Journal of Experimental Medicine Articles

Essential role of TNF receptor superfamily 25 (TNFRSF25) in the development of allergic lung inflammation

Fang, L., Adkins, B., Deyev, V., Podack, E. R. — 2008-05-12

We identify the tumor necrosis factor receptor superfamily 25 (TNFRSF25)/TNFSF15 pair as critical trigger for allergic lung inflammation, which is a cardinal feature of asthma. TNFRSF25 (TNFR25) signals are required to exert T helper cell 2 (Th2) effector function in Th2-polarized CD4 cells and co-stimulate interleukin (IL)-13 production by glycosphingolipid-activated NKT cells. In vivo, antibody blockade of TNFSF15 (TL1A), which is the ligand for TNFR25, inhibits lung inflammation and production of Th2 cytokines such as IL-13, even when administered days after airway antigen exposure. Similarly, blockade of TNFR25 by a dominant-negative (DN) transgene, DN TNFR25, confers resistance to lung inflammation in mice. Allergic lung inflammation–resistant, NKT-deficient mice become susceptible upon adoptive transfer of wild-type NKT cells, but not after transfer of DN TNFR25 transgenic NKT cells. The TNFR25/TL1A pair appears to provide an early signal for Th2 cytokine production in the lung, and therefore may be a drug target in attempts to attenuate lung inflammation in asthmatics.

TL1A-DR3 interaction regulates Th17 cell function and Th17-mediated autoimmune disease

2008-05-12

T helper type 17 (Th17) cells play an important pathogenic function in autoimmune diseases; their regulation, however, is not well understood. We show that the expression of a tumor necrosis factor receptor family member, death receptor 3 (DR3; also known as TNFRSF25), is selectively elevated in Th17 cells, and that TL1A, its cognate ligand, can promote the proliferation of effector Th17 cells. To further investigate the role of the TL1A–DR3 pathway in Th17 regulation, we generated a TL1A-deficient mouse and found that TL1A^–/– dendritic cells exhibited a reduced capacity in supporting Th17 differentiation and proliferation. Consistent with these data, TL1A^–/– animals displayed decreased clinical severity in experimental autoimmune encephalomyelitis (EAE). Finally, we demonstrated that during EAE disease progression, TL1A was required for the optimal differentiation as well as effector function of Th17 cells. These observations thus establish an important role of the TL1A–DR3 pathway in promoting Th17 cell function and Th17-mediated autoimmune disease.

Regulation of inflammatory responses by IL-17F

2008-05-12

Although interleukin (IL) 17 has been extensively characterized, the function of IL-17F, which has an expression pattern regulated similarly to IL-17, is poorly understood. We show that like IL-17, IL-17F regulates proinflammatory gene expression in vitro, and this requires IL-17 receptor A, tumor necrosis factor receptor–associated factor 6, and Act1. In vivo, overexpression of IL-17F in lung epithelium led to infiltration of lymphocytes and macrophages and mucus hyperplasia, similar to observations made in IL-17 transgenic mice. To further understand the function of IL-17F, we generated and analyzed mice deficient in IL-17F or IL-17. IL-17, but not IL-17F, was required for the initiation of experimental autoimmune encephalomyelitis. Mice deficient in IL-17F, but not IL-17, had defective airway neutrophilia in response to allergen challenge. Moreover, in an asthma model, although IL-17 deficiency reduced T helper type 2 responses, IL-17F–deficient mice displayed enhanced type 2 cytokine production and eosinophil function. In addition, IL-17F deficiency resulted in reduced colitis caused by dextran sulfate sodium, whereas IL-17 knockout mice developed more severe disease. Our results thus demonstrate that IL-17F is an important regulator of inflammatory responses that seems to function differently than IL-17 in immune responses and diseases.

Native and aspirin-triggered lipoxins control innate immunity by inducing proteasomal degradation of TRAF6

2008-05-12

Innate immune signaling is critical for the development of protective immunity. Such signaling is, perforce, tightly controlled. Lipoxins (LXs) are eicosanoid mediators that play key counterregulatory roles during infection. The molecular mechanisms underlying LX-mediated control of innate immune signaling are of interest. In this study, we show that LX and aspirin (ASA)-triggered LX (ATL) inhibit innate immune signaling by inducing suppressor of cytokine signaling (SOCS) 2–dependent ubiquitinylation and proteasome-mediated degradation of TNF receptor–associated factor (TRAF) 2 and TRAF6, which are adaptor molecules that couple TNF and interleukin-1 receptor/Toll-like receptor family members to intracellular signaling events. LX-mediated degradation of TRAF6 inhibits proinflammatory cytokine production by dendritic cells. This restraint of innate immune signaling can be ablated by inhibition of proteasome function. In vivo, this leads to dysregulated immune responses, accompanied by increased mortality during infection. Proteasomal degradation of TRAF6 is a central mechanism underlying LX-driven immune counterregulation, and a hitherto unappreciated mechanism of action of ASA. These findings suggest a new molecular target for drug development for diseases marked by dysregulated inflammatory responses.

Essential role of Notch signaling in effector memory CD8+ T cell-mediated airway hyperresponsiveness and inflammation

2008-05-12

Adoptive transfer of in vivo–primed CD8⁺ T cells or in vitro–generated effector memory CD8⁺ T (T_EFF) cells restores airway hyperresponsiveness (AHR) and airway inflammation in CD8-deficient (CD8^–/–) mice. Examining transcription levels, there was a strong induction of Notch1 in T_EFF cells compared with central memory CD8⁺ T cells. Treatment of T_EFF cells with a -secretase inhibitor (GSI) strongly inhibited Notch signaling in these cells, and after adoptive transfer, GSI-treated T_EFF cells failed to restore AHR and airway inflammation in sensitized and challenged recipient CD8^–/– mice, or to enhance these responses in recipient wild-type (WT) mice. These effects of GSI were also associated with increased expression of the Notch ligand Delta1 in T_EFF cells. Treatment of sensitized and challenged WT mice with Delta1-Fc resulted in decreased AHR and airway inflammation accompanied by higher levels of interferon in bronchoalveolar lavage fluid. These results demonstrate a role for Notch in skewing the T cell response from a T helper (Th)2 to a Th1 phenotype as a consequence of the inhibition of Notch receptor activation and the up-regulation of the Notch ligand Delta1. These data are the first to show a functional role for Notch in the challenge phase of CD8⁺ T cell–mediated development of AHR and airway inflammation, and identify Delta1 as an important regulator of allergic airway inflammation.

A role for sex chromosome complement in the female bias in autoimmune disease

2008-05-12

Most autoimmune diseases are more common in women than in men. This may be caused by differences in sex hormones, sex chromosomes, or both. In this study, we determined if there was a contribution of sex chromosomes to sex differences in susceptibility to two immunologically distinct disease models, experimental autoimmune encephalomyelitis (EAE) and pristane-induced lupus. Transgenic SJL mice were created to permit a comparison between XX and XY within a common gonadal type. Mice of the XX sex chromosome complement, as compared with XY, demonstrated greater susceptibility to both EAE and lupus. This is the first evidence that the XX sex chromosome complement, as compared with XY, confers greater susceptibility to autoimmune disease.

Bmi1 regulates memory CD4 T cell survival via repression of the Noxa gene

2008-05-12

The maintenance of memory T cells is central to the establishment of immunological memory, although molecular details of the process are poorly understood. In the absence of the polycomb group (PcG) gene Bmi1, the number of memory CD4⁺ T helper (Th)1/Th2 cells was reduced significantly. Enhanced cell death of Bmi1^–/– memory Th2 cells was observed both in vivo and in vitro. Among various proapoptotic genes that are regulated by Bmi1, the expression of proapoptotic BH3-only protein Noxa was increased in Bmi1^–/– effector Th1/Th2 cells. The generation of memory Th2 cells was restored by the deletion of Noxa, but not by Ink4a and Arf. Direct binding of Bmi1 to the Noxa gene locus was accompanied by histone H3-K27 methylation. The recruitment of other PcG gene products and Dnmt1 to the Noxa gene was highly dependent on the expression of Bmi1. In addition, Bmi1 was required for DNA CpG methylation of the Noxa gene. Moreover, memory Th2-dependent airway inflammation was attenuated substantially in the absence of Bmi1. Thus, Bmi1 controls memory CD4⁺ Th1/Th2 cell survival and function through the direct repression of the Noxa gene.

Virulent Shigella flexneri subverts the host innate immune response through manipulation of antimicrobial peptide gene expression

2008-05-12

Antimicrobial factors are efficient defense components of the innate immunity, playing a crucial role in the intestinal homeostasis and protection against pathogens. In this study, we report that upon infection of polarized human intestinal cells in vitro, virulent Shigella flexneri suppress transcription of several genes encoding antimicrobial cationic peptides, particularly the human β-defensin hBD-3, which we show to be especially active against S. flexneri. This is an example of targeted survival strategy. We also identify the MxiE bacterial regulator, which controls a regulon encompassing a set of virulence plasmid-encoded effectors injected into host cells and regulating innate signaling, as being responsible for this dedicated regulatory process. In vivo, in a model of human intestinal xenotransplant, we confirm at the transcriptional and translational level, the presence of a dedicated MxiE-dependent system allowing S. flexneri to suppress expression of antimicrobial cationic peptides and promoting its deeper progression toward intestinal crypts. We demonstrate that this system is also able to down-regulate additional innate immunity genes, such as the chemokine CCL20 gene, leading to compromised recruitment of dendritic cells to the lamina propria of infected tissues. Thus, S. flexneri has developed a dedicated strategy to weaken the innate immunity to manage its survival and colonization ability in the intestine.

C5 deficiency and C5a or C5aR blockade protects against cerebral malaria

2008-05-12

Experimental infection of mice with Plasmodium berghei ANKA (PbA) provides a powerful model to define genetic determinants that regulate the development of cerebral malaria (CM). Based on the hypothesis that excessive activation of the complement system may confer susceptibility to CM, we investigated the role of C5/C5a in the development of CM. We show a spectrum of susceptibility to PbA in a panel of inbred mice; all CM-susceptible mice examined were found to be C5 sufficient, whereas all C5-deficient strains were resistant to CM. Transfer of the C5-defective allele from an A/J (CM resistant) onto a C57BL/6 (CM-susceptible) genetic background in a congenic strain conferred increased resistance to CM; conversely, transfer of the C5-sufficient allele from the C57BL/6 onto the A/J background recapitulated the CM-susceptible phenotype. The role of C5 was further explored in B10.D2 mice, which are identical for all loci other than C5. C5-deficient B10.D2 mice were protected from CM, whereas C5-sufficient B10.D2 mice were susceptible. Antibody blockade of C5a or C5a receptor (C5aR) rescued susceptible mice from CM. In vitro studies showed that C5a-potentiated cytokine secretion induced by the malaria product P. falciparum glycosylphosphatidylinositol and C5aR blockade abrogated these amplified responses. These data provide evidence implicating C5/C5a in the pathogenesis of CM.

Bone sialoprotein plays a functional role in bone formation and osteoclastogenesis

2008-05-12

Bone sialoprotein (BSP) and osteopontin (OPN) are both highly expressed in bone, but their functional specificities are unknown. OPN knockout (^–/–) mice do not lose bone in a model of hindlimb disuse (tail suspension), showing the importance of OPN in bone remodeling. We report that BSP^–/– mice are viable and breed normally, but their weight and size are lower than wild-type (WT) mice. Bone is undermineralized in fetuses and young adults, but not in older (≥12 mo) BSP^–/– mice. At 4 mo, BSP^–/– mice display thinner cortical bones than WT, but greater trabecular bone volume with very low bone formation rate, which indicates reduced resorption, as confirmed by lower osteoclast surfaces. Although the frequency of total colonies and committed osteoblast colonies is the same, fewer mineralized colonies expressing decreased levels of osteoblast markers form in BSP^–/– versus WT bone marrow stromal cultures. BSP^–/– hematopoietic progenitors form fewer osteoclasts, but their resorptive activity on dentin is normal. Tail-suspended BSP^–/– mice lose bone in hindlimbs, as expected. In conclusion, BSP deficiency impairs bone growth and mineralization, concomitant with dramatically reduced bone formation. It does not, however, prevent the bone loss resulting from loss of mechanical stimulation, a phenotype that is clearly different from OPN^–/– mice.

The tumor suppressor semaphorin 3B triggers a prometastatic program mediated by interleukin 8 and the tumor microenvironment

2008-05-12

Semaphorins are a large family of evolutionarily conserved morphogenetic molecules originally identified for their repelling role in axonal guidance. Intriguingly, semaphorins have recently been implicated in cancer progression (Neufeld, G., T. Lange, A. Varshavsky, and O. Kessler. 2007. Adv. Exp. Med. Biol. 600:118–131). In particular, semaphorin 3B (SEMA3B) is considered a putative tumor suppressor, and yet we found that it is expressed at high levels in many invasive and metastatic human cancers. By investigating experimental tumor models, we confirmed that SEMA3B expression inhibited tumor growth, whereas metastatic dissemination was surprisingly increased. We found that SEMA3B induced the production of interleukin (IL) 8 by tumor cells by activating the p38–mitogen-activated protein kinase pathway in a neuropilin 1–dependent manner. Silencing the expression of endogenous SEMA3B in tumor cells impaired IL-8 transcription. The release of IL-8, in turn, induced the recruitment of tumor-associated macrophages and metastatic dissemination to the lung, which could be rescued by blocking IL-8 with neutralizing antibodies. In conclusion, we report that SEMA3B exerts unexpected functions in cancer progression by fostering a prometastatic environment through elevated IL-8 secretion and recruitment of macrophages coupled to the suppression of tumor growth.

T cell receptor-instructed {alpha}{beta} versus {gamma}{delta} lineage commitment revealed by single-cell analysis

Kreslavsky, T., Garbe, A. I., Krueger, A., von Boehmer, H. — 2008-05-12

β and T cell lineages develop in the thymus from a common precursor. It is unclear at which stage of development commitment to these lineages takes place and in which way T cell receptor signaling contributes to the process. Recently, it was demonstrated that strong TCR signals favor lineage development, whereas weaker TCR signals promote β lineage fate. Two models have been proposed to explain these results. The first model suggests that commitment occurs after TCR expression and TCR signaling directly instructs lymphocytes to adopt one or the other lineage fate. The second model suggests that commitment occurs before TCR expression and that TCR signaling merely confirms the lineage choice. By tracing the fate of single T cell precursors, this study shows that there is no commitment to either the β or lineage before TCR expression and that modulation of TCR signaling in progeny of a single TCR-expressing cell changes lineage commitment.

The stream of precursors that colonizes the thymus proceeds selectively through the early T lineage precursor stage of T cell development

Benz, C., Martins, V. C., Radtke, F., Bleul, C. C. — 2008-05-12

T cell development in the thymus depends on continuous colonization by hematopoietic precursors. Several distinct T cell precursors have been identified, but whether one or several independent precursor cell types maintain thymopoiesis is unclear. We have used thymus transplantation and an inducible lineage-tracing system to identify the intrathymic precursor cells among previously described thymus-homing progenitors that give rise to the T cell lineage in the thymus. Extrathymic precursors were not investigated in these studies. Both approaches show that the stream of T cell lineage precursor cells, when entering the thymus, selectively passes through the early T lineage precursor (ETP) stage. Immigrating precursor cells do not exhibit characteristics of double-negative (DN) 1c, DN1d, or DN1e stages, or of populations containing the common lymphoid precursor 2 (CLP-2) or the thymic equivalent of circulating T cell progenitors (CTPs). It remains possible that an unknown hematopoietic precursor cell or previously described extrathymic precursors with a CLP, CLP-2, or CTP phenotype feed into T cell development by circumventing known intrathymic T cell lineage progenitor cells. However, it is clear that of the known intrathymic precursors, only the ETP population contributes significant numbers of T lineage precursors to T cell development.

MR1 uses an endocytic pathway to activate mucosal-associated invariant T cells

2008-05-12

Like CD1d-restricted iNKT cells, mucosal-associated invariant T cells (MAITs) are "innate" T cells that express a canonical TCR chain, have a memory phenotype, and rapidly secrete cytokines upon TCR ligation. Unlike iNKT cells, MAIT cells require the class Ib molecule MHC-related protein I (MR1), B cells, and gut flora for development and/or expansion, and they preferentially reside in the gut lamina propria. Evidence strongly suggests that MAIT cell activation is ligand-dependent, but the nature of MR1 ligand is unknown. In this study, we define a mechanism of endogenous antigen presentation by MR1 to MAIT cells. MAIT cell activation was dependent neither on a proteasome-processed ligand nor on the chaperoning by the MHC class I peptide loading complex. However, MAIT cell activation was enhanced by overexpression of MHC class II chaperones Ii and DM and was strikingly diminished by silencing endogenous Ii. Furthermore, inhibiting the acidification of the endocytic compartments reduced MR1 surface expression and ablated MAIT cell activation. The importance of the late endosome for MR1 antigen presentation was further corroborated by the localization of MR1 molecules in the multivesicular endosomes. These findings demonstrate that MR1 traffics through endocytic compartments, thereby allowing MAIT cells to sample both endocytosed and endogenous antigens.

IL-15R{alpha} chaperones IL-15 to stable dendritic cell membrane complexes that activate NK cells via trans presentation

Mortier, E., Woo, T., Advincula, R., Gozalo, S., Ma, A. — 2008-05-12

Natural killer (NK) cells are innate immune effectors that mediate rapid responses to viral antigens. Interleukin (IL)-15 and its high affinity IL-15 receptor, IL-15R, support NK cell homeostasis in resting animals via a novel trans presentation mechanism. To better understand how IL-15 and IL-15R support NK cell activation during immune responses, we have used sensitive assays for detecting native IL-15 and IL-15R proteins and developed an assay for detecting complexes of these proteins. We find that IL-15 and IL-15R are preassembled in complexes within the endoplasmic reticulum/Golgi of stimulated dendritic cells (DCs) before being released from cells. IL-15R is required for IL-15 production by DCs, and IL-15 that emerges onto the cell surface of matured DCs does not bind to neighboring cells expressing IL-15R. We also find that soluble IL-15–IL-15R complexes are induced during inflammation, but membrane-bound IL-15–IL-15R complexes, rather than soluble complexes, support NK cell activation in vitro and in vivo. Finally, we provide in vivo evidence that expression of IL-15R specifically on DCs is critical for trans presenting IL-15 and activating NK cells. These studies define an unprecedented cytokine–receptor biosynthetic pathway in which IL-15R serves as a chaperone for IL-15, after which membrane-bound IL-15R–IL-15 complexes activate NK cells via direct cell–cell contact.

ATF4 is an oxidative stress-inducible, prodeath transcription factor in neurons in vitro and in vivo

2008-05-12

Oxidative stress is pathogenic in neurological diseases, including stroke. The identity of oxidative stress–inducible transcription factors and their role in propagating the death cascade are not well known. In an in vitro model of oxidative stress, the expression of the bZip transcription factor activating transcription factor 4 (ATF4) was induced by glutathione depletion and localized to the promoter of a putative death gene in neurons. Germline deletion of ATF4 resulted in a profound reduction in oxidative stress–induced gene expression and resistance to oxidative death. In neurons, ATF4 modulates an early, upstream event in the death pathway, as resistance to oxidative death by ATF4 deletion was associated with decreased consumption of the antioxidant glutathione. Forced expression of ATF4 was sufficient to promote cell death and loss of glutathione. In ATF4^–/– neurons, restoration of ATF4 protein expression reinstated sensitivity to oxidative death. In addition, ATF4^–/– mice experienced significantly smaller infarcts and improved behavioral recovery as compared with wild-type mice subjected to the same reductions in blood flow in a rodent model of ischemic stroke. Collectively, these findings establish ATF4 as a redox-regulated, prodeath transcriptional activator in the nervous system that propagates death responses to oxidative stress in vitro and to stroke in vivo.

The insulator factor CTCF controls MHC class II gene expression and is required for the formation of long-distance chromatin interactions

Majumder, P., Gomez, J. A., Chadwick, B. P., Boss, J. M. — 2008-04-14

Knockdown of the insulator factor CCCTC binding factor (CTCF), which binds XL9, an intergenic element located between HLA-DRB1 and HLA-DQA1, was found to diminish expression of these genes. The mechanism involved interactions between CTCF and class II transactivator (CIITA), the master regulator of major histocompatibility complex class II (MHC-II) gene expression, and the formation of long-distance chromatin loops between XL9 and the proximal promoter regions of these MHC-II genes. The interactions were inducible and dependent on the activity of CIITA, regulatory factor X, and CTCF. RNA fluorescence in situ hybridizations show that both genes can be expressed simultaneously from the same chromosome. Collectively, the results suggest a model whereby both HLA-DRB1 and HLA-DQA1 loci can interact simultaneously with XL9, and describe a new regulatory mechanism for these MHC-II genes involving the alteration of the general chromatin conformation of the region and their regulation by CTCF.

Either a Th17 or a Th1 effector response can drive autoimmunity: conditions of disease induction affect dominant effector category

2008-04-14

Experimental autoimmune uveitis (EAU) represents autoimmune uveitis in humans. We examined the role of the interleukin (IL)-23–IL-17 and IL-12–T helper cell (Th)1 pathways in the pathogenesis of EAU. IL–23 but not IL-12 was necessary to elicit disease by immunization with the retinal antigen (Ag) interphotoreceptor retinoid-binding protein (IRBP) in complete Freund's adjuvant. IL-17 played a dominant role in this model; its neutralization prevented or reversed disease, and Th17 effector cells induced EAU in the absence of interferon (IFN)-. In a transfer model, however, a polarized Th1 line could induce severe EAU independently of host IL-17. Furthermore, induction of EAU with IRBP-pulsed mature dendritic cells required generation of an IFN-–producing effector response, and an IL-17 response by itself was insufficient to elicit pathology. Finally, genetic deficiency of IL-17 did not abrogate EAU susceptibility. Thus, autoimmune pathology can develop in the context of either a Th17 or a Th1 effector response depending on the model. The data suggest that the dominant effector phenotype may be determined at least in part by conditions present during initial exposure to Ag, including the quality/quantity of Toll-like receptor stimulation and/or type of Ag-presenting cells. These data also raise the possibility that the nonredundant requirement for IL-23 in EAU may extend beyond its role in promoting the Th17 effector response and help provide a balance in the current Th1 versus Th17 paradigm.

The Th17-ELR+ CXC chemokine pathway is essential for the development of central nervous system autoimmune disease

Carlson, T., Kroenke, M., Rao, P., Lane, T. E., Segal, B. — 2008-04-14

The ELR⁺ CXC chemokines CXCL1 and CXCL2 are up-regulated in the central nervous system (CNS) during multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE). However, their functional significance and the pathways regulating their expression are largely unknown. We show that transfer of encephalitogenic CD4⁺ Th17 cells is sufficient to induce CXCL1 and CXCL2 transcription in the spinal cords of naive, syngeneic recipients. Blockade or genetic silencing of CXCR2, a major receptor for these chemokines in mice, abrogates blood–brain barrier (BBB) breakdown, CNS infiltration by leukocytes, and the development of clinical deficits during the presentation as well as relapses of EAE. Depletion of circulating polymorphonuclear leukocytes (PMN) had a similar therapeutic effect. Furthermore, injection of CXCR2⁺ PMN into CXCR2^–/– mice was sufficient to restore susceptibility to EAE. Our findings reveal that a Th17–ELR⁺ CXC chemokine pathway is critical for granulocyte mobilization, BBB compromise, and the clinical manifestation of autoimmune demyelination in myelin peptide–sensitized mice, and suggest new therapeutic targets for diseases such as MS.

OX40 triggering blocks suppression by regulatory T cells and facilitates tumor rejection

Piconese, S., Valzasina, B., Colombo, M. P. — 2008-04-14

Regulatory T (T reg) cells are the major obstacle to cancer immunotherapy, and their depletion promptly induces conversion of peripheral precursors into T reg cells. We show that T reg cells can be functionally inactivated by OX40 triggering. In tumors, the vast majority of CD4⁺ T cells are Foxp3⁺ and OX40^bright. However, intratumor injection of the agonist anti-OX40 monoclonal antibody (mAb) OX86, but not anti-CD25 mAb, induces tumor rejection in 80% of mice, an effect that is abrogated by CD8 depletion. Upon intratumor OX40 triggering, increased numbers of infiltrating dendritic cells (DCs) migrate to draining lymph nodes and generate a new wave of tumor-specific cytotoxic T lymphocytes, as detected by tetramer and CD44 staining of node CD8⁺ T lymphocytes. Tumor-bearing Rag1-knockout (KO) mice reconstituted with OX40-deficient T reg cells and wild-type (WT) effector T cells, or the reciprocal combination, showed that both T reg and effector T cells must be triggered via OX40 for the tumor to be rejected. Accordingly, WT but not OX40-KO mice receiving intratumor coinjection of OX86 and ovalbumin protein were able to revert tumor-induced tolerization of adoptively transferred OX40-competent OTII T lymphocytes. In conclusion, OX40-mediated inactivation of T reg cell function unleashes nearby DCs, allowing them to induce an adaptive immune response. In addition, the known OX40-dependent delivery of fitness signals to activated T cells is boosted by concurrent T reg cell inhibition. OX40 triggering thus has multiple effects that converge to mediate tumor rejection.

Polyomavirus BK with rearranged noncoding control region emerge in vivo in renal transplant patients and increase viral replication and cytopathology

2008-04-14

Immunosuppression is required for BK viremia and polyomavirus BK–associated nephropathy (PVAN) in kidney transplants (KTs), but the role of viral determinants is unclear. We examined BKV noncoding control regions (NCCR), which coordinate viral gene expression and replication. In 286 day–matched plasma and urine samples from 129 KT patients with BKV viremia, including 70 with PVAN, the majority of viruses contained archetypal (ww-) NCCRs. However, rearranged (rr-) NCCRs were more frequent in plasma than in urine samples (22 vs. 4%; P < 0.001), and were associated with 20-fold higher plasma BKV loads (2.0 x 10⁴/ml vs. 4.4 x 10⁵/ml; P < 0.001). Emergence of rr-NCCR in plasma correlated with duration and peak BKV load (R² = 0.64; P < 0.001). This was confirmed in a prospective cohort of 733 plasma samples from 227 patients. For 39 PVAN patients with available biopsies, rr-NCCRs were associated with more extensive viral replication and inflammation. Cloning of 10 rr-NCCRs revealed diverse duplications or deletions in different NCCR subregions, but all were sufficient to increase early gene expression, replication capacity, and cytopathology of recombinant BKV in vitro. Thus, rr-NCCR BKV emergence in plasma is linked to increased replication capacity and disease in KTs.

Phospholipase C-{gamma}2 and Vav cooperate within signaling microclusters to propagate B cell spreading in response to membrane-bound antigen

2008-04-14

B cell receptor (BCR) recognition of membrane-bound antigen initiates a spreading and contraction response, the extent of which is controlled through the formation of signaling-active BCR-antigen microclusters and ultimately affects the outcome of B cell activation. We followed a genetic approach to define the molecular requirements of BCR-induced spreading and microcluster formation. We identify a key role for phospholipase C-2 (PLC2), Vav, B cell linker, and Bruton's tyrosine kinase in the formation of highly coordinated "microsignalosomes," the efficient assembly of which is absolutely dependent on Lyn and Syk. Using total internal reflection fluorescence microscopy, we examine at high resolution the recruitment of PLC2 and Vav to microsignalosomes, establishing a novel synergistic relationship between the two. Thus, we demonstrate the importance of cooperation between components of the microsignalosome in the amplification of signaling and propagation of B cell spreading, which is critical for appropriate B cell activation.

Alum adjuvant boosts adaptive immunity by inducing uric acid and activating inflammatory dendritic cells

2008-04-14

Alum (aluminum hydroxide) is the most widely used adjuvant in human vaccines, but the mechanism of its adjuvanticity remains unknown. In vitro studies showed no stimulatory effects on dendritic cells (DCs). In the absence of adjuvant, Ag was taken up by lymph node (LN)–resident DCs that acquired soluble Ag via afferent lymphatics, whereas after injection of alum, Ag was taken up, processed, and presented by inflammatory monocytes that migrated from the peritoneum, thus becoming inflammatory DCs that induced a persistent Th2 response. The enhancing effects of alum on both cellular and humoral immunity were completely abolished when CD11c⁺ monocytes and DCs were conditionally depleted during immunization. Mechanistically, DC-driven responses were abolished in MyD88-deficient mice and after uricase treatment, implying the induction of uric acid. These findings suggest that alum adjuvant is immunogenic by exploiting "nature's adjuvant," the inflammatory DC through induction of the endogenous danger signal uric acid.

Distinct cell-specific control of autoimmunity and infection by Fc{gamma}RIIb

2008-04-14

FcRIIb is an inhibitory Fc receptor expressed on B cells and myeloid cells. It is important in controlling responses to infection, and reduced expression or function predisposes to autoimmunity. To determine if increased expression of FcRIIb can modulate these processes, we created transgenic mice overexpressing FcRIIb on B cells or macrophages. Overexpression of FcRIIb on B cells reduced the immunoglobulin G component of T-dependent immune responses, led to early resolution of collagen-induced arthritis (CIA), and reduced spontaneous systemic lupus erythematosus (SLE). In contrast, overexpression on macrophages had no effect on immune responses, CIA, or SLE but increased mortality after Streptococcus pneumoniae infection. These results help define the role of FcRIIb in immune responses, demonstrate the contrasting roles played by FcRIIb on B cells and macrophages in the control of infection and autoimmunity, and emphasize the therapeutic potential for modulation of FcRIIb expression on B cells in inflammatory and autoimmune disease.

IL-9- and mast cell-mediated intestinal permeability predisposes to oral antigen hypersensitivity

2008-04-14

Previous mouse and clinical studies demonstrate a link between Th2 intestinal inflammation and induction of the effector phase of food allergy. However, the mechanism by which sensitization and mast cell responses occurs is largely unknown. We demonstrate that interleukin (IL)-9 has an important role in this process. IL-9–deficient mice fail to develop experimental oral antigen–induced intestinal anaphylaxis, and intestinal IL-9 overexpression induces an intestinal anaphylaxis phenotype (intestinal mastocytosis, intestinal permeability, and intravascular leakage). In addition, intestinal IL-9 overexpression predisposes to oral antigen sensitization, which requires mast cells and increased intestinal permeability. These observations demonstrate a central role for IL-9 and mast cells in experimental intestinal permeability in oral antigen sensitization and suggest that IL-9–mediated mast cell responses have an important role in food allergy.

Interaction of CD44 and hyaluronan is the dominant mechanism for neutrophil sequestration in inflamed liver sinusoids

2008-04-14

Adhesion molecules known to be important for neutrophil recruitment in many other organs are not involved in recruitment of neutrophils into the sinusoids of the liver. The prevailing view is that neutrophils become physically trapped in inflamed liver sinusoids. In this study, we used a biopanning approach to identify hyaluronan (HA) as disproportionately expressed in the liver versus other organs under both basal and inflammatory conditions. Spinning disk intravital microscopy revealed that constitutive HA expression was restricted to liver sinusoids. Blocking CD44–HA interactions reduced neutrophil adhesion in the sinusoids of endotoxemic mice, with no effect on rolling or adhesion in postsinusoidal venules. Neutrophil but not endothelial CD44 was required for adhesion in sinusoids, yet neutrophil CD44 avidity for HA did not increase significantly in endotoxemia. Instead, activation of CD44–HA engagement via qualitative modification of HA was demonstrated by a dramatic induction of serum-derived HA-associated protein in sinusoids in response to lipopolysaccharide (LPS). LPS-induced hepatic injury was significantly reduced by blocking CD44–HA interactions. Administration of anti-CD44 antibody 4 hours after LPS rapidly detached adherent neutrophils in sinusoids and improved sinusoidal perfusion in endotoxemic mice, revealing CD44 as a potential therapeutic target in systemic inflammatory responses involving the liver.

Gene placement and competition control T cell receptor {gamma} variable region gene rearrangement

Xiong, N., Zhang, L., Kang, C., Raulet, D. H. — 2008-04-14

The production of distinct sets of T cell receptor (TCR) ⁺ T cells occurs in an ordered fashion in thymic development. The V3 and V4 genes, located downstream in the TCR C1 gene cluster, are expressed by the earliest waves of developing TCR⁺ T cells in the fetal thymus, destined for intraepithelial locations. Upstream V2 and V5 genes are expressed in later waves in the adult and constitute most TCR⁺ T cells in secondary lymphoid tissue. This developmental pattern is caused in part by a preference for rearrangements of the downstream V3 and V4 genes in the early fetal stage, which switches to a preference for rearrangements of the upstream V2 and V5 gene rearrangements in the adult. Our gene targeting studies show that the downstream V genes rearrange preferentially in the early fetal thymus because of their downstream location, independent of promoter or recombination signal sequences and unrelated to the extent of germline transcription. Remarkably, gene deletion studies show that the downstream V genes competitively inhibit upstream V rearrangements at the fetal stage. These data provide a mechanism for specialization of the fetal thymus for the production of T cells expressing specific V genes.

A minimal binding footprint on CD1d-glycolipid is a basis for selection of the unique human NKT TCR

2008-04-14

Although it has been established how CD1 binds a variety of lipid antigens (Ag), data are only now emerging that show how β T cell receptors (TCRs) interact with CD1-Ag. Using the structure of the human semiinvariant NKT TCR–CD1d–-galactosylceramide (-GalCer) complex as a guide, we undertook an alanine scanning mutagenesis approach to define the energetic basis of this interaction between the NKT TCR and CD1d. Moreover, we explored how analogues of -GalCer affected this interaction. The data revealed that an identical energetic footprint underpinned the human and mouse NKT TCR–CD1d–-GalCer cross-reactivity. Some, but not all, of the contact residues within the J18-encoded invariant CDR3 loop and Vβ11-encoded CDR2β loop were critical for recognizing CD1d. The residues within the V24-encoded CDR1 and CDR3 loops that contacted the glycolipid Ag played a smaller energetic role compared with the NKT TCR residues that contacted CD1d. Collectively, our data reveal that the region distant to the protruding Ag and directly above the F' pocket of CD1d was the principal factor in the interaction with the NKT TCR. Accordingly, although the structural footprint at the NKT TCR–CD1d–-GalCer is small, the energetic footprint is smaller still, and reveals the minimal requirements for CD1d restriction.

Leukocyte transmigration requires kinesin-mediated microtubule-dependent membrane trafficking from the lateral border recycling compartment

Mamdouh, Z., Kreitzer, G. E., Muller, W. A. — 2008-04-14

Diapedesis of leukocytes across endothelial cells is a crucial step in both the innate and adaptive immune responses. Surface molecules on leukocytes and endothelial cells critical for diapedesis have been identified, but the mechanisms underlying this process are not understood. Homophilic interaction between platelet/endothelial cell adhesion molecule (PECAM) on leukocytes and PECAM at the endothelial border triggers targeted recycling of membrane from a reticulum localized close to the endothelial cell lateral border. This membrane surrounds the transmigrating leukocyte (Mamdouh, Z., X. Chen, L.M. Pierini, F.R. Maxfield, and W.A. Muller. 2003. Nature. 421:748–753). How this process occurs and whether it is required for diapedesis independent of PECAM are not known. We now report that targeted recycling from this lateral border recycling compartment (LBRC) is required for diapedesis, is mediated by kinesin family molecular motors, and requires normally functioning endothelial microtubules. Selective disruption of microtubules or inhibition of kinesin motor domain blocked targeted recycling and diapedesis of monocytes. Furthermore, targeted recycling of membrane from the LBRC was required for transmigration of lymphocytes, which migrate independently of PECAM. Thus, trafficking of membrane from the LBRC to surround leukocytes may be a general requirement for migration of leukocytes across the endothelial cell border. Furthermore, these data provide the first demonstration of a role for endothelial microtubules and kinesins in promoting diapedesis, and a mechanism to explain targeted recycling.

Arthritis induced by posttranslationally modified (citrullinated) fibrinogen in DR4-IE transgenic mice

2008-04-14

Rheumatoid arthritis (RA) is a common autoimmune disease that afflicts the synovium of diarthrodial joints. The pathogenic mechanisms inciting this disease are not fully characterized, but may involve the loss of tolerance to posttranslationally modified (citrullinated) antigens. We have demonstrated that this modification leads to a selective increase in antigenic peptide affinity for major histocompatibility complex (MHC) class II molecules that carry the RA-associated shared epitope, such as HLA-DRB1*0401 (DR4). We describe the induction of arthritis in DR4-IE transgenic (tg) mice with citrullinated fibrinogen, a protein commonly found in inflamed synovial tissue and a frequent target of autoantibodies in RA patients. The disease induced in these mice was characterized by synovial hyperplasia followed by ankylosis, but lacked a conspicuous polymorphonuclear cell infiltrate. Immunological analysis of these mice through T cell epitope scanning and antibody microarray analysis identified a unique profile of citrulline-specific reactivity that was not found in DR4-IE tg mice immunized with unmodified fibrinogen or in wild-type C57BL/6 mice immunized with citrullinated fibrinogen, two conditions where arthritis was not observed. These observations directly implicate citrullinated fibrinogen as arthritogenic in the context of RA-associated MHC class II molecules.

The orthopoxvirus type I IFN binding protein is essential for virulence and an effective target for vaccination

2008-04-14

Nonliving antiviral vaccines traditionally target proteins expressed at the surface of the virion with the hope of inducing neutralizing antibodies. Orthopoxviruses (OPVs), such as the human smallpox virus and the mouse-equivalent ectromelia virus (ECTV; an agent of mousepox), encode immune response modifiers (IRMs) that can increase virulence by decreasing the host immune response. We show that one of these IRMs, the type I interferon (IFN) binding protein (bp) of ECTV, is essential for ECTV virulence and is a natural target of the antibody response. More strikingly, we demonstrate that immunization with recombinant type I IFN bp protects mice from lethal mousepox. Collectively, our experiments have important implications for our understanding of the role of IRMs in OPV virulence and of type I IFNs in OPV infections. Furthermore, our work provides proof of concept that effective antiviral vaccines can be made to prevent disease by targeting virulence factors as an alternative to the traditional approach that attempts to prevent infection by virus neutralization.

Enhancing therapeutic vaccination by blocking PD-1-mediated inhibitory signals during chronic infection

2008-03-17

Therapeutic vaccination is a potentially promising strategy to enhance T cell immunity and viral control in chronically infected individuals. However, therapeutic vaccination approaches have fallen short of expectations, and effective boosting of antiviral T cell responses has not always been observed. One of the principal reasons for the limited success of therapeutic vaccination is that virus-specific T cells become functionally exhausted during chronic infections. We now provide a novel strategy for enhancing the efficacy of therapeutic vaccines. In this study, we show that blocking programmed death (PD)-1/PD-L1 inhibitory signals on exhausted CD8⁺ T cells, in combination with therapeutic vaccination, synergistically enhances functional CD8⁺ T cell responses and improves viral control in mice chronically infected with lymphocytic choriomeningitis virus. This combinatorial therapeutic vaccination was effective even in the absence of CD4⁺ T cell help. Thus, our study defines a potent new approach to augment the efficacy of therapeutic vaccination by blocking negative signals. Such an approach may have broad applications in developing treatment strategies for chronic infections in general, and perhaps also for tumors.

DNA-PKcs and Artemis function in the end-joining phase of immunoglobulin heavy chain class switch recombination

Franco, S., Murphy, M. M., Li, G., Borjeson, T., Boboila, C., Alt, F. W. — 2008-03-17

The DNA-dependent protein kinase catalytic subunit (DNA-PKcs) and Artemis are classical nonhomologous DNA end-joining (C-NHEJ) factors required for joining a subset of DNA double-strand breaks (DSB), particularly those requiring end processing. In mature B cells, activation-induced cytidine deaminase (AID) initiates class switch recombination (CSR) by introducing lesions into S regions upstream of two recombining C_H exons, which are processed into DSBs and rejoined by C-NHEJ to complete CSR. The function of DNA-PKcs in CSR has been controversial with some reports but not others showing that DNA-PKcs–deficient mice are significantly impaired for CSR. Artemis-deficient B cells reportedly undergo CSR at normal levels. Overall, it is still not known whether there are any CSR-associated DSBs that require DNA-PKcs and/or Artemis to be joined. Here, we have used an immunoglobulin (Ig)H locus-specific fluorescent in situ hybridization assay to unequivocally demonstrate that both DNA-PKcs and, unexpectedly, Artemis are necessary for joining a subset of AID-dependent DSBs. In the absence of either factor, B cells activated for CSR frequently generate AID-dependent IgH locus chromosomal breaks and translocations. We also find that under specific activation conditions, DNA-PKcs^–/– B cells with chromosomal breaks are eliminated or at least prevented from progressing to metaphase via a p53-dependent response.

The AKT-mTOR axis regulates de novo differentiation of CD4+Foxp3+ cells

Haxhinasto, S., Mathis, D., Benoist, C. — 2008-03-17

CD4⁺Foxp3⁺ regulatory T (T reg) cells play an essential role in maintaining immunological tolerance via their suppressive function on conventional CD4⁺ T (Tconv) cells. Repertoire studies suggest that distinct T cell receptor signaling pathways lead to T reg differentiation, but the signals that regulate T reg specification are largely unknown. We identify AKT as a strong repressor of entry into the T reg phenotype in vitro and in vivo. A constitutively active allele of AKT substantially diminished TGF-β–induced Foxp3 expression in a kinase-dependent manner and via a rapamycin-sensitive pathway, implicating the AKT–mammalian target of rapamycin axis. The observed impairment in Foxp3 induction was part of a broad dampening of the typical T reg transcriptional signature. Expression of active AKT at a stage before Foxp3 turn on during normal T reg differentiation in the thymus selectively impaired differentiation of CD4⁺Foxp3⁺ cells without any alteration in the positive selection of Tconv. Activated AKT, in contrast, did not affect established Foxp3 expression in T reg cells. These results place AKT at a nexus of signaling pathways whose proper activation has a strong and broad impact on the onset of T reg specification.

Glutamate mediates platelet activation through the AMPA receptor

2008-03-17

Glutamate is an excitatory neurotransmitter that binds to the kainate receptor, the N-methyl-D-aspartate (NMDA) receptor, and the -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor (AMPAR). Each receptor was first characterized and cloned in the central nervous system (CNS). Glutamate is also present in the periphery, and glutamate receptors have been identified in nonneuronal tissues, including bone, heart, kidney, pancreas, and platelets. Platelets play a central role in normal thrombosis and hemostasis, as well as contributing greatly to diseases such as stroke and myocardial infarction. Despite the presence of glutamate in platelet granules, the role of glutamate during hemostasis is unknown. We now show that activated platelets release glutamate, that platelets express AMPAR subunits, and that glutamate increases agonist-induced platelet activation. Furthermore, we demonstrate that glutamate binding to the AMPAR increases intracellular sodium concentration and depolarizes platelets, which are important steps in platelet activation. In contrast, platelets treated with the AMPAR antagonist CNQX or platelets derived from GluR1 knockout mice are resistant to AMPA effects. Importantly, mice lacking GluR1 have a prolonged time to thrombosis in vivo. Our data identify glutamate as a regulator of platelet activation, and suggest that the AMPA receptor is a novel antithrombotic target.

Sustained expression of microRNA-155 in hematopoietic stem cells causes a myeloproliferative disorder

2008-03-17

Mammalian microRNAs are emerging as key regulators of the development and function of the immune system. Here, we report a strong but transient induction of miR-155 in mouse bone marrow after injection of bacterial lipopolysaccharide (LPS) correlated with granulocyte/monocyte (GM) expansion. Demonstrating the sufficiency of miR-155 to drive GM expansion, enforced expression in mouse bone marrow cells caused GM proliferation in a manner reminiscent of LPS treatment. However, the miR-155–induced GM populations displayed pathological features characteristic of myeloid neoplasia. Of possible relevance to human disease, miR-155 was found to be overexpressed in the bone marrow of patients with certain subtypes of acute myeloid leukemia (AML). Furthermore, miR-155 repressed a subset of genes implicated in hematopoietic development and disease. These data implicate miR-155 as a contributor to physiological GM expansion during inflammation and to certain pathological features associated with AML, emphasizing the importance of proper miR-155 regulation in developing myeloid cells during times of inflammatory stress.

The MHC class I peptide repertoire is molded by the transcriptome

2008-03-17

Under steady-state conditions, major histocompatibility complex (MHC) I molecules are associated with self-peptides that are collectively referred to as the MHC class I peptide (MIP) repertoire. Very little is known about the genesis and molecular composition of the MIP repertoire. We developed a novel high-throughput mass spectrometry approach that yields an accurate definition of the nature and relative abundance of unlabeled peptides presented by MHC I molecules. We identified 189 and 196 MHC I–associated peptides from normal and neoplastic mouse thymocytes, respectively. By integrating our peptidomic data with global profiling of the transcriptome, we reached two conclusions. The MIP repertoire of primary mouse thymocytes is biased toward peptides derived from highly abundant transcripts and is enriched in peptides derived from cyclins/cyclin-dependent kinases and helicases. Furthermore, we found that ~25% of MHC I–associated peptides were differentially expressed on normal versus neoplastic thymocytes. Approximately half of those peptides are derived from molecules directly implicated in neoplastic transformation (e.g., components of the PI3K–AKT–mTOR pathway). In most cases, overexpression of MHC I peptides on cancer cells entailed posttranscriptional mechanisms. Our results show that high-throughput analysis and sequencing of MHC I–associated peptides yields unique insights into the genesis of the MIP repertoire in normal and neoplastic cells.

Antagonism of FOG-1 and GATA factors in fate choice for the mast cell lineage

2008-03-17

The zinc finger transcription factor GATA-1 requires direct physical interaction with the cofactor friend of GATA-1 (FOG-1) for its essential role in erythroid and megakaryocytic development. We show that in the mast cell lineage, GATA-1 functions completely independent of FOG proteins. Moreover, we demonstrate that FOG-1 antagonizes the fate choice of multipotential progenitor cells for the mast cell lineage, and that its down-regulation is a prerequisite for mast cell development. Remarkably, ectopic expression of FOG-1 in committed mast cell progenitors redirects them into the erythroid, megakaryocytic, and granulocytic lineages. These lineage switches correlate with transcriptional down-regulation of GATA-2, an essential mast cell GATA factor, via switching of GATA-1 for GATA-2 at a key enhancer element upstream of the GATA-2 gene. These findings illustrate combinatorial control of cell fate identity by a transcription factor and its cofactor, and highlight the role of transcriptional networks in lineage determination. They also provide evidence for lineage instability during early stages of hematopoietic lineage commitment.

Functional and genomic profiling of effector CD8 T cell subsets with distinct memory fates

2008-03-17

An important question in memory development is understanding the differences between effector CD8 T cells that die versus effector cells that survive and give rise to memory cells. In this study, we provide a comprehensive phenotypic, functional, and genomic profiling of terminal effectors and memory precursors. Using killer cell lectin-like receptor G1 as a marker to distinguish these effector subsets, we found that despite their diverse cell fates, both subsets possessed remarkably similar gene expression profiles and functioned as equally potent killer cells. However, only the memory precursors were capable of making interleukin (IL) 2, thus defining a novel effector cell that was cytotoxic, expressed granzyme B, and produced inflammatory cytokines in addition to IL-2. This effector population then differentiated into long-lived protective memory T cells capable of self-renewal and rapid recall responses. Experiments to understand the signals that regulate the generation of terminal effectors versus memory precursors showed that cells that continued to receive antigenic stimulation during the later stages of infection were more likely to become terminal effectors. Importantly, curtailing antigenic stimulation toward the tail end of the acute infection enhanced the generation of memory cells. These studies support the decreasing potential model of memory differentiation and show that the duration of antigenic stimulation is a critical regulator of memory formation.

Loss of the BH3-only protein Bmf impairs B cell homeostasis and accelerates {gamma} irradiation-induced thymic lymphoma development

2008-03-17

Members of the Bcl-2 protein family play crucial roles in the maintenance of tissue homeostasis by regulating apoptosis in response to developmental cues or exogenous stress. Proapoptotic BH3-only members of the Bcl-2 family are essential for initiation of cell death, and they function by activating the proapoptotic Bcl-2 family members Bax and/or Bak, either directly or indirectly through binding to prosurvival Bcl-2 family members. Bax and Bak then elicit the downstream events in apoptosis signaling. Mammals have at least eight BH3-only proteins and they are activated in a stimulus-specific, as well as a cell type–specific, manner. We have generated mice lacking the BH3-only protein Bcl-2–modifying factor (Bmf) to investigate its role in cell death signaling. Our studies reveal that Bmf is dispensable for embryonic development and certain forms of stress-induced apoptosis, including loss of cell attachment (anoikis) or UV irradiation. Remarkably, loss of Bmf protected lymphocytes against apoptosis induced by glucocorticoids or histone deacetylase inhibition. Moreover, bmf^–/– mice develop a B cell–restricted lymphadenopathy caused by the abnormal resistance of these cells to a range of apoptotic stimuli. Finally, Bmf-deficiency accelerated the development of irradiation–induced thymic lymphomas. Our results demonstrate that Bmf plays a critical role in apoptosis signaling and can function as a tumor suppressor.

Contrasting roles of SPARC-related granuloma in bacterial containment and in the induction of anti-Salmonella typhimurium immunity

Rotta, G., Matteoli, G., Mazzini, E., Nuciforo, P., Colombo, M. P., Rescigno, M. — 2008-03-17

The role of matricellular proteins in bacterial containment and in the induction of pathogen-specific adaptive immune responses is unknown. We studied the function of the matricellular protein secreted protein, acidic and rich in cysteine (SPARC/osteonectin) in the dissemination of locally injected Salmonella typhimurium and in the subsequent immune response. We show that SPARC was required for the development of organized acute inflammatory reactions with granuloma-like (GL) features and for the control of bacterial spreading to draining lymph nodes (DLNs). However, SPARC-related GL also inhibited dendritic cell (DC) migration to the DLNs and limited the development of adaptive immune response, thus conferring increased susceptibility to the pathogen. In SPARC-deficient mice, both DC migration and antigen-specific responses were restored against bacteria, leading to protective anti–S. typhimurium immunity. This highlights a new function of matricellular proteins in bacterial infection and suggests that initial containment of bacteria can have drawbacks.

A gammaherpesvirus-secreted activator of V{beta}4+ CD8+ T cells regulates chronic infection and immunopathology

2008-03-17

Little is known about herpesvirus modulation of T cell activation in latently infected individuals or the implications of such for chronic immune disorders. Murine gammaherpesvirus 68 (MHV68) elicits persistent activation of CD8⁺ T cells bearing a Vβ4⁺ T cell receptor (TCR) by a completely unknown mechanism. We show that a novel MHV68 protein encoded by the M1 gene is responsible for Vβ4⁺ CD8⁺ T cell stimulation in a manner reminiscent of a viral superantigen. During infection, M1 expression induces a Vβ4⁺ effector T cell response that resists functional exhaustion and appears to suppress virus reactivation from peritoneal cells by means of long-term interferon- (IFN) production. Mice lacking an IFN receptor (IFNR^–/–) fail to control MHV68 replication, and Vβ4⁺ and CD8⁺ T cell activation by M1 instead contributes to severe inflammation and multiorgan fibrotic disease. Thus, M1 manipulates the host CD8⁺ T cell response in a manner that facilitates latent infection in an immunocompetent setting, but promotes disease during a dysregulated immune response. Identification of a viral pathogenecity determinant with superantigen-like activity for CD8⁺ T cells broadens the known repertoire of viral immunomodulatory molecules, and its function illustrates the delicate balance achieved between persistent viruses and the host immune response.

Caspase-8 is activated by cathepsin D initiating neutrophil apoptosis during the resolution of inflammation

2008-03-17

In the resolution of inflammatory responses, neutrophils rapidly undergo apoptosis. We describe a new proapoptotic pathway in which cathepsin D directly activates caspase-8. Cathepsin D is released from azurophilic granules in neutrophils in a caspase-independent but reactive oxygen species–dependent manner. Under inflammatory conditions, the translocation of cathepsin D in the cytosol is blocked. Pharmacological or genetic inhibition of cathepsin D resulted in delayed caspase activation and reduced neutrophil apoptosis. Cathepsin D deficiency or lack of its translocation in the cytosol prolongs innate immune responses in experimental bacterial infection and in septic shock. Thus, we identified a new function of azurophilic granules that is in addition to their role in bacterial defense mechanisms: to regulate the life span of neutrophils and, therefore, the duration of innate immune responses through the release of cathepsin D.

Allergic pulmonary inflammation in mice is dependent on eosinophil-induced recruitment of effector T cells

2008-03-17

The current paradigm surrounding allergen-mediated T helper type 2 (Th2) immune responses in the lung suggests an almost hegemonic role for T cells. Our studies propose an alternative hypothesis implicating eosinophils in the regulation of pulmonary T cell responses. In particular, ovalbumin (OVA)-sensitized/challenged mice devoid of eosinophils (the transgenic line PHIL) have reduced airway levels of Th2 cytokines relative to the OVA-treated wild type that correlated with a reduced ability to recruit effector T cells to the lung. Adoptive transfer of Th2-polarized OVA-specific transgenic T cells (OT-II) alone into OVA-challenged PHIL recipient mice failed to restore Th2 cytokines, airway histopathologies, and, most importantly, the recruitment of pulmonary effector T cells. In contrast, the combined transfer of OT-II cells and eosinophils into PHIL mice resulted in the accumulation of effector T cells and a concomitant increase in both airway Th2 immune responses and histopathologies. Moreover, we show that eosinophils elicit the expression of the Th2 chemokines thymus- and activation-regulated chemokine/CCL17 and macrophage-derived chemokine/CCL22 in the lung after allergen challenge, and blockade of these chemokines inhibited the recruitment of effector T cells. In summary, the data suggest that pulmonary eosinophils are required for the localized recruitment of effector T cells.

Age-associated decline in T cell repertoire diversity leads to holes in the repertoire and impaired immunity to influenza virus

2008-03-17

A diverse T cell repertoire is essential for a vigorous immune response to new infections, and decreasing repertoire diversity has been implicated in the age-associated decline in CD8 T cell immunity. In this study, using the well-characterized mouse influenza virus model, we show that although comparable numbers of CD8 T cells are elicited in the lung and lung airways of young and aged mice after de novo infection, a majority of aged mice exhibit profound shifts in epitope immunodominance and restricted diversity in the TCR repertoire of responding cells. A preferential decline in reactivity to viral epitopes with a low naive precursor frequency was observed, in some cases leading to "holes" in the T cell repertoire. These effects were also seen in young thymectomized mice, consistent with the role of the thymus in maintaining naive repertoire diversity. Furthermore, a decline in repertoire diversity generally correlated with impaired responses to heterosubtypic challenge. This study formally demonstrates in a mouse infection model that naturally occurring contraction of the naive T cell repertoire can result in impaired CD8 T cell responses to known immunodominant epitopes and decline in heterosubtypic immunity. These observations have important implications for the design of vaccine strategies for the elderly.

CD94-NKG2A recognition of human leukocyte antigen (HLA)-E bound to an HLA class I leader sequence

2008-03-17

The recognition of human leukocyte antigen (HLA)-E by the heterodimeric CD94-NKG2 natural killer (NK) receptor family is a central innate mechanism by which NK cells monitor the expression of other HLA molecules, yet the structural basis of this highly specific interaction is unclear. Here, we describe the crystal structure of CD94-NKG2A in complex with HLA-E bound to a peptide derived from the leader sequence of HLA-G. The CD94 subunit dominated the interaction with HLA-E, whereas the NKG2A subunit was more peripheral to the interface. Moreover, the invariant CD94 subunit dominated the peptide-mediated contacts, albeit with poor surface and chemical complementarity. This unusual binding mode was consistent with mutagenesis data at the CD94-NKG2A–HLA-E interface. There were few conformational changes in either CD94-NKG2A or HLA-E upon ligation, and such a "lock and key" interaction is typical of innate receptor–ligand interactions. Nevertheless, the structure also provided insight into how this interaction can be modulated by subtle changes in the peptide ligand or by the pairing of CD94 with other members of the NKG2 family. Differences in the docking strategies used by the NKG2D and CD94-NKG2A receptors provided a basis for understanding the promiscuous nature of ligand recognition by NKG2D compared with the fidelity of the CD94-NKG2 receptors.

iPLA2{beta}: front and center in human monocyte chemotaxis to MCP-1

Mishra, R. S., Carnevale, K. A., Cathcart, M. K. — 2008-02-18

Monocyte chemoattractant protein-1 (MCP-1) directs migration of blood monocytes to inflamed tissues. Despite the central role of chemotaxis in immune responses, the regulation of chemotaxis by signal transduction pathways and their in vivo significance remain to be thoroughly deciphered. In this study, we examined the intracellular location and functions of two recently identified regulators of chemotaxis, Ca²⁺-independent phospholipase (iPLA₂β) and cytosolic phospholipase (cPLA₂), and substantiate their in vivo importance. These enzymes are cytoplasmic in unstimulated monocytes. Upon MCP-1 stimulation, iPLA₂β is recruited to the membrane-enriched pseudopod. In contrast, cPLA₂ is recruited to the endoplasmic reticulum. Although iPLA₂β or cPLA₂ antisense oligodeoxyribonucleotide (ODN)–treated monocytes display reduced speed, iPLA₂β also regulates directionality and actin polymerization. iPLA₂β or cPLA₂ antisense ODN–treated adoptively transferred mouse monocytes display a profound defect in migration to the peritoneum in vivo. These converging observations reveal that iPLA₂β and cPLA₂ regulate monocyte migration from different intracellular locations, with iPLA₂β acting as a critical regulator of the cellular compass, and identify them as potential targets for antiinflammatory strategies.

Pulmonary arterial remodeling induced by a Th2 immune response

2008-02-18

Pulmonary arterial remodeling characterized by increased vascular smooth muscle density is a common lesion seen in pulmonary arterial hypertension (PAH), a deadly condition. Clinical correlation studies have suggested an immune pathogenesis of pulmonary arterial remodeling, but experimental proof has been lacking. We show that immunization and prolonged intermittent challenge via the airways with either of two different soluble antigens induced severe muscularization in small- to medium-sized pulmonary arteries. Depletion of CD4⁺ T cells, antigen-specific T helper type 2 (Th2) response, or the pathogenic Th2 cytokine interleukin 13 significantly ameliorated pulmonary arterial muscularization. The severity of pulmonary arterial muscularization was associated with increased numbers of epithelial cells and macrophages that expressed a smooth muscle cell mitogen, resistin-like molecule , but surprisingly, there was no correlation with pulmonary hypertension. Our data are the first to provide experimental proof that the adaptive immune response to a soluble antigen is sufficient to cause severe pulmonary arterial muscularization, and support the clinical observations in pediatric patients and in companion animals that muscularization represents one of several injurious events to the pulmonary artery that may collectively contribute to PAH.

Increased endothelial expression of Toll-like receptor 2 at sites of disturbed blood flow exacerbates early atherogenic events

2008-02-18

Toll-like receptors (TLRs) are pattern recognition receptors of innate immunity. TLRs initiate inflammatory pathways that may exacerbate chronic inflammatory diseases like atherosclerosis. En face laser scanning confocal microscopy (LSCM) of isolated aortic segments revealed the distribution of intimal TLR2 expression and the atheroprotective outcomes resulting from a TLR2 deficiency. TLR2 expression was restricted to endothelial cells in regions of disturbed blood flow, such as the lesser curvature region, in atherosclerosis-prone, low-density lipoprotein receptor–deficient (LDLr^–/–) mice. Diet-induced hyperlipidemia in LDLr^–/– mice increased this regional endothelial TLR2 expression. Bone marrow (BM) reconstitution of LDLr^–/– and LDLr^–/–TLR2^–/– mice created chimeric mice with green fluorescent protein (GFP) expression in BM-derived cells (BMGFP⁺). Lesser curvature BMGFP⁺ leukocyte accumulation, lipid accumulation, foam cell generation and endothelial cell injury were all increased by hyperlipidemia, whereas hyperlipidemic double mutant BMGFP⁺LDLr^–/–TLR2^–/– mice had reduced BMGFP⁺ leukocyte accumulation, lipid accumulation, foam cells, and endothelial cell injury. This is the first report of in vivo site-specific expression of endothelial cell TLR2. Expression of this receptor on endothelial cells contributed to early atherosclerotic processes in lesion-prone areas of the mouse aorta.

Ozone exposure in a mouse model induces airway hyperreactivity that requires the presence of natural killer T cells and IL-17

2008-02-18

Exposure to ozone, which is a major component of air pollution, induces a form of asthma that occurs in the absence of adaptive immunity. Although ozone-induced asthma is characterized by airway neutrophilia, and not eosinophilia, it is nevertheless associated with airway hyperreactivity (AHR), which is a cardinal feature of asthma. Because AHR induced by allergens requires the presence of natural killer T (NKT) cells, we asked whether ozone-induced AHR had similar requirements. We found that repeated exposure of wild-type (WT) mice to ozone induced severe AHR associated with an increase in airway NKT cells, neutrophils, and macrophages. Surprisingly, NKT cell–deficient (CD1d^–/– and J18^–/–) mice failed to develop ozone-induced AHR. Further, treatment of WT mice with an anti-CD1d mAb blocked NKT cell activation and prevented ozone-induced AHR. Moreover, ozone-induced, but not allergen-induced, AHR was associated with NKT cells producing interleukin (IL)-17, and failed to occur in IL-17^–/– mice nor in WT mice treated with anti–IL-17 mAb. Thus, ozone exposure induces AHR that requires the presence of NKT cells and IL-17 production. Because NKT cells are required for the development of two very disparate forms of AHR (ozone- and allergen-induced), our results strongly suggest that NKT cells mediate a unifying pathogenic mechanism for several distinct forms of asthma, and represent a unique target for effective asthma therapy.

Inhibition of histamine-mediated signaling confers significant protection against severe malaria in mouse models of disease

2008-02-18

From the inoculation of Plasmodium sporozoites via Anopheles mosquito bites to the development of blood-stage parasites, a hallmark of the host response is an inflammatory reaction characterized by elevated histamine levels in the serum and tissues. Given the proinflammatory and immunosuppressive activities associated with histamine, we postulated that this vasoactive amine participates in malaria pathogenesis. Combined genetic and pharmacologic approaches demonstrated that histamine binding to H1R and H2R but not H3R and H4R increases the susceptibility of mice to infection with Plasmodium. To further understand the role of histamine in malaria pathogenesis, we used histidine decarboxylase–deficient (HDC^–/–) mice, which are free of histamine. HDC^–/– mice were highly resistant to severe malaria whether infected by mosquito bites or via injection of infected erythrocytes. HDC^–/– mice displayed resistance to two lethal strains: Plasmodium berghei (Pb) ANKA, which triggers cerebral malaria (CM), and Pb NK65, which causes death without neurological symptoms. The resistance of HDC^–/– mice to CM was associated with preserved blood–brain barrier integrity, the absence of infected erythrocyte aggregation in the brain vessels, and a lack of sequestration of CD4 and CD8 T cells. We demonstrate that histamine-mediated signaling contributes to malaria pathogenesis. Understanding the basis for these biological effects of histamine during infection may lead to novel therapeutic strategies to alleviate the severity of malaria.

Oct2 enhances antibody-secreting cell differentiation through regulation of IL-5 receptor {alpha} chain expression on activated B cells

2008-02-18

Mice lacking a functional gene for the Oct2 transcriptional activator display several developmental and functional deficiencies in the B lymphocyte lineage. These include defective B cell receptor (BCR) and Toll-like receptor 4 signaling, an absence of B-1 and marginal zone populations, and globally reduced levels of serum immunoglobulin (Ig) in naive and immunized animals. Oct2 was originally identified through its ability to bind to regulatory regions in the Ig loci, but genetic evidence has not supported an essential role for Oct2 in the expression of Ig genes. We describe a new Oct2-mediated role in B cells. Oct2 augments the ability of activated B cells to differentiate to antibody-secreting plasma cells (ASCs) under T cell–dependent conditions through direct regulation of the gene encoding the chain of the interleukin (IL) 5 receptor. Ectopic expression of IL-5R in oct2-deficient B cells largely restores their ability to differentiate to functional ASCs in vitro but does not correct other phenotypic defects in the mutants, such as the maturation and specialization of peripheral B cells, which must therefore rely on distinct Oct2 target genes. IL-5 augments ASC differentiation in vitro, and we show that IL-5 directly activates the plasma cell differentiation program by enhancing blimp1 expression.

Altered development of the brain after focal herpesvirus infection of the central nervous system

2008-02-18

Human cytomegalovirus infection of the developing central nervous system (CNS) is a major cause of neurological damage in newborn infants and children. To investigate the pathogenesis of this human infection, we developed a mouse model of infection in the developing CNS. Intraperitoneal inoculation of newborn animals with murine cytomegalovirus resulted in virus replication in the liver followed by virus spread to the brain. Virus infection of the CNS was associated with the induction of inflammatory responses, including the induction of a large number of interferon-stimulated genes and histological evidence of focal encephalitis with recruitment of mononuclear cells to foci containing virus-infected cells. The morphogenesis of the cerebellum was delayed in infected animals. The defects in cerebellar development in infected animals were generalized and, although correlated temporally with virus replication and CNS inflammation, spatially unrelated to foci of virus-infected cells. Specific defects included decreased granular neuron proliferation and migration, expression of differentiation markers, and activation of neurotrophin receptors. These findings suggested that in the developing CNS, focal virus infection and induction of inflammatory responses in resident and infiltrating mononuclear cells resulted in delayed cerebellar morphogenesis.