Testing the relationship between preferences for infant-directed speech and vocabulary development: A multi-lab study.

From early on, infants show a preference for infant-directed speech (IDS) over adult-directed speech (ADS), and exposure to IDS has been correlated with language outcome measures such as vocabulary. The present multi-laboratory study explores this issue by investigating whether there is a link between early preference for IDS and later vocabulary size. Infants' preference for IDS was tested as part of the ManyBabies 1 project, and follow-up CDI data were collected from a subsample of this dataset at 18 and 24 months. A total of 341 (18 months) and 327 (24 months) infants were tested across 21 laboratories. In neither preregistered analyses with North American and UK English, nor exploratory analyses with a larger sample did we find evidence for a relation between IDS preference and later vocabulary. We discuss implications of this finding in light of recent work suggesting that IDS preference measured in the laboratory has low test-retest reliability.

Neutrophils with low production of reactive oxygen species are activated during immune priming and promote development of arthritis.

Rheumatoid arthritis (RA) is an inflammatory autoimmune disease mediated by immune cell dysfunction for which there is no universally effective prevention and treatment strategy. As primary effector cells, neutrophils are important in the inflammatory joint attack during the development of RA. Here, we used single-cell sequencing technology to thoroughly analyze the phenotypic characteristics of bone marrow-derived neutrophils in type II collagen (COL2)-induced arthritis (CIA) models, including mice primed and boosted with COL2. We identified a subpopulation of neutrophils with high expression of neutrophil cytoplasmic factor 1 (NCF1) in primed mice, accompanied by a characteristic reactive oxygen species (ROS) response, and a decrease in Ncf1 expression in boosted mice with the onset of arthritis. Furthermore, we found that after ROS reduction, arthritis occurred in primed mice but was attenuated in boosted mice. This bidirectional effect of ROS suggested a protective role of ROS during immune priming. Mechanistically, we combined functional assays and metabolomics identifying Ncf1-deficient neutrophils with enhanced migration, chemotactic receptor CXCR2 expression, inflammatory cytokine secretion, and Th1/Th17 differentiation. This alteration was mainly due to the metabolic reprogramming of Ncf1-deficient neutrophils from an energy supply pathway dominated by gluconeogenesis to an inflammatory immune pathway associated with the metabolism of histidine, glycine, serine, and threonine signaling, which in turn induced arthritis. In conclusion, we have systematically identified the functional and inflammatory phenotypic characteristics of neutrophils under ROS regulation, which provides a theoretical basis for understanding the pathogenesis of RA, to further improve prevention strategies and identify novel therapeutic targets.

Improved diagnosis of systemic lupus erythematosus with human-derived double-stranded DNA antigen.

Anti-double-stranded DNA antibodies (anti-dsDNA) serve as a crucial serological indicator for systemic lupus erythematosus (SLE). Chemiluminescent immunoassay (CIA) is mainly used in clinical diagnosis of SLE, but suffers from low specificity, partially because the use of dsDNA antigens of varied sources in current CIA kits that sometimes led to controversial results. On the basis that anti-dsDNA in healthy individuals tend to selectively bind with dsDNA originating from pathogens, whereas pathogenic anti-dsDNA in SLE patients bind all forms of dsDNA, here we proposed the use of dsDNA fragment derived from human genome as antigen (synthesized via PCR using the human genomic DNA as the template). A magnetic bead-based immunofluorescence assay (IFA) was thus developed for SLE diagnosis, which exhibited improved sensitivity and specificity over CIA using the WHO reference reagent (15/174) as standard. For clinical serum sample analysis (n = 590), IFA exhibited an accuracy of 71.9% that was higher than CIA (65.3%). Crucially, the IFA results exhibited stronger correlations with the activity of SLE, renal involvement, and its prognosis. Besides the improved clinical diagnosis, the proposed IFA also holds great promise in assay standardization due to the high homogeneity of the synthetic dsDNA.

Calpain-1 weakens the nuclear envelope and promotes the release of neutrophil extracellular traps.

The inducers of neutrophil extracellular trap (NET) formation are heterogeneous and consequently, there is no specific pathway or signature molecule indispensable for NET formation. But certain events such as histone modification, chromatin decondensation, nuclear envelope breakdown, and NET release are ubiquitous. During NET formation, neutrophils drastically rearrange their cytoplasmic, granular and nuclear content. Yet, the exact mechanism for decoding each step during NET formation still remains elusive. Here, we investigated the mechanism of nuclear envelope breakdown during NET formation. Immunofluorescence microscopic evaluation revealed a gradual disintegration of outer nuclear membrane protein nesprin-1 and alterations in nuclear morphology during NET formation. MALDI-TOF analysis of NETs that had been generated by various inducers detected the accumulation of nesprin-1 fragments. This suggests that nesprin-1 degradation occurs before NET release. In the presence of a calpain-1, inhibitor nesprin-1 degradation was decreased in calcium driven NET formation. Microscopic evaluation confirmed that the disintegration of the lamin B receptor (LBR) and the collapse of the actin cytoskeleton occurs in early and later phases of NET release, respectively. We conclude that the calpain-1 degrades nesprin-1, orchestrates the weakening of the nuclear membrane, contributes to LBR disintegration, and promoting DNA release and finally, NETs formation.

Integrin αVß6 - autoantigen and driver of epithelial remodeling in colon and bile ducts in primary sclerosing cholangitis and inflammatory bowel disease.

OBJECTIVE: Recently, autoantibodies directed against the epithelial adhesion protein integrin αVß6 have been identified which strongly associate with ulcerative colitis (UC). We aimed to elucidate whether anti-integrin αVß6 (anti- αVß6) is present in primary sclerosing cholangitis (PSC), its associated inflammatory bowel disease or other cholestatic liver diseases and their persistence after proctocolectomy. DESIGN: We detected anti- αVß6 by an enzyme-linked immunosorbent assay in sera collected at two German tertiary centers, including healthy controls (N=62), UC (N=36), Crohn's disease (CD, N=65), PSC-IBD (78 samples from N=41 patients), PSC without IBD (PSC, 41 samples from N=18 patients), primary biliary cholangitis (PBC, N=24), autoimmune hepatitis (AIH, N=32), secondary sclerosing cholangitis (SSC, N=12) and metabolic dysfunction-associated steatotic liver disease (MASLD, N=24). Additionally, sera after proctocolectomy were studied (44 samples / N= 10 patients). Immunofluorescent analyses were performed in tissue samples from liver, large bile duct from surgical resections and colon of PSC patients. RESULTS: Anti- αVß6 occurred in 91% of UC, 17% of CD, 73% of PSC-IBD, 39% of PSC, 4% of PBC, 14% of AIH, and 0% of healthy controls, SSC or MASLD. Integrin αVß6 is selectively expressed in disease-associated epithelia of both bile duct and colon. Anti- αVß6 levels correlate moderately with intestinal disease activity in PSC-IBD, but only weakly with biliary disease. CONCLUSION: Anti- αVß6 frequently occur in patients suffering from PSC, especially in PSC-IBD. Anti- αVß6 levels positively correlate to IBD activity in PSC-IBD, but may also occur in the absence of clinically manifest IBD in PSC.

Hidden Appendicoliths and Their Impact on the Severity and Treatment of Acute Appendicitis.

Background/Objectives: In patients diagnosed with uncomplicated acute appendicitis (UAA), the absence of calcified deposits or stones, called appendicoliths, often leads to consideration of non-operative treatment (NOT), despite the notable treatment failure rate associated with this approach. Previous research has indirectly estimated the prevalence of appendicoliths to range between 15% and 38% retrospectively by CT scan, intraoperative palpation, and pathology report, thereby potentially missing certain concrements. Our hypothesis proposes that this reported prevalence significantly underestimates the occurrence of appendicoliths, which could explain the high failure rate of 29% of patients with appendicitis observed with NOT. Methods: In our prospective study, conducted with a cohort of 56 adult patients diagnosed with acute appendicitis (AA), we employed intraoperative extracorporeal incisions of the vermiform appendix, in addition to standard diagnostic methods. Results: Our findings revealed 50% more appendicoliths by intraoperative incision (n = 36, p < 0.001) compared to preoperative imaging (n = 24). Appendicoliths were present in 71.4% (n = 40, p < 0.001) of AA patients. Conclusions: These results suggest that conventional diagnostic procedures plausibly underestimate the actual prevalence of appendicoliths, potentially elucidating the frequent treatment failures observed in NOT approaches applied to patients with UAA.

Sustained pacifier use is associated with smaller vocabulary sizes at 1 and 2 years of age: A cross-sectional study.

Pacifier use during childhood has been hypothesized to interfere with language processing, but, to date, there is limited evidence revealing detrimental effects of prolonged pacifier use on infant vocabulary learning. In the present study, parents of 12- and 24-month-old infants were recruited in Oslo (Norway). The sample included 1187 monolingual full-term born (without visual, auditory, or cognitive impairments) infants: 452 (230 girls; 222 boys) 12-month-olds and 735 (345 girls; 390 boys) 24-month-olds. Parents filled out an online Norwegian Communicative Development Inventory (CDI), which assesses the vocabulary in comprehension and production for 12-month-old infants and in production only for 24-month-old infants. CDI scores were transformed into age- and sex-adjusted percentiles using Norwegian norms. Additionally, parents retrospectively reported their child's daytime pacifier use, in hours, at 2-month intervals, from birth to the assessment date. Maternal education was used to control, in the analyses, for the socio-economic status. We found that greater pacifier use in an infant's lifespan was associated with lower vocabulary size. Pacifier use later in life was more negatively associated with vocabulary size than precocious use, and increased the odds of being a low language scorer. In sum, our study moves beyond the findings of momentary effects of experimentally induced "impairment" in articulators' movement on speech perception and suggests that, from 12 months of age, constraints on the infant's speech articulators (pacifier use) may be negatively associated with word comprehension and production. RESEARCH HIGHLIGHT: We examined the relationship between pacifier use and vocabulary sizes in production at 24 months of age and comprehension and production at 12 months of age. Lifespan Pacifier Use (LPU) was negatively correlated with vocabulary sizes in comprehension and production among 12-month-old infants and negatively correlated with production for 24-month-olds. Later pacifier use was found to be more negatively correlated with vocabulary size in infants, as compared to more precocious use. The amount of pacifier use in the 2 months prior to a child's second birthday was predictive of a higher prevalence of low vocabulary scores in 24-month-olds.

Suppression of neutrophils by sodium exacerbates oxidative stress and arthritis.

Introduction: Typical Western diet, rich in salt, contributes to autoimmune disease development. However, conflicting reports exist about the effect of salt on neutrophil effector functions, also in the context of arthritis. Methods: We investigated the effect of sodium chloride (NaCl) on neutrophil viability and functions in vitro, and in vivo employing the murine K/BxN-serum transfer arthritis (STA) model. Results and discussion: The effects of NaCl and external reactive oxygen species (H2O2) were further examined on osteoclasts in vitro. Hypertonic sodium-rich media caused primary/secondary cell necrosis, altered the nuclear morphology, inhibited phagocytosis, degranulation, myeloperoxidase (MPO) peroxidation activity and neutrophil extracellular trap (NET) formation, while increasing total ROS production, mitochondrial ROS production, and neutrophil elastase (NE) activity. High salt diet (HSD) aggravated arthritis by increasing inflammation, bone erosion, and osteoclast differentiation, accompanied by increased NE expression and activity. Osteoclast differentiation was decreased with 25 mM NaCl or 100 nM H2O2 addition to isotonic media. In contrast to NaCl, external H2O2 had pro-resorptive effects in vitro. We postulate that in arthritis under HSD, increased bone erosion can be attributed to an enhanced oxidative milieu maintained by infiltrating neutrophils, rather than a direct effect of NaCl.

IL-3 orchestrates ulcerative colitis pathogenesis by controlling the development and the recruitment of splenic reservoir neutrophils.

Inflammatory bowel diseases (IBDs) are a global health issue with an increasing incidence. Although the pathogenesis of IBDs has been investigated intensively, the etiology of IBDs remains enigmatic. Here, we report that interleukin-3 (Il-3)-deficient mice are more susceptible and exhibit increased intestinal inflammation during the early stage of experimental colitis. IL-3 is locally expressed in the colon by cells harboring a mesenchymal stem cell phenotype and protects by promoting the early recruitment of splenic neutrophils with high microbicidal capability into the colon. Mechanistically, IL-3-dependent neutrophil recruitment involves CCL5+ PD-1high LAG-3high T cells, STAT5, and CCL20 and is sustained by extramedullary splenic hematopoiesis. During acute colitis, Il-3-/- show, however, increased resistance to the disease as well as reduced intestinal inflammation. Altogether, this study deepens our understanding of IBD pathogenesis, identifies IL-3 as an orchestrator of intestinal inflammation, and reveals the spleen as an emergency reservoir for neutrophils during colonic inflammation.

Glutathione peroxidase 3 is a novel clinical diagnostic biomarker and potential therapeutic target for neutrophils in rheumatoid arthritis.

BACKGROUND: Neutrophils have a critical role in the pathogenesis of rheumatoid arthritis (RA) with immune system dysfunction. However, the molecular mechanisms of this process mediated by neutrophils still remain elusive. The purpose of the present study is to identify hub genes in neutrophils for diagnosis and treatment of RA utilizing publicly available datasets. METHODS: Gene expression profiles were downloaded from the Gene Expression Omnibus, and batch-corrected and normalized expression data were obtained using the ComBat package. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analysis were used to conduct significantly functional analysis and crucial pathways. The resulting co-expression genes modules and hub genes were generated based on the weighted gene co-expression network analysis and visualization by Cytoscape. Flow cytometry was conducted to detect reactive oxygen species (ROS) levels in neutrophils. RESULTS: Neutrophils underwent transcriptional changes in synovial fluid (SF) of RA patients, different from peripheral blood of healthy controls or patients with RA. Especially, glycolysis, HIF-1 signaling, NADH metabolism, and oxidative stress were affected. These hub genes were strongly linked with classical glycolysis-related genes (ENO1, GAPDH, and PKM) responsible for ROS production. The antioxidant enzyme glutathione peroxidase 3 (GPX3), a ROS scavenger, was first identified as a hub gene in RA neutrophils. Neutrophils from patients with autoinflammatory and autoimmune diseases had markedly enhanced ROS levels, most notably in RA SF. CONCLUSION: This research recognized hub genes and explored the characteristics of neutrophils in RA. Our findings suggest that the novel hub gene GPX3 is involved in the neutrophil-driven oxidative stress-mediated pathogenesis of RA. It has the potency to be a target for neutrophil-directed RA therapy.

Anti-DNA-IgM Favors the Detection of NET-Associated Extracellular DNA.

During inflammatory responses, neutrophils enter the sites of attack where they execute various defense mechanisms. They (I) phagocytose microorganisms, (II) degranulate to release cytokines, (III) recruit various immune cells by cell-type specific chemokines, (IV) secrete anti-microbials including lactoferrin, lysozyme, defensins and reactive oxygen species, and (V) release DNA as neutrophil extracellular traps (NETs). The latter originates from mitochondria as well as from decondensed nuclei. This is easily detected in cultured cells by staining of DNA with specific dyes. However, in tissues sections the very high fluorescence signals emitted from the condensed nuclear DNA hamper the detection of the widespread, extranuclear DNA of the NETs. In contrast, when we employ anti-DNA-IgM antibodies, they are unable to penetrate deep into the tightly packed DNA of the nucleus, and we observe a robust signal for the extended DNA patches of the NETs. To validate anti-DNA-IgM, we additionally stained the sections for the NET-markers histone H2B, myeloperoxidase, citrullinated histone H3, and neutrophil elastase. Altogether, we have described a fast one-step procedure for the detection of NETs in tissue sections, which provides new perspectives to characterize neutrophil-associated immune reactions in disease.

Formation and Clearance of NETs in Health and Disease.

Neutrophils are the most abundant innate immune cells in humans and the first line of defense against invading pathogens [...].

A Pleomorphic Puzzle: Heterogeneous Pulmonary Vascular Occlusions in Patients with COVID-19.

Vascular occlusions in patients with coronavirus diseases 2019 (COVID-19) have been frequently reported in severe outcomes mainly due to a dysregulation of neutrophils mediating neutrophil extracellular trap (NET) formation. Lung specimens from patients with COVID-19 have previously shown a dynamic morphology, categorized into three types of pleomorphic occurrence based on histological findings in this study. These vascular occlusions in lung specimens were also detected using native endogenous fluorescence or NEF in a label-free method. The three types of vascular occlusions exhibit morphology of DNA rich neutrophil elastase (NE) poor (type I), NE rich DNA poor (type II), and DNA and NE rich (type III) cohort of eleven patients with six males and five females. Age and gender have been presented in this study as influencing variables linking the occurrence of several occlusions with pleomorphic contents within a patient specimen and amongst them. This study reports the categorization of pleomorphic occlusions in patients with COVID-19 and the detection of these occlusions in a label-free method utilizing NEF.

Induction of Ocular Surface Inflammation and Collection of Involved Tissues.

Ocular surface diseases include a range of disorders that disturb the functions and structures of the cornea, conjunctiva, and the associated ocular surface gland network. Meibomian glands (MG) secrete lipids that create a covering layer that prevents the evaporation of the aqueous part of the tear film. Neutrophils and extracellular DNA traps populate MG and the ocular surface in a mouse model of allergic eye disease. Aggregated neutrophil extracellular traps (aggNETs) formulate a mesh-like matrix composed of extracellular chromatin that occludes MG outlets and conditions MG dysfunction. Here, a method for inducing ocular surface inflammation and MG dysfunction is presented. The procedures for collecting organs related to the ocular surface, such as the cornea, conjunctiva, and eyelids, are described in detail. Using established techniques for processing each organ, the major morphological and histopathological features of MG dysfunction are also shown. Ocular exudates offer the opportunity to assess the inflammatory state of the ocular surface. These procedures enable the investigation of topical and systemic anti-inflammatory interventions at the preclinical level.

Alpha-1 antitrypsin in autoimmune diseases: Roles and therapeutic prospects.

Alpha-1 antitrypsin (A1AT) is a protease inhibitor in the serum. Its primary function is to inhibit the activity of a series of proteases, including proteinase 3, neutrophil elastase, metalloproteases, and cysteine-aspartate proteases. In addition, A1AT also has anti-inflammatory, anti-apoptotic, anti-oxidative stress, anti-viral, and anti-bacterial activities and plays essential roles in the regulation of tissue repair and lymphocyte differentiation and activation. The overactivation of the immune system characterizes the pathogenesis of autoimmune diseases. A1AT treatment shows beneficial effects on patients and animal models with autoimmune diseases such as rheumatoid arthritis and systemic lupus erythematosus. This review summarizes the functions and therapeutic prospects of A1AT in autoimmune diseases.

Influenza Virus-like Particle-Based Hybrid Vaccine Containing RBD Induces Immunity against Influenza and SARS-CoV-2 Viruses.

Several approaches have produced an effective vaccine against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Since millions of people are exposed to influenza virus and SARS-CoV-2, it is of great interest to develop a two-in-one vaccine that will be able to protect against infection of both viruses. We have developed a hybrid vaccine for SARS-CoV-2 and influenza viruses using influenza virus-like particles (VLP) incorporated by protein transfer with glycosylphosphatidylinositol (GPI)-anchored SARS-CoV-2 RBD fused to GM-CSF as an adjuvant. GPI-RBD-GM-CSF fusion protein was expressed in CHO-S cells, purified and incorporated onto influenza VLPs to develop the hybrid vaccine. Our results show that the hybrid vaccine induced a strong antibody response and protected mice from both influenza virus and mouse-adapted SARS-CoV-2 challenges, with vaccinated mice having significantly lower lung viral titers compared to naive mice. These results suggest that a hybrid vaccine strategy is a promising approach for developing multivalent vaccines to prevent influenza A and SARS-CoV-2 infections.

Hypoxia Promotes Neutrophil Survival After Acute Myocardial Infarction.

Phagocytosis, degranulation, and neutrophil extracellular traps (NETs) formation build the armory of neutrophils for the first line of defense against invading pathogens. All these processes are modulated by the microenvironment including tonicity, pH and oxygen levels. Here we investigated the neutrophil infiltration in cardiac tissue autopsy samples of patients with acute myocardial infarction (AMI) and compared these with tissues from patients with sepsis, endocarditis, dermal inflammation, abscesses and diseases with prominent neutrophil infiltration. We observed many neutrophils infiltrating the heart muscle after myocardial infarction. Most of these had viable morphology and only few showed signs of nuclear de-condensation, a hallmark of early NET formation. The abundance of NETs was the lowest in acute myocardial infarction when compared to other examined diseases. Since cardiac oxygen supply is abruptly abrogated in acute myocardial infarction, we hypothesized that the resulting tissue hypoxia increased the longevity of the neutrophils. Indeed, the viable cells showed increased nuclear hypoxia inducible factor-1α (HIF-1α) content, and only neutrophils with low HIF-1α started the process of NET formation (chromatin de-condensation and nuclear swelling). Prolonged neutrophil survival, increased oxidative burst and reduced NETs formation were reproduced under low oxygen tensions and by HIF-1α stabilization in vitro. We conclude that nuclear HIF-1α is associated with prolonged neutrophil survival and enhanced oxidative stress in hypoxic areas of AMI.

Corrigendum: Low Dose Radiation Therapy Induces Long-Lasting Reduction of Pain and Immune Modulations in the Peripheral Blood - Interim Analysis of the IMMO-LDRT01 Trial.

[This corrects the article DOI: 10.3389/fimmu.2021.740742.].

Neutrophil extracellular traps drive epithelial-mesenchymal transition of human colon cancer.

Neutrophil extracellular traps (NETs) are extracellular structures, composed of nuclear DNA and various proteins released from neutrophils. Evidence is growing that NETs exert manifold functions in infection, immunity and cancer. Recently, NETs have been detected in colorectal cancer (CRC) tissues, but their association with disease progression and putative functional impact on tumourigenesis remained elusive. Using high-resolution stimulated emission depletion (STED) microscopy, we showed that citrullinated histone H3 (H3cit) is sufficient to specifically detect citrullinated NETs in colon cancer tissues. Among other evidence, this was supported by the close association of H3cit with de-condensed extracellular DNA, the hallmark of NETs. Extracellular DNA was reliably differentiated from nuclear condensed DNA by staining with an anti-DNA antibody, providing a novel and valuable tool to detect NETs in formalin-fixed paraffin-embedded tissues. Using these markers, the clinical association of NETs was investigated in a cohort of 85 patients with colon cancer. NETs were frequently detected (37/85, 44%) in colon cancer tissue sections and preferentially localised either only in the tumour centre or both in the tumour centre and the invasive front. Of note, citrullinated NETs were significantly associated with high histopathological tumour grades and lymph node metastasis. In vitro, purified NETs induced filopodia formation and cell motility in CRC cell lines. This was associated with increased expression of mesenchymal marker mRNAs (vimentin [VIM], fibronectin [FN1]) and epithelial-mesenchymal transition promoting transcription factors (ZEB1, Slug [SNAI2]), as well as decreased expression of the epithelial markers E-cadherin (CDH1) and epithelial cell adhesion molecule (EPCAM). These findings indicated that NETs activate an epithelial-mesenchymal transition-like process in CRC cells and may contribute to the metastatic progression of CRC. © 2021 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Neutrophils Orchestrate the Periodontal Pocket.

The subgingival biofilm attached to tooth surfaces triggers and maintains periodontitis. Previously, late-onset periodontitis has been considered a consequence of dysbiosis and a resultant polymicrobial disruption of host homeostasis. However, a multitude of studies did not show "healthy" oral microbiota pattern, but a high diversity depending on culture, diets, regional differences, age, social state etc. These findings relativise the aetiological role of the dysbiosis in periodontitis. Furthermore, many late-onset periodontitis traits cannot be explained by dysbiosis; e.g. age-relatedness, attenuation by anti-ageing therapy, neutrophil hyper-responsiveness, and microbiota shifting by dysregulated immunity, yet point to the crucial role of dysregulated immunity and neutrophils in particular. Furthermore, patients with neutropenia and neutrophil defects inevitably develop early-onset periodontitis. Intra-gingivally injecting lipopolysaccharide (LPS) alone causes an exaggerated neutrophil response sufficient to precipitate experimental periodontitis. Vice versa to the surplus of LPS, the increased neutrophil responsiveness characteristic for late-onset periodontitis can effectuate gingiva damage likewise. The exaggerated neutrophil extracellular trap (NET) response in late-onset periodontitis is blameable for damage of gingival barrier, its penetration by bacteria and pathogen-associated molecular patterns (PAMPs) as well as stimulation of Th17 cells, resulting in further neutrophil activation. This identifies the dysregulated immunity as the main contributor to periodontal disease.