Endogenous expression of thermo-sensitive ion channels ... · 1Institute of Physics Campus, Sachivalaya Marg, Bhubaneswar, Orissa, ... MATERIAL AND METHODS: 2.1 Ethical approval This

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Endogenous expression of thermo-sensitive ion channels TRPV1

and TRPV4 in immune tissues of avian species (duck, Anas

platyrhynchos)

Rakesh Kumar Majhi 1, 2, #, Apratim Maity 3, #, Manas Ranjan Senapati 3, #, Prakash

Chandra Behera3, Arun Kumar Mandal4, Sunil Chandra Giri5, Chandan Goswami 1, 2 *

1Institute of Physics Campus, Sachivalaya Marg, Bhubaneswar, Orissa, India of Biological Sciences,

National Institute of Science and Education Research, Bhubaneswar, India-751005.

2Homi Bhabha National Institute, Training School Complex, Anushakti Nagar, Mumbai 400094, India

3Department of Veterinary Biochemistry, College of Veterinary Science and Animal Husbandry, Orissa

University of Agriculture and Technology, Bhubaneswar, India-751003.

4Department of Veterinary Anatomy and Histology, College of Veterinary Science and Animal

Husbandry, Orissa University of Agriculture and Technology, Bhubaneswar, India-751003.

5Central Avian Research Institute, ICAR, Bhubaneswar,

# Equal contribution

*Corresponding authors: [email protected]

ABSTRACT:

Calcium signaling and body temperature are two important factors governing activation of

immune cells. However, molecular identities of major players involved in such critical regulations are

still unknown, especially in avian systems. In this work we explored the endogenous expression of

Transient Receptor Potential Vanilloid subtype 1 and subtype 4 (TRPV1 and TRPV4) channels, in the

immune cells and different tissues of duck (Anas platyrhynchos). TRPV1 and TRPV4 represent two

important non-selective ion channels which are also thermo sensitive in nature. Using confocal

microscopy we demonstrate that TRPV1 and TRPV4 are expressed endogenously in the duck immune

system such as thymus, spleen, caecum and bursa. In thymus, bursa of Fabricius and caecum, these

channels are differentially localized in the plasma membrane and intra-cellular regions. In addition,

using Flow cytometry we demonstrate very specific expression of these two channels in Peripheral

Blood Mononuclear Cells (PBMCs) and CD3+ve T cells. This result is the first report of endogenous

expression of TRPV channels in cells and tissues relevant for avian immune system. Such findings may

have importance in the context of immune functions and responses to pathological challenges faced by

PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.26614v1 | CC BY 4.0 Open Access | rec: 4 Mar 2018, publ: 4 Mar 2018

mailto:[email protected]

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birds. Presence of these two important ion channels in the duck immune system may also have

commercial importance in the context of livestock management, food supplement and production of

quality poultry products.

Keywords: Duck, TRPV1, TRPV4, immune tissue, poultry, Calcium signaling, T cells

Running title: Avian immune system

Author Comment

A large number of work has been done on mammalian immune system. However, not much work has

been done on the avian immune system. In this pre-print we submit the expression of thermosensitive

ion channels in specific tissue and cells that are part of avian immune system.


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1. INTRODUCTION:

Environmental temperature is an abiotic selection factor responsible for population dynamics

and spatial distribution of organisms. The transient receptor potential (TRP) channels are a group of

non-selective cationic channels which regulate important behavioral parameters such as avoiding high

and low temperatures and choosing the optimum temperature for survival (Lu et al., 2014). This is

mainly due to the ability of TRP ion channels to sense external environment and to discriminate the

minute changes. All animals have potentiality to sense the temperature of their surroundings, from

noxious cold to physiological temperature to noxious hot conditions and TRP ion channels play

important role in those sensory functions (Venkatachalam and Montell, 2007; Caterina, 2007). These

channels have been described as the molecular thermometers regulating animal behavior. Acting as

integral circuit of several stimuli and signaling pathways, dysfunction of these channels leads to several

pathological states (Huertas et al., 2014).

Studies, primarily from mammals suggest that Transient Receptor Potential Vanilloid (TRPV)

group of channels are activated by temperature, mechanical force, changes in osmotic pressure, different

electro-magnetic waves and other endogenous as well as exogenous compounds (Fowler and Montell,

2013). TRPV sub type 1 (TRPV1), also known as the “Capsaicin receptor” is a Ca2+-permeable non-

selective cation channel and the founder member of the TRPV subfamily (Caterina et al., 1997). So far,

TRPV1 gene has been sequenced from different species and its endogenous expression has been

detected in several tissues and cells of certain species representing primarily mammals (Planells-Cases

et al., 2005). Recently TRPV channels have been detected in mammalian immune cells where these

channels play important roles in antigen detection, clonal selection, cytokine production and immune

activation (Feske et al., 2012; Majhi et al., 2015). In addition, these channels are involved in multiple

physiological and sensory processes. As living habitats and environments encountered by different

species is tightly linked with detection of noxious stimuli and further recruitment of downstream

signaling, TRP channels provide examples of unique candidates that can be studied in the context of

molecular evolution (Sardar et al., 2012; Kumari et al., 2014). Involvement of TRPV1 in several

physiological conditions has prompted this channel as a potential pharmacological target for


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development of new drugs, especially in the context of different diseases including inflammation

(Gavva et al., 2007).

Transient receptor potential vanilloid sub type 4 (TRPV4) is also a member of TRP super

family. This polymodal receptor is also involved in cellular processes such as mechanosensation,

osmosensation and thermosensation (Liedtke et al., 2003; Goswami et al., 2010). In higher organisms

TRPV4 is endogenously expressed in dorsal root ganglion (DRG) neurons and in several other non-

neuronal tissues such as in skin, kidney and corneal epithelial cells (Pan et al., 2008), cerebral micro-

vascular endothelial cells (Ma et al., 2008), cortical astrocytes (Benfenati et al., 2007), and in tracheal

epithelial cells (Lorenzo et al., 2008). The widespread distribution of TRPV4 is indicative of its

involvement in various physiological functions.

In most cases, infection is followed by increment in body temperature, a physiological step

which is well known as an activator of immune system (Tournier et al., 2003). Though recent studies

have pointed that heat shock proteins (HSPs) are involved in temperature-mediated effects on the

immune cells (Jolesch et al., 2012), the extreme sensitivity to slight changes in the temperature and

precise temperature-dependent activity makes thermosensitive ion channels as ideal regulatory

candidates suitable for temperature-dependent immune modulations. The thermo-sensitivity and Ca2+

permeability in general suggests that this group of ion channels are ideal candidates that can act as

“molecular thermosensors” and are important for the temperature-induced immune activation relevant

in the context of infection and pathogenic challenges (Basu and Srivastava, 2005). Indeed, few reports

have suggested the physical and functional presence of thermosensitive TRP channels in different

immune cells, especially in T cells, macrophages and dendritic cells (Yamashiro et al., 2010). Recently

we have demonstrated the physical and functional presence of TRPV1 and TRPV4 in T cells and have

shown that these two channels are important for immune activation (Majhi et al., 2015). However, the

presence of functional TRPVs in cells and tissues relevant for immunity has been reported from

mammalian species only, and comprehensive information about the presence of TRPV channels in

avian immune systems is still lacking. In this work we have characterized the distribution of TRPV1


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and TRPV4 in different tissues and cells that provides immunity. This work is the first systemic report

demonstrating the specific presence of thermo-sensitive TRPV channels in the avian immune systems.

2. MATERIAL AND METHODS:

2.1 Ethical approval

This study was conducted with prior approval of the Institutional Animal Ethics Committee

(No: 433/ SPCEA/ OVC). The ducks were procured from Central Avian Research Institute (CARI),

Bhubaneswar, Odisha.

2.2 Sample collection

The study was carried out on six numbers of poultry birds (duck) of both sexes of 8-10 months

of age. These birds had no developmental disorders and detectable disease to avoid the abnormalities

of the histological architecture of lymphoid tissues. These birds were sacrificed by cervical sub-luxation

method and the immune tissues such as bursa of Fabricius, apical part of caecum, spleen and thymus

were collected through ventral abdominal dissection, which were free from pathological lesions. Prior

to collection of tissues, whole blood was collected in a sterilized heparinized tube in aseptic manner for

isolation of PBMCs (Peripheral Blood Mononuclear cells).

2.3 Isolation of PBMCs:

For probing the expression of TRP channels, PBMCs were isolated from whole blood as

described previously with some modification (Feldman and Mogelesky, 1987). Briefly, 3.0 ml of HiSep

LSM 1084 was aseptically transferred to 15 ml clean centrifuge tube followed by carefully overlay with

3.0 ml whole blood. Without mixing, the tube was centrifuged at 2300-2500 rpm (400 × g) for 30

minutes at room temperature. Plasma and platelet containing supernatant above the interface band was

aspirated. Using a clean glass Pasteur pipette, the mononuclear cell band i.e. opaque interface was

carefully aspirated and transferred it to a clean 15 ml centrifuge tube. Ten ml of isotonic phosphate

buffered saline (PBS) was added to the tube followed by centrifugation at 250 x g for 10 minutes. The


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supernatant was discarded then and the cell pellet was re-suspended with 0.5 ml of isotonic PBS

followed by proper mixing. Again the mixture was centrifuged at 250 x g for 10 minutes and the

supernatant was removed carefully. Two-three washes were typically required to remove any remaining

HiSep LSM 1084 from the mononuclear cells. The cells in the form of pellet was re-suspended after

the final wash in 0.5 ml PBS and transferred to 1.5 ml centrifuge tube and 0.5 ml of 4% Para-

formaldehyde (PFA) was added to it for fixation and kept at 4°C for further study.

2.4 Slide preparation of tissues

After collection, tissues were thoroughly washed with physiological saline and fixed with 4%

Paraformaldehyde (PFA). After 1 day of fixation, the tissues were transferred to 25% Sucrose and stored

at 4°C. Just before cryo-sectioning, the tissues were snap frozen in dry ice and were then mounted on

to the object plate holder of cryostat by embedding solution (Leica Biosystems). The object plate holder

was then attached to the object head maintained at -19°C. The chamber was maintained at -20°C.

Sections of 25µm thickness were cut using CM3050S cryostat (Leica Biosystems). The sections were

mounted onto slides pre-coated with 0.1% Ploy-L-Lysine (Sigma-Aldrich). The slides were kept frozen

at -20°C freezer till processing.

2.5 Immunohistochemistry of tissues

For immunohistochemistry (IHC), the slides were brought to room temperature and washed

thrice with 1X PBS. The sections were permeabilized with 0.5% Triton-X 100 (Sigma-Aldrich) for 30

minutes at room temperature, blocked with 5% BSA in PBS for 45 minutes and then incubated with

primary antibodies raised against TRPV channels (Alomone Labs, Jerusalem). Primary antibodies at

1:200 dilution in 2% BSA was incubated for overnight in moist chamber maintained at 4°C. These

slides were then washed thrice with 0.1% PBS-T (PBS with 0.1% Tween-20) for 5 minutes each and

then incubated with AlexaFluor 488 labelled anti-rabbit secondary antibody (Molecular Probes) at

1:750 dilution in 2% BSA for 2 h in moist chamber maintained at room temperature. The sections were

then washed thrice with 0.1% PBS-T and incubated with DAPI (5µg/ml) for 15 minutes minimum.


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After washing thrice with 0.1% PBS-T, the slices were layered with Fluoromount-G and covered by

coverslip (Fisher Scientific). After the samples were dried for 24 h at room temperature, the images

were acquired by LSM 780 Confocal microscope (Carl Zeiss, Germany) by using 63X oil immersion

objective. The images were processed using LSM image browser software.

2.6 Immunocytochemistry of PBMCs

PBMCs were isolated from Duck blood and fixed with 4% PFA at room temperature. The cells

were washed thrice with PBS and permeabilized with 0.1% Triton-X 100 (Sigma-Aldrich) for 5minutes

at room temperature, blocked with 5% BSA in PBS for 45 minutes and then incubated with primary

antibodies against TRP channels (Alomone Labs, Jerusalem) at 1:200 dilution in 2% BSA overnight in

0.7ml tubes at 4°C. The cells were then washed thrice with 0.1% PBS-T (PBS with 0.1% Tween20) for

5 minutes each and then incubated with AlexaFluor 488-labelled anti-rabbit secondary antibody

(Molecular Probes) at 1:1000 dilution in 2% BSA for 2 h in moist chamber at room temperature. Cells

were counter stained with DAPI and images were acquired as described before.

2.7 Flow cytometry analysis

For probing for TRPV channels expression, cells were stained with individual TRP channels-

specific antibodies (Alomone Lab) and anti CD3 antibody (Abcam) and subsequently flow cytometric

analysis was performed as described previously (Majhi et al. 2015). Briefly, Duck PBMCs were

permeabilized with 0.1% Triton-X100, blocked with 5%BSA for 30 minutes and incubated in with anti-

CD3 and anti-TRP channel antibodies overnight. Anti-Rabbit AlexaFluor 488 was used for labelling

TRP channel antibodies and anti-rat AlexaFluor 647 was used for labelling CD3 antibody. The cells

were re-suspended in FACS buffer (1X PBS, 1%BSA and 0.05% Sodium Azide) and were acquired

with FACS Calibur (BD Biosciences). 10000 cells were acquired within the gated region for live cells

and expressions of TRP channels were detected in FL-1 channel while CD3 signal was detected in FL-

4 channel. All CD3+ cells were gated and FL1 signal has been represented to show the expression of

TRP channels. Data was analyzed using Cell Quest Pro software (BD Biosciences). Percentages of cells

expressing the markers are presented in Dot Plots.


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3. RESULTS:

To visualize the expression pattern of temperature sensitive TRPV1 and TRPV4 channels in

immune tissues, we performed immune-localization followed by confocal microscopic analysis of bursa

of Fabricius, apical part of caecum, spleen and thymus of duck. When probed with TRPV1 and TRPV4

specific antibody (Alomone), we noted the epithelial lining of bursa showed maximum expression of

both TRPV1 (Fig. 1a) and TRPV4 (Fig. 2a) followed by cortex and medulla region. Similarly, TRPV1

specific immune-reactivity is observed primarily in the caecal mesothelium and some faint staining is

detected in the crypts and lamina propria regions too (Fig. 1b). However, TRPV4 expression pattern is

noticed in mucosal assisted lymphoid tissue of vilus of caecum (Fig. 2b). When spleen was probed with

TRPV1- and TRPV4-specific antibodies, these antibodies remain un-reactive to white pulp and red pulp

respectively (Fig. 1c and 2c). On the other hand, thymus show faint staining for TRPV1 (Fig. 1d), but

prominent expression for TRPV4 (Fig. 2d) in its medullary region. Next, we explored if the expression

of TRPV1 and TRPV4 is ubiquitous in all tissues. Therefore we used tissues from pancreas which does

not have any known role in immune function. We observed that TRPV1 antibody remains un-reactive

to pancreas whereas TRPV4 specific immune-reactivity is observed primarily among the insulin

secreting β-cells.

The Flow cytometry analysis of purified duck PBMC cells revealed that 83.24±11.07% and

42.41±1.42% of the total PBMCs under study were positive for presence of TRPV1 and TRPV4

respectively (Fig. 3a-b). However, among CD3 positive T cells, 74.26±1.97% and 44.28±7.25% cells

are expressing TRPV1 and TRPV4 ion channels respectively (Fig. 3d-e). Although TRPV1 is expressed

in higher number of cells than TRPV4, analysis of Mean Fluorescence Intensity (MFI) values indicated

that TRPV4 intensity per cell (both for PBMCs and CD3 positive T cells) was higher than that of

TRPV1. The PBMCs reveal MFI values of 14.71±3.99 for TRPV1 and 29.74±17.82 for TRPV4. The

CD3 positive T cells have MFI values of 33.38±4.55 for TRPV1 and 172.85±111.79 for TRPV4

respectively. Confocal imaging of PBMCs reveal punctate expression of TRPV1 and TRPV4

throughout the cells (Fig. 4). TRPV4 signal is more intense in comparison to that of TRPV1, thereby


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confirming the higher expression levels of TRPV4 in Duck PBMCs. The signal specific for TRPV1 and

TRPV4 is almost absent on pre-absorbing the antibodies with blocking peptide, thus confirming the

specificity of these antibodies (Fig. 4, right hand side). Taken together, our analysis suggest for the

endogenous expression of TRPV1 and TRPV4 in diverse tissues and cells related to avian immune

system.

4. DISCUSSION:

Poultry industry is being considered as the most rising and economically viable sector round

the globe owing to the small size, early maturity and fast growth of the species involved in it. But, this

industry faces problems due to high prevalence of infectious diseases and abiotic stress factors that

cumulatively affect the immune system resulting in poor production, mortality and serious economic

losses. Presence of several TRP channels and especially TRPV channels has been reported in

immunologically important tissues and cells from different mammalian species (Flockerzi and Nilius,

2014; Bertin et al., 2014; Majhi et al., 2015). Expression and specific localization of these thermo-

sensitive ion channels in such tissues suggest their involvement in immune systems per se. Indeed,

several reports suggest the importance of TRPV channels in immune functions, at least in mammalian

systems (Bertin et al., 2014; Majhi et al., 2015). However, similar studies have not been carried out till

date to study expression pattern of different endogenous TRP channels and/or other thermosensitive

channels in immune cells/tissues of avian species. Findings from this study can be exploited for different

applications such as improved immune response, vaccination strategy, immune-stimulation through

specific feed ingredients, reducing mortality rate, development of disease resistance variety in poultry

industry etc.

Environmental stresses are common to birds and such stresses influence the immune system

owing to susceptibility to diseases. Particularly, exposure of birds to heat or cold stress act as immune-

suppressive since heat stress modulates immunity by induction of HSPs in lymphocytes, heterophils

and macrophages, while cold stress do so by suppression and enhancement of plasma corticosterone

and thyroid hormone levels respectively (Dietert et al., 1994; Hangalapura et al., 2004). TRPV1 is

involved in the thermo-sensory pathways and has direct effects on HSP expression. It is reported that


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antagonism of TRPV1 can block the cellular HSP expression (Bromberg et al., 2013). Rise in body

temperature as well as inflammatory mediators like extracellular ATP, bradykinin, prostaglandins and

trypsin or tryptase have been reported to potentiate TRPV1 responses (Dai et al., 2004; Moriyama et

al., 2005).

In poultry birds, the thymus and Bursa of Fabricius are considered to be the primary lymphoid

organs whereas, the secondary lymphoid organs are the spleen, mucosal associated lymphoid tissues

(MALT) or gut associated lymphoid tissues (GALT) (Rezaian and Hamedi, 2007). The thymus is the

primary location for development and maturation of T-lymphocytes, particularly in the cortex region.

Endogenous expression of TRPV channels in that region can be correlated with their involvement in

the T cell functions in avian systems too. The “Bursa of Fabricius” is an organ unique to birds and is

the sole site of maturation and differentiation of B cells as birds are devoid of bone marrow. The cortex

of it contains large numbers of closely packed lymphocytes (Erf, 2004). The lymphocytes are exposed

to external antigens through cloacal drinking, with antigen presentation by specialized phagocytic

epithelial cells to the developing lymphocytes (Scott, 2004). Such a function can be linked with the

endogenous expression of both TRPV1 and TRPV4 in epithelial region followed by cortex and medulla.

The spleen, a lymphocyte predominated organ, is a major site of antigen processing and antibody

production in mature birds, dependent on both the thymus and bursa of Fabricius (Indu et al., 2000). It

contains lymphatic nodules and the white pulp within which small-, medium- and large-sized

lymphocytes and plasma cells were distributed diffusely; however, its red pulp region is mainly

occupied by erythrocytes (Sultana et al., 2011). Red pulp region contains several erythrocytes and this

specific region is mainly un-reactive to these antibodies suggesting that these region lacks endogenous

expression of both TRPV1 and TRPV4. The faint staining specific for TRPV4 may be due to the

presence of natural killer cells, monocytes and dendritic cells (Swirski et al., 2009; Igyarto et al., 2011;).

The caecal tonsil is the largest of the GALT centers and plays a role in antibody production in large

mass of diffuse and nodular lymphatic tissue in lamina propria and sub mucosa resulting in cell-

mediated immune functions (Mary, 2006; Firdous and Lucy. 2012). Well maintained immune defense

system with enormous lymphoid nodules throughout the mucosa provides protection against caecal

environment. This correlates well with endogenous expression of TRPV1 and TRPV4 channels in


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crypts and inside the villus region. Pancreas of poultry birds is mostly a non-immune tissue, consisting

of only exocrine and endocrine cells. In this context, we failed to detect TRPV1 in this tissue mostly

suggesting that the immune-staining are specific in nature. TRPV4 specific antibody detects

endogenous expression primarily among the insulin secreting β-cells. This is in agreement with studies

where similar pancreas-specific expression of TRPV4 in mouse is reported (Casas et al., 2008).

In the context of immunity development, both B- and T-lymphocytes are the principal cells

responsible for both humoral and cell-mediated immunity (Zekarias et al., 2002). Lymphocyte function

is regulated by a complex network of ion channels and transporters regulating calcium signaling which

function as second messenger to regulate crucial lymphocyte functions such as cytokine production,

differentiation and cytotoxicity. TRP ion channels, pore-forming transmembrane proteins that enable

the flow of ions down an electrochemical gradient play a significant role in lymphocyte function and

immunity (Feske et al., 2012; Bertin et al., 2014; Majhi et al., 2015). We have observed that TRPV1

and TRPV4 are expressed in majority of PBMC and T cell population. This indicates that both these

channels could have significant contribution to not only T cell mediated immunity in poultry birds but

also in non-T cell mediated (involving macrophages, dendritic cells etc.) immunity.

TRP ion channels are molecular detectors for physical and chemical stimuli such as noxious

hot and cold temperatures, infectious diseases and toxic substances which can cause potential tissue

damages. In this work, we demonstrate that like mammals in birds too, TRPV1 and TRPV4 channels

are also present in the immune cells and tissues and suggest for similar functions. These findings may

have potential implications in livestock management in poultry firms as well.

ACKNOWLEDGEMENTS:

This work was supported by Intramural funding from Central Lab, OUAT, Bhubaneswar and

from the Intramural funding from NISER, Bhubaneswar. We are thankful to the Confocal,

Flow cytometry and Cryostat facilities of NISER, Bhubaneswar for support.


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REFERENCES

Basu S and Srivastava P 2005 Immunological role of neuronal receptor vanilloid receptor 1 expressed on dendritic

cells. Proc. Nat. Acad. Sci. USA 102:5120-5125.

Benfenati V, Amiry-Moghaddam M, Caprini M, Mylonakou MN, Rapisarda C, Ottersen OP, Ferroni S. 2007.

Expression and functional characterization of transient receptor potential vanilloid-related channel 4 (TRPV4) in

rat cortical astrocytes. Neuroscience 148:876-892.

Bertin S, Aoki-Nonaka Y, de Jong PR, Nohara LL, Xu H, Stanwood SR, Srikanth S, Lee J, To K, Abramson L,

Yu T, Han T, Touma R, Li X, González-Navajas JM, Herdman S, Corr M, Fu G, Dong H, Gwack Y, Franco A,

Jefferies WA, Raz E 2014 The ion channel TRPV1 regulates the activation and proinflammatory properties of

CD4⁺ T cells. Nat. Immunol 15:1055-1063.

Bromberg Z, Goloubinoff P, Saidi Y, Weiss YG 2013 The membrane-associated transient receptor potential

vanilloid channel is the central heat shock receptor controlling the cellular heat shock response in epithelial cells.

PLoS One 8:e57149.

Casas S, Novials A, Reimann F, Gomis R, Gribble FM 2008 Calcium elevation in mouse pancreatic beta cells

evoked by extracellular human islet amyloid polypeptide involves activation of the mechanosensitive ion channel

TRPV4. Diabetologia 51:2252-2262.

Caterina MJ 2007 Transient receptor potential ion channels as participants in thermosensation and

thermoregulation. Am. J. Physiol 292:64-76.

Choi SI, Yoo S, Lim JY, Hwang SW 2014 Are Sensory TRP Channels Biological Alarms for Lipid Peroxidation?

Int. J. Mol. Sci 15:16430-16457.

Dai Y, Moriyama T, Higashi T, Togashi K, Kobayashi K, Yamanaka H, Tominaga M, Noguchi K 2004.

Proteinase-activated receptor 2-mediated potentiation of transient receptor potential vanilloid subfamily 1 activity

reveals a mechanism for proteinase-induced inflammatory pain. J. Neuroscience 24:4293-4299.


13

Dietert RR, Golemboski KA, Austic RE 1994 Environment-immune interactions. Poult. Sci. 73:1062-1076.

Erf GF 2004 Cell-mediated immunity in poultry. Poult. Sci. 83:580-590.

Feldman DL, Mogelesky TC 1987 Use of Histopaque for isolating mononuclear cells from rabbit blood. J

Immunol Meth 102:243-249.

Feske S, Skolnik EY, Prakriya M 2012 Ion channels and transporters in lymphocyte function and immunity. Nat.

Rev. Immunol 12:532-547.

Firdous AD and Lucy KM 2012 Caecal development in Kuttanad duck (Anas platyrhynchos domesticus). IOSR

J. Agricult.Vet. Sci 1:13-16.

Flockerzi V and Nilius B 2014 TRPs: Truly Remarkable Proteins. In: Mammalian Transient Receptor Potential

(TRP) Cation Channels; Nilius, B., Flockerzi, V., Eds.; Handbook of Experimental Pharmacology; Springer:

Berlin, Germany, 222:1-12.

Fowler MA and Montell C 2013. Drosophila TRP channels and animal behavior. Life Sci. 92:394-403.

Gavva NR, Bannon AW, Hovland DN Jr, Lehto SG, Klionsky L, Surapaneni S, Immke DC, Henley C, Arik L,

Bak A, Davis J, Ernst N, Hever G, Kuang R, Shi L, Tamir R, Wang J, Wang W, Zajic G, Zhu D, Norman MH,

Louis JC, Magal E, Treanor JJ 2007 Repeated administration of vanilloid receptor TRPV1 antagonists attenuates

hyperthermia elicited by TRPV1 blockade. J. Pharm. Exp. Therap. 323:128-137.

Goswami C, Kuhn J, Heppenstall PA, Hucho T 2010 Importance of Non-Selective Cation Channel TRPV4

Interaction with Cytoskeleton and Their Reciprocal Regulations in Cultured Cells. PLoS One 5:e11654.


14

Hangalapura BN, Nieuwland MG, de Vries Reilingh G, van den Brand H, Kemp B, Parmentier HK 2004

Durations of cold stress modulates overall immunity of chicken lines divergently selected for antibody responses.

Poult. Sci. 83:765-775.

Ferrandiz-Huertas C, Mathivanan S, Wolf CJ, Devesa I, Ferrer-Montiel A 2014 Trafficking of Thermo TRP

Channels. Membranes 4:525-564.

Igyártó BZ, Haley K, Ortner D, Bobr A, Gerami-Nejad M, Edelson BT, Zurawski SM, Malissen B, Zurawski G,

Berman J, Kaplan DH 2011 Skin-resident murine dendritic cell subsets promote distinct and opposing antigen-

specific T helper cell responses. Immunity 35:260-272.

Indu VR, Chungath JJ, Harshan KR, Lucy KM and Maya S 2000 Post natal development of spleen in the White

Pekin duck. Ind. J. Poult. Sci. 35:32-34.

Jolesch A, Elmer K, Bendz H, Issels RD, Noessner E 2012 Hsp70, a messenger from hyperthermia for the immune

system. Eur. J. Cell Biol. 91:48-52.

Liedtke W, Tobin DM, Bargmann CI, Friedman JM 2003 Mammalian TRPV4 (VR-OAC) directs behavioral

responses to osmotic and mechanical stimuli in Caenorhabditis elegans. Proc. Nat. Acad. Sci. USA. 100:14531-

14536.

Lorenzo IM, Liedtke W, Sanderson MJ, Valverde MA 2008 TRPV4 channel participates in receptor-operated

calcium entry and ciliary beat frequency regulation in mouse airway epithelial cells. Proc. Nat. Acad. Sci. USA.

105:12611-12616.

Lü ZC, Li Q, Liu WX, Wan FH 2014 Transient Receptor Potential Is Essential for High Temperature Tolerance

in Invasive Bemisiatabaci Middle East Asia Minor 1 Cryptic Species. PLoS One 9:e108428.


15

Ma YY, Huo HR, Li CH, Zhao BS, Li LF, Sui F, Guo SY, Jiang TL 2008 Effects of cinnamaldehyde on PGE2

release and TRPV4 expression in mouse cerebral microvascular endothelial cells induced by interleukin-1beta.

Biol. Pharm. Bull. 31:426-430.

Majhi RK, Sahoo SS, Yadav M, Pratheek BM, Chattopadhyay S, Goswami C 2015 Functional Expression of

TRPV Channels in T cells And Their Implications in Immune Regulation. FEBS J. 282:2661–2681

Mary HC 2006 The avian cecum. J. Exp. Zool. 283:441-447.

Moriyama T, Higashi T, Togashi K, Iida T, Segi E, Sugimoto Y, Tominaga T, Narumiya S, Tominaga M 2005.

Sensitization of TRPV1 by EP1 and IP reveals peripheral nociceptive mechanism of prostaglandins. Mol. Pain

1:3.

Pan Z, Yang H, Mergler S, Liu H, Tachado SD, Zhang F, Kao WW, Koziel H, Pleyer U, Reinach PS 2008.

Dependence of regulatory volume decrease on transient receptor potential vanilloid 4 (TRPV4) expression in

human corneal epithelial cells. Cell Calcium 44:374-385.

Planells-Cases R, Garcìa-Sanz N, Morenilla-Palao C, Ferrer-Montiel A 2005. Functional aspects and mechanisms

of TRPV1 involvement in neurogenic inflammation that leads to thermal hyperalgesia. Eur. J. Physiol. 451:151-

159.

Rezaian M and Hamedi S 2007 Histological study of the caecal tonsil in the caecum of 4-6 months of age White

Leghorn chicks. Am. J. Anim. Vet. Sci. 2:50-54.

Sardar P, Kumar A, Bhandari A, Goswami C 2012. Conservation of Tubulin-Binding Sequences in TRPV1

throughout Evolution. PLoS One 7:e31448.

Scott TR 2004 Our current understanding of humoral immunity in poultry. Poult. Sci. 83:574-579.


16

Sultana NM, Khan ZI, Wares MA, Masum MA 2011 Histomorphological study of the major lymphoid tissues in

indigenous ducklings of Bangladesh. Bang. J. Vet. Med. 9:53-58.

Swirski FK, Nahrendorf M, Etzrodt M, Wildgruber M, Cortez-Retamozo V, Panizzi P, Figueiredo JL, Kohler RH,

Chudnovskiy A, Waterman P, Aikawa E, Mempel TR, Libby P, Weissleder R, Pittet MJ 2009.Identification of

splenic reservoir monocytes and their deployment to inflammatory sites. Science 325:612-616.

Tournier JN, Hellmann AQ, Lesca G, Jouan A, Drouet E, Mathieu J 2003. Fever-like thermal conditions regulate

the activation of maturing dendritic cells. J. Leuk. Biol. 73:493-501.

Venkatachalam K and Montell C 2007 TRP channels. Ann. Rev. Biochem. 76:387-417.

Yamashiro K, Sasano T, Tojo K, Namekata I, Kurokawa J, Sawada N, Suganami T, Kamei Y, Tanaka H, Tajima

N, Utsunomiya K, Ogawa Y, Furukawa T 2010 Role of transient receptor potential vanilloid 2 in LPS-induced

cytokine production in macrophages. Biochem. Biophys. Res. Commun. 398:284-289.

Zekarias B, Ter Huurne AA, Landman WJ, Rebel JM, Pol JM, Gruys E 2002. Immunological basis of differences

in disease resistance in the chicken. Vet. Res. 33:109-125.


17

Figures and legends

TRPV1 DAPI Merge

a.

b.

c.

d.

e.


18

Figure 1. Expression of TRPV1 in different immune tissues. Confocal images of duck tissues

stained for TRPV1 (green) are shown for the following organs: a. Bursa, b. Caecum, c. Spleen, d.

Thymus, e. Pancreas. Specific regions of special importance are indicated as: 1. Epithelium, 2.

Cortex, 3. Medulla, 4. Mesothelium, 5. Crypts, 6. Lamina Propria, 7. White pulp, 8. Red pulp, 9.

Medulla, 10. Acini

TRPV4 DAPI Merge

a.

b.

c.

d.

e.


19


20

Figure 2. Expression of TRPV4 in different immune tissues. Confocal images of duck tissues

stained for TRPV4 (green) are shown for the following organs: a. Bursa, b. Caecum, c. Spleen, d.

Thymus, e. Pancreas, 1. Epithelium, 2. Cortex, 3. Medulla, 4. Villi, 5. Lymphocytes, 6. Blood

vessel, 7. Red pulp, 8. White pulp, 9. Medulla, 10. Lymphocytes, 11. Neural end, 12. β- Cells, 13.

Acinar cells.

Figure 3. Endogenous expression profile of TRPV1 and TRPV4 in avian PBMC. Flow cytometric

evaluation of TRP channels in Duck (a-c) Peripheral Blood Mononuclear Cells (PBMCs), (d-f) CD3+ve

T cells is shown. Representative dot plots showing the percentage of cells expressing the indicated TRP

channel in (a) PBMCs and (d) CD3+ve T cells. Average values of the percentage of cells expressing

the indicated TRP channel in (b) PBMCs and (e) CD3+ve T cells (N=3) is shown in the histograms.

The mean fluorescence intensity values indicating the abundance of TRP channels per cells is depicted

for the (c) PBMCs and (f) CD3+ve T cells (N=3). The P values are: ns, non-significant; *, < 0.05; **,

< 0.01; ***, < 0.001.


21

Figure 4. Localization of endogenous TRPV1 and TRPV4 in PBMC. Endogenous expression of (a)

TRPV1 and (b) TRPV4 in Duck PBMCs is shown as distinct puncta (green). Specificity of the

antibodies is depicted by loss of signal in peptide control set (using antibodies pre-adsorbed with their

antigenic peptide) shown in the extreme right.


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