Examining The Influence of

Seminal Plasma On The Female

Genital Tract Epithelial Cell

Barrier

Jessica Kafka

PhD Candidate

Supervisor: Dr. Charu Kaushic

McMaster University

Hamilton, Ontario

I have no conflicts of interest

The Epithelial Cell (EC) Barrier

Not to scale

Single layer of columnar epithelial cells

Tight junction proteins

Intestine

Upper Female Genital Tract (FGT)

An Inflammatory

Microenvironment Can Break the

EC Barrier

Not to scale

HIV-1

E.coli (EHEC)

Pro-inflammatory

cytokines

C. parvum

Immunoregulatory Cytokines Can

Strengthen the EC Barrier

Howe et al., 2005

• Interleukin-22 (Presented by Dr. Nazli)

• TGF-β strengthens the intestinal EC barrier by upregulating expression of

tight junction (TJ) proteins (Howe et al., 2005; Hering et al., 2011)

• TGF-β prevents intestinal EC barrier permeability mediated by:

• Pro-inflammatory cytokines IFN-γ and TNF-α (Planchon et al., 1994 and 1999)

• Intestinal pathogens E. coli (EHEC) and C. parvum (Howe et al., 2005; Roche et al., 2000)

TGF-β1

SP Cytokine Profiles from HIV Negative

and Positive, HAART-Naive Men

Kafka et al., 2012

• Many factors present in the mucosal microenvironment can affect the integrity of the EC barrier.

• Inflammation and certain pathogens can break the EC barrier.

• Immunoregulatory cytokines can maintain and strengthen the barrier.

• Exposure to HIV-1 can impair EC barrier integrity.

• Seminal plasma contains differing cytokine profiles.

• Acute SP – high concentration of pro-inflammatory cytokines + high VL

• Chronic SP – high concentration of TGF-β + low VL

• SP can affect GEC proinflammatory cytokine expression, but what effect does it have on the GEC barrier?

• Unclear how the balance between pro-inflammatory and immunoregulatory components in SP can influence the EC barrier in the FGT.

Study Rationale

1. What is the role of seminal plasma on FGT epithelial cell

barrier function?

2. What components of seminal plasma are influencing FGT

epithelial cell barrier function?

3. Do these components alone influence FGT epithelial

barrier function?

4. Does seminal plasma prevent HIV-mediated epithelial

cell barrier permeability?

Research Questions

(Kaushic et al., 2011; Methods)

Transwell Insert

Apical

Compartment

Basolateral

Compartment

Barrier Function Measured By:

• Trans-epithelial Electrical Resistance

(TER) as percent pretreatment

• Fluorescent microscopy of GECs

stained for tight junction proteins (green = ZO-1)

• Western Blot to measure expression of tight

junction proteins (in progress)

Measuring GEC

Barrier Function

SP (HIV- and HIV+)

TGF-β1 (20ng/ml)

HIV-1 (105 IU/ml)

TGF-β1 and seminal plasma from chronically

infected, HAART-naive men increase expression

of TJ proteins

ZO-1 Nucleus Combined

Untreated

(UnT)

TGF-β1

Chronic SP

*p<0.05

24h exposure

* *

Uninfected SP

Western blot analysis

in progress

TGF-β1 Increases the FGT Barrier in

the Presence of HIV-1

* *

Endometrial ECs Endocervical ECs

*p<0.05

**p<0.01

24h exposure

HIV-1 TGF-β1 +

HIV-1

TGF-β1 UnT

ZO-1

** ** * *

Example: SP from acutely infected, HAART-naive men

• low TGF-β

• High HIV-1

• High pro-inflammatory cytokines

Not to scale

HIV-1

TGF-β

TGF-β

HIV-1

Example: SP from chronically infected, HAART-naive

men

• High TGF-β

• Low HIV-1

• Low pro-inflammatory cytokines

Not to scale

• SP is another member of the FGT microenvironment that can impact the

EC barrier.

• Physiological concentrations of TGF-β increased EC barrier integrity by

increasing tight junction protein (ZO-1) expression.

• Comparable to seminal plasma from HIV-infected , HAART-naive men in

chronic stages of infection.

• TGF-β prevented GEC barrier permeability compared to HIV-1 alone.

• TGF-β1 will be replaced with seminal plasma from HIV negative and

positive, HAART-naive men along with HIV-1 to examine the balance

between them on EC barrier.

• Determine how TGF-β and seminal plasma influences the TNF pathway.

Conclusions & Future Work

Significance of Work

• Contribute to our understanding of the complexity of the

FGT microenvironment in the maintenance of the EC

barrier.

• Provide valuable knowledge regarding the role of seminal

plasma in the establishment of HIV infection in the FGT.

• Can lead to the design of therapies aimed at strengthening

the FGT EC barrier in the presence of inflammatory

conditions.

Acknowledgements

Kaushic Lab

Dr. Charu Kaushic

Dr. Aisha Nazli

Kristen Mueller

Victor Ferreira

Varun Anipindi

Kristy Roth

Philip Nguyen

Alexia Kim

Oscar Chan

Committee Members

Dr. Fiona Smaill

Dr. Brian Lichty

Collaborators

Dr. Rupert Kaul

Dr. Colin Kovacs

Brendan Osborne

CIHR Emerging

Team Grant HET85518

Thank You

Clinical Pathology Staff

Hysterectomy Tissue Donors

Seminal Plasma Donors

OHTN Conference Organizers

Funding Sources