Flagellin induces myeloid-derived suppressor cells: implications for Pseudomonas aeruginosa infection in cystic fibrosis lung disease.

Pseudomonas aeruginosa persists in patients with cystic fibrosis (CF) and drives CF lung disease progression. P. aeruginosa potently activates the innate immune system, mainly mediated through pathogen-associated molecular patterns, such as flagellin. However, the host is unable to eradicate this flagellated bacterium efficiently. The underlying immunological mechanisms are incompletely understood. Myeloid-derived suppressor cells (MDSCs) are innate immune cells generated in cancer and proinflammatory microenvironments and are capable of suppressing T cell responses. We hypothesized that P. aeruginosa induces MDSCs to escape T cell immunity. In this article, we demonstrate that granulocytic MDSCs accumulate in CF patients chronically infected with P. aeruginosa and correlate with CF lung disease activity. Flagellated P. aeruginosa culture supernatants induced the generation of MDSCs, an effect that was 1) dose-dependently mimicked by purified flagellin protein, 2) significantly reduced using flagellin-deficient P. aeruginosa bacteria, and 3) corresponded to TLR5 expression on MDSCs in vitro and in vivo. Both purified flagellin and flagellated P. aeruginosa induced an MDSC phenotype distinct from that of the previously described MDSC-inducing cytokine GM-CSF, characterized by an upregulation of the chemokine receptor CXCR4 on the surface of MDSCs. Functionally, P. aeruginosa-infected CF patient ex vivo-isolated as well as flagellin or P. aeruginosa in vitro-generated MDSCs efficiently suppressed polyclonal T cell proliferation in a dose-dependent manner and modulated Th17 responses. These studies demonstrate that flagellin induces the generation of MDSCs and suggest that P. aeruginosa uses this mechanism to undermine T cell-mediated host defense in CF and other P. aeruginosa-associated chronic lung diseases.

AAV8 induces tolerance in murine muscle as a result of poor APC transduction, T cell exhaustion, and minimal MHCI upregulation on target cells.

Following gene transfer of adeno-associated virus 2/8 (AAV2/8) to the muscle, C57BL/6 mice show long-term expression of a nuclear-targeted LacZ (nLacZ) transgene with minimal immune activation. Here, we show that pre-exposure to AAV2/8 can also induce tolerance to the more immunogenic AAV2/rh32.33 vector, preventing otherwise robust T-cell activation and allowing stable transgene expression. Depletion and adoptive transfer studies showed that a suppressive factor was not sufficient to account for AAV2/8-induced tolerance, whereas further characterization of the T-cell population showed upregulation of the exhaustion markers PD1, 2B4, and LAG3. Furthermore, systemic administration of Toll-like receptor (TLR) ligands at the time of AAV2/rh32.33-administration broke AAV2/8-induced tolerance, restoring T-cell activation and ß-gal clearance. As such, AAV2/8 transduction appears to lack the inflammatory signals necessary to prime a functional cytotoxic T-cell response. Inadequate T-cell priming could be explained upstream by AAV2/8's poor transduction and activation of antigen-presenting cells (APCs). Immunohistochemical analysis indicates that AAV2/8 transduction also fails to upregulate major histocompatibility complex class I (MHCI) expression on the surface of myocytes, rendering transduced cells poor targets for T-cell-mediated destruction. Overall, AAV2/8-induced tolerance in the muscle is multifactorial, spanning from poor APC transduction and activation to the subsequent priming of functionally exhausted T-cells, while simultaneously avoiding upregulation of MHCI on potential targets.

The structure of AAVrh32.33, a novel gene delivery vector.

The Adeno-associated viruses (AAVs) are being developed as gene delivery vectors for therapeutic clinical applications. However, the host antibody immune response directed against their capsid, prevalent in ∼40-70% of the general population, depending on serotype, negatively impacts efficacy. AAVrh32.33, a novel vector developed from rhesus macaques isolates, has significantly lower seroprevalence in human populations compared to AAV2 and AAV8, which are both in clinical use. To better understand the capsid determinants of this differential immune response to AAVrh32.33, its structure was determined by X-ray crystallography to 3.5 Å resolution. The capsid viral protein (VP) structure conserves the eight-stranded ß-barrel core and αA helix reported for other parvoviruses and the distinct capsid surface topology of the AAVs: a depression at the icosahedral twofold axis, three protrusions surrounding the threefold axis, and a depression surround a cylindrical channel at the fivefold axis. A comparison to AAV2, AAV4, and AAV8, to which AAVrh32.33 shares ∼61%, ∼81%, and ∼63% identity, respectively, identified differences in previously defined AAV VP structurally variable regions (VR-1 to VR-IX) which function as receptor attachment, transduction efficiency, and/or antigenic determinants. This structure thus provides a 3D platform for capsid engineering in ongoing efforts to develop AAVrh32.33, as well as other AAV serotypes, for tissue targeted gene-therapy applications with vectors that can evade pre-existing antibody responses against the capsid. These features are required for full clinical realization of the promising AAV gene delivery system.

Mapping the structural determinants responsible for enhanced T cell activation to the immunogenic adeno-associated virus capsid from isolate rhesus 32.33.

Avoiding activation of immunity to vector-encoded proteins is critical to the safe and effective use of adeno-associated viral (AAV) vectors for gene therapy. While commonly used serotypes, such as AAV serotypes 1, 2, 7, 8, and 9, are often associated with minimal and/or dysfunctional CD8(+) T cell responses in mice, the threshold for immune activation appears to be lower in higher-order species. We have modeled this discrepancy within the mouse by identifying two capsid variants with differential immune activation profiles: AAV serotype 8 (AAV8) and a hybrid between natural rhesus isolates AAVrh32 and AAVrh33 (AAVrh32.33). Here, we aimed to characterize the structural determinants of the AAVrh32.33 capsid that augment cellular immunity to vector-encoded proteins or those of AAV8 that may induce tolerance. We hypothesized that the structural domain responsible for differential immune activation could be mapped to surface-exposed regions of the capsid, such as hypervariable regions (HVRs) I to IX of VP3. To test this, a series of hybrid AAV capsids was constructed by swapping domains between AAV8 and AAVrh32.33. By comparing their ability to generate transgene-specific T cells in vivo versus the stability of transgene expression in the muscle, we confirmed that the functional domain lies within the VP3 portion of the capsid. Our studies were able to exclude the regions of VP3 which are not sufficient for augmenting the cellular immune response, notably, HVRs I, II, and V. We have also identified HVR IV as a region of interest in conferring the efficiency and stability of muscle transduction to AAVrh32.33.

Equipping Nurse Practitioner Students With Social Determinants of Health Competencies.

BACKGROUND: Social determinants of health (SDOH) cause significant burden on individuals living with acute and chronic disease. There are meaningful data to support screening for social needs, yet implementation limitations exist in the clinical setting. Incorporating SDOH education into nurse practitioner (NP) curriculum provides a framework for bringing these concepts into clinical practice. As NP education transitions from concept-based to competency-based instruction, NP programs across the nation are tasked with ensuring socially competent, practice-ready NPs. METHOD: To help students understand the impact of SDOH in an applied way, a multisemester clinical and didactic dyad approach to SDOH competency was implemented. RESULTS: NP students (N = 521) in three separate cohorts completed the SDOH project between 2019 and 2021. Statistically significant results (p > .001) demonstrated increased knowledge related to SDOH in the clinical setting. CONCLUSION: Equipping NP students with SDOH competencies empowers them to optimize health, improve patient outcomes, and promote health equity. [J Nurs Educ. 2024;63(X):XXX-XXX.].

Expression and regulation of interferon-related development regulator-1 in cystic fibrosis neutrophils.

A genome-wide association study identified interferon-related development regulator-1 (IFRD1), a protein expressed by neutrophils, as a key modifier gene in cystic fibrosis (CF) lung disease. Here, we investigated the expression and regulation of IFRD1 in CF neutrophils. IFRD1 expression was quantified in peripheral blood and airway neutrophils from patients with CF, patients with non-CF lung disease, and healthy control subjects. The regulation of IFRD1 expression was analyzed using isolated neutrophils and ex vivo stimulation assays with CF airway fluids. IFRD1 single-nucleotide polymorphisms (SNPs) were analyzed in a CF cohort (n = 572) and correlated with longitudinal lung function and IFRD1 expression. Patients with CF expressed higher protein levels of IFRD1 in peripheral blood neutrophils compared with healthy or non-CF disease control subjects. Within patients with CF, IFRD1 protein expression levels in neutrophils were lower in airway fluids compared with peripheral blood. High IFRD1 expression was positively associated with the production of reactive oxygen species (ROS) in CF neutrophils. In vitro regulation studies showed that CF airway fluid and the CF-characteristic chemokines CXCL8 and CXCL2 down-regulated IFRD1 expression in neutrophils, an effect that was mediated through CXCR2. Genetic analyses showed that three IFRD1 SNPs were associated with longitudinal declines in lung function, and modulated IFRD1 expression. These studies demonstrate that IFRD1 expression is systemically up-regulated in human CF neutrophils, is linked to the production of ROS, and is modulated by chemokines in CF airway fluids, depending on the IFRD1 genotype. Understanding the regulation of IFRD1 may pave the way for novel therapeutic approaches to target neutrophilic inflammation in CF.

Neutrophils express distinct RNA receptors in a non-canonical way.

RNAs are capable of modulating immune responses by binding to specific receptors. Neutrophils represent the major fraction of circulating immune cells, but receptors and mechanisms by which neutrophils sense RNA are poorly defined. Here, we analyzed the mRNA and protein expression patterns and the subcellular localization of the RNA receptors RIG-I, MDA-5, TLR3, TLR7, and TLR8 in primary neutrophils and immortalized neutrophil-like differentiated HL-60 cells. Our results demonstrate that both neutrophils and differentiated HL-60 cells express RIG-I, MDA-5, and TLR8 at the mRNA and protein levels, whereas TLR3 and TLR7 are not expressed at the protein level. Subcellular fractionation, flow cytometry, confocal laser scanning microscopy, and immuno-transmission electron microscopy provided evidence that, besides the cytoplasm, RIG-I and MDA-5 are stored in secretory vesicles of neutrophils and showed that RIG-I and its ligand, 3p-RNA, co-localize at the cell surface without triggering neutrophil activation. In summary, this study demonstrates that neutrophils express a distinct pattern of RNA recognition receptors in a non-canonical way, which could have essential implications for future RNA-based therapeutics.

CXCR1 and CXCR2 haplotypes synergistically modulate cystic fibrosis lung disease.

Cystic fibrosis (CF) lung disease severity is largely independent on the CF transmembrane conductance regulator (CFTR) genotype, indicating the contribution of genetic modifiers. The chemokine receptors CXCR1 and CXCR2 have been found to play essential roles in the pathogenesis of CF lung disease. Here, we determine whether genetic variation of CXCR1 and CXCR2 influences CF lung disease severity. Genomic DNA of CF patients in Germany (n = 442) was analysed for common variations in CXCR1 and CXCR2 using a single-nucleotide polymorphism (SNP) tagging approach. Associations of CXCR1 and CXCR2 SNPs and haplotypes with CF lung disease severity, CXCR1 and CXCR2 expression, and neutrophil effector functions were assessed. Four SNPs in CXCR1 and three in CXCR2 strongly correlated with age-adjusted lung function in CF patients. SNPs comprising haplotypes CXCR1_Ha and CXCR2_Ha were in high linkage disequilibrium and patients heterozygous for the CXCR1-2 haplotype cluster (CXCR1-2_Ha) had lower lung function compared with patients with homozygous wild-type alleles (forced expiratory volume in 1 s ≤ 70% predicted, OR 7.24; p = 2.30 × 10(-5)). CF patients carrying CXCR1-2_Ha showed decreased CXCR1 combined with increased CXCR2 mRNA and protein expression, and displayed disturbed antibacterial effector functions. CXCR1 and CXCR2 genotypes modulate lung function and antibacterial host defence in CF lung disease.

The complex and evolving story of T cell activation to AAV vector-encoded transgene products.

Original reports of adeno-associated virus (AAV) vector-mediated gene transfer to the muscle resulted in high-level ß-galactosidase (ß-gal) expression and the promise of a viral vector that was largely nonimmunogenic. Subsequent attempts to utilize these vectors for genetic vaccination, however, demonstrated that it was possible to activate cellular and humoral immunity to AAV-encoded antigens. These findings fueled years of investigation into factors impacting the immunogenicity of recombinant AAV-mediated gene delivery, including route of administration, dose, host species, capsid serotype, and transgene product. In cases where AAV vectors could avoid transgene-directed immunity, it became clear that mechanisms of tolerance were at work, varying between ignorance, anergy/deletion, or active suppression. Here, we follow the field of AAV gene therapy from inception, as investigators have worked to understand the delicate balance between AAV-mediated tolerance and the activation of immunity. This review discusses our current appreciation of AAV vector immunology, with a specific focus on the transgene-specific T cell response.

Summary of New Guidance on Selected Immunizations.

In 2022, the Centers for Disease Control and Prevention (CDC) updated its Adult Immunization Schedule Recommendations for Ages 19 Years or Older to provide the most current evidence-based recommendations following comprehensive reviews of data related to vaccines. In its report, the CDC highlighted the importance of health care professionals staying up to date on the latest evidence. During the novel coronavirus pandemic, the ability to provide routine vaccinations to the adult population was limited and even halted at times. As in-person health care visits continue to resume, it is imperative for nurses to refocus on and be familiar with the most up-to-date vaccine recommendations. Here, we summarize information on vaccine guidelines, safety, and special considerations for women, and we highlight changes to the 2022 adult immunization schedule. Keeping individuals free of vaccine-preventable diseases is one of the most effective and important public health interventions in health care.

A case of severe acute respiratory failure after elective abdominoplasty.

Surgical correction of abdominal muscle diastasis may decrease intra-abdominal volume and increase intra-abdominal pressure. The induced changes may ultimately lead to respiratory compromise. In abdominoplasty, one of the most frequently performed esthetic procedures, those changes are believed to be transient and clinically insignificant. We describe a case where acute change in respiratory physiology after abdominoplasty led to severe respiratory failure with significantly decreased pulmonary compliance in a young and otherwise healthy patient. In this case mechanical ventilation failed to improve compliance, and reversal of abdominoplasty was required to restitute pulmonary function.

Adeno-associated virus capsid structure drives CD4-dependent CD8+ T cell response to vector encoded proteins.

The immunological sequelae of adeno-associated virus (AAV)-mediated gene transfer in vivo is quite complex. In murine models, most AAV capsids are associated with minimal or dysfunctional T cell responses to antigenic transgene products. In this study we compared T cell activation against AAV2/8 and AAV2/rh32.33 vectors expressing nuclear-targeted LacZ (nLacZ), GFP, or firefly luciferase in murine skeletal muscle. We show that, unlike AAV8, AAVrh32.33 yields qualitatively and quantitatively robust T cell responses to both the capsid and transgene product. AAV2/rh32.33.CB.nLacZ, but not AAV2/8, drives a high degree of cellular infiltration and a loss of detectable transgene expression in C57BL/6 mice. However, cellular immunity to AAVrh32.33 is ablated in the absence of CD4, CD40L, or CD28, permitting stable beta-galactosidase expression. Treatment of CD40L(-/-) mice with the CD40 agonist, FGK45, failed to restore the CD8 response to AAV2/rh32.33.nLacZ, suggesting that additional factors are involved. Our results suggest that specific domains within the AAVrh32.33 capsid augment the adaptive response to both capsid and transgene Ags in a CD4-dependent pathway involving CD40L signaling and CD28 costimulation. Structural comparison of the AAV8 and rh32.33 capsids has identified key differences that may drive differential immunity by affecting tropism, Ag presentation or the activation of innate immunity. This murine model of AAV-mediated cytotoxicity allows us to delineate the mechanism of viral immune activation, which is relevant to the translation of AAV technology in higher order species.

Identification of the murine AAVrh32.33 capsid-specific CD8+ T cell epitopes.

BACKGROUND: Adeno-associated virus (AAV) is an ideal gene therapy vector and is non-immunogenic in many small animal models. The stable gene expression commonly seen in murine models does not necessarily translate to nonhuman primates and higher-order species, highlighting the need for a better understanding of immune activation to these vectors. One capsid variant, AAVrh32.33, demonstrates a unique phenotype in murine muscle, reminiscent of what is often seen in higher-order species. AAVrh32.33 generates a strong CD8+ T-cell response to both capsid and encoded transgene antigens in a manner independent of transgene product or major histocompatability complex haplotype, making it an ideal candidate for studying immune activation to AAV in the mouse. METHODS: To map the H-2b and H-2d dominant epitopes of the AAVrh32.33 capsid, C57BL/6 or Balb/C mice received an intramuscular injection of 1 x 10(11) genome copies of AAV2/rh32.33.CB.nLacZ. Three weeks later, splenocytes were harvested and stimulated in vitro with pooled or individual peptides from the AAVrh32.33 capsid peptide library and analysed by an interferon (IFN)-gamma enzyme-linked immunosorbent spot assay or intracellular cytokine staining. RESULTS: The immunodominant epitopes within the AAVrh32.33 capsid responsible for driving CD8+ T-cell responses to the capsid protein in C57BL/6 (SSYELPYVM) and Balb/C (KIPASGGNAL) mice were defined. CONCLUSIONS: Identification of dominant capsid epitopes will make it possible to monitor cellular responses to the AAV capsid in vivo, facilitating mechanistic studies critical to defining how cellular immunity to the AAV capsid arises and, ultimately, how the generation of capsid-specific T cells can be avoided to ensure safety in a gene therapy setting.

Identifying Geriatric Patients at Risk for Malnutrition: A Quality Improvement Project.

Malnourishment leads to poor outcomes in the geriatric surgical population and national guidelines recommend preoperative nutrition screening. However, care practices do not reflect current recommendations. As a quality-improvement project, a validated nutritional screening tool, the Mini Nutritional Assessment-Short Form (MNA-SF), was implemented in the preoperative clinic of a large academic health center to identify patients at-risk for malnutrition prior to elective surgery. Patients were screened during the nursing intake process and categorized as no nutritional risk; at-risk for malnourishment; or severely malnourished. During the four-week screening period, 413 patients met inclusion criteria with 67.8% (n = 280) screened. No nutritional risk was identified in 77.5% (n = 215) of patients, 18.2% (n = 51) were at-risk, and 4.3% (n = 12) were malnourished. This project will inform and guide a prehabilitation plan for nutrition optimization to improve healthcare quality, outcomes, and costs in the geriatric surgical population.

Modified Foxp3 mRNA protects against asthma through an IL-10-dependent mechanism.

Chemically modified mRNA is capable of inducing therapeutic levels of protein expression while circumventing the threat of genomic integration often associated with viral vectors. We utilized this novel therapeutic tool to express the regulatory T cell transcription factor, FOXP3, in a time- and site-specific fashion in murine lung, in order to prevent allergic asthma in vivo. We show that modified Foxp3 mRNA rebalanced pulmonary T helper cell responses and protected from allergen-induced tissue inflammation, airway hyperresponsiveness, and goblet cell metaplasia in 2 asthma models. This protection was conferred following delivery of modified mRNA either before or after the onset of allergen challenge, demonstrating its potential as both a preventive and a therapeutic agent. Mechanistically, FOXP3 induction controlled Th2 and Th17 inflammation by regulating innate immune cell recruitment through an IL-10-dependent pathway. The protective effects of FOXP3 could be reversed by depletion of IL-10 or administration of recombinant IL-17A or IL-23. Delivery of Foxp3 mRNA to sites of inflammation may offer a novel, safe therapeutic tool for the treatment of allergic asthma and other diseases driven by an imbalance in helper T cell responses.

Expression of therapeutic proteins after delivery of chemically modified mRNA in mice.

Current viral vectors for gene therapy are associated with serious safety concerns, including leukemogenesis, and nonviral vectors are limited by low gene transfer efficiency. Here we investigate the therapeutic utility of chemically modified mRNA as an alternative to DNA-based gene therapy. A combination of nucleotide modifications abrogates mRNA interaction with Toll-like receptor (TLR)3, TLR7, TLR8 and retinoid-inducible gene I (RIG-I), resulting in low immunogenicity and higher stability in mice. A single intramuscular injection of modified murine erythropoietin mRNA raises the average hematocrit in mice from 51.5% to 64.2% after 28 days. In a mouse model of a lethal congenital lung disease caused by a lack of surfactant protein B (SP-B), twice weekly local application of an aerosol of modified SP-B mRNA to the lung restored 71% of the wild-type SP-B expression, and treated mice survived until the predetermined end of the study after 28 days.

Adeno-associated virus-mediated gene transfer to nonhuman primate liver can elicit destructive transgene-specific T cell responses.

Gene transfer to murine liver with vectors based on novel adeno-associated virus (AAV) serotypes is efficient, stable, and safe even in the setting of antigenic transgene products. We undertook a study in cynomolgus macaques to evaluate the relevance of these findings to primates. The vectors were based on AAV serotype 7 and expressed green fluorescence protein (GFP) from the cytomegalovirus enhanced beta-actin promoter in both single-stranded and self-complementary genomes. Transduction efficiencies from the single-stranded vectors were similar to those observed in mice, although there was no advantage in primates with the self-complementary vectors. Primates elicited vibrant cytotoxic T cell responses to GFP that correlated with hepatitis and loss of transgene expression. There was no evidence of T cell activation in response to the AAV capsid. These studies indicate that under some conditions primates may activate more robust T cell responses to transgene products than is observed in mice.

Maintaining immunological tolerance with Foxp3.

Central tolerance in the thymus is the primary mechanism for deleting autoreactive T cells. Despite this, escape of self-reactive T lymphocytes into the periphery reveals the threat of autoimmunity. To compensate for its imperfection, the thymus also produces a naturally occurring subset of Foxp3+ CD4+ CD25+ regulatory T cells with suppressive function, capable of controlling autoreactive cells. Foxp3 (forkhead box P3), the lineage-specific marker for this subset of cells, is crucial to their thymic development and peripheral function, and yet the transcriptional program driven by Foxp3 was until now largely undefined. Emerging evidence has provided insight into its role: from the ability of Foxp3 to cooperate with other transcription factors such as NFAT, to the genome-wide characterization of target genes directly bound and regulated by Foxp3. Here we discuss the discovery of naturally occurring regulatory T cells - their phenotype, development, maintenance, and function - largely as they are defined by the lineage-specific marker, Foxp3.

Vaccines based on novel adeno-associated virus vectors elicit aberrant CD8+ T-cell responses in mice.

We recently discovered an expanded family of adeno-associated viruses (AAVs) that show promise as improved gene therapy vectors. In this study we evaluated the potential of vectors based on several of these novel AAVs as vaccine carriers for human immunodeficiency virus type 1 Gag. Studies with mice indicated that vectors based on AAV type 7 (AAV7), AAV8, and AAV9 demonstrate improved immunogenicity in terms of Gag CD8(+) T-cell and Gag antibody responses. The quality of these antigen-specific responses was evaluated in detail for AAV2/8 vectors and compared to results with an adenovirus vector expressing Gag (AdC7). AAV2/8 produced a vibrant CD8(+) T-cell effector response characterized by coexpression of gamma interferon and tumor necrosis factor alpha as well as in vivo cytolytic activity. No CD8(+) T-cell response generated by any of the AAVs was effectively boosted with AdC7, a result consistent with the finding of a relative lack of cells expressing interleukin-2 (IL-2) or a central memory phenotype at 3 months after the prime. The primary response to an AdC7 vaccine differed from that generated by AAVs in that the peak effector response evolved into populations of Gag-specific T cells expressing high levels of cytokines, including IL-2, and with effector memory and central memory phenotypes. A number of mechanisms could be considered to explain the aberrant activation of CD8(+) T cells by AAV, including insufficient inflammatory responses, CD4 help, and/or chronic antigen expression and T-cell exhaustion. Interestingly, the B-cell response to AAV-encoded Gag was quite vibrant and easily boosted with AdC7.