Azithromycin reduces hemoglobin-induced innate neuroimmune activation

Abstract

Neonatal intraventricular hemorrhage (IVH) releases blood products into the lateral ventricles and brain parenchyma. There are currently no medical treatments for IVH and surgery is used to treat a delayed effect of IVH, post-hemorrhagic hydrocephalus. However, surgery is not a cure for intrinsic brain injury from IVH, and is performed in a subacute time frame. Like many neurological diseases and injuries, innate immune activation is implicated in the pathogenesis of IVH. Innate immune activation is a pharmaceutically targetable mechanism to reduce brain injury and post-hemorrhagic hydrocephalus after IVH. Here, we tested the macrolide antibiotic azithromycin, which has immunomodulatory properties, to reduce innate immune activation in an in vitro model of microglial activation using the blood product hemoglobin (Hgb). We then utilized azithromycin in our in vivo model of IVH, using intraventricular blood injection into the lateral ventricle of post-natal day 5 rat pups. In both models, azithromycin modulated innate immune activation by several outcome measures including mitochondrial bioenergetic analysis, cytokine expression and flow cytometric analysis. This suggests that azithromycin, which is safe for neonates, could hold promise for modulating innate immune activation after IVH.

Keywords: Azithromycin; Innate immune; Intraventricular hemorrhage.

Figure 1.. Hgb increases microglial ROS and cytotoxicity.

Hgb (0–0.2 mg/mL) increased microglial A) reactive oxygen species (ROS) measured by intracellular DCF-DA cleavage, compared to vehicle control and B) % cytotoxicity measured by LDH media concentration relative to positive lysis control at 24 hrs. Data are mean ± SEM. n= 3. Significance determined with one-way ANOVA followed by Tukey’s post-hoc test. * p<0.05 and *** p<0.001 relative to vehicle.

Figure 2.. Hgb decreases mitochondrial respiration in microglia.

Hgb (0–0.1 mg/mL) decreased A) seahorse profile plot, B) basal respiration, C) maximal respiration and D) ATP production, but not E) non-mitochondrial respiration, measured via oxygen consumption rate (OCR), in microglia at 24 hrs. Data are mean ± SEM. n=4–8 wells. Significance determined with one-way ANOVA followed by Tukey’s post-hoc test. ** p<0.01, *** p<0.001, **** p<0.0001 relative to vehicle. ^ p<0.05 and ^^ p<0.01 relative to 0.025 mg/mL.

Figure 3.. Dose dependent effects of AZM on mitochondrial respiration in microglia.

High concentrations of AZM (100 and 250 μM) increased A) % cytotoxicity (via LDH media concentration relative to positive lysis control) at 24 hrs. AZM (0–20 μM) did not alter A) % cytotoxicity or oxygen consumption rate (OCR) measured by B) seahorse profile plot, C) basal respiration, D) maximal respiration, E) ATP production, and F) non-mitochondrial respiration in microglia at 24 hrs. n=4–8 wells. Data are mean ± SEM. Significance determined with one-way ANOVA followed by Tukey’s post-hoc test. * p<0.05 relative to vehicle.

Figure 4.. AZM decreases Hgb induced microglial ROS and cytotoxicity

Intracellular ROS, measured by DCF-DA, normalized to vehicle control at A) 3 hr and B) 24 hr in microglia treated ± Hgb (0.1 mg/mL) and ± AZM (20 μM). C) % cytotoxicity, via LDH media concentration relative to positive lysis control, at 24 hrs. n=3–4 per group for 3 hr data. n=7–10 for 24 hr data from 2 independent replications combined. Data are mean ± SEM. Significance determined with two-way ANOVA followed by Tukey’s post-hoc test. ** p<0.01 and *** p<0.001 relative to vehicle or vehicle control. # p<0.05 and ### p<0.001 relative to vehicle Hgb. An effect, **** p<0.0001, of injury (Hgb relative to control) and an effect, ^ p<0.05, of drug (AZM relative to vehicle). D) Representative images of microglial cell cultures treated with media control (i), Hgb 0.1 mg/ml (ii), AZM 20uM (iii), AZM 20 μM and Hgb 0.1 mg/ml (iv).

Figure 5.. Hemoglobin decreases mitochondrial respiration in microglia.

Changes in oxygen consumption rate (OCR) in microglia at 24 hrs measured by A) OCR profile plot, B) basal respiration, C) maximal respiration, D) ATP production, and E) non-mitochondrial respiration. Data are mean ± SEM. n=7–8 from 2 independent replications combined. Significance determined with two-way ANOVA followed by Tukey’s post-hoc test. ^^^^ p<0.0001 relative to control vehicle and control AZM. $ p<0.05 relative to Hgb vehicle. An effect, **** p<0.0001, of injury (Hgb relative to control).

Figure 6.. AZM reduces hemoglobin-induced increase in inflammatory signaling and cytokine production at 24 hrs.

A) Immunoblot analysis of Hgb-induced activation of p65 in presence of azithromycin. B) Densitometric analysis shows that azithromycin (20 μM) protects Hgb (0.1 mg/mL) induced increased in phospho-p65 relative to total p65 in microglia for 1 hr. n=6 from 2 independent replications. C) AZM decreased Hgb increase in IL-10 expression at 24 hrs in the ipsilateral brain. n=4 per group. D) Hgb increased TNFα expression at 24 hrs in the ipsilateral brain. n= 9 from 2 independent replications. Data are mean ± SEM. Significance determined with two-way ANOVA followed by Tukey’s post-hoc test. * p<0.05 relative to control + vehicle. ## p<0.01 relative to control + vehicle, and ### p<0.001 relative to Hgb + vehicle. **** p<0.0001 relative to vehicle control and ### p<0.001 relative to vehicle Hgb. An effect, ^^^^ p<0.0001, of injury (Hgb relative to control).

Figure 7.. AZM attenuates innate immune cell proliferation, activation status, and ROS production in vivo after IVH.

A) The bead-estimated concentration of brain-resident innate immune cells (CD45/CD11b⁺) was calculated and compared across groups. Representative histograms show the relative intensity of (B) forward scatter and (C) side scatter properties of cells, indicating AZM reduces cell size and granularity after IVH, respectively. D) The relative production of intracellular ROS was assessed by the dye indicator DHR123. For A-D, n=4–5/group. For E, data from two independent experiments were combined and expressed as a fold change from the control group (n= 6–7/group). Data were analyzed by one-way ANOVA with multiple comparisons test. Abbreviations: Max maximum, FSC forward scatter, MI mean intensity, SSC side scatter, DHR dihydrorhodamine. *= p<0.5, **= p<0.01, ***= p<0.001, and ****= p<0.0001.

Figure 8.. AZM reduces TNFα production in the brain after IVH.

TNFα in contralateral hemispheres of the same animals assessed in Figure 7 was assessed 24 hours after IVH. AZM alone has no significant effect on TNFα production. IVH led to an average of 96 pg/ml TNFα in brain homogenate and this was reduced to 16 pg/ml in animals that were pretreated with AZM. n=5–7 per group. Data were analyzed by one-way ANOVA with Tukey’s post-hoc test. ***=p<0.001