Expression, purification and investigation of antibacterial activity of a novel hybrid peptide LL37/hBD-129 by applied comprehensive computational and experimental approaches.

Antibiotic-resistant pathogens have become a great universal health concern. Antimicrobial peptides (AMPs) are small amphipathic and cationic polypeptides with high therapeutic potential against various microorganisms containing drug-resistant strains. Two major groups of these peptides, which have antibacterial activity against Gram-positive and Gram-negative bacteria, antiviral activity, and even antifungal activity, are defensins and cathelicidins. Hybridization of various AMPs is an appropriate approach to achieving new fusion AMPs with high antibacterial activity but low cellular toxicity. In the current research, the amino-acid sequence of human cathelicidin LL-37 (2-31) and Human beta-defensin (hBD)-129 were combined, and the fusion protein was evaluated by bioinformatics tool. The designed AMP gene sequence was commercially synthesized and cloned in the pET-28a expression vector. The LL-37/hBD-129 fusion protein was expressed in E.coli BL21-gold (DE3). The expression of the recombinant protein was evaluated using the SDS-PAGE method. The LL37/hBD-129 was successfully expressed as a recombinant hybrid AMP in E.coli BL21-gold (DE3) strain. Purification of the expressed AMP was performed by Ni-NTA column affinity chromatography, and the purified AMP was validated using the Western blot technic. Finally, the antimicrobial activity of the fusion AMP against Staphylococcus aureus and Escherichia coli bacteria was assessed. Based on the in silico analysis and experimental evaluations, the fusion AMP showed a significant antimicrobial effect on E. coli and Staphylococcus aureus bacteria.

Glyceryl butyrate attenuates enterotoxigenic Escherichia coli-induced intestinal inflammation in piglets by inhibiting the NF-κB/MAPK pathways and modulating the gut microbiota.

The aims of this study were to evaluate whether a diet supplemented with glyceryl butyrate could attenuate the immune-inflammatory response in piglets challenged with enterotoxigenic Escherichia coli (ETEC), and to explore the mechanisms of its regulation. Eighteen weaning piglets were assigned to three diets: basal diet (CON), antibiotics diet (ATB), and 0.5% glyceryl butyrate diet (GB group). Significantly lower concentrations of IL-1ß, IL-6 and TNF-α in the jejunum and IL-6 in the ileum were observed in the GB group than that in the CON group (P < 0.05). Moreover, a decreasing trend of IL-1ß (P = 0.075) and TNF-α (P = 0.070) was observed in the ileum in the GB group. Correspondingly, the GB group had significantly increased mRNA expression of porcine beta defensins (pBDs) in the jejunum (pBD1, pBD2, pBD114 and pBD129) and ileum (pBD2, pBD3, pBD114 and pBD129) (P < 0.05), and protein abundance of Claudin 1, Occludin, and ZO-1 in the jejunum and ileum (P < 0.05). Further research results showed that the improvement of beta defensins and tight junctions in the GB group was related to the decreased phosphorylation of the NFκB/MAPK pathway. In addition, the results of 16S rDNA sequencing showed that glycerol butyrate supplementation altered the ileal microbiota composition of piglets, increasing the relative abundance of Lactobacillus reuteri, Lactobacillus salivarius, and Lactobacillus agrilis. In summary, glyceryl butyrate attenuated the immune-inflammatory response in piglets challenged with ETEC by inhibiting the NF-κB/MAPK pathways and modulating the gut microbiota, and thus improved piglet intestinal health.

Age-Related Expression of IFN-λ1 Versus IFN-I and Beta-Defensins in the Nasopharynx of SARS-CoV-2-Infected Individuals.

SARS-CoV-2 coronavirus infection induces heterogeneous symptoms, ranging from asymptomatic to lethal forms. Severe forms usually occur in the elderly and/or individuals with comorbidities. Children generally remain asymptomatic to primary infection, suggesting that they may have an effective local innate immune response. IFN-I and -III have non-redundant protective roles against SARS-CoV-2, although sometimes damaging the host. The expression and role of anti-viral peptides during SARS-CoV-2 infection have thus far been little studied. We aimed to identify the innate immune molecules present at the SARS-CoV-2 entry point. We analyzed the mRNA levels of type I (IFN-α and -ß) and type III (IFN-λ1-3) interferons and selected antiviral peptides (i.e., ß-defensins 1-3, α-defensins [HNP1-3, HD5] pentraxin-3, surfactant protein D, the cathelicidin LL-37 and interleukin-26) in nasopharyngeal swabs from 226 individuals of various ages, either infected with SARS-CoV-2 (symptomatic or asymptomatic) or negative for the virus. We observed that infection induced selective upregulation of IFN-λ1 expression in pediatric subjects (≤15 years), whereas IFN-α, IFN-ß, IFN-λ2/λ3, and ß-defensin 1-3 expression was unaffected. Conversely, infection triggered upregulation of IFN-α, IFN-ß, IFN-λ2/λ3, and ß-defensin 1-3 mRNA expression in adults (15-65 years) and the elderly (≥ 65 years), but without modulation of IFN-λ1. The expression of these innate molecules was not associated with gender or symptoms. Expression of the interferon-stimulated genes IFITM1 and IFITM3 was upregulated in SARS-CoV-2-positive subjects and reached similar levels in the three age groups. Finally, age-related differences in nasopharyngeal innate immunity were also observed in SARS-CoV-2-negative subjects. This study shows that the expression patterns of IFN-I/-III and certain anti-viral molecules in the nasopharyngeal mucosa of SARS-CoV-2-infected subjects differ with age and suggests that susceptibility to SARS-CoV-2 may be related to intrinsic differences in the nature of mucosal anti-viral innate immunity.

Recombinant Avian ß-Defensin Produced by Food-Grade Lactococcus as a Novel and Potent Immunological Enhancer Adjuvant for Avian Vaccine.

In this study, we expressed rAvBD1-2-6-13 protein through Lactococcus lactis NZ3900, and the effects of the recombinant L. lactis NZ3900 as an immune enhancer and immune adjuvant were verified using in vivo and in vitro tests. In vitro tests revealed that recombinant L. lactis NZ3900 significantly activated the NF-κB signaling pathway and IRF signaling pathway in J774-Dual™ report cells and significantly increased the transcript levels of IL-10, IL-12p70, CD80, and CD86 in chicken PBMCs and chicken HD11 cells. In vivo experiments revealed that the immunized group supplemented with recombinant L. lactis NZ3900 as an adjuvant had significantly higher serum antibody titers and higher proliferative activity of PBMCs in the blood of the chickens immunized with NDV live and inactivated vaccines. Our study shows that the recombinant L. lactis NZ3900 has strong immunomodulatory activity both in vivo and in vitro and is a potential immune enhancer. Our work lays the foundation for the research and development of new animal immune enhancers for application in the poultry industry.

Steroid hormone modulates the production of cathelicidin and human ß-defensins in lung epithelial cells and macrophages promoting Mycobacterium tuberculosis killing.

Several studies have documented the interaction between the immune and endocrine systems as an effective defense strategy against tuberculosis, involving the production of several molecules and immunological processes. In this study, we determined the effect of cortisol and dehydroepiandrosterone (DHEA) on the production of antimicrobial peptides such as cathelicidin and human ß-defensin (HBD) -2, and HBD-3 and their effect on intracellular growth of Mycobacterium tuberculosis (Mtb) in lung epithelial cells and macrophages. Our results showed that DHEA promotes the production of these antimicrobial peptides in infected cells, correlating with the decrease of Mtb bacilli loads. These results suggest the use of exogenous DHEA as an adjuvant for tuberculosis therapy.

Mycoplasma hyopneumoniae Inhibits Porcine Beta-Defensin 2 Production by Blocking the Unfolded Protein Response To Facilitate Epithelial Adhesion and Infection.

Mycoplasma hyopneumoniae causes the disease porcine enzootic pneumonia, a highly contagious and chronic disease affecting pigs. Understanding the molecular mechanisms of its pathogenicity is critical for developing effective interventions to control this swine respiratory disease. Here, we describe a novel virulence mechanism by which M. hyopneumoniae interferes with the host unfolded protein response (UPR) and eventually facilitates bacterial adhesion and infection. We observed that M. hyopneumoniae infection suppressed the UPR target molecules GRP78 and CHOP by reducing PKR-like endoplasmic reticulum kinase/eukaryotic initiation factor 2 alpha (PERK/eIF2α) phosphorylation, ATF6 cleavage, and X-box binding protein 1 (XBP1) splicing. Interestingly, further analyses revealed that host UPR inhibition subsequently suppressed the NF-κB pathway, leading to the reduced production of porcine beta-defensin 2 (PBD-2), thus facilitating M. hyopneumoniae adherence and infection. This study provides new insights into the molecular pathogenesis of M. hyopneumoniae and sheds light upon its interactions with the host.

Improving the heterologous expression of human ß-defensin 2 (HBD2) using an experimental design.

At present, expressing antimicrobial peptides in bacterial models is considered a routine lab bench work. However, low expression yields of these types of proteins are usually obtained. In this work, the antimicrobial peptide human ß-defensin 2 (HBD2) was obtained in low expression yields in Escherichia coli BL21(DE3). To improve the expression yields of HBD2, some variables such as cell density, temperature, and length of induction, as well as the inducer concentration, were investigated using a 24-factorial design of experiments (DoE). This approach allowed us to identify the identification of critical variables (main effects and interactions among factors) affecting bacterial HBD2 expression. After the evaluation of 19 different combination, the best condition to express HBD2 had a pre-induction temperature of 37 °C, a cell density of 1.0 U (600 nm), an induction temperature of 20 °C and a 0.1 mM of gene expression inducer (IPTG) over four hours. Under such conditions, the expression yield of the HBD2 peptide was one order of magnitude higher than the peptide expression performed initially.

Interleukin-17A affects extracellular vesicles release and cargo in human keratinocytes.

Psoriasis is a chronic inflammatory systemic disease caused by deregulation of the interleukin-23/-17 axis that allows the activation of Th17 lymphocytes and the reprogramming of keratinocytes proliferative response, thereby inducing the secretion of cyto-/chemokines and antimicrobial peptides. Beside cell-to-cell contacts and release of cytokines, hormones and second messengers, cells communicate each other through the release of extracellular vesicles containing DNA, RNA, microRNAs and proteins. It has been reported the alteration of extracellular vesicles trafficking in several diseases, but there is scarce evidence of the involvement of extracellular vesicles trafficking in the pathogenesis of psoriasis. The main goal of the study was to characterize the release, the cargo content and the capacity to transfer bioactive molecules of extracellular vesicles produced by keratinocytes following recombinant IL-17A treatment if compared to untreated keratinocytes. A combined approach of standard ultracentrifugation, RNA isolation and real-time RT-PCR techniques was used to characterize extracellular vesicles cargo. Flow cytometry was used to quantitatively and qualitatively analyse extracellular vesicles and to evaluate cell-to-cell extracellular vesicles transfer. We report that the treatment of human keratinocytes with IL-17A significantly modifies the extracellular vesicles cargo and release. Vesicles from IL-17A-treated cells display a specific pattern of mRNA which is undid by IL-17A neutralization. Extracellular vesicles are taken up by acceptor cells irrespective of their content but only those derived from IL-17A-treated cells enable recipient cells to express psoriasis-associated mRNA. The results imply a role of extracellular vesicles in amplifying the pro-inflammatory cascade induced in keratinocyte by pro-psoriatic cytokines.

Human ß-Defensin 2 Expression in Oral Epithelium: Potential Therapeutic Targets in Oral Lichen Planus.

Human ß-defensin 2 (hBD-2) is a potent antimicrobial peptide that participates in defense against invading bacteria. We recently showed that bacterial components and histamine, through histamine H4 receptor (H4R), are involved in the pathogenesis of the potentially malignant lesion, oral lichen planus (OLP). However, the underlying mechanisms remain unknown. We, therefore, investigated the role of hBD2-histamine crosstalk signaling in promoting OLP pathology. Biopsies from OLP and oral tongue squamous cell carcinoma (OTSCC) patients, and healthy controls were used. Two OTSCC cell lines and normal human oral keratinocytes (HOKs) were used. HBD-2 and other targets were mapped by immunostaining and analyzed by ImageJ2 software. The highly sensitive droplet-digital PCR technology and qRT-PCR were utilized to study the clinically derived and in vitro samples, respectively. H4R was challenged with the specific agonist HST-10 and inverse agonist ST-1007. HBD-2 was highly induced in OLP lesions. In contrast, hBD2 expression was attenuated in OTSCC tissues, while very low levels of hBD-2 messenger RNA (mRNA) were observed in OTSCC cells. Together with tumor necrosis factor-α (TNF-α), histamine upregulated hBD-2 mRNA expression in HOKs. Activation of H4R seems to modulate the expression of epithelial hBD-2. These findings suggest the involvement of hBD-2 in the pathogenesis of OLP and may, thus, be harnessed for therapeutic interventions in OLP.

4-Hydroxybutyrate Promotes Endogenous Antimicrobial Peptide Expression in Macrophages.

IMPACT STATEMENT: This study evaluated the biological activity of hydroxylated derivatives of butyrate as inductors of antimicrobial peptides (AMPs) in murine bone marrow-derived macrophages in vitro. A differential modulation of AMP expression by the hydroxylated derivatives of butyrate is shown. The ability of sodium 4-hydroxybutyrate to upregulate AMP expression through a histone deacetylase inhibitory-independent mechanism, and to promote increased resistance to bacterial contamination in vivo are also shown. The findings provide an alternative for prevention of bacterial contamination of implanted biomaterials. Functionalization of biomaterials with hydroxylated derivatives of butyrate can enhance the endogenous antimicrobial activity of the immune system through increased production of AMPs by host cells, thus providing protection against bacterial contamination.

Long-term ethanol consumption promotes changes in ß-defensin isoform gene expression and induces structural changes and oxidative DNA damage to the epididymis of rats.

Chronic alcohol ingestion causes sexual dysfunction, impairs sperm motility and fertility, and changes semen quality. Considering the key role of epididymis in sperm development, the aim of the present study was to evaluate the effects of long-term ethanol consumption on epididymis changes, including alterations in ß-defensin isoform gene expression, oxidative stress, and pathological changes, such as cell proliferation and fibrosis in the epididymis of rats. In this study, male Wistar rats were equally divided into control and ethanol (4.5 g/kg BW) groups. After six weeks of treatment, the results revealed the proliferation of epididymis cells, fibrosis in the epididymis tissue, and a significant rise in the level of 8-OHdG and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase in the ethanol group, compared with the control group. Moreover, the ethanol group showed an increase in the gene expression of epididymal ß-defensin isoforms 15 and 21 and a reduction in the gene expression of ß-defensin isoforms 27 and 30, compared with the controls. These findings indicate that ethanol-induced epididymal damage and sperm abnormalities might be partly associated with changes in ß-defensin isoforms and epididymal structure, mediated by the increased activities of 8-OHdG and NADPH oxidase.

Suppression of IL-17F, but not of IL-17A, provides protection against colitis by inducing Treg cells through modification of the intestinal microbiota.

The cytokines IL-17A and IL-17F have 50% amino-acid identity and bind the same receptor; however, their functional differences have remained obscure. Here we found that Il17f-/- mice resisted chemically induced colitis, but Il17a-/- mice did not, and that Il17f-/- CD45RBhiCD4+ T cells induced milder colitis in lymphocyte-deficient Rag2-/- mice, accompanied by an increase in intestinal regulatory T cells (Treg cells). Clostridium cluster XIVa in colonic microbiota capable of inducing Treg cells was increased in both Il17f-/- mice and mice given transfer Il17f-/- T cells, due to decreased expression of a group of antimicrobial proteins. There was substantial production of IL-17F, but not of IL-17A, not only by naive T cells but also by various colon-resident cells under physiological conditions. Furthermore, antibody to IL-17F suppressed the development of colitis, but antibody to IL-17A did not. These observations suggest that IL-17F is an effective target for the treatment of colitis.

Effect of cytostatic agents on expression levels of human beta-defensins-1-4 in A431 and MCF-7 cell lines.

The aim of the study was to analyze an effect of cytostatic agents of different mechanism of action on expression levels of human beta-defensins-1-4 (hBD-1-4) in cultured human cancer cell lines. MATERIALS AND METHODS: Expression levels of hBD-1-4 mRNA were assessed using qPCR in human epidermoid carcinoma A431 cells and human breast adenocarcinoma MCF7 cells treated with cisplatin, methotrexate, doxorubicin or vincristine at the IC20 concentrations. RESULTS: The cytostatic agents with different mechanisms of action affected differently expression of hBDs, dependent on the cell line. Mostly, cytostatic agents suppressed significantly expression of hBDs. In contrast, vincristine caused significant up-regulation of hBD-1 (12 fold, p < 0.05) and hBD-4 (2 fold, p < 0.05) in MCF7, and doxorubicin significantly enhanced expression of hBD-3 (2 fold, p < 0.05) and hBD-4 (> 10 fold, p < 0.05) in A431 cells. CONCLUSION: The results of this pilot study show that expression levels of hBD-1-4 may be altered upon treatment with cytostatic agents depending on nature of cells.

Β-defensins - Underestimated peptides in influenza combat.

Defensins are a family of host defense peptides present in vertebrates, invertebrates and plants. They display broad antimicrobial activity and immunomodulatory functions. Herein, the natural anti-influenzal role of ß-defensins, as well as their potential usage as anti-influenza vaccine adjuvants and therapeutic agents, is reviewed. This article summarizes previously published information on ß-defensin modes of action, expression changes after influenza infection and vaccination, biotechnological usage and possible boosting of their production by dietary supplementation.

Bcl-3 induced by IL-22 via STAT3 activation acts as a potentiator of psoriasis-related gene expression in epidermal keratinocytes.

IL-22 induces STAT3 phosphorylation and mediates psoriasis-related gene expression. However, the signaling mechanism leading from pSTAT3 to the expression of these genes remains unclear. We focused on Bcl-3, which is induced by STAT3 activation and mediates gene expression. In cultured human epidermal keratinocytes, IL-22 increased Bcl-3, which was translocated to the nucleus with p50 via STAT3 activation. The increases in CXCL8, S100As and human ß-defensin 2 mRNA expression caused by IL-22 were abolished by siRNA against Bcl-3. Although CCL20 expression was also augmented by IL-22, the knockdown of Bcl-3 increased its level. Moreover, the combination of IL-22 and IL-17A enhanced Bcl-3 production, IL-22-induced gene expression, and the expression of other psoriasis-related genes, including those encoding IL-17C, IL-19, and IL-36γ. The expression of these genes (except for CCL20) was also suppressed by the knockdown of Bcl-3. Bcl-3 overexpression induced CXCL8 and HBD2 expression but not S100As expression. We also compared Bcl-3 expression between psoriatic skin lesions and normal skin. Immunostaining revealed strong signals for Bcl-3 and p50 in the nucleus of epidermal keratinocytes from psoriatic skin. The IL-22-STAT3-Bcl-3 pathway may be important in the pathogenesis of psoriasis.

Skin Wound Healing Is Accelerated by a Lipid Mixture Representing Major Lipid Components of Chamaecyparis obtusa Plant Extract.

In chronic nonhealing wounds, the healing process is disrupted and wounds are often infected with bacteria. About 85% of lower extremity amputations in diabetes are attributed to deep infection of foot ulcers. Therefore, infection control is critical for wound care. In this study, we analyzed lipid composition of Chamaecyparis obtusa extract, and we describe the wound-healing properties of its combination of 10 major lipid components. A 10-lipid mixture up-regulated HBD-3 and LL-37 through the olfactory receptor 2AT4 and induced phosphorylation of extracellular signal-regulated kinases and p38 mitogen-activated protein kinases in primary human keratinocytes. In addition, the 10-lipid mixture had direct bactericidal effects against Staphylococcus aureus and Streptococcus pyogenes and protected against staphylococcal α-toxin-induced keratinocyte cell death. In an animal model, the 10-lipid mixture accelerated skin wound healing and was also effective in healing wounds superinfected with S. aureus. We suggest that the 10-lipid mixture, because of its wound-healing and antimicrobial properties, can be beneficial for wound treatment.

Direct effects of fermented cow's milk product with Lactobacillus paracasei CBA L74 on human enterocytes.

Cow's milk fermented with Lactobacillus paracasei CBA L74 (FM-CBAL74) exerts a preventive effect against infectious diseases in children. We evaluated if this effect is at least in part related to a direct modulation of non-immune and immune defence mechanisms in human enterocytes. Human enterocytes (Caco-2) were stimulated for 48 h with FM-CBAL74 at different concentrations. Cell growth was assessed by colorimetric assay; cell differentiation (assessed by lactase expression), tight junction proteins (zonula occludens1 and occludin), mucin 2, and toll-like receptor (TRL) pathways were analysed by real-time PCR; innate immunity peptide synthesis, beta-defensin-2 (HBD-2) and cathelicidin (LL-37) were evaluated by ELISA. Mucus layer thickness was analysed by histochemistry. FMCBA L74 stimulated cell growth and differentiation, tight junction proteins and mucin 2 expression, and mucus layer thickness in a dose-dependent fashion. A significant stimulation of HBD-2 and LL-37 synthesis, associated with a modulation of TLR pathway, was also observed. FM-CBAL74 regulates non-immune and immune defence mechanisms through a direct interaction with the enterocytes. These effects could be involved in the preventive action against infectious diseases demonstrated by this fermented product in children.

Influence of gender on epithelial host defence peptide gene expression under non-infected and infected conditions: A basic medical research study.

Bacterial resistance against conventional antibiotics is increasing. This introduces challenges, for example, in the treatment of infected surgical wounds. Host defence peptides (HDP), which are endogenous peptide antibiotics, show broad-spectrum antimicrobial effectiveness. They protect the organism against pathological microorganisms. Synthetic HDP might supplement or even become alternatives to conventional antibiotics. Knowledge of their quantities under physiological and pathophysiological conditions is therefore required. The influence of gender on HDP expression is unknown. This study evaluates whether gender influences HDP expression in infected or healthy epithelium. Expression levels of HDP human beta-defensin (hBD)-1, -2 and -3 and psoriasin (S100A7) were analysed, by using real-time polymerase chain reaction, in samples of epithelium from infected surgical wounds (n = 20) and healthy epithelium (n = 14) from the neck in a basic medical research study (analytic observational design). The results demonstrated a significantly elevated expression of hBD-2, hBD-3 and psoriasin (P = 0.001 each) in infected epithelium compared with healthy epithelium. No difference in HDP expression levels was evident between samples from female and male patients, either within infected samples or within healthy epithelium samples. Thus, gender does not affect the cutaneous expression of the investigated HDP. This is fundamental knowledge for the study and potential use of HDP derivates as alternative antibiotic substances.

Exposure to fermentation supernatant of Staphylococcus aureus accelerated dedifferentiation of chondrocytes and production of antimicrobial peptides.

Staphylococcus aureus (S. aureus) is the most popular pathogen found in septic arthritis. Despite bacteria was eradicated from joint cavity during acute infection, destruction of articular cartilage often continues for years, leading to permanent joint damage. The mechanism responsible for this consistent catabolic reaction in septic arthritis remains unclear. Here, we found that fermentation supernatant (FS) of S. aureus accelerated dedifferentiation of chondrocytes and induced expression of catabolic factors including A Disintegrin-like and Metalloproteinase with Thrombospondin-1 motifs 5, NO synthase 2, matrix metalloproteinase-3, -13. In response to FS of S. aureus stimulation, expression of antimicrobial peptides (AMPs) including ß-defensin-1, -2, -3, -4, cathelicidin antimicrobial peptide (CAMP) in dedifferentiated chondrocytes was significantly higher than that in chondrocytes which maintained their differentiated phenotype. Among AMPs detected, expression of CAMP in dedifferentiated chondrocytes was observed to increase 170 times higher than that in differentiated ones. When exposed to FS of S. aureus, expression of interleukin (IL)-1ß, IL-17F, and IL-22 were remarkably increased in dedifferentiated chondrocytes. These results indicated that dedifferentiation of chondrocytes caused by exposure to S. aureus might be responsible for secondary osteoarthritis (OA) after acute S. aureus infection in joint. While, one potential benefit of dedifferentiation resulted from S. aureus exposure is that chondrocytes initiates a self-protective responsiveness by producing more AMPs against bacterial infection. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:443-451, 2018.

Comparison of IL-1ß, TNF-α, hBD-2, and hBD-3 Expression in the Dental Pulp of Smokers Versus Nonsmokers.

INTRODUCTION: To date, the endodontic literature lacks research on the effect of smoking on cytokine and defensin expression in the dental pulp. Therefore, the aim of this study was to investigate the expression of interleukin (IL)-1ß, tumor necrosis factor (TNF)-α, human beta defensin (hBD)-2 and hBD-3 in the dental pulp of smokers and compare them with nonsmokers. We hypothesized that cytokine and defensin expression would be reduced in smokers as compared with nonsmokers. METHODS: Thirty-two smokers and 37 nonsmokers with endodontic pulpal diagnoses of normal, symptomatic irreversible pulpitis and asymptomatic irreversible pulpitis were included in this cross-sectional study. Samples from pulp chambers were collected and stored in phosphate-buffered saline at -80°C. Luminex was used to measure IL-1ß and TNF-α levels. The levels of hBD-2 and hBD-3 were measured using enzyme-linked immunosorbent assay. Marker levels were normalized to protein concentrations and data were analyzed using Kruskal-Wallis test, Mann-Whitney U test, and 2-way analysis of variance (α = 0.05). RESULTS: Pulpal concentrations of TNF-α and hBD-2 were significantly lower among smokers (P < .01), whereas no significant difference was observed for IL-1ß, or hBD-3. Two-way analysis of covariance revealed that smoking status (P < .001), not endodontic diagnosis (pulpal status), significantly affected TNF-α and hBD-2 levels. CONCLUSIONS: This study reported that smokers are immunologically deficient in TNF-α and hBD-2, suggesting that dental pulps of smokers possess limited defense mechanisms, affecting their endodontic prognosis and indicating a cause for their reported inferior outcome.