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Interleukin6playsamultivariateroleinresponsetorespiratorysyncytialvirusinfection

MollyBrazil
Abstract
Respiratorysyncytialvirus(RSV)isoneoftheleadingcausesforpatientadmissionsin
ournationshospitals,andgloballyitisthemostcommoncauseofseverelowerrespiratorytract
infectionsinyoungchildren.Theviruscausesapproximatelyfourmilliondeathsannually,but
thereisnocurrentvaccine.Interleukin6(IL6)isapluripotentcytokineproducedbymost
nucleatedcellsandispartoftheacutephaseresponse;previousresearchhasfoundIL6tobean
importantcontributortothedevelopmentofTh17adaptiveimmunity(Korn).Thepurposeofthis
studywastoexplorehowIL6influenceshostimmuneresponsetoRSVinfectionthrough
interactionswithTh2andTh17pathways.Bronchoalveolarlavage(BAL)andquantitativereal
timepolymerasechainreaction(QPCR)oflungtissuewereusedtodetermineleukocyte
differentiationandIL6,IL17aandmucusproducinggeneexpression.Ourdataindicatesthatin
thepresenceofRSV,IL6drivestheproductionofIL17a,anothertypeofcytokine,leadingto
increasedmucusproduction,whileatthesametimeplayingaprotectiverolebylimiting
neutrophilrecruitment.TheseresultssuggestthatIL6playsamultivariateroleinhostdefense.
Introduction
Respiratorysyncytialvirus(RSV)isanenvelopedRNApneumovirusbelongingtothe
familyParamyxoviridae.Itissoprevalentthatmostinfantshavebeeninfectedbytheageof
two,anditistheleadingcauseofbronchiolitisandpneumoniainchildrenundertheageofone
intheUnitedStates(Nair).Inadditiontoyoungchildren,theelderlypopulationisnow

recognizedasahighriskdemographicforsevereinfection(Branche).RSVisalsoasignificant
burdenglobally;theWorldHealthOrganization(WHO)reportsthatitresultsinapproximately
fourmilliondeathsannually.Foralloftheaforementionedreasons,developingavaccinefor
RSVisconsideredahighpriority.
RSV,however,hasmanyuniqueandmajorhurdlestoovercomeinordertodevelopa
successfulvaccine.First,naturalinfectionresultsinincompleteimmunityresultingin
reoccurringinfectionsseasonafterseason(Ohuma).Secondly,theyoungageofinfection
imposesatimebarrier;therehastobeabalancebetweenwaitinguntilitissafetovaccinatebut
notwaitinguntilitistoolate(Anderson).Lastly,thereisahistoryofvaccineenhanceddisease;
aformalininactivatedwholevirusvaccinewasdevelopedinthe1960s,butclinicaltrials
revealedthatthevaccineledtoamoreseveresecondaryresponsewhenlaterexposedtoRSV
(Graham).
InflammationandtheoverproductionofmucusarechiefcontributorstoRSVdisease
pathology(Jafri).Togetherthesesymptomscauserestrictedairways,especiallyininfants,
leadingtolaboredbreathingandshortnessofbreath(Jafri).PreviousresearchfoundthatIL17,
producedbyThelper17(Th17)cells,playsapathogenicroleinRSVinfectionbyinducing
mucushypersecretion(Aujla).ResearchersalsofoundincreasedlevelsofIL6,apro
inflammatorycytokineproducedbymostnucleatedcells,intheairwaysofinfectedmice,and
determinedthatitplaysaroleindrivingtheTh17pathway(Korn).Thegoalofourstudywasto
furtherinvestigatetheroleofIL6ininhostdefenseduringRSVinfection using in vitro and in
vivo models. A clearer understanding of the role IL-6 plays in influencing host immune response
through the Th1, Th2 and Th17 pathways will help determine the next steps in developing the

vaccine, as well as determine whether targeting IL-6 could be therapeutically useful during RSV
infection.
Results
IL6andIL17Interaction
WhenwesensitizedCD45.1micewithRSVinfecteddendriticcells,thewildtype
transfergroupsignificantlyincreasedIL17aexpressioncomparedtotheuninfectedcontrol
(p=0.01),withatrendofincreasedexpressionabovetheothertwogroupsaswell(Fig.1).All
transferssignificantlyincreasedtheGob5expressioncomparedwiththenavegroup.Between
thetwotransfergroups,theIL6/transfertrendedtowardslowerIL17aandGob5expression
inlungtissuethanthewildtypetransfergrouppostRSVinfection(Fig.1).Thesedatasuggest
thattheabilityofdendriticcellstoproduceIL6isimportantforpromotingIL17aandmucus
production.
WenextdeterminedwhetherIL6aloneplayedaroleinpromotingIL17a.Tothisend,
weadministeredrecombinantIL6directlytothelungs.AcohortofmicereceivingIL6also
receivedRSVinfection.BothRSVinfectedgroupsshowedatrendtowardsincreasedIL6and
IL17aexpressionovercontrols(Fig.2A).Additionally,micethatreceivedbothRSVand
recombinantIL6demonstratedaclosetosignificant(p=0.08)increaseinIL6expression,as
wellasasignificantincreaseinIL17aexpression,comparedtouninfectedcontrols.Theyalso
exhibitedatrendtowardsincreasedIL17a,relativetothemicewhoonlyreceivedRSV(Fig.
2A).However,nosignificantdifferenceinviralclearancewasobservedbetweenthetwoRSV

infectedgroups(Fig.2B).ThesedatasuggestthatIL6playsadirectroleinenhancingIL17a
productioninthelungsinresponsetoRSVinfection.

RoleofIL17AinLungImmunopathology
ToinvestigatetheroleofIL17aindrivingmucusproduction,weadministeredIL17a
neutralizingantibodies.Theimmunoglobulin(Ig)treatedcontrolmiceexhibitedsignificantly
moreGob5andMuc5acexpressioninlungtissue(Fig.3A)andmoreairwaymucussecretion
(Fig.3B)inresponsetoRSVinfectionthantheantiIL17treatedmice.Boththeuninfected
controlandtheRSVinfectedwildtypemicehadlowerproportionsofneutrophilsand
eosinophilsintheirairwaysthantheRSVinfectedIL6/mice(Fig.4).Insummary,theresults
suggestthatinRSVinfectedmice,thepresenceofIL6increasesIL17aexpressionwhile
limitingneutrophilandeosinophilrecruitment.Furthermore,thepresenceofIL17leadsto
hypersecretionofmucus.
Discussion
Our results indicate that in RSV infected mice, IL-6 plays a multivariate role in immune
response. The presence of IL-6 stimulates the Th17 pathway leading to increased IL-17a
expression and hypersecretion of mucus, while at the same time playing a protective role by
limiting neutrophil and eosinophil recruitment to the airways.

Evidence for IL-6 stimulating the Th17 pathway comes from the association of IL-17a
expression with IL-6 expression. The mice in the IL-6-/- transfer group showed less IL-17a
expression than the wild type transfer mice (Fig. 1). Additionally, the IL-6 -/- mice who received
recombinant IL-6 before and during RSV infection showed higher IL-17a expression than the IL6 -/- mice who only received RSV (Fig. 2A). These results suggest that IL-6 influences the Th17
pathway and drives the expression of IL-17a. Our data also indicate that the Th17 pathway is
involved in mucus production, because when we treated mice with anti-IL-17 antibodies, Gob5
and Muc5ac expression (Fig. 3A), as well as mucus secretion (Fig. 3B), were lower than they
were in the control mice. This indicates that IL-17, and therefore the Th17 pathway, drives the
hypersecretion of mucus in RSV infection. These results are consistent with previous studies
findings on IL-6 influencing the differentiation of the Th17 lineage (Korn, Aujla), as well as IL17 inducing mucus hypersecretion (Mukherjee).
The protective role of IL-6 is demonstrated through the lower proportions of neutrophils
and eosinophils in the airways of the wild type control mice than the IL-6-/- mice (Fig. 4). This
suggests that IL-6 plays a role in limiting granulocyte recruitment. However, this contradicts
previous research that suggests IL-6 exacerbates granulocytic airway inflammation in asthma
(Chu). This contradiction could be due to the effects of RSV proteins on granulocyte recruitment,
because researchers demonstrated that RSV particles and the RSV Fusion protein can induce a
Neutrophil Extracellular Trap (Funchal).
Although we identified IL-6 and its influence on the Th17 pathway as an important
contributor to the mucus pathology of RSV infection, providing support for the existing data, it is
important to note that in the in vivo study, administering recombinant IL-6 did not have a
significant effect on IL-6 expression or viral clearance. This indicates that manipulating IL-6

expression through recombinant IL-6 does not appear to be important in effectively increasing
viral clearance. However, this could be due to the amount of recombinant IL-6 given to the mice,
and we might see a different in viral clearance if we administered at a higher concentration. An
additional complication in the development of an RSV vaccine is the protective role IL-6 plays.
Because IL-6 contributes to RSV pathology via mucus hypersecretion while simultaneously
protecting the host against inflammation, there is no clear-cut solution for mediating RSV
infection by targeting IL-6 alone. Future research in related pro-inflammatory cytokines such as
IL-23 and IL-12 are necessary to gain a complete understanding of host immune response to
RSV infection.
Methods
Mice
B6 wild-type (WT) mice were purchased from The Jackson Laboratory (Sacramento,
CA). IL-6 -/- mice on a B6 background were originally generated by Kopf et al. and
subsequently bred at SCRI via Jesus Lopez-Guiza. All animal work was performed in accordance
with the Seattle Childrens Research Institute Institution Animal Use and Care Committee
(IACUC). Mice were euthanized in accordance with IACUC, AAALAC, and other guidelines.
Respiratory syncytial virus (RSV)
Our laboratory utilizes the antigenic subgroup A strain of RSV, referred to as Line 19.
This isolate was obtained from a sick infant at the University of Michigan and has been
demonstrated in animal models to mimic human infection by stimulating mucus production using
an inoculum of 1 105 PFU/mouse by intratracheal or oropharyngeal administration (OP).

Quantitative PCR (QPCR)


The smallest lobe was removed and homogenized in 1 ml Trizol reagent (Invitrogen,
Carlsbad, CA). RNA was isolated as described (Invitrogen), and 5 g was reverse-transcribed to
assess gene expression. Detection of cytokine mRNA in lung samples was determined using predeveloped primer/probe sets (Applied Biosystems, Foster City, CA) and analyzed using an ABI
Prism 7500 Sequence Detection System (Applied Biosystems). Transcript levels of Muc5ac,
Gob5, IL-6 and IL-17 cytokines were determined using custom primers or those supplied by
Invitrogen. Gapdh was analyzed as an internal control and gene expression was normalized to
Gapdh. Fold changes in gene expression levels were calculated by comparison with the gene
expression in uninfected mice, which were assigned an arbitrary value of 1.
Lung and lymph node leukocyte isolation
Lung leukocytes were isolated from enzyme-dispersed lung tissue. Lung tissue was
excised, washed in PBS, minced, and digested enzymatically for 45 min in 15 ml/lung digestion
buffer (RPMI, 5% FCS, 1 mg/ml collagenase [Roche Applied Science, Indianapolis, IN], and 30
g/ml DNase [Sigma-Aldrich, St. Louis, MO]). Lung-associated lymph nodes (LALNs) were
dispersed similar to lungs, except that only 5 ml digestion buffer was used. Following
erythrocyte lysis using ammonium chloride (NH4Cl) buffer, cells were washed and resuspended
in media (RPMI, 5% FCS). Total lung leukocyte numbers were assessed in the presence of
Trypan blue using a hemocytometer; viability was >85%. Cell differentials were performed by a
trained investigator, using slides prepared from bronchoalveolar lavage (BAL) and mounted on
slides by cytospin. The Diff-quick system was used to aid differentiation of cell types.
Generation of bone marrow-derived dendritic cells (BMDCs)

Bone marrow was harvested from mice and seeded in tissue-culture flasks in RPMI 1640based complete media with 10 ng GM-CSF/ml (R&D Systems, Minneapolis, MN). Cells were
fed every 3 days, and loosely adherent cells were collected after 10 days. The majority of the
cells, >85%, were CD11c+ bone marrow-derived DCs (BMDCs) and subsequently were infected
with RSV (MOI ~0.5). The cells were then assessed for gene and protein expression as well as
used for flow cytometric analysis.
BMDC Transfer
After BMDCs were infected with RSV, they were used to sensitize live CD45.1 mice via
OP administration two days later. The mice were then RSV challenged eight days later. On day 8
post-infection, bronchoalveolar lavage (BAL) was collected and the lungs were harvested and
analyzed for gene and protein expression.
Recombinant IL-6
On day 0, IL-6 -/- mice were RSV infected using OP administration. On day 3, the IL-6
and RSV/IL-6 groups were given recombinant IL-6 via intranasal administration.
Bronchoalveolar lavage was collected and lungs were harvested day 9 post-infection and
analyzed for gene and protein expression.
Anti-IL-17
Syngeneic bone marrow transplanted CD45.1 mice were treated with anti-IL-17
antibodies or control immunoglobulin (Ig) on day 0. On day 1 the mice were RSV infected using
OP administration, and then received a boost of anti-IL-17 antibodies or Ig on day 2 and 4.
Lungs were harvested on day 9 (d8 post-infection).

Histology
Right lobes of the lungs were isolated and immediately fixed in 10% neutral buffered
formalin. Lung samples were subsequently processed, embedded in paraffin, sectioned, and
placed on L-lysine-coated slides, and stained using standard histological techniques using
Hemotoxylin and Eosin (H&E) and Periodic-acid Schiff (PAS). PAS staining was done to
identify mucus and mucus-producing cells.
Statistics
Data was analyzed using Prism software (GraphPad, San Diego, CA). Unless otherwise
specified, data shown are representative of two or more experiments. Statistical significance in
all experiments was determined by one-way ANOVA, followed by a Newman-Keuls post test.
Significant differences were regarded as p < 0.05. Statistical comparisons of quantitative PCR
(QPCR) data were determined from normalized cycle threshold values, pre-conversion to fold
increases.

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Figures

**

*
*
*
Fig. 1: Mice were sensitized (OP) with RSV infected BMDCs and then challenged with live RSV. The
expression of Th2 and Th17 cytokines and the mucus-producing gene Gob5 was assessed from lung RNA
via QPCR. Higher levels of IL-17a and lower levels of IL-6 were observed in the WT transfer group.

Columns represent the mean fold over uninfected controls for each group +/- SEM. * = p<0.05 versus
nave **= p<0.01 versus naive

IL-6

p=0.08

8
7
6
5
Fold +
over 4
uninfected
3
2
1
0
RSV

RSV/IL6

IL6

UC

IL-17a

18
16
14
12
Fold + 10
over
8
uninfected
6
4
2
0

RSV

RSV/IL6

IL6

UC

RSV F

100000

10000

copies per 10^51000


GAPDH
100

10

RSV

RSV/IL6

IL6

UC

Fig. 2: IL-6-/- mice were given recombinant IL-6 and then a cohort was challenged with live RSV. In (A),
columns represent the mean fold over uninfected controls for each group +/- SEM, determined from lung
RNA via QPCR. In (B), viral load was assessed via QPCR of RSV transcripts obtained from lung RNA.
Increased levels of IL-6 and IL-17 were observed in the group that received RSV and recombinant IL-6.
No change in viral clearance was observed. *= p<0.05 versus uninfected controls (UC)

Gob5

160.00
140.00
120.00
100.00
Fold+
over
80.00
uninfected

60.00
40.00
20.00
0.00
Naive

Ctrl Ig

Anti-IL17

Muc5ac

2.50

2.00

1.50
Fold+
over
uninfected
1.00

0.50

0.00
Naive

Ctrl Ig

Anti-IL17

B
Ctrl Ig

Anti-IL-17

Fig. 3: IL-17 promotes mucus production during RSV infection. Syngeneic bone marrow transplanted
mice were treated with anti-IL-17 antibodies or control Ig (Ctrl Ig) beginning at day 0. In (A), the

expression of mucus related genes Muc5ac and Gob5 were determined by QPCR. In (B), representative
lung sections stained with Periodic Acid Schiffs reagent (PAS) depict reduced mucus production, shown
in magenta in anti-IL-17 treated mice compared to the control Ig mice. * = p<0.05 versus Ctrl Ig.

NEUT

EOS
9.00%
8.00%
7.00%
6.00%
5.00%
4.00%
3.00%
2.00%
1.00%
0.00%
IL6-/- UC

IL6-/- RSV

WT UC

WT RSV

Fig. 4: IL-6 limits airway neutrophil and eosinophil recruitment. IL-6-/- and wild type mice were infected
with live RSV. IL-6 -/- mice show higher mean airway neutrophils and eosinophils. The percentage of
neutrophils and eosinophils per group was determined via differential staining of BAL cytospins using the
Diff-quick system. * = p<0.05 versus uninfected controls. ** = p< 0.05 versus all other groups.