Transglutaminase 2 (TG2) expression was examined in macrophages in response to Toxoplasma gondii infection and stress. TG2 was expressed in RAW macrophages and intestinal epithelial cells. TG2 expression increased at 3 hours post-infection but decreased at 8 hours. TG2 expression was induced by the stress hormone corticosterone, norepinephrine, and T. gondii infection, with maximum expression observed when infection and norepinephrine were combined. Increased TG2 expression may be detrimental to the host by decreasing anti-inflammatory transforming growth factor beta from macrophages and exacerbating inflammation.

1. Transglutaminase 2 Expression in Macrophages in Response to
Toxoplasma gondii Infection and Stress
Adam Bradley1, Inez Pabian2, Fernando Monroy2, PhD
1Coconino Community College, 2Department of Biological Sciences, Northern Arizona University, Flagstaff, AZ 86001
References
1. Fésüs, L. & Szondy, Z. (2005). Transglutaminase 2 in the balance of cell death and survival. Febs Letters, 579(15), 3297-3302.
2. Gratchev, A. A., Kzhyshkowska, J. J., Utikal, J. J. & Goerdt, S. S. (2005). Interleukin-4 and Dexamethasone Counterregulate Extracellular Matrix Remodelling and Phagocytosis in
Type-2 Macrophages. Scandinavian Journal of Immunology, 61(1), 10-17.
3. Gopal, R. R., Birdsell, D. D. & Monroy, F. P. (2011). Regulation of chemokine responses in intestinal epithelial cells by stress and Toxoplasma gondii infection. Parasite
Immunology, 33(1), 12-24.
4. Mehta, K., Kumar, A. & Kim H.I. (2010). Transglutaminase 2: A multi-tasking protein in the complex circuitry of inflammation and cancer. Biochemical Pharmacology, 80:1921-
1929.
5. Paolella, G., Caputo, I., Marabotti, A., Lepretti, M., Salzano, A., Scaloni, A. & Esposito, C. (2013). Celiac Anti-Type 2 Transglutaminase Antibodies Induce Phosphoproteome
Modification in Intestinal Epithelial Caco-2 Cells. Plos ONE, 8(12), 1-9.
6. Sommerville, C., Richardson, J. M., Williams, R. M., Mottram, J. C., Roberts, C. W., Alexander, J., & Henriquez, F. L. (2013). Biochemical and Immunological Characterization of
Toxoplasma gondii Macrophage Migration Inhibitory Factor. Journal of Biological Chemistry, 288(18), 12733-12741.
7. Yanagawa, Y., Hiraide, S., Matsumoto, M., Shimamura, K. & Togashi, H. (2014) Enhanced transglutaminase 2 expression in response to stress-related catecholamines in
macrophages. Immunobiology. 2014 Sep;219(9):680-6.
8. Zhang X, Goncalves R, Mosser DM (2008) The isolation and characterization of murine macrophages. Curr Protoc Immunol, 14: 14 11-1433.
Mouse RAW264.7 macrophages (MΦ) were grown in a T-25 flask to 90%
confluency.
Macrophages were seeded into a 6-well plate (500,000 MΦ cells/well) then
exposed to T. gondii (2 million parasites/well) in the presence or absence of nor-EPI.
Macrophage RNA was collected at 3 and 8 hr. post-infection, quantitated using
NanoDrop, and converted to cDNA (Reverse Transcriptase).
TG2 was amplified using specific primers by semi- and quantitative polymerase
chain reaction (qPCR) using the Bio-Rad MyiQ Real-Time PCR System.
Methods
Introduction
Macrophages are phagocytes distributed throughout the body and contribute
to tissue healing and homeostasis. They participate in innate and adaptive
immune responses. Macrophages remove foreign molecules including infectious
agents. Toxoplasma gondii is an intracellular protozoan parasite that can survive
within macrophages by altering the macrophage proteolytic and inflammatory
pathways. The end products of stress (epinephrine [EPI], nor-EPI, and
corticosterone [CORT]) are widely accepted to be immunosuppressive in part by
altering the function of macrophages. Our results show that both mouse
macrophages and the intestinal epithelial cell line MODE-K expressed TG2.
Expression of TG2 was induced by nor-EPI while T. gondii infection seemed to be
a better inducer. When both infection and nor-EPI were combined we observed
the highest expression of TG2. We observed that the stress hormone CORT was
also capable of inducing TG2. In this study I report for the first time induction of
TG2 expression by infection which was increased in the presence of stress
products. This neuro-immune crosstalk may be important in situations where
stress and infection occur to exacerbate inflammatory diseases. A potential
mechanism may be the suppression of macrophage-transforming growth factor
(TGF)- leading to host immune suppression and increase inflammatory
responses.
Abstract
•TG2 was expressed in both RAW macrophages and the intestinal epithelial cell line MODE-K.
•TG2 expression was increased by infection at 3 hr. post-infection but suppressed at 8 hr.
•TG2 expression was induced by CORT, nor-EPI and T. gondii infection. Maximum expression was observed
when infection and nor-EPI were combined.
•Increased TG2 expression may be detrimental to the host as TG2 is known to decrease macrophage
transforming growth factor (TGF)-which is crucial in regulating inflammation.
Conclusions
Funding was provided by the National Institutes of Health Bridges to Baccalaureate program Grant No.R25 GM 102788 and the
National Cancer Institute Native American Cancer Prevention program. Thanks to Victor Jimenez for passing on knowledge and
discussing science.
Acknowledgements
Results
Con CORT.
3 hr.
nor-EPI
3 hr.
inf.
3 hr.
nor-EPI +
inf.
8 hr.
nor-EPI
8 hr.
inf.
8 hr.
nor-EPI +
inf.
AIM 3
Is TG2 expression
altered when
RAW macrophages are
infected and exposed
to stress?
MΦ
GAPDH
Macrophages are phagocytes that function in innate and adaptive immunity8. Transglutaminase 2
(TG2) is expressed by macrophages and its expression is upregulated in response to cellular damage
to protect cells from stressors and contributes to inflammatory response2,4,,5,7. Its participation in
breast cancer, colon cancer, and Crohn’s disease is well documented; often referred to as a cell
component of tissue defense mechanisms, but the role of TG2 during infection is unknown1,7.
Toxoplasma gondii (T. gondii) is an obligate intracellular protozoan parasite and is one of the
most common parasites in humans. Infection in humans can occur by consuming raw or undercooked
meat, water, soil, or vegetables containing T. gondii oocysts. T. gondii can survive within phagocytic
cells by preventing fusion of the phagolysosome. It also prevents infected cells from undergoing
death (apoptosis) by decreasing expression of pro-inflammatory cytokines and pro-apoptotic
proteins3,8.
The goal of our study was to determine if TG2 expression was affected by T. gondii infection and
the stress product norepinephrine (nor-EPI)3. The end products of stress (epinephrine [EPI], nor-EPI,
and corticosterone [CORT]) are widely accepted to be immunosuppressive in part by altering the
function of macrophages4. However, stress seems to promote inflammation in diseases such as
inflammatory bowel disease, rheumatoid arthritis and psoriasis which are associated with increase
secretion of nor-EPI and its effects on macrophages5,7. It was hypothesized that T. gondii infection
will decrease TG2 expression and its expression should increase in the presence of nor-EPI.
AIM 1
Is TG2 expressed in Mouse
RAW264.7 macrophages ?
100 bp
Ladder RAW (MΦ) MODE-KControl
100 bp
Ladder
3 hr.
Con
3 hr.
inf. 1
3 hr.
inf. 2
8 hr.
Con
8 hr.
inf. 1
8 hr.
inf. 2AIM 2
Is TG2 expression in RAW
macrophages altered by
infection (inf.)?
MΦ
TG2
Figure 1: Agarose gel electrophoresis for Aim 1
Figure 2: Agarose gel electrophoresis for Aim 2
Figure 3: Agarose gel electrophoresis for Aim 3
Mouse RAW264.7
macrophages
6-well plate cDNA Synthesis
Real-time PCR
Agarose gel
electrophoresis
AIM 3
Is TG2 expression altered when
RAW macrophages are infected
and exposed to stress?