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. 2019 Sep;19(9):2421-2433.
doi: 10.1111/ajt.15376. Epub 2019 May 14.

A clinically relevant murine model unmasks a "two-hit" mechanism for reactivation and dissemination of cytomegalovirus after kidney transplant

Zheng Zhang  1   2 , Longhui Qiu  1 , Shixian Yan  1 , Jiao-Jing Wang  1 , Paul M Thomas  3 , Manoj Kandpal  1   4 , Lihui Zhao  1   4 , Andre Iovane  1 , Xue-Feng Liu  1   2 , Edward B Thorp  5 , Qing Chen  5 , Mary Hummel  1   2   6 , Yashpal S Kanwar  5   7 , Michael M Abecassis  1   2   6
Affiliations

A clinically relevant murine model unmasks a "two-hit" mechanism for reactivation and dissemination of cytomegalovirus after kidney transplant

Zheng Zhang et al. Am J Transplant. 2019 Sep.

Erratum in

  • Erratum.
    [No authors listed] [No authors listed] Am J Transplant. 2020 Mar;20(3):907. doi: 10.1111/ajt.15593. Epub 2019 Sep 19. Am J Transplant. 2020. PMID: 32090475 No abstract available.

Abstract

Reactivation of latent cytomegalovirus remains an important complication after transplant. Although immunosuppression (IS) has been implicated as a primary cause, we have previously shown that the implantation response of a kidney allograft can lead to early transcriptional activation of latent murine cytomegalovirus (MCMV) genes in an immune-competent host and to MCMV reactivation and dissemination to other organs in a genetically immune-deficient recipient. We now describe a model that allows us to separately analyze the impact of the implantation effect vs that of a clinically relevant IS regimen. Treatment with IS of latently infected mice alone does not induce viral reactivation, but transplant of latently infected allogeneic kidneys combined with IS facilitates MCMV reactivation in the graft and dissemination to other organs. The IS regimen effectively dampens allo-immune inflammatory pathways and depletes recipient anti-MCMV but does not affect ischemia-reperfusion injury pathways. MCMV reactivation similar to that seen in allogeneic transplants combined with also occurs after syngeneic transplants. Thus, our data strongly suggest that while ischemia-reperfusion injury of the implanted graft is sufficient and necessary to initiate transcriptional reactivation of latent MCMV ("first hit"), IS is permissive to the first hit and facilitates dissemination to other organs ("second hit").

Keywords: animal models: murine; basic (laboratory) research/science; immunosuppression/immune modulation; immunosuppressive regimens; infection and infectious agents - viral: Cytomegalovirus (CMV); infectious disease; ischemia reperfusion injury (IRI); kidney transplantation/nephrology; signaling/signaling pathways; translational research/science.

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Conflict of interest statement

DISCLOSURE

The authors of this manuscript have no conflicts of interest to disclose as described by the American Journal of Transplantation.

Figures

FIGURE 1
FIGURE 1
Immunosuppression (IS) treatment promoted mouse cytomegalovirus (MCMV) reactivation and systemic dissemination after D+-to-R– allogeneic transplants (allografts). Viral DNA in kidneys, lungs, and salivary glands (SGs) were quantified by quantitative PCR at postoperative days (POD)7-28 after allogeneic vascularized transplantation of D+ kidneys from B6 mice into R– BALB/c recipients (n = 5-6/ time/group). A, Schematic of experimental setup. B, Viral DNA copy numbers in kidneys at POD0 (donor contralateral controls) and the kidney allografts treated with IS (w/IS) or without IS (wo/IS) at PODs 7, 14, and 28. C, Viral DNA copy numbers in recipient lungs (r-lung) and salivary gland (r-sg) from transplant recipients at PODs 7, 14, and 28
FIGURE 2
FIGURE 2
Effect of IS on immune phenotypes in spleens and kidney allograft. Kidneys from BALB/c (CD45.1) were transplanted into B6 recipients (CD45.2) with immunosuppression (IS) (w/IS) or without IS (wo/IS) (n = 4-6/group). Cells isolated from spleens and renal grafts were analyzed by flow cytometry. Frequencies of recipients’ T cells (gated on CD45.2+CD3+ cells) and myeloid cells (gated on CD45.2+CD11b+ cells), including dendritic cells (DCs) (CD11c+MHCII+ DCs), Mac (CD11b+F4/80+ macrophages), Mono (CD11b+Ly6C+ monocytes), and Granu (Ly6G+ granulocytes) are calculated based on total number of live cells and normalized by tissue weight (mg). Data in bar graphs are expressed as mean value with SEM. (*P ≤.05 or **P <.01, wo/IS vs w/IS). A, Representative dot-plot demonstrating percentages of recipients’ T cells and myeloid cells and absolute number of subtypes in spleens at POD2. B, Representative dot-plot demonstrating percentages of recipients (CD45.2+) vs donor cells (CD45.1+) and their absolute numbers in the kidney allografts at POD2. C, Representative dot-plot demonstrating percentages of recipients’ T cells and myeloid cells and absolute number of subtypes (above mentioned) in the kidney allografts at POD2
FIGURE 3
FIGURE 3
Immunosuppression (IS) suppresses host antiviral T cell responses following transplant. Cells from D+/R− kidney allografts with IS (w/IS) or without IS (wo/IS) (n = 4-6/group) were isolated and analyzed at postoperative (POD)7 for the frequency of viral specific T cells MHC-mouse cytomegalovirus (MCMV) by using m38 tetramer (Kb-m38) staining. A, Representative dot-plot showing the percentage of MCMV-specific CD8 T cells (gated on CD3+CD44+ cells). B, Absolute number in the transplanted kidneys wo/IS or w/IS; **P <.01. Naive B6 kidneys were controls
FIGURE 4
FIGURE 4
Transcriptome profiling on kidney allografts. Kidney tissues or plasma samples were collected from D+-to-R− kidney allografts treated with immunosuppression (IS) (w/IS) or without IS (wo/IS) at the indicated endpoints. The contralateral latent donor kidney (con) was recovered at the time of the transplant (postoperative [POD]0). Pathways with –log P value >1.3 (dashed line) are statistically significant. A, Heat-map of deferentially expressed genes (DGF) in the kidney allografts (n > 4/group) at 48 hours posttransplant and clustered based on the expression levels. B, Ingenuity pathway analysis of selected genes in cluster IV (heat-map). C, Volcano plot showing changes of genes associated with DNA damage pathways in the transplants either wo/IS (left) and w/IS (right)
FIGURE 5
FIGURE 5
Quantification of cellular histone posttranslational modifications after transplant. Kidney tissues samples were collected from D+-to-R− kidney allografts (n = 3/group/time) treated with immunosuppression (IS) (w/IS; red) or without IS (wo/IS; black) at 3, 24, and 48 hours posttransplant. Contralateral kidneys from the donor were controls (0 hours). Histones were extracted and analyzed with liquid chromatography-tandem mass spectrometry. The relative abundance of each histone modification is determined by calculating the peptide peak area for a peptide of interest and dividing by the sum of the peak areas for all peptides with that sequence, based on the mean of 3 technical replicates with error bars representing the standard deviation. Data shown as percent of given modification in total pool of given peptide that was quantified. *P <.05; **P <.01 compared with 0 hours controls
FIGURE 6
FIGURE 6
Proteome profiling on kidney allografts and plasma protein analysis. Kidney tissues or plasma samples were collected from D+-to-R− kidney allografts treated with immunosuppression (IS) (w/IS) or without IS (wo/IS) at the indicated endpoints. The contralateral latent donor kidney (con) was recovered at the time of the transplant (postoperative [POD]0). Pathways with –log P value >1.3 (dashed line) are statistically significant. A, Mass spectometry analysis for proteins extracted from the kidney tissues at 3, 24, and 48 hours posttransplant (n = 3/group/time), followed by ingenuity pathway analysis. B, Multiplex proteomic analysis on plasma samples collected from D+/R− kidney transplants at 48 hours posttransplant (n = 6-8/group) and naive B6 (con). NS, not significant
FIGURE 7
FIGURE 7
Treatment with immunosuppression (IS) regimen alone failed to induced reactivation and dissemination of mouse cytomegalovirus (MCMV) after transplant of latently infected kidneys. BALB/c mice infected with Δm157 mcmv virus were treated with the same IS regimen (with IS [w/IS]) or PBS (without IS [wo/IS]) at 4 months after the infection. Selected organs were recovered at POD28. A, Schematic of experimental setup. B, DNA copy numbers in kidney, salivary gland (SG), lung, liver, and spleen determined by real-time PCR
FIGURE 8
FIGURE 8
Transplant ischemia-reperfusion injury (IRI) is sufficient for induction of mouse cytomegalovirus (MCMV) reactivation and dissemination. BALB/c mice infected with Δm157 mcmv virus were treated with the same immunosuppression (IS) regimen (with IS [w/IS]) or PBS (without IS [wo/IS]) at 6 months after the infection. DNA copy numbers in kidney, salivary gland, and lung were determined at day 28 by real-time PCR. A, DNA abundance in postoperative day (POD)0 control kidney (con) and POD28 after syngeneic D+/R− kidney grafts treated w/IS or wo/ IS. B, Representative section (periodic acid–Schiff staining, ×600 magnification) of kidney graft w/IS showing an inclusion body (arrow). C, DNA abundance at day 28 posttransplant recipients’ salivary gland (r-sg) and lungs (r-lung). D, Transcriptome profiling and pathway analyses were performed on untreated D+/R− syngeneic kidney grafts (Syn) or allogeneic (Allo) kidney grafts at POD2 (n = 3/group) as described in Figure 4. Pathways with –log P value >1.3 (dashed line) are statistically significant
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