Salvianolic acid B alleviates autoimmunity in Treg-deficient mice via inhibiting IL2-STAT5 signaling.

Regulatory T cell (Treg) deficiency leads to immune dysregulation, polyendocrinopathy, enteropathy, and X-linked (IPEX) syndrome, which is a CD4+ T cell-driven autoimmune disease in both humans and mice. Despite understanding the molecular and cellular characteristics of IPEX syndrome, new treatment options have remained elusive. Here, we hypothesized that salvianolic acid B (Sal B), one of the main active ingredients of Salvia miltiorrhiza, can protect against immune disorders induced by Treg deficiency. To examine whether Sal B can inhibit Treg deficiency-induced autoimmunity, Treg-deficient scurfy (SF) mice with a mutation in forkhead box protein 3 were treated with different doses of Sal B. Immune cells, inflammatory cell infiltration, and cytokines were evaluated by flow cytometry, hematoxylin and eosin staining and enzyme-linked immunosorbent assay Kits, respectively. Moreover, RNA sequencing, western blot, and real-time PCR were adopted to investigate the molecular mechanisms of action of Sal B. Sal B prolonged lifespan and reduced inflammation in the liver and lung of SF mice. Moreover, Sal B decreased plasma levels of several inflammatory cytokines, such as IL-2, IFN-γ, IL-4, TNF-α, and IL-6, in SF mice. By analyzing the transcriptomics of livers, we determined the signaling pathways, especially the IL-2-signal transducer and activator of transcription 5 (STAT5) signaling pathway, which were associated with Treg deficiency-induced autoimmunity. Remarkably, Sal B reversed the expression of gene signatures related to the IL-2-STAT5 signaling pathway in vitro and in vivo. Sal B prolongs survival and inhibits lethal inflammation in SF mice through the IL-2-STAT5 axis. Our findings may inspire novel drug discovery efforts aimed at treating IPEX syndrome.

Foxp3+ T Regulatory Cells as a Potential Target for Immunotherapy against Primary Infection with Echinococcus multilocularis Eggs.

Alveolar echinococcosis (AE) is a lethal disease caused by infection with the metacestode stage of the helminth Echinococcus multilocularis, which develops into a tumorlike mass in susceptible intermediate hosts. The growth potential of this parasite stage is directly linked to the nature of the surrounding periparasitic immune-mediated processes. In a first step (experiment 1), mice were orally infected with E. multilocularis eggs, to be used for assessing the hepatic expression profiles of 15 selected cytokine and chemokine genes related to acquired immunity from 21 to 120 days postinfection. The early stage of infection in immunocompetent animals was marked by a mixed Th1/Th2 immune response, as characterized by the concomitant presence of gamma interferon (IFN-γ) and interleukin-4 (IL-4) and their related chemokines. At the late stage of AE, the profile extended to a combined tolerogenic mode including Foxp3, IL-10, and transforming growth factor beta (TGF-ß) as key components. In a second step (experiment 2), the effect of T regulatory cell (Treg) deficiency on metacestode growth was assessed in E. multilocularis-infected DEREG (depletion of regulatory T cells) mice upon induction of Treg deficiency with diphtheria toxin (DT). The parasite lesions were significantly smaller in the livers of treated mice than in corresponding control groups. Foxp3+ Tregs appear to be one of the key players in immune-regulatory processes favoring metacestode survival by affecting antigen presentation and suppressing Th1-type immune responses. For these reasons, we suggest that affecting Foxp3+ Tregs could offer an attractive target in the development of an immunotherapy against AE.

Probiotic-Derived Ecto-5'-Nucleotidase Produces Anti-Inflammatory Adenosine Metabolites in Treg-Deficient Scurfy Mice.

Probiotic Limosilactobacillus reuteri DSM 17938 (DSM 17938) prolongs the survival of Treg-deficient scurfy (SF) mice and reduces multiorgan inflammation by a process requiring adenosine receptor 2A (A2A) on T cells. We hypothesized that L. reuteri-derived ecto-5'-nucleotidase (ecto-5'NT) activity acts to generate adenosine, which may be a central mediator for L. reuteri protection in SF mice. We evaluated DSM 17938-5'NT activity and the associated adenosine and inosine levels in plasma, gut, and liver of SF mice. We examined orally fed DSM 17938, DSM 17938Δ5NT (with a deleted 5'NT gene), and DSM 32846 (BG-R46) (a naturally selected strain derived from DSM 17938). Results showed that DSM 17938 and BG-R46 produced adenosine while "exhausting" AMP, whereas DSM 17938∆5NT did not generate adenosine in culture. Plasma 5'NT activity was increased by DSM 17938 or BG-R46, but not by DSM 17938Δ5NT in SF mice. BG-R46 increased both adenosine and inosine levels in the cecum of SF mice. DSM 17938 increased adenosine levels, whereas BG-R46 increased inosine levels in the liver. DSM 17938Δ5NT did not significantly change the levels of adenosine or inosine in the GI tract or the liver of SF mice. Although regulatory CD73+CD8+ T cells were decreased in spleen and blood of SF mice, these regulatory T cells could be increased by orally feeding DSM 17938 or BG-R46, but not DSM 17938Δ5NT. In conclusion, probiotic-5'NT may be a central mediator of DSM 17938 protection against autoimmunity. Optimal 5'NT activity from various probiotic strains could be beneficial in treating Treg-associated immune disorders in humans.

An Antibiotic-Impacted Microbiota Compromises the Development of Colonic Regulatory T Cells and Predisposes to Dysregulated Immune Responses.

Antibiotic exposure early in life and other practices impacting the vertical transmission and ordered assembly of a diverse and balanced gut microbiota are associated with a higher risk of immunological and metabolic disorders such as asthma and allergy, autoimmunity, obesity, and susceptibility to opportunistic infections. In this study, we used a model of perinatal exposure to the broad-spectrum antibiotic ampicillin to examine how the acquisition of a dysbiotic microbiota affects neonatal immune system development. We found that the resultant dysbiosis imprints in a manner that is irreversible after weaning, leading to specific and selective alteration of the colonic CD4+ T-cell compartment. In contrast, colonic granulocyte and myeloid lineages and other mucosal T-cell compartments are unaffected. Among colonic CD4+ T cells, we observed the most pronounced effects on neuropilin-negative, RORγt- and Foxp3-positive regulatory T cells, which are largely absent in antibiotic-exposed mice even as they reach adulthood. Immunomagnetically isolated dendritic cells from antibiotic-exposed mice fail to support the generation of Foxp3+ regulatory T cells (Tregs) from naive T cells ex vivo The perinatally acquired dysbiotic microbiota predisposes to dysregulated effector T-cell responses to Citrobacter rodentium or ovalbumin challenge. The transfer of the antibiotic-impacted, but not healthy, fecal microbiota into germfree recipients recapitulates the selective loss of colonic neuropilin-negative, RORγt- and Foxp3-positive Tregs. The combined data indicate that the early-life acquisition of a dysbiotic microbiota has detrimental effects on the diversity and microbial community composition of offspring that persist into adulthood and predisposes to inappropriate T-cell responses that are linked to compromised immune tolerance.IMPORTANCE The assembly of microbial communities that populate all mucosal surfaces of the human body begins right after birth. This process is prone to disruption as newborns and young infants are increasingly exposed to antibiotics, both deliberately for therapeutic purposes, and as a consequence of transmaternal exposure. We show here using a model of ampicillin administration to lactating dams during their newborn offspring's early life that such exposures have consequences that persist into adulthood. Offspring acquire their mother's antibiotic-impacted microbiota, which compromises their ability to generate a colonic pool of CD4+ T cells, particularly of colonic regulatory T cells. This Treg deficiency cannot be corrected by cohousing with normal mice later and is recapitulated by reconstitution of germfree mice with microbiota harvested from antibiotic-exposed donors. As a consequence of their dysbiosis, and possibly of their Treg deficiency, antibiotic-impacted offspring generate dysregulated Th1 responses to bacterial challenge infection and develop more severe symptoms of ovalbumin-induced anaphylaxis.

Antibiotic-modulated microbiome suppresses lethal inflammation and prolongs lifespan in Treg-deficient mice.

BACKGROUND: Regulatory T cell (Treg) deficiency leads to IPEX syndrome, a lethal autoimmune disease, in Human and mice. Dysbiosis of the gut microbiota in Treg-deficient scurfy (SF) mice has been described, but to date, the role of the gut microbiota remains to be determined. RESULTS: To examine how antibiotic-modified microbiota can inhibit Treg deficiency-induced lethal inflammation in SF mice, Treg-deficient SF mice were treated with three different antibiotics. Different antibiotics resulted in distinct microbiota and metabolome changes and led to varied efficacy in prolonging lifespan and reducing inflammation in the liver and lung. Moreover, antibiotics altered plasma levels of several cytokines, especially IL-6. By analyzing gut microbiota and metabolome, we determined the microbial and metabolomic signatures which were associated with the antibiotics. Remarkably, antibiotic treatments restored the levels of several primary and secondary bile acids, which significantly reduced IL-6 expression in RAW macrophages in vitro. IL-6 blockade prolonged lifespan and inhibited inflammation in the liver and lung. By using IL-6 knockout mice, we further identified that IL-6 deletion provided a significant portion of the protection against inflammation induced by Treg dysfunction. CONCLUSION: Our results show that three antibiotics differentially prolong survival and inhibit lethal inflammation in association with a microbiota-IL-6 axis. This pathway presents a potential avenue for treating Treg deficiency-mediated autoimmune disorders.

Calcium/Calmodulin Kinase IV Controls the Function of Both T Cells and Kidney Resident Cells.

Calcium calmodulin kinase IV (CaMK4) regulates multiple processes that significantly contribute to the lupus-related pathology by controlling the production of IL-2 and IL-17 by T cells, the proliferation of mesangial cells, and the function and structure of podocytes. CaMK4 is also upregulated in podocytes from patients with focal segmental glomerulosclerosis (FSGS). In both immune and non-immune podocytopathies, CaMK4 disrupts the structure and function of podocytes. In lupus-prone mice, targeted delivery of a CaMK4 inhibitor to CD4+ T cells suppresses both autoimmunity and the development of nephritis. Targeted delivery though to podocytes averts the deposition of immune complexes without affecting autoimmunity in lupus-prone mice and averts pathology induced by adriamycin in normal mice. Therefore, targeted delivery of a CaMK4 inhibitor to podocytes holds high therapeutic promise for both immune (lupus nephritis) and non-immune (FSGS) podocytopathies.