MASTITIS is a condition which
often emerges as a result of
immunological dysregulation.
This was the view that emerged
from the recent Immunity Science
Symposium hosted by Elanco Animal
Health in which six professors from
around the world described how the
transition cow represents a significant
challenge when it comes to immunity.
Describing
and reviewing
recent
literature,
the speakers
provided a
fresh look at
mastitis, with
a focus on
how the dairy cow responds differently
to different pathogens and the impact
of immunological mechanisms on the
presentation of clinical disease.
Pathogen responses
Professor Shpigel from Israel began
by looking at the molecular basis
for mastitis. This provided useful
explanations for the tendency of
mastitis to recur in individual animals
and for the sometimes poor response
to antibiotics.
He described how pattern
recognition receptors (PRRs) on
macrophages in the udder include
TLR4 (toll like receptor 4) and CD14.
The PRRs on these cells of the
innate immune system are able
to recognise specific areas on the
pathogen – pathogen-associated
molecular patterns (PAMPS). Only a
few of these sentinel macrophages are
likely to be present and mastitis is also
characterised by massive infiltration of
neutrophils.
Lipopolysaccaride (LPS)
fragments from bacteria such as E.
coli are recognised by TLR4 on the
macrophages – not the epithelial cells
– and trigger release of the cytokine
tumour necrosis factor alpha (TNFα).
This LPS-provoked signalling
prompts activation of “nuclear
factor kappa-light-chain-enhancer of
activated B cells” (NF-кB) in epithelial
cells.
NF-кB controls DNA transcription
and results in the release of additional
interleukins and cyclo-oxygenases;
in turn triggering an inflammatory
response and neutrophil recruitment.
This is followed by a bystander’s effect,
where adjacent epithelial cells are
recruited, starting a chain reaction.
Other bacterial components in E. coli
can trigger mastitis in non-TLR4 mice, so it is known that this is not the only
response occurring in the presence of
E. coli. Bacterial lipoproteins (BLP)
also signal via TLR2 (receptors) on the
epithelial cell.
In infected cells there may be an
absence of inflammatory response
due to alterations in signalling. Some
pathogens, including E. coli, may
survive within neutrophils (and
epithelial cells) allowing them to act as
“Trojan horses”, providing protection
from antibiotics and allowing resurgent
cases of mastitis to occur.
Macrophages usually control
neutrophil lifespan. As there are few
macrophages in the alveolus of the
teat, disposal of apoptotic neutrophils
and regulation of the inflammatory
response can be problematic.
An alternative mechanism may
exist in the close partnership
between epithelium and neutrophil
in the induction and resolution of
inflammation.
Recurrent and
resurgent mastitis
Professor Hans-Martin Seyfert of the
University of Rostock in Germany
picked up on the theme of resurgent
cases of mastitis and explored it in
more depth.5
While E. coli produces a very severe
mastitis, even shutting down casein
synthesis, S. aureus produces signs
that are subclinical but result in high somatic cell counts and
disease is often chronic in
nature.
This difference has been
attributed to inappropriate
calibration of the immune
response. E. coli tends to
provoke a strong immune
response both locally and
systemically and mastitis
due to the pathogen is
associated with fever and
udder necrosis. Mastitis due to S. aureus lasts for seven months
on average and 30-40% of cases are
not cured by antibiotics: the immune
response is weak.
There are over 100 different genes
controlling antibacterial peptides in the
cow compared to just two in humans,
which goes some way to explaining the
complexity of response.
Specifically in E. coli mastitis, TLRs
are upregulated and there is faster,
stronger induction of cytokines such as
IL8 and TNFα.
In S. aureus a more belated, weaker
response means that there is a low
level of cytokine response and no beta
defensins are induced; some quarters
may respond, but others will not. This
pathogen specific modulation of the
immune response in the udder explains
the big differences between how these
cases present.
Immunity processes
Both E. coli and S. aureus activate toll-
like receptors but S. aureus does not
activate NF-кB in mammary epithelial
cells. NF-кB, as already discussed, can
be thought of as the “master switch”
for immune gene expression.
Delayed and weaker regulation of
fewer genes in comparison to E. coli
means that S. aureus results in reduced
expression of pro-inflammatory
cytokines such as TNFα and IL6.
There are likely to be two processes
going on in these mammary epithelial
cells – one dependent on the MγD88
gene and one independent of MγD88. Induction of TNFα and IL1 is
dependent on MγD88, induction of
IL6 is independent of MγD88.
In the presence of E. coli there is
a MγD88 dependent response, early
induction of TNFα and IL1 and a
further late response triggered by these
cytokines as they recruit additional
cytokines that are inflammatory,
antimicrobial and anti-apoptotic.
Both E. coli and S. aureus can induce
a MγD88 independent response that
results in IL6 production. In turn, this
activates mitogen-activated protein
kinase (MAPK) and Janus kinase
(JAK) signalling, resulting in only
weak in ammation and no pathogen
clearance.
This means that pathogen clearance
for S. aureus, relying only on this
weaker response, is much reduced and sets the scene for more
long-standing chronic
infections.
Provoking adaptive
responses
Professor Dirk Werling
from the RVC talked
about how these processes mean that the udder heavily
relies on innate immune responses,
with adaptive immunity (and therefore
“memory”) playing no major role in
mounting a response to the disease.
He highlighted how specific
pathogens are recognised by specific
pathogen recognition receptors (PRRs)
and prompt particular responses by
the innate immune system – whether
that might be macrophage activation,
phagocytosis or complement
activation.
In other diseases, an innate response
can trigger a further response based on
adaptive immunity but in mastitis this
doesn’t occur – there is “no back-up
plan”.
The presence of a body-udder
barrier and barriers between the
quarters imply that acquired immunity
may not extend between systems. S.
aureus, in particular, has developed
a number of mechanisms to avoid
recognition by the immune system and
block T cell activation.Adjuvants may
hold one key as to how this could be
overcome.
Adjuvants were referred to by
Janeway (1989) as “the immunologist’s
dirty little secret”: it is known that they
work, but not precisely how they work.
Different adjuvants work at different
sites which can include antigen uptake
and presentation by antigen presenting
cells, or on the TLRs of the cells of
the innate immune system.
There are many new adjuvants being
used in human vaccines that have been tested on vaccines
against S. aureus. Some
of these have increased
the antibody titre in
cattle and reduced
somatic cells counts
but some side-effects
occurred.
There is, however,
a need to differentiate
between an antibody response and a protective response as
both a T cell and humoral response is
required. This is perhaps not surprising
given that adaptive immunity is not a
feature of the natural host response to
mastitis.
The characteristics of the pathogen,
the effect of the environment and
various host factors can all influence
the extent to which a vaccine can ever offer protection, so clearly there
is some way to go before mastitis
can ever be completely addressed by
vaccination.
Delegates left the seminar with a
clearer understanding of mastitis and
host responses at a molecular level.
Different manifestations of the disease
attributed to different pathogens
are largely occurring because of
differences in how the host immune
system interacts with these pathogens.
The health of the innate immune
system and the factors that adversely
affect or enhance immune function and
expression are likely to have a major
impact upon the performance and
welfare of the transition cow during
the vital 90 days around calving.
1. Shpigel, N. Y. (2013) Neutrophils – not
just killing machines. Facilitator of bacterial
colonisation and in ammation control.
Elanco Science Immunity Symposium:
Vienna.
2. Gonen, E., Vallon-Eberhard, A., Elazar,
S., Harmelin, A., Brenner, O., Rosenshine,
I., Jung, S. and Shpigel, N. Y. (2007) Toll
like receptor 4 is needed to restrict the
invasion of Escherichia coli P4 into mammary
gland epithelial cells in a murine model of
acute mastitis. Cellular Microbiology 9 (12):
2,826-2,838.
3. Kasper, C. A., Sorg, I., Schmutz, C.,
Tschon, T., Wischnewski, H., Kim, M.
L. and Arrieumerlou, C. (2010) Cell-Cell
Propagation of NF-кB Transcription
Factor and MAP Kinase Activation
Ampli es Innate Immunity against
Bacterial Infection. Immunity 33: 804-816.
4. Mintz, M., Mintz, D., Ilia-Ezra, R. and
Shpigel, N. Y. (2013) Pam3CSK4/TLR2
signaling elicits neutrophil recruitment and
restricts invasion of Escherichia coli P4 into
mammary gland epithelial cells in a murine
mastitis model. Veterinary Immunology 152:
168-175.
5. Seyfert, H.-M. (2013) Innate immunity:
activation of the initial immune response
during pathogen-speci c mastitis. Elanco
Science Immunity Symposium: Vienna.
6. Yang, W., Zerbe, H., Petzl, W., Brunner,
R. M., Günther, J., Draing, C., von Aulock,
S., Schuberth, H.-J. and Seyfert, H.-M.
(2008) Bovine TLR2 and TLR4 properly
transduce signals from S. aureus and E.
coli, but S. aureus fails to both activate
NF-кB in mammary epithelial cells and to
quickly induce TNFα and Interleukin-8
(CXCL8) expression in the udder. Molecular Immunology 45 (5): 1,385-1,397.
7. Günther, J., Poschadel, N., Petzl, W.,
Zerbe, H., Mitterhuemer, S., Blum, H. and
Seyfert, H.-M. (2011) Comparative kinetics
of E. coli v. S. aureus speci c activation
of key immune pathways in mammary
epithelial cells: S. aureus elicits a delayed
response dominated by IL-6, but not by
IL-1A or TNFα. Infection and Immunity 79:
695-707.
8. Schukken, Y. H., Günther, J., Fitzpatrick,
J., Fontaine, M. C. et al (2011) Host- response patterns of intramammary
infections in dairy cows. Veterinary
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9. Janeway, C. A. Jr. (1989) Review:
Approaching the asymptote? Evolution and revolution in immunology. Cold Spring
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10. Werling, D. (2013) Innate Immunity.
Communication between innate and
acquired: how this may affect vaccines.
Elanco Science Immunity Symposium:
Vienna.
