Carolyn Lee

Segmented Filamentous Bacteria Antigens Presented by Intestinal Dendritic Cells Drive Mucosal Th17 Cell Differentiation

How commensal microbiota contributes to immune cell homeostasis at barrier surfaces is poorly understood. Lamina propria (LP) T helper 17 (Th17) cells participate in mucosal protection and are induced by commensal segmented filamentous bacteria (SFB). Here we show that MHCII-dependent antigen presentation of SFB antigens by intestinal dendritic cells (DCs) is crucial for Th17 cell induction. Expression of MHCII on CD11c(+) cells was necessary and sufficient for SFB-induced Th17 cell differentiation. Most SFB-induced Th17 cells recognized SFB in an MHCII-dependent manner. SFB primed and induced Th17 cells locally in the LP and Th17 cell induction occurred normally in mice lacking secondary lymphoid organs. The importance of other innate cells was unveiled by the finding that MHCII deficiency in group 3 innate lymphoid cells (ILCs) resulted in an increase in SFB-independent Th17 cell differentiation. Our results outline the complex role of DCs and ILCs in the regulation of intestinal Th17 cell homeostasis.

Functionally Distinct Isoforms of STAT5 Are Generated by Protein Processing

The interleukin-3 family of cytokines, which play an important role in the development of myeloid lineages, transduce signals through the JAK-STAT pathway. Previous studies demonstrate that this process entails the activation of four distinct isoforms of STAT5, where two shorter isoforms are activated in a distinct population of cells. We now demonstrate that the shorter isoforms represent carboxy-terminal truncations. Moreover, these truncations are not generated by RNA processing, but by a specific proteolytic activity. Consistent with the notion that truncated STAT5 isoforms transduce distinct signals, they fail to promote the activation of several known interleukin-3 target genes. These studies suggest that the activity of a specific protease may play a critical role in defining the biological responses transduced by STAT5.

Dendritic Cell (DC)-Specific Targeting Reveals Stat3 as a Negative Regulator of DC Function

Dendritic cells (DCs) must achieve a critical balance between activation and tolerance, a process influenced by cytokines and growth factors. IL-10, which transduces signals through Stat3, has emerged as one important negative regulator of DC activation. To directly examine the role Stat3 plays in regulating DC activity, the Stat3 gene was targeted for deletion with a CD11c-cre transgene. Stat3 CKO mice developed cervical lymphadenopathy as well as a mild ileocolitis that persisted throughout life and was associated with impaired weight gain. Consistent with this, Stat3-deficient DCs demonstrated enhanced immune activity, including increased cytokine production, Ag-dependent T-cell activation and resistance to IL-10–mediated suppression. These results reveal a cell-intrinsic negative regulatory role of Stat3 in DCs and link increased DC activation with perturbed immune homeostasis and chronic mucosal inflammation.

Interferons direct an effective innate response to Legionella pneumophila infection

Legionella pneumophila remains an important opportunistic pathogen of human macrophages. Its more limited ability to replicate in murine macrophages has been attributed to redundant innate sensor systems that detect and effectively respond to this infection. The current studies evaluate the role of one of these innate response systems, the type I interferon (IFN-I) autocrine loop. The ability of L. pneumophila to induce IFN-I expression was found to be dependent on IRF-3, but not NF-κB. Secreted IFN-Is then in turn suppress the intracellular replication of L. pneumophila. Surprisingly, this suppression is mediated by a pathway that is independent of Stat1, Stat2, Stat3, but correlates with the polarization of macrophages toward the M1 or classically activated phenotype.

Deletion of the murine scavenger receptor CD68

Scavenger receptors (ScRs) are a structurally unrelated family of receptors with the ability to bind modified low density lipoprotein (LDL) as well as a broad range of polyanionic ligands. CD68, whose expression is restricted to mononuclear phagocytes, is a unique ScR family member, owing to its lysosome associated membrane protein (LAMP)-like domain and predominant endosomal distribution. Knockout (ko) mice were generated to directly evaluate the role murine CD68 may play in oxidized LDL (Ox-LDL) uptake. However, CD68−/− macrophages took up Ox-LDL robustly. Likewise, no defects were observed in the ability of CD68−/− mononuclear phagocytes to take up or mount an effective innate response against a number of microbes. Curiously, CD68−/− mononuclear phagocytes exhibited a trend toward enhanced antigen presentation to CD4+ T-cells, raising the possibility that CD68 may function either to negatively regulate antigen uptake, loading, or major histocompatibility complex class II (MHC-II) trafficking.

Characterization of the Stat5 Protease

Immature myeloid cells have been shown to transduce signals through a carboxyl-terminally truncated isoform of Stat5. This functionally distinct signal transducer and activator of transcription isoform is generated through a unique protein-processing event. Evaluation of numerous cell lines has determined that there is a direct correlation between the expression of truncated Stat5 and protease activity. Moreover, protease activity is found only in the myeloid and not in lymphoid progenitors. To further characterize the protease small quantities have been purified to near homogeneity. Studies on this purified material indicate that the protease has an apparent molecular mass of ∼25 kDa and is active over a wide range of pH values. The protease will also cleave both activated (i.e. tyrosine-phosphorylated) and inactivate Stat5. Although this activity is sensitive to phenylmethylsulfonyl fluoride, it is notably not sensitive to several other serine protease inhibitors. Additional studies have led to the identification of the unique site where the protease cleaves Stat5. Mutagenesis of this site renders Stat5 resistant to cleavage. Consistent with the model that Stat5 cleavage is important for early myeloid development, introduction of a “non-cleavable” isoform of Stat5 into FDC-P1 cells (a myeloid progenitor line) leads to significant phenotypic changes.

Stat2-Dependent Regulation of MHC Class II Expression

MHC type II (MHC II) expression is tightly regulated in macrophages and potently induced by IFN-γ (type II IFN). In contrast, type I IFNs (IFN-Is), which are far more widely expressed, fail to induce MHC II expression, even though both classes of IFNs direct target gene expression through Stat1. The unexpected finding that IFN-Is effectively induce MHC II expression in Stat2−/− macrophages provided an opportunity to explore this conundrum. The ensuing studies revealed that deletion of Stat2, which uniquely transduces signals for IFN-Is, leads to a loss in the IFN-I-dependent induction of suppressor of cytokine signaling-1. Impairment in the expression of this important negative regulator led to a striking prolongation in IFN-I-dependent Stat1 activation, as well as enhanced expression of the target gene, IFN-regulatory factor-1. The prolonged activity of these two transcription factors synergized to drive the transcription of CIITA, the master regulator of MHC II expression, analogous to the pattern observed in IFN-γ-treated macrophages. Thus, IFN-I-dependent suppressor of cytokine signaling-1 expression plays an important role in distinguishing the biological response between type I and II IFNs in macrophages.

A conserved IFN-alpha receptor tyrosine motif directs the biological response to type I IFNs.

Mammalian type I IFNs (IFN-Is) mediate their potent biological activities through an evolutionarily conserved IFN-α receptor (IFNAR), consisting of IFNAR1 and IFNAR2. These two chains direct the rapid activation of two founding members of the STAT family of transcription factors, STAT1 and STAT2. To understand how IFN-Is direct the recruitment and activation of STATs, a series of mutant murine IFNAR1 and IFNAR2 receptors were generated and evaluated in IFNAR1 and IFNAR2 knockout cells. These studies reveal that a single conserved IFNAR2 tyrosine, Y510, plays a critical role in directing the IFN-I-dependent activation of STAT1 and STAT2, both in murine fibroblasts and macrophages. A second IFNAR2 tyrosine, Y335, plays a more minor role. Likewise, Y510 > Y335 play a critical role in the induction of genes and antiviral activity traditionally associated with IFN-Is.

Different STAT Transcription Complexes Drive Early and Delayed Responses to Type I IFNs

IFNs, which transduce pivotal signals through Stat1 and Stat2, effectively suppress the replication of Legionella pneumophila in primary murine macrophages. Although the ability of IFN-γ to impede L. pneumophila growth is fully dependent on Stat1, IFN-αβ unexpectedly suppresses L. pneumophila growth in both Stat1- and Stat2-deficient macrophages. New studies demonstrating that the robust response to IFN-αβ is lost in Stat1-Stat2 double-knockout macrophages suggest that Stat1 and Stat2 are functionally redundant in their ability to direct an innate response toward L. pneumophila. Because the ability of IFN-αβ to signal through Stat1-dependent complexes (i.e., Stat1-Stat1 and Stat1-Stat2 dimers) has been well characterized, the current studies focus on how Stat2 is able to direct a potent response to IFN-αβ in the absence of Stat1. These studies reveal that IFN-αβ is able to drive the formation of a Stat2 and IFN regulatory factor 9 complex that drives the expression of a subset of IFN-stimulated genes, but with substantially delayed kinetics. These observations raise the possibility that this pathway evolved in response to microbes that have devised strategies to subvert Stat1-dependent responses.