Lose appetite, lose control: integrins and noncanonical autophagy regulate germinal center reactions

Jeremy S. Leventhal

J Clin Invest. 2018. https://doi.org/10.1172/JCI122766.

Commentary

T cell–dependent germinal center (GC) reactions are the pinnacle of adaptive immune responses, with profound effects on human health and disease. It has long been known that ligands of an innate immune pattern recognition receptor subgroup, TLRs, amplify antibody responses; however, the mechanisms regulating this phenomenon are poorly understood. In this issue of the JCI, Raso et al. demonstrate that αvβ3 integrins regulate the magnitude and speed of TLR-augmented GC reactions, limiting both short- and long-term humoral immunity. This phenomenon is dependent on a noncanonical form of the autophagy pathway and Rubicon, a noncanonical autophagy-associated protein. B cell–specific deletion of the gene encoding αvβ3 integrin enhanced GC responses in mice and conferred a dramatic survival advantage compared with controls after influenza infection, confirming that B cell integrin manipulation represents a potential and exciting target for augmenting or inhibiting GC reactions.

Find the latest version: https://jci.me/122766/pdf The Journal of Clinical Investigation COMMENTARY Lose appetite, lose control: integrins and noncanonical autophagy regulate germinal center reactions

Jeremy S. Leventhal Division of Nephrology, Department of Medicine, and Transplant Research Institute, Icahn School of Medicine at Mount Sinai, New York, New York, USA. Renal Division, James J. Peters VA Medical Center, Bronx, New York, USA.

imentally can be complex, given the reliance on LC3 for most readouts and the overlap of T cell–dependent germinal center (GC) reactions are the pinnacle of adaptive this marker with both forms of autophagy. immune responses, with profound effects on human health and disease. It Molecular analysis of noncanonical autoph- has long been known that ligands of an innate immune pattern recognition agy complexes identified the essential pro- receptor subgroup, TLRs, amplify antibody responses; however, the tein RUN and cysteine-rich domain contain- mechanisms regulating this phenomenon are poorly understood. In this ing beclin 1 interacting protein (Rubicon). issue of the JCI, Raso et al. demonstrate that αvβ3 integrins regulate the Rubicon is involved only in noncanonical magnitude and speed of TLR-augmented GC reactions, limiting both short- autophagy and has the potential to inhibit and long-term humoral immunity. This phenomenon is dependent on a canonical autophagy (6). Although the first noncanonical form of the autophagy pathway and Rubicon, a noncanonical observations of noncanonical autophagy autophagy-associated protein. B cell–specific deletion of the gene encoding were in the setting of pathogen clearance

αvβ3 integrin enhanced GC responses in mice and conferred a dramatic and effector immune responses, subse- survival advantage compared with controls after influenza infection, quent observations noted that noncanonical­ confirming that B cell integrin manipulation represents a potential and autophagy also plays a role in maintaining exciting target for augmenting or inhibiting GC reactions. humoral tolerance. Mice with myeloid cells lacking canonical autophagy proteins that overlap with LAP and mice lacking Rubicon, which are unable to process phagocytosed apoptotic cells, were shown to develop a Canonical versus noncanonical core autophagy machinery to deliver mem- lupus-like syndrome. Conversely, mice autophagy brane-bound cargo to lysosomes (3). This lacking proteins, such as Unc-51–like kinase Autophagy is an intracellular degradation phenomenon was first observed in macro- 1 (ULK1), only found in the canonical auto- pathway that provides a powerful mecha- phages, where aggregates of fluorescently phagy pathway did not develop lupus-like nism for maintaining cellular homeostasis, labeled LC3, visualized by areas of punctate disease (7). and dysfunctional autophagy is implicated fluorescence commonly considered evi- in the pathogenesis of several human diseas- dence of autophagosome formation, occur­ Canonical and noncanonical es (1). Autophagy is accomplished through a red during phagocytosis of yeast or zymo- autophagy and B cell function complex interaction of proteins that orches- san particles (4). Further inspection of the The story of autophagy in B cells is complex trate the sequestration of cytoplasmic con- resulting compartments revealed single- and depends on the subset of B cells stud- stituents within a de novo–formed organelle membrane phagosomes, in contrast with ied. Our lab built on the seminal findings of dubbed the “autophagosome.” Among auto- the double-membrane autophagosomes Chen et al. and demonstrated that allospe- phagy proteins, only the lipidated form of that occur during starvation, the prototyp- cific memory B cells (Bmem) are dependent microtubule-associated protein light chain ical stimulus for autophagy (5). Because on autophagy (8, 9). Neither group differ- 3 (LC3, also known as LC3-II) contributes of the involvement of the autophagy pro- entiated between involvement of canonical to extension of the nascent autophagosome tein LC3, this process was dubbed LC3- and noncanonical autophagy; however, the and remains part of fully formed autophago- associated phagocytosis (LAP), but has also abnormalities in oxidative stress and mito- somes, making its presence the basis of most become known as noncanonical autophagy, chondrial fitness in ATG7-null Bmem point experimental autophagy measurements (2). while response to stressors, such as star- to a deficiency of canonical autophagy (8). In recent years, the nomenclature of vation, that stimulates formation of dou- Conversely, during germinal center (GC) autophagy has been further complicated by ble-membrane autophagosomes is referred formation, canonical autophagy is relative- the identification, by several different labs, to as canonical autophagy. Discriminating ly suppressed and noncanonical autophagy of a degradation process that uses some between the two forms of autophagy exper- is upregulated in B cells (10). Using chloro- quine and bafilomycin A1 to measure the Related Article: https:/doi.org/10.1172/JCI99597 proportion of autophagy that is canonical versus noncanonical, Martinez-Martin et al. Conflict of interest: The author has declared that no conflict of interest exists. demonstrated that deletion or mutation of Reference information: J Clin Invest. https://doi.org/10.1172/JCI122766. canonical autophagy protein WD repeat

jci.org 1 COMMENTARY The Journal of Clinical Investigation domain, phosphoinositide interacting 2 cally IgG2c isotype), but GC cells undergo helper cells, as observed in B cells from mice (WIPI2), had two measurable effects: incre­ increased affinity maturation, as evidenced lacking liver kinase B1 (LKB1) (15). Regard- ased noncanonical autophagy and decreased­ by a significantly higher number of muta- less, the findings by Raso et al. are a major antigen-specific GC B cell formation (10). tions in sequenced IgG heavy chains (11). leap forward in our understanding of how The question remained, is there a Consistent with this, genes involved in GC responses, integrins, innate immune relationship between noncanonical auto- somatic hypermutation, IgG2c class switch, receptors, and noncanonical autophagy phagy and GC responses? Interestingly, and plasma cell differentiation were more interact to shape the immune responses at

Martinez-Martin and colleagues gener- highly expressed in αvβ3-deficient, antigen- the heart of human health. ated GC responses using viral particles or specific GC B cells (11). Impressively, the Address correspondence to: Jeremy TLR9 ligand CpG molecules as a stimulus; associated increase in mutations produced S. Leventhal, Icahn School of Medicine at therefore, it could be hypothesized that antibodies with higher affinity and greater Mount Sinai, 1 Gustave L. Levy Place, Box WIPI2 deficiency impairs GC responses breadth of antigen diversity, and mice with 1243, New York, New York 10029, USA. due to a concurrent increase in noncanon- αvβ3-deficient B cells produced higher titers Phone: 212.241.8539; Email: Jeremy.leven- ical autophagy. If this were true, impairing of anti-HA to both PR/8 and Cal-09 antigens [email protected]. noncanonical autophagy in B cells would compared with control mice (11). The exag- 1. Choi AM, Ryter SW, Levine B. Autophagy impair GC responses. In this issue, Raso gerated GC response not only increased anti- in human health and disease. N Engl J Med. 2013;368(7):651–662. et al. definitively answer this question by body titers, but also generation of antigen- 2. Klionsky DJ, et al. Guidelines for the use and demonstrating accentuated GC responses specific long-lived memory cells. Raso et al. interpretation of assays for monitoring autophagy in mice reconstituted with Rubicon- also demonstrated enhanced memory recall (3rd edition). Autophagy. 2016;12(1):1–222. deleted B cells (11). Moreover, these data are responses that are independent of TLR adju- 3. Codogno P, Mehrpour M, Proikas-Cezanne T. supported further by observations of a pref- vants in response to secondary challenge, Canonical and non-canonical autophagy: varia- tions on a common theme of self-eating? Nat Rev erential increase of noncanonical autopha- suggesting an intrinsic difference in GC Mol Cell Biol. 2011;13(1):7–12. gy in GC B cells compared with follicular B reactions of αvβ3-null B cells that affect short- 4. Sanjuan MA, et al. Toll-like receptor signalling cells, which did not increase noncanonical and long-lived humoral responses. in macrophages links the autophagy pathway to autophagy in response to TLR9 or TLR4 phagocytosis. Nature. 2007;450(7173):1253–1257. stimulation. This finding builds on previous Conclusions 5. Takeshige K, Baba M, Tsuboi S, Noda T, Ohsumi Y. work and more clearly defines how TLR The work of Raso et al. has tremendous Autophagy in yeast demonstrated with proteinase-deficient mutants and conditions for ligands and noncanonical autophagy work implications for clinical medicine, while also its induction. J Cell Biol. 1992;119(2):301–311. to shape B cell responses. raising additional mechanistic questions. 6. Zhong Y, et al. Distinct regulation of autophagic For years, models of autoimmune disease activity by Atg14L and Rubicon associated with TLR ligands, integrins, and GC in mice have been generated by injecting a Beclin 1-phosphatidylinositol-3-kinase complex. reactions bacterial extract, CFA, along with the self- Nat Cell Biol. 2009;11(4):468–476. 7. Martinez J, et al. Noncanonical autophagy inhibits It has been established that B cell responses peptide target, to generate disease pheno- the autoinflammatory, lupus-like response to are amplified by concurrent innate immune types. Could autoimmunity in humans arise dying cells. Nature. 2016;533(7601):115–119. receptor and B cell receptor (BCR) signal- from inherited or acquired problems with 8. Chen M, et al. Essential role for autophagy in the maintenance of immunological memory against ing, which link innate and adaptive immune the αvβ3 integrin in the setting of bacterial systems (12). In 2016, work from Acharya et infection? More to the point, could we limit influenza infection. Nat Med. 2014;20(5):503–510. 9. Fribourg M, Ni J, Nina Papavasiliou F, Yue Z, al. established that TLR-induced increases unwanted B cell responses by affecting the Heeger PS, Leventhal JS. Allospecific memory B in antibody production and proliferation αvβ3 integrin or, farther downstream, nonca- cell responses are dependent on autophagy. Am J by marginal zone (MZ) and B-1 B cells are nonical autophagy? In their work, Raso et al. Transplant. 2018;18(1):102–112. 10. Martinez-Martin N, et al. A switch from canon- regulated by integrin αvβ3 (13). Integrins are demonstrate the converse potential of their membrane-bound proteins that can trans- delineated mechanism; naive mice lacking ical to noncanonical autophagy shapes B cell responses. Science. 2017;355(6325):641–647. mit signals from extracellular to intracellu- αvβ3 in B cells survived, whereas control mice 11. Raso F, et al. αv Integrins regulate germinal center lar compartments through interactions with died, after influenza infection. Inhibiting B cell responses through noncanonical autophagy. the cytoskeleton and signal transduction αvβ3 or noncanonical autophagy in patients https:/doi.org/10.1172/JCI99597. molecules (14). In MZ B cells, loss of the with sepsis may provide a life-preserving 12. Leadbetter EA, Rifkin IR, Hohlbaum AM, Beau- dette BC, Shlomchik MJ, Marshak-Rothstein A. integrin αvβ3 prolongs TLR downstream sig- boost to the adaptive immune system. These naling through aborted LC3 and autophagy- possibilities are attractive and exciting, Chromatin-IgG complexes activate B cells by dual engagement of IgM and Toll-like receptors. related 5–dependent (ATG5-dependent) but require further understanding of the Nature. 2002;416(6881):603–607. degradation­ of TLR-containing endosomes integrin–B cell phenomenon. For example, 13. Acharya M, et al. αv Integrins combine with LC3 (13). This produces a robust but unregulated how does increased NF-κΒ activity lead to and atg5 to regulate Toll-like receptor signalling B cell response that is accompanied by gen- increased B cell responses? The effect may in B cells. Nat Commun. 2016;7:10917. eration of autoantibodies (13). be intrinsic to the B cell or involve complex 14. Hynes RO. Integrins: bidirectional, allosteric signaling machines. Cell. 2002;110(6):673–687. Raso et al. determined that a sim- interactions with other immune cells. One 15. Walsh NC, et al. LKB1 inhibition of NF-κB in B ilar phenomenon also occurs in T cell– possibility is that increased NF-κΒ activa- cells prevents T follicular helper cell differentia- dependent GC responses. In this case, not tion mediates IL-6 production that in turn tion and germinal center formation. EMBO Rep. only are antibody levels higher (specifi- encourages differentiation of T follicular 2015;16(6):753–768.

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