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Measurement of immune mediators and antimicrobial activity in female genital tract secretions may provide biomarkers predictive of risk for HIV-1 acquisition and surrogate markers of microbicide safety. However, optimal methods for sample collection do not exist. This study compared collection methods. Secretions were collected from 48 women (24 with bacterial vaginosis [BV]) using vaginal and endocervical Dacron and flocked swabs. Cervicovaginal lavage (CVL) was collected with 10 mL of Normosol-R (n = 20), saline (n = 14), or water (n = 14). The concentration of gluconate in Normosol-R CVL was determined to estimate the dilution factor. Cytokine and antimicrobial mediators were measured by Luminex or ELISA and corrected for protein content. Endogenous anti-HIV-1 and anti-E. coli activity were measured by TZM-bl assay or E. coli growth. Higher concentrations of protein were recovered by CVL, despite a 10-fold dilution of secretions, as compared to swab eluents. After protein correction, endocervical swabs recovered the highest mediator levels regardless of BV status. Endocervical and vaginal flocked swabs recovered significantly higher levels of anti-HIV-1 and anti-E. coli activity than Dacron swabs (P<0.001). BV had a significant effect on CVL mediator recovery. Normosol-R tended to recover higher levels of most mediators among women with BV, whereas saline or water tended to recover higher levels among women without BV. Saline recovered the highest levels of anti-HIV-1 activity regardless of BV status. Endocervical swabs and CVL collected with saline provide the best recovery of most mediators and would be the optimal sampling method(s) for clinical trials.

Defensins combat pathogenic bacteria invading the mammalian intestine. Salzman and co-workers find that defensins influence the composition of the small intestinal commensal microbiota and the presence of interleukin 17–producing T cells in the lamina propria. Antimicrobial peptides are important effectors of innate immunity throughout the plant and animal kingdoms. In the mammalian small intestine, Paneth cell α-defensins are antimicrobial peptides that contribute to host defense against enteric pathogens. To determine if α-defensins also govern intestinal microbial ecology, we analyzed the intestinal microbiota of mice expressing a human α-defensin gene ( DEFA5 ) and in mice lacking an enzyme required for the processing of mouse α-defensins. In these complementary models, we detected significant α-defensin-dependent changes in microbiota composition, but not in total bacterial numbers. Furthermore, DEFA5 -expressing mice had striking losses of segmented filamentous bacteria and fewer interleukin 17 (IL-17)-producing lamina propria T cells. Our data ascribe a new homeostatic role to α-defensins in regulating the makeup of the commensal microbiota.

Defensins are antimicrobial peptides that contribute broadly to innate immunity, including protection of mucosal tissues. Human α-defensin (HD) 6 is highly expressed by secretory Paneth cells of the small intestine. However, in contrast to the other defensins, it lacks appreciable bactericidal activity. Nevertheless, we report here that HD6 affords protection against invasion by enteric bacterial pathogens in vitro and in vivo. After stochastic binding to bacterial surface proteins, HD6 undergoes ordered self-assembly to form fibrils and nanonets that surround and entangle bacteria. This self-assembly mechanism occurs in vivo, requires histidine-27, and is consistent with x-ray crystallography data. These findings support a key role for HD6 in protecting the small intestine against invasion by diverse enteric pathogens and may explain the conservation of HD6 throughout Hominidae evolution.

Sepsis claims an estimated 30 million episodes and 6 million deaths per year, and treatment options are rather limited. Human neutrophil peptides 1–3 (HNP1–3) are the most abundant neutrophil granule proteins but their neutrophil content varies because of unusually extensive gene copy number polymorphism. A genetic association study found that increased copy number of the HNP-encoding gene DEFA1/DEFA3 is a risk factor for organ dysfunction during sepsis development. However, direct experimental evidence demonstrating that these risk alleles are pathogenic for sepsis is lacking because the genes are present only in some primates and humans. Here, we generate DEFA1/DEFA3 transgenic mice with neutrophil-specific expression of the peptides. We show that mice with high copy number of DEFA1/DEFA3 genes have more severe sepsis-related vital organ damage and mortality than mice with low copy number of DEFA1/DEFA3 or wild-type mice, resulting from more severe endothelial barrier dysfunction and endothelial cell pyroptosis after sepsis challenge. Mechanistically, HNP-1 induces endothelial cell pyroptosis via P2X7 receptor-mediating canonical caspase-1 activation in a NLRP3 inflammasome-dependent manner. Based on these findings, we engineered a monoclonal antibody against HNP-1 to block the interaction with P2X7 and found that the blocking antibody protected mice carrying high copy number of DEFA1/DEFA3 from lethal sepsis. We thus demonstrate that DEFA1/DEFA3 copy number variation strongly modulates sepsis development in vivo and explore a paradigm for the precision treatment of sepsis tailored by individual genetic information.

The neutrophil is the major phagocyte and the final effector cell of the innate immunity, with a primary role in the clearance of extracellular pathogens. Using the broad array of cytokines, extracellular traps, and effector molecules as the humoral arm, neutrophils play a crucial role in the host defense against pathogen infections. On the other hand, the pathogen has the capacity to overcome neutrophil-mediated host defense to establish infection causing human disease. Pathogens, such as S. aureus, have the potential to thwart neutrophil chemotaxis and phagocytosis and thereby succeed in evading killing by neutrophils. Furthermore, S. aureus surviving within neutrophils promotes neutrophil cytolysis, resulting in the release of host-derived molecules that promote local inflammation. Here, we provide a detailed overview of the mechanisms by which neutrophils kill the extracellular pathogens and how pathogens evade neutrophils degradation. This review will provide insights that might be useful for the development of novel therapies against infections caused by antibiotic resistant pathogens.

Acinetobacter baumannii is the causative agent of a wide range of nosocomial and community-acquired infections that remain extremely difficult to treat due largely to its antibiotic resistance contributed, in part, by biofilm formation. We find that the prototypic human neutrophil α-defensin HNP1, present in the bronchoalveolar lavage fluids from Acinetobacter baumannii -infected patients, promotes Acinetobacter baumannii biofilm formation through interactions with the bacterial outer membrane protein OmpA. As a result of HNP1-enhanced biofilm formation, Acinetobacter baumannii becomes more tolerant to antibiotics and more readily colonizes host cells and tissues. These unexpected findings contrast the protective roles HNP1 plays in innate immunity against microbial infection, showcasing an example of the host-pathogen arms race where a host defense peptide is exploited by a microbe for pathogenicity. Acinetobacter baumannii is an opportunistic pathogen that causes nosocomial and community-acquired infections. Here, the authors determine that human neutrophil α-defensin HNP1 can promote A. baumannii biofilm formation.

Antiviral immunity compromises the efficacy of adeno-associated virus (AAV) vectors used for gene therapy. This is well understood for the adaptive immune response. However, innate immune effectors like alpha-defensin antimicrobial peptides also block AAV infection, although their mechanisms of action are unknown. To address this gap in knowledge, we investigated AAV2 and AAV6 neutralization by human neutrophil peptide 1 (HNP1), a myeloid alpha-defensin, and human defensin 5 (HD5), an enteric alpha-defensin. We found that both defensins bind to AAV2 and inhibit infection at low micromolar concentrations, similar to our prior studies of AAV6. While HD5 prevents AAV2 and AAV6 from binding to cells, HNP1 does not. However, AAV2 and AAV6 exposed to HD5 after binding to cells are still neutralized, indicating an additional block to infection. Accordingly, both HD5 and HNP1 inhibit externalization of the VP1 unique domain of both AAV2 and AAV6, which contains a phospholipase A 2 enzyme required for endosome escape and nuclear localization signals required for nuclear entry. Consequently, both defensins prevent AAV from reaching the nucleus. Disruption of intracellular trafficking of the viral genome to the nucleus is reminiscent of how alpha-defensins neutralize other non-enveloped viruses, suggesting a common mechanism of inhibition. These results will inform the development of vectors capable of overcoming these hurdles to improve the efficiency of gene therapy.

Neutrophilic inflammation and a high level of free α-defensins are main features of chronic airway inflammation in alpha-1 antitrypsin-deficient (AATD) individuals. Despite the antimicrobial activities of α-defensins by direct bacterial killing and by modulation of immune responses, AATD individuals are paradoxically burdened by recurrent exacerbation triggered by bacterial infections, frequently with nontypeable Haemophilus influenzae (NTHi). Previous studies demonstrated that high, rather than low α-defensin level could modulate the local pro-inflammatory milieu of bronchial epithelial cells and macrophages promoting chronic inflammation and lower pathogen phagocytosis. IgG-mediated phagocytosis and NTHi adherence, engulfment and phagocytosis were measured in human alveolar macrophages and monocyte-derived macrophages (MDM) isolated from patients with AATD and from healthy individuals. A high concentration of free α-defensins induced NTHi adherence to MDMs but decreased IgG-mediated phagocytosis by MDMs. The decreased phagocytosis was associated with TLR4 activation, downstream signaling via NF-κB p65 and marked increased secretion of inflammatory cytokines, CXCL8, IL-1b, and TNFα by the α-defensin-treated and NTHi-infected MDMs. Exogenous AAT treatment and TLR4 inhibitor decreased TNFα expression in α-defensin-treated cells. Dampening the downstream effects of a high concentration of α-defensins may render AAT and TLR4 inhibitors as potential therapies to decrease NTHi colonization and increase its clearance by phagocytosis in AATD individuals.

Background Postnatal development of the mammalian mucosal immune system is crucial for responding to the rapid colonization by commensal bacteria and possible exposure to pathogens. This study analyzed expression patterns for mRNAs and their relationship with microRNAs (miRNAs) in the bovine small intestine during the critical neonatal period (0 to 42 days). This analysis revealed molecular mechanisms regulating the postnatal development of the intestinal mucosal immune system. Results Small intestine samples (jejunum and ileum) were collected from newborn male, Holstein calves immediately post-partum ( n  = 3) and at 7 ( n  = 5), 21 ( n  = 5), and 42 ( n  = 5) days of age and the transcriptomes were profiled using RNA-Seq. When analyzing all time points collectively, greater expression of genes encoding the complement functional pathway, as well as lower expression of genes encoding Toll-like receptors and NOD-like receptors were observed in the jejunum when compared to the ileum. In addition, significant changes in the expression of immune-related genes were detected within the first week post-partum in both jejunum and ileum. For example, increased expression of genes encoding tight junction proteins (claudin 1, claudin 4 and occludin), an antimicrobial peptide (Regenerating Islet-Derived 3-γ), NOD-like receptors (NACHT, LRR and PYD domain-containing protein 3), regulatory T cell marker (forkhead box P3), and both anti-inflammatory (interleukin 10) and pro-inflammatory (interleukin 8) cytokines was observed throughout the small intestine of 7-day-old calves when compared to newborn calves. Moreover, the expression of mucosal immune-related genes were either positively or negatively correlated with total bacterial population depending on both intestinal region and age. The integrated analysis of miRNAs and mRNAs supported the conclusion that miRNAs may regulate temporal changes in the expression of genes encoding tight junction proteins (miR-335), cytokines (miR-335) and bacterial recognition (miR-100) during the first week of small intestine development. Conclusion The rapid development of transcriptional differences between jejunum and ileum reveal that these two intestinal regions make distinct contributions to the intestinal mucosal immune system during the early neonatal period. In addition, transcriptome analysis indicates that the first week after birth is a very dynamic developmental period for the intestinal mucosal immune system and these changes may be regulated by both miRNAs and microbial colonization. Findings from this study indicate that a detailed analysis of both the abundance and diversity of the colonizing microbiome may be necessary to understand factors regulating the rapid development of the mucosal immune system during the first week of life.

Alcohol use among older adults is on the rise. This increase is clinically relevant as older adults are at risk for increased morbidity and mortality from many alcohol-related chronic diseases compared to younger patients. However, little is known regarding the synergistic effects of alcohol and age. There are intriguing data suggesting that aging may lead to impaired intestinal barrier integrity and dysbiosis of the intestinal microbiome, which could increase susceptibility to alcohol's negative effects. To study the effects of alcohol in age we exposed aged and young mice to 3 days of moderate ethanol and evaluated changes in gut parameters. We found that these levels of drinking do not have obvious effects in young mice but cause significant alcohol-induced gut barrier dysfunction and expression of the pro-inflammatory cytokine TNFα in aged mice. Ethanol-induced downregulation of expression of the gut-protective antimicrobial peptides Defa-rs1, Reg3b, and Reg3g was observed in aged, but not young mice. Analysis of the fecal microbiome revealed age-associated shifts in microbial taxa, which correlated with intestinal and hepatic inflammatory gene expression. Taken together, these data demonstrate that age drives microbiome dysbiosis, while ethanol exposure in aged mice induces changes in the expression of antimicrobial genes important for separating these potentially damaging microbes from the intestinal lumen. These changes highlight potential mechanistic targets for prevention of the age-related exacerbation of effects of ethanol on the gut. •Aged mice display increased sensitivity to the gastrointestinal effects of ethanol.•Three days of ethanol binge increase intestinal barrier dysfunction and TNFα expression in aged, but not young mice.•Ethanol exposure in aged mice induces downregulation of intestinal antimicrobial peptides.•Age-specific changes in the fecal microbiome correlate with altered expression of cytokines and antimicrobial peptides.