Resurrection of endogenous retroviruses in antibody-deficient mice

Abstract

The mammalian host has developed a long-standing symbiotic relationship with a considerable number of microbial species. These include the microbiota on environmental surfaces, such as the respiratory and gastrointestinal tracts, and also endogenous retroviruses (ERVs), comprising a substantial fraction of the mammalian genome. The long-term consequences for the host of interactions with these microbial species can range from mutualism to parasitism and are not always completely understood. The potential effect of one microbial symbiont on another is even less clear. Here we study the control of ERVs in the commonly used C57BL/6 (B6) mouse strain, which lacks endogenous murine leukaemia viruses (MLVs) able to replicate in murine cells. We demonstrate the spontaneous emergence of fully infectious ecotropic MLV in B6 mice with a range of distinct immune deficiencies affecting antibody production. These recombinant retroviruses establish infection of immunodeficient mouse colonies, and ultimately result in retrovirus-induced lymphomas. Notably, ERV activation in immunodeficient mice is prevented in husbandry conditions associated with reduced or absent intestinal microbiota. Our results shed light onto a previously unappreciated role for immunity in the control of ERVs and provide a potential mechanistic link between immune activation by microbial triggers and a range of pathologies associated with ERVs, including cancer.

Figure 1. eMLV activation in antibody-deficient mice

a, Significantly upregulated (>4-fold) genes in CD11b⁺MHC-II^hiB220⁻Gr1⁻ macrophages from Rag1^−/− mice compared with macrophages from WT mice. Triplicate microarrays from cells isolated from 40 mice are shown. b, eMLV spliced env mRNA expression in the same cells as in a. Each symbol represents macrophages from 20 mice (p=0.024; paired Student’s t-test). c, eMLV spliced env mRNA expression in indicated organs from WT or Rag1^−/− mice (spleen: p=0.020; small intestine: p=0.032; large intestine: p=0.004; lung: p= 0.001; muscle: p=0.016; and kidney: p=0.009; unpaired Student’s t-test). d, e, MLV SU expression in splenocytes (d) or in indicated cell types (e) from WT or Rag1^−/− mice. f, eMLV spliced env mRNA expression in the spleens of the indicated strains (p<0.001 between WT and either Ighm^−/− or Ighm^−/− MD4 mice; one-way ANOVA). g, MLV SU expression in splenic lymphocytes from WT or Ighm^−/− MD4 mice. h, eMLV spliced env mRNA expression in the spleens of the indicated strains (p<0.001 between WT and either Myd88^−/− or Tlr7^−/− mice; one-way ANOVA). In c, f and h, each symbol is an individual mouse. In d, e and g, plots are representative of 4 mice per group. In f and h, values above 10³ were considered high and are indicated with red-filled symbols.

Figure 2. Retroviremia and leukaemias/lymphomas in antibody-deficient mice

a, Detection of infectious MLV (RARV-5/XG7) from the plasma of a representative Rag1^−/− mouse by restoring infectivity of the green fluorescent protein (GFP)-expressing XG7 retroviral vector in the indicated cell type. Numbers within the plots denote the percentage of retrovirally-transduced (GFP⁺) cells. b, Fv1 tropism of RARVs isolated from 6- (R2-R4) or 25- (R5-R8) week-old healthy Rag1^−/− mice, shown as the ratio of infectivity in B-3T3 to N-3T3 cells (B:N ratio). B- and N-tropic strains of Friend MLV (F-MLV) are shown for comparison. c, Amino acid residues of capsid positions 105-113 deduced from the nucleotide sequence of Emv2 and the same RARVs as in b. Dots indicate identities. d, Phylogenetic tree of the same RARVs as in b. The scale indicates the probability of base substitution per site. e, eMLV spliced env mRNA expression in the spleens of Rag1^−/− mice or vertically-infected Rag1^−/−Emv2^−/− mice. Each dot is an individual mouse (p<0.001; unpaired Student’s t-test). Values above 10³ were considered high and are indicated with red-filled symbols. f, MLV SU expression in splenocytes from Rag1^−/− or vertically-infected Rag1^−/−Emv2^−/− mice (representative of 9 mice per group). g, eMLV DNA copy numbers per haploid genome, determined by qPCR for the pol or ecotropic env gene, in DNA from the spleens of healthy Rag1^−/− (right) or vertically-infected Rag1^−/− Emv2^−/− mice (left). Symbols represent individual mice, grouped according to their age. The sensitivity limit of this PCR method was determined as a median of 0.0003 copies per haploid genome, using Emv2^−/− mice. eMLV DNA copy numbers for Rag1^−/− mice include Emv2 (1/N). h, Tumour (leukaemias/lymphomas) incidence in cohorts of WT (n=37), Rag1^−/− (n=38) or vertically-infected Rag1^−/−Emv2^−/− mice (n=23) at the NIMR SPF facility (p<0.000001 between WT and Rag1^−/− mice; p=0.00025 between WT and Rag1^−/−Emv2^−/− mice; Log-Rank Survival analysis).

Figure 3. Murine ERV activation by microbial products

a, ERV/RE-reporting probeset (Supplementary Table 3) signals in a publicly-available Affymetrix HT Mouse Genome 430A microarray dataset (E-GEOD-17721) of WT B6 bone marrow-derived dendritic cells following stimulation with microbial products. Black arrows indicate the probesets that are significantly regulated (p<0.05) more than 2-fold by at least one stimulus. Mela (Emv2)-specific probesets are also indicated by grey arrows for comparison. b, Mean log₂ fold-change in MLV-reporting probeset in the same dataset. c, MLV SU expression in WT or Emv2^−/− splenocytes before (open histograms) and after stimulation with 10 μg/ml LPS for 48 hrs (grey-shaded histograms), according to Forward scatter (Fsc) and CD19 expression. Numbers with the plots denote the percentage of cells within each gate and represent 2 donors each analysed in duplicate.

Figure 4. eMLV activation in antibody-deficiency depends on husbandry conditions

a, eMLV spliced env mRNA expression in the spleens of Rag1^−/− mice on neutral pH (SPF) or acidified water (pH 2.5) at NIMR, on acidified water (pH 2.8) at JAX, on neutral pH at RCHCI or in germ-free (GF) facilities at UMICH (p<0.016 between Rag1^−/− mice at NIMR SPF and all other groups; one-way ANOVA). b, eMLV spliced env mRNA expression in the spleens of Ighm^−/−, Myd88^−/− or Tlr7^−/− mice on neutral pH water (SPF) at NIMR or on acidified water (pH 2.8) at JAX (p=0.005 and p=0.029 for Ighm^−/− and Tlr7^−/− mice, respectively; unpaired Student’s t-test). Each dot is an individual mouse and values above 10³ were considered high and are indicated with red-filled symbols.