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Microbial biofilm formation creates a persistent and resistant environment in which microorganisms can survive, contributing to antibiotic resistance and chronic inflammatory diseases. Increasingly, biofilms are caused by multi-drug resistant microorganisms, which, coupled with a diminishing supply of effective antibiotics, is driving the search for new antibiotic therapies. In this respect, antimicrobial peptides (AMPs) are short, hydrophobic, and amphipathic peptides that show activity against multidrug-resistant bacteria and biofilm formation. They also possess broad-spectrum activity and diverse mechanisms of action. In this comprehensive review, 150 publications (from January 2020 to September 2023) were collected and categorized using the search terms 'polypeptide antibiotic agent', 'antimicrobial peptide', and 'biofilm'. During this period, a wide range of natural and synthetic AMPs were studied, of which LL-37, polymyxin B, GH12, and Nisin were the most frequently cited. Furthermore, although many microbes were studied, Staphylococcus aureus and Pseudomonas aeruginosa were the most popular. Publications also considered AMP combinations and the potential role of AMP delivery systems in increasing the efficacy of AMPs, including nanoparticle delivery. Relatively few publications focused on AMP resistance. This comprehensive review informs and guides researchers about the latest developments in AMP research, presenting promising evidence of the role of AMPs as effective antimicrobial agents.

Introduction: Sepsis is characterized by a dysregulated innate immune response. It is a leading cause of morbidity and mortality in newborns, in particular for newborns that are born premature. Although previous literature indicate that the pro-inflammatory response may be impaired in preterm newborns, serum levels of monocyte-derived cytokines, such as TNF-α and IL-6, vary highly between newborns and can reach adult-like concentrations during sepsis. These contradictory observations and the severe consequences of neonatal sepsis in preterm newborns highlight the need for a better understanding of the pro-inflammatory cytokine response of preterm newborns to improve sepsis-related outcomes., Methods and Results: Using an in vitro model with multiple read outs at the transcriptional and protein level, we consistently showed that the monocyte-derived cytokine response induced by sepsis-related bacteria is comparable between preterm newborns, term newborns and adults. We substantiated these findings by employing recombinant Toll-like receptor (TLR) ligands and showed that the activation of specific immune pathways, including the expression of TLRs, is also similar between preterm newborns, term newborns and adults. Importantly, we showed that at birth the production of TNF-α and IL-6 is highly variable between individuals and independent of gestational age., Discussion: These findings indicate that preterm newborns are equally capable of mounting a pro-inflammatory response against a broad range of bacterial pathogens that is comparable to term newborns and adults. Our results provide a better understanding of the pro-inflammatory response by preterm newborns and could guide the development of interventions that specifically modulate the pro-inflammatory response during sepsis in preterm newborns., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Jans, van Dun, Gorissen, Pieterman, Voskamp, Schoenmakers, Taal and Unger.)

Background: Mycoplasma pneumoniae is a common cause of community-acquired pneumonia in school-aged children and can be preceded by asymptomatic carriage. However, its role in recurrent respiratory tract infections is unclear. We studied the prevalence of M.pneumoniae carriage in children with recurrent respiratory infections and identified associated factors., Methods: We tested M.pneumoniae carriage by qPCR in children with recurrent infections and their healthy family members in a cross-sectional study. Serum and mucosal total and M.pneumoniae-specific antibody levels were measured by ELISA and nasopharyngeal microbiota composition was characterized by 16S-rRNA sequencing., Findings: Prevalence of M.pneumoniae carriage was higher in children with recurrent infections (68%) than their family members without infections (47% in siblings and 27% in parents). M.pneumoniae carriage among family members appeared to be associated with transmission within the household, likely originating from the affected child. In logistic regression corrected for age and multiple comparisons, IgA (OR 0.16 [0.06-0.37]) and total IgG deficiency (OR 0.15 [0.02-0.74]) were less prevalent in M.pneumoniae carriers (n = 78) compared to non-carriers (n = 36). In multivariable analysis, the nasopharyngeal microbiota of M.pneumoniae carriers had lower alpha diversity (OR 0.27 [0.09-0.67]) and a higher abundance of Haemophilus influenzae (OR 45.01 [2.74-1608.11]) compared to non-carriers., Interpretation: M.pneumoniae carriage is highly prevalent in children with recurrent infections and carriers have a less diverse microbiota with an overrepresentation of disease-associated microbiota members compared to non-carriers. Given the high prevalence of M.pneumoniae carriage and the strong association with H. influenzae, we recommend appropriate antibiotic coverage of M.pneumoniae and H. influenzae in case of suspected pneumonia in children with recurrent respiratory tract infections or their family members., Funding: Wilhelmina Children's Hospital Research Fund, 'Christine Bader Stichting Irene KinderZiekenhuis', Sophia Scientific Research Foundation, ESPID Fellowship funded by Seqirus, Hypatia Fellowship funded by Radboudumc and The Netherlands Organisation for Health Research and Development (ZonMW VENI grant to LM Verhagen)., Competing Interests: Declaration of interests None of the authors have a conflict of interest., (Copyright © 2023 The Author(s). Published by Elsevier B.V. All rights reserved.)

Background: Cystic fibrosis (CF) airway disease is characterized by chronic inflammation, featuring neutrophil influx to the lumen. Airway macrophages (AMs) can promote both inflammation and resolution, and are thus critical to maintaining and restoring homeostasis. CF AM functions, specifically scavenging activity and resolution of inflammation, have been shown to be impaired, yet underlying processes remain unknown. We hypothesized that impaired CF AM function results from an altered expression of receptors that mediate or regulate scavenging, and set out to investigate changes in expression of these markers during the early stages of CF lung disease., Methods: Bronchoalveolar lavage fluid (BALF) was collected from 50 children with CF aged 1, 3 or 5 years. BALF cells were analyzed using flow cytometry. Expression levels of surface markers on AMs were expressed as median fluorescence intensities (MFI) or percentage of AMs positive for these markers. The effect of age and neutrophilic inflammation, among other variables, on marker expression was assessed with a multivariate linear regression model., Results: AM expression of scavenger receptor CD163 decreased with age (p = 0.016) and was negatively correlated with BALF %neutrophils (r = -0.34, p = 0.016). AM expression of immune checkpoint molecule SIRPα also decreased with age (p = 0.0006), but did not correlate with BALF %neutrophils. Percentage of AMs expressing lipid scavenger CD36 was low overall (mean 20.1% ± 16.5) and did not correlate with other factors. Conversely, expression of immune checkpoint PD-1 was observed on the majority of AMs (mean PD-1 pos 72.9% ± 11.8), but it, too, was not affected by age or BALF %neutrophils. Compared to matched blood monocytes, AMs had a higher expression of CD16, CD91, and PD-1, and a lower expression of CD163, SIRPα and CD36., Conclusion: In BALF of preschool children with CF, higher age and/or increased neutrophilic inflammation coincided with decreased expression of scavenger receptors on AMs. Expression of scavenging receptors and regulators showed a distinctly different pattern in AMs compared to blood monocytes. These findings suggest AM capacity to counter inflammation and promote homeostasis reduces during initiation of CF airway disease and highlight new avenues of investigation into impaired CF AM function., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Slimmen, Giacalone, Schofield, Horati, Manaï, Estevão, Garratt, Peng, Tirouvanziam, Janssens and Unger.)

The worldwide increase in the incidence of antibiotic resistance of the atypical bacterium Mycoplasma pneumoniae (MP) challenges the treatment of MP infections, especially in children. Therefore, alternative strategies for the treatment of MP infections are warranted. Galacto- and fructo-oligosaccharides (GOS and FOS) are a specific group of complex carbohydrates that were recently shown to possess direct anti-pathogenic properties. In this study, we assessed whether GOS and FOS exert anti-microbial and anti-infective effects against MP and, especially, macrolide-resistant MP (MRMP) in vitro. The MIC values of GOS for MP and MRMP were 4%. In contrast, the MIC values of FOS for both MP and MRMP were 16%. A time-kill kinetic assay showed that FOS possess bacteriostatic properties, while for GOS, a bactericidal effect against MP and MRMP was observed after 24 h at a concentration of 4x MIC. In co-cultures with human alveolar A549 epithelial cells, GOS killed adherent MP and MRMP and also concentration-dependently inhibited their adherence to A549 cells. Further, GOS suppressed (MR)MP-induced IL-6 and IL-8 in A549 cells. None of the aforementioned parameters were affected when FOS were added to these co-cultures. In conclusion, the anti-infective and anti-microbial properties of GOS could provide an alternative treatment against MRMP and MP infections.

Mycoplasma pneumoniae and Streptococcus pneumoniae are the most common bacterial causes of pneumonia in children. The clinical characteristics of pneumonia differ significantly between the two bacteria. We aimed to elucidate the differences in pathogenesis between M. pneumoniae and S. pneumoniae by characterizing the respiratory epithelial cell immune response to both pathogens. Using primary human bronchial epithelial cells in air-liquid interface cultures, we observed lower production of the proinflammatory cytokines interleukin-6 (IL-6) and IL-8 in response to M. pneumoniae than to S. pneumoniae. In contrast to the differences in proinflammatory cytokine production, Toll-like receptor 2 (TLR2)-mediated signaling in response to M. pneumoniae was stronger than to S. pneumoniae. This difference largely depended on TLR1 and not TLR6. We found that M. pneumoniae, but not S. pneumoniae, also induced signaling of TLR10, a coreceptor of TLR2 that has inhibitory properties. M. pneumoniae-induced TLR10 signaling on airway epithelial cells was partially responsible for low IL-8 production, as blocking TLR10 by specific antibodies increased cytokine production. M. pneumoniae maintained Th2-associated cytokine production by epithelial cells, which concurs with the known association of M. pneumoniae infection with asthma. M. pneumoniae left IL-33 levels unchanged, whereas S. pneumoniae downregulated IL-33 production both under homeostatic and Th2-promoting conditions. By directly comparing M. pneumoniae and S. pneumoniae, we demonstrate that M. pneumoniae avoids induction of proinflammatory cytokine response despite its ability to induce robust TLR2 signaling. Our new findings suggest that this apparent paradox may be partially explained by M. pneumoniae-induced signaling of TLR2/TLR10.

Enzyme replacement therapy (ERT) is the current standard treatment for Pompe disease, a lysosomal storage disorder caused by deficiency of the lysosomal enzyme acid alpha-glucosidase (GAA). ERT has shown to be lifesaving in patients with classic infantile Pompe disease. However, a major drawback is the development of neutralizing antibodies against ERT. Hematopoietic stem and progenitor cell-mediated lentiviral gene therapy (HSPC-LVGT) provides a novel, potential lifelong therapy with a single intervention and may induce immune tolerance. Here, we investigated whether ERT can be safely applied as additional or alternative therapy following HSPC-LVGT in a murine model of Pompe disease. We found that lentiviral expression at subtherapeutic dose was sufficient to induce tolerance to the transgene product, as well as to subsequently administered ERT. Immune tolerance was established within 4-6 weeks after gene therapy. The mice tolerated ERT doses up to 100 mg/kg, allowing ERT to eliminate glycogen accumulation in cardiac and skeletal muscle and normalizing locomotor function. The presence of HSPC-derived cells expressing GAA in the thymus suggested the establishment of central immune tolerance. These findings demonstrate that lentiviral gene therapy in murine Pompe disease induced robust and long-term immune tolerance to GAA either expressed by a transgene or supplied as ERT., Competing Interests: A.T.v.d.P. has received consulting fees from Sanofi Genzyme and has provided consulting services, participated in advisory board meetings, and received grants for premarketing studies and research from industries via agreements between Erasmus MC and the industry. N.P.v.T. is an employee of AVROBIO (Cambridge, MA)., (© 2022 The Author(s).)

Background: Mycoplasma pneumoniae is the most common bacterial cause of pneumonia in children hospitalised for community-acquired pneumonia (CAP). Prevention of infection by vaccines may be an important strategy in the presence of emerging macrolide-resistant M. pneumoniae. However, knowledge of immune responses to M. pneumoniae is limited, complicating vaccine design., Methods: We studied the antibody response during M. pneumoniae respiratory tract infection and asymptomatic carriage in two different cohorts., Results: In a nested case-control study (n=80) of M. pneumoniae carriers and matched controls we observed that carriage by M. pneumoniae does not lead to a rise in either mucosal or systemic M. pneumoniae- specific antibodies, even after months of persistent carriage. We replicated this finding in a second cohort (n=69) and also found that during M. pneumoniae CAP, mucosal levels of M. pneumoniae- specific IgA and IgG did increase significantly. In vitro adhesion assays revealed that high levels of M. pneumoniae- specific antibodies in nasal secretions of paediatric patients prevented the adhesion of M. pneumoniae to respiratory epithelial cells., Conclusions: Our study demonstrates that M. pneumoniae- specific mucosal antibodies protect against bacterial adhesion to respiratory epithelial cells, and are induced only during M. pneumoniae infection and not during asymptomatic carriage. This is strikingly different from carriage with bacteria such as Streptococcus pneumoniae where mucosal antibodies are induced by bacterial carriage., Competing Interests: Conflict of interest: None declared., (Copyright ©The authors 2022.)

Bacterial respiratory tract infections (RTIs) are a major global health burden, and the role of antigen-presenting cells (APCs) in mounting an immune response to contain and clear invading pathogens is well-described. However, most encounters between a host and a bacterial pathogen do not result in symptomatic infection, but in asymptomatic carriage instead. The fact that a pathogen will cause infection in one individual, but not in another does not appear to be directly related to bacterial density, but rather depend on qualitative differences in the host response. Understanding the interactions between respiratory pathogens and airway APCs that result in asymptomatic carriage, will provide better insight into the factors that can skew this interaction towards infection. This review will discuss the currently available knowledge on airway APCs in the context of asymptomatic bacterial carriage along the entire respiratory tract. Furthermore, in order to interpret past and futures studies into this topic, we propose a standardized nomenclature of the different stages of carriage and infection, based on the pathogen's position with regard to the epithelium and the amount of inflammation present.

Injection of antigenic peptides has been widely used as a vaccine strategy to boost T cell immunity. However, the poor immunogenicity of single peptides can potentially be strengthened through modification of the tertiary structure and the selection of the accompanying adjuvant. Here, we generated antigenic peptides into non-linear trimers by solid phase peptide synthesis, thereby enhancing antigen presentation by dendritic cells to CD8 + T cells in vitro and in vivo. CD8 + T cells from mice vaccinated with trimers showed an KLRG1 + effector phenotype and were able to recognize and kill antigen-expressing tumor cells ex vivo. Importantly, trimers outperformed synthetic long peptide in terms of T cell response even when equal number of epitopes were used for immunization. To improve the synthesis of trimers containing difficult peptide sequences, we developed a novel small molecule that functions as conjugation platform for synthetic long peptides. This platform , termed Antigen MAtriX (AMAX) improved yield, purity and solubility of trimers over conventional solid phase synthesis strategies. AMAX outperformed synthetic long peptides in terms of both CD8 + and CD4 + T cell responses and allowed functionalization with DC-SIGN-binding carbohydrates for in vivo dendritic cell targeting strategies, boosting T cell responses even further. Moreover, we show that agonistic CD40 antibody combined with MF59 (AddaVax) emulsion synergistically improves the antigen-specific T cell response of the AMAX in vivo. Also, tumor-associated antigens and neo-antigens could be incorporated in AMAX for tumor-specific CD8 + T cell responses. Importantly, immunization with a mix of neoantigen AMAX could reduce tumor growth in a pre-clinical syngeneic mouse model. Hence, we provide pre-clinical support for the induction of effector CD8 + T cells through the adaptable AMAX platform as easy implementable peptidic vaccination strategy against any antigen of choice, including neoantigens for anti-tumor immunity., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Outer membrane vesicles (OMVs) are vesicular nano-particles produced by Gram-negative bacteria that are recently being explored as vaccine vector. The fact that OMVs can be efficiently produced by a hypervesiculating Salmonella typhimurium strain, are packed with naturally-occurring adjuvants like lipopolysaccharides (LPS), and can be engineered to express any antigen of choice, makes them ideal candidates for vaccinology. However, it is unclear whether OMVs induce dendritic cell (DC)-mediated antigen-specific T cell responses and how immune activation is coordinated. Here, we show that OMVs induce maturation of human monocyte-derived DCs, murine bone marrow-derived DCs and CD11c+ splenic DCs. OMV-induced DC maturation was dependent on the presence of LPS and the myeloid differentiation primary response 88 (MyD88) adapter protein downstream of toll-like receptor signaling. Importantly, OMVs did not induce pyroptosis/cell death, but instead provided a significant survival benefit in DCs over non-stimulated DCs. OMVs displaying a sizeable ovalbumin fragment at the vesicle surface induce potent cross-presentation in BMDCs and splenic CD11c + DCs to OTI CD8 + T cells, dependent on MyD88. Interestingly, the OMV-induced preference to cross-presentation was only partly dependent on the BATF3-dependent CD8a + professional cross-presenting DC subset. Hence, an OMV-specific programming of DCs that induces maturation and provides a survival benefit for antigen presentation to T cells is identified. Additionally, for the first time, antigen-specific and potent cross-presentation of antigen-loaded OMVs to CD8 + T cells is demonstrated. These data provide mechanistical insight into the processes needed for the DC-mediated cross-presentation of OMV-derived antigens to CD8 + T cells with implications for therapeutic strategies. STATEMENT OF SIGNIFICANCE: Bacteria are primarily known to cause disease. However, recent research has focused on using engineered bacteria and its byproducts as vaccine agents. In particular, outer membrane vesicles (OMVs) have shown promise in eliciting potent immunity against a variety of pathogens. While most vaccines rely on the generation of antibodies, the control of viral replication and tumor growth is driven by cytotoxic CD8 + T cells induced by dendritic cells (DCs). As such, there is a dire need for vaccines that use DCs to elicit CD8 + T cell responses. Studying OMVs as engineered biomaterial and its interaction with DCs allows tailored induction of immunity. This study includes important findings on OMV-dendritic cell interactions and for the first time supports OMVs as vehicles for the induction of antigen-specific CD8 + T cell responses. Additionally, important mechanistical insight into the molecular pathways needed for the cross-presentation of OMV-derived antigens to CD8 + T cells is provided., (Copyright © 2019 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Antibody responses to Mycoplasma pneumoniae correlate with pulmonary M. pneumoniae clearance. However, M. pneumoniae -specific IgG antibodies can cross-react with the myelin glycolipid galactocerebroside (GalC) and cause neurological disorders. We assessed whether antiglycolipid antibody formation is part of the physiological immune response to M. pneumoniae We show that antibodies against M. pneumoniae proteins and glycolipids arise in serum of M. pneumoniae -infected children and mice. Although antibodies to M. pneumoniae glycolipids were mainly IgG, anti-GalC antibodies were only IgM. B-1a cells, shown to aid in protection against pathogen-derived glycolipids, are lacking in Bruton tyrosine kinase (Btk)-deficient mice. M. pneumoniae -infected Btk-deficient mice developed M. pneumoniae -specific IgG responses to M. pneumoniae proteins but not to M. pneumoniae glycolipids, including GalC. The equal recovery from M. pneumoniae infection in Btk-deficient and wild-type mice suggests that pulmonary M. pneumoniae clearance is predominantly mediated by IgG reactive with M. pneumoniae proteins and that M. pneumoniae glycolipid-specific IgG or IgM is not essential. These data will guide the development of M. pneumoniae -targeting vaccines that avoid the induction of neurotoxic antibodies., (Copyright © 2019 American Society for Microbiology.)

CD14(+) dendritic cells (DCs) present in the dermis of human skin represent a large subset of dermal DCs (dDCs) that are considered macrophage-like cells with poor antigen (cross)-presenting capacity and limited migratory potential to the lymph nodes. CD14(+) dDC highly express DC-specific ICAM-3-grabbing non-integrin (DC-SIGN), a receptor containing potent endocytic capacity, facilitating intracellular routing of antigens to major histocompatibility complex I and II (MHC-I andII) loading compartments for the presentation to antigen-specific CD8(+) and CD4(+) T cells. Here we show using a human skin explant model that the in situ targeting of antigens to DC-SIGN using glycan-modified liposomes enhances the antigen-presenting capacity of CD14(+) dDCs. Intradermal vaccination of liposomes modified with the DC-SIGN-targeting glycan Lewis(X), containing melanoma antigens (MART-1 or Gp100), accumulated in CD14(+) dDCs and resulted in enhanced Gp100- or MART-1-specific CD8(+) T-cell responses. Simultaneous intradermal injection of the cytokines GM-CSF and IL-4 as adjuvant enhanced the migration of the skin DCs and increased the expression of DC-SIGN on the CD14(+) and CD1a(+) dDCs. These data demonstrate that human CD14(+) dDCs exhibit potent cross-presenting capacity when targeted in situ through DC-SIGN.