Patent Application
20240230642
The present description relates to a method and kits for diagnosing or determining the progression of systemic autoimmune rheumatic diseases, comprising determining levels of autoantibodies specific to one or more mitochondrial proteins.
The present description refers to a number of documents, the contents of which are herein incorporated by reference in their entirety.
Among the various subcellular localizations, the mitochondrion is characterized by a peculiar structure, comprised within two membranes. This organelle varies in size and shape and is organized in a network whose morphology is determined by the metabolic needs of the cell [1-3]. Mitochondria also contain multiple copies of a double-stranded circular genome (i.e. mitochondrial DNA, mtDNA) that encodes only 13 of the estimated 1,100 to 1,900 proteins of the organelle's proteome, with the remainder being encoded by nuclear DNA [4-6]. The mitochondrial transcriptome also comprises 16S ribosomal RNA molecules, transfer and small noncoding antisense mtRNA molecules.
In regard to their evolutive origin, mitochondria bear resemblances to bacteria, with features such as the presence of cardiolipin, N-formylated peptides, and hypomethylated CpG motifs that elicit pro-inflammatory responses upon their recognition by cells from the innate immune system [7]. Genomic studies also indicated that mtDNA shares similarities with the genome of Rickettsia prowazekii, a modern-era bacterium responsible for typhus [8]. This observation supports the theory suggesting that mitochondria are derived from the endosymbiosis between an α-proteobacterium and a primitive eukaryotic cell.
Despite their cytosolic nature, whole mitochondria and/or mitochondrial components may be released into the extracellular milieu in conditions of necrosis or tissue damage [9-12] or the activation of various cell lines [13-20]. Furthermore, cells may release intact and functional mitochondria that could be recaptured, allowing the recipient cell to rescue its damaged mitochondrial network [21,22]. Conversely, damaged mitochondria may be jettisoned by cells to preserve their functions [23,24]. The release of biomolecules (e.g. cytochrome c, mtDNA, ATP, reactive oxygen species, etc.) from the mitochondria, into the cytosol or the extracellular milieu, is also recognized as mitochondrial damage associated molecular patterns (mtDAMPs) skewing innate immunity toward a pro-inflammatory response [25].
Mitochondrial antigens may also be targeted by the adaptive immune system as indicated by the presence of a humoral response, comprised of various types of anti-mitochondrial autoantibodies (AMA) [26-29], in various inflammatory and autoimmune conditions. Although mitochondrial antigens were detected in association with both the major histocompatibility complex (MHC) molecules-I and -II [30-34], the precise pathophysiological pathway leading to the production of AMA is still unclear. In addition, the mitochondrial antigens targeted by several AMA remain to be characterized [28].
Systemic autoimmune rheumatic diseases (SARDs) include several diseases and disorders such as mixed connective tissue disease (MCTD), rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), Sjögren's syndrome (SjS), systemic sclerosis (SSc), the group of the idiopathic inflammatory myositis (previously polymyositis (PM), and dermatomyositis (DM)), mixed connective tissue disease (MCTD) and the overlap and undifferentiated connective tissue diseases. SLE is a complex autoimmune disease in which the immune system generates autoantibodies recognizing self-epitopes. Antibodies directed against DNA and nuclear components are hallmarks of SLE [35-37]. Nevertheless, the appearance of antibodies directed against phospholipids and phospholipid-binding proteins (broadly referred to as antiphospholipid antibodies [aPL], as well as ribonucleoproteins have also been strongly associated with SLE. Furthermore, aPL are also present in patients with antiphospholipid syndrome, a syndrome which can also be diagnosed in patients with SLE (i.e., secondary APS). To date, there is a considerable obstacle in the diagnosis and classification of SARDs patients. In SLE, for example, certain existing diagnostic measures such as the American College of Rheumatology (ACR) criteria, Systemic Lupus International Collaborating Clinics (SLICC), European League Against Rheumatism (EULAR), and Systemic Lupus Erythematosus Disease Activity Index (SLEDAI) lack sensitivity and specificity. New measures for diagnosing these diseases are therefore needed.
Neutrophils and platelets are known sources of extracellular mitochondria in SLE [14,15,17,20,38,39]. Thus, antibodies directed against mitochondrial components may contribute to the disease pathogenesis through the formation of circulating antibody-antigen scaffolds called immune complexes. While the presence of antibodies against the mitochondrial phospholipid cardiolipin is well recognized, studies have also revealed that antibodies target mtDNA and mtRNA in SLE [27-29]. The 60-kDa heat-shock protein (HSP60), a chaperonin implicated in mitochondrial protein import, is encoded by mRNA from the nucleus. Although both healthy individuals and SLE patients present similar levels of antibodies against HSP60, the presence of these autoantibodies, in combination with increased antiphospholipids, is associated with arterial thrombosis [40,41].
Studies have revealed that antibodies could target mitochondria in SLE. However, the mitochondrial epitopes expressed on the outer membrane remains to be identified [26,27]. Herein, we aim to characterize the antigenic protein repertoire recognized by anti-mitochondrial antibodies in SLE patients.
In some embodiments described herein, is a method for identifying a subject having systemic lupus erythematosus (SLE) by detecting whether mitochondrial autoantibodies specific to one or more mitochondrial autoantigenic polypeptide are present; wherein said method comprises:
In some embodiments described herein, is a method for the treatment of systemic lupus erythematosus (SLE) in an SLE subject in need thereof, wherein said method comprises:
In some embodiments described herein, is a method for diagnosing or determining a progression of systemic lupus erythematosus (SLE) in a subject, said method comprising:
In some embodiments described herein, is a method of detecting one or more autoantibodies specific to one or more mitochondrial autoantigenic polypeptide in a biological sample from a human subject suspected of having systemic lupus erythematosus (SLE); said method comprising:
In some embodiments described herein, is a kit for use in diagnosing or determining the progression of systemic lupus erythematosus (SLE) in a subject, said kit comprising one or more reagents for detecting autoantibodies specific to one or more mitochondrial autoantigenic polypeptides in a biological sample from the subject, wherein said one or more mitochondrial autoantigenic polypeptide is or comprises mitofusin-1 (Mfn-1) or C1qBP.
In some embodiments described herein, is a method of producing a complex comprising one or more mitochondrial autoantigenic polypeptides and one or more autoantibodies specific to the respective one or more mitochondrial autoantigenic polypeptides; wherein said method comprises:
In some embodiments described herein, is a complex for use in the identification, diagnosis, or determination of progression of systemic lupus erythematosus (SLE) in a subject, wherein the complex comprises or is formed by one or more mitochondrial autoantigenic polypeptides and one or more autoantibodies specific to the respective one or more mitochondrial autoantigenic polypeptides, wherein said one or more mitochondrial autoantigenic polypeptide is or comprises mitofusin-1 (Mfn-1) or C1qBP.
In some embodiments described herein, is a complex for use as a research tool in the detection of one or more autoantibodies specific to the one or more mitochondrial autoantigenic polypeptides in a biological sample, wherein the complex comprises one or more mitochondrial autoantigenic polypeptides and one or more autoantibodies specific to the respective one or more mitochondrial autoantigenic polypeptides, wherein said one or more mitochondrial autoantigenic polypeptide is or comprises mitofusin-1 (Mfn-1) or C1qBP.
In some embodiments described herein, is a kit for use in the detection of autoantibodies in a biological sample, wherein the kit comprises autoantigens specific to the autoantibodies and reagents for the detection of the autoantibodies, wherein at least one of the autoantibodies are one or more autoantibodies specific to one or more mitochondrial autoantigenic polypeptides, and wherein at least one of the autoantigens are one or more mitochondrial autoantigenic polypeptides, wherein said one or more mitochondrial autoantigenic polypeptide is or comprises mitofusin-1 (Mfn-1) or C1qBP.
In some embodiments described herein, is a kit for use in the detection of one or more autoantibodies specific to a respective one or more mitochondrial autoantigenic polypeptides in a biological sample, wherein the kit comprises one or more mitochondrial autoantigenic polypeptides specific to the respective one or more autoantibodies and reagents for the detection of the one or more autoantibodies, wherein said one or more mitochondrial autoantigenic polypeptide is or comprises mitofusin-1 (Mfn-1) or C1qBP.
In some embodiments described herein, is a method of manufacturing the kit defined herein, said method comprising:
In some embodiments described herein, is a method of manufacturing the kit defined herein, said method comprising:
In some embodiments described herein, is a method for producing or modifying a test for detecting systemic lupus erythematosus, the method comprising adding or integrating into said test quantifying a panel of mitochondrial autoantibodies, the panel comprising mitochondrial autoantibodies specific to one or more mitochondrial autoantigenic polypeptide, wherein said one or more mitochondrial autoantigenic polypeptide is or comprises mitofusin-1 (Mfn-1) or C1qBP.
In the appended drawings:
Headings, and other identifiers, e.g., (a), (b), (i), (ii), etc., are presented merely for ease of reading the specification and claims. The use of headings or other identifiers in the specification or claims does not necessarily require the steps or elements be performed in alphabetical or numerical order or the order in which they are presented.
The use of the word “a” or “an” when used in conjunction with the term “comprising” in the claims and/or the specification may mean “one” but it is also consistent with the meaning of “one or more”, “at least one”, and “one or more than one”.
The term “about”, when used herein, indicates that a value includes the standard deviation of error for the device or method being employed in order to determine the value. In general, the terminology “about” is meant to designate a possible variation of up to 10%. Therefore, a variation of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10% of a value is included in the term “about”. Unless indicated otherwise, use of the term “about” before a range applies to both ends of the range.
As used in this specification and claim(s), the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.
As used herein, “protein” or “polypeptide” or “peptide” means any peptide-linked chain of amino acids, which may or may not comprise any type of modification (e.g., chemical or post-translational modifications such as acetylation, phosphorylation, glycosylation, sulfatation, sumoylation, prenylation, ubiquitination, etc.).
As used herein, “autoantibody” refers to an antibody produced by the immune system of a subject and that is directed to a self-antigen.
As used herein “mitochondrial autoantibodies specific to one or more mitochondrial autoantigenic polypeptide” refers to an autoantibody that specifically binds the target autoantigenic mitochondrial polypeptide or a fragment thereof.
As used herein, “autoantigenic mitochondrial polypeptide” refers to a polypeptide that is at any point located in or bound to the mitochondria, including but not limited to polypeptides synthesized in the mitochondria or derived from mitochondrial DNA or RNA. A mitochondrial polypeptide may be localized in any compartment of the mitochondria, including but not limited to the mitochondrial outer membrane (MOM), inner membrane (MIM), intermembrane space (IMS), crystal membranes, intracrystal space, and matrix (MM). Mitochondrial outer membrane (MOM) proteins may include but are not limited to proteins that are constitutively or transiently expressed on the surface of the mitochondria, polypeptides that are bound to the surface of the mitochondria, or polypeptide that can bind to the surface of the mitochondria. Autoantigenic mitochondrial polypeptides include those recited in
As used herein, “systemic autoimmune rheumatic diseases (SARDs)” refers to an autoimmune disease or disorder that involves or is mediated by autoantibodies, including but not limited to rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), Sjögren's syndrome (SjS), systemic sclerosis (SSc), the group of the idiopathic inflammatory myositis (IIM) (previously polymyositis [PM] and dermatomyositis [DM]), mixed connective tissue disease (MCTD) and the overlap and undifferentiated connective tissue diseases, or related diseases thereof.
As used herein, “subject” refers to a mammal, including but not limited to a human, mouse, rat, or rabbit. The subject may be a human patient, such as a patient having or suspected of having a SARD. The subject may also be a “healthy”, “reference” or “control” subject, which may not suffer from a SARD or does not present symptoms associated with a SARD and may be a healthy subject, which may be used as a control or reference in the determination of the presence, diagnosis, prognosis, classification, or progression of a SARD. Symptoms associated with a SARD may include but are not limited to a rash (such as a malar or butterfly rash), dry skin, shortness of breath, dermatitis, muscle or joint pain, swelling, or stiffness, inflammation, muscle weakness, fever, chest pain, hair loss, sun or light sensitivity, kidney malfunction, sores, blood clotting, fatigue, anemia, low blood cell counts, low white blood cells, low platelets, neurologic problem (such as seizures, strokes or psychosis), calcium deposits, diarrhea, constipation, Raynaud's phenomenon, and/or arrhythmia.
As used herein, “sample” or “biological sample” refers to any sample comprising or being tested for the presence of antibodies. Samples can include but are not limited to cellular extracts, extracellular fluid, fluid harvested from the body of a subject, culture media, blood, bone marrow, plasma, serum, biopsy, or any organ.
In one aspect described herein is a method for identifying, prognosing, classifying, or diagnosing a subject having an autoimmune disease comprising detecting one or more autoantibodies specific to one or more mitochondrial proteins. According to some aspects, the autoimmune disease includes but not limited to rheumatological diseases. According to some aspects, the rheumatological diseases include but are not limited to systemic autoimmune rheumatoid diseases (SARDs). According to some aspects, the method may further classify or determine the progression of the autoimmune disease.
In one aspect, described herein is a method for identifying a subject having a systemic autoimmune rheumatoid disease (SARD) in a biological sample from a subject comprising antibodies; wherein said method comprises providing one or more ligand specific to one or more mitochondrial proteins; contacting the biological sample from the subject with said one or more ligand specific to one or more mitochondrial proteins; detecting one or more autoantibodies specific to the one or more mitochondrial proteins; and identifying a patient having a SARD when one or more autoantibody specific to the one or more mitochondrial proteins is/are detected. The method described herein optionally includes measuring the levels of one or more autoantibodies specific to one or more mitochondrial proteins; and identifying a patient having a SARD when one or more autoantibody specific to the one or more mitochondrial proteins relative to a control present.
In one aspect, described herein is a method for diagnosing or determining the progression of a systemic autoimmune rheumatoid disease (SARD) in a subject, said method comprising providing a biological sample from the subject; detecting one or more autoantibody levels specific to one or more mitochondrial proteins; and diagnosing the subject as having the SARD or determining the progression of the SARD. The method optionally includes diagnosing the subject as having the SARD or determining the progression of the SARD by observing significantly increased autoantibody levels of specific to the one or more mitochondrial proteins as compared to a healthy subject or a subject not having SARD.
In one aspect, described herein is a method for identifying, prognosing, diagnosing, classifying, or determining a progression of a systemic autoimmune rheumatoid disease (SARD) in a subject, said method comprising: providing a biological sample from the subject; detecting autoantibodies specific to C1qBP and/or mitofusin 1 (Mfn1); optionally, measuring the levels autoantibodies specific to C1qBP and/or mitofusin 1 (Mfn1); and identifying, prognosing, diagnosing, classifying, or determining the progression of the subject having a SARD when one or more autoantibody specific to the one or more mitochondrial proteins relative to a control present.
In one aspect described herein, is a method for the treatment of a systemic autoimmune rheumatoid disease (SARD) in a SARD subject in need thereof; wherein said subject is identified as having a SARD by the methods described herein, and wherein the SARD subject is treated with anti-SARD therapy. Anti-SARD or anti-SLE therapy may include but is not limited to anti-inflammatory or non-steroidal anti-inflammatory drugs, corticosteroids, corticosteroid-sparing agents, analgesics, antimalarial drugs (such as hydroxychloroquine), immunosuppressants, biologics (such as belimumab, and rituximab), intravenous immunoglobulins, chemotherapies, disease-modifying antirheumatic drugs, sunscreens, transplantation, and/or surgery.
In some aspects, the methods described herein include identifying an SLE patient having a specific clinical manifestation (e.g., lupus nephritis, thrombosis). In some aspects, the method includes treating specific manifestations of SLE in a subject, such manifestations may include but are not limited to lupus nephritis and arterial/venous thrombosis or those shown in
In one aspect, described herein is a method of detecting, screening, or panning of one or more antibodies or autoantibodies to one or more mitochondrial proteins.
In one aspect, described herein a kit comprising instructions to perform the methods described herein.
In one aspect, there is described a kit comprising one or more reagents for detecting autoantibodies specific to one or more mitochondrial proteins in a biological sample from the subject. The kit may further include a plate (such as a microplate), tube, or any suitable support, upon which a protein (such as an autoantigen) is or may be immobilized. Reagents for the kits described herein may include but are not limited to reagents commonly used in an immunoassay, such as in an enzyme-linked immunosorbent assay (ELISA). For example, the reagents may include a detecting antibody for the detection of autoantibodies bound to an immobilized autoantigen.
In one aspect, described herein is a kit for use in the diagnosis, prognosis, identification, classification, or determination of the progression of an autoimmune disease. According to some aspects, the autoimmune disease includes but not limited to rheumatological diseases. According to some aspects, the rheumatological diseases include but are not limited to systemic autoimmune rheumatoid diseases (SARDs).
In one aspect, described herein is a kit for use in the diagnosis, prognosis, identification, classification, or determination of progression of a SARD in a subject, said kit comprising one or more reagents for detecting autoantibodies specific to one or more mitochondrial proteins in a biological sample from the subject.
In one aspect, the one or more mitochondrial autoantigenic polypeptides and one or more autoantibodies specific to the respective one or more mitochondrial autoantigenic polypeptides (for example a heterologous polypeptides) can form a complex. The complex can also be isolated. The complex can be an immunocomplex.
According to some aspects, the one or more mitochondrial proteins may be a mitochondrial outer membrane protein, a mitochondrial inner membrane protein, a mitochondrial matrix protein, a mitochondrial intermembrane space protein, a protein involved in the urea cycle, a protein involved in electron transfer and/or oxidative phosphorylation (OxPhos) pathways, a mitochondrial metabolism protein, or any combination thereof. The one or more mitochondrial proteins belonging to any networks listed in
According to some aspects, the one or more mitochondrial proteins is/are mitchondrial outer membrane proteins. According to some aspects, the one or more mitochondrial protein is/are C1qBP and/or mitofusin 1 (Mfn1).
According to some aspects, the SARD may be selected from but is not limited by the group consisting of mixed connective tissue disease (MCTD), rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), Sjögren's syndrome (SjS), systemic sclerosis (SSc), polymyositis (PM), and dermatomyositis (DM).
According to some aspects, the SARD is systemic lupus erythematosus (SLE). In some aspects, systemic lupus erythematosus (SLE) is characterized as lupus, discoid lupus, drug-induced lupus, neonatal lupus, acute or subacute cutaneous lupus erythematosus, cutaneous lupus, and/or lupus nephritis.
In some aspects, SLE may be accompanied by a secondary syndrome or disorder, including but not limited to antiphospholipid syndrome.
According to some aspects, the detection of one or more autoantibody/autoantibodies is determined using commonly known methods for the detection of antibodies, including but not limited to an immunoassay. Immunoassays may include an enzyme-linked immunosorbent assay (ELISA), such as a direct binding or sandwich ELISA.
According to some aspects, the methods or kits described herein may further comprises a step of combining with levels of one or more autoantibodies known to be associated with the identification, diagnosis or progression of said SARD. Examples of these known autoantibodies include but are not limited to anti-nuclear antibodies, anti-double stranded DNA antibodies, lupus anticoagulant antibodies, antiphospholipid antibodies, anticardiolipin antibodies, anti-D2 Glycoprotein I antibodies, anti-ribonucleoprotein antibodies, anti-Smith (Sm) antibodies, anti-small nuclear ribonucleoprotein antibodies, anti-Ro (SS/A) antibodies, anti-La (SS/B) antibodies, anti-mitochondrial DNA, anti-whole mitochondria, anti-mitochondrial RNA antibodies, or any combination thereof.
According to some aspects, the methods or kits described herein further comprises a step of combining with one or more criteria selected from one or more known diagnostic or classification measures of systemic autoimmune rheumatoid disease (SARD). In some aspects, the one or more known diagnostic or classification measures may be but are not limited to the American College of Rheumatology (ACR) classification criteria, Systemic Lupus International Collaborating Clinics (SLICC) classification criteria, Systemic Lupus Erythematosus Disease Activity Index (SLEDAI), lupus severity index (LSI), Systemic Lupus International Collaborating Clinics/American College of Rheumatology (SLICC/ACR) Damage Index (SDI), or European League Against Rheumatism (EULAR) classification criteria.
According to some aspects, the methods or kits described herein are combined with one or more criteria and/or measurements selected from
In one aspect, described herein is a method for producing or modifying a test for detecting systemic lupus erythematosus (SLE), the method comprising adding or integrating into said test quantifying a panel of mitochondrial autoantibodies, the panel comprising mitochondrial autoantibodies specific to one or more mitochondrial autoantigenic polypeptide. In some aspects, the one or more mitochondrial autoantigenic polypeptides comprise C1qBP and/or mitofusin 1 (Mfn1). In some aspects, the test for detecting SLE may include existing autoantibody tests for diagnosing SLE. Examples of these known autoantibodies include but are not limited to anti-nuclear antibodies, anti-double stranded DNA antibodies, lupus anticoagulant antibodies, antiphospholipid antibodies, anticardiolipin antibodies, anti-D2 Glycoprotein I antibodies, anti-ribonucleoprotein antibodies, anti-Smith (Sm) antibodies, anti-small nuclear ribonucleoprotein antibodies, anti-Ro (SS/A) antibodies, anti-La (SS/B) antibodies, anti-mitochondrial DNA, anti-whole mitochondria, anti-mitochondrial RNA antibodies, or any combination thereof. In some aspects, the test for detecting SLE may include one or more criteria selected from one or more known diagnostic or classification measures of systemic autoimmune rheumatoid disease (SARD). In some aspects, the one or more known diagnostic or classification measures may be but are not limited to the American College of Rheumatology (ACR) classification criteria, Systemic Lupus International Collaborating Clinics (SLICC) classification criteria, Systemic Lupus Erythematosus Disease Activity Index (SLEDAI), lupus severity index (LSI), Systemic Lupus International Collaborating Clinics/American College of Rheumatology (SLICC/ACR) Damage Index (SDI), or European League Against Rheumatism (EULAR) classification criteria.
In a further aspect, the following embodiments are provided:
Embodiment 1. A method for identifying a subject having a systemic autoimmune rheumatoid disease (SARD) by detecting whether mitochondrial autoantibodies specific to one or more mitochondrial autoantigenic polypeptide are present; wherein said method comprises:
Embodiment 2. A method for the treatment of a systemic autoimmune rheumatoid disease (SARD) in a SARD subject in need thereof, wherein said method comprises:
Embodiment 3. A method for diagnosing or determining a progression of a systemic autoimmune rheumatoid disease (SARD) in a subject, said method comprising:
Embodiment 4. The method of embodiment 1, 2 or 3, wherein the one or more mitochondrial autoantigenic polypeptide is/are:
Embodiment 5. The method of any one of embodiments 1 to 4, wherein the one or more autoantigenic polypeptide is/are mitochondrial outer membrane polypeptide (s).
Embodiment 6. The method of any one of embodiments 1 to 4, wherein the one or more autoantigenic polypeptide is/are the one or more autoantigenic polypeptide listed in
Embodiment 7. The method of any one of embodiments 1 to 6, wherein two or more mitochondrial autoantigenic polypeptide are detected.
Embodiment 8. The method of any one of embodiments 1 to 6, wherein the one or more mitochondrial autoantigenic polypeptide is/are C1qBP and/or mitofusin 1 (Mfn1).
Embodiment 9. The method of any one of embodiments 1 to 8, wherein said system autoimmune rheumatoid disease is selected from the group consisting of mixed connective tissue disease (MCTD), rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), Sjögren's syndrome (SjS), systemic sclerosis (SSc), polymyositis (PM), and dermatomyositis (DM).
Embodiment 10. The method of any one of embodiments 1 to 8, wherein said system autoimmune rheumatoid disease is systemic lupus erythematosus (SLE).
Embodiment 11. The method of embodiment 9 or 10, wherein the systemic lupus erythematosus (SLE) is accompanied by a secondary syndrome or disorder.
Embodiment 12. The method of embodiment 11, wherein the secondary syndrome or disorder is antiphospholipid syndrome.
Embodiment 13. The method of any one of embodiments 1 to 12, wherein the detecting of one or more autoantibody/autoantibodies is determined using an immunoassay.
Embodiment 14. The method of any one of embodiments 1 to 13, wherein the subject is a human.
Embodiment 15. The method of any one of embodiments 1 to 14, wherein the sample is blood, serum, or plasma.
Embodiment 16. The method of any one of embodiments 1 to 15, wherein the method further comprises a step of detecting or identifying the levels of one or more autoantibodies selected from the group consisting of anti-nuclear antibodies, anti-double stranded DNA antibodies, lupus anticoagulant antibodies, antiphospholipid antibodies, anticardiolipin antibodies, anti-D2 Glycoprotein I antibodies, anti-ribonucleoprotein antibodies, anti-small nuclear ribonucleoprotein antibodies, anti-Smith (Sm) anti-Ro (SS/A) antibodies, anti-La (SS/B) antibodies, anti-mitochondrial DNA, anti-whole mitochondria, anti-mitochondrial RNA antibodies, and any combination thereof.
Embodiment 17. The method of any one of embodiments 1 to 16, wherein the method further comprises a step of determining whether one or more criteria selected from the group consisting of American College of Rheumatology (ACR) classification criteria, Systemic Lupus International Collaborating Clinics (SLICC) classification criteria, Systemic Lupus Erythematosus Disease Activity Index (SLEDAI), lupus severity index (LSI), Systemic Lupus International Collaborating Clinics/American College of Rheumatology (SLICC/ACR) Damage Index (SDI), European League Against Rheumatism (EULAR) classification criteria, and criteria selected from
Embodiment 18. A method of detecting one or more autoantibodies specific to one or more mitochondrial autoantigenic polypeptide in a biological sample from a human subject suspected of having a systemic autoimmune rheumatoid disease (SARD); said method comprising:
Embodiment 19. The method of embodiment 18, wherein one or more autoantibodies as defined in embodiment 16 are present in the human subject suspected of having the systemic autoimmune rheumatoid disease (SARD).
Embodiment 20. The method of embodiment 18 or 19, wherein one or more criteria as defined in embodiment 17 are present in the human subject suspected of having the systemic autoimmune rheumatoid disease (SARD).
Embodiment 21. The method of any one of embodiments 18 to 20, wherein two or more mitochondrial autoantigenic polypeptide are detected.
Embodiment 22. The method of any one of embodiments 18 to 21, further comprising one or more features as defined in any one of embodiments 4 to 17.
Embodiment 23. A kit for use in diagnosing or determining the progression of a systemic autoimmune rheumatoid disease (SARD) in a subject, said kit comprising one or more reagents for detecting autoantibodies specific to one or more mitochondrial autoantigenic polypeptides in a biological sample from the subject.
Embodiment 24. The kit of embodiment 23, wherein the one or more mitochondrial proteins is/are:
Embodiment 25. The kit of embodiment 23 or 24, wherein the one or more mitochondrial proteins is/are mitochondrial outer membrane protein(s).
Embodiment 26. The kit of any one of embodiments 23 to 25, wherein the one or more mitochondrial proteins is/are the one or more mitochondrial proteins listed in
Embodiment 27. The kit of any one of embodiments 23 to 26, wherein the one or more mitochondrial protein is/are C1qBP and/or mitofusin 1 (Mfn1).
Embodiment 28. The kit of any one of embodiments 23 to 27, wherein said system autoimmune rheumatoid disease is selected from the group consisting of mixed connective tissue disease (MCTD), rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), Sjögren's syndrome (SjS), systemic sclerosis (SSc), polymyositis (PM), and dermatomyositis (DM).
Embodiment 29. The kit of embodiment 23 to 28, wherein said system autoimmune rheumatoid disease is systemic lupus erythematosus (SLE).
Embodiment 30. The kit of embodiment 28 or 29, wherein the systemic lupus erythematosus (SLE) is accompanied by a secondary syndrome or disorder.
Embodiment 31. The kit of embodiment 30, wherein the secondary syndrome or disorder is antiphospholipid syndrome.
Embodiment 32. The kit of any one of embodiments 23 to 31, wherein the detecting of autoantibody levels is determined using an immunoassay.
Embodiment 33. The kit of any one of embodiments 23 to 32, wherein the subject is a human.
Embodiment 34. The kit of any one of embodiments 23 to 33, wherein the sample is blood, serum, or plasma.
Embodiment 35. The kit for use of any one of embodiments 23 to 34, further comprising reagents for detecting one or more autoantibodies known to be associated with the diagnosis or progression of said SARD.
Embodiment 36. The kit for use of any one of embodiments 23 to 35, wherein the kit is used in combination with the detection of one or more autoantibodies known to be associated with the identification, diagnosis or progression of said SARD.
Embodiment 37. The kit of embodiment 35 or 36, wherein the one or more autoantibodies known to be associated with the diagnosis or progression of said SARD is/are selected from the group consisting of anti-nuclear antibodies, anti-double stranded DNA antibodies, lupus anticoagulant antibodies, antiphospholipid antibodies, anticardiolipin antibodies, anti-D2 Glycoprotein I antibodies, anti-ribonucleoprotein antibodies, anti-small nuclear ribonucleoprotein antibodies, anti-Ro (SS/A) antibodies, anti-La (SS/B) antibodies, anti-mitochondrial DNA, anti-whole mitochondria, anti-mitochondrial RNA antibodies, and any combination thereof.
Embodiment 38. The kit for use of any one of embodiments 23 to 37, wherein the kit is used in combination with one or more criteria selected from one or more known diagnostic or classification measures of systemic autoimmune rheumatoid disease (SARD).
Embodiment 39. The kit of embodiment 38, wherein the one or more known diagnostic or classification measures is/are selected from the group consisting of American College of Rheumatology (ACR) classification criteria, Systemic Lupus International Collaborating Clinics (SLICC) classification criteria, Systemic Lupus Erythematosus Disease Activity Index (SLEDAI), lupus severity index (LSI), Systemic Lupus International Collaborating Clinics/American College of Rheumatology (SLICC/ACR) Damage Index (SDI), European League Against Rheumatism (EULAR) classification criteria, and criteria selected from
Embodiment 40. A method of producing a complex comprising one or more mitochondrial autoantigenic polypeptides and one or more autoantibodies specific to the respective one or more mitochondrial autoantigenic polypeptides; wherein said method comprises:
Embodiment 41. The method of embodiment 40, further comprising one or more features as defined in any one of embodiments 3 to 17.
Embodiment 42. A complex for use in the identification, diagnosis, or determination of progression of a systemic autoimmune rheumatoid disease (SARD) in a subject, wherein the complex comprises or is formed by one or more mitochondrial autoantigenic polypeptides and one or more autoantibodies specific to the respective one or more mitochondrial autoantigenic polypeptides.
Embodiment 43. A complex for use as a research tool in the detection of one or more autoantibodies specific to the one or more mitochondrial autoantigenic polypeptides in a biological sample, wherein the complex comprises one or more mitochondrial autoantigenic polypeptides and one or more autoantibodies specific to the respective one or more mitochondrial autoantigenic polypeptides.
Embodiment 44. A kit for use in the detection of autoantibodies in a biological sample, wherein the kit comprises autoantigens specific to the autoantibodies and reagents for the detection of the autoantibodies, wherein at least one of the autoantibodies are one or more autoantibodies specific to one or more mitochondrial autoantigenic polypeptides, and wherein at least one of the autoantigens are one or more mitochondrial autoantigenic polypeptides.
Embodiment 45. A kit for use in the detection of one or more autoantibodies specific to a respective one or more mitochondrial autoantigenic polypeptides in a biological sample, wherein the kit comprises one or more mitochondrial autoantigenic polypeptides specific to the respective one or more autoantibodies and reagents for the detection of the one or more autoantibodies.
Embodiment 46. A method of manufacturing the kit as defined in any one of embodiments 23 to 39, or 45, said method comprising:
Embodiment 47. A method of manufacturing the kit as defined in embodiment 44, said method comprising:
Embodiment 48. A method for producing or modifying a test for detecting systemic lupus erythematosus, the method comprising adding or integrating into said test quantifying a panel of mitochondrial autoantibodies, the panel comprising mitochondrial autoantibodies specific to one or more mitochondrial autoantigenic polypeptide.
Embodiment 49. The method of embodiment 48, wherein the one or more mitochondrial autoantigenic polypeptides comprise C1qBP and/or mitofusin 1 (Mfn1).
Embodiment 50. The method of embodiment 48 or 49, wherein the test is a blood, serum, or plasma test.
Other objects, advantages and features of the present description will become more apparent upon reading of the following non-restrictive description of specific embodiments thereof, given by way of example only with reference to the accompanying drawings.
Human Serum Samples—Healthy Donors and Lupus Patients from the Systemic Autoimmune Rheumatic Disease Biobank and Database Repository (SARD-BDB)
This study was approved by the research ethics board of the CHUL (#B13-06-1243 and #B14-08-2108). Healthy volunteers, devoid of illnesses, infections or ongoing medications were recruited at the CHUde Québec—Université Laval (CHUL). Every patient met the ACR classification criteria for SLE [28]. Serum samples were obtained from peripheral blood punctures performed at the time of inclusion. In accordance with the Declaration of Helsinki, written consent was provided by each subject, and their clinical information and biological specimen were associated with an anonymized reference number. A convenience sample was used and no sample size calculation was done
Information concerning sociodemographic (e.g. age, sex, ethnicity), clinical (e.g. classification criteria, clinical indexes, clinical manifestations, co-morbidities), laboratory (e.g. serology) and medication variables was gathered as previously reported [28]. These values are presented in
Intact mitochondria were isolated from C57BL/6J mouse livers, following previously published procedures [20]. Freshly isolated mitochondria were used for the panning of antibodies targeting the mitochondrial outer membrane (i.e., anti-whole mitochondria antibodies, AwMA). Dry-pelleted mitochondria were stored at −80° C. until needed. Frozen mitochondria were resuspended in hypotonic lysis buffer (10 mM HEPES, 2 mM KCl, 0.1% CHAPS, pH7.2) with protease inhibitors (cOmplete™ protease inhibitor cocktail; Roche, Basel, Switzerland) and underwent three cycles, alternating between thawing in a water bath (37° C.) and freezing in an ethanol-dry ice bath, followed by a 15 min sonication in an ice bath. Unbroken mitochondria were pelleted at 7,000×g for 10 min at 4° C. and discarded. Supernatant protein (i.e., mitochondrial lysates) content was determined by the bicinchoninic acid method (BCA protein assay kit. Thermo Fisher Scientific, Waltham, MA, USA). Nucleic acids were digested by addition of benzonase nuclease (Sigma-Aldrich, St-Louis, MO, USA. 100 U/mL, 30 min, 37° C.). Lysates were stored at −80° C. until use.
Optical densities at 405 nm (OD405 nm) for 1:150 dilutions of serum samples from the SARD-BDB were previously assessed with our direct AwMA-ELISA [21] and a cut-off value for positivity to AwMA-IgG was calculated for OD405 nm values>0.30. To account for inter-personal variabilities in autoantibody production, equal volumes of sera from either ten healthy donors (OD405 nm values ranging from 0.08 to 0.17), or from ten SLE patients positive for AwMA (OD405 nm values ranging from 0.68 to 3.00) were pooled and their IgG concentrations were determined by Human IgG total uncoated ELISA kit (Thermo Fisher Scientific). Experiments were performed in triplicate.
Immunoprecipitations: All incubations and 5-min washings used gentle rotary mixing, and were performed at ambient temperature (about 21° C.)—unless indicated. In all experiments, 3 mg of Dynabeads®-ProteinG were used. Before use, beads were washed three times with 1 mL PBS 1× (Wisent, Montréal, Qc, Canada). For retention of the beads in the tubes, a DynaMag™-2 (Thermo Fisher Scientific) magnetic strip was used.
Enrichment in mitochondrial outer membrane (MOM) antigens by panning: Intact mitochondria (0.5 mg as quantitated by BCA assay) were incubated overnight at 4° C. with diluted 1 mL sera (10%, in PBS with protease inhibitor cocktail) from pooled samples of healthy individuals or lupus patients. Non-bound serum components were removed by three 7000×g centrifugal washing, each performed for 10 min at 4° C., with 1.5 ml PBS. AwMA, bound with their outer membrane mitochondrial antigens were released by lysis in 1 mL lysis buffer with protease inhibitor, overnight at 4° C. to ensure the capture of IgGs by Dynabeads®-Protein G. Three washings were performed in PBS containing protease inhibitors, followed by two final washes with PBS devoid of protease inhibitors. Samples were resuspended in 100 μL ammonium bicarbonate buffer, pH 8.0 and stored at −80° C.
Negative controls: Dynabeads®-Protein G were incubated for 2 hrs an irrelevant monoclonal IgG (Clone IV.3, i.e. targeting FcγRIIa). Dynabeads®-bound IgG were then washed three times in 1.5 mL PBS and once in the same volume of lysis buffer. Samples were incubated overnight 4° C. with 1.5 ml of mitochondrial lysate brought to a protein concentration of 3 mg/mL. 1.5 mL washing were then performed as follows: thrice in protease inhibitors-containing lysis buffer, and twice in PBS. Beads were then resuspended in 100 μL of 75 mM ammonium bicarbonate pH 8.0 and stored at −80° C. until required.
On-bead proteolysis: isolated samples were resuspended in 200 μL of 75 mM ammonium bicarbonate pH8.0 and supplemented with 2 g of a trypsin/Lys-C mix (Promega, Madison, WI, USA). Proteins in the samples were digested overnight at 37° C. on a rotating mixer. The digested products were acidified with trifluoroacetic acid and the peptides arising from the proteolytic digestion were isolated and washed on C18 tips (Thermo Fisher Scientific) according to the manufacturer's instructions. Finally, the purified peptides were dried by vacuum centrifugation and stored at −80° C.
LC-MS/MS analysis: Trypsin/Lys-C-digested peptides were separated on a Dionex Ultimate 3000 nano HPLC system coupled to a Q Exactive™ OrbiTrap™ mass spectrometer (Thermo Fisher). 10 μl of sample (a total of 1.5 μg) resuspended in 1% (vol/vol) formic acid were loaded with a constant flow of 4 l/min onto an Acclaim PepMap100 C18 column (0.3 mm id×5 mm; Dionex Corporation, Sunnyvale, CA, USA). After trap enrichment, peptides were eluted onto an EasySpray™ PepMap™ C18 nano column (75 μm×50 cm; Dionex Corporation) with a linear gradient of 8-40% solvent B (80% acetonitrile with 0.1% formic acid) over 240 min with a constant flow of 200 nl/min. The HPLC system was coupled to the mass spectrometer via an EasySpray™ source. The spray voltage was set to 2.0 kV and the temperature of the column set to 40° C. Full scan MS survey spectra (m/z 350-1600) in profile mode were acquired in the Orbitrap with a resolution of 70,000 after accumulation of 1,000,000 ions. The ten most intense peptide ions from the preview scan in the Orbitrap were fragmented by collision-induced dissociation (normalized collision energy 25% and resolution of 17,500) after the accumulation of 50,000 ions. Maximal filling times were 250 ms for the full scans and 60 ms for the MS/MS scans. Precursor ion charge state screening was enabled and all unassigned charge states as well as single, 7 and 8 charged species were rejected. The dynamic exclusion list was restricted to a maximum of 500 entries with a maximum retention period of 40 seconds and a relative mass window of 10 ppm. The lock mass option was enabled for survey scans to improve mass accuracy. Data were acquired using the XCalibur™ software (Thermo Fisher Scientific).
Mass spectrometry data analysis: Mass spectra data (.RAW files) were loaded into MaxQuant™ version 1.6.17.0 and searched against a protein database generated by merging the Homo sapiens and Mus musculus reference proteomes (Uniprot, versions 01-29-2021: 77,027 human and 55,470 mouse proteins) complemented with a list of common contaminants maintained by MaxQuant and concatenated with the reversed version of all sequences (decoy mode). The minimum peptide length was set to 7 amino acids and trypsin was specified as the protease allowing up to two missed cleavages. The mass tolerance was set to 7 ppm for the precursor ions and 20 ppm for the fragment ions. The following parameters were used: fixed carbamidomethylation of cysteine (+57.0214 Da), oxidation of methionine (+15.9949 Da) as a variable modification and a peptide-spectrum and protein match false-discovery rate of 0.01.
Intensity-based label-free quantification (LFQ) values were used to estimate the relative abundance of proteins in each replicate groups, which correspond to the sum of all peak intensities over all tandem mass spectra assigned to a particular protein. Missing LFQ data imputation was estimated by using a noise value corresponding to the lowest 1% percentile of the LFQ distribution. This noise value was imputated for each sample when the intensity value is missing (i.e., undetected proteins) and selected as the minimum LFQ intensity for low abundance protein identifications for which LFQ values fall below the 1% percentile background.
Filtering and data analysis of proteomic data: Matches to reverse proteins, proteins with zero intensity values in all datasets, proteins with negative MaxQuant scores and common contaminants were removed from the final protein repertoire. Identified proteins were cross-referenced using their Uniprot ID for their cellular localization and, when the information was available, for their mitochondrial sublocalization. Pathways involving the mitochondrial proteins identified were analyzed, using the Reactome pathway knowledge base and the Database for Annotation, Visualization and Integrated Discovery (DAVID).
Identification of Mitochondrial Antigens Recognized by AMA
Detection of IgG targeting several mtAgs were performed as previously described [20-22] with the following modification: microplates were coated overnight at 4° C. with 225 ng protein in 25 μL PBS 1× (i.e. 9 ng/μL). The following proteins were tested: C1qBP (Novus Biologicals, Centennial, CO, USA), Mfn1 (Origene Technologies, Rockville, MD, USA), ATP5F1B (Cusabio, Wuhan, China) and OTC. Other mitochondrial proteins were also tested (healthy: n=10; SLE n=30): CPS 1, NAGS, ETFA (Origene Technologies), GOT2 (BioVision Inc., Milpitas, California, USA), ALDH2 (RayBiotech Life, Peachtree Corners, GA, USA). OTC, CPS 1 and NAGS were provided by Dr. Rubio [29,30].
Sociodemographic, clinical and laboratory values are presented as either median±inter-quartile range (IQR), or n (%), or as mean±SD. Comparisons between groups were performed using the Wilcoxon-Mann-Whitney test. Spearman correlations were calculated to see associations between AMA and antibodies assessed in clinical serologies. Youden's index was determined to set a cut-off value for positivity to anti-C1qBP and anti-Mfn1. Logistic regressions, adjusted for sex and age were performed and expressed as their adjusted odds ratio (aOR), 95% confidence interval (95% CI) and p-value. Statistical analyses were performed with Prism 9 software (GraphPad Software Inc., La Jolla, CA, USA), SAS version 9.4 (SAS Institute Inc., Cary, NC, USA), and figures were assembled with Photoshop CC 2019 version 20.0.4 (Adobe Systems Inc.).
We used whole (i.e., intact) mitochondria to capture AMA recognizing components from the mitochondrial outer membrane (MOM). An irrelevant antibody, targeting a protein absent in mitochondria, was also utilized as control, to appreciate both the non-specific binding and potential endogenous proteins interacting with IgG (e.g., the complement pathway). These approaches are illustrated in
The complement component 1 Q subcomponent-binding protein (C1qBP) is a mitochondrial protein belonging to the C1q network that is stored within the mitochondrion and can be then addressed to the surface of the plasma membrane and/or shed into the extracellular space [31]. Thus, although C1qBP is not strictly a mitochondrial protein, its enrichment in SLE samples, identification in our proteomic analyses and its relevance to mitochondria and complement stimulated further analyses (
While antibodies in primary biliary cirrhosis (PBC) target proteins from the mitochondria inner membrane (MIM), inter-membrane space (IMS) or the matrix (MM) [19,32-34], previous findings determined that antibodies could recognize components on the surface of the outer membrane in SLE [20,22,35-37]. A protein from the MOM, associated to the PDC but distinct from the antigenic targets of AMA-M2 and presenting an elevated score in our panning approach was selected—mitofusin-1 (Mfn1) (
Interestingly, anti-Mfn-1 and anti-C1qBP IgG antibody levels were not significantly elevated in other autoimmune diseases compared to healthy controls, such as antiphospholipid syndrome (APS) and PBC (
Next, we determined if the autoantibodies to either of these two components, C1qBP and Mfn-1, were associated with a definable cytoplasmic immunofluorescent staining pattern on routine HEp-2 substrates, as seen with antibodies to pyruvate dehydrogenase complex in PBC serum (28, 51). Indirect immunofluorescence labelling of HEp-2 cells with a commercial antibody against C1qBP produced intense speckled staining of the nuclear region and a lower signal in the cytosol. Conversely, commercial anti-Mfn-1 displayed reticular staining of the cytoplasm, typical of AMAs (
We then investigated the biostatistical associations of C1qBP and Mfn-1 with various disease parameters. When associated to SLE autoantibodies routinely assessed by clinical laboratories (
When matched with levels of other AMA, levels of anti-C1qBP were associated with both AwMA-IgG (rs=0.319; p=0.003) and AmtDNA-IgG (rs=0.234; p=0.029). Moreover, while anti-Mfn1 also displayed strong correlations with AwMA-IgG (rs=0.657; p<0.0001) and AmtDNA-IgG (rs=0.518; p<0.0001), they also correlated with all the AMA assessed in the SARD-BDB. Of note, levels of anti-C1qBP also correlated with those of anti-Mfn1 (rs=0.49; p>0.0001).
Taken together, these data suggest the potential of anti-Mfn-1 and anti-C1qBP autoantibodies as potent and specific biomarkers for diagnosing SLE, as well as for determining SLE disease severity and progression.
Patients with SLE display antibodies from various subclasses (e.g., IgG, IgM, IgA) against a wide array of self-antigens [38]. The epitopes targeted by these autoantibodies comprise, but are not limited to, phospholipids [e.g., aPL, aCL, LA] [39], phospholipid-protein complexes [e.g., anti-β2 glycoprotein I (anti-β2 GPI)] [40], nucleic acids (e.g., dsDNA, AmtDNA, AmtRNA) [20,21] and associated proteins [e.g., anti-nuclear antibodies (ANA), transcription factors and ribonucleoproteins] [41] among others [42]. During the course of the disease, cell death and/or activation was reported to trigger the release of mitochondrial structures (e.g., mtDNA, cardiolipin) or even whole functional organelles [7,9,10,12]. The pro-inflammatory influence of mitochondrial DAMPs on the innate immune system is extensively documented in homeostasis and SLE pathophysiology [18]. Furthermore, distinct autoantibodies targeting various types of mitochondrial biomolecules such as phospholipids (i.e., aCL targeting cardiolipin) [43], nucleic acids (i.e., mtDNA, mtRNA) [20,21] and antigens whose precise nature remains to be characterized (i.e., AMA-M5) were reported in SLE [37].
While various proteins involved in the mitochondrial processing of pyruvate (e.g., PDC-E2), sulfite oxidase and glycogen phosphorylase are mitochondrial proteins known to be targeted by anti-mitochondrial antibodies (i.e., respectively by AMA-M2, -M4 and -M9) in primary biliary cirrhosis [19], limited knowledge is available concerning the extent of the mitochondrial proteome targeted by AMA in SLE [2]. To date, the only mitochondrial protein with autoantibodies associated with disease manifestations in SLE is HSP60 [44]. Herein, we used several approaches to enrich mitochondrial antigens in sample in our mass spectrometry analyses. In the experimental design, we also included a control samples that included an irrelevant antibody intended to identify non-specific binding. These approaches indicated the presence of the complement pathways (i.e., known to be targeted by circulating autoantibodies in SLE while also interacting with IgG and IgM [45]), suggesting that, one should consider these entities with caution as—additionally to their potential antigenicities—they can be co-isolated with autoantibodies. It is noteworthy that our analyses highlighted the presence of non-mitochondrial proteins (e.g., ficolin-3, serpins) that may be of interest in SLE. However, we restricted the focus of or study on the proteins assigned to the mitochondrial proteome. While we tested a handful of mitochondrial proteins, systematic characterization of patients' immunoreactivities to large subsets of mitochondrial antigens would be enhanced by the use of high-throughput screening methods such as nucleic acid programmable protein array (NAPPA). In addition, the present study focuses on IgG while other antibody classes such as IgM or IgA may also have an impact on SLE pathophysiology. These autoantibodies may be studied by using similar approaches with optimized isolation steps, depending on the antibody class assessed.
Two proteins with significant immunogenicities stood out from the various candidates assessed due to their increase in SLE samples. First, C1qBP, a protein stored within the mitochondrion and dispatched to the MOM and/or the cell membrane, or secreted into the circulation [48,49], where it may be targeted by circulating AMA. Second, Mfn1, a protein embedded in the MOM that could be recognized by AMA upon the release of whole mitochondria into the extracellular space. We speculate that C1qBP potentially constitutes a circulating autoantigen enabling the formation of protein-protein scaffolds with C1q and/or IgGs allowing pro-inflammatory signaling (e.g., respectively through the classical complement pathway and signaling by Fcγ receptors). Levels of anti-C1qBP were associated with those of IgGs targeting whole mitochondria, mtDNA and Mfn1, while anti-mitofusin 1 were correlated with all AMA assessed. In addition, the strong correlation between anti-Mfn1 and AwMA suggests that mitofusin 1 might represent one of the main mtAg of the MOM. Moreover, our previous work on AMA in SLE and the correlations of AMA with circulating mtDNA or anticardiolipin showcase the mitochondrion as a reservoir of various immunogenic biomolecules [14,20-22].
Patients with SLE display antibodies from various subclasses (e.g. IgG, IgM, and IgA) against a wide array of self-antigens [60]. The epitopes targeted by these autoantibodies comprise, but are not limited to, phospholipids (e.g. aPL, aCL, lupus anticoagulant [LA]) [61-63], phospholipid-protein complexes (e.g. anti-β2 glycoprotein I [anti-β2GPI]) [64], nucleic acids (e.g. anti-double-stranded DNA [dsDNA], AmtDNA, and AmtRNA) [27,28] and associated proteins (e.g. anti-nuclear antibodies [ANA], transcription factors, and ribonucleoproteins) [65] among others [66]. During the course of the disease, cell death and/or activation was reported to trigger the release of mitochondrial structures (e.g. mtDNA, cardiolipin) or even whole functional organelles [9-18]. The pro-inflammatory influence of mitochondrial DAMPs on the innate immune system is extensively documented in homeostasis and SLE pathophysiology [25,67-71]. Furthermore, distinct autoantibodies targeting various types of mitochondrial biomolecules such as phospholipids (i.e. aCL targeting cardiolipin) [72,73], nucleic acids (i.e. mtDNA, mtRNA) [27,28], and antigens whose precise nature remains to be characterized (i.e. AMA-M5) were reported in SLE [54,55].
Due to the nature of the patient recruitment, the samples available were collected at the time of patient inclusion into the cohort, which does not necessarily reflect the pattern of antibodies prevalent at the time of diagnosis. Moreover, the ethnic distribution of the patients and the relatively low clinical scores of the patients at the time of the phlebotomy may have influenced the various data assessed. Thus, future studies using samples from large inception cohorts should be dedicated to assessing the associations between anti-C1qBP-IgG and anti-Mfn1-IgG with disease manifestations in order to appreciate their qualities as biomarkers in SLE.
In conclusion, anti-mitochondrial autoantibodies and their specific antigens constitute potential candidates for the development of novel assays allowing improvements to diagnosis, prognosis and/or patient stratification in SLE with IgGs against C1qBP and Mfn1 as promising candidates.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CA2022/050849 | 5/26/2022 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63202080 | May 2021 | US |
