Immunology

The human host defence peptide LL-37 mediates pleiotropic immunomodulatory functions that can be both pro- and anti-inflammatory. Under inflammatory conditions in the lungs, LL-37 is susceptible to the post-translational modification (PTM) citrullination. The impact of this PTM on LL-37's immunomodulatory functions is not fully understood. Therefore, we characterized the transcriptome and cytokine profile in response to LL-37 and citrullinated-LL-37 (citLL-37), in human bronchial epithelial cells (HBEC). Cells were stimulated with either LL-37 or citLL-37 (0.5 M), or a scrambled peptide (sLL-37). RNA after 4 hours (h) was analyzed with NanoString nCounter Host Response Panel, and 96 cytokines were examined (after 24 h) in tissue culture supernatants (Luminex platform). Genes and cytokines with a Log2 fold-change [&ge;]0.5 with p<0.05 compared to unstimulated cells were considered differentially expressed (DE). These studies revealed overlapping and distinct biosignatures. There were 118 DE genes with LL-37 and 71 with citLL-37, of which magnitude of change was significantly different for 32 genes. 16 out of 17 DE genes altered by both peptides were directly related to inflammation, and these were significantly less (by ~45%) with citLL-37 compared to LL-37. Similarly, inflammatory cytokines enhanced in response to LL-37 were significantly less (by 37-94%) with citLL-37. Overall, our findings indicate that citrullination of LL-37 does not abrogate cellular response in HBEC, instead dampens the inflammatory biosignature induced by LL-37.

Introduction: Mast cells are central effectors of IgE-mediated allergic responses and reside in barrier tissues, where they encounter diverse environmental compounds. How naturally occurring bioactive compounds influence mast cell function under resting versus allergic conditions remains poorly defined. Methods: We compared the effects of cholera toxin, aconitine, and sennoside A on viability, degranulation, and cytokine production in murine bone marrow-derived mast cells. We then examined the acute physiological effects of oral sennoside A in an ovalbumin-induced mouse model of food allergy. Results: None of the compounds reduced mast cell viability at the concentrations tested. Sennoside A, but not cholera toxin or aconitine, induced modest degranulation in resting mast cells without increasing TNF- or IL-6 secretion. In contrast, sennoside A markedly suppressed IgE-dependent antigen-induced degranulation, TNF- and IL-6 secretion, and Il1b expression. Acute oral sennoside A induced dose-dependent diarrhea and hypothermia and increased colonic Tnfa, Il1b, Il33, and Hdc expression. In allergic mice, sennoside A attenuated allergen-induced hypothermia, reduced duodenal Tpsab1 expression, and decreased circulating total IgE, OVA-specific IgE, and OVA-specific IgG1, while increasing Il5 expression. Conclusion: Sennoside A differentially regulates mast cell responses according to activation state and attenuates features of experimental food allergy. These findings identify a previously unrecognized immunomodulatory effect of sennoside A on IgE-driven mast cell activation. However, the mechanisms and the contribution of mast cells to the in vivo phenotype remain to be defined.

BackgroundPremature ovarian insufficiency (POI) is a condition affecting female fertility e.g., in the context of autoimmune disease. A recent proof-of-concept study showed that B-cell depletion with rituximab (RTX) temporarily restored ovarian function in patients with autoimmune POI. However, the underlying immunological mechanisms behind this response is unknown. ObjectivesTo characterize B- and T-cell phenotypes before and after RTX therapy in the above-mentioned patients and associations with autoantibody levels and treatment response. MethodsCryopreserved cell samples were available from six autoimmune patients with POI, four of whom were positive for autoantibodies against steroidogenic enzymes (21OH, 17OH and/or SCC). Patients received RTX and blood samples were collected at baseline, second infusion (2 weeks), 3, 8 and 12 months. Peripheral B- and T-cell phenotypes were analysed using spectral flow cytometry and correlated with autoantibody levels. ResultsAt baseline, patients displayed signs of B-cell activation by alteration in subset composition compared to matched controls. Upon closer analysis, patients positive for steroidogenic enzyme autoantibodies had significantly increased frequency of CXCR3+switched memory (SWM) B-cells than autoantibody-negative patients, which correlated positively with anti-17OH and-SCC autoantibody levels. In parallel, increased Th1 cell frequencies were also observed in the same patient group. Longitudinal analyses showed expected B-cell depletion followed by repopulation after RTX. Changes in T-cell subsets were observed at 8 months with reduced frequencies of Th1 and Tfh cells. ConclusionAutoimmune POI displayed altered B- and T-cell subsets implicating cell crosstalk, immune cell infiltration to sites of inflammation such as ovaries, and presence of Th1-driven immunopathogenesis. Key messagesO_LIIncreased CXCR3+SWM B cells and Th1 cells were found in patients with autoimmune POI who were positive for autoantibodies against steroidogenic enzymes. C_LIO_LICXCR3 expression on SWM B cells positively associated with anti-17OH and anti-SCC autoantibody levels. C_LIO_LIRTX induced rapid B-cell depletion followed by repopulation and a delayed T-cell effect at 8 months with reduced Th1 and Tfh cell frequencies. C_LI Capsule summaryRTX has recently been shown to restore fertility in autoimmune POI. Lymphocyte profiling identified Th1 bias accompanied by CXCR3+memory B cells that correlate with autoantibody levels. B-cell depletion appears to temporarily alleviate ovarian autoimmunity and ameliorate physiological function.

The skull bone marrow axis has recently emerged as an important source of immune cells during neuroinflammation, including in the experimental autoimmune encephalomyelitis (EAE) , the mouse model of multiple sclerosis. The cribriform plate bone marrow (cpBM), is a portion of the ethmoid bone of the skull of mice, and is positioned next to nasal regions. At the cribriform plate olfactory nerves pass through the skull and recent evidence supports that it represents an important immune niche with links to CSF outflow and connections to cpBM. Previous data supports cellular composition of the ethmoid bone marrow in mice is altered in neuroinflammation suggesting it senses inflammatory cues, but the exact nature of these changes is still unclear. Here using scRNAseq and flow cytometry of the ethmoid bone marrow region we test how the region responds to EAE. In addition we also compare the cpBM region with systemic bone marrow in the femur to understand region specific differences. We report that neuroinflammation creates a regulatory response at these sites and implicates the cpBM in controlling these effects. A better understanding of the cribriform niche may offer new treatments for neuroinflammatory disease

Background: Neutrophils migrate to the respiratory epithelium during rhinovirus (RV) infection, but whether RV directly activates neutrophils or whether activation arises from epithelial responses remains unclear. Objective: To determine whether RV directly activates human neutrophils or indirectly activates them through RV infected airway epithelial cells. Methods: Human neutrophils were exposed to purified RV, and activation was assessed by myeloperoxidase activity, CD11b/CD66b expression, and reactive oxygen species (ROS) production. Neutrophils were co-cultured with RV-infected air liquid interface (ALI) bronchial epithelium evaluated for effects on epithelial integrity. At 48 to 72 hours post-infection, we used live-cell imaging to measure ROS responses and neutrophil-tracking assays to measure migration velocity and ROS responses to basolateral supernatants, clarified apical washes, and apical debris from RV-infected epithelial cells. Plaque assays were used to assess for RV infectivity after incubation with apical neutrophils. Results: RV did not directly activate neutrophils. Adding neutrophils to the apical surface of airway epithelial cells increased neutrophil ROS production at 48 to 72 hrs post-infection but did not alter epithelial cytotoxicity. Conversely, basolateral supernatants from infected cells enhanced neutrophil migration but not ROS, consistent with cytokine-mediated priming. Clarified apical washes had no effect on migration or ROS. By contrast, apical debris from infected cultures triggered a robust ROS response. Basolateral fractions from medium control or RV infected cultures did not induce ROS. Infectivity is not impacted by apical neutrophil presence. Conclusion: RV does not directly activate neutrophils. Instead, RV infected epithelial cells release basolateral factors that enhance neutrophil migration, while apical debris from infected cells drives ROS activation. These findings indicate that epithelial injury, not RV itself, is the dominant trigger of neutrophil activation during infection.

Background Vitiligo is an acquired depigmenting disorder characterised by melanocyte loss. While immune responses against melanocytes are implicated in its pathogenesis, melanocyte detachment, melanocyte senescence and aberrant epidermal differentiation are increasingly characterised as additional pathological features. Granzyme B (GzmB), classically recognised as an intracellular effector of perforin-dependent cytotoxicity, is increased in vitiligo lesions; however, its extracellular contribution to vitiligo pathology remains unclear. Objectives To determine whether extracellular GzmB contributes to depigmentation and epidermal pathological features relevant to vitiligo, and to define its cellular source and underlying mechanisms. Methods Human vitiligo skin samples, murine depigmentation models and cultured human epidermal cells were analysed. The functional effects of extracellular GzmB were examined by subcutaneous administration of recombinant GzmB in mice. A rhododendrol (RD)-induced leukoderma model was used to evaluate the disease relevance of GzmB and the therapeutic effect of topical GzmB inhibition with VTI-1002. Results GzmB-positive cells were markedly increased in active vitiligo lesions and were predominantly associated with tryptase-positive cells, with limited co-distribution with CD8 or perforin. Vitiligo lesions also showed melanocyte detachment, increased p16INK4A-positive melanocytes and aberrant keratinocyte differentiation. These pathological features were recapitulated in vivo by extracellular GzmB administration, accompanied by focal depigmentation. In cultured melanocytes, GzmB exerted no cytotoxic effects but reduced attachment strength and induced a senescence-associated phenotype characterised by decreased extracellular matrix- and adhesion-related gene expression, increased p16INK4A level, elevated senescence-associated {beta}-galactosidase activity and activation of transforming growth factor-{beta}/SMAD signalling. In keratinocytes, extracellular GzmB promoted aberrant differentiation associated with activation of p53 signalling. Topical inhibition of GzmB in RD-induced leukoderma attenuated depigmentation progression, melanocyte detachment, melanocyte senescence and abnormal epidermal differentiation. Conclusions Extracellular GzmB promotes depigmentation associated with inducing melanocyte detachment, melanocyte senescence and aberrant keratinocyte differentiation. These findings identify extracellular GzmB as a previously underrecognised pathogenic mediator and potential therapeutic target in vitiligo-associated depigmentation.

The longitudinal effects of initial influenza exposure on the magnitude, persistence, and antigen specificity of antibody responses during infancy remain incompletely understood. We investigated humoral antibody responses following primary influenza infection or seasonal vaccination in a prospective US birth cohort with weekly respiratory surveillance for influenza infection. Influenza-specific serum IgG binding to H1 and H3 haemagglutinin (HA) antigens, HA inhibition (HAI), and live-virus neutralizing antibody responses were assessed longitudinally. To minimize influence of maternally derived antibodies, analyses of primary exposure responses were restricted to infants for whom influenza-specific maternal antibodies had declined below the assay detection limit before exposure. Antibody responses were compared following primary infection and vaccination and examined after subsequent annual vaccination. We found that maternal influenza-specific IgG declined rapidly during early infancy. Among infants without detectable pre-exposure influenza-specific antibodies, primary influenza infection was associated with greater post-exposure antibody responses and less measurable decline over follow-up than primary vaccination. These groups differed in age, calendar season, and exposure characteristics, limiting direct attribution of these differences to route of exposure. In infants with previous influenza infection followed by vaccination, responses after subsequent vaccination initially showed greater binding to the previously infecting subtype among the tested HA antigens. With repeated vaccination, responses became more distributed across the tested antigens. Our findings reveal differences in the magnitude, persistence, and antigen specificity of antibody responses according to the nature and sequence of influenza exposure. By prospectively capturing early-life exposures, this study provides longitudinal evidence linking exposure history to subsequent humoral antibody responses.

Autoreactive B cells must escape immune tolerance mechanisms to drive autoimmune disease, potentially through alternative paths to memory and antibody-secreting cell formation. "Atypical" B cells represent one such alternative pathway and are expanded in several human autoimmune diseases. Atypical B cells display phenotypic heterogeneity, but how this connects with differences in transcriptional programming, isotype switching and somatic hypermutation is incompletely understood in type 1 diabetes (T1D). Screening efforts to detect T1D prior to clinical diabetes onset affords an opportunity to study B cells early in immunopathogenesis, in which they primarily function as antigen-presenting cells. Prior evidence points to atypical B cell populations as reservoirs for islet autoreactivity in human T1D. We therefore developed sorting strategies to enrich for atypical and other autoreactive-prone B cell populations from the peripheral blood of early-stage T1D individuals and performed single-cell RNA/BCR-sequencing. We identified four transcriptionally defined atypical B cell clusters, each showing differential upregulation of atypical B cell defining genes. Additional gene expression changes were noted in broadly transcriptionally similar, but phenotypically distinct atypical B cell populations, with differences noted in the average extent of isotype switching and somatic hypermutation. Some atypical B cell populations showed extensive clonal sharing with classical memory or plasmablast subsets, consistent with relationships amongst these subsets. B cells reactive against a key islet autoantigen, insulin, were enriched in select atypical B cell populations. These findings highlight specific atypical B cell populations and features with potential value as biomarkers of disease progression or response to immunotherapy in T1D, and potentially other autoimmune diseases.

Core fucosylation (alpha1,6-fucosylation) of N-glycans, catalyzed solely by fucosyltransferase 8 (FUT8), plays important roles in development, immunity, and antibody effector functions. Here we report the generation of Fut8 floxed mice using a CRISPR/Cas9-based Extreme Genome Editing (EGE) system, in which loxP sites flank exon 8 of the Fut8 gene and a LoxP-STOP-LoxP-RFP cassette was knocked in downstream of the promoter, rendering the targeted allele functionally null before Cre-mediated recombination. Crossing floxed mice with CMV-Cre transgenic mice yielded complete knockout mice carrying the Cre-recombined allele. A PCR-based genotyping method was established that unambiguously distinguishes the wild-type, heterozygous, and homozygous genotypes (+/+, delta/+, and delta/delta). Intercrossing of heterozygous mice revealed that homozygous knockout mice die during the early postnatal period, consistent with previous reports, whereas heterozygous mice are viable. Flow cytometric analysis of AAL binding to splenic B cells and AAL lectin blotting both demonstrated markedly reduced core fucosylation in heterozygous mice (60.0 +/- 6.8% and approximately 48% of wild-type levels, respectively). These results show that disruption of a single Fut8 allele substantially reduces core fucosylation, and the mouse model described here provides a practical tool for functional studies of core fucosylation.

Background and aims. Inflammatory Bowel Disease (IBD) is a complex immune-mediated condition with a strong genetic contribution. The genetics of IBD are highly complex, with inflammation, autoimmunity, and mucosal immunity pathways involved in its pathogenicity. GenePy is a gene-level disease burden metric. The integration of clinical and genetic data and the application of machine learning methods can aid patient stratification for personalised care. Methods. Lists of consensus IBD pathway genes were curated. GenePy scores were computed from whole exome sequencing data. A K-Means algorithm was optimised using clustering quality and stability metrics on the GenePy scores of genes in each IBD pathway, and the results replicated in three independent IBD cohorts (SPARC IBD, MSCCR IBD, and Southampton IBD). Chi-square tests assessed the association between cluster assignment and phenotypic features, and were multiple test corrected via Benjamini-Hochberg FDR. Results. A strong association was replicated for NOD signalling genes driving IBD subtype, Crohns disease behaviour, and IBD surgery across all cohorts, as well as granuloma in Southampton IBD. This was mainly driven by NOD2. Similar genetic clustering patterns were observed across all cohorts, with additional phenotypic enrichment in clusters driven by NLRP1 (surgery in MSCCR IBD), and nominally significant enrichment for disease behaviour linked to BIRC2 (B2 in Southampton IBD and B2B3 in SPARC IBD). Conclusions. This analysis identified a common genetic architecture across NOD signalling genes across IBD cohorts, replicated a signal for NOD2 as a monogenic driver of disease with a strong phenotypic enrichment, and identified other putative genetic drivers of IBD, such as the NLRP1 gene.

Background. Persistent SARS-CoV-2 antigen is a leading hypothesis for post-acute sequelae of SARS-CoV-2 infection (PASC), commonly known as Long COVID. Impaired Fc-mediated antiviral antibody function could contribute to inefficient clearance of infected cells and viral antigen despite preserved antibody levels. Therefore, we tested whether PASC is associated with altered Fc{gamma} receptor-mediated antibody function and differences in IgG1 Fc fucosylation, a major regulator of Fc effector function. Methods. Serum samples from RECOVER-Adult participants with prior SARS-CoV-2 infection (99 PASC, 139 non-PASC) were analyzed for anti-SARS-CoV-2 spike receptor-binding domain (RBD) IgG and subclasses, Fc{gamma}RIIIa- and Fc{gamma}RIIa-mediated reporter activity as surrogate measures of antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP), anti-RBD IgG1 Fc glycosylation, and circulating fucose-modifying enzymes. Results. Despite higher anti-RBD IgG and IgG1 abundance, PASC sera showed lower ADCC- and ADCP-associated Fc{gamma}R reporter activity, including after normalization for antibody abundance. Anti-RBD IgG1 showed greater fucosylation in PASC samples (30 PASC, 34 non-PASC). Serum FUCA2 was lower in PASC samples (24 PASC, 34 non-PASC), whereas FUCA1 and soluble CD16a levels did not differ between the two groups. Conclusion. PASC is associated with impaired antiviral IgG Fc function, increased IgG1 Fc fucosylation, and reduced circulating FUCA2, identifying a glycoimmune axis that may contribute to impaired Fc-dependent antigen clearance and represents a potentially targetable pathway in Long COVID.

The transcriptional and cell repertoire changes of lymph node (LN) T cells following influenza vaccination have been under-investigated across different ethnicities. Draining (dLN) and non-draining (ndLN) axillary LN cells from 17 participants with genotypically varied African or Asian ancestry (ISRCTN13657999) were collected by fine needle aspiration (FNA) before, 5 days and 42 days after adjuvanted influenza vaccination and analysed by single-cell RNA-sequencing (scRNA-seq). The early dLN transcriptional profile and cellular dynamics were distinct from the ndLN; BACH2 expressing T cells were highly induced, exhibiting stem-like and unique quiescence signatures. Proliferating T follicular helper cells (Tfh) were enriched in dLN and Th1-like Tfh predominated with highly induced transcriptional changes. The BACH2+ T cell state and Th1-like Tfh cells, transcriptionally distinct from germinal centre (GC) T cells, were co-localised with LAMP3+ dendritic cells in the T cell zone of publicly available LN datasets, a region that was also distinct from GC Tfh niches. Together, these findings indicate that the induction of a robust, proliferative, and transcriptionally diverse Tfh response, alongside a discrete subset of BACH2+ T cells, represents a marked feature of the early T cell zone immune response to adjuvanted influenza vaccine in the dLN.

Background: Common variable immunodeficiency is frequently underrecognized due to its heterogeneous clinical presentation, resulting in diagnostic delays that increase disease-related complications, with each year of delay increasing mortality risk by 4% for the most common inborn error of immunity. Objective: In individuals with established common variable immunodeficiency, we sought to recognize patterns in patient diagnostic trajectories to identify opportunities for interventions to reduce time to diagnosis. Methods: We analyzed retrospective electronic health records data from 139 individuals with physician-confirmed common variable immunodeficiency across five academic health systems representing >10million patients. We evaluated diagnosing specialties and international classification of disease code trajectories in the two years prior to and at common variable immunodeficiency confirmation. Additionally, we construct specialty-specific prediction on a 1:50 case: control cohort using a gradient boosting algorithm. Results: The leading diagnoses at common variable immunodeficiency determination were respiratory (35% of individuals), and individuals with respiratory symptoms were more likely to be diagnosed by Allergy / Immunology than by other specialties (odds ratio 2.9, standard error 0.35, p-value 1.9x10-3) and had a higher frequency of visits to pulmonary disease in the two years prior to diagnosis. Individuals with neoplasms were 50% more likely than other patients (OR 2.0, SE 0.29, p-value 0.02) to receive a diagnosis at each visit, reducing their diagnostic delay by up to a year. We find that a larger ratio of number of specialties visited to the total number of visits is significantly correlated with an increased time to diagnosis. A trajectory-informed specialty-specific prediction improved detection with an area under the precision-recall curve of fifty times the value expected under the null. Conclusions: Individuals referred to Allergy / Immunology have shorter time to diagnosis, and a specialty-specific prediction could improve detection of common variable immunodeficiency. Clinical Implications: Overall, we find that phenotype-specialty interactions may inform targeted interventions to improve common variable immunodeficiency recognition across specialties, reducing diagnostic odyssey and disease-related complications.

Background: Moderate-to-severe asthma affects 15-30% of asthma patients, but accounts for over 50% of healthcare costs and disproportionate morbidity. However, the genetic mechanisms underlying moderate-to-severe versus mild asthma remain poorly understood. Methods: We performed separate pooled multi-ancestry GWAS of mild (N = 14,372) and moderate-to-severe asthma (N = 7,096) versus non-asthma controls (N = 28,816) among adult participants in the NIH All of Us Research Program (version 8). European, African, and Latino/admixed ancestry-specific GWAS were combined using fixed-effect meta-analysis. Lung expression quantitative trait loci (eQTL) and pathway analyses were performed to identify candidate genes and biological pathways across asthma phenotypes. Results: Mild and moderate-to-severe asthma shared susceptibility loci at IL1RL1, IKZF3, and the GTF3AP1-IL33, GTF3AP1-RANBP6, LINC02757-EMSY and HLA-DRB1-HLA-DQA1 regions. Shared lung eQTLs implicated IL18R1, IL18RAP, HLA genes, IL33, LRRC32, GRB7, MIEN1 and GSDMB. In contrast, the phenotypes exhibited distinct genetic architectures. Mild asthma was characterized by broader HLA class II signals and associations at SMAD3 and GSDMB, with enrichment of antigen presentation, T-helper cell differentiation, and TGF-{beta} regulation, consistent with adaptive immune mechanisms. Moderate-to-severe asthma was characterized by WDR36 and PTCH1, with lung eQTLs implicating TSLP, CAMK4, and FANCC. Pathway analysis identified enrichment of IL-13, IL-6, and IL-10 production, myeloid leukocyte differentiation, and oxidative stress responses, consistent with innate inflammation and pathways implicated in steroid resistance. The IL33 signals showed a severity-gradient effect, with stronger associations in moderate-to-severe asthma. Conclusion: Mild and moderate-to-severe asthma exhibit shared and distinct genetic architectures that support existing biologic targets and suggest novel therapeutic candidates.

Type 1 diabetes (T1D) is a chronic autoimmune disease characterized by T-cell mediated destruction of pancreatic islet {beta}-cells with genetic, environmental, and molecular triggers involved in disease pathogenesis. Patients with T1D have elevated serum levels of microRNA146a (miR146a). Polymorphisms in the miR146a gene that result in reduced expression of miR146a are associated with protection from T1D. We studied physiological regulators of miR146a expression and found that both hyperglycemia and elevated O-GlcNAcylation increased miR146a expression in T cells. Peripheral blood mononuclear cells (PBMCs) from T1D patients showed increased miR146a and O-GlcNAc transferase (OGT) expression, suggesting increased O-GlcNAcylation may promote miR146a expression in T1D patients. To determine the genetic and developmental role of miR146a in T1D, we generated miR146a-knockout (KO) non-obese diabetic (NOD) mice and found that absence of miR146a significantly protected NOD mice from spontaneous autoimmune diabetes. While we found no impact of miR146a knockout on general hematopoietic parameters and immune cell populations, remarkably, immune cell infiltration into the pancreas was significantly attenuated. Protection from autoimmune diabetes in miR146a-KO NOD mice was associated with increased regulatory T (Treg) cells in the spleen and pancreatic lymph node. Mechanistically, absence of miR146a increased NF-{kappa}B c-Rel expression in Treg cells and enhanced c-Rel binding at the Forkhead box protein P3 (FOXP3) promoter, which positively regulated Treg cell development and suppressor function, offering protection from T1D in miR146a-KO NOD mice. Our findings reveal miR146a as a key regulator of Treg cell-mediated immune tolerance through controlling NF-{kappa}B c-Rel-dependent FOXP3 expression and suggest targeting miR146a as a potential strategy to restore peripheral tolerance in T1D.

Asthma severity and immune responses are regulated by circadian rhythms, yet the role of circadian timing in fungal allergen-induced airway inflammation remains poorly defined. Alternaria alternata, a clinically relevant fungal allergen, is strongly associated with severe asthma exacerbations and persistent airway dysfunction. Using a preclinical mouse model, we investigated whether pulmonary responses to Alternaria exposure are influenced by time of day and sex. We demonstrate that Alternaria induces allergic airway inflammation, airway hyperresponsiveness (AHR), and epithelial barrier disruption in a circadian- and sex-dependent manner. Female mice exhibited enhanced eosinophilic, neutrophilic, interstitial macrophage, and dendritic cell infiltration predominantly at Zeitgeber Time 0 (ZT0), whereas males showed stronger inflammatory responses at ZT12. Alternaria exposure increased IL-33 release and promoted ILC2 and Th2 cell accumulation, with distinct temporal patterns between sexes. Cytokine and chemokine profiling revealed robust Th2, proinflammatory, and Th17 responses that varied with exposure time, with higher responses at ZT12 in females and ZT0 in males. Markers of tissue injury, including lactate dehydrogenase and uric acid, followed similar circadian-dependent trends. Functionally, Alternaria-exposed mice displayed greater AHR during the resting phase, particularly in males. Additionally, Alternaria disrupted epithelial junctional proteins (E-cadherin and ZO-1) and altered expression of core circadian clock proteins CLOCK, BMAL1, and REV-ERB. Mechanistically, peak inflammation coincided with enhanced STAT1, STAT3, and STAT6 activation. Collectively, these findings identify circadian timing and sex as key determinants of fungal allergen-induced airway inflammation and suggest chronotherapeutic approaches may improve asthma outcomes.

Background: Recurrent ocular herpetic disease is a leading infectious cause of corneal blindness and is associated with psychological distress, anxiety, and social withdrawal. No ocular herpes vaccine or immunotherapy currently exists. Hypothesis: A prime/pull/keep (PPK) T-cell immunotherapy, built on HSV-1 epitopes preferentially recognized by CD8+/CD4+ T cells from naturally protected asymptomatic (ASYMP) individuals, would reduce viral reactivation, shedding, recurrent corneal disease, and associated neurobehavioral deficits. Methods: Immunodominant HLA-restricted CD8+/CD4+ epitopes from HSV-1 gD/gB, tegument proteins VP11/12/VP13/14, and helicase UL9 were delivered via topical ocular AAV8 vector (prime) to latently infected HLA-A*0201/DRB1 mice, combined with chemokine CXCL11 (pull) and IL-2/IL-15 cytokines to sustain TRM cells (keep), then UVB-exposed to trigger reactivation and recurrent corneal disease. Results: PPK immunotherapy conferred robust, near-complete protection against recurrent HSV-1 disease and improved survival after UVB-induced reactivation, unlike its individual components. This protection was associated with site-specific accumulation of HSV-1-specific CD8+/CD4+ TRM cells in the cornea and trigeminal ganglia (TG), but not in the spleen, with peak retention from combined IL-2/IL-15, alongside durable central/effector memory populations. PPK-induced T cells showed enhanced polyfunctionality (cytokine production, cytotoxicity, proliferation), reduced exhaustion markers, and a TCF-1+ self-renewing phenotype. PPK also elicited robust, Th1-biased HSV-1-specific IgG responses complementing mucosal immunity, and restored spatial memory, reduced depression-like behavior, and reversed social withdrawal in mice with recurrent ocular herpes. Conclusion: PPK immunotherapy induces potent, durable antiviral TRM cells at primary sites of HSV-1 latency and reactivation, protecting against recurrent ocular herpetic disease and alleviating associated neurobehavioral deficits, supporting its translational potential in humans.

Despite its relative anti-inflammatory nature, IgG4 is involved in a variety of pathological conditions such as autoimmune disease and cancer. To investigate whether the IgG4 repertoire presents any abnormalities in diseases where IgG4 autoantibodies predominate, we charted longitudinal IgG4 repertoires in health, in an IgG4-mediated autoimmune disease (AID; muscle-specific kinase myasthenia gravis (MuSK MG)), and in IgG1/IgG3 mediated AIDs (acetylcholine receptor (AChR) MG and Lambert-Eaton myasthenic syndrome (LEMS). In total, we assessed 218 samples of 69 donors using mass spectrometry-based antibody repertoire analysis, covering a timespan of up to eight years. Despite being superficially inconspicuous in clonal diversity and abundances, we detected a slightly elevated number of IgG4 clones in MuSK MG patients compared to healthy donors. Notably, this observation did not coincide with drastic repertoire changes during treatment, suggesting that a large portion of the IgG4 repertoire is not significantly affected by common MuSK MG treatment regimens. Comparing the IgG4 antibody repertoires of all donors, we found that they were exceptionally unique for each individual. Against what would be expected in the case of a constantly adapting antibody repertoire, a large portion of the IgG4 clones remained stable without any further maturation. This observation even extended to patients receiving B cell depleting or IgG modulating treatments. Our findings suggest that a dominating fraction of the IgG4 pool may be produced by long-lived plasma cells, which are unaffected by disease or medical interventions.

Background. The cholinergic anti-inflammatory pathway regulates inflammation through vagus signaling that activates the 7 nicotinic acetylcholine receptor (7nAChR) on leukocytes. Suppression of macrophage inflammatory cytokine production through 7nAChR-mediated inhibition of NF-{kappa}B activation is considered a central mechanism of this pathway. However, the effects of cholinergic activation of 7nAChR on neutrophils remain poorly understood. Here, we investigated whether and how activation of 7nAChR in neutrophils regulates neutrophil migration, defined the underlying molecular mechanism, and evaluated its contribution to anti-inflammatory protection during endotoxemia. Results. The ex vivo swarming assays revealed that activation of 7nAChR with the selective agonists PNU-282987 and AR-R17779 significantly inhibited human and mouse neutrophil migration. The inhibitory effect of PNU-282987 was abolished in 7nAChR-deficient neutrophils, confirming receptor specificity. Mechanistically, 7nAChR activation reduced integrin M{beta}2 activation, decreased F-actin polymerization, and impaired neutrophil polarization. These effects required M{beta}2, as PNU-282987 failed to inhibit actin remodeling in M-deficient neutrophils. Modulation of M{beta}2 activation with an activating antibody (MEM48) demonstrated that 7nAChR regulates neutrophil migration by limiting excessive M{beta}2 activation and maintaining an optimal adhesive state. In vivo, adoptively transferred fluorescently labeled PNU-282987-treated neutrophils exhibited reduced recruitment to the lungs compared with vehicle-treated neutrophils during LPS-induced endotoxemia. Furthermore, neutrophil depletion markedly diminished the protective effect of PNU-282987 on survival, demonstrating that neutrophils contribute to 7nAChR-mediated protection during systemic inflammation. Conclusions. Our findings identify neutrophils as an important cellular target of the cholinergic anti-inflammatory pathway and demonstrate that 7nAChR activation limits neutrophil migration by suppressing integrin M{beta}2 activation and actin cytoskeletal remodeling. Together, these findings establish M{beta}2 as a critical downstream effector of 7nAChR signaling in neutrophils and identify regulation of integrin activation as a previously unrecognized mechanism of the cholinergic anti-inflammatory pathway.

BackgroundPost-acute COVID-19 syndrome (PACS) can cause persistent disability involving fatigue, post-exertional malaise, neurocognitive symptoms, and autonomic manifestations. A clinically overlapping condition has been reported following COVID-19 vaccination and termed post-acute COVID-19 vaccination syndrome (PACVS), but its underlying mechanisms remain poorly understood. We investigated whether immune dysregulation, autoreactivity, antigen persistence, and viral reactivation--mechanisms implicated in PACS--are also present in PACVS. MethodsThis cross-sectional study included 28 PACS patients, 29 PACVS patients, and 16 convalescent controls (CCG). Clinical manifestations were assessed using ME/CFS-related symptom questionnaires and the Bell Score. T-cell phenotypes and T cells reactive to SARS-CoV-2 Spike, {beta}1/{beta}2-adrenergic receptor, and M3/M4 muscarinic receptor antigens were analyzed by multicolor flow cytometry. Circulating Spike protein and GPCR autoantibodies were quantified by ELISA, and EBV load by qPCR. ResultsPACS and PACVS showed substantial clinical overlap, although several symptoms were more severe in PACS. GPCR autoantibody profiles were comparable between patient groups, while {beta}2-adrenergic receptor autoantibodies were increased compared with CCG. Only three of 57 conventional immunophenotyping parameters differed between PACS and PACVS in unadjusted analyses, whereas both groups showed multiple alterations compared with CCG. Circulating Spike protein was detected in both patient groups, with the highest prevalence in PACVS. {beta}1/{beta}2- and M3/M4-reactive CD4+ T cells were increased, whereas Spike-specific CD4+ T cells were reduced compared with CCG. EBV reactivation was rare. ConclusionsPACS and PACVS exhibit substantial clinical and immunological overlap, including GPCR-directed autoreactivity and altered receptor-reactive T-cell responses. These shared immune-mediated features suggest overlapping pathophysiological mechanisms and warrant investigation of whether therapeutic strategies currently explored for PACS may also be relevant to PACVS.