A therapeutic vaccine strategy to prevent Pneumocystis pneumonia in an immunocompromised host in a non-human primate model of HIV and Pneumocystis co-infection.

Introduction: Pneumocystis is a ubiquitous fungal pathogen that causes pneumonia (PCP) and pulmonary sequelae in HIV-infected individuals and other immunocompromised populations. With the success of anti-retroviral therapy for HIV-infected individuals the frequency of PCP in that population has decreased, however, PCP remains a significant cause of morbidity and mortality in individuals with hematologic and solid malignancies, and in individuals treated with immunosuppressive therapies for autoimmune diseases, and following bone marrow and solid organ transplantation. Despite the clinical need, there is no approved vaccine to prevent PCP in vulnerable populations. The ultimate goal of the field is to develop an effective vaccine that can overcome immune deficits in at risk populations and induce long-lasting protective immunity to Pneumocystis. Toward this goal, our laboratory has established a model of PCP co-infection in simian immunodeficiency virus (SIV)-infected non-human primates (NHP) and identified a recombinant protein sub-unit vaccine, KEX1, that induces robust anti-Pneumocystis immunity in immune-competent macaques that is durable and prevents PCP following simian immunodeficiency virus (SIV)-induced immunosuppression. Type I, or invariant natural killer T (iNKT) cells have the potential to provide B cell help under conditions of reduced CD4+ T cell help. Methods: In the present study, we used the SIV model of HIV infection to address whether therapeutic vaccination with the iNKT cell-activating adjuvant α-galactosylceramide (α-GC) and KEX1 (α-GC+KEX1) can effectively boost anti-Pneumocystis humoral immunity following virus-induced immunosuppression. Results: Immunization of antigen-experienced NHPs with α-GC+KEX1 during the early chronic phase of SIV-infection significantly boosted anti-Pneumocystis humoral immunity by increasing memory B cells and antibody titers, and enhanced titer durability during SIV-induced immunosuppression. This therapeutic vaccination strategy boosted anti-Pneumocystis immune responses during SIV-infection and contributed to protection against Pneumocystis co-infection in KEX1-vaccinated macaques. Conclusion: These studies present a novel strategy for stimulating durable anti-Pneumocystis humoral immunity in the context of complex, chronic SIV-induced immunosuppression and may be further applied to immunization of other immunosuppressed populations, and toward other common recall antigens.

Immunogenicity and protective efficacy of a pan-fungal vaccine in preclinical models of aspergillosis, candidiasis, and pneumocystosis.

Invasive fungal infections cause over 1.5 million deaths worldwide. Despite increases in fungal infections as well as the numbers of individuals at risk, there are no clinically approved fungal vaccines. We produced a "pan-fungal" peptide, NXT-2, based on a previously identified vaccine candidate and homologous sequences from Pneumocystis, Aspergillus,Candida, and Cryptococcus. We evaluated the immunogenicity and protective capacity of NXT-2 in murine and nonhuman primate models of invasive aspergillosis, systemic candidiasis, and pneumocystosis. NXT-2 was highly immunogenic and immunized animals had decreased mortality and morbidity compared to nonvaccinated animals following induction of immunosuppression and challenge with Aspergillus, Candida, or Pneumocystis. Data in multiple animal models support the concept that immunization with a pan-fungal vaccine prior to immunosuppression induces broad, cross-protective antifungal immunity in at-risk individuals.

Vaccine-Induced Protection in Two Murine Models of Invasive Pulmonary Aspergillosis.

Life-threatening, invasive fungal infections (IFIs) cause over 1.5 million deaths worldwide and are a major public health concern with high mortality rates even with medical treatment. Infections with the opportunistic fungal pathogen, Aspergillus fumigatus are among the most common. Despite the growing clinical need, there are no licensed vaccines for IFIs. Here we evaluated the immunogenicity and protective efficacy of an A. fumigatus recombinant protein vaccine candidate, AF.KEX1, in experimental murine models of drug-induced immunosuppression. Immunization of healthy mice with AF.KEX1 and adjuvant induced a robust immune response. Following AF.KEX1 or sham immunization, mice were immunosuppressed by treatment with either cortisone acetate or hydrocortisone and the calcineurin inhibitor, tacrolimus. To test vaccine efficacy, immunosuppressed mice were intranasally challenged with A. fumigatus conidia (Af293) and weight and body temperature were monitored for 10 days. At study termination, organism burden in the lungs was evaluated by quantitative PCR and Gomori's methanamine silver staining. In both models of immunosuppression, AF.KEX1 vaccinated mice experienced decreased rates of mortality and significantly lower lung organism burden compared to non-vaccinated controls. The lung fungal burden was inversely correlated with the peak anti-AF.KEX1 IgG titer achieved following vaccination. These studies provide the basis for further evaluation of a novel vaccine strategy to protect individuals at risk of invasive aspergillosis due to immunosuppressive treatments.

Statin treatment prevents the development of pulmonary arterial hypertension in a nonhuman primate model of HIV-associated PAH.

Pulmonary arterial hypertension (PAH) is a life-threatening disease characterized by pulmonary vascular remodeling, elevated pulmonary arterial pressure, and right heart failure. Human immunodeficiency virus (HIV)-infected individuals have a higher incidence of PAH than the non-HIV infected population and evidence suggests a role for systemic and pulmonary inflammation in the pathogenesis of HIV-associated PAH. Due to their pleiotropic effects, including immune-modulatory and anti-inflammatory effects, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins) have been considered for the treatment of PAH, with conflicting results. The effects of statins on HIV-associated PAH have not been specifically evaluated. We have developed a non-human primate (NHP) model of HIV-associated PAH that closely mimics HIV-PAH using simian immunodeficiency virus (SIV)-infected rhesus macaques (Macaca mulatta). We determined that treatment of healthy macaques with atorvastatin prior to and throughout SIV infection prevented the development of SIV-associated PAH. Additionally, SIV-infected macaques that initiated atorvastatin treatment during the early chronic disease stage had reduced incidence of PAH compared to untreated animals. Statin treatment reduced inflammatory mediators TGF-ß, MIP-1α, and TNF-α and the numbers of CD14dimCD16+ non-classical monocytes, and CD14+CCR7-CD163-CD206+ alveolar macrophages previously shown to be associated with SIV-PAH. These results support the concept that statins reduce inflammatory processes that contribute to PAH and may provide a safe and effective prophylactic strategy for the prevention of PAH in HIV-infected individuals.

Immunization with Pneumocystis recombinant KEX1 induces robust and durable humoral responses in immunocompromised non-human primates.

Infection with the opportunistic fungal pathogen, Pneumocystis jirovecii causes life-threatening pneumonia in immunocompromised individuals. In addition to HIV-1 infected patients, individuals at risk of Pneumocystis infection include those receiving immunosuppressive therapies due to transplantation, cancer or autoimmune disease. Antibiotic treatment is not always successful, and it does not prevent obstructive lung disease after clearance of the pathogen. Therefore, it is essential to develop therapeutic alternatives that are more effective against PCP. We reported that Pneumocystis recombinant protein KEX1 induces protective immunity against the development of PCP in a non-human primate model of HIV-induced immunosuppression. In this study, we tested the immunogenicity KEX1 immunization of healthy rhesus macaques and the durability of these responses during drug-induced immunosuppression using tacrolimus (FK506) and methylprednisolone. We observed that vaccination with KEX1 prior to the start of the immunosuppressive regimen generated a robust and long-lasting antibody response that was maintained throughout the immunosuppressive treatment. Furthermore, boosting with KEX1 during immunosuppression induced recall of memory responses against recombinant KEX1. The durability of the anti-KEX1 response and the ability to induce a recall response during immunosuppressive therapy provide a proof-of-concept data supporting further investigation of the KEX1 as a prophylactic vaccine to prevent PCP in drug-induced immunosuppression. This approach provides fundamental knowledge for the elaboration of therapeutic and prophylactic alternatives for PCP in patients undergoing severe immunosuppressive therapy.

IL-15 Enables Septic Shock by Maintaining NK Cell Integrity and Function.

Interleukin 15 is essential for the development and differentiation of NK and memory CD8+ (mCD8+) T cells. Our laboratory previously showed that NK and CD8+ T lymphocytes facilitate the pathobiology of septic shock. However, factors that regulate NK and CD8+ T lymphocyte functions during sepsis are not well characterized. We hypothesized that IL-15 promotes the pathogenesis of sepsis by maintaining NK and mCD8+ T cell integrity. To test our hypothesis, the pathogenesis of sepsis was assessed in IL-15-deficient (IL-15 knockout, KO) mice. IL-15 KO mice showed improved survival, attenuated hypothermia, and less proinflammatory cytokine production during septic shock caused by cecal ligation and puncture or endotoxin-induced shock. Treatment with IL-15 superagonist (IL-15 SA, IL-15/IL-15Rα complex) regenerated NK and mCD8+ T cells and re-established mortality of IL-15 KO mice during septic shock. Preventing NK cell regeneration attenuated the restoration of mortality caused by IL-15 SA. If given immediately prior to septic challenge, IL-15-neutralizing IgG M96 failed to protect against septic shock. However, M96 caused NK cell depletion if given 4 d prior to septic challenge and conferred protection. IL-15 SA treatment amplified endotoxin shock, which was prevented by NK cell or IFN-γ depletion. IL-15 SA treatment also exacerbated septic shock caused by cecal ligation and puncture when given after the onset of sepsis. In conclusion, endogenous IL-15 does not directly augment the pathogenesis of sepsis but enables the development of septic shock by maintaining NK cell numbers and integrity. Exogenous IL-15 exacerbates the severity of sepsis by activating NK cells and facilitating IFN-γ production.

A Distinct Lung-Interstitium-Resident Memory CD8(+) T Cell Subset Confers Enhanced Protection to Lower Respiratory Tract Infection.

The nature and anatomic location of the protective memory CD8(+) T cell subset induced by intranasal vaccination remain poorly understood. We developed a vaccination model to assess the anatomic location of protective memory CD8(+) T cells and their role in lower airway infections. Memory CD8(+) T cells elicited by local intranasal, but not systemic, vaccination with an engineered non-replicative CD8(+) T cell-targeted antigen confer enhanced protection to a lethal respiratory viral challenge. This protection depends on a distinct CXCR3(LO) resident memory CD8(+) T (Trm) cell population that preferentially localizes to the pulmonary interstitium. Because they are positioned close to the mucosa, where infection occurs, interstitial Trm cells act before inflammation can recruit circulating memory CD8(+) T cells into the lung tissue. This results in a local protective immune response as early as 1 day post-infection. Hence, vaccine strategies that induce lung interstitial Trm cells may confer better protection against respiratory pathogens.

Peripheral tolerance can be modified by altering KLF2-regulated Treg migration.

Tregs are essential for maintaining peripheral tolerance, and thus targeting these cells may aid in the treatment of autoimmunity and cancer by enhancing or reducing suppressive functions, respectively. Before these cells can be harnessed for therapeutic purposes, it is necessary to understand how they maintain tolerance under physiologically relevant conditions. We now report that transcription factor Kruppel-like factor 2 (KLF2) controls naive Treg migration patterns via regulation of homeostatic and inflammatory homing receptors, and that in its absence KLF2-deficient Tregs are unable to migrate efficiently to secondary lymphoid organs (SLOs). Diminished Treg trafficking to SLOs is sufficient to initiate autoimmunity, indicating that SLOs are a primary site for maintaining peripheral tolerance under homeostatic conditions. Disease severity correlates with impaired Treg recruitment to SLOs and, conversely, promotion of Tregs into these tissues can ameliorate autoimmunity. Moreover, stabilizing KLF2 expression within the Treg compartment enhances peripheral tolerance by diverting these suppressive cells from tertiary tissues into SLOs. Taken together, these results demonstrate that peripheral tolerance is enhanced or diminished through modulation of Treg trafficking to SLOs, a process that can be controlled by adjusting KLF2 protein levels.

Transcription factor KLF2 regulates homeostatic NK cell proliferation and survival.

Natural killer (NK) cells are innate lymphocytes that recognize and lyse virally infected or transformed cells. This latter property is being pursued in clinics to treat leukemia with the hope that further breakthroughs in NK cell biology can extend treatments to other cancers. At issue is the ability to expand transferred NK cells and prolong their functionality within the context of a tumor. In terms of NK cell expansion and survival, we now report that Kruppel-like factor 2 (KLF2) is a key transcription factor that underpins both of these events. Excision of Klf2 using gene-targeted mouse models promotes spontaneous proliferation of immature NK cells in peripheral tissues, a phenotype that is replicated under ex vivo conditions. Moreover, KLF2 imprints a homeostatic migration pattern on mature NK cells that allows these cells to access IL-15-rich microenvironments. KLF2 accomplishes this feat within the mature NK cell lineage via regulation of a subset of homing receptors that respond to homeostatic ligands while leaving constitutively expressed receptors that recognize inflammatory cytokines unperturbed. Under steady-state conditions, KLF2-deficient NK cells alter their expression of homeostatic homing receptors and subsequently undergo apoptosis due to IL-15 starvation. This novel mechanism has implications regarding NK cell contraction following the termination of immune responses including the possibility that retention of an IL-15 transpresenting support system is key to extending NK cell activity in a tumor environment.

IL-15 Superagonist-Mediated Immunotoxicity: Role of NK Cells and IFN-γ.

IL-15 is currently undergoing clinical trials to assess its efficacy for treatment of advanced cancers. The combination of IL-15 with soluble IL-15Rα generates a complex termed IL-15 superagonist (IL-15 SA) that possesses greater biological activity than IL-15 alone. IL-15 SA is considered an attractive antitumor and antiviral agent because of its ability to selectively expand NK and memory CD8(+) T (mCD8(+) T) lymphocytes. However, the adverse consequences of IL-15 SA treatment have not been defined. In this study, the effect of IL-15 SA on physiologic and immunologic functions of mice was evaluated. IL-15 SA caused dose- and time-dependent hypothermia, weight loss, liver injury, and mortality. NK (especially the proinflammatory NK subset), NKT, and mCD8(+) T cells were preferentially expanded in spleen and liver upon IL-15 SA treatment. IL-15 SA caused NK cell activation as indicated by increased CD69 expression and IFN-γ, perforin, and granzyme B production, whereas NKT and mCD8(+) T cells showed minimal, if any, activation. Cell depletion and adoptive transfer studies showed that the systemic toxicity of IL-15 SA was mediated by hyperproliferation of activated NK cells. Production of the proinflammatory cytokine IFN-γ, but not TNF-α or perforin, was essential to IL-15 SA-induced immunotoxicity. The toxicity and immunological alterations shown in this study are comparable to those reported in recent clinical trials of IL-15 in patients with refractory cancers and advance current knowledge by providing mechanistic insights into IL-15 SA-mediated immunotoxicity.

KLF2 is a rate-limiting transcription factor that can be targeted to enhance regulatory T-cell production.

Regulatory T cells (Tregs) are a specialized subset of CD4(+) T cells that maintain self-tolerance by functionally suppressing autoreactive lymphocytes. The Treg compartment is composed of thymus-derived Tregs (tTregs) and peripheral Tregs (pTregs) that are generated in secondary lymphoid organs after exposure to antigen and specific cytokines, such as TGF-ß. With regard to this latter lineage, pTregs [and their ex vivo generated counterparts, induced Tregs (iTregs)] offer particular therapeutic potential because these cells can be raised against specific antigens to limit autoimmunity. We now report that transcription factor Krüppel-like factor 2 (KLF2) is necessary for the generation of iTregs but not tTregs. Moreover, drugs that limit KLF2 proteolysis during T-cell activation enhance iTreg development. To the authors' knowledge, this study identifies the first transcription factor to distinguish between i/pTreg and tTreg ontogeny and demonstrates that KLF2 is a therapeutic target for the production of regulatory T cells.

In vitro induction of regulatory CD4+CD8α+ T cells by TGF-ß, IL-7 and IFN-γ.

In vitro CD4(+) T cell differentiation systems have made important contributions to understanding the mechanisms underlying the differentiation of naive CD4(+) T cells into effector cells with distinct biological functions. Mature CD4(+) T cells expressing CD8αα homodimers are primarily found in the intestinal mucosa of men and mice, and to a lesser extent in other tissues such as peripheral blood. Although CD4(+)CD8α(+) T cells are easily identified, very little is known about their development and immunological functions. It has been reported, however, that CD4(+)CD8α(+) T cells possess regulatory properties. In this report, we present a novel in vitro differentiation system where CD4(+) T cells are stimulated to become CD4(+)CD8α(+) T cells in the presence of TGF-ß, IL-7 and IFN-γ, resulting in cells with very similar features as CD4(+)CD8α(+) intraepithelial lymphocytes. This novel in vitro differentiation culture should provide a powerful and tractable tool for dissecting the differentiation and biological functions of CD4(+)CD8α(+) T cells.

Aberrant macrophages mediate defective kidney repair that triggers nephritis in lupus-susceptible mice.

CSF-1, required for macrophage (Mø) survival, proliferation, and activation, is upregulated in the tubular epithelial cells (TECs) during kidney inflammation. CSF-1 mediates Mø-dependent destruction in lupus-susceptible mice with nephritis and, paradoxically, Mø-dependent renal repair in lupus-resistant mice after transient ischemia/reperfusion injury (I/R). We now report that I/R leads to defective renal repair, nonresolving inflammation, and, in turn, early-onset lupus nephritis in preclinical MRL/MpJ-Faslpr/Fas(lpr) mice (MRL-Fas(lpr) mice). Moreover, defective renal repair is not unique to MRL-Fas(lpr) mice, as flawed healing is a feature of other lupus-susceptible mice (Sle 123) and MRL mice without the Fas(lpr) mutation. Increasing CSF-1 hastens renal healing after I/R in lupus-resistant mice but hinders healing, exacerbates nonresolving inflammation, and triggers more severe early-onset lupus nephritis in MRL-Fas(lpr) mice. Probing further, the time-related balance of M1 "destroyer" Mø shifts toward the M2 "healer" phenotype in lupus-resistant mice after I/R, but M1 Mø continue to dominate in MRL-Fas(lpr) mice. Moreover, hypoxic TECs release mediators, including CSF-1, that are responsible for stimulating the expansion of M1 Mø inherently poised to destroy the kidney in MRL-Fas(lpr) mice. In conclusion, I/R induces CSF-1 in injured TECs that expands aberrant Mø (M1 phenotype), mediating defective renal repair and nonresolving inflammation, and thereby hastens the onset of lupus nephritis.

Distinct roles of CSF-1 isoforms in lupus nephritis.

Colony-stimulating factor-1 (CSF-1), the principal growth factor for macrophages, is increased in the kidney, serum, and urine of patients with lupus nephritis, and eliminating CSF-1 suppresses lupus in MRL-Fas(lpr) mice. CSF-1 has three biologically active isoforms: a membrane-spanning cell surface glycoprotein (csCSF-1), a secreted proteoglycan (spCSF-1), and a secreted glycoprotein (sgCSF-1); the role of each isoform in the circulation and kidney in autoimmune disease is not well understood. Here, we constructed mutant MRL-Fas(lpr) mice that only express csCSF-1 or precursors of the spCSF-1 and sgCSF-1 isoforms. Both csCSF-1 and spCSF-1 shifted monocytes toward proinflammatory, activated populations, enhancing their recruitment into the kidney during lupus nephritis. With advancing lupus nephritis, spCSF-1 was the predominant isoform responsible for increasing circulating CSF-1 and, along with the csCSF-1 isoform, for increasing intrarenal CSF-1. Thus, csCSF-1 appears to initiate and promote the local activation of macrophages within the kidney. Intrarenal expression of csCSF-1 and spCSF-1 increases with advancing nephritis, thereby promoting the intrarenal recruitment of monocytes and expansion of Ly6C(hi) macrophages, which induce apoptosis of the renal parenchyma. Taken together, these data suggest that the three CSF-1 isoforms have distinct biologic properties, suggesting that blocking both circulating and intrarenal CSF-1 may be necessary for therapeutic efficacy.

Circulating CSF-1 promotes monocyte and macrophage phenotypes that enhance lupus nephritis.

Macrophages mediate kidney disease and are prominent in a mouse model (MRL-Fas(lpr)) of lupus nephritis. Colony stimulating factor-1 (CSF-1) is the primary growth factor for macrophages, and CSF-1 deficiency protects MRL-Fas(lpr) mice from kidney disease and systemic illness. Whether this renoprotection derives from a reduction of macrophages and whether systemic CSF-1, as opposed to intrarenal CSF-1, promotes macrophage-dependent lupus nephritis remain unclear. Here, we found that increasing systemic CSF-1 hastened the onset of lupus nephritis in MRL-Fas(lpr) mice. Using mutant MRL-Fas(lpr) strains that express high, moderate, or no systemic CSF-1, we detected a much higher tempo of kidney disease in mice with the highest level of CSF-1. Furthermore, we uncovered a multistep CSF-1-dependent systemic mechanism central to lupus nephritis. CSF-1 heightened monocyte proliferation in the bone marrow (SSC(low)CD11b(+)), and these monocytes subsequently seeded the circulation. Systemic CSF-1 skewed the frequency of monocytes toward "inflammatory" (SSC(low)CD11b(+)Ly6C(high)) and activated populations that homed to sites of inflammation, resulting in a more rapid accumulation of intrarenal macrophages (CD11b(+)CSF-1R(+) or CD68(+)) that induced apoptosis of tubular epithelial cells, damaging the kidney. In humans, we found increased levels of CSF-1 in the serum, urine, and kidneys of patients with lupus compared with healthy controls. Furthermore, serum and urine CSF-1 levels correlated with lupus activity, and intrarenal CSF-1 expression correlated with the histopathology activity index of lupus nephritis. Taken together, circulating CSF-1 is a potential therapeutic target for lupus nephritis.

CSF-1 signals directly to renal tubular epithelial cells to mediate repair in mice.

Tubular damage following ischemic renal injury is often reversible, and tubular epithelial cell (TEC) proliferation is a hallmark of tubular repair. Macrophages have been implicated in tissue repair, and CSF-1, the principal macrophage growth factor, is expressed by TECs. We therefore tested the hypothesis that CSF-1 is central to tubular repair using an acute renal injury and repair model, ischemia/reperfusion (I/R). Mice injected with CSF-1 following I/R exhibited hastened healing, as evidenced by decreased tubular pathology, reduced fibrosis, and improved renal function. Notably, CSF-1 treatment increased TEC proliferation and reduced TEC apoptosis. Moreover, administration of a CSF-1 receptor-specific (CSF-1R-specific) antibody after I/R increased tubular pathology and fibrosis, suppressed TEC proliferation, and heightened TEC apoptosis. To determine the contribution of macrophages to CSF-1-dependent renal repair, we assessed the effect of CSF-1 on I/R in mice in which CD11b+ cells were genetically ablated and determined that macrophages only partially accounted for CSF-1-dependent tubular repair. We found that TECs expressed the CSF-1R and that this receptor was upregulated and coexpressed with CSF-1 in TECs following renal injury in mice and humans. Furthermore, signaling via the CSF-1R stimulated proliferation and reduced apoptosis in human and mouse TECs. Taken together, these data suggest that CSF-1 mediates renal repair by both a macrophage-dependent mechanism and direct autocrine/paracrine action on TECs.

Sunlight triggers cutaneous lupus through a CSF-1-dependent mechanism in MRL-Fas(lpr) mice.

Sunlight (UVB) triggers cutaneous lupus erythematosus (CLE) and systemic lupus through an unknown mechanism. We tested the hypothesis that UVB triggers CLE through a CSF-1-dependent, macrophage (Mø)-mediated mechanism in MRL-Fas(lpr) mice. By constructing mutant MRL-Fas(lpr) strains expressing varying levels of CSF-1 (high, intermediate, none), and use of an ex vivo gene transfer to deliver CSF-1 intradermally, we determined that CSF-1 induces CLE in lupus-susceptible MRL-Fas(lpr) mice, but not in lupus-resistant BALB/c mice. UVB incites an increase in Møs, apoptosis in the skin, and CLE in MRL-Fas(lpr), but not in CSF-1-deficient MRL-Fas(lpr) mice. Furthermore, UVB did not induce CLE in BALB/c mice. Probing further, UVB stimulates CSF-1 expression by keratinocytes leading to recruitment and activation of Møs that, in turn, release mediators, which induce apoptosis in keratinocytes. Thus, sunlight triggers a CSF-1-dependent, Mø-mediated destructive inflammation in the skin leading to CLE in lupus-susceptible MRL-Fas(lpr) but not lupus-resistant BALB/c mice. Taken together, CSF-1 is envisioned as the match and lupus susceptibility as the tinder leading to CLE.

Programmed death ligand 1 regulates a critical checkpoint for autoimmune myocarditis and pneumonitis in MRL mice.

MRL/MpJ-Fas(lpr) (MRL-Fas(lpr)) mice develop a spontaneous T cell and macrophage-dependent autoimmune disease that shares features with human lupus. Interactions via the programmed death 1/programmed death ligand 1 (PD-1/PD-L1) pathway down-regulate immune responses and provide a negative regulatory checkpoint in mediating tolerance and autoimmune disease. Therefore, we tested the hypothesis that the PD-1/PD-L1 pathway suppresses lupus nephritis and the systemic illness in MRL-Fas(lpr) mice. For this purpose, we compared kidney and systemic illness (lymph nodes, spleen, skin, lung, glands) in PD-L1 null (-/-) and PD-L1 intact (wild type, WT) MRL-Fas(lpr) mice. Unexpectedly, PD-L1(-/-);MRL-Fas(lpr) mice died as a result of autoimmune myocarditis and pneumonitis before developing renal disease or the systemic illness. Dense infiltrates, consisting of macrophage and T cells (CD8(+) > CD4(+)), were prominent throughout the heart (atria and ventricles) and localized specifically around vessels in the lung. In addition, once disease was evident, we detected heart specific autoantibodies in PD-L1(-/-);MRL-Fas(lpr) mice. This unique phenotype is dependent on MRL-specific background genes as PD-L1(-/-);MRL(+/+) mice lacking the Fas(lpr) mutation developed autoimmune myocarditis and pneumonitis. Notably, the transfer of PD-L1(-/-);MRL(+/+) bone marrow cells induced myocarditis and pneumonitis in WT;MRL(+/+) mice, despite a dramatic up-regulation of PD-L1 expression on endothelial cells in the heart and lung of WT;MRL(+/+) mice. Taken together, we suggest that PD-L1 expression is central to autoimmune heart and lung disease in lupus-susceptible (MRL) mice.

SIFamide peptides in clawed lobsters and freshwater crayfish (Crustacea, Decapoda, Astacidea): a combined molecular, mass spectrometric and electrophysiological investigation.

Recently, we identified the peptide VYRKPPFNGSIFamide (Val(1)-SIFamide) in the stomatogastric nervous system (STNS) of the American lobster Homarus americanus using matrix-assisted laser desorption/ionization-Fourier transform mass spectrometry (MALDI-FTMS). Given that H. americanus is the only species thus far shown to possess this peptide, and that a second SIFamide isoform, Gly(1)-SIFamide, is broadly conserved in other decapods, including another astacidean, the crayfish Procambarus clarkii, we became interested both in confirming our identification of Val(1)-SIFamide via molecular methods and in determining the extent to which this isoform is conserved within other members of the infraorder Astacidea. Here, we present the identification and characterization of an H. americanus prepro-SIFamide cDNA that encodes the Val(1) isoform. Moreover, we demonstrate via MALDI-FTMS the presence of Val(1)-SIFamide in a second Homarus species, Homarus gammarus. In contrast, only the Gly(1) isoform was detected in the other astacideans investigated, including the lobster Nephrops norvegicus, a member of the same family as Homarus, and the crayfish Cherax quadricarinatus, P. clarkii and Pacifastacus leniusculus, which represent members of each of the extant families of freshwater astacideans. These results suggest that Val(1)-SIFamide may be a genus (Homarus)-specific isoform. Interestingly, both Val(1)- and Gly(1)-SIFamide possess an internal dibasic site, Arg(3)-Lys(4), raising the possibility of the ubiquitously conserved isoform PPFNGSIFamide. However, this octapeptide was not detected via MALDI-FTMS in any of the investigated species, and when applied to the isolated STNS of H. americanus possessed little bioactivity relative to the full-length Val(1) isoform. Thus, it appears that the dodeca-variants Val(1)- and Gly(1)-SIFamide are the sole bioactive isoforms of this peptide family in clawed lobsters and freshwater crayfish.