Fig 1.
Coinfection with H. polygyrus increases ex vivo reactivation independently of STAT6.
(A) HP was infected by oral gavage 1 week prior to MHV68 infection. HP-infected or uninfected mice were challenged with 106 PFU of MHV68 intraperitoneally. PECs and spleen were isolated at day 28–31 of MHV68 infection. Approximate timepoint outlined by red box. Partially created using BioRender. (B) Limiting dilution assays were performed from C57BL/6 PECs. Data pooled from 4 independent experiments (3 mice pooled/group). (C) Limiting dilution assays were performed from C57BL/6 splenocytes. Data pooled from 4 independent experiments (3 mice pooled/group). (D) Stat6-/- or littermate control mice were infected and LDAs were performed at day 28–29 of MHV68 infection. Data are pooled from 2 independent experiments (3 mice pooled/group). Dotted line represents Poisson distribution.
Fig 2.
Coinfected mice have increased infection in tissue-resident macrophages during latency.
(A) Timeline of infections with HP by oral gavage and MHV68 by intraperitoneal injection. The timepoints shown in (B-G) are outlined by a red box. Partially created in BioRender. (B) Quantification of flow cytometric analysis of the number of LPMs and SPMs at day 28 of MHV68 infection. Data are pooled from 3 independent experiments (n = 11-13/group, mean ± standard deviation). Each dot represents an individual mouse. (C-F) Quantification of flow cytometric analysis of MHV68-infected CD11b+ PECs at day 28 of MHV68 infection. CD11b+ cells were isolated with CD11b+ beads and MACs columns before staining. LPMs were gated as CD19- CD11bhi ICAM-2hi. SPMs were gated as CD19- CD11b+ ICAM-2lo. B cells were gated as CD19+. Data are pooled from 3 independent experiments (n = 11-13/group, mean ± standard deviation). Each dot represents an individual mouse. (C) Total number of MHV68-infected CD11b+ PECs. (D) Representative flow plots of MHV68+ LPMs. (E) Quantification of number of MHV68-infected macrophages and B1 B cells. (F) Proportion of MHV68-infected cells out of the parent populations. (G) Total PECs from days 28–31 of MHV68 infection were subjected to limiting dilution PCR analysis to detect frequency of viral genomes. Data are pooled from 4 independent experiments (3 mice pooled/ group, mean± standard deviation). (H) LPMs were sorted from mice at days 28 and 29 of MHV68 infection as CD19- SiglecF- Ly6G- F4/80+ CD11bhi ICAM-2hi. Sorted cells were subjected to limiting dilution PCR analysis to detect frequency of viral genomes. Data are pooled from 2 independent experiments (4–8 mice were pooled for each group before sorting, mean± standard deviation). (B, D, E) P-values, 2-way ANOVA, Tukey’s multiple comparisons. (C) P-values, unpaired t-test. (G, H) Dotted line represents Poisson distribution. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001.
Fig 3.
LPM expansion during intestinal parasite infection increases viral infection in the peritoneal cavity.
(A) C57BL/6J mice were infected with 100 L3 larvae of HP by oral gavage. Quantification of flow cytometric analysis of LPMs in C57BL/6 mice at the indicated days post HP infection. LPMs were gated as CD3- CD19- Siglec F- CD11bhi ICAM-2hi. Data are pooled from 3 independent experiments (n = 9-10/group, mean ± standard deviation). (B) C57BL/6J mice were left untreated and uninfected (Uninfected), intraperitoneally injected with sterile PBS (Vehicle) or intraperitoneally infected with 106 PFU of MHV68 (MHV68+). PECs were collected on day 4 of MHV68 infection and the number of LPMs was quantified by flow cytometry. LPMs were gated as CD3- CD19- Siglec F- CD11bhi ICAM-2hi. Data are pooled from 2 independent experiments (n = 10/group, mean ± standard deviation) (C) Timeline of infections with HP by oral gavage and MHV68 by intraperitoneal injection (106 plaque forming units (PFU)). The time points shown in (D-K) are outlined by a red box. Partially created in BioRender. (D) Quantification of flow cytometric analysis of LPMs in C57BL/6 mice at days 2, 4, and 7 post MHV68 infection. LPMs were gated as CD19- CD11bhi ICAM-2hi. Data are pooled from 3 independent experiments (n = 10–11 MHV68+ groups, n = 4 HP+ only group, n = 2 uninfected group, mean ± standard deviation). (E-J) Mice were infected as in (C) with 106 PFU of MHV68.ORF73β-lactamase virus. Quantification of flow cytometric analysis of MHV68-infected PECs at days 2, 4, and 7 post MHV68 infection. Data are pooled from 3 independent experiments (n = 10-11/group, mean ± standard deviation). (E) Representative flow plots of the total β-lactamase-positive (MHV68+) PECs. (F) Quantification of the total number of MHV68-infected PECs. (G) Number of MHV68-infected LPMs. (H) Proportion of LPMs that are MHV68-infected. (I) Number of MHV68-infected SPMs. SPMs were gated as CD19- CD11b+ ICAM-2lo. (J) Number of MHV68-infected B cells. B cells were gated as CD19+. Each dot represents an individual mouse. (K) MHV68 titers determined by plaque assay of PECs at day 1–7 of infection. Data are pooled from 5 independent experiments (n = 6–7 day 1, n = 11–14 days 2 and 4, n = 3–4 day 7, mean ± standard deviation). P-values, 2-way ANOVA, Tukey’s multiple comparisons * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001.
Fig 4.
IL-4c-induced LPM expansion is sufficient to increase viral infection in the peritoneal cavity during acute MHV68 infection.
(A) Timeline of intraperitoneal IL-4 complex (IL-4c), thioglycolate (Thio) treatments and MHV68.ORF73β-lactamase intraperitoneal infection (106 plaque forming units (PFU)). 5μg of IL-4 and 25 μg α-IL-4 were complexed and injected for LPM expansion. 3.8% sterile thioglycolate broth was used to expand SPMs. The time point shown in (B-H) is outlined by a red box. (B) Quantification of flow cytometric analysis of LPMs, SPMs, and B cells at 2 days post MHV68 infection. LPMs were gated as CD19- CD11bhi ICAM-2hi. SPMs were gated as CD19- CD11b+ ICAM-2lo. B cells were gated as CD19+. Data are pooled from 3 independent experiments (n = 10-12/group, mean ± standard deviation). (C) Representative flow plots of the macrophage populations with the different treatments. All mice were infected with MHV68.ORF73β-lactamase. (D-F) Mice were infected as in A with 106 PFU of MHV68.ORF73β-lactamase virus. Quantification of flow cytometric analysis of MHV68-infected PECs at day 2 post MHV68 infection. Data are pooled from 3 independent experiments (n = 10-12/group, mean ± standard deviation). (D) Quantification of the total number of MHV68-infected PECs. (E) Proportion of total MHV68-infected PECs for each treatment. (F) Number of MHV68-infected LPMs, SPMs, and B cells. (G) Proportion of MHV68-infected LPMs and SPMs out of the LPM and SPM populations, respectively. (H) Proportion of MHV68-infected LPMs and SPMs out of the LPM and SPM populations, respectively. 2-way ANOVA analysis compared proportion of LPMs to SPMs for each treatment. Each dot represents an individual mouse. (B, F, G, H) P-values, 2-way ANOVA, Tukey’s multiple comparisons * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001 (D, E) P-values, Ordinary 1-way ANOVA, Tukey’s multiple comparisons * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001.
Fig 5.
HP-induced increased MHV68 infection is dependent on vitamin A during the acute stage of MHV68 infection.
Mice were raised on a vitamin A deficient diet or a control diet and infected with HP followed by the MHV68.ORF73β-lactamase reporter virus. PECs were collected on day 2 and day 4 of MHV68 infection. (A) Timeline of infections with HP by oral gavage and MHV68 by intraperitoneal injection (106 plaque forming units (PFU)). The time points shown in (B-H) are outlined by a red box. Partially created in BioRender. (B) Representative flow plots of LPMs (ICAM-2hi CD11bhi CD19-) and SPMs (CD11b+ ICAM-2lo CD19-) in each of the infection states and diets at day 2 of MHV68 infection. (C) Quantification of flow cytometric analysis of LPMs at days 2 and 4 of MHV68 infection. LPMs were gated as CD19- CD11bhi ICAM-2hi. (D) Quantification of flow cytometric analysis of SPMs at days 2 and 4 of MHV68 infection. SPMs were gated as CD19- CD11b+ ICAM-2lo. Data are pooled from 2 independent experiments (n = 7-12/group, mean ± standard deviation). Each dot represents an individual mouse. P-values, 2-way ANOVA, Tukey’s multiple comparisons. (E-H) Quantification of flow cytometric analysis of MHV68-infected PECs at day 2 and 4 of MHV68 infection. Data are pooled from 2 independent experiments (n = 7-12/group, mean ± standard deviation). Each dot represents an individual mouse. (E) Total number of MHV68-infected PECs. (F) Number of MHV68-infected LPMs. (G) Proportion of MHV68-infected LPMs out of total LPMs. (H) Number of MHV68-infected SPMs. (C-F) P-values, 2-way ANOVA, Tukey’s multiple comparisons. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001.
Fig 6.
Early HP-independent expansion of LPMs and SPMs is not sufficient to cause increased latent infection.
(A) Timeline of intraperitoneal IL-4 complex (IL-4c), thioglycolate (Thio) treatments and MHV68.ORF73β-lactamase intraperitoneal infection (106 plaque forming units (PFU)). 5μg of IL-4 and 25 μg α-IL-4 were complexed and i.p. injected for LPM expansion. 3.8% sterile thioglycolate broth was i.p. injected to expand SPMs. The time point shown in (B-E) is outlined by a red box. (B) Quantification of flow cytometric analysis of LPMs and SPMs 28 days post MHV68 infection. LPMs were gated as CD19- CD11bhi ICAM-2hi. SPMs were gated as CD19- CD11b+ ICAM-2lo. Data are pooled from 2 independent experiments (n = 9/group, mean ± standard deviation). (C-E) Mice were infected as in A with 106 PFU of MHV68.ORF73β-lactamase virus. CD11b+ PECs were isolated to enrich for MHV68+ cells. Quantification of flow cytometric analysis of MHV68-infected PECs at day 28 post MHV68 infection. Data are pooled from 2 independent experiments (n = 9/group, mean ± standard deviation). (C) Quantification of the total number of MHV68-infected CD11b+ PECs. P-values, 1-way ANOVA, Tukey’s multiple comparisons. (D) Number of MHV68-infected LPMs and SPMs. (E) Proportion of MHV68-infected LPMs and SPMs of their respective parent populations. Each dot represents an individual mouse. (B, D, E) P-values, 2-way ANOVA, Tukey’s multiple comparisons * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001.
Fig 7.
HP-induced increased latent MHV68 infection is dependent on vitamin A.
(A) Mice were raised on a vitamin A deficient diet or a control diet and infected with HP followed by the MHV68.ORF73β-lactamase reporter virus, as in the timeline. PECs were collected on day 28 or 29 of MHV68 infection, as marked by the red box on the timeline. CD11b+ cells were isolated by MACs columns before staining. (B) Representative flow plots of the macrophage populations with the control diet or VAD at day 29 of MHV68 infection. Flow plots are from the single cell gate. (C) Quantification of flow cytometric analysis of LPMs and SPMs at day 29 of MHV68 infection. Data are pooled from 2 independent experiments (n = 9-13/group, mean ± standard deviation). Each dot represents an individual mouse. P-values, 2-way ANOVA, Tukey’s multiple comparisons. (D-F) Quantification of flow cytometric analysis of MHV68-infected CD11b+ PECs at day 28 or 29 of MHV68 infection. Data are pooled from 2 independent experiments (n = 9-13/group, mean ± standard deviation). Each dot represents an individual mouse. (D) Total number of MHV68-infected CD11b+ PECs. (E) Number of MHV68-infected macrophages. (F) Proportion of MHV68-infected cells out of the parent populations. (G-H) Mice on VAD or control diet were infected and PECs collected on day 28 of MHV68 infection. LDA’s and LD-PCR were performed on the total PECs to determine ex vivo reactivation (G) and frequency of viral genomes (H). Data are pooled from 2 independent experiments (3–5 mice pooled/group). Dotted line represents Poisson distribution. (C-F) P-values, 2-way ANOVA, Tukey’s multiple comparisons. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001.
Fig 8.
Comparison of MHV68/HP and HP/MHV68 coinfection models and mechanisms.
(A) Model of HP challenge during MHV68 latency inducing reactivation through IL-4 and IL-13 signaling. (B) HP infection prior to MHV68 coinfection. HP-induced increased acute MHV68 infection is driven by LPM expansion. (C) HP infection prior to MHV68 infection. HP infection maintains the LPM population and possibly induces LPM-intrinsic changes that drive increased latent infection and increased ex vivo reactivation. Model created in BioRender.