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Background Sepsis remains a leading cause of death in most intensive care units. Many deaths in sepsis are due to nosocomial infections in patients who have entered the immunosuppressive phase of the disorder. One cause of immunosuppression in sepsis is T-cell exhaustion mediated by programmed cell death-1 (PD-1) interaction with its ligand (PD-L1). Studies demonstrated that blocking the interaction of PD-1 with PD-L1 with knockout mice or inhibitory antibodies reversed T-cell dysfunction and improved sepsis survival. This study assessed the efficacy of a novel short-acting peptide (compound 8) that inhibits PD-1:PD-L1 signaling in a clinically relevant second-hit fungal sepsis model. Methods Mice underwent cecal ligation and puncture to induce peritonitis. Three days later, mice received intravenous injection of Candida albicans. Forty-eight hours after Candida infection, mice were treated with compound 8 or inactive peptide. The effect of Candida infection on expression of coinhibitory molecules, PD-1, and PD-L1 were quantitated by flow cytometry on CD4+ cells, CD8+ cells, natural killer (NK) cells, and natural killer T-cells (NKT). The effect of compound 8 on survival was also examined. Results Four days after fungal infection, PD-1 and PD-L1 expressions were markedly increased on CD4+, NK, and NKT cells in septic versus sham-operated mice (%PD-1 on CD4+, 11.9% versus 2.8%; and %PD-L1 on NKT, 14.8% versus 0.5%). Compared with control, compound 8 caused a 2-fold increase in survival from 30% to 60%, P Conclusions Compound 8 significantly improved survival in a clinically relevant immunosuppressive model of sepsis. These results support immunoadjuvant therapy targeting T-cell exhaustion in this lethal disease.

Sepsis is a highly lethal disorder characterized by widespread apoptosis-induced depletion of immune cells and the development of a profound immunosuppressive state. IL-7 is a potent antiapoptotic cytokine that enhances immune effector cell function and is essential for lymphocyte survival. In this study, recombinant human IL-7 (rhIL-7) efficacy and potential mechanisms of action were tested in a murine peritonitis model. Studies at two independent laboratories showed that rhIL-7 markedly improved host survival, blocked apoptosis of CD4 and CD8 T cells, restored IFN-γ production, and improved immune effector cell recruitment to the infected site. Importantly, rhIL-7 also prevented a hallmark of sepsis (i.e., the loss of delayed-type hypersensitivity), which is an IFN-γ– and T cell-dependent response. Mechanistically, rhIL-7 significantly increased the expression of the leukocyte adhesion markers LFA-1 and VLA-4, consistent with its ability to improve leukocyte function and trafficking to the infectious focus. rhIL-7 also increased the expression of CD8. The potent antiapoptotic effect of rhIL-7 was due to increased Bcl-2, as well as to a dramatic decrease in sepsis-induced PUMA, a heretofore unreported effect of IL-7. If additional animal studies support its efficacy in sepsis and if current clinical trials continue to confirm its safety in diverse settings, rhIL-7 should be strongly considered for clinical trials in sepsis.

Sepsis in mice possessing a functional human immune system causes extensive apoptotic death of human immune effector cells and increases in both pro- and anti-inflammatory human cytokines. Sepsis is the leading cause of death in critically ill patients in the United States with over 210,000 deaths annually. One stumbling block to an effective therapy of sepsis has been the lack of a clinically relevant animal model. There are important distinctions in the mouse versus human immune system regarding the host response to invading pathogens. These differences may explain the disappointing results in many sepsis clinical trials despite the clear efficacy of these agents in mouse models of sepsis. The purpose of the present study was to develop a “humanized” mouse model of sepsis and to determine if the model recapitulated the major findings of lymphocyte apoptosis and cytokine response that exist in patients with sepsis. Two-day-old NOD-scid IL2rγnull mice received an adoptive transfer of hCD34+ hematopoietic cord blood stem cells. These mice acquired a functional human innate and adaptive immune system, as evidenced by the development of all lineages of human immune cells as well as by mounting a DTH response. Eight weeks post-transfer, mice were made septic using the highly clinical relevant CLP model of sepsis, and sepsis induced marked elevations in human pro- and anti-inflammatory cytokines as well as a dramatic increase in human T and B cell apoptosis. Collectively, these results show that the humanized mouse model recapitulates many of the classic findings in patients with sepsis. Therefore, it represents an advanced, clinically relevant model for mechanistic studies of sepsis and testing of novel therapies.

This study examined the fate of dendritic cells (DCs) and macrophages (M Phi) in vivo in a murine model of sepsis. Wild-type, knockout, and transgenic mice were used to examine the role of Bcl-2 family members on the regulation of splenic DCs and M Phi survival. Bim knockout (Bim) mice and mice overexpressing Bcl-2 in selected hematopoietic cells were used: (a) overexpression of Bcl-2 in all hematopoietic cells using a vav promoter (Vav-Bcl-2) and (b) overexpression of Bcl-2 in all Major histocompatibility complex (MHC) class I cells (H-2K-Bcl-2). Mice underwent sham surgery or cecal ligation and puncture, and absolute numbers of splenic DCs and M Phi were determined. Importantly, two distinct M Phi populations, that is, well-differentiated "mature" M Phi population and a less differentiated "immature," "monocyte-like" (IM Phi) population were identified that demonstrated differential susceptibility to apoptosis. In wild-type mice, sepsis induced a 64% +/- 7% and a 77% +/- 3% decrease in absolute cell numbers of splenic DCs and IM Phi, respectively (n = 7, P < 0.05). Mature M Phi were not depleted in sepsis. No significant cell depletion was evident in Vav-Bcl-2, H-2K-Bcl-2, or Bim mice. We conclude that sepsis induces a major depletion of developing M Phi as well as DCs, and this depletion may be an important mechanism of immune suppression in sepsis.

Isolated lymphoid follicles (ILFs) are organized intestinal lymphoid structures whose formation can be induced by luminal stimuli. ILFs have been demonstrated to act as inductive sites for the generation of immune responses directed toward luminal stimuli; however, the phenotype of the immune response initiated within ILFs has largely been uninvestigated. To gain a better understanding of the immune responses initiated within ILFs, we examined phenotypic and functional aspects of the largest cellular component of the murine ILF lymphocyte population, B lymphocytes. We observed that murine ILF B lymphocytes are composed of a relatively homogenous population of follicular B-2 B lymphocytes. Consistent with their proximity to multiple stimuli, ILF B lymphocytes displayed a more activated phenotype compared with their counterparts in the spleen and Peyer's patch (PP). ILF B lymphocytes also expressed higher levels of immunomodulatory B7 and CD28 family members B7X and programmed death-1 compared with their counterparts in the spleen and PP. ILF B lymphocytes preferentially differentiate into IgA-producing plasma cells and produce more IL-4 and IL-10 and less interferon-γ compared with their counterparts in the spleen. Immunoglobulin repertoire analysis from individual ILFs demonstrated that ILFs contain a polyclonal population of B lymphocytes. These findings indicate that murine ILFs contain a polyclonal population of follicular B-2 B lymphocytes with a phenotype similar to PP B lymphocytes and that, in unchallenged animals, ILFs promote immune responses with a homeostatic phenotype.

World conditions place large populations at risk from ionizing radiation (IR) from detonation of dirty bombs or nuclear devices. In a subgroup of patients, ionizing radiation exposure would be followed by a secondary infection. The effects of radiation combined injury are potentially more lethal than either insult in isolation. The purpose of this study was to determine mechanisms of mortality and possible therapeutic targets in radiation combined injury. Mice were exposed to IR with 2.5 Gray (Gy) followed four days later by intratracheal methicillin-resistant Staphylococcus aureus (MRSA). While either IR or MRSA alone yielded 100% survival, animals with radiation combined injury had 53% survival (p = 0.01). Compared to IR or MRSA alone, mice with radiation combined injury had increased gut apoptosis, local and systemic bacterial burden, decreased splenic CD4 T cells, CD8 T cells, B cells, NK cells, and dendritic cells, and increased BAL and systemic IL-6 and G-CSF. In contrast, radiation combined injury did not alter lymphocyte apoptosis, pulmonary injury, or intestinal proliferation compared to IR or MRSA alone. In light of the synergistic increase in gut apoptosis following radiation combined injury, transgenic mice that overexpress Bcl-2 in their intestine and wild type mice were subjected to IR followed by MRSA. Bcl-2 mice had decreased gut apoptosis and improved survival compared to WT mice (92% vs. 42%; p

In the adult intestine, luminal microbiota induce cryptopatches to transform into isolated lymphoid follicles (ILFs), which subsequently act as sites for the generation of IgA responses. The events leading to this conversion are incompletely understood. Dendritic cells (DCs) are components of cryptopatches (CPs) and ILFs and were therefore evaluated in this process. We observed that the adult murine intestine contains clusters of DCs restricted to the CP/ILF continuum. A numerical and cell associative hierarchy in the adult intestine and a chronologic hierarchy in the neonatal intestine demonstrated that these clusters form after the coalescence of CD90 + cells to form CPs and before the influx of B220 + B lymphocytes to form ILFs. Cluster formation was dependent on lymphotoxin and the lymphotoxin β receptor and independent of lymphocytes. The ILF DC population was distinguished from that of the lamina propria by the absence of CD4 + CD11c + cells and an increased proportion of CD11c + B220 + cells. The formation of clusters was not limited by DC numbers but was induced by luminal microbiota. Moreover, in the absence of the chemokine CXCL13, CP transformation into ILF was arrested. Furthermore, ILF DCs express CXCL13, and depletion of DCs resulted in regression of ILFs and disorganization of CPs. These results reveal DC participation in ILF transformation and maintenance and suggest that in part this may be due to CXCL13 production by these cells.

Cell penetrating peptides (CPPs) have been used to deliver the anti-apoptotic Bcl-xL-derived BH4 peptide to prevent injury-induced apoptosis both in vitro and in vivo . Here we demonstrate that the nuclear localization sequence (NLS) from the SV40 large T antigen has favorable properties for BH4 domain delivery to lymphocytes compared to sequences based on the HIV-1 TAT sequence. While both TAT-BH4 and NLS-BH4 protected primary human mononuclear cells from radiation-induced apoptotic cell death, TAT-BH4 caused persistent membrane damage and even cell death at the highest concentrations tested (5–10 μM) and correlated with in vivo toxicity as intravenous administration of TAT-BH4 caused rapid death. The NLS-BH4 peptide has significantly attenuated toxicity compared to TAT-BH4 and we established a dosing regimen of NLS-BH4 that conferred a significant survival advantage in a post-exposure treatment model of LD90 total body irradiation.

Immune suppression is a major cause of morbidity and mortality in the patients with sepsis. Apoptotic loss of immune effector cells such as CD4 T and B cells is a key component in the loss of immune competence in sepsis. Inhibition of lymphocyte apoptosis has led to improved survival in animal models of sepsis. Using quantitative real-time polymerase chain reaction of isolated splenic CD4 T and B cells, we determined that Bim and PUMA, two key cell death proteins, are markedly upregulated during sepsis. Lymphocytes have been notoriously difficult to transfect with small interfering RNA (siRNA). Consequently a novel, cyclodextrin polymer-based, transferrin receptor-targeted, delivery vehicle was used to coadminister siRNA to Bim and PUMA to mice immediately after cecal ligation and puncture. Antiapoptotic siRNA-based therapy markedly decreased lymphocyte apoptosis and prevented the loss of splenic CD4 T and B cells. Flow cytometry confirmed in vivo delivery of siRNA to CD4 T and B cells and also demonstrated decreases in intracellular Bim and PUMA protein. In conclusion, Bim and PUMA are two critical mediators of immune cell death in sepsis. Use of a novel cyclodextrin polymer-based, transferrin receptor-targeted siRNA delivery vehicle enables effective administration of antiapoptotic siRNAs to lymphocytes and reverses the immune cell depletion that is a hallmark of this highly lethal disorder.

The alpha(4) integrins alpha(4)beta(7) and alpha(4)beta(1), and their ligands mucosal vascular addressin cell adhesion molecule 1 (MAdCAM-1) and VCAM-1, have diverse functions, including roles in the formation of secondary lymphoid tissues at early time points during the colonization and clustering of the fetal lymphoid tissue inducer (LTi) cells and at later time points during the recruitment of lymphocytes. In this study, we evaluated the role of alpha(4) integrins in the development of a recently appreciated class of intestinal lymphoid tissues, isolated lymphoid follicles (ILFs). We observed that diverse ILF cellular populations express alpha(4)beta(7) and alpha(4)beta(1), including the LTi-like cells and lymphocytes, while ILF stromal cells and vessels within ILFs express VCAM-1 and MAdCAM-1, respectively. Evaluation of adult and neonatal beta(7)(-/-) mice and adult and neonatal mice given blocking Abs to alpha(4)beta(7), MAdCAM-1, or VCAM-1 did not identify a role for alpha(4) integrins in cryptopatch (CP) development; however, these studies demonstrated that alpha(4)beta(7) and MAdCAM-1 are required for the transitioning of CP into lymphoid tissues containing lymphocytes or ILFs. Competitive bone marrow transfers demonstrated that beta(7)(-/-) LTi-like cells had a reduced but not significantly impaired ability to localize to CP. Bone marrow transfers and adoptive transfers of B lymphocytes revealed that beta(7) expression by B lymphocytes was essential for their entry into the developing ILFs. These findings demonstrate an essential role for alpha(4)beta(7)/MAdCAM-1 in ILF development corresponding to the influx of beta(7)-expressing lymphocytes and a nonessential role for beta(7)-localizing LTi-like cells to the small intestine.

To assess the degree of lymphocyte apoptosis and survival in mice treated with small interfering RNA (siRNA) targeted to Bim, a proapoptotic molecule from the Bcl-2 family, within a clinically relevant model of sepsis. C57BL/6 mice were treated with a single dose of Bim siRNA complexed in cationic liposomes via tail vein injection. Approximately 24 h later, mice were subjected to either cecal ligation and puncture (CLP) or sham surgery. Animals were killed at 20 h postsurgery, and spleens were harvested for fluorescence-activated cell sorting analysis using terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling as a marker for apoptosis. A second cohort of mice was followed for survival for 7 days. The degree of lymphocyte apoptosis in Bim siRNA-treated mice was markedly decreased compared with controls. Fluorescent activated cell sorter analysis demonstrated 13.1% +/- 1.2% B-cell apoptosis and 11.5% +/- 1.5% T-cell apoptosis in control mice compared with 2.7% +/- 0.4% B-cell apoptosis and 3.9% +/- 0.3% T-cell apoptosis in Bim siRNA-treated mice after CLP (P < 0.001 and P < 0.01, respectively). This striking difference in lymphocyte apoptosis correlated with a significant survival advantage in Bim siRNA-treated mice. At 7 days, there was 90% overall survival in Bim siRNA-treated septic mice compared with 50% overall survival in control septic mice (P < 0.05). Treatment with Bim siRNA in vivo has the potential to be an effective therapy in the treatment of sepsis.

Isolated lymphoid follicles (ILFs) are organized lymphoid structures that facilitate the efficient interaction of antigen, antigen-presenting cells, and lymphocytes to generate controlled adaptive immune responses within the intestine. Because CC chemokine receptor 6 (CCR6) deficiency affects the generation of mucosal immune responses, we evaluated a potential role for CCR6 in the development of ILFs. We observed that CCR6 and its ligand CCL20 are highly expressed within ILFs and that B lymphocytes are the largest CCR6-expressing population within ILFs. ILF development was profoundly arrested in the absence of CCR6. Concordant with a block in ILF development at a stage corresponding to the influx of B lymphocytes, we observed that CCR6-deficient mice had a diminished population of intestinal B lymphocytes. Bone marrow reconstitution studies demonstrated that ILF development is dependent on CCR6-sufficient B lymphocytes, and adoptive transfers demonstrated that CCR6 −/− B lymphocytes were inefficient at localizing to intestinal lymphoid structures. Paralleling these findings, we observed that CCR6-deficient mice had a reduced proportion of Peyer's patch B lymphocytes and an associated re-duction in the number and size of Peyer's patch follicular domes. These observations define an essential role for CCR6 expression by B lymphocytes in localizing to intestinal lymphoid structures and in ILF development.

Isolated lymphoid follicles (ILFs) are recently appreciated members of the mucosal immune system. The architecture, composition, and inducible nature of these structures indicates that these structures are tertiary lymphoid structures. The process leading to the formation of tertiary lymphoid structures, lymphoid neogenesis, has been observed in a number of inflammatory and autoimmune conditions. Given this association, there is considerable interest in identifying the factors promoting lymphoid neogenesis, and understanding the steps in this process. Using murine ILF formation as a model, we have examined the roles of different cellular sources of lymphotoxin (LT) and the adaptive immune response in lymphoid neogenesis. In this study, we report that, although other cellular sources of LT may supplant B lymphocytes in the formation of immature ILFs (loosely organized clusters of B lymphocytes), LT-sufficient B lymphocytes are required for the progression of immature ILFs to mature ILFs (organized lymphoid aggregates with a follicle-associated epithelium). ILF formation occurs in the absence of T lymphocytes and Ag-specific B lymphocyte responses, and ILF B lymphocytes express elevated levels of LT in the absence of antigenic stimulation. Consistent with a role for chemokines inducing LT expression in Ag-naive B lymphocytes, and a chemokine-driven positive-feedback loop driving mature ILF formation, mature ILFs express elevated levels of B lymphocyte chemoattractant in the absence of Ag-specific B lymphocyte stimulation. These observations indicate that ILFs contain Ag-naive lymphocytes, and suggest that events occurring within ILFs shape subsequent immune responses mediated by these lymphocytes.

Lymphotoxin (LT), a cytokine belonging to the TNF family, has established roles in the formation of secondary lymphoid structures and in the compartmentalization of T and B lymphocyte areas of the spleen. In this study, we examine the role of LT in directing the composition of intestinal lymphocytes. We report that mice deficient in LT have a normal composition of intestinal lamina propria (LP) T lymphocytes, and an absence of intestinal LP B lymphocytes. We further refine this observation to demonstrate that the interaction of LT with the LTβR is essential for the presence LP B lymphocytes. The LT/LTβR-dependent events relevant for the presence of LP B lymphocytes occur after birth, do not require the presence of Peyer’s patches, lymph nodes, or the spleen; and therefore, are distinct and independent from the previously identified roles of LT/LTβR. The LT-dependent signal relevant for the presence of LP B lymphocytes is optimally supplied by a LT-sufficient B lymphocyte, and requires a LTβR-sufficient radio-resistant, non-bone marrow-derived cell. Based upon the severity of the deficit of LP B lymphocytes we observed, these novel LT/LTβR-dependent events are of primary importance in directing the entry and residence of LP B lymphocytes.

The mechanisms allowing the gastrointestinal immune system to avoid an inappropriate inflammatory response to nonpathogenic luminal Ags are poorly understood. We have previously described a role for cyclooxygenase (COX)-2-dependent arachidonic acid metabolites produced by the murine small intestine lamina propria in controlling the immune response to a dietary Ag. To better understand the role of COX-2-dependent arachidonic acid metabolites produced by the lamina propria, we examined the pattern of expression and the cellular source of COX-2 and COX-2-dependent PGE2. We now demonstrate that non-bone marrow-derived lamina propria stromal cells have basal COX-2 expression and that COX-2-dependent PGE2 production by these cells is spontaneous and continuous. The other mucosal and nonmucosal lymphoid compartments examined do not share this phenotype. In contrast to the majority of descriptions of COX-2 expression, COX-2 expression by lamina propria stromal cells is not dependent upon exogenous stimuli, including adhesion, LPS signaling via Toll-like receptor 4, or the proinflammatory cytokines TNF-α, IFN-γ, and IL-1β. These findings, in conjunction with the known immunomodulatory capacities of PGs, suggest that COX-2 expression by the small intestine lamina propria is a basal state contributing to the hyporesponsiveness of the intestinal immune response.

We have assayed the ability of human adenoviruses from heterologous subgroups to complement early temperature-sensitive mutants of the C group virus Ad5 by analyzing coinfections at the restrictive temperature for serotype-specific DNA and late protein synthesis. Our results indicate that the B group virus AM, does not complement either ts125 or the N complementation group of Ad5 (ts36, ts69, and ts149). In contrast, studies with Ad12, an A group virus, and these mutants, indicate a differential complementation in that Ad12 complements all of the mutants in the Ad5 N complementation group but does not complement ts125. Failure to complement the ts125 defect in coinfected cells has been shown to be due to the inability of the heterologous wt gene product to substitute for the ts125 gene product directly at the level of DNA replication and not at some earlier event in the virus growth cycle.