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BACKGROUND Integrin α4β7 mediates lymphocyte trafficking to the intestinal lamina propria (LP), a process central to gut immune homeostasis. Therapeutic blockade of this pathway by vedolizumab, a monoclonal antibody against α4β7, has proven effective in the treatment of inflammatory bowel disease (IBD). We have shown that IgA-producing B cells rely on β7-dependent homing to migrate to LP, where they mature to professional IgA-producing resident plasma cells to maintain barrier integrity and regulate the microbiota. In previous work, we demonstrated the critical role of the α4β7-MAdCAM-1 pathway for homeostasis between the host and its microbiota during chronic colitis in the IL-10-/- model. Given that homing determinants to the ileum and colon may be only partially overlapping (e.g., CCR9), we investigated how α4β7 contributes to B-cell gut homing, ileitis severity and ileal microbial communities in a model of chronic ileitis (ie, TNFΔARE model). METHODS We generated β7 deficient TNFΔARE mice (TNFΔARE+/- Itgb7-/-) and compared ileitis severity and disease progression with that of β7-sufficient TNFΔARE controls (TNFΔARE+/- Itgb7+/+) using a semi-quantitative histological scoring system. Ileal memory B cells and IgA+ plasma cells were isolated from the LP and quantified by flow cytometry. Fecal IgA levels were determined by ELISA. Stool microbial composition was profiled by 16S rRNA sequencing, and downstream bioinformatic analyses performed in R of TNFΔARE mice, TNFΔARE+/- Itgb7-/- mice compared with that of TNFΔARE+/- IgA-/- mice. RESULTS TNFΔARE+/- Itgb7-/- mice developed worse ileitis across the disease time course compared with TNFΔARE+/- Itgb7+/+ controls (n = 20, p < 0.05). This was accompanied by a marked reduction in ileal lamina propria B cells (n = 10, p < 0.001), particularly IgA+ plasma cells (n = 11, p < 0.05). Consistently, fecal IgA levels were significantly decreased (n = 14, p < 0.0001). Metagenomic analysis revealed distinct alterations in community composition, including separation of groups in principal coordinate analysis (PCoA; n = 12) in which the β7- and IgA-deficient clustered most closely. CONCLUSIONS Itgb7-deficit results in worse ileitis, decreased LP IgA+ antibody secreting cells, (ASC) lower luminal IgA and shifts in microbial communities in stool of TNFΔARE+/- Itgb7-/- mice that recapitulate those of IgA deficiency. Thus, α4β7-MAdCAM-1 interactions are required for ASC homing to ilea, maintenance of luminal IgA and homeostasis between the microbiota and its host.

Background/Aims Crohn’s disease involvement may be restricted to the terminal ileum (ileitis), colon (colitis) or both (ileocolitis). Mutations in the NOD2 gene, and development of antibacterial antibodies are more prevalent in ileal CD suggesting that ileitis may represent a distinct disease entity. The determinants of this regional distribution are not known but are likely bacterial in origin. MDR1a-/- housed at our facility develop not only colitis but also ileitis. However, the colony from which they are derived at Taconic does not develop ileitis or colitis. Here, we characterize the time course of ileocolitis in MDR1a-/- mice at UCSD and examine the effect of cohousing with non-colitic MDR1α−/− mice from Taconic on ileocolitis severity and ileal and colonic microbial communities. METHODS The severity of ileal and colonic histopathological involvement was analyzed at 5, 12, 20, and >30 weeks old by a pathologist in a blinded fashion. Relevant cellular subsets of mesenteric lymph nodes (MLN) in inflamed and non-inflamed mice were examined via mass cytometry. Mice originating from colonies with and without colitis were cohoused, after which their ilea and colon were harvested for histopathologic evaluation. Metagenomic analyses (shotgun) were performed in ileum contents and stool before and after cohousing. RESULTS Mice develop ileitis between 5 and 12 weeks (n > 7 for all time points, p < 0.01), and its severity remains relatively stable afterwards. Colitis was first observed at 12-weeks-of age, remained stable through 20 weeks and worsened in 30-week-old mice (p < 0.01). Cellular composition of MLN showed expansion of central memory (TCM (CD44+/CD62L+) CD8+ T cells) in inflamed mice from our colony compared with non-colitic mice from Taconic (n = 4, p < 0.005). Cohousing experiments showed attenuation of ileitis in ileocolitic mice (UCSD colony, n = 4, p < 0.05), when co-housed with non-colitic mice from the Taconic colony, which were not affected. Cohousing did not trigger ileitis or colitis in mice from Taconic. After cohousing, Taconic mice had a loss of verrucomicrobiales and increased campylobacterales, whereas in UCSD mice, desulfovibrionales increased along with overall diversity and lactobacillales decreased. Evaluation of the ileal microbiota is ongoing. CONCLUSIONS MDR1α -/- mice at our vivarium develop not only colitis but also ileitis (ileocolitis) between 5 and 12 weeks-of age with expansion of TCM in their MLN. Cohousing of ileocolitic mice (UCSD) with non-inflamed mice from Taconic carrying an identical MDR1α mutation led to attenuation of ileitis but not colitis. Thus, mice at Taconic appear to have transmissible beneficial (anti-inflammatory) elements in their microbiota able to attenuate ileitis in their ileocolitic MDR1α -/- counterparts at our facility.

Abstract Background Sphingosine-1-phospate (S1P) receptor agonists (eg, ozanimod) desensitize migrating lymphocytes by irreversibly binding to S1P receptors (S1PR) and triggering their proteasomal degradation. Desensitized lymphocytes cannot sense S1P, therefore, halting lymphocyte recirculation. The S1P lyase (SPL) irreversibly degrades S1P and its inhibition disrupts the S1P gradient. We previously found that systemic SPL inhibitors induce central immunosuppression. Here, we examined whether SPL inhibition may attenuate colitis without systemic immunotoxicity. Methods We first analyzed SPL expression and localization in mice using qRT-PCR and immunohistochemistry. SPL inhibitors 4-deoxypyridoxine hydrochloride (DOP) and 2-acetyl-4-(tetrahydroxybutyl) imidazole (THI) were used to inhibit SPL systemically, whereas a conditional intestinal epithelial cell (IEC)-specific SPL-deficient mouse was used to evaluate the effects of IEC-specific SPL inhibition on survival, disease activity, histological severity of dextran sulfate sodium-induced colitis, S1P levels, and intestinal permeability. Results Sgpl1 mRNA transcripts and protein were ubiquitously expressed in gastrointestinal (GI) tract leukocytes and IEC. Systemic SPL inhibitors did not induce colitis by themselves but depleted CD4+ and CD8+ T cells from blood. However, contrary to its therapeutic effects on ileitis, systemic inhibition reduced survival, accelerated weight loss, worsened histopathological inflammation indices, and tissue damage. We then examined the effects of IEC-specific inhibition on peripheral cell counts and severity of colitis. We found that while it spared peripheral immunity, it similarly hastened colitis. Finally, we examined whether colitis acceleration was due to epithelial barrier compromise after disruption of the S1P gradient. We found that not only systemic but also IEC-specific SPL inhibition increased local S1P levels and led to IEC barrier compromise. Conclusion Homeostatic intestinal S1P levels are critical for the regulation of IEC barrier function. Further studies using adaptive immunity-based inflammatory bowel diseases (IBD) models are required to assess the translational value of IEC-specific SPL inhibition as a therapeutic target for human IBD. Lay Summary Inhibition of the Sphingosine-1-phospate (S1P) lyase systemically and specifically in intestinal epithelium increased colonic S1P levels and intestinal permeability, which aggravated dextran sulfate sodium-induced acute colitis. Thus, homeostatic S1P levels are required for normal intestinal epithelial barrier function.

Abstract Background Tumor necrosis factor (TNF) is a pleiotropic cytokine that plays a critical role in the pathogenesis of immune-mediated diseases including inflammatory bowel disease (IBD). The stability of its mRNA transcript, determined in part by destabilizing sequences in its AAUU repeats (ARE) gene region, is an important regulator of its tissue and systemic levels. A deletion in the ARE region of the gene resulted in IBD and arthritis in mice and pigs, supporting a critical role for the cytokine in human IBD and several human arthritides. A mutation in the same area of the mouse genome by Genentech scientists (T.Y., M.K.) resulted in a similar but not identical phenotype. Methods Here, we compare histopathological, cellular, and molecular features of the strains and propose reasons for their distinct phenotypes. First, while homozygous TNFΔARE mice develop severe arthritis and die after weaning, homozygous Genentech TNFΔARE (ΔG/ΔG) mice have normal lifespans, and males are often fertile. Results We found that while the ileitic phenotype had peaked at 12 weeks of age in all mice, colitis progressed mostly after 20 weeks of age in heterozygous mice. Their variably penetrant arthritic phenotype progressed mostly after 20 weeks, also in heterozygous mice from both strains. There was expansion of central memory T and B cells in lymphoid organs of TNF-overproducing strains and their transcriptional profile shared well-known pathogenetic pathways with human IBD. Finally, we found differences in the mutated sequences within the ARE regions of the TNF gene and in their microbiota composition and genetic background. These differences likely explain their phenotypic differences. Conclusions In summary, we describe a different strain of TNF-overproducing mice with an overlapping, yet not identical phenotype, which may have differential applications than the original strain.

Background Fishes are the one of the most diverse groups of animals with respect to their modes of sex determination, providing unique models for uncovering the evolutionary and molecular mechanisms underlying sex determination and reversal. Here, we have investigated how sex is determined in a species of both commercial and ecological importance, the Siamese fighting fish Betta splendens . Results We conducted association mapping on four commercial and two wild populations of B. splendens . In three of the four commercial populations, the master sex determining (MSD) locus was found to be located in a region of ~ 80 kb on LG2 which harbours five protein coding genes, including dmrt1 , a gene involved in male sex determination in different animal taxa. In these fish, dmrt1 shows a male-biased gonadal expression from undifferentiated stages to adult organs and the knockout of this gene resulted in ovarian development in XY genotypes. Genome sequencing of XX and YY genotypes identified a transposon, drbx1 , inserted into the fourth intron of the X-linked dmrt1 allele. Methylation assays revealed that epigenetic changes induced by drbx1 spread out to the promoter region of dmrt1 . In addition, drbx1 being inserted between two closely linked cis -regulatory elements reduced their enhancer activities. Thus, epigenetic changes, induced by drbx1 , contribute to the reduced expression of the X-linked dmrt1 allele, leading to female development. This represents a previously undescribed solution in animals relying on dmrt1 function for sex determination. Differentiation between the X and Y chromosomes is limited to a small region of ~ 200 kb surrounding the MSD gene. Recombination suppression spread slightly out of the SD locus. However, this mechanism was not found in the fourth commercial stock we studied, or in the two wild populations analysed, suggesting that it originated recently during domestication. Conclusions Taken together, our data provide novel insights into the role of epigenetic regulation of dmrt1 in sex determination and turnover of SD systems and suggest that fighting fish are a suitable model to study the initial stages of sex chromosome evolution.

Since severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2)-specific T cells have been found to play essential roles in host immune protection and pathology in patients with coronavirus disease 2019 (COVID-19), this study focused on the functional validation of T cell epitopes and the development of vaccines that induce specific T cell responses. A total of 120 CD8+ T cell epitopes from the E, M, N, S, and RdRp proteins were functionally validated. Among these, 110, 15, 6, 14, and 12 epitopes were highly homologous with SARS-CoV, OC43, NL63, HKU1, and 229E, respectively; in addition, four epitopes from the S protein displayed one amino acid that was distinct from the current SARS-CoV-2 variants. Then, 31 epitopes restricted by the HLA-A2 molecule were used to generate peptide cocktail vaccines in combination with Poly(I:C), R848 or poly (lactic-co-glycolic acid) nanoparticles, and these vaccines elicited robust and specific CD8+ T cell responses in HLA-A2/DR1 transgenic mice as well as wild-type mice. In contrast to previous research, this study established a modified DC-peptide-PBL cell coculture system using healthy donor PBMCs to validate the in silico predicted epitopes, provided an epitope library restricted by nine of the most prevalent HLA-A allotypes covering broad Asian populations, and identified the HLA-A restrictions of these validated epitopes using competitive peptide binding experiments with HMy2.CIR cell lines expressing the indicated HLA-A allotype, which initially confirmed the in vivo feasibility of 9- or 10-mer peptide cocktail vaccines against SARS-CoV-2. These data will facilitate the design and development of vaccines that induce antiviral CD8+ T cell responses in COVID-19 patients.

Alzheimer's disease (AD) and Osteoarthritis (OA) have been shown to have a close association in previous studies, but the pathogenesis of both diseases are unclear. This study explores the potential common molecular mechanisms between AD and OA through bioinformatics analysis, providing new insights for clinical treatment strategies. The AD and OA-related datasets were downloaded from the gene expression database GEO. The datasets were analyzed to obtain differentially expressed gene (DEG) datasets for OA and AD, respectively. The intersection of these DEGs was analyzed to identify common DEGs (Co-DEGs). Subsequently, the Co-DEGs were enriched, and a protein-protein interaction network was constructed to identify core genes. The expression of these genes was validated in a separate dataset, and their diagnostic value for the diseases was analyzed. In addition, the core genes were analyzed using gene set enrichment analysis and single-gene genome variation analysis. Analysis of DEGs on gene chips from OA and AD patients revealed significant changes in gene expression patterns. Notably, EFEMP2 and TSPO, genes associated with inflammatory responses, showed lower expression levels in both AD and OA patients, suggesting a downregulation in the pathological backgrounds of these diseases. Additionally, GABARAPL1, which is crucial for the maturation of autophagosomes, was found to be upregulated in both conditions. These findings suggest the potential of these genes as diagnostic biomarkers and potential therapeutic targets. However, to confirm the effectiveness of these genes as therapeutic targets, more in-depth mechanistic studies are needed in the future, particularly to explore the feasibility and specific mechanisms of combating disease progression by regulating the expression of these genes. This study suggests that AD and OA shares common molecular mechanisms. The identification of EFEMP2, GABARAPL1, and TSPO as key target genes highlights potential common factors in both diseases. Further investigation into these findings could lead to new candidate targets and treatment directions for AD and OA, offering promising avenues for developing more effective and targeted therapeutic interventions.

Background The clinical routine test of HBV-specific T cell reactivity is still limited due to the high polymorphisms of human leukocyte antigens (HLA) in patient cohort and the lack of universal detection kit, thus the clinical implication remains disputed. Methods A broad-spectrum peptide library, which consists of 103 functionally validated CD8 + T-cell epitopes spanning overall HBsAg, HBeAg, HBx and HBpol proteins and fits to the HLA polymorphisms of Chinese and Northeast Asian populations, was grouped into eight peptide pools and was used to establish an ELISpot assay for enumerating the reactive HBV-specific T cells in PBMCs. Totally 294 HBV-infected patients including 203 ones with chronic hepatitis B (CHB), 13 ones in acute resolved stage (R), 52 ones with liver cirrhosis (LC) and 26 ones with hepatocellular carcinoma (HCC) were detected, and 33 CHB patients were longitudinally monitored for 3 times with an interval of 3–5 months. Results The numbers of reactive HBV-specific T cells were significantly correlated with ALT level, HBsAg level, and disease stage (R, CHB, LC and HCC), and R patients displayed the strongest HBV-specific T cell reactivity while CHB patients showed the weakest one. For 203 CHB patients, the numbers of reactive HBV-specific T cells presented a significantly declined trend when the serum viral DNA load, HBsAg, HBeAg or ALT level gradually increased, but only a very low negative correlation coefficient was defined (r = − 0.21, − 0.21, − 0.27, − 0.079, respectively). Different Nucleotide Analogs (NUCs) did not bring difference on HBV-specific T cell reactivity in the same duration of treatment. NUCs/pegIFN-α combination led to much more reactive HBV-specific T cells than NUCs monotherapy. The dynamic numbers of reactive HBV-specific T cells were obviously increasing in most CHB patients undergoing routine treatment, and the longitudinal trend possess a high predictive power for the hepatitis progression 6 or 12 months later. Conclusion The presented method could be developed into an efficient reference method for the clinical evaluation of cellular immunity. The CHB patients presenting low reactivity of HBV-specific T cells have a worse prognosis for hepatitis progression and should be treated using pegIFN-α to improve host T-cell immunity.

Although host T cell immune responses to hepatitis B virus (HBV) have been demonstrated to have important influences on the outcome of HBV infection, the development of T cell epitope-based vaccine and T cell therapy and the clinical evaluation of specific T cell function are currently hampered markedly by the lack of validated HBV T cell epitopes covering broad patients. This study aimed to screen T cell epitopes spanning overall HBsAg, HBeAg, HBx and HBpol proteins and presenting by thirteen prevalent human leukocyte antigen (HLA)-A allotypes which gather a total gene frequency of around 95% in China and Northeast Asia populations. 187 epitopes were in silico predicted. Of which, 62 epitopes were then functionally validated as real-world HBV T cell epitopes by ex vivo IFN-γ ELISPOT assay and in vitro co-cultures using peripheral blood mononuclear cells (PBMCs) from HBV infected patients. Furthermore, the HLA-A cross-restrictions of each epitope were identified by peptide competitive binding assay using transfected HMy2.CIR cell lines, and by HLA-A/peptide docking as well as molecular dynamic simulation. Finally, a peptide library containing 105 validated epitopes which cross-binding by 13 prevalent HLA-A allotypes were used in ELISPOT assay to enumerate HBV-specific T cells for 116 patients with HBV infection. The spot forming units (SFUs) was significantly correlated with serum HBsAg level as confirmed by multivariate linear regression analysis. This study functionally validated 62 T cell epitopes from HBV main proteins and elucidated their HLA-A restrictions and provided an alternative ELISPOT assay using validated epitope peptides rather than conventional overlapping peptides for the clinical evaluation of HBV-specific T cell responses.

Hepatocellular carcinoma (HCC) is a malignant tumor with high mortality, but lacks effective treatments. Carcinoembryonic antigen glypican-3 (GPC3) is a tumor-associated antigen overexpressed in HCC but rarely expressed in healthy individuals and thus is one of the most promising therapeutic targets. T cell epitope-based vaccines may bring light to HCC patients, especially to the patients at a late stage. However, few epitopes from GPC3 were identified to date, which limited the application of GPC3-derived epitopes in immunotherapy and T cell function detection. In this study, a total of 25 HLA-A0201 restricted GPC3 epitopes were in silico predicted and selected as candidate epitopes. Then, HLA-A0201+/GPC3+ HCC patients’ PBMCs were collected and co-stimulated with the candidate epitope peptides in ex vivo IFN-γ Elispot assay, by which five epitopes were identified as real-world epitopes. Their capacity to elicit specific CD8+ T cells activation and proliferation was further confirmed by in vitro co-cultures of patients’ PBMCs with peptide, in vitro co-cultures of healthy donors’ PBLs with DCs and peptide, T2 cell binding assay as well as HLA-A2 molecule stability assay. Moreover, the in vivo immunogenicity of the five validated epitopes was confirmed by peptides cocktail/poly(I:C) vaccination in HLA-A0201/DR1 transgenic mice. Robust epitope-specific CD8+ T cell responses and cytotoxicity targeting HepG2 cells were observed as detected by IFN-γ Elispot, intracellular IFN-γ staining and cytolysis assay. This study provided novel GPC3 CTL epitopes for the development of T cell epitope vaccines and evaluation of GPC3 specific T cell responses.