Reduction of IKKα Expression Promotes Chronic Ultraviolet B Exposure-Induced Skin Inflammation and Carcinogenesis

Ultraviolet B light (UVB) is a common cause of human skin cancer. UVB irradiation induces mutations in the tumor suppressor p53 gene as well as chronic inflammation, which are both essential for UVB carcinogenesis. Inhibitor of nuclear factor κB kinase-α (IKKα) plays an important role in maintaining skin homeostasis, and expression of IKKα was found to be down-regulated in human and murine skin squamous cell carcinomas. However, the role of IKKα in UVB skin carcinogenesis has not been investigated. Thus, here we performed UVB carcinogenesis experiments on Ikkα^+/+ and Ikkα^+/− mice. Ikkα^+/− mice were found to develop a twofold greater number of skin tumors than Ikkα^+/+ mice after chronic UVB irradiation. In addition, tumor latency was significantly shorter and tumors were bigger in Ikkα^+/− than in Ikkα^+/+ mice. At an early stage of carcinogenesis, an increase in UVB-induced p53 mutations as well as macrophage recruitment and mitogenic activity, and a decrease in UVB-induced apoptosis, were detected in Ikkα^+/− compared with those in Ikkα^+/+ skin. Also, reduction of IKKα levels in keratinocytes up-regulated the expression of monocyte chemoattractant protein-1 (MCP-1/CCL2), TNFα, IL-1, and IL-6, and elevated macrophage migration, which might promote macrophage recruitment and inflammation. Therefore, these findings suggest that reduction of IKKα expression orchestrates UVB carcinogen, accelerating tumorigenesis.Ultraviolet B (UVB) irradiation induces DNA damage. The tumor suppressor gene p53 is an UVB target, and human cutaneous squamous cell carcinoma (SCC) cells contain p53 mutations.¹ Skin cells can harbor UVB-induced p53 mutations for decades before the onset of human SCC, however,^2,3 underscoring the importance of coactivators in skin tumorigenesis. In mice, p53 mutations are an early genetic event in UVB skin carcinogenesis, which recapitulates the process of human SCC development.^4,5 The p53 mutations have been proposed to be important for UVB carcinogenesis because prevention of these mutations prevents skin tumor development, and the number of p53 mutation-positive keratinocytes correlates with the number of skin tumors in mice.^6,7,8,9 UVB exposure also induces chronic inflammation, cell proliferation, oxidant stress, and immunosuppression, which essentially facilitate UVB carcinogenesis.^10,11 Particularly, chronic inflammation can create a microenvironment that is prone to cell proliferation and DNA damage, thereby promoting tumor development.¹²On the other hand, UVB exposure also elicits protective responses, such as cell cycle arrest, DNA repair, and apoptosis, which reduce UVB-induced damage.^7,10,11 Previously, it was reported that mice lacking p53 were defective in inducing apoptosis after UVB irradiation and thus had more skin tumors than wild-type mice did.^13,14 Mice lacking Fas ligand had defects in apoptosis, which increased numbers of cells containing UVB-induced p53 mutations, and the mutant mice were more susceptible to UVB-induced skin tumors than wild-type mice.^5,8 Therefore, the different defects in inducing protective responses against UVB-induced damage can amplify the severity of the cancer cause, thereby accelerating carcinogenesis.Inhibitor of nuclear factor κB kinase-α (IKKα) is required for embryonic skin development in mice.^15,16 Several laboratories have reported the down-regulated expression and altered localization of IKKα protein as well as deletions and mutations in the Ikkα gene in human SCCs of the skin, lung, esophagus, and head and neck.^{17,18,19,20,21} In particular, IKKα deletion in keratinocytes was found to elevate an autocrine loop activity of epidermal growth factor receptor (EGFR), Ras, extracellular signal-related kinase (ERK), EGFR ligands, and the ligands’ activators and induced spontaneous skin SCCs in mice.²² Reduction of IKKα expression was found to promote chemical carcinogen-induced skin tumorigenesis.²³ Also, IKKα is an UVB-induced gene, and the defect in IKKα function was linked to inflammation.^24,25 UVB is a very common cause of human skin cancer; however, the role of IKKα in skin UVB carcinogenesis is largely unknown. Thus, here we examined the effect of reduced IKKα expression on UVB skin carcinogenesis in Ikkα^+/− and Ikkα^+/+ mice. Ikkα^+/− mice were significantly susceptible to UVB skin carcinogenesis than were Ikkα^+/+ mice. Because the tumor latency was significantly shorter and many more tumors were found in Ikkα^+/− mice than in Ikkα^+/+ mice, we analyzed the early events during UVB skin carcinogenesis. This study provided the first evidence showing the importance of IKKα in UVB skin carcinogenesis.     

Resistance of CD1d-/- mice to ultraviolet-induced skin cancer is associated with increased apoptosis

Inhibition of p53-induced epidermal apoptosis, generation of p53 mutations, and suppressor T cells are the critical events responsible for the induction and development of UV-induced skin cancers. Recently, we demonstrated that CD1d knockout mice were resistant to UV-induced immunosuppression, prompting us to further address the role of CD1d in regulating UV carcinogenesis. We, therefore, investigated the response of wild-type (WT) and CD1d-/- mice to UV carcinogenesis. We found that although 100% of WT mice developed skin tumors after 45 weeks of UV irradiation, only 60% of CD1d-/- mice developed skin tumors. Surprisingly, keratinocytes and fibroblasts from CD1d-/- mice were more sensitive to UV-induced apoptosis and persisted longer than cells derived from WT mice. In addition, epidermis and dermis taken from chronically UV-irradiated CD1d-/- mice harbored significantly fewer p53 mutations than WT mice. Our findings identify an unexpected and novel function for CD1d as a critical molecule regulating UV carcinogenesis, by inhibiting apoptosis to prevent elimination of potentially malignant keratinocytes and fibroblasts.     

Up-Regulation of p21WAF1 by Phorbol Ester and Calcium in Human Keratinocytes through a Protein Kinase C-Dependent Pathway

Terminal differentiation in a variety of cell types has been associated with p53-independent up-regulation of p21^WAF1. p21^WAF1 mRNA and protein are expressed at low levels in normal human skin, but overexpression of p21^WAF1 has been observed in differentiating keratinocytes in involved psoriatic epidermis and in human squamous cell carcinoma. In this study we investigated by immunohistochemistry and Western blotting whether calcium and the phorbol ester 12-O-tetradecanoylphorbol-13-acetate, well characterized differentiation signals, induce p21^WAF1 in cultured normal human keratinocytes and whether induction of p21^WAF1 in this system depends on protein kinase C activation or functional p53. Phorbol ester induced p21^WAF1 expression, which was maximal at 4 to 8 h with reduction back to baseline by 24 to 48 h. In contrast, increasing the extracellular Ca²⁺ concentration from 70 μmol/L to 1.5 mmol/L resulted in up-regulation of p21^WAF1 expression with a slower time course, with peak induction at 18 to 24 h. No parallel increase in p53 expression was observed in normal human keratinocytes. Up-regulation of p21^WAF1 was also observed in response to phorbol ester in HaCaT cells, which carry homozygous and inactivating mutations for p53. Induction of p21^WAF1 by phorbol ester and Ca²⁺ was inhibited by the specific protein kinase C inhibitor Ro 31-8220. The results demonstrate a differential time course of p21^WAF1 protein up-regulation in response to phorbol ester and Ca²⁺, signals that result in keratinocyte differentiation, and suggest that induction of p21^WAF1 in differentiating human keratinocytes occurs through protein kinase C-dependent and p53-independent mechanisms.     

The scaffold protein JLP plays a key role in regulating ultraviolet B‐induced apoptosis in mice

The ultraviolet B (UVB) component of sunlight can cause severe damage to skin cells and even induce skin cancer. Growing evidence indicates that the UVB‐induced signaling network is complex and involves diverse cellular processes. In this study, we investigated the role of c‐Jun NH₂‐terminal kinase‐associated leucine zipper protein (JLP), a scaffold protein for mitogen‐activated protein kinase (MAPK) signaling cascades, in UVB‐induced apoptosis. We found that UVB‐induced skin epidermal apoptosis was prevented in Jlp knockout (KO) as well as in keratinocyte‐specific Jlp KO mice. Analysis of the repair of UVB‐induced DNA damage over time showed no evidence for the involvement of JLP in this process. In contrast, UVB‐stimulated p38 MAPK activation in the skin was impaired in both Jlp KO and keratinocyte‐specific Jlp KO mice. Moreover, topical treatment of UVB‐irradiated mouse skin with a p38 inhibitor significantly suppressed the epidermal apoptosis in wild‐type mice, but not in Jlp KO mice. Our findings suggest that JLP in skin basal keratinocytes plays an important role in UVB‐induced apoptosis by modulating p38 MAPK signaling pathways. This is the first study to show a critical role for JLP in an in vivo response to environmental stimulation.     

CD4+ T-cell dependence of primary CD8+ T-cell response against vaccinia virus depends upon route of infection and viral dose

CD4⁺ T-cell help (CD4 help) plays a pivotal role in CD8⁺ T-cell responses against viral infections. However, the role in primary CD8⁺ T-cell responses remains controversial. We evaluated the effects of infection route and viral dose on primary CD8⁺ T-cell responses to vaccinia virus (VACV) in MHC class II^−/− mice. CD4 help deficiency diminished the generation of VACV-specific CD8⁺ T cells after intraperitoneal (i.p.) but not after intranasal (i.n.) infection. A large viral dose could not restore normal expansion of VACV-specific CD8⁺ T cells in i.p. infected MHC II^−/− mice. In contrast, dependence on CD4 help was observed in i.n. infected MHC II^−/− mice when a small viral dose was used. These data suggested that primary CD8⁺ T-cell responses are less dependent on CD4 help in i.n. infection compared to i.p. infection. Activated CD8⁺ T cells produced more IFN-γ, TNF-α and granzyme B in i.n. infected mice than those in i.p. infected mice, regardless of CD4 help. IL-2 signaling via CD25 was not necessary to drive expansion of VACV-specific CD8⁺ T cells in i.n. infection, but it was crucial in i.p. infection. VACV-specific CD8⁺ T cells underwent increased apoptosis in the absence of CD4 help, but proliferated normally and had cytotoxic potential, regardless of infection route. Our results indicate that route of infection and viral dose are two determinants for CD4 help dependence, and intranasal infection induces more potent effector CD8⁺ T cells than i.p. infection.     

The alternative activation pathway and complement component C3 are critical for a protective immune response against Pseudomonas aeruginosa in a murine model of pneumonia

Pseudomonas aeruginosa is a leading cause of hospital-acquired pneumonia, and approximately 80% of patients with cystic fibrosis are infected with this bacterium. To investigate the overall role of complement and the complement activation pathways in the host defense against P. aeruginosa pulmonary infection, we challenged C3-, C4-, and factor B-deficient mice with P. aeruginosa via intranasal inoculation. In these studies, C3^−/− mice had a higher mortality rate than C3^+/+ mice. Factor B^−/− mice, but not C4^−/− mice, infected with P. aeruginosa had a mortality rate similar to that of C3^−/− mice, indicating that in this model the alternative pathway of complement activation is required for the host defense against Pseudomonas infection. C3^−/− mice had 6- to 7-fold more bacteria in the lungs and 48-fold more bacteria in the blood than did C3^+/+ mice at 24 h postinfection. In vitro, phagocytic cells from C3^+/+ or C3^−/− mice exhibited a decreased ability to bind and/or ingest P. aeruginosa in the presence of C3-deficient serum compared to phagocytic cells in the presence of serum with sufficient C3. C3^−/− mice displayed a significant increase in neutrophils in the lungs and had higher levels of interleukin-1β (IL-1β), IL-6, IL-10, KC, and MIP-2 in the lungs at 24 h postinfection than did C3^+/+ mice. Collectively, these results indicate that complement activation by the alternative pathway is critical for the survival of mice infected with P. aeruginosa and that the protection provided by complement is at least in part due to C3-mediated opsonization and phagocytosis of P. aeruginosa.     

Susceptibility to Salmonella typhimurium Infection and Effectiveness of Vaccination in Mice Deficient in the Tumor Necrosis Factor Alpha p55 Receptor

Mice defective in the ability to produce the tumor necrosis factor alpha p55 receptor (TNFαp55R) were orally challenged with a number of Salmonella typhimurium HWSH derivatives that differ in virulence. In comparison to TNFαp55R^+/+ mice, TNFαp55R^−/− mice succumbed earlier to challenge with wild-type S. typhimurium HWSH and S. typhimurium HWSH purE. In contrast, TNFαp55R^−/− mice were able to control an S. typhimurium HWSH aroA challenge, although greater numbers of Salmonella organisms were present in the tissues for a longer time period than was observed with TNFαp55R^+/+ mice. Vaccination of normal and TNFαp55R knockout animals with S. typhimurium HWSH aroA showed that TNFαp55R^−/− mice, unlike TNFαp55R^+/+ mice, were not protected against a virulent S. typhimurium HWSH challenge. Splenocytes from TNFαp55R^−/− mice exhibited a reduced ability to proliferate in the presence of S. typhimurium antigen compared to TNFαp55R^+/+ mice. Thus, TNFαp55R is essential for controlling Salmonella growth in tissues and for recall of immunity in murine salmonellosis.     

TLR4 inactivation protects from graft-versus-host disease after allogeneic hematopoietic stem cell transplantation

Graft-versus-host disease (GVHD) is the most common complication after hematopoietic stem cell transplantation. To clarify the role of Toll-like receptor 4 (TLR4), which is a major receptor for bacterial lipopolysaccharides (LPS), in the development of acute GVHD, we used a TLR4-knockout (TLR4^−/−) mouse GVHD model and analyzed the underlying immunological mechanisms. When TLR4^−/− mice were used as bone marrow and splenocyte cell graft donors or recipients, GVHD symptom occurrence and mortality were delayed compared to wild-type (TLR4^+/+) mice. In addition, histopathological analyses revealed that in TLR4^−/−→BALB/c chimeras, liver and small intestine tissue damage was reduced with minimal lymphocytic infiltration. In contrast to TLR4^+/+, TLR4^−/− mice dendritic cells did not express CD80, CD86, CD40, MHC-II or IL-12 during LPS induction and remained in an immature state. Furthermore, the ability of TLR4^−/− mice spleen dendritic cells to promote allogeneic T-cell proliferation and, in particular, T-helper cell 1 (Th1) development was obviously attenuated compared with TLR4^+/+ mice dendritic cells, and the levels of interferon-γ (IFN-γ) and IL-10, Th2-cell specific cytokines, were significantly higher in the serum of TLR4^−/−→BALB/c than in TLR4^+/+→BALB/c chimeric mice. Overall, our data revealed that TLR4 may play a role in the pathogenesis of GVHD and that targeted TLR4 gene therapy might provide a new treatment approach to reduce the risk of GVHD.     

Decreased DNA repair but normal apoptosis in ultraviolet-irradiated skin of p53-transgenic mice.

p53 tumor suppressor plays a vital role in the cellular responses to genotoxic stress. It is believed that p53 regulates the cell cycle by activating the G1 checkpoint after exposure to agents like ionizing radiation, ultraviolet (UV) radiation, or genotoxic chemicals. Recently, it is conjectured that p53 may have additional functions in DNA repair and apoptosis. Previously, we demonstrated that p53-transgenic mice that carry mutant alleles of a p53 gene developed twice as many skin tumors as control mice after UV exposure. To elucidate the molecular mechanisms of mutant p53 in skin cancers, we studied DNA repair efficiency and the rate of apoptosis in murine keratinocytes after UV irradiation. In this report, we show that mutant p53-transgenic mouse skin has reduced repair of UV-induced DNA damage in both in vivo and in vitro radioimmunoassays. In control mice, DNA repair is associated with increased amounts of wild-type P53 protein. Unexpectedly, mutant p53-transgenic mice had slightly increased apoptosis after UV irradiation, suggesting that the wild-type p53 protein in the cells still functions in inducing apoptosis, or that this cell death results from p53-independent mechanisms. These results suggest that mutant p53 interferes with wild-type p53 in the repair of UV-induced DNA damage but not in apoptosis.     

Type 1 interferon-induced IL-7 maintains CD8+ T-cell responses and homeostasis by suppressing PD-1 expression in viral hepatitis

Type 1 interferon (IFN-I) promotes antigen-presenting cell maturation and was recently shown to induce hepatic IL-7 production during infection. Herein, we further explored the underlying mechanisms used by IFN-I to orchestrate antiviral immune responses in the liver. Acute viral hepatitis was induced by i.v. injection of adenovirus (Ad) in IFN-α receptor knockout (IFNAR^−/−) and control mice. To disrupt signaling, monoclonal antibodies (mAbs) against IL-7 receptor alpha (IL-7Rα) or PD-L1 were i.p. injected. We found that CD8⁺ T cells in IFNAR^−/− mice were less effective than those in control mice. The reduced T-cell function was accompanied by increased levels of PD-1 expression, apoptosis and decreased IFN-γ production. The lack of IFN-I signaling also impaired the expression of accessory molecules in both intrahepatic dendritic cell (DCs) and hepatocytes. PD-L1 was comparably and highly expressed on hepatocytes in both IFNAR^−/− and control mice. Injection of PD-L1-specific mAb in IFNAR^−/− mice reversed the compromised immune responses in the liver. Further investigation showed that hepatic IL-7 elevation was less pronounced in IFNAR^−/− mice compared to the controls. A treatment with recombinant IL-7 suppressed PD-1 expression on CD8⁺ T cells in vitro. Accordingly, blocking IL-7R signaling in vivo resulted in increased PD-1 expression on CD8⁺ T cells in Ad-infected mice. Collectively, the results suggest that IFN-I-induced hepatic IL-7 production maintains antiviral CD8⁺ T-cell responses and homeostasis by suppressing PD-1 expression in acute viral hepatitis.     

Human mesenchymal stromal cells enhance the immunomodulatory function of CD8+CD28? regulatory T cells

One important aspect of mesenchymal stromal cells (MSCs)-mediated immunomodulation is the recruitment and induction of regulatory T (Treg) cells. However, we do not yet know whether MSCs have similar effects on the other subsets of Treg cells. Herein, we studied the effects of MSCs on CD8⁺CD28⁻ Treg cells and found that the MSCs could not only increase the proportion of CD8⁺CD28⁻ T cells, but also enhance CD8⁺CD28⁻T cells'' ability of hampering naive CD4⁺ T-cell proliferation and activation, decreasing the production of IFN-γ by activated CD4⁺ T cells and inducing the apoptosis of activated CD4⁺ T cells. Mechanistically, the MSCs affected the functions of the CD8⁺CD28⁻ T cells partially through moderate upregulating the expression of IL-10 and FasL. The MSCs had no distinct effect on the shift from CD8⁺CD28⁺ T cells to CD8⁺CD28⁻ T cells, but did increase the proportion of CD8⁺CD28⁻ T cells by reducing their rate of apoptosis. In summary, this study shows that MSCs can enhance the regulatory function of CD8⁺CD28⁻ Treg cells, shedding new light on MSCs-mediated immune regulation.     

Chlamydial Infection in Inducible Nitric Oxide Synthase Knockout Mice

Type 1 CD4⁺-T-cell-mediated immunity is crucial for the resolution of chlamydial infection of the murine female genital tract. Previous studies demonstrating a correlation between CD4⁺-T-cell-mediated inhibition of chlamydial growth and gamma interferon (IFN-γ)-mediated induction of nitric oxide synthase suggested a potential role for the nitric oxide (NO) effector pathway in the clearance of Chlamydia from genital epithelial cells by the immune system. To clarify the role of this pathway, the growth levels of Chlamydia trachomatis organisms in normal (iNOS^+/+) mice and in genetically engineered mice lacking the inducible nitric oxide synthase (iNOS) gene (iNOS^−/− mice) were compared. There was no significant difference in the course of genital chlamydial infections in iNOS^+/+ and iNOS^−/− mice as determined by recovery of Chlamydia organisms shed from genital epithelial cells. Dissemination of Chlamydia to the spleen and lungs occurred to a greater extent in iNOS^−/− than in iNOS^+/+ mice, which correlated with a marginal increase in the susceptibility of macrophages from iNOS^−/− mice to chlamydial infection in vitro. However, infections were rapidly cleared from all affected tissues, with no clinical signs of disease. The finding of minimal dissemination in iNOS^−/− mice suggested that activation of the iNOS effector pathway was not the primary target of IFN-γ during CD4⁺-T-cell-mediated control of chlamydial growth in macrophages because previous reports demonstrated extensive and often fatal dissemination of Chlamydia in mice lacking IFN-γ. In summary, these results indicate that the iNOS effector pathway is not required for elimination of Chlamydia from epithelial cells lining the female genital tract of mice although it may contribute to the control of dissemination of C. trachomatis by infected macrophages.     

Proapoptotic Bcl-2 family member Bim promotes persistent infection and limits protective immunity

Following the peak of the T-cell response, most of the activated effector T cells die by apoptosis driven by the proapoptotic Bcl-2 family member Bim (Bcl-2-interacting mediator of death). Whether the absence of Bim-mediated T-cell apoptosis can affect protective immunity remains unclear. Here, we used a mouse model of Leishmania major infection, in which parasite persistence and protective immunity are controlled by an equilibrium reached between parasite-specific gamma interferon (IFN-γ)-producing effector T cells and interleukin-10 (IL-10)-producing CD4⁺ CD25⁺ T regulatory cells. To further understand the role of Bim-mediated apoptosis in persistent infection and protective immunity, we infected Bim^−/− mice with L. major. We found that the initial parasite growth and lesion development were similar in Bim^−/− and wild-type mice after primary L. major infection. However, at later times after infection, Bim^−/− mice had significantly increased L. major-specific CD4⁺ T-cell responses and were resistant to persistent infection. Interestingly, despite their resistance to primary L. major infection, Bim^−/− mice displayed significantly enhanced protection against challenge with L. major. Increased resistance to challenge in Bim^−/− mice was associated with a significant increase in the number of L. major-specific IFN-γ-producing CD4⁺ T cells and a lack of IL-10 production at the challenge site. Taken together, these data suggest that Bim limits protective immunity and that the absence of Bim allows the host to bypass antigen persistence for maintenance of immunity against reinfection.     

Inhibition of Pim2-prolonged skin allograft survival through the apoptosis regulation pathway

Recently, apoptosis has been considered to be an important regulator for allograft survival. The serine/threonine kinase Pim2 has been implicated in many apoptotic pathways. In a previous study, we found that pim2 was highly expressed in CD4⁺ T cells in an allograft model. Here, we further investigated the effects of Pim2 on allograft survival and the underlying mechanisms associated with apoptosis. The results showed that pim2 was overexpressed in grafts and spleens, particularly in spleen CD4⁺ T cells when acute allorejection occurred, and correlated positively with the extent of rejection. In T cells from the spleens of naive BALB/c mice treated with 5 µM 4a (a specific inhibitor of Pim2) for 24 h, the apoptosis rate increased and the phosphorylation of BAD was decreased. Furthermore, adoptive transfer of CD4⁺ T cells treated with 4a in vitro to allografted severe combined immunodeficiency (SCID) mice effectively prolonged allograft survival from 19.5±1.7 days to 31±2.3 days. Moreover, the results demonstrated that the CD4⁺CD25⁻ effector T-cell subset was the predominate expresser of the pim2 gene as compared with the CD4⁺CD25⁺ regulatory T (Treg) cell subset. Alloantigen-induced CD4⁺CD25⁺ T cells displayed less Foxp3 expression and a low suppression of apoptosis compared with effector CD4⁺CD25⁻ T cells treated with 4a. Collectively, these data revealed that Pim2 facilitated allograft rejection primarily by modulating the apoptosis of effector T cells and the function of Treg cells. These data suggested that Pim2 may be an important target for in vivo anti-rejection therapies and for the ex vivo expansion of CD4⁺CD25⁺ T cells.     

Gamma interferon-independent effects of interleukin-12 on immunity to Salmonella enterica serovar Typhimurium

Interleukin-12 (IL-12) and IL-18 are both central to the induction of gamma interferon (IFN-γ), and various roles for IL-12 and IL-18 in control of intracellular microbial infections have been demonstrated. We used IL-12p40^−/− and IL-18^−/− mice to further investigate the role of IL-12 and IL-18 in control of Salmonella enterica serovar Typhimurium. While C57BL/6 and IL-18^−/− mice were able to resolve attenuated S. enterica serovar Typhimurium infections, the IL-12p40^−/− mice succumbed to a high bacterial burden after 60 days. Using ovalbumin (OVA)-specific T-cell receptor transgenic T cells (OT-II cells), we demonstrated that following oral infection with recombinant S. enterica serovar Typhimurium expressing OVA, the OT-II cells proliferated in the mesenteric lymph nodes of C57BL/6 and IL-18^−/− mice but not in IL-12p40^−/− mice. In addition, we demonstrated by flow cytometry that equivalent or increased numbers of T cells produced IFN-γ in IL-12p40^−/− mice compared with the numbers of T cells that produced IFN-γ in C57BL/6 and IL-18^−/− mice. Finally, we demonstrated that removal of macrophages from S. enterica serovar Typhimurium-infected C57BL/6 and IL-12p40^−/− mice did not affect the bacterial load, suggesting that impaired control of S. enterica serovar Typhimurium infection in the absence of IL-12p40 is not due to reduced macrophage bactericidal activities, while IL-18^−/− mice did rely on the presence of macrophages for control of the infection. Our results suggest that IL-12p40, but not IL-18, is critical to resolution of infections with attenuated S. enterica serovar Typhimurium and that especially the effects of IL-12p40 on proliferative responses of CD4⁺ T cells, but not the ability of these cells to produce IFN-γ, are important in the resolution of infection by this intracellular bacterial pathogen.     

Dendritic Cell and NK Cell Reciprocal Cross Talk Promotes Gamma Interferon-Dependent Immunity to Blood-Stage Plasmodium chabaudi AS Infection in Mice

Dendritic cells (DCs) are important accessory cells for promoting NK cell gamma interferon (IFN-γ) production in vitro in response to Plasmodium falciparum-infected red blood cells (iRBC). We investigated the requirements for reciprocal activation of DCs and NK cells leading to Th1-type innate and adaptive immunity to P. chabaudi AS infection. During the first week of infection, the uptake of iRBC by splenic CD11c⁺ DCs in resistant wild-type (WT) C57BL/6 mice was similar to that in interleukin 15^−/− (IL-15^−/−) and IL-12p40^−/− mice, which differ in the severity of P. chabaudi AS infection. DCs from infected IL-15^−/− mice expressed costimulatory molecules, produced IL-12, and promoted IFN-γ secretion by WT NK cells in vitro as efficiently as WT DCs. In contrast, DCs from infected IL-12p40^−/− mice exhibited alterations in maturation and cytokine production and were unable to induce NK cell IFN-γ production. Coculture of DCs and NK cells demonstrated that DC-mediated NK cell activation required IL-12 and, to a lesser extent, IL-2, as well as cell-cell contact. In turn, NK cells from infected WT mice enhanced DC maturation, IL-12 production, and priming of CD4⁺ T-cell proliferation and IFN-γ secretion. Infected WT mice depleted of NK cells, which exhibit increased parasitemia, had impaired DC maturation and DC-induced CD4⁺ Th1 cell priming. These findings indicate that DC-NK cell reciprocal cross talk is critical for control and rapid resolution of P. chabaudi AS infection and provide in vivo evidence for the importance of this interaction in IFN-γ-dependent immunity to malaria.     

Ambiguous Role of Interleukin-12 in Yersinia enterocolitica Infection in Susceptible and Resistant Mouse Strains

Endogenous interleukin-12 (IL-12) mediates protection against Yersinia enterocolitica in C57BL/6 mice by triggering gamma interferon (IFN-γ) production in NK and CD4⁺ T cells. Administration of exogenous IL-12 confers protection against yersiniae in Yersinia-susceptible BALB/c mice but exacerbates yersiniosis in resistant C57BL/6 mice. Therefore, we wanted to dissect the different mechanisms exerted by IL-12 during Yersinia infections by using different models of Yersinia-resistant and -susceptible mice, including resistant C57BL/6 mice, susceptible BALB/c mice, intermediate-susceptible wild-type 129/Sv mice, 129/Sv IFN-γ-receptor-deficient (IFN-γR^−/−) mice and C57BL/6 tumor necrosis factor (TNF) receptor p55 chain-deficient (TNFR p55^−/−) mice. IFN-γR^−/− mice turned out to be highly susceptible to infection by Y. enterocolitica compared with IFN-γR^+/+ mice. Administration of IL-12 was protective in IFN-γR^+/+ mice but not in IFN-γR^−/− mice, suggesting that IFN-γR-induced mechanisms are essential for IL-12-induced resistance against yersiniae. BALB/c mice could be rendered Yersinia resistant by administration of anti-CD4 antibodies or by administration of IL-12. In contrast, C57BL/6 mice could be rendered more resistant by administration of transforming growth factor β (TGF-β). Furthermore, IL-12-triggered toxic effects in C57BL/6 mice were abrogated by coadministration of TGF-β. While administration of IL-12 alone increased TNF-α levels, administration of TGF-β or TGF-β plus IL-12 decreased both TNF-α and IFN-γ levels in Yersinia-infected C57BL/6 mice. Moreover, IL-12 did not induce toxicity in Yersinia-infected TNFR p55^−/− mice, suggesting that TNF-α accounts for IL-12-induced toxicity. Taken together, IL-12 may induce different effector mechanisms in BALB/c and C57BL/6 mice resulting either in protection or exacerbation. These results are important for understanding the critical balance of proinflammatory and regulatory cytokines in bacterial infections which is decisive for beneficial effects of cytokine therapy.     

Activated T Lymphocytes Disappear from Circulation during Endotoxemia in Humans

Seventeen volunteers received an intravenous bolus of endotoxin (2 ng/kg of body weight). Endotoxin-induced lymphopenia was constituted mainly by cells with an immature phenotype (CD45RA⁺ CD45RO⁻) that were less likely to undergo apoptosis (CD28⁺), whereas cells with the highest rates of disappearance were characterized by an activated phenotype (CD45RA⁻ CD45RO⁺) as well as a phenotype linked to apoptosis (CD95⁺ CD28⁻). In conclusion, endotoxin-induced lymphopenia reflects the disappearance from the circulation of activated lymphocytes prone to undergo apoptosis.     

Developmental regulation of p53-dependent radiation-induced thymocyte apoptosis in mice

The production of T cell receptor αβ⁺ (TCRαβ⁺) T lymphocytes in the thymus is a tightly regulated process that can be monitored by the regulated expression of several surface molecules, including CD4, CD8, cKit, CD25 and the TCR itself, after TCR genes have been assembled from discrete V, D (for TCR-β) and J gene segments by a site-directed genetic recombination. Thymocyte differentiation is the result of a delicate balance between cell death and survival: developing thymocytes die unless they receive a positive signal to proceed to the next stage. This equilibrium is altered in response to various physiological or physical stresses such as ionizing radiation, which induces a massive p53-dependent apoptosis of CD4⁺CD8⁺ double-positive (DP) thymocytes. Interestingly, these cells are actively rearranging their TCR-α chain genes. To unravel an eventual link between V(D)J recombination activity and thymocyte radio-sensitivity, we analysed the dynamics of thymocyte apoptosis and regeneration following exposure of wild-type and p53-deficient mice to different doses of γ-radiation. p53-dependent radio-sensitivity was already found to be high in immature CD4⁻CD8⁻ (double-negative, DN) cKit⁺CD25⁺ thymocytes, where TCR-β gene rearrangement is initiated. However, TCR-αβ⁻CD8⁺ immature single-positive thymocytes, an actively cycling intermediate population between the DN and DP stages, are the most radio-sensitive cells in the thymus, even though their apoptosis is only partially p53-dependent. Within the DP population, TCR-αβ⁺ thymocytes that completed TCR-α gene recombination are more radio-resistant than their TCR-αβ⁻ progenitors. Finally, we found no correlation between p53 activation and thymocyte sensitivity to radiation-induced apoptosis.