Transcript The interplay of infection, stress and the immune response

The interplay of infection, stress
and the immune response
Abraham J Pellissery
29th April, 2015
Transition Cows, Immunosuppression and
Disease susceptibility
• Transition/Periparturient period in cows
• Periparturient period – Dry cow and Lactating cow
• Homeostasis vs Homeorhesis
• Negative Energy Balance and Metabolic stress
• Effects of Non-esterified fatty acids on the innate immune system
• Effects of Ketone bodies on the innate immune system
• Bovine Mastitis and Ketosis
Transition cow
• Transition period of dairy cows – defined as a period ~3 weeks
prepartum through 3 weeks post-partum
• Significant physiologic and immunologic changes –
• Shifts from a gestational non-lactating state to the onset of lactation
(Grummer, 1995)
• Period of hormonal change and stress – gestational and lactation
• Metabolic and infectious diseases – mastitis, metritis, ketosis and
displaced abomasum (Le Blanc et al, 2006)
Periparturient period – Dry cow and Lactating cow
90-Day “Transition”
Far Dry Period
-60
Close-up Dry Period
-21
Calving
0
Fresh/ Early Lactation
30
305
days
Adapted from Transition Management of Dairy Cattle by Michael Overton – Elanco Knowledge Solutions
Reasons for stress during transition period
• Changes in nutrient requirements that occur at the time of calving –
• Glucose and amino acid requirements increase in order to support fetal
development
• Milk synthesis and secretion – increased demands for energy, protein and
calcium
Sordillo and Mavangira, 2014
Homeostasis vs. Homeorhesis
• Living systems are in a permanent physiological stable state Homeostasis
• Living systems seem to be characterized by changing energy dynamicsHomeorhesis
De la Fuente et al, 2014
Homeorhetic adaptation
Negative Energy Balance (NEB) and Metabolic
stress
650
550
21
DMI (kg/d)
19
450
17
350
15
13
250
11
150
9
7
50
-21 -18 -15 -12 -9
-6
-3
0
3
6
9
Day relative to calving
12 15 18 21
NEFA, mEq/L
23
DMI
NEFA
Strucken et al, 2015
• Rapid increase of milk production immediately after calving
• Require more energy for maintenance, milk production and growth
• Mild physiological NEB observed
• Cows fail to adapt to rapid foetal growth, calving, high-energy
demands of lactation and the consequent perturbations in nutrient
metabolism  Metabolic stress
• Managing energy balance and immune function helps mitigate impact
of problems arising during transition phase in cows
Suriyasathaporn et al, 2000
• 7–9 week period of lactation – peak production is attained between
4th and 6th week
• Body reserves utilized  potential increase of ketone bodies in milk
• Suboptimal feed quality, feed quantity, or the management of feed
supply  severe, decompensated NEB  Ketonaemia  Clinical
ketosis.
Suriyasathaporn et al, 2000
• Predisposing factors for a decompensated NEB –
• Cows calving in fat conditions  Lower feed intake
• Excess of rumen degradable protein in the ration requires extra energy for the
removal of surplus ammonia from the rumen  higher incidence of clinical
ketosis
• Insufficiency of ruminal microbes for digestion of concentrates after calving –
Dry cow feeding vs. Lactation feeding regimes
Suriyasathaporn et al, 2000
Lean et al., 1994; Rukkawamsuk et al., 1998
Periparturient Energy Metabolism, Immune
Function and Disease
Hypocalcemia (clinical or
subclinical)
Severe or Poor Response to
Negative Energy Balance
(i.e., low DMI)
Reduced Function of Neutrophils
And/or Lymphocytes
Increased Disease Susceptibility
Mastitis
• Incidence
• Severity
• Duration
Retained
Placenta
Uterine Infections
• Endometritis
• Chronic infections?
Bovine Mastitis and Ketosis
• Bovine Mastitis –
• Mastitis is the inflammatory response of the mammary gland (MG) tissue to
physiological and metabolic changes, traumas, and allergies and, most
frequently, to injuries caused by various microorganisms
• Common disease, and the economic loss due to mastitis in dairy cattle is
estimated at $185/cow/year annually in the US This totals more than 2 billion
dollars annually in the US alone
• Losses are in the form of discarded abnormal milk from clinically infected
quarters and as the result of antibiotic therapy, cull cow replacement costs,
extra labor to handle mastitic cows, antibiotic and other treatment costs,
veterinary services, and most importantly, reduced milk production in
subclinically infected cows, which contributes to two-thirds of mastitis losses
Akers and Nickerson, 2011
• Bovine Ketosis• Blood levels of BHBA and its metabolite acetoacetate are elevated
• Results from an impaired glucose homeostasis
• Associated with increased risk of infectious diseases - Mastitis
Grinberg et al, 2008
Effects of Non-esterified fatty acids (NEFA’s) on the
innate immune system
• Effects are inconclusive or poorly understood
• Lipids activate leuckocytes –
• De Vries et al. (2015)- examined leukocyte activation between T2D and
hyperlipidemia (FHC) patients where FHC patients experienced elevated
leukocyte activation when compared with controls.
• Circulating NEFA prepartum were considered a better risk factor for the
development of metritis, milk fever and retained placenta than plasma BHB
and glucose or calculated energy balance (Moyes et al., 2013)
• In general, unsaturated fatty acids (e.g. C18:1, C18:2 and C20:4) impair the
immune response whereas saturated fatty acids (e.g. C12:0, C14:0 and C16:0)
improve the immune response (Lee et al., 2001; Lee et al., 2004; Lee and
Hwang, 2006).
• Saturated fatty acids may also be responsible for stimulation of toll-like
receptor (TLR) signaling (Moyes et al., 2010; Sordillo et al., 2009).
• During the normal course of lipolysis after parturition, the circulating NEFA
pool becomes enriched with the major fatty acids of adipose tissue, such as
16:0 (Douglas et al., 2007).
• The TLR are activated by lipids including lauric acid (12:0), myristic acid (14:0),
and 16:0 (Lee and Hwang, 2006).
• Brassard et al., 2007; Lacetera et al., 2004 - identified negative relationships
between elevated circulating NEFA and immunosuppression
• Scalia et al. (2006) reported increased in vitro phagocytosis-associated
oxidative burst activity whereas viability was reduced in bovine blood
neutrophils.
• Matrix Metalloproteinases 8 & 9/ Tissue Inhibitor of
Metalloproteinases (TIMP) 
• Burton et al, 2004; Madsen et al, 2004 and Ferris et al, 2006 - observed a
tremendous increase in neutrophil counts and expression of MMP-8 and
MMP-9 during labor, delivery, and 1 to 2 days after calving. In addition, there
was inhibited expression of TIMP genes and genes that normally keep TIMP
expression at normal levels during this period.
• HIgh MMP activity in the blood serum of these cows, suggesting that
neutrophils become activated into a highly pro-inflammatory state with
increased tissue destroying capacity around calving
Effects of Ketone bodies on the innate
immune system
• BHB, have been shown to negatively impact the immune response
including reduced trap formation, chemotaxis and phagocytosis of
neutrophils and reduced lymphocyte blastogenesis (Grinberg et al.,
2008; Ingvartsen and Moyes, 2013).
• Immune cells do not use ketone bodies as a fuel cell source
(Newsholme et al., 1987) nor ketones enhance immunity in vitro
(Ingvartsen and Moyes, 2013).
Introduction and Aim of the study
• Ketosis poses a significant risk for Mastitis
• Hyperketonemia in an E.coli mastitis model showed increased
severity of disease which was attributed to neutrophil dysfunction
• Effect of beta hydroxybutyrate(BHBA) on NET formation
Neutrophil Extracellular Traps
• Neutrophils eliminate
extracellular microbes by
releasing NETs
• Core DNA element + Histones +
Lactoferrin, Cathepsins + MPO,
elastase
• Helps immobilize pathogens and
prevents them from spreading
• Facilitates phagocytosis
• Able to kill pathogens directly by
means of antimicrobial histones
and proteases
Kolaczkowska and Kubes, 2013
Materials and methods
• Isolation of blood neutrophils – Diff-Quik staining
• E.coli strain P4 serotype O32:H37
• pSA11 carrying lacIq and GFP gene under the regulation of the tac
promoter – GFP expression was achieved by IPTG/Lactose
• Neutrophil phagocytosis and bactericidal activity assays
RPMI + Bacteria with
no neutrophils
RPMI + Bacteria with
neutrophils
RPMI + Bacteria with
neutrophils + 20%
heat inactivated
serum
RPMI + Bacteria with
neutrophils + 20% heat
inactivated serum +
cytochalasin D
RPMI + Bacteria
with neutrophils +
20% heat
inactivated serum +
100U/ml DNase
• Plates were incubated for 5, 10, 20 and 30 minutes at 37°C in a humidified
CO2 incubator and thereafter kept on ice for further processing
• Plates centrifuged
• At each time point 
• Supernatant from control and treatment wells were collected
• Neutrophil pellets were suspended in PBS containing 50μg/ml gentamicin to kill
any adherent extracellular bacteria  neutrophils were lysed with Triton to
release intracellular bacteria
• All samples obtained were cultured in LB agar to determine the CFU count
• Percentage of extracellular and intracellular killing was calculated all treatments
• NET formation by stimulation with bacteria
• Neutrophils were seeded on coverslips treated with 0.001% poly-L-lysine and
placed in 24 well plates
• Plates were centrifuged and cells were allowed to adhere by incubation at the
same conditions for 1hr
• Neutrophils were infected with P4 and incubated for 5, 10, 20, and 30min in
the same incubation conditions
• In replicated control wells, infected neutrophils were treated with 100U/ml
DNase or left uninfected
• Cells were washed with PBS and stained with 5μM Sytox Orange in the dark
for 15min at room temperature
• Slide mounted and view under a epifluorescence microscope
• Effect of BHBA on neutrophil phagocytosis and bactericidal acitivity
• Neutrophils were preincubated for 40min at 37°C (in CO2 incubator)
containing 0, 0.1, 1.4 or 8 mmol/L BHBA
• Phagocytic and bactericidal, as well NET formation was tested as explained
above
Conclusions
• Demonstrated a strong negative effect of BHBA on NET formation and
bactericidal activity
• Indicates that NET formation would be impaired in hyperketonaemic
animals and hence would render them more sensitive to infection
• NETs are extremely sensitive to the destructive effect of DNase –
these are mostly seen as virulence factors
• Signaling mechanisms of NETosis during hyperketonaemia need to be
elucidated
Introduction
• Neutrophils are the first line of host defense
• One of the first cells to migrate from the blood into injured or
infected tissues
• Exert defensive roles by• Oxidative mechanisms – ROS production
• Non-oxidative mechanisms – release of granules that contain proteolytic
proteins – MMP
DMI
NEFA
650
550
21
19
450
17
350
15
13
250
11
150
9
7
50
-21 -18 -15 -12 -9
-6 -3
0
3
6
9 12 15 18 21
Day relative to calving
NEFA, mEq/L
23
DMI (kg/d)
• FFA regulate immune and
inflammatory responses
• FA markedly increase
intracellular and extracellular
ROS production in rat and
human neutrophils in vitro
• Bovine neutrophils – C18:1
increases intracellular
superoxide in an intracellular Ca
dependent manner
• C18:2 and C18:1 rapidly increase MMP-9 activity in a Ca dependent
manner
• MMP-9 secretion in breast cancer cells occurs through PKC, Src and
EGFR-dependent pathways
Free fatty acid receptors
• Seven transmembrane domain receptors
• FFAR1 or GPR40 (G Protein coupled receptor 40) – is a receptor for
MCFA and LCFA like C22:6, C20:5, C18:2 and C18:1
• FFAR1 mediates insulin secretion from pancreatic cells – Insulin
mediates lipogenesis
• FFAR1 has a role in cellular proliferation and innate immunity – it is
seen in bovine neutrophils and has a role in its activation
Aim of the study
• Cloned bovine cDNA of bovine FFAR1 receptor and conducted
sequence analysis
• Functional studies were conducted by expressing the receptor in
CHO-K1 cells and in bovine neutrophils
• Intracellular MMP-9 release and ROS production in relation to FFAR1
receptor activation and inhibition
Materials and methods
• Neutrophils were isolated from 5 healthy Holstein heifers
• Total RNA extraction – RT-PCR using bFFAR1 specific primers
• Cloned gene using pGEM-T vector
• Sequence analysis and homology modelling – BLAST, Clustal Omega,
EMBOSS Transeq, MODELLER
• pcDNA3.1 vector used for expression of bFFAR1 in CHO-K1 cells using
FuGene 6 reagent
• Measurement of i/c Ca levels in CHO-K1/b using Fura-2AM
fluorescent indicator dye – using FA’s, agonist and antagonists
• Measurement of i/c Ca levels in bovine neutrophils using Fluo-4AM
• MMP-9 activity – cells treated with agonists and/or antagonists were
screened for the presence of gelatinase activity by zymography
• ROS production - Neutrophils treated with HE is allowed to react with
different antagonist and/or agonists
Conclusion
• Confirmed the presence and identity of FFAR1 receptor in bovine
neutrophils which are activated by C18:1 and C18:2 and not C3 fatty
acids
• PLC-PKC signaling controls the release of MMP-9 granules and ROS
production via FFAR1 activation
Future studies
• To identify hydroxycarboxylic acid receptor-2 in bovine neutrophils specific for
ketone bodies (eg. BHBA) and assess whether Raf-MEK-ERK pathway is
downregulated during NET formation
Thank you
Questions ?????