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The present study is about [Relationship between NLRs and autophagy, which is crucial for the induction of adaptive immunity to infections, plays an important role in balancing inflammatory responses].

Review body

Since this is a Review article, authors should cover much of the articles published and be up-to-dated. Following references should be included: NLR proteins 1. A new eye on NLR proteins: focused on clarity or diffused by complexity? Current Opinion Immunology. 2012.

Inflammasomes 1. Activation and regulation of the inflammasomes. Nat Rev Immunol. 2013 Jun;13(6):397-411. doi: 10.1038/nri3452.

NLRP3 inflammasomes 1. Inflammasome-derived IL-1β production induces nitric oxide-mediated resistance to Leishmania. Nat Med. 2013 Jul;19(7):909-15. doi: 10.1038/nm.3221. Epub 2013 Jun 9

IPAF and Naip5 1. Crystal structure of NLRC4 reveals its autoinhibition mechanism. Science. 2013 Jul 12;341(6142):172-5. doi: 10.1126/science.1236381. Epub 2013 Jun 13. 2. Cytosolic flagellin-induced lysosomal pathway regulates inflammasome-dependent and -independent macrophage responses. Proc Natl Acad Sci U S A. 2013 Aug 13. [Epub ahead of print] 3. Activation of inflammasome signaling mediates pathology of acute P. aeruginosa pneumonia. J Clin Invest. 2013 Apr 1;123(4):1630-7. doi: 10.1172/JCI66142. Epub 2013 Mar 8.

NLRX1 1. Lei Y, Wen H, Ting JP. The NLR protein, NLRX1, and its partner, TUFM, reduce type I interferon, and enhance autophagy. Autophagy. 2013 Mar;9(3):432-3. doi: 10.4161/auto.23026. Epub 2013 Jan 15. 2. Lei Y, Wen H, Yu Y et al. The mitochondrial proteins NLRX1 and TUFM form a complex that regulates type I interferon and autophagy. Immunity. 2012 Jun 29;36(6):933-46. doi: 10.1016/j.immuni.2012.03.025. 3. Magalhaes JG, Sorbara MT, Girardin SE, Philpott DJ. What is new with Nods? Curr Opin Immunol. 2011 Feb;23(1):29-34. doi: 10.1016/j.coi.2010.12.003. Epub 2010 Dec 27.

NLRP4 1. Jounai N, Kobiyama K, Shiina M, Ogata K, Ishii KJ, Takeshita F. NLRP4 negatively regulates autophagic processes through an association with beclin1. J Immunol. 2011 Feb 1;186(3):1646-55. doi: 10.4049/jimmunol.1001654. Epub 2011 Jan 5. 2. Cui J, Li Y, Zhu L, Liu D, et al. NLRP4 negatively regulates type I interferon signaling by targeting the kinase TBK1 for degradation via the ubiquitin ligase DTX4. Nat Immunol. 2012 Mar 4;13(4):387-95. doi: 10.1038/ni.2239. 3. Higa N, Toma C, Koizumi Y, et al. Vibrio parahaemolyticus effector proteins suppress inflammasome activation by interfering with host autophagy signaling. PLoS Pathog. 2013 Jan;9(1):e1003142. doi: 10.1371/journal.ppat.1003142. Epub 2013 Jan 24.

Auotphagy 1. Choi AM, Ryter SW, Levine B. N Engl J Med. Autophagy in human health and disease. 2013 Feb 14;368(7):651-62. doi: 10.1056/NEJMra1205406. 2. Yang Z, Klionsky DJ.Eaten alive: a history of macroautophagy. Nat Cell Biol. 2010 Sep;12(9):814-22. doi: 10.1038/ncb0910-814.

The machinery for autophagosome biogenesis 1. Ragusa MJ, Stanley RE, Hurley JH. Architecture of the Atg17 complex as a scaffold for autophagosome biogenesis. Cell. 2012 Dec 21;151(7):1501-12. doi: 10.1016/j.cell.2012.11. 028. Epub 2012 Dec 6. 2. Cebollero E, van der Vaart A, Reggiori F. Understanding phosphatidylinositol-3-phosphate dynamics during autophagosome biogenesis. Autophagy. 2012 Dec;8(12):1868-70. doi: 10.4161/auto.22162. Epub 2012 Sep 19.

The core autophagy pathway 1. Chang TK, Shravage BV, Hayes SD, Powers CM, Simin RT, Wade Harper J, Baehrecke EH. Uba1 functions in Atg7- and Atg3-independent autophagy. Nat Cell Biol. 2013 Jul 21. doi: 10.1038/ncb2804 2. Quidville V, Alsafadi S, Goubar A, et al. Targeting the deregulated spliceosome core machinery in cancer cells triggers mTOR blockade and autophagy. Cancer Res. 2013 Apr 1;73(7):2247-58. doi: 10.1158/0008-5472 3. Besteiro S. Role of ATG3 in the parasite Toxoplasma gondii: autophagy in an early branching eukaryote. Autophagy. 2012 Mar;8(3):435-7. doi: 10.4161/auto.19289. Epub 2012 Feb 24. 4. Kraft C, Kijanska M, Kalie E, Siergiejuk E, Lee SS, Semplicio G, Stoffel I, Brezovich A, Verma M, Hansmann I, Ammerer G, Hofmann K, Tooze S, Peter M. Binding of the Atg1/ULK1 kinase to the ubiquitin-like protein Atg8 regulates autophagy. EMBO J. 2012 Sep 12;31(18):3691-703. doi: 10.1038/emboj.2012.225.

Control of autophagy 1. Vyas AR, Hahm ER, Arlotti JA, Watkins S, Beer-Stolz D, Desai D, Amin S, Singh SV. Chemoprevention of Prostate Cancer by D,L-Sulforaphane Is Augmented by Pharmacological Inhibition of Autophagy. Cancer Res. 2013 Aug 6. [Epub ahead of print]. 2. Linares JF, Duran A, Yajima T, Pasparakis M, Moscat J, Diaz-Meco MT. K63 Polyubiquitination and Activation of mTOR by the p62-TRAF6 Complex in Nutrient-Activated Cells. Mol Cell. 2013 Aug 8;51(3):283-96 3. Lamoureux F, Zoubeidi A. Dual inhibition of autophagy and the AKT pathway in prostate cancer. Autophagy. 2013 May 6;9(7). 4. Alers S, Löffler AS, Paasch F, Dieterle AM, Keppeler H, Lauber K, Campbell DG, Fehrenbacher B, Schaller M, Wesselborg S, Stork B. Atg13 and FIP200 act independently of Ulk1 and Ulk2 in autophagy induction. Autophagy. 2011 Dec;7(12):1423-33.

Autophagy and immunity to infections 1. Dupont N, Lacas-Gervais S, Bertout J, Paz I, Freche B, Van Nhieu GT, van der Goot FG, Sansonetti PJ, Lafont F. Shigella phagocytic vacuolar membrane remnants participate in the cellular response to pathogen invasion and are regulated by autophagy. Cell Host Microbe. 2009 Aug 20;6(2):137-49. doi: 10.1016/j.chom.2009.07.005. 2. Jae-Min Yuk, Tamotsu Yoshimori, and Eun-Kyeong Jo. Autophagy and bacterial infectious diseases. Exp Mol Med. 2012 February 29; 44(2): 99–108. 3. Kayath CA, Hussey S, El hajjami N, Nagra K, Philpott D, Allaoui A. Escape of intracellular Shigella from autophagy requires binding to cholesterol through the type III effector, IcsB. Microbes Infect. 2010 Nov;12(12-13):956-66. doi: 10.1016/j.micinf.2010.06.006. Epub 2010 Jul 3. 4. Methods to monitor autophagy of Salmonella enterica serovar Typhimurium. Birmingham CL, Brumell JH. Methods Enzymol. 2009;452:325-43. doi: 10.1016/S0076-6879(08)03620-3.

NLR mediated autophagy and infection 1. Eran Elinav, Till Strowig, Jorge Henao-Mejia, Richard A. Flavell. (2011) Regulation of the Antimicrobial Response by NLR Proteins. Immunity. 2. Lei Y, Wen H, Ting JP. (2013) The NLR protein, NLRX1, and its partner, TUFM, reduce type I interferon, and enhance autophagy. Autophagy.

Autophagy dependent control of NLR dependent inflammation 1. Beth Levine, Noboru Mizushima & Herbert W. Virgin (2011). Autophagy in immunity and inflammation. Nature Immunology. 469. doi:10.1038/nature09782

Autophagy and inflammasomes: A role of ROS 1. Mary A. Rodgers, James W. Bowman, Qiming Liang, and Jae U. Jung (2013). Regulation Where Autophagy Intersects the Inflammasome. Antioxydants & Redox Signaling. doi: 10.1089/ars.2013.5347 2. Salminen A, Kaarniranta K, Kauppinen A (2013). Beclin 1 interactome controls the crosstalk between apoptosis, autophagy and inflammasome activation: impact on the aging process. Ageing Res Rev. 12(2):520-34. doi: 10.1016/j.arr.2012.11.004. 3. Byrne BG, Dubuisson JF, Joshi AD, Persson JJ, Swanson MS (2013). Inflammasome components coordinate autophagy and pyroptosis as macrophage responses to infection. MBio. 4(1). doi:e00620-12. doi: 10.1128/mBio.00620-12.

[In the first round of review] General comments 1. Authors should provide full description for the Abbreviation used for the first time. 2. Professional editorial work on English by native tongue is needed.

Major comments 1. Lines 229-248: make these concept as a Table format would be easy to understand. 2. Authors indicated at the end of Introduction that ‘Several families of PRMs have been recently implicated in the control of autophagy, an adaptive cellular response to environmental and microbial-induced stress.’ It seems highly required to provide a Table for recent development on these relationship with references. 3. Lines 351-369: Figure for the processing of autophagy formation would be helpful.

Minor comments 1. Too often, References are not properly stated (missing or appeared with title) throughout the text (A few examples are lines 164, 253, 279, 300, 335, 351, 478, 590). 2. Line 503-507: make two sentences into one. 3. Line 571: rephrase the title to deliver clear meaning. 4. Authors should include ‘HMGB1’ as a DAMP. 5. Concluding remarks should be re-written based on what was described in the main text. 6. The last section about ‘ubiquitin’ does not seem to fly well in here although it is important in degradation of autophagosome.

Specific comments Line 56: ~ NLRs and autophagy, an evolutionary conserved mechanism that is crucial for homeostasis maintenance and that has been recently shown to be involved in the response to infections. -> ~ NLRs and autophagy, an evolutionarily conserved mechanism that is crucial for homeostasis and recently shown to be involved in the protective response against infections. Line 110: which are molecules that represent a signature of ~ -> which represent a signature of ~ Line 133: NF-κB -> give a full name Line 150: three BIR (baculovirus inhibitors of the apoptosis protein repeat domain. -> three BIRs ((baculovirus inhibitors of the apoptosis protein repeat domain). Line 158: immunioadjuvants ?? Line 160 : DAP -> state a full name Line 164: subsequent studies that demonstrated that -> subsequent studies demonstrated that Line 168 : virus -> viruses ? Line 170: Helicobacter pylori -> Helicobacter pylori, (add comma) Line 173: it looked inappropriate as there is a title of the reference. Line 178-179 : “Silva et al., reported that” -> omit Line 180: Nod1 is possibly involved in PG-independent microbial sensing ~ -> should explain more. Line 189: virus -> viruses? Line 191: ssRNA (single strand RNA) -> full name when first appeared. Line 194: Together the studies of Sabbah et al. and Shaw et al. expand the role of Nod2 as a key regulator of immune responses. -> omit. Line 209: contain- ing -> containing Line 213: NLRP3 (NALP3) and NLRC4 (IPAF) -> unclear!! Line 221: MSU (monosodium urate), CPPD (calcium pyrophosphate dihydrate) -> full name should come in front of abbreviation Lines 232-248: what are these? References? Texts? Clarify/correct them. Line 269: check the reference. Line 279: regarding other NLR proteins such as Nod2 and NLRP3, little is know about ~ -> regarding other NLR proteins, little is known about ~ Line 311: The term autophagy -> the term, autophagy Line 370: unclear, vague; rephrase or omit. Line 385: when target of rapamycin (TOR) in yeast and mammalian target of rapamycin (mTOR) were discovered, ~ -> when target of rapamycin (TOR) in yeast and mammal was discovered, ~ Line 396: Under, nutrient optimal conditions autophagy ~ -> Under optimal nutrient conditions, autophagy ~ Line 399: phosphorilation -> phosphorylation Line 413: contamination of the cytosolic compartment -> unclear, vague. Line 417: rephrase. Line 411: Autophagy and immunity to infections -> title is too broad, yet the content is restricted mainly to bacterial autophagy escaping. Line 454: VSV -> indicate full name Line 541: has also been been shown -> has also been shown Line 550: “knockout mice” -> indicate what knockout mice was and details on what the experiment was about. Line 551: “MAVS” -> need more explanation. Line 580: disccuss -> discuss Line 580: bellow -> below Line 603: IL-1β seems to be the cytokine whose production in more dramatically affected by autophagy. -> more than what? Unclear, rephrase. Line 612: omit. Line 618: However, it remains to be ~ -> vague, rephrase ! Lines 702-707: omit. Line 721: The NLR and autophagy fields are two of the most promising research areas in biology ~ -> is it? How so? Rephrase!

[In the second round] 1. It seems better now. But authors should be aware the fact that there are substantial numbers of typos and errors. 2. It would be helpful for readers if authors provide Table on 'escaping mechanism' (Autophagy and immunity to infections Section)

[Specific comments] line 113: HGMB1 -> HMGB1 line 145: a N -> an N line 178: is can be involved -> rephrase line 189: In a recent study, Sabbah et al. demonstrated that Nod2 mediated the in vitro production of type I IFN in cells stimulated with single stranded RNA (ssRNA) or infected with various RNA viruses. These results support the observations, made in the same study, that Nod2-deficient mice are more susceptible to respiratory syncytial virus (RSV) (Sabbah et al., 2009). -> Sabbah et al. (2009) demonstrated that Nod2 mediated the in vitro production of type I IFN in cells stimulated with single stranded RNA (ssRNA) or infected with various RNA viruses. These results support the observations, made in the same study, that Nod2-deficient mice are more susceptible to respiratory syncytial virus (RSV). line 198: were provided by Hugot et al., -> omit line 259: considering -> omit line 260: that -> omit line 261: more recent studies -> it was line 267: cathepsin B and D dependent-manner -> cathepsin B- and D-dependent manner line 275: Differently -> Different line 280: Arnoult et al. -> Arnoult et al. (2009) line 281: Moore et al. -> Moore et al. (2008) line 282: membrane (Arnoult et al., 2009; Moore et al., 2008). -> membrane. line 284: Tattoli et al. -> Tattoli et al. (2008) line 286: Moore et al. -> Moore et al. (2008) line 288: in vitro (Tattoli et al., 2008; Moore et al., 2008). -> in vitro. line 373: sumarize -> summarize line 398: (Takeuchi et al., 2005). This negative regulation of autophagy by mTOR has been recently shown to require -> (Takeuchi et al., 2005), which requires the line 446: induction autophagy by -> induction of autophagy by line 446: In one of them -> unclear !! lines 449-451: this part should combined with T3SS that mentioned before in the same Section. line 544: In contrast, in another study, the same group has demonstrated -> In contrast, another study by the same group demonstrated line 554: NLRX1 has been shown to enhance autophagy through. -> incomplete sentence, omit ! lines 563-4: rephrase ! line 576: qhere -> where ?

Overall categorization

This is a good piece of the article. Promising. As best as you can assess, this paper appears possibly significant and presents a breakthrough in research or the promise of one.