http://www.kidney-international.org review & 2010 International Society of Nephrology

Innate immune receptors and autophagy: implications for autoimmune kidney injury Hans-Joachim Anders1 and Detlef O. Schlondorff2

1Department of Nephrology, University of Munich, Munich, Germany and 2Department of Medicine, Mount Sinai Medical Center, New York, New York, USA

Inflammationistheimmunesystem’sresponsetoinfectious The integrity and survival of multicellular organisms depends or noninfectious sources of danger. Danger recognition is on the initiation of an immediate host defense against facilitated by various innate immune receptor families infectious agents.1 Immediate pathogen recognition is including the Toll-like receptors (TLRs), which detect danger guaranteed by innate pathogen recognition receptors, which signals in extracellular and intracellular compartments. It is can either activate complement-mediated killing or the an evolving concept that renal damage triggers intrarenal activation of parenchymal and immune cells to produce inflammation by immune recognition of molecules that are cytokines and other mediators of inflammation designed to being released by dying cells. Such danger-associated contain the infection (Table 1). Although most receptors are molecules act as immunostimulatory agonists to TLRs and surprisingly specific for chemically defined pathogen-asso- other innate immune receptors and induce cytokine and ciated molecular patterns (PAMPs), the sum of the different chemokine secretion, leukocyte recruitment, and tissue receptor classes covers the entire spectrum of pathogens. remodeling. As a new entry to this concept, autophagy Recently, it has been shown that endogenous molecules that allows stressed cells to reduce intracellular microorganisms, are generated during tissue injury and labeled as danger- aggregates, and cellular organelles by moving and associated molecular pattern (DAMP) molecules, can also subsequently digesting them in autophagolysosomes. Within activate pattern recognition receptors similarly to PAMPs, the autophagolysosome, endogenous molecules and danger- thereby offering a novel understanding of sterile types of associated molecules may be presented to TLRs or loaded inflammation (Table 2).2 DAMPs can originate from onto the major histocompatibility complex and presented as intracellular sources or can be generated from extracellular autoantigens. Here we discuss the current evidence for the matrix degradation. As such DAMPs would normally not be danger signaling concept in autoimmune kidney injury and available for presentation to immune sensors (Figure 1). propose that autophagy-related processing of self- However, DAMPs can be generated and released during cell provides a source of immunostimulatory molecules and stress, apoptosis, or necrosis due to traumatic, ischemic, autoantigens. A better understanding of danger signaling toxic, or inflammatory tissue injuries. It is likely that DAMPs should enable us to unravel yet unknown triggers for renal function as danger signals and that DAMP-mediated immune immunopathology and progressive kidney disease. activation developed during evolution to aid danger control Kidney International (2010) 78, 29–37; doi:10.1038/ki.2010.111; and tissue repair. If such a process escapes the normal control published online 28 April 2010 and/or suppression of an adaptive immune response to KEYWORDS: autoimmunity; autophagy; danger signaling; glomerulonephritis; endogenous molecules, the recognition of PAMP and DAMP molecular mimicry; Toll-like receptor by receptors of the innate immune system could contribute to an autoimmune response. In this review we provide a conceptual outline by discussing examples from the field of autoimmune kidney disease: first, we introduce the concept that DAMP release from dying or generation by stressed cells can induce renal inflammation inside the kidney. Second, we discuss the possibility, that during an initial renal cell immune injury against foreign antigens, endogenous neoantigens may be generated. This can result in a process of antigen spreading, eventually provoking an autoimmune response contributing Correspondence: Detlef O. Schlondorff, Department of Medicine, Mount Sinai Medical Center, Box 1243, One Gustave Levy Place, New York, New York to the progression of renal disease by humoral and cellular 10029, USA. E-mail: [email protected] immune mechanisms. Third, we discuss the concept of Received 3 February 2010; revised 5 March 2010; accepted 23 March molecular mimicry of endogenous nucleic acids with viral 2010; published online 28 April 2010 nucleic acids with the potential of promoting lupus nephritis

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Table 1 | Classes of innate and adaptive pattern recognition Table 2 | Endogenous molecules proposed to function as molecules DAMPs by activating TLRs or other receptors Innate recognition Adaptive recognition DAMP Receptor Compartment molecules molecules HMGB1 TLR2, TLR4, RAGE, RIG Secreted to Pentraxins (CRP, SAP, IgA, IgM, IgG, IgE Heat shock proteins TLR2, TLR4 extracellular fluids pentraxin-3) Hyaluronates, biglycan TLR2, TLR4 Complement factors Heparan sulfate TLR4 Mannose-binding lectin Fibrinogen TLR4 Defensins TLR4 Cell surface T-cell receptors U1snRNP-IgG TLR7 (FcR/BCR) Scavenger receptors B-cell receptors (Ig) DNA-nucleosomes-IgG TLR9 (FcR/BCR) Complement receptors Antigen-presenting Adenosine A1/A2A/A2B/A3 molecules ATP P1/P2X/P2Y Fc receptors S100 proteins RAGE Toll-like receptors dsDNA AIM2 – IL-1R Dectins Cathepsin-B NALP3 – IL-1R Uric acid crystals NALP3 – IL-1R Intracellular Toll-like receptors MHC I, MHC II Abbreviations: AIM, absent in melanoma; BCR, B-cell receptor; DAMP, damage- endosomes associated molecular pattern; HMGB, high-mobility group B; Ig, immunoglobulin; IL, interleukin; NALP, Nacht domain-leucine-rich repeat- and PYD-containing protein; Intracellular cytosol RIG-like helicases RAGE, receptor for advanced glycation end products; TLR, Toll-like receptor. NOD-like receptors Inflammasome-activating molecules tuft, histopathologically referred to as necrotizing glomer- 6 Abbreviations: CRP, C-reactive protein; Ig, immunoglobulin; MHC, major histo- ulonephritis. Podocytes and parietal epithelia are activated 7 compatibility complex; NOD, nucleotide-binding oligomerization domain; to proliferate leading to crescentic glomerulonephritis. Both RIG, retinoic-acid-inducible protein; SAP, serum amyloid P. types of lesions involve the local production of chemokines, which enhance the influx of antigen-specific T cells and macrophages into the glomerular compartment or the through Toll-like receptors (TLRs) at the systemic level. periglomerular space. Resident cells, as well as infiltrating Forth, we summarize data on the role of TLRs in mediating cells, also produce proinflammatory cytokines such as IL-12, infection-associated flares of immune complex glomerulone- TNF-a, and interferon-g (IFN-g). These cytokines will drive phritis both at the systemic and the tissue levels. Finally, we T-cell responses toward the Th1 phenotype and the resulting discuss the potential role of autophagy in many of the above cellular immune response. Such T-cell responses are usually processes and in the eventual development of autoimmunity. counterbalanced by regulatory T cells in secondary lymphoid organs as well as at the tissue level. However, in experimental low DANGER-ASSOCIATED MOLECULAR PATTERNS CAN DRIVE anti-GBM disease CD4 þ CD25 þ Foxp3 þ CD69-CD45RB RENAL AUTOIMMUNE TISSUE INJURY regulatory T cells were less potent in suppressing nephritis as The triggering events for the loss of tolerance, for example, compared with regulatory T cells from nonnephritic mice.8 against glomerular basement membrane (GBM) collagen Crescentic glomerulonephritis has therefore been classified as components in anti-GBM disease, neutrophil or lysosomal the renal manifestation of a Th1-like delayed type of -associated membrane protein 2 (LAMP2) antigens hypersensitivity reaction.9 Antineutrophil cytoplasmic anti- in antineutrophil cytoplasmic antibody–associated vasculitis, body–associated vasculitis is associated with comparable or chromatin in systemic lupus erythematosus (SLE), remain glomerular pathology, even though termed pauci-immune, complex and difficult to sort out in individual patients. that is, few, if any, glomerular immune complex deposits can However, it is clear, that genetic and environmental factors be seen.6 In antineutrophil cytoplasmic antibody–associated need to overcome several check points before autoreactive vasculitis neutrophils disrupt the endothelium in the lymphocyte clones are allowed to expand, so that rising microvasculature, causing focal glomerular necrosis by a serum concentrations of the respective autoantibodies combination of the release of toxic granule contents, become detectable by enzyme-linked immunosorbent assay.3 ischemia, and complement activation.6 The novel danger Although suspected for a long time, only recently has signaling adds to these mechanisms and highlights the evidence been provided for molecular mimicry between proinflammatory role of dying cells.10 Although early phases infectious and endogenous antigens such as LAMP2 as a of apoptotic cell death with the rapid removal of apoptotic trigger for pathogenic antibodies in causing pauci immune bodies may avoid the release of intracellular DAMPs and a glomerulonephritis.4 In addition, there is little doubt that subsequent inflammatory response, late apoptotic or necrotic autoantibodies against the a3NC1 domain of collagen IV are cells release the content into the extracellular space.2 Here pathogenic in anti-GBM disease, because they bind to the they can bind to pattern recognition receptors either directly intrarenal autoantigen and cause in situ immune complex on the cell surface or, after endocytosis, in the endosomal formation, which activate FcgR and complement factors.5 compartment of immune and nonimmune cells (Figure 1). This process usually leads to focal necrosis of the glomerular Furthermore, the local activation of matrix-degrading

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Stranger Danger Avoiding immune TLR activation modulates the immunization phase and the model signal activation adaptive immune response, which affects kidney disease by Impaired additional extrarenal mechanisms. Although the role of TLR2 Pathogen clearance and TLR4 in crescentic glomerulonephritis is consistent with Necrotic Matrix Apoptotic their role for sterile inflammation in postischemic acute renal cell degradation cell failure,15–17 a potential role of endogenous RNA in activating PAMPs DAMPs Hidden DAMPs TLR3 during crescentic glomerulonephritis is less likely, as serum nephritis could be induced in mice deficient in TRIF, the adapter protein required for TLR3 signaling.18 Necrotic APC Phagocytosis cells activate primary mouse mesangial cells preferentially APC activation through TLR2/MyD88 rather than TLR3/TRIF, making –Il-10 endogenous dsRNA as a relevant DAMP for TLR3 ligation 18 –TGF-β less likely. One may speculate that HMGB1, hyaluronic acid, biglycan fragments, or fibrinogen function as endogen- ous TLR2 and TLR4 agonists in glomerular cells, as shown +Costimuli Lymphocyte for fibrinogen activating TLR4 in podocytes.19 Although self- T cell RNA does not seem to trigger TLR3/TRIF signaling in anti- GBM nephritis, nuclear HMGB proteins can exert their effect Activated Phagocyte as cofactors for RNA and DNA recognition through TLR7 APC and TLR9 as well as TLR-independent nucleic acid recogni- Anergy 20 Apoptosis tion pathways. Whether such a potential process contri- Clonal proliferation Ignorance butes to anti-GBM nephritis has not yet been formally examined. However, this mechanism could serve as an Figure 1 | Immune recognition of extrinsic and intrinsic dangers. Pathogens release pathogen-associated molecular explanation for the intraglomerular induction of type I IFNs, patterns (PAMPs) that activate antigen-presenting cells (APCs) and which promote glomerular inflammation in autologous nonimmune cells through pattern recognition receptors. The nephrotoxic serum nephritis.21 Thus, glomerular cell injury maturation of, e.g., APC-like dendritic cells leads to antigen and extracellular matrix modification have the potential to presentation in the presence of costimulatory molecules that set off adaptive immune responses involving clonal expansion of activate innate immune responses through the release of antigen-specific T and B cells. Necrotic cells release intracellular intracellular DAMPs, which can activate pattern recognition molecules that can activate the same classes of immune receptors receptors such as TLR2 and TLR4 on adjacent glomerular and thereby act as danger-associated molecular patterns (DAMPs). immune and nonimmune cells. The nature of these This mechanism can explain sterile types of inflammation that present clinically like infectious diseases such as a gout attack glomerular DAMPs remains to be defined in detail, but data (DAMP ¼ uric acid crystal) or postischemic tissue inflammation. In from other areas provide sufficient evidence for the danger- SLE nuclear particles containing immunostimulatory nucleic acids signaling hypothesis as a whole.2,10 function as adjuvant-like DAMPs in addition to their role as autoantigens. Apoptotic cell death avoids DAMP release and inappropriate immune activation. Vice versa, genetic defects in EPITOPE SPREADING AS A MECHANISM FOR AN IMMUNE apoptosis or the rapid clearance of apoptotic cells by phagocytes RESPONSE IN RENAL DISEASE predispose to chronic inflammatory autoimmune diseases such as The role of T cells and specifically CD8 þ T cells in SLE because secondary necrosis of apoptotic cells causes DAMP release. autoimmune-mediated glomerular and tubulointerstitial disease has been discussed for decades.22 Lately, clear experimental evidence for the significance and mechanisms enzymes around apoptotic and necrotic cells can generate of T-cell involvement in glomerular and tubulointerstitial fragments of extracellular matrix, such as hyaluronates and disease has been forthcoming, as recently reviewed by Sung biglycans, which can serve as DAMPs.2 and Bolton.22 This process may also link glomerular Many studies of the heterologous model of nephrotoxic pathology and proteinuria to progressive interstitial disease. serum nephritis in mice lacking TLR2 or TLR4support the Subsequent to injury in the glomerulus or in the tubuloin- role of DAMPs in glomerular disease. In these knockout mice terstitium neoantigens can be generated leading to epitope full-blown crescentic glomerulonephritis is prevented by spreading with generation of neoantigens, which are then reduced activation of proinflammatory mediators in the presented by dendritic cells to T cells either within the kidney glomerular compartment.11,12 Some studies have reported or in draining lymph nodes.23–26 Potentially, such neoanti- similar results by using the autologous serum nephritis in gens could at the same time exert their effect as DAMPs, and TLR-deficient mice or by co-injecting TLR agonists during activate the innate immune response through TLRs on the immunization phase.13,14 Furthermore, there is evidence mesangial, epithelial, interstitial, and endothelial cells as well from chimeric mice that TLR2 on both intrinsic glomerular as on macrophages and dendritic cells. The combined cells and on leukocytes is involved in this process.12 The activation of an innate and adaptive immune response would interpretation of these studies must take into account that lead to a propagation and spreading of the disease from the

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glomerulus to the tubular and interstitial compartment. biopsies, which may originate from either glomerular Recently, Macconi et al.27 showed, in a model of proteinuria leukocytes or intrinsic renal cells.35 In fact, complexed RNA secondary to reduction in kidney mass, that filtered albumin or dsDNA can trigger the production of large amounts of was partially processed by tubular epithelial cells and IFN-a and IFN-b as well as multiple IFN-dependent in subsequently by dendritic cells, resulting in major histocom- glomerular endothelial cells and mesangial cells.36–38 As patibility complex (MHC) I-mediated antigen presentation endothelial cells and mesangial cells lack TLR7 and TLR9, to CD8 þ T cells in the regional lymph nodes. Cytotoxic these responses rather involve TLR-independent nucleic acid CD8 þ T cells specific for renal antigens then propagated recognition pathways.36,38 Nevertheless, self-RNA recognition renal damage. This mechanism provides a novel explanation by TLR7 has a dominant role in mediating SLE disease for the progression of renal diseases by autoimmune renal activity.31,39 TLR7 deficiency40,41 or TLR7 blockade42 pre- injury. This concept is further supported by a study using a vents proliferative lupus nephritis in mice by specifically transgenic mouse model with podocyte-specific expression of impairing the production of RNA autoantibodies. Further- a neoantigen.28 Thus, intrarenal epitope spreading involving more, lupus nephritis becomes more severe when TLR7 intrinsic renal cells, dendritic cells, and T cells could signaling is enhanced, for example, by TLR7 duplica- contribute to an autoimmune type of kidney injury. The tion43 or by genetic elimination of TLR7 inhibitors such as potential roles of the innate immune system and TLRs in this SIGIRR44 or TLR9.45 Hence, self-nucleic acid-induced process, as well as the contribution of autolysosomal ‘pseudoantiviral’ immunity translates into autoimmune processing of neoantigens, have not been examined as yet. tissue damage. The functional importance of type I IFN was, also shown by studies, that observed almost complete ENDOGENOUS NUCLEIC ACIDS DRIVE LUPUS NEPHRITIS BY suppression of lupus nephritis in mice deficient in the type I TRIGGERING ANTIVIRAL IMMUNITY IFN receptor.46–49 Can this concept be translated to human Lupus nephritis is characterized by a loss of tolerance and a lupus? Type I IFN expression levels are elevated in SLE polyclonal autoimmune response against multiple nuclear patients with active disease and dropped on immunosup- and chromatin-related autoantigens, which are ubiquitous, pressive therapy.50,51 Furthermore, type I IFN cause ultra- albeit hidden inside cells. Hence, nuclear autoantigens reach structural changes in lymphocytes and endothelial cells, that the extracellular space only when apoptotic cells are not is, the tubuloreticular structures.52 These have only been properly removed and undergo secondary necrosis. This observed in three cohorts of patients: (1) patients with viral process will foster the formation of immune complexes hepatitis treated with IFN-a;53 (2) in renal biopsies of containing autoantibodies and nuclear autoantigens that patients with HIVAN;54 (3) and in blood lymphocytes of SLE 29 contain immunostimulatory nucleic acids. Circulating patients.55 In lupus nephritis biopsies these structures are also immune complexes tend to deposit in glomeruli and activate commonly noted in glomerular endothelial cells and are FcgR and complement, either along the inside or the outside referred to as ‘lupus inclusions’.56 Lupus inclusions seem to the filtration barrier as well as in the mesangium, causing a represent a specific type of IFN-a-induced protein assembly 29 spectrum of different histopathological manifestations. in the cytosol of cells. As such they do not only serve as a Especially, diffuse proliferative lupus nephritis is associated diagnostic tool57 but also as a hint for the pathogenic role of with mixed macrophage, T-cell, and B-cell infiltrates due to IFN induction in lupus immunopathology. RNA and DNA local expression of proinflammatory cytokines and chemo- lupus autoantigens have additional, adjuvant-like, immunos- kines. timulatory properties by binding to viral nucleic acid A new entry into the pathogenesis of lupus nephritis is the recognition receptors comparable to viral particles. This concept that the immune dysregulation in SLE is largely may explain the overlapping clinical presentations of viral homologous to antiviral immunity. For example, transcrip- infection and SLE, and suggest a new pathogenic concept of tome profiles of peripheral blood monocytes from lupus lupus nephritis.58 patients show a profound induction of type I IFN and IFN- 30 related genes, recalling a classical antiviral response pattern. HOW INFECTIONS CAN TRIGGER FLARES OF AUTOIMMUNE Immune complexes containing nuclear lupus autoantigens KIDNEY DISEASE have the potential to activate TLR7 and TLR9 on plasma- It is a common clinical observation that various infections cytoid dendritic cells and B cells initiating a response induce flares of lupus nephritis, of renal vasculitis, of IgA program comparable to that during viral infection.30,31 This nephritis, or of other forms of immune complex glomer- was shown for immune complexes containing chromatin or ulonephritis. Recently, a direct molecular mimicry between a hypomethylated CpG-DNA that activate TLR932 as well as for pathogen-derived protein and a lysosome chaperone LAMP2 immune complexes containing U1snRNP that activate protein in endothelial cells was identified, and showed to TLR7.33,34 In addition, nuclear HMGB proteins can function function as an autoantigen, thereby triggering a pauci as cofactors for RNA and DNA recognition through TLR- immune focal necrotizing glomerulonephritis.4 Molecular dependent as well as -independent nucleic acid recognition mimicry had been discussed as a mechanism for vasculitis for pathways.20 The same IFN signature was found by tran- many years, but had never been clearly shown. Besides such scriptome profiling of glomerular isolates of human renal direct and specific activation of the immune system by

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Table 3 | Effects of transiently exposing MRL(Fas)lpr mice with lupus-like immune complex glomerulonephiritis to various TLR agonists PRR Ligand TLR2 lipoprotein TLR3 dsRNA TLR4 LPS TLR7 ssRNA TLR9 CpG-DNA ?, DAI dsDNA RIG-I 3P-RNA Mesangiolysis + Proteinuria +++ + + + + + + Macrophages + + ++ + ++ + + Inflammation ++ ++ ++ ++ +++ ++ ++ Anti-dsDNA + + + +++ + + Glom. C3 + ++ ++ + + Lymphoprol. + + + +++ +++ Abbreviations: C3, complement factor 3; ds, double stranded; glom., glomerular; lymphoprol., lymphoproliferation; 3P-RNA, 50-triphosphate RNA; PRR, pattern recognition receptor; ss, single stranded. molecular mimicry, other potential mechanisms for infec- mice. Agonists for TLR2, TLR3, TLR4, TLR7, RIG-I, and tion-associated activation of the immune response and cytosolic DNA sensors had a similar potential to aggravate resultant renal immunopathology exist. For example, pha- glomerular inflammation (Table 3).63,71–74 The TLR9 agonist, gocytosis of infected apoptotic cells by dendritic cells triggers unmethylated CpG-DNA, which may derive from viruses or the expansion of autoimmunity-related Th17 T cells, whereas bacteria, was unique in inducing crescentic glomerulone- phagocytosis of noninfected apoptotic cells favors the phritis and renal vasculitis in nephritic MRL(Fas)lpr mice,74 expansion of immunosuppressive regulatory T cells.59 During and was the only TLR agonist that could trigger the onset of systemic infection circulating PAMPs activate immune cells glomerulonephritis in young MRL(Fas)lpr mice that had not and nonimmune cells throughout the body, which affects yet developed SLE-like autoimmunity.75 The latter is immune responses at the systemic as well as at the local level. remarkable because most reports are consistent in that For example, vascular leakage is a hallmark of sepsis or TLR9 expression is restricted to extrarenal plasmacytoid noninfectious tissue injury, because PAMPs increase vascular dendritic cells, macrophages, and B cells in healthy mice.76 permeability by activating TLRs on vascular endothelial Obviously, the activation of immune cells outside the kidney cells.60,61 The same mechanism enhances proteinuria during contributes to the onset of renal disease. In fact, CpG-DNA immune complex glomerulonephritis as shown for systemic and—to a lower extent most other PAMPs—enhanced LPS exposure from Gram-negative bacteria11,62,63 or lipo- immune complex disease by increasing serum cytokine levels, protein from Gram-positive bacteria.63 These bacterial cell autoantibody levels, and glomerular immune complex wall components enhance the permeability of glomerular deposits in MRL(Fas)lpr mice (Table 3). Only the TLR3 endothelial cell and podocyte monolayers by activating agonist dsRNA aggravated lupus nephritis by activating surface TLR2 and TLR4.63,64 Viral nucleic acid complexes mesangial cells rather than by affecting immune complex have a similar effect when they reach the intracellular cytosol disease.72 Thus circulating PAMPs will not only activate an and activate cytosolic viral nucleic acid sensors.36 These antigen-specific adaptive immune response, but will at the experimental findings may explain why endotoxinemia or same time serve as nonspecific adjuvants, thereby enhancing sepsis produces a mild and short-lasting proteinuria in the response of preexisting B- and T-cell clones fueling the humans.65 Such PAMPs may produce stronger and longer- autoimmune diseases. In addition, circulating PAMPs can lasting renal dysfunction in patients with preexisting activate renal parenchymal cells and intrarenal immune cells glomerular diseases and a preactivated and thereby hyperre- to enhance renal inflammation. sponsive glomerular endothelium. In addition, other PAMPs can activate TLRs and other AUTOPHAGY, A POTENTIAL LINK TO AUTOIMMUNITY receptors of the innate immune system on glomerular cells to Autophagy may be an as-yet-underappreciated link between secrete proinflammatory cytokines such as IL-6 and TNF-a, the innate and adaptive immune responses and danger thereby enhancing glomerular inflammation.66–69 As men- signaling. Autophagy was initially recognized as a pathway to tioned earlier, viral nucleic acids may also trigger glomerular salvage cellular proteins during stress, such as starvation, to expression of type I IFN, which adds to the proinflammatory guarantee cell survival.77,78 As such, autophagy represents an microenvironment.37,38 The PAMPs as well as the secreted early step in the organism’s strategy to promote survival, that, mediators also activate resident dendritic cells as well as the if it fails, will result in apoptosis or even necrosis. During the leukocytic cell infiltrate, which is usually present in chronic previous years it has become ever more apparent that nephropathies.70 These activated intrinsic renal parenchymal autophagy is also broadly associated with many steps of cells and intrarenal immune cells also produce proinflam- immune responses.79–81 matory chemokines such as MCP-1/CCL2, CCL5/RANTES, and CXCL10/IP-10, which will foster additional leukocyte Pathways of autophagy recruitment and renal damage. The potential of TLR agonists All mammalian cells undergo constitutive autophagy to some to trigger disease activity of murine lupus-like immune extent, that is, they engulf and digest endogenous cytosolic complex glomerulonephritis was assessed in MRL(Fas)lpr proteins, membrane particles, or intracellular organelles.77

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MHC class I cross-presentation has been provided for tumor PI3 Kinase Autophagosome TLR antigens84,85 as well as for intracellular pathogens.86–90 Within Microautophagy Rapamycin Lysosome the autolysosomes pathogens can interact with membrane- Chaperone- TOR mediated bound and cytosolic receptors of the innate immune system, autophagy Atg proteins which reside in endosomal compartments (such as TLR3, -4, Chaperone LAMP-2A -7, -9). Furthermore, cytosolic receptors such as retinoic TLR Unfolded Macroautophagy substrate acid-inducible gene I (RIG-I), such as helicase receptors, and Cytoplasm protein other cytosolic receptors for RNA and DNA and NOD Parts of Lysosomal receptors can interact with components of autophagy.86,91–93 membranes hydrolase intracellular Developing Potentially, autophagy pathways may also deliver DAMPs, organelles (e.g. MHC II Autolysosome mitochondria) autophagosome generated by stressed or dying cells, to innate immune receptors, such as TLRs present in autolysosomes.20,94–96 However, activation of a number of these receptors can in Figure 2 | Schematic illustration of macro-, micro-, and chaperone-mediated autophagy and the formation of turn enhance autophagy thereby further propagating the autolysosomes. Under normal conditions and in the presence of process.80,92,97 The activation of receptors of the innate growth factors, PI3 kinase will be active and stimulate TOR (target immune system would then result in a proinflammatory of rapamycin), which will inhibit ATGs and thereby autophagy. Decreased PI3 kinase activity would result in less TOR activity, less response as described above. Thereby, autophagy will ATG inhibition, and enhanced autophagy. Rapamycin as an establish a connection between innate and adaptive immune inhibitor of TOR would also activate ATGs and thereby autophagy. responses by delivering either infectious agents or modified self-proteins and/or DNA or RNA to autolysosomes for processing as foreign or self-antigens. During starvation macro-autophagy is initiated by dimin- Autophagy also influences B- and T-cell homeostasis, ished phosphatidylinositol-3 kinase (PI3K) activation due to including Th1/Th2 polarization, and tolerance.79,81,95,98,99 loss of growth factors. PI3K normally activates the mamma- Under normal conditions autophagy is involved in the lian target of rapamycin. Decreased mammalian target of delivery to and presentation of self-antigens by MHC II, rapamycin activity secondary to diminished PI3K activity contributing to the induction and maintenance of CD4 þ T or inhibition of mammalian target of rapamycin by, for cell tolerance.79,83,90 Potentially, enhanced autophagy during example, rapamycin will induce autophagy,77 which may cell stress together with a change in the cytokine milieu may account for beneficial or detrimental effects in the kidney. lead to a weakening or breakdown of tolerance and thereby to The formation of macro-autophagosomes proceeds by the development of autoimmunity.81,95 In this context, the invagination of newly formed double membranes to form description by Kain et al.4 of LAMP2 as a novel autoantigen intracellular vesicles. This involves a number of autophagy- in pauci immune vasculitis is of interest. This ‘autoantigen’ is related gene (Atg) products.77 During the formation of the a major component of chaperone-mediated autophagy for autophagosome portions of the cytosol as well as entire the delivery of proteins to the autolysosome.77 Kain et al.4 organelles, such as intracellular membrane vesicles or showed that in pauci immune vasculitis LAMP2 becomes an mitochondria, can be engulfed. The autophagosome then autoantigen. The authors attribute this to molecular mimicry fuses with , forming the autolysosome (Figure 2). with a bacterial antigen. Alternatively, one might speculate In chaperone-mediated autophagy specific cytosolic proteins that as both the bacterial antigen and Lamp2 will end up in are recognized by chaperone complexes, containing compo- the autolysosome during infection, the local control of nents of the heat shock cognate protein HSC70 and LAMPs, tolerance might be overwhelmed in the autolysosome, enabling docking to lysosomes and transfer of the captured resulting in processing not only of the bacterial antigen, proteins for lysosomal degradation (Figure 2). Within the but also of the LAMP2 self-antigen.4 This would then cause lumen of the autolysosome, membrane structures and an adaptive immune response not only to the invading proteins are degraded by lysosomal proteases, which are bacteria, but also to the autophagosomal LAMP2. Obviously activated by the acid pH generated by proton pump ATPases this only represents a hypothesis at present. of autolysosomes.77 In B cells autophagy facilitates the interaction of the B-cell receptor with TLR9 in an autolysosome-like compart- AUTOPHAGY AS PART OF THE IMMUNE RESPONSE ment.79,81,95,98,99 This results in enhanced production of In dendritic cells macro-autophagy and chaperone-mediated antibodies to DNA antigens, especially in autoimmune autophagy can deliver both foreign and self-proteins to diseases such as systemic lupus.100 The ligand for the TLR9 autolysosomes, where they are processed and may eventually activation could consist of DNA complexed with HMGBs, come in contact with and bind to MHC class II in a both being released from dying cells.2 Potentially, such specialized compartment.82 In support of this concept is the mechanisms could also contribute to an autoimmune observation that more than 50% of autophagosomes merge response during progressive tissue damage. For example, and colocalize with the MHC II loading compartment.83 protein and lipid overload of tubular epithelial cells during Initial evidence for macro-autophagy also contributing to proteinuria or prolonged cell stress of epithelial and

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endothelial cells due to hypoxia/ischemia and endoplasmic autophagy. Perhaps we have already, but unknowingly, benefited reticulum stress could enhance the formation of autophago- from the modulation of autophagy by rapamycin therapy. Thus, somes and autolysosomes, and generation and release of research in autophagy and immune-mediated renal disease may DAMPs. In adjacent dendritic cells, autolysosomal proteolysis already have started under a different label. of DAMPs could potentially generate neoantigens binding to MHC class I or II molecules for surface presentation and DISCLOSURE subsequent T-cell activation.79 This may represent an The authors declared no competing interests. intriguing possibility linking cellular protein overload, endoplasmic reticulum stress, hypoxia, and other mechan- ACKNOWLEDGMENTS isms of local cell injury during kidney disease to autophagy HJA was supported by grants from the Deutsche and a resulting autoimmune response as part of progression Forschungsgemeinschaft (AN372/9-12 and GRK 1202). DS was supported by a grant from the National Institutes of Health, USA of renal diseases. Recently, epitope spreading was identified as (R01 DK 081420). 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