Role of Interleukin-17 in the Adaptive Immune Response in Lyme Arthritis

81 Pages·2016·0.99 MB·English

by  Emily Siebers

University of Wisconsin Milwaukee
UWM Digital Commons
Theses and Dissertations
August 2012
Role of Interleukin-17 in the Adaptive Immune
Response in Lyme Arthritis
Emily Siebers
University of Wisconsin-Milwaukee
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ROLE OF INTERLEUKIN-17 IN THE ADAPTIVE
IMMUNE RESPONSE IN LYME ARTHRITIS

by

Emily M. Siebers

A Thesis Submitted in
Partial Fulfillment of the
Requirements for the Degree of
Master of Science
in Biomedical Sciences
at
The University of Wisconsin-Milwaukee
August 2012
ABSTRACT

ROLE OF INTERLEUKIN-17 IN THE ADAPTIVE
IMMUNE RESPONSE IN LYME ARTHRITIS
by

Emily M. Siebers

The University of Wisconsin-Milwaukee, 2012
Under the Supervision of Dr. Dean T. Nardelli

Lyme arthritis is a devastating symptom of Lyme borreliosis that causes severe
inflammation of the synovial joints. Interleukin-17 (IL-17) plays a role in the
pathogenesis of various arthritides, including, possibly, Lyme arthritis, by causing
the expression of genes involved in the production of inflammatory cytokines by
synoviocytes. However, the cellular sources of IL-17 in the context of Borrelia
burgdorferi infection are unknown, as are the effects of these cells on the
development of arthritis and stimulation of humoral immunity through the
production of borreliacidal antibodies. Using multiple models of Lyme arthritis, the
hypothesis that IL-17 produced by CD4+ cells contributes to the inflammation
associated with B. burgdorferi infection and is associated with an increase in
borreliacidal antibody production was tested. Serum IL-17 levels were increased
during Borrelia infection, especially at the time of peak swelling. Neutralization of
interferon-gamma increased these IL-17 levels, and the addition of anti-CD4
antibodies to cultures of stimulated cells isolated during peak swelling reduced
IL-17 levels. In addition, a role for IL-10 in the production of IL-17 was
demonstrated. Furthermore, increased IL-17 was associated with an increase of
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borreliacidal antibodies. These findings suggest CD4+ cells (possibly T cells)
contribute to the production of IL-17 following infection with B. burgdorferi and
that levels of this cytokine may affect borreliacidal antibody production.

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TABLE OF CONTENTS

Title Page .............................................................................................................. i

Abstract ................................................................................................................. ii

Table of Contents ................................................................................................. iv

List of Figures ...................................................................................................... vii

List of Abbreviations ............................................................................................. ix

Acknowledgements .............................................................................................. xi

Chapter 1: Introduction ......................................................................................... 1

I. Background .................................................................................................... 1

1. Lyme Borreliosis ............................................................................................... 1

2. Stages of Disease, Diagnosis, and Therapeutic Measures .............................. 2

3. Lyme Arthritis .................................................................................................... 5

4. Innate Immune Events of Lyme Arthritis .................................................... 6

5. T Cell Involvement in Lyme Arthritis ................................................................. 7

6. Animal Models of Lyme Arthritis ..................................................................... 10

7. Borreliacidal Antibody Response .................................................................... 12

II. Hypothesis and Specific Aims ..................................................................... 13

Chapter 2: Materials and Methods ...................................................................... 16

I. Mice .............................................................................................................. 16
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II. Vaccination of Mice ........................................................................................ 16

III. Infection of Mice ......................................................................................... 17

IV. Antibody Treatment ....................................................................................... 17

V. Assessment of Inflammation ....................................................................... 18

VI. Cell Culture ................................................................................................... 18

VII. ELISA ........................................................................................................ 19

VIII. Borreliacidal Antibody Assay ....................................................................... 19

IX. Statistics..................................................................................................... 20

Chapter 3: Results .............................................................................................. 21

I. Specific Aim 1: Establish CD4+ cells as a source of IL-17 following infection
with B. burgdorferi. .......................................................................................... 21

1. Effects of anti-IFN-γ antibody administration on hind paw swelling ................ 21

2. Serum levels of IL-17 following treatment with anti-IFN-γ antibodies ............ 22

3. Role of sustained infection on production of IL-17 .......................................... 23

4. Assessment of IL-17 production in different models of Lyme arthritis ............. 25

5. Assessment of Borrelia-induced IL-17 production in models of Lyme arthritis in
IL-10-deficient mice ............................................................................................ 27

6. Effect of IL-10 deficiency on IL-17 production in models of Lyme arthritis ...... 29

7. Anti-CD4 antibody modulation of IL-17 in models of Lyme arthritis ................ 31

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II. Specific Aim 2: Establish a relationship between IL-17 levels and
borreliacidal antibody production. .................................................................... 34

1. Borreliacidal antibody titers following sustained infection with B. burgdorferi . 34

2. Effect of IL-10 on borreliacidal antibody titers in models of Lyme arthritis ...... 35

Chapter 4: Discussion ........................................................................................ 37

I. Specific Aim 1: Establish CD4+ cells as a source of IL-17 following infection
with B. burgdorferi. .......................................................................................... 37

II. Specific Aim 2: Establish a relationship between IL-17 levels and
borreliacidal antibody production. ................................................................... 48

Chapter 5: Conclusion and Future Directions ..................................................... 54

References ......................................................................................................... 60

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LIST OF FIGURES

Figure 1. Administration of anti-IFN-γ antibody does not affect paw swelling in B.
burgdorferi-infected C3H mice. Mice were infected with B. burgdorferi 297 and
treated with anti-IFN-γ antibody (n=10) or an isotype control antibody (n=10) for 7
days. Non-infected control mice (n=10) were treated with PBS. Error bars
indicate the SEM. ............................................................................................... 22

Figure 2. Concentrations of IL-17 in serum of non-infected C3H mice (n=5) and
C3H mice infected with B. burgdorferi 297, with (n=5) or without (n=5)
administration of anti-IFN-γ antibodies, at day 12 after infection. * denotes a
significant difference (P<0.05). Error bars indicate the SEM. ............................. 23

Figure 3. Hind paw swelling of Borrelia-vaccinated C3H mice injected with live
(n=16) or heat-killed (n=12) B. burgdorferi 297 (Bb). A control group consisted of
vaccinated mice that were not infected (n=8). * denotes a significant (P<0.05)
difference in paw swelling. Error bars indicate SEM. .......................................... 24

Figure 4. Concentrations of IL-17 from culture supernatants of lymph node cells
from Borrelia-vaccinated C3H mice injected with live (n=3) or heat-killed (n=4) B.
burgdorferi 297 (Bb) or BSK medium (n=2). Cells were obtained at day 10 after
infection and restimulated with B. burgdorferi 297. Error bars indicate SEM. .... 25

Figure 5. Concentrations of IL-17 from culture supernatants of spleen cells
obtained from naïve (n=7), Borrelia-infected (n=8), Borrelia-vaccinated (n=6), and
Borrelia-vaccinated and –infected (n=6) wild-type (WT) C57BL/6 mice. Cells were
obtained at peak swelling (8 days after the time of infection) and then stimulated
with B. burgdorferi in vitro for 6 or 24 hours. * denotes a significant difference
(P<0.05). ND, not detected. Error bars indicate SEM. ........................................ 27

Figure 6. Concentrations of IL-17 from culture supernatants of spleen cells
obtained from naïve (n=6), Borrelia-infected (n=8), Borrelia-vaccinated (n=10),
and Borrelia-vaccinated and –infected (n=10) IL-10 knock-out (KO) C57BL/6
mice. Cells were obtained at peak swelling (8 days after the time of infection) and
then stimulated with B. burgdorferi in vitro for 6 or 24 hours. * denotes a
significant difference (P<0.05). Error bars indicate SEM. ................................... 29

Figure 7. Concentrations of IL-17 from culture supernatants of spleen cells
obtained from wild-type (WT) and IL-10-deficient (KO), naïve (n=7) and Borrelia-
infected (n=8) C57BL/6 mice. Cells were obtained at peak swelling (day 8 after
the time of infection) and were stimulated with B. burgdorferi in vitro for 6 or 24
hours. * denotes a significant difference (P<0.05). ND, not detected. Error bars
indicate SEM. ..................................................................................................... 30

Figure 8. Concentrations of IL-17 from culture supernatants of spleen cells
obtained from wild-type (WT) and IL-10-deficient (KO), Borrelia-vaccinated (n=8)
and Borrelia-vaccinated and –infected (n=8) C57BL/6 mice. Cells were obtained
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at peak swelling (day 8 after the time of infection) and were stimulated with B.
burgdorferi in vitro for 6 or 24 hours. * denotes a significant difference (P<0.05).
ND, not detected. Error bars indicate SEM. ........................................................ 31

Figure 9. Concentrations of IL-17 from culture supernatants of spleen cells from
naïve (n=6), Borrelia-infected (n=8), Borrelia-vaccinated (n=6), and Borrelia-
vaccinated and -infected (n=6) wild-type (WT) C57BL/6 mice. Cells were
obtained at peak swelling (day 8 after the time of infection) and then stimulated
with B. burgdorferi 297 (Bb), with or without anti-CD4 antibody (αCD4). * denotes
a significant difference (P<0.05). ND, not detected. Error bars indicate SEM. ... 32

Figure 10. Concentrations of IL-17 from culture supernatants of spleen cells from
naïve (n=6), Borrelia-infected (n=8), Borrelia-vaccinated (n=10), and Borrelia-
vaccinated and –infected (n=10) IL-10 deficient (KO) C57BL/6 mice. Cells were
obtained at peak swelling (day 8 after the time of infection) and then stimulated
with B. burgdorferi 297 (Bb), with or without anti-CD4 antibody (αCD4). * denotes
a significant difference (P<0.05). Error bars indicate SEM. ................................ 33

Figure 11. Borreliacidal antibody titers from culture supernatants of lymph node
cells from Borrelia-vaccinated C3H mice injected with live (n=8) or heat-killed
(n=6) B. burgdorferi 297 or BSK medium (n=4). Cells were obtained at the peak
of swelling (day 10 after the time of injections) and stimulated with B. burgdorferi
297 in vitro for 24 hours. * denotes a significantly greater (4-fold) titer than that of
cells from non-infected mice. .............................................................................. 35

Figure 12. Borreliacidal antibody titers obtained from sera of naïve (n=4),
Borrelia-infected (n=4), Borrelia-vaccinated (n=4), and Borrelia-vaccinated and –
infected (n=4) wild-type (WT) and IL-10-deficient (KO) C57BL/6 mice at the peak
of hind paw swelling (day 8 after the time of infection). * denotes a significantly
greater (4-fold) titer than wild-type counterparts. ................................................ 36

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LIST OF ABBREVIATIONS

αCD4: anti-CD4

ANOVA: analysis of variance

Bb: Borrelia burgdorferi

BSK: Barbour-Stoenner-Kelly

CIA: collagen-induced arthritis

ELISA: enzyme-linked immunosorbent assay

EM: erythema migrans

IFN-γ: interferon-gamma

IL-1β: interleukin-1-beta

IL-6: interleukin-6

IL-10: interleukin-10

IL-17: interleukin-17

IL-21: interleukin-21

IL-23: interleukin-23

IL-35: interleukin-35

KO: knock-out

NK: natural killer

OspA: outer surface protein A

OspC: outer surface protein C

PBS: phosphate buffered saline

SEM: standard error of the mean

TGF-β: transforming growth factor-beta

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