Overview of Lupus Pathogenesis

SECTION 2  Pathogenesis

4  Overview of Lupus Pathogenesis Bevra Hannahs Hahn

OUTLINE Phases of SLE: Evolution of Disease in Susceptible Persons, 44 Overview: The Major Immune Pathways Favoring Autoantibody Production, 44 Stimulation of Innate and Adaptive Immune Responses by Foreign and Autoantigens, 44 Autoantibodies and Immune Complexes of SLE, 45 Regulatory Mechanisms Fail to Control Autoimmune Responses, 45

Abnormalities in T and B Lymphocytes in SLE, 45 Cytokines/Chemokines and SLE, 47 Genetics and Epigenetics, 47 Gender Influences, 50 Environmental Factors, 51 Tissue Damage in SLE, 51 Current Approved and Investigational Therapies for SLE, 52

The purpose of this brief chapter is to review how systemic lupus erythematosus (SLE) evolves and is sustained. Ideas reflect the author’s opinions, which are based largely on the information provided throughout this book. References are restricted to recent review articles because each topic is addressed in detail in other chapters.

Stimulation of Innate and Adaptive Immune Responses by Foreign and Autoantigens

PHASES OF SLE: EVOLUTION OF DISEASE IN SUSCEPTIBLE PERSONS As shown in Fig. 4.1, the development of SLE occurs in a series of steps. There is a long period of predisposition to autoimmunity, conferred by genetic susceptibility, gender, and environmental exposures, and then (in a small proportion of those predisposed) development of autoantibodies, which usually precede clinical symptoms by months to years. A proportion of individuals with autoantibodies demonstrate clinical SLE, often starting with involvement of a small number of organ systems or abnormal laboratory values, and then evolving into enough clinical and laboratory abnormalities to be classified as SLE. Finally, over a period of many years, most individuals with clinical SLE experience intermittent disease flares and improvements (usually not complete remission), and compile organ damage and comorbidities related to genetic predisposition, chronic inflammation, activation of pathways that damage organs (such as endothelial cells [ECs] and glomerular podocytes), and/or induce fibrosis, to therapies, and to aging.

OVERVIEW: THE MAJOR IMMUNE PATHWAYS FAVORING AUTOANTIBODY PRODUCTION These pathways are summarized in Fig. 4.2.

44

Antigenic stimulation (Ag) of the innate and adaptive immune responses is provided by many sources, including infectious agents (i.e., providing ssRNA, dsRNA, and DNA); autologous cells undergoing apoptosis (which present autoantigens such as nucleosome and Ro in surface blebs, and phosphatidyl serine on outer surfaces of membranes); or necrosis, which releases cell components that form neoantigens under the influence of oxidation, phosphorylation, and cleavage. Microorganisms have antigenic protein sequences that cross-react with human autoantigens and induce immune responses, such as antiribonucleoprotein (anti-RNP) and anti-Ro (SSA). In addition many autologous cells release microparticles that contain immunogenic DNA. Antigen-presenting cells (APCs), such as dendritic cells (DCs), monocytes/macrophages (M/Ms), and B lymphocytes, process and present such Ags. In addition, cells of innate immunity (DC, M/M) are activated via internal toll-like receptors (TLRs) by DNA/protein and RNA/protein, which can be provided by dying cells, particularly polymorphonuclear neutrophils (PMNs) undergoing NETosis, by SLE immune complexes (ICs) via Fc receptors, and by infectious agents activating surface receptors that recognize dangerous patterns. The net result of activation of DCs from tolerogenic to proinflammatory cells secreting inflammatory cytokines (including the lupus-promoting interferon alpha [IFN-α]), and of M/Ms to proinflammatory cells secreting tumor necrosis factor alpha (TNF-α), and interleukins IL-1, IL-12, and IL-23, is activation of effector T cells that help B cells make immunoglobulin (Ig) G autoantibodies, infiltrate tissues, and be cytotoxic for some tissue cells such as podocytes in the kidney. B lymphocytes, which are activated directly by DNA/protein

CHAPTER 4  Overview of Lupus Pathogenesis Step 1: Genes and Gender and Environment

45

Step 2: Autoantibodies Antigen Antigen-binding site

DNA

Figure 4.1  Overview of the pathogen-

Antibody

Epigenetics

esis of systemic lupus erythematosus (SLE). SLE develops in an individual in a process that may take decades. At birth the individual is predisposed by multiple genes/gene copies/epigenetic changes and by a permissive gender (usually female). Exposure to environmental stimuli such as ultraviolet B (UVB) light and silica and infections such as Epstein–Barr virus (EBV) stimulate immune responses and additional epigenetic changes. Over time, persistent autoantibodies appear; they are usually present for a few years before the first symptom of disease. In some autoantibody-positive individuals, clinical SLE develops, which is shown here as polyarthritis. Within that group, some have chronic irreversible damage. End-stage renal disease with sclerotic glomeruli and tubular loss.

and RNA/protein via their TLRs, by antigens recognized by their B-cell surface receptors, and by IFN-α, can also be helped in their secretion of autoantibodies by T cells, and in their survival and maturation to plasmablasts by B-lymphocyte stimulator (BLyS)/B-cell–activating factor (BAFF), IL-6, and other cytokines. T cells of the adaptive immune system are activated by cytokines and by protein sequences in autoantibodies presented in human leukocyte antigen (HLA) molecules of APCs. In patients with SLE these processes escape normal regulatory mechanisms, which are listed in Box 4.1. Thus autoantibodies induce the first phase of clinical disease (organ inflammation of joints, skin, glomeruli, destruction of platelets, etc.) because (1) the autoantibodies and the ICs they form persist, (2) they are quantitatively high, (3) they contain subsets that bind target tissues, (4) they form ICs that are trapped in basement membranes or bound on cell surfaces, (5) charges on antibodies or ICs favor nonspecific binding to tissues, and (6) their complexes activate complement. And yet, in spite of this deluge of autoantibodies and ICs attacking tissue, mouse models suggest that susceptibility to clinical disease requires more; there are several examples of autoantibody formation, abundant Ig deposition in glomeruli, and even complement fixation without development of clinical nephritis.

Autoantibodies and Immune Complexes of SLE Autoantibodies are the main effectors of the onset of disease in SLE. In humans, they are probably necessary for disease but not sufficient. That is, their deposition must be followed by activation of complement and/ or other mediators of inflammation; a series of events that includes chemotaxis for lymphocytes and phagocytic mononuclear cells; and release of cytokines, chemokines, and proteolytic enzymes, as well as oxidative damage, which must occur for organ inflammation and damage to be severe. In nearly 85% of patients with SLE, autoantibodies precede the first symptom of disease by an average of 2 to 3 years and sometimes as long as 9 years. The autoantibodies appear in a temporal hierarchy, with antinuclear antibodies (ANAs) first, then anti-DNA and antiphospholipid, and finally anti-Sm and anti-RNP. These observations imply

Step 3: Clinical Disease Step 4: Chronic Damage

that immunoregulation of potentially pathogenic autoantibodies can occur for a sustained period, and that only in individuals whose regulation becomes “exhausted” does disease appear. Among autoantibodies, some are clearly pathogenic, including subsets of anti-DNA that cause nephritis on transfer to healthy animals. Antibodies to receptors on neurons (anti-N-methyl-aspartate receptor, a subset of anti-DNA) can cause neuronal death. Antibodies to platelets and erythrocytes can cause the cells to be phagocytized and destroyed. Antibodies to Ro/La (SSA/SSB) can damage cells in the fetal cardiac conduction system and cause heart block. Human antibodies to phospholipids can cause fetal loss in mice and in humans, probably because of their effects on the clotting system that promote thrombosis. In addition, autoantibodies generate selfperpetuating cycles. The autoantibodies contain amino acid sequences that are T-cell determinants; these peptides activate T helper cells to further expand autoantibody production. Mechanisms of pathogenicity are discussed in detail in other chapters, and for many autoantibodies the mechanisms are not entirely known. Pathogenic ICs in patients with SLE are dominated by soluble complexes that avoid clearance by phagocytic mononuclear cells, and both size and charge of the complexes can cause them to be trapped in tissue, rather than continuing to circulate. In addition, complement products in ICs are bound by complement receptors. Ig in ICs is bound by FcRγ; thus, the ICs can fix to cells and tissues by those interactions. Defects in clearing the complexes characteristic of SLE are probably major causes of their persistence and enhance their quantities and potentially harmful properties.

Regulatory Mechanisms Fail to Control Autoimmune Responses As shown in Box 4.1, several mechanisms that downregulate active immune responses are defective in SLE.

Abnormalities in T and B Lymphocytes in SLE B- and T-cell interactions in SLE play a major role in the production of IgG and complement-fixing autoreactive antibodies. It is likely that

46

SECTION 2 Pathogenesis Antigen sources: Apoptotic cells Necrotic cells

Neoantigens Infections Microparticles

x

ple

Ab un

NUC RNAp PS

BCR

PMN NET DNA/prot

IFNa

TLR

B

m Im

0

,IL1

IL6

Autoab

pDC

PC

A S/B

TH1,2

Tfh

a

F y TN BL 2, 1 , IL IL1

M/M

TH17

R

FF

IFNa

TC

TLR

e

m co

R

Fc

mDC

Cytokines (IL2), IFNg, IL17, IL23

pDC

DNA/RNA in bacteria viruses, immune complexes

Figure 4.2  Interactions between innate and acquired immune systems. Antigen/cell interactions that drive autoimmune responses in systemic lupus erythematosus (SLE). Antigens containing nucleosomal DNA, RNA/ protein, phospholipids presented by apoptotic cells, neoantigens generated from necrotic cells and inflammatory cell debris, and RNA/protein are shown; DNA/protein in the neutrophil extracellular trap (NET)-like structures of polymorphonuclear neutrophils (PMNs) and immune complexes set up immune responses that characterize human SLE. Plasmacytoid dendritic cells (pDCs) and B lymphocytes are activated on engagement of these antigens by their toll-like receptors (TLRs); pDCs generate interferon alpha (IFN-α), and B cells produce autoantibodies and cytokines. The IFN-α activates PMNs to die by NETosis; the NETs they secrete contain DNA and DNA-binding proteins that further engage TLRs in B cells, with more B-cell activation. Both pDC and myeloid DC (mDC) subsets present autoantigens and cytokines to T lymphocytes, resulting in T-cell activation with pushing of T cells to helper/effector subsets that include IFN-γ–producing T helper 1 (TH1) and tissue-damaging TH17 cells (Teffectors). SLE T and B cells are intrinsically abnormal and hyperrespond to stimuli. Multiple “hits” drive B cells, which at this level of maturation are prone to hyperactivation. The hits include T-cell help, exposure to increased quantities of apoptotic materials and neoantigens recognized by their B-cell receptors, and exposure to activated DCs and pools of activating cytokines. Green indicates molecules, antigens, and pathways that promote the hyperimmune responses of SLE. Cytokine receptors on cell surfaces (green diamonds), TLRs in pDCs and B cells (black bars), B-cell receptors or T-cell receptors, respectively (red circles or crescents), and B-cell receptors (pink ovoids). B, B lymphocyte; M/M, monocyte/ macrophage; NUC, DNA-containing nucleosome; PS, phosphatidylserine, which is the phospholipid presented to the immune system on the outer surface of cells undergoing apoptosis; RNAp, RNA bound to a protein that in complex can be recognized by the immune system; Teff, effector (helper), which can be CD4+ TH1, or TH2, or TH17, or follicular T-cell helper (TFH) that secretes IL-17.

activation of T-cells and/or B-cells must be precise: hyperactivation promotes SLE by making higher quantities of autoantibodies and proinflammatory cytokines, and hypoactivation allows autoreactive B and T cells to escape apoptosis. Thus tweaking of the T-cell/B-cell activation immunostat away from the “norm” promotes autoimmunity. B-cell surface antigen receptors (BCRs) are assembled from various combinations of Ig heavy and light chains in bone marrow; the vast majority of BCRs and their autoantibodies in people with SLE are assembled from a variety of Ig genes and combinations that do not differ from normal protective antibody assembly. The SLE autoantibody response has somewhat limited clonality (not different from antibody responses to external antigens) and somatic hypermutation, indicating that cells have been stimulated by antigens. A major difference between people with SLE and healthy individuals is abnormalities of B-cell

tolerance. The end result is elevated quantities of activated B cells, of memory B cells, and of plasma cells in patients with active SLE. There are several defects that permit survival of autoreactive B-cell subsets in SLE. The usual tolerance processes (apoptosis, anergy, ignorance, BCR editing, and external suppression) are blunted, allowing survival and maturation of dangerous autoreactive B cells. After normal B cells exit the bone marrow, they go through a series of checkpoints that normally remove autoreactive cells. There are defects in several of these checkpoints in SLE, including entry of early immature B cells to mature B cells and of transitional B cells to mature B cells, entry into germinal centers (GCs) in which B cells in the marginal zone can move to follicular zones and acquire T-cell help, and naive B cell to activated B-cell maturation. In addition, some patients have defective expression of FcγRIIB in memory B cells, which is a molecule that suppresses

CHAPTER 4  Overview of Lupus Pathogenesis BOX 4.1  Mechanisms of Downregulation

of the Immune Response That Are Defective in SLE 1. Disposal of ICs and ACs: defective phagocytosis, transport by complement receptors, and binding by Fcγ receptors. Can be caused by macrophage defects intrinsic to SLE; low levels of complement-binding CR1 receptors, or occupied receptors; and FcγRs that are occupied, downregulated, or genetically low-binding of the Ig in ICs. Early components of complement or MBL protein also participate in solubilizing and transporting IC. They may be missing or defective. 2. Defective idiotypic networks: because of low production of antiidiotypic antibodies, there is defective regulation of T helper cells by T-regulator cells that recognize idiotypes in their TCRs. 3. Inadequate production and/or function of regulatory cells that kill or suppress autoreactive B cells, T helper cells, and other effector cells: this includes CD8+ cytotoxic cells that kill autoreactive B, regulatory CD4+CD25+Foxp3+ T cells that normally target both T helper cells and autoreactive B cells, inhibitory CD8+Foxp3+ T cells that suppress both T helper and B cells, regulatory B cells, tolerogenic DCs, and possibly NK cell defects. 4. Low production of IL-2 by T cells: survival of regulatory T cells requires IL-2, and effector T cells in SLE make decreased quantities of IL-2. IL-2 is also required for activation-induced death in lymphocytes. 5. Defects in apoptosis that permit survival of effector T and autoreactive B cells; these are usually genetically determined. AC, Apoptotic cell; DC, dendritic cell; FcR, Fc-receptor; IC, immune complex; Ig, immunoglobulin; IL, interleukin; MBL, mannose-binding lectin; NK, natural killer; SLE, systemic lupus erythematosus; TCR, T-cell receptor.

B-cell development. Thus defects allow persistence of autoreactive cells that would be inactive or deleted in healthy individuals. The majority of patients with SLE have abnormally high levels of BLyS/BAFF cytokine, which promotes survival of B cells from the late transitional stage through mature activated and memory B cells. Genetic polymorphisms predisposing to SLE include several that affect signaling through the BCR, such as PTPN22 and BLK. Abnormally high quantities of Ca2+ are mobilized intracellularly after BCR activation in SLE. Overall, memory and activated B cells, as well as plasma cells, are increased in numbers in SLE. They require smaller than normal stimuli to be activated, and many pathways from BCR signaling to nuclear factor kappa B (NF-κB) activation may be altered. Elevated numbers of plasma cells and a plasma cell “signature” in upregulated gene expression are associated with clinical disease activity. Normally, in GCs, nonautoreactive B cells migrate into T-zone (follicular) areas, in which they contact CD4+ T helper cells and follicular helper cells (Tfh), which drive them into activated and memory subsets, with subsequent Ig class switching and plasma cell production. This process results in protective antibody responses. In the GCs of SLE patients there is a tolerance defect that allows T-cell help for production of potentially harmful autoantibodies. Normal and SLE B cells can also produce autoantibodies with class switching and maturation independent of T-cell help via activation of B-cell TLRs. In SLE this process may be enhanced probably by autoantigens in ICs, in Igs, and in the protein nets produced by neutrophils undergoing NETosis. This environmental exposure of B cells to autoantigens is probably influenced by the SLE genetic variants that promote activation of innate immunity and high IFN production by innate immune cells. T cells in SLE are also abnormal. Like B cells, they respond to lesser stimuli than are required for healthy T cells. A major abnormality of SLE CD4+ T cells is assembly of an abnormal signaling apparatus after T-cell

47

receptor (TCR) activation. Fig. 4.3 shows some of these abnormalities. In health, TCR stimulation results in assembly of the CD3ζ chain into the surface activation cluster. In SLE, the FcRγ chain is substituted for CD3ζ, resulting in a different activation pathway. The end results are an increased release of intracellular calcium, which promotes translocation of calcium/calmodulin-dependent protein kinase IV (CaMK4) to the nucleus, and upregulation of transcription repressor cyclic adenosine monophosphate (AMP) response–element modulator alpha (CREMα), which on binding to promoter regions of DNA suppresses IL-2 production and enhances IL-17 production. Abnormally low secretion of IL-2 by T cells impairs production of regulatory T cells, whereas increased production of IL-17 promotes inflammation and T-cell help in GCs. Causes of the downregulation of CD3ζ include antibodies to T lymphocytes and mTOR activation in T cells resulting from increased levels of nitric oxide (NO), which are related to elevated transmembrane potentials and oxidation in the mitochondria of SLE T cells. SLE T-cell subsets have many other abnormalities: CD8+ cytotoxic T cells may be defective, adding to the persistence of autoreactive B cells. Regulatory T cells of CD4+ and CD8+ phenotypes also are abnormal in quantities and/ or functions. Double-negative (DN) T cells (CD3+CD4−CD8−), which probably derive from CD8+ T cells, infiltrate tissue and secrete IL-17. Help is derived from classical TH1 and TH2 cells and from Tfh in GCs in which B cells are contacted. Lupus nephritis (LN) biopsy specimens contain large numbers of B cells, plasma cells, CD4+ T cells and CD8+ T cells, and DN T cells, as well as monocyte/macrophages and DCs. These are discussed in more detail in the section on tissue damage.

Cytokines/Chemokines and SLE Actions of cytokines and chemokines in SLE are complex, with some properties favoring autoimmunity and others opposing it. Table 4.1 lists some of these proteins that are thought to play a major role in the pathogenesis of SLE. The end of the Table 4.1 lists some of the proteins that are excreted in higher quantities in the urine of patients with SLE, especially those with nephritis, than in the urine of controls.

Genetics and Epigenetics Genetic predisposition is probably the single most important factor in development and progression of SLE. The risk for SLE is approximately 10-fold higher in monozygotic than in dizygotic twins, and 8- to 20-fold higher in siblings of patients with SLE than in the healthy population. However, concordance for SLE in monozygotic twins is approximately 40%, suggesting that nongenetic and epigenetic factors play a major role in disease susceptibility. Some of the gene polymorphisms or mutations associated with increased risk for SLE are shown in Fig. 4.4, placed within the cellular networks they influence. The vast majority of patients with SLE inherit multiple predisposing genes that are common in the population, with each gene associated with odds ratios of 1.1 to 2.5. Rare exceptions in which 25% to 95% of people with single gene mutations develop clinical SLE include homozygous deficiencies of early complement components (especially C1q); mutations in TREX1 or DNASE1 genes that regulate destruction of genomic DNA; and ACP5 polymorphisms, which result in overactivation of IFN-α. For SLE polygenic disease, the current understanding is that predisposing gene polymorphisms, copy numbers, mutations, and gene–gene interactions account for at most 50% of genetic predisposition to SLE. Many predisposing genetic elements have been identified. The highest signal for genome-wide associations with SLE is in the HLA/major histocompatibility complex (MHC) region. This is not surprising because the extended MHC region occupies 7.6 Mb of DNA, and the gene products are responsible for antigen presentation and for some components of complement. Within HLA DR3 and DR2 have consistently

48

SECTION 2 Pathogenesis

C Aggregated lipid rafts

Antigen TCR CD4 FcR-γ

CD44

CD3

ERM P Syk

B D

A Increase in intracellular calcium CaMK4 CaMK4

CREM-α

X

Interleukin-2

CREM-α

Interleukin-17

strong associations with susceptibility to SLE in European and EuroAmerican Caucasians, with each gene in a heterozygotic person conferring an odds ratio of 1.2 to 1.5 and in a homozygote of 1.8 to 2.8. Approximately 75% of patients with SLE in all ethnic groups have at least one HLA gene that increases risk (primarily subsets of DR2, DR3, DR4, or DR8). A stronger association for several SLE-predisposing genes is with autoantibody production instead of disease. For example, there is a strong association with DR3 and DQ2 (which are in strong disequilibrium) and antibodies to Ro(SSA) and La(SSB), and of DR4 with antibodies to phospholipids. Many SLE-predisposing genes influence the pathways to disease shown in Fig. 4.2. These include disposal of ICs/apoptotic cells (C1Q, C2, C4, CR2, FcγR-2A, FcγR-3A, FcγR-2B), activation/regulation of the innate immune pathway (TLR7 copy numbers, IRF5, STAT4, IRF7, TNFAIP3), regulation of adaptive immunity (PTPN22, TNFS4, BLK, BANK1, LYN, ETS1, IL-10, IL-21), and migration/ adhesion to target tissues (ITGAM/CD11B). In some cases, altered copy numbers of a given gene, such as complement C4 and Tlr7, confer predisposition to SLE rather than the gene itself. Many polymorphisms in predisposing genes differ between populations, particularly racial groups (e.g., HLA D3 in Caucasians), whereas others are found in SLE patients of multiple races (e.g., IRF7,TLR7/8, TNFS4, IL-10 in Asians, Mexicans, African Americans, and Europeans). Gene–gene interactions are also known to increase susceptibility and/or disease severity, such as HLA + CTLA4 + ITGAM + IRF5, or IRF5 + STAT4. Some of these genes and/or interactions are associated with earlier disease, anti-DNA, and nephritis, such as certain single nucleotide

ROCK

Figure 4.3  Abnormalities of Tlymphocyte activation in patients with systemic lupus erythematosus (SLE). After T-cell stimulation, SLE T cells have abnormal signaling; when compared with healthy T cells, IL-2 production is decreased (IL-2 is required for production/maintenance of regulatory T cells) and proinflammatory IL-17 production is increased. (A) The process starts with replacement of the usual CD3ζ chain with FcRγ (which signals via Syk) in the surface signaling complex. (B and C) Aggregation of lipid rafts occurs. (D) The rafts contain aggregated T-cell receptors (TCRs) and additional signaling molecules, including CD44, which is an adhesion molecule facilitating homing of T cells to target tissues, such as kidneys. CD44 signals via ERM (ezfin, radixin, moesin) and is phosphorylated by Rho kinase (ROCK). The increased intracellular calcium concentrations that result in activated SLE B and T cells promote translocation of protein kinase IV (CaMK4) to the nucleus. CaMK4 facilitates binding of the transcriptional repressor cyclic adenosine monophosphate (cAMP) response–element modulator alpha (CREM-α) to the promoter for IL-2, suppressing its expression. At the same time, binding of CREM-α to the promoter for IL-17 enhances its transcription. (From Tsokas GC: Systemic lupus erythematosus. N Eng J Med 365(22): 2110–2121, 2011.)

polymorphisms (SNPs) of STAT4. Some of the individual “lupus” genes/SNPs are also associated with other autoimmune diseases, such as inflammatory bowel disease, psoriasis, type 1 diabetes, and multiple sclerosis. Thus it is possible that some individuals are predisposed genetically to autoimmunity, and other genes determine exactly which clinical autoimmune disease will develop. There is one report of a gene that confers protection from SLE (a polymorphism for TLR5), which reduces the levels of proinflammatory cytokines, such as TNF-α, IL-1β, and IL-6, released from cells stimulated by bacterial flagellin. One of the reasons that discovery of predisposing genes, gene copies, and gene interactions fails to fully account for susceptibility to SLE is the role of epigenetics in gene expression. Epigenetics refers to alterations in DNA that are inheritable. The ability to transcribe DNA into messenger RNA (mRNA) and then into proteins, or posttranslational modifications in mRNA, may be altered by DNA methylation and histone modulation (especially acetylation, but also ubiquitination, phosphorylation, etc.). These epigenetic changes alter gene transcription into protein similar to binding of transcription regions by microRNA (miRNA, miR). All of these processes can be altered in people with SLE. Within DNA, islands of CpG are sites of methylation by methyltransferases, with 70% to 90% of somatic cell DNA methylated in healthy individuals. DNA from T cells of patients with SLE is hypomethylated, resulting in upregulated expression of surface molecules, such as lymphocyte-function–associated antigen 1 (LFA-1), which is associated with T-cell autoreactivity. Factors that promote hypomethylation of DNA include ultraviolet (UV) light,

49

CHAPTER 4  Overview of Lupus Pathogenesis TABLE 4.1  Summary of Cytokines and Chemokines Known to Influence SLE Cytokine/ Chemokine

Source

Actions

Observations in SLE Patients

IFN-α

pDC

IFN-α–inducible genes increased in many cell types in majority Serum IFN-α activity Increased in 40%–50% High levels are an inheritable trait Target for current clinical trials

IFN-γ

NK, TH1

Antiviral Promotes DC maturation Stimulates B-cell differentiation to Ig-secreting plasma cells Increases expression of TLRs 7/9 Enhances CD8+ T-cell production of granzyme B and perforin Expression enhanced by IL-12 + IL-27 Signature cytokine of TH1 cells

IL-1

Proinflammatory in tissues

IL-2

Activated mononuclear phagocytic cells, T cells Lymphocytes, T cells

IL-4

T cells

IL-6

T and B lymphocytes, monocytes, fibroblasts, endothelial cells, epithelial cells Monocytes, T and B cells

Signature cytokine of TH2 cells May protect from tissue fibrosis (mice) In combination with IL-2 and TGF-β promotes differentiation of TH17 cells Promotes differentiation of B cells to terminal cells secreting Ig Promotes Ig synthesis by B cells, also mediates suppression by some Tregs Signature cytokine of TH17 cells Proinflammatory Cells also make IL-21 and IL-22 TH17 cells require IL-23 for maintenance Required to generate TH1 and NK cells Proinflammatory in tissue and tissue fluids

IL-10

IL-17

TFH, DN T cells

IL-12, IL-18 TNF-α

Activated macrophages Macrophages, DCs

TGF-β

NK and other cells

BLyS/BAFF

Myeloid lineage cells

Growth factor for lymphocytes, especially T cells Required for generation of Tregs

Required to generate regulatory T cells (with IL-2) Participates in generation of TH17 cells (with IL-2 + IL-6 or IL-1) Can downregulate autoimmune responses Also contributes to tissue fibrosis Maintains B cells and required for maturation to Ig-secreting cells

Present in renal tissue in human LN Induces apoptosis in renal parenchymal cells IL-27 levels low in SLE sera Necessary for nephritis in several murine lupus strains Potential target for clinical trials High serum levels associated with disease activity Low levels associated with LN Levels in PBMCs from SLE patients are low; low levels suppress activationinduced cell death in T helper cells (increasing apoptotic autoantigen load) and prevent generation of Tregs (allowing persistence of autoreactive T and B cells) Several murine lupus strains also develop low IL-2 levels in serum and PBMCs before disease onset Administration of low levels may suppress chronic graft-versus-host response in humans and vasculitis associated with hepatitis C IL-4+ T cells increased in PBMCs of SLE patients

Increased serum levels in SLE patients Expressed in target tissues, including kidney

Increased serum levels in many patients with SLE

Found in target tissue in human and murine SLE, including kidneys Potential target for clinical trials

Increased serum levels in patients with SLE Found in renal tissue of patients with LN Elevated serum levels in some patients Therapy with TNF inhibitors not yet proved to have good efficacy/toxicity ratio PBMCs from SLE patients secrete abnormally low levels Serum levels are low in some patients

High serum levels in SLE patients; levels correlate positively with disease activity in some studies Found in target tissues, especially dermis, also in kidney Targeted by anti-BLyS (belimumab), which is approved for treatment of patients with SLE Continued

50

SECTION 2 Pathogenesis

TABLE 4.1  Summary of Cytokines and Chemokines Known to Influence SLE—cont’d Cytokine/ Chemokine

Source

Chemokines in Serum IL-8, IP-10, Endothelial and other MCP-1, cells fractalkine

Actions

Observations in SLE Patients

Chemotactic for PMNs, T cells, monocytes

Increased in renal tissue in LN Increased in sera of patients, especially those with LN

Chemokines and Other Molecules Increased in Urine TWEAK Activated monocyte/ TNF superfamily member, macrophages TWEAK-R binds Fn14 on endothelial cells and vascular smooth muscle May mediate renal tissue damage by causing proliferation of mesangial cells, damage to podocytes and renal tubular cells Induces IP-10, MCP-1, MIP, ICAM-1, VCAM-1, MMP-1, and MMP-9 Induces apoptosis in human monocytes NGAL Mesangial cells Iron-bearing protein Induces apoptosis via activation of caspase 3, increases expression of proinflammatory genes in renal tissue CXCL16 Expressed on DCs and Recruits T and NK cells to tissues, monocytes mates with CXCR6 IP-10 Endothelial cells, IFN-γ–inducible protein 10 fibroblasts, Mates with CXCR3 monocytes Attracts lymphocytes

Elevations in urine have 50% sensitivity and 90% specificity for active GN in LN May come into use to predict flare and response to treatment in LN

Increase in urine excretion correlates with flare of LN Knockout in mice protects from nephrotoxic nephritis

Increased urine excretion correlated with active GN and renal SLEDAI scores Increased urine excretion in some patients with LN

BAFF, B-cell–activating factor; BLyS, B-lymphocyte stimulator; CXCL16, chemokine C-X-C motif ligand 16; CXCR, receptor for C-X-C motif chemokine; DC, dendritic cell; DN, double-negative; GN, glomerulonephritis; ICAM-1, intercellular adhesion molecule 1; IFN, interferon; Ig, immunoglobulin; IL, interleukin; IP, inducible protein; LN, lupus nephritis; MCP-1, monocyte chemotactic protein 1; MIP, macrophage inflammatory protein; MMP, matrix metalloproteinase; NGAL, neutrophil gelatinase–associated lipocalin; NK, natural killer; PBMC, peripheral blood mononuclear cell; pDC, plasmacytoid dendritic cell; PMN, polymorphonuclear neutrophils; SLE, systemic lupus erythematosus; SLEDAI, Systemic Lupus Erythematosus Disease Activity Index; TFH, T follicular helper cells; TGF, transforming growth factor; TH1/2/17, T helper 1/2/17; TLR, toll-like receptor; TNF, tumor necrosis factor; Treg, T-regulator cell; TWEAK, TNF-like weak inducer of apoptosis; VCAM-1, vascular cell adhesion molecule 1.

SLE-inducing drugs, aging, and altered expression of certain miRNAs. For example, increases in miR-148a and miR-21 inhibit expression of DNA methyltransferase 1 (DMNT1), with resultant hypomethylation of target DNA. Nucleosomal DNA exists as 146 base pairs of DNA wrapped around an octamer of histones. Alteration of the histones can change DNA transcription and repair. Deacetylation of histones seems to promote autoimmunity. There has been great interest in observations that histone deacetylase inhibitors alter TLR signaling as well as cytokine production in CD4+ T cells; in animal models, treatment with these inhibitors prevents development of SLE. MiRNAs are endogenous noncoding small RNAs (19–25 nucleotides in length) that regulate gene expression at posttranscriptional levels, primarily by binding to mRNA regions that encode proteins. At least 1000 unique miRNAs have been identified in humans, and approximately 45% of immune response genes contain miRNA binding sites. The miRNAs can alter target gene expression or mRNA translation via levels of miRNA expression or via polymorphisms in the sequence of individual

miRs. For example, miR-155 is an essential regulator of responses in both innate and adaptive immunity. Expression of miR-182 in T cells inhibits Foxo1 activation, decreasing synthesis of IL-2. All the known lupus susceptibility genes in humans and mice can be targeted by miRNAs. In the early studies currently available, differential expression of miRNA in SLE in comparison with normal tissues has yielded different results. However, there is good evidence that activation of TLRs 2, 4, and 5 leads to upregulation of miR-146a, which increases expression of TRAF6, IRAK1, IRF5, and STAT1, with subsequent enhancement of innate immune cell signaling and increase in production of IFN-α (a hallmark cytokine in many patients with SLE). Over the next few years, an explosion of information is expected on how epigenetics influences susceptibility to SLE and its clinical severity.

Gender Influences Gender influences on disease susceptibility must be of major importance because there are nine women for every man with SLE. The most important

51

CHAPTER 4  Overview of Lupus Pathogenesis A

Environmental/infectious/ endogenous trigger TREX1

ITGAM FCGR

Apoptotic cell

TREX1 Phagocyte Clearance defects

C1q C2 C4

Release of danger signal

Figure 4.4  Summary of putative human genes with polymorphisms (or duplications or mutations) that increase susceptibility to systemic lupus erythematosus (SLE). Genes are presented in the cell networks known to be activated in patients with SLE. (A) Genes that affect cell apoptosis (or DNA breakdown) and genes that influence clearing of apoptotic cells, and immune complexes. (B) Genes that influence the response of plasmacytoid dendritic cells (pDCs) to binding of surface and endosomal TLRs by external danger signals and by RNA and DNA in infectious agents and in lupus immune complexes, with resultant increase in IFN-α. (C) Genes influencing the response of T cells, B cells, and plasma cells to activation by DC (and other antigen-presenting cells) with ultimate production of autoantibodies and the immune complexes they form with antigens. (From Deng Y, Tsao BP: Genetic susceptibility to SLE in the genomic era. Nat Rev Rheumatol 6(12): 683–692, 2010.)

Dendritic cell

TNFSF4

STAT4

Plasmacytoid dendritic cell

FCGR2B HLA

Immune complexes

BCR

STAT4 PTPN22 TCR Cytokines IL10 T cell Adaptive immune response

Environmental factors that predispose to SLE are undoubtedly important, although few have been identified in a definitive manner. UV light (UVB in particular) exacerbates disease in a majority of people with SLE, and in some people the clinical onset of disease is preceded by

TLR3, TLR7–TLR9 Endosome

ITGAM

TNFRSF4

Environmental Factors

FCGR2A

TNFAIP3 RNA, DNA IRAK1, TNIP1 PHRF1/IRF7? IRF5 STAT4 ↑Cytokines ↑IFN-α

FCGR

C

influence may be hormonal because sex differences in susceptibility are the largest during reproductive years. Estradiol probably prolongs the life of autoreactive B and T lymphocytes. Women exposed to oral contraceptives, or to hormone replacement therapy regimens containing estrogenic compounds, have a small but statistically significant increased risk for the development of SLE. Prospective, randomized, blinded, controlled trials showed that administration of one hormone replacement therapy containing conjugated estrogens and a progesterone significantly raised the risk of mild/moderate disease flare in women with established SLE. There are many experiments in some murine SLE strains showing that an increase in levels of estrogen or progesterone worsens disease, whereas male hormones are protective. However, other features of female gender may also be important in predisposing to SLE. For example, most women after pregnancy have circulating stem cells from their fetuses (microchimerism), which might set up lupus-like graft-versus-host type immune reactions. The X chromosome and its loci and methylation status may be important in predisposing a person to SLE. Women may be predisposed to SLE because their inactive X chromosome is enriched in hypomethylated regions. The CpG in these regions can be bound by TLR9, activating innate immune responses and increasing risk for autoimmunity. Lupuspredisposing genes located on the X chromosome include TLR7/9 (where copy number seems important), IRAK1, and TREX1. Whether their location on the X chromosome in humans is important in their effects remains to be determined. Additional evidence for the importance of the X chromosome in SLE includes the observation that phenotypic men with an extra X (XXY, Klinefelter syndrome) have a significantly higher prevalence of SLE than men who are XY.

B

Innate immune response

BANK1 BLK PRDM1 LYN ETS1 IKZF1 B cell

PRDM1 ETS1 Plasma cell

Autoantibodies

unusually large exposure. Mechanisms include alteration of the structure of DNA in the dermis to render it more immunogenic (i.e., neoantigen formation) and induction of apoptosis in keratinocytes and other dermal cells, presenting higher quantities of self-antigens to the immune system. Infections have long been suspect as inducers and enhancers of SLE. Work from several laboratories has linked infection with Epstein–Barr virus (EBV) to SLE. EBV infection activates B lymphocytes, which might cause a genetically predisposed person to make large quantities of autoantibodies, overwhelming regulatory mechanisms. The EBNA-1 molecule of EBV has molecular mimicry with a sequence in the Ro particle; immunization with that sequence can induce multiple autoantibodies and lupus-like disease in animals. Evidence has now implicated exposure to silica dust as predisposing to SLE, especially in African American women. Exposure can occur in agricultural or industrial settings. Many potential toxins in the environment may initiate and influence immune and inflammatory responses, but so far there is no consistent evidence for many that have been implicated, such as exposure to dogs and wearing of lipstick.

Tissue Damage in SLE Initiation of SLE by tissue deposition/binding of pathogenic subsets of autoantibodies and ICs is only the beginning of the story. Many other processes are required to initiate inflammation and the tissue damage that ultimately destroys quantity and quality of life in this chronic disease. Inflammation and damage begin with complement activation. The 30 plasma and membrane-bound proteins in the complement system, through sequential serine protease–mediated cleavage events, release biologically active fragments. In the first stage early complement components are cleaved to ultimately form C3 convertases; in the second stage proinflammatory activation products such as C3a and C5a form, as well as a lytic complex containing terminal complement components C5b-9. Initiation of the cascade begins with (1) binding of the Fc portion of Ig in ICs by C1q (classical

52

SECTION 2 Pathogenesis

pathway); (2) binding of factors B, D, or properdin by interaction with carbohydrates, lipid, and proteins on surfaces of microbial pathogens or apoptotic cells, with subsequent C3 activation (the alternative pathway); and (3) binding of lectins such as mannose-binding lectin (MBL)/protein to carbohydrate moieties on microorganisms, with changes in MBL that cleave C4 and then C3 (the lectin pathways). Several other proteins control complement activation, including factor I carboxypeptidase, factor H (a membrane cofactor protein), and protease and convertase inhibitors (C1-inhibitor, C4-binding protein). The membrane protein, protectin (CD59), can prevent formation of the lytic complex within plasma membranes. C3a, C4a, and C5a recruit leukocytes into sites of IC deposition, activate them, and cause inflammation. C4b and C3b bind to ICs and facilitate their clearance, including transport by CR1 on erythrocytes and phagocytosis by cells with FcR in the reticuloendothelial system. However, when CR1 transport systems are overloaded, as in SLE, IC clearance is impaired and the system tilts toward complement activation by persistent ICs, with resultant persistent inflammation. Thus, as in other systems, balance must be maintained between complement activation to remove pathogens, ICs, and apoptotic cells/debris, and dysregulated persistent activation that promotes inflammation. Hereditary deficiencies of early complement components or MBL predispose to SLE. Some patients with SLE make antibodies to C1q, to C1-INH, or to the convertase BbC3b; each of these autoantibodies may promote SLE. Generally, quantitatively low plasma levels of C3, C4, and C1q and functionally low quantities of total hemolytic complement have a statistically significant association with SLE disease activity, particularly with nephritis. Increased excretion of C3d in urine is associated with active disease, and rising levels of complement have correlated with clinical improvement in high-quality clinical trials, both in SLE and in LN. However, positive and negative predictive values for these measures in general clinical use are not strong. Methods identifying erythrocyte-bound C4d (high in active SLE), a breakdown product of complement activation, can differentiate between painful SLE and fibromyalgia; they may prove to be useful in identifying impending disease flares. Erythrocyte display of CR1 receptor (low in active SLE) has better positive and negative predictive values than complement levels. The last two methods are not yet widely used, but that may change over the next few years. The imperfect ability to correlate SLE activity with complement activation may reflect failure to reflect subsequent tissue damage initiated by postcomplement fixation pathways. The extensive number of cells and structures that are assaulted in LN are illustrated in Fig. 4.5. Some of the proteins excreted in the urine that reflect processes beyond IC/complement fixation are listed in Table 4.1. In the Fig. 4.5 insert the autoantibodies and ICs (shown as green Ys) binding to capillaries in glomeruli not only fix complement (shown as black stars) but also activate ECs to secrete chemokines, such as monocyte chemotactic protein 1 (MCP-1), and mesangial cells to initiate proliferation pathways. The process also results in podocyte injury, initiating pathways that lead to the podocyte fusion characteristic of patients with membranous features of LN. During the nephritic process, ECs are damaged in vessels outside glomeruli, leading to ischemia of renal tubules; pathways promoting thrombosis are initiated; and renal tubule epithelial cells are activated, initiating pathways that can lead to renal tubular atrophy. The soluble mediators released by tissue cells (such as metalloproteinases) and infiltrating cells activate tissue-resident mononuclear phagocytic cells (variably regarded as tissue-fixed macrophages or DCs) that attract circulating monocytes and T and B cells into tissues. Thus damage is driven by immune pathway cells that are partially understood and by nonimmune pathway cells that may take over the process of chronic inflammation and damage. In

the most unfortunate patients, pathways that promote fibrosis (with known participation by TGF-β and IL-4 as well as many other growth factors), with resultant glomerulosclerosis and interstitial fibrosis, have the highest chance of progressing to renal failure. Although chronic inflammation may initiate the ischemia/EC damage/podocyte damage, and so on, other processes that occur in tissue, such as chronic oxidative damage and metalloproteinase release, probably continue to drive at least some of these pathways. The accelerated atherosclerosis characteristic of patients with SLE is another example of a tissue in which an initial attack by the immune system leads to serious chronic disease mediated by nonimmune cells. Risk for atherosclerotic disease is 5- to 10-fold higher in SLE patients than in age-matched non-SLE populations. ICs, complement split products, and some autoantibodies directly activate ECs in arteries. This activation sets in motion the release of chemokines and cytokines from the ECs and infiltration of the artery wall with monocytes and lymphocytes. As with LN, monocyte infiltration and activation of mononuclear phagocytic cells is an initiator of tissue damage. In the arteries, the activated monocyte becomes the nidus of plaque formation because it phagocytizes oxidized lipids, particularly oxLDL, to become a foam cell. The continuing process of simmering SLE with chronic oxidation, chronic inflammation, release of metalloproteinases, and long-term activation of ECs leads to plaque formation, smooth muscle proliferation, activated cell surfaces that trap platelets, fibrosis in late lesions, and narrowing and occlusion that presage myocardial infarcts. Damage to ECs also results in altered pathways of repair; in SLE, replacement of damaged ECs with progenitor cells is impaired. This finding brings up the possibility that stem cells of patients with SLE have inherent abnormalities. It is equally possible that because of the local “toxic” environment in arteries, veins, glomeruli, pulmonary capillaries, synovium, and other vascular tissue assaulted by SLE, stem cells cannot support development of normal ECs, mesangial cells, and so on. Currently, therapies are directed primarily at suppressing the initiating autoantibody/immune attack on tissues. It is likely that more attention needs to be given to other involved cells and processes that lead to tissue damage. For example, it is disappointing that one study has not shown a reduction in the rate of end-stage renal disease in LN, in spite of the fact that there are better therapies for reducing disease activity, maintaining improvement, and reducing damage from hypertension and proteinuria. In later high-quality clinical trials, sustained remission of LN in patients treated with cyclophosphamide or mycophenolate plus glucocorticoids plus antimalarials occurred in only a minority of patients over a period of 6 to 36 months. On the other hand, recent reports suggested excellent short-term results with combination therapies with mycophenolate plus tacrolimus plus steroids or mycophenolate plus rituximab without long-term steroids. In responders to induction therapies, long-term maintenance with mycophenolate also shows good long-term results, with 85% of those responders maintaining improvement for at least 3 years. Our mandate is to move quickly to understand and use activation or inhibition of the other tissuedamaging pathways that mediate SLE damage so that patients who are poor responders to current therapies, or who do not maintain initial responses, can be protected from the damage of the disease. It is hoped that the next edition of this text will be able to recommend such strategies.

CURRENT APPROVED AND INVESTIGATIONAL THERAPIES FOR SLE Fig. 4.6 illustrates therapies for SLE associated with their effects on various portions of innate/adaptive immunity.

53

CHAPTER 4  Overview of Lupus Pathogenesis

Podocyte injury

Mesangial cell activation Glomeruli

Endothelial cell activation

**

Soluble mediators

Podocyte Thrombosis

Lymphocytic infiltration

Dendritic cell

Collecting duct

Figure 4.5  Cells and substrates in a target organ (the kidney) that are all subject to damage in patients with lupus nephritis. (From Davidson A, Aranow C: Lupus nephritis: lessons from murine models. Nat Rev Rheumatol 6(1): 13–20, 2010.)

Endothelial activation and death

Autoantibody

Dendritic cell activation

*

Loop of Henle

Tubular atrophy

Complement proteins Mesangial cell

Fibrosis

Lymphocyte

Targeted Therapies in SLE Hydroxychloroquine, Laquinimod, OGN for TLR 7/9

MP/DC HLA

LJP394

Edratide, Lupuzor Treg TCR

Figure 4.6  Targets of current and experimental therapies for patients with systemic lupus erythematosus (SLE). Treatments are presented as affecting specific cell types, and many have multiple effects in addition to what is shown. Treatments that are standard of care in the management of SLE (2018) are surrounded by bold black boxes. Others listed have either failed to be better than placebo in recent clinical trials (red) or are currently in active clinical trials (black lettering). Nonspecific therapies that are used in standard therapy of SLE include glucocorticoids and cyclophosphamide. See chapters 53 and 54.

Y

CTLA4 Ig

T Cell

CD28 AMG5571

IMPDH Inosinic acid

B7

ICOS

purines

CD40L

B7RP1 CD 40

Anti-CD40L Cytokines/inhibitors Mycophenolate anti-IL12/23 IFNγ inh IFN type 1 inh Low dose IL2 Anti-IL6 IMPDH= inosine monophosphate dehydrogenase

SUGGESTED READING Davidson A. What is damaging the kidney in lupus nephritis? Nat Rev Rheumatol. 2016;12(3):143–153. Mouton VR, Suarez-Fueyo A, Meidan E, et al. Pathogenesis of human systemic lupus erythematosus: a cellular perspective. Trends Mol Med. 2017;23(7):617–635. Perl A. Metabolic control of immune system activation in rheumatic diseases. Arthritis Rheumatol. 2017;69(12):2259–2270.

DNAbinding BCR

CD20 CD22

B Cell IMPDH Inosinic acid

Rituximab SBI087 Epratuzumab

BCMA TACI purines BAFF-R

aBLyS TACI Ig

BLyS Mycophenolate

MP/DC

By BH Hahn

Sinicato NA, Postal M, Appenzeller S, et al. Defining biological subsets in systemic lupus erythematosus: progress toward personalized therapy. Pharmaceut Med. 2017;31(2):81–88. Tsokos GC, Lo MS, Costa Reis P, et al. New insights into the immunopathogenesis of systemic lupus erythematosus. Nat Rev Rheumatol. 2016;12(12):716–730. Zharkova O, Celhar T, Cravens PD, et al. Pathways leading to an immunological disease: systemic lupus erythematosus. Rheumatology (Oxford). 2017;56(suppl):i55–i66.