The interplay of infection, stress and the immune

responseAbraham 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 Close-up Dry Period Calving Fresh/ Early Lactation

Adapted from Transition Management of Dairy Cattle by Michael Overton – Elanco Knowledge Solutions

-60 -21 0 30

305 days

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 dynamics-

Homeorhesis

De la Fuente et al, 2014

Homeorhetic adaptation

Negative Energy Balance (NEB) and Metabolic stress

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FA

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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 DiseaseSevere or Poor Response to

Negative Energy Balance(i.e., low DMI)

Reduced Function of NeutrophilsAnd/or Lymphocytes

Increased Disease Susceptibility

Mastitis• Incidence• Severity• Duration

Retained Placenta

Uterine Infections• Endometritis• Chronic infections?

Hypocalcemia (clinical or subclinical)

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

• 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

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-21 -18 -15 -12 -9 -6 -3 0 3 6 9 12 15 18 21

Day relative to calving

DM

I (k

g/d

)

50

150

250

350

450

550

650

NE

FA

, mE

q/L

DMI

NEFA

• 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 ?????