WO2012092382A2 - Role of microrna in t cell immune response - Google Patents

Abstract

The present disclosure provides methods for increasing or decreasing T cell activation. The methods include contacting a T cell with an miRNA nucleic acid or an inhibitor of an miRNA. In some embodiments, the disclosed methods for increasing T cell activation include activating a T cell and contacting the activated T cell with an effective amount of an miRNA nucleic acid (such as one or more of miR-21, miR-101a, miR-377, let-7b, let-7c, or miR-574); or an inhibitor of an miRNA (for example, an inhibitor of one or more of miR-99a, miR-467b, miR- 467d, miR-199a, or miR-192), thereby increasing T cell activation, for example as compared to a control. In other embodiments, the disclosed methods for decreasing T cell activation include contacting a T cell (such as an activated T cell) with an effective amount of an inhibitor of an miRNA (for example, an inhibitor of one or more of miR-21, miR-101a, miR-377, let-7b, let-7c, or miR-574); or an miRNA nucleic acid (such as one or more of miR-99a, miR-467b, miR-467d, miR-199a, or miR-192), thereby decreasing the T cell activation, for example as compared to a control.

Description

ROLE OF MICRORNA IN T CELL IMMUNE RESPONSE

CROSS REFERENCE TO RELATED APPLICATION

This claims the benefit of U.S. Provisional Application No. 61/428,112, filed December 29, 2010, which is incorporated herein by reference in its entirety.

FIELD

This disclosure relates to immunology, particularly methods for increasing or decreasing T cell activation, for example with microRNAs, microRNA mimics, or microRNA inhibitors.

ACKNOWLEDGMENT OF GOVERNMENT SUPPORT

This invention was made with government support under VA Merit Review awarded by the Department of Veterans Affairs; NCC 2-1361 and NAG- 1286 awarded by the National Aeronautics and Space Administration; and

1HU2AG037628-01 awarded by the National Institutes of Health. The government has certain rights in the invention.

BACKGROUND

Discovered in nematodes in 1993, microRNAs (miRNAs) are non-coding

RNAs that are related to small interfering RNAs (siRNAs), the small RNAs that guide RNA interference (RNAi). miRNAs sculpt gene expression profiles during plant and animal development and may regulate as many as one-third of human genes. miRNAs are found only in plants and animals, and in the viruses that infect them. As of the recent Sanger release of the miRNA repository (miRBase v. 12.0), 866 human miRNAs have been annotated, and this number continues to increase. Each miRNA may have hundreds of targets due to imperfect base pairing.

miRNAs function very much like siRNAs, but these two types of small RNAs can be distinguished by their distinct pathways for maturation (Du and Zamore, Development (Cambridge) 132:4645-4652, 2005). miRNA is made from larger pri-miRNA which is much longer than the processed mature miRNA molecule. Pri-miRNA has a cap and poly-A tail and is processed to short 70- nucleotide stem loop structures in the cell nucleus. The pri-miRNA is processed by Drosha and made into pre-miRNA. The pre-miRNA is then exported to the cytoplasm by exportin-5 and processed further by the enzyme Dicer into mature miRNA. miRNA in the cytoplasm then combines to form a complex miRISC, which is guided to its mRNA target by the miRNA strand to match RNA targets.

Most miRNAs are 20-25 nucleotides long and are non-protein coding. The majority regulate gene expression by binding to the 3' untranslated region (UTR) of target mRNAs inducing translational repression of RNA and protein through mechanisms not fully understood. It has recently been discovered that at least some miRNAs may increase gene expression through a process called RNA activation. In these cases, the miRNA appears to target a sequence in the gene promoter (e.g., Li et al, Proc. Natl. Acad. Sci. USA 103: 17337-17342, 2006; Janowski et al, Nature Chem. Biol. 3: 166-173, 2007; Schwartz et al., Nature Struct. Mol. Biol. 15:842-848, 2008).

Over 50% of the Apollo astronauts had bacterial or viral infections during flight, or within one week of landing. Apollo 7 marked humanity's first experience with spaceflight infection when all three crewmembers contracted head colds during their mission and on Apollo 13, one astronaut contracted Pseudomonas aeruginosa and suffered from intense chills and fever (Hawkins and Zeiglchmid, In Biomedical Results of Apollo (Johnston et al, eds.), pp. 43-81, NASA, 1975). P. aeruginosa is an opportunistic pathogen and rarely causes disease unless the person suffers from a break in epithelia or from immune suppression. As a result, the U.S. and Russian programs implemented pre-flight quarantine programs. Even with the precautions, one of the astronauts working on the International Space Station (ISS) had full body shingles while in orbit. Experiments from Skylab and Shuttle have confirmed that T-cells have a suppressed immune response (in vivo and in vitro) with lower T cell proliferation/activation, lower IL-2 synthesis and severely reduced IL-2Ra expression (RNA and protein); these blunted immune responses are also seen in the immunosuppressed elderly (Merck Manual of Geriatrics, 3 rd edition and online addition, 2005). miRNAs may be up-or down-regulated in spaceflight (microgravity conditions) or during the aging process and may present targets for modulating immune responses.

SUMMARY

The present disclosure provides methods for increasing or decreasing T cell activation. The methods include contacting a T cell (such as an activated T cell) with an miRNA nucleic acid or an inhibitor of an miRNA.

In some embodiments, the disclosed methods for increasing T cell activation include activating a T cell and contacting the activated T cell with an effective amount of an miRNA nucleic acid that activates T cells (such as one or more of miR-21, miR-lOla, miR-377, let-7b, let-7c, and miR-574); or an inhibitor of an miRNA that inhibits T cell activity (for example, wherein the miRNA includes one or more of miR-99a, miR-467b, miR-467d, miR-199a, and miR-192), thereby increasing T cell activation, for example as compared to a control.

In other embodiments, the disclosed methods for decreasing T cell activation include contacting a T cell (such as an activated T cell) with an effective amount of an inhibitor of an miRNA that activates T cells (for example, wherein the miRNA includes one or more of miR-21, miR-lOla, miR-377, let-7b, let-7c, and miR-574); or an miRNA nucleic acid that inhibits T cell activity (such as one or more of miR- 99a, miR-467b, miR-467d, miR-199a, and miR-192), thereby decreasing the T cell activation, for example as compared to a control.

In some examples, the disclosed methods are performed in vitro or ex vivo, for example in a sample from a subject that includes T cells. In particular examples, T cells are contacted with one or more of the disclosed miRNAs or inhibitors of miRNAs and then administered to a subject. In some examples, the subject has decreased immune system function (such as a subject infected with human immunodeficiency virus (HIV) or a subject which has been exposed to

micro gravity). In other examples, the subject has an inflammatory or autoimmune disorder (such as rheumatoid arthritis). The foregoing and other features of the disclosure will become more apparent from the following detailed description, which proceeds with reference to the accompanying figures. BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a series of bar graphs showing fold increase in expression of the indicated genes in activated T cells in normal gravity (lg) or microgravity ^g) during spaceflight conditions as compared to non-activated (NA) T cells, as determined by qRTPCR. * p<0.05; ** p<0.001.

FIG. 2 is a bar graph showing MiR-21 expression in activated T cells in normal gravity (lg), one-half normal gravity (0.5g), or microgravity ^g) during spaceflight as determined by qRTPCR. * p<0.05; ** p<0.001.

FIG. 3 is a series of bar graphs showing fold increase in expression of the indicated genes in activated T cells in normal gravity (lg) or simulated microgravity ^g) conditions as compared to non-activated (NA) T cells as determined by qRTPCR. * p<0.01; ** p<0.005; *** p<0.001.

FIG. 4 is a digital image of a Western blot (top) and a graph showing band intensity (bottom) of TAGAP protein in activated human T cells in normal gravity (lg) or simulated microgravity (μ^) conditions as compared to non-treated (NT) T cells. Bars represent mean + SD of each set of samples (n=3; *p<0.05 with two- tailed Student's t-test).

FIG. 5A shows a schematic diagram of a predicted pathway of gene expression and down-regulation by MiR-21 during T cell activation. FIG. 5B shows predicted promoter regions of interleukin-2 (IL-2), IL-2 receptor a (IL-2Ra), Pre- MiR-21 (MiPPR-21), FASLG, SPRY2, and TAGAP.

FIGS. 6A-D are a series of bar graphs showing mRNA expression of IL-2 (FIG. 6A), IL2ra (FIG. 6B), IFNy (FIG. 6C), and CCL3 (FIG. 6D) in T cells from C57BL/6 mice kept in normal gravity (ground) or exposed to microgravity for 15 days in spaceflight (flight). T cells were activated with T cell activation beads (BA) or Concanavalin A (ConA) and anti-CD28. mRNA expression was normalized to cyclophilin (CPHI) expression. NT, non-treated controls. SEQUENCE LISTING

The nucleic acid and amino acid sequences listed in the accompanying sequence listing are shown using standard letter abbreviations for nucleotide bases, and three letter code for amino acids, as defined in 37 C.F.R. 1.822. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood as included by any reference to the displayed strand. In the

accompanying sequence listing:

SEQ ID NO: 1 is an exemplary pri-miR-21 nucleic acid sequence.

SEQ ID NO: 2 is an exemplary pre-miR-21 nucleic acid sequence.

SEQ ID NO: 3 is an exemplary mature miR-21 nucleic acid sequence.

SEQ ID NO: 4 is a nucleic acid sequence of a Fas ligand 3' untranslated region (UTR).

SEQ ID NO: 5 is a nucleic acid sequence of a sprouty homolog 2 3' UTR. SEQ ID NO: 6 is a nucleic acid sequence of a T-cell activation Rho GTPase activating protein 3' UTR.

DETAILED DESCRIPTION

Abbreviations

ConA Concanavalin A

FASLG Fas ligand

HIV human immunodeficiency virus

IL interleukin

IFN interferon

miRNA microRNA

miR-21 microRNA-21

qRTPCR quantitative real-time PCR

SPRY2 sprouty homolog 2 (Drosophila)

TAGAP T-cell activation Rho GTPase activating protein II. Terms

Unless otherwise noted, technical terms are used according to conventional usage. Definitions of common terms in molecular biology may be found in

Benjamin Lewin, Genes V, published by Oxford University Press, 1994 (ISBN 0-19- 854287-9); Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology : a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8).

Unless otherwise explained, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. The singular terms "a," "an," and "the" include plural referents unless context clearly indicates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise. As used herein, "comprises" means "includes." Thus, "comprising A or B," means

"including A, B, or A and B," without excluding additional elements. It is further to be understood that all base sizes or amino acid sizes, and all molecular weight or molecular mass values, given for nucleic acids or polypeptides are approximate, and are provided for description. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety for all purposes. All GenBank Accession Nos. and miRBase Accession Nos. mentioned herein are incorporated by reference in their entirety as present on December 29, 2010.

Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. In case of conflict, the present specification, including explanations of terms, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

In order to facilitate review of the various embodiments of the invention, the following explanations of specific terms are provided: Autoimmune disorder: A disorder in which the immune system produces an immune response (e.g., a B cell or a T cell response) against an endogenous antigen, with consequent injury to tissues. The injury may be localized to certain organs, such as thyroiditis, or may involve a particular tissue at different locations, such as Goodpasture's disease, or may be systemic, such as lupus erythematosus.

In some examples, autoimmune diseases include systemic lupus

erythematosus, Sjogren's syndrome, rheumatoid arthritis, type I diabetes mellitus, Wegener's granulomatosis, inflammatory bowel disease, polymyositis,

dermatomyositis, multiple endocrine failure, Schmidt's syndrome, autoimmune uveitis, Addison's disease, adrenalitis, Graves' disease, thyroiditis, Hashimoto's thyroiditis, autoimmune thyroid disease, pernicious anemia, gastric atrophy, chronic hepatitis, lupoid hepatitis, atherosclerosis, presenile dementia, demyelinating diseases, multiple sclerosis, subacute cutaneous lupus erythematosus,

hypoparathyroidism, Dressier' s syndrome, myasthenia gravis, autoimmune thrombocytopenia, idiopathic thrombocytopenic purpura, hemolytic anemia, pemphigus vulgaris, pemphigus, dermatitis herpetiformis, alopecia areata, pemphigoid, scleroderma, progressive systemic sclerosis, CREST syndrome (calcinosis, Raynaud's phenomenon, esophageal dysmotility, sclerodactyly, and telangiectasia), adult onset diabetes mellitus (Type II diabetes), male and female autoimmune infertility, ankylosing spondylitis, ulcerative colitis, Crohn's disease, mixed connective tissue disease, polyarteritis nedosa, systemic necrotizing vasculitis, juvenile onset rheumatoid arthritis, glomerulonephritis, atopic dermatitis, atopic rhinitis, Goodpasture's syndrome, Chagas' disease, sarcoidosis, rheumatic fever, asthma, recurrent abortion, anti-phospholipid syndrome, farmer's lung, erythema multiforme, post cardiotomy syndrome, Cushing' s syndrome, autoimmune chronic active hepatitis, bird-fancier's lung, allergic disease, allergic

encephalomyelitis, toxic epidermal necrolysis, alopecia, Alport's syndrome, alveolitis, allergic alveolitis, fibrosing alveolitis, interstitial lung disease, erythema nodosum, pyoderma gangrenosum, transfusion reaction, leprosy, malaria, leishmaniasis, trypanosomiasis, Takayasu's arteritis, polymyalgia rheumatica, temporal arteritis, schistosomiasis, giant cell arteritis, ascariasis, aspergillosis, Sampter's syndrome, eczema, lymphomatoid granulomatosis, Behcet's disease, Caplan's syndrome, Kawasaki's disease, dengue, encephalomyelitis, endocarditis, endomyocardial fibrosis, endophthalmitis, erythema elevatum et diutinum, psoriasis, erythroblastosis fetalis, eosinophilic faciitis, Shulman's syndrome, Felty's syndrome, filariasis, cyclitis, chronic cyclitis, heterochronic cyclitis, Fuch's eye litis, IgA nephropathy, Henoch-Schonlein purpura, glomerulonephritis, graft versus host disease, transplantation rejection, human immunodeficiency virus infection, echovirus infection, cardiomyopathy, Alzheimer's disease, parvovirus infection, rubella virus infection, post vaccination syndromes, congenital rubella infection, Hodgkin' s and Non-Hodgkin' s lymphoma, renal cell carcinoma, multiple myeloma, Eaton-Lambert syndrome, relapsing polychondritis, malignant melanoma, cryoglobulinemia, Waldenstrom's macroglobulemia, Epstein-Barr virus infection, rubulavirus, and Evan's syndrome.

Chemokine (chemoattractant cytokine): A type of cytokine (a soluble molecule that a cell produces to control reactions between other cells) that specifically alters the behavior of leukocytes (white blood cells). Chemokines include CC chemokines, CXC chemokines, C chemokines, and CX₃C chemokines. Examples include, but are not limited to, CCL3 and XCL2, and the like.

Complementarity and percentage complementarity: Molecules with complementary nucleic acids form a stable duplex or triplex when the strands bind, (hybridize), to each other by forming Watson-Crick, Hoogsteen or reverse

Hoogsteen base pairs. Stable binding occurs when an oligonucleotide remains detectably bound to a target nucleic acid sequence under the required conditions.

Complementarity is the degree to which bases in one nucleic acid strand base pair with the bases in a second nucleic acid strand. Complementarity is

conveniently described by percentage, e.g., the proportion of nucleotides that form base pairs between two strands or within a specific region or domain of two strands. For example, if 10 nucleotides of a 15-nucleotide oligonucleotide form base pairs with a targeted region of a DNA molecule, that oligonucleotide is said to have 66.67% complementarity to the region of DNA targeted. In the present disclosure, "sufficient complementarity" means that a sufficient number of base pairs exist between the oligonucleotide and the target sequence to achieve detectable binding. When expressed or measured by percentage of base pairs formed, the percentage complementarity that fulfills this goal can range from as little as about 50% complementarity to full (100%) complementary. In general, sufficient complementarity is at least about 50%, about 75%

complementarity, about 90% or 95% complementarity, about 98%, 99%, or even 100% complementarity.

A thorough treatment of the qualitative and quantitative considerations involved in establishing binding conditions that allow one skilled in the art to design appropriate oligonucleotides for use under the desired conditions is provided by Beltz et al. Methods Enzymol 100:266-285, 1983, and by Sambrook et al. (ed.), Molecular Cloning: A Laboratory Manual, 2nd ed., vol. 1-3, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989.

Contacting: Placement in direct physical association, including for example, a solid or liquid form. Contacting can occur in vitro or ex vivo with isolated cells or tissue or in vivo by administering to a subject (for example, administering a compound to a subject to achieve a desired concentration for a desired time at a target cell type in the body, for example, T cells).

Control: A "control" refers to a sample or standard used for comparison with an experimental sample. In some embodiments, the control is an activated T cell that has not been contacted with an miRNA or an miRNA inhibitor, such as those disclosed herein. In other embodiments, the control is a historical control or standard reference value or range of values (such as a previously tested control sample, such as a measure of T cell activation that represents baseline or normal values, such as a measure of T cell activation in a population or group, such as individuals with a particular condition or disorder).

Cytokine: Proteins made by cells that affect the behavior of other cells, such as lymphocytes. In one embodiment, a cytokine is a chemokine, a molecule that affects cellular trafficking. The term "cytokine" is used as a generic name for a diverse group of soluble proteins and peptides that act as humoral regulators generally at nanomolar to picomolar concentrations and which, either under normal or pathological conditions, modulate the functional activities of individual cells and tissues. These proteins also mediate interactions between cells directly and regulate processes taking place in the extracellular environment. Examples of cytokines include, but are not limited to, tumor necrosis factor a (TNFa), interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-6 (IL-6), interleukin-10 (IL-10), interleukin-12 (IL- 12), interleukin-21 (IL-21), macrophage inflammatory protein 2 (MIP-2), keratinocyte derived cytokine (KC), and interferon-γ (INF- γ).

Effective amount: A quantity of an agent or compound sufficient to achieve a desired effect in a subject or a cell being treated. For instance, this can be the amount of an agent or compound necessary to increase or decrease T cell activation. The effective amount of the agent or compound will be dependent on several factors, including, but not limited to the subject or cells being treated and the manner of administration of the agent or compound. In some instances, a "therapeutically effective amount" is a quantity of an agent or compound sufficient to prevent advancement, delay progression, or to cause regression of a disease, or which is capable of reducing symptoms caused by a disease, such as an inflammatory or autoimmune disease or disorder.

Immunocompromised: An immunocompromised subject is a subject who is incapable of developing or unlikely to develop a robust immune response, usually as a result of disease, malnutrition, or immunosuppressive therapy. An

immunocompromised immune system is an immune system that is functioning below normal. Immunocompromised subjects are more susceptible to opportunistic infections, for example viral, fungal, protozoan, or bacterial infections, prion diseases, and certain neoplasms.

In some examples, those who are considered to be immunocompromised include, but are not limited to, subjects with AIDS (or HIV positive), subjects with severe combined immune deficiency (SCID), diabetics, subjects who have had transplants and who are taking immunosuppressives, and those who are receiving chemotherapy for cancer. Immunocompromised individuals also include subjects with most forms of cancer (other than skin cancer), sickle cell anemia, cystic fibrosis, those who do not have a spleen, subjects with end stage kidney disease (dialysis), and those who have been taking corticosteroids on a frequent basis by pill or injection within the last year. Subjects with severe liver, lung, or heart disease also may be immunocompromised.

Inflammatory disease: A primary inflammation disorder is a disorder that is caused by inflammation itself. A secondary inflammation disorder is

inflammation that is the result of another disorder. Inflammation can lead to inflammatory diseases, such as rheumatoid arthritis, osteoarthritis, inflammatory lung disease (including chronic obstructive pulmonary lung disease), inflammatory bowl disease (including ulcerative colitis and Crohn's Disease), periodontal disease, polymyalgia rheumatica, atherosclerosis, systemic lupus erythematosus, systemic sclerosis, Sjogren's Syndrome, asthma, allergic rhinitis, and skin disorders

(including dermatomyositis and psoriasis) and the like.

Inflammation is a localized protective response elicited by injury to tissue that serves to sequester the inflammatory agent. Inflammation is orchestrated by a complex biological response of vascular tissues to harmful stimuli, such as pathogens, damaged cells, or irritants. It is a protective attempt by the organism to remove the injurious stimuli as well as initiate the healing process for the tissue. An inflammatory response is characterized by an accumulation of white blood cells, either systemically or locally at the site of inflammation. The inflammatory response may be measured by many methods well known in the art, such as the number of white blood cells, the number of polymorphonuclear neutrophils (PMN), a measure of the degree of PMN activation, such as luminol enhanced- chemiluminescence, or a measure of the amount of cytokines present. C-reactive protein is a marker of a systemic inflammatory response.

Microgravity: A state in which there is very little net gravitational force, for example, gravity less than about 0.1 x g. Microgravity conditions exist in space, for example, aboard the Space Shuttle, the International Space Station, a satellite, or a rocket while in flight outside the Earth's atmosphere. Simulated microgravity is microgravity which is simulated by a set of Earth-based conditions that mimic microgravity, such as by balancing gravity with equal and opposite forces (for example, shear force, centripetal force, Coriolus forces, buoyancy, and/or magnetic field). In one example, simulated microgravity may be generated by use of a clinostat, such as a rotating wall vessel (RWV). In another example, simulated microgravity may be generated by a random positioning machine (RPM). The term "microgravity conditions" and "microgravity" are used synonymously herein.

Normal gravity is the gravity normally experienced on Earth, such as on the surface of the Earth and/or in its atmosphere (for example, in aircraft in the atmosphere of the Earth). Gravity is measured in terms of acceleration due to gravity, denoted by g. The strength (or apparent strength) of Earth's gravity varies with latitude, altitude, local topography, and geology. In some examples, normal gravity (such as

1 x g) is about 9-10 m/s 2 , for example, about 9.7-9.9 m/s 2. In particular preferred embodiments, normal gravity is that experienced on the surface of the Earth under normal gravity at that location on the Earth.

MicroRNA (miRNA, miR): Single- stranded RNA molecules that regulate gene expression. MicroRNAs are generally 20-25 nucleotides in length.

MicroRNAs are processed from primary transcripts known as pri-miRNA to short stem-loop structures called precursor (pre)-miRNA and finally to functional, mature microRNA. Mature microRNA molecules are partially complementary to one or more messenger RNA molecules, and their primary function is to down-regulate gene expression. MicroRNAs regulate gene expression through the RNAi pathway.

MicroRNA sequences are publicly available. For example, miRBase (mirbase.org) includes a searchable database of annotated miRNA sequences.

miRNA sequences are also available through other databases known to one of skill in the art, including the National Center for Biotechnology Information

(ncbi.nlm.nih.gov). One of skill in the art can also identify targets for specific miRNAs utilizing public databases and algorthims, for example at MicroCosm Targets (ebi . ac.uk/enright- srv/microco sm/htdoc s/targets/) , TargetScan

(targetscan.org), and PicTar (pictar.mdc-berlin.de).

MicroRNA-21 (miR-21): A small non-coding RNA located on human chromosome 17. MicroRNA-21 is also known as miR-21, miRNA21 and hsa-mir- 21. The expression of miR-21 has been linked to inflammatory responses. miR-21 expression is increased following lipopolysaccharide-induced inflammation and increased miR-21 expression occurs during T-cell differentiation. Interleukin 6 (IL- 6), a proinflammaotory cytokine, can drive miR-21 expression through a STAT3 dependent mechanism.

miR-21 sequences are publicly available, for example, GenBank Accession

Nos. NR_029493, NC_000017 (nucleotides 57918627-57918698), AC004686, BC053563, AF480524, AJ421741, and AY699265, and miRBase Accession Nos. MI0000077, MIMAT0000076, and MIMAT000449, each of which are incorporated by reference herein as present in GenBank or miRBase on December 29, 2010. One skilled in the art will appreciate that miR-21 nucleic acid molecules can vary from those publicly available, such as polymorphism resulting in one or more

substitutions, deletions, insertions, or combinations thereof, while still retaining miR-21 biological activity (e.g., hybridization to a target sequence).

Pharmaceutically acceptable carrier: The pharmaceutically acceptable carriers (vehicles) useful in this disclosure are conventional. Remington: The

Science and Practice of Pharmacy, The University of the Sciences in Philadelphia, Editor, Lippincott, Williams, & Wilkins, Philadelphia, PA, 21^st Edition (2005), describes compositions and formulations suitable for pharmaceutical delivery of one or more therapeutic compounds, molecules or agents.

In general, the nature of the carrier will depend on the particular mode of administration being employed. For instance, parenteral formulations usually comprise injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle. For solid compositions (for example, powder, pill, tablet, or capsule forms), conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. In addition to biologically-neutral carriers, pharmaceutical compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate. Sequence identity: The similarity between two nucleic acid sequences, or two amino acid sequences, is expressed in terms of the similarity between the sequences, otherwise referred to as sequence identity. Sequence identity is frequently measured in terms of percentage identity (or similarity or homology); the higher the percentage, the more similar the two sequences are. For example, homologs or orthologs of a nucleic acid molecule will possess a relatively high degree of sequence identity when aligned using standard methods. This homology will be more significant when the orthologous nucleic acids are derived from species that are more closely related (e.g., human and chimpanzee sequences), compared to species more distantly related (e.g., human and C. elegans sequences).

Methods of alignment of sequences for comparison are well known in the art. Various programs and alignment algorithms are described in: Smith &

Waterman, Adv. Appl. Math. 2: 482, 1981; Needleman & Wunsch, J. Mol. Biol. 48: 443, 1970; Pearson & Lipman, Proc. Natl. Acad. Sci. USA 85: 2444, 1988; Higgins & Sharp, Gene, 73: 237-244, 1988; Higgins & Sharp, CABIOS 5: 151-153, 1989;

Corpet et al, Nucl. Acids Res. 16, 10881-90, 1988; Huang et al, Computer Appls. in the Biosciences 8, 155-65, 1992; and Pearson et al, Meth. Mol. Bio. 24:307-31, 1994. Altschul et al. (J. Mol. Biol. 215:403-410, 1990) presents a detailed

consideration of sequence alignment methods and homology calculations.

The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al, J.

Mol. Biol. 215:403-410, 1990) is available from several sources, including the National Center for Biotechnology Information (NCBI, Bethesda, MD) and on the Internet, for use in connection with the sequence analysis programs blastp, blastn, blastx, tblastn and tblastx. By way of example, for comparisons of amino acid sequences of greater than about 30 amino acids, the Blast 2 sequences function is employed using the default BLOSUM62 matrix set to default parameters (gap existence cost of 11, and a per residue gap cost of 1). When aligning short peptides (fewer than around 30 amino acids), the alignment is performed using the Blast 2 sequences function, employing the PAM30 matrix set to default parameters (open gap 9, extension gap 1 penalties). An alternative indication that two nucleic acid molecules are closely related is that the two molecules hybridize to each other under stringent conditions.

Stringent conditions are sequence-dependent and are different under different environmental parameters. Generally, stringent conditions are selected to be about 5°C to 20°C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The T_m is the temperature (under defined ionic strength and pH) at which 50% of the target sequence remains hybridized to a perfectly matched probe or complementary strand. Conditions for nucleic acid hybridization and calculation of stringencies can be found in Sambrook et al. (In Molecular Cloning: A Laboratory Manual, CSHL, New York, 1989) and Tijssen

{Laboratory Techniques in Biochemistry and Molecular Biology— Hybridization with Nucleic Acid Probes Part I, Chapter 2, Elsevier, New York, 1993).

Nucleic acid sequences that do not show a high degree of sequence identity may nevertheless encode similar amino acid sequences, due to the degeneracy of the genetic code. It is understood that changes in nucleic acid sequence can be made using this degeneracy to produce multiple nucleic acid molecules that all encode substantially the same protein.

Subject: Living multi-cellular vertebrate organisms, a category that includes both human and non-human mammals. Subjects include veterinary subjects, including livestock such as cows and sheep, rodents (such as mice and rats), and non-human primates.

T Cell: A white blood cell critical to the immune response. T cells include, but are not limited to, CD4⁺ T cells and CD8⁺ T cells. A CD4⁺ T lymphocyte is an immune cell that carries a marker on its surface known as "cluster of differentiation 4" (CD4). These cells, also known as helper T cells, help orchestrate the immune response, including antibody responses as well as killer T cell responses. CD8⁺ T cells carry the "cluster of differentiation 8" (CD8) marker. In one embodiment, a CD8⁺ T cell is a cytotoxic T lymphocyte. In another embodiment, a CD8⁺ cell is a suppressor T cell.

As used herein, "allogeneic" encompasses a genetically different phenotype present in non-identical individuals of the same species. Cells, tissues, organs, and the like from, or derived from, a non-identical individual of the same species are "allogeneic." An "alloantigen" encompasses any antigen recognized by different individuals of the same species. Organisms, cells, tissues, organs, and the like from, or derived from, a single individual, or from a genetically identical individual are "autologous."

T cell activation: To initiate or increase the action or function of a T cell. T cells are activated by binding of an antigen (such as an antigen bound to an MHC molecule, for example, on an antigen presenting cell or infected cell) to a T cell receptor on the cell surface. Activation of a T cell also requires a co-stimulatory signal in addition to T cell receptor signaling. In some examples, the co- stimulatory signal is provided by CD28. In particular examples, activation of a T cell is detected by an increase in cell proliferation and/or expression or secretion of a cytokine (such as IL-2, IL-4, IL-6, IFNy, or TNFcc) as compared to a control. In some examples, activation of a CD8+ T cell is detected by an increase in cytolytic activity as compared to a control.

III. Overview of Several Embodiments

Disclosed herein are methods for increasing or decreasing T cell activation (such as T cell proliferation and/or cytokine expression or secretion). In some examples, the methods include activating a T cell and contacting the activated T cell with an effective amount of an miRNA nucleic acid. In other examples, the methods include contacting an activated T cell with an effective amount of an miRNA inhibitor. In particular examples, the methods are performed in vitro or ex vivo.

In some embodiments an activated T cell, which has had its activation increased by contacting with an miRNA nucleic acid or miRNA inhibitor, is administered to a subject in need of increased T cell activation, for example, a subject with decreased immune function, such as an immunocompromised subject. In some examples, the subject is infected with human immunodeficiency virus (HIV) or has been exposed to, is exposed to, or will be exposed to microgravity. In other embodiments, a T cell which has had its activation decreased by contacting with an miRNA nucleic acid or miRNA inhibitor is administered to a subject in need of decreased T cell activation, for example, a subject having an inflammatory or autoimmune disorder (such as rheumatoid arthritis).

The disclosed methods include activating T cells and contacting the activated T cell with an miRNA nucleic acid or miRNA inhibitor, or contacting an activated T cell with an miRNA or an miRNA inhibitor. miRNAs and inhibitors of miRNAs are discussed in detail below (Section IV). Methods of activating T cells are known to one of skill in the art. In some examples, T cells are present in a subject (such as a human subject, a non-human primate, or a rodent). In other examples, T cells are in a sample from the subject, such as a blood sample (for example, an apheresis sample). In further examples, T cells are isolated T cells from a subject. The T cells may be activated in vivo or in vitro. The T cells may be CD4+ T cells, CD8+ T cells, or a combination thereof. The activated T cells may also be naive T cells, memory T cells, or a combination thereof.

In some examples, T cells are activated by contacting the cells with an antigen bound to MHC and a co-stimulatory molecule. In other examples, T cells are activated by contacting the cells with antibodies or other molecules that bind a T cell receptor and co- stimulatory receptors on the surface of a T cell. In one particular example, T cells are activated by contacting the T cells with Concanavalin A (ConA) and anti-CD28. In further examples, T cells are activated by contacting the T cells with anti-CD3 and anti-CD28, with anti-CD3, anti-CD28, and anti- CD137, or with anti-CD2, anti-CD3, and anti-CD28. In some examples, the molecules required to produce T cell activation are coated on the surface of a bead that is contacted with the T cells. Such beads are commercially available, for example DynaBeads® T- Activator from In vitro gen/Life Technologies (Carlsbad, CA) or MACSiBead™ particles from Miltenyi Biotec (Auburn, CA).

T cell activation can be detected by any means known to one of skill in the art. In one example, CD8⁺ T cell activation is detected by evaluating cytolytic activity. In another example, CD8⁺ T cell activation and/or CD4⁺ T cell activation is detected by cell proliferation. Activation of a T cell is also detected by the start of or an increase in expression or secretion of a substance from the T cell, such as one or more cytokines, including but not limited to interferon (IFN)-y, IL-2, IL-4, IL-6, IL-17, granulocyte-macrophage colony-stimulating factor (GM-CSF; also known as CSF2), or TNF-cc. In other examples, activation of a T cell is detected by the start of or an increase in expression or secretion of one or more chemokines by the T cell, including but not limited to CCL3 or XCL2. In one example, the substance (such as a cytokine or chemokine) can be detected by allowing it to bind to a specific binding agent and then measuring the presence of the specific binding agent/substance complex. The specific binding agent is typically an antibody, such as polyclonal or monoclonal antibodies that binds the substance, such as the cytokine or chemokine. Antibodies to cytokines and chemokines are commercially available, or can be made using standard techniques.

In other examples, T cell activation is detected by expression of one or more genes that are upregulated during T cell activation. These genes include BCL2- related Al, B cell translocation gene 2 (BTG2), T cell activation Rho GTPase activating protein (TAGAP), sprouty homolog 2 (SPRY2), Fas ligand (FASLG), MYC proto-oncogene, inducible T cell co-stimulator (ICOS), IFNy, signal transducer and activator of transcription 3 (STAT3), nuclear factor of kappa light polypeptide gene enhancer in B cells (NFKBIA), or CD40 ligand (CD40LG). In particular examples, the genes include those that include an miRNA target sequence. In one example, the genes include an miR-21 target sequence, including but not limited to BCL2A1, BTG2, MYC, TAGAP, SPRY2, and FASLG.

In some examples, T cell activation is compared to a control. An increase in T cell activation includes an increase in one or more measure of T cell activation (such as T cell proliferation, cytokine expression or secretion, or cytolytic activity) compared to a control, such as an increase of at least 10% (such as 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or more) as compared to the control. A decrease in T cell activation includes a decrease in in one or more measure of T cell activation (such as T cell proliferation, cytokine expression or secretion, or cytolytic activity) compared to a control, such as a decrease of at least 10% (such as 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more) as compared to the control. In some examples, a control is a T cell that has not been treated with an miRNA or miRNA inhibitor, such as an activated T cell that has not been contacted with an miRNA or miRNA inhibitor disclosed herein. In other examples, the control is a historical control or standard reference value or range of values (such as a previously tested control sample, such as a measure of T cell activation that represents baseline or normal values, such as a measure of T cell activation in a population or group, such as individuals with a particular condition or disorder).

A. Methods for Increasing T Cell Activation

The disclosure includes methods for increasing T cell activation. In some examples, increasing T cell activation is of use to treat a subject with decreased immune function or response, such as a subject infected with HIV or a subject who has been, is, or will be exposed to microgravity (for example, during spaceflight). In other examples, a subject with decreased immune function or response is a subject at least 50 years of age (such as at least 55, 60, 65, 70, 75, or more years of age). In one example, the subject is at least 65 years of age.

In some examples, a subject with decreased immune function (such as decreased T cell function) is identified as having altered miRNA expression as compared to a control (such as a subject or population with normal immune function). In some examples, decreased T cell function includes decreased levels of one or more of miR-21, miR-lOla, miR-377, let-7b, let-7c, or miR-574 upon T cell activation as compared to a control, such as at least a 10% decrease (such as 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more) as compared to the control. In other examples, decreased T cell function includes increased levels of one or more of miR-99a, miR-467b, miR-467d, miR-199a, or miR-192 as compared to a control, such as at least a 10% increase (such as 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more) as compared to the control.

In some embodiments, the disclosed methods for increasing T cell activation include activating a T cell and contacting the activated T cell with an effective amount of an miRNA, an miRNA mimic, or an miRNA inhibitor, thereby increasing the T cell activation. In some examples, the miRNA is one or more of miR-21 or an miRNA listed in Table 4. In particular examples, the miRNA includes one or more of miR-21, miR-lOla, miR-377, let-7b, let-7c, or miR-574. In further examples, the miRNA mimic is a mimic of miR-21, miR-lOla, miR-377, let-7b, let-7c, or miR- 574. In other examples, the inhibitor of an miRNA includes an inhibitor of one or more of miR-99a, miR-467b, miR-467d, miR-199a, or miR-192. In some examples, the activated T cell is contacted with more than one miRNA (such as 2, 3, 4, 5, or more miRNAs). In other examples, the activated T cell is contacted with more than one miRNA inhibitor (such as 2, 3, 4, 5, or more miRNA inhibitors).

In some embodiments, an miRNA (such as miR-21, miR-lOla, miR-377, let- 7b, let-7c, or miR-574) or an miRNA inhibitor (such as an inhibitor of miR-99a, miR-467b, miR-467d, miR-199a, or miR-192) is administered to a subject. The miRNA or miRNA inhibitor may be included in a composition including one or more pharmaceutically acceptable carrier and/or adjuvant. One of skill in the art can determine dosages and route of administration, for example, based on the potency of the specific formulation, the age, weight, sex and physiological condition of the subject.

In other embodiments, the method can be used for increasing T cell activation in vitro or ex vivo. Following the in vitro or ex vivo method, the T cells may be introduced to a subject in need of increased T cell activation (such as a subject infected with HIV or a subject which is exposed to or will be exposed to micro gravity). In some examples, the T cells are autologous to the subject. A sample (such as a blood sample) including T cells is obtained from the subject. In some examples, T cells are isolated from the sample. The T cells (either isolated T cells or T cells present in the sample from the subject) are incubated with agents to activate the T cells (such as an antigen presenting cell with an MHC molecule bound to an antigen, anti-CD28 and ConA, or anti-CD3 antibodies (if the subject is human)) for a sufficient amount of time to activate the T cells in the sample, such as at least 10 minutes (for example, at least 15 minutes, 20 minutes, 30 minutes, 40 minutes, 1 hour, 1.5 hours, 2 hours, 3 hours, or more). In some examples, the T cells are activated for about 2-24 hours. The activated T cells in the sample are contacted with an miRNA inhibitor (such as an inhibitor of one or more of miR-99a, miR-467b, miR-467d, miR-199a, or miR-192) or an miRNA (such as one or more of miR-21, miR-lOla, miR-377, let-7b, let-7c, and miR-574) that increases activation of the T cells. The activated T cells are contacted with the miRNA or miRNA inhibitor for at least 10 minutes (for example, at least 15 minutes, 20 minutes, 30 minutes, 40 minutes, 1 hour, 1.5 hours, 2 hours, 3 hours, 4 hours, 8 hours, 12 hours, 18 hours, 24 hours, 36 hours, 48 hours, 72 hours, or more). In some examples, the activated T cells are contacted with one or more miRNAs, miRNA mimics, or miRNA inhibitors for about 1-48 hours. In some examples, the resulting T cells with increased activation are then introduced into a subject in need of increased T cell activation. In other examples, the resulting T cells with increased activation are then re-introduced into the same subject from which they originated. In some examples, the T cells with increased activation treat or inhibit at least one symptom of the subject (for example, decreased immune response in a subject infected with HIV).

Agents that increase immune function (such as T cell function) can be identified by monitoring changes in miRNA expression in an activated T cells. In one example, an activated T cell is contacted with a test compound and the level of one or more miRNA is measured (for example utilizing an miRNA microarray). In some examples, an increase in expression of one or more of miR-21, miR-lOla, miR-377, let-7b, let-7c, or miR-574 as compared to a control (such as an activated T cell which is not contacted with the test compound) indicates that the test compound increases T cell function. In other examples, a decrease in expression of one or more of miR-99a, miR-467b, miR-467d, miR-199a, or miR-192 as compared to a control (such as an activated T cell which is not contacted with the test compound) indicates that the test compound increases T cell function.

B. Methods for Decreasing T Cell Activation

The disclosure includes methods for decreasing T cell activation. In some examples, decreasing T cell activation is of use to treat a subject with an

inflammatory or autoimmune disorder. In some examples, the disclosed methods include decreasing T cell activation in a subject with an inflammatory disorder such as rheumatoid arthritis, chronic obstructive pulmonary lung disease, inflammatory bowel disease, or systemic lupus erythematosus. In other examples, the disclosed methods include decreasing T cell activation in a subject with an autoimmune disorder such as multiple sclerosis, ankylosing spondylitis, celiac disease, Crohn's disease, Graves' disease, Hashimoto's thyroiditis, or autoimmune uveitis.

In some examples, the subject may be undergoing or at risk of a cytokine storm (uncontrolled cytokine production in response to an antigen). In some examples, a subject with autoimmune disease (such as rheumatoid arthritis) may experience a cytokine storm. In other examples, a subject with graft versus host disease, acute respiratory distress syndrome, sepsis, avian influenza, smallpox, or systemic inflammatory response syndrome may experience a cytokine storm. A cytokine storm may be treated by decreasing the T cell response of the subject, thereby decreasing cytokine production. In some examples, the methods disclosed herein may be effective to treat or inhibit a cytokine storm in a subject by decreasing T cell activation.

In some embodiments, the disclosed methods for decreasing T cell activation include contacting an activated T cell with an effective amount of an miRNA or an miRNA inhibitor, thereby decreasing the T cell activation, for example as compared to a control. In some examples, the miRNA inhibitor includes an inhibitor of one or more of miR-21, miR-lOla, miR-377, let-7b, let-7c, and miR-574. In other examples, the miRNA includes one or more of miR-99a, miR-467b, miR-467d, miR-199a, and miR-192. In some examples, the activated T cell is contacted with more than one miRNA (such as 2, 3, 4, 5, or more miRNAs). In other examples, the activated T cell is contacted with more than one miRNA inhibitor (such as 2, 3, 4, 5, or more miRNA inhibitors).

In some embodiments, an miRNA (such as miR-99a, miR-467b, miR-467d, miR-199a, or miR-192) or an miRNA inhibitor (such as an inhibitor of miR-21, miR-lOla, miR-377, let-7b, let-7c, and miR-57) is administered to a subject. The miRNA or miRNA inhibitor may be included in a composition including one or more pharmaceutically acceptable carrier and/or adjuvant. One of skill in the art can determine dosages and route of administration, for example, based on the potency of the specific formulation, the age, weight, sex and physiological condition of the subject.

In other embodiments, the method can be used for decreasing T cell activation in vitro or ex vivo. Following the in vitro or ex vivo method, the T cells may be introduced to a subject in need of decreased T cell activation (such as a subject with an inflammatory or autoimmune disorder or a subject having or a risk of a cytokine storm). In some examples, the T cells are autologous to the subject. A sample (such as a blood sample) including T cells (such as activated T cells) is obtained from the subject. The T cells in the sample are contacted with an miRNA inhibitor (such as an inhibitor of one or more of of miR-21, miR-lOla, miR-377, let- 7b, let-7c, and miR-574), an miRNA (such as miR-99a, miR-467b, miR-467d, miR- 199a, or miR-192), or an miRNA mimic (such as a mimic of miR-99a, miR-467b, miR-467d, miR-199a, or miR-192) that decreases activation of the T cells. The activated T cells are contacted with one or more miRNAs, miRNA mimics, or miRNA inhibitors for at least at least 10 minutes (for example, at least 15 minutes, 20 minutes, 30 minutes, 40 minutes, 1 hour, 1.5 hours, 2 hours, 3 hours, 4 hours, 8 hours, 12 hours, 18 hours, 24 hours, 36 hours, 48 hours, 72 hours, or more). In some examples, the activated T cells are contacted with one or more miRNAs or miRNA inhibitors for about 1-48 hours). In some examples, the resulting T cells with decreased activation are then introduced into a subject in need of decreased T cell activation. In other examples, the resulting T cells with decreased activation are then re-introduced into the same subject from which they originated. The T cells with decreased activation treat or inhibit at least one symptom of the subject (for example, decreased cytokine production in a subject having or at risk of a cytokine storm).

Agents that decrease immune function (such as T cell function) can be identified by monitoring changes in miRNA expression in an activated T cell. In one example, an activated T cell is contacted with a test compound and the level of one or more miRNA is measured (for example utilizing an miRNA microarray). In some examples, a decrease in expression of one or more of miR-21, miR-lOla, miR- 377, let-7b, let-7c, or miR-574 as compared to a control (such as an activated T cell which is not contacted with the test compound) indicates that the test compound decreases T cell function. In other examples, an increase in expression of one or more of miR-99a, miR-467b, miR-467d, miR-199a, or miR-192 as compared to a control (such as an activated T cell which is not contacted with the test compound) indicates that the test compound decreases T cell function.

IV. MicroRNAs

The miRNAs of use in the disclosed methods include miRNAs which are differentially regulated in activated T cells that have been exposed to microgravity as compared to activated T cells that have not been exposed to microgravity. In some examples, the T cells are exposed to microgravity (either in vivo or in vitro) and are activated in microgravity conditions. In other examples, the T cells are exposed to microgravity (either in vivo or in vitro) and are activated upon return to normal gravity.

MicroRNAs of use in the disclosed methods include the miRNAs provided in Tables 1 and 4, below. In some examples, the miRNAs are miR-21, miR-99a, miR-lOla, miR-377, let-7b, let-7c, miR-467b, miR-467d, miR-199a, miR-574, and miR-192. In some examples, the miRNA is a mouse miRNA or a human miRNA. miRNA sequences are publicly available. In some examples, the miRNAs include those with the miRBase Accession numbers listed in Table 1. One of skill in the art can identify the sequences of the listed miRNAs, or other miRNAs (such as those listed in Tables 1 and 4) or homologs or orthologs thereof, for example using publicly available databases such as the National Center for Biotechnology

Information (ncbi.nlm.nih.gov) and miRBase (mirbase.org).

Table 1. Exemplary miRNA Accession Numbers

miRNA miRBase Accession Numbers

miR-21 MI0000077; MI0000569

miR-99a MI0000101; MI0000146

miR-lOla MI0000148; MI0000103

miR-377 MI0000785; MI0000794

Let-7b MI0000063; MI0000558

miR-467b MI0004671

As disclosed herein, an miRNA nucleic acid includes precursor miRNAs, as well processed or mature miRNA nucleic acids. For example, an miRNA nucleic acid may be a pri-miRNA, a pre-miRNA, or a mature miRNA nucleic acid, One of skill in the art can identify miRNA precursors, as well as processed or mature miRNAs,

In some examples, the miRNA nucleic acid of use in the methods disclosed herein has a sequence at least 90%, identical to the nucleic acid sequence of one of the miRNAs listed in Tables 1 and 4. For example, the miRNA nucleic acid includes or consists of a nucleic acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the nucleic acid sequence of one of the miRNAs listed in Tables 1 and 4. Exemplary sequences can be obtained using computer programs that are readily available on the internet and the nucleic acid sequences set forth herein. In one example, the miRNA nucleic acid retains a function of the miRNA, such as hybridization to an miRNA target sequence.

In additional examples, an miRNA nucleic acid includes an miRNA nucleic acid that is slightly longer or shorter than the nucleotide sequence of any one of the miRNAs listed in Tables 1 and 4, as long as the miRNA nucleic acid retains a function of the particular miRNA, such as hybridization to an miRNA target sequence. For example, an miRNA nucleic acid can include a few nucleotide deletions or additions at the 5'- or 3'-end of the nucleotide sequence of an miRNA listed in Tables 1 and 4, such as addition or deletion of 1, 2, 3, 4, or more nucleotides from the 5'- or 3'-end, or combinations thereof (such as a deletion from one end and an addition to the other end). In particular examples, a mature miRNA nucleic acid is about 17 to 25 nucleotides in length (for example, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length).

In other examples, an miRNA nucleic acid includes an miRNA mimic. miRNA mimics are small double-stranded RNA molecules designed to mimic endogenous mature miRNA when introduced into cells. In particular examples, an miRNA mimic is a RNA that mimics one or more of the miRNAs listed in Table 1 (for example, miR-99a, miR-467b, miR-467d, miR-199a, miR-192, miR-21, miR- 101a, miR-377, let-7b, let-7c, or miR-57). One of skill in the art can design and produce an miRNA mimic for a desired miRNA. miRNA mimics are also commercially available, for example MISSION® miRNA mimics from Sigma- Aldrich (St. Louis, MO) or miRIDIAN® miRNA mimics (Thermo Scientific/Dharmacon RNAi Technologies, Lafayette, CO).

In some embodiments, the disclosed methods include an miRNA inhibitor. In some exmaples, an miRNA inhibitor includes or consists of a nucleic acid molecule that is at least about 90% complementary to an miRNA nucleic acid, such as the nucleic acid sequence of an miRNA listed in Tables 1 and 4. In some examples, an miRNA inhibitor includes or consists of a nucleic acid molecule that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% complementary to nucleic acid sequence of an miRNA listed in Tables 1 and 4. In particular examples, an miRNA inhibitor is about 17 to 25 nucleotides in length (for example, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length). One of skill in the art can design and produce an miRNA inhibitor for a desired miRNA. miRNA inhibitors are also commercially available, for example Anti-miR™ miRNA inhibitors (Ambion, Austin, TX), miRIDIAN® microRNA hairpin inhibitors (Thermo Scientific/Dharmacon, Lafayette, CO), or miScript® miRNA inhibitors (Qiagen, Carlsbad, CA).

In one particular, non-limiting example, the miRNA is an miR-21 nucleic acid. An miR-21 nucleic acid includes mature miR-21 and miR-21 precursor molecules, such as pri-miR-21 and pre-miR-21. In particular examples, miR-21 is human miR-21. One of skill in the art can identify miR-21 nucleic acids of use in the disclosed methods. For example, miR-21 nucleic acids include miR-21 sequences provided in miRBase (mirbase.org), such as miRBase Accession Nos. MI0000077, MIMAT0000076, and MIMAT0004494. In other examples, miR-21 nucleic acids include miR-21 sequences provided in GenBank, such as GenBank Accession Nos. NR_029493, NC_000017 (nucleotides 57918627-57918698), AC004686, BC053563, AF480524, AJ421741, and AY699265.

In some examples, an miR-21 nucleic acid includes a full-length miR-21 gene (pri-miR-21), for example, a nucleic acid that includes or consists of: ATAAACCAAGGCTCTTACCATAGCTGAACTTTAAAACTTAGACTGTCTTTTCTGTAAACGATTCTGAG GCAAAGGGAAATGACTAGAAGAGGATGAGTAAACAATAACCTGAAATGGGAAACTCGAGGGAAGCACA GGTTTTTTTTGTTTTGTTTTGTTTGGTTCGTTTTTTGTTCTTTGGGGTTTTTTTGAGACAGAATTTCG CTCTCGTTGCCCAAGTTGGAGTGCAATGGCGCGATCTTGGCTCACTGCAACCTCCGCCTCCCGGGTTC AAGCGATTCTCCTGCCTCAGCCTCCCAAGTAGCTGTGATTCCAGGCACGTGCCACCACACCAGCTAAT TTTTTGTATTTTAATAGAAACAGGGTTTCACCGTGTTAGCCAGGCTGGTCTCAAACTGACCTCAGATG ATCCGCCCGCCTTGGCCTCCCAAAGTGCTGGGATTACAGATGTGAGCCACCGCGCCCGGCCAGAGCAC TGTTTTTTTTAATGGCCTTGCACTCTTCTTATGGACCTTTGCTGCCCTCAGTTGACCAAACATGACAT CAGAAACAGATACATTTGTGTGTTTTAAAAACAGCTCCTAATACTGGAACAAAAATATTTAACTGTCT TGACAATACTCATGAGTATCTGCATGGCGACTTCAGAGTTGAGTTTAATCAAAGAGTTTATTCTTAGG TCCTAGTAGAAGAGCTAACCTCACACTCATCCCATTCTAAACTATGTGATTCAACACTGATTTTACAT CCCACAAAGTGAAATCTTGATAGTTGGGTGTAAAAAGGAGAGTAATGGAGATTTCAGAGTAGTTGGGG TTGCTTACTTTTCATTTTTAATTCTTTAGGTTTTGTAAGTTACACACTTCAAGCATTATAGATGATCC TCTTTTTACTACTGAACTAATGAAGCCTTTTTCATTGCATTGTTCTGCATTTATTTCTACAGGGAGAA AACTGGTTGTCCTGGATGTTTGAAAAGTTGGTCGTTGTCATGGTGTGTTACTTCATCCTATCTATCAT TAACTCCATGGCACAAAGTTATGCCAAACGAATCCAGCAGCGGTTGAACTCAGAGGAGAAAACTAAAT AAGTAGAGAAAGTTTTAAACTGCAGAAATTGGAGTGGATGGGTTCTGCCTTAAATTGGGAGGACTCCA AGCCGGGAAGGAAAATTCCCTTTTCCAACCTGTATCAATTTTTACAACTTTTTTCCTGAAAGCAGTTT AGTCCATACTTTGCACTGACATACTTTTTCCTTCTGTGCTAAGGTAAGGTATCCACCCTCGATGCAAT CCACCTTGTGTTTTCTTAGGGTGGAATGTGATGTTCAGCAGCAAACTTGCAACAGACTGGCCTTCTGT TTGTTACTTTCAAAAGGCCCACATGATACAATTAGAGAATTCCCACCGCACAAAAAAAGTTCCTAAGT ATGTTAAATATGTCAAGCTTTTTAGGCTTGTCACAAATGATTGCTTTGTTTTCCTAAGTCATCAAAAT GTATATAAATTATCTAGATTGGATAACAGTCTTGCATGTTTATCATGTTACAATTTAATATTCCATCC TGCCCAACCCTTCCTCTCCCATCCTCAAAAAAGGGCCATTTTATGATGCATTGCACACCCTCTGGGGA AATTGATCTTTAAATTTTGAGACAGTATAAGGAAAATCTGGTTGGTGTCTTACAAGTGAGCTGACACC ATTTTTTATTCTGTGTATTTAGAATGAAGTCTTGAAAAAAACTTTATAAAGACATCTTTAATCATTCC AAAATTGTGTCCGTTTTCTTGAGCGTTTTGATTTTTTACTTTTAGCTTATACCAGCTGAATGGCAGCC TTGCCTAATCCACCTACAACAAGAATTTCTTAAGCTTTCTTTTATTTGCATGAGAGAGCCACTACCAA GGCATGTTTTGTTATGCTGAAACTGGGCTGCTGCATACTGCTAAATGGCACCTCTGGGATTGGCCTAC CTGGGGATTTCTTGGTTTGTGAAAACAGGAGAGGAGAAATATCTCATACAAGTGAAAGGATACTGGAG AGAGAAATTACCCATTTCTAAAAAAAAACCACACTCTGTCGTATCTGTGTTAATGTTTTCTAGCATGT ACTCTGGTTTCAACAGACACAAATTTATATGTTAACCCAGTTTTCTTGCCGTTCTGTAAGTGTTTTAT TCTTAGTGTGATTTTTTTCCATTGGGATGTTTTTGATTGAACTTGTTCATTTTGTTTTGCTTGGGAGG AAAATAAACAATTTTACTTTTTTCCTTTAGGAGCATTATGAGCATTATGTCAGAATAGAATAGAATTG GGGTTCGATCTTAACAGGCCAGAAATGCCTGGGTTTTTTTTGGTTTGTTTTTGTTTTTGTTTTTTTAT CAAATCCTGCCTGACTGTCTGCTTGTTTTGCCTACCATCGTGACATCTCCATGGCTGTACCACCTTGT CGGGTAGCTTATCAGACTGATGTTGACTGTTGAATCTCATGGCAACACCAGTCGATGGGCTGTCTGAC ATTTTGGTATCTTTCATCTGACCATCCATATCCAATGTTCTCATTTAAACATTACCCAGCATCATTGT TTATAATCAGAAACTCTGGTCCTTCTGTCTGGTGGCACTTAGAGTCTTTTGTGCCATAATGCAGCAGT ATGGAGGGAGGATTTTATGGAGAAATGGGGATAGTCTTCATGACCACAAATAAATAAAGGAAAACTAA GCTGCATTGTGGGTTTTGAAAAGGTTATTATACTTCTTAACAATTCTTTTTTTCAGGGACTTTTCTAG CTGTATGACTGTTACTTGACCTTCTTTGAAAAGCATTCCCAAAATGCTCTATTTTAGATAGATTAACA TTAACCAACATAATTTTTTTTAGATCGAGTCAGCATAAATTTCTAAGTCAGCCTCTAGTCGTGGTTCA TCTCTTTCACCTGCATTTTATTTGGTGTTTGTCTGAAGAAAGGAAAGAGGAAAGCAAATACGAATTGT ACTATTTGTACCAAATCTTTGGGATTCATTGGCAAATAATTTCAGTGTGGTGTATTATTAAATAGAAA AAAAAATTTTGTTTCCTAGGTTGAAGGTCTAATTGATACGTTTGACTTATGATGACCATTTATGCACT TTCAAATGAATTTGCTTTCAAAATAAATGAAGAGCAGCTGTCCTTCTTTCCTCTTTTAAGTGTTCAGC TGTGGCATGCTCAGAGGTTCCTGCTGGATTCCAGCTGGAGCGGTGTGATACCCTTCTTTTTCAGCTGT TCGTGCCTTCCTTTCTTGTGTCCACCAAAGTGGAGACAAATACATGATCTCAAAGATACACAGTACCT ACTTAATTCCAGCTGATGGGAGACCAAAGAATTTGCAAGTGGATGGTTTGGTATCACTGTAAATAAAA AGAGGGCCTGGGAATTCTTGCGATTCCATCTCTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA ( SEQ ID NO: 1) In other examples, an miR-21 nucleic acid includes a precursor miR-21 (pre- miR-21) nucleic acid, for example, a double-stranded nucleic acid that includes or consists of the nucleic acid set forth as: UGUCGGGUAGCUUAUCAGACUGAUGUUGACUGUUGAAUCUCAUGGCAACACCAGUC GAUGGGCUGUCUGACA ( SEQ I D NO : 2 )

In other examples, an miR-21 nucleic acid includes a mature miR-21 nucleic acid, for example a nucleic acid including or consisting of the nucleic acid set forth as:

UAGCUUAUCAGACUGAUGUUGA ( SEQ I D NO : 3 )

In some examples, a miR-21 nucleic acid specifically binds to a target gene or sequence and regulates gene expression (such as increasing or decreasing RNA or protein expression). In some examples, an miR-21 nucleic acid target may include the oncogenes Homo sapiens v-ski sarcoma viral oncogene homolog (SKI), RAB6A (member RAS oncogene family), RAB6C (member RAS oncogene family), and RAS homolog gene family member B (RHOB); transforming growth factor-beta- induced protein (TGFBI); transforming growth factor beta receptor II (TGFBR2); RAS p21 protein activator (RASA1); B-cell CLL/lymphoma 2 (BCL2); and the apoptosis-related gene, programmed cell death 4 (PDCD4). See, e.g., Yan et ah, RNA 14:2348-2360, 2008. In other examples, an miR-21 nucleic acid target gene may include Fas ligand (FASLG), sprouty homolog 2 (SPRY2), T cell activation Rho GTPase activating protein (TAGAP), Myc proto-oncogene, B cell translocation gene 2 (BTG2), and BCL2-related protein Al (BCL2A1).

In some examples, the miR-21 nucleic acid of use in the methods disclosed herein has at least 90% sequence identity to the nucleic acid sequence set forth in one of SEQ ID NOs: 1-3. For example, the miR-21 nucleic acid can have an nucleic acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to one of the nucleic acid sequences set forth in SEQ ID NOs: 1-3.

Exemplary sequences can be obtained using computer programs that are readily available on the internet and the nucleic acid sequences set forth herein. In one example, the miR-21 nucleic acid retains a function of miR-21, such as

hybridization to an miR-21 target sequence.

In additional examples, an miR-21 nucleic acid includes an miR-21 nucleic acid that is slightly longer or shorter than the nucleotide sequences shown in any of SEQ ID NOs: 1-3, as long as such the miR-21 nucleic acid retains a function of miR-21, such as hybridization to an miR-21 target sequence. For example, an miR- 21 nucleic acid can include a few nucleotide deletions or additions at the 5'- or 3'- end of the nucleotide sequences shown in any of SEQ ID NOs: 1-3, such as addition or deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more nucleotides from the 5'- or 3'-end, or combinations thereof (such as a deletion from one end and an addition to the other end). In particular examples, a mature miR-21 nucleic acid is about 17 to 25 nucleotides in length (for example, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length).

In some embodiments, the disclosed methods include an miR-21 inhibitor.

An miR-21 inhibitor includes or consists of a nucleic acid molecule that is at least about 90% complementary to an miR-21 nucleic acid, such as the nucleic acid sequences shown in any of SEQ ID NOs: 1-3. In some examples, an miR-21 inhibitor includes or consists of a nucleic acid molecule that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% complementary to an miR-21 nucleic acid, such as the nucleic acid sequences shown in any of SEQ ID NOs: 1-3. In particular examples, an miR-21 inhibitor is about 17 to 25 nucleotides in length (for example, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length). The present disclosure is illustrated by the following non-limiting Examples.

EXAMPLES

Example 1

Gene Expression in Activated T Cells in Spaceflight This example describes alterations in gene expression in T cells activated in microgravity conditions during spaceflight. T cells from three individual donors were launched from Baikonur,

Kazakhstan to the International Space Station (ISS). Some cells were activated in an onboard centrifuge that provided lg or 0.5 g controls, while other cells were activated in microgravity. CD4+ T cells were activated with Concanavalin A (ConA) and anti-CD28 for 1.5 hours. The cells were then fixed with RNALater and the mRNA was preserved for analysis upon return to Earth.

The mRNA samples were loaded on Affymetrix gene arrays (U133) and analyzed using GeneSpring® software (Agilent Technologies, Santa Clara, CA). The microarray data was analyzed in two different ways to generate a list of genes that were upregulated with T cell activation in lg, but not in microgravity. In the first method, a post-hoc analysis was performed on 617 differentially expressed genes generated by ANOVA in which the background normalization was set to the average level of expression in microgravity non-activated T cells. Genes were filtered for those significantly upregulated in lg- activated T cells compared to non- activated T cells and in which expression was 2-fold or greater increased or decreased compared to microgravity- activated T cells. This analysis resulted in 47 genes.

Since donor-to-donor variability may mask more subtle, but significant, differences in gene expression an analysis was also performed in which each donor sample was normalized to itself as background. Genes were filtered for those with 2-fold or greater expression in lg-activated T cells compared to micro gravity- activated T cells. A total of 109 genes exhibiting sensitivity to gravity (significance of <0.05 and at least 1.5-fold change in expression) are shown in Table 2. Of these 109 genes, nine are potential MiR-21 targets.

Table 2. Differentially regulated genes in T cells activated in lg and microgravity

Fold

Gene

difference Description GO cellular component

Symbol

1 g vs.

nuclear receptor subfamily 4, group

23.09 NR4A3 5634 nucleus

A, member 3

tumor necrosis factor (TNF 16021 integral to membrane;

15.24 TNF

superfamily, member 2) 5625 soluble fraction

5622 intracellular; 5634

13.18 EGR2 early growth response 2

nucleus

5622 intracellular; 5634

12.82 EGR1 early growth response 1

nucleus

5622 intracellular; 5634

11.35 EGR3 early growth response 3

nucleus

11.01 NR4A2 nuclear receptor SF4,GRA,M 2 5634 nucleus

nuclear receptor subfamily 4, group

10.11 NR4A1 5634 nucleus

A, member 1

8.892 NR4A2 nuclear receptor SF4,GRA,M 2 5634 nucleus

8.312 NR4A2 nuclear receptor SF4,GRA,M 2 5634 nucleus

8.107 NR4A1 nuclear receptor s SF4,GRA,M 1 5634 nucleus

pleckstrin homology-like domain,

7.298 PHLDA1 5634 nucleus

family A, member 1

7.075 TA-NFKBH T-cell activation NFKB-like protein No notation

16021 integral to membrane;

6.659 IER3 immediate early response 3

16020 membrane

6.365 TA-NFKBH T-cell activation NFKB-like protein No notation

6.284 CCL3 chemokine (C-C motif) ligand 3 5615 extracellular space

Nuclear receptor subfamily 4, group

6.143 NR4A2 5634 nucleus

A, member 2

5. 17 BCE2A I B( Ί ;2-reliited protein Λ t 5622 intracellular

5615 extracellular space; tumor necrosis factor (ligand)

5.171 TNFSF14 16021 integral to membrane;

superfamily, member 14

16020 membrane

4,956 MGI BTG family, member 2 No notation

4.835 CIAS 1 cold autoinflammatory syndrome 1 5737 cytoplasm

CD69 antigen (p60, early T-cell 5887 integral to plasma

4.817 CD69

activation antigen) membrane; 16020 membrane

4.62 mm v-myc myel cyt mat sis viral

5634 nucleus: 5 19 spindle on ogen homolog (avian)

pleckstrin homology-like domain,

4.609 PHLDAl 5634 nucleus

family A, member 1

4.382 FOSL2 FOS-like antigen 2 5634 nucleus

5737 cytoplasm; 5634

4.337 SNF1LK SNFl-like kinase; SNFl-like kinase

nucleus cytotoxic T-lymphocyte-as sociated 5887 integral to plasma

4.154 CTLA4

protein 4 membrane; 16020 membrane

5887 integral to plasma

CD40 ligand (TNF superfamily,

4.109 CD40LG membrane; 5625 soluble member 5, hyper-IgM syndrome)

fraction

Fold

Gene

difference Description GO cellular component

Symbol

1 g vs.

5634 nucleus; 5681

1.96 RBM8A RNA binding motif protein 8A

spliceosome zinc finger protein 36, C3H type-like 5737 cytoplasm; 5634

1.957 ZFP36L1

1 nucleus

1.93 LOC440918 No notation

16021 integral to membrane;

1.927 IL21R interleukin 21 receptor

1602 membrane

1.912 CREM cAMP responsive element modulator 5634 nucleus

CDC28 protein kinase regulatory

1.842 CKS2 No notation

subunit 2

16021 integral to membrane; cytotoxic T-lymphocyte-as sociated

1.815 CTLA4 5887 integral to plasma protein 4

membrane ; 16020 membrane nuclear factor of activated T-cells, 5737 cytoplasm; 5634

1.798 NFATC1

cytoplasmic, calcineurin-dependent 1 nucleus

1.781 TMEM2 transmembrane protein 2 16021 integral to membrane interferon induced transmembrane 16021 integral to membrane;

1.78 IFITM1

protein 1 (9-27) 5886 plasma membrane chromosome 10 open reading frame

1.774 C10orf22 No notation

22

1.767 CST7 cystatin F (leukocystatin) No notation

CDNA: FLJ21531 fis, clone

1.764 FLJ21531 No notation

COL06036

interferon induced transmembrane 16021 integral to membrane;

1.712 IFITM3

protein 3 (1-8U) 5886 plasma membrane

Phosphatidylinositol transfer protein,

1.705 PITPNB 5622 intracellular beta

microtubule associated

1.672 MICAL2 monoxygenase, calponin and LIM 5856 cytoskeleton domain containing 2

5887 integral to plasma

1.653 IL4R interleukin 4 receptor

membrane; 16020 membrane

1.649 FLJ 11773 hypothetical protein FLJ 11773 No notation

microtubule associated

1.644 MAST4 serine/threonine kinase family No notation

member 4

5737 cytoplasm; 5634

1.631 NARG1 NMDA receptor regulated 1 nucleus; 5667 transcription factor complex

1.629 RNF19 ring finger protein 19 5813 centrosome

5737 cytoplasm; 5634

1.629 ZFP36L1 zinc finger protein 36, C3H type

nucleus Fold

Gene

difference Description GO cellular component

Symbol

1 g vs.

16021 integral to membrane;

1.625 SFXN1 sideroflexin 1 16020 membrane;

5739(mitochondrion) family with sequence similarity 53,

1.618 FAM53C No notation

member C

fatty acid binding protein 5 (psoriasis-

1.6 FABP5 5737 cytoplasm

associated)

1.592 NCDN neurochondrin No notation

PWP2 periodic tryptophan protein

1.59 PWP2H 5634 nucleus

homolog

MADS box transcription enhancer

1.556 MEF2B factor 2, polypeptide B (myocyte 5634 nucleus

enhancer factor 2B)

1.553 FLJ35630 hypothetical protein FLJ35630 No notation

ras homolog gene family, member G 5622 intracellular; 16020

1.533 RHOG

(rho G) membrane

5737 cytoplasm; 5634

1.527 ZCSL2 zinc finger, CSL-type containing 2

nucleus

5887 integral to plasma

1.527 DAF CD55, Cromer blood group system membrane; 16020 membrane;

5625 soluble fraction

786 nucleosome; 5634

1.51 H2AFY H2A histone family, member Y

nucleus ankyrin repeat and KH domain

1.508 ANKHD1 No notation

containing 1

16021 (integral to membrane);

1.506 CD7 CD7 antigen (p41)

5886(plasma membrane) splicing factor, arginine/serine-rich 10

1.505 SFRS 10 5634 nucleus

(transformer 2 homolog,

Myeloid/lymphoid or mixed-lineage

0.664 MLL3 5634 nucleus

leukemia 3

HNRPA3P1 ;

heterogeneous nuclear 5634 nucleus; 5681

0.655 HNRPA3;

ribonucleoprotein A3 spliceosome LOC387933

cell division cycle 2-like 6 (CDK8-

0.642 CDC2L6

like)

Cut-like 1, CCAAT displacement 16021 integral to membrane;

0.641 CUTL1

protein 5634 nucleus similar to hypothetical protein

0.636

MGC45438

0.635

0.628 ARRDC3 arrestin domain containing 3

CDNA FLJ 12091 fis, clone

0.625

HEMBB 1002582

0.624 ATP11B ATPase, Class VI, type 1 IB 16021 integral to membrane

CDNA FLJ42980 fis, clone

0.616

BRTHA2006735 Fold

Gene

difference Description GO cellular component

Symbol

1 g vs.

RBM14; RNA binding motif protein 14; 5667 transcription factor

0.606

MGC15912 hypothetical protein MGC15912 complex

0.576 TEX27 Testis expressed sequence 27

0.551 ANKRD11 Ankyrin repeat domain 11 5634 nucleus

0.545 DDX17 DEAD (Asp-Glu-Ala-Asp) box ppl7 5634 nucleus

0.542 PHC3 polyhomeotic like 3 (Drosophila) 5634 nucleus

0.536 LOC284058 LOC284058 protein 5634 nucleus

0.509 CDNA clone IMAGE:3950788

0.477 KIAA0265 KIAA0265 protein

Nuclear autoantigenic sperm protein

0.47 NASP 5634 nucleus

(histone-binding)

Highlighted genes are potential MiR-21 targets, μg, microgravity qRTPCR was used to analyze potential Mir-21 targets BTG2, TAGAP, SPRY2, and FASLG in samples that were activated and fixed in orbit. NFKBIA and CD40LG were also analyzed. Each of the MiR-21 targets BTG2, TAGAP, SPRY2, and FASLG were upregulated after T cell activation under lg conditions, but were not upregulated in microgravity (FIG. 1). NFKBIA and CD40LG were also upregulated after T cell activation under lg conditions, but were not upregulated in microgravity (FIG. 1).

Example 2

Confirmation of Down-Regulation of MiR-21 Expression in Microgravity

This example describes confirmation of MiR-21 downregulation seen in microarray studies.

T cells were activated in spaceflight as described in Example 1. Cells were placed in microgravity ^g), 0.5g, or lg for 3 hours prior to activation. After activation for 1.5 hours, RNA was stabilized with RNALater. Total RNA was isolated.

MiR-21 was detected and quantified using mirVana™ qRT-PCR miRNA detection kit (Ambion/Life Technologies, Austin, TX) according to the

manufacturer's protocol. Total RNA (100 ng) was added to 10 μΐ reverse transcriptase (RT) reaction buffer containing mirVana™ RT buffer, mirVana™ RT primer (mir21 or 5S internal standard), and ArrayScript® enzyme mix. The RT reaction was incubated at 37 °C for 10 minutes, then 95 °C for 10 minutes to inactivate. A no template control was also done for each primer set.

cDNA from the RT reaction (10 μΐ) was added to a total of 25 μΐ real-time quantitative polymerase chain reaction (qPCR) mixture containing 5 μΐ of 50X ROX, 0.5 μΐ mirVana™ PCR primers (mir21 or 5S internal standard), and 1 U of SuperTaq™ polymerase (Applied Biosystems, Foster City, CA). PCR was carried out in a Bio-Rad MyiQ™ single-color real-time PCR detection system (Bio-Rad, Hercules, CA) with a thermal profile of 50°C for 2 minutes, denaturation at 95°C for 3 minutes, followed by 40 amplification cycles of 95°C for 15 seconds and 60°C for 30 seconds. Fluorescence was measured and used for quantitative purposes. At the end of the amplification period, melting curve analysis was performed to confirm the specificity of the amplicon. RNA samples were normalized to 5S rRNA internal standard. Relative quantification of gene expression was calculated by 2^{"(Ct gene T~Ct}

5St)- (Ct gene μ_§ act - Ct 5S μ_§ act) _{where ( 1Q γ}, _{represents me ca}1_cul_ated threshold _Cycle

(Ct) of a time point of each sample other than microgravity ^g) activated. All data derived using qRTPCR was from independent biological samples (n=4-8).

MiR-21 was upregulated in activated T cells in lg conditions. However, MiR-21 was not upregulated in activated T cells in microgravity (FIG. 2).

Fractional gravity (0.5g) did not suppress MiR-21 expression, and even resulted in increased upregulation as compared to lg (FIG. 2).

Example 3

Alteration in Gene Expression in Simulated Microgravity

This example describes alteration in gene expression in activated T cells in simulated microgravity conditions.

Alterations in gene expression in activated human CD4+ T cells were also analyzed in lg and a simulated microgravity environment created utilizing a rotating wall vessel (RWV). In the RWV, cells are placed in a cylinder rotating at about 12- 20 RPM. This keeps the cells in suspension and simulates microgravity conditions. In this study, expression of BTG2, TAGAP, SPRY2, NFKB1A, FASLG and CD40LG were examined. Genes were selected from the list of significantly altered gene expressions in μg that were found in the spaceflight experiment gene array data (Example 1). BTG2 is a known target of Mir-21 and a cell cycle regulator (Liu et al, Cell Res. 19:828-837, 2009). SPRY2 is an inducible inhibitor of tyrosine kinase and downstream signals Ras/Raf/MAPK and is a known target of MiR-21. TAGAP is a Rho GTPase- activating protein and is a predicted target of MiR-21. Expression of interferon γ (IFNy), inducible T cell co-stimulator (ICOS), and STAT3 was also analyzed. IFNy is a cytokine which is critical for innate and adaptive immunity against viral and bacterial infection. ICOS is expressed in activated T cells; ICOS is a receptor belonging to the same family as CD28 and CTLA4, which regulate T- lymphocyte activation during the immune response. Stat3 is phosphorylated by tyrosine receptor kinases in response to cytokines during the immune response; Stat3 forms homo- or heterodimers that translocate to the nucleus to act as transcription factors.

As shown in FIG. 3, BTG2, TAGAP, IFNy, ICOS, and STAT3 were upregulated in lg activated T-cells, however, in simulated micro gravity activation, the induction of these genes was significantly blunted. There was no change in gene induction of cyclophilin in simulated microgravity.

Example 4

Simulated Microgravity Reduces TAGAP Protein Synthesis

This example describes the effect of simulated microgravity on TAGAP protein levels.

Human T-cells were isolated from a Leukocyte Reduction Chamber (LRC) using a ficoll plaque gradient and T cell isolation beads (Stem Cell). Once isolated, T-cells were loaded into 10 ml vessels and incubated at 37°C and 5% C0₂. The microgravity samples were placed on a Rotary system to simulate microgravity, controls were in the same incubator, but were in lg. After incubation for 4 hours, cells were activated with ConA and anti-CD28 and incubated at 37°C and 5% C0₂ for 1.5 hours. Western blots were accomplished with 1-D gel electrophoresis, transferred to nitrocellulose using iBlot® blotting system (Life Technologies, Grand Island, NY), and then visualized using primary and then secondary antibodies.

As shown in FIG. 4, TAGAP protein levels were significantly decreased in activated T-cells in simulated micro gravity as compared to activated T-cells in lg conditions.

Example 5

Potential MiR-21 Regulatory Mechanism

This example describes a potential mechanism of regulation of MiR-21 and its targets in early T-cell activation.

Bioinformatics software including Match™ program on the TRANSFAC® Pro and oPOSSUM was used and target genes were predicted by TargetScan software, Match regRNA, and Pictar matching promoter units to the seed sequence of Mir-21. With the informatics, four genes were identified as potential MiR-21 targets (BTG2, TAGAP, SPRY2, and FASLG). At 1.5 hours after activation, these four genes were upregulated in lg and expression was blunted in microgravity (Examples 1 and 3). All four have 3' UTR seed sequences for a Mir-21

downregulation mechanism (ATAAGCT; Table 3). These four target genes were paradoxically upregulated in the presence of increased levels of MiR-21 in lg onboard controls.

Table 3. MiR-21 seed sequences in 3' UTR of genes upregulated during T cell activation

^a Capital letters indicate exon sequence, lower case letters indicate UTR sequence; MiR-21 seed sequence is bold.

Initially, miRNA activation was believed to be taking place, however, final analysis of the 5 'UTR region of these genes showed no MiR-21 seed sequences in the 5' UTR promoter region (2000 bp upstream) in the upregulated target genes. The 3' UTR of all four of the genes have a MiR-21 seed sequence; however, these genes are upregulated in the initial activation of the T cells in lg and reduced in samples activated in microgravity. Without being bound by theory, it is possible that these genes are upregulated by the activation of API during the initial exposure to ConA and anti-CD28 and are co-upregulated in with MiR-21. Taken together, the early activation of T-cells and the gene induction of MiR-21 and four of its computational targets share a common pathway of upregulation by AP-1. Of the 109 genes found to be significantly induced by T cell activation, several additional potential targets (discovered in the gene array data of 210 dysregulated genes in microgravity) are SPRY2, STAT3, CCD25A, BCL2, and NF1B.

API is a key regulator of MiR-21 gene induction. Fujita et al. (J. Mol. Biol. 378:492-504, 2008) demonstrated that MiR-21 is regulated after phorbol ester treatment of HL-60 cells; studies by Talotta et al. (Oncogene 28:73-84, 2009) demonstrated that RAS activation of MiR-21 is regulated by the AP-1 protein.

Taken together, the early activation of T-cells and the gene induction of MiR-21 and four of its computational targets share a common pathway of regulation. While not being bound by theory, it is hypothesized that in early regulation T-cell activation induced gene expression is regulated by API and later by PKC. Later, as MiR-21 is expressed for a longer time, it most likely inhibits translation of MiR-21 targets like BTG2, TAGAP, SPRY2, and FASLG. Our previous experiments have

demonstrated that API and NFkB can account for 37% of all significant induction of gene expression four hours after T cell activation. The predicted pathways responsible for early T cell activation of NFkB and API are shown in FIG. 5A and 5B. This illustration was deduced from prior work (Boonyaratanakornkit et al., FASEB J. 10:2020-2022, 2005; Hughes-Fulford et al., Adv. Cell Signal. 17: 1111- 1124, 2005) and data obtained in Examples 1 and 3, above. Mir-21 targets may include known targets BTG2 and SPRY2 (Liu et al, Cell Res. 19:828-837, 2009; Chow et al, J. Biol. Chem. 284: 19623-19636, 2009), as well as predicted targets (from the 210 gene list from gene arrays of the spaceflight experiment described in Example 1) FASL, TAGAP, Stat3, BC12, and CDC25A that are upregulated in early T cell activation, most likely by transcription factors API and NFkB

Example 6

T Cell Activation in Mice Exposed to Microgravity This example describes the effect of whole animal microgravity exposure on

T cell activation.

C57BL/6J female mice were exposed to microgravity for 15 days on STS- 131. Splenocytes were harvested within 2-3 hours post-flight, cells were isolated and activated (about 9 hours after landing) with Concanavalin A and anti-CD28 or anti-CD3/CD28 beads (Invitrogen, Carlsbad, CA) for 2.5 hours. Cells were harvested (11.5 hours post landing) and stored in RNALater® (Qiagen, Valencia, CA) at -80°C. Total RNA was isolated from the activated samples with RNeasy® (Qiagen).

T-cell activation markers interleukin-2 (IL-2), IL-2 receptor alpha (IL2Ra), interferon γ (IFNy), TNFa, and IL-17 were measured using quantitative real time

RT-PCR. Expression of IL-2, IL-2Ra, IFNy, CCL-3, TNFa and IL-17 mRNA levels were significantly decreased 2-10 fold in the activated flight group as compared to ground controls (FIG. 6A-D). Expression of cyclophilin, and VEGF was unchanged. Protein synthesis of IL-2, IL-2Ra, IFNy, CCL-3, TNFa and IL-17 was decreased in the flight cell supernatants when compared to ground controls. The observed down-regulation of induction of T-cell activation genes combined with changes of synthesis of these proteins show that animal immune cells require gravity and that the spaceflight induced changes in T-cell function remains after landing. Also, these data correspond with previous findings of human immune function in astronauts after return to earth and, isolated human T-cells in spaceflight {e.g., Example 1), and support the conclusion that a physiological dependence of the immune system on gravity.

Example 7

MicroRNA Expression in Mice Exposed to Microgravity

This example describes the effect of whole animal microgravity exposure on miRNA expression in activated T cells.

Mice were exposed to microgravity and T cells were bead-activated as described in Example 6. RNA was isolated after 2.5 hours of bead activation and analyzed on an miRNA microarray. Total RNA was isolated using the Qiagen microRNA kit (Qiagen, Valencia, CA) and processed according to the

manufacturer's instructions. 300 ng of RNA was added to 30 μΐ of MultiScribe™ reverse transcriptase reaction buffer (Applied Biosystems, Carlsbad, CA) according to the manufacturer's instructions.

RNA was analyzed by the Agilent 2100 Bioanalyzer (Agilent, Santa Clara,

CA) and hybridized on the miRNA Array (Affymetrix, Santa Clara, CA) and processed at the Gladstone Institute. Microarray data were analyzed using the GeneSpring® software (Agilent) and the GC-RMA algorithm. Only flag positive genes were analyzed and p>0.05 was selected for significance. Statistics were performed by ANOVA or Student' s t-test.

Forty miRNAs were significantly differentially expressed (p<0.05) in bead- activated T cells following microgravity exposure of the mice. The miRNAs and fold-change are shown in Table 4. Table 4. miRNAs Significantly Differentially Regulated in Activated T Cells after Spaceflight

mmu-miR- 504_st 0.48 DOWN 0.23 DOWN 2.06 HIGHER mmu-miR- 199b_st 0.48 DOWN 0.14 DOWN 3.53 HIGHER mmu-miR-

101a- star_st 0.49 DOWN 1.07 UP 0.46 LOWER mmu-miR- 377_st 0.53 DOWN 1.48 UP 0.35 LOWER mmu-miR- 193b_st 0.56 DOWN 0.25 DOWN 2.26 HIGHER mmu-miR- 669j_st 0.57 DOWN 0.28 DOWN 2.01 HIGHER mmu-miR- 331-5p_st 0.61 DOWN 0.28 DOWN 2.20 HIGHER mmu-let- 7b_st 0.72 DOWN 1.45 UP 0.50 LOWER mmu-miR- 467g_st 0.72 DOWN 0.25 DOWN 2.87 HIGHER mmu-miR- 467b_st 0.73 DOWN 0.05 DOWN 14.61 HIGHER mmu-miR- 497_st 0.74 DOWN 0.14 DOWN 5.43 HIGHER mmu-miR- 322_st 0.75 DOWN 0.30 DOWN 2.48 HIGHER mmu-miR- 127_st 0.78 DOWN 0.19 DOWN 4.04 HIGHER mmu-let- 7c_st 0.78 DOWN 1.58 UP 0.49 LOWER mmu-miR- 715_st 0.85 DOWN 0.31 DOWN 2.68 HIGHER mmu-miR- 467d_st 0.88 DOWN 0.32 DOWN 2.80 HIGHER mmu-miR- 378-star_st 0.91 DOWN 0.33 DOWN 2.78 HIGHER mmu-miR- 224_st 0.92 DOWN 0.34 DOWN 2.71 HIGHER mmu-miR- NO

202-3p_st 1.00 CHANGE 0.46 DOWN 2.19 HIGHER mmu-miR- NO

293_st 1.00 CHANGE 0.47 DOWN 2.14 HIGHER mmu-miR- NO

294_st 1.00 CHANGE 0.44 DOWN 2.29 HIGHER mmu-miR- NO

216a_st 1.00 CHANGE 0.45 DOWN 2.23 HIGHER mmu-miR- NO

297b-3p_st 1.00 CHANGE 0.26 DOWN 3.81 HIGHER mmu-miR- NO

220_st 1.00 CHANGE 0.50 DOWN 2.02 HIGHER mmu-miR- NO

669k_st 1.00 CHANGE 0.48 DOWN 2.10 HIGHER mmu-miR- NO

290-5p_st 1.00 CHANGE 0.41 DOWN 2.47 HIGHER mmu-miR- 183_st 1.02 UP 0.31 DOWN 3.29 HIGHER mmu-miR- 31-star_st 1.04 UP 0.51 DOWN 2.04 HIGHER mmu-miR-

297c- star_st 1.04 UP 0.25 DOWN 4.09 HIGHER mmu-miR- 376b_st 1.06 UP 0.42 DOWN 2.55 HIGHER mmu-miR- 216b_st 1.11 UP 0.31 DOWN 3.53 HIGHER mmu-miR- 199a-3p_st 1.37 UP 0.11 DOWN 12.42 HIGHER mmu-miR- 574-5p_st 1.41 UP 2.91 UP 0.48 LOWER mmu-miR- 107_st 1.44 UP 0.69 DOWN 2.08 HIGHER mmu-miR- 151-5p_st 1.46 UP 0.65 DOWN 2.25 HIGHER mmu-miR- 103_st 1.51 UP 0.76 DOWN 2.00 HIGHER mmu-miR- 192_st 3.76 UP 0.14 DOWN 27.16 HIGHER mmu-miR- 706_st 8.80 UP 0.80 DOWN 11.06 HIGHER

CA, ConA activation, FLT, flight animals, GRD, ground animals, NT, non-activated controls.

Example 8

Increasing T Cell Activation in a Subject with Decreased Immune Response

In this example, a sample including T cells (such as a blood sample) is removed from a subject, for example a subject with decreased immune response, such as a subject infected with HIV. In some examples, T cells are isolated from the sample. The T cells (either isolated T cells or T cells present in the sample from the subject) are incubated with agents to activate the T cells (such as an antigen presenting cell with an MHC molecule bound to an antigen, anti-CD3 antibodies (if the subject is human), or anti-CD28 and ConA (if the subject is a mouse) for a sufficient amount of time to activate the T cells in the sample. The activated T cells are further incubated in vitro with an effective amount of an miRNA (such as miR- 21, miR-lOla, miR-377, let-7b, let-7c, or miR-374) or an miRNA inhibitor (such as an inhibitor of miR-99a, miR-467b, miR-467d, miR-199a, or miR-192), such that the activation of the T cells is increased (for example as compared to the activated T cells that have not been incubated with the miRNA or miRNA inhibitor). The resulting T cells with increased activation are re-introduced to the subject with the decreased immune response, in an amount sufficient to treat the symptoms of the decreased immune response. Example 9

Decreasing T Cell Activation in a Subject with Inflammatory or Autoimmune

Disorder

In this example, a sample including T cells (such as a blood sample) is removed from a subject, for example a subject with an inflammatory or autoimmune disorder, such as a subject with rheumatoid arthritis. In some examples, the sample includes activated T cells (for example, a subject undergoing a cytokine storm). In some examples, activated T cells are isolated from the sample. The activated T cells (either isolated T cells or T cells present in the sample from the subject) are incubated in vitro with an effective amount of an miRNA inhibitor (such as an inhibitor of miR-21, miR-lOla, miR-377, let-7b, let-7c, or miR-374) or an miRNA (such as miR-99a, miR-467b, miR-467d, miR-199a, or miR-192), such that the activation of the T cells is decreased (for example as compared to the activated T cells that have not been incubated with the miRNA or miRNA inhibitor). The resulting T cells with decreased activation are re-introduced to the subject with the inflammatory or autoimmune disorder, in an amount sufficient to treat the symptoms of the inflammatory or autoimmune disorder.

In view of the many possible embodiments to which the principles of the disclosure may be applied, it should be recognized that the illustrated embodiments are only examples and should not be taken as limiting the scope of the invention. Rather, the scope of the invention is defined by the following claims. We therefore claim as our invention all that comes within the scope and spirit of these claims.

Claims

We claim:

1. A method of decreasing T cell activation, comprising contacting an activated T cell with an effective amount of:

a) an inhibitor of a microRNA, wherein the microRNA is selected from the group consisting of miR-21, miR-lOla, miR-377, let-7b, let-7c, and miR-574; or b) a microRNA nucleic acid selected from the group consisting of miR-99a, miR-467b, miR-467d, miR-199a, and miR-192,

thereby decreasing the T cell activation as compared to a control.

2. The method of claim 1, wherein decreasing the T cell activation comprises decreasing T cell proliferation, decreasing T cell cytokine production, decreasing T cell chemokine production, or a combination of two or more thereof.

3. The method of claim 2, wherein the cytokine comprises interleukin (IL)-

2, IL-4, IL-6, IL-17, tumor necrosis factor (TNF)-cc, interferon (IFN)-y, granulocyte- macrophage colony stimulating factor, or a combination of two or more thereof and/or wherein the chemokine comprises CCL3, XCL2, or a combination thereof.

4. The method of any one of claims 1 to 3, wherein the miRNA inhibitor comprises a nucleic acid at least 90% complementary to a mature miRNA nucleic acid.

5. The method of claim 4, wherein the miRNA inhibitor comprises a nucleic acid at least 90% complementary to the nucleotide sequence of any one of SEQ ID

NOS: 1-3.

6. The method of claim 5, wherein the miRNA inhibitor comprises a nucleic acid complementary to the nucleotide sequence of any one of SEQ ID NOS: 1-3.

7. The method of claim 6, wherein the miRNA inhibitor consists of a nucleic acid complementary to the nucleotide sequence of any one of SEQ ID NOS: 1-3.

8. The method of any one of claims 1 to 7, wherein the microRNA nucleic acid molecule comprises a pri-miRNA nucleic acid, a pre-miRNA nucleic acid or a mature miRNA nucleic acid.

9. The method of any one of claims 1 to 8, wherein the miRNA nucleic acid molecule is a human miR-21 nucleic acid molecule.

10. The method of claim 9, wherein the miR-21 nucleic acid comprises the sequence as set forth in any one of SEQ ID NOS: 1-3.

11. The method of claim 10, wherein the miR-21 nucleic acid consists of the sequence as set forth in any one of SEQ ID NOS: 1-3.

12. The method of any one of claims 1 to 11 wherein contacting an activated T cell with an effective amount of the miRNA nucleic acid or miRNA inhibitor is performed in vitro or ex vivo.

13. The method of claim 12, further comprising administering the activated T cell to a subject after contacting the activated T cell with the miRNA nucleic acid or miRNA inhibitor.

14. The method of claim 13, wherein the subject has an inflammatory or autoimmune disorder.

15. The method of claim 14, wherein the inflammatory disorder comprises rheumatoid arthritis, chronic obstructive pulmonary lung disease, inflammatory bowel disease, or systemic lupus erythematosus.

16. The method of claim 14, wherein the autoimmune disorder comprises multiple sclerosis, ankylosing spondylitis, celiac disease, Crohn's disease, Graves' disease, Hashimoto's thyroiditis, or autoimmune uveitis.

17. A method for increasing T cell activation comprising:

activating a T cell; and

contacting the activated T cell with an effective amount of:

a) a microRNA (miRNA) nucleic acid selected from the group consisting of miR-21, miR-lOla, miR-377, let-7b, let-7c, and miR-574; or

b) an inhibitor of an miRNA, wherein the miRNA is selected from the group consisting of miR-99a, miR-467b, miR-467d, miR-199a, and miR-192, thereby increasing T cell activation as compared to a control.

18. The method of claim 17, wherein the T cell activation comprises T cell proliferation, T cell cytokine production, T cell chemokine production, or a combination of two or more thereof.

19. The method of claim 18, wherein the cytokine comprises interleukin (IL)-2, IL-4, IL-6, IL-17, tumor necrosis factor (TNF)-cc, interferon (IFN)-y, granulocyte-macrophage colony stimulating factor, or a combination of two or more thereof and/or wherein the chemokine comprises CCL3, XCL2, or a combination thereof.

20. The method of claim 17, wherein the T cell activation comprises increased expression of at least one of B cell translocation gene 2 (BTG2), T cell activation Rho GTPase activating protein (TAGAP), sprouty homolog 2 (SPRY2), Fas ligand (FASLG), inducible T cell co-stimulator (ICOS), interferon-γ (IFNy), signal transducer and activator of transcription 3 (STAT3), nuclear factor of kappa light polypeptide gene enhancer in B cells (NFKBIA), and CD40 ligand (CD40LG).

21. The method of claim 20, wherein the T cell activation comprises increased expression of TAGAP, SPRY2, and FASLG.

22. The method of any one of claims 17 to 21, wherein the T cell is a CD4+ T cell, a CD8+ T cell, or a combination thereof.

23. The method of any one of claims 17 to 22, wherein the microRNA nucleic acid molecule comprises a pri-miRNA nucleic acid, a pre-miRNA nucleic acid or a mature miRNA nucleic acid.

24. The method of any one of claims 17 to 23, wherein the miRNA nucleic acid molecule is a human miR-21 nucleic acid molecule.

25. The method of claim 24, wherein the miR-21 nucleic acid comprises the sequence as set forth in any one of SEQ ID NOS: 1-3.

26. The method of claim 25, wherein the miR-21 nucleic acid consists of the sequence as set forth in any one of SEQ ID NOS: 1-3.

27. The method of any one of claims 17 to 22, wherein the miRNA inhibitor comprises a nucleic acid at least 90% complementary to a mature miRNA nucleic acid.

28. The method of claim 27, wherein the miRNA inhibitor comprises a nucleic acid at least 90% complementary to the nucleotide sequence of any one of

SEQ ID NOS: 1-3.

29. The method of claim 28, wherein the miRNA inhibitor comprises a nucleic acid complementary to the nucleotide sequence of any one of SEQ ID NOS: 1-3.

30. The method of claim 29, wherein the miRNA inhibitor consists of a nucleic acid complementary to the nucleotide sequence of any one of SEQ ID NOS: 1-3.

31. The method of any one of claims 17 to 30, wherein activating the T cell comprises contacting the T cell with CD28 and Concanavalin A.

32. The method of any one of claims 17 to 31 wherein activating the T cell and/or contacting the activated T cell with an effective amount of the miRNA nucleic acid or the miRNA inhibitor is performed in vitro or ex vivo.

33. The method of claim 32, further comprising administering the activated T cell to a subject after contacting the activated T cell with the miRNA nucleic acid or the miRNA inhibitor.

34. The method of claim 33, wherein the subject is infected with human immunodeficiency virus or the subject has been or will be exposed to microgravity.