The drug monosodium luminol (GVT) preserves crypt-villus epithelial organization and allows survival of intestinal T cells in mice infected with the ts1 retrovirus

Abstract

Of the cytopathic retroviruses that affect mammals, including HIV-1, many selectively infect CD4+ T cells and cause immunosuppressive syndromes. These diseases destroy both the thymus and the small and large intestines, after infecting and killing T-lineage cells in both tissues. A mutant of the murine leukemia retrovirus MoMuLV-TB, called ts1, causes this syndrome in susceptible strains of mice. In FVB/N strain mice that are infected at birth, thymic atrophy, CD4+ T cell loss, intestinal collapse, body wasting, and death occur by ∼30–40 days postinfection (dpi). Apoptosis of ts1-infected T-lineage cells, in the thymus, peripheral lymphoid system and intestines is caused by accumulation of the ts1 mutant viral envelope preprotein gPr80^env, which is inefficiently cleaved into the mature viral proteins gp70 and PrP15E. We show here that ts1 infection in the small intestine is followed by loss of intestinal epithelial cell (IEC) thyroid-stimulating hormone (TSH) and cell cycling gradients (along the crypt-villus axes), accumulation of gPr80^env in intestinal cells, apoptosis of developing T cells in the lamina propria (LP), and intestinal collapse by ∼30 dpi. In infected mice treated with the antioxidant drug monosodium luminol (GVT^®), however, normal intestinal epithelial cell gradients are still in place at 30 dpi, and IECs covering both the crypts and villi contain large amounts of the antioxidant transcription factor Nrf2. In addition, no apoptotic cells are present, and accumulated gpr80^env is absent from the tissue at this time. We conclude that GVT treatment can make ts1 a noncytopathic virus for intestinal lymphoid cells, as it does for thymocytes [25]. As in the thymus, GVT may protect the intestine by reducing oxidant stress in infected intestinal T cells, perhaps by prevention of gPr80^env accumulation via Nrf2 upregulation in the IECs. These results identify GVT as a potential therapy for intestinal diseases or inflammatory conditions, including HIV-AIDS, in which oxidative stress is a triggering or exacerbating factor.

Introduction

The murine retrovirus MoMuLV causes thymic leukemias by 8–10 months of age in susceptible mice that are infected at birth [1]. A mutant of MoMuLV, called ts1, causes a fulminant disease with a much shorter timecourse, leading to death of infected animals by 30 days after infection (dpi) [2]. The ts1 mutant has a mutation in its env gene, so that productively infected cells contain an abnormal gPr80^env preprotein [3], [4]. This abnormality interferes with the normal cleavage of the gPr80^env to the mature viral envelope proteins gp70 and PrP15E. Without efficient cleavage, gPr80^env accumulates in certain infected target cells, including astrocytes in the central nervous system (CNS), thymocytes and T cells [5], [6]. In glial cells of the central nervous system, this accumulation initiates an unfolded protein response, endoplasmic reticulum stress, mitochondrial stress, oxidative stress and apoptosis [7], [8], [9], [10], [11], [12], [13], [14], [15], [16], [17], [18]. Similar responses occur in T-lineage cells of infected mice, which accumulate gpr80^env and die as a consequence of oxidative stress-induced apoptosis [19].

The systemic consequences of ts1 infection are similar to HIV-AIDS at many levels [6], [8], [20]. Studies in this paper were designed to identify specific effects of ts1 infection on the mouse small intestine, whose symptomatology is similar to that induced by HIV-1, the primate retrovirus SIV, and the feline immunodeficiency virus FIV [21], [22], [23], [24]. The intestines are significant in retroviral diseases for another reason. Like the thymus, they can receive bone marrow T-cell progenitors and host their maturation into mature T cells (see below). This makes this tissue an important target for retroviral infection and cellular damage, because production of naïve T cells normally continues in these tissues throughout life, and because intestinal lymphoid cells in the LP govern the health and functions of the mucosal epithelium above. We therefore conducted this study in parallel with investigations of thymus tissues in ts1-infected mice [25].

Fig. 1 shows that mouse small intestines are composed of finger-like villi facing the gut lumen and of crypts buried in the connective tissue. Both regions are covered with a continuous layer of epithelium, with the pluripotent stem cells for the entire epithelium residing in the lower crypts [26], [27], [28]. These stem cells give rise to several cell types found on the villi: to absorptive enterocytes covering the villi (the predominant cell type in the mucosa), to mucus-producing goblet cells and enteroendocrine cells scattered over the villi between the enterocytes (specific cell types are not shown in the figure). Crypt stem cells also give rise to defensin-producing Paneth cells, which form a cup at the bottom of the crypts, and which do not move upward toward the villi [27], [28].

The progeny of the crypt epithelial stem cells are transit amplifying cells and differentiating cells, which move upward, as they divide, toward the crypt-villus junction [CVJ]. Once differentiated, these cells stop dividing, so that the epithelium covering the villi, above the CVJ, contains mostly quiescent enterocytes, goblet cells and enterendocrine cells nudging each other passively upward to the tips of the villi. When the IECs arrive at the tips of the villi, they undergo apoptosis and are shed, together with any T cells that have inserted themselves between the cells (top of Fig. 1). The CVJ thus is a sharp boundary that separates dividing from quiescent cells.

As noted above, the small and large intestines of mammals can host the differentiation of bone marrow T-precursor cells into mature T cells [29], [30], [31], [32]. The idea that the intestine might host T cell differentiation was put forward more than 10 years ago (reviewed in [32]). However, this claim was controversial until recent laser-capture confocal microscopy studies established clonal identity between early T cell progenitors in aggregates in the crypts, called cryptopatches (CPs) and mature T cells in the distal villi [33].

Fig. 1 shows T-cell progenitors differentiating in the lamina propria (LP) connective tissue beneath the CMJ, and mature T cells above it. This reflects the continuous arrival in the crypts of bone marrow stem cells, which are c-kit-positive (red). These begin differentiation to become mitotic T-cell progenitor progeny in the crypt LP (purple). The maturing and fully mature descendants of these intestine-born T cells are CD8-positive T cells that reside in the LP of the villi [29], [30], [31], [32]. CD4+ cells are also present, but these arrive from the blood as mature T cells that have already completed their differentiation in the thymus [34]. These are scattered throughout the crypt and villous LP. As in the thymus, therefore, the CVJ boundary separates distinct epithelia whose adjacent T cells are at distinct developmental stages.

In the normal thymus, the cortical and medullary epithelial cells express two distinct cytokeratin types [35]. Unlike the thymus however, the intestinal epithelium is a simple epithelium throughout; and all of its cells express CK8 only [36], as do cortical epithelial cells of the thymus. However, two other differences distinguish the epithelial cells of the intestinal crypts from those of the villi. One is these is the constitutive expression of TSH by crypt epithelial cells, and not by epithelial cells of the villi [37], [38], [39], [40]. The other is the presence of dividing epithelial cells exclusively in crypts, but not in the villi [26]. The presence and maintenance of these two standing gradients in the epithelial intestine reflects the presence of normally differentiating T-cell progenitors in the LP beneath, just as the presence of a healthy corticomedullary epithelial separation does for thymocytes [25]. Like thymocytes, intestinal T cells undergo sequential developmental checkpoints that depend upon their being at the right place, at the right time, in relation to the epithelium above [35].

In mice infected with ts1 (ts1-only), we show here that intestinal epithelial cell gradients are dysregulated, that extensive T cell apoptosis occurs, and that the intestines collapse by 30 dpi. In mice infected with ts1 and also treated with GVT (ts1-GVT), however, crypt-villus cytoarchitecture is intact at 30 dpi, intestinal epithelial cell gradients are in place, no T cell apoptosis is occurring, and the gPr80^env protein is not accumulated by any cell type in the tissue. In the intact small intestines of GVT-protected mice, most epithelial cells are negative for gp70, and thus do not appear to be infected, but they do contain large amounts of the transcription factor Nrf2, which orchestrates protective antioxidant responses [41], [42]. These effects of ts1-GVT treatment are remarkably similar to those occurring in the thymi of ts1-GVT mice, whose epithelial cells also become loaded with Nrf2 [25].