Maxime K Collard

Julien Tourneur-Marsille

Mathieu Uzzan

Miguel Albuquerque

Maryline Roy

Anne Dumay

Jean-Noël Freund

Jean-Pierre Hugot

Nathalie Guedj

Xavier Treton

Yves Panis

Dr Eric Ogier-Denis
email: eric.ogier-denis@inserm.fr

The appendix orchestrates T-cell mediated immunosurveillance in colitis-associated cancer

Keywords: azoxymethane, CAC: colitis-associated cancer, CRC: colorectal cancer, DSS: dextran sodium sulfate, IBD: inflammatory bowel disease, UC: ulcerative colitis Appendectomy, Appendicitis, Ulcerative Colitis, Inflammatory Bowel Disease

Appendectomy suppresses a major site of T-cell priming in the colon resulting in a reduced colitis-associated colorectal cancer immunosurveillance. The fundamental mechanism identified emphasizes that precautions will be necessary if appendectomy becomes an accepted treatment of ulcerative colitis.

Introduction

In 1871, Charles Darwin has described the appendix as a rudimentary and useless vestige, that appeared as a consequence of a progressive shrinking of the cecum due to diet changes in our distant ancestors [START_REF] Ogier-Denis | The Descent of Man and Selection in Relation to Sex[END_REF]. This historical conception has been refuted by a modern analysis of the evolutive history showing that the appendix has been positively selected among mammals for at least 80 million years and has made multiple independent appearances without any association with diet changes or cecum shrinking [START_REF] Smith | Comparative anatomy and phylogenetic distribution of the mammalian cecal appendix[END_REF][START_REF] Smith | Morphological evolution of the mammalian cecum and cecal appendix[END_REF], pointing out a potential benefit of this structure, the function of which is still almost uncertain [START_REF] Girard-Madoux | The immunological functions of the Appendix: An example of redundancy?[END_REF].

An important question has been raised regarding whether removing the normal appendix is safe or not over time. This question is especially important given the recent improvements in the treatment of ulcerative colitis (UC). The incidence of this inflammatory bowel disease (IBD) can reach up to 465 cases per 100,000 inhabitants in developed countries [START_REF] Khalil | Incidence Rates and Predictors of Colectomy for Ulcerative Colitis in the Era of Biologics: Results from a Provincial Database[END_REF] and it is characterized by chronic inflammation of the rectum and colon. Additionally, the risk of developing colorectal cancer (CRC), referred to as colitis-associated cancer (CAC), is increased in UC patients [START_REF] Liu | Distinct clinicohistologic features of inflammatory bowel disease-associated colorectal adenocarcinoma: in comparison with sporadic microsatellite-stable and Lynch syndrome-related colorectal adenocarcinoma[END_REF]. A history of appendicitis is rare in UC patients and a reduced incidence of UC has been observed in families with a history of appendicitis [START_REF] Andersen | Reduced risk of UC in families affected by appendicitis: a Danish national cohort study[END_REF]. This suggests either a protective effect of appendectomy for colitis [START_REF] Andersson | Appendectomy and protection against ulcerative colitis[END_REF] or that appendicitis and UC involve alternative inflammatory responses [START_REF] Beaugerie | Appendicitis, not appendectomy, is protective against ulcerative colitis, both in the general population and first-degree relatives of patients with IBD[END_REF].

Interestingly, a history of appendectomy in subjects younger than 20-year-old reduces the risk of developing UC in the general population, only in case of actual appendix inflammation [START_REF] Andersson | Appendectomy and protection against ulcerative colitis[END_REF][START_REF] Frisch | Appendicitis, mesenteric lymphadenitis, and subsequent risk of ulcerative colitis: cohort studies in Sweden and Denmark[END_REF]. In contradiction with these findings, the protective effect of preemptive appendectomy without appendicitis has been investigated as a potential therapy for refractory UC and a clinical improvement has been shown in up to 50% of patients [START_REF] Stellingwerf | Prospective cohort study of appendicectomy for treatment of therapy-refractory ulcerative colitis[END_REF]. The possible effect of appendectomy on UC clinical outcomes could be a promising therapeutic option [START_REF] Felice | Therapeutic Role of Appendectomy in Ulcerative Colitis: A Tangible Perspective?[END_REF][START_REF] Gardenbroek | The ACCURE-trial: the effect of appendectomy on the clinical course of ulcerative colitis, a randomised international multicenter trial (NTR2883) and the ACCURE-UK trial: a randomised external pilot trial (ISRCTN56523019)[END_REF] but so far, evidence is lacking to use elective appendectomy as a routine therapeutic procedure in UC.

Data on the use of appendectomy as a therapeutic option are contradictory. While some studies have shown a decreased colectomy rate after appendectomy in UC patients, especially when performed after the onset of the disease [START_REF] Stellingwerf | A nationwide database study on colectomy and colorectal cancer in ulcerative colitis: what is the role of appendectomy?[END_REF], other studies have suggested an increased risk of CAC after appendectomy and no significant change in colectomy rate despite a significant reduction in colorectal inflammation [START_REF] Stellingwerf | The Risk of Colectomy and Colorectal Cancer After Appendectomy in Patients With Ulcerative Colitis: A Systematic Review and Meta-analysis[END_REF][START_REF] Harnoy | Effect of appendicectomy on colonic inflammation and neoplasia in experimental ulcerative colitis[END_REF]. Since it is commonly accepted that CAC is related to colitis severity and extent [START_REF] Flores | Impact of mucosal inflammation on risk of colorectal neoplasia in patients with ulcerative colitis: a systematic review and metaanalysis[END_REF], this last finding is counterintuitive and the mechanisms of this paradoxical effect remain to be investigated.

We have previously shown that appendectomy performed in a mouse model of UC led to a spontaneous onset of colonic tumors [START_REF] Harnoy | Effect of appendicectomy on colonic inflammation and neoplasia in experimental ulcerative colitis[END_REF]. To further decipher the underlying mechanisms involved in appendectomy-induced CAC tumorigenesis, we investigated appendectomy consequences on immunosurveillance and lymphocyte trafficking in a mouse model of CAC.

We then confirmed these results in colorectal tumors from patients with UC.

Results

Appendectomy increases tumorigenesis of CAC without worsening colitis

We first investigated the impact of appendectomy on CAC development in the AOM/DSS model. This AOM/DSS murine model aims to induce colon tumors in the setting of chronic colitis via the administration of azoxymethane (AOM), a carcinogenic agent, and of dextran sodium sulfate (DSS), a colitogenic agent, as shown in figure 1A. A significantly increased number of colorectal tumors was macroscopically observed in the appendectomy group (24.5 [20.0-31.8] tumors) compared to the control group (sham laparotomy) (15.0 [11.8-22.3] tumors, P=0.0028, figure 1B-C). This increase was confirmed microscopically (13.5 [12.3-19.8] tumors versus 9.0 [7.5-10.3] tumors per slide, P <0.0001, figure 1D-E).

As chronic inflammation is the primary cause of CAC, we first explored the impact of appendectomy on chronic colitis severity after the third DSS cycle without initial injection of AOM according to the "DSS-only protocol". This "DSS-only protocol" consisted in colitis evaluation at the end of the third cycle of DSS independently of CAC in order to assess the effect of appendectomy only on chronic colitis (figure 1F). Body weight changes during each DSS cycle and length of the colon did not significantly differ between the appendectomy and control groups (figure 1G-H). However, the histological assessment revealed that appendectomy significantly reduced the extent of colitis compared to controls (P=0.0453, figure 1I), suggesting that appendectomy could moderately decrease the severity of chronic inflammation.

Reduced colonic inflammation following appendectomy has also been reported in patients with UC [START_REF] Stellingwerf | Prospective cohort study of appendicectomy for treatment of therapy-refractory ulcerative colitis[END_REF]. However, it is classically admitted that the risk of developing CAC correlates with colitis severity in UC while we observed a concomitant paradoxical increase in the number of colonic tumors. To explore this counterintuitive result, mice were exposed to the AOM/DSS protocol at four different DSS concentrations from 0.5% to 2.0%. We found a positive correlation between the DSS concentration and colitis severity (figure 2A), and the number of colonic tumors (figure 2B-C), confirming a role of inflammation in tumorigenesis and thus suggesting that appendectomy increased tumorigenesis through a mechanism other than inflammation.

To assess whether appendectomy induced specific tumorigenic pathways, we profiled the molecular landscape of tumors from appendectomized and control mice. The comparative transcriptome analysis of tumors revealed a similar gene expression profile between both groups. Among the 23,517 RNA transcripts analyzed, only 21 (0.1%) were differentially expressed due to appendectomy (figure 3A). In addition, tumor proliferation assessed by immunohistochemistry staining of PCNA was similar in both groups (figure 3B). Thus, no specific tumor molecular pattern was identified in appendectomized mice.

The appendix may be instrumental in maintaining the intestinal microbiome [START_REF] Bollinger | Biofilms in the large bowel suggest an apparent function of the human vermiform appendix[END_REF] and gut dysbiosis could be a relevant hypothesis to explain the increased tumorigenesis.

The non-spore-forming Gram-negative bacteria, Fusobacterium nucleatum, have been associated with both acute appendicitis [START_REF] Swidsinski | Acute appendicitis is characterised by local invasion with Fusobacterium nucleatum/necrophorum[END_REF], mild colitis in UC [START_REF] Tahara | Fusobacterium detected in colonic biopsy and clinicopathological features of ulcerative colitis in Japan[END_REF], and more severe forms of sporadic CRC in humans [START_REF] Mima | Fusobacterium nucleatum in colorectal carcinoma tissue and patient prognosis[END_REF]. To date, the specific consequences of this bacteria on the CAC are unknown in human, nevertheless a recent published study in mice reported that this bacteria may accelerate the progression of CAC in the AOM-DSS model [START_REF] Yu | Fusobacterium nucleatum Accelerates the Progression of Colitis-Associated Colorectal Cancer by Promoting EMT[END_REF]. Thus, we assessed the intra-tumor infiltration of Fusobacterium nucleatum by qPCR in individual tumors.

Fusobacterium nucleatum was detected in 1 out of 7 and in 1 out of 5 mice (figure 4A and4B) in the appendectomy and control groups, respectively, indicating that Fusobacterium nucleatum was not directly involved in the tumorigenic mechanisms.

We further investigated the overall composition of the fecal microbiota one week after surgery and before DSS treatment. This time point was selected to identify the specific effects of appendectomy, avoiding the indirect effects of inflammation [START_REF] Hakansson | Immunological alteration and changes of gut microbiota after dextran sulfate sodium (DSS) administration in mice[END_REF] and tumorigenesis on the microbiota composition. 16s rRNA sequencing of the fecal microbiota revealed a similar abundance of the 7 most represented bacterial phyla between control and appendectomized mice (figure 3C). Both alpha diversity, assessed as the observed richness (Chao1) and diversity (Shannon), (figure 3D) and beta diversity, measured based on the Bray-Curtis or Jaccard indexes, did not significantly differ between both groups (figure 3E-F). LEfSe (Linear discriminant analysis Effect Size) analysis on the fecal microbiota composition did not shown any differences between the two groups, except for the genus Roseburia (figure 3G). All in one, our microbiota analysis suggests, in the limits of our explorations, a comparable profile of the fecal microbiota 1 week after the surgery between the appendectomy and the control groups.

Appendectomy reduces intra-tumor T-cell infiltration

To determine the importance of the immunological function of the cecal patch (the murine equivalent of the lymphoid structures of the human appendix) in colitis-associated tumorigenesis, we focused on the anti-tumor immunity driven by CD3+ and CD8+ T cells [START_REF] Pages | International validation of the consensus Immunoscore for the classification of colon cancer: a prognostic and accuracy study[END_REF].

Immunohistochemistry showed a significant decrease in CD3+ and CD8+ T-cell tumor infiltration after appendectomy (P=0.0001 figure 5A-B and P=0.031 figure 5C-D, respectively).

We further characterized T cells infiltrating CAC by multiparameter flow cytometry (figure 5E-N). The proportion of intra-tumor memory T cells (CD3+ and CD44 high cells among CD3+ cells) (figure 5E) was not affected by appendectomy (P=0.5382) even in the subgroups of CD4+ T cells (P=0.4059) (figure 5H) and CD8+ T cells (P=0.3203) (figure 5K). Among memory T cells (CD3+ and CD44 high cells), the ratio between effector (CD62L low ) and central (CD62L high ) memory T cells was significantly decreased after appendectomy (P=0.0398) (figures 5F and 5N). Accordingly, it is noteworthy that the SELL mRNA encoding CD62L was overexpressed in tumors after appendectomy (see figure 3A). Percentages of PD1 high T-cells among CD3+ T cells and CD8+ T cells were reduced in the appendectomy group, suggesting a lower proportion of intra-tumor exhausted T cells (figure 5G and 5M, respectively). However, the functional evaluation of intra-tumor T cells stimulated with PMA-ionomycin did not confirm this hypothesis (figure 5O).

Of note, at the DSS treatment endpoint (DSS-only protocol), CD3+ and CD8+ T-cell densities in the lamina propria were significantly reduced after appendectomy (figure 6A-D), in line with the observation of less severe colitis. In contrast, one week after surgery without AOM/DSS treatment (Surgery-only protocol), CD3+ and CD8+ T-cell densities in the lamina propria were low and did not differ between groups (figure 6E-H). The number of isolated lymphoid follicles in the colon was also unchanged (figure 6I-J). These findings suggested that the immunological impact of appendectomy was mainly observed in case of colitis.

Overall, in tumors from appendectomized mice, CD8 + T-cell infiltration was decreased and the cell phenotype was switched from effector memory T cells to central memory T cells. This finding suggested that the increased CAC development after appendectomy was associated with decreased immune induction and surveillance.

Inhibiting lymphocyte trafficking mitigates the pro-tumor effect of appendectomy

As a key inducer site, the appendix could orchestrate anti-tumor immunity in the colon in the context of chronic inflammation and antigenic stimulation. To test this hypothesis, intestinal homing of lymphocytes from the bloodstream to the gut was reduced with an anti-α4β7 integrin antibody in the one hand and lymphocyte egress from mesenteric lymph nodes and the appendix was inhibited with FTY720 on the other hand. Both blockades were supposed to mimic in part the pro-tumor effect of appendectomy although their own mechanism of action on lymphocyte trafficking into the appendix is unknown.

As expected, anti-α4β7 integrin treatment eliminated the macroscopic and microscopic differences in tumor number between the AOM/DSS appendectomy + anti-α4β7 integrin and anti-α4β7 integrin only groups (figure 7A-B). Furthermore, anti-α4β7 integrin treatment reduced intra-tumor CD3+ and CD8+ T-cell densities that became similar between appendectomy + anti-α4β7 integrin and anti-α4β7 integrin only groups (figure 7C-D). FTY720 treatment led to identical findings (figure 7E-H). Together, these experiments supported a defect of protective T-cell trafficking to the gut in the mechanism of appendectomy-associated tumorigenesis.

Appendicitis protects against CAC and induces intra-tumor T-cell infiltration

We then assumed that appendicitis could have effects on tumorigenesis opposite to those induced by appendectomy, through the activation of immune surveillance and lymphocytes.

Therefore, we explored the potential benefit of inducing neo-appendicitis (figure 8A), on antitumor immune protection. As part of the AOM/DSS protocol, significant decreases in macroscopic (P=0.0075) and microscopic (P=0.0035) tumor numbers were detected in the appendicitis group compared to the control group (figure 8B-C). As expected, CD3+ and CD8+ T-cell infiltration was significantly increased in CAC after appendicitis (figure 8D-E). Of note as part of the DSS-only protocol, no significant difference in body weight change after each DSS cycle was observed between the appendicitis and control groups (figure 8F). However, the pathological analysis showed that appendicitis increased colitis extent throughout the colon compared to the control group (P=0.0453, figure 8G). Thus, appendicular inflammation could worsen colitis and induce a phenotype opposite to that observed after appendectomy.

Systemic injection of CD3+ or CD8+ T cells activated by appendicitis protects against CAC and induces intra-tumor T-cell infiltration

To further validate the role of circulating T cells, neo-appendicitis was surgically induced in 27 donor mice and appendicular cells were isolated and purified one week later. T-cell depleted immune cells (CD45+ CD3-cells) or purified CD3+ T cells or CD8+ T cells were injected into the retro-orbital venous sinus of recipient mice that were further treated with AOM/DSS. Mice injected with CD3+ or CD8+ T cells showed a significant reduction in the number of colonic tumors compared to mice injected with CD45+ CD3-cells (figure 9A-B

The reduced intra-tumor CD3+ and CD8+ T-cell infiltration observed in mice after appendectomy is confirmed in CAC samples from UC patients

To confirm our results obtained in mice in humans, we analyzed colonic tumors from 21 consecutive UC patients who underwent colorectal resection for CAC between January 2006 and December 2017. Five patients (24%) had a history of appendectomy and 16 patients (76%) did not have any history of appendectomy. Clinical and oncological characteristics are summarized in table 1.

More than one CAC (synchronous tumors) was found in the surgical specimen in one patient with a history of appendectomy (2 tumors) and in one patient without history of appendectomy (3 tumors). Thus, we assessed by immunohistochemistry 6 tumors in the appendectomy group and 18 tumors in the control group. The analysis of intra-tumor T cells confirmed that patients with a history of appendectomy had significantly lower CD3+ and CD8+ T-cell densities compared to patients without history of appendectomy (respectively P=0.0044, figure 10A-B and P=0.0472, figure 10C-D).

Discussion

Our results showed that appendectomy was associated with a reduced CD3+ and CD8+ T-cell infiltration in CAC in both mice and humans. In animal models, appendectomy increased colitis-associated tumorigenesis by preventing T-cell trafficking and the subsequent decrease in immune surveillance of cancer. As a mirror image, neo-appendicitis reinforced the protection against CAC through an increase in CD3+ and CD8+ T-cell immunosurveillance. The appendix thus appeared as a major inducer site for T-cell priming of colonic T lymphocytes and could also prime intra-tumor T cells in the context of CAC.

The negative impact of low intra-tumor CD3+ and CD8+ T-cell densities on the prognosis of sporadic CRC is well established [START_REF] Galon | Type, density, and location of immune cells within human colorectal tumors predict clinical outcome[END_REF]. The immunoscore based on CD3+ and CD8+ T-cell densities in sporadic colorectal tumors and in their invasive margins is a powerful prognostic marker for tumor evolution [START_REF] Pages | International validation of the consensus Immunoscore for the classification of colon cancer: a prognostic and accuracy study[END_REF]. Intra-tumor CD3+ and CD8+ T-cell densities in CAC have been reported in two contradictory studies. Michael-Robinson et al. have highlighted higher densities in CAC [START_REF] Michael-Robinson | Characterization of tumour-infiltrating lymphocytes and apoptosis in colitis-associated neoplasia: comparison with sporadic colorectal cancer[END_REF], whereas Seung Soh et al. have observed lower densities [START_REF] Soh | Immunoprofiling of Colitis-associated and Sporadic Colorectal Cancer and its Clinical Significance[END_REF]. Whatever, low intra-tumor CD3+ and CD8+ density was associated with a poorer tumor prognosis in CAC as in sporadic CRC [START_REF] Soh | Immunoprofiling of Colitis-associated and Sporadic Colorectal Cancer and its Clinical Significance[END_REF]. Here, we demonstrate in a mouse model of CAC that lower densities of these cell populations were associated with a higher number of tumors, suggesting a protective role of intra-tumor CD3+ and CD8+ T cells. Interestingly, both the transcriptome analysis and the proliferation of CAC were similar between control and appendectomy groups suggesting that the protective role of T-cell immunity conferred by the presence of the appendix might be mainly involved in the protection against tumor initiation rather than tumor progression.

After appendectomy, the number of CD3+ and CD8+ T cells was reduced in both human and mouse with CAC, raising the question of the role of the appendix in cancer immunosurveillance. However, the limited number of CAC analyzed in human did not allow to go further in the exploration of the data with a statistical analysis adjusted on the confounding factors. Noteworthy, the lower intra-tumor CD3+ and CD8+ T-cell densities in mice were associated with a change in T-cell phenotype characterized by a reduced CD62L low /CD62L high ratio among memory T cells (CD3+ and CD44 high ). CD3+/CD44 high /CD62L low and CD3+/CD44 high /CD62L high cells are effector (Tem-cell) and central (Tcm-cell) memory T cells, respectively [START_REF] Sallusto | Two subsets of memory T lymphocytes with distinct homing potentials and effector functions[END_REF]. The proportion of Tem-cells and Tcm-cells generated after antigen presentation to naïve T cells is not constant and depends on the intensity of the antigen exposure: the differentiation into Tem-cells requires a strong antigen exposure while it is the opposite for Tcm-cells [START_REF] Wherry | Lineage relationship and protective immunity of memory CD8 T cell subsets[END_REF][START_REF] Marzo | Initial T cell frequency dictates memory CD8+ T cell lineage commitment[END_REF]. The appendix is a lymphoid structure where intestinal microbiota and environmental antigens are sampled and presented to the immune system [START_REF] Gebbers | Bacterial translocation in the normal human appendix parallels the development of the local immune system[END_REF].

The appendix is thus a major priming site in the colon, able to locally induce Tem-cells and its surgical resection is likely to reduce T-cell education with a significantly decreased Temcell/Tcm-cell ratio.

Appendectomy is also associated with a decreased expression of PD1 by T cells in tumors. Intra-tumor exhausted T cells are characterized by a high PD1 expression and an impaired effector function [START_REF] Ahmadzadeh | Tumor antigen-specific CD8 T cells infiltrating the tumor express high levels of PD-1 and are functionally impaired[END_REF]. Indeed, the capacity of these exhausted T cells to proliferate and produce effector cytokines is reduced. Here, we found a low proportion of PD1 high T cells after appendectomy but similar cytokine levels between the appendectomy and control groups. This finding suggests that PD1 high T cells are not exhausted T cells. Beswick et al. have shown that PD1 is upregulated in inflamed colonic mucosa [START_REF] Beswick | Expression of Programmed Death-Ligand 1 by Human Colonic CD90(+) Stromal Cells Differs Between Ulcerative Colitis and Crohn's Disease and Determines Their Capacity to Suppress Th1 Cells[END_REF]. In line with our results, Yassin et al. [START_REF] Yassin | Upregulation of PD-1 follows tumour development in the AOM/DSS model of inflammation-induced colorectal cancer in mice[END_REF] have shown a progressive increase in PD1 expression over time in the mucosal T-cell subset using the AOM/DSS model. Further CAC treatment with antibodies directed against PD1 has failed to reactivate PD1 high T cells directed against the tumors [START_REF] Yassin | Upregulation of PD-1 follows tumour development in the AOM/DSS model of inflammation-induced colorectal cancer in mice[END_REF]. Recently, we have shown that using anti-PD1 treatment in the AOM/DSS model led to an increased tumor proliferation [START_REF] Collard | Immune-checkpoint inhibitor anti-PD1 aggravates colitis-associated colorectal cancer without enhancing intestinal inflammation[END_REF]. Therefore, PD1 upregulation could reflect previous colonic inflammation rather than an exhausting state of intra-tumor T cells.

CAC is a consequence of DSS-induced colonic inflammation. In the DSS-only model of colitis, colitis extent was reduced and the CD3+ and CD8+ T-cell densities in the lamina propria were significantly reduced after appendectomy. These findings are consistent with a loss of T-cell priming in the appendix even if other mechanisms could be involved [START_REF] Cheluvappa | Experimental appendicitis and appendectomy modulate the CCL20-CCR6 axis to limit inflammatory colitis pathology[END_REF][START_REF] Cheluvappa | Autophagy suppression by appendicitis and appendectomy protects against colitis[END_REF][START_REF] Cheluvappa | The Role of Specific Chemokines in the Amelioration of Colitis by Appendicitis and Appendectomy[END_REF].

T-cell trafficking blockade with an anti-α4β7 integrin antibody or FTY720 mimicked the effect of the appendectomy on the median number of tumors in the AOM/DSS model of CAC. The loss of differences in tumor number and the reduced intra-tumor CD3+/CD8+ T-cell densities between the appendectomy and appendectomy after trafficking blockade suggested that the effect of appendectomy could be mediated by lymphocyte recirculation towards the colonic lamina propria and tumors. Finally, a protection against CAC induced by intra-tumor CD3+/CD8+ T cells primed in the appendix was strongly supported by the T-cell transfer experiments. Indeed, intravenous injection of intra-appendicular CD3+/CD8+ T cells obtained from donor mice in which neo-appendicitis was induced increased tumor infiltration by T cells and limited tumor number in recipient mice. This demonstrates that CD8+ T cells can acquire an efficient immunity against tumorigenesis through an initial priming in the appendix that does not imply exposition to a specific tumor antigen.

Importantly, our results do not suggest that a systematic increased risk of CAC would be inherent to all immunosuppressive or immunomodulatory treatments given to patients with UC.

Two parameters should be considered to assess this risk, which are the treatment efficiency on the colonic inflammation and the mechanism of action of the treatment. As these two parameters differ between each treatment, our results regarding the impact of appendectomy on the risk of CAC can in no way be extrapolated to the impact of other treatments. To warrant this point, a significant lower risk of developing a CAC in IBD patients treated by immunosuppressive therapy such as thiopurines [START_REF] Zhu | Reduced Risk of Inflammatory Bowel Disease-associated Colorectal Neoplasia with Use of Thiopurines: a Systematic Review and Meta-analysis[END_REF] or by anti-TNFα [START_REF] Alkhayyat | Lower Rates of Colorectal Cancer in Patients With Inflammatory Bowel Disease Using Anti-TNF Therapy[END_REF] has been reported. A higher risk of CAC despite a decrease of UC severity is not only reported in the specific context of the appendectomy but also in patients with UC with a concomitant primary sclerosing cholangitis (PSC) [START_REF] Aranake-Chrisinger | Primary sclerosing cholangitis associated colitis: Characterization of clinical, histologic features, and their associations with liver transplantation[END_REF][START_REF] Zheng | Increased risk of colorectal neoplasia in patients with primary sclerosing cholangitis and inflammatory bowel disease: a meta-analysis of 16 observational studies[END_REF]. As we know up to now, the toxicity of bile acids is suspected of being implicated in the increased risk of CAC in patients with PSC. It is assumed that liver cholestasis observed during PSC would decrease the absorption of bile acids in the digestive tract [START_REF] Hruz | Adaptive regulation of the ileal apical sodium dependent bile acid transporter (ASBT) in patients with obstructive cholestasis[END_REF] leading to an elevation of bile acid concentration in the colon, and specifically of the secondary bile acids which are known to be carcinogenic for the colon [START_REF] Bernstein | Carcinogenicity of deoxycholate, a secondary bile acid[END_REF].

Thus, despite a similar phenotype between patients with UC with a concomitant PSC and patients with UC with a history of appendectomy, the mechanism involved seems to be totally different.

If the appendix plays a crucial role in CAC, why is the oncological immunosurveillance provided by CD8+ T cells primed in the appendix not effective in sporadic CRC? In industrialized countries, about 10% of the population will undergo appendectomy during their life [START_REF] Anderson | Examining a common disease with unknown etiology: trends in epidemiology and surgical management of appendicitis in California, 1995-2009[END_REF]. Despite a high incidence of sporadic CRC, a history of appendectomy is not known as a risk factor [START_REF] Lee | The Risk of Colorectal Cancer After Cholecystectomy or Appendectomy: A Population-based Cohort Study in Korea[END_REF]. However, in most cases, appendectomy is performed for acute appendicitis. Indeed, a Taiwanese national cohort study has confirmed that a history of appendectomy for appendicitis was not associated with a risk of cancer (hazard ratio (HR): 1.02, 95%CI [0.90-1.16]) but a history of incidental appendectomy without appendicitis was significantly associated with a risk of sporadic CRC (HR: 2.90, 95%CI [2.24-3.75]) [START_REF] Wu | Association between appendectomy and subsequent colorectal cancer development: an Asian population study[END_REF]. This finding suggests that the appendix could also play a role in sporadic CRC.

In conclusion, using a mouse model of CAC, we showed that appendectomy induced a protumorigenic effect mediated by intra-tumor T-cell infiltration. Blocking cell egress from the appendix or T-cell homing to the colon mimicked the appendectomy-associated phenotype whereas re-injecting appendix-primed T cells increased intra-tumor T-cell infiltration. In UC patients with CAC, appendectomy was associated with a decreased intra-tumor T-cell infiltration. The fundamental mechanism identified in this study emphasizes that precautions will be necessary if appendectomy becomes an accepted therapeutic option for the treatment of refractory UC.

Methods

Animal models

All experimental procedures were approved by our local Animal Ethics Committee and the French Ministry of Research in accordance with the European legislation (APAFIS n° 14004-2018030914101923v5 and 24604-2020030518127896v3).

Five-week-old male BALB/c mice were purchased from Charles River and housed in ventilated cages with free access to water and food under controlled temperature (22 ± 2°C) and humidity (50 ± 10%). Mice were randomly assigned to experimental and control groups and the azoxymethane/dextran sodium sulfate (AOM/DSS) model of CAC was induced [START_REF] Tanaka | A novel inflammationrelated mouse colon carcinogenesis model induced by azoxymethane and dextran sodium sulfate[END_REF]. Mice were injected intraperitoneally with AOM (10 mg/kg of body weight; Sigma-Aldrich) at day 0.

One week later, mice were treated with DSS (MW=36,000-50,000; MP Biomedicals) in sterile drinking water according to the following sequence: 1.5% DSS for 7 days (first DSS cycle), sterile drinking water for 7 days, 1.5% DSS for 7 days (second cycle), sterile drinking water for 14 days and 1.5% DSS for 4 days (third cycle). Between the end of the third DSS cycle and sacrifice, mice had free access to DSS-free drinking water. Mice were weighed once a week all along the AOM/DSS protocol and the clinical severity of colitis was assessed during each DSS cycle based on the percentage of weight change. Mice were sacrificed 12 weeks after AOM injection. To induce chronic colitis without CAC, mice underwent 3 DSS cycles without prior injection of AOM (DSS-only protocol). Colonic specimens were collected for blinded histological evaluation and further biological and biochemical analyzes.

Surgical procedures

Surgery was performed under general anesthesia (intraperitoneal injection of buprenorphine at 0.1 mg/kg and inhalation of 3% isoflurane during the induction, and then 1.5% isoflurane during the procedure). Three different surgical procedures were performed: appendectomy, appendicitis induction and sham laparotomy (thereafter referred to as "control"). All mice underwent a single surgical procedure. After initiation of anesthesia, the skin was shaved and prepped with 70% ethanol. In the left iliac fossa, 1-cm paramedian laparotomy was performed, and the cecum was externalized from the peritoneal cavity. The cecal patch was identified as a 2-mm white ovoid structure on the antimesenteric side of the cecum. The appendectomy procedure consisted in the resection of the cecal patch. This structure is a major lymphoid structure in the colon of mice, located at the end of the cecum, and corresponds to the human appendix lymphoid structure [START_REF] Harnoy | Effect of appendicectomy on colonic inflammation and neoplasia in experimental ulcerative colitis[END_REF][START_REF] Li | Murine Appendectomy Model of Chronic Colitis Associated Colorectal Cancer by Precise Localization of Caecal Patch[END_REF]. Bacterial translocation occurs in the murine cecal patch via microfold (M) cells [START_REF] Kimura | Visualization of the entire differentiation process of murine M cells: suppression of their maturation in cecal patches[END_REF] as observed in the human appendix [START_REF] Gebbers | Bacterial translocation in the normal human appendix parallels the development of the local immune system[END_REF] recapitulating its immune function. Nevertheless, the cecal patch is a flat surface that does not have the cul-de-sac shape of the human appendix, thus does not reproducing the microbiota sanctuary function. To perform the resection of the cecal patch, this structure was suctioned with a 1-ml plastic syringe, ligated at its base with Corolene ® 8/0 thread and resected. Then, the cecum was reintegrated into the abdomen and the abdominal wall was closed with two layers (muscles and skin) of Filapeau ® 6/0 continuous sutures. To induce experimental appendicitis, the cecal patch was ligated at its base but not resected, leading to local inflammation that resolved spontaneously without any antibiotics or secondary appendectomy. For the sham procedure, the cecum was externalized and reintegrated into the abdomen without any ligation of the cecal patch (control group). Buprenorphine (0.1 mg/kg) could be administered postoperatively in case of apparent pain but no other treatments such as anti-inflammatory drugs or antibiotics were administered.

As part of the AOM/DSS protocol, surgery was performed at the same time as the AOM injection, i.e., 1 week before starting the first DSS cycle. AOM was injected into the peritoneal cavity at the end of the procedure, after abdomen closure. Similarly, as part of the DSS-only protocol, surgery was performed 1 week before starting the first DSS cycle. A third protocol called "surgery-only" was used to assess the effect of surgery in the absence of colitis and CAC.

As part of this protocol, mice underwent surgery (appendectomy, appendicitis or sham laparotomy) and were sacrificed 1 week later without any administration of AOM or DSS.

Human samples

From January 2006 to December 2017, all UC patients (n=21) who underwent surgical resection for CAC in our institution (Beaujon hospital, Clichy, France) were included. Patients with Crohn's disease or unclassified IBD or with dysplastic lesions only were excluded.

Patients' clinical characteristics were retrospectively collected. Paraffin-embedded CAC samples were collected for pathological analysis and immunohistochemistry. Any history of appendectomy was obtained from the pathology report of (sub-)total colectomy or coloproctectomy describing the presence or the absence of an appendix on the surgical specimen. Paraffin-embedded blocks with colorectal tumor fragments were collected for immunohistochemistry.

This study was approved by our local Ethics Committee and the French Ministry of Research (n° 12.739) in accordance with the European legislation.

Pathology

After sacrifice of mice, the entire colon was removed, opened in the longitudinal axis and macroscopically-visible tumors were counted. Then, swiss rolls of colons were fixed for 24h in 10% formalin and embedded in paraffin to observe the full-length organ. Paraffin-embedded sections (5 µm) were deparaffinized, stained with H&E reagent and the number of microscopically-visible tumors was determined. The colitis severity was assessed by the histological extent of colitis [START_REF] Cheung | TIGAR is required for efficient intestinal regeneration and tumorigenesis[END_REF]. Tissue sections from mice that underwent the DSS-only protocol were digitized (Scanscope AT turbo, Leica). Histological lesions of colitis were delineated with Aperio ImageScope software to calculate the percentage of inflamed colonic epithelium surface within the entire colonic epithelium for each mouse.

Immunohistochemistry

Paraffin-embedded colonic sections (5 µm) from each mouse were prepared for immunohistochemistry using antibodies directed against CD3 (ab16669, Abcam, 1/150 dilution) and CD8 (ab209775, Abcam, 1/1,000 dilution). Tissue slides from AOM/DSS-treated mice were also stained with antibodies directed against PCNA (Sc-56, Biotechnology, 1/100 dilution) to assess tumor cell proliferation. Tumor sections from human samples were analyzed by immunohistochemistry using antibodies directed against CD3 (A0452, Dako, 1/50 dilution) and CD8 (M7103, Dako, 1/50 dilution). Immunostained slides were digitized with Scanscope AT turbo, Leica. Using Aperio ImageScope software, human and mouse tumors were delineated. Tumor surfaces and the number of cells stained with the specific antibodies were automatically quantified. The median intra-tumor CD3+ and CD8+ T-cell densities were then calculated. This method was thus observer-independent. The lamina propria was also delineated on slides from mice that underwent the Surgery-only and DSS-only protocols. The surface and number of cells stained with the specific antibodies were quantified to assess the median CD3+ and CD8+ T-cell densities.

Isolation of intra-tumor T cells

Colonic tumors obtained at the end of the AOM/DSS protocol were collected, taking care to not remove the adjacent healthy colon. All tumors from the same colon were pooled in 10 mL of RPMI 1640 medium with GlutaMAX (61870-010, Gibco) supplemented with 10 mg of type IV Collagenase (LS004188 Serlabo), 0.5% of fetal bovine serum (FBS) and 10 mg of DNase (DN25-100MG Sigma). Fresh tumors were transferred to gentleMACS tubes (130-096-334, Miltenyi). Digestion was performed with gentleMACS Dissociator for 36 minutes at 37°C and with subsequent centrifugation at 930 rpm (37C_m_LIDK_1 program). After collagenase digestion and mechanical disruption, a single-cell suspension was obtained after filtration with 100-μm and then 40-μm cell strainers and washed twice with RPMI. To increase T-cell concentration, intra-tumor T cells were selected using Mouse CD90.2 MicroBeads (130-121-278, Miltenyi) according to the manufacturer's instructions. Magnetic separation was performed with the MultiMACs Separator Plus (130-098-637, Miltenyi).

Flow cytometry

Cell suspensions were Fc-blocked (FcR Blocking mouse, 130-092-575 Miltenyi) and dead cells were stained with LIVE/DEAD Fixable Aqua Dead Cell Stain Kit-AmCyan (L34957, Thermofisher). Then, cells were incubated for 20 minutes at 4°C in the dark with a cocktail of

T-cell stimulation and cytokine measurement by ELISA

For each mouse, 100,000 intra-tumor T cells were transferred to 96-well plates with 200 μL of cell culture medium in the presence or in the absence of a stimulation cocktail of PMA and ionomycin (00-4970-93; 1/500 dilution; Thermofisher). Cell culture medium included RPMI 1640 with GlutaMAX, 10% of FBS and 1% of antibiotic-antimycotic (15240096, Thermofisher). After 16 hours in a humidified 37°C incubator with 5% CO2, supernatants were collected and stored at -20°C for cytokine assay. Concentrations of TNF-α and INF-γ were measured by ELISA according to the manufacturer's instructions (respectively 88-7324-22 and 88-7314-22; Thermofisher). Detection ranges were 8-1,000 pg/mL for TNF-α and 15-2,000 pg/mL for INF-γ.

Transcriptome analyzes

Total RNAs were extracted from fresh colonic tumors using the RNAble Kit (Eurobio) and quantified with a nanodrop-1000 spectrophotometer (Thermofisher). Microarray processing was performed by a genomic platform (genom'IC, Cochin Institute, Paris, France). After validation of the RNA quality with the Bioanalyzer 2100 (using the Agilent RNA6000 nano chip kit), 100 ng of total RNAs were reverse transcribed using the GeneChip® WT Plus Reagent Kit (Thermofisher). Briefly, the resulting double-strand cDNA was used for in vitro transcription with T7 RNA polymerase (all these steps are included in the WT cDNA synthesis and amplification kit from Thermofisher). After purification according to Thermofisher protocol, 5.5 μg of Sens Target DNA were fragmented, biotin labelled and controlled using the Bioanalyzer 2100. cDNAs were then hybridized to GeneChip® MouseGene2.0ST (Affymetrix) at 45°C for 17 hours, and the chips were washed on the FS450 fluidics station (Affymetrix) and scanned using the GCS3000 7G. Scanned images were then analyzed with Expression Console software (Affymetrix) to obtain raw data (CEL files) and metrics for Quality Controls. No apparent outlier value was detected. CEL files were normalized by Robust Multi-array Averaging (RMA) in the Bioconductor R with the Brain Array custom CDF vs 23. Statistical analyzes were performed with Partek® GS. A t-test was used to explore differences in expressed genes between the appendectomy and control groups. Only genes with p-values <0.05 and expression fold-changes >1.5 were considered differentially expressed between both groups.

Fecal microbiota characterization

Mouse fecal samples were collected one week after surgery and frozen at -80°C. DNA was extracted using the QIAamp Fast DNA Stool Mini Kit (51604, Qiagen). A mechanical lysis with FastPrep (MP Biomedicals) was added to the protocol before thermal lysis. DNA concentration was measured using the Qubit dsDNA High Sensitivity Assay Kit (Q32851, Thermofisher) and adjusted for each sample to 5 ng/µL. 16S rRNA genes were amplified by PCR with universal primers amplifying the V4 variable region (515F: GTGCCAGCMGCCGCGGTAA and 806R: GGACTACHVGGGTWTCTAAT) [START_REF] Caporaso | Global patterns of 16S rRNA diversity at a depth of millions of sequences per sample[END_REF].

Barcodes and Illumina sequencing adapters were attached using the Nextera XT Index Kit (Illumina). Amplicons were purified using Agencourt AMPure (Beckman Coulter), quantified by qPCR using the KAPA Library Quantification Kit (Roche), pooled in equimolar concentration and diluted to 5.5 pM for sequencing. Sequencing was performed on the Illumina Miseq (600 cycles, 2x300 bp, paired sequences). Sequencing data was processed in R (version 4.1.2) using the DADA2 pipeline [START_REF] Callahan | DADA2: High-resolution sample inference from Illumina amplicon data[END_REF]. Quality profiles of the reads were analysed, filtered and trimmed. Forward and reverse denoised reads were merged together. The amplicon sequence variant (ASV) table was constructed with the full denoised sequences. After the removal of chimeras (method="consensus"), taxonomy was assigned from 16S rRNA database Silva 138.1 [START_REF] Quast | The SILVA ribosomal RNA gene database project: improved data processing and webbased tools[END_REF]. Data obtained were assembled and converted into a phyloseq object [START_REF] Mcmurdie | phyloseq: an R package for reproducible interactive analysis and graphics of microbiome census data[END_REF]. Abundance, alpha and beta diversity measurements were performed with the Shiny Migale script (https://shiny.migale.inrae.fr/app/easy16S/). Alpha diversity indices (Chao1 and Shannon) were calculated for each sample and compared between the appendectomy and control groups using an ANOVA analysis. A principal coordinates analysis based on the distance matrix of the beta diversity indices (Bray Curtis and Jaccard) was used to visualize differences in microbial composition between groups. Significance was assessed using a PERMANOVA (Permutational Multivariate Analysis of Variance Using Distance Matrices) test. A P-value <0.05 was considered significant.

A LEfSe (Linear discriminant analysis Effect Size) analysis was conducted to assess the bacterial taxa enriched in the feces of the appendectomy and of the control groups [START_REF] Segata | Metagenomic biomarker discovery and explanation[END_REF]. To do so, first we added a refseq slot to the phyloseq object (Microbiomeutilities package) , then LefSe analysis was run based on p-value <0.05 and on a LDA >4. Results were plotted in a cladogram.

Quantitative PCR for the detection of Fusobacterium nucleatum DNA Genomic DNA was extracted from fresh colonic tumors using the DNeasy Blood and Tissue kit (Qiagen). DNA was quantified using a Qubit 3.0 Fluorometer (Thermo Fisher Scientific).

Real-time qPCR was performed with 10 ng or 40 ng of DNA sample, 10 M of primers and MesaBlue qPCR MasterMix (Eurogentec). Fusobacterium nucleatum DNA was detected using the following primers: forward 5'-CCAACCATTACTTTAACTCTACCATGTTCA-3' and reverse 5'-GTTGACTTTACAGAAGGAGATTATGTAAAAATC-3' [START_REF] Repass | Registered report: Fusobacterium nucleatum infection is prevalent in human colorectal carcinoma[END_REF]. To detect the presence of bacterial DNA in samples, a 16S rDNA non-specific PCR was performed using the primers U968 5'-GAACGCGAAGAACCTTAC-3' and L1401 5'-GCGTGTGTACAAGACCC-3' [START_REF] Iebba | Higher prevalence and abundance of Bdellovibrio bacteriovorus in the human gut of healthy subjects[END_REF]. PCR were carried out using a LightCycler 480 instrument (Roche Diagnostics). Initial denaturation was performed at 95°C for 10 min, followed by 45 cycles consisting of 95°C for 15s and 60°C for 45s. A dissociation step was added, and dissociation curves were analyzed to confirm amplification fidelity. Positive PCR products were sent for sequencing to Eurofins Genomics, and sequences were analyzed through BLAST program (NCBI) to confirm Fusobacterium nucleatum-specific amplification.

Blocking lymphocyte trafficking

An anti-integrin α4-β7 antibody and FTY720 (a sphingosine-1-phosphate receptor agonist)

were independently used to limit lymphocyte trafficking to the colon. From Day 1 of the first DSS cycle (i.e., 1 week after appendectomy or sham surgery and after the AOM injection) until sacrifice, 125 μg/100μL of InVivoMAb anti-mouse LPAM-1 (integrin α4β7) (BE0034, Bioxcell) or 60 μg/100μL of FTY720 (SML0700, Sigma-Aldrich) were intraperitoneally administered twice a week. Regarding the control group, 100μL of sterile PBS was injected intraperitoneally according to the same chronology as the anti-integrin α4-β7 antibody and FTY720.

Transfers of systemic immune cells isolated from inflamed appendices

One week after appendicitis induction, 27 mice were sacrificed and inflamed appendices were resected and pooled in gentleMACS tubes (130-096-334, Miltenyi) containing 10 mL of RPMI 1640 with GlutaMAX (61870-010, Gibco) supplemented with 10 mg of type IV Collagenase (LS004188 Serlabo), 0.5% of FBS and 10 mg of DNase (DN25-100MG Sigma). Cell digestion and dissociation were performed using the gentleMACS Dissociator for 36 minutes at 37°C

with subsequent centrifugation at 930 rpm. After incubation, the single-cell suspension was obtained after filtration with 100-μm and 40-μm cell strainers and washed twice with RPMI. At this step, 2.1x10 8 living cells were obtained and then divided into two different tubes (tubes A and B). Tube A was used to isolate CD8+ T cells from inflamed appendices using the Mouse CD8a+ T-cell Isolation Kit (130-104-075, Miltenyi). Magnetic separation was performed using the MultiMACs Separator Plus (130-098-637, Miltenyi) according to the manufacturer's instructions. Tube B was used to isolate CD3+ T cells using Mouse CD90.2 MicroBeads (130-121-278, Miltenyi). Appendicular cells depleted in CD3+ T cells were then incubated with Mouse CD45 MicroBeads (130-052-301, Miltenyi). Cell isolation quality was controlled by flow cytometry. Among CD8+ filtered T cells, 87% of living cells were CD8+ and 0.007% were CD4+. Among CD3+ filtered T cells, 80% of living cells were CD3+, 38% were CD4+ and 39% were CD8+. Among CD45+ cells depleted in CD3+ T cells, 99% of living cells were CD45+ and 92% were CD3-. Comparisons of multiple groups were performed using the Kruskal-Wallis's test. A p-value <0.05 was considered statistically significant. Cladogram generated by LEfSe (Linear discriminant analysis Effect Size) assessing differences in taxa of fecal microbiota between appendectomy and control. Regions in red indicate taxa that were enriched in the appendectomy group, white regions show no statistical differences and green regions would have referred to taxa enriched in the control group, but no significant differences found on this comparison. A p-value <0.05 was considered statistically significant.

  ). A parallel increase in intra-tumor CD3+ and CD8+ T-cell densities was observed (figure 9C-D). No difference in tumor number or intra-tumor T-cell infiltration was noted between mice that received a CD3+ or CD8+ T-cell systemic injection.

  antibodies directed against CD3 (APC-Vio770, 130-119-793 Miltenyi), CD4 (FITC, 130-118-692, Miltenyi), CD8 (PE-Vio770, 130-119-123, Miltenyi), CD62L (APC, 130-112-837, Miltenyi), CD44 (PE, 130-118-694, Miltenyi) and PD1 (PE-CF594, 562523 BD). Samples were washed twice with PBS (phosphate buffered saline). Samples were acquired using LSRFortessa (BD) and analyzed with FlowJo v10.

Figures: titles and legendsFigure 1 :

 1 Figures: titles and legends

Figure 2 :

 2 Figure 2: Relationship between the severity of DSS-induced colitis and the number of colonic

Figure 3 :

 3 Figure 3: Transcriptome analysis of tumors, and assessment of tumor proliferation and fecal

Figure 4 :

 4 Figure 4: Fusobacterium nucleatum intra-tumor infiltration is not influenced by

Figure 5 :

 5 Figure 5: Appendectomy significantly alters intra-tumor T-cell immunity in mice.

Figure 6 :

 6 Figure 6: Effect of appendectomy on T-cell density after 3 DSS cycles (DSS-only protocol)

Figure 8 :

 8 Figure 8: Appendicitis provides a partial protection against CAC and increases intra-tumor T-

Figure 9 :

 9 Figure 9: Injecting purified appendicular CD3+ or CD8+ T cells activated by appendicitis

Acknowledgements

We thank Nathalie Colnot, Raphaelle Liquard, Ian Morilla, Arielle Pierre, Olivier Thibaudeau and Laure Wingertsmann for their substantial contribution to this project. We are grateful to the INRAE MIGALE bioinformatics facility (MIGALE, INRAE, 2020. Migale bioinformatics Facility, doi: 10.15454/1.5572390655343293E12) for providing help and computing and storage resources.

Grant support: This work was supported by the recurrent operating grant of the French Institute of Health (INSERM), Métropole, Investissements d'Avenir programme (ANR-11-IDEX-0005-02), Sorbonne Paris Cité, Laboratoire d'Excellence Labex Inflamex, Fondation de l'Avenir (AP-RM-17-008), la Ligue contre le Cancer (LCC) and Association François Aupetit (AFA).

was considered statistically significant (2-sided tests). Statistical analyzes were performed using Prism v8.2.1 (GraphPad Software).

All authors had access to the study data and had reviewed and approved the final manuscript.

Representative CD3+ cell staining of a colon after appendectomy or sham laparotomy (control). M-stage: 0 / 1 6 (100)/0 (0) 18 (100)/0 (0) 1.000 1 number of patients (percent); 2 median (interquartile range); 3 according to the 8 th TNM classification


Satoshi Munakata
email: munakata.satosi@fujitsu.com

Caterina Urban
email: caterina.urban@inria.fr

Haruki Yokoyama

Koji Yamamoto

Kazuki Munakata

Verifying Attention Robustness of Deep Neural Networks against Semantic Perturbations

In this paper, we propose the first verification method for attention robustness, i.e., the local robustness of the changes in the saliency-map against combinations of semantic perturbations. Specifically, our method determines the range of the perturbation parameters (e.g., the amount of brightness change) that maintains the difference between the actual saliencymap change and the expected saliency-map change below a given threshold value. Our method is based on linear activation region traversals, focusing on the outermost boundary of attention robustness for scalability on larger deep neural networks.

I. BACKGROUND Classification Robustness. Deep neural networks (DNN) are dominant solutions in image classification; however, quality assurance is essential when DNNs are used in safety-critical systems [START_REF] Ashmore | Assuring the machine learning lifecycle: Desiderata, methods, and challenges[END_REF]. Reference [START_REF] Gao | Fuzz testing based data augmentation to improve robustness of deep neural networks[END_REF] reported that despite input images can be perturbed in the real world by various mechanisms, such as brightness change and translation; such semantic perturbations can unexpectedly change the classification labels for DNNs. Therefore, it is essential to verify classification robustness (CR) against semantic perturbations to be tolerable from an assurance point of view. Several methods have already been proposed to compute the range of perturbation parameters (e.g., the amount of brightness change and translation) that do not change classification labels [START_REF] Balunovic | Certifying geometric robustness of neural networks[END_REF], [START_REF] Mohapatra | Towards verifying robustness of neural networks against a family of semantic perturbations[END_REF]. They defined the property of CR by the following predicate:

CR(x; Θ) def = ∀θ ∈ Θ.F g(θ, x) = F (x) (1)
where x ∈ X ⊆ R d X is an d X -dimensional real vector of an input image, Θ ⊂ R dΘ is the range of d Θ -dimensional perturbation parameters to be tolerable, g : Θ × X → X is the image perturbation function, and

F (x) def = arg max 1≤j≤d O f j (x)
is the classification function to map an image into the classification label (i.e., the dimension) that corresponds to the maximum output value of DNN f : X → R d O . Namely, the property CR(x; Θ) is satisfied iff the classification label remains the same no matter how image x is perturbed within the range Θ. Saliency-map. It is known that DNNs classify an input image by paying particular attention to certain specific pixels in the image; a graphical representation of the magnitude of attention to each pixel, like a heatmap, is called saliency-map [START_REF] Simonyan | Deep inside convolutional networks: Visualising image classification models and saliency maps[END_REF]. A saliency-map of each classification label can be obtained from the gradients of each DNN output for an input image [START_REF] Simonyan | Deep inside convolutional networks: Visualising image classification models and saliency maps[END_REF]; it is defined by the image-to-heatmap function

map j (x) def = ∂fj (x) ∂x1 , . . . , ∂fj (x) ∂x d X
, where x i is the i-th pixel value of image x. That is, pixels with higher values of map j (x) can contribute more to the classification of image x to label j. Classification Validity. Saliency-maps are used to check the validity of the classification decision basis. For instance, if a DNN classifies the subject type by paying attention to a background rather than the subject to be classified in an input image, it is not a valid basis for classification. We believe such low-validity classification should not be accepted in safety-critical situations, even if the correct classification labels. Semantic perturbations can significantly change the saliency-maps [START_REF] Montavon | Methods for interpreting and understanding deep neural networks[END_REF], [START_REF] Guo | Visual attention consistency under image transforms for multi-label image classification[END_REF]. For example, Fig. 1 shows the changes in MNIST image "8" and the actual saliency-map when the brightness is gradually changed; the collapsed saliency-maps indicate that the DNN does not pay proper attention to text "8" in each image. However, existing robustness verification methods only target changes in the classification labels, not the saliency-maps [START_REF] Huang | A survey of safety and trustworthiness of deep neural networks: Verification, testing, adversarial attack and defence, and interpretability[END_REF], [START_REF] Urban | A review of formal methods applied to machine learning[END_REF].

II. OUR ON-GOING WORK

Attention Robustness. We define the new property of attention robustness (AR) -the local robustness of the changes in the saliency-map against combinations of semantic perturbation -by the following predicate:

AR(x; Θ, δ) def = ∀θ ∈ Θ.ai(x; θ) ≤ δ (2) ai(x; θ) def = j∈Y map j g(θ, x) -g θ, map j (x) 2
where δ ∈ R is a given threshold value, • 2 denotes L2norm, and g : Θ × R d X → R d X is the heatmap perturbation function that corresponds to the image perturbation function g. Namely, the property AR(x; Θ, δ) is satisfied iff ai(x; θ) remains less than or equal to threshold δ no matter how image x is perturbed within the range Θ. ai(x; θ) represents attention inconsistency, i.e., the difference between (a) the actual saliency-map change map j g(θ, x) and (b) the expected saliency-map change g θ, map j (x) . Regarding the latter (b), brightness change keeps the saliency-map unchanged, whereas translation moves one along with the image; cf. columns (B) and (T) in Fig. 2. Although the concept of such difference is the same as saliency-map consistency used in semi-supervised learning [START_REF] Guo | Visual attention consistency under image transforms for multi-label image classification[END_REF], for the sake of verification, it is necessary to calculate the maximum value of ai within Θ.

Verifying AR. Therefore, we propose the first verification method for the property AR. We focus on feed-forward DNNs with rectified linear units (ReLU-FNN) and two facts;

(i) ReLU-FNN output is linear for its input within each activation region where the activation statuses of all ReLUs are fixed [START_REF] Hanin | Deep relu networks have surprisingly few activation patterns[END_REF] and (ii) when x is fixed at ẋ, many semantic perturbation functions g(θ, ẋ) can be interpreted as a ReLU-FNN g ẋ(θ) [START_REF] Mohapatra | Towards verifying robustness of neural networks against a family of semantic perturbations[END_REF], similarly for g. That is, within each activation region η ⊆ Θ of synthesized DNN f (g ẋ(θ)), (a) the actual saliency-map change map j g ẋ(θ) is constant because Traversing Outermost Boundary By traversing all activation regions η ⊆ Θ following the existing method [START_REF] Jordan | Provable certificates for adversarial examples: Fitting a ball in the union of polytopes[END_REF], we can finally determine AR( ẋ; Θ, δ) for small ReLU-FNNs. In practice, to represent the trend of the weakness against semantic perturbations for larger ReLU-FNNs, we propose the method to traverse activation regions near the outermost boundary of AR. More formally, the outermost boundary is the connected-space that (1) lays on both AR, ¬AR and (2) contains the farthest point from the origin 0 ∈ Θ through only regions that satisfy AR. The results of traversing the outermost boundaries of CR and AR (cf. Fig. 3); the shapes of the boundaries indicate the existence of regions that satisfy CR but not AR, and the DNN can misclassify the perturbed image with a thin patch and middle brightness in ¬AR regions. Preliminary Evaluation We implemented our method in a python tool for evaluation. The ReLU-FNN with 200 neurons had a 58% probability of complete verification within 2 hours in the case of traversing all activation regions, and the ReLU-FNN with 800 (2,028) neurons had a 51% (100%) probability of complete verification within 2 hours in the case of traversing outermost boundaries of AR (CR). For further details of our work, please refer to https://arxiv.org/abs/2207.05902.

  ẋ(θ))/∂θs ∂g ẋ i (θ)/∂θs and (b) the expected saliencymap change gmapj( ẋ) (θ) is a linear function; thus we can efficiently compute ai ẋ η def = max θ∈η ai( ẋ; θ) by convex optimization and then determine AR( ẋ; η, δ) by whether ai ẋ η ≤ δ.

Fig. 1 .

 1 Fig. 1. Perturbation-induced changes in images (first row), the actual saliencymap changes for label 8 (second row), and the values of attention consistency (third row). For threshold δ = 3, the perturbation range up to the third column from the left satisfies attention robustness.


H Lambert

S Corn

R Léger

P Ienny

P Slangen

M Fages

Ceramic bonding interface under shear-compression stress: UHSI contribution

Keywords: imaging, biomaterials, adhesives, silanes, biomechanics, Finite Element Analysis (FEA) 1

The aim of this study is to visualize and characterize by ultra-high-speed imaging (UHSI) the failure phenomena at the resin-ceramic bonding interface of lithium disilicate (LiSi2) samples bonded with gold-standard protocol (Monobond Plus [MB]) and the nontoxic one (Monobond Etch & Prime [MEP]) subjected to mechanical loading. Unprecedented frame rate, image resolution, and recording time were reached by using the most advanced UHSI camera. The finite element analysis (FEA) of the proposed mechanical test confirmed that the specific design of our samples enables a combined shear and compression stress state, prone to test the bonding interface while being close to physiological stresses. Ten LiSi2 samples were pretreated by gold standard (MB, n = 5) and self-etching primer (MEP, n = 5). Axial compression loading gradually increased until catastrophic failure was performed. As shown by the FEA, the angle between the bonding interface and load direction leads to shear-compression stresses at the resin-ceramic bonding interface. Failure was recorded by UHSI at 300,000 fps. All recorded images were analyzed to segregate events and isolate the origin of fracture. For the first time, thanks to the image recording setup, it was observed that debonding is the first event before breakage, highlighting that sample fracture occurs by interfacial rupture followed by slippage and cohesive failure of materials. Failure mode could be described as mixed. MEP and MB showed similar results and behavior.

Introduction:

Ceramic adhesive restorations (CARs) are widely used for aesthetic treatment. Their good optical properties, translucency, biocompatibility, and high mechanical properties have expanded the range of their indications [START_REF] Guimarães | Simplified surface treatments for ceramic cementation: use of universal adhesive and self-etching ceramic primer[END_REF]. CARs are indicated for veneers as well as endodontically treated teeth, notably on severely damaged posterior teeth [START_REF] Fages | The endocrown: a different type of all-ceramic reconstruction for molars[END_REF][START_REF] Decerle | Evaluation of Cerec endocrowns: a preliminary cohort study[END_REF]Fages, Raynal, et al. 2017). The success of CARs on posterior teeth has already been demonstrated (Magne et al. 1999;Fages, Raynal, et al. 2017). Nevertheless, the bonding interface, involving ceramic and resin cement, seems to be the main trigger causing the majority of encountered failures [START_REF] Magne | Porcelain veneers: dentin bonding optimization and biomimetic recovery of the crown[END_REF][START_REF] Breschi | Dental adhesion review: aging and stability of the bonded interface[END_REF]Yu and Wang 2008;[START_REF] Lafuente | SEM analysis of hybrid layer and bonding interface after chlorhexidine use[END_REF][START_REF] Fages | Glass ceramic CAD/CAM crowns and severely altered posterior teeth: a three levels study[END_REF]Yao et al. 2019). The literature is rich about ceramic fracture and ceramic debonding under mechanical stress [START_REF] Burke | Fracture resistance of teeth restored with dentin-bonded crowns constructed in a leucitereinforced ceramic[END_REF][START_REF] Pineda-Vásquez | Shear bond strength of debonded ceramic restorations re-cemented by means of a cleaning and retreatment protocol[END_REF][START_REF] Venturini | Fatigue performance of adhesively cemented glass-, hybrid-and resin-ceramic materials for CAD/CAM monolithic restorations[END_REF]). In most of these studies, crack initiation is not detected with sufficient accuracy. Shear-compression test appears to be close to the clinical situation where shear may occur at interfaces angled from the loading direction. Detecting the cause of crack initiation may help to better understand the failure phenomena of CARs.

Ultra-high-speed imaging (UHSI) appears as the best solution to segregate events and isolate the first one happening. Resin-dentin interface failure under different mechanical stresses had been observed with UHSI but without providing those answers [START_REF] Sidhu | Failure of resin-modified glass-ionomers subjected to shear loading[END_REF][START_REF] Griffiths | Video rate confocal microscopic imaging of dentin/adhesive interfacial failure under load[END_REF].

The latest improvements of UHSI for both higher frame rate (320,000 fps) and image quality (resolution and dynamic range) will allow isolating and characterizing the first event and those following until catastrophic failure.

For the second interrogation, shear and tensile bond strength tests (macro-or micro-test; SBS, TBS, µSBS, µTBS) are usually static tests. They generate a more or less homogeneously distributed stress field that could deliver an indicative interfacial adhesion load. According to Guggenbül et al. (2021), "µSBT could be more suitable for testing the adhesion of resin-based materials to ceramics." Shear stresses are believed to be major stresses involved in in vivo bonding failures in CARs [START_REF] Guggenbühl | Adhesion of resin-resin and resin-lithium disilicate ceramic: a methodological assessment[END_REF]. In this sense, we demonstrated by a preliminary finite element analysis presented in this study the interest of our original lithium disilicate (LiSi2) bonded sample and the benefit of a 45° resin bonding interface. The resulting shear-compression stress field induced to the resin bonding interface is closer to clinical reality than other interfacial mechanical tests (TBS, µTBS, SBS, µSBS tests) [START_REF] Burke | Fracture resistance of teeth restored with dentin-bonded crowns constructed in a leucitereinforced ceramic[END_REF][START_REF] Blatz | Resin-ceramic bonding: a review of the literature[END_REF][START_REF] Chi | Aspects of adhesion tests on resin-glass ceramic bonding[END_REF][START_REF] Pineda-Vásquez | Shear bond strength of debonded ceramic restorations re-cemented by means of a cleaning and retreatment protocol[END_REF][START_REF] Venturini | Fatigue performance of adhesively cemented glass-, hybrid-and resin-ceramic materials for CAD/CAM monolithic restorations[END_REF][START_REF] Guggenbühl | Adhesion of resin-resin and resin-lithium disilicate ceramic: a methodological assessment[END_REF]).

Concomitantly, with clinical consideration, we decided to compare 2 ceramic pretreatments. Classically, the ceramic intaglio is conditioned with hydrofluoric acid (HF), allowing a selective elimination of the vitreous matrix causing a roughness of the ceramic and more surface energy [START_REF] Siqueira | Effect of self-etching primer associated to hydrofluoric acid or silane on bonding to lithium disilicate[END_REF]. Then, a primer containing organo-silane is applied to promote bifunctional and durable adhesion [START_REF] Blatz | Resin-ceramic bonding: a review of the literature[END_REF][START_REF] Dejak | Three-dimensional finite element analysis of strength and adhesion of composite resin versus ceramic inlays in molars[END_REF][START_REF] Lise | Microshear bond strength of resin cements to lithium disilicate substrates as a function of surface preparation[END_REF]. However, HF is toxic for the human body and the environment, and its use has been prohibited in several countries [START_REF] Özcan | Possible hazardous effects of hydrofluoric acid and recommendations for treatment approach: a review[END_REF][START_REF] Kalavacharla | Influence of etching protocol and silane treatment with a universal adhesive on lithium disilicate bond strength[END_REF][START_REF] Maier | New approaches in bonding to glass-ceramic: self-etch glass-ceramic primer and universal adhesives[END_REF][START_REF] Dapieve | Onestep ceramic primer as surface conditioner: effect on the load-bearing capacity under fatigue of bonded lithium disilicate ceramic simplified restorations[END_REF]. A recent nontoxic self-etching glass-ceramic primer, Monobond Etch & Prime (MEP; Ivoclar-Vivadent), was proposed to secure and simplify the bonding protocol. Ammonium polyfluoride contained in MEP is milder than HF and generates a less rough surface (El-Damanhoury and Gaintantzopoulou 2018).

However, this single-component product allows etching and silanizing in 1 step, with the same application time regardless of the glass ceramic [START_REF] Maier | New approaches in bonding to glass-ceramic: self-etch glass-ceramic primer and universal adhesives[END_REF].

The purpose of the present study was to visualize and characterize by UHSI the failure phenomena at the resin bonding interface by shear-compression test of LiSi2 samples bonded with gold-standard protocol and the new nontoxic alternative.

2

Material and method

Samples

Ten discs (diameter 18.5 mm, thickness 4 mm) of LiSi2 (IPS E.max press; Ivoclar-Vivadent) were diametrically cut in half with a laboratory saw (Isomet 2000; Buehler). Samples were divided in 2 groups. Intaglio pretreatment and bonding protocol were achieved according to the manufacturer recommendations. For the first group (n = 5), surfaces were etched for 20 s using 5% HF (IPS Ceramic Etching Gel; Ivoclar-Vivadent), rinsed off, and thoroughly dried. Then a thin coat of silane agent (Monobond Plus [MB]; Ivoclar-Vivadent) was applied using a microbrush. The silane was allowed to react for 60 s, and then a strong stream of air was used to disperse any remaining excess. The second group (n = 5) received the MEP (Ivoclar-Vivadent) 1-step ceramic primer. The MEP was applied on the adhesive surface using a microbrush and agitated into the surface for 20 s. It was left to react for another 40 s. Then the MEP was thoroughly rinsed off with water and the restoration was dried with a strong jet of water-and oil-free air for 10 s. Bonding protocol was the same for each sample, regardless of the conditioning treatment. Samples parts were bonded (Variolink Esthetic DC Neutral; Ivoclar Vivadent), and resin was light cured 1 min with a 1,200-W light-polymerizing unit (Bluephase Style 20i; Ivoclar-Vivadent) on each face. Ceramic resin bonding interface thickness was measured as 600 µm. Then, bonded discs were cut to obtain 2 parallel surfaces at the top and bottom of each sample and a 45° inclined bonding resin interface according to the finite element analysis (FEA) presented hereafter (Fig. 1).

Mechanical test

As illustrated in Figure 1, samples were submitted to a compression loading performed at a crosshead speed of 1 mm/min on a Zwick ZH010 testing machine (ZwickRoell GmbH) equipped with a 10-kN load cell. This test allows the bonded layer to be submitted to a shear-compression stress state. A 20-N preload was applied to the sample before the test. During the test, force and displacement were continuously recorded up to failure. The stiffness of samples in kN/mm is also evaluated between 0.3 and 0.5 mm of displacement.

Figure 1. Scheme of the mechanical and optical setup.

Ultra-High-Speed Imaging

The fracture phenomena were recorded with a UHS Phantom TMX7510 camera (Phantom TMX7510; Vision Research). This camera has 76 Gpix/s throughput, offering 1,280 × 800 back-side illuminated (BSI; 18.5 µm)

CMOS pixels at 76 kfps, which was equipped with a 105-mm magnification macro lens (Nikon) and using image resolution of 640 by 320 pixels, a frame interval of 3.33 µs, at a maximum rate of 300,000 fps at this resolution mode thanks to 2 × 2-pixel binning. This mode ensures sensitivity and leads to shorter exposure time. Binning also reduces resolution in order to deal with ultra-high-speed imaging frame rates. The BSI CMOS of the TMX 7510 with 512-GB onboard memory can then tackle frame rates and resolution of in situ image storage (ISIS) cameras but with huge image storage in a rolling memory buffer. Additional light source (Veritas Constellation 120; IDT Europe) delivered 80,000 lux of cold light at 1 m to obtain the best-recorded image by allowing a 2-µs exposure time to freeze the object displacement (Fig. 1). During the test, the camera was continuously recording images in onboard 512-GB memory. When memory gets "full," recording continues by cycling back to the beginning, constantly overwriting itself (FIFO rolling buffer mode until the camera is triggered). For these tests, the trigger stopped the record and enabled saving all required frames present in the rolling memory buffer before and after the trigger event (e.g., a sudden decrease of the force at break or a crack sound). All the captured images were examined thanks to the dedicated software (Phantom Camera Control [PCC]; Vision Research) to chronologically identify different events until catastrophic failure.

Finite Element Analysis (FEA)

A numerical model was developed using Comsol Multiphysics Finite Element software, in order to calculate and analyze the stress state inside a sample subjected to the proposed mechanical test, with a particular interest at the resin-ceramic bonding interface. The modeled geometry corresponds to a ceramic disk (diameter 18.5 mm, thickness 4 mm) diametrically split in 2 halves separated by a 600-µm resin joint and cropped to a 15-mm total height. The mechanical behaviors of both ceramic and resin were assumed linear and elastic before failure.

Displacement continuity at the interfaces was considered in the model, which corresponds to perfectly adhesive interfaces. According to average values of the literature, the Young's modulus of the ceramic (respectively, the resin) was set to 80 GPa (respectively, 8 GPa) and the Poisson's ratio to 0.2 (respectively, 0.3). As illustrated in Figure 2A, upper and bottom flat surfaces are subjected to a 10-kN evenly distributed axial compression load (bottom surface being fixed for stability). The joint angle (α) was defined as a variable design parameter in the finite element (FE) model in order to assess its influence on the stress state inside the sample. According to the plane stress hypothesis, a 2-dimensional FE mesh has been produced. A mesh convergence study has been carried out, leading to 7,914 elements (6,314 triangles, 1,600 quadrangles), with a denser mesh near the resinceramic interfaces, as displayed in Figure 2B (for α = 45°). The FE parametric resolutions confirmed that the gradual variation of the joint angle from 0 to 45 degrees enables increasing the amount of shear stress inside the resin (from zero up to a value higher than compression stress), as plotted in Figure 2A. Thus, because higher shear stresses are prone to trial the bonding interface (for a given axial load value), the joint angle was subsequently set at 45° for the samples designed in that study. A FE plot of the shear strain distribution in the sample for α = 45° (Fig. 2C) corroborates that the shear strain is discontinuous at the bonding interface and is higher in the resin than in the ceramic. For all tested samples, visual observations revealed an adhesive failure where 1 face is covered by bonding resin while the opposite side is raw ceramic. The first event observed for MEP6 was a sudden bleaching that occurred in the resin bonding interface near the center of the sample (labeled t3.3 µs in Fig. 4). The previous frame was defined as the reference time (t0). Then, in the upper right corner, the resin bonding interface bleached and a first crack appeared in ceramic (t6.6 µs). At t9.9 µs, the identified bleached zone increased and a relative displacement (slippage) of the 2 ceramic parts and resin occurred in the lower left corner. For t13.2 µs, the displacement stepped up and the resin bonding interface bleached in the same area. The following images rom 16,5 µs to 46,2 µs (in Fig. 4) show the catastrophic failure resulting from the initial rupture.

The time code was defined similarly for all samples. For the MB6 sample (Figure 5), the first event observed occurred in the lower left corner. A slight slippage was visualized (labeled t3.3 µs in Fig. 5), becoming larger at t6.6 µs, when a ceramic crack appeared near the center of the sample. From t9.9 µs to t13.2 µs, resin bonding interface bleached in the upper right corner and slippage still extended. The following images from 16,5 µs to 46,2 µs show the catastrophic failure resulting from the initial rupture.

Discussion

The observation in Figure 3 reveals that the sample preparation protocol has a negligible impact on the overall shape of the force-displacement curves. The stiffness in each sample (Fig. 3), for both MB and MEP, is around 16 kN/mm with a low standard deviation. Such a result was expected as the bonding procedure has no influence on the macro-homogeneous response of the material. Despite the mechanical test being able to assess the impact of the bonding protocol on the shear-compression strength, results evidenced no significant differences between MB and MEP samples for both strength and stiffness as expressed by the high P values (0.34; respectively, 0.49) calculated with a 2-tailed Student's test (the level of significant difference being set at 0.05). The meticulous analysis of UHSI images by high digital zoom and slow motion scrutiny makes it possible to characterize and decompose the failure mechanism. As shown in Figures 4 and5, it is already possible to observe that the first occurring event is a modification inside the resin bonding interface followed by catastrophic failure.

Then, the succession of images shows that, even if secondary cracks can occur in the ceramic or in the bonded layer, the principal crack, leading to the catastrophic failure of the whole sample, propagates at the interface between ceramic and the bonded layer (see Figures 4 and5 and multimedia components). Thanks to the extreme frame rate, the speed failure was calculated. The slippage speed was about 10 m/s while the crack propagation can reach about 150 m/s for the crack, appearing up to 1,000 m/s when the crack develops. The FEA put forward the combined shear and compressive stress at the interface (Fig. 2). The shear stress led to the slippage of the materials at rupture while the compressive stress prevented their separation, resulting in a micro-displacement of the upper half-disk downward (Figs. 4 and5). Secondary cracks appeared subsequently by strength transfer initiated by debonding in the resin bonding interface. For all tested samples, cracks started at the interface toward press jaws. Results for MB and MEP (Fig. 3 and Appendix Table ) suggest that the MEP treatment slightly improves the bonding strength of the specimens while reducing dispersion of the results. Clinicians seek materials with the easiest protocol leading to repeatable results in order to ensure a serene daily activity. In this study, the intaglio pretreatment did not seem to have any significant impact on the macro-homogeneous mechanical response; the mean shear-compression strength was 7,850 N (SD = 1,420 N) for MB and 8,690 N (SD = 860 N) for MEP (Fig. 3 and Appendix Table ). These results seem consistent with the literature [START_REF] Román-Rodríguez | Bonding to silicate ceramics: conventional technique compared with a simplified technique[END_REF][START_REF] Wille | Durability of resin bonding to lithium disilicate and zirconia ceramic using a self-etching primer[END_REF][START_REF] Dönmez | Novel ceramic primer vs. conventional treatment methods: effects on roughness and bond strength of all-ceramic restorations[END_REF][START_REF] Lyann | Effect of different surface treatments on the tensile bond strength to lithium disilicate glass ceramics[END_REF][START_REF] Prado | Ceramic surface treatment with a single-component primer: resin adhesion to glass ceramics[END_REF][START_REF] Lopes | Does a self-etching ceramic primer improve bonding to lithium disilicate ceramics? Bond strengths and FeSEM analyses[END_REF][START_REF] Vichi | Comparison between hydrofluoric acid and single-component primer as conditioners on resin cement adhesion to lithium silicate and lithium disilicate glass ceramics[END_REF]

Figure 2 .

 2 Figure 2. FE Analysis (A) Compressive stress (in MPa) at resin central point versus joint angle (α, in degrees) for a 10-kN compression load. Green triangles (respectively, blue squares) correspond to direction along (respectively, across) the joint. Red circles correspond to the shear component of stress. (B) Finite element (FE) mesh (displayed for α = 45°). (C) FE plot of the shear strain in the sample (displayed for α = 45°), highlighting the highest shear strain inside the resin. 5

Figure 3 .

 3 Figure 3. Load/displacement curves of each Monobond Plus (MB) and Monobond Etch & Prime (MEP) sample submitted to compression/shear test.

Figure 4 .

 4 Figure 4. Sequence of successive images recorded by ultra-high-speed imaging (delay between each image is 3.3 µs) for Monobond Etch & Prime sample until failure (failure load: 7,560 N). Focus on the failure mechanism: (A) first visible event: bleaching at resin bonding interface, (B) crack initiation highlighted by Edge Laplacian filtering, (C) slippage of the ceramic, (D) crack propagation and resin bonding bleaching, (E) following slippage, and (F) crack propagation highlighted by Edge Laplacian filtering.

Figure 5 .

 5 Figure 5. Sequence of successive images recorded by ultra-high-speed imaging (delay between each image is 3.3 µs) for Monobond Plus sample until failure (failure load: 7,435 N). Focus on the failure mechanism: (A) initial situation, (B) first visible event: slippage of the ceramic, (C) crack initiation highlighted by Edge Laplacian filtering, (D) following slippage, (E) bleaching at resin bonding interface, (F) following slippage, (G) following displacement and bleaching at resin bonding interface, and (H) crack propagation highlighted by Edge Laplacian filtering.

  Such events have never been clearly and entirely recorded and described. UHSI studies on dental material behavior are unusual.Griffith et al. (2000) first used UHSI coupled to a mechanical test. Composite restoration bonded on dentin was submitted to SBS without drawing any conclusion on the bonding interface failure mechanism.[START_REF] Forquin | Application of microtomography and image analysis to the quantification of fragmentation in ceramics after impact loading[END_REF] used UHSI to analyze the quantification of fragmentation in ceramics after impact loading.[START_REF] Hosaka | Ultra-high-speed videography of resin-dentin interface failure dynamics under tensile load[END_REF] observed with USHI the bonding interface of dentin and restorative composite in µTBS. They concluded, "UHS videography with even higher frame rate and resolution can help in understanding the mechanism of fracture dynamics."In this study, the used camera had a much higher frame rate and resolution than those used by Hosaka. Moreover, a shear compression test was operated to get closer to physiologic and pathologic (bruxism) stresses. With this objective, shear and compression cannot be dissociated due to the mastication functionality to reduce the alimentary bolus.

  . Using SBS tests,[START_REF] Román-Rodríguez | Bonding to silicate ceramics: conventional technique compared with a simplified technique[END_REF] obtained higher data for MB with greater homogeneity than MEP without a statistically significant difference.[START_REF] Alrahlah | Effect of self etching ceramic rimer and universal adhesive on bond strength of lithium disilicate ceramic[END_REF], also with SBS tests, combined with universal adhesive, obtained higher values for MEP than MB without a statistically significant difference. Slight differences between MEP and MB bond strength tests are reported in the literature but without statistically significant differences.[START_REF] Vichi | Comparison between hydrofluoric acid and single-component primer as conditioners on resin cement adhesion to lithium silicate and lithium disilicate glass ceramics[END_REF] obtained similar results for µSBS with LiSi2 conditioned by HF and HF + Silane and questioned the usefulness of applying silane after HF etching in clinical situations. Furthermore, for these authors, MEP and MB performed comparable bond strength values, and the choice should "be based on other factors."5 ConclusionThanks to UHSI technological evolutions, this study shows that failure of bonded LiSi2 submitted to compression-shear strength is first due to debonding followed by catastrophic failure. Higher frame rate associated with sufficient camera resolution allowed us to visualize for the first time the failure mechanism. This 9

work highlights the usefulness of UHSI in dental research and begins to provide concrete answers to a major clinical prosthodontic problem. FEA indicated the interest of our original 45° resin bonding LiSi2 sample. The resulting shear-compression stress induced to the resin bonding interface draws closer from the clinical situation than other mechanical tests. Moreover, within the limits of this study, the nontoxic alternative pretreatment (MEP) shows a similar mechanical behavior to the gold standard (MB). Catastrophic failure results with no significant difference indicate MEP as an alternative to the gold standard in clinical practice. Thus, UHSI appears as a first-rate investigation protocol for a study of dental material mechanical behavior. It paves the way to new research possibilities in dental mechanics and biomechanics. New investigations are in progress with dental sample-bonded CARs.

Acknowledgments

The ceramic, conditioning, and bonding materials were generously supplied by the manufacturer (Ivoclar Vivadent AG). The authors thank Ametek Phantom France for lending the Phantom TMX7510 high-speed camera during the test period. They express their thanks and appreciation to Guillaume Ounoughi for his help with laboratory procedures.

Funding

The authors received no financial support for the research, authorship, and/or publication of this article.

ORCID iD

H. Lambert https://orcid.org/0000-0002-4160-9491

Author Contributions

H. Lambert, S. Corn, R. Léger, P. Slangen, M. Fages, contributed to conception and design, data acquisition, analysis, and interpretation, drafted and critically revised the manuscript; P. Ienny, contributed to conception and design, data analysis, and interpretation, critically revised the manuscript. All authors gave their final approval and agree to be accountable for all aspects of the work.

Declaration of Conflicting Interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.


Hugo Talibart

François Coste

Mathilde Carpentier

PPalign: optimal alignment of Potts models representing proteins with direct coupling information

Introduction

To assign structural and functional annotations to the ever increasing amount of sequenced proteins, the main approach relies on sequence-based homology search methods, e.g. BLAST or the current state-of-the-art methods based on profile Hidden Markov Models, which rely on significant alignments of query sequences to annotated proteins or protein families. While powerful, these approaches do not take coevolution between residues into account. Taking advantage of recent advances in the field of contact prediction, our approach, recently published in BMC Bioinformatics [START_REF] Talibart | PPalign: optimal alignment of Potts models representing proteins with direct coupling information[END_REF], proposes to represent proteins by Potts models, which model direct couplings between positions in addition to positional composition, and to compare proteins by aligning these models. Due to non-local dependencies, the problem of aligning Potts models is hard and remains the main computational bottleneck for their use.

Inference of Potts models

As introduced in Direct Coupling Analysis [START_REF] Weigt | Identification of direct residue contacts in protein-protein interaction by message passing[END_REF], a Potts model for a multiple sequence alignment (MSA) of homologous sequences can be defined as a statistical model whose probability distribution maximizes Shannon entropy and generates the empirical single and double frequencies of the MSA as marginals.

Its probability distribution has the following form:

P(x|w, v) = 1 Z exp L-1 i=1 L j=i+1 w ij (x i , x j ) + L i=1 v i (x i ) Probability of sequence x = x 1 , . . . , x L

Normalization constant

Couplings Fields Its parameters can be assigned a practical interpretation:

• v = {v i } i=1,••• ,L are positional parameters termed "fields". (v i ∈ R q ) v i (a)
∼ propensity of letter a to be found at position i.

• w = {w ij } i,j=1,••• ,L are pairwise "coupling" parameters. (w ij ∈ R q×q ) w ij (a, b)
∼ compatibility of letters a and b at positions i and j

From protein sequence to Potts model: One can get a Potts model for a sequence by inferring it on a MSA of its close homologs. In this study, homologs were retrieved using HHblits [START_REF] Steinegger | HH-suite3 for fast remote homology detection and deep protein annotation[END_REF], then MSAs were processed by filtering at 80% identity, setting a depth threshold at 1000, trimming columns with > 50% gaps, and fed to CCMpredpy [START_REF] Vorberg | Synthetic protein alignments by CCMgen quantify noise in residue-residue contact prediction[END_REF] to infer Potts models.

Alignment as an ILP problem

We built on the work of Wohlers et al. [START_REF] Wohlers | DALIX: optimal DALI protein structure alignment[END_REF], initially dedicated to protein structure alignment, to propose an Integer Linear Programming formulation for the alignment of two Potts models A and B of parameters (v A , w A ) and (v B , w B ).

max

L A i=1 L B k=1 ⟨v A i , v B k ⟩x ik + L A -1 i=1 L A j=i+1 L B -1 k=1 L B l=k+1 ⟨w A ij ,
y ikrs j ∈ [i + 1, L A ], i ∈ [1, L A -1], k ∈ [1, L B -1]
x ik ≥ r.s∈col ik (l)

y ikrs l ∈ [k + 1, L B ], i ∈ [1, L A -1], k ∈ [1, L B -1]
x ik ≥ r.s∈row ik (j)

y rsik j ∈ [1, i -1], i ∈ [2, L A ], k ∈ [2, L B ]
x ik ≥ r.s∈col ik (l)

y rsik l ∈ [1, k -1], i ∈ [2, L A ], k ∈ [2, L B ] x ik ≤ r.s∈row ik (j) s(A ri ,B sk )≤0 (y rsik -x rs ) + 1 j ∈ [1, i -1], i ∈ [2, L A ], k ∈ [2, L B ] k l=1 x il + i-1 j=1 x jk ≤ 1 i ∈ [1, L A ], k ∈ [1, L B ]
x, y binary alignment constraints:

one-to-one and non-crossing Similarity score based on scalar product:

• ⟨v A i , v B k ⟩ = a v A i (a)v B k (a) • ⟨w A ij , w B kl ⟩ = a b w A ij (a, b)w B kl (a, b)
Using their efficient solver, the exact solution of this ILP within a chosen epsilon range can be computed in tractable time.

PPalign improves alignment quality of remote homologs

We assessed the quality of PPalign's alignments on a benchmark of low sequence identity (4-18%) pairwise sequence alignments based on reference structural alignments from SISYPHUS [START_REF] Andreeva | SISYPHUS-structural alignments for proteins with non-trivial relationships[END_REF] using the F 1 score metric:

Ongoing work and perspectives

Our current work now focuses on the inference of Potts models more suitable for pairwise comparison, which was not their original purpose. By improving their robustness to sampling variations and seeking a more canonical form, we are hoping to improve these already encouraging results and to better assess the contribution of direct couplings.

This research provides ground work for future exciting applications such as a homology search package which would take coevolution into account, or Potts model annotation databases (e.g. for viral proteins).

  sequence HHblits MSA trim filter train MSA CCMpredPy Potts model Alignment of Potts models Potts models provide an interesting alternative to pHMMs for sequence comparison since they can model pairwise dependencies in addition to positional conservation. To investigate on their performances in alignment-based homology detection, we introduced PPalign, a pairwise Potts model alignment method. PPalign can provide in tractable time an alignment of two protein sequences which takes both positional composition and pairwise dependencies into account by aligning Potts models representing them.

  Decision variables: x, y binary • x ik = 1 iff node i and node k aligned • y ikjl = 1 iff edges (i, j) and (k, l) aligned

F 1 =

 1 2P R P +R where P = # correctly aligned pairs # aligned pairs in computed alignment , R = # correctly aligned pairs # aligned pairs in reference alignment PPalign's alignments achieve a better mean F 1 score than HHalign's [3] alignments of pHMMs built on the same MSAs (0.600 vs 0.578), while BLAST [7] fails to align most sequences (mean F 1 score of 0.113). PPalign outperforms HHalign in 12/22 alignments (4 significantly •), with better F 1 scores when sequence identity is lower. It is mostly outperformed when MSAs have more gaps (•).

Conclusion•

  PPalign initiates a new approach for remote homology search • Similarly to HHalign from HHsuite... • ...with the addition of long distance sequence correlations reflecting higher order constraints • Tractable time (1 ′ 37 ′′ on average in this study) despite computationally hard • Encouraging results in terms of alignment quality Code and benchmark: https://www-dyliss.irisa.fr/ppalign