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

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

Muscle-infiltrating CD4⁺ and CD8⁺ T cells isolated from LysMcre;Arg1^F/F mice express enhanced IFN-γ transcript.

<p>(A) Representative flow plots demonstrating the gating strategy to FACS-sort CD19^-CD3⁺CD4⁺CD8^- and CD19^-CD3⁺CD8⁺CD4^- T cells isolated from quadriceps muscles of RRV-infected WT or LysMcre;Arg1^F/F mice at 10 dpi and the post-sort purities. RT-qPCR analysis of (B) IFN-γ, (C) TNF-α, (D) IL-10, and (E) IL-2 expression in FACS-sorted T cells from the spleen of a mock-inoculated mouse (n = 1) or from the quadriceps muscle of RRV-infected WT (n = 3) or LysMcre;Arg1^F/F mice (n = 3) at 10 dpi. Data are normalized to 18S rRNA levels and are expressed as the relative expression (<i>n</i>-fold increase) over expression in spleen T cells from a mock-inoculated mouse. Data are represented as the arithmetic mean ± SEM. Data are representative of two independent experiments. <i>P</i>-values determined by two-tailed, unpaired <i>t</i>-tests with or without Welch’s correction. nd, not detected.</p

Similar frequencies and total number of CD4⁺ and CD8⁺ T cells in inflamed muscle tissues of WT and LysMcre;Arg1^F/F mice at 7, 10, and 14 dpi.

<p>Three-to-four week-old WT and LysMcre;Arg1^F/F mice were mock-inoculated (n = 3) or inoculated with 10³ PFU of RRV (n = 7–8 per time point per genotype). At 7, 10, and 14 dpi, quadriceps muscles were dissected, enzymatically digested, and the infiltrating leukocytes were isolated for FACS analysis. (A) Representative flow plots indicating the gating strategy to identify lymphocytes, CD3⁺CD8⁺ T cells versus CD3⁺CD8^- T cells, and CD3⁺CD4⁺ T cells in the muscle tissue of mock (top panel), RRV-inoculated WT (middle panel), and RRV-inoculated LysMcre;Arg1^F/F (bottom panel) mice at 10 dpi. Frequency (top panel) and total number (bottom panel) of (B) CD3⁺CD4⁺ T cells and (C) CD3⁺CD8⁺ T cells in muscle tissue of RRV-inoculated WT and LysMcre;Arg1^F/F mice compared with mock-inoculated mice. Data are represented as the arithmetic mean ± SEM and combined from two independent experiments. Each time point was individually evaluated for statistical difference by a two-tailed, unpaired <i>t</i>-test, and all were found to be not significant (<i>P</i> > 0.05).</p

LysMcre;Arg1^F/F mice have significantly more virus-specific CD8⁺ T cells in inflamed muscle tissue than WT mice at 10 dpi.

<p>Three-to-four week-old WT and LysMcre;Arg1^F/F mice were inoculated with 10³ PFU of WT RRV (n = 3) or recombinant RRV-LCMV (n = 18 for WT mice; n = 19 for LysMcre;Arg1^F/F mice). At 10 dpi, leukocytes from (A-E) spleens and (F-J) quadriceps muscles (following enzymatic digestion) were isolated for FACS analysis of virus-specific CD8⁺ T cells. Muscle-infiltrating leukocytes from a mouse inoculated with WT RRV was used as a control for gp33 tetramer staining. (A, F) Representative flow plots demonstrating the gating strategy to identify gp33⁺CD8⁺CD4^- T cells in the spleen (A) and muscle (F) tissue at 10 dpi. Frequency (B, G) and total number (C, H) of CD8⁺CD4^- T cells in the spleen (B, C) and muscle (G, H) tissue of WT RRV-inoculated mice, RRV-LCMV-inoculated WT mice, and RRV-LCMV-inoculated LysMcre;Arg1^F/F mice at 10 dpi. Frequency (D, I) and total number (E, J) of gp33⁺CD8⁺CD4^- T cells in the spleen (D, E) and muscle (I, J) tissue of WT RRV-inoculated mice, RRV-LCMV-inoculated WT mice, and RRV-LCMV-inoculated LysMcre;Arg1^F/F mice at 10 dpi. Data are represented as the arithmetic mean ± SEM and combined from five independent experiments. *** <i>P</i> < 0.001 as determined by two-tailed, unpaired <i>t</i>-tests with or without Welch’s correction (WT versus LysMcre;Arg1^F/F).</p

IFN-γ expression by T cells is required to control RRV infection in <i>Rag1</i>^-/- mice.

<p>T cells were isolated from the spleens of 6–8 week-old WT and <i>Ifng</i>^-/- mice via negative selection and 2.5 x 10⁶ T cells were adoptively transferred into three-to-four week-old <i>Rag1</i>^-/- mice i.p. one day prior to inoculation with 10³ PFU of RRV. (A-C) At 14 dpi, spleen and left quadriceps muscle tissues were harvested for flow cytometric analysis of the presence of T cells. (A) Representative flow plots showing the frequency of CD3⁺ and CD4⁺ or CD8⁺ T cells in the spleen and muscle tissues of <i>Rag1</i>^-/- mice that received either WT T cells or <i>Ifng</i>^-/- T cells. Quantification of the (B) frequency and (C) total number of CD4⁺CD3⁺ or CD8⁺CD3⁺ T cells in the mice that received T cells. Horizontal bars indicate the mean. (D) At 14 dpi, the right quadriceps muscle was dissected, total RNA was isolated, and RRV genomes were quantified by absolute RT-qPCR. Horizontal bars indicate the mean. Data are combined from 2 independent experiments. <i>P</i>-value was determined by a two-way, unpaired <i>t-</i>test.</p

T cell depletion from LysMcre;Arg1^F/F mice restores RRV loads to those in depleted WT mice at 14 dpi in muscle tissue.

<p>Three-to-four week-old WT or LysMcre;Arg1^F/F mice were inoculated with 10³ PFU of RRV and treated with anti-CD4 and anti-CD8α antibodies (WT, n = 7; LysMcre;Arg1^F/F, n = 10) or a control antibody (n = 10 per genotype) on days 7 and 12 pi. At 14 dpi, spleen cells were stained for CD3, CD4, and CD8β to determine the efficiency of T cell depletion. (A) Representative flow plots demonstrating efficient depletion of CD8⁺ (top panel) and CD4⁺ (bottom panel) T cells compared to the control antibody-treated mice. (B) Frequency of CD3⁺CD8⁺ (left panel) and CD3⁺CD4⁺ (right panel) T cells in the spleens of WT and LysMcre;Arg1^F/F mice. Data are represented as the arithmetic mean ± SEM. (C) At 14 dpi, the right quadriceps muscle was dissected, total RNA was isolated, and RRV genomes were quantified by absolute RT-qPCR. Horizontal bars indicate the mean. Data are combined from 2–3 independent experiments. <i>P</i>-value was determined by one-way ANOVA followed by Tukey’s multiple comparison test.</p

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 significantly induced in PBMCs collected from CHIKV-infected patients and expression correlates with viral load.

<p>(A) PBMCs isolated from CHIKV-infected patients (n = 23) were analyzed for Arg1 gene expression compared to healthy controls (n = 8). At each time point analyzed, Arg1 expression was found to be statistically different from healthy controls (**** <i>P</i> < 0.001), as determined by performing a two-tailed Mann Whitney <i>U</i> test. Each dot represents a patient, and the horizontal line represents the mean. (B) Based on their viremia during the acute and early convalescent phase of infection, patients were grouped into either a low viral load group (LVL; n = 11) or a high viral load group (HVL; n = 12). Arg1 expression was compared between the two groups via two-tailed Mann Whitney <i>U</i> test. * <i>P</i> < 0.05. (C and D) Spearman’s correlation analysis between transcriptional profiles of Arg1 expression in PBMCs of CHIKV-infected patients (n = 23) and (C) acute plasmatic C-reactive protein (CRP) and IP-10 levels, and IL-4 plasma levels during the early convalescent phase as well as (D) IFN-α, IFN-β, IL-6, IP-10, IL-10, and Nox1 genes during acute disease. The level of Arg1 gene expression was expressed relative to healthy controls (n = 8) after normalization to GAPDH. Early Conv., early convalescent phase.</p

CHIKV-infected HS 633T fibroblast co-culture and supernatant induces <i>Arg1</i> expression in human monocytes.

<p>(A) CHIKV adsorption (MOI 10) on HS 633T cells was performed for 1.5 h before virus overlay was removed; human monocytes were then co-cultured with the HS 633T cells for 24h. The level of gene expression was expressed as fold change compared to monocytes co-cultured with mock-infected cells after normalization to GAPDH. Experiments were performed in triplicate and data are mean ± SEM of two independent donors. Pair-wise comparison was performed using a two-tailed Mann Whitney <i>U</i> test. (B) HS 633T cells were infected with CHIKV (MOI 10) for 24h and 48h. Cell-free supernatant were collected and overlaid onto human monocytes for 24h. The level of gene expression was expressed as fold change compared to monocytes inoculated with cell-free supernatant from mock-infected cell lines after normalization to GAPDH. Experiments were performed in triplicate, and data are represented as mean ± SEM of two independent donors. Pair-wise comparison was performed using two-tailed Mann Whitney <i>U</i> test (** <i>P</i> < 0.01).</p