Suzanne Faure-Dupuy

David Durantel

Dr Julie Lucifora
email: julie.lucifora@inserm.fr

Kupffer cells: friend or foe during hepatitis B infection? 1

Keywords: granulocyte myeloid derived suppressor cell 30 Hepatitis B virus, Kupffer cells, innate immunity inhibition, tolerance 58 59

The Hepatitis B virus chronically infects the liver of 250 million people worldwide.

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Over the past decades, major advances have been made in the understanding of 50 Hepatitis B virus life cycle in hepatocytes. Beside these parenchymal cells, the liver 51 also contains resident and infiltrating cells involved in immune responses to 52 pathogens and much less is known about their interplay with Hepatitis B virus. In this 53 review, we summarized and discussed the current knowledge of the role of Kupffer 54 cells, the liver resident macrophages, in HBV infection.

• Hepatitis B virus can be recognized by macrophages in specific conditions (i.e. hepatocytes cell death).

• Hepatitis B virus inhibits pro-inflammatory responses in macrophages that are described to have an anti-viral effect.

• Hepatitis B virus favours anti-inflammatory responses macrophages and liver tolerance.

• Kupffer cells participate in hepatitis B virus associated pathogenesis through activation of hepatic stellate cells and through inhibition of T cell responses by anti-inflammatory secretions.

• Understanding the mechanisms by which hepatitis B virus inhibits macrophages responses could be the first step towards development of new therapies for chronically infected patients. In solid organs, MΦ can have two origins. Resident macrophages comes from the 83 yolk sac whereas infiltrating macrophages, which are recruited upon pathogen 84 contamination and/or injury, are derived from the differentiation of peripheral blood 85 circulating monocytes, themselves primarily coming from the bone marrow [START_REF] Ginhoux | Tissue-Resident Macrophage Ontogeny and Homeostasis[END_REF] .

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Depending on the tissue, it is not always possible to distinguish resident MΦ from 87 infiltrating ones. For example, in the human liver, no specific marker has been 88 identified to differentiate these two types of MΦ. Recognition Receptors (PRR) [START_REF] Hume | The Many Alternative Faces of Macrophage Activation[END_REF] . Upon activation, MΦ can secrete a large spectrum of 96 pro-inflammatory cytokines involved in different activities such as lymphocyte 97 polarization (IL-1, IL-12, IFN-γm) or neutrophil recruitment (IL-8, TNFαm), and also 98 anti-inflammatory cytokines involved in inflammation regulation (IL-10 and TGFβ), as 99 well as chemokines involved in leukocyte recruitment to the sites of injury 100 (CXCL8m) [START_REF] Martins-Green | Chemokines and Their Receptors Are Key Players in the 530 Orchestra That Regulates Wound Healing[END_REF] and others factors, such as VEGF implicated in angiogenesis [START_REF] Pollard | Macrophages define the invasive microenvironment in breast cancer[END_REF] .

101 MΦ can have a large variety of phenotypes that can be classified by specific marker 102 expression and/or secreted cytokines [START_REF] Hume | The Many Alternative Faces of Macrophage Activation[END_REF][START_REF] Murray | Macrophage activation and polarization: nomenclature and 534 experimental guidelines[END_REF] . Several classifications have been proposed 103 over the years, but a consensus is yet to be found. Nevertheless, they can be divided 104 into two major phenotypes: pro-and anti-inflammatory. Of note, the pro-/anti-105 inflammatory dichotomy is a simplified view since each MΦ expresses a set of genes 106 that on its own could define a new type of MΦ [START_REF] Hume | The Many Alternative Faces of Macrophage Activation[END_REF] . [START_REF] Crispe | The liver as a lymphoid organ[END_REF][START_REF] Seki | The liver as a crucial organ in the first line of host defense: the 556 roles of Kupffer cells, natural killer (NK) cells and NK1.1 Ag+ T cells in T helper 1 immune responses[END_REF] . KC are localized in liver blood vessels, seeded on liver sinusoidal 144 endothelial cells and their fenestration, where they can phagocytose debris, aging 145 blood cells or pathogens. After phagocytosis, pathogens can be processed into 146 phagolysosomes and antigens can be presented to lymphocytes to activate or recall 147 a local immune response [START_REF] Crispe | The liver as a lymphoid organ[END_REF][START_REF] Liaskou | Innate immune cells in liver inflammation[END_REF] . As resident MΦ, KC express all common MΦ markers 148 (i.e. CD68, CD14m) [START_REF] Ginhoux | Tissue-Resident Macrophage Ontogeny and Homeostasis[END_REF] and have more precisely been defined as CD14 + , HLA-DE + , 149 HLA-ABC + , CD86 + , DC-SIGN + with low expression of CD1b, CD40 and CD83 [START_REF] Tu | TLR-dependent cross talk between 561 human Kupffer cells and NK cells[END_REF] . As 150 previously mentioned, in humans, no specific marker has been identified to 151 differentiate KC from infiltrating macrophages, whereas in the mouse (i.e. Clec4F) [START_REF] Scott | Bone marrow-derived monocytes give rise to self-563 renewing and fully differentiated Kupffer cells[END_REF] 152 and rat (i.e. ED-1 and ED-2) [START_REF] Zeng | A new method to isolate and culture rat kupffer cells[END_REF] markers have been described. In addition, KC have a 153 long life-span (more than a year) and are self-renewing cells with a slow replicating 154 activity. Thus the liver MΦ pool can be renewed, unlike monocyte derived 155 macrophages which have a limited life-span [START_REF] Naito | Development, differentiation, and maturation of Kupffer 567 cells[END_REF] . Interestingly, a recent study by Scott 159 KC play a critical role in liver tolerance. Indeed, the liver is at the crossroad between 160 systemic and enteric circulation, with 80% of the blood reaching the liver coming from 161 the hepatic portal vein. Thus, liver cells are constantly exposed to pathogens. Indeed, 162 lipopolysaccharide (LPS) endotoxin is found in measurable amounts (few nano 163 grams) in the enteric circulation [START_REF] Prytz | Portal venous and systemic endotoxaemia in 569 patients without liver disease and systemic endotoxaemia in patients with cirrhosis[END_REF] . This phenomenon should theoretically lead to a 164 constant inflammation within the liver but none is observed in healthy individuals. The 165 liver is indeed described as an immune-tolerant organ [START_REF] Crispe | The liver as a lymphoid organ[END_REF] . This tolerance has been 166 defined as a systemic or "oral" tolerance of enteric pathogens, which depends on the 167 normal connection between the liver and gut [START_REF] Yang | Intestinal venous drainage through the liver is a 572 prerequisite for oral tolerance induction[END_REF] . Yet, the full role of KC in this 168 tolerance phenomenon still need further investigation, especially by assessing KC 169 phenotype/state in healthy liver. Indeed, the anti-or pro-inflammatory phenotype of 170 KC at steady state is yet to be properly determined. Depending on their localization in 171 the liver, MΦ may have different phenotypes. It could be hypothesised that near the 172 enteric circulation, where the exposition to bacteria is constant, KC will be more likely 173 to be M2 MΦ, the MΦ implicated in tolerance, through the secretion of immune-174 regulatory mediators [START_REF] Ju | Tolerogenic role of Kupffer cells in immune-mediated adverse drug reactions[END_REF] . In contrast, near the systemic circulation, where the presence 175 of pathogens is a sign of infection, KC would be more likely to be in a pre-pro-176 inflammatory state in order to quickly eliminate any blood infections. Moreover, other 177 factors could play a role in the fate of KC phenotype. The oxygen gradient in the liver 178 is linked to the expression of hypoxia regulated genes (HIF family), among which is 179 HIF1α, a factor linked to the accumulation of IL-1β in M1 MΦ [START_REF] Kietzmann | Metabolic zonation of the liver: The oxygen gradient revisited[END_REF] .

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As previously described, even in this tolerant environment, KC are able to respond to 181 the detection of specific pathogens [START_REF] Seki | The liver as a crucial organ in the first line of host defense: the 556 roles of Kupffer cells, natural killer (NK) cells and NK1.1 Ag+ T cells in T helper 1 immune responses[END_REF][START_REF] Tu | TLR-dependent cross talk between 561 human Kupffer cells and NK cells[END_REF][START_REF] Knolle | Human Kupffer 578 cells secrete IL-10 in response to lipopolysaccharide (LPS) challenge[END_REF] . KC express a large spectrum of PRR, among which, TLR1/2, TLR2/6, TLR3, TLR4, TLR8 and RIGI/MDA5 [START_REF] Sun | Expression of toll-like receptors in hepatic cirrhosis and 580 hepatocellular carcinoma[END_REF][START_REF] Faure-Dupuy | Characterisation of Pattern Recognition Receptors (PRR) 582 expression and functionality in liver primary cells and derived cell lines[END_REF] , leading, 183 after stimulation, to the secretion of a large spectrum of immune factors such as 184 ROS, cytokines (IL-6, IL-10, IL-12, TNFαm), and chemokines (CCL3, CCL5m) 185 among others [START_REF] Tu | TLR-dependent cross talk between 561 human Kupffer cells and NK cells[END_REF][START_REF] Knolle | Parenchymal and nonparenchymal liver cells and their 584 interaction in the local immune response[END_REF][START_REF] Tacke | Targeting hepatic macrophages to treat liver diseases[END_REF][START_REF] Sica | Macrophage plasticity and polarization in liver homeostasis and 587 pathology[END_REF] . KC have also been described to be functional for several 186 inflammasomes (AIM2, NLRP3), which, upon stimulation, induce the production, 187 maturation and secretion of IL-1β and IL-18 [START_REF] Zannetti | Characterization of the Inflammasome in Human Kupffer 589 Cells in Response to Synthetic Agonists and Pathogens[END_REF][START_REF] Yu | HBV inhibits LPS-induced NLRP3 inflammasome activation and IL-1β 592 production via suppressing the NF-κB pathway and ROS production[END_REF] . Chemokines and cytokines secretion 188 will further recruit and activate monocytes from the circulating blood that will locally 189 differentiated and further reinforce the MΦ population and inflammation [START_REF] Tacke | Targeting hepatic macrophages to treat liver diseases[END_REF] . Thus in 190 order to chronically infect the liver, pathogens (such as hepatitis viruses) must have 191 evolved strategies to escape or prevent recognition by the immune system and liver 192 inflammation.

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In summary, the liver is both an immune-tolerant [START_REF] Crispe | Cellular and molecular 594 mechanisms of liver tolerance[END_REF] and immune-competent organ [START_REF] Crispe | The liver as a lymphoid organ[END_REF] be find in peripheral blood mononuclear cells [START_REF] Loustaud-Ratti | Distribution of total DNA and cccDNA in serum and 596 PBMCs may reflect the HBV immune status in HBsAg+ and HBsAg-patients coinfected or not with HIV 597 or HCV[END_REF][START_REF] Shi | Impact of HBV replication in peripheral blood mononuclear cell on HBV 599 intrauterine transmission[END_REF][START_REF] Gao | Compartmental HBV evolution and replication in liver and 601 extrahepatic sites after nucleos/tide analogue therapy in chronic hepatitis B carriers[END_REF] , yet, HBV is not able to replicate 207 inside KC (i.e. no secretion of HBeAg and HBsAg, and no detection of HBV RNA) 208 (our unpublished data). It has been described that HBV infection can modify the 209 functions of several immune cells such as natural killer (NK) cells, T cells, and 210 granulocytic myeloid derived suppressive cells (gMDSC) [START_REF] Pallett | Metabolic regulation of hepatitis B immunopathology by 604 myeloid-derived suppressor cells[END_REF] . We will now focus on the Indeed, several studies have shown that in animal models, primary exposition to HBV 228 elicits little to no immune responses [START_REF] Wieland | Genomic analysis of the host response to hepatitis B 522 virus infection[END_REF][START_REF] Fletcher | Transcriptomic analysis of the woodchuck model of chronic 524 hepatitis B[END_REF] . Even though the authors did not specifically 229 analyse the responses in macrophages, the lack of immune responses in liver 230 biopsies may be also interpreted as an absence of response in macrophages. block the production of IL-1β, which has been recently described as the most efficient 306 cytokine, amongst several tested (including IFNs) against the replication of HBV in 307 vitro [START_REF] Isorce | Antiviral activity of various interferons and pro-633 inflammatory cytokines in non-transformed cultured hepatocytes infected with hepatitis B virus[END_REF] .

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In addition, HBV has been shown to prevent pro-inflammatory cytokine secretion by 309 influencing PRR expression in MΦ. A down-regulation of TLR2 and TLR3 was found 310 in peripheral blood mononuclear cells (PBMC) and KC of chronically HBV-infected 311 patients compared to healthy controls [START_REF] Huang | Reduced Toll-like receptor 3 expression in chronic hepatitis B 636 patients and its restoration by interferon therapy[END_REF][START_REF] Visvanathan | Regulation of Toll-like receptor-2 expression in 638 chronic hepatitis B by the precore protein[END_REF] (Figure 2, path#3). As TLR2 and TLR3 312 have been shown to, respectively, recognize HBc protein [START_REF] Cooper | Cytokine induction by the hepatitis B virus capsid in 612 macrophages is facilitated by membrane heparan sulfate and involves TLR2[END_REF] and HBV genome 313 replication in non-parenchymal cells [START_REF] Real | Hepatitis B virus genome replication triggers toll-like receptor 3-625 dependent interferon responses in the absence of hepatitis B surface antigen[END_REF] , a down-regulation of their expression in 314 hepatocytes, KC and PBMCs may contribute to the lack of an efficient immune 315 response against HBV [START_REF] Real | Hepatitis B virus genome replication triggers toll-like receptor 3-625 dependent interferon responses in the absence of hepatitis B surface antigen[END_REF] . However, another study showed a higher secretion of IFN-α 316 and -β, as well as IL-6 and IL-10 upon TLR3 stimulation in PBMC from HBV patients 317 compared to healthy controls, contradicting the results of a down-regulation of TLR3 318 in monocytes [START_REF] Jiang | Toll-like receptor-mediated immune responses are 630 attenuated in the presence of high levels of hepatitis B virus surface antigen[END_REF] . Moreover, in HBV patients, TLR2 expression on KC and peripheral 319 monocytes was reduced in HBeAg-positive chronic HBV patients (i.e. immune-320 tolerant/immune active patients) compared to controls individuals, whereas it was 321 significantly increased in HBeAg-negative chronic HBV patients (e.g., in inactive HBV 322 carrier) compared to controls [START_REF] Visvanathan | Regulation of Toll-like receptor-2 expression in 638 chronic hepatitis B by the precore protein[END_REF] . This modification of expression was specific to TLR2, 323 as TLR4 expression did not differ between groups. It appears that the effect of HBV [START_REF] Wu | Toll-like receptor-mediated control of HBV replication by 643 nonparenchymal liver cells in mice[END_REF] . 337 However, this antiviral effect was lost when KC were pre-incubated with HBV [START_REF] Wu | Hepatitis B virus suppresses toll-like receptor-mediated innate 627 immune responses in murine parenchymal and nonparenchymal liver cells[END_REF]

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(Figure 2, path#5). Among the mediators inhibiting HBV infection, IFN-α and IFN-γ 339 were identified and shown to directly inhibit HBV replication in patients [START_REF] Isorce | Immune-modulators to combat hepatitis B virus 645 infection: From IFN-α to novel investigational immunotherapeutic strategies[END_REF] . Moreover, a 340 recent study showed that HBV virions hijack enzymes involved in lipid metabolism to 341 infect hepatocytes [START_REF] Esser | Lipase inhibitor orlistat prevents hepatitis B virus infection 648 by targeting an early step in the virus life cycle[END_REF] . It could be hypothesised that the same pathways could be also 342 hijack by HBV in MΦ to enter or pass-through these cells to be delivered to there is an up-regulation of Galectin-9 on KC as well as an up-regulation of HBV 376 specific Tim3 + CD8 T cell [START_REF] Nebbia | Upregulation of the Tim-3/galectin-9 pathway of T cell 664 exhaustion in chronic hepatitis B virus infection[END_REF] (Figure 2, path#8). This Tim-3/Galectin-9 pathway up-377 regulation could be the cause of T cell exhaustion during chronic infection [START_REF] Nebbia | Upregulation of the Tim-3/galectin-9 pathway of T cell 664 exhaustion in chronic hepatitis B virus infection[END_REF] .

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Finally, Programmed Death-1 (PD-1) and its ligands (PD-L1), proteins implicated in 379 tolerance [START_REF] Wang | PD-L1 expression in human cancers and its association with clinical 666 outcomes[END_REF] , have been described to be essential to balance antiviral immunity and inflammation in acute HBV patients [START_REF] Kassel | Chronically inflamed livers up-668 regulate expression of inhibitory B7 family members[END_REF] . A study carried-out with 32 chronic HBV 381 patients, showed an up-regulation of PD-1 and PD-L1 in liver biopsies compared to 382 healthy controls [START_REF] Xie | Intrahepatic PD-1/PD-L1 up-regulation closely correlates with 670 inflammation and virus replication in patients with chronic HBV infection[END_REF] (Figure 2, path#9). The same results were found in a chronic 383 carrier mouse model [START_REF] Tian | Maternal-Derived Hepatitis B Virus e Antigen Alters 673 Macrophage Function in Offspring to Drive Viral Persistence after Vertical Transmission[END_REF] . PD-1/PD-L1 up-regulation correlated with hepatic 384 inflammation and increased alanine transaminase levels in patients [START_REF] Xie | Intrahepatic PD-1/PD-L1 up-regulation closely correlates with 670 inflammation and virus replication in patients with chronic HBV infection[END_REF] and with 385 impaired cytotoxic T lymphocytes response in mice [START_REF] Tian | Maternal-Derived Hepatitis B Virus e Antigen Alters 673 Macrophage Function in Offspring to Drive Viral Persistence after Vertical Transmission[END_REF] . However, PD-L1 expression 386 was lower in liver residential antigen presenting cells, including KC [START_REF] Xie | Intrahepatic PD-1/PD-L1 up-regulation closely correlates with 670 inflammation and virus replication in patients with chronic HBV infection[END_REF] , during the 387 inactive carrier phase. Moreover, in mice, PD-L1 blockade or KC depletion led to an 388 inhibition of HBV viral parameters, as well as an increase of IFN-γ + CD8 + cells [START_REF] Tian | Maternal-Derived Hepatitis B Virus e Antigen Alters 673 Macrophage Function in Offspring to Drive Viral Persistence after Vertical Transmission[END_REF] .

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Another study further showed that PD-L1 expression was up-regulated in CD14+ 390 circulating cells during chronic HBV infection, liver cirrhosis and hepatocellular 391 carcinoma [START_REF] Huang | Clinical Significance of Dynamics of Programmed Death Ligand-1 676 Expression on Circulating CD14+ Monocytes and CD19+ B Cells with the Progression of Hepatitis B 677 Virus Infection[END_REF] . PD-1/PD-L1 interaction may therefore play an important role in 392 balancing liver damage during chronic HBV infection [START_REF] Xie | Intrahepatic PD-1/PD-L1 up-regulation closely correlates with 670 inflammation and virus replication in patients with chronic HBV infection[END_REF] .

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In summary (Figure 2), HBV seems to block pro-inflammatory responses in KC and 394 peripheral monocytes/macrophages, correlating with a poor elimination of the virus.

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On the other hand, HBV strongly activates anti-inflammatory cytokines secretion (IL-396 10 and TGF-β) as well as inhibitory checkpoint factors (e.g., PD-1/PD-L1). Overall, it 397 seems that HBV concomitantly inhibits a pro-inflammatory/anti-viral M1-like 398 phenotype, and activates an anti-inflammatory/pro-tolerogenic M2-like phenotype. [START_REF] Tan-Garcia | Intrahepatic CD206+ macrophages contribute to 690 inflammation in advanced viral-related liver disease[END_REF] . This population has been described as 454 resistant to LPS tolerization. Another population of CD205 + macrophages has been 455 described in the liver of HBV transgenic mice and chronic hepatitis B patients [START_REF] Yong | Identification of pro-inflammatory CD205+ macrophages in livers of 692 hepatitis B virus transgenic mice and patients with chronic hepatitis B[END_REF] . This 456 population presented a higher expression of activation markers, pro-inflammatory 457 cytokines, chemokines/chemokine receptors, and phagocytosis related genes [START_REF] Yong | Identification of pro-inflammatory CD205+ macrophages in livers of 692 hepatitis B virus transgenic mice and patients with chronic hepatitis B[END_REF] .

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Moreover, in patients with high ALT levels, a large decrease of CD8+ T lymphocytes 459 and increase of Fas-L positive cells, especially KC, were shown [START_REF] Tang | The role of intrahepatic immune effector cells in 694 inflammatory liver injury and viral control during chronic hepatitis B infection[END_REF] . This observation 460 led the authors to the hypothesis that during chronic infection, liver injury could not be 461 the result of CD8+ T cell response but an increase of Fas-L expression on KC and, 462 through an increased cytolytic activity [START_REF] Tang | The role of intrahepatic immune effector cells in 694 inflammatory liver injury and viral control during chronic hepatitis B infection[END_REF] (Figure 3, path#5). Another study performed 463 in liver biopsies from 74 chronically infected patients revealed that iNOS intrahepatic 464 expression was significantly higher in patients with chronic HBV [START_REF] Wu | Increased expression of inducible nitric oxide synthase in chronic 697 hepatitis B patients is correlated with histopathological grading and staging[END_REF] . In these samples, 465 Kupffer cells stained positive for iNOS, a marker of pro-inflammatory MΦ, which level 466 correlated with ALT level, and with the grading of liver inflammation and staging of 467 liver fibrosis [START_REF] Wu | Increased expression of inducible nitric oxide synthase in chronic 697 hepatitis B patients is correlated with histopathological grading and staging[END_REF] (Figure 3, path#6). Moreover, as previously described, peroxisome 468 proliferator-activated receptor-β/δ (PPARβ/δ) activation was shown to inhibit 469 steatosis and inflammation, well-known cancer risks factors [START_REF] Nagasawa | Effects of bezafibrate, PPAR pan-agonist, and GW501516, 701 PPARdelta agonist, on development of steatohepatitis in mice fed a methionine-and choline-702 deficient diet[END_REF] . In a model of HBV 470 transgenic mice, activation of PPARβ/δ reduced steatosis and tumour multiplicity [START_REF] Balandaram | Ligand activation of peroxisome proliferator-activated 704 receptor-β/δ suppresses liver tumorigenesis in hepatitis B transgenic mice[END_REF] .

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PPARβ/δ activation was also correlated with reduced Tnfα mRNA levels, as well as, 472 reduced ALT level but increased apoptotic signalling [START_REF] Balandaram | Ligand activation of peroxisome proliferator-activated 704 receptor-β/δ suppresses liver tumorigenesis in hepatitis B transgenic mice[END_REF] related to the increased of IL-10 secretion described by several teams [START_REF] Li | Kupffer Cells Support Hepatitis B Virus-Mediated CD8+ T Cell Exhaustion 654 via Hepatitis B Core Antigen-TLR2 Interactions in Mice[END_REF][START_REF] Xu | Kupffer cell-derived IL-10 plays a key role in maintaining 657 humoral immune tolerance in hepatitis B virus-persistent mice[END_REF][START_REF] Dunn | Temporal analysis of early immune responses in patients with 660 acute hepatitis B virus infection[END_REF] . Indeed, a 478 tolerogenic environment in the liver could lead to a non-efficient lymphocyte response 479 and consequently, to cancer progression [START_REF] Trehanpati | Immune Regulation by T Regulatory Cells in Hepatitis B Virus-Related 707 Inflammation and Cancer[END_REF] (Figure 3, path#8). However, a study by HBV-infected animal model [START_REF] Wieland | Genomic analysis of the host response to hepatitis B 522 virus infection[END_REF][START_REF] Fletcher | Transcriptomic analysis of the woodchuck model of chronic 524 hepatitis B[END_REF] , despite the fact that liver macrophages are capable of 496 recognising HBV [START_REF] Hösel | Not interferon, but interleukin-6 controls early gene 608 expression in hepatitis B virus infection[END_REF][START_REF] Cheng | Hepatitis B virus evades innate immunity of hepatocytes but 610 activates cytokine production by macrophages[END_REF][START_REF] Cooper | Cytokine induction by the hepatitis B virus capsid in 612 macrophages is facilitated by membrane heparan sulfate and involves TLR2[END_REF][START_REF] Boltjes | Kupffer cells interact with hepatitis B surface antigen 617 in vivo and in vitro, leading to proinflammatory cytokine production and natural killer cell function[END_REF][START_REF] Hou | CD205-TLR9-IL-12 axis contributes to CpG-induced oversensitive 620 liver injury in HBsAg transgenic mice by promoting the interaction of NKT cells with Kupffer cells[END_REF][START_REF] Real | Hepatitis B virus genome replication triggers toll-like receptor 3-625 dependent interferon responses in the absence of hepatitis B surface antigen[END_REF] , raises the possibility of an active HBV-driven immune

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  Tissue resident MΦ represent the first line of defence against pathogens such as 90 bacteria and viruses, and are therefore an important component of the innate 91 immune response to infections. In addition to their physiologic phagocytic capacity, 92 which allows the elimination of physiologically aging, or infected, or cancerous cells, 93 MΦ can importantly also contribute to adaptive immunity by processing and 94 presenting antigens after pathogen engulfment and their recognition through Pattern95

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  Pro-inflammatory MΦ, called M1, have been defined as mediators of the defence 108 against bacterial and viral pathogens. They are implicated in inflammation, tumour 109 resistance and killing of intracellular pathogens 11 . M1 are commonly described as 110 STAT1 + , IRF5 + , and iNOS + , and can secrete a large panel of pro-inflammatory 111 cytokines (IL-6, IL-1 family, IL-12, IL-23, TNFαm), chemokines (CXCL9, CXCL10, 112 CXCL11, CCL3, CCL5m), and inflammatory mediators (i.e. reactive oxygen species 113 or ROS) 11 . M1 MΦ have a "broken Krebs cycle" leading to the accumulation of citrate 114 and subsequently NO production, as well as the accumulation of succinate, HIF1α 115 and IL-1β secretion 12 . M1 MΦ are reported to be able to activate tumour-killing 116 mechanisms and to amplify Th1 responses, inducing a positive feedback in the anti-117 tumour response 13 . Indeed, M1 macrophages can present antigens to induce an 118 adaptive immune response but can also reactivate lymphocytes locally, at the 119 injury/infection site. Although pro-inflammatory MΦ are mainly implicated in the 120 elimination of noxious signals or microorganism invaders, M1-like macrophage-121 derived cytokine production have also been identified as key factors in several 122 autoimmune and chronic inflammatory diseases, including Crohn's disease, multiple 123 sclerosis, and autoimmune hepatitis 14,15 . 124 Anti-inflammatory MΦ, commonly called M2, are mainly implicated in the resolution of 125 inflammation and wound healing, but also, in some circumstances and by 126 mechanisms not fully understood, in the defence against parasites 16 . M2 are 127 commonly described as STAT6 + , IRF4 + , and CD163 + , and can secrete anti-128 inflammatory cytokines (IL-10, TGFβ), chemokines (CCL1, CCL17, CCL24m) and 129 growth/angiogenic factors (PDGF, VEGF, EGF). In contrast to M1, M2 MΦ have a 130 "functional Krebs cycle" allowing production of ATP and glycosylation of M2-131 associated receptors 12 . M2 macrophages are mandatory for the resolution of 132 tissue repair after an injury and/or cytotoxic activity 11 . However, M2 133 are also associated with tumour progression through the secretion of negative 134 immuno-modulators, and are called, in the tumour micro-environment, TAM for 135 "tumour associated macrophages" 17 . TAM have been associated to the progression 136 of numerous cancers and as such are potential new target for therapeutic purposes 137 17-19 . 138 139 Kupffer cells: the liver macrophages 140 Kupffer cells (KC), the liver resident MΦ, was first described by Fahimi in 1970 as 141 peroxidase positive cells 20 . KC are the largest MΦ population in the human body as 142 they represent 80% of the total human MΦ count under normal physiological 143 conditions

  156and colleagues described that in mice monocyte derived MΦ can, with time, acquire 157 KC phenotype (including self-renewal capacity) after depletion of the resident MΦ in 158 a model of KC expressing the diphtheria toxin receptor[START_REF] Scott | Bone marrow-derived monocytes give rise to self-563 renewing and fully differentiated Kupffer cells[END_REF] .

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  , 194 in which Kupffer cells play a central role in preventing the circulation in the blood of 195 pathogens and aberrant cells.

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  The study of HBV infection often focuses on the hepatocytes in which the virus 199 replicates. Viral antigens are defined as secreted viral components which can be 200 protein (hepatitis B e antigen; HBeAg), subviral particles (hepatitis B surface antigen 201 embed in lipid membrane; HBsAg), and virions containing an icosahedral capsid 202 enveloped by surface protein (empty virions, containing no viral genome or Dane 203 particles, the infectious particles). However, the effect of the production of infectious 204 particles and subviral particles in the liver and in the systemic circulation is still largely 205 unknown. To note, several studies have shown that HBV replicative intermediate can 206

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  interaction of HBV and macrophages from four different angles: i) Can macrophages 212 recognize HBV? ii) Can HBV modify the phenotype and function of MΦ? iii) Do MΦ 213 play a role in the establishment or persistence of the infection? iv) Do MΦ play a role 214 in the development of pathologies associated with HBV infection? 215 216 Can liver macrophages recognize hepatitis B virus? 217 Liver MΦ are equipped to recognize pathogens and mount an efficient pro-218 inflammatory response if needed to prevent, control and delay infections that could 219 become chronic, as described above. A study in Pekin duck revealed that minutes 220 after inoculation HBV was taken up by KC, highlighting their interaction in vivo 45 . As a 221 DNA virus with RNA intermediates, HBV could theoretically be recognized by several 222 PRR expressed by KC such as TLR3 or RIGI/MDA5 as well as some cytosolic DNA 223 sensors 34 . However, the recognition of HBV nucleic acids may be impaired by (i) the 224 physical separation of encapsidated rcDNA (relaxed circular DNA, a partially double 225 stranded DNA) from cytosolic DNA sensors, and (ii) the resemblance between HBV 226 RNAs and host mRNAs as viral RNAs are transcribed by the host polymerase.

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  In the KC, the situation may be different as although they do not support viral 232 replication (i.e. no neo formation of HBV RNAs), viral components could be present 233 after phagocytosis and viral epitopes could be recognised by immune receptors. 234 Indeed, after purification of different subsets of human liver cells, Hösel et al. showed 235 that non-parenchymal cells (i.e., all liver cells except hepatocytes) and especially KC 236 were activated after a 3h of exposure to HBV. They observed a transient activation of 237 nuclear factor kappa B (NF-κB) and the secretion of pro-inflammatory cytokines (IL-6,238IL-8, TNF-α, and IL-1β) without any induction of an interferon response[START_REF] Hösel | Not interferon, but interleukin-6 controls early gene 608 expression in hepatitis B virus infection[END_REF] (Figure1, 239 path#1). This results were recently confirmed by Cheng and colleagues who showed 240 that in monocyte derived macrophages exposed to high quantities of HBV, a 241 transient (between 0.5 to 6h) increase of IL-6, IL-1β, and TNF-α mRNAs was 242 observed[START_REF] Cheng | Hepatitis B virus evades innate immunity of hepatocytes but 610 activates cytokine production by macrophages[END_REF] (Figure1, path#1).243Unexpectedly, TLR2, a PRR implicated in the recognition of bacterial components, 244 has been described to recognize HBV capsid/core protein (HBc)[START_REF] Cooper | Cytokine induction by the hepatitis B virus capsid in 612 macrophages is facilitated by membrane heparan sulfate and involves TLR2[END_REF] . Cooper and 245 colleagues found that, in a monocyte cell line, THP1, a full-length HBc was bound to 246 monocyte receptors, leading to their differentiation into MΦ. Moreover, this binding 247 strongly induced the production of the pro-inflammatory cytokines TNF-α, IL-6, and 248 IL-12p40[START_REF] Cooper | Cytokine induction by the hepatitis B virus capsid in 612 macrophages is facilitated by membrane heparan sulfate and involves TLR2[END_REF] (Figure1, path#2). These inductions were dependent of NF-κB, ERK-1/2, 249 and p38 MAPK activation. The authors demonstrated the necessity of binding 250 between the arginine-rich C terminus of HBc and membrane heparin sulphate for a 251 recognition by monocytes[START_REF] Cooper | Cytokine induction by the hepatitis B virus capsid in 612 macrophages is facilitated by membrane heparan sulfate and involves TLR2[END_REF] . However, the relevance of this model is to be questioned 252 as capsid proteins are normally enveloped in the virion when circulating outside of 253 the hepatocytes, which should prevent recognition by TLR2 ligands, and MΦ 254 activation. Nevertheless, in the case of cell death, it may be possible that naked 255 capsid could be released out of the hepatocytes. For example, a study by Arzberger 256 al, showed that apoptosis of HBV infected hepatocytes led to the release of naked 257 nucleocapsid[START_REF] Arzberger | Apoptosis of hepatitis B virus-infected hepatocytes prevents 615 release of infectious virus[END_REF] which could theoretically induce TLR2-mediated MΦ activation 258 (Figure1, path#2'). In addition, a study performed with samples from HBV-positive 259 patients showed that KC stained positive for HBsAg and were more activated than 260 those from control livers[START_REF] Boltjes | Kupffer cells interact with hepatitis B surface antigen 617 in vivo and in vitro, leading to proinflammatory cytokine production and natural killer cell function[END_REF] . Internalization of HBsAg was also observed in vitro using 261 purified KC or monocyte-derived MΦ and led to a strong induction of pro-262 inflammatory cytokines such as IL-6, TNF-α, and IL-15 as well as the anti-263 inflammatory cytokine IL-10, the chemokines CCL4 and CXCL8[START_REF] Boltjes | Kupffer cells interact with hepatitis B surface antigen 617 in vivo and in vitro, leading to proinflammatory cytokine production and natural killer cell function[END_REF] (Figure1, path#3) 264 and the subsequent activation of IFN-γ production by NK[START_REF] Boltjes | Kupffer cells interact with hepatitis B surface antigen 617 in vivo and in vitro, leading to proinflammatory cytokine production and natural killer cell function[END_REF] (Figure1, path#4).265Moreover, a study in HBsAg-transgenic mice showed that KC secretes IL-12 upon 266 TLR9 ligand inducing liver injury caused by NK[START_REF] Hou | CD205-TLR9-IL-12 axis contributes to CpG-induced oversensitive 620 liver injury in HBsAg transgenic mice by promoting the interaction of NKT cells with Kupffer cells[END_REF] . In this model, an increase of Fas 267 ligand (FasL) and Fas proteins were observed respectively on NK and PHH, leading 268 to an induction of a cytotoxic response against PHH[START_REF] Hou | CD205-TLR9-IL-12 axis contributes to CpG-induced oversensitive 620 liver injury in HBsAg transgenic mice by promoting the interaction of NKT cells with Kupffer cells[END_REF] (Figure1, path#2'). 269 Some studies have shown that HBV could also induce an interferon-dependent 270 immune response, which should have a direct antiviral effect on HBV infection in 271 hepatocytes, indeed Peg-IFN is currently used to treat HBV infection 52 . Using a 272 transgenic mouse model allowing high levels of HBV replication, Real et al. recently 273 showed that HBV replication lead to a TLR3 dependent interferon response in non-274 parenchymal liver cells 53 (Figure 1, path#5). A significant up-regulation of IFN-β1 and 275 subsequently ISG15 and LFIT1 mRNAs was observed in mouse model in which the 276 small HBsAg is not produced (Figure 1, path#5'). Of note, HBsAg has previously 277 been described to attenuate immune responses facilitating chronic infection (see 278section on HBV modification of KC phenotype)[START_REF] Zannetti | Characterization of the Inflammasome in Human Kupffer 589 Cells in Response to Synthetic Agonists and Pathogens[END_REF][START_REF] Wu | Hepatitis B virus suppresses toll-like receptor-mediated innate 627 immune responses in murine parenchymal and nonparenchymal liver cells[END_REF][START_REF] Jiang | Toll-like receptor-mediated immune responses are 630 attenuated in the presence of high levels of hepatitis B virus surface antigen[END_REF] .279In summary (Figure1), contradictory results regarding the potential activation or lack 280 of activation of an immune response in MΦ by HBV have been reported. These 281 from the differences in model (chimpanzees, woodchuck, 282 transgenic micem) and virus (genotypes, purification, naked nucleocapsidm) used in 283 different experimental approaches. The lack of responses in HBV-infected animal 284 models[START_REF] Wieland | Genomic analysis of the host response to hepatitis B 522 virus infection[END_REF][START_REF] Fletcher | Transcriptomic analysis of the woodchuck model of chronic 524 hepatitis B[END_REF] , compared to results demonstrating that macrophages seems to be 285 equipped to recognize HBV[START_REF] Hösel | Not interferon, but interleukin-6 controls early gene 608 expression in hepatitis B virus infection[END_REF][START_REF] Cheng | Hepatitis B virus evades innate immunity of hepatocytes but 610 activates cytokine production by macrophages[END_REF][START_REF] Cooper | Cytokine induction by the hepatitis B virus capsid in 612 macrophages is facilitated by membrane heparan sulfate and involves TLR2[END_REF][START_REF] Boltjes | Kupffer cells interact with hepatitis B surface antigen 617 in vivo and in vitro, leading to proinflammatory cytokine production and natural killer cell function[END_REF][START_REF] Hou | CD205-TLR9-IL-12 axis contributes to CpG-induced oversensitive 620 liver injury in HBsAg transgenic mice by promoting the interaction of NKT cells with Kupffer cells[END_REF][START_REF] Real | Hepatitis B virus genome replication triggers toll-like receptor 3-625 dependent interferon responses in the absence of hepatitis B surface antigen[END_REF] , raises the possibility of an active HBV-driven 286 evasion mechanism which could contribute to the establishment and persistence of 287 infections in vivo. 288 289 Can HBV modify macrophage phenotype and function? 290 Several studies have described inhibitory mechanisms used by HBV to block the 291 induction of a potential pro-inflammatory/anti-viral response, including inflammasome 292 inhibition, immune receptor down-regulation, pro-inflammatory secretion inhibition, 293 increase of anti-inflammatory secretion, and negative immune checkpoint induction. 294 First, HBV has been shown to interfere with inflammasome activation. Zannetti et al. 295 determined that KC production of IL-1β by AIM2 inflammasome engagement was 296 blocked by HBV, through an HBsAg-dependent mecanism 38 (Figure 2, path#1). This 297 inhibition seemed to be specific to the AIM2 inflammasome, as HBV was not capable 298 of blocking IL-1β production driven by other inflammasomes, including NLRP3 38 . 299 However, Yu et al. recently showed that HBV could also inhibit NLRP3 induced IL-1β 300 secretion by KC and that this inhibition was HBeAg dependent 39 (Figure 2, path#2). 301 This difference in results may come from differences in the HBV inocula used and 302 especially the quantity of HBeAg in the viral preparations. Although mechanisms 303 involved may differ, both studies described an inhibition of IL-1β secretion by KC 304 after exposure to HBV components. HBV has likely evolved several strategies to 305

  324on TLR expression may be differentially regulated during the different phases of HBV 325 chronic infection that would partially explain the difference of immune responses 326 observed during those phases.327HBV may also directly target pro-inflammatory cytokines secretion. For example, in 328 CHB patients, HBsAg induced an inhibition of IL-12 secretion by 329 monocytes/macrophages after TLR2 stimulation through the interference of HBV with 330 MAPK pathway 59 (Figure 2, path#4). To further decipher what happens in 331 the liver, the team of Pr Schlaak purified murine KC and exposed them to some TLR-332 ligands in the presence or absence of HBsAg, HBeAg or HBV virions. Upon 333 exposition to any of these viral components, expression of interferon sensitive genes 334 and pro-inflammatory cytokines was inhibited in KC 55 (Figure 2, path#5). In addition, 335 Wu et al. have shown that stimulating KC with TLR3 or TLR4 ligands induced the 336 production of IFNβ and the inhibition of viral replication in hepatocytes in mice

344

  Aiming at the same goal, others studies have shown that in addition to preventing 345 pro-inflammatory responses, HBV also increases anti-inflammatory responses. In 346 purified rat KC exposed to different quantities of HBV virions, no morphological 347 changes were observed on KC, but, compared to non-infected animals, TGF-β 348 secretion was increased from 5.38 at day-3 to 7.75 fold at day-7 post-exposure[START_REF] Li | Hepatitis B virus particles preferably induce Kupffer cells to 651 produce TGF-β1 over pro-inflammatory cytokines[END_REF] 349(Figure2, path#6). In this study, no modification in the levels of pro-inflammatory 350 cytokines IL-1, IL-6 and TNF-α was observed compared to non-HBV-exposed cells. 351 However, the authors neither indicated the secretion level of those cytokines in both 352 controls and HBV exposed KC nor did they examine if the pro-inflammatory cytokines 353 could be secreted by KC after TLR stimulation and modified by incubation with 354 HBV 63 . In addition, Jiang et al. demonstrated that in non-parenchymal liver cells 355 to HBV, the expression of anti-inflammatory cytokines (IL-10 and TGFβ) 356 was enhanced 55 (Figure 2, path#6). This phenotype was associated with an inhibition 357 of NF-κB, IRF-3 and MAPKs pathways in these cells. Specific T-cell activation by KC 358 was also impaired when cells were exposed to HBsAg, but this phenotype could be 359 reverted by a treatment with IL-10 antibodies, suggesting that the effect of HBV on 360 KC impairment of T cell response was mediated by this cytokine 55 (Figure 2, 361 path#6'). Furthermore, in an established HBV carrier mouse model, Li et al. showed 362 that TLR2 deficiency improved HBV elimination, thus indicating that TLR2 could 363 modulate HBV persistence 64 . Liver MΦ were shown to have an increased TLR2 364 expression in HBV-carrier mice and to produce more IL-10 upon TLR2 activation by 365 an HBcAg stimulation leading to a poor induction of CD8+ T cells 64,65 (Figure 2, 366 path#7). Indeed, IL-10 deficiency or anti-IL-10R treatment resulted in a functional 367 CD8+ T cell response and HBV elimination in this mice model 64,65 . To get further 368 insights into the context of a primary infection, Dunn et al. analysed a cohort of 21 369 patients with acute HBV, including 8 patients analysed before the peak of viremia, 370 when the infection is still being established 66 . At the peak of viremia, during the 371 inhibition of lymphocyte activation, a peak of IL-10 was observed in the serum. 372 Moreover, in chronically infected HBV patients an increase of CD68 + CD86 + MΦ was 373 observed, promoting a specific Th2 response, thus promoting of phagocytic-374 independent inflammation 67 . Lastly, Nebbia et al. described that in CHB patients 375

  380

  the establishment or persistence of the infection? 401 As the first line of defence against pathogens, KC should counteract infection of 402 hepatocytes by HBV. A study performed in ducks infected with DHBV (duck hepatitis 403 B virus) showed that endotoxin stimulation could inhibit DHBV replication in primary 404 hepatocytes (i.e., accumulation of viral proteins and amplification of the nuclear 405 extrachromosomal DHBV DNA templates) 74 . The authors further observed that this 406 inhibition was due to non-parenchymal cells (NPC)-mediated cytokine secretion, and 407 more probably to KC, which contaminates isolated hepatocytes. Nevertheless, HBV 408 is able to chronically infect the liver, and counteract/escape these immune 409 responses. The role of KC in the establishment and persistence of the infection 410 remains unclear. 411 Interestingly, in a mouse model the depletion of KC prior to HBV infection prevented 412 the establishment of a chronic infection 65 (Figure 3, path#1). This effect was 413 correlated with the secretion of IL-10 by KC, which impaired the humoral response 414 against HBV infected hepatocytes 65 . Moreover, Chen et al. have shown that the 415 matrix metalloproteinase 9 (MMP-9) is up-regulated in PBMCs of chronic hepatitis B 416 patients and can be activated by HBV in PBMCs and macrophages in vitro 75 . This 417 up-regulation may favour the infection as the authors further showed that MMP-9 418 enhanced HBV replication in hepatocytes through binding to IFNAR1 (IFNα receptor 419 1), inducing its ubiquitinylation and degradation 75 (Figure 3, path#2). IFNAR1 420 degradation prevents the binding of type I IFN and the associated antiviral effects. 421 In contrast, some studies have shown that KC may play a role in HBV elimination. 422 Hösel et al. showed that KC exposed to HBV secreted more IL-6, inducing the 423 activation of the mitogen-activated protein kinases (MAPK) in PHH, leading to the 424 inhibition of hepatocytes nuclear factor (HNF) 1α and 4α, two transcriptional factors 425 essential for HBV replication 46 (Figure 3, path#3). Another study conducted with 426 HepG2 cells, showed that a treatment with TGF-β1 (cytokine secreted by KC 427 exposed to HBV 63 ) could supress HBV replication 76 . TGF-β1 specifically reduced 428 activity, and, subsequently, viral pre-genomic RNA, core protein, 429 and HBV replication, through repression of HNF 4α expression 76 (Figure 3, path#4). 430 In summary (Figure 3), it is still unclear if KC plays a role in the establishment and 431 persistence of the infection and further studies are required. The discrepancies 432 between results may be due to the phenotype (pro-or anti-inflammatory) of KC 433 during primary infection and could be one of the key factors involved in the infection 434 outcome. 435 436 Do macrophages play a role in the development of pathologies associated with 437 HBV infection? 438 KC have been described to play a role in several liver associated pathologies 439 including aberrant responses 22,30 and may be associated with of HBV-associated liver 440 pathologies including fibrosis, cirrhosis and the progression to hepatocellular 441 carcinoma 77 . 442 As described above, Li et al. have shown an increase of TGF-β1 secretion by KC in 443 the presence of HBV 63 . TGF-β1 can directly promote fibrosis by inducing the 444 differentiation of hepatic stellate cells into myo-fibroblasts, subsequently stimulating 445 the production of extracellular matrix components 78 (Figure 3, path#4'). 446 In addition to liver fibrosis, KC plays an important role in liver inflammation/tolerance 447 and injury associated to uncontrolled inflammation. Different populations of 448 macrophages have recently been described in the liver of chronically infected animal 449 models and patients. A study showed that end-stage HBV liver disease is associated 450 with an accumulation of CD14 + HLA-DR hi CD206 + macrophages 79 . These cells 451 expressed spontaneously pro-inflammatory markers (TNF-α, GM-CSF, Caspase 1m) low expression of anti-inflammatory markers (arginine 2, PPARGm) with 453 the exception of IL-10 which was increased

  (Figure 3, path#7). Ligand 473 activation of PPARβ/δ inhibited LPS signalling and though Tnfα expression, which 474 could be prevented in a model of KC non-expressing-PPARβ/δ 84 . This data 475 suggested a role of KC in liver inflammation during chronic HBV infection through 476 TNF-α secretion. Finally, tumour progression in chronic HBV infection could be 477

480

  Sitia et al. showed that in a chronic HBV carrier mouse model, KC, through 481 scavenging receptors, removed apoptotic PHH, blocking the release of high-mobility 482 group box 1 (HMGB1) and neutrophil infiltration in the liver[START_REF] Sitia | Kupffer cells hasten resolution of liver immunopathology in 709 mouse models of viral hepatitis[END_REF] . The authors 483 hypothesized that this may prevent liver inflammation and damage during chronic 484 infection. 485 Taken together these data show that HBV may modify KC phenotype in favour of the 486 development of liver-associated pathologies such as fibrosis, particularly through 487 TGF-β1 secretion, and, ultimately, HCC, through aberrant expression of factors 488 implicated in carcinogenesis. Moreover, KC could play an important role in liver 489 inflammation during chronic HBV and prevent a functional humoral response by 490 largely increasing IL-10 secretion by macrophages. been published with respect to the lack or activation of 494 immune responses in MΦ during HBV infection. The absence of responses seen in 495

497

  evasion mechanism, which could contribute to the establishment and persistence of 498 infections in vivo. Several studies have concluded that HBV does modulate MΦ 499 phenotypes by: i) inhibiting pro-inflammatory responses in KC and peripheral 500 monocytes/macrophages, ii) promoting anti-inflammatory cytokines secretion (IL-10 501 β), and/or iii) increasing inhibitory checkpoint factors expression (e.g., PD-502 1/PD-L1). Taken together, HBV would concomitantly inhibit the pro-inflammatory/anti-503 viral M1-like phenotype, and promote an anti-inflammatory/pro-tolerogenic M2-like 504 differentiation. This phenotype switch may initially favour viral replication but in the 505 long term may also promote the pathogenesis of liver disease, such as fibrosis, 506 cirrhosis and, ultimately, HCC as collateral damage. 507 A specific modulation of the function of macrophages to restore more favourable T-508 cell-mediated immune responses against HBV should be tested within the 509 development of complex immune therapeutic strategies to defeat HBV 87 . Therefore a 510 substantial effort should be made to better understand the complex interplays 511 between liver macrophages and HBV infections in order to determine whether a 512 therapeutic intervention aiming at reversing the phenotype switch induced by HBV 513 could favour the inhibition of HBV infection (i.e. by direct mechanisms for example 514 through IL-1β secretion 56 , or by indirect mechanisms through the activation of a

Figure 1 :

 1 Figure 1: Detection of HBV by macrophages. Detailed mechanisms are given in

  717

Figure 2 :Figure 3 :Figure 1 :Figure 2 :

 2312 Figure 2: Modification of macrophages phenotype by HBV. Detailed mechanisms


Richard P Binzel

Brent W Barbee

Olivier S Barnouin

Jhu Apl

ASU James F Bell

Mirel Birlan

Paris Obs Aaron Boley

William Bottke

Marina Swri

JPL Joshua T Brozović

Jhu Cahill

Humberto Apl

Campins

Andy Cheng

JPL Paul W Chodas

Terik Daly

William C Danchi

Nasa Gsfc

Joseph V Demartini

Univ Maryland

MIT Francesca Demeo

Daniel Durda

Anthony Swri

JPL Masatoshi Freeman

Hirabayashi

Yaeji Kim

NASA GSFC Lucy F Lim

MIT Michael Marsset

Patrick Michel

Obs Cote D'azure

Nicholas Moskovitz

Lowell Observatory

Univ Michael C Nolan

Joseph A Arizona

NASA GSFC Ryan S Nuth

JPL Alex H Park

SWRI Carol A Parker

JPL Kim R Raymond

JPL Derek Reh

Univ Richardson

Paul Maryland

Sava

Daniel J Scheeres

Jean Souchay

Patrick A Taylor

Anne Virkki

JPL Donald K Yeomans

Decadal Opportunity for the Science of Planetary Defense

1

Apophis 2029: Decadal Opportunity for the Science of Planetary Defense Executive Summary:

Many advancements in planetary science are achieved by seizing upon the opportunity created by rare natural events (e.g. comet Shoemaker-Levy 9 Jupiter impact; 1994) and by planned physical interactions (e.g. Deep Impact into comet Tempel 1; 2005). A forthcoming opportunity to seize upon a natural physical "experiment" occurs on April 13, 2029 with the unprecedentedly close Earth encounter by the large 340m asteroid (99942) Apophis. On that date, nature is performing the "experiment" of subjecting the physical body of Apophis to Earth's tidal torques as it approaches to within 31,000 km of Earth's surface, a distance that is closer than orbiting geosynchronous satellites.

A potentially hazardous asteroid (PHA) as large as Apophis encountering Earth this closely (within 0.1 lunar distances) is, on average, a once-per-thousand year event. (In context, Apophis is 7 times larger and 350 times more massive than the Tunguska 1908 impactor; 5000 times more massive than Chelyabinsk 2013.) Because of this event's incredible rarity, knowledge gained through measurements and outcomes of the Apophis 2029 "natural experiment" are clearly a decadal, if not millennial, opportunity for planetary science. Most specifically, this knowledge opportunity is for the science supporting planetary defense. Further, on April 13, 2029 all of Earth will be watching: Apophis will be visible to the naked eye speeding across the evening sky for an estimated 2 billion people spanning western Europe and northern Africa.

In this White Paper we outline our current best understanding, and uncertainties, for scientific advances in the physical study of potentially hazardous asteroids that may be achievable by measuring physical changes of Apophis' spin, surface structure, and/or shape configuration in response to Earth's tidal torques. If tidal torques themselves, or surface configuration changes induce any measurable seismic vibration signal inside Apophis, a new field of asteroid seismology has the potential to be born. Over six decades of planetary science, seismology has been achieved beyond Earth for only two planetary worlds: Moon and Mars.

With this White Paper, we take no position on how to implement specific investigations capable of achieving the science advances offered by the Apophis 2029 opportunity. Instead we advocate that competitive selection of investigations, be they theoretical or observational (ground-based, space-based, or in situ), be executed under the direction of NASA's Planetary Defense Coordination Office, with a projected and perhaps necessarily augmented budget capable of supporting them. International collaboration is strongly encouraged.

Thus, summarizing in four specific points, we urge the framers of the Decadal Survey to:

• Recognize the decadal, if not millennial, opportunity for the science of planetary defense presented by the Apophis 2029 once-per-thousand-year "natural experiment."

• Prioritize as a top-level planetary defense science goal modeling and measuring the physical outcome on Apophis exerted by Earth's tidal torques so as to achieve the greatest possible new insights into the physical nature, including the internal structure, of PHAs.

• Recognize that time is of the essence for defining and implementing investigations of physical effects on Apophis, particularly if in situ measurements are to be considered. An Apophis 2029 Science Definition Team may be prescribed.

• Recognize that the achievable knowledge of PHAs presented by the Apophis 2029 opportunity could have immeasurable benefits to the future of humanity, in the highly unlikely, but not impossible necessity to mitigate a future impact threat.

Apophis 2029: Decadal Opportunity for the Science of Planetary Defense 1 Asteroid Close Encounters: Separating Fact from 'Media Sensation'

Weekly, if not daily, news stories declare warnings of impending asteroid "close encounters" with Earth. While tabloids are easily dismissed, mainstream media outlets are not immune, even for objects passing more than 10 lunar distances away (Figure 1). The cacophony of asteroid sensationalism by the media dulls the context for truly spectacular close encounter opportunities.

Figure 2 displays a timespan of two centuries to give context for the frequency of known asteroid encounters within less than one-half lunar distance. Objects a few meters in size pass inside the lunar distance weekly, and the modern uptick in their discovery efficiency is apparent in the data. However, over this two-century window, one event spectacularly stands out.

2

Apophis April 13, 2029: A Decadal If Not Millennial Close Encounter Opportunity As shown in Figure 2, the 340m asteroid Apophis is the largest known object encountering Earth within 0.1 lunar distance over the span of two centuries [START_REF] Brozović | Goldstone and Arecibo radar observations of (99942) Apophis in 2012-2013[END_REF]. Apophis is 7 times larger and 350 times more massive than estimates for the Tunguska 1908 impacting body [START_REF] Robertson | Hydrocode simulations of asteroid airbursts and constraints for Tunguska[END_REF] and 5000 times more massive than Chelyabinsk [START_REF] Popova | Chelyabinsk airburst, damage assessment, meteorite recovery, and characterization[END_REF]).

On April 13, 2029 (a Friday), Apophis approaches within 31,000 km of Earth's surface, a miss distance that is closer than orbiting geosynchronous satellites. Time averaged, an object as large as Apophis approaches Earth within 0.1 lunar distance approximately once-per-thousand years, making Apophis 2029 a once-per-millennium type of science opportunity. In fact, we can also expect that all of Earth will be watching. In the early evening hours of April 13, 2029 Apophis' rapid trek across the sky (brightening to 3 rd magnitude) will be visible to the unaided eye for an estimated 2 billion people spanning western Europe and northern Africa. The science opportunity of the Apophis 2029 encounter arises not only because of the proximity for Earth observers. The Apophis 2029 encounter presents an unprecedented science opportunity because of the Earth's physical interactions on the body of Apophis itself. At its geocentric miss distance of six Earth radii, Apophis will pass well outside the canonical Roche limit (~2 planetary radii). Recall however, that the Roche limit describes destruction of a passing body. At a miss distance of six Earth radii, significant tidal torques will be exerted on the body of Apophis. The consequences of Earth's tidal torques on Apophis include altering its rotation state, with the possibility of creating internal structural alteration within the asteroid itself, possibly resulting in measurable seismic waves and real-time surface disturbances.

As discussed in Section 5, considerable uncertainty remains in predicting which, if any, of these observable effects will occur on Apophis and be measurable within the precision limits of applicable technology. The greatest uncertainty factor for the outcome is the lack of knowledge of the internal structure of potentially hazardous asteroids. Thus, we urge the framers of the Decadal Survey to recognize the Apophis 2029 encounter as a "natural experiment" whose measurable outcomes could be transformative in understanding the internal structure for PHAs.

Herein we argue that the Apophis 2029 encounter event has an historical planetary science parallel: The 1994 Jupiter impact by comet Shoemaker-Levy 9 (SL9). Significant resources were allocated and extensive observational campaigns were organized for reasons that included: i) the event was unprecedented to modern science and modern measurement technology; ii) the SL9 impact was a "natural experiment" whose outcome was unpredictable and ultimately surprising, see Table I; iii) detailed observations and measurements of the impact consequences promised (and delivered) interdisciplinary advances in understanding the structure of comets, the structure and chemistry of Jupiter's atmosphere, and impact physics. Further, the "impact" of SL9 on public and Congressional awareness of asteroid hazards cannot be understated.

Herein we also point out precedents for planetary science mission expenditures up to the level of Discovery Class missions for conducting active experiments: Deep Impact into Tempel 1; Double Asteroid Redirection Test (DART). For each, the mission motivation was driven by the potential new knowledge to be gained by resolving the wide-ranging uncertainty of the outcome. Thus, we argue for Apophis 2029: The uncertainty of the outcome creates a strong motivation to seize upon this unprecedented learning opportunity for insights into the physical characteristics of potentially hazardous asteroids. This argument also revolves around a central motivation for planetary defense itself: In the highly unlikely, but not impossible necessity to mitigate a future asteroid threat, the physical insights gained from the Apophis 2029 encounter could be one of the most important science products ever delivered to humanity by planetary science investigations.

Implementation of Apophis 2029 Investigations

Within this White Paper, we take no position on how to implement specific prediction or observational investigations capable of achieving measurement and science advances offered by the Apophis 2029 opportunity. (Rationale for in situ investigation are given by Barbee 2020; Cheng 2020. Taylor 2020 describes the essential role of radar; Haynes 2020 describes the potential of radar tomography.) As outlined in Section 5, predictions for measurable outcomes are highly uncertain at this time. We advocate the knowledge potential of the Apophis 2029 opportunity merits a directed Science Definition Team study. We note particularly that time is of the essence for considering in situ measurements. For example, a student-led study (MIT Project Apophis, 2017) identified low cost mission opportunities opening as early as 2026.

While planetary science expertise and techniques enable the path forward, we argue that the new knowledge gained has its greatest value to the science of planetary defense. In colloquial terms, Apophis is the "Poster Child for Planetary Defense." For this reason, we advocate that Apophis 2029 investigations be supported under NASA's Planetary Defense budget, in accordance with a specific recommendation of the Melosh (2019) National Academy Report:

"Missions meeting high-priority planetary defense objectives should not be required to compete against missions meeting high-priority science objectives." We thus propose as a Decadal Report recommendation: Competitively selected or directed Apophis science investigations, be they theoretical or observational, ground-based, space-based, or in situ, be executed under the direction of NASA's Planetary Defense Coordination Office (PDCO). Commensurate with this recommendation, an appropriate budget must be allocated within the NASA Science Mission Directorate that is capable of specifically supporting Apophis investigations. International collaboration is strongly encouraged. We note that the PDCO is currently supporting, and should be encouraged to continue support, international planning efforts toward the Apophis 2029 opportunity.

Apophis 2029 Model Predictions, Science Implications, Measurements and Uncertainties

While Apophis is coming for certain, and it will miss the Earth for certain, the actual physical consequences of the encounter on Apophis itself are uncertain. As noted in Section 3, the Apophis 2029 miss distance of 6 Earth radii is outside the canonical Roche limit (for destruction), but still close enough to subject the physical body of Apophis to tidal torques.

Table I summarizes a variety of physical effects that have been studied to date by a variety of independent models. {Covid-19 Note: A more detailed compilation was to be a product of a planned Apophis T-9 Workshop in April 2020, postponed to November 2020.} As a first table entry, the 2029 encounter will result in Apophis undergoing a definitive orbit change (semimajor axis altered from 0.9 AU to >1 AU; [START_REF] Farnocchia | Yarkovsky-driven impact risk analysis for asteroid (99942)[END_REF]. Current models are also certain for a change occurring in Apophis' rotational properties (e.g. [START_REF] Scheeres | Abrupt alteration of Asteroid 2004 MN4's [Apophis] spin state during its 2029 Earth flyby[END_REF]; see Table I). Apophis is presently in a tumbling non-principal axis state with precession and rotation periods of 27.38h and 263h, respectively [START_REF] Pravec | The tumbling spin state of (99942) Apophis[END_REF][START_REF] Brozović | Goldstone and Arecibo radar observations of (99942) Apophis in 2012-2013[END_REF]. The tumbling state of Apophis may be a result of torques exerted during one or more previous (but not calculable with precision) Earth encounters. As Apophis experiences new torques on its angular momentum vectors during the 2029 encounter, the physical (and observable) consequences of the new body axis misalignments remain uncertain (e.g. [START_REF] Yu | Numerical predictions of surface effects during the 2029 close approach of Asteroid 99942[END_REF]). In the most substantial case, any re-arrangement of Apophis' (presumed) interior rubble pile structure could result in a gross reconfiguration of its shape. Models to date (e.g. [START_REF] Scheeres | The Abrupt Alteration of Apophis' Spin State and its Implications[END_REF], however, deem that a gross reconfiguration is unlikely for such a "far" encounter outside of the canonical Roche limit.

Minor internal reconfigurations, or just the body's adjustment to a new rotation state, has the potential to spur downslope movement of regolith and/or generate seismic waves inside of Apophis [START_REF] Souchay | Rotational changes of the asteroid 99942 Apophis during the 2029 close encounter with Earth[END_REF][START_REF] Souchay | Changes of spin axis and rate of the asteroid (99942) Apophis during the 2029 close encounter with Earth: A constrained model[END_REF][START_REF] Yu | Numerical predictions of surface effects during the 2029 close approach of Asteroid 99942[END_REF][START_REF] Demartini | Using a discrete element method to investigate seismic response and spin change of 99942 Apophis during its 2029 tidal encounter with Earth[END_REF]. Beyond Earth, natural seismic waves have been measured for only two planetary bodies in the history of solar system exploration: Moon by Apollo and Mars by InSight. In the context of priorities set by past Decadal Surveys for seismological investigations, and the considerable planetary mission expense allocated toward achieving these seismic measurements, we propose a specific finding:

The potential for measuring seismic waves in Apophis, thereby providing the first direct insight into the internal structure of a potentially hazardous asteroid, is the most enticing and transformative outcome that may be achievable through Apophis 2029 investigations.

Observational evidence supports finding that planetary encounters outside of the Roche limit generate enough (presumably) seismic activity to rearrange the upper surface layer [START_REF] Binzel | Earth encounters as the origin of fresh surfaces on near-Earth asteroids[END_REF]. [START_REF] Terentjeva | Meteorite producing fragment on the Apophis' orbit. WGN[END_REF] suggests a possibly associated meteor stream. "Refreshing" by Earth encounters was suggested by [START_REF] Nesvorny | Evidence for asteroid space weathering from the Sloan Digital Sky Survey[END_REF] and subsequently modeled by [START_REF] Nesvorny | Do planetary encounters reset surfaces of near-Earth asteroids?[END_REF]. The 'seismic limit' inside which the encounter is close enough to effect surface changes is uncertain, with [START_REF] Binzel | Earth encounters as the origin of fresh surfaces on near-Earth asteroids[END_REF] finding spectrally fresh surfaces consistent with a limiting distance inside of ~15 Earth radii. More detailed theoretical modeling by [START_REF] Nesvorny | Do planetary encounters reset surfaces of near-Earth asteroids?[END_REF] places the 'seismic limit' inside of ~5 Earth radii. We note that the Apophis 2029 encounter is inside or very close to these suggested thresholds for tidally induced seismicity.

As noted in Section 4, within this White Paper we take no position on any methodology that should be employed or any architecture for executing Earth-based and/or in situ investigations. I are purely notional in order to demonstrate the range of potential options. Detailed efforts moving forward are needed, both in terms of physical prediction and technological capabilities, to create science definitions for what may be achieved through Apophis 2029 measurements. The Decadal Survey report may consider prescribing the formation of an Apophis 2029 Science Definition Team to discern the achievable objectives. As argued in Section 3, uncertainties in the outcome is a strong driver for making direct measurements of a completely new phenomenon. However, because the uncertainties may include the possibility of no measurable outcome, we recommend science definitions of measurement objectives should include a science value assessment for meaningful constraints gained on the physical nature of PHAs in the event that any attempted investigation yields a null result (signal below the limit of measurement sensitivity). The lower sections in Table I recall the state of predictions in advance of previous "experiments:" the 1994 Jupiter impact of comet Shoemaker-Levy 9 and the 2005 Deep Impact penetration of Tempel 1. The parallels to Apophis 2029 are striking in the considerable range of uncertainties in outcomes up until the moment each "experiment" occurred. The lesson is that only through dedicated planning and measurement can the true outcome be learned, and from that outcome, considerable new insights be gained into the physical nature of planetary bodies. Apophis is coming. Will we be ready? It's up to you to make Apophis 2029 a Decadal priority.

Entries on methodology in Table

TABLE I:

Summary of predicted outcomes for the Apophis 2029 encounter, placed in an historical context with predicted outcomes for other "natural" and induced experiments.

3

  Apophis 2029: Decadal Opportunity for the Science of Planetary Defense

6

  Precedents and Lessons in Planetary Science: Applications to Apophis 2029 Planning

a

  Table References:[A] Scheeres 2005; [B] Holsapple 2006; [C] Souchay 2014; [D] Yu 2014; [E] Garcia 2015 [F] Farnochia 2013; [G] Souchay 2018; [H] DeMartini 2019; [J] Scheeres 2020; [K] Zhang 2020; [L] Weissman 1994; [M] Boslough 1994; [P] Neff 2010; [Q] A'Hearn 2005; [R] Schultz 2005. b Methodologies listed are notional. No specific recommendations on methodologies, or their value assessment, are intended by this White Paper. A dedicated Science Definition Team may be prescribed to produce recommendations.

Acknowledgements

We thank Mainzer et al. (2020) for setting the broad context of Planetary Defense for the 2023-2032 Decadal Survey.

Physical Effects on Apophis

Degree of Certainty a

Implications of Measurement Possible Measurement Methods b

Earth-based

In situ