Interleukin-6 Plays a Multivariate Role In Respiratory

Syncytial Virus Infection

Molly Brazil, Maria White, Jesus Lopez-Guisa and Dennis Lindell
Results

Introduction
Respiratory syncytial virus (RSV) is one of the
leading causes for child admissions at our
nations hospitals
There is currently no vaccine
Inflammation and the over-production of
mucus are chief contributors to RSV disease
pathology
Interleukin 17 (IL-17) induces mucus
hypersecretion during RSV infection
IL-6 plays a role in driving the T-helper 17 cell
(Th17) pathway

Figure 1
50

35
30

200.00

IL-6

Gob5

150.00

25
20

15

100.00

WT xfer

IL6-/- xfer no xfer

nave

50.00

WT xfer

IL6-/- xfer

no xfer

nave

0.00

WT xfer

IL6-/- xfer

no xfer

nave

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

Figure 3

Figure 2
A

16

160.00

14

IL-6

18

p=0.08

140.00

IL-17

12

120.00

10

Fold+ 4
over
3
uninfected

40.00

RSV

RSV/IL6

IL6

Copies per
10^5
GAPDH

nave

Gob5

2.00

Muc5ac

1.50

1.00

0.50

20.00

RSV

RSV/IL6

IL6

0.00

nave

Naive

100000

2.50

100.00

Fold+
80.00
over
uninfected 60.00

10000

BMDC transfer: Bone marrow was isolated from

WT and IL-6-/- mice and RSV infected on day 10.
Infected BMDCs were used to sensitize WT
CD45.1 mice. Mice were RSV challenged 8 days
later. Lungs were harvested d8 post infection.
Recombinant IL-6: IL-6-/- mice were RSV
infected d0. IL-6 and IL-6/RSV groups were
given recombinant IL-6 d3. Lungs were harvested
d9 post-infection.
Anti-IL-17: Mice were treated with anti-IL-17
antibodies or control Ig d0 and RSV infected d1.
They were then given antibody boosters d2 and
d4. Lungs were harvested d8 post-infection.
QPCR: RNA was isolated from lower left lobes
of lungs using Trizol then reverse transcribed to
cDNA. QPCR performed using custom designed
primers. Gene expression normalized to GAPDH.
Histology: Right lobes isolated and fixed in 10%
neutral buffered formalin then embedded in
paraffin, sectioned and placed on L-lysine-coated
slides and stained using Hemotoxylin and Eosin
and Periodic-acid Schiff.
All animal work performed in accordance with
the Seattle Childrens Research Institute Institution
of Animal Use and Care Committee (IACUC).

IL-17a

10

Methods

40

Fold+
over
uninfected

250.00

**

45

Purpose
Further explore how IL-6 influences host
immune response to RSV infection through
interactions with Th2 and Th17 pathways
Deter mine the next steps in vaccine
development as well as whether or not
targeting IL-6 would be therapeutically useful

Results

Ctrl Ig

Anti-IL17

Ctrl Ig

0.00

Naive

Ctrl Ig

Anti-IL17

Anti-IL-17

RSV F

1000
100

IL-6 stimulates Th17 pathway

IL-6-/- transfer group showed less IL-17a
expression than the wild type transfer mice
(Fig. 1)
IL-6 -/- mice who received recombinant IL-6
before and during RSV infection showed
higher IL-17a expression than the IL-6 -/mice who only received RSV
Th17 pathway induces mucus hypersecretion
Mice treated with anti-IL-17 antibodies,
exhibited lower Gob5 and Muc5ac expression
(Fig. 3A), as well as mucus secretion (Fig. 3B),
than in the control mice
IL-6 plays a protective role
Lower proportions of neutrophils and
eosinophils in the airways of the wild type
control mice than the IL-6-/- mice (Fig. 4)
Other findings and implications
Administering recombinant IL-6 did not have
a significant effect on IL-6 expression or viral
clearance
Redo studies with greater number of animals
and higher dose of recombinant IL-6
Future research in related pro-inflammatory
cytokines such as IL-23 and IL-12 is necessary
to gain a complete understanding of host
immune response to RSV infection

Conclusions

10
1

RSV

RSV/IL6

IL6

nave

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 Schiff s 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.

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 nave.

Figure 4
12.00%

25.00%

**

10.00%

EOS

8.00%

**

20.00%

NEUT

In RSV infected mice, IL-6 plays a multivariate

role in immune response
The presence of IL-6 drives the Th17 pathway
leading to increased IL-17a expression
Th17 pathway drives hypersecretion of mucus
IL-6 plays a protective role by limiting
neutrophil and eosinophil recruitment to the
airways
Targeting IL-6 alone is not sufficient for
mediating RSV infection

15.00%

6.00%
10.00%

4.00%

2.00%
0.00%

IL6-/- UC

IL6-/- RSV

WT UC

WT RSV

Acknowledgements

5.00%
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.

These studies were supported by Seattle

Childrens Research Foundation and The
National Institutes of Health (R01HL111589 and
K22AI077712).