Impacts of GFP-FoxP3+ regulatory T cells on lupus hallmarks differ by genetic background and type of GFP knock-in.

FoxP3 reporter mice expressing green fluorescence protein (GFP) have been used as a very convenient tool to investigate the impact of regulatory T (Treg) cells on pathogenesis in autoimmune diseases. Here, we found that GFP-FoxP3+ knock-in (KI) mice showed alterations in the production of anti-nuclear autoantibodies (ANAs) and nephritis with different extent, depending on the presence or absence of lupus susceptibility gene locus 1 (Sle1) and KI method: contrasting with B6.Sle1.fGFP-FoxP3 mice, expressing GFP via N-terminal insertion, B6.Sle1.iGFP-FoxP3, expressing GFP via bicistronic internal ribosome entry site-driven promotion, exhibited significantly lower penetrance of serum ANA, comparing to control B6.Sle1 mice. Moreover, B6.Sle1.GFP-FoxP3+ mice reduced the Sle1-induced splenomegaly and B-cell expansion independently of the KI method employed, mainly by reducing the numbers of transitional 1 (T1) B cells and CD21-CD23- B cells, including plasmablasts and plasma cells. The absolute numbers of both splenic CD4+ T cells and Treg cells from B6.Sle1.GFP-FoxP3 KI mice were significantly reduced but their proportion was not changed, compared to B6.Sle1 mice. Although the glomerular basement membranes were thickened in both B6.Sle1 and B6.Sle1.iGFP-FoxP3 mice, they were thinner in B6.Sle1.fGFP-FoxP3 mice. The latter mice expressed more nephrophilic autoantibodies and deposited more complement component 3 in glomeruli compared to B6.iGFP-FoxP3 mice. FoxP3+ Treg cells may modulate B-cell tolerance in lupus-prone B6.Sle1 mice, presumably by modulating pathogenic, nephrophilic autoantibody production and nephritis.

Taurodeoxycholate Increases the Number of Myeloid-Derived Suppressor Cells That Ameliorate Sepsis in Mice.

Bile acids (BAs) control metabolism and inflammation by interacting with several receptors. Here, we report that intravenous infusion of taurodeoxycholate (TDCA) decreases serum pro-inflammatory cytokines, normalizes hypotension, protects against renal injury, and prolongs mouse survival during sepsis. TDCA increases the number of granulocytic myeloid-derived suppressor cells (MDSCLT) distinctive from MDSCs obtained without TDCA treatment (MDSCL) in the spleen of septic mice. FACS-sorted MDSCLT cells suppress T-cell proliferation and confer protection against sepsis when adoptively transferred better than MDSCL. Proteogenomic analysis indicated that TDCA controls chromatin silencing, alternative splicing, and translation of the immune proteome of MDSCLT, which increases the expression of anti-inflammatory molecules such as oncostatin, lactoferrin and CD244. TDCA also decreases the expression of pro-inflammatory molecules such as neutrophil elastase. These findings suggest that TDCA globally edits the proteome to increase the number of MDSCLT cells and affect their immune-regulatory functions to resolve systemic inflammation during sepsis.

Diagnostic value of peripheral blood immune profiling in colorectal cancer.

PURPOSE: Little is known about the clinical value of peripheral blood immune profiling. Here, we aimed to identify colorectal cancer (CRC)-related peripheral blood immune cells and develop liquid biopsy-based immune profiling models for CRC diagnosis. METHODS: Peripheral blood from 131 preoperative patients with CRC and 174 healthy controls was analyzed by flow cytometry and automated hematology. CRC-related immune factors were identified by comparing the mean values of immune cell percentages and counts. Subsequently, CRC diagnostic algorithms were constructed using binary logistic regression. RESULTS: Significant differences were observed in percentages and counts of white blood cells, lymphocytes, neutrophils, regulatory T cells, and myeloid-derived suppressor cells (MDSCs) of patients and controls. The neutrophil/lymphocyte and Th1/Th2 ratios were also significantly different. Likewise, the percentages and counts of peripheral blood programed death 1, cytotoxic T lymphocyte antigen 4, B-and T-lymphocyte attenuator, and lymphocyte activation gene-3 were higher in patients with CRC. The binary logistic regression model included 12 variables, age, CD3+%, NK%, CD4+CD279+%, CD4+CD25+%, CD4+CD152+%, CD3+CD366+%, CD3+CD272+%, CD3+CD223+%, CD158b-CD314+CD3-CD56+%, Th2%, and MDSCs cells/µL, for the prediction of cancer. Results of retrospective and prospective evaluation of the area under the curve, sensitivity, and specificity were 0.980 and 0.940, 91.53% and 85.80%, and 93.50% and 86.20%, respectively. CONCLUSION: Peripheral blood immune profiling may be valuable in evaluating the immunity of CRC patients. Our liquid biopsy-based immune diagnostic method and its algorithms may serve as a novel tool for CRC diagnosis. Future largescale studies are needed for better characterization of its diagnostic value and potential for clinical application.

Cardiolipin activates antigen-presenting cells via TLR2-PI3K-PKN1-AKT/p38-NF-kB signaling to prime antigen-specific naïve T cells in mice.

Mitochondrial defects and antimitochondrial cardiolipin (CL) antibodies are frequently detected in autoimmune disease patients. CL from dysregulated mitochondria activates various pattern recognition receptors, such as NLRP3. However, the mechanism by which mitochondrial CL activates APCs as a damage-associated molecular pattern to prime antigen-specific naïve T cells, which is crucial for T-cell-dependent anticardiolipin IgG antibody production in autoimmune diseases is unelucidated. Here, we show that CL increases the expression of costimulatory molecules in CD11c+ APCs both in vitro and in vivo. CL activates CD11c+ APCs via TLR2-PI3K-PKN1-AKT/p38MAPK-NF-κB signaling. CD11c+ APCs that have been activated by CL are sufficient to prime H-Y peptide-specific naïve CD4+ T cells and OVA-specific naïve CD8+ T cells. TLR2 is necessary for anti-CL IgG antibody responses in vivo. Intraperitoneal injection of CL does not activate CD11c+ APCs in CD14 KO mice to the same extent as in wild-type mice. CL binds to CD14 (Kd = 7 × 10-7 M). CD14, but not MD2, plays a role in NF-kB activation by CL, suggesting that CD14+ macrophages contribute to recognizing CL. In summary, CL activates signaling pathways in CD11c+ APCs through a mechanism similar to gram (+) bacteria and plays a crucial role in priming antigen-specific naïve T cells.

Differentially Expressed Potassium Channels Are Associated with Function of Human Effector Memory CD8+ T Cells.

The voltage-gated potassium channel, Kv1.3, and the Ca2+-activated potassium channel, KCa3.1, regulate membrane potentials in T cells, thereby controlling T cell activation and cytokine production. However, little is known about the expression and function of potassium channels in human effector memory (EM) CD8+ T cells that can be further divided into functionally distinct subsets based on the expression of the interleukin (IL)-7 receptor alpha (IL-7Rα) chain. Herein, we investigated the functional expression and roles of Kv1.3 and KCa3.1 in EM CD8+ T cells that express high or low levels of the IL-7 receptor alpha chain (IL-7Rαhigh and IL-7Rαlow, respectively). In contrast to the significant activity of Kv1.3 and KCa3.1 in IL-7Rαhigh EM CD8+ T cells, IL-7Rαlow EM CD8+ T cells showed lower expression of Kv1.3 and insignificant expression of KCa3.1. Kv1.3 was involved in the modulation of cell proliferation and IL-2 production, whereas KCa3.1 affected the motility of EM CD8+ T cells. The lower motility of IL-7Rαlow EM CD8+ T cells was demonstrated using transendothelial migration and motility assays with intercellular adhesion molecule 1- and/or chemokine stromal cell-derived factor-1α-coated surfaces. Consistent with the lower migration property, IL-7Rαlow EM CD8+ T cells were found less frequently in human skin. Stimulating IL-7Rαlow EM CD8+ T cells with IL-2 or IL-15 increased their motility and recovery of KCa3.1 activity. Our findings demonstrate that Kv1.3 and KCa3.1 are differentially involved in the functions of EM CD8+ T cells. The weak expression of potassium channels in IL-7Rαlow EM CD8+ T cells can be revived by stimulation with IL-2 or IL-15, which restores the associated functions. This study suggests that IL-7Rαhigh EM CD8+ T cells with functional potassium channels may serve as a reservoir for effector CD8+ T cells during peripheral inflammation.

Modulation of gut microbiota and delayed immunosenescence as a result of syringaresinol consumption in middle-aged mice.

Age-associated immunological dysfunction (immunosenescence) is closely linked to perturbation of the gut microbiota. Here, we investigated whether syringaresinol (SYR), a polyphenolic lignan, modulates immune aging and the gut microbiota associated with this effect in middle-aged mice. Compared with age-matched control mice, SYR treatment delayed immunosenescence by enhancing the numbers of total CD3+ T cells and naïve T cells. SYR treatment induced the expression of Bim as well as activation of FOXO3 in Foxp3+ regulatory T cells (Tregs). Furthermore, SYR treatment significantly enhanced the Firmicutes/Bacteroidetes ratio compared with that in age-matched controls by increasing beneficial bacteria, Lactobacillus and Bifidobacterium, while reducing the opportunistic pathogenic genus, Akkermansia. In addition, SYR treatment reduced the serum level of lipopolysaccharide-binding protein, an inflammatory marker, and enhanced humoral immunity against influenza vaccination to the level of young control mice. Taken together, these findings suggest that SYR may rejuvenate the immune system through modulation of gut integrity and microbiota diversity as well as composition in middle-aged mice, which may delay the immunosenescence associated with aging.

The lupus susceptibility locus Sle1 facilitates the peripheral development and selection of anti-DNA B cells through impaired receptor editing.

Systemic lupus erythematosus is characterized by the spontaneous production of IgG autoantibodies in patients and lupus-prone mice. In this study, we investigated the effect of the Sle1 lupus susceptibility locus on the peripheral development of 56R(+) anti-DNA transgenic B cells by tracking 56R(+) B cells in mice without (B6.56R) or with (B6.Sle1.56R) the Sle1 locus. Compared with B6.56R mice, B6.Sle1.56R mice exhibited increased class-switched IgG2a anti-DNA Abs in their serum, encoded by the transgene. Interestingly, within the spleen, Sle1 facilitated the development of these cells into clusters of IgG2a class-switched B cells juxtaposed to CD4(+) T cells within extrafollicular sites. Through sequence analysis of B cell hybridomas, we also found that B cells from B6.Sle1.56R mice are inefficient at Ig H and L chain editing. Thus, the Ig H chains in Sle1.56R(+) B cells are partnered more often with cationic L chains that facilitate DNA binding. Taken together, these findings indicate that the Sle1 lupus-susceptibility locus may facilitate the emergence of anti-DNA B cells by subduing BCR revision and possibly by shaping the extrafollicular development of effector B cells, although the precise molecular mechanisms await further study.

Anti-nuclear antibody reactivity in lupus may be partly hard-wired into the primary B-cell repertoire.

When monoclonal ANAs and non-ANAs generated from a genetically simplified mouse model of lupus, B6.Sle1, were recently compared, the ANAs exhibited three sequence motifs in their immunoglobulin heavy chains, including increased cationicity in CDR3 ("motif A"), reduced anionicity in CDR2 ("motif B") and increased aspartate at H50 ("motif C"). The present study was designed to elucidate the extent to which these ANA-associated sequence motifs might be hard-wired into the primary B-cell repertoire in lupus. The immunoglobulin heavy chain sequence of total splenic B-cells, follicular B-cells and marginal zone B-cells from B6.Sle1 congenic mice and C57BL/6 controls were amplified by single-cell PCR and compared. Analysis of the primary immunoglobulin heavy chain repertoire indicated that the first two sequence motifs "A" and "B" were already encoded in the naïve repertoire of B6.Sle1(z) mice, whereas the third motif "C" was introduced in part by somatic mutation. Site-directed mutagenesis confirmed that non-anionic CDR2 and cationic CDR3 residues in the immunoglobulin heavy chain facilitated nuclear antigen binding in concert, whereas aspartate at H50 strongly vetoed DNA-binding, while preserving nucleosome reactivity. Hence, anti-nuclear antibodies appear to arise as a consequence of two distinct processes-genetically programmed selection of specific CDR charge motifs into the primary immunoglobulin repertoire, with secondary contribution from somatic mutation. Polymorphisms in the lupus susceptibility gene Ly108 that impair central B-cell tolerance may be mechanistically responsible for these early repertoire differences in lupus.