Insights into the enigma of oral streptococci in carcinogenesis.

SUMMARYThe genus Streptococcus consists of a taxonomically diverse group of Gram-positive bacteria that have earned significant scientific interest due to their physiological and pathogenic characteristics. Within the genus Streptococcus, viridans group streptococci (VGS) play a significant role in the oral ecosystem, constituting approximately 80% of the oral biofilm. Their primary role as pioneering colonizers in the oral cavity with multifaceted interactions like adherence, metabolic signaling, and quorum sensing contributes significantly to the complex dynamics of the oral biofilm, thus shaping oral health and disease outcomes. Perturbations in oral streptococci composition drive oral dysbiosis and therefore impact host-pathogen interactions, resulting in oral inflammation and representing VGS as an opportunistic pathogen. The association of oral streptococci in tumors across distant organs, spanning the esophagus, stomach, pancreas, and colon, illuminates a potential association between oral streptococci, inflammation, and tumorigenesis. This finding emphasizes the need for further investigations into the role of oral streptococci in mucosal homeostasis and their involvement in carcinogenesis. Hence, here, we review the significance of oral streptococci in biofilm dynamics and how the perturbation may impact mucosal immunopathogenesis in the context of cancer, with a vision of exploiting oral streptococci for cancer intervention and for the development of non-invasive cancer diagnosis.

Oral streptococci S. anginosus and S. mitis induce distinct morphological, inflammatory, and metabolic signatures in macrophages.

Oral streptococci, key players in oral biofilm formation, are implicated in oral dysbiosis and various clinical conditions, including dental caries, gingivitis, periodontal disease, and oral cancer. Specifically, Streptococcus anginosus is associated with esophageal, gastric, and pharyngeal cancers, while Streptococcus mitis is linked to oral cancer. However, no study has investigated the mechanistic links between these Streptococcus species and cancer-related inflammatory responses. As an initial step, we probed the innate immune response triggered by S. anginosus and S. mitis in RAW264.7 macrophages. These bacteria exerted time- and dose-dependent effects on macrophage morphology without affecting cell viability. Compared with untreated macrophages, macrophages infected with S. anginosus exhibited a robust proinflammatory response characterized by significantly increased levels of inflammatory cytokines and mediators, including TNF, IL-6, IL-1ß, NOS2, and COX2, accompanied by enhanced NF-κB activation. In contrast, S. mitis-infected macrophages failed to elicit a robust inflammatory response. Seahorse Xfe96 analysis revealed an increased extracellular acidification rate in macrophages infected with S. anginosus compared with S. mitis. At the 24-h time point, the presence of S. anginosus led to reduced extracellular itaconate, while S. mitis triggered increased itaconate levels, highlighting distinct metabolic profiles in macrophages during infection in contrast to aconitate decarboxylase expression observed at the 6-h time point. This initial investigation highlights how S. anginosus and S. mitis, two Gram-positive bacteria from the same genus, can prompt distinct immune responses and metabolic shifts in macrophages during infection.IMPORTANCEThe surge in head and neck cancer cases among individuals devoid of typical risk factors such as Human Papilloma Virus (HPV) infection and tobacco and alcohol use sparks an argumentative discussion around the emerging role of oral microbiota as a novel risk factor in oral squamous cell carcinoma (OSCC). While substantial research has dissected the gut microbiome's influence on physiology, the oral microbiome, notably oral streptococci, has been underappreciated during mucosal immunopathogenesis. Streptococcus anginosus, a viridans streptococci group, has been linked to abscess formation and an elevated presence in esophageal cancer and OSCC. The current study aims to probe the innate immune response to S. anginosus compared with the early colonizer Streptococcus mitis as an important first step toward understanding the impact of distinct oral Streptococcus species on the host immune response, which is an understudied determinant of OSCC development and progression.

An injectable subcutaneous colon-specific immune niche for the treatment of ulcerative colitis.

As a chronic autoinflammatory condition, ulcerative colitis is often managed via systemic immunosuppressants. Here we show, in three mouse models of established ulcerative colitis, that a subcutaneously injected colon-specific immunosuppressive niche consisting of colon epithelial cells, decellularized colon extracellular matrix and nanofibres functionalized with programmed death-ligand 1, CD86, a peptide mimic of transforming growth factor-beta 1, and the immunosuppressive small-molecule leflunomide, induced intestinal immunotolerance and reduced inflammation in the animals' lower gastrointestinal tract. The bioengineered colon-specific niche triggered autoreactive T cell anergy and polarized pro-inflammatory macrophages via multiple immunosuppressive pathways, and prevented the infiltration of immune cells into the colon's lamina propria, promoting the recovery of epithelial damage. The bioengineered niche also prevented colitis-associated colorectal cancer and eliminated immune-related colitis triggered by kinase inhibitors and immune checkpoint blockade.

Interferon activated gene 204 protects against bone loss in experimental periodontitis.

BACKGROUND: Periodontal destruction can be the result of different known and yet-to-be-discovered biological pathways. Recent human genetic association studies have implicated interferon-gamma inducible protein 16 (IFI16) and absent in melanoma 2 (AIM2) with high periodontal interleukin (IL)-1ß levels and more destructive disease, but mechanistic evidence is lacking. Here, we sought to experimentally validate these observational associations and better understand IFI16 and AIM2's roles in periodontitis. METHODS: Periodontitis was induced in Ifi204-/- (IFI16 murine homolog) and Aim2-/- mice using the ligature model. Chimeric mice were created to identify the main source cells of Ifi204 in the periodontium. IFI16-silenced human endothelial cells were treated with periodontal pathogens in vitro. Periodontal tissues from Ifi204-/- mice were evaluated for alveolar bone (micro-CT), cell inflammatory infiltration (MPO+ staining), Il1b (qRT-PCR), and osteoclast numbers (cathepsin K+ staining). RESULTS: Ifi204-deficient mice> exhibited >20% higher alveolar bone loss than wild-type (WT) (P < 0.05), while no significant difference was found in Aim2-/- mice. Ifi204's effect on bone loss was primarily mediated by a nonbone marrow source and was independent of Aim2. Ifi204-deficient mice had greater neutrophil/macrophage trafficking into gingival tissues regardless of periodontitis development compared to WT. In human endothelial cells, IFI16 decreased the chemokine response to periodontal pathogens. In murine periodontitis, Ifi204 depletion elevated gingival Il1b and increased osteoclast numbers at diseased sites (P < 0.05). CONCLUSIONS: These findings support IFI16's role as a novel regulator of inflammatory cell trafficking to the periodontium that protects against bone loss and offers potential targets for the development of new periodontal disease biomarkers and therapeutics.

Type 2 innate lymphoid cells treat and prevent acute gastrointestinal graft-versus-host disease.

Acute graft-versus-host disease (aGVHD) is the most common complication for patients undergoing allogeneic stem cell transplantation. Despite extremely aggressive therapy targeting donor T cells, patients with grade III or greater aGVHD of the lower GI tract, who do not respond to therapy with corticosteroids, have a dismal prognosis. Thus, efforts to improve understanding of the function of local immune and non-immune cells in regulating the inflammatory process in the GI tract during aGVHD are needed. Here, we demonstrate, using murine models of allogeneic BMT, that type 2 innate lymphoid cells (ILC2s) in the lower GI tract are sensitive to conditioning therapy and show very limited ability to repopulate from donor bone marrow. Infusion of donor ILC2s was effective in reducing the lethality of aGVHD and in treating lower GI tract disease. ILC2 infusion was associated with reduced donor proinflammatory Th1 and Th17 cells, accumulation of donor myeloid-derived suppressor cells (MDSCs) mediated by ILC2 production of IL-13, improved GI tract barrier function, and a preserved graft-versus-leukemia (GVL) response. Collectively, these findings suggest that infusion of donor ILC2s to restore gastrointestinal tract homeostasis may improve treatment of severe lower GI tract aGVHD.

NLRX1 suppresses tumorigenesis and attenuates histiocytic sarcoma through the negative regulation of NF-κB signaling.

Histiocytic sarcoma is an uncommon malignancy in both humans and veterinary species. Research exploring the pathogenesis of this disease is scarce; thus, diagnostic and therapeutic options for patients are limited. Recent publications have suggested a role for the NLR, NLRX1, in acting as a tumor suppressor. Based on these prior findings, we hypothesized that NLRX1 would function to inhibit tumorigenesis and thus the development of histiocytic sarcoma. To test this, we utilized Nlrx1-/- mice and a model of urethane-induced tumorigenesis. Nlrx1-/- mice exposed to urethane developed splenic histiocytic sarcoma that was associated with significant up-regulation of the NF-κB signaling pathway. Additionally, development of these tumors was also significantly associated with the increased regulation of genes associated with AKT signaling, cell death and autophagy. Together, these data show that NLRX1 suppresses tumorigenesis and reveals new genetic pathways involved in the pathobiology of histiocytic sarcoma.

The Innate Immune Receptor NLRX1 Functions as a Tumor Suppressor by Reducing Colon Tumorigenesis and Key Tumor-Promoting Signals.

NOD-like receptor (NLR) proteins are intracellular innate immune sensors/receptors that regulate immunity. This work shows that NLRX1 serves as a tumor suppressor in colitis-associated cancer (CAC) and sporadic colon cancer by keeping key tumor promoting pathways in check. Nlrx1(-/-) mice were highly susceptible to CAC, showing increases in key cancer-promoting pathways including nuclear factor κB (NF-κB), mitogen-activated protein kinase (MAPK), signal transducer and activator of transcription 3 (STAT3), and interleukin 6 (IL-6). The tumor-suppressive function of NLRX1 originated primarily from the non-hematopoietic compartment. This prompted an analysis of NLRX1 function in the Apc(min/+) genetic model of sporadic gastrointestinal cancer. NLRX1 attenuated Apc(min/+) colon tumorigenesis, cellular proliferation, NF-κB, MAPK, STAT3 activation, and IL-6 levels. Application of anti-interleukin 6 receptor (IL6R) antibody therapy reduced tumor burden, increased survival, and reduced STAT3 activation in Nlrx1(-/-)Apc(min/+) mice. As an important clinical correlate, human colon cancer samples expressed lower levels of NLRX1 than healthy controls in multiple patient cohorts. These data implicate anti-IL6R as a potential personalized therapy for colon cancers with reduced NLRX1.

NLRP12 provides a critical checkpoint for osteoclast differentiation.

The alternative or noncanonical nuclear factor kappa B (NF-κB) pathway regulates the osteoclast (OC) response to receptor activator of nuclear factor kappa B ligand (RANKL) and thus bone metabolism. Although several lines of evidence support the emerging concept that nucleotide-binding leucine-rich repeat and pyrin domain-containing receptor 12 (NLRP12) impedes alternative NF-κB activation in innate immune cells, a functional role for NLRP12 outside an inflammatory disease model has yet to be reported. Our study demonstrates that NLRP12 has a protective role in bone via suppression of alternative NF-κB-induced osteoclastogenesis and is down-modulated in response to osteoclastogenic stimuli. Here, we show that retroviral overexpression of NLRP12 suppressed RelB nuclear translocation and OC formation. Conversely, genetic ablation of NLRP12 promoted NIK stabilization, RelB nuclear translocation, and increased osteoclastogenesis in vitro. Using radiation chimeras, we demonstrated these in vitro observations dovetail with our in vivo findings that NLRP12 deficiency leads to enhanced OC numbers accompanied by a significant decline in bone mass under physiological conditions. Consistent with the basal bone phenotype, we also observed an enhanced osteolytic response following RANKL injection over the calvaria of NLRP12-deficient chimeric mice compared with wild-type control mice. Thus, modulation of NLRP12 levels controls alternative NF-κB signaling in OC precursors, altering bone homeostasis and osteolytic responses.

Inflammasome-independent role of AIM2 in suppressing colon tumorigenesis via DNA-PK and Akt.

The inflammasome activates caspase-1 and the release of interleukin-1ß (IL-1ß) and IL-18, and several inflammasomes protect against intestinal inflammation and colitis-associated colon cancer (CAC) in animal models. The absent in melanoma 2 (AIM2) inflammasome is activated by double-stranded DNA, and AIM2 expression is reduced in several types of cancer, but the mechanism by which AIM2 restricts tumor growth remains unclear. We found that Aim2-deficient mice had greater tumor load than Asc-deficient mice in the azoxymethane/dextran sodium sulfate (AOM/DSS) model of colorectal cancer. Tumor burden was also higher in Aim2(-/-)/Apc(Min/+) than in APC(Min/+) mice. The effects of AIM2 on CAC were independent of inflammasome activation and IL-1ß and were primarily mediated by a non-bone marrow source of AIM2. In resting cells, AIM2 physically interacted with and limited activation of DNA-dependent protein kinase (DNA-PK), a PI3K-related family member that promotes Akt phosphorylation, whereas loss of AIM2 promoted DNA-PK-mediated Akt activation. AIM2 reduced Akt activation and tumor burden in colorectal cancer models, while an Akt inhibitor reduced tumor load in Aim2(-/-) mice. These findings suggest that Akt inhibitors could be used to treat AIM2-deficient human cancers.

G Protein signaling modulator-3 inhibits the inflammasome activity of NLRP3.

Inflammasomes are multi-protein complexes that regulate maturation of the interleukin 1ß-related cytokines IL-1ß and IL-18 through activation of the cysteine proteinase caspase-1. NOD-like receptor family, pyrin domain containing 3 (NLRP3) protein is a key component of inflammasomes that assemble in response to a wide variety of endogenous and pathogen-derived danger signals. Activation of the NLRP3-inflammasome and subsequent secretion of IL-1ß is highly regulated by at least three processes: transcriptional activation of both NLRP3 and pro-IL-1ß genes, non-transcriptional priming of NLRP3, and final activation of NLRP3. NLRP3 is predominantly expressed in cells of the hematopoietic lineage. Using a yeast two-hybrid screen, we identified the hematopoietic-restricted protein, G protein signaling modulator-3 (GPSM3), as a NLRP3-interacting protein and a negative regulator of IL-1ß production triggered by NLRP3-dependent inflammasome activators. In monocytes, GPSM3 associates with the C-terminal leucine-rich repeat domain of NLRP3. Bone marrow-derived macrophages lacking GPSM3 expression exhibit an increase in NLRP3-dependent IL-1ß, but not TNF-α, secretion. Furthermore, GPSM3-null mice have enhanced serum and peritoneal IL-1ß production following Alum-induced peritonitis. Our findings suggest that GPSM3 acts as a direct negative regulator of NLRP3 function.

Characterization of NLRP12 during the in vivo host immune response to Klebsiella pneumoniae and Mycobacterium tuberculosis.

The majority of nucleotide binding domain leucine rich repeats-containing (NLR) family members has yet to be functionally characterized. Of the described NLRs, most are considered to be proinflammatory and facilitate IL-1ß production. However, a newly defined sub-group of NLRs that function as negative regulators of inflammation have been identified based on their abilities to attenuate NF-κB signaling. NLRP12 (Monarch-1) is a prototypical member of this sub-group that negatively regulates both canonical and noncanonical NF-κB signaling in biochemical assays and in colitis and colon cancer models. The role of NLRP12 in infectious diseases has not been extensively studied. Here, we characterized the innate immune response of Nlrp12(-/-) mice following airway exposure to LPS, Klebsiella pneumoniae and Mycobacterium tuberculosis. In response to E. coli LPS, Nlrp12(-/-) mice showed a slight decrease in IL-1ß and increase in IL-6 production, but these levels were not statistically significant. During K. pneumoniae infection, we observed subtle differences in cytokine levels and significantly reduced numbers of monocytes and lymphocytes in Nlrp12(-/-) mice. However, the physiological relevance of these findings is unclear as no overt differences in the development of lung disease were observed in the Nlrp12(-/-) mice. Likewise, Nlrp12(-/-) mice demonstrated pathologies similar to those observed in the wild type mice following M. tuberculosis infection. Together, these data suggest that NLRP12 does not significantly contribute to the in vivo host innate immune response to LPS stimulation, Klebsiella pneumonia infection or Mycobacterium tuberculosis.

Francisella tularensis elicits IL-10 via a PGE2-inducible factor, to drive macrophage MARCH1 expression and class II down-regulation.

Francisella tularensis is a bacterial pathogen that uses host-derived PGE2 to subvert the host's adaptive immune responses in multiple ways. Francisella-induced PGE2 acts directly on CD4 T cells to blunt production of IFN-γ. Francisella-induced PGE2 can also elicit production of a >10 kDa soluble host factor termed FTMØSN (F. tularensismacrophage supernatant), which acts on IFN-γ pre-activated MØ to down-regulate MHC class II expression via a ubiquitin-dependent mechanism, blocking antigen presentation to CD4 T cells. Here, we report that FTMØSN-induced down-regulation of MØ class II is the result of the induction of MARCH1, and that MØ expressing MARCH1 "resistant" class II molecules are resistant to FTMØSN-induced class II down-regulation. Since PGE2 can induce IL-10 production and IL-10 is the only reported cytokine able to induce MARCH1 expression in monocytes and dendritic cells, these findings suggested that IL-10 is the active factor in FTMØSN. However, use of IL-10 knockout MØ established that IL-10 is not the active factor in FTMØSN, but rather that Francisella-elicited PGE2 drives production of a >10 kDa host factor distinct from IL-10. This factor then drives MØ IL-10 production to induce MARCH1 expression and the resultant class II down-regulation. Since many human pathogens such as Salmonella typhi, Mycobacterium tuberculosis and Legionella pneumophila also induce production of host PGE2, these results suggest that a yet-to-be-identified PGE2-inducible host factor capable of inducing IL-10 is central to the immune evasion mechanisms of multiple important human pathogens.

NLRP12 suppresses colon inflammation and tumorigenesis through the negative regulation of noncanonical NF-κB signaling.

In vitro data suggest that a subgroup of NLR proteins, including NLRP12, inhibits the transcription factor NF-κB, although physiologic and disease-relevant evidence is largely missing. Dysregulated NF-κB activity is associated with colonic inflammation and cancer, and we found Nlrp12(-/-) mice were highly susceptible to colitis and colitis-associated colon cancer. Polyps isolated from Nlrp12(-/-) mice showed elevated noncanonical NF-κB activation and increased expression of target genes that were associated with cancer, including Cxcl13 and Cxcl12. NLRP12 negatively regulated ERK and AKT signaling pathways in affected tumor tissues. Both hematopoietic- and nonhematopoietic-derived NLRP12 contributed to inflammation, but the latter dominantly contributed to tumorigenesis. The noncanonical NF-κB pathway was regulated upon degradation of TRAF3 and activation of NIK. NLRP12 interacted with both NIK and TRAF3, and Nlrp12(-/-) cells have constitutively elevated NIK, p100 processing to p52 and reduced TRAF3. Thus, NLRP12 is a checkpoint of noncanonical NF-κB, inflammation, and tumorigenesis.

Francisella tularensis induces ubiquitin-dependent major histocompatibility complex class II degradation in activated macrophages.

The intracellular bacterium Francisella tularensis survives and replicates within macrophages, ultimately killing the host cell. Resolution of infection requires the development of adaptive immunity through presentation of F. tularensis antigens to CD4+ and CD8+ T cells. We have previously established that F. tularensis induces macrophage prostaglandin E2 (PGE2) production, leading to skewed T-cell responses. PGE2 can also downregulate macrophage major histocompatibility complex (MHC) class II expression, suggesting that F. tularensis-elicited PGE2 may further alter T-cell responses via inhibition of class II expression. To test this hypothesis, gamma interferon (IFN-gamma)-activated reporter macrophages were exposed to supernatants from F. tularensis-infected macrophages, and the class II levels were measured. Exposure of macrophages to infection supernatants results in essentially complete clearance of surface class II and CD86, compromising the macrophage's ability to present antigens to CD4 T cells. Biochemical analysis revealed that infection supernatants elicit ubiquitin-dependent class II downregulation and degradation within intracellular acidic compartments. By comparison, exposure to PGE2 alone only leads to a minor decrease in macrophage class II expression, demonstrating that a factor distinct from PGE2 is eliciting the majority of class II degradation. However, production of this non-PGE2 factor is dependent on macrophage cyclooxygenase activity and is induced by PGE2. These results establish that F. tularensis induces the production of a PGE2-dependent factor that elicits MHC class II downregulation in IFN-gamma-activated macrophages through ubiquitin-mediated delivery of class II to lysosomes, establishing another mechanism for the modulation of macrophage antigen presentation during F. tularensis infection.

Francisella tularensis-infected macrophages release prostaglandin E2 that blocks T cell proliferation and promotes a Th2-like response.

Francisella tularensis is a highly infectious bacterial pathogen, and is likely to have evolved strategies to evade and subvert the host immune response. In this study, we show that F. tularensis infection of macrophages alters T cell responses in vitro, by blocking T cell proliferation and promoting a Th2-like response. We demonstrate that a soluble mediator is responsible for this effect and identify it as PGE(2). Supernatants from F. tularensis-infected macrophages inhibited IL-2 secretion from both MHC class I and MHC class II-restricted T cell hybridomas, as well as enhanced a Th2-like response by inducing increased production of IL-5. Furthermore, the soluble mediator blocked proliferation of naive MHC class I-restricted T cells when stimulated with cognate tetramer. Indomethacin treatment partially restored T cell proliferation and lowered IL-5 production to wild-type levels. Macrophages produced PGE(2) when infected with F. tularensis, and treatment of infected macrophages with indomethacin, a cyclooxygenase-1/cyclooxygenase-2 inhibitor, blocked PGE(2) production. To further demonstrate that PGE(2) was responsible for skewing of T cell responses, we infected macrophages from membrane PGE synthase 1 knockout mice (mPGES1(-/-)) that cannot produce PGE(2). Supernatants from F. tularensis-infected membrane PGE synthase 1(-/-) macrophages did not inhibit T cell proliferation. Furthermore, treatment of T cells with PGE(2) recreated the effects seen with infected supernatant. From these data, we conclude that F. tularensis can alter host T cell responses by causing macrophages to produce PGE(2). This study defines a previously unknown mechanism used by F. tularensis to modulate adaptive immunity.