Phagocytosis and the Immune System: Developing Protection from Infectious Diseases

Sarah Mathes Biology 113: MicrobiologyNovember 2010

Introduction:
Figure 1. Presence of TLR2 on phagosomes containing yeast particles. TLR2
In his book The Microbe Hunters, published in 1926, Paul de Kruif elaborates
was epitope-tagged and detected via immunofluorescence microscopy (a).
on the studies of the biologists whose work has established the realm of
After incubation with zymosan (yeast cell wall particle) for 5 min, zymosan
microbiology today. The common thread of every story is the the individual’s
particles were visible both inside and outside phagosome (b). TLR2 is shown
devotion to his work and determination to answer a specific scientific
in high concentration (bright fluorescence) during all stages of internalization
question. From Leeuwenhoek in the mid-1600s to Ehrlich in the early 1900s,
of zymosan into cell (c).
de Kruif discusses the development of the first microscope, the advent of
aseptic technique, Koch’s postulates to establish a microbe as pathogenic and
able to be isolated, and the discovery of phagocytes and their role in killing Is TLR2 able to signal production of TNF-alpha when phagocytosing other
microbes. pathogens besides yeast, like the Gram-positive Staphylococcus aureus?
What about the Gram-negative Salmonella minnesota?
Because the knowledge base of microbiology has greatly expanded since the
publishing of the book, this research question focuses on a theme that runs
throughout the book: the microbes that cause infectious diseases, their Figure 2. Presence of TLR2 on phagosomes. Cells were analyzed for TNF-
transmission, and why an organism may not develop the disease– whether alpha production: thin lines indicate TNF-alpha production in low-GFP (low-
due to an attenuated microbe, a lower dose, or the development of immunity TLR) cells; bold lines indicate TNF-alpha production in high-GFP (TLR-
to this disease after a primary inoculation. expressing) cells. (a) putative mutation for TLR2, (b) Wild-type TLR2, (c)
putative mutation for TLR4.
Research question:
How is immunity developed in an organism after exposure to or
inoculation with an infectious disease-causing microbe?
Figure 3. TLR presence in macrophage phagosome and
Background: signal pathway for antigen presentation. From initial
Macrophages are cells involved in the immune response that have various engulfment of the pathogen, the TLR is bound and can send
transmembrane receptors called Toll-like Receptors (TLRs), each of which are signals for production of TNF-alpha to present foreign
thought to give some specificity to the innate immune response. They peptide in MHC.
recognize PAMPs, or Pathogen-Associated Molecular Patterns to turn on the
appropriate effector response, which initiates a signaling pathway, which
leads to white blood cell localization, ininflammation and increased
phagocytosis at the site of recognition The topic of this paper is macrophage
phagosomes, or vacuoles formed around the pathogen being taken up by the
macrophage. Results:

TLR2 is normally recruited to macrophages that phagocytose yeast, but this
recruitment can be disrupted by a point mutation in the receptor. This can be
seen in the macrophage uptake of zymosan, a yeast cell-wall particle that will
start the inflammatory response.
TNF-alpha is a cytokine that helps to contain inflammation at the point of
recognition (keeps infection from becoming systemic).
TLR2 expression partially inhibited zymosan and S. aureus-induced TNF-alpha production, but had no
effect on S. minnesota and LPS (lipopolysaccharide)-induced TNF-alpha production.
Yeast and gram-positive bacteria are affected by TLR2 expression, but gram-negative bacteria are not
affected.
TLR2 thus does not participate in macrophage response to Gram-negative bacteria.

Methods:
To see if TLR2 is present on phagosomes during phagocytosis, the receptor
was tagged with an epitope and the macrophage is allowed to incubate with
the pathogen (zymosan). Cells are then analyzed for localization of the TLRs.
Mutate TLR2 so it will not produce TNF-alpha, and then have an equivalent
mutation for TLR4 as the two controls. Incubate these along with WT TLR2
and analyze for TNF-alpha expression.
Conclusion:
• Macrophages, a type of phagocyte, helps the organism to mount an innate immune response
• First phagocytose pathogens and sample peptide from these pathogens
• Then develop an appropriate inflammatory response.
• Host organism uses Toll-like receptors (TLRs) to discriminate between pathogens to mediate the most
effective defense.
•Different TLRs are shown to implicate different responses
• includes anti-fungal, anti-Gram-positive bacteria, or anti-Gram-negative bacteria defense.

Implications for Metchnikoff and phagocytes as of the beginning of the 1900s:

References:
Underhill, David M., Adrian Ozinsky, Adeline M. Hajjar, Anne Stevens, Christopher B. Wilson, Michael Bassetti, and Alan Aderem. "The Toll-like Receptor 2 Is Recruited to Macrophage Phagosomes and
Discriminates between Pathogens." Nature. 21 Oct. 1999. Web. 3 Nov. 2010.
Todar, Kenneth. “Immune Defense Against Bacterial Pathogens: Innate Immunity.” Todar’s Online Textbook of Bacteriology. 2008. http://www.textbookofbacteriology.net/innate.html.
Murphy, Kenneth, Paul Travers, Mark Walport. Janeway’s Immunobiology, 7th Edition. 2008.
Blander, J Magarian. “Phagocytosis and Antigen Presentation: a Partnership Initiated by Toll-like Receptors.” Annals of the Rheumatic Diseases. 10 Jul 2008.
http://ard.bmj.com/content/67/Suppl_3/iii44.abstract