IL-1α and IL-1β protein levels after ingenol mebutate treatment.

<p>(A, B) C57BL/6 and MyD88^-/- mice with B16 tumours were treated topically with ingenol mebutate day 0 and 1, treatment sites were excised and IL-1α and IL-1β levels were measured in extracts using BD BD™ Cytometric Bead Array (n = 6 mice per group and time point). Statistics by Kolmogorov-Smirnov tests (differences in variance >4). (C) Cultured adult human keratinocytes were treated with the indicated concentration of ingenol mebutate for 16 hours and the supernatants analysed by Western using an anti-IL-1α antibody. Arrow indicates the position of the 18 kDa bioactive form of IL-1α.</p

Relapse and survival following ingenol mebutate treatment of B16 tumours grown in MyD88^-/- and C57BL/6 mice.

<p>(A) Relapse rates following (i) ingenol mebutate treatment of B16 tumours grown in MyD88^-/- mice (n = 25), (ii) ingenol mebutate treatment of B16 tumours grown in C57BL/6 mice (n = 21), (iii) placebo treatment of B16 tumours grown in MyD88^-/- mice (n = 18) and (iv) placebo treatment of B16 tumours grown in C57BL/6 mice (n = 19). Mice were scored positive when a tumour was clearly visible (≥1–2 mm in diameter). Data from two independent experiments. Ingenol mebutate treatment groups were significantly different p = 0.021, log-rank (Mantel-Cox) test. (B) Survival rates of the same mice described in A; mice were euthanized when tumours reached 100 mm². Ingenol mebutate treatment groups were significantly different p = 0.018, log-rank (Mantel-Cox) test.</p

Relapse and survival following ingenol mebutate treatment of B16 tumours grown in C57BL/6 mice treated with anakinra.

<p>(A) Relapse rates after C57BL/6 mice bearing B16 tumours were treated with ingenol mebuate or placebo, and received daily injections of PBS or anakinra, days 1–7. (n = 9–12 mice per group). Mice were scored positive when a tumour was clearly visible (≥1–2 mm in diameter). Statistics compared + anakinra with + PBS in ingenol mebutate treated groups using the log-rank (Mantel-Cox) test. (B) Survival of the mice described in A; mice were euthanized when tumours reached 100 mm². Statistics as in A.</p

Neutrophil recruitment and apoptosis.

<p>(A) Neutrophil recruitment to treatments sites (left bar chart) and to within ≈200 μm of tumour (right bar chart). Treatment sites; day 2 post initiation of ingenol mebutate treatment, treatment sites were excised and processed for immunohistochemistry and stained with the neutrophil marker, anti-Ly6G. Slides were scanned and analysed by Aperio Pixel count software for brown staining (default settings) excluding areas containing tumour (as melanosomes provide a false positive signal). Two sections per mouse, 6 mice per group. Statistics by Kolmogorov-Smirnov tests (differences in variance between groups was >4). Within ≈200 μm of tumour; in sections where the tumour mass could be readily identified (by the presence of black melanosomes), brown staining surrounding the tumour (within ≈200 μm) was quantitated as above. One section per mouse, 4–5 mice per group. Statistics by Mann Whitney U test (non-parametric data distribution and differences in variance <4). (B) Images illustrating the reduced density of anti-Ly6G staining neutrophils (brown stain) within ≈200 μm of the tumour mass in anakinra versus PBS treated mice. The tumours are delineated by white lines and identified by the presence of black melanosomes. Sections are oriented with the skin (not shown) at the top, with the tumours located in the dermis. (C) ApoTag staining of the sections described in A. Six mice per group, 2/3 sections per mouse, statistics by 2 way ANOVA (parametric data distribution and differences in variance <4, drug and mouse as fixed factors, 2/3 sections per mouse as dependent variables). (Examples of the staining are shown in Figure F in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0153975#pone.0153975.s001" target="_blank">S1 File</a>).</p

CHIKV neutralization and IgG-isotyping.

<p>A) Serum of immunized mice were collected and tested for their neutralizing ability based on >95% protection against CHIKV induced CPE in Vero cells. BEI corresponds to the inactivated CHIKV positive control. B–C) Immunoglobulin-G1 and -2c isotypes were determined using ELISA upon serial dilution. Statistical analysis shows that all CHIKV antigen immunized groups are significantly different from the control groups.</p

CHIKV VLP vaccination and CHIKV challenge.

<p>A) At least 6 weeks old mice were vaccinated with 0.1 µg VLPs, 1 µg VLPs and 1 µg VLPs adjuvanted with Quil A (QA). PBS and GFP were used as a negative control and inactivated CHIKV virus as a positive control (n = 6 per group). Mice were challenged with the Rèunion Island isolate 5 w post infection. Viraemia levels were determined over 6 days. B) Foot size in mm² (width×height) was determined over 16 days post challenge. Statistical analysis shows that mice vaccinated with inactivated CHIKV virus, 1 µg VLPs and 1 µg VLPs adjuvanted with Quil A display significant lack of viraemia or lack of footswelling at times of peak viraemia (1–3 dpi) and peak foot swelling (6–8 dpi) in the control groups, respectively.</p

CHIKV-E1 and –E2 detection on the surface of Ac-S27 infected <i>Sf</i>21 cells.

<p>Sf21 cells were infected with Ac-S27 and subjected to immunostaining using α-E1 and α-E2 antibodies. Cells were analysed by fluorescent microscopy where positive staining indicates E1 or E2 surface exposure.</p

Lower temperatures reduce type I interferon activity and promote alphaviral arthritis

<div><p>Chikungunya virus (CHIKV) belongs to a group of mosquito-borne alphaviruses associated with acute and chronic arthropathy, with peripheral and limb joints most commonly affected. Using a mouse model of CHIKV infection and arthritic disease, we show that CHIKV replication and the ensuing foot arthropathy were dramatically reduced when mice were housed at 30°C, rather than the conventional 22°C. The effect was not associated with a detectable fever, but was dependent on type I interferon responses. Bioinformatics analyses of RNA-Seq data after injection of poly(I:C)/jetPEI suggested the unfolded protein response and certain type I interferon responses are promoted when feet are slightly warmer. The ambient temperature thus appears able profoundly to effect anti-viral activity in the periphery, with clear consequences for alphaviral replication and the ensuing arthropathy. These observations may provide an explanation for why alphaviral arthropathies are largely restricted to joints of the limbs and the extremities.</p></div

The role of the type I interferon response.

<p>(A) Viremia in IRF3/7^-/- mice housed at 30°C or 22°C after CHIKV infection s.c. into the feet (n = 9/12 mice per group). (B) Foot swelling in IRF3/7^-/- mice housed at 30°C or 22°C and infected s.c. in the feet. (Statistics by t test, n = 14–18 feet from 7–9 mice per group). (C) Survival of IRF3/7^-/- mice housed at 30°C or 22°C and infected s.c. in the feet. (Statistics by Mantel-Cox Log Rank test, p = 0.071, n = 7–9 mice per group). (Data for A-C was obtained from 2 independent experiments). (D) As for A except viremia in IFNAR^-/- mice (n = 5/6 mice per group). (E) As for B except foot swelling in IFNAR^-/- mice. (Statistics by t test, n = 12 feet from 6 mice per group). (F) As for C except survival of IFNAR^-/- mice (n = 11/12 animals per group; data obtained from 2 independent experiments). (G) Serum IFNβ and IFNα levels after s.c. injection of 5 μg poly(I:C)/jetPEI into feet. Statistics by t test except for 6 hours where a Mann Whitney U test was used due to non-normal data distribution (n = 6 mice). (H) Vero cell were incubated at the indicated temperatures and treated with IFNα for 4 hrs followed by infection with CHIKV (MOI = 0.05). Cytopathic effect (CPE) was measured after 3 days. (I) Vero cells at the indicated temperatures were infected with CHIKV at the indicated multiplicity of infection (MOI), were washed, and viral titers in the supernatants assessed at the indicated hours after washing. Data generated from 3 replicate wells.</p

Affect of ambient temperature in the RRV model of alphaviral arthropathy.

<p>Mice were housed at 22°C or 30°C and were infected s.c. in the pectoral region with RRV. (A) Viremia. (Statistics by repeat measures ANOVA for days 3–6, n = 5 mice per group). (B) Feet titers. (Statistics by Mann-Whitney U test, n = 5 feet from 5 mice per group). (C) Disease severity score. This is primarily a score of hind limb paralysis. (Statistics by repeat measures ANOVA, n = 5–10 mice per group, day 0–21).</p