Cystic fibrosis (CF) is a genetic disease in which the battle between pulmonary infection and inflammation becomes the major cause of morbidity and mortality. We have previously shown that human MSCs (hMSCs) decrease inflammation and infection in the in vivo murine model of CF. The studies in this paper focus on the specificity of the hMSC antimicrobial effectiveness using Pseudomonas aeruginosa (gram negative bacteria) and Staphylococcus aureus (gram positive bacteria). Our studies show that hMSCs secrete bioactive molecules which are antimicrobial in vitro against Pseudomonas aeruginosa, Staphylococcus aureus, and Streptococcus pneumonia, impacting the rate of bacterial growth and transition into colony forming units regardless of the pathogen. Further, we show that the hMSCs have the capacity to enhance antibiotic sensitivity, improving the capacity to kill bacteria. We present data which suggests that the antimicrobial effectiveness is associated with the capacity to slow bacterial growth and the ability of the hMSCs to secrete the antimicrobial peptide LL-37. Lastly, our studies demonstrate that the tissue origin of the hMSCs (bone marrow or adipose tissue derived), the presence of functional cystic fibrosis transmembrane conductance regulator (CFTR: human, Cftr: mouse) activity, and response to effector cytokines can impact both hMSC phenotype and antimicrobial potency and efficacy. These studies demonstrate, the unique capacity of the hMSCs to manage different pathogens and the significance of their phenotype in both the antimicrobial and antibiotic enhancing activities.

BackgroundTumor-associated macrophages (TAMs) constitute a significant part of infiltrating inflammatory cells that are frequently correlated with progression and poor prognosis of a variety of cancers. Tumor cell-produced human β-defensin-3 (hBD-3) has been associated with TAM trafficking in oral cancer; however, its involvement in tumor-related inflammatory processes remains largely unknown.MethodologyThe relationship between hBD-3, monocyte chemoattractant protein-1 (MCP-1), TAMs, and CCR2 was examined using immunofluorescence microscopy in normal and oral carcinoma in situ biopsy specimens. The ability of hBD-3 to chemoattract host macrophages in vivo using a nude mouse model and analysis of hBD-3 on monocytic cell migration in vitro, applying a cross-desensitization strategy of CCR2 and its pharmacological inhibitor (RS102895), respectively, was also carried out.Conclusions/FindingsMCP-1, the most frequently expressed tumor cell-associated chemokine, was not produced by tumor cells nor correlated with the recruitment of macrophages in oral carcinoma in situ lesions. However, hBD-3 was associated with macrophage recruitment in these lesions and hBD-3-expressing tumorigenic cells induced massive tumor infiltration of host macrophages in nude mice. HBD-3 stimulated the expression of tumor-promoting cytokines, including interleukin-1α (IL-1α), IL-6, IL-8, CCL18, and tumor necrosis factor-α (TNF-α) in macrophages derived from human peripheral blood monocytes. Monocytic cell migration in response to hBD-3 was inhibited by cross-desensitization with MCP-1 and the specific CCR2 inhibitor, RS102895, suggesting that CCR2 mediates monocyte/macrophage migration in response to hBD-3. Collectively, these results indicate that hBD-3 utilizes CCR2 to regulate monocyte/macrophage trafficking and may act as a tumor cell-produced chemoattractant to recruit TAMs. This novel mechanism is the first evidence of an hBD molecule orchestrating an in vivo outcome and demonstrates the importance of the innate immune system in the development of tumors.

Human beta-defensins (hBDs, −1, 2, 3) are a family of epithelial cell derived antimicrobial peptides (AMPs) that protect mucosal membranes from microbial challenges. In addition to their antimicrobial activities, they possess other functions; e.g., cell activation, proliferation, regulation of cytokine/chemokine production, migration, differentiation, angiogenesis, and wound healing processes. It has also become apparent that defensin levels change with the development of neoplasia. However, inconsistent observations published by various laboratories make it difficult to reach a consensus as to the direction of the dysregulation and role the hBDs may play in various cancers. This is particularly evident in studies focusing on oral squamous cell carcinoma (OSCC). By segregating each hBD by cancer type, interrogating methodologies, and scrutinizing the subject cohorts used in the studies, we have endeavored to identify the “take home message” for each one of the three hBDs. We discovered that (1) consensus-driven findings indicate that hBD-1 and−2 are down- while hBD-3 is up-regulated in OSCC; (2) hBD dysregulation is cancer-type specific; (3) the inhibition/activation effect an hBD has on cancer cell lines is related to the direction of the hBD dysregulation (up or down) in the cancer from which the cell lines derive. Therefore, studies addressing hBD dysregulation in various cancers are not generalizable and comparisons should be avoided. Systematic delineation of the fate and role of the hBDs in a specific cancer type may lead to innovative ways to use defensins as prospective biomarkers for diagnostic/prognostic purposes and/or in novel therapeutic modalities.

Background: Human ␤-defensins (hBDs) are epithelial cell-derived antimicrobial and immunoregulatory cationic peptides. Our objective was to establish an analytical tool to quantify inducible hBD-2 and -3 in body fluids. Methods: We developed sandwich ELISAs using commercially available capture and detection antibodies and determined optimal assay conditions (with 250 mmol/L CaCl 2 ) to overcome masking by endogenous components of body fluids. We used recombinant hBD as calibrators and for recovery testing. Results: hBD-2 and -3 detection limits were ϳ75 ng/L and ϳ3 g/L, respectively. Mean (SD range) values in saliva samples from healthy donors (n ‫؍‬ 60) were 9.5 (1.2-21) g/L for hBD-2 and 326 (50 -931) g/L for hBD-3. We did not detect hBD-3 in suction blister fluid (BF; n ‫؍‬ 10) or bronchoalveolar lavage (BAL; n ‫؍‬ 5) from healthy participants. We detected low hBD-2 peptide concentrations in BF and BAL, 0.16 (0.03-0.32) and 0.04 (0 -0.049) g/g total protein, respectively. We observed no correlation of hBD-2 in BF and saliva or BAL and saliva from the same person. In vaginal swabs from healthy women (n ‫؍‬ 2), mean hBD-2 and -3 concentrations were 3.42 and 103 g/g total protein, respectively. Cervicovaginal lavage from the same women contained mean concentrations of 1.46 and 55.5 g/g total protein.Conclusion: These ELISA assays can measure inducible hBD peptide concentrations in body fluids by overcom-

We previously identified a cell wall-associated protein from Fusobacterium nucleatum, a Gram-negative bacterium of the oral cavity, that induces human beta defensin 2 (hBD-2) in primary human oral epithelial cells (HOECs) and designated it FAD-I (Fusobacterium-associated defensin inducer). Here, we report differential induction of hBD-2 by different strains of F. nucleatum; ATCC 25586 and ATCC 23726 induce significantly more hBD-2 mRNA than ATCC 10953. Heterologous expression of plasmid-borne fadI from the highly hBD-2-inducing strains in a ⌬fadI mutant of ATCC 10953 resulted in hBD-2 induction to levels comparable to those of the highly inducing strains, indicating that FAD-I is the principal F. nucleatum agent for hBD-2 induction in HOECs. Moreover, anti-FAD-I antibodies blocked F. nucleatum induction of hBD-2 by more than 80%. Recombinant FAD-I (rFAD-I) expressed in Escherichia coli triggered levels of hBD-2 transcription and peptide release in HOECs similar to those of native FAD-I (nFAD-I) isolated from F. nucleatum ATCC 25586. Tandem mass spectrometry revealed a diacylglycerol modification at the cysteine residue in position 16 for both nFAD-I and rFAD-I. Cysteine-to-alanine substitution abrogated FAD-I's ability to induce hBD-2. Finally, FAD-I activation of hBD-2 expression was mediated via both Toll-like receptor-1/2 (TLR-1/2) and TLR-2/6 heterodimerization. Microbial molecules like FAD-I may be utilized in novel therapeutic ways to bolster the host innate immune response at mucosal surfaces.T he epithelial surfaces of the oral cavity are sites of active bacterial colonization. While colonizing, certain bacteria promote activation of human beta defensin (hBD) expression in the oral mucosa (1-3). By virtue of their antimicrobial and immunoregulatory properties, these epithelial-cell-derived innate response peptides contribute to the homeostasis between the bacterium and the host (4). Human beta defensin 2 (hBD-2) and hBD-3 are the two inducible members of the hBD peptide family that we and others have described in the oral cavity (1, 5-10). Interestingly, while hBD-3 is associated with the highly proliferating, nondifferentiated stratum basale of the oral mucosa, hBD-2 is compartmentalized in the more superficial stratum spinosum and stratum granulosum; i.e., nonproliferating yet differentiating regions of the oral mucosa (11,12). This, along with other results showing that hBD-2 is induced as a result of inflammation via MAPK or NF〉 (5) while hBD-3 is activated through epidermal growth factor receptor (13, 14), strongly suggests that the latter is more involved in wound healing while the former plays a more active role in inhibiting microbial invasion during mucosal-barrier disruption (15-17). Moreover, in addition to their antimicrobial properties (18, 19), both peptides have been shown to act as chemokines in recruiting lymphoid and myeloid cells from the bloodstream (20).Fusobacterium nucleatum, a ubiquitous Gram-negative bacterium of the human oral cavity, has been extensively studied for its propert...

New approaches to complement vaccination are needed to combat the spread of SARS-CoV-2 and stop COVID-19 related deaths and medical complications. Human beta defensin 2 (hBD-2) is a naturally occurring epithelial cell derived host defense peptide that has antiviral properties. Our comprehensive in-silico studies demonstrate that hBD-2 binds the site on the CoV-2-RBD that docks with the ACE2 receptor. Biophysical measurements confirm that hBD-2 indeed binds to the CoV-2-receptor binding domain (RBD) (K D ∼ 2μM by surface plasmon resonance), preventing it from binding to ACE2 expressing cells. Importantly, hBD-2 shows specificity by blocking CoV-2/spike pseudoviral infection, but not VSVG mediated infection, of ACE2 expressing human cells with an IC 50 of 2.8 + 0.4 μM. These promising findings offer opportunities to develop hBD-2 and/or its derivatives and mimetics to safely and effectively use as agents to prevent SARS-CoV-2 infection.

Cystic fibrosis (CF) is a genetically inherited disease which is characterized by excessive inflammation and inability to resolve infection with pathogens such as Pseudomonas aeruginosa. Treatment options have improved with correctors and potentiators, but a cure remains elusive. Human mesenchymal stem cells (hMSCs) have the potential to be both anti-inflammatory and anti-microbial, which makes them ideal candidates for exploration as an innovative new therapeutic for CF. Using a sublethal CF mouse model of chronic Pseudomonas aeruginosa infection, we show that hMSCs and wild type bone marrow derived macrophages (BMM) have the capacity to attenuate inflammation while at the same time improving the ability to resolve infection. Animals infected with bacteria and treated with hMSCs and BMM had less weight lost, decreased pro-inflammatory cytokines, decreased severity of gross lung pathology as well as clinical score. Importantly, even though the inflammation was decreased in vivo, both BMM and hMSC treatment resulted in significant decrease in lung bacterial load. The improvement in the CF model was consistent with hMSC induced anti-inflammatory and anti-microbial activity which may involve the cathelicidin LL-37. These studies suggest that both healthy MSCs and BMM may provide important new direction toward cell based therapies in CF.

Background Antimicrobial peptides (AMPs) maintain a sterile environment in intestinal crypts, limiting microbial colonization and invasion. Decreased AMP expression is proposed to increase the risk for inflammatory bowel disease. Expression and function of inducible AMPs, human β-defensin 2 and 3 (hBD-3), remain poorly characterized in healthy and chronically inflamed intestine. Methods hBD-2 and hBD-3 peptide concentrations in serum and intestinal biopsies of ulcerative colitis, Crohn's Disease (CD), and healthy subjects were measured by ELISA. HBD-2 and hBD-3 mRNA was quantified by qPCR in biopsies from the terminal ileum (TI) of CD patients and healthy controls. HBD-3 peptide localization in the TI was visualized by confocal microscopy. Results Immunoreactive hBD-3 peptide is present in the TI and colon in healthy subjects. In the TI of CD patients, hBD-3, but not hBD-2 peptide, is increased four-fold, whereas hBD-2 peptide is elevated in the serum. hBD-3 mRNA in the CD TI remains unchanged and does not correlate with hBD-3 peptide expression. hBD-3 is localized to Paneth cell granules and the apical surface of the healthy columnar epithelium. In CD, hBD-3 peptide location switches to the basolateral surface of the columnar epithelium and is diffusely distributed within the lamina propria. Conclusion hBD-3 peptide throughout the healthy gastrointestinal tract suggests a role in maintaining balance between host defenses and commensal microbiota. Increased and relocalized secretion of hBD-3 toward the lamina propria in the CD TI indicates possible local immunomodulation during chronic inflammation, while increased serum hBD-2 in CD implicates its systemic antimicrobial and immunomodulatory role.