Transglutaminase 2 Expression in Macrophages in Response to Toxoplasma gondii Infection and Stress

Adam Bradley1, Inez Pabian2, Fernando Monroy2, PhD1Coconino Community College, 2Department of Biological Sciences, Northern Arizona University, Flagstaff, AZ 86001

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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)-β 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)-β 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-EPI3 hr.

inf.

3 hr. nor-EPI +

inf. 8 hr.

nor-EPI8 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 1Is 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 3Is TG2 expression altered whenRAW macrophages are infected

and exposed to stress?

Control

CORT

3 hr. nor-E

PI

3 hr. nor-E

PI + in

f.

3 hr. inf.

8 hr. nor-E

PI

8 hr. inf.

8 hr. nor-E

PI + in

f.