JAKs and STATs from a Clinical Perspective: Loss-of-Function Mutations, Gain-of-Function Mutations, and Their Multidimensional Consequences.

The JAK/STAT signaling pathway plays a key role in cytokine signaling and is involved in development, immunity, and tumorigenesis for nearly any cell. At first glance, the JAK/STAT signaling pathway appears to be straightforward. However, on closer examination, the factors influencing the JAK/STAT signaling activity, such as cytokine diversity, receptor profile, overlapping JAK and STAT specificity among non-redundant functions of the JAK/STAT complexes, positive regulators (e.g., cooperating transcription factors), and negative regulators (e.g., SOCS, PIAS, PTP), demonstrate the complexity of the pathway's architecture, which can be quickly disturbed by mutations. The JAK/STAT signaling pathway has been, and still is, subject of basic research and offers an enormous potential for the development of new methods of personalized medicine and thus the translation of basic molecular research into clinical practice beyond the use of JAK inhibitors. Gain-of-function and loss-of-function mutations in the three immunologically particularly relevant signal transducers STAT1, STAT3, and STAT6 as well as JAK1 and JAK3 present themselves through individual phenotypic clinical pictures. The established, traditional paradigm of loss-of-function mutations leading to immunodeficiency and gain-of-function mutation leading to autoimmunity breaks down and a more differentiated picture of disease patterns evolve. This review is intended to provide an overview of these specific syndromes from a clinical perspective and to summarize current findings on pathomechanism, symptoms, immunological features, and therapeutic options of STAT1, STAT3, STAT6, JAK1, and JAK3 loss-of-function and gain-of-function diseases.

Integrin ß1 regulates marginal zone B cell differentiation and PI3K signaling.

Marginal zone (MZ) B cells represent innate-like B cells that mediate a fast immune response. The adhesion of MZ B cells to the marginal sinus of the spleen is governed by integrins. Here, we address the question of whether ß1-integrin has additional functions by analyzing Itgb1fl/flCD21Cre mice in which the ß1-integrin gene is deleted in mature B cells. We find that integrin ß1-deficient mice have a defect in the differentiation of MZ B cells and plasma cells. We show that integrin ß1-deficient transitional B cells, representing the precursors of MZ B cells, have enhanced B cell receptor (BCR) signaling, altered PI3K and Ras/ERK pathways, and an enhanced interaction of integrin-linked kinase (ILK) with the adaptor protein Grb2. Moreover, the MZ B cell defect of integrin ß1-deficient mice could, at least in part, be restored by a pharmacological inhibition of the PI3K pathway. Thus, ß1-integrin has an unexpected function in the differentiation and function of MZ B cells.

Cochaperone Mzb1 is a key effector of Blimp1 in plasma cell differentiation and ß1-integrin function.

Plasma cell differentiation involves coordinated changes in gene expression and functional properties of B cells. Here, we study the role of Mzb1, a Grp94 cochaperone that is expressed in marginal zone (MZ) B cells and during the terminal differentiation of B cells to antibody-secreting cells. By analyzing Mzb1-/-Prdm1+/gfp mice, we find that Mzb1 is specifically required for the differentiation and function of antibody-secreting cells in a T cell-independent immune response. We find that Mzb1-deficiency mimics, in part, the phenotype of Blimp1 deficiency, including the impaired secretion of IgM and the deregulation of Blimp1 target genes. In addition, we find that Mzb1-/- plasmablasts show a reduced activation of ß1-integrin, which contributes to the impaired plasmablast differentiation and migration of antibody-secreting cells to the bone marrow. Thus, Mzb1 function is required for multiple aspects of plasma cell differentiation.

MZB1 is a GRP94 cochaperone that enables proper immunoglobulin heavy chain biosynthesis upon ER stress.

MZB1 (pERp1) is a B-cell-specific and endoplasmic reticulum (ER)-localized protein implicated in antibody secretion and integrin-mediated cell adhesion. Here, we examine the role of MZB1 in vivo by conditional gene inactivation in the mouse germline and at different stages of B lymphopoiesis. Deletion of MZB1 impairs humoral immune responses and antibody secretion in plasma cells that naturally undergo ER stress. In addition, we found that experimental induction of ER stress by tunicamycin injections in mice results in a block of pro-B-cell to pre-B-cell differentiation specifically in Mzb1(-/-) mice. A similar developmental block was observed in Mzb1(fl/fl)mb1(Cre) mice, whereby a Cre recombinase-induced genotoxic stress unmasks a role for MZB1 in the surface expression of immunoglobulin µ heavy chains (µHCs). MZB1 associates directly with the substrate-specific chaperone GRP94 (also called HSP90B1 or gp96) in an ATP-sensitive manner and is required for the interaction of GRP94 with µHCs upon ER stress. Thus, MZB1 seems to act as a substrate-specific cochaperone of GRP94 that enables proper biosynthesis of µHCs under conditions of ER stress.

IFNß produced by TLR4-activated tumor cells is involved in improving the antitumoral immune response.

Toll-like receptor (TLR) ligands may be a valuable tool to promote antitumor responses by reinforcing antitumor immunity. In addition to their expression in immune cells, functional TLRs are also expressed by many cancer cells, but their significance has been controversial. In this study, we examined the action of TLR ligands on tumor pathophysiology as a result of direct tumor cell effects. B16 murine melanoma cells were stimulated in vitro with a TLR4 ligand (LPS-B16) prior to inoculation into TLR4-deficient mice (Tlr4 (lps-del)). Under such conditions, B16 cells yielded smaller tumors than nonstimulated B16 cells. The apoptosis/proliferation balance of the cells was not modified by TLR ligand treatment, nor was this effect compromised in immunocompromised nude mice. Mechanistic investigations revealed that IFNß was the critical factor produced by TLR4-activated tumor cells in mediating their in vivo outgrowth. Transcriptional analysis showed that TLR4 activation on B16 cells induced changes in the expression of type I IFN and type I IFN-related genes. Most importantly, culture supernatants from LPS-B16 cells improved the maturation of bone marrow-derived dendritic cells (BMDC) from TLR4-deficient mice, upregulating the expression of interleukin-12 and costimulatory molecules on those cells. BMDC maturation was blunted by addition of an IFNß-neutralizing antibody. Moreover, tumor growth inhibition observed in LPS-B16 tumors was abrogated in IFNAR1-deficient mice lacking a functional type I IFN receptor for binding IFN. Together, our findings show that tumor cells can be induced through the TLR4 pathway to produce IFN and positively contribute to the antitumoral immune response.

Expression of Toll-like receptor 4 in the prostate gland and its association with the severity of prostate cancer.

BACKGROUND: Chronic inflammation has been postulated to be an important driving force to prostate carcinoma. Toll-like receptors (TLRs) compose a family of receptors mainly expressed on immune cells. Recently, functional TLRs have been shown to be also expressed in numerous cancer cells, but their significance has only recently begun to be explored. The purpose of this study was to investigate the putative role of TLR4 expression in prostate carcinoma. METHODS: To determine if there is an association between TLR4 expression and the malignancy of the tumor, 35 prostate carcinoma samples showing different Gleason grades were analyzed by immunohistochemistry. Also, to explore the functionality of the receptors expressed on the epithelium, we analyzed the type of cytokine response elicited and the signaling pathways involved after TLR4 triggering in the human prostate adenocarcinoma cell line, DU-145. RESULTS: TLR4 is expressed in the normal prostate gland in both stroma and epithelium. TLR4 expression significantly drops to negative values as the Gleason grade augments in both, stroma and epithelium. Moreover, DU-145 cells also exhibit TLR4 expression and respond to TLR4 agonists, activating the transcription factor NF-kappaB and increasing the expression of pro-inflammatory mediators. Inhibition of the molecular adaptors MyD88 and MAL by overexpression of dominant-negative mutants diminished LPS-induced activation of NF-kappaB, showing that DU-145 cells activate the NF-kappaB through MyD88-dependent signaling pathways. CONCLUSIONS: We hypothesize that TLR4 in prostate cells could synergize with innate immune cells contributing to an eventual inflammatory process, which in genetically prone individuals could promote carcinogenesis. Prostate 69: 1387-1397, 2009. (c) 2009 Wiley-Liss, Inc.

Activation of Toll-like receptor 4 on tumor cells in vitro inhibits subsequent tumor growth in vivo.

Although an eruption of information on the role of Toll-like receptor 4 (TLR4), the main receptor for bacterial lipopolysaccharide, in activating macrophages and dendritic cells has emerged, very little is known about the role of TLR4 present on epithelial cells from sterile environments like tumors. The main goal of this work was to investigate the consequences of TLR4 activation present on tumor cells in two different animal models of cancer: the Dunning rat prostate cancer and the B16 murine melanoma models. We show that (a) activating TLR4 signaling in two different tumor cell lines in vitro modifies the tumor outgrowth in vivo; (b) this effect is not due to a direct consequence of TLR4 signaling on the proliferation/apoptosis balance of the tumor cells; (c) the T-cell compartment is somehow involved in the described phenomenon because the inhibitory effect observed is not seen in athymic nude mice; and (d) tumor-infiltrating lymphocytes purified from tumors induced by TLR4-activated cells show strong induction of IFN gamma transcript in detriment of interleukin-10 transcript, suggesting a change in their functionality. We hypothesize that TLR4 signaling in tumor cells in vitro induces the expression of proinflammatory mediators, which could dramatically alter the maturation state of dendritic cells present at the site of inoculation, switching the type of immune response elicited against the tumor. These results open up new avenues for understanding the role of TLR4 in tumor cells and for identifying potential new therapy strategies for cancer.