The Comparison of Adipose Tissue Immune Response against

Bacterial and Viral Infection in Fish (Teleost): a Review

Mochamad Aji Purbayu

Abstract

In general, adipose tissue is prevalent in the vertebrates. The adipose tissue is where the lipid
stored and has a responsibility for the source of energy, insulator, support body metabolism
and immune response function. Mammals and human adipose tissue have well defined to have
the capability of providing an immune response against the foreign pathogen. They can release
immune response components such as hormones, immune response cells, and cytokines. There
are still a few studies which are focused on fish adipose tissue in their immunological activities.
Previous study of the adipose tissue immunologically response against bacterial-source antigen
described that the immunologic activity in the fish adipose tissue was significant increases as
indicated by upregulation of several primary immune response genes (such as Acute Phase
Protein, Cytokines, and immune cells). Recent study of fish adipose tissue in response to viral
infection revealed the role of fish adipose tissue during viral infection and replication. Viral
infection triggered upregulation of some immune genes especially those that express proteins
specific to counteract viral properties (such as Interferon, Chemokines, Immunoglobulin, and
Toll-Like Receptors). In conclusion, both bacterial and viral infection in fish definitely will
activate fish adipose tissue to give immune response through immune elements which have a
unique characteristic when the infection came from bacteria or viruses.

Introduction

A. Fish immune system

The immune system is defined as a defense of the body through biological components against
largely various foreign infiltrator including the viral, bacterial, and parasitic agent. The immune
system can recognize the differences of the foreign agents and parts of the body (Rauta et al.,
2012). The immune system is prevalent in organisms from a very simple system to complex
immune system (Beck and Gail, 2007). The system is known to have a cascade of defense
against foreign substances. The outer layer which has a direct contact with the environment is
considered as a physical barrier (Bruce et al., 2002; Boyton and Openshaw, 2002). During the
infiltrate period, pathogen sometimes can pass through the first barrier and enter the body.
When this happens, the body gives a response by releasing nonspecific response to handle the
pathogen (Litman et al., 2005).

The fish immune system generally resemble the higher vertebrate, even though in certain part
the system has it specifies (Uribe et al., 2011). Fish skin is very different with other vertebrates.
The skin has special structure and characteristic (Fast et al., 2002). The skin of fish plays a key

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role in their immune defense against any invader by releasing mucus (Salinas et al., 2011). This
specific function occurred the fish adaptation to an environment which contains a large number
of a dissolved substance such as physical, chemical and also pathogens (Magnadottir, 2010).
A review by Esteban (2012) regarded that fish skin plays as an envelope analog which hub the
outer environment with the body internal organ and at the same time provide protection against
any harmful agents. In general, fish skin not only established as a physical barrier in term of
immune response, but it also regulate osmoregulation, respiration, and excretion (Ostrander,
2000).

The second layer of defense against foreign agents’ infiltration is called innate immune system
(Litman et al., 2005). In fish, the innate immune system can also recognize any pathogens in
general way (Rauta et al., 2012). A report by Kurtz (2005) mentioned that the innate immune
response has an ability to memorize the infection.

The latter response in the cascade of fish immune response is an adaptive acquired immune
system (Rauta et al., 2012). The adaptive immune response is well-known as immune reaction
triggered by the help of another cell containing the memory of pathogen/part of the pathogen
(Mayer, 2017). This adaptive immune response also involving antigen-antibody reaction such
as T-cell and immunoglobulin (Ellis, 2001; Swain, 2006). Those type of immune response in
fish will provide a protection optimally through the element of the immune response. The
elements are molecular-structure specific depending on the type of immune cascade (Rauta et
al., 2012). The outer layer of immune response or physical barrier has elements such as lectins,
antibacterial peptide and IgM (Saurabh & Sahoo, 2008).

While the innate-immune response involving some elements such as lysozyme, which attack
peptidoglycan layer of bacterial cell wall (Hjelmeland, 1983), lectin that act to agglutinate cells
(Arason, 1996), acute phase protein which has some essential functions such as protease
blocker, eliminate microbe, and support tissue recovery (Bayne and Gerwick, 2001), and the
complement system which establish a network between innate and adaptive immune response
in fish (Carrol, 2004). Studies by Yin et al. (1997) and also Tafalla et al. (2001) reported that
Tumor Necrosis Factor (TNF) is an important element of innate-immune response in the
activation of macrophage in some fish (teleost). In the meantime, another study by Robertson
(2006) found that cytokines (prior to Interferon (INF)-α & β) are very important as an innate-
immune response against viral infection. The fish Interleukin (IL) also found as innate-immune
response element that analog to mammalian IL-1 β (Magnadottir, 2010).

The fish adaptive-immune response is regarded to has a lower level than those in mammals
(such simple types T and β cell found in teleost) (Fischer et al., 2006). Fish also demonstrated
to have antibodies IgM class (Acton et al., 1971) and Immunoglobulin D as the next antibody
that can be identified in fish (Wilson et al., 1997).

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Table 1. The comparison between teleost and mammalian immune system (Tort et al., 2003)
Comparison Teleost Mammal
Body temperature range -2 to 35 0C 36.5 to 37.5 0C
Main environment Water Air
Metabolism Poikilothermic Homeothermic
External interface Mucous skin, gill Respiratory tree
Immunoglobulin isotypes IgM, IgD IgM, IgA, IgD, IgE, IgG
Antibody affinity Low High
Antibody response Slow Fast
Memory response Weak Strong
Affinity maturation Low or absent High

B. Fish adipose tissue

Lipid plays an important role to provide energy for fish. Those lipid can be found in some fish
body parts such as visceral, subcutaneous lipid and intramuscular fat (Weil et al, 2012). The
main fat depots in fish are in the peri-visceral muscle and liver (Leaver et al, 2008).
Anatomically the visceral fat or known with abbreviation “Vis” occupied mainly in the
abdominal cavity surrounding the tractus digestivus. The vis-fat take a part of 2-25% of fish
weight. Subcutaneous fat (also well-known with abbreviation SC) are covering the fish body
and has more densely mass in the dorsal or ventral aspect of salmonid species. The vis-fat
structurally fulfilled with the adipocytes in large abundance. While in the white and red muscle
the adipocytes are wrapped within a connective tissue called myoseptum. The portion of lipid
in the myoseptum is 40% of white muscle (Nanton et al., 2007).

Fish adipocyte is unique with many variations of diameters. The cell size of fish adipocyte
dependent on nutritional and water temperature (Fauconneau et al., 1997). For many years,
adipose tissue is well-known to have an immunological function, since the tissue is resident for
immune cells (Kaminski and Randall, 2010). In the meantime, the immunological role of fish
adipose tissue was first recognized when there was an expression of immune genes after
exposure to bacterial antigen (Skugor et al., 2010). A report by Pignatelli et al. (2014) further
described the prevalent of resident leucocyte within fish adipose tissue near tractus digestivus.

The ability of the fish adipose tissue to support immune response against foreign pathogen has
well described since 1990’s when Afonso et al. (1997) found that the cavity of the peritoneum
in fish actually harbored some immune cells such as lymphocytes, macrophages, and
granulocytes. A challenge injection of antigen into fish peritoneum cavity demonstrated the
activity of fish adipose tissue to hold antigen in the cavity (Mutoloki et al., 2004). While in the
study Noia et al. (2014) proved that the existence of foreign agents inside the cavity stimulated
the rising of immune cells. Recent report obviously showed fish adipose tissue can isolate
foreign agents which infiltrated peritoneum (Pignatelli et al., 2014). A comprehensive study of
the role of fish adipose tissue in the immune response of rainbow trout (Onchorynchuss
myskiss) finally opened a good understanding by showing Immunoglobulin M and
Immunoglobulin T activities within interspace of the adipocyte in visceral fat (Ballestros et al.,
2013).

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The bacterial fish pathogen is various and wide-spread. Fish are also susceptible to many
species of bacteria pathogens. However, they only can infect fish in very unfortunate
conditions, poor diet, and unstandardized fish care (Tendencia and Pitogo, 2004). Among
bacterial fish pathogens worldwide, the aerobic Gram-negative rod has been occupying the
most number of them (Abdelsalam, 2017). The viral fish disease is also widespread and rapidly
growing. Until nowadays fish viruses have been attacking various fish species. The fish virus
is well adapted to the temperature and sometimes triggering latency (Petty, 2018).

Fish Adipose Tissue Immune Response in Viral Infection

The limited study only available in the field of fish (teleost) adipose tissue immune response
against virus infection. The study obviously revealed the activity of Immunoglobulin M and
Immunoglobulin G activity within the space among adipocyte of rainbow trout after challenged
through vaccination (Ballesteros et al., 2013). The study was regarded as the first scientific
report which demonstrated how the fish adipose tissue gave response towards virus infection
((Pignatelli et al., 2014). The adipose tissue strongly revealed a key role in the expression of
viral immune response elements during viral infection (Castro et al., 2014). The current
research that took priority to investigate the specific immune response of fish adipose tissue
against viral infection was conducted using Viral Hemorrhagic Septicemia Virus (VHSV)
challenging infection. During VHSV infection, vis-fat of rainbow trout (Onchorynchuss
myskiss) revealed the content of Immunoglobulin M+, Immunoglobulin T+ and
Immunoglobulin D+ lymphocyte cells and Major Histocompatibility Complex (MHC) as well
(Castro et al., 2014).

The amount number of specific antibody in rainbow trout is limited in both lymphocytes B and
CD8+ cells, therefore, those specific cells have a prominent function in the immune response.
Comparing to the total leucocyte that prevalent in the fish adipose tissue, B cells occupied
about 50% with expressing Immunoglobulin M, Immunoglobulin T and Immunoglobulin D
(Zhang et al., 2010). The type of Immunoglobulin M and Immunoglobulin D in fish are similar
to those in mammalian B lymphocyte (Fillatreau et al., 2013). In a scientific report by Edholm
et al. (2010), there was a fact that fish B cells also harbored Immunoglobulin IgM+IgD- and
IgD+IgM-. In the current study of fish adipose tissue, Pignatelli et al. (2014) found that most
leucocytes within fish adipose tissue were having a shape similar to lymphocyte which (a few
of them) looked like macrophage and another cell was found to have morphology as a large
cell with a small core and irregular shape. The viral infection challenge in rainbow trout
stimulated the rising of Immunoglobulin M about 30%, Immunoglobulin T about 10%, and
Immunoglobulin D about 10% of total leucocyte in the fish adipose tissue. There was also
CD8α+ found during the viral infection challenge (Pignatelli et al., 2014).

An interesting study by Castro et al. (2014) found that B lymphocyte of fish (rainbow trout)
has differences with that lymphocyte in mammals. Instead contains Immunoglobulin M and
Immunoglobulin D, fish B lymphocyte harbored Immunoglobulin IgD+IgM- cells and
IgM+IgD- cells. The B cells with IgD+IgM- and IgM+IgD- take apart about 88% of the total

4
B lymphocyte (Pignatelli et al., 2014). Interestingly, during infection course with VHSV, the
fish adipose tissue was proven to have an ability as a precursor of B lymphocyte. Those
capability as confirmed by the expression of terminal deoxynucleotidyl transferase (TDT).
According to Hansen (1997), the expression of TDT was considered as a sign of B cell
lymphopoiesis.

The behavior of fish adipose tissue as a response towards virus infiltration and replication was
studied by Pignatelli et al. (2014). They resulted interesting finding that fish adipose tissue
could absorb virus particle, isolate and then blocking viral replication as confirmed by the
absence of viral N-protein. Besides these findings, the study found that virus infection in teleost
will induce the activation of many genes encoded immune response elements such as Mx, IFN-
γ, Galectin-9, IgM, IgD, IgT, CD8α, CD8β, CK3, CK5B, CK10, CK12, CXCR4 and TLR. The
expression of TLR-3 within fish adipose tissue proved that the adipose tissue gave a response
to counteract virus infection since TLR-3 has a specific ability to recognize dsRNA which can
be detected in a viral infection process (Palti, 2011). The upregulation of several chemokines
during viral course infection modelled by VHSV (Pignatelli et al., 2014) was resemble the
mammalian response against viral infection, for example, CK5B in fish which has similarity
with RANTES/CXCL 5 in human (Peatman and Liu, 2007). The response of fish adipose tissue
during viral infection has obviously happened as indicated by the fall down of the number of
lymphocytes which contains Immunoglobulin M on their surface. In the other hands, the
number of CD8α+ was observed to raise in a certain level (Pignatelli et al., 2014).

Table 2. Summary of characteristic of fish adipose tissue response against viral infection
(Pignatelli et al., 2014)
Variable of changes
Lymphocyte characteristics
within adipose tissue
+
IgM B lymphocyte in the
adipose tissue
Lymphopoiesis
Molecular genomic activities High level of transcription several important genes such as
The changes during viral infection
 The rising number of IgM+, IgT+ and IgD+
lymphocytes
 The existence of cells expressing Major
Histocompatibility Complex Class II (MHC-II)
 Most of the leucocyte shaped lymphocyte-like
morphology
B lymphocytes harbored a number of IgD+IgM- cells and
also IgM+IgD- cells (in mammals we can find both IgM and
IgD on the surface of B2 lymphocyte)
The fish adipose tissue was proven has a capability as B
cell lymphopoiesis
Interferon (IFN) response protein (Mx, IFN-γ and galectin
9), pro-inflammatory components (IL-1β), IL-6, tumor
necrosis factor α (TNF- α), various leucocyte types
(secreted IgM (sIgM), sIgT, sIgD, CD8 α, CD8β),
chemokines (CK1, CK3, CK5B, CK6A, CK7A, CK9,
CK10, CK11, CK12, IL-8, CXCL1 and CXCL_F1),
chemokines receptors (CCR6, CCR7, CCR9, CCR13, and
CXCR1, CXCR3A, CXCR3B and CXCR4), Toll-like
receptors (TLR1, TLR2, TLR5, TLR7, TLR8a2, TLR9, and
TLR22).

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 Viral replication within fish The virus can replicate within adipose tissue as indicated by
adipose tissue the increase of viral mRNA day 2 p.i. (VHSV case)
Changes the quantity of Cells with IgM decrease (transformed to plasmablast), and
leucocyte cells with the CD8α+ increase

Picture 1. Diagram shows the upregulation of immune response genes in the infected group
after infection with VHSV compare with naïve group in rainbow trout (Pignatelli et
al., 2014)

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 Fish Adipose Tissue Immune Response in Bacterial Infection

As described previously that fish adipose tissue had the ability to release pro-inflammatory
substances such as cytokine and IFN (Todorcevic et al., 2010; Pinatelli et al., 2014). Besides
fish adipose tissue has been proved to have immune response capability against VHSV
(Pignatelli et al., 2014), the tissue also gave a response during bacterial infection (Fedorko et
al., 1971 and Rangel-Moreno et al., 2009). The response of fish adipose tissue against bacterial
infection was investigated by some studies and resulted in a fact that the presence of bacterial
antigen inside the peritoneum cavity triggered the changes of the number of cells and or
substances in the cavity (Korytar et al., 2013 and Noia et al., 2014).

Veenstra et al. (2011) injected rainbow trout with a Gram-negative bacteria, Aeromonas
salmonicida (Kipp et al., 2018) intra peritoneal and they found that there was a significant high
level of Interleukin, Interferon (IFN) and chemokines detected in the rainbow trout adipose
tissue compared to those naïve fish. In the meantime, several Interleukins (such as Il-10B, IL-
17A/FI-3, IL-17N), Secreted Aspartyl Proteinase (SAP1 and SAP2), Tumor Necrosis Factor
(TNF-α3), Cathelicidin-1 and GATA-3 showed that their level did not be triggered to raise
after bacterial infection. Those findings confirmed to the previous report in a human
experiment that Lipopolysaccharide (LPS) (as apart of Gram-negative bacterial structure) can
trigger a response of immune cells to produce immune elements (Chandra et al., 2001).

In a study by Starr et al. (2009), the LPS of Gram-negative bacteria showed to give an
inflammatory reaction in mice adipose tissue, especially by the release of Interleukins (IL-6).
The IL-6 can also be detected in trout and showed to cause the rising of Adenosine
Monophosphate (AMPs) (prior to Chatelicidin-2 and Hepcidin) excluded Cathelicidin-1 (Costa
et al., 2011).

Fish adipose tissue also gave different response against different types of antigen. For example,
the immune response genes in rainbow trout will be upregulated after infection challenge with
A. salmonicida compare to stimulation with recombinant flagellin of Yersinia ruckeri (rYRF)
(Veenstra et al., 2018). In general, fish adipose tissue was able to counteract the infiltration of
bacterial antigen by the stimulation of released cytokines through bacterial PAMP (Pathogen-
associated Molecular Pattern) (Hynes et al., 2011). Nevertheless, in some cases the response
might be different because the different type of antigens have largely likelihood to trigger
distinguished anti-pathogen response depend on the specify of antigen (Wangkahart et al.,
2016).

A scientific report by Veenstra et al. (2017) described on how cytokine IL-β plays key to
activate initial immune response and capability of trout adipose tissue in counteracting the
infiltration of a pathogen. In another fish adipose tissue study using recombinant IL-1β of trout,
the study reported that the recombinant Interleukin made the transcription level of IL-1β, COX-
2, and TNF-α and MHC II stayed in a higher level than the control group (Laing et al., 2002).

7
Table 2. Summary of characteristic of fish adipose tissue response against bacterial infection
(Veenstra et al., 2017)
Variable of changes The changes during viral infection
Antibody response Antibody reached higher titer compare to control group after
fish were injected with bacterial antigen (Vaccine and
antigen of Aeromonas salmonicida)
Molecular genomic activities The upregulation of immune response elements genes such
as cytokines IL-1β, IL-6A, IL-6B, IL-8, IL-11, IL-18, TNF-
α1, TNF-α2, TNF-α3, APP and AMP, COX-2, CATH2 and
Hepcidin genes

Picture 2. Diagram shows the upregulation of immune response genes in the infected group
after infection with Aeromonas salmonicida antigen in rainbow trout (Veenstra et
al., 2017)

Conclusion

Fish adipose tissue was proven to play important role in fish immune response against the
pathogen. Fish visceral adipose tissue which occupies near digestive tract is the most
immunologically active part of the adipose tissue, unlike in mammal and or human that take
place in the omentum. In general, fish adipose tissue showed active response against pathogen
infiltration by the increasing level of various types of lymphocytes, antibodies, cytokines, and
interferon. Viral and bacterial infection also stimulate a response in molecular level by
triggering upregulation of several genes which encoded immune response elements.

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 Future work

There are still a few studies to explore the role of fish adipose tissue in immunological
activities. Recent research has described the deep understanding how the adipose tissue
immune response counteracts viral (Pignatelli et al., 2014) and bacterial (Veenstra et al., 2017)
infection. Those both studies can be regarded as an important principle to conduct further
follow up research. There are some future works that can be conducted to deeply elaborate the
fish adipose tissue immune response.

a. A study to investigate the response of fish adipose tissue against Gram positive and
Gram negative bacteria (comparison)
b. Research that focused on the reaction of fish adipose tissue against long period-type
virus infection such a fish herpesvirus. Fish herpesvirus, similar to those which infect
mammals and human, they have the ability to do latency.
c. Research that focused on the ability fish adipose tissue as virus reservoir for some
important viruses in the fish world, and the ability of those viruses to replicate within
the fish adipose tissue.
d. Research that focused on the comparison of fish adipose tissue reaction during
vaccination by several bacterial, viral antigen including parasitic antigen prior to the
disease that important to aquaculture
e. Research that focused on the relationship among fat diet, adipose tissue, and pathogen
infection to learn the effect of fish food to support their immune system.

9
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