The fish immune system

The fish immune system is tasked with protecting the organism against a wide range of pathogens. The fish immune system, as in mammals, is comprised of an innate and an acquired component. An effective immune response must be able to rapidly determine self from non-self and induce a proportional response to the pathogen. The first line of defence consists of the surface barrier which is in constant contact with the environment. In fish, the barrier consists of the skin, gills and gut, which are coated in a layer of mucus containing antimicrobial peptides (MAREL et al. 2012).

However, once the physical barrier is breached the pathogen must first be identified by pattern recognition receptors (PRRs). PRRs recognise pathogen associated molecular patterns (PAMPs) and damage associated molecular patterns (DAMPs) and are located either on the membrane or in the cytoplasm of the cell (BIANCHI 2007). There is a wide range of receptors present in fish and more are being discovered. However, there are large differences between mammalian and fish and between fish species (PALTI 2011). There are currently 21 Toll like receptors (TLRs) identified in fish, with 7 unique to teleosts (TLR 5s, 14, 19, 20, 21, 22 and 23). Each receptor is responsible for detecting a specific group of pathogens, for instance mammalian TLR 4 has been shown to bind to ligands such as LPS found on bacteria such as Aeromonas hydrophila in rare minnow (Gobiocypris rarus) (SU et al. 2009; AOKI et al. 2013). TLR 3 and TLR 9 have been described in carp and have been shown to detect virus associated dsRNA and dsDNA respectively in teleosts (YANG u. SU 2010; AOKI et al. 2013).

Once a potential pathogen has been identified, specific cells and organs are tasked with

inducing a specific response. In mammals, antigens will be presented in the lymphoid tissues

and new immune competent cells are produced in the bone marrow. However, as fish lack

bone marrow, the head kidney (also known as pronephros), trunk kidney and the thymus are

the main sites of pluripotent stem cell production (ZAPATA et al. 2006).

Chapter 1 Introduction

11

The pronephros plays a major role in the acquired immune system, where macrophages phagocytose antigens and thus plays an important role in immunological memory (URIBE et al. 2011). Similarly the thymus also plays an important role and has been described as a site for T cell production (URIBE et al. 2011).

Fish also have an active and important acquired immune system. Antibody producing B cells are produced in the kidney (URIBE et al. 2011) and thus fish can utilise antibodies in their immune repertoire against pathogens. Natural antibodies are present in significant concentrations in teleost plasma and IgM and IgD immunoglobulins. Recently IgT has been identified in teleost mucus (ZHANG et al. 2010).

1.4.1 Innate immune system

The teleost innate immune system is, like in mammals, comprised of cellular and humoral components. However, as fish are evolutionary precursors to mammals, the acquired immune system does not appear to have developed to the same level as seen in mammals.

Although fish have the equivalent immune organ homologues to mammals, they exhibit lower levels of structural complexity, which potentially limits the capability of their acquired immune response (L. TORT 2003).

The innate immune system comprises of a cellular and humoral response. Fish, like mammals, contain a large repertoire of cytokines, which act as signal conductors within the immune system (URIBE et al. 2011) and are divided into pro- and anti-inflammatory cytokines. Antimicrobial peptides (AMPs) have been shown to be very important in the immune defence against bacteria and viruses (ELLIS 2001; MAIER et al. 2008). AMPs such as defensins have shown to be involved in the immune defence of Chinese loach (Paramisgurnus dabryanus) against bacteria, including Aeromonas hydrophila (CHEN et al.

2013). Furthermore, β defensins have been described in carp and they have also been shown to be tissue specific and up-regulated by β-glucans (MAREL et al. 2012).

Fish have a strong arsenal of immuno-competent cell types including macrophages, mast

cells and polymorphonuclear (PMNs) leukocytes. Macrophages are able to phagocytose

bacteria, and along with neutrophils are the two most prominent cell types which are

involved in phagocytosis in fish (SECOMBES u. FLETCHER 1992). Furthermore, phagocytosis

is even more important to poikilotherms as this process is relatively not so adversely

12

affected by changes in temperature (BLAZER 1991; LANGE u. MAGNADOTTIR 2003;

MAGNADOTTIR et al. 2005).

Mast-like cells have been identified in zebrafish (S. DA'AS et al. 2011; S. I. DA'AS et al. 2012), however, there is still some controversy due to differing tissue distribution and staining profiles compared to their mammalian counterparts (REITE u. EVENSEN 2006).

Finally, PMNs such as neutrophils are a critical component of the innate immune system and can perform four main functions; degranulation (PALIC et al. 2005), cytokine release (KASAMA et al. 2005), phagocytosis (W. L. LEE et al. 2003b) and the production of neutrophil extracellular traps (BRINKMANN et al. 2004).

1.4.2 Neutrophil extracellular traps

Neutrophils are an important component of the innate immune system in fish. Neutrophil extracellular traps (NETs) have recently been identified as a novel important host innate immune defence mechanism against pathogens in mammals such as human (BRINKMANN et al. 2004), mice (BUCHANAN et al. 2006), cats (WARDINI et al. 2010) and zebrafish (PALIC et al. 2007b). NETs consist of a nuclear DNA backbone associated with antimicrobial peptides and stabilising proteins such as histones, which are released during a kind of programmed cell death, known as NETosis. NETs have been shown to be responsible for the extracellular entrapment and in some cases killing of invading pathogens (FUCHS et al. 2007).

NETs in fish have been described in zebrafish and fathead minnow (PALIC et al. 2007a;

JOVANOVIC et al. 2011). Kidney derived neutrophils were shown to produce NETs when

incubated in vitro with β-glucan. However, fish NETs have not as yet been shown to be

functionally active. Therefore, the role of carp derived NETs in response to the bacterium

Aeromonas hydrophila was investigated, with focus on the entrapment and killing ability

(chapter 2) and the host evasion strategies employed by the bacterium and how β-glucan is

able to stabilise and protect against the host evasion strategy employed (chapter 3).

Chapter 1 Introduction

13

Figure 1. Diagram showing the process of NET formation. Annotations in blue have been described in fish (PALIC et al. 2007a). Black annotations have only been described in mammals at the time of writing (FUCHS et al. 2007; VON KOCKRITZ-BLICKWEDE u. NIZET 2009).

As this new immune mechanism has only recently been discovered, large areas in the

understanding of NETosis still remain unanswered. NETosis was first described as a

programmed cell death mechanism which involved the release of DNA fibres bound to

14

specific antimicrobial proteins, however, recent research has shown that after neutrophils have released their DNA, they are still able to simultaneously function by crawling towards a stimulus and phagocytising bacteria (YIPP et al. 2012).

The mechanism of NET formation is a complicated and not fully understood process. Firstly a neutrophil must be activated by a cytokine such as IL-8 or IFNα/γ, or a PAMP such as LPS from bacteria such as

Staphylococcus aureus (PILSCZEK et al. 2010). Stimulation of

neutrophils will lead to the activation of NADPH oxidases which will catalyse the conversion of NADPH to NADPH

⁺

+H

⁺

N and the release of reactive oxygen species (ROS). ROS signalling is commonly described as a prerequisite with the start of NETosis, although ROS independent NETosis has also been observed (MARCOS et al. 2010) and also presented in this thesis in chapter 4. The next step is the disruption of the nuclear membrane and decondensation of the chromatin leading to the mixing of nuclear DNA and proteins, mostly histones. Interestingly the majority of the proteins comprising the NETs do not originate in the cytoplasm, with the greatest percentage of proteins originating from the nucleus, including histones 2A (26.29 %), 2B (23.95 %) and H3 (14.50%) (URBAN et al. 2009). Finally, the DNA strands encrusted with stabilising proteins is released into the external environment and thus producing an extracellular trap cable of entrapping and in some cases killing bacteria.

Interestingly, the role of lipids in NET formation has only tentatively been explored. Firstly,

Oh H

et al. demonstrated that higher levels of cholesterol led to slower rolling behaviour,

which was attributed to an increase in the length of tethers produced and an increase in cell

deformity leading to an increase in contact surface area (OH et al. 2009). Furthermore,

Chow

et al. (CHOW et al. 2010), demonstrated that treatment of isolated neutrophils with

the cholesterol synthase inhibitor Mevastatin induced the formation of NETs. These results

show that cholesterol plays an important role in neutrophils function. However, detailed

insight into the mechanism underlying NET-formation is still missing, e.g. it is still completely

unclear how a membrane remodelling is involved in the process.

Im Dokument Cell-pathogen interactions in common carp (Cyprinus carpio L.) (Seite 18-22)