Post by: Haley Lucian
Some background for you:
The human macrophage plays
such a significant role in immunity and immune responses that understandably,
it’s been at the center of a wealth of research. Residing in tissues and
flowing through our bloodstream, this high functioning cell type patrols for invading
parasites and microbes. Defensively, this large white blood cell, has an
amazing ability to adeptly phagocytose its prey, including invading bacteria.
Upon identification of an invader, the macrophage cell deftly engulfs the
bacteria, removing it as a possible danger. The impressive rapid identification
and engulfment of the bacteria is controlled by a highly malleable plasma
membrane, quick reorganization of the actin cytoskeleton, and sophisticated
signaling molecules.
A macrophages engulfs, or phagocytoses, a bacteria
So, how are macrophages
able to find invaders in the large swamp of the human body? These hardworking
cells actually possess multiple ways of locating invaders, including a truly
fascinating method of “sensing” quorum sensing, that is, detecting the
production of quorum sensing molecules. Quorum sensing is a way in which
bacteria communicate with each other to regulate expression of proteins and
population. The bacteria Pseudomonas aeruginosa uses quorum sensing to
control production of virulence factors and harmful biofilm development present
in individuals with infections, compromised immune systems, and most notably,
cystic fibrosis. To better understand processes involved in this disease and
others, the authors chose to investigate the mechanics of how this bacteria, P.
aeruginosa, influences both macrophage deployment and ability to identify
bacteria. Ultimately, the remarkable mechanisms of the macrophage contribute to
the overall outcome of infection, any resulting inflammation and the prevention
of pathogenesis.
Why macrophages and P. aeruginosa?:
First on the to-do list for these investigators was
figuring out how the P. aeruginosa quorum sensing gene products, LasI
and RhlI, contribute to the process of macrophage phagocytosis and macrophage
morphology. The P. aeruginosa, a gram-negative pathogen, has
three known quorum sensing systems: two LuxI/LuxR type systems and one
quinoline type system. LuxI/LuxR systems and protein products differ between
bacteria. For the purposes of this blog, only the LuxI/LuxR system in P.
aeruginosa will be considered to illustrate the general process. In the
first system, LuxI/LuxR, the protein products, LasI and RhlI, synthesize the
essential quorum sensing molecule, 3O-C12-HSL, that is subsequently recognized
by cytoplasmic receptor lasR. In the second LuxI/LuxR system, protein
products of the two genes synthesize the quorum sensing molecule, C4-HSL, to
later be recognized by cytoplasmic receptor RH1R. Together these two cytoplasmic
receptors control the transcriptional and translational activity of roughly 300
genes in the P. aeruginosa genome. These gene products control the
development of biofilms and extracellular virulence factors targeting the host
organism that consequently can lead to inflammation and infection.
In real time though, the
macrophage has quite a large feat to engulf these invaders. For the macrophage
to successfully phagocytose bacteria, it must promptly alter cellular
morphology and motility, shape and volume. These notable tasks are controlled
by the flexible plasma membrane, rapid reorganization of the actin
cytoskeleton, and perhaps most importantly, transport of water into and out of
the cell. Though water molecules freely diffuse, transport is expedited by pore-forming
membrane proteins called aquaporins. The influx of water, controlled by these
aquaporins, then causes the membrane to be pushed outward, promoting a change
in cell shape. The next task then was to figure out how these quorum sensing
genes produced by the bacteria, P. aeruginosa, specifically
aquaglyceroporin AQP9, actually affect aquaporin distribution and expression in
the macrophage.
How they did it:
Since aquaporins are key to
macrophage function, let’s delve into the investigation of aquaporin
distribution and expression to reveal details about their purpose. As a first
step, the investigators explored macrophage response to pathogenic strains
varying in quorum sensing proteins, either the wild-type strain of the P.
aeruginosa or an altered P. aeruginosa strain, a LasI/RhlI mutant.
Note that the LasI-/RhlI- mutant lacks the key quorum sensing proteins and
virulence factors. The group found that the wild-type strain was more easily
targeted by the macrophages for phagocytosis than the mutant strain. The figure
below illustrates this difference, allow me to take you through it.
In (A) the graphs show macrophages that have
been infected with wild-type and/or mutant P. aeruginosa both containing
GFP, a fluorescent tag used to visualize
cellular distribution. The fluorescent tag is visualized under laser scanning
confocal microscopy (LSCM). The white box of the GFP only graph shows a
location where bacteria have been engulfed by macrophages. The white arrows in
the GFP only and GFP+ P. aeruginosa show recognition of the
bacteria. Now in (B), the percentage of phagocytic-positive macrophages is
compared between the wild-type and mutant strains and similarly in (C), the
percentage of macrophages containing bound/ingested bacteria is quantified both
showing greater phagocytosis of the wild-type cells than of the mutant cells.
So why is this important? It shows that P. aeruginosa with an intact
quorum sensing repertoire, are more easily targeted by macrophages for
destruction.
Now that the investigators
have identified a mutant strain eliciting a phagocytic macrophage response
different from the response for the wild-type strain, they next looked at how P.
aeruginosa infection affects aquaporin expression. Specifically they sought
to determine the quantity of AQP9 in the macrophage. Remember aquaporins affect
cell morphology allowing macrophages to successfully engulf their prey
therefore, more aquaporins would mean more influx of water and greater
propensity for cell elongation. This group found that AQP9 protein expression
levels were increased in macrophages during P. aeruginosa infection.
Furthermore, expression
levels of AQP9 were higher in the wild-type strain compared to the mutant P.
aeruginosa strain. They further show that P. aeruginosa infection
not only affected AQP9 expression, but that this higher expression promotes re-localization
of AQP9 to the polar regions of the macrophage thereby increasing cell area and
length. Bottom line, greater cell area and length created by the increased
aquaporins means that the macrophage is better able to engulf invading
bacteria. More aquaporins improve macrophage function.
What it all means:
So, why is all of this
important anyway? As you may recall, the bacterial strain, P. aeruginosa,
has been implicated in harmful biofilm development in patients with cystic
fibrosis, multiple types of infections, and in immunocompromised or autoimmune
compromised individuals as well. Since typically
the macrophage engulfs pathogenic bacteria to minimize the accumulation of a
harmful biofilm, it is important to understand what factors influence their
ability to perform their phagocytic job properly. The skill and aptitude
displayed by properly functioning macrophages should make us all feel lucky to
have these scavengers in our defensive immune system arsenal. Now, since the immune cells often work cooperatively
to keep pathogens at bay, one excellent line of study we can next tackle
is to study how other immune system cells behave in the presence of P.
aeruginosa. For example, if P. aeruginosa has a unifying effect on
many or all of our white blood cells, quorum sensing could be implicated as a
viable target for infection control!
Want to know a little more?
If you’re interested in learning more about the diseases
integral to this research, as an example, please visit the link posted below
about Cystic Fibrosis (CF). CF is a disease commonly under siege by a P.
aeruginosa infection: https://www.cff.org/What-is-CF/About-Cystic-Fibrosis/
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