Inhibition of SOCS1-/- lethal autoinflammatory disease correlated to enhanced peripheral Foxp3+ regulatory T cell homeostasis.

Suppressor of cytokine signaling 1-deficient (SOCS1(-/-)) mice, which are lymphopenic, die <3 wk after birth of a T cell-mediated autoimmune inflammatory disease characterized by leukocyte infiltration and destruction of vital organs. Notably, Foxp3(+) regulatory T cells (Tregs) have been shown to be particularly potent in inhibiting inflammation-associated autoimmune diseases. We observed that SOCS1(-/-) mice were deficient in peripheral Tregs despite enhanced thymic development. The adoptive transfer of SOCS1-sufficient Tregs, CD4(+) T lymphocytes, or administration of SOCS1 kinase inhibitory region (KIR), a peptide that partially restores SOCS1 function, mediated a statistically significant but short-term survival of SOCS1(-/-) mice. However, the adoptive transfer of SOCS1-sufficient CD4(+) T lymphocytes, combined with the administration of SOCS1-KIR, resulted in a significant increase in the survival of SOCS1(-/-) mice both short and long term, where 100% death occurred by day 18 in the absence of treatment. Moreover, the CD4(+)/SOCS1-KIR combined therapy resulted in decreased leukocytic organ infiltration, reduction of serum IFN-γ, and enhanced peripheral accumulation of Foxp3(+) Tregs in treated mice. These data show that CD4(+)/SOCS1-KIR combined treatment can synergistically promote the long-term survival of perinatal lethal SOCS1(-/-) mice. In addition, these results strongly suggest that SOCS1 contributes to the stability of the Foxp3(+) Treg peripheral population under conditions of strong proinflammatory environments.

Inhibition of type 1 diabetes correlated to a Lactobacillus johnsonii N6.2-mediated Th17 bias.

Although it is known that resident gut flora contribute to immune system function and homeostasis, their role in the progression of the autoimmune disease type 1 diabetes (T1D) is poorly understood. Comparison of stool samples isolated from Bio-Breeding rats, a classic model of T1D, shows that distinct bacterial populations reside in spontaneous Bio-Breeding diabetes-prone (BBDP) and Bio-Breeding diabetes-resistant animals. We have previously shown that the oral transfer of Lactobacillus johnsonii strain N6.2 (LjN6.2) from Bio-Breeding diabetes-resistant to BBDP rodents conferred T1D resistance to BBDP rodents, whereas Lactobacillus reuteri strain TD1 did not. In this study, we show that diabetes resistance in LjN6.2-fed BBDP rodents was correlated to a Th17 cell bias within the mesenteric lymph nodes. The Th17 bias was not observed in the non-gut-draining axillary lymph nodes, suggesting that the Th17 bias was because of immune system interactions with LjN6.2 within the mesenteric lymph node. LjN6.2 interactions with the immune system were observed in the spleens of diabetes-resistant, LjN6.2-fed BBDP rats, as they also possessed a Th17 bias in comparison with control or Lactobacillus reuteri strain TD1-fed rats. Using C57BL/6 mouse in vitro assays, we show that LjN6.2 directly mediated enhanced Th17 differentiation of lymphocytes in the presence of TCR stimulation, which required APCs. Finally, we show that footpad vaccination of NOD mice with LjN6.2-pulsed dendritic cells was sufficient to mediate a Th17 bias in vivo. Together, these data suggest an interesting paradigm whereby T1D induction can be circumvented by gut flora-mediated Th17 differentiation.

Early Ovariectomy Results in Reduced Numbers of CD11c+/CD11b+ Spleen Cells and Impacts Disease Expression in Murine Lupus.

Ninety percent of those diagnosed with systemic lupus erythematosus are female, with peak incidence between the ages of 15 and 45, when women are most hormonally active. Despite significant research effort, the mechanisms underlying this sex bias remain unclear. We previously showed that a functional knockout of estrogen receptor alpha (ERα) resulted in significantly reduced renal disease and increased survival in murine lupus. Dendritic cell (DC) development, which requires both estrogen and ERα, is impacted, as is activation status and cytokine production. Since both estrogen and testosterone levels have immunomodulating effects, we presently studied the phenotype of NZM2410 lupus-prone mice following post- and prepubertal ovariectomy (OVX) ± estradiol (E2) replacement to determine the impact of hormonal status on disease expression and DC development in these mice. We observed a trend toward survival benefit in addition to decreased proteinuria and improved renal histology in the early OVX, but not late OVX- or E2-repleted WT mice. Interestingly, there was also a significant difference in splenic DC subsets by flow cytometry. Spleens from NZM mice OVX'd early had a significant decrease in proinflammatory CD11c+CD11b+ DCs (vs. unmanipulated WTs, late OVX- and E2-repleted mice). These early OVX'd animals also had a significant increase in tolerogenic CD11c+CD8a+ DCs vs. WT. These data join a growing body of evidence that supports a role for hormone modulation of DCs that likely impacts the penetrance and severity of autoimmune diseases, such as lupus.

Isolation and th17 differentiation of naïve CD4 T lymphocytes.

Th17 cells are a distinct subset of T cells that have been found to produce interleukin 17 (IL-17), and differ in function from the other T cell subsets including Th1, Th2, and regulatory T cells. Th17 cells have emerged as a central culprit in overzealous inflammatory immune responses associated with many autoimmune disorders. In this method we purify T lymphocytes from the spleen and lymph nodes of C57BL/6 mice, and stimulate purified CD4+ T cells under control and Th17-inducing environments. The Th17-inducing environment includes stimulation in the presence of anti-CD3 and anti-CD28 antibodies, IL-6, and TGF-ß. After incubation for at least 72 hours and for up to five days at 37 °C, cells are subsequently analyzed for the capability to produce IL-17 through flow cytometry, qPCR, and ELISAs. Th17 differentiated CD4+CD25- T cells can be utilized to further elucidate the role that Th17 cells play in the onset and progression of autoimmunity and host defense. Moreover, Th17 differentiation of CD4+CD25- lymphocytes from distinct murine knockout/disease models can contribute to our understanding of cell fate plasticity.