Katja Merches

Involvement of Toso in activation of monocytes, macrophages, and granulocytes

Rapid activation of immune responses is necessary for antibacterial defense, but excessive immune activation can result in life-threatening septic shock. Understanding how these processes are balanced may provide novel therapeutic potential in treating inflammatory disease. Fc receptors are crucial for innate immune activation. However, the role of the putative Fc receptor for IgM, known as Toso/Faim3, has to this point been unclear. In this study, we generated Toso-deficient mice and used them to uncover a critical regulatory function of Toso in innate immune activation. Development of innate immune cells was intact in the absence of Toso, but Toso-deficient neutrophils exhibited more reactive oxygen species production and reduced phagocytosis of pathogens compared with controls. Cytokine production was also decreased in Toso−/− mice compared with WT animals, rendering them resistant to septic shock induced by lipopolysaccharide. However, Toso−/− mice also displayed limited cytokine production after infection with the bacterium Listeria monocytogenes that was correlated with elevated presence of Listeria throughout the body. Accordingly, Toso−/− mice succumbed to infections of L. monocytogenes, whereas WT mice successfully eliminated the infection. Taken together, our data reveal Toso to be a unique regulator of innate immune responses during bacterial infection and septic shock.

Stimulation of the glucose carrier SGLT1 by JAK2

JAK2 (Janus kinase-2) overactivity contributes to survival of tumor cells and the (V617F)JAK2 mutant is found in the majority of myeloproliferative diseases. Tumor cell survival depends on availability of glucose. Concentrative cellular glucose uptake is accomplished by Na(+) coupled glucose transport through SGLT1 (SLC5A1), which may operate against a chemical glucose gradient and may thus be effective even at low extracellular glucose concentrations. The present study thus explored whether JAK2 activates SGLT1. To this end, SGLT1 was expressed in Xenopus oocytes with or without wild type JAK2, (V617F)JAK2 or inactive (K882E)JAK2 and electrogenic glucose transport determined by dual electrode voltage clamp experiments. In SGLT1-expressing oocytes but not in oocytes injected with water or JAK2 alone, the addition of glucose to the extracellular bath generated a current (I(g)), which was significantly increased following coexpression of JAK2 or (V617F)JAK2, but not by coexpression of (K882E)JAK2. Kinetic analysis revealed that coexpression of JAK2 enhanced the maximal transport rate without significantly modifying the affinity of the carrier. The stimulating effect of JAK2 expression was abrogated by preincubation with the JAK2 inhibitor AG490. Chemiluminescence analysis revealed that JAK2 enhanced the carrier protein abundance in the cell membrane. The decline of I(g) during inhibition of carrier insertion by brefeldin A was similar in the absence and presence of JAK2. Thus, JAK2 fosters insertion rather than inhibiting retrieval of carrier protein into the cell membrane. In conclusion, JAK2 upregulates SGLT1 activity which may play a role in the effect of JAK2 during ischemia and malignancy.

CEACAM1 induces B-cell survival and is essential for protective antiviral antibody production

B cells are essential for antiviral immune defence because they produce neutralizing antibodies, present antigen and maintain the lymphoid architecture. Here we show that intrinsic signalling of CEACAM1 is essential for generating efficient B-cell responses. Although CEACAM1 exerts limited influence on the proliferation of B cells, expression of CEACAM1 induces survival of proliferating B cells via the BTK/Syk/NF-κB-axis. The absence of this signalling cascade in naive Ceacam1−/− mice limits the survival of B cells. During systemic infection with cytopathic vesicular stomatitis virus, Ceacam1−/− mice can barely induce neutralizing antibody responses and die early after infection. We find, therefore, that CEACAM1 is a crucial regulator of B-cell survival, influencing B-cell numbers and protective antiviral antibody responses.

Stimulation of the amino acid transporter SLC6A19 by JAK2.

JAK2 (Janus kinase-2) is expressed in a wide variety of cells including tumor cells and contributes to the proliferation and survival of those cells. The gain of function mutation (V617F)JAK2 mutant is found in the majority of myeloproliferative diseases. Cell proliferation depends on the availability of amino acids. Concentrative cellular amino acid uptake is in part accomplished by Na(+) coupled amino acid transport through SLC6A19 (B(0)AT). The present study thus explored whether JAK2 activates SLC6A19. To this end, SLC6A19 was expressed in Xenopus oocytes with or without wild type JAK2, (V617F)JAK2 or inactive (K882E)JAK2 and electrogenic amino acid transport determined by dual electrode voltage clamp. In SLC6A19-expressing oocytes but not in oocytes injected with water or JAK2 alone, the addition of leucine (2mM) to the bath generated a current (I(le)), which was significantly increased following coexpression of JAK2 or (V617F)JAK2, but not by coexpression of (K882E)JAK2. Coexpression of JAK2 enhanced the maximal transport rate without significantly modifying the affinity of the carrier. Exposure of the oocytes to the JAK2 inhibitor AG490 (40μM) resulted in a gradual decline of I(le). According to chemiluminescence JAK2 enhanced the carrier protein abundance in the cell membrane. The decline of I(le) following inhibition of carrier insertion by brefeldin A (5μM) was similar in the absence and presence of JAK2 indicating that JAK2 stimulates carrier insertion into rather than inhibiting carrier retrival from the cell membrane. In conclusion, JAK2 up-regulates SLC6A19 activity which may foster amino acid uptake into JAK2 expressing cells.

AMPKα1-sensitivity of Orai1 and Ca(2+) entry in T - lymphocytes.

Background/Aims: T-lymphocyte activation and function critically depends on Ca²⁺ signaling, which is regulated by store operated Ca²⁺ entry (SOCE). Human and mouse T lymphocytes express AMP activated kinase AMPKα1, which is rapidly activated following elevation of cytosolic Ca²⁺ concentration ([Ca²⁺]_i) by treatment of the cells with Ca²⁺ ionophore or following inhibition of endosomal Ca²⁺ ATPase with thapsigargin. AMPK is further activated by triggering of the T cell antigen receptor (TCR). The present study explored whether AMPK influences Ca²⁺ entry and Ca²⁺-sensitive regulation of T-lymphocyte function. Methods: T-lymphocytes were isolated and cultured from AMPKα1-deficient (ampk^-/-) mice and from their wildtype (ampk^+/+) littermates. The phenotype of the cells was analysed by flow cytometry, [Ca²⁺]_i estimated from Fura-2 fluorescence, SOCE from increase of [Ca²⁺]_i following thapsigargin treatment (1 µM), and cell function analysed by measuring cytokine secretion and western blotting. Results: Expression of surface markers in CD4⁺ and CD8⁺ T-cells were similar in ampk^-/- and ampk^+/+ T-lymphocyte blasts. Moreover, total STIM1 protein abundance was similar in ampk^-/- and ampk^+/+ T-lymphocyte blasts. However, Orai1 cell membrane protein abundance was significantly higher in ampk^-/- than in ampk^+/+ T-lymphocyte blasts. SOCE and increase of [Ca²⁺]_i following TCR activation by triggering TCR with anti-CD3 and cross-linking secondary antibody were both significantly more pronounced in ampk^-/- than in ampk^+/+ T-lymphocyte blasts. The difference of Ca²⁺ entry between ampk^-/- and ampk^+/+ T-lymphocytes was abrogated by Orai1 inhibitor 2-aminoethoxydiphenyl borate (2-APB, 50 µM). Proliferation of unstimulated ampk^-/- lymphocytes was higher than proliferation of ampk^+/+ T-lymphocytes, a difference reversed by Orai1 silencing. Conclusions: AMPK downregulates Orai1 and thus SOCE in T-lymphocytes and thus participates in negative feed-back regulation of cytosolic Ca²⁺ activity.

Reply to Honjo et al.: Functional relevant expression of Toso on granulocytes

Functional relevant expression of Toso on granulocytes. (A) Toso expression. Splenocytes from WT and Toso−/− mice were immunostained with anti-CD19 (B cells) and anti-Gr1 (granulocytes) antibody in combination with DAPI and anti-Toso antibody after pretreatment with Fc receptor block. One representative staining of cells gated on Gr1hi DAPI− cells (Left) or CD19+ DAPI− cells is shown (Right). (B) Quantification of FACS stainings derived from A (n = 4/group). (C–E) WT recipient mice were irradiated and reconstituted with BM from WT mice (CD45.1; group 1), Toso−/− mice (CD45.2; group 2), or a 1:1 mixture of WT and Toso−/− BM (group 3). At 30 d posttransplantation, peripheral blood cells were stimulated with different concentrations of fMLP (Formyl-Methionyl-Leucyl-Phenylalanine) and granulocytes (Gr1+ cells) were analyzed after 30 min for CD45.1 expression (anti-CD45.1), CD45.2 expression (anti-CD45.2), reactive oxygen species production (dihydrorhodamine staining), and degranulation (side scatter). Representative FACS …

Virus-Induced Type I Interferon Deteriorates Control of Systemic Pseudomonas Aeruginosa Infection.

Background: Type I interferon (IFN-I) predisposes to bacterial superinfections, an important problem during viral infection or treatment with interferon-alpha (IFN-α). IFN-I-induced neutropenia is one reason for the impaired bacterial control; however there is evidence that more frequent bacterial infections during IFN-α-treatment occur independently of neutropenia. Methods: We analyzed in a mouse model, whether Pseudomonas aeruginosa control is influenced by co-infection with the lymphocytic choriomeningitis virus (LCMV). Bacterial titers, numbers of neutrophils and the gene-expression of liver-lysozyme-2 were determined during a 24 hours systemic infection with P. aeruginosa in wild-type and Ifnar-/- mice under the influence of LCMV or poly(I:C). Results: Virus-induced IFN-I impaired the control of Pseudomonas aeruginosa. This was associated with neutropenia and loss of lysozyme-2-expression in the liver, which had captured P. aeruginosa. A lower release of IFN-I by poly(I:C)-injection also impaired the bacterial control in the liver and reduced the expression of liver-lysozyme-2. Low concentration of IFN-I after infection with a virulent strain of P. aeruginosa alone impaired the bacterial control and reduced lysozyme-2-expression in the liver as well. Conclusion: We found that during systemic infection with P. aeruginosa Kupffer cells quickly controlled the bacteria in cooperation with neutrophils. Upon LCMV-infection this cooperation was disturbed.

Toso regulates differentiation and activation of inflammatory dendritic cells during persistence-prone virus infection.

During virus infection and autoimmune disease, inflammatory dendritic cells (iDCs) differentiate from blood monocytes and infiltrate infected tissue. Following acute infection with hepatotropic viruses, iDCs are essential for re-stimulating virus-specific CD8+ T cells and therefore contribute to virus control. Here we used the lymphocytic choriomeningitis virus (LCMV) model system to identify novel signals, which influence the recruitment and activation of iDCs in the liver. We observed that intrinsic expression of Toso (Faim3, FcμR) influenced the differentiation and activation of iDCs in vivo and DCs in vitro. Lack of iDCs in Toso-deficient (Toso–/–) mice reduced CD8+ T-cell function in the liver and resulted in virus persistence. Furthermore, Toso–/– DCs failed to induce autoimmune diabetes in the rat insulin promoter-glycoprotein (RIP-GP) autoimmune diabetes model. In conclusion, we found that Toso has an essential role in the differentiation and maturation of iDCs, a process that is required for the control of persistence-prone virus infection.

AKT/SGK-sensitive phosphorylation of GSK3 in the regulation of L-selectin and perforin expression as well as activation induced cell death of T-lymphocytes.

Survival and function of T-lymphocytes critically depends on phosphoinositide (PI) 3 kinase. PI3 kinase signaling includes the PKB/Akt and SGK dependent phosphorylation and thus inhibition of glycogen synthase kinase GSK3α,β. Lithium, a known unspecific GSK3 inhibitor protects against experimental autoimmune encephalomyelitis. The present study explored, whether Akt/SGK-dependent regulation of GSK3 activity is a determinant of T cell survival and function. Experiments were performed in mutant mice in which Akt/SGK-dependent GSK3α,β inhibition was disrupted by replacement of the serine residue in the respective SGK/Akt-phosphorylation consensus sequence by alanine (gsk3(KI)). T cells from gsk3(KI) mice were compared to T cells from corresponding wild type mice (gsk3(WT)). As a result, in gsk3(KI) CD4(+) cells surface CD62L (L-selectin) was significantly less abundant than in gsk3(WT) CD4(+) cells. Upon activation in vitro T cells from gsk3(KI) mice reacted with enhanced perforin production and reduced activation induced cell death. Cytokine production was rather reduced in gsk3(KI) T cells, suggesting that GSK3 induces effector function in CD8(+) T cells. In conclusion, PKB/Akt and SGK sensitive phosphorylation of GSK3α,β is a potent regulator of perforin expression and activation induced cell death in T lymphocytes.