Src-family kinase-Cbl axis negatively regulates NLRP3 inflammasome activation.

Activation of the NLRP3 inflammasome is crucial for immune defense, but improper and excessive activation causes inflammatory diseases. We previously reported that Pyk2 is essential for NLRP3 inflammasome activation. Here we show that the Src-family kinases (SFKs)-Cbl axis plays a pivotal role in suppressing NLRP3 inflammasome activation in response to stimulation by nigericin or ATP, as assessed using gene knockout and gene knockdown cells, dominant active/negative mutants, and pharmacological inhibition. We reveal that the phosphorylation of Cbl is regulated by SFKs, and that phosphorylation of Cbl at Tyr371 suppresses NLRP3 inflammasome activation. Mechanistically, Cbl decreases the level of phosphorylated Pyk2 (p-Pyk2) through ubiquitination-mediated proteasomal degradation and reduces mitochondrial ROS (mtROS) production by contributing to the maintenance of mitochondrial size. The lower levels of p-Pyk2 and mtROS dampen NLRP3 inflammasome activation. In vivo, inhibition of Cbl with an analgesic drug, hydrocotarnine, increases inflammasome-mediated IL-18 secretion in the colon, and protects mice from dextran sulphate sodium-induced colitis. Together, our novel findings provide new insights into the role of the SFK-Cbl axis in suppressing NLRP3 inflammasome activation and identify a novel clinical utility of hydrocortanine for disease treatment.

Natural T-cell ligands that are created by genetic variants can be transferred between cells by extracellular vesicles.

CD4 T cells play a central role as helper cells in adaptive immunity. Presentation of exogenous antigens in MHC class II by professional antigen-presenting cells is a crucial step in induction of specific CD4 T cells in adaptive immune responses. For efficient induction of immunity against intracellular threats such as viruses or malignant transformations, antigens from HLA class II-negative infected or transformed cells need to be transferred to surrounding antigen-presenting cells to allow efficient priming of naive CD4 T cells. Here we show indirect antigen presentation for a subset of natural HLA class II ligands that are created by genetic variants and demonstrated that (neo)antigens can be transferred between cells by extracellular vesicles. Intercellular transfer by extracellular vesicles was not dependent on the T-cell epitope, but rather on characteristics of the full-length protein. This mechanism of (neo)antigen transfer from HLA class II-negative cells to surrounding antigen-presenting cells may play a crucial role in induction of anti-tumor immunity.

The nonreceptor protein tyrosine kinase Pyk2 promotes the turnover of monocytes at steady state.

Monocytes are short-lived myeloid cells that perform functions essential for tissue homeostasis and disease resolution. However, the cellular mechanisms controlling the maintenance and turnover of monocyte populations are largely undefined. Proline-rich tyrosine kinase 2 (Pyk2) is a nonreceptor tyrosine kinase that regulates numerous immune cell functions, but its role in monocytes is currently unknown. In this study, we sought to characterize the expression and function of Pyk2 in lineage-committed monocyte populations. Here, we report that Pyk2 protein expression is increased in the Ly6C- monocyte population. Using a Pyk2 knockout mouse model (Pyk2-/-), we show that Pyk2 regulates the relative proportion of monocyte subsets normally represented in the bone marrow (BM) at steady state. In support of this conclusion, a similar phenotype was observed in the peripheral blood and spleen. Data from reciprocal BM chimera experiments indicate that the alterations in monocyte populations exhibited by Pyk2-/- mice are due to factors intrinsic to the monocytes. Lineage-tracing of monocyte populations suggests that Pyk2 promotes apoptosis in BM monocytes, thereby acting as an important homeostatic regulator of turnover in these short-lived, innate immune cells.

Pyk2 activates the NLRP3 inflammasome by directly phosphorylating ASC and contributes to inflammasome-dependent peritonitis.

The inflammasome adaptor protein, ASC, contributes to both innate immune responses and inflammatory diseases via self-oligomerization, which leads to the activation of the protease, caspase-1. Here, we report that the cytosolic tyrosine kinases, FAK and Pyk2, are differentially involved in NLRP3 and AIM2 inflammasome activation. The inhibition of FAK and Pyk2 with RNA interference or chemical inhibitors dramatically abolished ASC oligomerization, caspase-1 activation, and IL-1ß secretion in response to NLRP3 or AIM2 stimulation. Pyk2 is phosphorylated by the kinase Syk and relocalizes to the ASC specks upon NLRP3 inflammasome activation. Pyk2, but not FAK, could directly phosphorylate ASC at Tyr146, and only the phosphorylated ASC could participate in speck formation and trigger IL-1ß secretion. Moreover, the clinical-trial-tested Pyk2/FAK dual inhibitor PF-562271 reduced monosodium urate-mediated peritonitis, a disease model used for studying the consequences of NLRP3 activation. Our results suggest that although Pyk2 and FAK are involved in inflammasome activation, only Pyk2 directly phosphorylates ASC and brings ASC into an oligomerization-competent state by allowing Tyr146 phosphorylation to participate ASC speck formation and subsequent NLRP3 inflammation.

Pyk2 Controls Integrin-Dependent CTL Migration through Regulation of De-Adhesion.

Protein tyrosine kinase 2 (Pyk2) is required for T cell adhesion to ICAM-1; however, the mechanism by which it regulates adhesion remains unexplored. Pyk2 function in murine CTL clones and activated ex vivo CD8(+) T cells was disrupted by pharmacological inhibition, knockdown of expression with small interfering RNA, or expression of the dominant-negative C-terminal domain. We found that Pyk2 is not absolutely required for adhesion of CTL to ICAM-1, but rather delays the initial adhesion. Disruption of Pyk2 function caused cells to display an unusual elongated appearance after 1 h on ICAM-1, consistent with abnormally strong adhesion. Furthermore, the random mobility of CTL on ICAM-1 was severely compromised using all three methods of disrupting Pyk2 function. Live-cell imaging studies revealed that the decreased migration is the result of a defect in the detachment from ICAM-1 at the trailing edge when Pyk2 function is inhibited. Examination of Pyk2 tyrosine phosphorylation in normal polarized cells demonstrated that Pyk2 phosphorylated at Y579 and Y580 preferentially localizes to the leading edge, whereas Y881-phosphorylated Pyk2 is enriched at the trailing edge, suggesting that the tyrosine phosphorylation of Pyk2 is spatially regulated in migrating CTL. Additionally, inhibition of Pyk2 caused cells to form multiple LFA-1-rich tails at the trailing edge, most likely resulting from a defect in LFA-1 release required for forward movement. Our results show that Pyk2 contributes to CTL migration by regulating detachment of CTL at the trailing edge, which could explain why Pyk2 is important for chemotactic and migratory responses.

Micro-adhesion rings surrounding TCR microclusters are essential for T cell activation.

The immunological synapse (IS) formed at the interface between T cells and antigen-presenting cells represents a hallmark of initiation of acquired immunity. T cell activation is initiated at T cell receptor (TCR) microclusters (MCs), in which TCRs and signaling molecules assemble at the interface before IS formation. We found that each TCR-MC was transiently bordered by a ring structure made of integrin and focal adhesion molecules in the early phase of activation, which is similar in structure to the IS in microscale. The micro-adhesion ring is composed of LFA-1, focal adhesion molecules paxillin and Pyk2, and myosin II (MyoII) and is supported by F-actin core and MyoII activity through LFA-1 outside-in signals. The formation of the micro-adhesion ring was transient but especially sustained upon weak TCR stimulation to recruit linker for activation of T cells (LAT) and SLP76. Perturbation of the micro-adhesion ring induced impairment of TCR-MC development and resulted in impaired cellular signaling and cell functions. Thus, the synapse-like structure composed of the core TCR-MC and surrounding micro-adhesion ring is a critical structure for initial T cell activation through integrin outside-in signals.

Galectin-1 regulates tissue exit of specific dendritic cell populations.

During inflammation, dendritic cells emigrate from inflamed tissue across the lymphatic endothelium into the lymphatic vasculature and travel to regional lymph nodes to initiate immune responses. However, the processes that regulate dendritic cell tissue egress and migration across the lymphatic endothelium are not well defined. The mammalian lectin galectin-1 is highly expressed by vascular endothelial cells in inflamed tissue and has been shown to regulate immune cell tissue entry into inflamed tissue. Here, we show that galectin-1 is also highly expressed by human lymphatic endothelial cells, and deposition of galectin-1 in extracellular matrix selectively regulates migration of specific human dendritic cell subsets. The presence of galectin-1 inhibits migration of immunogenic dendritic cells through the extracellular matrix and across lymphatic endothelial cells, but it has no effect on migration of tolerogenic dendritic cells. The major galectin-1 counter-receptor on both dendritic cell populations is the cell surface mucin CD43; differential core 2 O-glycosylation of CD43 between immunogenic dendritic cells and tolerogenic dendritic cells appears to contribute to the differential effect of galectin-1 on migration. Binding of galectin-1 to immunogenic dendritic cells reduces phosphorylation and activity of the protein-tyrosine kinase Pyk2, an effect that may also contribute to reduced migration of this subset. In a murine lymphedema model, galectin-1(-/-) animals had increased numbers of migratory dendritic cells in draining lymph nodes, specifically dendritic cells with an immunogenic phenotype. These findings define a novel role for galectin-1 in inhibiting tissue emigration of immunogenic, but not tolerogenic, dendritic cells, providing an additional mechanism by which galectin-1 can dampen immune responses.

Proline-rich tyrosine kinase 2 controls PI3-kinase activation downstream of the T cell antigen receptor in human T cells.

TCR-induced signaling controls T cell activation that drives adaptive immunity against infections, but it can also induce dysfunctional T cell responses that promote pathologic disease. The PI3K pathway regulates many downstream effector responses after TCR stimulation. However, the molecular mechanisms that induce PI3K function downstream of the TCR are not fully understood. We have previously shown that Pyk2 is activated downstream of the TCR in a PI3K-independent manner. Although Pyk2 controls adhesion, proliferation, and cytokine production in T cells, the mechanisms by which it controls these processes are not known. In this study, we generated Pyk2-deficient human T cells to elucidate further the role that this kinase plays in TCR-induced effector functions and signaling. We observed that Pyk2 localized with the p85 regulatory subunit of PI3K at the LAT complex and that PI3K-dependent signaling was impaired in Pyk2-deficient T cells. Likewise, functions downstream of PI3K, including IFN-γ production and proliferation, were also suppressed in human T cells deficient in Pyk2. Collectively, these data demonstrate that Pyk2 is a critical regulator of PI3K function downstream of the TCR.

Functions of the FAK family kinases in T cells: beyond actin cytoskeletal rearrangement.

T cells control the focus and extent of adaptive immunity in infectious and pathological diseases. The activation of T cells occurs when the T cell antigen receptor (TCR) and costimulatory and/or adhesion receptors are engaged by their ligands. This process drives signaling that promotes cytoskeletal rearrangement and transcription factor activation, both of which regulate the quality and magnitude of the T cell response. However, it is not fully understood how different receptor-induced signals combine to alter T cell activation. The related non-receptor tyrosine kinases focal adhesion kinase (FAK) and proline-rich tyrosine kinase 2 (Pyk2) are phosphorylated downstream of the TCR and several costimulatory and adhesion receptors. FAK family proteins integrate receptor-mediated signals that influence actin cytoskeletal rearrangement and effector T cell responses. In this review, we summarize the receptor-specific roles that FAK and Pyk2 control to influence T cell development and activation.

Control of dendritic cell migration, T cell-dependent immunity, and autoimmunity by protein tyrosine phosphatase PTPN12 expressed in dendritic cells.

Dendritic cells (DCs) capture and process antigens in peripheral tissues, migrate to lymphoid tissues, and present the antigens to T cells. PTPN12, also known as PTP-PEST, is an intracellular protein tyrosine phosphatase (PTP) involved in cell-cell and cell-substratum interactions. Herein, we examined the role of PTPN12 in DCs, using a genetically engineered mouse lacking PTPN12 in DCs. Our data indicated that PTPN12 was not necessary for DC differentiation, DC maturation, or cytokine production in response to inflammatory stimuli. However, it was needed for full induction of T cell-dependent immune responses in vivo. This function largely correlated with the need of PTPN12 for DC migration from peripheral sites to secondary lymphoid tissues. Loss of PTPN12 in DCs resulted in hyperphosphorylation of the protein tyrosine kinase Pyk2 and its substrate, the adaptor paxillin. Pharmacological inhibition of Pyk2 or downregulation of Pyk2 expression also compromised DC migration, suggesting that Pyk2 deregulation played a pivotal role in the migration defect caused by PTPN12 deficiency. Together, these findings identified PTPN12 as a key regulator in the ability of DCs to induce antigen-induced T cell responses. This is due primarily to the role of PTPN12 in DC migration from peripheral sites to secondary lymphoid organs through regulation of Pyk2.

Development of a coordinated allo T cell and auto B cell response against autosomal PTK2B after allogeneic hematopoietic stem cell transplantation.

It is well known that allo-reactive T cells play a crucial role in graft-versus-leukemia and graft-versus-host disease after allogeneic hematopoietic stem cell transplantation (alloSCT). Allo-reactive CD4(+) T cells can mediate direct cytolysis, but may also stimulate production of IgG antibodies as helper cells. Immune complexes may subsequently be processed and presented by professional antigen presenting cells and stimulate induction of specific CD8(+) T cells. As such, proteins targeted in coordinated T- and B-cell responses may represent a class of immunodominant antigens in clinical responses after alloSCT. We previously identified LB-PTK2B-1T as HLA class II restricted polymorphic antigen in a patient treated with donor lymphocyte infusion for relapsed chronic myeloid leukemia after HLA-matched alloSCT. Since PTK2B has also been described as antibody target, we here investigated whether a coordinated T- and B-cell response against PTK2B was induced. Patient serum before and after alloSCT and donor lymphocyte infusion (DLI) was screened for antibodies, and we indeed observed development of a humoral immune response against PTK2B. Antibodies against PTK2B were only found after DLI and, in contrast to the CD4(+) T cells, recognized a monomorphic region of the protein. To our knowledge, this is the first description of a coordinated allo-reactive CD4(+) T-cell and auto-reactive antibody response against an autosomal antigen.

SHP-1-Pyk2-Src protein complex and p38 MAPK pathways independently regulate IL-10 production in lipopolysaccharide-stimulated macrophages.

The role of tyrosine phosphatase Src homology region 2 domain-containing phosphatase (SHP)-1 in LPS-activated cytokine production and inflammation was investigated by determining TNF-α and IL-10 production in splenic macrophages employing SHP-1-null (me/me) mouse model. LPS-stimulated me/me splenic macrophages secreted significantly less IL-10 with concomitantly elevated levels of TNF-α compared with wild-type (WT) macrophages irrespective of LPS dose and duration of stimulation. IL-10 significantly inhibited LPS-induced TNF-α production in both me/me and WT macrophages. The critical requirement for SHP-1 in regulating LPS-induced IL-10 and TNF-α production was confirmed by interfering with SHP-1 expression in WT macrophages and by reconstituting me/me macrophages with the SHP-1 gene. To delineate the role of SHP-1 in positive regulation of LPS-induced IL-10 production, signaling proteins representing SHP-1 targets were examined. The results reveal that tyrosine kinases Src and proline-rich tyrosine kinase 2 (Pyk2) regulate SHP-1-dependent LPS-induced IL-10 production and infer that optimal LPS-induced IL-10 production requires an assembly of a protein complex consisting of SHP-1-Pyk2-Src proteins. Moreover, LPS-induced IL-10 production also requires activation of the p38 MAPK independent of SHP-1 function. Overall, to our knowledge our results show for the first time that SHP-1 acts as a positive regulator of LPS-induced IL-10 production in splenic macrophages through two distinct and independent SHP-1-Pyk2-Src and p38 MAPK pathways.

Bifurcation of Arabidopsis NLR immune signaling via Ca²âº-dependent protein kinases.

Nucleotide-binding domain leucine-rich repeat (NLR) protein complexes sense infections and trigger robust immune responses in plants and humans. Activation of plant NLR resistance (R) proteins by pathogen effectors launches convergent immune responses, including programmed cell death (PCD), reactive oxygen species (ROS) production and transcriptional reprogramming with elusive mechanisms. Functional genomic and biochemical genetic screens identified six closely related Arabidopsis Ca²âº-dependent protein kinases (CPKs) in mediating bifurcate immune responses activated by NLR proteins, RPS2 and RPM1. The dynamics of differential CPK1/2 activation by pathogen effectors controls the onset of cell death. Sustained CPK4/5/6/11 activation directly phosphorylates a specific subgroup of WRKY transcription factors, WRKY8/28/48, to synergistically regulate transcriptional reprogramming crucial for NLR-dependent restriction of pathogen growth, whereas CPK1/2/4/11 phosphorylate plasma membrane-resident NADPH oxidases for ROS production. Our studies delineate bifurcation of complex signaling mechanisms downstream of NLR immune sensors mediated by the myriad action of CPKs with distinct substrate specificity and subcellular dynamics.

Altered Toll-like receptor 9 signaling in Mycobacterium avium subsp. paratuberculosis-infected bovine monocytes reveals potential therapeutic targets.

Mycobacterium avium subsp. paratuberculosis is the causative agent of Johne's disease in cattle. The complex, multifaceted interaction of M. avium subsp. paratuberculosis with its host includes dampening the ability of infected cells to respond to stimuli that promote M. avium subsp. paratuberculosis clearance. By disrupting host defenses, M. avium subsp. paratuberculosis creates an intracellular environment that favors the establishment and maintenance of infection. Toll-like receptors (TLRs) are important sensors that initiate innate immune responses to microbial challenge and are also immunotherapeutic targets. For example, TLR9 contributes to host defense against M. avium subsp. paratuberculosis, and its agonists (CpG oligodeoxynucleotides [ODNs]) are under investigation for treatment of Johne's disease and other infections. Here we demonstrate that M. avium subsp. paratuberculosis infection changes the responsiveness of bovine monocytes to TLR9 stimulation. M. avium subsp. paratuberculosis inhibits classical TLR9-mediated responses despite a 10-fold increase in TLR9 expression and maintained uptake of CpG ODNs. Other TLR9-mediated responses, such as oxidative burst, which occur through noncanonical signaling, remain functional. Kinome analysis verifies that classic TLR9 signaling is blocked by M. avium subsp. paratuberculosis infection and that signaling instead proceeds through a Pyk2-mediated mechanism. Pyk2-mediated signaling does not hinder infection, as CpG ODNs fail to promote M. avium subsp. paratuberculosis clearance. Indeed, Pyk2 signaling appears to be an important aspect of M. avium subsp. paratuberculosis infection, as Pyk2 inhibitors significantly reduce the number of intracellular M. avium subsp. paratuberculosis bacteria. The actions of M. avium subsp. paratuberculosis on TLR9 signaling may represent a strategy to generate a host environment which is better suited for infection, revealing potential new targets for therapeutic intervention.

Slit2N/Robo1 inhibit HIV-gp120-induced migration and podosome formation in immature dendritic cells by sequestering LSP1 and WASp.

Cell-mediated transmission and dissemination of sexually-acquired human immunodeficiency virus 1 (HIV-1) in the host involves the migration of immature dendritic cells (iDCs). iDCs migrate in response to the HIV-1 envelope protein, gp120, and inhibiting such migration may limit the mucosal transmission of HIV-1. In this study, we elucidated the mechanism of HIV-1-gp120-induced transendothelial migration of iDCs. We found that gp120 enhanced the binding of Wiskott-Aldrich Syndrome protein (WASp) and the Actin-Related Protein 2/3 (Arp2/3) complex with ß-actin, an interaction essential for the proper formation of podosomes, specialized adhesion structures required for the migration of iDCs through different tissues. We further identified Leukocyte-Specific Protein 1 (LSP1) as a novel component of the WASp-Arp2/3-ß-actin complex. Pretreating iDCs with an active fragment of the secretory glycoprotein Slit2 (Slit2N) inhibited HIV-1-gp120-mediated migration and podosome formation, by inducing the cognate receptor Roundabout 1 (Robo1) to bind to and sequester WASp and LSP1 from ß-actin. Slit2N treatment also inhibited Src signaling and the activation of several downstream molecules, including Rac1, Pyk2, paxillin, and CDC42, a major regulator of podosome formation. Taken together, our results support a novel mechanism by which Slit2/Robo1 may inhibit the HIV-1-gp120-induced migration of iDCs, thereby restricting dissemination of HIV-1 from mucosal surfaces in the host.

DOCK8 functions as an adaptor that links TLR-MyD88 signaling to B cell activation.

The adaptors DOCK8 and MyD88 have been linked to serological memory. Here we report that DOCK8-deficient patients had impaired antibody responses and considerably fewer CD27(+) memory B cells. B cell proliferation and immunoglobulin production driven by Toll-like receptor 9 (TLR9) were considerably lower in DOCK8-deficient B cells, but those driven by the costimulatory molecule CD40 were not. In contrast, TLR9-driven expression of AICDA (which encodes the cytidine deaminase AID), the immunoglobulin receptor CD23 and the costimulatory molecule CD86 and activation of the transcription factor NF-κB, the kinase p38 and the GTPase Rac1 were intact. DOCK8 associated constitutively with MyD88 and the tyrosine kinase Pyk2 in normal B cells. After ligation of TLR9, DOCK8 became tyrosine-phosphorylated by Pyk2, bound the Src-family kinase Lyn and linked TLR9 to a Src-kinase Syk-transcription factor STAT3 cascade essential for TLR9-driven B cell proliferation and differentiation. Thus, DOCK8 functions as an adaptor in a TLR9-MyD88 signaling pathway in B cells.

Hypophosphorylated and inactive Pyk2 associates with paxillin at the microtubule organizing center in hematopoietic cells.

Pyk2 is a non-receptor tyrosine kinase that regulates cellular adhesion. We generated antibodies to a peptide corresponding to the N-terminus (NT) of Pyk2 and another to a portion of the C-terminal (CT) domain. Only the CT antiserum recovered paxillin-associated Pyk2. These antibodies recognized overlapping but biochemically distinct molecular species of Pyk2 since the CT antiserum recovered Pyk2 after NT antibody immunodepletion. Furthermore, the CT antibody could not immunoblot NT antibody-captured Pyk2. Phosphorylation partially accounts for the differential binding of these antibodies as dephosphorylation of Pyk2 recovered with the NT antibodies allows for recognition by the CT antibody. Additionally, Pyk2 recovered with the NT antibody displays increased serine/threonine phosphorylation. We suggest that the NT epitope is inaccessible to the antibody because Pyk2 is in a closed confirmation in association with paxillin. Upon induction of serine and/or threonine phosphorylation of Pyk2, it opens to a confirmation that allows for antibody binding to the NT epitope but at the same time no longer binds paxillin or the CT antiserum. These antibodies also display differential staining of Pyk2 in both T cells and macrophages. Pyk2 recognized by the CT antibody, but not the NT antibody, colocalized with paxillin at the microtubule-organizing center (MTOC). The MTOC-bound Pyk2 was not tyrosine phosphorylated upon T cell activation. We hypothesize that a reservoir of primarily inactive Pyk2 associates with paxillin at the MTOC, which may allow for rapid delivery of Pyk2 to specific sites of adhesion.

Proline-rich tyrosine kinase-2 is critical for CD8 T-cell short-lived effector fate.

T-cell interactions with antigen-presenting cells are important for CD8 T-cell effector or memory fate determination. The integrin leukocyte function-associated antigen-1 (LFA-1) mediates T-cell adhesion but the contribution of LFA-1-induced signaling pathways to T-cell responses is poorly understood. Here we demonstrate that proline-rich tyrosine kinase-2 (PYK2) deficiency impairs CD8 T-cell activation by synergistic LFA-1 and T-cell receptor stimulation. Furthermore, PYK2 is essential for LFA-1-mediated CD8 T-cell adhesion and LFA-1 costimulation of CD8 T-cell migration. During lymphocytic choriomeningitis virus infection in vivo, PYK2 deficiency results in a specific loss of short-lived effector CD8 T cells but does not affect memory-precursor CD8 T-cell development. Similarly, lack of LFA-1 primarily impairs the generation of short-lived effector cells. Thus, PYK2 facilitates LFA-1-dependent CD8 T-cell responses and promotes CD8 T-cell short-lived effector fate, suggesting that PYK2 may be an interesting therapeutic target to suppress exacerbated CD8 T-cell responses.