Immunobiology. 2014 Jan;219(1):64-77.

Fas/FasL pathway participates in resolution of mucosal inflammatory response early during HSV-2 infection.

Krzyzowska M, Baska P, Grochowska A, Orlowski P, Nowak Z, Winnicka A.

Department of Regenerative Medicine, Military Institute of Hygiene and Epidemiology, Warsaw, Poland; Division of Immunology, Department of Preclinical Sciences, Faculty of Veterinary Medicine, Warsaw University of Life Sciences, Warsaw, Poland. Electronic address: krzyzowskam@yahoo.com.

 

Abstract

Apoptotic cell death is critical for maintaining integrity of the epithelia as well as for removal of the virus infected cells. We assessed the role of Fas/FasL-dependent pathway in apoptosis of genital epithelium during HSV-2 infection using a murine model of HSV-2 infection applied to C57BL6, MRL-Fas(lpr)/J (Fas-/-) and C3-Fasl(gld)/J (FasL-/-) mice and an in vitro model of HSV-2 infection in monocyte RAW 264.7 and keratinocyte 291.03C cell cultures and peritoneal macrophages. In contrast to keratinocyte in vitro cultures, HSV-2 infection of the monocytic cell cultures led to early induction of apoptosis. HSV-2 infection of peritoneal macrophages isolated from Fas- and FasL-deficient mice showed decreased activation of apoptosis, which could be further blocked by caspase-9 inhibitor. Infection of Fas and FasL-deficient mice increased the percentage of apoptotic cells and activation of caspase-9 in the vaginal tissue in comparison to C57BL6 wild type strain. Furthermore, Fas and FasL-deficient mice showed increased infiltration of neutrophiles in the vaginal mucosal epithelium at 3 and 7 day of infection in contrast to HSV-2 infected wild-type mice. Our results show that while the Fas/FasL pathway during HSV-2 infection of the vaginal epithelium plays an important role in controlling early local inflammatory response, mitochondrial apoptotic pathway also becomes activated by the inflammatory reaction. Copyright © 2013 Elsevier GmbH.

KEYWORDS: Apoptosis, Caspase-9, Fas/FasL, HSV-2, ICP10, NETs, NK cells, ROS, STD, TNF, Vaginal epithelium, herpes simplex virus type 2, iNOS, inducible nitric oxide synthase, natural killer cells, neutrophil extracellular traps, reactive oxygen species, ribonucleotide reductase R1, sexually transmitted diseases, tumour necrosis factor

PMID: 24028839

 

SUPPLEMENT:

Genital herpes, caused primarily by herpes simplex virus type 2 (HSV-2) is one of the most prevalent sexually transmitted diseases in the world and is also a major risk factor for human immune deficiency  virus type 1 (HIV-1) acquisition, transmission and infection progression. The intact vaginal mucosa consists of a multilayered squamous epithelium providing mechanical protection against invading pathogens. During the life cycle, epithelial cells of vagina migrate from the germinal basal layer, becoming flattened and keratinized cells.

There are several types of cell death, of which apoptosis is a physiological process necessary for normal development and homeostatic maintenance of multicellular organisms, also for vaginal epithelium turnover. Fas and other receptors from the TNF family upon interaction with their ligands, (e.g. FasL) trigger so called death-receptor pathway of apoptosis. The Fas/FasL system plays an important role in skin and epithelial homeostasis, carcinogenesis and inflammatory skin diseases. The role of apoptosis  in development of lesions in sexually transmitted diseases (STD), such as genital herpes, and involvement of different both pro-apoptotic and anti-apoptotic proteins in compromising the vaginal mucosa integrity has not been extensively studied so far.

Our previously published [1] results showed that inflammatory lesions within the HSV-2 infected epithelium of C57BL6 mice consisted of infected cells up-regulating Fas and FasL, uninfected cells up-regulating Fas and neutrophils expressing both Fas and FasL. Apoptosis was detected in HSV-2 infected cells and to even higher extent, in non-infected cells surrounding HSV-2 infection sites. However, infection of mice lacking Fas or FasL expression resulted in the increased apoptosis and strong recruitment of neutrophils within the vaginal infection sites. Interestingly, either HSV-2 infection or triggering of Fas receptor on keratinocytes in vitro led to up-regulated expression of pro-inflammatory chemokines- CXCL1 and CXCL10- and cytokines which may participate in infiltration of neutrophils at infected vaginal sites.  This led us to the conclusion that  Fas pathway participates in regulation of inflammatory response in the vaginal epithelium at the initial stage of HSV-2 infection [1].

In this study [2], we further explored apoptotic pathways  detected during HSV-2 infection in vitro and in vivo. Expression analysis of 84 key apoptotic genes using the RT2-PCR technique for the murine vaginal tissue isolated at 3 day of HSV-2 infection showed up-regulation of mRNA for the genes from TNF superfamily (Fas, FasL, TNF-alpha, TRAF 1) and for the genes involved in the mitochondrial pathway of apoptosis, such as Apaf-1, Bnip3, Bak and Bax, but also anti-apoptotic cIAP-2, Bcl-2 and Bcl-xl genes.

To test whether Fas/FasL apoptotic pathway is solely responsible for apoptosis induction in the vaginal epithelium  during HSV-2 infection, we infected mice lacking Fas or FasL expression together with the control background strain (C57BL/6). The vaginal tissues lacking Fas or FasL expression showed significantly increased numbers of cells with active caspase- 9 form at 3 d post infection (p.i.). At the same time, we observed a significant decrease in  the total percentage of cells with anti-apoptotic Bcl-2 expression in comparison to wild-type mice.

Professional phagocytes (neutrophil granulocytes and monocytes/macrophages) constitute an important first line of defence against microbial intruders, including HSV infection. Following infection, neutrophils are recruited to tissues by chemotactic factors presented in a temporally and spatially defined manner. Increased accumulation of neutrophils in the vaginal tissue of Fas- and FasL-deficient mice and delayed neutrophil clearance was related with decreased apoptosis. Additionally, we used the in vitro model of co-culture, consisting of HSV-2 infected keratinocytes incubated for 12 h with monocytes or neutrophils isolated from wild, Fas- and FasL-deficient mice.  Monocytes and neutrophils without Fas expression showed significantly decreased apoptosis in contact with FasL-expressing HSV-2 infected keratinocytes.

Increased  accumulation of inflammatory cells is related with the increased production of multiply inflammatory mediators, such as nitric oxide (NO) and free oxygen radicals (ROS). These mediators are known inducers of apoptosis through mitochondrial pathway. Accumulation of neutrophils in Fas- and FasL-deficient mice in our study was accompanied by  significantly increased expression of iNOS, an inducible nitric oxide synthase responsible for NO production, which may explain increased apoptosis via mitochondrial pathway, observed in the vaginal tissue of Fas- and FasL-deficient mice in comparison to the wild type strain.

In this context, it is important to notice that apoptosis induction during HSV-2 infection is also cell-type dependent. When comparing apoptosis induction during HSV-2 infection in the murine RAW 264.7 cell line and keratinocyte 291.03C cell line, we found that HSV-2-infected monocytes showed signs of apoptosis early after infection (6 h p.i.)  in comparison to keratinocytes. In another study [3] we showed that infected cells of epithelial origin activated “apoptosis suppression window” related with expression of anti-apoptotic proteins such as Bcl-2 and protection from Fas-induced apoptosis. During in vivo HSV-2 infection, Fas/FasL-dependent apoptosis of monocytes led to development of the local chemokine and cytokine milieu, necessary for mounting proper anti-viral response [3]. HSV-2 infection of Fas and FasL- deficient mice was accompanied by decreased production of CXCL9, CXCL10 and TNF-α  and impaired recruitment of NK, CD4+ and CD8+ T cells within the infection sites in comparison to HSV-2 infected wild-type mice strain. The decreased recruitment of immune competent cells resulted in delayed virus clearance from the infected tissue.

Altogether, our results show that Fas/FasL – dependent apoptotic pathway plays an important role  not only in elimination of local infection, but also in regulation of inflammatory response to HSV-2 infection.

These results are important for the development of effective vaginal microbicides, based on the possibility of influencing the local immune inflammatory responses by modulation of Fas/FasL expression and/or Fas mediated apoptosis.

Role of Fas/FasL in mucosal inflammatory response early during HSV-2 infection:

fig1

Acknowledgements:  This study was supported by the Polish National Science Centrum grant no. N N401 178839 and ERG FP7-PEOPLE-2010-RG fellowship no. 276907.

 

References:

  1. Krzyzowska M, Shestakov A, Eriksson K, Chiodi F. 2011. Role of Fas/FasL in regulation of inflammation in vaginal tissue during HSV-2 infection. Cell Death Dis. 2:e132.
  2. Krzyzowska M, Baska P, Grochowska A, Orlowski P, Nowak Z, Winnicka A. 2014. Fas/FasL pathway participates in resolution of mucosal inflammatory response early during HSV-2 infection. Immunobiology. 219(1):64-77.
  3. Krzyzowska M, Baska P, Orlowski P, Zdanowski R, Winnicka A, Eriksson K, Stankiewicz W. 2013. HSV-2 regulates monocyte inflammatory response via the Fas/FasL pathway. PLoS One. 8(7):e70308.
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