maranickel_UROproposal

Overexpressed microRNA-146a in myeloid cells decreases effects of rheumatoid arthritis

Mara Nickel

Sophomore-The Ohio State University

Overview: Arthritis is a disease that causes inflammation in joints. A specific type of arthritis, rheumatoid

arthritis, occurs when the inflammation of the joints show symmetry, i.e. both hands, both wrists or both knees

(1). Along with its symmetric progression, it can also cause inflammation in the skin, eyes, heart, lungs, blood

or nerves of the patient (1). Rheumatoid arthritis causes severe joint pain and swelling, stiffness and fatigue in

the individual. It is an autoimmune disease; the immune system becomes deregulated in certain cells, causing

them to release certain chemical that attack the healthy cartilage of the joints and bone (2). Rheumatoid arthritis

affects approximately 1.3 million Americans, seventy-five percent of which are female (3). With the pain and

swelling associated with the disease, it brings a compromised quality of life, disabilities, and in some cases,

premature death (2). There is no cure for the arthritis, only medications that hope to bring the disease to a

remission state, one where inflammation is gone or very low (3).

Ostrowski’s lab has been researching the mechanism of the differentiation of osteoclast, a type of bone

cell that functions in the remolding of the bone, specifically its degradation. The pathway of two cytokines,

RANKL and CSF-1, have been studied for their role in osteoclast differentiation. It has been found that their

signaling on myeloid precursors triggers a cascade of events that leads to the differentiation (4). Ostrowski’s lab

has previously examined the relationship between RANKL/CSF-1 signaling and osteoclast differentiation using

in vitro techniques. Mouse bone marrow precursors were grown in the present of CSF-1 for three days on

plastic dishes to enrich myeloid precursors. These precursors were then exposed to 50ng/mL of RANKL in

addition to 25ng/mL of CSF-1 and left to grow for three days. After these three days, histochemical staining

showed high levels of TRAP positive multinuclear osteoclasts. Additional two days of CSF-1 RANKL

treatment enriched these differentiated multinuclear osteoclasts in the culture (not published) (Figure 1).

Figure 1:Differentiation of osteoclast in bone marrow derived macrophage precursors after days (indicated at top) of exposure to CSF-1/RANKL cytokines.

While osteoclasts are important in normal bone function, in patients of RA, osteoclast differentiation is

hyper activated, causing the bone erosion associated with the disease (5). In a study where researchers inhibited

osteoclast by denosumab, an antibody that selectively binds RANKL, in humans with RA, periarticular bone

loss and erosions were halted (5).

While much of the mechanism for the effect of RANKL and CSF-1 stimulation in myeloid precursors

has been established, recent research has identified the significance of microRNA in the differentiation process.

Regulation of miRNA is also associated with the RANKL/CSF-1 signaling pathway, but less is known of the

multitude of effects these microRNA have on the production of osteoclast (6). Because of the plasticity in their

regulation, looking further into their effects may allow insight to the fine-tuning and control of the

differentiation process in cells, potentially leading to a mechanism for mediating overproduction.

MiRNA are a class of non-coding RNA, usually around 20-25 nucleotides in length (7). Each miRNA is

complimentary to one or more mRNA; their main function with the mRNA is to regulate gene expression, often

times through translation repression, mRNA cleavage and deadenylation (7). Because of their large role in gene

regulation, it is suggested that finding the mechanisms and targets of these miRNA in biological pathways could

open the doors to effective therapeutic target treatments (7). Specifically to osteoclast differentiation,

Ostrowski’s lab has discovered 43 miRNAs differentially expressed in myeloid precursors with the treatment of

CSF-1 and RANKL, 38 upregulated and five downregulated (not published) (Figure 2).

One specific upregulated miRNA, miR-146a, also found to be in high levels in the RA synovial fluid,

has caught the attention of the Ostrowski’s lab. It was discovered to control the downregulation of TRAF6 and

IRAK1, two proteins critical in osteoclast differentiation as well as rheumatoid arthritis (8). Synovial fluid of

Figure 2: MiRNA downregulated (right) and samples of miRNA upregulated (right) by RANKL/CSF-1 signaling in myeloid precursors

RA contains several cell types, such as stromal fibroblasts, T cells, macrophages and monocytes, so it is

difficult to evaluate which compartment of these cell types is responsible for the upregulation of miR-146a (9).

Since RA synovial tissue has abundance of RANKL, one plausible theory is that upon exposure to RANKL, the

myeloid lineage of cells present in RA synovium overexpress miR-146a as a negative feed back loop

mechanism (5). To evaluate whether upregulation of miR-146a in myeloid precursors has any effect on

osteoclast differentiation, members of Ostrowski’s lab over-expressed miR-146 in myeloid precursors and

treated them with RANKL and CSF-1. TRAP staining multinucleated osteoclast-like cells showed that there

was a significant reduction in differentiation. This process was repeated using antago mir-146, and with this

there was a dramatic increase in differentiation compared to the control, scrambled miRNA (not published)

(Figure 3).

They next validated that the reason for the decrease in differentiation is because miR-146 targets two

proteins necessary for osteoclast differentiation: TRAF6 and IRAK1. Using cells expressing the scrambled

miRNA and cells with overexpression of mir-146, they analyzed the expression of the proteins using

immunofluorescence analysis and immunoblotting (Figure 4). Their results validated their theory;

overexpression of miR -146a caused a noticeable decrease in TRAP6 and IRAK1 (not published).

Figure 3: TRAP staining of osteoclast-like cells after expression of scrambled miRNA (control), over-expression of miR-146, and

antagonist miR-146 after three days of treatment with CSF-1 and RANKL.

Figure 4: Immunofluorescence of IRAK1 and TRAF6 after treatment with scrambled miRNA (control) and

overexpression of mir-146a.

The RA synovial tissue has an abundant supply of cytokines and cells that may have a role in

upregulation of miR-146a. In my research, I will examine the effect of over-expressed miR-146a in myeloid

cells in a RA model. Since RA is a manifestation of several cell types and it is difficult to mimic the interaction

of these cell types in vitro, I will use the genetic mouse models where miR-146a is deleted in all cell types, with

overexpression possible in only myeloid cell compartment. Hyperactive osteoclast differentiation of myeloid

precursors leads to many of the symptoms associated with RA (5). By studying the effect of miR-146a in

myeloid cells throughout the progression of RA, it could allow for a potential compartment for targeted

microRNA treatment.

Hypothesis: Overexpression of miR-146a in myeloid compartment will lead to lower osteoclast activity,

therefore lower inflammation and bone erosion in a preclinical model of rheumatoid arthritis.

Specific Aim: Evaluate the extent of rheumatoid arthritis and concurrent bone destruction in presence or

absence of miR-146a in myeloid cells in a mouse model of RA.

Methodology: I will use a genetic mouse model designed to control the regulation of miR-146a. I was involved

in the generation of a genetic mouse model with a systemic knockdown of miR-146a (miR-146a KO) combined

with a genetic knockin of miR-146a (miR-146a KI). The knock-in model removes the Loxp-stop-loxp cassette

using myeloid-specific Cre, which functions when stimulated by tamoxifen (Figure 5). This allows for the

transcription of miR-146a in the myeloid lineage. I have been crossing the mir-146a KO mice with the miR-

146a KI and FmsERt-cre mice to obtain the required genotypes: miR-146a-KO/KO;miR-146a Ki/Ki, used as a

control, and miR-146a-KO/KO;mir-146a-KI/KI:FmsERT-cre, used for experimental purposes.

Also being utilized is the methylated bovine serum albumin (mBSA) and Interleukin-1 beta (IL-1 beta)

induced RA model. To create a model of RA, mice will start with one injection of mBSA into their right knee.

This will be followed on the same day with an injection of IL-1 beta to the right foot of the mouse. The mBSA

is an autoantigen that allows the immune system to mimic an autoimmune response found in RA. IL-1 beta

promotes this simulation. In the consecutive two days, IL-1 injections will continue to be administered.

To look at the extent of the effects of miRNA-146a on RA, I will express miR-146a in the myeloid cells

at two time points: pre-RA model injections and post-RA model injections. This will be controlled by when

tamoxifen is induced into the model. The timeline describing the administration of tamoxifen through injections

and food is described in Table 1. This will be done for the control, experimental, and wild type genotypes. For

seven days after the injection of the mBSA/IL-1 beta, the mice will be clinically scored for inflammation at the

knee joints. At the end of ten days, the mice will be euthanized and the knee joints will be harvested. Coronal

section of the knee joints embedded in plastic will be analyzed for the pathology of RA. They will be stained for

Figure 5: Schematic representation of miR-146a knock-in strategy. In the presence of cre the Lox-STOP-Lox sequences are recombined to excise the 3Xpoly A, enabling the transcription of miR-146 (top panel). I will use a transgenic cre-ER fusion that utilizes the fms promotor,

expressed in the myeloid compartment. In absence of tamoxifen, cre is localized to the cytoplasm, rendering it ineffective. In presence of tamoxifen cre is translocated to the nucleus

and able to recombine the loxp sites, excising the STOP sequences (bottom right).

cartilage with Safronin O, infiltration of immune cells with hematoxylin and eosin staining, osteoclast activity

with TRAP staining, and bone erosion using alizarin red and alacian blue staining.

*intra peritoneal injection

Expected outcomes: I would expect that overexpression of miR-146a in the myeloid compartment prior to the

induction of RA would have an effect on recruitment of osteoclast precursors, and thus less bone erosion and

inflammation. Expressing miRNA-146a after inducing RA in the mouse model may not cause a decrease in the

recruitment of osteoclast or bone erosion, as the bone marrow precursors with knockout of miR-146a may have

already moved to the cartilage. If this is the case, I plan to lengthen the timeline following the induced RA

model to see if that allows time for miR-146a to have an effect.

Significance: MiRNA-146a has shown to decrease the differentiation of osteoclast of myeloid precursors when

stimulated with RANKL and CSF-1. If it can be found that miR-146a functions similarly in RA, and has a large

effect on the symptoms of RA, it has the potential to be utilized as a targeted treatment. Confirming this

compartment is critical to development of the treatment. Further, for targeted treatments to be efficient, they

must be administered at the proper timing. My research could give insight on when the treatment must be

administered to obtain the full effect.

Days   1   2   3   4   5   6   7   8   9   10  

Tam  ip*   +   +   +   -­‐   -­‐   -­‐   -­‐   -­‐   -­‐   -­‐  Tam  Food   -­‐   - - + + + + + + + Tam  ip   -­‐   - - - - - + + + - Tam  Food   -­‐   - - - - - - - - - mBSA   -­‐   - - + - - - - - - IL1  beta   -­‐   -­‐   -­‐   +   +   +   -­‐   -­‐   -­‐   -­‐  

Table 1: Treatments of mouse models. Yellow indicates tamoxifen administration to mice where miR-146a is expressed before RA, blue indicates miR-146a expressed after RA.

References:

1. "What Is Rheumatoid Arthritis? - WebMD." WebMD. WebMD, n.d. Web. 11 Feb. 2014.

2. "Rheumatoid Arthritis." Centers for Disease Control and Prevention. Centers for Disease Control and

Prevention, 19 Nov. 2012. Web. 12 Feb. 2014. 3. Rheumatoid Arthritis | American College of Rheumatology | ACR." Rheumatoid Arthritis | American College

of Rheumatology | ACR. N.p., n.d. Web. 12 Feb. 2014. 4. Sharma, S. M., Bronisz, A., Hu, R., Patel, K., Mansky, K. C., Sif, S., and Ostrowski, M. C. (2007) MITF and

PU.1 recruit p38 MAPK and NFATc1 to target genes during osteoclast differentiation. J Biol Chem 282, 15921-15929

5. Geusens, P. (2012) The role of RANK ligand/osteoprotegerin in rheumatoid arthritis. Ther Adv Musculoskelet Dis. 4, 225-233. 6. Sagatani, T., Hruska, K. A. (2007). MicroRNA-233 is a key factor in osteoclast differentiation. J Bio Chem

101, 996-999.

7. "MiRNA (microRNA) Introduction." Sigma-Aldrich. N.p., n.d. Web. 12 Feb. 2014. 8. Nakasa, T., Shibuya, H., Nagata, Y., Niimoto, T. Ochi, M. (2011) The Inhibitory Effect of MicroRNA-146a

Expression on Bone Destruction in Collagen-Induced Arthritis. Arthritis Rheum 63, 1582-1590. 9. Nakasa, T., Miyaki, S., Okubo, A., Hashimoto, M., Nashida, K., Ochi, M., Asahara, H. (2008) Expression of

microRNA-146 in rheumatoid arthritis synovial tissue. Athritis Rheum 5, 1284-1292.