Phenotypic complementation of genetic immunodeficiency by chronic herpesvirus infection
- Washington University School of Medicine, United States
- Howard Hughes Medical Institute, United States
- Rockefeller University, United States
- Institut Pasteur, Centre National de la Recherche Scientifique, Unité de Recherche Associée, France
- Necker Hospital for Sick Children, France
- Necker Hospital for Sick Children, Imagine Institute, INSERM UMR 1163, France
- Paris Descartes University, France
- Kyoto University, Japan
Figures
Figure 1 with 5 supplements
HOIL-1 KO mice are highly susceptible to acute infection with Listeria monocytogenes, Toxoplasma gondii and Citrobacter rodentium.
(A) Survival of control (blue circles) and HOIL-1 KO (red squares) mice following i.p. inoculation with 105 (left panel; control n = 35, HOIL-1 KO n = 19), 104 (middle panel; control n = 15, HOIL-1 KO n = 15) or 103 (right panel; control n = 15, HOIL-1 KO n = 15) CFU Listeria strain EGD. (B) Listeria CFU in spleen and liver from control (blue circles) and HOIL-1 KO (red squares) mice infected with 105 CFU i.p. for 1 day (left panel), 3 days (middle panel) or 6 days (right panel). Each symbol represents an individual mouse and the mean log10 CFU is indicated. The dashed line indicates the limit of detection. (C) Survival of control (blue circles) and HOIL-1 KO (red squares) mice following inoculation with 5000 (left panel; control n = 17, HOIL-1 KO n = 5) or 100 (middle panel; control n = 10, HOIL-1 KO n = 10) tachyzoites T. gondii strain Pru-luc. (D) Log10 total flux (luciferase activity; photons per second) as a measure of parasite burden 8 days post-infection with 100 tachyzoites. Each symbol represents an individual mouse and the mean log10 is indicated. (E,F) Survival (E) and weight (F) of control (blue circles) and HOIL-1 KO (red squares) mice following oral gavage with 2 × 109 CFU C. rodentium. n = 20/group for survival and n = 10/group for weight. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001. Statistical analyses were performed using logrank Mantel–Cox test (A, C and E), Mann–Whitney test (B), or t-test (D).
Figure 1—figure supplement 1
Comparison of RBCK1/HOIL1 alleles from RBCK1/HOIL1-mutant patients.
Npl4 zinc finger ubiquitin binding domain, RING; really interesting new gene E3 ligase domain, IBR; Inbetween-RING domain. HOIL1L/RBCK1 isoform 2 (reference sequences NM_031229.2/NP_112506.2) was used for annotation.
Figure 1—figure supplement 2
Myocardium from aged HOIL-1 KO mice contains amylopectin-like deposits.
Representative PAS (top), PAS plus diastase digestion (middle) and H&E-stained sections of myocardium from 18 month-old HOIL-1 KO (right) and control (left) mice. The scale bar (inset) represents 100 µm.
Figure 1—figure supplement 3
Analysis of hematopoietic cell populations from naïve HOIL-1 KO mice.
(A–C) Flow cytometric analysis of cell populations in the spleen (A), thymus (B) and peritoneum (C) of HOIL-1 KO (red squares) and control (blue circles) mice. DP; CD4, CD8 double-positive. (D) Complete blood counts from HOIL-1 KO (red squares) and control (blue circles) mice. WBC, white blood cells, ×103/mm3, RBC, red blood cells, ×106/mm3; HGB, hemoglobin, g/dl; HCT, hematocrit, %; MCV, mean corpuscular volume, µm3; MCH, mean corpuscular hemoglobin, pg; MCHC, mean corpuscular hemoglobin concentration, %; Seg Neu, segmented neutrophils, %; Lymphos, lymphocytes, %, Monos, monocytes, %. Eosinophils, basophils or band neutrophils were not detected. Each symbol represents an individual mouse and the mean is indicated.
Figure 1—figure supplement 4
Hoil1/Rbck1 and neighboring gene (Trib3 and Tbc1d20) transcript expression in control and HOIL-1 KO bone marrow derived macrophages.
Data represent the mean ± SEM from cells derived from four pairs of mice each analyzed in triplicate. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001. Statistical analyses were performed using t-test.
Figure 1—figure supplement 5
Pathology of HOIL-1 KO mice during Listeria infection.
Alanine aminotransferase (ATL; left panel) and aspartate aminotransferase (AST; right panel) levels in serum from control (blue circles) and HOIL-1 KO (red squares) mice 6 days after infection with 105 CFU Listeria. Each symbol represents an individual mouse and the mean is indicated. *p ≤ 0.05, **p ≤ 0.01; t-test with Welch's correction.
Figure 2 with 2 supplements
HOIL-1 is required in an innate immune cell compartment during Listeria infection.
(A) Survival of control and HOIL-1 KO reciprocal bone marrow chimeric mice following infection with 105 CFU Listeria. *p ≤ 0.0083; logrank Mantel–Cox test corrected for multiple comparisons. (B) Survival of RAG1 KO HOIL-1 WT (blue circles; n = 12) and RAG1 KO HOIL-1 KO (red squares; n=11) mice following infection with 104 CFU Listeria. (C) Listeria CFU in spleen and liver from RAG1 KO HOIL-1 WT (blue circles) and RAG1 KO HOIL-1 KO (red squares) mice infected with 104 CFU for 3 days. Each symbol represents an individual mouse and the mean log10 is indicated. For B and C, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001; logrank Mantel–Cox test and Mann–Whitney test, respectively.
Figure 2—figure supplement 1
Confirmation of hematopoietic reconstitution of bone marrow chimeric mice.
Percent Hoil1/Rbck1+/+ (Hoil1/Rbck1 intron 7; top panel) and percent Hoil1/Rbck1−/− (neomycin-resistance cassette; bottom panel) genomic DNA (gDNA) in peripheral blood from control and HOIL-1 KO reciprocal bone marrow chimeric mice determined by qPCR.
Figure 2—figure supplement 2
HOIL-1 KO mice are capable of generating an adaptive immune response to Listeria.
Listeria titers in spleen and liver of naïve (circles) or pre-immunized (103 CFU for 28 days, squares) control (blue symbols) and HOIL-1 KO (red symbols) mice challenged with 106 CFU Listeria for 3 days. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001. Statistical analyses were performed using two-way ANOVA with Holm-Sidak's multiple comparison test.
Figure 3 with 3 supplements
HOIL-1 is important for induction of pro-inflammatory cytokines following Listeria infection.
(A) TNFα (6 hr), IL-6 (6 hr) and IL-12p70 (24 hr) protein in macrophage culture supernatants following infection with Listeria (LM) ± IFNγ co-treatment. (B) Induction of Tnf, Il6 and Il12b transcripts in macrophages over 12 hr following infection with Listeria plus IFNγ. Data represent the mean ± SEM of macrophages derived from two mice per genotype analyzed in triplicate and are representative of at least three independent experiments. (C) Induction of cytokine transcripts in peritoneal cells over 12 hr following infection of control (blue circles) and HOIL-1 KO (red squares) mice with 105 Listeria. Each symbol represents an individual mouse. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001. Statistical analyses were performed using t-test.
Figure 3—figure supplement 1
HOIL-1 is not required for listericidal activity of bone marrow-derived macrophages.
Growth and killing of Listeria in untreated or IFNγ-pre-treated control and HOIL-1 KO macrophages at 0 and 6 hr post-infection. Data are from two independent experiments performed in duplicate.
Figure 3—figure supplement 2
Analysis of peritoneal cell populations following Listeria infection.
Flow cytometric analysis of peritoneal cell populations in control (blue circles; 0 hr n = 20, 6 hr n = 10, 12 hr n = 7) and HOIL-1 KO mice (red squares; 0 hr n = 9, 6 hr n = 12, 12 hr n = 7) over 12 hr after infection with 105 CFU i.p. Data represent the mean ± SEM. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001. Statistical analyses were performed using t-test.
Figure 3—figure supplement 3
HOIL-1 is important for induction of pro-inflammatory cytokines by innate cells following Listeria infection in vivo.
Induction of cytokine transcripts in peritoneal cells from uninfected (0 hr) RAG1 KO HOIL-1 WT (blue circles) and RAG1 KO HOIL-1 KO (red squares) mice or 3 hr after infection with 104 Listeria. Each symbol represents an individual mouse.
Figure 4 with 2 supplements
Enhanced inflammatory response and control of MHV68 and M. tuberculosis by HOIL-1 KO mice.
(A) Limiting dilution assay of peritoneal cells from control (blue circles) and HOIL-1 KO (red squares) mice infected with MHV68 for 28 days onto mouse embryonic fibroblast monolayers to measure the frequency of cells capable of MHV68 reactivation. The dashed line indicates 63.2%, which was used to determine the frequency of cells reactivating virus by the Poisson distribution. Data represent the mean from three independent experiments each with cells combined from three mice/group. *p ≤ 0.05. Statistical analyses were performed by calculating the number of control and HOIL-1 KO cells required for 63.2% of wells to contain complete cytopathic effect for each individual experiment by non-linear regression, then comparing these values by paired t-test. Preformed virus was not detected in disrupted samples (not shown). (B) M. tuberculosis titers in the lung and spleen of HOIL-1 KO (red squares) and control (blue circles) mice 70 days post-infection. *p ≤ 0.05. Statistical analyses were performed using t-test. (C) TNFα, IL-6, IL-12/IL-23p40 and IFNγ protein detected in serum from naïve or latently-infected (28 days) control (blue circles) and HOIL-1 KO (red squares) mice. Each symbol represents an individual mouse and the mean is indicated. *p ≤ 0.05, t-test with Welch's correction (IL-12/IL-23p40) or Mann Whitney test (TNFα, IL-6, IFNγ). (D) TNFα, IL-6, IL-12/IL-23p40 and IFNγ protein in serum from mice from (B). Each symbol represents an individual mouse. Data are combined from two independent experiments. *p ≤ 0.05, **p ≤ 0.01. Statistical analyses were performed using t-test (TNFα, IL-12p40) with Welch's correction (IFNγ) or Mann Whitney test (IL-6).
Figure 4—figure supplement 1
Acute MHV68 replication in vitro and in vivo is minimally affected by HOIL-1-deficiency.
(A) MHV68 growth in HOIL-1 KO (red symbols) and control (blue symbols) bone marrow-derived macrophages with (squares) or without (circles) IFNγ pre- and post-treatment. Data represent the mean ± SEM from three independent experiments performed in triplicate. (B) MHV68 titers in spleen during acute infection of HOIL-1 KO (red squares) and control (blue circles) mice. **p ≤ 0.01, Mann Whitney test. The dashed line indicates the limit of detection.
Figure 4—figure supplement 2
Establishment of MHV68 latency is similar in control and HOIL-1 KO mice.
Limiting dilution PCR to determine the frequency of peritoneal cells from latently infected (28 days) HOIL-1 KO (red squares) and control (blue circles) mice containing MHV68 genomes. The dashed line indicates 63.2%, which was used to determine the frequency of cells containing viral genome by the Poisson distribution. Data represent the mean from three independent experiments each with cells combined from three mice/group. Statistical analyses were performed by calculating the number of control and HOIL-1 KO cells required for 63.2% of reactions to be positive for viral genome for each individual experiment by non-linear regression, then comparing these values by paired t-test.
Figure 5 with 3 supplements
MHV68 latency rescues HOIL-1 KO, IL-6, Caspase-1 and Caspase-1;Caspase-11-deficient mice from Listeria-induced lethality.
(A) Survival of control (blue symbols; mock n = 9, MHV68 n = 15) and HOIL-1 KO (red symbols; mock n = 10, MHV68 n = 20) mice challenged with 106 CFU Listeria 28 days following mock infection (circles) or infection with 106 PFU MHV68 (squares). *p ≤ 0.0083; logrank Mantel–Cox test corrected for multiple comparisons. (B) Survival of control (blue symbols) and HOIL-1 KO (red symbols) mice challenged with 106 CFU Listeria 28 days following intranasal mock infection (circles) or infection with 5 × 104 PFU wild-type (squares) or ORF73.stop (triangles) MHV68. Significantly different groups were: control mock infected and control MHV68wt infected, control mock infected and HOIL-1 KO MHV68wt infected, control mock infected and HOIL-1 KO MHV68orf73.stop infected, HOIL-1 KO mock infected and control MHV68wt infected, HOIL-1 KO mock infected and HOIL-1 KO MHV68wt infected, control MHV68wt infected and control MHV68orf73.stop infected, control MHV68wt infected and HOIL-1 KO MHV68orf73.stop infected, HOIL-1 KO MHV68wt infected and control MHV68orf73.stop infected, HOIL-1 KO MHV68wt infected and HOIL-1 KO MHV68orf73.stop infected, control MHV68orf73.stop infected and HOIL-1 KO MHV68orf73.stop infected. *p ≤ 0.0033; logrank Mantel–Cox test corrected for multiple comparisons. (C) Cytokine transcript levels in peritoneal cells from mock (circles) and MHV68-infected (squares) control (blue symbols) and HOIL-1 KO (red symbols) mice (28 days post-infection). (D) Induction of cytokine transcripts in peritoneal cells from mock (circles) and MHV68-infected (squares) control (blue symbols) and HOIL-1 KO (red symbols) mice (28 days) 3 hr after infection with 105 Listeria. Each symbol represents an individual mouse. For (C) and (D), *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001. Statistical analyses were performed using one-way ANOVA. (E) Survival of control (blue symbols) and Il6−/− (purple symbols) mice challenged with 106 CFU Listeria 28 days following mock infection (circles) or infection with 106 PFU MHV68 (squares). *p ≤ 0.0083; logrank Mantel–Cox test corrected for multiple comparisons. (F) Survival of control (blue symbols), Caspase-1;Caspase-11 (orange symbols) and Caspase-1 (green symbols) –deficient mice challenged with 106 CFU Listeria 28 days following mock infection (circles) or infection with 106 PFU MHV68 (squares). *p ≤ 0.0033; logrank Mantel–Cox test corrected for multiple comparisons.
Figure 5—figure supplement 1
MHV68 latency-induced cross-protection is maintained for at least 6 months.
Survival of control (blue symbols) and HOIL-1 KO (red symbols) mice challenged with 106 CFU Listeria 6 months after mock infection (circles) or infection with 106 PFU MHV68 (squares). Statistical analyses were performed by logrank Mantel–Cox test corrected for multiple comparisons, with p ≤ 0.0083 considered significant. Control mock vs HOIL-1 KO mock, p = 0.1824; control mock vs control MHV68 infected, p = 0.0408; control mock vs HOIL-1 KO MHV68 infected, p = 0.0070; HOIL-1 KO mock vs control MHV68 infected, p = 0.0028; HOIL-1 KO mock vs HOIL-1 KO MHV68 infected, p = 0.0003; control MHV68 infected vs HOIL-1 KO MHV68 infected, p = 0.9303.
Figure 5—figure supplement 2
MHV68 latency enhances the listericidal activity of peritoneal macrophages.
Listeria CFU in control (blue symbols) and HOIL-1 KO (red symbols) ex vivo cultures of peritoneal macrophages from mock (circles) or MHV68-infected (32 days, squares) mice at 0 and 6 hr post-infection. Data are combined from two independent experiments. Each symbol represents an individual mouse analyzed in duplicate. Cells from the same mouse were used for both 0 hr and 6 hr time points. Statistical analyses were performed by one-way ANOVA with Holm-Sidak's multiple comparison test for each time point.
Figure 5—figure supplement 3
Il6−/− mice have increased susceptibility to Listeria infection.
Survival of control (blue circles) and Il6−/− (purple squares) mice following i.p. inoculation with 105 CFU Listeria. *p ≤ 0.0083; logrank Mantel–Cox test corrected for multiple comparisons.
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Phenotypic complementation of genetic immunodeficiency by chronic herpesvirus infection
eLife 4:e04494.
https://doi.org/10.7554/eLife.04494
