Photodynamic inactivation of microorganisms (aPDI) is an excellent method to destroy antibiotic-resistant microbial isolates. The use of an exogenous photosensitizer or irradiation of microbial cells already equipped with endogenous photosensitizers makes aPDI a convenient tool for treating the infections whenever technical light delivery is possible. Currently, aPDI research carried out on a vast repertoire of depending on the photosensitizer used, the target microorganism, and the light delivery system shows efficacy mostly on in vitro models. The search for mechanisms underlying different responses to photodynamic inactivation of microorganisms is an essential issue in aPDI because one niche (e.g., infection site in a human body) may have bacterial subpopulations that will exhibit different susceptibility. Rapidly growing bacteria are probably more susceptible to aPDI than persister cells. Some subpopulations can produce more antioxidant enzymes or have better performance due to efficient efflux pumps. The ultimate goal was and still is to identify and characterize molecular features that drive the efficacy of antimicrobial photodynamic inactivation. To this end, we examined several genetic and biochemical characteristics, including the presence of individual genetic elements, protein activity, cell membrane content and its physical properties, the localization of the photosensitizer, with the result that some of them are important and others do not appear to play a crucial role in the process of aPDI. In the review, we would like to provide an overview of the factors studied so far in our group and others that contributed to the aPDI process at the cellular level. We want to challenge the question, is there a general pattern of molecular characterization of aPDI effectiveness? Or is it more likely that a photosensitizer-specific pattern of molecular characteristics of aPDI efficacy will occur?

One of the factors determining efficient antimicrobial photodynamic inactivation (aPDI) is the accumulation of a light-activated compound, namely, a photosensitizer (PS). Targeted PS recognition is the approach based on the interaction between the membrane receptor on the bacterial surface and the PS, whereas the compound is efficiently accumulated by the same mechanism as the natural ligand. In this study, we showed that gallium mesoporphyrin IX (Ga3+MPIX) provided dual functionalityiron metabolism disruption and PS properties in aPDI. Ga3+MPIX induced efficient (>5log10 reduction in CFU/mL) bacterial photodestruction with excitation in the area of Q band absorption with relatively low eukaryotic cytotoxicity and phototoxicity. The Ga3+MPIX is recognized by the same systems as haem by the iron-regulated surface determinant (Isd). However, the impairment in the ATPase of the haem detoxification efflux pump was the most sensitive to the Ga3+MPIX-mediated aPDI phenotype. This indicates that changes within the metalloporphyrin structure (vinyl vs ethyl groups) did not significantly alter the properties of recognition of the compound but influenced its biophysical properties.

Neutrophils and macrophages differentiate from common myeloid progenitors in the bone marrow, where they undergo nuclear morphologic changes during maturation. During this process, both cell types acquire critical innate immune functions that include phagocytosis of pathogens, and for neutrophils the release of nuclear material called nuclear extracellular traps (NETs). Primary cells used to study these functions are typically purified from mature mouse tissues, but bone marrow-derived ex vivo cultures provide more abundant numbers of progenitors and functionally mature cells. Routine analyses of these cells use conventional microscopy and flow cytometry, which present limitations; microscopy is laborious and subjective, whereas flow cytometry lacks spatial resolution. Here we describe methods to generate enriched populations of neutrophils or macrophages from cryopreserved mouse bone marrow cultured ex vivo, and to use imaging flow cytometry that combines the resolution of microscopy with flow cytometry to analyze cells for morphologic features, phagocytosis, and NETosis.

Neutrophils mediate critical innate immune responses by migrating to sites of infection or inflammation, phagocytosing microorganisms, and releasing an arsenal of antimicrobial agents, including reactive oxygen species. These functions are shared by other innate immune cell types, but an interesting feature of neutrophils is their hallmark lobulated nuclei. Although why this bizarre nuclear shape forms is still being elucidated, studies of two intermediate filament proteins that associate with the nuclear envelope, lamin A and C, indicate that expression levels of these proteins govern nuclear maturation. These A-type lamins also modulate nuclear stiffness, the loss of which may be critical to the migration of not only neutrophils but also cancer cells that become prone to metastasis. We investigated whether increased expression of either lamin A or C affects neutrophil nuclear morphologic maturation, but more importantly we tested whether overexpression of either lamin also affects neutrophil functional responses, using two mouse myeloid progenitor models that can be induced toward functionally responsive neutrophil-like cells. Collectively, our results demonstrate that overexpression of either lamin A or C not only disrupts nuclear lobulation but also causes aberrant functional responses critical to innate immunity, including chemotaxis, phagocytosis, and reactive oxygen species production. Moreover, the lamin A-overexpressing cells exhibit decreased expression of a critical NADPH oxidase complex factor, gp91 phox , and transcriptomic profiling demonstrated differential expression of a number of myeloid differentiation and functional pathway components. Taken together, these data demonstrate that A-type lamin expression levels modulate not only nuclear morphologic features but also gene expression changes as neutrophils mature. ImmunoHorizons, 2022, 6: 16-35.

No abstract

Background: Streptococcus agalactiae, referred to as Group B Streptococcus (GBS), is a prominent bacterium causing life-threatening neonatal infections. Although antibiotics are efficient against GBS, growing antibiotic resistance forces the search for alternative treatments and/or prevention approaches. Antimicrobial photodynamic inactivation (aPDI) appears to be a potent alternative non-antibiotic strategy against GBS. Methods: The effect of rose bengal aPDI on various GBS serotypes, Lactobacillus species, human eukaryotic cell lines and microbial vaginal flora composition was evaluated. Results: RB-mediated aPDI was evidenced to exert high bactericidal efficacy towards S. agalactiae in vitro (>4 log10 units of viability reduction for planktonic and >2 log10 units for multispecies biofilm culture) and in vivo (ca. 2 log10 units of viability reduction in mice vaginal GBS colonization model) in microbiological and metagenomic analyses. At the same time, RB-mediated aPDI was evidenced to be not mutagenic and safe for human vaginal cells, as well as capable of maintaining the balance and viability of vaginal microbial flora. Conclusions: aPDI can efficiently kill GBS and serve as an alternative approach against GBS vaginal colonization and/or infections.

Neutrophils differentiate from common myeloid progenitors in the bone marrow where they form uniquely lobulated nuclei while acquiring multiple functions critical to their innate immune responses. We are investigating the roles of nuclear envelope (NE)-associated proteins, including the lamin B receptor (Lbr) and the nuclear lamina proteins lamin A/C, in regulating these nuclear changes, along with determining if these proteins are important to neutrophil functions including chemotaxis, phagocytosis, or reactive oxygen species (ROS) production. We previously reported that Lbr expression increases during neutrophil differentiation while lamin A/C expression decreases. We also used immortalized mouse myeloid progenitors to show that either loss of Lbr or increased lamin A/C expression caused nuclear hypolobulation similar to Pelger-Huët anomaly in humans, plus aberrant chemotaxis. Interestingly, cells lacking Lbr produced less ROS, whereas those overexpressing the lamins exhibited deficient phagocytosis. These studies suggest that the functions of these nuclear proteins go beyond simply supporting nuclear structural changes. Since our previous studies relied on the use of immortalized cell lines, which may not exhibit the full spectrum of normal neutrophil functional responses, we have focused on using techniques to derive and characterize neutrophils with aberrant NE protein expression from ex vivo cultured bone marrow. We also examined whether loss of both A-type lamins affects myeloid progenitor growth and differentiation. Here we show that functionally active neutrophils can be generated by ex vivo culture of bone marrow progenitors from cryopreserved stocks. Imaging flow cytometry, which combines the spatial resolution of fluorescence microscopy with the high throughput format of flow cytometry, was used to demonstrate that the ex vivo cultures at progressive stages of neutrophil maturation exhibit characteristic changes to cell surface marker expression. Customized gating and masking strategies were applied using the image analysis software to quantitatively measure phagocytosis of Escherichia coli particles and release of neutrophil extracellular traps (NETs). We also identified a method to evaluate NET release as a function of cell activation during phagocytosis. We next transduced our ex vivo cultured progenitors with retroviral vectors providing ectopic lamin A expression, and found that derived neutrophils exhibited hypolobulated nuclei morphologically resembling our in vitro models. We also knocked out Lmna expression in the immortalized myeloid progenitors by CRISPR-Cas9 mediated gene disruption, and identified defective growth of the progenitors plus aberrant ROS production in derived neutrophils, similar to the phenotypes observed in cells lacking Lbr. The transcriptional control of the Lmna gene during neutrophil differentiation also was investigated. In contrast to our previous report showing that the ETS factor GABP cooperates with PU.1 or C/EBPε to regulate the Lbr promoter, none of these factors affected background levels of Lmna promoter activities in COS cell reporter assays despite identified ETS and C/EBP consensus binding sites. However, addition of C/EBPα expression caused a greater than 50% decrease in Lmna promoter activities. Furthermore, quantitative ChIP assays demonstrated that C/EBPα most efficiently bound to identified C/EBP binding sequences in the Lmna promoter, indicating that C/EBPα inhibits Lmna expression in differentiating neutrophils. Combined our results demonstrate that precisely regulated levels of NE-associated proteins are critical to neutrophil nuclear maturation and their acquisition of certain functional responses. Our continued studies will utilize imaging flow cytometry to characterize the functions of ex vivo culture-derived neutrophils with aberrant A-type lamin expression, as well as identify roles of additional NE-associated proteins during neutrophil maturation. We are also poised to further characterize the control of genes encoding such NE proteins by various combinations of ETS and C/EBP transcription factors known to regulate myelopoiesis. We predict these studies will provide important insight into the molecular mechanisms governing neutrophil nuclear changes, and why aberrant lobulation is commonly associated with certain hematopoietic disorders. Disclosures No relevant conflicts of interest to declare.

Ingestion of tart cherry extract (TCE), a rich source of polyphenolic anti-oxidant/anti-inflammatory compounds, improves skeletal muscle (SM) recovery following exercise-induced muscle damage. This response may be due in part to attenuated oxidative/pro-inflammatory activities of recruited neutrophils and macrophages during SM regeneration. How TCE promotes the molecular mechanisms regulating SM regeneration or attenuates those controlling myeloid cell pro-inflammatory responses is unknown. Herein, we describe how TCE affects two murine cell models optimized to recapitulate in vivo interactions between regenerating SM and recruited pro-inflammatory myeloid cells: 1) strained skeletal muscle tissue derived from C2C12 myoblasts, and 2) mature myeloid lineages derived from ex vivo-cultured primary bone marrow. We show that TCE treatment may improve SM regeneration by increasing MHC protein expression plus MCP-1 and HGF secretion. Importantly, TCE treatment modestly reduced myoblast proliferation but without affecting viability, and did not affect proliferation or differentiation-specific protein expression profiles (via imaging flow cytometry) of myeloblasts or derived progenitors. Moreover, TCE treatment reduced levels of reactive oxygen species (ROS) released by activated neutrophils. Our continued analyses will identify how TCE either independently affects damaged SM regeneration or myeloid cell functional responses, or dependently affects the interactions between these disparate cell types upon muscle injury. Our studies may reveal that this natural compound not only improves muscle recovery following damage but also attenuates pro-inflammatory responses of recruited myeloid cells.