In this chapter I established the bead-based assays to measure the phagocytosis ability
of macrophage as well as its antimicrobial activities including acidification and
proteolysis. At first I successfully generated the beads coated with ligands and
line to use it as a model to set up and optimize the bead-based assays. In such models,
the assays were shown to work well giving consistent results. I observed the decrease in
phagosomal pH and the occurrence of proteolysis in macrophage treated with beads. I
found that stimulation of macrophages with TDM or β-glucan led to a significantly
decreases in proteolytic activities in comparison to resting macrophage. Interaction
between macrophages and bacteria could be investigated by infection of macrophages
with Mtb expressing mCherry. Our assays also worked in the hMDM model which
displayed variations in antimicrobial activities of macrophages from different subjects.
The bead-based assays were originally developed to study the physiological changes in
phagosome of macrophage that can help to explain its plastic functions in both
homeostasis and during immune response. Therefore, it is suggested that this new
approach can be applied in clinical settings to identify the macrophage-associated
pathologies275,273,226,299. The bead-based measurement of antimicrobial activities of AM
in HIV patients have suggested the possible association of the impairment in AM
activities and the increasing susceptibility to upper respiratory disease274. Furthermore,
this new approach has helped to provide the insight to the global modification in
hMDM activities in response to Mtb infection239.
The beads used in these studies were only opsonized with IgG to facilitate the uptake by
macrophages. It remains unclear whether different routes of entry into macrophages
may lead to differences in subsequent events within the host such as signal transduction,
immune activation, and intracellular survival of Mtb as well as outcome of infection14.
The beads developed in our study were novel since they were coated with specific
contribution of the signaling pathways specific for these ligands in controlling
macrophage antimicrobial activities in response to Mtb infection.
By exploiting the beads labeled with pH-sensitive fluorochrome carboxyfluorescein SE,
I have seen a rapid redution in phagosomal pH, which had been observed in previous
studies239, from neutral to 5.5 within 1 h in resting macrophage. The bulk protease
activity of macrophages was assessed by the beads coupled with DQ green BSA. I
observed an increase in fluorescence signal generated by degradation of this substrate
overtime, indicating the occurrence of proteolysis in phagosome. J774 macrophages
treated with TDM- or β-glucan- coated beads showed a reduced proteolysis activity
compared with resting macrophage. The beads are coated with an excess amount of
ligands (IgG/TDM or β-glucan) so that they are optimal for recognition and phagocytosis which is further indicated by the comparable phagocytic ability of
macrophages towards IgG, TDM or β-glucan beads. Therefore, the variations in proteolysis activity of macrophages treated with different ligands-coated beads could
come from the nature of ligands. Different ligands mediate their specific pathways that
would induce different macrophage responses. The decreased activity in macrophages
treated with TDM- or β-glucan beads was similar to that observed previously upon
activation of macrophages with LPS272. These results suggest a shift in macrophage
function from its role in homeostasis to the role in immunity. More clearly, the
homeostatic function of macrophages is to remove dead cells or cell debris, hence their
hydrolytic activity is programmed to occur with high efficiency for complete
degradation of those materials. Whereas, under activation by cytokines or bacterial
and efficient antigen processing for presenting to T cells. The consistency in our data
and previous data also provided a validation for the reliability of our bead-based assays.
The Mtb strain expressing mCherry provided a sensitive and rapid tool in a study of
macrophage-mycobacterial interaction. As other reporter Mtb strains generated in
previous study, our strain showed a strong correlation between mCherry intensity and
bacteria viability276. mCherry signal was very sensitive, so the signal could be detected
at a very low cell density of Mtb. It has been revealed that the fate of Mtb-infected
macrophage is different due to the intracellular burden of bacteria300, cells with heavily
burden progressing to necrosis. The reporter fluorescence in our study facilitated the
assessment the correlation of intracellular growth of Mtb and the macrophage lysis
when bacteria were infected to murine and human macrophage cell lines at different
MOI. In consistent with the previous study290, I also observed the rapid increase of
intracellular Mtb growth at MOI 2 and 3 that leads to early host cell lysis in comparison
to MOI 1 in both J774 and THP1 models. Because of slow bacterial growth and delay in
host cell lysis, I propose that the infection of macrophage at MOI 1 is applicable for
examining the host-pathogen interactions in detail.
In summary I have successfully established the assays to examine the phagosomal
antimicrobial activities as well as killing ability of macrophages; these assays showed
consistent results during experimental replications. In the next chapter, I apply these
assays developed here to study the association of macrophage antimicrobial activities