Prolonged CHIKV viremia with age and evidence of persistent infection.

<p>(A) Serum and (B) CHIKV-inoculated feet were harvested on indicated days post-infection and assayed for viral titer by plaque assay. (A) Serum viral titers on days 1–4 post-infection and (B) viral titer of CHIKV feet on days 3 and 9 post-infection (<i>n</i> = 7–8 per group). (C) Genome copies of virus in CHIKV feet on day 60 post-infection (<i>n</i> = 16–18 per group). (D) CHIKV-inoculated feet were harvested at day 90 p.i. and assayed for the presence of fluorescent infectious virus by co-culture on C6/36 insect cells (n = 8–10 per group). Dashed line indicates limit of detection for all assays. Horizontal lines indicate the median. Statistical significance determined on log-transformed data by unpaired student’s <i>t</i>-test. *<i>P</i>< 0.05; **<i>P</i>< 0.01; ***<i>P</i>< 0.001.</p

Age-related impairment of adaptive immune response to CHIKV.

<p>(A) Popliteal LNs collected and quantified from either naïve or CHIKV-infected A and O mice at day 3, 7, or 9 post-infection. The LN draining from the CHIKV-inoculated foot is indicated as dLN and from the non-inoculated foot as ndLN. Table under graph indicates the average fold-increase from naïve for each age in either the dLN or ndLN (<i>n</i> = 6–8 per group). Horizontal lines indicate the median. Statistical significance determined by student’s <i>t</i>-test. (B-C) Lymphocytes from popliteal LNs on d7 post-infection were stimulated with CHIKV peptides in the presence of protein transport inhibitor. Total number of IFNγ⁺ CD4 T cells (B) and frequency (C) for each age. Data are mean ± SEM (<i>n</i> = 10 per group). Statistical significance determined by unpaired Student’s <i>t</i>-test. (D) CHIKV-specific IgM and (E) IgG2c in serum determined by ELISA at the indicated day post-infection. Data are mean (<i>n</i> = 4–24 per group). Statistical significance was determined by two-way ANOVA with Bonferroni post-test. (F) Plaque reduction neutralizing test on serum from days 9 and 60 post-infection. Data are mean + SEM (<i>n</i> = 12 per group). Statistical significance was determined by two-way ANOVA with Bonferroni post-test. (G) Serum samples collected from patients experiencing acute CHIKV-disease were evaluated by plaque reduction neutralizing test. Data are mean + SEM (<i>n</i> = 24 young and 15 aged). Statistical significance was evaluated by unpaired student’s <i>t</i>-test. In all panels black indicates A, red indicates O; * <i>P</i>< 0.05; ** <i>P</i>< 0.01; *** <i>P</i>< 0.001.</p

Blocking TGFβ prevents acute CHIKV-induced disease in O mice.

<p>A and O B6 mice were inoculated and treated with 100ug of anti-TGFβ antibody or isotype control and swelling was measured daily as described in Methods. Data are mean ± SEM (n = 8 per group). Statistical significance was determined using mixed model, repeated measures analyses of variance (ANOVA) as detailed in Statistics. Red stars indicate reduction of swelling in O mice from αIgG1 to αTGFβ treated; black stars indicate reduction of swelling in A mice from αIgG1 to αTGFβ treated.</p

Age increases acute CHIKV-induced joint swelling.

<p>(A) A (12 weeks) and O (18–20 months) B6 mice were inoculated via f.p. and swelling was measured daily as described in Methods. Data are mean ±SEM (<i>n</i> = 10–16 per group). Statistical significance was determined using mixed model, repeated measures analyses of variance (ANOVA) as detailed in Statistics. ***<i>P</i>< 0.001.</p

Dysregulated cytokine production with age.

<p>Serum was collected from A and O mice and assayed by ELISA for (A) CXCL9 or (B) TGFβ concentration at days 2 or 9 and 30, respectively. Data are mean + SEM (<i>n</i> = 3 naïve and 7–8 infected per age). (C) Human samples from IgM-positive CHIKV patients or age and sex-matched controls were assayed for Free-active TGFβ cytokine by ELISA. Data are mean + SEM (<i>n</i> = 39 each group). Statistical significance was evaluated by unpaired student’s <i>t</i>-test. * <i>P</i>< 0.05; ** <i>P</i>< 0.01; *** <i>P</i>< 0.001.</p