PGE₂ is overproduced in BMT mice.

<p>BMT mice (BALB/c donor, C57BL/6 recipient) and untransplanted BALB/c and C57BL/6 controls were infected i.n. with MAV-1 or mock infected with conditioned media. ELISA was used to quantify PGE₂ concentrations in BALF at the indicated time points. Combined data from n = 3–4 mice per group are presented as means ± S.E.M. Statistical comparisons were made using two-way ANOVA followed by Bonferroni’s multiple comparison tests. ***<i>P</i><0.001.</p

Type I IFN production in BMT mice.

<p>A) BMT mice (BALB/c donor, C57BL/6 recipient) and untransplanted BALB/c and C57BL/6 controls were infected i.n. with MAV-1, and lungs were harvested at the indicated time points. RT-qPCR was used to quantify IFN-β mRNA levels. Combined data from n = 7–8 mice per group (n = 3 per group at day 0) are presented as means ± S.E.M. Statistical comparisons were made using two-way ANOVA followed by Bonferroni’s multiple comparison tests. B) Untransplanted C57BL/6 and IFNAR^-/- mice were infected i.n. with MAV-1. DNA was extracted from lungs harvested at the indicated time points. qPCR was used to quantify MAV-1 genome copies in lung DNA. DNA viral loads are expressed as copies of MAV-1 genome per 100 ng of input DNA. Individual circles represent values for individual mice and horizontal bars represent means for each group. Statistical comparisons were made using two-way ANOVA followed by Bonferroni’s multiple comparison tests.</p

Primers and probes used for real-time PCR analysis.

<p>Primers and probes used for real-time PCR analysis.</p

Virus-induced pulmonary inflammation in BMT mice.

<p>Allogeneic BMT mice (BALB/c donor, C57BL/6 recipient) and untransplanted BALB/c and C57BL/6 mice were infected i.n. with MAV-1 or mock infected at 5 weeks post BMT. Lungs were harvested and hematoxylin-and-eosin-stained sections were prepared from paraffin-embedded specimens. Representative images are shown from mice before infection (A-C) and from infected mice at the indicated time points (D-L). Scale bars, 100 μm. M) Pathology index scores were generated to quantify cellular inflammation in the lungs of mock-infected and infected mice. Data from 2 to 3 mock-infected mice and 3 to 6 infected mice per group are presented as means and standard errors of the means at each time point. Statistical comparisons were made using two-way ANOVA followed by Bonferroni’s multiple comparison tests. *<i>P</i><0.05, **<i>P</i><0.01, and ***<i>P</i><0.001.</p

Prostaglandin E₂ Production and T Cell Function in Mouse Adenovirus Type 1 Infection following Allogeneic Bone Marrow Transplantation

<div><p>Adenovirus infections are important complications of bone marrow transplantation (BMT). We demonstrate delayed clearance of mouse adenovirus type 1 (MAV-1) from lungs of mice following allogeneic BMT. Virus-induced prostaglandin E₂ (PGE₂) production was greater in BMT mice than in untransplanted controls, but BMT using PGE₂-deficient donors or recipients failed to improve viral clearance, and treatment of untransplanted mice with the PGE₂ analog misoprostol did not affect virus clearance. Lymphocyte recruitment to the lungs was not significantly affected by BMT. Intracellular cytokine staining of lung lymphocytes demonstrated impaired production of INF-γ and granzyme B by cells from BMT mice, and production of IFN-γ, IL-2, IL-4, and IL-17 following ex vivo stimulation was impaired in lymphocytes obtained from lungs of BMT mice. Viral clearance was not delayed in untransplanted INF-γ-deficient mice, suggesting that delayed viral clearance in BMT mice was not a direct consequence of impaired IFN-γ production. However, lung viral loads were higher in untransplanted CD8-deficient mice than in controls, suggesting that delayed MAV-1 clearance in BMT mice is due to defective CD8 T cell function. We did not detect significant induction of IFN-β expression in lungs of BMT mice or untransplanted controls, and viral clearance was not delayed in untransplanted type I IFN-unresponsive mice. We conclude that PGE₂ overproduction in BMT mice is not directly responsible for delayed viral clearance. PGE₂-independent effects on CD8 T cell function likely contribute to the inability of BMT mice to clear MAV-1 from the lungs.</p></div

Impaired IFN-γ production in BMT mice.

<p>A) BMT mice (BALB/c donor, C57BL/6 recipient) and untransplanted BALB/c controls were infected i.n. with MAV-1 or mock infected with conditioned media, and lung leukocytes were isolated at 7 dpi. Lung leukocytes were stimulated overnight with anti-CD3 antibody and ELISA was used to measure IFN-γ concentrations in supernatants. Combined data from n = 3–8 mice per group are presented as means ± S.E.M. Statistical comparisons were made using one-way ANOVA followed by Tukey’s multiple comparison tests. **<i>P</i><0.01 and *<i>P</i><0.05 comparing mock to MAV-1. ††<i>P</i><0.01 and †<i>P</i><0.05 comparing BALB/c to BMT mice. B) A) IFN-γ^+/+ and IFN-γ^-/- mice were infected i.n. with MAV-1. DNA was extracted from lungs harvested at 14 dpi. qPCR was used to quantify MAV-1 genome copies in lung DNA. DNA viral loads are expressed as copies of MAV-1 genome per 100 ng of input DNA. Individual circles represent values for individual mice and horizontal bars represent means for each group. Statistical comparisons were made using the Mann-Whitney test.</p

T cells in lungs of BMT mice.

<p>BMT mice (BALB/c donor, C57BL/6 recipient) and untransplanted BALB/c and C57BL/6 controls were infected i.n. with MAV-1 or mock infected with conditioned media. Lung leukocytes isolated at 7 dpi were stimulated with PMA/ionomycin and stained to quantify the number of A) TCRβ⁺CD4⁺ T cells, B) TCRβ⁺CD8⁺ T cells, C) IFN-γ⁺ T cells, and D) GzmB⁺ T cells per lung. Combined data from n = 3–4 mice per group are presented as means ± S.E.M. Statistical comparisons were made using one-way ANOVA followed by Bonferroni’s multiple comparison tests. **<i>P</i><0.01 and *<i>P</i><0.05 comparing mock to MAV-1. ††<i>P</i><0.01 and †<i>P</i><0.05 comparing BALB/c to BMT mice.</p

Myeloid Cell Arg1 Inhibits Control of Arthritogenic Alphavirus Infection by Suppressing Antiviral T Cells

<div><p>Arthritogenic alphaviruses, including Ross River virus (RRV) and chikungunya virus (CHIKV), are responsible for explosive epidemics involving millions of cases. These mosquito-transmitted viruses cause inflammation and injury in skeletal muscle and joint tissues that results in debilitating pain. We previously showed that arginase 1 (Arg1) was highly expressed in myeloid cells in the infected and inflamed musculoskeletal tissues of RRV- and CHIKV-infected mice, and specific deletion of Arg1 from myeloid cells resulted in enhanced viral control. Here, we show that Arg1, along with other genes associated with suppressive myeloid cells, is induced in PBMCs isolated from CHIKV-infected patients during the acute phase as well as the chronic phase, and that high Arg1 expression levels were associated with high viral loads and disease severity. Depletion of both CD4 and CD8 T cells from RRV-infected Arg1-deficient mice restored viral loads to levels detected in T cell-depleted wild-type mice. Moreover, Arg1-expressing myeloid cells inhibited virus-specific T cells in the inflamed and infected musculoskeletal tissues, but not lymphoid tissues, following RRV infection in mice, including suppression of interferon-γ and CD69 expression. Collectively, these data enhance our understanding of the immune response following arthritogenic alphavirus infection and suggest that immunosuppressive myeloid cells may contribute to the duration or severity of these debilitating infections.</p></div

Loss of myeloid cell Arg1 results in activation of virus-specific CD8⁺ T cells in inflamed muscle tissue.

<p>Three-to-four week-old WT (n = 8) and LysMcre;Arg1^F/F (n = 10) mice were inoculated with 10³ PFU of RRV-LCMV. At 10 dpi, leukocytes from quadriceps muscles (following enzymatic digestion) were isolated for flow cytometric analysis of CD11a and CD69 expression on virus-specific CD8⁺ T cells. Muscle-infiltrating leukocytes that were left unstained were utilized as a control for CD11a and CD69 staining. (A) Representative histograms demonstrating CD11a (left) and CD69 (right) expression on CD44⁺gp33⁺CD8⁺CD4^- T cells at 10 dpi. (B) Frequency of CD11a⁺ or CD69⁺ cells (of CD44⁺gp33⁺CD8⁺CD4^- T cells) in the muscle tissue of RRV-LCMV-inoculated WT mice and LysMcre;Arg1^F/F mice at 10 dpi. Data are represented as the arithmetic mean ± SEM and combined from two independent experiments. * <i>P</i> < 0.05 as determined by two-way ANOVA followed by a Bonferroni multiple comparison test.</p

Arg1 is induced in inflamed musculoskeletal tissues and in blood leukocytes but not lymphoid tissues of RRV- and CHIKV-infected mice.

<p>WT mice were mock-inoculated (n = 5) or inoculated with 10³ PFU of (A) RRV (n = 6) or (B) CHIKV (n = 6). At 10 dpi the gastrocnemius muscle (“Muscle”), ankle/foot joint (“Joint”), circulating blood leukocytes (“Blood”), bone marrow (“BM”) from the femur, the draining popliteal LN (“pLN”), and the spleen were harvested for RT-qPCR analysis of <i>Arg1</i> expression. Data are combined from two independent experiments, normalized to 18S rRNA levels, and are expressed as the relative expression (<i>n</i>-fold increase) over expression in each tissue from mock-inoculated mice. Each data point represents the arithmetic mean ± SEM. ** <i>P</i> < 0.01, * <i>P</i> < 0.05 as determined by significance test of greater than one.</p