Fig 2: Suppressing CXCL1 signaling by AZD5069 treatment improved survival and clinical scores that correlated with reduced HEVA71 viral load in infected mice.a Survival rates and b mean clinical scores of mice infected with S41 or UI with or without AZD5069 treatment, respectively. Arrowheads indicate time points of AZD5069 administration. S41-infected mice displayed mortality and high clinical scores from 4 dpi. AZD5069 administration to S41-infected mice delayed mortality and high clinical scores until 8 dpi. UI-infected mice were healthy till 15 dpi. c AZD5069 treatment lowered viral loads in brains of S41-infected mice from 4 to 7 dpi. d, e CXCL1 levels were slightly elevated in d serum but highly elevated in e CSF of S41-infected mice with or without AZD5069 but not in UI-infected mice. f Brain lysates of S41- or UI-infected mice with or without AZD5069 treatment were subjected to immunoblotting analyses at 7 dpi. AZD5069 treatment delays the expression of HEVA71 viral proteins. Immunoblot representative of 3 biological repeats is shown. Bar charts below show quantifications for each protein. g VP0 and VP2 levels decreased at 7 dpi with AZD5069 treatment. Exact P values for: VP0 = 0.0006 and VP2 = 0.0009. h pERK1/2 and ERK1/2 levels were reduced in AZD5069-treated mice. Exact P values for: ERK1 = 0.0002, ERK2 = 0.0013, pERK1 = 0.0001 and pERK2 = 0.0007. i JAK2 levels persisted in CXCR2-suppressed mice with S41 infection. Exact P values for: JAK2 = 0.001. j AZD5069 treatment increased phosphorylation of STAT3 at pT705 but decreased pS727 levels at 7 dpi. Exact P values for: pT705 = 0.0007. k Cleaved caspase 3 was detected only in S41-infected mice without AZD5069 treatment at 7 dpi. Exact P values for: caspase 3 = 0.0013 and cleaved caspase 3 = 0.0015. ß-actin was used as a loading control for immunoblotting experiments. Data are expressed as the average with error bars representing ±SD from 3 independent experiments (n = 3). Statistical analyses were performed with two-way ANOVA corrected using Bonferroni post-test. (**P < 0.01, ***P < 0.001 and ****P < 0.0001). Source data are provided as a Source Data file.

Fig 3: CXCL1 signaling activates downstream ERK1/2 via CXCR2 to enhance viral replication in neuronal cultures.a HEVA71 viral titers increase in CXCL1-pretreated DIV7 neuronal cultures. Exact P values for: 25 U/ml at 24 hpi = 0.0033 and 48 hpi = 0.0009. b Immunoblotting of cell lysates from CXCL1-pretreated or untreated S41-infected DIV7 neuronal cultures against VP0, VP2, ERK1/2, pERK1/2, JAK2, pS727 (STAT3), pT705 (STAT3) and STAT3. CXCL1 pre-treatment accelerates the appearance HEVA71 viral proteins. Elevated pERK1/2 and ERK1/2 levels also emerged earlier. Immunoblot representative of 3 biological repeats is shown. Bar charts below show the quantifications for each protein. c High levels of VP0 and VP2 were detectable at 12 hpi only with exposure to CXCL1. Exact P values for VP0: 25 U/ml at 24 hpi = 0.0195, 50 U/ml at 24 hpi = 0.0001 and 100 U/ml at 24 hpi = 0.002. Exact P values for VP2: 25 U/ml at 24 hpi = 0.001 and 100 U/ml at 24 hpi = 0.0215. d pERK1/2 to ERK1/2 ratios were higher in pretreated cultures versus controls in a CXCL1 dose-dependent manner at all timepoints. Exact P value for pERK1 to ERK1 ratio: 25 U/ml at 48 hpi = 0.0094. ß-actin was used as a loading control for immunoblotting experiments. MOI of 1 was used for all experiments. Data are expressed as the average with error bars representing ±SD from 3 independent experiments (n = 3). Statistical analyses were performed with two-way ANOVA corrected using Bonferroni post-test. (*P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001). Source data are provided as a Source Data file.