Hyaluronan provokes inflammation but suppresses phagocytotic function in macrophages

Abstract

Background: The extracellular matrix (ECM) provides structural and functional support for the myocardium, but myocardial infarction (MI) changes the composition of the ECM. One of the chief components of the ECM, hyaluronan (HA), accumulates after MI; however, specific biological actions of HA-particularly at the level of infiltrating immune cells and implications of such interactions on ventricular remodeling-have not been explored.

Goal: Because acute accumulation of HA coincides with macrophage infiltration after MI, we assessed the impact of HA on macrophage function.

Results: Compared to SHAM hearts, HA levels were elevated in both the infarct and remote regions of infarcted hearts. Because acute accumulation of HA coincides with macrophage infiltration after MI, we explored the implication of HA accumulation on various endpoints of macrophage function, including macrophage activation, phagocytosis, and efferocytosis. Our data suggests that exposing macrophages to HA^HMW pushes macrophages toward a more pro-inflammatory phenotype as indicated by increased secretion of pro-inflammatory signals such as IL-2, IL-17, and IP-10. Our data also suggests that in the presence of HA, both macrophage efferocytosis and Fc-receptor dependent phagocytosis are suppressed. These results are unique to treatment with HA^HMW, as similar results were not observed when cells were treated with HA^LMW. Using macrophages from Cd44^-/- mice, we determined that while the impact of HA^HMW on cytokine secretion does seem to be dependent in part on Cd44 expression, the impact on macrophage phagocytosis is independent. Since macrophage efferocytosis of dying cardiomyocytes and cellular debris is critical following MI, we believe that this response will prolong the resolution of inflammation and lead to maladaptive remodeling.

Conclusion: HA accumulates post-MI and may promote a pro-inflammatory phenotype in macrophages. Future studies will explore the extent to which post infarct HA accumulation regulates cardiac macrophage dynamics and function in vivo.

Keywords: Efferocytosis; Extracellular matrix; Immune cells; Phagocytosis.

Figure 1:. High molecular weight hyaluronan (HA^HMW) accumulates early after myocardial infarction (MI).

C57BL/6J mice were subjected to myocardial infarction (MI), and HA levels were determined by hyaluronan binding protein (HABP) fluorescent staining. A) HABP stained SHAM and MI hearts 2 days post-MI. B) Quantification of HABP stained SHAM (n=7) and MI (n=5) hearts 2 days post MI. C) HABP stained SHAM and MI hearts at 7 days post MI. D) Quantification of HABP stained SHAM (n=7) and MI (n=3) hearts 7 days post MI. E) HA levels in the remote and infarct zone of SHAM (n=6) and MI (n=8) hearts were quantified using ELISA. F) HA was isolated from heart tissue 24 hours post-MI and run on an agarose sizing gel to assess size. An unpaired Student’s t test was used to determine significant differences between SHAM and MI groups.

Figure 2:. HA^HMW suppresses polarized macrophage efferocytosis.

To assess the impact of HA^HMW on polarized macrophage efferocytosis, naïve (M0), pro-inflammatory (M1), and reparative macrophage (M2) macrophages were exposed to either vehicle (PBS) or HA^HMW and efferocytosis was assessed by flow cytometry. A) Efferocytosis in M0 macrophages. B) Quantification of M0 efferocytosis. C) Efferocytosis in M1 macrophage. D) Quantification of M1 efferocytosis. E) Efferocytosis in M2 macrophage. F) Quantification of M2 efferocytosis. A paired Student’s t test was used to determine significant differences between vehicle and HA^HMW-treated macrophages (n=3).

Figure 3:. HA^HMW suppresses Fc-receptor dependent phagocytosis independent of CD44.

To assess the impact of HA^HMW on macrophage phagocytosis, naïve and polarized macrophages from wildtype (WT) or Cd44^−/− mice were exposed to either vehicle control (PBS) or HA^HMW and phagocytosis was assessed by flow cytometry. A-B) Representative flow plot and quantification of FITC positive naive WT cells (n=4). C-D) Representative flow plot and quantification of FITC positive naive Cd44^−/− cells (n=4). E-F) Representative flow plot and quantification of FITC positive M1 polarized WT cells (n=3). G-H) Representative flow plot and quantification of FITC positive M1 polarized Cd44^−/− cells (n=3). I-J) Representative flow plot and quantification of FITC positive M2 polarized WT cells(n=3). K-L) Representative flow plot and quantification of FITC positive M2 polarized Cd44^−/− cells(n=3). A paired Student’s t test was used to assess differences between vehicle and HA^HMW-treated groups. Red data points indicate that <10,000 flow events were collected.

Figure 4:. HA inhibits phagocytosis in a size dependent manner.

Naïve macrophages were exposed to either vehicle control (PBS), HA^LMW (equal mass) or HA^HMW and phagocytosis was assessed using confocal microscopy. A) Representative images acquired using confocal microscopy. B) Percentage of cells associated with FITC signal compared to the total number of cells. A repeated measures one-way ANOVA was used to determine significant differences between HA treated groups and the vehicle control group (n=6).

Figure 5:. HA^HMW treatment alters macrophage cytokine profile.

To assess the impact of HA^HMW on cytokine secretion, naïve macrophages from WT or Cd44^−/− mice (n=3 per group) were treated with either a vehicle control (PBS), HA^LMW, or HA^HMW and conditioned media was collected and analyzed using a cytokine discovery assay. Targets where significant changes were observed are shown above. A) G-CSF B) IL-2 C) IP-17 D) IP-10 E) MIP-3α F) RANTES G) IFNβ−1 H) IL-11 I) IL-6 J) IL-20 K) MIP-3β. If a value was not detected it was excluded, for this reason some treatment groups may have n<3. A two-way ANOVA was used to determine statistical significance. Outputs determined to be significantly different (p<0.05) have been highlighted in red.