Role of the programmed Death-1 pathway in the suppressive activity of alternatively activated macrophages in experimental cysticercosis

Abstract

We characterised a population of macrophages potentially involved in the immunoregulation induced by experimental cysticercosis. Following Taenia crassiceps infection, macrophages recruited in the peritoneal cavity were isolated and co-cultured at different ratios with T cells from naïve mice previously stimulated with anti-CD3/CD28 antibodies; these macrophages inhibited naïve T cell proliferation. This suppressive effect was Interleukin (IL)-10, Interferon-gamma (IFN-γ), and nitric oxide (NO) independent. In contrast, macrophage-T cell contact was necessary to maintain anergy of T cells. Reverse transcriptase-PCR analysis of these macrophages showed higher transcripts of IL-10, chitinases Fizz1 and Ym1, and arginase-1 compared with naïve macrophages; by contrast, IL-12p40, and inducible nitric oxide synthase (iNOS) transcripts were undetected, whereas C-C chemokine ligand 5 (CCL5) was unchanged. Analysis of the membrane molecules expressed on Taenia-induced macrophages showed an up-regulation of several markers, mainly programmed death ligand 1 (PD-L1) and PD-L2. Blockade of PD-L1, PD-L2 or their receptor PD-1, but not of another marker, eliminated their ability to inhibit T-cell proliferation. Parallel experiments using ovalbumin (OVA)-peptide as a model antigen displayed similar results. Additionally, the same mechanism appears to be functional in splenocytes of T. crassiceps-infected mice given that blockade of PD-1, PD-L1 or PD-L2 re-established their ability to proliferate in response to parasite antigens. Moreover, Taenia-induced macrophages were able to suppress a mixed lymphocyte reaction in a PD-1-dependent manner. Thus, cestode infections induce macrophages alternatively activated with strong suppressive activity involving the PD-1/PD-L's pathway.

Introduction

Parasitic helminths have developed complex mechanisms to evade their host's responses (Gause et al., 2003). Helminths and their antigens are now recognised to have important immunomodulatory activities (Maizels and Yazdanbakhsh, 2003). Commonly, helminth infections induce Th2-biased immune responses in their hosts that have been associated with impaired proliferative cell responses to parasitic antigens or unrelated antigens (Holland et al., 2000, Gause et al., 2003). Moreover, helminth infections can alter the immunity and susceptibility to other pathogens (Cox, 2001) or reduce the effectiveness of vaccines, such as tetanus vaccination during lymphatic filariasis (Nookala et al., 2004). How helminth infections modulate the host immune system remains unclear.

With few exceptions (Spolski et al., 2000, Hernandez-Mendoza et al., 2005), research on mechanisms of immunomodulation during cestode infections has been largely neglected. We have recently examined the immunomodulatory activities of the cestode Taenia crassiceps. As in most helminth infections, experimental cysticercosis caused by T. crassiceps induces Th2-type biased immune responses (Villa and Kuhn, 1996, Terrazas et al., 1998, Toenjes et al., 1999) as well as anergy of splenocytes in response to both antigen-specific and polyclonal stimuli (Sciutto et al., 1995, Villa and Kuhn, 1996, Rodriguez-Sosa et al., 2003). Besides, increased susceptibility to Trypanosoma cruzi (Rodriguez et al., 1999) and to vaccinia virus (Spolski et al., 2002) was observed in mice previously infected with T. crassiceps. Thus, in addition to activating Th2-type biased responses, infection with T. crassiceps can regulate other lymphocyte functions in vivo, suggesting that this infection may induce strong modulation of the immune response.

Classical activation of T cells involves interaction between membranes of both antigen presenting cells (APC) and T cells (Sharpe and Freeman, 2002) where the interaction of major histocompatibility complex (MHC) and T-cell receptor (TCR), as well as co-stimulatory molecules such as B7-1 and B7-2 with CD28 activate T cell responses. On the other hand, inhibitory interactions can be induced if cytotoxic T lymphocyte antigen 4 (CTLA-4) on the surface of activated T cells binds to B7-1 or B7-2 on APC (Sharpe and Freeman, 2002). Thus, accessory molecules can stimulate or inhibit T cells. In the last few years, new members of the B7 family have been identified. Two of the most relevant have been programmed death ligand 1 (PD-L1) and PD-L2 (Khoury and Sayegh, 2004, Greenwald et al., 2005). These molecules bind to the receptor programmed death 1 (PD-1) which is expressed on activated T cells (B cells and myeloid cells also) and their interactions result in down-modulation of T cell responses (Latchman et al., 2004).

In an attempt to dissect out mechanisms that lead to immune alteration in experimental murine cysticercosis, we examined the role of macrophages induced in response to T. crassiceps infection in modulating T cell proliferation to different classes of stimuli such as, polyclonal activation by anti-CD3/CD28 mAb, antigen-specific stimulation to parasite or unrelated antigens and, CD4⁺ cell proliferative response in a mixed lymphocyte reaction (MLR). We found that Taenia-induced macrophages had the ability to suppress T cell proliferation in vitro in a cell contact-dependent manner via an increased expression of PD-L1 and PD-L2.