Decidualization renders the endometrium transiently receptive to an implanting blastocyst although the underlying mechanisms remain incompletely understood. Here we show that human endometrial stromal cells (HESCs) rapidly release IL-33, a key regulator of innate immune responses, upon decidualization. In parallel, differentiating HESCs upregulate the IL-33 transmembrane receptor ST2L and other pro-inflammatory mediators before mounting a profound anti-inflammatory response that includes downregulation of ST2L and increased expression of the soluble decoy receptor sST2. We demonstrate that HESCs secrete factors permissive of embryo implantation in mice only during the pro-inflammatory phase of the decidual process. IL-33 knockdown in undifferentiated HESCs was sufficient to abrogate this pro-inflammatory decidual response. Further, sequential activation of the IL-33/ST2L/sST2 axis was disordered in decidualizing HESCs from women with recurrent pregnancy loss. Signals from these cultures prolonged the implantation window but also caused subsequent pregnancy failure in mice. Thus, Il-33/ST2 activation in HESCS drives an autoinflammatory response that controls the temporal expression of receptivity genes. Failure to constrain this response predisposes to miscarriage by allowing out-of-phase implantation in an unsupportive uterine environment.

Mycobacterium tuberculosis resides and replicates within host phagocytes by modulating host microbicidal responses. In addition, it suppresses the production of host protective cytokines to prevent activation of and antigen presentation by M. tuberculosis-infected cells, causing dysregulation of host protective adaptive immune responses. Many cytokines are regulated by microRNAs (miRNAs), a newly discovered class of small noncoding RNAs, which have been implicated in modulating host immune responses in many bacterial and viral diseases. Here, we show that miRNA-99b (miR-99b), an orphan miRNA, plays a key role in the pathogenesis of M. tuberculosis infection. We found that miR-99b expression was highly up-regulated in M. tuberculosis strain H37Rv-infected dendritic cells (DCs) and macrophages. Blockade of miR-99b expression by antagomirs resulted in significantly reduced bacterial growth in DCs. Interestingly, knockdown of miR-99b in DCs significantly up-regulated proinflammatory cytokines such as IL-6, IL-12, and IL-1β. Furthermore, mRNA and membrane-bound protein data indicated that inhibition of miR-99b augments TNF-α and TNFRSF-4 production. Thus, miR-99b targets TNF-α and TNFRSF-4 receptor genes. Treatment of anti-miR-99b-transfected DCs with anti-TNF-α antibody resulted in increased bacterial burden. Thus, our findings unveil a novel host evasion mechanism adopted by M. tuberculosis via miR-99b, which may open up new avenues for designing miRNA-based vaccines and therapies.

Despite its relatively poor efficacy, Bacillus Calmette-Guérin (BCG) has been used as a tuberculosis (TB) vaccine since its development in 1921. BCG induces robust T helper 1 (Th1) immune responses but, for many individuals, this is not sufficient for host resistance against Mycobacterium tuberculosis (M. tb) infection. Here we provide evidence that early secreted antigenic target protein 6 (ESAT-6), expressed by the virulent M. tb strain H37Rv but not by BCG, promotes vaccine-enhancing Th17 cell responses. These activities of ESAT-6 were dependent on TLR-2/MyD88 signalling and involved IL-6 and TGF-β production by dendritic cells. Thus, animals that were previously infected with H37Rv or recombinant BCG containing the RD1 region (BCG::RD1) exhibited improved protection upon re-challenge with virulent H37Rv compared with mice previously infected with BCG or RD1-deficient H37Rv (H37RvΔRD1). However, TLR-2 knockout (TLR-2^-/-) animals neither showed Th17 responses nor exhibited improved protection in response to immunization with H37Rv. Furthermore, H37Rv and BCG::RD1 infection had little effect on the expression of the anti-inflammatory microRNA-146a (miR146a) in dendritic cells (DCs), whereas BCG and H37RvΔRD1 profoundly induced its expression in DCs. Consistent with these findings, ESAT-6 had no effect on miR146a expression in uninfected DCs, but dramatically inhibited its upregulation in BCG-infected or LPS-treated DCs. Collectively, our findings indicate that, in addition to Th1 immunity induced by BCG, RD1/ESAT-6-induced Th17 immune responses are essential for optimal vaccine efficacy. Tuberculosis is a global health problem, with one-third of the global population infected with tubercle bacteria. Numerous studies have shown that Th1 cell responses are indispensable for protective immunity against TB. However, while the vaccine strain BCG induces sufficient Th1 cell response, this response does not appear to be sufficient for immune protection in many individuals. Here, we provide evidence for the first time that Th17 cell responses in the lung play a critical role for enhanced protection against TB. Surprisingly, the virulent M. tb strain H37Rv induced Th17 cell responses in the lung. Consequently, antibiotic-treated animals that were previously infected with H37Rv, as compared with similarly treated BCG-infected mice, generated improved protective immune responses against infection with virulent M. tb. We also provide evidence that the ESAT-6 protein, which is absent in BCG but present in H37Rv, induces IL-6 and TGF-β in dendritic cells in a TLR-2 and MyD88-dependent manner, which generates an environment that is conducive for the differentiation of Th17 cells in the lung. Our findings indicate that, in addition to Th1 cells, Th17 cells play a critical role in conferring optimal protection against TB.

A significant enrichment of CD4⁺Foxp3⁺ T cells (regulatory T cells, Treg) is frequently observed in murine and human carcinomas. As Tregs can limit effective antitumor immune responses, thereby promoting tumor progression, it is important that the mechanisms underpinning intratumoral accumulation of Tregs are identified. Because of evidence gathered mostly in vitro, the conversion of conventional T cells (Tconv) into Tregs has been proposed as one such mechanism. We assessed the contribution of conversion in vivo by analyzing the TCR (T-cell receptor) repertoires of Tconvs and Tregs in carcinogen-induced tumors in mice. Our results indicate that the TCR repertoires of Tregs and Tconvs within tumor-infiltrating lymphocytes (TIL) are largely distinct. Indeed, the cell population with the greatest degree of repertoire similarity with tumor-infiltrating Tregs was the Treg population from the tumor-draining lymph node. These findings demonstrate that conversion of Tconvs does not contribute significantly to the accumulation of tumor-infiltrating Tregs; rather, Tconvs and Tregs arise from different populations with unique TCR repertoires. Enrichment of Tregs within TILs most likely, therefore, reflects differences in the way that Tregs and Tconvs are influenced by the tumor microenvironment. Elucidating the nature of these influences may indicate how the balance between tumor-infiltrating Tregs and Tconvs can be manipulated for therapeutic purposes. Cancer Res; 71(3); 736–46. ©2010 AACR.

CD4⁺ regulatory T cells (Tregs) are essential for controlling immune responses and preventing autoimmunity. Their development requires regulation of gene expression by microRNAs (miRNAs). To understand miRNA function in Treg development, we searched for important miRNAs and their relevant target genes. Of the more abundantly expressed miRNAs in Tregs, only miR-15b/16, miR-24, and miR-29a impacted the production of in vitro–induced Tregs (iTregs) in overexpression and blocking experiments. miRNA mimics for these significantly enhanced the induction of iTregs in Dicer^−/− CD4⁺ T cells. Furthermore, the overexpression of miR-15b/16 in conventional CD4⁺ T cells adoptively transferred into Rag2^−/− mice increased the in vivo development of peripheral Tregs and diminished the severity of autoimmune colitis. In searching for targets of miR-15b/16, we observed that the mammalian target of rapamycin (mTOR) signaling pathway was enhanced in Dicer^−/− CD4⁺ T cells, and its pharmacological inhibition restored induction of iTregs. Suppression of mTOR signaling is essential for induction of iTregs from naive CD4⁺ T cells, and the mTORC2 component, Rictor, contained a functional target site for miR-15b/16. Rictor was more abundantly expressed in Dicer^−/− T cells as was mTOR, and their expression was downregulated by the overexpression of miR-15b/16. This led to a reduction in mTOR signaling, as measured by phosphorylation of the downstream target, ribosomal protein S6. Finally, knockdown of Rictor by small interfering RNAs enhanced Treg induction in Dicer^−/− CD4⁺ T cells. Therefore, an important mechanism of miRNA regulation of Treg development is through regulation of the mTOR signaling pathway.

Relatively little is known about regulatory T (Treg) cells and their functional responses in dogs. We have used the cross-reactive anti-mouse/rat Foxp3 antibody clone FJK-16s to identify a population of canine CD4⁺ FOXP3^high T cells in both the peripheral blood (PB) and popliteal lymph node (LN). FOXP3⁺ cells in both PB and LN yielded positive staining with the newly developed anti-murine/human Helios antibody clone 22F6, consistent with the notion that they were naturally occurring Treg cells. Stimulation of mononuclear cells of LN origin with concanavalin A (Con A) in vitro yielded increased proportions and median fluorescence intensity of FOXP3 expression by both CD4⁺ and CD8⁺ T cells. Removal of the Con A and continued culture disclosed a CD4⁺ FOXP3^high population, distinct from the CD4⁺ FOXP3^intermediate T cells; very few CD8⁺ FOXP3^high T cells were observed, though CD8⁺ FOXP3^intermediate cells were present in equal abundance to CD4⁺ FOXP3^intermediate cells. The CD4⁺ FOXP3^high T cells were thought to represent activated Treg cells, in contrast to the FOXP3^intermediate cells, which were thought to be a more heterogeneous population comprising predominantly activated conventional T cells. Co-staining with interferon-γ (IFN-γ) supported this notion, because the FOXP3^high T cells were almost exclusively IFN-γ⁻, whereas the FOXP3^intermediate cells expressed a more heterogeneous IFN-γ phenotype. Following activation of mononuclear cells with Con A and interleukin-2, the 5% of CD4⁺ T cells showing the highest CD25 expression (CD4⁺ CD25^high) were enriched in cells expressing FOXP3. These cells were anergic in vitro, in contrast to the 20% of CD4⁺ T cells with the lowest CD25 expression (CD4⁺ CD25⁻), which proliferated readily. The CD4⁺ CD25^high FOXP3^high T cells were able to suppress the proliferation of responder CD4⁺ T cells in vitro, in contrast to the CD4⁺ CD25⁻ cells, which showed no regulatory properties.