The LPS-induced acute-phase reaction and its associated systemically metabolic, clinical and immunological alterations

The acute-phase reaction (APR) is an early, non-specific, non-adaptive defence response of the innate immune system aiming at rapid re-establishment of homeostasis (GRUYS et al.

2005; O’REILLY and ECKERSALL 2014). Depending on their severity, APR induction such as bacterial infection, trauma, neoplasms and other lesions causing cell damage can enlarge the early local immune response to a systemic event with marked metabolic, endocrine and immunological alterations (CHAMANZA et al. 1999; HUMPHREY and KLASING 2004;

GRUYS et al. 2005). In poultry the APR can be induced by local or systemic LPS application experimentally (XIE et al. 2000; CHENG et al. 2004; SHINI et al. 2008; TAN et al. 2014).

At the site of lesion tissue resident sentinel cells such as macrophages and dendritic cells are activated through the interaction of PRR and PAMP that cause the release of diverse mediators (VAN MIERT 1995; MEDZHITOV and JANEWAY 2000; TOMAS-COBOS et al.

2008; HE et al. 2006). These molecules include vasoactive substances (e.g. histamine, prostaglandins, leukotrienes, NO), pain promoting mediators (e.g. bradykinin, serotonin, PGE2) as well as pro-inflammatory cytokines (1β, 6, TNF-α-like), chemokines (e.g. IL-8) and pathogens directly killing RNI and ROI (TIZARD 2009; MURPHEY 2012; JUUL-MADSEN et al. 2014). In particular, chemokines are of crucial importance for attracting further leukocytes from peripheral blood and lymphoid organs to enter the infected tissue via diapedesis and sustain inflammatory response (BAGGIOLINI 1998; LUSTER et al. 1998).

In order to facilitate diapedesis, released vasoactive molecules and TNF-α activate endothelial cells to express adhesion molecules (e.g. selectins), dilate blood vessels, slow blood flow down and increase endothelial permeability (STRIETER et al. 1989; LAMAS et al. 1991;

HARRIS et al. 2002). Thereafter, plasma components (e.g. complement factors) and cells (e.g. leukocytes, thrombocytes, and erythrocytes) leave the blood, enter the site of infection and trigger inflammatory response (GRUYS et al. 2005; JUUL-MADSEN et al. 2014). The

BACKGROUND

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complement factors improve phagocytosis via opsonising bacteria, perform chemotaxis and direct cytolysis of bacteria as well as they enhance T and B cell immunity (HARMON 1998;

CARROLL 2004; RUS et al. 2005).

As a result of early inflammatory response, severe alterations in peripheral leukocyte proportions occur. During the first 24 hours, systemic leukopenia comprising heterophilia, lymphopenia and thrombocytopenia occurs, peaks at 12 hours after induction and changes to a leukocytosis associated with lymphocytosis and monocytosis at 48 to 72 hours (GROSS and SIEGEL 1983; WANG et al. 2003; SHINI et al. 2008; BOWEN et al. 2009).

During the first 6 to 12 hours, heterophils form the first line defence in chickens since they are attracted to the site of infection at first (ANDREASEN et al. 1993; HARMON 1998;

GENOVESE et al. 2013). In contrast to mammalian neutrophils, chicken heterophils lack catalase and myeloperoxidase required for a sufficient oxidative burst (HARMON 1998;

GENOVESE et al. 2013). However, these cells kill bacteria effectively by phagocytosis, degranulation and the synthesis of lysozyme and different kinds of antimicrobial peptides (e.g. cathelicidin-like proteins, defensins; VAN DIJK et al. 2009; GENOVESE et al. 2013).

A further difference to mammalian immune response is formed by chicken thrombocytes, which act as important immune cells supporting innate immune response by phagocytosing bacteria and releasing pro-inflammatory cytokines IL-1β and IL-6 as well as PGE₂ and NO after TLR4 activation (FERDOUS et al. 2008; SCOTT and OWENS 2008; ST. PAUL et al.

2012). After heterophils and thrombocytes, blood monocytes also enter the site of infection, differentiate into macrophages and sustain inflammation further by chemotaxis, phagocytosis, killing bacteria and pro-inflammatory cytokine, NO and PGE2 production (QURESHI et al.

2000; QURESHI 2003; JUUL-MADSEN et al. 2014).

In case of severe APR induction or prolonged exposure to pathogens, a large amount of pro-inflammatory cytokines enter the blood circulation and induce severe metabolic, endocrine and immunological alterations systemically (BESEDOVSKY and DEL REY 1997, 2001;

WIGLEY and KAISER 2003; GRUYS et al. 2005). This process is accompanied by the gradual transition from innate to acquired immunity implemented by the cell-mediated (T and B lymphocytes) and humoral immune response (JUUL-MADSEN et al. 2014). The majority of such systemic alterations results from cytokines’ action on the hypothalamus and its subordinated hypothalamic-pituitary-adrenal axis (CURTIS et al. 1980; KLASING and

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JOHNSTONE 1991). IL-1β primary, and IL-6 and TNF-α secondary induce fever and pain response (JOHNSON 1993; FRAIFELD et al. 1995, 1998; TAKAHASHI et al. 1995), unspecific sickness behaviour, characterised by somnolence, lethargy and anorexia (KLASING and KORVER 1997; XIE et al. 2000; CHENG et al. 2004), and reduced somatotropin secretion (GRUYS et al. 1999; SCANES 2009).

As pro-inflammatory cytokines are rapidly cleared from circulation, the APR can be sustained over a prolonged time by specific plasma proteins, the so-called acute-phase proteins (APP;

CHAMANZA et al. 1999; GRUYS et al. 2005). For that reason, hepatocytes’ protein synthesis and secretion of specific plasma proteins is activated by released IL-6 directly and hypothalamic-pituitary-adrenal-axis through glucocorticoids indirectly (AMRANI et al. 1986;

KLASING and JOHNSTONE 1991). The APP can increase or decrease in their concentration and are termed positive or negative APP, respectively (KLASING and JOHNSTONE 1991;

GRUYS et al. 1994; GRUYS and LANDMAN 1997). While the concentration of albumin, the major negative APP in chicken, decreases to 50 to 75 % of its physiological baseline (ADLER et al. 2001), the concentration of positive APP increases up to 2 to 1000 fold of their basal levels depending on the class of positive APP (GRUYS et al. 2005). These classes are:

minor (e.g. ceruloplasmin, fibrinogen; peak at 24 to 72 hours), moderate (e.g. α1-acid-glycoprotein; peak at 24 to 48 hours) and major (e.g. serum amyloid A; peak at 7 to 10 days) positive APP (CHAMANZA et al. 1999; O’REILLY and ECKERSALL 2014). The functions of APP comprise opsonisation of microbes, chemotaxis of leukocytes and binding plasma cooper, iron and zinc ions to prevent their uptake by microbes (LAURIN and KLASING 1987, TAKAHASHI et al. 1997; WEINBERG 1999).

In addition to immunological changes, the sickness-induced anorexia and metabolic activation of the immune system further dysregulate metabolic homeostasis during early inflammation (ELSASSER et al. 2000; HUMPHREY et al. 2002). The maintenance of certain metabolic pathways and the sufficient realization of fever response require adequate amounts of energy from lipid and protein breakdown (BARACOS et al. 1987; CHIOLÉRO et al. 1997). As the synthesis of protective factors (e.g. complement, APP, cytokines, antibodies) and leukocyte proliferation need increased quantities of amino acids (KLASING and AUSTIC 1984; DAHN et al. 1995; BIOLO et al. 1997), especially aromatic ones (REEDS et al. 1994), the uptake of amino acids by the liver increases severely, in lymphoid organs moderately and decreases in

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skeletal muscular strongly (BARNES et al. 2002; HUMPHREY and KLASING 2004). The cytokine-induced anorexia restricts the dietary input of amino acids further and makes endogenous protein breakdown necessary (KLASING and AUSTIC 1984; KLASING 1988).

As skeletal muscles represents the largest labile pool of amino acids (HENTGES et al. 1984;

TIAN and BARACOS 1989), TNF-α coordinates in mammals the required protein degradation and following release of amino acids from skeletal muscles and the reduced hepatic synthesis of negative APPs (ROSENBLATT et al. 1983; SAX et al. 1988; COONEY et al. 1997; GRUYS et al. 2005). As a result of these severe alterations in protein and amino acid metabolism, the whole body nitrogen balance expresses strong changes (BIOLO et al.

1997; BREUILLE et al. 1999; DICKERSON et al. 2001; BRUINS et al. 2002). In chronic stages of inflammation, IL-1β-induced anorexia and TNF-α-induced protein catabolism can lead to severe cachexia well visible by declines in growth rate and protein accretion (BENSON et al. 1993; WEBEL et al. 1998).

SCOPE OF THE THESIS

20 SCOPE OF THE THESIS

The present thesis aimed to test the following hypotheses derived from scientific literature referred to in introduction and background:

1. A marginal dietary Arg supply induces adaptive difficulties in growth and laying performance which are less pronounced in genetically low performing layer-type chickens than in high performing ones.

2. The metabolic and immunological response of genetically high performing layer-type chickens to a LPS-induced acute-phase reaction is more pronounced by a dietary Arg supply beyond the requirement for optimal growth and performance when compared to low performing strains.

These hypotheses were tested using a chicken model (Figure 6) that contrasted four purebred layer lines differing in their performance level (high vs. low) and phylogenetic origin (white vs. brown layer) each. In a first step genotypes’ suitability for investigating both hypotheses named above were verified by characterising female chickens of these genotypes in their growth and performance potential from hatch to the 74^th week of age under commercial feeding conditions (Paper I).

Figure 6. Depiction of the established chicken model contrasting four different genotypes.

In a second step two experiments were conducted to examine genotypes’ adaptability and sensibility to varying concentrations of dietary Arg. For that reason, female chickens of each

SCOPE OF THE THESIS

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genotype were long-termly supplied with graded dietary Arg equivalent to 70, 100 and 200 % of recommended age-dependent Arg level (NRC 1994). From hatch to the 41^st week of age the effects of dietary Arg on growth and performance traits of genotypes were investigated (Paper II). In a parallel study female chickens of each genotype were reared under the same conditions described in Paper II to examine the effects of dietary Arg on growth of internal organs from hatch to the 18^th week of age (Paper III).

Furthermore, in two separate experiments the interacting effects of a long-term graded dietary Arg supply and a single E.coli LPS injection on the metabolic, clinical and immune response in chickens of different genetic backgrounds were studied until 48 hours post-injection.

Therefore, 12-week-old cockerels (Paper IV) and 18-week-old pullets (Paper V) reared under the same conditions, described in the preceding studies (Paper II and III), were one-time treated with 2 mg LPS/ kg BW i.m.. In differently Arg supplied cockerels the LPS-induced alterations in haematology, body temperature, feed intake and weight gain were investigated until 48 hours post-injection (Paper IV). Additionally, the LPS-induced changes in metabolic and clinical response were closer studied in differently Arg supplied pullets by examining nitrogen balance, plasma amino acids, clinical scoring and core body temperature until 48 hours post-injection (Paper V).

The sequential course of studies carried out in this thesis is given in Figure 7.

Figure 7. Schematic presentation of studies carried out in the present thesis (I – V: Paper of the related studies).

PAPER I

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PAPER I

Phylogenic versus selection effects on growth development, egg laying and egg quality in purebred laying hens

M.-A. Lieboldt¹, I. Halle¹, J. Frahm¹, L. Schrader², U. Baulain³, M. Henning³, R. Preisinger⁴, S. Dänicke¹ and S. Weigend³

1 Institute of Animal Nutrition, Friedrich-Loeffler-Institute, Federal Research Institute for Animal Health, Braunschweig, Germany

2 Institute of Animal Welfare and Animal Husbandry, Friedrich-Loeffler-Institute, Federal Research Institute for Animal Health, Celle, Germany

3 Institute of Farm Animal Genetics, Friedrich-Loeffler-Institute, Federal Research Institute for Animal Health, Neustadt-Mariensee, Germany

4 Lohmann Tierzucht GmbH, Cuxhaven, Germany