Page 32 - Biennial Report 2018-20 Jun 2021

However, the mechanistic basis of defective efferocytosis in advanced atherosclerosis is not
understood.

Based on literature evidence, it was hypothesized that chronic endoplasmic reticulum (ER) stress
might lead to defective efferocytosis. To model the in vivo conditions in vitro,
pathophysiologically relevant compounds such as palmitate (a saturated fatty acid) and 7-
ketocholesterol (a modified form of cholesterol which is enriched in atherosclerotic plaques)
were used to induce ER stress in macrophages. Consistent with the hypothesis, ER stressed
macrophages demonstrated defective clearance of apoptotic cells (efferocytosis). Since
palmitate and 7-ketocholesterol have pleiotropic functions, ER stress was also modulated using
specific chemical chaperone 4- phenylbutyric acid (4-PBA). Interestingly, 4-PBA reversed the ER
stress-induced defective efferocytosis in macrophages demonstrating the causal role of ER stress
in mediating defective efferocytosis in macrophages.

Furthermore, from a mechanistic point of view, the ATF4 arm of the ER stress signaling pathway
plays a dominant role in mediating defective macrophage efferocytosis by i) decreasing the
ability of macrophages to recognize apoptotic cells via downregulation of key efferocytosis
receptors, and ii) decreasing focal exocytosis of endomembranes to the nascent phagosome
thereby preventing apoptotic cell engulfment and phagosome maturation. Whether relieving ER
stress promotes efferocytosis in vivo during atherogenic dyslipidemia is currently under
investigation in a pre-clinical animal model of atherosclerosis.
Interestingly, the specific molecular mechanism by which efferocytosis coordinates
inflammation resolution is poorly understood. To test the hypothesis that efferocyte-derived
exosomes promote inflammation resolution responses in vitro and in vivo and to identify the
specific molecular cargo in exosomes promoting the inflammation resolution response.
Exosomes are vesicular structures that contain several proteins and non-coding RNAs, and are
actively released from cells. In vitro culture supernatants can be used to study exosomes, but in
vivo exosomes are found in plasma. Exosomes can carry cargo from one cell to another
effectively creating a systemic signaling system in disease conditions.

In a DBT-Wellcome Trust India Alliance funded project, Manikandan Subramanian’s group
optimized the process of isolation of exosomes from macrophage culture supernatants using a
polyethylene glycol-mediated precipitation protocol. The isolated vesicles were in the range of
80-120 nm which is the characteristic size range of exosomal vesicles. To test the hypothesis that
macrophages that have engulfed an apoptotic cell (henceforth called an efferocyte) released
exosomes which promote inflammation resolution, exosomes from either control macrophages
or efferocytes were incubated with naïve macrophages exposed to an inflammatory stimulus.
Consistent with the hypothesis, efferocyte-derived exosomes increased the expression of
arginase (Arg1), a marker of proresolving M2 macrophage and suppressed the expression of
iNOS, a marker of proinflammatory M1 macrophage. Interestingly, this effect was specific to
efferocyte-derived exosomes, since exosomes isolated from macrophages engulfing necrotic
cells were unable to mediate these phenotypic changes. Besides promoting the generation of
M2-type macrophages, efferocyte-derived exosomes also suppressed the LPS-induced release
of pro-inflammatory cytokine TNF-α and increased the efferocytosis efficiency of naïve
macrophages. Interestingly, these effects were also observed only with efferocyte-derived
exosomes and not with exosomes isolated from macrophages engulfing necrotic cells. In vitro
and in vivo validation of the functional effects of the identified exosomal cargo using a simple
zymosan-peritonitis model and a complex atherosclerosis model is also planned.

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