CCR2 deficiency promotes exacerbated chronic erosive neutrophil-dominated chikungunya virus arthritis

Chikungunya virus (CHIKV) is a member of a globally distributed group of arthritogenic alphaviruses that cause weeks to months of debilitating polyarthritis/arthralgia, which is often poorly managed with current treatments. Arthritic disease is usually characterized by high levels of the chemokine CCL2 and a prodigious monocyte/macrophage infiltrate. Several inhibitors of CCL2 and its receptor CCR2 are in development and may find application for treatment of certain inflammatory conditions, including autoimmune and viral arthritides. Here we used CCR2 mice to determine the effect of CCR2 deficiency on CHIKV infection and arthritis. Although there were no significant changes in viral load or RNA persistence and only marginal changes in antiviral immunity, arthritic disease was substantially increased and prolonged in CCR2 mice compared to wild-type mice. The monocyte/macrophage infiltrate was replaced in CCR2 mice by a severe neutrophil (followed by an eosinophil) infiltrate and was associated with changes in the expression levels of multiple inflammatory mediators (including CXCL1, CXCL2, granulocyte colony-stimulating factor [G-CSF], interleukin-1β [IL-1β], and IL-10). The loss of anti-inflammatory macrophages and their activities (e.g., efferocytosis) was also implicated in exacerbated inflammation. Clear evidence of cartilage damage was also seen in CHIKV-infected CCR2 mice, a feature not normally associated with alphaviral arthritides. Although recruitment of CCR2+ monocytes/macrophages can contribute to inflammation, it also appears to be critical for preventing excessive pathology and resolving inflammation following alphavirus infection. Caution might thus be warranted when considering therapeutic targeting of CCR2/CCL2 for the treatment of alphaviral arthritides

Multiple immune factors are involved in controlling acute and chronic chikungunya virus infection

The recent epidemic of the arthritogenic alphavirus, chikungunya virus (CHIKV) has prompted a quest to understand the correlates of protection against virus and disease in order to inform development of new interventions. Herein we highlight the propensity of CHIKV infections to persist long term, both as persistent, steady-state, viraemias in multiple B cell deficient mouse strains, and as persistent RNA (including negative-strand RNA) in wild-type mice. The knockout mouse studies provided evidence for a role for T cells (but not NK cells) in viraemia suppression, and confirmed the role of T cells in arthritis promotion, with vaccine-induced T cells also shown to be arthritogenic in the absence of antibody responses. However, MHC class II-restricted T cells were not required for production of anti-viral IgG2c responses post CHIKV infection. The anti-viral cytokines, TNF and IFNγ, were persistently elevated in persistently infected B and T cell deficient mice, with adoptive transfer of anti-CHIKV antibodies unable to clear permanently the viraemia from these, or B cell deficient, mice. The NOD background increased viraemia and promoted arthritis, with B, T and NK deficient NOD mice showing high-levels of persistent viraemia and ultimately succumbing to encephalitic disease. In wild-type mice persistent CHIKV RNA and negative strand RNA (detected for up to 100 days post infection) was associated with persistence of cellular infiltrates, CHIKV antigen and stimulation of IFNα/β and T cell responses. These studies highlight that, secondary to antibodies, several factors are involved in virus control, and suggest that chronic arthritic disease is a consequence of persistent, replicating and transcriptionally active CHIKV RNA

Antibodies were unable to clear virus from persistently infected Rag1^−/− and µMT mice, and IFNγ, TNF and IL-6 levels were persistently elevated in persistently infected Rag1^−/− mice.

<p>(A) Rag1^−/− mice day 485 post infection (n = 4) and µMT mice day 550 post infection (n = 4) were given 200 µl immune serum (neutralization titer 1/2560) i.p. on day 0 and viraemia determined on the indicated days. (B) Serum cytokine levels measured in Rag1^−/− mice at the indicated times post-infection (n = 3/4 mice per time point). (Data for IFNα day 1 & 2 and day 60 & 120, and day 200 & 265 for the other cytokines were combined, as n = 2 for each one of these times).</p

Upstream regulator analysis of 192 up-regulated genes from mouse feet at day 30 post CHIKV infection compared to day 0.

<p>All upstream regulators with a p value <10e-9 are listed. A positive z-score indicates that the identified regulator is activated, since it normally up-regulates specific genes found in the 192 gene set. Similarly, a negative z-score indicates that the identified regulator is inhibited, since it normally down-regulates specific genes found in the 192 gene set. The full data set from this analysis with associated lists of up-regulated genes is shown in S4 Table in S1 Text.</p><p>Upstream regulator analysis of 192 up-regulated genes from mouse feet at day 30 post CHIKV infection compared to day 0.</p

Persistent CHIKV RNA, up-regulation of ISG54 and inflammatory infiltrates in C57BL/6 mice.

<p>(A–C) RNA was isolated from feet of infected mice at the indicated time points and analyzed by qRT-PCR. (A) CHIKV RNA levels determined by standard qRT PCR using primers specific for E1 normalized to RPL13A mRNA levels (n = 3–7 feet from independent mice per time point). Statistics by Mann-Whitney U tests comparing RNA levels at the indicated times post infection with levels at time 0. (B) Negative strand-specific qRT PCR using primers specific for nsP1 (n and statistics as for A). (C) ISG54 RNA levels as determined by standard qRT PCR, normalized to RPL13A mRNA levels (n and statistics as for A). (D) H & E staining of feet day 0 and day 30 post CHIKV infection. Infiltrates are shown for day 30 in muscle tissue (bottom left, while circles) and in the synovial membrane (bottom right, black arrows). (E) Aperio Positive Pixel Count determination of the ratio of blue (nuclear) to red (cytoplasmic) staining areas in whole foot sections at the indicated times post-infection. Leukocytes tend to have a high nuclear/cytoplasmic area ratio, so elevated ratios in whole foot sections indicate the presence of leukocyte infiltrates. (n = 3/4 feet from independent mice per time point, statistics by t test).</p

Viraemias in different mouse strains; B cell deficiency results in persistent “set point” viraemias.

<p>The viraemias for the indicated mouse strains at the indicated times are shown (strains ranked best to worst in terms of ability to control the viraemia); C57BL/6 (n = 43–50 mice before day 6, 3–9 thereafter; data from 8 independent experiments; not all time points were tested in each experiment), NOD (n = 6, except day 44 where n = 4, data from 2 independent experiments), MHCII^Δ/Δ (CD4 T cell deficient) (n = 9–16 day 0–6, n = 4–7 thereafter; data from 4 independent experiments), µMT (B cell deficient) (n = 6–10; data from 2 independent experiments), Rag1^−/− (B and T cell deficient) (n = 7–16; data from 3 independent experiments), Rag2/Il2rg (B, T and NK cell deficient) (n = 4), NRG (B, T and NK cell deficient on a NOD background) (n = 5–13 day 0–37, n = 3–9 thereafter; data from 3 independent experiments). Set-point viraemia levels for each mouse strain are indicated (bold, top right) and represent the mean (log₁₀CCID₅₀/ml of serum <u>+</u>SD) of all viraemia measurements taken ≥10 days post infection. For B cell deficient mice (bottom 4 panels), statistical comparisons (by t test) of set-point viraemia levels (<i>e.g.</i> p = 3.8E–11 for comparison of Rag1^−/− with NRG) used all viraemia measurements taken ≥10 days post infection. All mice were on a C57BL/6 background except the NOD and NRG mice.</p

The interferon signature.

<p>(A) Expression of IFNα6 and IFNβ as determined by qRT-PCR in different tissues on day 2 post CHIKV infection (n = 3 mice), the time of the peak IFNα/β response [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006155#ppat.1006155.ref028" target="_blank">28</a>]. Values are normalized to RPL13a mRNA levels and expressed as fold induction relative to mock infected controls (n = 3). CHIKV titres in the tissues were determined as described [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006155#ppat.1006155.ref028" target="_blank">28</a>]. Spearman’s correlation tests showed a significant relationship between IFN mRNA levels and viral titres (IFNα6 Spearman’s rho = 0.829 p = 0.042, IFNβ Spearman’s rho = 0.943 p = 0.005). (B) For all samples the up- and down-regulated DEGs (for which FC>2, q<0.01 and FPKM>1, see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006155#ppat.1006155.s010" target="_blank">S1 Table</a>) were analyzed using Interferome and the percentage of these DEGs that are interferon regulated genes (IRGs) is shown. (Interferome does not distinguish between genes directly or indirectly stimulated by IFNs, and some type I and/or II IRGs may not be identified by Interferome). (C) Heat map of FPKM values for all IFN genes identified by the RNA-Seq analysis (the same data is plotted as bar chart in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006155#ppat.1006155.s004" target="_blank">S4 Fig</a>). (D) Transcription factors associated with IFN responses in feet. Fold change of indicated transcription factors with vertical numbers representing the mean FKPM values (for the 3 biological replicates). Horizontal bold numbers represent the activation Z scores for the indicated transcription factor as determined by the direct function of the upstream regulator analysis of IPA; corresponding p values are provided in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006155#ppat.1006155.s005" target="_blank">S5 Fig</a>. (E) CiiiDER analysis of putative transcription factor site enrichment in the up-regulated type II IRGs in feet (as identified by Interferome). Color and size of circles reflect p values of the enrichment. Calculations for x and y values and the input/output data for the labeled transcription factors are provided in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006155#ppat.1006155.s007" target="_blank">S7 Fig</a>.</p

CHIKV genome.

<p>(A) The percentage of all the reads that aligned to the mouse genome for each tissue and time point. Read alignment numbers are provided in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006155#ppat.1006155.s001" target="_blank">S1D Fig</a>. (B) Examples of alignments of RNA-Seq reads from 3 foot samples mapped to the CHIKV genome (map quality threshold 20) viewed using Integrated Genomics Viewer (IGV version 2.3.34). The CHIKV genome is shown at the top for reference (arrow represents position of the sub-genomic promoter). Upper graphs (dark grey) show sequence coverage (log scale) for each nucleotide position in the CHIKV genome with y axis scale ranges (e.g. 0–300,000) shown in the left hand corners. The bottom graphs are “squished” views of the 100 bp reads aligned to the CHIKV genome (each grey horizontal bar represents one read). Black spots represent deletions/insertions within each read.</p

Concordance of up-regulated genes identified by RNA-Seq in the current study of CHIKV infected mice and previously published protein and mRNA expression studies in human CHIKV patients.

<p>For studies showing protein expression of mouse mediators see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006155#ppat.1006155.s011" target="_blank">S2 Table</a>.</p