Sandra Beer-Hammer

Murine Guanylate Binding Protein 2 (mGBP2) controls Toxoplasma gondii replication

IFN-γ orchestrates the host response against intracellular pathogens. Members of the guanylate binding proteins (GBP) comprise the most abundant IFN-γ–induced transcriptional response. mGBPs are GTPases that are specifically up-regulated by IFN-γ, other proinflammatory cytokines, toll-like receptor agonists, as well as in response to Listeria monocytogenes and Toxoplasma gondii infection. mGBP2 localizes at the parasitophorous vacuole (PV) of T. gondii; however, the molecular function of mGBP2 and its domains in T. gondii infection is not known. Here, we show that mGBP2 is highly expressed in several cell types, including T and B cells after stimulation. We provide evidence that the C-terminal domain is sufficient and essential for recruitment to the T. gondii PV. Functionally, mGBP2 reduces T. gondii proliferation because mGBP2-deficient cells display defects in the replication control of T. gondii. Ultimately, mGBP2-deficient mice reveal a marked immune susceptibility to T. gondii. Taken together, mGBP2 is an essential immune effector molecule mediating antiparasitic resistance.

Primary cilium migration depends on G-protein signalling control of subapical cytoskeleton

In ciliated mammalian cells, the precise migration of the primary cilium at the apical surface of the cells, also referred to as translational polarity, defines planar cell polarity (PCP) in very early stages. Recent research has revealed a co-dependence between planar polarization of some cell types and cilium positioning at the surface of cells. This important role of the primary cilium in mammalian cells is in contrast with its absence from Drosophila melanogaster PCP establishment. Here, we show that deletion of GTP-binding protein alpha-i subunit 3 (Gαi3) and mammalian Partner of inscuteable (mPins) disrupts the migration of the kinocilium at the surface of cochlear hair cells and affects hair bundle orientation and shape. Inhibition of G-protein function in vitro leads to kinocilium migration defects, PCP phenotype and abnormal hair bundle morphology. We show that Gαi3/mPins are expressed in an apical and distal asymmetrical domain, which is opposite and complementary to an aPKC/Par-3/Par-6b expression domain, and non-overlapping with the core PCP protein Vangl2. Thus G-protein-dependent signalling controls the migration of the cilium cell autonomously, whereas core PCP signalling controls long-range tissue PCP.

Intranasal Delivery of Bone Marrow-Derived Mesenchymal Stem Cells, Macrophages, and Microglia to the Brain in Mouse Models of Alzheimer's and Parkinson's Disease

In view of the rapid preclinical development of cell-based therapies for neurodegenerative disorders, traumatic brain injury, and tumors, the safe and efficient delivery and targeting of therapeutic cells to the central nervous system is critical for maintaining therapeutic efficacy and safety in the respective disease models. Our previous data demonstrated therapeutically efficacious and targeted delivery of mesenchymal stem cells (MSCs) to the brain in the rat 6-hydroxydopamine model of Parkinsons disease (PD). The present study examined delivery of bone marrow-derived MSCs, macrophages, and microglia to the brain in a transgenic model of PD [(Thy1)-h[A30P] aS] and an APP/PS1 model of Alzheimers disease (AD) via intranasal application (INA). INA of microglia in naive BL/6 mice led to targeted and effective delivery of cells to the brain. Quantitative PCR analysis of eGFP DNA showed that the brain contained the highest amount of eGFP-microglia (up to 2.1 × 104) after INA of 1 × 106 cells, while the total amount of cells detected in peripheral organs did not exceed 3.4 × 103. Seven days after INA, MSCs expressing eGFP were detected in the olfactory bulb (OB), cortex, amygdala, striatum, hippocampus, cerebellum, and brainstem of (Thy1)-h[A30P] aS transgenic mice, showing predominant distribution within the OB and brainstem. INA of eGFP-expressing macrophages in 13-month-old APP/PS1 mice led to delivery of cells to the OB, hippocampus, cortex, and cerebellum. Both MSCs and macrophages contained Iba-1-positive population of small microglia-like cells and Iba-1-negative large rounded cells showing either intracellular amyloid β (macrophages in APP/PS1 model) or α-synuclein [MSCs in (Thy1)-h[A30P] aS model] immunoreactivity. Here, we show, for the first time, intranasal delivery of cells to the brain of transgenic PD and AD mouse models. Additional work is needed to determine the optimal dosage (single treatment regimen or repeated administrations) to achieve functional improvement in these mouse models with intranasal microglia/macrophages and MSCs. This manuscript is published as part of the International Association of Neurorestoratology (IANR) special issue of Cell Transplantation.

Depletion of Dendritic Cells Enhances Innate Anti-Bacterial Host Defense through Modulation of Phagocyte Homeostasis

Dendritic cells (DCs) as professional antigen-presenting cells play an important role in the initiation and modulation of the adaptive immune response. However, their role in the innate immune response against bacterial infections is not completely defined. Here we have analyzed the role of DCs and their impact on the innate anti-bacterial host defense in an experimental infection model of Yersinia enterocolitica (Ye). We used CD11c-diphtheria toxin (DT) mice to deplete DCs prior to severe infection with Ye. DC depletion significantly increased animal survival after Ye infection. The bacterial load in the spleen of DC-depleted mice was significantly lower than that of control mice throughout the infection. DC depletion was accompanied by an increase in the serum levels of CXCL1, G-CSF, IL-1α, and CCL2 and an increase in the numbers of splenic phagocytes. Functionally, splenocytes from DC-depleted mice exhibited an increased bacterial killing capacity compared to splenocytes from control mice. Cellular studies further showed that this was due to an increased production of reactive oxygen species (ROS) by neutrophils. Adoptive transfer of neutrophils from DC-depleted mice into control mice prior to Ye infection reduced the bacterial load to the level of Ye-infected DC-depleted mice, suggesting that the increased number of phagocytes with additional ROS production account for the decreased bacterial load. Furthermore, after incubation with serum from DC-depleted mice splenocytes from control mice increased their bacterial killing capacity, most likely due to enhanced ROS production by neutrophils, indicating that serum factors from DC-depleted mice account for this effect. In summary, we could show that DC depletion triggers phagocyte accumulation in the spleen and enhances their anti-bacterial killing capacity upon bacterial infection.

The catalytic PI3K isoforms p110γ and p110δ contribute to B cell development and maintenance, transformation, and proliferation

Class I PI3K‐dependent signaling regulates cell proliferation, differentiation, and survival. Analysis of gene‐deficient mice revealed specific roles for the hematopoietically expressed PI3K catalytic subunits, p110γ and p110δ, in development and function of T and B lymphocytes. However, the functional redundancy between these two PI3K isoforms in the B cell lineage remains unclear. Here, we demonstrate that p110δ and p110γ are expressed in B cells at early developmental stages. Normal B cell differentiation requires both isoforms, as p110γ/p110δ double deficiency causes an increased percentage of CD43hi/B220+/CD19− cells as compared with single deficiency. Interestingly, initial transformation efficiency of B cell precursors was strongly reduced in double‐deficient cells following transformation by p185 bcr‐abl or v‐abl oncogenes as compared with single‐deficient cells. The requirement of p110γ and p110δ in B cell development is underlined by reduced splenic B cell numbers of p110γ/p110δ double‐deficient mice and of lethally irradiated wild‐type mice reconstituted with double‐deficient BM. Moreover, the peripheral maintenance of p110γ/p110δ double‐deficient T and B cells was highly impaired following adoptive transfer of double‐deficient splenocytes into wild‐type mice. Functionally, LPS stimulation of splenocytes revealed proliferation defects resulting in decreased survival of p110γ/p110δ double‐deficient B cells, which correlated with impaired induction of D‐type cyclins and Bcl‐XL. Surprisingly, this was not observed when purified B cells were analyzed, indicating a contribution of likely cell‐extrinsic factor(s) to the impaired proliferation of double‐deficient B cells. Thus, we provide novel evidence that p110γ and p110δ have overlapping and cell‐extrinsic roles in the development, peripheral maintenance, and function of B cells.

Staphylococcus aureus Phenol-Soluble Modulin Peptides Modulate Dendritic Cell Functions and Increase In Vitro Priming of Regulatory T Cells

The major human pathogen Staphylococcus aureus has very efficient strategies to subvert the human immune system. Virulence of the emerging community-associated methicillin-resistant S. aureus depends on phenol-soluble modulin (PSM) peptide toxins, which are known to attract and lyse neutrophils. However, their influences on other immune cells remain elusive. In this study, we analyzed the impact of PSMs on dendritic cells (DCs) playing an essential role in linking innate and adaptive immunity. In human neutrophils, PSMs exert their function by binding to the formyl peptide receptor (FPR) 2. We show that mouse DCs express the FPR2 homolog mFPR2 as well as its paralog mFPR1 and that PSMs are chemoattractants for DCs at noncytotoxic concentrations. PSMs reduced clathrin-mediated endocytosis and inhibited TLR2 ligand-induced secretion of the proinflammatory cytokines TNF, IL-12, and IL-6, while inducing IL-10 secretion by DCs. As a consequence, treatment with PSMs impaired the capacity of DCs to induce activation and proliferation of CD4+ T cells, characterized by reduced Th1 but increased frequency of FOXP3+ regulatory T cells. These regulatory T cells secreted high amounts of IL-10, and their suppression capacity was dependent on IL-10 and TGF-β. Interestingly, the induction of tolerogenic DCs by PSMs appeared to be independent of mFPRs, as shown by experiments with mice lacking mFPR2 (mFPR2−/−) and the cognate G protein (p110γ−/−). Thus, PSMs from highly virulent pathogens affect DC functions, thereby modulating the adaptive immune response and probably increasing the tolerance toward the pathogen.

Cell-intrinsic and -extrinsic control of Treg-cell homeostasis and function revealed by induced CD28 deletion

While the requirement for CD28 and its ligands for the generation and function of “natural” (n)Treg cells is well established, it has not been possible yet to investigate cell‐intrinsic effects after interrupted CD28 expression. Here, we demonstrate a selective loss of Treg cells after disruption of the CD28 gene. The decline in Treg‐cell number was accompanied by reduced homeostatic proliferation, probably due to lack of costimulation during self‐antigen recognition, and by impaired Treg‐cell function including downregulation of CTLA‐4. The decline in Treg‐cell number was unaffected by thymectomy or by the presence of CD28 expressing T cells within the same animal, indicating that impairment of peripheral homeostasis and function of nTreg cells by CD28 deletion is cell‐intrinsic. In contrast, downregulation of CD25, the α chain of the IL‐2R, did not occur in the presence of WT T cells, indicating that its expression does not depend on CD28 signals in cis.

Defective Macrophage Migration in Gαi2- but Not Gαi3-Deficient Mice

Various heterotrimeric Gi proteins are considered to be involved in cell migration and effector function of immune cells. The underlying mechanisms, how they control the activation of myeloid effector cells, are not well understood. To elucidate isoform-redundant and -specific roles for Gαi proteins in these processes, we analyzed mice genetically deficient in Gαi2 or Gαi3. First, we show an altered distribution of tissue macrophages and blood monocytes in the absence of Gαi2 but not Gαi3. Gαi2-deficient but not wild-type or Gαi3-deficient mice exhibited reduced recruitment of macrophages in experimental models of thioglycollate-induced peritonitis and LPS-triggered lung injury. In contrast, genetic ablation of Gαi2 had no effect on Gαi-dependent peritoneal cytokine production in vitro and the phagocytosis-promoting function of the Gαi-coupled C5a anaphylatoxin receptor by liver macrophages in vivo. Interestingly, actin rearrangement and CCL2- and C5a anaphylatoxin receptor-induced chemotaxis but not macrophage CCR2 and C5a anaphylatoxin receptor expression were reduced in the specific absence of Gαi2. Furthermore, knockdown of Gαi2 caused decreased cell migration and motility of RAW 264.7 cells, which was rescued by transfection of Gαi2 but not Gαi3. These results indicate that Gαi2, albeit redundant to Gαi3 in some macrophage activation processes, clearly exhibits a Gαi isoform-specific role in the regulation of macrophage migration.

Gαi2 Is the Essential Gαi Protein in Immune Complex–Induced Lung Disease

Heterotrimeric G proteins of the Gαi family have been implicated in signaling pathways regulating cell migration in immune diseases. The Gαi-protein–coupled C5a receptor is a critical regulator of IgG FcR function in experimental models of immune complex (IC)–induced inflammation. By using mice deficient for Gαi2 or Gαi3, we show that Gαi2 is necessary for neutrophil influx in skin and lung Arthus reactions and agonist-induced neutrophilia in the peritoneum, whereas Gαi3 plays a less critical but variable role. Detailed analyses of the pulmonary IC-induced inflammatory response revealed several shared functions of Gαi2 and Gαi3, including mediating C5a anaphylatoxin receptor–induced activation of macrophages, involvement in alveolar production of chemokines, transition of neutrophils from bone marrow into blood, and modulation of CD11b and CD62L expression that account for neutrophil adhesion to endothelial cells. Interestingly, C5a-stimulated endothelial polymorphonuclear neutrophil transmigration, but not chemotaxis, is enhanced versus reduced in the absence of neutrophil Gαi3 or Gαi2, respectively, and knockdown of endothelial Gαi2 caused decreased transmigration of wild-type neutrophils. These data demonstrate that Gαi2 and Gαi3 contribute to inflammation by redundant, overlapping, and Gαi-isoform–specific mechanisms, with Gαi2 exhibiting unique functions in both neutrophils and endothelial cells that appear essential for polymorphonuclear neutrophil recruitment in IC disease.

p87 and p101 Subunits Are Distinct Regulators Determining Class IB Phosphoinositide 3-Kinase (PI3K) Specificity

Background: p87 and p101 represent non-catalytic subunits of class IB PI3Kγ. Results: Expression and activity of PI3Kγ is modified differently by p87 and p101 in vitro and in living cells. Conclusion: Non-catalytic subunits of PI3Kγ represent two different regulators in the absence of Gβγ or Ras. Significance: p87 and p101 determine diversity within class IB PI3Kγ and allow integration in distinct PI3Kγ signaling pathways. Class IB phosphoinositide 3-kinase γ (PI3Kγ) comprises a single catalytic p110γ subunit, which binds to two non-catalytic subunits, p87 or p101, and controls a plethora of fundamental cellular responses. The non-catalytic subunits are assumed to be redundant adaptors for Gβγ enabling G-protein-coupled receptor-mediated regulation of PI3Kγ. Growing experimental data provide contradictory evidence. To elucidate the roles of the non-catalytic subunits in determining the specificity of PI3Kγ, we tested the impact of p87 and p101 in heterodimeric p87-p110γ and p101-p110γ complexes on the modulation of PI3Kγ activity in vitro and in living cells. RT-PCR, biochemical, and imaging data provide four lines of evidence: (i) specific expression patterns of p87 and p101, (ii) up-regulation of p101, providing the basis to consider p87 as a protein forming a constitutively and p101 as a protein forming an inducibly expressed PI3Kγ, (iii) differences in basal and stimulated enzymatic activities, and (iv) differences in complex stability, all indicating apparent diversity within class IB PI3Kγ. In conclusion, expression and activities of PI3Kγ are modified differently by p87 and p101 in vitro and in living cells, arguing for specific regulatory roles of the non-catalytic subunits in the differentiation of PI3Kγ signaling pathways.