antiviral defense. \emph{Science.}
1994;264(5167):1918-1921.

21. Johnston CJ, Robertson E, Harcus Y, et al. Cultivation of
Heligmosomoides polygyrus: an immunomodulatory nematode parasite and its
secreted products. \emph{J Vis Exp.}2015(98):e52412.

22. Currie SM, Findlay EG, McHugh BJ, et al. The human cathelicidin
LL-37 has antiviral activity against respiratory syncytial virus.
\emph{PLoS One.} 2013;8(8):e73659.

23. Plantinga M, Guilliams M, Vanheerswynghels M, et al. Conventional
and monocyte-derived CD11b(+) dendritic cells initiate and maintain T
helper 2 cell-mediated immunity to house dust mite allergen.
\emph{Immunity.} 2013;38(2):322-335.

24. Hawley CA, Rojo R, Raper A, et al. Csf1r-mApple Transgene Expression
and Ligand Binding In Vivo Reveal Dynamics of CSF1R Expression within
the Mononuclear Phagocyte System.\emph{J Immunol.}
2018;200(6):2209-2223.

25. Tacke F, Randolph GJ. Migratory fate and differentiation of blood
monocyte subsets.\emph{Immunobiology.} 2006;211(6-8):609-618.

26. Ingersoll MA, Spanbroek R, Lottaz C, et al. Comparison of gene
expression profiles between human and mouse monocyte subsets.
\emph{Blood.} 2010;115(3):e10-19.

27. Yona S, Kim KW, Wolf Y, et al. Fate mapping reveals origins and
dynamics of monocytes and tissue macrophages under homeostasis.
\emph{Immunity.} 2013;38(1):79-91.

28. Hettinger J, Richards DM, Hansson J, et al. Origin of monocytes and
macrophages in a committed progenitor.\emph{Nat Immunol.}
2013;14(8):821-830.

29. Ali NM, Behnke JM, Manger BR. The pattern of peripheral blood
leucocyte changes in mice infected with Nematospiroides dubius. \emph{J
Helminthol.} 1985;59(1):83-93.

30. Baker NF. The nature and etiology of the leukocytic response of
Webster mice infected with Nematospiroides dubius. \emph{J Parasitol.}
1962;48:438-441.

31. Dick SA, Wong A, Hamidzada H, et al. Three tissue resident
macrophage subsets coexist across organs with conserved origins and life
cycles. \emph{Sci Immunol.}2022;7(67):eabf7777.

32. Desai P, Diamond MS, Thackray LB. Helminth-virus interactions:
determinants of coinfection outcomes.\emph{Gut Microbes.}
2021;13(1):1961202.

33. Chowaniec W, Wescott RB, Congdon LL. Interaction of Nematospiroides
dubius and influenza virus in mice.\emph{Exp Parasitol.}
1972;32(1):33-44.

34. Furze RC, Hussell T, Selkirk ME. Amelioration of influenza-induced
pathology in mice by coinfection with Trichinella spiralis. \emph{Infect
Immun.} 2006;74(3):1924-1932.

35. Scheer S, Krempl C, Kallfass C, et al. S. mansoni bolsters
anti-viral immunity in the murine respiratory tract. \emph{PLoS One.}
2014;9(11):e112469.

36. Santos MP, Goncalves-Santos E, Goncalves RV, et al. Doxycycline
aggravates granulomatous inflammation and lung microstructural
remodeling induced by Schistosoma mansoni infection. \emph{Int
Immunopharmacol.} 2021;94:107462.

37. Reese TA, Wakeman BS, Choi HS, et al. Helminth infection reactivates
latent gamma-herpesvirus via cytokine competition at a viral promoter.
\emph{Science.} 2014;345(6196):573-577.

38. Mylonas KJ, Jenkins SJ, Castellan RF, et al. The adult murine heart
has a sparse, phagocytically active macrophage population that expands
through monocyte recruitment and adopts an 'M2' phenotype in response to
Th2 immunologic challenge.\emph{Immunobiology.} 2015;220(7):924-933.

39. Ogger PP, Garcia Martin M, Michalaki C, et al. Type I interferon
receptor signalling deficiency results in dysregulated innate immune
responses to SARS-CoV-2 in mice.\emph{Eur J Immunol.}
2022;52(11):1768-1775.

40. Channappanavar R, Fehr AR, Vijay R, et al. Dysregulated Type I
Interferon and Inflammatory Monocyte-Macrophage Responses Cause Lethal
Pneumonia in SARS-CoV-Infected Mice. \emph{Cell Host Microbe.}
2016;19(2):181-193.

41. Garcia-Sastre A, Durbin RK, Zheng H, et al. The role of interferon
in influenza virus tissue tropism.\emph{J Virol.} 1998;72(11):8550-8558.

42. Price GE, Gaszewska-Mastarlarz A, Moskophidis D. The role of
alpha/beta and gamma interferons in development of immunity to influenza
A virus in mice. \emph{J Virol.}2000;74(9):3996-4003.

43. Lee AJ, Chen B, Chew MV, et al. Inflammatory monocytes require type
I interferon receptor signaling to activate NK cells via IL-18 during a
mucosal viral infection. \emph{J Exp Med.} 2017;214(4):1153-1167.

44. D'Souza SS, Zhang Y, Bailey JT, et al. Type I Interferon signaling
controls the accumulation and transcriptomes of monocytes in the aged
lung. \emph{Aging Cell.}2021;20(10):e13470.

45. Bosteels C, Neyt K, Vanheerswynghels M, et al. Inflammatory Type 2
cDCs Acquire Features of cDC1s and Macrophages to Orchestrate Immunity
to Respiratory Virus Infection. \emph{Immunity.} 2020;52(6):1039-1056
e1039.

46. Aegerter H, Lambrecht BN, Jakubzick CV. Biology of lung macrophages
in health and disease.\emph{Immunity.} 2022;55(9):1564-1580.

47. T'Jonck W, Bain CC. The role of monocyte-derived macrophages in the
lung: It's all about context.\emph{Int J Biochem Cell Biol.}
2023;159:106421.

48. Han S, Zhuang H, Lee PY, et al. Differential Responsiveness of
Monocyte and Macrophage Subsets to Interferon. \emph{Arthritis
Rheumatol.} 2020;72(1):100-113.

49. Pogue SL, Preston BT, Stalder J, Bebbington CR, Cardarelli PM. The
receptor for type I IFNs is highly expressed on peripheral blood B cells
and monocytes and mediates a distinct profile of differentiation and
activation of these cells.\emph{J Interferon Cytokine Res.}
2004;24(2):131-139.

50. Dickensheets HL, Donnelly RP. Inhibition of IL-4-inducible gene
expression in human monocytes by type I and type II interferons. \emph{J
Leukoc Biol.} 1999;65(3):307-312.

51. Tong Y, Zhou L, Yang L, et al. Concomitant type I IFN and M-CSF
signaling reprograms monocyte differentiation and drives pro-tumoral
arginase production.\emph{EBioMedicine.} 2019;39:132-144.

52. Fraser E, Denney L, Antanaviciute A, et al. Multi-Modal
Characterization of Monocytes in Idiopathic Pulmonary Fibrosis Reveals a
Primed Type I Interferon Immune Phenotype.\emph{Front Immunol.}
2021;12:623430.

53. Simon HU, Seelbach H, Ehmann R, Schmitz M. Clinical and
immunological effects of low-dose IFN-alpha treatment in patients with
corticosteroid-resistant asthma.\emph{Allergy.} 2003;58(12):1250-1255.

54. van Boxel-Dezaire AH, Rani MR, Stark GR. Complex modulation of cell
type-specific signaling in response to type I interferons.
\emph{Immunity.} 2006;25(3):361-372.

55. Winkler ES, Shrihari S, Hykes BL, Jr., et al. The Intestinal
Microbiome Restricts Alphavirus Infection and Dissemination through a
Bile Acid-Type I IFN Signaling Axis.\emph{Cell.} 2020;182(4):901-918
e918.

56. Erttmann SF, Swacha P, Aung KM, et al. The gut microbiota prime
systemic antiviral immunity via the cGAS-STING-IFN-I axis.
\emph{Immunity.} 2022;55(5):847-861 e810.

57. Kennedy MHE, Brosschot TP, Lawrence KM, et al. Small Intestinal
Levels of the Branched Short-Chain Fatty Acid Isovalerate Are Elevated
during Infection with Heligmosomoides polygyrus and Can Promote Helminth
Fecundity.\emph{Infect Immun.} 2021;89(12):e0022521.

58. Reynolds LA, Smith KA, Filbey KJ, et al. Commensal-pathogen
interactions in the intestinal tract: lactobacilli promote infection
with, and are promoted by, helminth parasites. \emph{Gut Microbes.}
2014;5(4):522-532.

59. Di Scala M, Gil-Farina I, Vanrell L, et al. Chronic exposure to
IFNalpha drives medullar lymphopoiesis towards T-cell differentiation in
mice. \emph{Haematologica.}2015;100(8):1014-1022.

60. Siakaeva E, Pylaeva E, Spyra I, et al. Neutrophil Maturation and
Survival Is Controlled by IFN-Dependent Regulation of NAMPT Signaling.
\emph{Int J Mol Sci.} 2019;20(22).

61. Nguyen-Jackson H, Panopoulos AD, Zhang H, Li HS, Watowich SS. STAT3
controls the neutrophil migratory response to CXCR2 ligands by direct
activation of G-CSF-induced CXCR2 expression and via modulation of CXCR2
signal transduction.\emph{Blood.} 2010;115(16):3354-3363.

62. Grainger JR, Wohlfert EA, Fuss IJ, et al. Inflammatory monocytes
regulate pathologic responses to commensals during acute
gastrointestinal infection. \emph{Nat Med.}2013;19(6):713-721.

63. Buechler MB, Akilesh HM, Hamerman JA. Cutting Edge: Direct Sensing
of TLR7 Ligands and Type I IFN by the Common Myeloid Progenitor Promotes
mTOR/PI3K-Dependent Emergency Myelopoiesis. \emph{J Immunol.}
2016;197(7):2577-2582.

64. Chenery AL, Antignano F, Hughes MR, Burrows K, McNagny KM, Zaph C.
Chronic Trichuris muris infection alters hematopoiesis and causes
IFN-gamma-expressing T-cell accumulation in the mouse bone marrow.
\emph{Eur J Immunol.} 2016;46(11):2587-2596.

65. Askenase MH, Han SJ, Byrd AL, et al. Bone-Marrow-Resident NK Cells
Prime Monocytes for Regulatory Function during Infection.
\emph{Immunity.} 2015;42(6):1130-1142.

66. Schoenemeyer A, Barnes BJ, Mancl ME, et al. The interferon
regulatory factor, IRF5, is a central mediator of toll-like receptor 7
signaling. \emph{J Biol Chem.}2005;280(17):17005-17012.

67. Corbin AL, Gomez-Vazquez M, Berthold DL, et al. IRF5 guides
monocytes toward an inflammatory CD11c(+) macrophage phenotype and
promotes intestinal inflammation.\emph{Sci Immunol.} 2020;5(47).

68. Progatzky F, Shapiro M, Chng SH, et al. Regulation of intestinal
immunity and tissue repair by enteric glia. \emph{Nature.}
2021;599(7883):125-130.

69. Michalska A, Blaszczyk K, Wesoly J, Bluyssen HAR. A Positive
Feedback Amplifier Circuit That Regulates Interferon (IFN)-Stimulated
Gene Expression and Controls Type I and Type II IFN Responses.
\emph{Front Immunol.} 2018;9:1135.

70. Nasr N, Maddocks S, Turville SG, et al. HIV-1 infection of human
macrophages directly induces viperin which inhibits viral production.
\emph{Blood.} 2012;120(4):778-788.

71. Lee D, Le Pen J, Yatim A, et al. Inborn errors of OAS-RNase L in
SARS-CoV-2-related multisystem inflammatory syndrome in children.
\emph{Science.}2023;379(6632):eabo3627.

72. Fagone P, Nunnari G, Lazzara F, et al. Induction of OAS gene family
in HIV monocyte infected patients with high and low viral load.
\emph{Antiviral Res.} 2016;131:66-73.

73. Teng TS, Foo SS, Simamarta D, et al. Viperin restricts chikungunya
virus replication and pathology.\emph{J Clin Invest.}
2012;122(12):4447-4460.

\emph{Figure Legends:}

\textbf{Fig 1.} \emph{H. polygyrus}infection induces an expansion of
lung mononuclear phagocytes. Female BALB/c mice were administered an
equal volume of either sterile dH\textsubscript{2}O as infection control
(i) or 200 \emph{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\textsuperscript{+},
Ly6G\textsuperscript{-}, SigF\textsuperscript{+},
CD64\textsuperscript{+}) and mononuclear phagocytes
(CD45\textsuperscript{+}, Ly6G\textsuperscript{-},
SigF\textsuperscript{-}, CD11b\textsuperscript{+},
CD64\textsuperscript{+}). (B-C) Absolute numbers of (B) mononuclear
phagocytes and (C) alveolar macrophages at the indicated time points
post \emph{H. polygyrus} infection. (D) Representative expression of
Ly6C and CD11c by lung SigF\textsuperscript{-},
CD11b\textsuperscript{+}, CD64\textsuperscript{+} cells obtained from
\emph{H. polygyrus} infected mice at 10 dpi. (E-F) Absolute numbers of
(E) Ly6C\textsuperscript{+}CD11c\textsuperscript{--} and (F)
Ly6C\textsuperscript{--}CD11c\textsuperscript{+} 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. *\emph{P} \textless{}0.05, ***\emph{P}
\textless{}0.001.

\textbf{Fig 2.} \emph{H. polygyrus} infection induces circulatory
monocytosis. Female BALB/c mice were administered an equal volume of
either sterile dH\textsubscript{2}O as infection control (i) or 200
\emph{H. polygyrus}(H. poly) L3 larvae (ii) by oral gavage. (A) Flow
cytometry to identify monocytes (CD45\textsuperscript{+},
Lineage\textsuperscript{-}, CD11b\textsuperscript{+},
Ly6G\textsuperscript{-}, CD115\textsuperscript{+}), and the monocytic
subsets -- classical (Ly6C\textsuperscript{+},
Treml4\textsuperscript{-}) intermediate (Ly6C\textsuperscript{+},
Treml4\textsuperscript{+}), and non-classical (Ly6C\textsuperscript{-},
Treml4\textsuperscript{+}) (B) Monocyte numbers per mL blood at
indicated time points post \emph{H. polygyrus}infection. (C) Numbers of
blood monocyte subtypes 10 dpi with \emph{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. **\emph{P} \textless{}0.01, ***\emph{P}
\textless{}0.001.

\textbf{Fig 3.} \emph{H. polygyrus} infection induces bone marrow
monopoiesis. Female BALB/c mice were administered an equal volume of
either sterile dH\textsubscript{2}O as infection control (i) or 200
\emph{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\textsuperscript{-},
CD16/CD32\textsuperscript{+}, CD117\textsuperscript{-},
CD115\textsuperscript{+}, Ly6C\textsuperscript{+}). (D) Numbers of bone
marrow monocytes were assessed throughout \emph{H. polygyrus} infection.
Symbols represent individual mice n=4 for control and n=6 for \emph{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. *\emph{P} \textless{}0.05, **\emph{P}
\textless{}0.01.

\textbf{Fig 4.} IFNAR signalling is essential \emph{for H.
polygyrus}induced blood monocyte and lung mononuclear phagocyte
expansion but not bone marrow monopoiesis. Female C57BL/6 or
\emph{Ifnar1-/-} mice were administered 200 \emph{H. polygyrus} L3
larvae by oral gavage or an equal volume of sterile dH\textsubscript{2}O
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\textsuperscript{+} 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 *\emph{P}
\textless{}0.05, ** \emph{P} \textless{}0.01, ***\emph{P}
\textless{}0.001, ****\emph{P} \textless{}0.0001.

\textbf{Fig 5.} \emph{H. polygyrus} infection accelerates accumulation
of mononuclear phagocytes in early RSV infection. (A) Female BALB/c mice
were ntranasally administered 10\textsuperscript{5} 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 \emph{H.
polygyrus} (H. poly) or dH\textsubscript{2}O infection control by oral
gavage and ten days later infected intranasally with
10\textsuperscript{5} pfu RSV. Eight hours after RSV infection lungs
were analysed by flow cytometry to assess numbers of (B) lung
mononuclear phagocytes, (C) Ly6C\textsuperscript{+} macrophages and (D)
CD11c\textsuperscript{+} macrophages. (E) Macrophage subsets were also
assessed 96 hours post RSV administration with and without prior\emph{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. *\emph{P}
\textless{}0.05, *\emph{P} \textless{}0.0001.

\textbf{Fig 6.} Circulatory monocytes are necessary and sufficient for
the anti-RSV effects of \emph{H. polygyrus} infection. Female BALB/c
mice were administered \emph{H. polygyrus} (H. poly) or
dH\textsubscript{2}O as a control by oral gavage. At 7 and 9 dpi
the\emph{H. 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\textsuperscript{+} macrophages and (D)
CD11C\textsuperscript{+} macrophages in the lung. All remaining mice
were infected intranasally with 10\textsuperscript{5} 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
with\emph{H. polygyrus} or dH\textsubscript{2}O 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. *\emph{P} \textless{}0.05, ** \emph{P}
\textless{}0.01, ****\emph{P} \textless{}0.0001. Counts in (B-D) log
transformed prior to statistical analysis.

\textbf{Fig S1. Alveolar macrophages in \emph{H. polygyrus} and
respiratory syncytial virus infections.} Female BALB/c mice were
administered \emph{H. polygyrus} (H. poly) or dH\textsubscript{2}O
infection control by oral gavage and ten days later infected
intranasally with 10\textsuperscript{5} pfu RSV. Alveolar macrophage
numbers assessed at (A) 8 hours and (B) 96 hours post RSV infection.
Female BALB/c mice were administered \emph{H. polygyrus} (H. poly) or
dH\textsubscript{2}O as a control by oral gavage. At 7 and 9 dpi the
\emph{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.

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