Opportunistic
Bacterial Infections
in Inflammatory
Bowel Disease
By: Christina Philips
Overview
• Background on Opportunistic Bacterial
Infections & IBD
• Paper #1
• Paper #2
• Specific Aim
Background
• Opportunistic Bacterial Infections in
Inflammatory Bowel Disease can be either
non-therapy related or therapy related.
• Some infections that occur due to therapies
are:
• Viruses such as CMV (cytomegalovirus) & HPV
(human papillomavirus)
• Bacterial infections such as C.difficile
• Fungus such as Candida
Therapies
• Some IBD Therapies:
• Immunomodulators such as 6-MP, Imuran, and
Methotrexate
• Anti-TNF Therapies such as Infliximab and
Adalimumab
• Risks with Anti-TNF Therapies:
• patients stop responding to treatment over time
• May develop a bacterial infection
How Anti-TNF Therapy Works
http://journals.prous.com/journals/dot/20044
004/html/dt400281/images/etan_f05.jpg
Paper 1
• Objective: To observe the effects of anti-IL23
mAb on active colitis in a T-cell mediated
mouse model.
• Model: C3H/HeJBir mouse model (obtained
from Jackson Labratories)
• This model is highly susceptible to colitis under
certain conditions
Figure 1 – Cytokine
Expression in Colonic
Tissue
• Researchers tested IL-23 and
related cytokines in order to find
out the role that these cytokines
play in colitis.
• SCID mouse models were injected
with either the pathogenic CBA –
specific Bir14 T cells or the control
group (anti-CD3 activated CD 4+ T
cells)
• In graph A, an increase in these
cytokines was seen in the mice
injected with the pathogenic Bir14
T cells when compared to the
control.
• In graph B, both IL-17 & IFNgamma showed increased levels
of mRNA expression.
Figure 2 – Th17 Cell
Detection During Colitis
• The T cells were stimulated with
PMA & ionomycin after a resting
period. The Bir14 T cells showed
a higher level of IL-17 rather than
IFN-gamma. IL-4 & and IL-10
were not expressed but TNFalpha and IL-6 were expressed.
• Mesenteric lymph nodes (MLN)
and Lamina propria cells (LPL)
from the Bir14 T cells and the
control (anti-CD3 T cells) were
studied. The IL-17 and IFNgamma production were
analyzed via flow cytometry and
an increase in these cytokines
was seen in the Bir14 T cell but
not in the control groups.
Figure 3 – Expression of CD 4+ T
cell IL-17 response in the
presence of IL-23 and IL-12 after
stimulation with CBA
• As seen from the graph, CD4+ T
cell producing IL-17 increases in
IL-23 while the production of CD
4+ T cells by IL-12 is inhibited.
• The graph the shows the CD4+ T
cells production IFN-gamma
shows the opposite effect.
Figure 4
• In order to test whether the
production of IL-23 and IL-12p70
were driven by the CD 4+ T cells,
the cytokines were plated in
cultures that contained
dendritic cell and the
pathogenic CD 4+ T cell strain.
• The production of IL-23 and IL12p70 was expressed in large
amounts in the culture that
contained both the Bir14 CD 4+ T
cells and the CBA – pulsed
BMDCs. This shows that
production of these cytokines
are dependent on T-cells and
antigens.
Figure 5 – Th 17 Cells that are
Mediators of Colitis
•
•
•
•
Graph A shows the two different cell lines
that were generated after culturinf Bir14
CD4+ T cells with CBA-APC in the presence
of IL-23, anti-IL-12, anti-IFN-gamma or IL-12,
IFN-gamma, and anti-IL-23. One cell line is a
Th17 cell line that produces IL-17 while the
other is Th1 cell that produces IFN-gamma.
Graph B shows the effects that each cell
line has inducing colitis. Only a small
amount of Th17 cells were needed to induce
colitis whereas a larger amount of Th1 cells
was needed to induce colitis.
Graph C shows that IL-17 production
increased greatly after Th17 cells were
transferred. IFN-gamma was barely
produced.
Graph D is the histopathology between a
normal colon, one that has a TH17 cells
transferred in, and a colon that has Th1 cells
transferred. The Th17 recipient shows more
signs of inflammation than Th1 recipient.
Figure 6
• Graph A shows that mice that
were given the control antibody
had colitis but the mice that
were given the anti-IL23p19
antibody has little to no effects
of the disease.
• In graph B, the histopathology
between the two antibodies
were observed. The control
shows more signs of
inflammation than the anti-IL-23
antibody.
• Graph C shows the expression
on genes as analyzed by PCR.
This graph shows that gene
expression is down regulated in
mice that were injected with the
anti-IL23p19 antibody.
Figure 7
• Graph A shows that mice that had
Bir14 CD 4+ T cells transferred and
were given the control Abs
developed colitis while the group
given the anti-IL23 did not.
• These three groups were tested for
gene expression and the mice
given the anti-IL23 Abs had down
regulated gene expression.
• Figures E and F show the
histopathology of the two
antibodies. The anti-Il23 antibody
shows less effects of colitis.
Figure 8
• This graph shows the effects of
the blockage of Il-23 and
whether it an induce apoptosis
of Th17 cells.
• The Th17 cells were reduced in
the presence of anti-Il23p19 but
Th1 cell were not reduced in the
presence of anti-Il12p70.
Conclusion
• Overall, IL-12 stimulates IFN-gamma and Th1
cells while IL-23 effects memory T cells and
Th17 cells.
• CD4+ Th17 cells is an effector of colitis
• Anti-Il23p19 Abs could be used as a therapy to
reduce the effects of colitis.
Paper 2
• Objective: To investigate the role of the
chemokine CXCL10 and its receptor, CXCR 3 in
acute colitis.
Figure 1
• Graph A shows the mRNA expression of
CACL10 and CXCR3 in colonic tissue. The
mRNA levels of CXCL10 increased on day
1 and peaked on Day 3.
• In B, the CXCL10 is expressed at day 0
and graph C shows that the basal crypt
level grew from 1/3 o ½ by day 3 with the
addition of DSS.
• Non-epithelial cells that produced
CXCL10 were seen in the epithelium but
not the lamina propria.
• Graph D and E shows the expression on
CXCL3 in the luminal surface epithelium
and a little in the basal crypts. When DSS
was added, the CXCL3 cells vanished
from the luminal surface but increased in
the basal crypts.
• F and G were controls
Figure 2
• Testing the effects of anti-CXCL10
mAb on DSS induced colitis vs a
control mAb. Graph A shows that the
mice that were injected with the
control experienced weight loss and
B shows the decrease in colon length
due to the weight loss. The mice that
were injected with the anti-CXCL mAb
showed well preserved mucosal
architecture.
• I shows that the blockage of CXCL10
did not effect cell infiltration into the
lamina propria.
Figure 3
•
•
•
Effect of anti-CXCL10 mAB on Cell
traffic to the colon.
Slide A-E are with the control Ab while
slides B – F are with anti-CXCL10 mAB.
A & B are immunostains for CD4 cells,
C & D are immunostains for F4/80 and
E & F are immunostains for CD11c. The
cells that are stained brown are
positive cells.
Figure 4
• This Graph shows that mucosal
infiltration was characterized by
apoptotic cells since there
weren’t any significant
differences in MAsCAM and
vicia villosa lectin. The addition
of anti-CXCL10 mAbs reduced
the number of apoptotic cells.
Figure 5
•
•
•
•
•
•
•
Effect of Anti-CXCL10 mAb restricted to
damage of epithelial cells.
5-Bromo-2’ deoxyuridine (BrdU) is used to
label the cells before they are observed.
A – Distal colon tissue from mice injected
with control mAb
B – Distal Colon tissue from mice injected
with anti-CXCL10 mAb.
BrdU cells are stained in brown.
These cells are seen in the bottom 1/3 of
crypts in normal colon cells but cells that
were exposed to DSS, there is a significant
reduction in BrdU cells (A). This shows that
the crypt cell growth was impeded. In
contrast, B shows that the BrdU+ cells are
seen in the replicative zone of the intestinal
crypts.
Graph C - BrdU+ cells increased in mice that
were injected with anti-CXCL10 mAb
Figure 6
• Whether CXCL10 directly inhibits reepithelialization or promotes epithelial
apoptosis.
• Looking at A, mice were pretreated
with PBS (vehicle) before injection
with BrdU. In B, mice were pretreated
with rCXCL10 before injection with
BrdU. Graph C shows that there was a
significant decrease of BrdU+ cells in
the mice that were treated with
rCXCL10.
• The location of BrdU + cells after 72
hours is seen in D-E. In the control
mice (D), these cells disappeared from
the epithelium and some were seen in
the luminal surface. In the mice
treated with rCXCL10, the cells
continued to remain in the
proliferative zone.
• Overall, rCXCL10 inhibits crypt cell
proliferation and migration.
Conclusions
• CXCL10 inhibits crypt cell proliferation and
migration.
• CXCL10 and the receptor CXCR3 interaction is
enhanced in the epithelium in the proliferative
zone after DSS induced colitis.
• The blockage of CXCL10 increases BrdU+ cells
after DSS induced colitis and prevents acute
colitis
Specific Aim
• To determine whether the blocking of
chemokines (such as CXCL10) or the
development of Anti-Il23p19 Abs therapy
would be more effective therapy and decrease
the bacterial infections associated with antiTNF therapies.