Reversal of IKZF1-induced glucocorticoid resistance by dual targeting of AKT and ERK signaling pathways.

Although long-term survival in pediatric acute lymphoblastic leukemia (ALL) currently exceeds 90%, some subgroups, defined by specific genomic aberrations, respond poorly to treatment. We previously reported that leukemias harboring deletions or mutations affecting the B-cell transcription factor IKZF1 exhibit a tumor cell intrinsic resistance to glucocorticoids (GCs), one of the cornerstone drugs used in the treatment of ALL. Here, we identified increased activation of both AKT and ERK signaling pathways as drivers of GC resistance in IKZF1-deficient leukemic cells. Indeed, combined pharmacological inhibition of AKT and ERK signaling effectively reversed GC resistance in IKZF1-deficient leukemias. As inhibitors for both pathways are under clinical investigation, their combined use may enhance the efficacy of prednisolone-based therapy in this high-risk patient group.

Increased intra-articular granzyme M may trigger local IFN-λ1/IL-29 response in rheumatoid arthritis.

OBJECTIVES: Granzymes are serine proteases involved in eliminating tumour cells and virally infected cells. In addition, extracellular granzyme levels are elevated in inflammatory conditions, including several types of infection and autoimmune diseases, such as rheumatoid arthritis (RA). While GrA and GrB have been associated with RA, a role for the other three granzymes (GrH, GrK, and GrM) in this disease remains unclear. Here, we aimed to investigate the presence and role of GrM and GrK in serum and synovial fluid of patients with RA, psoriatic arthritis, and osteoarthritis. METHODS: Granzyme levels were determined in serum, synovial fluid, peripheral blood mononuclear cells (PBMCs) and synovial fluid mononuclear cells (SFMCs) of RA patients and relevant control groups. In addition, the link between GrM and inflammatory cytokines in synovial fluid was investigated. RESULTS: Serum GrM and GrK levels were not affected in RA. GrM, but not GrK, levels were elevated in synovial fluid of RA patients. GrM was mainly expressed by cytotoxic lymphocytes in SFMCs with a similar expression pattern as compared with PBMCs. Intra-articular GrM expression correlated with IL-25, IL-29, XCL1, and TNFα levels. Intriguingly, purified GrM triggered the release of IL-29 (IFN-λ1) from human fibroblasts in vitro. CONCLUSIONS: These data indicate that GrM levels are increased in RA synovial fluid and that GrM can stimulate proinflammatory IL-29 release from fibroblasts, suggesting a role of GrM in the pathogenesis of RA.

Proline-serine-threonine phosphatase interacting protein 1 (PSTPIP1) controls immune synapse stability in human T cells.

BACKGROUND: Proline-serine-threonine phosphatase interacting protein 1 (PSTPIP1) is a cytosolic adaptor protein involved with T-cell activation, differentiation, and migration. On cognate T-cell contact, PSTPIP1 is recruited to surface-expressed CD2, where it regulates F-actin remodeling. An immune synapse (IS) is thereby rapidly formed, consisting of T-cell receptor clusters surrounded by a ring of adhesion molecules, including CD2. OBJECTIVE: From genetic screening of patients with primary immunodeficiencies, we identified 2 mutations in PSTPIP1, R228C and T274M, which we further characterized in the primary patients' T cells. METHODS: F-actin dynamics were assessed in primary T cells from the patients and control subjects by using fluorescence-activated cell sorting. HEK293T and Jurkat cells were transfected with R228C, T274M, and wild-type PSTPIP1 to visualize F-actin in IS formation. CD2-PSTPIP1 association was quantified through immunoprecipitation assays. RESULTS: The patients presented with immunodeficiency without signs of autoinflammation. The patient with the R228C mutation had expansion of mostly naive phenotype T cells and few memory T cells; the patient with the T274M mutation had 75% reduction in CD4 T cells that were predominantly of the memory subset. We observed F-actin polymerization defects in T cells from both patients with PSTPIP1, most notably the patient with the T274M mutation. Capping of CD2-containing membrane microdomains was disrupted. Analysis of IS formation using Jurkat T-cell transfectants revealed a reduction in F-actin accumulation at the IS, again especially in cells from the patient with the T274M PSTPIP1 mutation. T cells from the patient with the T274M mutation migrated spontaneously at increased speed, as assessed in a 3-dimensional collagen matrix, whereas T-cell receptor cross-linking induced a significantly diminished calcium flux. CONCLUSIONS: We propose that PSTPIP1 T-cell differentiation defects are caused by defective control of F-actin polymerization. A preactivated polymerized F-actin status, as seen in T cells from patients with the PSTPIP1 T274M mutation, appears particularly damaging. PSTPIP1 controls IS formation and cell adhesion through its function as an orchestrator of the F-actin cytoskeleton.

Granzyme M and K release in human experimental endotoxemia.

Granzymes are serine proteases involved in killing of tumor cells and virally infected cells. However, granzymes are also upregulated in blood under inflammatory conditions and contribute to cytokine release and processing. Here, we show that granzyme M (GrM) and to a lesser extent GrK are transiently elevated in the circulation following LPS administration in humans. GrM is released upon stimulation of whole blood with LPS or the gram-negative bacteria Escherichia coli BL21, Pseudomonas aeruginosa, and Neisseria meningitidis. GrK is only released upon stimulation with P. aeruginosa. Thus, GrM and GrK are differentially released in response to LPS and gram-negative bacteria.

Reduced serpinB9-mediated caspase-1 inhibition can contribute to autoinflammatory disease.

Patients who suffer from autoinflammatory disease (AID) exhibit seemingly uncontrolled release of interleukin (IL)-1ß. The presence of this inflammatory cytokine triggers immune activation in absence of pathogens and foreign material. The mechanisms that contribute to 'sterile inflammation' episodes in AID patients are not fully understood, although for some AIDs underlying genetic causes have been identified. We show that the serine protease inhibitor B9 (serpinB9) regulates IL-1ß release in human monocytes. SerpinB9 function is more commonly known for its role in control of granzyme B. SerpinB9 however also serves to restrain IL-1ß maturation through caspase-1 inhibition. We here describe an autoinflammatory disease-associated serpinB9 (c.985G>T, A329S) variant, which we discovered in a patient with unknown AID. Using patient cells and serpinB9 overexpressing monocytic cells, we show the A329S variant of serpinB9 exhibits unobstructed granzyme B inhibition, but compromised caspase-1 inhibition. SerpinB9 gene variants might contribute to AID development.

A novel FcγRIIa Q27W gene variant is associated with common variable immune deficiency through defective FcγRIIa downstream signaling.

We identified a novel Q27W FcγRIIa variant that was found more frequently in common variable immunodeficiency (CVID) or CVID-like children. We analyzed the possible functional consequence of the Q27W FcγRIIa mutation in human cells. We used peripheral blood mononuclear cells from Q27W FcγRIIa patients and healthy controls, and cultured cells that overexpress the Q27W and common FcγRIIa variants. The Q27W FcγRIIa mutation does not disrupt FcγRIIa surface expression in peripheral blood mononuclear cells. Mononuclear cells express multiple FcγR, precluding careful analysis of Q27W FcγRIIa functional deviation. For functional analysis of FcγRIIa function, we therefore overexpressed the Q27W FcγRIIa and common FcγRIIa variant in IIA1.6 cells that are normally deficient in FcγR. We show that FcγRIIa triggering-induced signaling is obstructed, as measured by both decrease in calcium flux and defective MAPK phosphorylation. In conclusion, we here describe a novel Q27W FcγRIIa variant that causes delayed downstream signaling. This variant may contribute to CVID.

Defects in mitochondrial clearance predispose human monocytes to interleukin-1ß hypersecretion.

Most hereditary periodic fever syndromes are mediated by deregulated IL-1ß secretion. The generation of mature IL-1ß requires two signals: one that induces synthesis of inflammasome components and substrates and a second that activates inflammasomes. The mechanisms that mediate autoinflammation in mevalonate kinase deficiency, a periodic fever disease characterized by a block in isoprenoid biosynthesis, are poorly understood. In studying the effects of isoprenoid shortage on IL-1 ß generation, we identified a new inflammasome activation signal that originates from defects in autophagy. We find that hypersecretion of IL-1ß and IL-18 requires reactive oxygen species and is associated with an oxidized redox status of monocytes but not lymphocytes. IL-1ß hypersecretion by monocytes involves decreased mitochondrial stability, release of mitochondrial content into the cytosol and attenuated autophagosomal degradation. Defective autophagy, as established by ATG7 knockdown, results in prolonged cytosolic retention of damaged mitochondria and increased IL-1ß secretion. Finally, activation of autophagy in healthy but not mevalonate kinase deficiency patient cells reduces IL-1ß secretion. Together, these results indicate that defective autophagy can prime monocytes for mitochondria-mediated NLRP3 inflammasome activation, thereby contributing to hypersecretion of IL-1ß in mevalonate kinase deficiency.

Synthesis of multivalent Streptococcus suis adhesion inhibitors by enzymatic cleavage of polygalacturonic acid and 'click' conjugation.

A galabiose disaccharide building block was synthesized by an efficient pectinase cleavage of polygalacturonic acid and subsequent chemical functional group transformations. Besides the disaccharide, the corresponding trisaccharide was also obtained and modified. The compounds were subsequently conjugated to dendrimers with up to eight end groups using 'click' chemistry. The compounds were evaluated as inhibitors of adhesion of the pathogen Streptococcus suis in a hemagglutination assay and strong inhibition was observed for the tetra- and octavalent galabiose compound with MIC values in the low nanomolar range. The corresponding octavalent trisaccharide was a ca. 20-fold weaker inhibitor.