Bacillus subtilis spore induces efficient generation of memory T cells via ICAM-1 expression on dendritic cells

The intestinal mucosa is the primary exposure and entry site of infectious organisms. Tissue-resident memory T cells (Trms) is an important first line of defense against infection in mucosal tissues, their function in intestinal immunization remains to be investigated. Here, we reported that the levels of local mucosal and systemic immune responses were enhanced through oral immunization with H9N2 whole inactivated virus (H9N2 WIV) plus spore. Subsequently, H9N2 WIV plus spore led to the generation of CD103+CD69+ Trms, which was independent of circulating T cells during the immune period. Meanwhile, we also found that Bacillus subtilis spore can stimulate Acrp30 expression in 3T3-L1 adipocytes. Moreover, adipocyte supernatant or spore also upregulated intercellular adhesion molecule-1 (ICAM-1) expression on dendritic cells (DCs) (P<0.01). Furthermore, the proportion of HA-tetramer+cells was severely curtailed when ICAM-1 expression was suppressed, which was also dependent on HA-loaded DCs. Taken together, our data demonstrated that spore promoted the immune response by stimulating Trms, which were associated with activation of ICAM-1 in DCs. Author summary Taken together, Bacillus subtilis spore combined with H9N2 WIV enhanced the mucosal antibody response and induced efficient intestinal-resident memory T cells. Then we demonstrated that spore can induce memory T cell formation through an ICAM-1-mediated contact of a dendritic cell (DC)-derived mechanism. Further, our findings indicated that Acrp30 from adipocytes induced by spore might increase ICAM-1 expression on DCs, which might provide new insight into the significance of adipocyte metabolism related molecules in regulating immunological memory T cells.

especially in modulating intestinal mucosal immunity through evoking tissue-resident 48 memory T cell (Trm) [7][8][9]. 49 Previous studies on Bacillus subtilis spore found that it can stimulate the 50 secretion of cytokines as innate immune signaling, which is indispensable for efficient 51 induction of adaptive immune responses during primary immunization [10]. Recent 52 study found that mucosal immunization with Spore-FP1increased CD69 + CD103 + Trm 53 in the lung parenchyma [11]. Meanwhile, our previous study also confirmed Bacillus 54 subtilis spore, as advantages of mucosal delivery, could regulate memory T cells in 55 the intestine of piglets [12]. As we known, T cells is important for cell immunity,  60 and effector memory T cells (Tems) [14]. Moreover, Trms mediate rapid clearance of 61 and heterosubtypic protection against secondary IAV infections in mice [15,16]. 62 Further analyses have revealed that Trms were also detected in the intestinal mucosa, 63 leading to tissue-specific influences [17][18][19]. Until recently, Trms, which express high 64 levels of C-type lectin CD69 and low levels of the sphingosine-1-phosphate (S1P) 65 receptor S1PR1, are thought to be phenotypically and functionally distinct from 66 circulating memory T cells [20]. Furthermore, establishment of long-term and resident 67 memory depends on the maintenance of CD103 and CD69 expression in T cells [21]. 68 Thus, the features and processes of memory cells are involved in the retention and 69 persistence of T cells in mucosal tissue, thereby promoting long-term protection for 70 viral clearance [19]. 71 Our study provided further insight into the potential immunopotentiator ability of 72 Bacillus subtilis spore to assist PEDV WIV in the potentiation of immunity by 73 upregulating memory T cells via oral immunization in piglets [12]. However, the 74 specific mechanism in memory cell formation remains to be further studied. Previous DCs are essential for the generation of T-cell immunity after mucosal immunization 92 [25]. To investigate whether spore had the capacity to recruit submucosal DCs to form 93 transepithelial dendrites (TEDs) for viral capture, we assessed TED formation in vitro 94 and in vivo. Initially, in a DC/epithelial cell (EC) coculture system (Fig. 1A), we 95 observed spore, but not medium, induced DCs to form TEDs across ECs at 30 min in 96 cross-sectional images (Fig. 1B). Then, Ligated loop experiments at 0.5 h after spore 97 adminstrated found DCs were apparently gathering to the laminal propria of ECs, 98 which were significantly increased by spore compared with the control (Fig. 1C). 99 Moreover, we found spore had the powerful capacity to increase the expression of     Significance was tested against the unstimulated control by one-way ANOVA, *P <     208 We also investigated antigen-specific Trms after mucosal immunization. To detect 209 whether FTY720 treatment could inhibit lymphocyte circulation, FTY720 was 210 administered to inhibit the circulation of T cells six weeks after the primary 211 vaccination, as illustrated in Figure 4A. In addition, mice were injected i.v. with  Data are represented as the mean ± SEM (n=6). *P < 0.05, **P < 0.01. One  Since we have demonstrated that spore could enhance the Acrp30 level in intestine 293 (Fig. 5A). In order to determine stimulation effects of spore treatment on 294 differentiated 3T3-L1 cells, we performed the induction culture assay of adipocytes.

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Initially, cells displayed a fibroblast phenotype. Then, cell morphology changed and 296 cells accumulated lipid droplets internally during the process of differentiation. After 297 12 days, when the adipocytes were mature, almost the entire cell volume was stained 298 red by red oil (Fig. 7A). We subsequently determined the effects of spore on Acrp30 299 protein expression. Spore at 10 6 CFU/ml was sufficient to elicit up-regulation of the 300 Acrp30. Treatment of 10 7 CFU/ml of spore led to a 2-fold increase in the Acrp30 301 protein amount in comparison with the control without spore (Fig. 7B). The extent of with spore treatment, and the treatment of ICAM-1 inhibitor A-205804 suppressed the 331 CD44 + CD69 + phenotype induced by spore ( Fig. 8A and B)(P < 0.01).

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To assess whether HA-tetramer + specific T cells persisted in the intestine at 6 333 weeks after immunization, antigen-loaded DCs with or without ICAM-1 inhibitor 334 were incubated with IMALs isolated from immunized mice at 37°C for 3 d (Fig. 8D).

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Flow cytometric results showed that spore plus H9N2 WIV induced more 336 HA-tetramer + specific cells than H9N2 WIV alone at the present of antigen-pulsed 337 DCs ( Fig. 3E and F). Taken together, these results indicated that DCs partially require 338 the expression of ICAM-1 for the generation of antigen-specific Trms, which were 339 altered in the presence of ICAM-1 inhibitor added in DCs.  antigens. Furthermore, we demonstrated that SF produced by Bacillus subtilis spore 415 also induced more antibodies through the Th2 response, thus differing from spore. 416 Overall, these findings may reflect a detection limitation in our assays or at least some 417 potential metabolites of Bacillus subtilis spore as active components of mucosal 418 immune enhancement. 419 Here, spore was able to induce a dramatically larger percentage of proliferating  Agriculture University. In this study, the medium was taken from 3T3-L1 adipocytes 520 treated with spore varying from 10 6 and 10 7 CFU/ml for 24 h. Then cell culture 521 supernatant from adipocyte was added to DCs for another 24 h. 522 523 FTY720 treatments and tetramer staining 524 To inhibit circulation of memory T cells, FTY720 (Sigma) 1 mg/kg in PBS was 525 administered intraperitoneally (i.p.), daily for 10 d. In addition, to assess the 526 protective efficacy of the vaccines, the mice were immunized with the same vaccine.

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Samples were analyzed with a mouse XL cytokine array kit (R&D Systems), 574 according to the manufacturer's instructions [49]. Immunospots were captured with 575 an Odyssey Fc Imager (LI-COR), and data were analyzed with ImageJ software.

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Histology and immunohistochemistry 578 Immunohistochemistry detection was performed with the SABC kit (Boster 579 Bioscience). Intrinsic peroxidase in samples was inactivated using 3% hydrogen   The fixed filters were permeabilized in 0.5% Triton X-100 in PBS for 5 min and 588 blocked with 5% bovine serum albumin (BSA) in PBS for 2 h. Then the filters were 589 stained with primary antibodies Armenian hamster anti-CD11c (N418) and rabbit 590 anti-ICAM-1 (1A29, Abcam) overnight at 4°C, followed by incubation with 591 secondary antibodies for 2 h at room temperature. For the in vivo model, cryosections 592 were treated as described above. The filters were identified using confocal laser 593 scanning microscopy (CLSM) (LSM 710; Zeiss, Oberkochen, Germany).  Western blot assay 610 The cells were lysed with RIPA buffer containing a 1% protease inhibitor cocktail on 611 ice for 20 min. After removing debris by centrifugation at 4°C, supernatant protein 612 was collected, and the total concentration was determined by a BCA protein assay kit.  The asterisks indicate significant differences between H9N2 WIV plus spore and 626 H9N2 WIV. P values < 0.05 were considered to be statistically significant (* P < 0.05, 627 **P < 0.01).