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.

Tumor-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. The 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. MCP-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 β-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 CaCl₂) 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 ∼75 ng/L and ∼3 μg/L, respectively. Mean (SD range) values in saliva samples from healthy donors (n = 60) were 9.5 (1.2–21) μg/L for hBD-2 and 326 (50–931) μg/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) μg/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 μg/g total protein, respectively. Cervicovaginal lavage from the same women contained mean concentrations of 1.46 and 55.5 μg/g total protein. Conclusion: These ELISA assays can measure inducible hBD peptide concentrations in body fluids by overcoming masking effects of anionic molecules. This approach may therefore be applicable for quantifying these peptides in health and disease.

We previously identified a cell wall-associated protein fromFusobacterium 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 (F usobacterium-associateddefensininducer). Here, we report differential induction of hBD-2 by different strains ofF. nucleatum; ATCC 25586 and ATCC 23726 induce significantly more hBD-2 mRNA than ATCC 10953. Heterologous expression of plasmid-bornefadIfrom the highly hBD-2-inducing strains in a ΔfadImutant of ATCC 10953 resulted in hBD-2 induction to levels comparable to those of the highly inducing strains, indicating that FAD-I is the principalF. nucleatumagent for hBD-2 induction in HOECs. Moreover, anti-FAD-I antibodies blockedF. nucleatuminduction of hBD-2 by more than 80%. Recombinant FAD-I (rFAD-I) expressed inEscherichia colitriggered levels of hBD-2 transcription and peptide release in HOECs similar to those of native FAD-I (nFAD-I) isolated fromF. nucleatumATCC 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.

More than one million new cases of sexually transmitted diseases (STDs) occur each day. The immune responses and inflammation induced by STDs and other frequent non-STD microbial colonizations (i.e. Candida and bacterial vaginosis) can have serious pathologic consequences in women including adverse pregnancy outcomes, infertility and increased susceptibility to infection by other pathogens. Understanding the types of immune mediators that are elicited in the lower genital tract by these infections/colonizations can give important insights into the innate and adaptive immune pathways that are activated and lead to strategies for preventing pathologic effects. 32 immune mediators were measured by multiplexed immunoassays to assess the immune environment of the lower genital tract mucosa in 84 women attending an urban STD clinic. IL-3, IL-1ß, VEGF, angiogenin, IL-8, ß2Defensin and ß3Defensin were detected in all subjects, Interferon-α was detected in none, while the remaining mediators were detected in 40% to 93% of subjects. Angiogenin, VEGF, FGF, IL-9, IL-7, lymphotoxin-α and IL-3 had not been previously reported in genital mucosal fluid from women. Strong correlations were observed between levels of TNF-α, IL-1ß and IL-6, between chemokines IP-10 and MIG and between myeloperoxidase, IL-8 and G-CSF. Samples from women with any STD/colonization had significantly higher levels of IL-8, IL-3, IL-7, IL-1ß, lactoferrin and myeloperoxidase. IL-1ß and lactoferrin were significantly increased in gonorrhea, Chlamydia, cervicitis, bacterial vaginosis and trichomoniasis. These studies show that mucosal fluid in general appears to be an environment that is rich in immune mediators. Importantly, IL-1ß and lactoferrin are biomarkers for STDs/colonizations providing insights into immune responses and pathogenesis at this mucosal site.