Gut commensal bacteria enhance pathogenesis of a tumorigenic murine retrovirus

Abstract

The influence of the microbiota on viral transmission and replication is well appreciated. However, its impact on retroviral pathogenesis outside of transmission/replication control remains unknown. Using murine leukemia virus (MuLV), we found that some commensal bacteria promoted the development of leukemia induced by this retrovirus. The promotion of leukemia development by commensals is due to suppression of the adaptive immune response through upregulation of several negative regulators of immunity. These negative regulators include Serpinb9b and Rnf128, which are associated with a poor prognosis of some spontaneous human cancers. Upregulation of Serpinb9b is mediated by sensing of bacteria by the NOD1/NOD2/RIPK2 pathway. This work describes a mechanism by which the microbiota enhances tumorigenesis within gut-distant organs and points at potential targets for cancer therapy.

Keywords: CP: Immunology; gut commensal bacteria; negative immune regulators; retroviruses.

Figure 1.. Dependence of MuLV-induced leukemia development on commensal bacteria

Adult BALB/cJ females from different experimental groups were injected with MuLV and bred (G0 mice). Their offspring (G1 mice) were monitored for leukemia. Diseased mice removed from the cohorts and mice surviving up to 150 days were examined according to a leukemia scoring system based on histological analysis of the spleen (Figure S1C). (A) Comparison of viral load (number of infectious centers [ICs]) in preleukemic SPF-, GF-, and Abx-treated animals with score 1. (B) Survival curves of G1 mice from the same groups. (C) Spleen weights of infected and uninfected SPF and GF mice at age 4–5 months. Dotted horizontal line indicates 0.35 g (all mice with spleen weight ≥ 0.35 g have leukemia, Figures S1D–S1F). (D) Survival curves of MuLV-infected SPF- and ASF-colonized BALB/cJ mice observed for 97 days. (E) Survival of GF, SPF, and gnotobiotic BALB/cJ mice colonized with single bacterial lineages monitored for 150 days. (F) Final assessment of leukemia development in mice from these groups at day 150. Abx, antibiotic. n, number of mice used per group. p values were calculated using an unpaired t test (A and C), Fisher’s exact test (F) and Mantel-Cox test (B, D, and E). Error bars indicate standard error of the mean.

Figure 2.. Comparison of leukemia development in SPF and GF BALB/c mice

(A) Increase in extramedullary hematopoiesis in the spleens of infected mice. Nucleated splenocytes were stained with anti-mouse Sca-1 mAb and analyzed by FACS. Uninfected GF and SPF BALB/cJ mice were used as negative controls. (B and C) Frequency of HSCs in the bone marrow (BM) of GF and SPF mice measured by colony formation in the spleens of lethally irradiated SPF BALB/cJ recipients. Splenic colonies were counted 9 days after transfer of 2 × 10⁶ BM cells. CFUs, colony forming units per 2 × 10⁶ BM cells. n, number of recipients. Combined data from two independent experiments (B). Fixed spleens were sectioned and stained with hematoxylin and eosin to visualize differentiated cells within colonies. Representative images of one of five recipient mice per group. Red and white pulp indicated by white arrows. Cells of the different hematopoietic lineages are identified by yellow arrows. M, megakaryocyte; E, erythroid precursor; L, lymphocyte (C). (D) Determination of a specific developmental stage at which erythroid cells are stalled in infected SPF mice. The stages of erythrocyte development from the megakaryocyte-erythrocyte progenitor stage to the final red blood cell stage (R1-R5) were determined as described (Zhang et al., 2003) by staining nucleated spleen cells with a mixture of anti-mouse CD71/anti-mouse Ter119 mAbs. R1 corresponds to erythroid burst-forming units and erythroid colony forming units; R2 corresponds to proerythroblasts and basophilic erythroblasts; R3 is made up of early and late basophilic erythroblasts; R4 corresponds to polychromatophilic and orthochromatophilic erythroblasts and R5 contains reticulocytes and erythrocytes. Staining profiles of splenocytes from the indicated mice. Of the 18 SPF leukemic mice analyzed, 15/18 mice exhibited CD71^highTER19^low leukemias and 3/18 mice exhibited CD71^highTER19^high leukemias. (E) A quantitative comparison of cell numbers arrested at R1 (left) and R2 (right) see (D) stage in SPF and GF mice. n, number of mice used. p values calculated using unpaired t test. Error bars indicate standard error of the mean.

Figure 3.. Comparison of leukemia susceptibility of immunosufficient- and immunodeficient-infected GF and SPF BALB/cJ mice

(A) Survival of IL-6-sufficient and IL-6-deficient MuLV-infected SPF BALB/cJ mice. (B) Serum cytokine concentration of IL-6 was measured in uninfected (score 0), infected preleukemic (score 1), and infected leukemic (scores 2–3) BALB/cJ mice using a flow cytometry bead-based assay. (C) Survival of infected RAG1-sufficient SPF, RAG1-deficient SPF, RAG1-sufficient GF, and RAG1-deficient GF BALB/cJ mice during 150 days. (D) Total leukemia incidence of infected indicated mice at 150 days. (E) Comparison of the viral burden (frequency of infected cells per 10⁶ splenocytes) in indicated preleukemic mice (score 1). (F) MuLV-specific ELISA to detect anti-virus antibodies. I/LnJ mice fostered on MuLV-infected BALB/cJ females are included as a positive control as they produce virus-neutralizing antibodies (Case et al., 2008). n, number of mice used. p values calculated using Mantel-Cox test (A and C), Fisher’s exact test (D), or unpaired t test (B, E, and F). Error bars indicate standard error of the mean.

Figure 4.. Induction of negative regulators of the immune response by commensal bacteria and the virus

RNA isolated from spleens of preleukemic mice (score 1) of four groups (SPF ± MuLV infection and GF ± MuLV infection) was subjected to high-throughput sequencing. (A) Diagram detailing the series of operations taken to identify genes differentially expressed in spleens of mice from four groups. See explanation in the text. (B) Heatmap of gene expression found to be significantly upregulated in SPF MuLV-infected mice compared with mice from all other groups. Red arrows indicate established negative regulators of adaptive immunity. (C) Real-time quantitative PCR (qPCR) measurement of expression of the negative regulators of immune response in SPF and GF mice. Mice were either uninfected (score 0) or infected pre-leukemic (score 1). n, number of mice used. (D) qPCR with RNA isolated from spleens of uninfected (score 0) and infected pre-leukemic (score 1) mice colonized with L. murinus, P. goldsteinii, or B. theta. Data are represented as log₂ fold change compared with uninfected controls, normalized to the endogenous control (β-actin). Two-way ANOVA was used to identify genes upregulated in infected SPF mice compared with uninfected SPF, uninfected GF, and infected GF mice (A and B). p values calculated using unpaired t test (C and D). Error bars indicate standard error of the mean.

Figure 5.. Resistance of Rnf128-deficient and Serpinb9b-deficient SPF mice to MuLV-induced leukemia

MuLV-infected BALB/cJ mice deficient in VSig4, Serpinb9b, or Rnf128 were monitored for leukemia development. (A) Survival curves for up to 150 days are shown. (B) Final leukemia assessment at 150 days. (C and D) Expression of Rnf128 (C) or Serpinb9b (D) in the MACS sorted splenocytes from WT uninfected and infected mice analyzed by qPCR. (E) Serpinb9b expression in RNA isolated from spleens of SPF RAG1^−/− uninfected (score 0), pre-leukemic (score 1), and leukemic (scores 2–3) mice analyzed via qPCR. qPCR data are represented as log₂ fold change compared with uninfected controls and normalized to the endogenous control β-actin. n, number of mice used. p values calculated using Mantel-Cox test (A), Fisher’s exact test (B), or unpaired t test (C–E). Error bars indicate standard error of the mean.

Figure 6.. NOD1/2 adaptor RIPK2 contributes to MuLV-induced leukemia via upregulation of Serpinb9b

Leukemia development was monitored in RLMuLV-infected SPF BALB/cJ mice deficient in either TLR2, TLR4, TLR2/TLR4, or RIPK2. (A) Survival curves up to 150 days. (B) Total leukemia incidence at 150 days. (C) Expression of Serpinb9b in splenic RNA from RIPK2^−/− and control RIPK2^+/− uninfected and infected mice analyzed by qPCR. Data are represented as log₂ fold change compared with uninfected controls, normalized to the endogenous control (β-actin). n, number of mice used. Error bars indicate standard error of the mean. p values calculated using Mantel-Cox test (A), Fisher’s exact test (B) or unpaired t test (C).

Figure 7.. Hypothetical model of Serpinb9b- and Rnf128-mediated suppression of the adaptive immune response

Upregulation of Serpinb9b, likely in tumor cells, potentially promotes tumor development through inactivation of cytotoxic granzymes. Rnf128 upregulation in CD4⁺ T cells stimulates unresponsiveness, inhibiting their immune function.