Brent Berwin, Ph'D'

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• Brent Berwin, Ph.D.
• Dartmouth Medical Center
• Norris Cotton Cancer Center

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Body Snatching Germs!

• Bacteria
• Viruses
• and a variety of other micro-organisms,
  including fungi, unicellular eukaryotic
  pathogens, etc.

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Virus
Needs host cellular machinery to replicate
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Bacteria
Unicellular organism
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INTERACTION WITH CILIATED HOST CELLS
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Jogging your memories Bacteria can be quite
harmful pathogens

• Black death/Bubonic Plague
• Caused by bacteria Yersinia pestis
• 14th century outbreak killed 40 of Europe
• Inspired the hit song
• Ring around the Rosey Pocket full of
  Posey Ashes,
• Ashes, They all Fall Down

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More recently Diphtheria

• Caused by the bacterium C. diphtheriae
• As recently as the 1920s it was killing 20,000
  people / year in the USA
• Success story largely eradicated in USA with use
  of DPT vaccine (DiphtheriaPertussisTetanus)

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In 1925 Nome, Alaska was stricken with a
Diphtheria outbreak and anti-toxin couldnt be
flown in20 mushers brought the anti-toxin 700
miles to Nome by dogsled in 5 days this was the
inspiration for The annual Iditarod dogsled
race from Anchorage to Nome
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Outline for this evening Host Immune Responses
to Bacterial Pathogens

• Vaccination
• Immune responses to infection
• How do our cells/immune systems recognize we have
  a bacterial infection?
• How do we repel a bacterial infection
• The role of antibodies
• Phagocytosis of bacteria
• Cytokine responses
• Antibiotics/Treatment

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A model host response Vaccination against
BORDETELLA PERTUSSIS

• DISCOVERED IN 1906 BY BORDET AND GENGOU
• CAUSE OF WHOOPING COUGH (PERTUSSIS)
• SMALL GRAM-NEGATIVE COCCOBACILLUS

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Before Development of a Vaccine

• PERTUSSIS WAS A MAJOR CHILDHOOD KILLER PRE-1940
• CAUSED MORE INFANT DEATHS THAN MEASLES,
  DIPHTHERIA, POLIO SCARLET FEVER COMBINED
• THE FIRST VACCINE WAS INTRODUCED IN THE UK IN
  1937, AND ROUTINE USE IN THE US OCCURRED BY 1944
• VACCINATION HAS DECREASED THE NUMBER OF PERTUSSIS
  CASES BY gt100-FOLD

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Pertussis The Early Vaccine

• HEAT-KILLED BACTERIA
• EFFICACY AS LOW AS 20
• A NUMBER OF DETRIMENTAL SIDE EFFECTS
• CONVULSIONS
• NEUROLOGICAL SIDE EFFECTS
• HIGH TEMPERATURE (FEVER)
• IF USING ATTENUATED VACCINE gt POSSIBLE EFFECTS ON
  IMMUNOCOMPROMISED HOSTS

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IMMUNITY NEW COMPONENT VACCINE

• CONTAINS COMPONENTS OF B. PERTUSSIS
• PERTUSSIS TOXOID
• FHA
• PERTACTIN (SURFACE PROTEIN)
• TWO TYPES OF FIMBRAE

SO WHY DOES THIS WORK? 1) AVOIDS THE USE OF
BACTERIAL CELL WALL PRODUCTS 2) USES CELL
SURFACE OR SECRETED PROTEINS important for
antibody response
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Generation of Antibody Responses
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Figure 1-24 part 2 of 3
Antibody Activities
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THE ESSENCE OF VACCINATION A MEMORY RESPONSE

• 2 RESPONSE
• QUICKER
• LARGER
• MORE POTENT

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IMMUNITY NEW COMPONENT VACCINE

• CONTAINS COMPONENTS OF B. PERTUSSIS
• PERTUSSIS TOXOID
• FHA
• PERTACTIN (SURFACE PROTEIN)
• TWO TYPES OF FIMBRAE

Y
Y
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The immune response to vaccination is analogous
to bacterial infection

• The immune system recognizes bacteria or
  bacterial products as non-self
• In the right context, B cells in the immune
  system produce antibodies
• These antibodies hopefully neutralize the
  bacterial toxins and/or aid in the clearance of
  the bacteria
• The 2nd time the immune system sees the same
  bacteria (due to vaccination or a subsequent
  infection) the memory response will be potent and
  efficient

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But What if we arent vaccinated against a
particular bacteriaOr what if the bacteria
infects us at an anatomical location inaccessible
to antibodiesOr what other immunological
mechanisms can hold the bacteria at bay prior to
antibodies?
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THE EARLY IMMUNE RESPONSE
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A good eater
Kobayashi
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Our white blood cells Great Eaters
Inflammatory Cytokines
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Bacterial Recognition by the Immune System
TOLL-LIKE RECEPTORS
-Trafficking Scavenger Receptor-A traffics
(phagocytosis) bacteria into cells -Signaling
Toll-Like Receptors recognize bacterial cell wall
products and elicit inflammatory cytokine
responses
TLR2
BACTERIA
TLR4

• Both Scavenger Receptors and Toll-Like
• Receptors recognize bacteria / bacterial
• products as non-self and initiate
• immune responses
• The bacterial products are common to
• many bacteria, not easily mutated

SCAVENGER RECEPTORS
PHAGOCYTOSIS
INFLAMMATION
KILLING OF MICROBES
CYTOKINES
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Local Produce the Role of Scavenger Receptor-A
during Bacterial Infection

• We can quantitatively measure the ability
• of cells to internalize/phagocytose bacteria
• We can compare cells that express
• different levels of particular receptors
• for their ability to phagocytose bacteria
• Specifically we can deduce the
• contribution of SRA if we use cells that
• are deficient for SRA

4ºC
WGA
E. coli
37ºC
WGA
E. Coli
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SRA-deficient (SRA-/-) Phagocytes are Impaired in
their Ability to Clear E. coli
E. coli
Pseudomonas aeruginosa
Relative uptake of bacteria
MOI
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in vivo SRA-/- mice are impaired in their
ability to clear an E. coli challenge
Relative Clearance of E. coli
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Loss of SRA leads to Susceptibility to Death from
Bacterial Challenge (Body Snatchers!)
Haworth et al., J Exp. Med 186, 1997 Thomas et
al., J. Exp. Med., 191, 2000
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So everybodys on the same page

• SRA is a receptor expressed on our white blood
  cells
• Important for binding to (some) bacteria,
  internalizing the bacteria into the cells for
  clearance/degradation
• Loss of SRA expression can predispose the host to
  susceptibility to bacterial infection and even
  death caused by the bacteria

However, in the data I just showed you, the
death is caused by cytokines, not bacterial
virulence
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Cytokines

• Cytokines are signaling molecules that allow
  cells to communicate with eachother
• There are many different cytokines, with
  differing functionssome inflammatory, some
  suppressive, some that help recruit other
  cellsvital players in a proper immune response
• Toll-Like Receptors are key players in the
  recognition of bacteria and the initiation of a
  pro-inflammatory cytokine response

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Cytokines targeting shown to help disease

• Chemokines recruit cells to sites
• Proinflammatory cytokines (innate/adaptive)
• TNF, IL-1, IL-6, IL-12/IL-23, IL-17
• 3. Anti-inflammatory cytokines
• TGF-beta, IL-10
• B cell growth factors
• BAFF/Blys, APRIL, BCMA,

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Enhanced Bacterially-Elicited Cytokine Responses
in SRA-/- Mice
TLR2
Bacteria
TLR4
x
SR-A
x
PHAGOCYTOSIS
INFLAMMATION
CYTOKINES
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The Goldilocks Aspect of Cytokines

• Upon bacterial infection
• Too little of an inflammatory response
  insufficient immune response against bacteria
• The right amount of inflammatory response
  recruitment of anti-bacterial cells and products
• Too much inflammatory response harmful to the
  host
• Toxic Shock Syndrome

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The Porridge was Too Hot
TOLL-LIKE RECEPTORS
TLR2
Bacteria
TLR4
x
SCAVENGER RECEPTORS
x
PHAGOCYTOSIS
INFLAMMATION
CYTOKINES
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So, in a perfect world (our research goal,
naturally)

• Our epithelial barriers (skin, mucosa) keep the
  majority of bacteria from infecting us
• Our white blood cells (phagocytes) and antibody
  responses (from B cells) fight off the bacteria
  that make it in
• We produce cytokine
• responses appropriate to the infection/insult

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but its not a perfect world

• A major goal of our research is to be able to
  manipulate/stimulate our immune system to
  efficiently and effectively clear bacterial
  infections
• Context
• People whose immune system is fine but the
  bacteria are winning a war of infection
  nonetheless
• People whose immune system is compromised

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Home cooking ongoing collaborative research at
DHMC

• Cystic Fibrosis mutation in a chloride channel
• Results in bacterial infection and chronic
• inflammation in the lungs that is a major cause
  of
• CF-associated morbidity and mortality
• A common outcome is lung failure
• Largely unknown why the CF lung cant clear
  bacterial infections

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• Polymicrobial
• Temporal P.a. dominance
• High rates of morbidity and mortality
• are associated with P.a. infection

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Your taxpayer at workwhat were doing right
now

• Why is the CF lung prone to bacterial infection?
• How does CF compromise the immune response?
• A Pseudomonas focus
• How does Pseudomonas colonize the CF lung?
• How does it evade immune clearance?
• Why does Pseudomonas predominate?
• Working both ends of the equation
• How can we manipulate the lung
• and the immune system to fight off infection?
• How can we manipulate Pseudomonas
• and its bacterial products to ameliorate the
• disease and improve the expectations and
• outcomes of CF individuals?

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Conspirators
Dartmouth Deb Hogan Dean Madden Bruce
Stanton George OToole Karl Griswold UVM Matt
Poynter Dan Weiss Laurie Whittaker Duke
University Soman Abraham Harvard Les Kobzik

• Berwin Lab
• Eyal Amiel
• Pete Bak
• Kevin Hart
• Julie Acker
• Ryan Collins
• Yi Zhang
• Rustin Lovewell

• Funding
• NIH (COBRE) P20 RR16437
• NIH RO1 AI067405
• Dartmouth CTSA (Berwin/Weiss/Griswold)
• CF-RDP
• NIH Immunology Training Grant (EA)
• Prouty Research Grant

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Phenomenology of CF lung infection

• Infection becomes predominantly P.A.

• Poly-microbial infection
• Microbes not effectively cleared

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DHMC researchers involved in CF host/bacterial
pathogen interactions
Stanton
Hogan
Griswold
OToole
Enelow
Schwartzman
Madden
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Things are getting betterAdvances in CF
treatment

• In the 1950s, few children with cystic fibrosis
• lived to attend elementary school.
• Many people with the disease can now
• expect to live into their 40s and beyond.
• Control of bacterial infection and chronic
• inflammation that is a major cause of
• CF-associated morbidity and mortality