Osteoarthritis (OA) is the most prevalent joint disease, and it is characterized by cartilage degeneration, synovitis, and bone sclerosis, resulting in swelling, stiffness, and joint pain. TAM receptors (Tyro3, Axl, and Mer) play an important role in regulating immune responses, clearing apoptotic cells, and promoting tissue repair. Here, we investigated the anti-inflammatory effects of a TAM receptor ligand, i.e., growth arrest-specific gene 6 (Gas6), in synovial fibroblasts from OA patients. TAM receptor expression was determined in synovial tissue. Soluble Axl (sAxl), a decoy receptor for the ligand Gas6, showed concentrations 4.6 times higher than Gas6 in synovial fluid of OA patients. In OA fibroblast-like synoviocytes (OAFLS) exposed to inflammatory stimuli, the levels of sAxl in the supernatants were increased, while the expression of Gas6 was downregulated. In OAFLS under TLR4 stimulation by LPS (Escherichia coli lipopolysaccharide), the addition of exogenous Gas6 by Gas6-conditioned medium (Gas6-CM) reduced pro-inflammatory markers including IL-6, TNF-α, IL-1β, CCL2, and CXCL8. Moreover, Gas6-CM downregulated IL-6, CCL2, and IL-1β in LPS-stimulated OA synovial explants. Pharmacological inhibition of TAM receptors by a pan inhibitor (RU301) or by a selective Axl inhibitor (RU428) similarly abrogated Gas6-CM anti-inflammatory effects. Mechanistically, Gas6 effects were dependent on Axl activation, determined by Axl, STAT1, and STAT3 phosphorylation, and by the downstream induction of the suppressors of the cytokine signaling family (SOCS1 and SOCS3). Taken together, our results showed that Gas6 treatment dampens inflammatory markers of OAFLS and synovial explants derived from OA patients associated with SOCS1/3 production.

Colonic bacterial biofilms are frequently present in ulcerative colitis [UC] and may increase dysplasia risk through pathogens expressing oncotraits. This prospective cohort study aimed to determine [1] the association of oncotraits and longitudinal biofilm presence with dysplasia risk in UC, and [2] the relation of bacterial composition with biofilms and dysplasia risk. Faeces and left- and right-sided colonic biopsies were collected from 80 UC patients and 35 controls. Oncotraits [FadA of Fusobacterium, BFT of Bacteroides fragilis, colibactin [ClbB] and Intimin [Eae] of Escherichia coli] were assessed in faecal DNA with multiplex quantitative polymerase chain reaction [qPCR]. Biopsies were screened for biofilms [n = 873] with 16S rRNA fluorescent in situ hybridiation. Shotgun metagenomic sequencing [n = 265], and ki67-immunohistochemistry were performed. Associations were determined with a mixed-effects regression model. Biofilms were highly prevalent in UC patients [90.8%] with a median persistence of 3 years (interquartile range [IQR] 2–5 years). Biofilm-positive biopsies showed increased epithelial hypertrophy [p = 0.025] and a reduced Shannon diversity independent of disease status [p = 0.015], but were not significantly associated with dysplasia in UC: adjusted odds ratio [aOR] 1.45, 95% confidence interval [CI] 0.63–3.40. In contrast, ClbB independently associated with dysplasia [aOR 7.16, 95% CI 1.75–29.28], and FadA and Fusobacteriales were associated with a decreased dysplasia risk in UC [aOR 0.23, 95% CI 0.06–0.83, p <0.01]. Biofilms are a hallmark of UC; however, because of their high prevalence are a poor biomarker for dysplasia. In contrast, colibactin presence and FadA absence independently associate with dysplasia in UC and might therefore be valuable biomarkers for future risk stratification and intervention strategies.

Degradation of mucins at the host-microbial mucus interphase involves glycosidases that release monosaccharides from O-glycans and mucinases that cleave the mucin protein backbone. Mucinases recognize and cleave peptide bonds at specific sequence motifs with varying O-glycan structures required and/or permissible. Mucinases that digest mucins with intact O-glycans can potentially destroy the protective mucus, while mucinases that only digest mucins with partially degraded O-glycans may serve at a later stage of nutrient sourcing from mucins. Here, we discovered nine CBM-bearing M60-like mucinases across gut commensals and opportunists, including a conserved Bacteroides fragilis mucinase denoted HC11. We also investigated the previously described Bacteroides thetaiotaomicron mucinase BT4244, which together delineates two functional classes with distinct preferences: BT4244 for bis-Tn (GalNAcα1-O-Ser/Thr) and HC11 for bis-T (Galβ1-3GalNAcα1-O-Ser/Thr) O-glycans. Both mucinases harbor carbohydrate-binding modules (CBM32) that bind their cognate O-glycan motifs and are required - together with the catalytic domains - for efficient cleavage of extended mucin domains, which is consistent with cooperative engagement, but are not required for the cleavage of short glycopeptides. We show B. fragilis strains secrete HC11 and degrade mucins only after the removal of sialic acids. Together, these findings expand the mucinase repertoire by nine enzymes spanning commensals and opportunists, demonstrate that CBM32 domains are essential for efficient cleavage of extended mucin substrates likely by promoting multivalent engagement and substrate positioning, and nominateidentify CBM-catalytic cooperation as a mechanism and intervention point for controlling mucus turnover and barrier integrity.

Biofilms are polymeric matrices containing bacteria that can express oncotraits and are frequently present in ulcerative colitis (UC). Oncotraits can impact colon epithelial cells directly and may increase dysplasia risk. This study aimed to determine (1) the association of oncotraits and longitudinal biofilm presence with dysplasia risk in UC, and (2) the relation of bacterial composition with biofilms and dysplasia risk. In this prospective cohort study, feces and left- and right-sided colonic biopsies were collected from 80 UC patients and 35 controls. Oncotraits (FadA of Fusobacterium , BFT of Bacteroides fragilis , Colibactin (ClbB) and Intimin (Eae) of Escherichia coli ) in fecal DNA were assessed with multiplex qPCR. Biopsies were analyzed for biofilms (n=873) with 16S rRNA fluorescent in situ hybridization and shotgun metagenomic sequencing (n=265), and ki67-immunohistochemistry for cell proliferation. Associations were determined with a regression (mixed) model. ClbB significantly associated with dysplasia in UC (aOR 7.16, (95%CI 1.75-29.28, p<0.01)), while FadA was inversely associated (aOR 0.23, (95%CI 0.06-0.83, p=0.03)). Patients with UC had a significantly lower Shannon diversity compared to controls (p=0.0009), as well as patients with a biofilm (p=0.015) independent of disease status. The order Fusobacteriales was significantly correlated with a decreased dysplasia risk only in right-sided colonic biopsies (p<0.01). Longitudinal biofilms were not significantly associated with dysplasia (aOR 1.45 (95% CI0.63-3.40, p=0.38)), however, biofilm-positive biopsies showed increased epithelial hypertrophy (p=0.025). Colibactin and FadA impact dysplasia risk in UC, in contrast to biofilms. These oncotraits are valuable targets for future risk classification and intervention studies. What is already known on this topic: Bacterial biofilms sometimes contain bacteria with oncogenic traits (oncotraits) and have been associated with colon carcinogenesis in mice and humans. It is yet unknown whether biofilms and oncotraits are involved in early carcinogenesis and could be used as a risk factor for dysplasia in ulcerative colitis patients. What this study add: Bacterial biofilms associated with lower bacterial diversity and epithelial cell hypertrophy, but did not predict dysplasia. Moreover, in agreement to piling evidence suggesting a role of colibactin in human colorectal cancer, we provide the missing clinical evidence that this oncotrait actually associates with risk for (early) carcinogenesis in human patients. Additionally, dysplasia in UC patients was predicted by absence of Fusobacterium adhesin. How this study might affect research, practice or policy: This prospective cohort study indicates a putative role of bacterial oncotraits in early carcinogenesis, suggesting them as promising targets for future risk classification and intervention studies in ulcerative colitis patients. Lay summary: Patients with ulcerative colitis have an increased risk for colorectal cancer. This study found that bacterial factors in fecal material can predict the development of cancer precursors in these patients. Abstract Figure:

Siglec-15 has recently emerged as an important regulator at the interface of glyco-immunology and cancer-immunology. The transmembrane immune receptor links cancer-associated glycosylation to immune suppression in the tumor microenvironment (TME). Siglec-15 signals through the adapter protein DAP12, using its immunoreceptor tyrosine-based activation motif to induce signaling. While typically expressed by osteoclasts, increased expression of Siglec-15 holds critical implications for tumor-associated macrophages (TAMs). Current evidence suggests that Siglec-15 modulates anti-tumor immunity via reprogramming of TAMs toward an M2-like, protumorigenic phenotype characterized by direct suppression of effector T cells through interleukin-10 and transforming growth factor-β production. Identification of its ligands is ongoing, but Siglec-15 has been shown to interact both with N-linked and O-linked sialoglycans and binding can be enhanced by glycan sulfation. In addition, ligand specificity appears to be determined by glycan presentation on specific glycoprotein scaffolds, among others found in CD44 and CD11b. Siglec-15 expression is detected in many solid and hematological cancers and has been shown to negatively correlate with programmed cell death protein 1/programmed death-ligand 1 expression in several studies, providing rationale for targeting this Siglec. Defining the physiological and cancer-associated ligands of Siglec-15 is critical for understanding its signaling and the functional consequences thereof. Blocking antibodies for Siglec-15 show promising effects in (pre)clinical studies. Here, we provide an overview of current insights into the glycan ligands of Siglec-15, discuss its role in controlling TAMs phenotypes, and functions in the TME. Moreover, we discuss current therapeutic strategies aimed at targeting Siglec-15 and its glycan ligands in cancer immunotherapy.