"Find Me" and "Eat Me" signals: tools to drive phagocytic processes for modulating antitumor immunity.

Phagocytosis, a vital defense mechanism, involves the recognition and elimination of foreign substances by cells. Phagocytes, such as neutrophils and macrophages, rapidly respond to invaders; macrophages are especially important in later stages of the immune response. They detect "find me" signals to locate apoptotic cells and migrate toward them. Apoptotic cells then send "eat me" signals that are recognized by phagocytes via specific receptors. "Find me" and "eat me" signals can be strategically harnessed to modulate antitumor immunity in support of cancer therapy. These signals, such as calreticulin and phosphatidylserine, mediate potent pro-phagocytic effects, thereby promoting the engulfment of dying cells or their remnants by macrophages, neutrophils, and dendritic cells and inducing tumor cell death. This review summarizes the phagocytic "find me" and "eat me" signals, including their concepts, signaling mechanisms, involved ligands, and functions. Furthermore, we delineate the relationships between "find me" and "eat me" signaling molecules and tumors, especially the roles of these molecules in tumor initiation, progression, diagnosis, and patient prognosis. The interplay of these signals with tumor biology is elucidated, and specific approaches to modulate "find me" and "eat me" signals and enhance antitumor immunity are explored. Additionally, novel therapeutic strategies that combine "find me" and "eat me" signals to better bridge innate and adaptive immunity in the treatment of cancer patients are discussed.

The impact of the gut microbiome on tumor immunotherapy: from mechanism to application strategies.

Immunotherapy is one of the fastest developing areas in the field of oncology. Many immunological treatment strategies for refractory tumors have been approved and marketed. Nevertheless, much clinical and preclinical experimental evidence has shown that the efficacy of immunotherapy in tumor treatment varies markedly among individuals. The commensal microbiome mainly colonizes the intestinal lumen in humans, is affected by a variety of factors and exhibits individual variation. Moreover, the gut is considered the largest immune organ of the body due to its influence on the immune system. In the last few decades, with the development of next-generation sequencing (NGS) techniques and in-depth research, the view that the gut microbiota intervenes in antitumor immunotherapy through the immune system has been gradually confirmed. Here, we review important studies published in recent years focusing on the influences of microbiota on immune system and the progression of malignancy. Furthermore, we discuss the mechanism by which microbiota affect tumor immunotherapy, including immune checkpoint blockade (ICB) and adoptive T-cell therapy (ACT), and strategies for modulating the microbial composition to facilitate the antitumor immune response. Finally, opportunity and some challenges are mentioned to enable a more systematic understanding of tumor treatment in the future and promote basic research and clinical application in related fields.

The claim of primacy of human gut Bacteroides ovatus in dietary cellobiose degradation.

A demonstration of cellulose degrading bacterium from human gut changed our view that human cannot degrade the cellulose. However, investigation of cellulose degradation by human gut microbiota on molecular level has not been completed so far. We showed here, using cellobiose as a model that promoted the growth of human gut key members, such as Bacteroides ovatus (BO), to clarify the molecular mechanism. Our results showed that a new polysaccharide utilization locus (PUL) from BO was involved in the cellobiose capturing and degradation. Further, two new cellulases BACOVA_02626GH5 and BACOVA_02630GH5 on the cell surface performed the degradation of cellobiose into glucose were determined. The predicted structures of BACOVA_02626GH5 and BACOVA_02630GH5 were highly homologous with the cellulase from soil bacteria, and the catalytic residues were highly conservative with two glutamate residues. In murine experiment, we observed cellobiose reshaped the composition of gut microbiota and probably modified the metabolic function of bacteria. Taken together, our findings further highlight the evidence of cellulose can be degraded by human gut microbes and provide new insight in the field of investigation on cellulose.

CXCL10-armed oncolytic adenovirus promotes tumor-infiltrating T-cell chemotaxis to enhance anti-PD-1 therapy.

Resistance remains an obstacle to anti-programmed cell death protein 1 (PD-1) therapy in human cancer. One critical resistance mechanism is the lack of T cell chemotaxis in the tumor microenvironment (TME). CXCL10-CXCR3 signaling is required for T cell tumor infiltration and tumor immunotherapy. Oncolytic viruses (OVs), including oncolytic adenoviruses (AdVs), induce effective T cell immunity and tumor infiltration. Thus, arming OV with CXCL10 would be an attractive strategy to overcome resistance to anti-PD1 therapy. Here, we successfully constructed a novel recombinant oncolytic adenovirus encoding murine CXCL10, named Adv-CXCL10. Through intratumoural injection, the continuous expression of the functional chemokine CXCL10 in the TME is realized to recruit more CXCR3+ T cells into the TME to kill tumor cells, and the recombinant adenovirus shows great power to 'fire up' the TME and enhance the antitumour efficiency of PD-1 antibodies.

Discrete genetic loci in human gut Bacteroides thetaiotaomicron confer pectin metabolism.

Although the polysaccharide utilization loci (PULs) activated by pectin have been defined, due to the complex of side-chain structure, the degradative mechanisms still remain vague. Thus, we hypothesize that there may have other specific PULs to target pectin. Here, we characterize loci-encoded proteins expressed by Bacteroides thetaiotaomicron (BT) that are involved in the pectin capturing, importation, de-branching and degradation into monosaccharides. Totally, four PULs contain ten enzymes and four glycan binding proteins which including a novel surface enzyme and a surface glycan binding protein are identified. Notably, PUL2 and PUL3 have not been reported so far. Further, we show that the degradation products support the growth of other Bacteroides spp. and probiotics. In addition, genes involved in this process are conservative in other Bacteroides spp. Our results further highlight the contribution of Bacteroides spp. to metabolism the pectic network.

Crataegus pinnatifida polysaccharide alleviates colitis via modulation of gut microbiota and SCFAs metabolism.

Inflammatory bowel disease (IBD) afflicted individual and most medications have side-effects. Crataegus pinnatifida (Hawthorn), which is a safe medicine and food homolog plant, has been reported to prevent colitis in murine. Yet the bioactivity component and the underlying molecular mechanism remain unclear. Here, we established a direct link between colitis induced by dextran sulphate sodium (DSS) in mice and polysaccharide HAW1-2 isolated from hawthorn. Our results showed HAW1-2 restored the pathological lesions in colon and inhibited the expression of inflammatory cytokines including IL-1ß, IL-6 and TNF-α. Meanwhile, IKKα/ß, IκBα, NF-κB and the phosphorylation levels were inhibited significantly. These findings suggested HAW1-2 could alleviate the inflammation of colon. Further, we found the composition of gut microbiota was modified and Bacteroides including Alistipes and Odoribacter were significantly enriched. Besides, we showed Alistipes and Odoribacter were positively co-related with acetic acid and propionic acid while were negatively co-related with inflammatory cytokines. Finally, we demonstrated the anti-inflammation activity of HAW1-2 might be induced by acetic acid. Together, the present data revealed HAW1-2 could directly modify the gut microbiota, especially for Bacteroides, and generate SCFAs to inhibit colitis. It also implies microbiota-directed intervention in IBD patients should be particularly given more attention.

Enhanced antitumor efficacy of a novel oncolytic vaccinia virus encoding a fully monoclonal antibody against T-cell immunoglobulin and ITIM domain (TIGIT).

BACKGROUND: Oncolytic virotherapy with vaccinia virus (VV) can lead to effective anti-tumor immunity by turning "cold" tumors into "hot" tumors. However, its therapeutic potential is affected by the tumor's local immunosuppressive tumor microenvironment (TME). Therefore, it is necessary to explore the use of immune checkpoint inhibitors to arm oncolytic VVs to enhance their anti-tumor efficacy. METHODS: A novel recombinant oncolytic VV, VV-α-TIGIT, which encoded a fully monoclonal antibody against T-cell immunoglobulin and ITIM domain (TIGIT) was generated by homologous recombination with a shuttle plasmid. The anti-tumor efficacy of the VV-α-TIGIT was investigated in several subcutaneous and ascites tumor models. FINDINGS: The functional α-TIGIT was sufficiently produced and secreted by tumor cells infected with VV-α-TIGIT, which effectively replicated in tumor cells leading to significant oncolysis. Intratumoral injection of VV-α-TIGIT improved anti-tumor efficacy in several murine subcutaneous tumor models compared to VV-Control (without α-TIGIT insertion). Intraperitoneal injection of VV-α-TIGIT achieved approximately 70% of complete tumor regression in an ascites tumor model. At the same time, treatment with VV-α-TIGIT significantly increased the recruitment and activation of T cells in TME. Moreover, the in vivo anti-tumor activity of VV-α-TIGIT was largely dependent on CD8+ T cell-mediated immunity. Finally, the tumor-bearing mice cured of VV-α-TIGIT treatment resisted rechallenge with the same tumor cells, suggesting a long-term persistence of tumor-specific immunological memory. INTERPRETATION: The recombinant oncolytic virus VV-α-TIGIT successfully combines the advantages of oncolytic virotherapy and intratumorally expression of immune checkpoint inhibitor against TIGIT. This novel strategy can provide information on the optimal design of novel antibody-armed oncolytic viruses for cancer immunotherapy. FUNDING: This work was supported by the National Natural Science Foundation of China (81773255, 81472820, and 81700037), the Science and Technology Innovation Foundation of Nanjing University (14913414), and the Natural Science Foundation of Jiangsu Province of China (BK20171098).

Nostoc sphaeroids Kütz polysaccharide and powder enrich a core bacterial community on C57BL/6j mice.

Gut microbiota is the collection of microbes that lives in the host. Glycan is the major factor to shape the composition of microbial community. Nostoc sphaeroids Kütz (NSK) has been used as food and medicine for thousands of years in Asian countries while the bioactivity on gut microbiota is unclear till now. Here, we used NSK polysaccharide and NSK powder to investigate the bioactivity on the gut microbiota of C57BL/6j mice, respectively. By 16S ribosomal RNA gene sequencing, we found the composition of gut microbiota had been changed and differed from each other. However, the abundance of Bacteroides, Parabacteroides, Escherichia-Shigella and Parasutterella on genus level were significantly increased by NSK polysaccharide and NSK powder. In addition, Akkermansia and Rikenellaceae were enriched by NSK powder. Moreover, we found the IL-1ß and IL-6 decreased significantly while TNF-α and IL-10 increased significantly especially in NSK powder group. Intriguingly, the increased microbes were significantly positively co-related with TNF-α and IL-10 while negatively co-related with IL-1ß and IL-6 by co-relation and network analysis. The above results suggested that Nostoc sphaeroids Kütz may selectively enrich a "core bacterial community" and add new evidence to discover how Nostoc sphaeroids Kütz has biological function.

The homogenous polysaccharide SY01-23 purified from leaf of Morus alba L. has bioactivity on human gut Bacteroides ovatus and Bacteroides cellulosilyticus.

Function of mulberry leaf (Morus alba L.) polysaccharide has been reported on antitumor, immunostimulatory and anti-inflammatory effects. However, the bioactivity on human gut microbiota is unclear so far. Here, three homogenous polysaccharides named SY01-21, SY01-22, SY01-23 were isolated from mulberry leaf with molecular weight 57 kDa, 25 kDa and 7.2 kDa, respectively. The monosaccharide composition of SY01-21 contained rhamnose, galactose and arabinose in a molar ratio of 7.60:43.52:48.88. SY01-22 contained rhamnose, galacturonic acid, glucose, galactose, xylose and arabinose in a molar ratio of 14.61:9.06:1.35:34.65:2.99:37.34. SY01-23 contained rhamnose, glucuronic acid, galacturonic acid, glucose, galactose, xylose and arabinose in a molar ratio of 23.00:4.12:24.60:5.74:17.28:1.12:24.13. Bioactivity test showed SY01-21 promoted the growth of Bacteroides cellulosilyticus (BC) while SY01-22 benefited the growth of Bacteroides ovatus (BO). Interestingly, SY01-23 boosted the growth of both BO and BC. However, Bacteroides thetaiotamicron (BT) only grew on 5 mg/mL SY01-21. Intriguingly, the growth of co-culture of BT with BO or BC was better than monoculture. This suggested that cross-feeding might exist between them. Besides, we found BO and BC generated acetate and propionate by utilizing SY01-23. The above results suggested that SY01-23 might modify human gut microbiota by driving colonization of Bacteroides in the gut to improve wellness.

Isolation and structure characterization of a polysaccharide from Crataegus pinnatifida and its bioactivity on gut microbiota.

A glucose rich heteroglycan named HAW1-2 was isolated and purified from fruit of Crataegus pinnatifida by hot-water extraction, ethanol precipitation, DEAE-cellulose anion exchange and gel permeation chromatography. HAW1-2 was proved as a homogenous polysaccharide with a molecular weight (Mw) of 8.94 kDa. Monosaccharide composition results indicated that HAW1-2 was composed of arabinose, galactose and glucose. Methylation analysis and NMR spectrum showed that HAW1-2 contained →4)-ß-d-Glcp-(1→, →4)-ß-d-Galp-(1→, α-L-Araf-(1→, →5)-α-L-Araf-(1→, ß-d-Glcp-(1→ and →4,6)-ß-d-Glcp-(1→. Bioactivity test showed that HAW1-2 could significantly promote the growth of Bacteroides thetaiotamicron (BT), Bacteroides ovatus (BO) and Bifidobacterium longum (BL), which showed competition with the Bacteroides spp. under co-culture. Interestingly, Bacteroides spp. generated more acetic acids and propionic acids while BL only generated acetic acids. These results imply that polysaccharide HAW1-2 may be useful for human by modulating intestinal bacteria and producing short chain fatty acids.

Intestinal microbes derived butyrate is related to the immunomodulatory activities of Dendrobium officinale polysaccharide.

Although immunomodulatory activities of Dendrobium officinale polysaccharide has been investigated for many years, yet the potential contribution of its metabolite derived from intestinal microbes on immunoregulation effect has not been reported. In this study, polysaccharide DOW-5B with average molecular weight of 39.4 kDa was isolated from the stem of Dendrobium officinale Kimura et Migo. The carbohydrate content was 91.97% and no protein was detected. The monosaccharide analysis showed this polysaccharide was composed of glucuronic acid and glucose at a molar ratio (M/G) of 1.2:19.4. Animal test indicated DOW-5B increased the diversity of gut microbiota on mice. Beneficial microbes such as Ruminococcus, Eubacterium, Clostridium, Bifidobacterium, Parabacteroides and Akkermansiamuciniphila increased while harmful bacteria in Proteobacteria decreased. Surprisingly, DOW-5B promoted gut microbes to generate more butyrate and mainly produced by Parabacteroides_sp_HGS0025. Further, we found the health of large intestine as well as immunity response of mice was improved. In addition, Parabacteroides_sp_HGS0025 positively correlated with butyrate, IgM, IL-10, and TNF-α products in intestine and mice blood, respectively. The data suggested that Dendrobium officinale polysaccharide has function on immunity may be mediated by butyrate. It adds new evidence to support the basis of how herbal polysaccharides affect immunity.

Blocking ATP-sensitive potassium channel alleviates morphine tolerance by inhibiting HSP70-TLR4-NLRP3-mediated neuroinflammation.

BACKGROUND: Long-term use of morphine induces analgesic tolerance, which limits its clinical efficacy. Evidence indicated morphine-evoked neuroinflammation mediated by toll-like receptor 4 (TLR4) - NOD-like receptor protein 3 (NLRP3) inflammasome was important for morphine tolerance. In our study, we investigated whether other existing alternative pathways caused morphine-induced activation of TLR4 in microglia. We focused on heat shock protein 70 (HSP70), a damage-associated molecular pattern (DAMP), which was released from various cells upon stimulations under the control of KATP channel and bound with TLR4-inducing inflammation. Glibenclamide, a classic KATP channel blocker, can improve neuroinflammation by inhibiting the activation of NLRP3 inflammasome. Our present study investigated the effect and possible mechanism of glibenclamide in improving morphine tolerance via its specific inhibition on the release of HSP70 and activation of NLRP3 inflammasome induced by morphine. METHODS: CD-1 mice were used for tail-flick test to evaluate morphine tolerance. The microglial cell line BV-2 and neural cell line SH-SY5Y were used to investigate the pharmacological effects and the mechanism of glibenclamide on morphine-induced neuroinflammation. The activation of microglia was accessed by immunofluorescence staining. Neuroinflammation-related cytokines were measured by western blot and real-time PCR. The level of HSP70 and related signaling pathway were evaluated by western blot and immunofluorescence staining. RESULTS: Morphine induced the release of HSP70 from neurons. The released HSP70 activated microglia and triggered TLR4-mediated inflammatory response, leading to the phosphorylation of p38 mitogen-activated protein kinase (MAPK) and nuclear factor-κB (NF-κB) p65 and the activation of NLRP3 inflammasome. Moreover, anti-HSP70 neutralizing antibody partly attenuated chronic morphine tolerance. The secretion of HSP70 was under the control of MOR/AKT/KATP/ERK signal pathway. Glibenclamide as a classic KATP channel blocker markedly inhibited the release of HSP70 induced by morphine and suppressed HSP70-TLR4-NLRP3 inflammasome-mediated neuroinflammation, which consequently attenuated morphine tolerance. CONCLUSIONS: Our study indicated that morphine-induced extracellular HSP70 was an alternative way for the activation of TLR4-NLRP3 in analgesic tolerance. The release of HSP70 was regulated by MOR/AKT/KATP/ERK pathway. Our study suggested a promising target, KATP channel and a new leading compound, glibenclamide, for treating morphine tolerance.