Dysregulated TGF-β Production Underlies the Age-Related Vulnerability to Chikungunya Virus

Abstract

Chikungunya virus (CHIKV) is a re-emerging global pathogen with pandemic potential, which causes fever, rash and debilitating arthralgia. Older adults over 65 years are particularly susceptible to severe and chronic CHIKV disease (CHIKVD), accounting for >90% of all CHIKV-related deaths. There are currently no approved vaccines or antiviral treatments available to limit chronic CHIKVD. Here we show that in old mice excessive, dysregulated TGFβ production during acute infection leads to a reduced immune response and subsequent chronic disease. Humans suffering from CHIKV infection also exhibited high TGFβ levels and a pronounced age-related defect in neutralizing anti-CHIKV antibody production. In vivo reduction of TGFβ levels minimized acute joint swelling, restored neutralizing antibody production and diminished chronic joint pathology in old mice. This study identifies increased and dysregulated TGFβ secretion as one key mechanism contributing to the age-related loss of protective anti-CHIKV-immunity leading to chronic CHIKVD.

Fig 1. Age increases acute CHIKV-induced joint swelling.

(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 (n = 10–16 per group). Statistical significance was determined using mixed model, repeated measures analyses of variance (ANOVA) as detailed in Statistics. ***P< 0.001.

Fig 2. Prolonged CHIKV viremia with age and evidence of persistent infection.

(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 (n = 7–8 per group). (C) Genome copies of virus in CHIKV feet on day 60 post-infection (n = 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 t-test. *P< 0.05; **P< 0.01; ***P< 0.001.

Fig 3. Age-related impairment of adaptive immune response to CHIKV.

(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 (n = 6–8 per group). Horizontal lines indicate the median. Statistical significance determined by student’s t-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 (n = 10 per group). Statistical significance determined by unpaired Student’s t-test. (D) CHIKV-specific IgM and (E) IgG2c in serum determined by ELISA at the indicated day post-infection. Data are mean (n = 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 (n = 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 (n = 24 young and 15 aged). Statistical significance was evaluated by unpaired student’s t-test. In all panels black indicates A, red indicates O; * P< 0.05; ** P< 0.01; *** P< 0.001.

Fig 4. Dysregulated cytokine production with age.

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 (n = 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 (n = 39 each group). Statistical significance was evaluated by unpaired student’s t-test. * P< 0.05; ** P< 0.01; *** P< 0.001.

Fig 5. Blocking TGFβ prevents acute CHIKV-induced disease in O mice.

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.

Fig 6. Blocking TGFβ prevents chronic CHIKV-induced disease and restores neutralizing antibody titers in old mice.

Blinded H&E stained histology sections were evaluated by an anatomic pathologist for arthritis and metatarsal muscle Inflammation (MMI). (A) Representative metatarsal muscle inflammation. Astrisks indicate areas of inflammatory cells, muscle cell degeneration and pallor. H&E 40X magnification, bar is 100uM. (B) Representative arthritis. Arrow indicates area of articular cartilage erosion with infiltrate by neutrophils. Asterisk is an area of inflammation of the joint capsule characterized by the infiltration of neutrophils and mild edema. JO = Joint. H&E 20X magnification. (C) Mice were scored as having either metatarsal muscle inflammation (MMI), arthritis, or both. (D) Plaque reduction neutralizing antibody titers were determined at day 90 post-infection. Data are mean ± SEM (n = 11–12 per group). Statistical significance evaluated by unpaired student’s t-test * P< 0.05; ** P< 0.01; *** P< 0.001.