Knockout mice reveal a role for protein tyrosine phosphatase H1 in cognition

<p>Abstract</p> <p>Background</p> <p>The present study has investigated the protein tyrosine phosphatase H1 (PTPH1) expression pattern in mouse brain and its impact on CNS functions.</p> <p>Methods</p> <p>We have previously described a PTPH1-KO mouse, generated by replacing the PTP catalytic and the PDZ domain with a LacZ neomycin cassette. PTPH1 expression pattern was evaluated by LacZ staining in the brain and PTPH1-KO and WT mice (n = 10 per gender per genotype) were also behaviorally tested for CNS functions.</p> <p>Results</p> <p>In CNS, PTPH1 is expressed during development and in adulthood and mainly localized in hippocampus, thalamus, cortex and cerebellum neurons. The behavioral tests performed on the PTPH1-KO mice showed an impact on working memory in male mice and an impaired learning performance at rotarod in females.</p> <p>Conclusion</p> <p>These results demonstrate for the first time a neuronal expression of PTPH1 and its functionality at the level of cognition.</p

C1q-Mediated Complement Activation and C3 Opsonization Trigger Recognition of Stealth Poly(2-methyl-2-oxazoline)-Coated Silica Nanoparticles by Human Phagocytes

Poly(2-methyl-2-oxazoline) (PMOXA) is an alternative promising polymer to poly(ethylene glycol) (PEG) for design and engineering of macrophage-evading nanoparticles (NPs). Although PMOXA-engineered NPs have shown comparable pharmacokinetics and in vivo performance to PEGylated stealth NPs in the murine model, its interaction with elements of the human innate immune system has not been studied. From a translational angle, we studied the interaction of fully characterized PMOXA-coated vinyltriethoxysilane-derived organically modified silica NPs (PMOXA-coated NPs) of approximately 100 nm in diameter with human complement system, blood leukocytes, and macrophages and compared their performance with PEGylated and uncoated NP counterparts. Through detailed immunological and proteomic profiling, we show that PMOXA-coated NPs extensively trigger complement activation in human sera exclusively through the classical pathway. Complement activation is initiated by the sensing molecule C1q, where C1q binds with high affinity (Kd = 11 \ub1 1 nM) to NP surfaces independent of immunoglobulin binding. C1q-mediated complement activation accelerates PMOXA opsonization with the third complement protein (C3) through the amplification loop of the alternative pathway. This promoted NP recognition by human blood leukocytes and monocyte-derived macrophages. The macrophage capture of PMOXA-coated NPs correlates with sera donor variability in complement activation and opsonization but not with other major corona proteins, including clusterin and a wide range of apolipoproteins. In contrast to these observations, PMOXA-coated NPs poorly activated the murine complement system and were marginally recognized by mouse macrophages. These studies provide important insights into compatibility of engineered NPs with elements of the human innate immune system for translational steps

The Soluble Recombinant Neisseria meningitidis Adhesin NadAΔ351–405 Stimulates Human Monocytes by Binding to Extracellular Hsp90

The adhesin NadA favors cell adhesion/invasion by hypervirulent Neisseria meningitidis B (MenB). Its recombinant form NadAΔ351–405, devoid of the outer membrane domain, is an immunogenic candidate for an anti-MenB vaccine able to stimulate monocytes, macrophages and dendritic cells. In this study we investigated the molecular mechanism of NadAΔ351–405 cellular effects in monocytes. We show that NadAΔ351–405 (against which we obtained polyclonal antibodies in rabbits), binds to hsp90, but not to other extracellular homologous heat shock proteins grp94 and hsp70, in vitro and on the surface of monocytes, in a temperature dependent way. Pre-incubation of monocytes with the MenB soluble adhesin interfered with the binding of anti-hsp90 and anti-hsp70 antibodies to hsp90 and hsp70 at 37°C, a condition in which specific cell-binding occurs, but not at 0°C, a condition in which specific cell-binding is very diminished. Conversely, pre-incubation of monocytes with anti-hsp90 and anti-hsp70 antibodies did not affected NadAΔ351–405 cell binding in any temperature condition, indicating that it associates to another receptor on their plasma membrane and then laterally diffuses to encounter hsp90. Consistently, polymixin B interfered with NadAΔ351–405 /hsp90 association, abrogated the decrease of anti-hsp90 antibodies binding to the cell surface due to NadAΔ351–405 and inhibited adhesin-induced cytokine/chemokine secretion without affecting monocyte-adhesin binding. Co-stimulation of monocytes with anti-hsp90 antibodies and NadAΔ351–405 determined a stronger but polymixin B insensitive cell activation. This indicated that the formation of a recombinant NadA/hsp90/hsp70 complex, although essential for full monocyte stimulation, can be replaced by anti-hsp90 antibody/hsp90 binding. Finally, the activation of monocytes by NadAΔ351–405 alone or in the presence of anti-hsp90 antibodies were both inhibited by neutralizing anti-TLR4 antibodies, but not by anti-TLR2 antibodies. We propose that hsp90-dependent recruitment into an hsp90/hsp70/TLR4 transducing signal complex is necessary for the immune-stimulating activity of NadAΔ351–405 anti-MenB vaccine candidate

Variations of the corona HDL:albumin ratio determine distinct effects of amorphous SiO2 nanoparticles on monocytes and macrophages in serum.

Aim: We investigated monocyte and macrophage death and cytokine production induced by amorphous silica nanoparticles (SiO2-NPs) to clarify the role of defined serum corona proteins. Materials & methods: The cytotoxic proinflammatory effects of SiO2-NPs on human monocytes and macrophages were characterized in no serum, in fetal calf serum and in the presence of purified corona proteins. Results: In no serum and in fetal calf serum above approximately 75 \ub5g/ml, SiO2-NPs lysed monocytes and macrophages by plasma membrane damage (necrosis). In fetal calf serum below approximately 75 \ub5g/ml, SiO2-NPs triggered an endolysosomal acidification and caspase-1-dependent monocyte death (pyroptosis). The corona high-density lipoproteins:albumin ratio accounted for the features of the SiO2-NPs in serum. Discussion: Corona high-density lipoproteins are a major determinant of the differential cytotoxic action of SiO2-NPs on monocytes and macrophage

The functional dissection of the plasma corona of SiO2-NPs spots histidine rich glycoprotein as a major player able to hamper nanoparticle capture by macrophages

A coat of strongly-bound host proteins, or hard corona, may influence the biological and pharmacological features of nanotheranostics by altering their cell-interaction selectivity and macrophage clearance. With the goal of identifying specific corona-effectors, we investigated how the capture of amorphous silica nanoparticles (SiO2-NPs; 8 = 26 nm; zeta potential = -18.3 mV) by human lymphocytes, monocytes and macrophages is modulated by the prominent proteins of their plasma corona. LC MS/MS analysis, western blotting and quantitative SDS-PAGE densitometry show that Histidine Rich Glycoprotein (HRG) is the most abundant component of the SiO2-NP hard corona in excess plasma from humans (HP) and mice (MP), together with minor amounts of the homologous Kininogen-1 (Kin-1), while it is remarkably absent in their Foetal Calf Serum (FCS)-derived corona. HRG binds with high affinity to SiO2-NPs (HRG Kd 3c2 nM) and competes with other plasma proteins for the NP surface, so forming a stable and quite homogeneous corona inhibiting nanoparticles binding to the macrophage membrane and their subsequent uptake. Conversely, in the case of lymphocytes and monocytes not only HRG but also several common plasma proteins can interchange in this inhibitory activity. The depletion of HRG and Kin-1 from HP or their plasma exhaustion by increasing NP concentration (>40 \u3bcg ml-1 in 10% HP) lead to a heterogeneous hard corona, mostly formed by fibrinogen (Fibr), HDLs, LDLs, IgGs, Kallikrein and several minor components, allowing nanoparticle binding to macrophages. Consistently, the FCS-derived SiO2-NP hard corona, mainly formed by hemoglobin, \u3b12 macroglobulin and HDLs but lacking HRG, permits nanoparticle uptake by macrophages. Moreover, purified HRG competes with FCS proteins for the NP surface, inhibiting their recruitment in the corona and blocking NP macrophage capture. HRG, the main component of the plasma-derived SiO2-NPs' hard corona, has antiopsonin characteristics and uniquely confers to these particles the ability to evade macrophage capture

Polymixin B inhibits the induction of cytokine/chemokine by NadA_Δ351–405 in monocytes.

<p>Human monocytes were stimulated for 24 hours with the indicated concentrations of NadA_Δ351–405 in the absence (open circles) or in the presence of 10 µg/ml polymixin B in DMEM plus 10% FCS. Cytokines/chemokines were subsequently quantified in the cells extracellular medium using a BioPlex assay. Data are from a representative experiment of three, run in duplicate and bars are ranges.</p

NadA_Δ351–405 binds to human hsp90 in an overlay-assay.

<p>A) Samples of total monocytes extracts obtained with 1% TX-100 were separated by SDS-PAGE and challenged or not as indicated with 1 µM NadA_Δ351–405 after blotting on nitrocellulose. After extensive washings, the polypeptide bands able to retain NadA were detected with antibodies to the whole adhesin or to NadA 52–70, as indicated. The picture is from a representative experiment out of four and arrows point to the NadA-interacting ∼90 KDa protein. B) Monocytes detergent extracts prepared as in A) were separated by SDS-PAGE, blotted on nitrocellulose and subjected to western blot with anti- grp94 and hsp90 antibodies or to an overlay assay with NadA/anti-NadA antibodies. The mobility of the NadA interacting protein and of hsp90 or grp94 was compared. C) Purified human hsp90 were subjected to overlay assay with (1 µM) or without NadA_Δ351–405 and HP-NAP as indicated. D) Whole TX-100 extracts from human monocytes were incubated with protein A sepharose-bound anti-NadA IgG in the presence or not of NadA_Δ351–405. After washings, beads were treated with LSB containing 4%SDS and 10% βme and analyzed by western blot after SDS-PAGE using anti-hsp90 antibodies and alkaline phosphatase-conjugated anti-IgG secondary antibodies. Arrow points to hsp90, H and L indicate antibody heavy and light chains respectively.</p