RETRACTED ARTICLE: Pharmacological perturbation of CXCL1 signaling alleviates neuropathogenesis in a model of HEVA71 infection

Hand, foot and mouth disease (HFMD) caused by Human Enterovirus A71 (HEVA71) infection is typically a benign infection. However, in minority of cases, children can develop severe neuropathology that culminate in fatality. Approximately 36.9% of HEVA71-related hospitalizations develop neurological complications, of which 10.5% are fatal. Yet, the mechanism by which HEVA71 induces these neurological deficits remain unclear. Here, we show that HEVA71-infected astrocytes release CXCL1 which supports viral replication in neurons by activating the CXCR2 receptor-associated ERK1/2 signaling pathway. Elevated CXCL1 levels correlates with disease severity in a HEVA71-infected mice model. In humans infected with HEVA71, high CXCL1 levels are only present in patients presenting neurological complications. CXCL1 release is specifically triggered by VP4 synthesis in HEVA71-infected astrocytes, which then acts via its receptor CXCR2 to enhance viral replication in neurons. Perturbing CXCL1 signaling or VP4 myristylation strongly attenuates viral replication. Treatment with AZD5069, a CXCL1-specific competitor, improves survival and lessens disease severity in infected animals. Collectively, these results highlight the CXCL1-CXCR2 signaling pathway as a potential target against HFMD neuropathogenesis.


Reporting for specific materials, systems and methods
We require information from authors about some types of materials, experimental systems and methods used in many studies. Here, indicate whether each material, system or method listed is relevant to your study. If you are not sure if a list item applies to your research, read the appropriate section before selecting a response. All relevant sample sizes are described in the legend to each figure and/or in the Methods section. In general, no calculations were done to determine sample size. Sample size was determined based on standards for experimental biology and animal studies, attempting to have a minimum of N = 3 biological replicates with sufficient reproducibility. In most instances, sample sizes were determined based on previous experience with each of the experimental systems. In experiments involving animals, N = 3 with further experimental details on sample (brain, plasma, CSF) collection and analyses are included in the Methods section. Statistical tests were not used to predetermine sample size.
No data were excluded from analysis.
All experiments were performed 3 independent times with 3 or more technical replicates each time. All attempts at replication of results were successful.
Biological cell and animals samples were allocated into groups based on treatment regimens. Patient samples were allocated into groups by disease severity.
In general, data obtained from cellular models could not be blinded as only one investigator (the first author) was performing all experiments and analyses. Experiments with mice or harvested mouse tissue mostly did not employ blinding since readouts were quantitative and not prone to subjective judgment of investigators. Only imaging and analysis for immunohistochemistry staining of mice brains were blinded as data collection and analysis was performed by different investigators, using arbitrary sample codes whenever possible, to minimise bias. Blinding is not applicable to human studies as involved participants and respective clinicians are aware that the respective participants are suffering from that disease based on their symptoms.

Human research participants
Policy information about studies involving human research participants Population characteristics Tau (314004) detects Tau corresponding to AA 3 to 214 from mouse Tau-D. Specific for Tau. Sequence used for immunisation is present in all splice variants. The antibody binds phosphorylated and non-phosphorylated Tau. Species Reactivity: Rat and mouse. Weaker signal is detected in human. No signal is detected in chicken. Applications: WB, IF and IHC. GFAP (173002) detects AA 1 to 432 of the human GFAP. Specific fro GFAP, detects all isoforms. This antibody is more sensitive for WB and suitable as detector antibody fro sandwich-ELISA with 173011 as capture antibody. Species Reactivity: Human, rat, mouse, chicken, zebrafish. Other species not tested yet. Applications: WB, IF, IHC and ELISA. MAP2 (M4403) reacts with all isoforms of MAP2, namely MAP2a, MAP2b and MAP2c. Does not cross react with other MAPs or tubulin. By IHC, the antibody shows selective labeling of dendritic trees throughout the brain. Species Reactivity: Rat, Mouse, Chicken, Human, Bovine and Quail. Applications: WB and IHC. CXCL1 antibody (PA546932) is derived from a E.coli derived recombinant human CXCL1 and is purified by affinity chromatography. In direct ELISAs, less than 50% cross reactivity with recombinant human CXCL1 is observed, less than 25% cross reactivity with MIP-2 is observed, less than 15% cross reactivity with rat CINC-1 and IL-8 is observed. Species Reactivity: Rat, Mouse, Human. Applications: WB, IHC and ELISA. Ly6G-FITC (BD551460) is a antibody that only binds to Ly6g on neutrophils. Species Reactivity: Rat, Mouse, Human. Applications: ELISA and flow cytometry. CD45-PE-Cy7 (25-0453-82) antibody reacts with all isoforms of mouse CD45, also known as the Leukocyte Common Antigen (LCA). Species Reactivity: Mouse, Human. Applications: ELISA, IHC, IF and flow cytometry. Calcein violet AM antibody (C34858) is a commercial dye that converts to the flueorscent version in live cells with esterase activity. The dye is well-retained in live cells. Species Reactivity: Mouse, Human. Applications: Flow cytometry.
Source of cell lines are indicated in the Methods section. We used human RD (CCL-136, ATCC) and HEK-293 cells (CRL-1573, ATCC) that were purchased from ATCC approximately 20 years ago. These lines has been passaged in our laboratory since.
None of the cell lines were authenticated.
Mycoplasma contamination results were negative.
No commonly misidentified cell line were used in this study.
For primary neurons and/or astrocytes cell culture, we used the brains harvested from postnatal day 0 pups. Strain: MPF HanTAc: WH (Wistar). Sex: We have not determined the sex of the pups. Infectious virus experiments and compound studies were conducted in 0 or 1-day-old BALB/cAnNTac suckling mice. Sex: We have not determined the sex of the pups. Mice and rats are housed at a dedicated facility, with regulated environment, temperature in the range 22 to 24ºC, a 12h/12h dark/light cycle and 30 to 70% humidity. They had access to food and water.
The study did not involve wild animals.
The study did not involve samples collected from the field.