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Possible interaction of sympathetic nerves

with macrophages via fractalkine-CX3CR1

ligation in pancreatic islets

Yuexi Wang

Bachelor’s Degree Project IBG214, 15 hp, 2021

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Abstract

Disturbing the interaction between sympathetic nerves and macrophages in islets has been shown to protect from insulitis development. To study the intra-islet neuroimmune communications, we explored the interactions between macrophages and nerves in the islets by looking at their juxta-positioning in the islets of wild type and CX3CR1 knock out mice. We

also cultured M1/M2 bone marrow-derived macrophages in order to understand basic expression profile changes during catecholaminergic stimulation. The results showed weaker colocalization between intra-islet macrophages and sympathetic nerves in CX3CR1-deficient

mice as compared to the wild type showing that CX3CR1 played an important role in

nerve-macrophage interactions. Also, low concentration of norepinephrine might induce pro-inflammatory effects in M1, as implied in how intra-islet macrophages responded to nerve signal excitation in type 1 diabetes development.

Abbreviations used in this scientific report: Tregs, regulatory T cells; T1D, type 1 diabetes; IFN, interferon; GM-SCF, granulocyte-macrophage colony-stimulating factor; TNF, tumor necrosis factor; IL, interleukin; ROS, reactive oxygen species; NF-κb, nuclear factor κb; TAMs, tumor-associated macrophages; TGF-β, transforming growth factor-β; Arg-1, arginase-1; CX3CL1, fractalkine; PNS,peripheral nervous system; CNS, central nervous system; RIP-GP, rat insulin promoter–lymphocytic choriomeningitis virus glycoprotein; M-CSF, macrophage colony stimulating factor; M0, bone marrow-derived macrophages; RT-qPCR, real-time quantitative polymerase chain reaction

Introduction

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3 autoimmune diseases, such as systemic lupus erythematosus, rheumatoid arthritis, and type 1 diabetes (T1D) are under intense investigation (Christoffersson and von Herrath 2019).

Type 1 Diabetes

The islet of Langerhans forms the endocrine tissue of the pancreas and is mainly composed of α,β,δ and pancreatic polypeptide cells, while the exocrine part consists of glands to produce and release various enzymes required for protein and lipid breakdown in digestion. Islets account for only 1-2% of the volume of the pancreas, and they are innervated and highly vascularized (Martinic and von Herrath 2008).

T1D is caused by the destruction of β cells in the islets thus requiring exogenous insulin for glucose metabolism (A et al. 2014). Genetic elements as well as environmental factors are possible causes for T1D development (Tremblay and Hamet 2019). In T1D, the infiltration of immune cells such as autoreactive lymphocytes, macrophages, and dendritic cells into the islets results in the loss of β cells, leading to hyperglycemia, and subsequent ketoacidosis and death if left untreated(Warshauer et al. 2020). However, due to limitations of monotherapy of insulin injection, normal glucose levels are often difficult to control, causing vascular and neurological comorbidities like stroke and blindness (Marro et al. 2019). Since understanding of T1D pathogenesis and technology have improved over time, novel immunotherapies to replace the causal autoreactive T cells with MHC matched naïve CD8 T cells has been proposed as one of the possible approaches (Faustman and Davis 2009). Additionally, not fully understood innate immune counterparts in T1D may provide new insights into diagnosis and treatment. Recently, the islet-infiltrated macrophages were proven to represent an important relay in neuro-immune communication, which might be a novel target in rescuing insulitis deterioration (Rodriguez-Calvo et al. 2015, Christoffersson et al. 2020).

Macrophages in Islets

M1 Macrophages: Macrophages are professional phagocytic cells and reside across the whole

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4 macrophages, are described as pro-inflammatory, assisting in the clearing of pathogens and tumor cells (Orecchioni et al. 2019). Interferon (IFN)-γ, granulocyte-macrophage colony-stimulating factor (GM-SCF) and tumor necrosis factor (TNF)-α induce differentiation and infiltration of M1 macrophages, which then express TNF-α, interleukin (IL)-1β, and reactive oxygen species (ROS) to promote an inflammatory environment (Feduska and Tse 2018). Consequently following exposure to inflammatory cytokines, nuclear factor κb (NF-κb) and STAT1 signal pathways in β-cells are activated, leading to β-cell death by the action of intracellular caspase1/3/9/12(Van Gassen et al. 2015). Besides, M1 macrophages also present islet autoantigens to CD8+T cells concomitant with T cell maturation and differentiation, thus

indirectly inducing β cell cytotoxicity(Jun et al. 1999) Also, macrophages express IL-1β and IL-6 to proliferate TH17 cells, which produce IL-17 to induce expansion of effector T cells and

a reduction of regulatory T cells, further deteriorates β-cells loss(Van Gassen et al. 2015). M2 macrophages: On the other hand, M2 macrophages, tumor-associated macrophages (TAMs) and myeloid-derived suppressor cells are anti-inflammatory cells, alternatively activated by IL-4, IL-10 and transforming growth factor-β (TGF-β)(Chávez-Galán et al. 2015). Therefore, M2 macrophages inhibit host defense action and promote wound healing through expression of TGF-β, IL-10 and arginase-1 (Arg-1)(Padgett et al. 2015). Notably, the particular subtype of an individual macrophage is not predetermined, which is to say, they may be reprogrammed from one subtype into another, in response to disparate resident microenvironments (Murray and Wynn 2011).

Macrophages in Pancreas: In the healthy state, macrophages present in islets are the only immune cell population(Zakharov et al. 2020) . It is known that islet-resident macrophages are M1 subtypes while in acinar stroma M2 macrophages are dominant(Calderon et al. 2015). Evidence has shown that transplanting M2 macrophages or elevating their proportion by depleting NADPH oxidase-derived ROS in nonobese diabetic mice could halt T1D progression (Parsa et al. 2012)(Padgett et al. 2015).

Fraktalkine-CX3CR1 system

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5 CX3CR1, receptor of CX3CL1, is expressed on tissue resident macrophages, natural killer cells

and subsets of T cells. CX3CL1-CX3CR1 ligation induces cell adhesion, contributing to

inflammatory initiation, progression and wound healing(Echigo et al. 2004)(Ishida et al. 2008). It is known that some intra-islet macrophages express CX3CR1, and the expression of this

receptor has been shown to be involved in neuro-immune interactions in brain and intestine (Bosmans et al. 2019)(Cardona et al. 2006).

Sympathetic Nerves and Macrophages

Neuro-immune interactions, which regulate tissue homeostasis and disease progression, such as cancer, multiple sclerosis and autism are becoming more intensively investigated (Veiga-Fernandes and Artis 2018). The peripheral nervous system (PNS) is comprised of the somatosensory system and the autonomic system, which works to sustain internal homeostasis (Ordovas-Montanes et al. 2015). Sympathetic nerves, belonging to the autonomic system, deliver commands from central nervous system (CNS) to peripheral effector tissues, such as pancreas-draining lymph nodes, to control immune cell activation (Veiga-Fernandes and Artis 2018). Besides, sympathetic neurons also innervate islets to control for example cytoarchitecture development and functional maturation(Borden et al. 2013).

In donated pancreas from T1D patients, loss of sympathetic neurons has been observed (Mundinger et al. 2016), however, whether it is the cause or the result of T1D remains to be clarified. Notably, researchers found that through inhibition of α1 adrenoceptors, surgical or

chemical denervation of sympathetic nerves in rat insulin promoter–lymphocytic choriomeningitis virus glycoprotein (RIP-GP) mouse model of T1D could dampen autoimmune function of macrophages (Christoffersson et al. 2020). The reason for which is that islet macrophages receive low concentration of catecholaminergic signals from sympathetic neurons to induce inflammation via α1 adrenoceptors, although β adrenoceptors exert the

immunosuppressive function. Therefore, blocking sympathetic signaling in T1D would protect β cells from destruction by autoreactive CD8+ T cells and then prevent hyperglycemia (Christoffersson et al. 2020).

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6 macrophages receive neurotransmitters from neurons to execute immune function while also impacting innervation. For example, sympathetic neuron-associated macrophages uptake excess norepinephrine secreted from sympathetic nerves to induce inflammation in obesity (Pirzgalska et al. 2017). In brown adipose tissue, CX3CR1+ macrophages affect autonomic

innervation thus influence adipocytes homeostasis and normal function (Wolf et al. 2017). In the tumor microenvironment, β-adrenoceptors receive catecholamine signals from sympathetic pathway thus helping breast cancer progression (Powe et al. 2010). As for a molecular mechanism, synapse-like interactions between noradrenergic fibers and lymphocytes have been observed in spleen(Felten and Olschowka 1987).

Aim of study

Here, we conduct research to test the hypothesis that fractalkine-CX3CR1 ligation in islet plays

an important role in sympathetic neurons innervation, macrophage interaction and insulitis development. Also, we identify the basic expression profile of M1/M2 macrophages and observe variations after norepinephrine stimulation.

Materials and methods

Immunofluorescent staining

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7 at RT for 1 hour. After washing twice with 0.2% PBST, 100 μl of Hoechst 33342 was applied to mark nuclear DNA (1X PBST and 1:10,000 Hoechst 33342) for 10 minutes at room temperature followed by wash in 0.2% PBST buffer. Extra water was wiped off and 50 mm coverslips were mounted using ProLong Gold Antifade Mountant (squeeze out bubbles by forceps). Slides were left to cure overnight prior to image acquisition using Leica TCS SP8 confocal microscope.

Primary Antibodies: Biolegend Alexa Fluor 555 rabbit anti-mouse TH; Biolegend Alexa Fluor 647 rat anti-mouse CD68.

Secondary Antibodie:Biolegend Alexa Fluor 555 donkey anti-rabbit IgG.

Bone marrow macrophages isolation

Mice were anesthetized by isoflurane inhalation and sacrificed by cervical dislocation. PBS was used to wash out bone marrow cells from tibia and femur. To clear away red cells, ACK lysis buffer was added to cell tube and separated debris from intact cells by centrifugation. Macrophage colony stimulating factor (M-CSF) (20 µg/ml) was added to RPMI cell culture media to generate bone marrow-derived macrophages (M0).

M1, M2 polarization

The resultant M0 cells were split into6-well plates at 8×105 cells per well. LPS (100 ng/ml) and

IFN-γ (20 ng/ml) were added to generate M1 while IL-4 (20 ng/ml) was used to induce to M2. Also, M-CSF was present to support macrophage activation as a growth factor. The cells were stimulated for approximately 12 hours.

Norepinephrine stimulation

10-8 M norepinephrine was added to M0, M1 and M2 cells to mimic the natural NE stimulation,

therefore activating pro-inflammatory α1 adrenoceptors in macrophages. After 4 h incubation

at 37℃, M1/M2 macrophages were scraped off the plates and mRNA was extracted with the help of a single cell RNA purification kit (Norgen Biotek).

Real-time quantitative polymerase chain reaction (RT-qPCR)

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8 was added into the cDNA reaction mix. The cDNA Master Mix (Thermo Fisher Scientific) consisted of 10× RT buffer for 2 μL, 25× dNTPs for 0. 8μL, Random Primers for 2 μL, Multiscribe RT for 1 μL and RNase Inhibitor for 1 μL. Lastly, DEPC-water was added into the reaction mix to make up 20 μL.

Real-time quantitative polymerase chain reaction: The RT-qPCR Master Mix consisted of

DEPC-water for 2.8 µl, Fast Syber Green (Thermo Fisher Scientific) for 5 µl and 30 µM Forward/Reverse Primer for 1 µl each. Then, 2 µl cDNA templates were added to the qPCR reaction mix to make total volume of 10 µl per well. The program was run using preset cycles in QuantStudio5 (Thermo Fisher Scientific) and comparative expression levels were analyzed. Primer sequences are listed below:

Gene Primer Sequence (5’→3’)

Mrc-1 F- GCAAATGGAGCCGTCTGTGC R- CTCGTGGATCTCCGTGACAC Arg-1 F- CAGAAGAATGGAAGAGTCAG R- CAGATATGCAGGGAGTCACC Emr1 (F4/80) F- TTTCCTCGCCTGCTTCTTC R- CCCCGTCTCTGTATTCAACC Il10 F- CAGAGCCACATGCTCCTAGA R- TGTCCAGCTGGTCCTTTGTT Tnfa F- GAGAAAGTCAACCTCCTCTCTG R- GAAGACTCCTCCCAGGTATATG Plx F-GCGGATTGAACCAGAATGGAGC R-TTCCTCCATGCTTGGCACGGAT Adra1a F- CTGCCATTCTTCCTCGTGAT R- GCTTGGAAGACTGCCTTCTG Adra2b F- GCTGATCTGGTCATGGGATT R- CGTCACACACAGCACATCTA Actb F- GCCCTGAGGCTCTTTTCCAG R- TGCCACAGGATTCCATACCC

Flow Cytometry

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9 surface markers for15 min at 4°C and then washed in FACS buffer. The cells were fixed and permeabilized followed by intracellular staining. The cells were washed with perm buffer before being suspended in FACS buffer for acquisition. Flow data was analyzed using FlowJo (TreeStar).

Antibodies used: Arginase-1, iNOS, CD206, CD80, CD86

Data analysis

Comparison between control and experiment group is performed with Student-t test. ANOVA is used to compare selected group. A P value <0.05 was considered significant.

Results

CX

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CR1 deficiency affects sympathetic innervation in islets.

It was seen that intra-islet macrophages were in close proximity with sympathetic nerves. (Fig.1, A to B). Christofferson et al have found that intra-islet macrophages were reactive to catecholamines stimulation in vitro. Thus, we speculated islet-infiltrated macrophages could communicate with sympathetic nerves. Also, we saw the similar juxtaposition in heart tissue, which was usually highly innervated by sympathetic nerves (Fig.2A).

Macrophage-nerve colocalization in exocrine tissue was weaker than in islet (Fig.1C). But, there were some interactions, probably due to their preferred location near the vasculature (Christoffersson et al. 2020).

Antigen retrieval impacted both nerve pattern and macrophage staining quality (Fig.1, D to E). As an important step to keep epitopes active in immunofluorescent staining, antigen retrieval could improve TH but weaken CD68 staining. After optimizing experimental conditions for several times, we figured out that non-antigen retrieval plus overnight primary staining was be the best solution to get both TH and CD68 clear and bright.

Images showed that GFP was expressed in a majority of islet-resident macrophage in CX3CR1GFP/GFP mouse (Fig.1, F to H). And CX3CR1 deficiency disrupted sympathetic nerves

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10 A B C

D E F

G H I

Fig.1. CX3CR1 deficiency affects sympathetic innervation in islets. (A) Non-antigen

retrieval islet displayed self-reflection when performed on confocal microscope, which was a straightforward way to look for islets in pancreas section. (B) WT islet was highly infiltrated by macrophages (red, CD68) and sympathetic nerves (cyan, tyrosine hydroxylase). Arrows show colocalization. (C) Less colocalization was present in exocrine tissue. Arrowheads indicate macrophage location. (D-E) Antigen retrieval increased nerve staining pattern yet weakened macrophage staining. (F-H) Majority of islet-infiltrated macrophages were CX3CR1GFP/GFP. (I) In CX3CR1-deficient islets, sympathetic nerves exhibited less axon-like

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A B

Fig.2. Sympathetic innervation decreased in hearts of CX3CR1 knockout mice. (A-B)

Macrophages and sympathetic nerves were stained in heart tissues from WT and CX3CR1GFP/GFP

mice. Decreases of nerves were observed in CX3CR1 knock-out mice.

Bone marrow-derived M1/M2 macrophages exhibit different expression profile

under NE presence and/or absence.

M1 and M2 macrophages were induced from bone marrow progenitors (Fig.3A). We used IL-4 to polarize BMDMs to M2 cells and added both LPS and TNF-α to get M1 cells. The classic M2 polarization markers—Mrc-1 and Arg-1—were highly expressed in M2 control group (M2+PBS) while they were expressed less in the M1 control group (M1+PBS). Meanwhile, expression of Emr1 (F4/80), an immunostimulatory and pan-macrophage molecule, was largely similar between the two.

Neurotransmitter expression levels were diverse between M1 and M2 (Fig.3B). We found α1

adrenoceptor (Adra1a) was expressed 4-times more in M1 control group than in M2 control group and NE stimulation did not change the expression level significantly. However, compared with the M2 control group, the β2 adrenoreceptor (Adrb2b) had half the expression in M1

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12 The cytokine expression profile was seen to be different in M1 vs. M2. We found that TNF-α was highly expressed in the M1 control group yet slightly decreased after NE stimulation. However, this pro-inflammatory cytokine maintained lower expression in the M2 macrophages (Fig.3C). Counterintuitively, IL-10, generally recognized as an anti-inflammatory cytokine, displayed a 10-fold higher expression level in M1 control group than in M2 control group (Fig.3D). This phenomenon was observed in our lab for several times in different IL-10 quantitative experiments of in vitro cultured macrophages. Besides, NE treatment inhibited IL-10 in M1 group while α adrenoreceptor was mainly responsible for mediating this decrease. Also, we collected supernatants from cell culture media to quantify immune biomarkers (Fig.3E). Proinflammatory cytokines, such as IL-12, IL-1β, KC/GRO and TNF-α, had higher expression in M1 group. However, as mentioned before, IL-10 expressed more in M1 rather than in M2 group.

Polarization into M1 and M2 phenotypes was clearly observed from our results apart from showing that NE stimulation can skew the M1 into a more pro inflammatory type by α1

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13 A B

C D

E

Fig.3. Bone marrow derived M1/M2 exhibit different expression profile under NE

presence and/or absence. (A-D) 2-ΔCt was calculated from RT-qPCR data with Actb as the

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M1 and M2 express different levels of intracellular markers and plasma

membrane markers indicating diverse inflammatory status.

To detect how NE manipulation changed the M1/M2 polarization status, we conducted flow cytometry. Mean fluorescence intensity (MFI) can be used to indicate antigen expression level across various samples. In this case, pro-inflammatory and anti-inflammatory markers were measured, and the NE stimulation effect was evaluated.

We found that Arginase-1 and CD206, as classic anti-inflammatory markers, had a higher expression level in M2 control than in M1 control, while NE stimulation decreased Arg-1 expression in M2 cells. Meanwhile, inflammatory markers iNOS and CD86 were highly expressed in the M1 group compared to the M2 group. Also, iNOS increased a little in M1 macrophages after incubating with NE (Fig.4, A to C).

Overall, MFI data verified that bone marrow-derived M1/M2 groups matched M1/M2 characterizations respectively. However, we suspected that 4 h of 10-8 NE co-culturing was not

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15 A

B

C

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Discussion

Sympathetic nerve-macrophage communication is indispensable in both maintaining functional islets and inducing type 1 diabetes (Saravia and Homo-Delarche 2003)(Pirzgalska et al. 2017). In this project, we observed nerve-macrophage colocalization in both WT and CX3CR1

knockout islets. And we also aimed at providing baseline of macrophage reactivity to NE while the final goal is to understand the mechanism behind macrophage-nerve communication. Christofferson et al have reported (2020) that macrophages could be an important relay where sympathetic nerves affect the inflammatory condition of islets in healthy and diabetic mice. Therefore, we continued exploring how macrophages and nerves were positioned in islet and exocrine tissue. Images showed a close proximity between islet-infiltrated macrophages and sympathetic nerves. However, lacking CX3CR1 in macrophages disrupted this kind of

juxtaposition. We speculate that CX3CR1 plays an important role in intra-islet macrophage and

sympathetic nerve communication. Besides, when conducting immunofluorescence staining experiments, we tried to optimize the experiment protocol to get satisfying images. We had previously used F4/80 as a macrophage marker, yet turned to CD68 due to brighter and more stable staining results, which allowed us to detect macrophage-nerve colocalization more easily. The usage of antigen retrieval is to be debated especially while considering macrophage marker identification. Sympathetic nerves showed ideal continuous axons rather than unsatisfied dot-like shape under confocal microscope after antigen retrieval, however, CD68 staining performed better without antigen retrieval. Hence, this trade-off determined the image quality and data credibility. Finally, we decided to incubate primary antibodies overnight and to not conduct antigen retrieval, which enabled us to optimize both TH and CD68 staining in the same slide.

We analyzed bone marrow-derived M1/M2 macrophages expression profile with or without NE stimulation in order to collect a basal reference of macrophage reactivity to NE(Vasamsetti et al. 2018). The slight decrease of IL-10 after 10-8 NE treatment in M1 group indicated the

tendency that M1 macrophages might skew to become more proinflammatory, which corresponded to α1 adrenoceptor activation by low concentration of NE. Nonetheless, the reason

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17 not observe any clear shift of cytokine expression in M1 or M2 after NE stimulation, we suspect that 4 h NE incubation was insufficient to cause significant change inside the cells, especially with less adrenoceptors in BMDMs compared with intra-islet macrophages. Thus, the NE stimulation time should be extended to 24 h in future experiments. Also, flow data showed that there was no distinct difference with NE presence or absence. Due to that, we have confirmed the islet-infiltrated macrophage did respond to catecholamines on protein level (Christoffersson et al. 2020), hence, an increase of NE incubation time might be required to detect changes.

Future perspectives

We will conduct the experiment with exact settings in more than 3 mice in both wild-type and CX3CR1GFP/GFP to obtain sufficient data to perform statistical tests. In addition, blocking

cytoskeletal rearrangement will be performed before antibody staining in flow cytometry sample preparation to prevent cytokines to be released from cells. Next, we will isolate and culture macrophages from islets and exocrine tissues in wild-type and CX3CR1GFP/GFP mice to

repeat the above-mentioned experiments. Also, we will use diverse concentrations of fractalkine to stimulate these macrophages. This will give us more direct observations about how intra-islet macrophages respond to sympathetic signals and whether this communication is executed by fractalkine-CX3CR1 ligation.

Acknowledgement

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References

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In addition, the interaction between B cells and dendritic cells in IgE-mediated immune enhancement were studied in mice immunized with antigens alone or

IgE-antigen complex is captured by CD23 receptor on follicular B cell in the circulation and (a) is transported into spleen, where follicular B cell transfers the antigen on DC (b)..

A test of monocytes derived from the bone marrow of the mice suggested that two factors, one responsible for blood vessel survival and one responsible for facilitating migration

We were interested to know if MZB cells can stimulate T-cells to produce specific cytokines that aid in breakage of tolerance and induction of inflammatory response in mouse model