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Macrophages play a central role in innate immunity via the phagocytosis process that is

initiated by recognizing and internalizing the invading pathogen into its phagosome.

The recognition can be promoted either directly by utilizing conserved structures on the

pathogen surface, such as LPS or peptidoglycan, or indirectly by opsonization of the

pathogen with serum complement, and immunoglobulins203. Once formed after

pathogen engulfment, the phagosome undergoes a process termed maturation, in which

both its membrane and lumen contents were modified by the means of fission and

fusion with other cellular compartments203. During this process, phagosome develops

into a toxic environment by acidifying, acquiring a range of hydrolytic enzymes and

producing toxic radical compounds. Phagosome acidification is prerequisite to optimal

function of hydrolytic enzyme and degradation of phagosomal content. Furthermore,

acidic pH establishes a hostile environment for an internalized pathogen. Acidification

commences early in phagosome maturation and predominantly results from recruitment

of V-ATPase to the phagosomal membrane. The hydrolytic enzymes can directly kill

bacteria by interrupting the integrity of mycobacterium membrane but also help in

degrading the pathogen. Antigen generated from such degradation is presented to T

lymphocyte via MHC class II molecule, bridging the innate and adaptive immunity.

It is reported that an impairment in phagocytic capacity of macrophages could lead to

the development of several pulmonary diseases that are caused by Haemophilus

influenza, Streptococcus pneumoniae282,283,284, or Coxiella burnetii285. Several

professional intracellular bacteria such as Mtb, Listeria monocytogenes, and Legionella

the outcome of infection depends on the innate interaction between bacteria and

macrophage286; any defects in the phagosomal function of macrophage could have

major impact on the establishment of the infection. Therefore, it is important to assess

the macrophage phagosomal function, which may help to understand not only the

contribution of macrophage in host defense and susceptibility to the disease but also the

macrophage manipulation by the pathogen.

Many assays have been developed to examine the phagosome maturation within

macrophages. Most approaches use immunofluorescence colocalization with well-

characterized markers285,287,288,289. The most common markers are Ras-related proteins

(Rab5 or Rab7 for early and late endosomal markers), LAMP-1, LAMP-2 (phagosomal

markers), and Cathepsin D (lysosomal marker). The expression of these markers in a

compartment indicates how aggressive this compartment might be towards an invading

microbe. These methods have been widely used, however they are very subjective, have

low sensitivity and do not completely reflect the environment within the phagosome

205,290 .

To overcome these disadvantages, Russell and colleagues have developed the platform

of different assays to measure directly and quantitatively functionally-relevant

parameters of phagosomal luminal biology, allowing researchers to design their own

experiments to address the interesting biological questions on phagosomal maturation in

response to microbial infection271,273,275,291. These assays exploit the model of beads

coated with microbe-derived ligands that facilitate particle uptake and also mimic the

conditions of infection in vivo. Different phagosomal activities in live macrophages can

access phagosomal acidification while DQ-BSA that is highly sensitive to the digestion

of various proteases is used to detect the proteolysis. The fluorescent output can be

assessed on different analytical platforms, including (i) flow cytometry, (ii) confocal

microscopy, and (iii) fluorescence microplate; each has both strengths and limitations.

The flow cytometry provides detail at individual cell level within and between cell

populations. Visualization by microscope provides excellent spatial and temporal details

at the level of single phagosome. These methods are time-consuming, lacking statistical

power, and only handle limited experiment conditions at a time; whereas the microplate

reader can allow monitoring of multiple conditions with many replicates. Depending on

the research question and the existing infrastructure, single platforms can be used or

different platforms can be combined to complement one another for speed, sensitivity,

and resolution271.

The efficiency of phagosomal functions of macrophages could be also evaluated by

measuring the bacterial killing ability of macrophages upon infection292; hence counting

the viable bacterial numbers becomes an important read-out for the host response.

Viable bacteria have the capacity to form colonies, hence the most commonly used

method for measuring the viable bacterial numbers after infection is by plating and

culturing defined volumes of lysed cells followed by the counting of colony-forming

units (CFU) on agar plates. To analyze colony-forming units, bacteria need to replicate

multiple times before colonies start to appear. However, this becomes a major drawback

when studying very slow-growing bacteria such as Mtb, since it takes 2-3 weeks for a

colony of Mtb to appear. Furthermore, CFU plating is rather labor-intensive and it is

bacteria can be used as a valuable alternative tool in intracellular growth studies because

of rapid detection, high sensitivity and real time detection276.

In order to study the association of macrophage phagosomal functions and susceptibility

to TB disease, in this chapter I generated the beads and established the bead-based

assays to measure the macrophages’ activities including phagocytosis (uptake ability),

acidification and bulk proteolysis. I also generated the Mtb reporter strain that expresses

fluorescent protein mCherry to study the outcome of interaction between macrophages

and bacteria to measure the bacterial survival.