Viral and bacterial co-infection in severe pneumonia triggers innate immune responses and specifically enhances IP-10: a translational study

Abstract

Mixed viral and bacterial infections are widely described in community-acquired pneumonia; however, the clinical implications of co-infection on the associated immunopathology remain poorly studied. In this study, microRNA, mRNA and cytokine/chemokine secretion profiling were investigated for human monocyte-derived macrophages infected in-vitro with Influenza virus A/H1N1 and/or Streptococcus pneumoniae. We observed that the in-vitro co-infection synergistically increased interferon-γ-induced protein-10 (CXCL10, IP-10) expression compared to the singly-infected cells conditions. We demonstrated that endogenous miRNA-200a-3p, whose expression was synergistically induced following co-infection, indirectly regulates CXCL10 expression by targeting suppressor of cytokine signaling-6 (SOCS-6), a well-known regulator of the JAK-STAT signaling pathway. Additionally, in a subsequent clinical pilot study, immunomodulators levels were evaluated in samples from 74 children (≤5 years-old) hospitalized with viral and/or bacterial community-acquired pneumonia. Clinically, among the 74 cases of pneumonia, patients with identified mixed-detection had significantly higher (3.6-fold) serum IP-10 levels than those with a single detection (P = 0.03), and were significantly associated with severe pneumonia (P < 0.01). This study demonstrates that viral and bacterial co-infection modulates the JAK-STAT signaling pathway and leads to exacerbated IP-10 expression, which could play a major role in the pathogenesis of pneumonia.

Figure 1. Single and mixed infection of human monocyte derived-macrophages (MDMs) by IAV and SP.

(a) MDMs were infected with IAV at an MOI of 0.01, 0.1 or 1 and total RNA was extracted at 0, 6, 18, 24, 48 and 72 hours post-infection. The IAV A/H1N1 hemagglutinin (HA) gene was amplified by real-time qRT-PCR. (b) Influenza A/H1N1 viral load (TCID₅₀) in the supernatant of single IAV-infected MDMs or IAV-infected MDMs following subsequent challenge with SP. (c) Confocal imaging of IAV and SP in mixed-infected MDMs at 8 hours post-IAV infection and 4 hours post-SP infection. DAPI, nuclear stain, blue. IAV, Influenza A virus nucleoprotein stain, green. SP, S. pneumoniae stain, red. Scale bar = 5 μM. (d) Impact of single and mixed IAV and SP infection on MDM cell viability. Statistical analyses were performed using two-way ANOVA with Tukey’s post-hoc test; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Figure 2. Investigation of host mRNA, miRNA and protein expression levels during IAV and/or SP infection of MDMs or HAEC.

(a,b,c) THP-1 MDMs were infected with IAV (MOI = 1) for 4 h before SP (MOI = 1) was added. Total cellular mRNA (a) or miRNA (b) were extracted 24 h post-infection, reverse-transcribed and amplified using specific RT² profiler PCR Arrays. Major differentially expressed mRNA or miRNAs are shown. (c–e) THP-1 MDMs, primary human MDMs or primary airway epithelial cells (HAEC) were infected with IAV (MOI = 1) for 4 h before SP (MOI = 1) was added. The concentration of IP-10 in cell supernatant (24 h post-infection) was determined by a multiplex immunoassay on a FlexMap3D Luminex platform. Values represent mean ± SEM of four (c) biological replicates, four (d) independent experiments with different donors, three (e) biological replicates from one donor. Statistical analyses for each panel of experiments (c–e) were performed using a Kruskal-Wallis test (non-parametric, one-way ANOVA with Dunn’s post-hoc test); *P < 0.05; **P < 0.01.

Figure 3. Functional analysis of the synergistic induction of miR-200a-3p in MDMs after mixed infection with IAV and SP.

(a) Preceding IAV infection elevates miR-200a-3p expression after mixed infection with SP. MDMs were infected with IAV for 4 hours, then infected with SP, incubated for 20 h and total endogenous miRNAs were purified. A specific PCR-probe assay targeting miR-200a-3p was used to assess the fold changes in miR-200a-3p expression in mock-transfected and infected cells. (b) In silico predictive target alignment showing that miR-200a-3p targets the 3′ UTRs of both TGFB2 and SOCS6. (c) TGFB2, (d) CXCL10 and (e) SOCS6 expression profiles in MDMs transfected with negative transfection control (NTC), miR-200a mimic (MIM-200a) or miR-200a inhibitor (INH-200a). At 18 h after transfection, the MDMs were singly or mixed infected as described previously. At 8 h post-IAV and/or SP infection, total mRNA was extracted and amplified by PCR using specific primers for the indicated genes. Values represent median ± IQR (a, c) or mean ± SEM (d, e) of three biological replicates. Statistical analyses were performed using a Kruskal-Wallis test (non-parametric, one-way ANOVA with Dunn’s post-hoc test) for data presented in (a, c). An ordinary two-way ANOVA (with Tukey’s post-hoc multiple comparison test) was used for data presented in (d, e). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. (f) Western blot analysis of SOCS-6, SOCS-3, IAV nucleoprotein (NP) and β-actin expression in MDMs transfected with negative transfection control (NTC), miR-200a mimic (MIM-200a) or miR-200a inhibitor (INH-200a), cultured for 18 h, and then infected as described previously. Cell lysates were harvested 24 hours post-infection.

Figure 4. IAV and SP co-infection of MDMs exacerbates induction of IP-10: putative role for miR-200a-3p in regulating the JAK-STAT signaling pathway.

Both the in vitro and clinical studies indicated IP-10 is associated with mixed viral/bacterial infection in pediatric community-acquired pneumonia. In vitro, miR-200a-3p was synergistically induced following mixed IAV/SP infection of human MDMs and found to indirectly regulate CXCL10 (IP-10) expression by targeting the suppressor of cytokine signaling-6 gene (SOCS6), a well-known repressor of the JAK-STAT signaling pathway. In vivo, IP-10 was significantly elevated in the serum samples of pediatric patients with mixed viral/bacterial severe pneumonia compared to patients with single detection (P = 0.03) and non-severe pneumonia (P < 0.01).