Threonine phosphorylation of STAT1 restricts interferon signaling and promotes innate inflammatory responses.

Since its discovery over three decades ago, signal transducer and activator of transcription 1 (STAT1) has been extensively studied as a central mediator for interferons (IFNs) signaling and antiviral defense. Here, using genetic and biochemical assays, we unveil Thr748 as a conserved IFN-independent phosphorylation switch in Stat1, which restricts IFN signaling and promotes innate inflammatory responses following the recognition of the bacterial-derived toxin lipopolysaccharide (LPS). Genetically engineered mice expressing phospho-deficient threonine748-to-alanine (T748A) mutant Stat1 are resistant to LPS-induced lethality. Of note, T748A mice exhibited undisturbed IFN signaling, as well as total expression of Stat1. Further, the T748A point mutation of Stat1 recapitulates the safeguard effect of the genetic ablation of Stat1 following LPS-induced lethality, indicating that the Thr748 phosphorylation contributes inflammatory functionalities of Stat1. Mechanistically, LPS-induced Toll-like receptor 4 endocytosis activates a cell-intrinsic IκB kinase-mediated Thr748 phosphorylation of Stat1, which promotes macrophage inflammatory response while restricting the IFN and anti-inflammatory responses. Depletion of macrophages restores the sensitivity of the T748A mice to LPS-induced lethality. Together, our study indicates a phosphorylation-dependent modular functionality of Stat1 in innate immune responses: IFN phospho-tyrosine dependent and inflammatory phospho-threonine dependent. Better understanding of the Thr748 phosphorylation of Stat1 may uncover advanced pharmacologically targetable molecules and offer better treatment modalities for sepsis, a disease that claims millions of lives annually.

Celastrol suppresses humoral immune responses and autoimmunity by targeting the COMMD3/8 complex.

Celastrol, a bioactive molecule extracted from the Tripterygium wilfordii plant, has been shown to exhibit anti-inflammatory properties. However, its mechanism of action has not been fully elucidated. Here, we show that celastrol suppresses humoral immune responses and autoimmunity by disabling a protein complex consisting of copper metabolism MURR1 domain-containing (COMMD) 3 and COMMD8 (COMMD3/8 complex), a signaling adaptor for chemoattractant receptors. Having demonstrated the involvement of the COMMD3/8 complex in a mouse model of rheumatoid arthritis, we identified celastrol as a compound that covalently bound to and dissociated the COMMD3/8 complex. Celastrol inhibited B cell migration, reduced antibody responses, and blocked arthritis progression, recapitulating deficiency of the COMMD3/8 complex. These effects of celastrol were abolished in mice expressing a celastrol-resistant mutant of the COMMD3/8 complex. These findings establish that celastrol exerts immunosuppressive activity by targeting the COMMD3/8 complex. Our study suggests that the COMMD3/8 complex is a potentially druggable target in autoimmune diseases and points to celastrol as a lead pharmacologic candidate in this capacity.

A sex-biased imbalance between Tfr, Tph, and atypical B cells determines antibody responses in COVID-19 patients.

Sex-biased humoral immune responses to COVID-19 patients have been observed, but the cellular basis for this is not understood. Using single-cell proteomics by mass cytometry, we find disrupted regulation of humoral immunity in COVID-19 patients, with a sex-biased loss of circulating follicular regulatory T cells (cTfr) at a significantly greater rate in male patients. In addition, a male sex-associated cellular network of T-peripheral helper, plasma blasts, proliferating and extrafollicular/atypical CD11c+ memory B cells was strongly positively correlated with neutralizing antibody concentrations and negatively correlated with cTfr frequency. These results suggest that sex-specific differences to the balance of cTfr and a network of extrafollicular antibody production-associated cell types may be a key factor in the altered humoral immune responses between male and female COVID-19 patients.

Isolation and Characterization of Both Human and Mouse Tfh/Tfr Cells.

Regulatory T cells (Tregs) expressing the transcription factor Foxp3 have a critical role for the control of immune homeostasis. The Treg subgroup T follicular regulatory cells (Tfr) have a specialized function to travel to the B cell follicle and control antibody responses. While Tfr may be identified by their protein or gene expression profiles, the use of in vitro functional assays to determine their suppressive capacity is important to further characterize these cells. Here we present methods for the identification and purification of Tfr from both mice and humans followed by co-culture with B cells and T follicular helper cells (Tfh). The suppressive activity of the Tfr is then assessed by the ability to prevent Tfh-dependent B cell class switching and plasma blast formation measured by flow cytometry and immunoglobulin production in culture supernatants measured by enzyme-linked immunosorbent assay. These assays will also provide in-depth characterization of the functional suppressive capacity of any isolated Tfr or Treg population. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Isolation of murine T follicular regulatory cells Basic Protocol 2: Measurement of murine T follicular regulatory cell suppressive function Basic Protocol 3: Isolation of human T follicular regulatory cells Basic Protocol 4: Measurement of human T follicular regulatory cell suppressive function.

Probiotics Prevents Sensitization to Oral Antigen and Subsequent Increases in Intestinal Tight Junction Permeability in Juvenile-Young Adult Rats.

Increased intestinal permeability is thought to underlie the pathogenesis of food allergy. We explore the mechanism responsible for changes in the morphology and function of the intestinal barrier using a rat model of food allergy, focusing on the contribution of intestinal microbiota. Juvenile-young adult rats were sensitized with ovalbumin and treated with antibiotics or probiotics (Clostridium butyricum and Lactobacillus reuteri), respectively. The serum ovalbumin-IgE levels, intestinal permeability, histopathological features, tight junction (TJ)-associated proteins, Th2 cytokines, and gut microbiota in feces were analyzed in each group. Sensitized rats showed an increase in ovalbumin-IgE levels and intestinal permeability with gut mucosal inflammation, whereas rats that received probiotics were only mildly affected. Rats given ovalbumin, but not those given probiotics, showed a reduction in both TJ-related protein expression and localization. Th2 cytokine levels were increased in the sensitized rats, but not in those given probiotics. TJs in rats treated with ovalbumin and antibiotics were disrupted, but those in rats administered probiotics were undamaged. Clostridiaceae were increased in the probiotics groups, especially Alkaliphilus, relative to the ovalbumin-sensitized group. Gut microbiota appears to play a role in regulating epithelial barrier function, and probiotics may help to prevent food sensitization through the up-regulation of TJ proteins.

Aggregate formation analysis of GFAPR416W found in one case of Alexander disease.

Alexander disease (AxD) is a neurodegenerative disease in astrocytes caused by a mutation in the gene encoding glial fibrillary acidic protein, GFAP. We herein present the case of a 12-year-old girl who showed intermittent exotropia at 3 years of age and central precocious puberty at 7 years of age. The periventricular and medulla oblongata showed high signal intensity on T2-weighted magnetic resonance imaging. The patient was diagnosed with AxD after direct sequencing revealing a de novo recurrent mutation, c.1246C>T (p.R416W) in GFAP. The transient expression of GFAPR416W in cells resulted in the significant formation of aggregates, which recapitulated the hallmark of AxD. We firstly utilized In Cell analyzer to prove the tendency of aggregate formation by mutants of GFAP.

A case of severe Alexander disease with de novo c. 239 T > C, p.(F80S), in GFAP.

Alexander disease (AxD) is a progressive neurodegenerative disease caused by a mutation in the glial fibrillary acid protein (GFAP) gene. A 4-year-old boy presented several times with hemiclonic seizures with eye deviation for a few minutes at 28 days after birth. Electroencephalogram showed independent sharp waves in the right and left temporal area. Magnetic resonance imaging showed high intensity T1-weighted images in the white matter of the frontal lobe and basal ganglia. He showed no head control at 4 years of age, and his weight gain was insufficient. He did not show macrocephaly. At 4 years of age, he died of bacterial pneumonia and septic shock. He was diagnosed with AxD, and direct sequencing revealed a de novo known mutation, c. 239 T > C, p.(F80S), in GFAP. Hela and U2-OS cells transfected with GFAP cDNA with c. 239 T > C showed dot-like cytoplasmic aggregation, similar to R239C, a common mutation found in severe infantile AxD. Aggregation in the cytoplasm caused by a GFAP mutation is a hallmark of AxD. Although there is only one previous report of a patient with an F80S mutation, our data support that F80S can cause the severe, infantile form of AxD.

Failure of Fecal Microbiota Transplantation in a Three-Year-Old Child with Severe Refractory Ulcerative Colitis.

Fecal microbiota transplantation (FMT) is a treatment designed to correct gut dysbiosis by administration of feces from a healthy volunteer. It is still unclear whether FMT for children with ulcerative colitis (UC) is effective or hazardous. Here we describe a young patient to have received FMT for UC. A three-year-old girl was admitted to our hospital with severe active UC, and treated with aminosalicylates and various immunosuppressive drugs. As remission was not achieved, we decided to try FMT before colectomy. We administered donor fecal material a total of six times by retention enema (×2) and via a nasoduodenal tube (×4) within 10 days. The patient developed abdominal pain and pyrexia after each FMT session. Analyses revealed the transferred donor fecal microbiota had not been retained by the patient, who ultimately underwent colectomy. The severity of the UC and/or timing of FMT may have partly accounted for the poor outcome.

Mesalazine allergy in a boy with ulcerative colitis: clinical usefulness of mucosal biopsy criteria.

5-Aminosalicylic acid preparations have been used as first-line drugs for treatment of ulcerative colitis (UC). However, some patients with UC present with exacerbation of symptoms because of allergy to mesalazine. Diagnosis of mesalazine allergy in active UC may be challenging because its symptoms mimic those of UC. Here we describe a 13-year-old boy with mesalazine allergy who achieved remission when his medication was changed from mesalazine to salazosulfapyridine. During his clinical course mesalazine was prescribed twice, and on each occasion exacerbation of the symptoms occurred. We considered a diagnosis of mesalazine allergy, and this was confirmed by a drug lymphocyte stimulation test; the result for salazosulfapyridine was negative. On the basis of criteria involving simple mucosal biopsy combined with endoscopy for predicting patients with UC who would ultimately require surgery, we considered that the UC in this case might be susceptible to steroid treatment, and we therefore treated the patient with salazosulfapyridine and prednisolone. Shortly afterwards, remission was achieved and the patient has remained in good condition on salazosulfapyridine alone. When treating patients with mesalazine, the possibility of allergy should always be borne in mind, especially when the clinical course is inconsistent with the results of biopsy.