Maternal gut bacteria drive intestinal inflammation in offspring with neurodevelopmental disorders by altering the chromatin landscape of CD4+ T cells.

Children with autism spectrum disorders often display dysregulated immune responses and related gastrointestinal symptoms. However, the underlying mechanisms leading to the development of both phenotypes have not been elucidated. Here, we show that mouse offspring exhibiting autism-like phenotypes due to prenatal exposure to maternal inflammation were more susceptible to developing intestinal inflammation following challenges later in life. In contrast to its prenatal role in neurodevelopmental phenotypes, interleukin-17A (IL-17A) generated immune-primed phenotypes in offspring through changes in the maternal gut microbiota that led to postnatal alterations in the chromatin landscape of naive CD4+ T cells. The transfer of stool samples from pregnant mice with enhanced IL-17A responses into germ-free dams produced immune-primed phenotypes in offspring. Our study provides mechanistic insights into why children exposed to heightened inflammation in the womb might have an increased risk of developing inflammatory diseases in addition to neurodevelopmental disorders.

Different molecular complexes that mediate transcriptional induction and repression by FoxP3.

FoxP3 conditions the transcriptional signature and functional facets of regulatory T cells (Treg cells). Its mechanism of action, whether as an activator or a repressor, has remained unclear. Here, chromatin analysis showed that FoxP3 bound active enhancer elements, not repressed chromatin, around loci over- or under-expressed in Treg cells. We evaluated the impact of a panel of FoxP3 mutants on its transcriptional activity and interactions with DNA, transcriptional cofactors and chromatin. Computational integration, confirmed by biochemical interaction and size analyses, showed that FoxP3 existed in distinct multimolecular complexes. It was active and primarily an activator when complexed with the transcriptional factors RELA, IKZF2 and KAT5. In contrast, FoxP3 was inactive when complexed with the histone methyltransferase EZH2 and transcription factors YY1 and IKZF3. The latter complex partitioned to a peripheral region of the nucleus, as shown by super-resolution microscopy. Thus, FoxP3 acts in multimodal fashion to directly activate or repress transcription, in a context- and partner-dependent manner, to govern Treg cell phenotypes.

Maternal inflammation and its ramifications on fetal neurodevelopment.

Exposure to heightened inflammation in pregnancy caused by infections or other inflammatory insults has been associated with the onset of neurodevelopmental and psychiatric disorders in children. Rodent models have provided unique insights into how this maternal immune activation (MIA) disrupts brain development. Here, we discuss the key immune factors involved, highlight recent advances in determining the molecular and cellular pathways of MIA, and review how the maternal immune system affects fetal development. We also examine the roles of microbiomes in shaping maternal immune function and the development of autism-like phenotypes. A comprehensive understanding of the gut bacteria-immune-neuro interaction in MIA is essential for developing diagnostic and therapeutic measures for high-risk pregnant women and identifying targets for treating inflammation-induced neurodevelopmental disorders.

Murine Coronavirus Disease 2019 Lethality Is Characterized by Lymphoid Depletion Associated with Suppressed Antigen-Presenting Cell Functionality.

The disease severity of coronavirus disease 2019 (COVID-19) varies considerably from asymptomatic to serious, with fatal complications associated with dysregulation of innate and adaptive immunity. Lymphoid depletion in lymphoid tissues and lymphocytopenia have both been associated with poor disease outcomes in patients with COVID-19, but the mechanisms involved remain elusive. In this study, human angiotensin-converting enzyme 2 (hACE2) transgenic mouse models susceptible to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection were used to investigate the characteristics and determinants of lethality associated with the lymphoid depletion observed in SARS-CoV-2 infection. The lethality of Wuhan SARS-CoV-2 infection in K18-hACE2 mice was characterized by severe lymphoid depletion and apoptosis in lymphoid tissues related to fatal neuroinvasion. The lymphoid depletion was associated with a decreased number of antigen-presenting cells (APCs) and their suppressed functionality below basal levels. Lymphoid depletion with reduced APC function was a specific feature observed in SARS-CoV-2 infection but not in influenza A infection and had the greatest prognostic value for disease severity in murine COVID-19. Comparison of transgenic mouse models resistant and susceptible to SARS-CoV-2 infection revealed that suppressed APC function could be determined by the hACE2 expression pattern and interferon-related signaling. Thus, we demonstrated that lymphoid depletion associated with suppressed APC function characterizes the lethality of COVID-19 mouse models. Our data also suggest a potential therapeutic approach to prevent the severe progression of COVID-19 by enhancing APC functionality.

New insights into the microbiota of wild mice.

Laboratory mice have long been an invaluable tool in biomedical science and have made significant contributions in research into life-threatening diseases. However, the translation of research results from mice to humans often proves difficult due to the incomplete nature of laboratory animal-based research. Hence, there is increasing demand for complementary methods or alternatives to laboratory mice that can better mimic human physiological traits and potentially bridge the translational research gap. Under these circumstances, the natural/naturalized mice including "wild", "dirty", "wildling", and "wilded" systems have been found to better reflect some aspects of human pathophysiology. Here, we discuss the pros and cons of the laboratory mouse system and contemplate how wild mice and wild microbiota are able to help in refining such systems to better mimic the real-world situation and contribute to more productive translational research.

FoxP3 scanning mutagenesis reveals functional variegation and mild mutations with atypical autoimmune phenotypes.

FoxP3+ regulatory T cells (Tregs) are a central element of immunological tolerance. FoxP3 is the key determining transcription factor of the Treg lineage, interacting with numerous cofactors and transcriptional targets to determine the many facets of Treg function. Its absence leads to devastating lymphoproliferation and autoimmunity in scurfy mutant mice and immunodysregulation polyendocrinopathy enteropathy X-linked (IPEX) patients. To finely map transcriptionally active regions of the protein, with respect to disease-causing variation, we performed a systematic alanine-scan mutagenesis of FoxP3, assessing mutational impacts on DNA binding and transcriptional activation or repression. The mutations affected transcriptional activation and repression in a variegated manner involving multiple regions of the protein and varying between different transcriptional targets of FoxP3. There appeared to be different modalities for target genes related to classic immunosuppressive function vs. those related to atypical or tissue-Treg functions. Relevance to in vivo Treg biology was established by introducing some of the subtle Foxp3 mutations into the mouse germline by CRISPR-based genome editing. The resulting mice showed Treg populations in normal numbers and exhibited no overt autoimmune manifestations. However, Treg functional defects were revealed upon competition or by system stress, manifest as a strikingly heightened susceptibility to provoked colitis, and conversely by greater resistance to tumors. These observations suggest that some of the missense mutations that segregate in human populations, but do not induce IPEX manifestations, may have unappreciated consequences in other diseases.

Correction to: Cinnamon extract induces tumor cell death through inhibition of NFκB and AP1.

Following publication of the original article [1], the authors have re-evaluated the authorship for this article. The updated author group is.

Unilateral Mastication Evaluated Using Asymmetric Functional Tooth Units as a Risk Indicator for Hearing Loss.

BACKGROUND: Some previous studies reported hearing ability can be reduced by impaired masticatory ability, but there has been little evidence reported of an association between hearing loss and unilateral mastication. Therefore, this study aimed to investigate the relationship between unilateral mastication (UM), estimated from individual functional tooth units (FTUs), and hearing loss in a representative sample of Korean adults. METHODS: The analyzed data were obtained from 1,773 adults aged 40-89 years who participated in Korean national survey. Hearing loss was defined as a pure-tone average of >25 dB at frequencies of 0.5, 1, 2, and 4 kHz in either ear. In each subject, UM was calculated as the difference in the sums of the FTU scores, which is an index of posterior tooth occlusion, on the two sides of the oral cavity. The scores were used to classify the UM into low, moderate, and high. The adjusted odds ratios (aORs) and their 95% confidence intervals (CIs) were calculated in multivariable logistic regression analyses. RESULTS: When controlling for sociodemographic factors, the aOR for hearing loss was 3.12 (95% CI, 1.21-8.03) for high UM relative to low UM. This association remained in a fully-adjusted model containing factors related to noise exposure (aOR 2.88; 95% CI, 1.12-7.46). CONCLUSION: Adults with high UM as measured using FTUs showed a higher occurrence of hearing loss than those with low UM.

Unstable FoxP3+ T regulatory cells in NZW mice.

Regulatory T (Treg) cells that express the transcription factor FoxP3 play a key role in self-tolerance and the control of inflammation. In mice and humans, there is a wide interindividual range in Treg frequency, but little is known about the underlying genetic or epigenetic mechanisms. We explored this issue in inbred strains of mice, with a special focus on the low proportion of Treg cells found in NZW mice. Mixed bone marrow chimera experiments showed this paucity to be intrinsic to NZW Treg cells, a dearth that could be tied to poor stability of the Treg pool and of FoxP3 expression. This instability was not a consequence of differential epigenetic marks, because Treg-specific CpG hypomethylation profiles at the Foxp3 locus were similar in all strains tested. It was also unrelated to the high expression of IFN signature genes in NZW, as shown by intercross to mice with an Ifnar1 knockout. NZW Tregs were less sensitive to limiting doses of trophic cytokines, IL-2 and -33, for population homeostasis and for maintenance of FoxP3 expression. Gene-expression profiles highlighted specific differences in the transcriptome of NZW Tregs compared with those of other strains, but no single defect could obviously account for the instability. Rather, NZW Tregs showed a general up-regulation of transcripts normally repressed in Treg cells, and we speculate that this network-level bias may account for NZW Treg instability.

Explaining the Red Fluorescence Evident on the Surface of Failed Dental Implants: Case Reports.

INTRODUCTION: It is important to identify the reasons for implant failure when planning future dental treatment. No studies have distinguished the factors that affect implant failure by evaluating the appearance of failed fixtures. PURPOSE: This study investigated these factors by evaluating the surface of implant fixtures using quantitative light-induced fluorescence-digital (QLF-D), which involves detecting the red fluorescence emitted by porphyrin metabolized by oral bacteria in a mature biofilm. MATERIALS AND METHODS: The areas of red fluorescence in QLF-D images obtained from all aspects of the fixture surface were then analyzed using quantitative analysis software. RESULTS: Red fluorescence was evident on the surface of implants that failed after at least 2 years of occlusal loading and exhibited severe bone loss. Implants with no fluorescence exhibited a clean surface in blue-light images, and the bone loss could not be observed radiographically. CONCLUSIONS: The cases presented that failed dental implant surfaces caused by periimplantitis can be detected by the red fluorescence evident as QLF-D.

Prevention of dental erosion of a sports drink by nano-sized hydroxyapatite in situ study.

AIM: To evaluate the inhibitory effects of the sports drink containing nano-sized hydroxyapatite (nano-HA) on dental erosion in situ. DESIGN: The study had a single-blind, two-treatment crossover design. The two treatment groups were a control group (CG; Powerade only) and an experimental group (EG; 0.25% wt/vol nano-HA was added to Powerade). Ten subjects wore removable palatal appliances containing bovine enamel specimens. The appliances were immersed in each drink for 10 mins, 4 times a day for 10 days. The tooth surface microhardness (SMH) was tested, and the erosion depth and the morphology of the tooth surface were observed. The data were analysed by repeated measures anova and t-test. RESULTS: Between the baseline and the 10th day, SMH was decreased by 80% in the specimens of the CG (P < 0.001), whereas there was only a 6% decrease in the SMH of the specimens in the EG. An erosion depth of 12.70 ± 4.66 µm and an irregular tooth surface were observed on the 10th day in the specimens of the CG. No dental erosions, however, was observed in the specimens of the EG. CONCLUSIONS: The sports drink containing 0.25% nano-HA was effective in preventing dental erosion in situ.

Interaction of Ets-1 with HDAC1 represses IL-10 expression in Th1 cells.

IL-10 is a multifunctional cytokine that plays a crucial role in immunity and tolerance. IL-10 is produced by diverse immune cell types, including B cells and subsets of T cells. Although Th1 produce IL-10, their expression levels are much lower than Th2 cells under conventional stimulation conditions. The potential role of E26 transformation-specific 1 (Ets-1) transcription factor as a negative regulator for Il10 gene expression in CD4(+) T cells has been implicated previously. In this study, we investigated the underlying mechanism of Ets-1-mediated Il10 gene repression in Th1 cells. Compared with wild type Th1 cells, Ets-1 knockout Th1 cells expressed a significantly higher level of IL-10, which is comparable with that of wild type Th2 cells. Upregulation of IL-10 expression in Ets-1 knockout Th1 cells was accompanied by enhanced chromatin accessibility and increased recruitment of histone H3 acetylation at the Il10 regulatory regions. Reciprocally, Ets-1 deficiency significantly decreased histone deacetylase 1 (HDAC1) enrichment at the Il10 regulatory regions. Treatment with trichostatin A, an inhibitor of HDAC family, significantly increased Il10 gene expression by increasing histone H3 acetylation recruitment. We further demonstrated a physical interaction between Ets-1 and HDAC1. Coexpression of Ets-1 with HDAC1 synergistically repressed IL-10 transcription activity. In summary, our data suggest that an interaction of Ets-1 with HDAC1 represses the Il10 gene expression in Th1 cells.

Alterations in Plasma Cytokine Levels in Korean Children with Autism Spectrum Disorder.

PURPOSE: Numerous studies have supported the role of the immune dysfunction in the pathogenesis of autism spectrum disorder (ASD); however, to our knowledge, no study has been conducted on plasma cytokine levels in children with ASD in South Korea. In this study, we aimed to analyze the immunological characteristics of Korean children with ASD through plasma cytokine analysis. MATERIALS AND METHODS: Blood samples were collected from 94 ASD children (mean age 7.1; 81 males and 13 females) and 48 typically developing children (TDC) (mean age 7.3; 30 males and 18 females). Plasma was isolated from 1 mL of blood by clarifying with centrifugation at 8000 rpm at 4℃ for 10 min. Cytokines in plasma were measured with LEGENDplex HU Th cytokine panel (BioLegend, 741028) and LEGENDplex HU cytokine panel 2 (BioLegend, 740102). RESULTS: Among 25 cytokines, innate immune cytokine [interleukin (IL)-33] was significantly decreased in ASD children compared with TDC. In acute phase proteins, tumor necrosis factor α (TNF-α) was significantly increased, while IL-6, another inflammation marker, was decreased in ASD children compared with TDC. The cytokines from T cell subsets, including interferon (IFN)-γ, IL-5, IL-13, and IL-17f, were significantly decreased in ASD children compared to TDC. IL-10, a major anti-inflammatory cytokine, and IL-9, which modulates immune cell growth and proliferation, were also significantly decreased in ASD children compared to TDC. CONCLUSION: We confirmed that Korean children with ASD showed altered immune function and unique cytokine expression patterns distinct from TDC.

Tacrolimus-loaded chitosan-based nanoparticles as an efficient topical therapeutic for the effective treatment of atopic dermatitis symptoms.

Atopic dermatitis (AD) is a chronic cutaneous disease with a complex underlying mechanism, and it cannot be completely cured. Thus, most treatment strategies for AD aim at relieving the symptoms. Although corticosteroids are topically applied to alleviate AD, adverse side effects frequently lead to the withdrawal of AD therapy. Tacrolimus (TAC), a calcineurin inhibitor, has been used to treat AD, but its high molecular weight and insolubility in water hinder its skin permeability. Herein, we developed and optimized TAC-loaded chitosan-based nanoparticles (TAC@CNPs) to improve the skin permeability of TAC by breaking the tight junctions in the skin. The prepared nanoparticles were highly loadable and efficient and exhibited appropriate characteristics for percutaneous drug delivery. TAC@CNP was stable for 4 weeks under physiological conditions. CNP released TAC in a controlled manner, with enhanced skin penetration observed. In vitro experiments showed that CNP was non-toxic to keratinocyte (HaCaT) cells, and TAC@CNP dispersed in an aqueous solution was as anti-proliferative as TAC solubilized in a good organic solvent. Importantly, an in vivo AD mouse model revealed that topical TAC@CNP containing ~1/10 of the dose of TAC found in commercially used Protopic® Ointment exhibited similar anti-inflammatory activity to that of the commercial product. TAC@CNP represents a potential therapeutic strategy for the management of AD.

A longitudinal molecular and cellular lung atlas of lethal SARS-CoV-2 infection in K18-hACE2 transgenic mice.

BACKGROUND: The global pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has led to approximately 500 million cases and 6 million deaths worldwide. Previous investigations into the pathophysiology of SARS-CoV-2 primarily focused on peripheral blood mononuclear cells from patients, lacking detailed mechanistic insights into the virus's impact on inflamed tissue. Existing animal models, such as hamster and ferret, do not faithfully replicate the severe SARS-CoV-2 infection seen in patients, underscoring the need for more relevant animal system-based research. METHODS: In this study, we employed single-cell RNA sequencing (scRNA-seq) with lung tissues from K18-hACE2 transgenic (TG) mice during SARS-CoV-2 infection. This approach allowed for a comprehensive examination of the molecular and cellular responses to the virus in lung tissue. FINDINGS: Upon SARS-CoV-2 infection, K18-hACE2 TG mice exhibited severe lung pathologies, including acute pneumonia, alveolar collapse, and immune cell infiltration. Through scRNA-seq, we identified 36 different types of cells dynamically orchestrating SARS-CoV-2-induced pathologies. Notably, SPP1+ macrophages in the myeloid compartment emerged as key drivers of severe lung inflammation and fibrosis in K18-hACE2 TG mice. Dynamic receptor-ligand interactions, involving various cell types such as immunological and bronchial cells, defined an enhanced TGFß signaling pathway linked to delayed tissue regeneration, severe lung injury, and fibrotic processes. INTERPRETATION: Our study provides a comprehensive understanding of SARS-CoV-2 pathogenesis in lung tissue, surpassing previous limitations in investigating inflamed tissues. The identified SPP1+ macrophages and the dysregulated TGFß signaling pathway offer potential targets for therapeutic intervention. Insights from this research may contribute to the development of innovative diagnostics and therapies for COVID-19. FUNDING: This research was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (2020M3A9I2109027, 2021R1A2C2004501).

Immune Cells Are Differentially Affected by SARS-CoV-2 Viral Loads in K18-hACE2 Mice.

Viral load and the duration of viral shedding of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are important determinants of the transmission of coronavirus disease 2019. In this study, we examined the effects of viral doses on the lung and spleen of K18-hACE2 transgenic mice by temporal histological and transcriptional analyses. Approximately, 1×105 plaque-forming units (PFU) of SARS-CoV-2 induced strong host responses in the lungs from 2 days post inoculation (dpi) which did not recover until the mice died, whereas responses to the virus were obvious at 5 days, recovering to the basal state by 14 dpi at 1×102 PFU. Further, flow cytometry showed that number of CD8+ T cells continuously increased in 1×102 PFU-virus-infected lungs from 2 dpi, but not in 1×105 PFU-virus-infected lungs. In spleens, responses to the virus were prominent from 2 dpi, and number of B cells was significantly decreased at 1×105 PFU; however, 1×102 PFU of virus induced very weak responses from 2 dpi which recovered by 10 dpi. Although the defense responses returned to normal and the mice survived, lung histology showed evidence of fibrosis, suggesting sequelae of SARS-CoV-2 infection. Our findings indicate that specific effectors of the immune response in the lung and spleen were either increased or depleted in response to doses of SARS-CoV-2. This study demonstrated that the response of local and systemic immune effectors to a viral infection varies with viral dose, which either exacerbates the severity of the infection or accelerates its elimination.

Amelioration of experimental autoimmune encephalomyelitis by probiotic mixture is mediated by a shift in T helper cell immune response.

The immunomodulatory effect of probiotics has been shown mainly in gastro-intestinal immune disorders and little information is available on the inflammation of central nervous system. Recently we reported that IRT5 probiotics, a mixture of 5 probiotics, could suppress diverse experimental inflammatory disorders. In this study, we evaluated the prophylactic and therapeutic effects of IRT5 probiotics in experimental autoimmune encephalomyelitis (EAE), a T cell mediated inflammatory autoimmune disease of the central nervous system. Pretreatment of IRT5 probiotics before disease induction significantly suppressed EAE development. In addition, treatment with IRT5 probiotics to the ongoing EAE delayed the disease onset. Administration of IRT5 probiotics inhibited the pro-inflammatory Th1/Th17 polarization, while inducing IL10(+) producing or/and Foxp3(+) regulatory T cells, both in the peripheral immune system and at the site of inflammation. Collectively, our data suggest that IRT5 probiotics could be applicable to modulate T cell mediated neuronal autoimmune diseases, including multiple sclerosis.

Stat6 and c-Jun mediate Th2 cell-specific IL-24 gene expression.

TCR signaling regulates multiple aspects of T cell function by controlling expression of various cytokine genes. IL-24 is a multifunctional cytokine belonging to the IL-10 family. It displays anticancer effects in diverse cancer cells and regulates immunopathology of psoriasis and rheumatoid arthritis. IL-24 also plays an important role in B cell differentiation. Mouse IL-24 gene is selectively expressed in activated Th2 cells upon TCR stimulation. However, the molecular mechanisms by which TCR stimulation induces IL-24 gene expression are still unclear. In this study, to elucidate the mechanism of Th2 cell-specific expression of IL-24, we identified a proximal promoter region (-157/+95 bp) that plays critical role in activating the IL-24 gene in Th2 cells. This region has a Th2 cell-specific open chromatin structure along with permissive histone modifications. In vivo binding of Stat6 and AP-1 (c-Jun) to the IL-24 promoter locus in Th2 cells synergistically transactivated the IL-24 promoter. Stat6 and c-Jun proteins were found to physically cooperate with each other and upregulated IL-24 gene transcription. Knockdown of either Stat6 or c-Jun suppressed endogenous IL-24 gene expression in Th2 cells. In summary, TCR stimulation induces IL-24 expression in Th2 cells by the coordinate action of Stat6 and c-Jun transcription factors at the transcriptional level.

Generation of regulatory dendritic cells and CD4+Foxp3+ T cells by probiotics administration suppresses immune disorders.

The beneficial effects of probiotics have been described in many diseases, but the mechanism by which they modulate the immune system is poorly understood. In this study, we identified a mixture of probiotics that up-regulates CD4(+)Foxp3(+) regulatory T cells (Tregs). Administration of the probiotics mixture induced both T-cell and B-cell hyporesponsiveness and down-regulated T helper (Th) 1, Th2, and Th17 cytokines without apoptosis induction. It also induced generation of CD4(+)Foxp3(+) Tregs from the CD4(+)CD25(-) population and increased the suppressor activity of naturally occurring CD4(+)CD25(+) Tregs. Conversion of T cells into Foxp3(+) Tregs is directly mediated by regulatory dendritic cells (rDCs) that express high levels of IL-10, TGF-beta, COX-2, and indoleamine 2,3-dioxygenase. Administration of probiotics had therapeutical effects in experimental inflammatory bowel disease, atopic dermatitis, and rheumatoid arthritis. The therapeutical effect of the probiotics is associated with enrichment of CD4(+)Foxp3(+) Tregs in the inflamed regions. Collectively, the administration of probiotics that enhance the generation of rDCs and Tregs represents an applicable treatment of inflammatory immune disorders.