Their last will and testament: dying immune cells protect the urinary system with extracellular DNA traps.

Like most epithelial organs, the bladder and kidney can be directly accessed by bacteria evolved for invasion. Epithelia and immune cells attempt to stymie this infection with biophysical and chemical mechanisms. Goldspink et al. connected the Na+ gradient in the kidney medulla with an immune defense mounted by dead cells (namely, the explosive death of neutrophils and macrophages), resulting in extracellular DNA traps. The pathway from Na+ concentration to immune death is depicted.

Timing is everything: impact of development, ageing and circadian rhythm on macrophage functions in urinary tract infections.

The bladder supports a diversity of macrophage populations with functional roles related to homeostasis and host defense, including clearance of cell debris from tissue, immune surveillance, and inflammatory responses. This review examines these roles with particular attention given to macrophage origins, differentiation, recruitment, and engagement in host defense against urinary tract infections (UTIs), where these cells recognize uropathogens through a combination of receptor-mediated responses. Time is an important variable that is often overlooked in many clinical and biological studies, including in relation to macrophages and UTIs. Given that ageing is a significant factor in urinary tract infection pathogenesis and macrophages have been shown to harbor their own circadian system, this review also explores the influence of age on macrophage functions and the role of diurnal variations in macrophage functions in host defense and inflammation during UTIs. We provide a conceptual framework for future studies that address these key knowledge gaps.

Selection and immune recognition of HIV-1 MPER mimotopes.

The membrane proximal external region (MPER) of HIV-1 gp41 is targeted by several neutralizing antibodies (NAbs) and is of interest for vaccine design. In this study, we identified novel MPER peptide mimotopes and evaluated their reactivity with HIV + plasma antibodies to characterize the diversity of the immune responses to MPER during natural infection. We utilized phage display technology to generate novel mimotopes that fit antigen-binding sites of MPER NAbs 4E10, 2F5 and Z13. Plasma antibodies from 10 HIV + patients were mapped by phage immunoprecipitation, to identify unique patient MPER binding profiles that were distinct from, and overlapping with, those of MPER NAbs. 4E10 mimotope binding profiles correlated with plasma neutralization of HIV-2/HIV-1 MPER chimeric virus, and with overall plasma neutralization breadth and potency. When administered as vaccines, 4E10 mimotopes elicited low titer NAb responses in mice. HIV mimotopes may be useful for detailed analysis of plasma antibody specificity.

Long-lived self-renewing bone marrow-derived macrophages displace embryo-derived cells to inhabit adult serous cavities.

Peritoneal macrophages are one of the most studied macrophage populations in the body, yet the composition, developmental origin and mechanisms governing the maintenance of this compartment are controversial. Here we show resident F4/80(hi)GATA6(+) macrophages are long-lived, undergo non-stochastic self-renewal and retain cells of embryonic origin for at least 4 months in mice. However, Ly6C(+) monocytes constitutively enter the peritoneal cavity in a CCR2-dependent manner, where they mature into short-lived F4/80(lo)MHCII(+) cells that act, in part, as precursors of F4/80(hi)GATA6(+) macrophages. Notably, monocyte-derived F4/80(hi) macrophages eventually displace the embryonic population with age in a process that is highly gender dependent and not due to proliferative exhaustion of the incumbent embryonic population, despite the greater proliferative activity of newly recruited cells. Furthermore, although monocyte-derived cells acquire key characteristics of the embryonic population, expression of Tim4 was impaired, leading to cumulative changes in the population with age.

Designing the epitope flanking regions for optimal generation of CTL epitopes.

The flanking amino acids that surround epitopes are critical for effective antigen processing and maintenance of epitope integrity. In the present study, the frequency and characteristics of each amino acid that flanked the peptides generated from the proteasomal degradation of three different subtypes of HIV-1 Gag-p24 were determined. Synthetic flanking regions were designed based on the highest and the lowest frequencies of amino acid with the ideal characteristics at positions upstream and downstream of the proteasomal cleavage site. Peptides were synthesized that contained known CD8+ CTL-epitopes from HIV-1 Gag, CMV pp65, and vaccinia proteins HRP-2, and C16, flanked by amino acid sequences specifically designed to either generate or inhibit the known CD8+ CTL-epitopes. As predicted, the known CD8+ CTL-epitopes were effectively generated from the peptides with synthetic flanking regions specifically designed to promote epitope generation in a proteasome-dependent manner. The majority of the proteasome-generated epitopes were cleaved immediately after the C-terminal amino acid of the specific CTL-epitope. The synthetic peptide sequences containing known CD8+ CTL-epitopes with the flanking regions that promote epitope generation were effectively processed and presented to epitope specific CD8+ T-cells resulting in the production of IFN-γ. These results highlight the importance of flanking regions in promoting efficient antigen processing and presentation. This concept can have important implications in vaccine design and development strategies.

Liposomes containing lipid A: an effective, safe, generic adjuvant system for synthetic vaccines.

Liposomes containing monophosphoryl lipid A (MPLA) have previously exhibited considerable potency and safety in human trials with a variety of candidate vaccines, including vaccines to malaria, HIV-1 and several different types of cancer. The long history of research and development of MPLA and liposomal MPLA as vaccine adjuvants reveals that there are numerous opportunities for creation and development of generic (nonproprietary) adjuvant system formulations with these materials that are not only highly potent and safe, but also readily available as native materials or as synthetic compounds. They are easily manufactured as potentially inexpensive and easy to use adjuvant systems and might be effective even with synthetic peptides as antigens.

HIV-1 envelope resistance to proteasomal cleavage: implications for vaccine induced immune responses.

BACKGROUND: Antigen processing involves many proteolytic enzymes such as proteasomes and cathepsins. The processed antigen is then presented on the cell surface bound to either MHC class I or class II molecules and induces/interacts with antigen-specific CD8+ and CD4+ T-cells, respectively. Preliminary immunological data from the RV144 phase III trial indicated that the immune responses were biased towards the Env antigen with a dominant CD4+ T-cell response. METHODS: In this study, we examined the susceptibility of HIV-1 Env-A244 gp120 protein, one of the protein boost subunits of the RV144 Phase III vaccine trial, to proteasomes and cathepsins and identified the generated peptide epitope repertoire by mass spectrometry. The peptide fragments were tested for cytokine production in CD4(+) T-cell lines derived from RV144 volunteers. RESULTS: Env-A244 was resistant to proteasomes, thus diminishing the possibility of the generation of class I epitopes by the classical MHC class I pathway. However, Env-A244 was efficiently cleaved by cathepsins generating peptide arrays identified by mass spectrometry that contained both MHC class I and class II epitopes as reported in the Los Alamos database. Each of the cathepsins generated distinct degradation patterns containing regions of light and dense epitope clusters. The sequence DKKQKVHALF that is part of the V2 loop of gp120 produced by cathepsins induced a polyfunctional cytokine response including the generation of IFN-γ from CD4(+) T-cell lines-derived from RV144 vaccinees. This sequence is significant since antibodies to the V1/V2-loop region correlated inversely with HIV-1 infection in the RV144 trial. CONCLUSIONS: Based on our results, the susceptibility of Env-A244 to cathepsins and not to proteasomes suggests a possible mechanism for the generation of Env-specific CD4(+)T cell and antibody responses in the RV144 vaccinees.

Liposome-encapsulated HIV-1 Gag p24 containing lipid A induces effector CD4+ T-cells, memory CD8+ T-cells, and pro-inflammatory cytokines.

Liposomal lipid A is an effective adjuvant for the delivery of antigens and for the induction of both cellular and humoral immunity. In this study, we demonstrate that following the third immunization with HIV-1 Gag p24 encapsulated in liposomes containing lipid A [L(p24+LA)], central memory CD8+ T-cells were localized in the spleen and lymph nodes of mice while effector memory CD8+ T-cells and effector CD4+ T-cells were found in the PBMC. Effector CD4+ T-cells were also detected in the spleen and lymph nodes. The predominant cytokine secreted from splenic lymphocytes and lymph nodes was IFN-gamma. In contrast, IL-6 and IL-10 were the major cytokines produced from PBMC. The peptide stimulation indicated that the cytokine responses observed were T-cell specific. The results demonstrate the importance of the adjuvant liposomal lipid A for the induction of HIV-1 Gag p24 -specific CD8+ T-cells, effector CD4+ T-cells, and cytokines with a Th-1 type profile after immunization with L(p24+LA).

Human immunodeficiency virus type 1 Gag p24 alters the composition of immunoproteasomes and affects antigen presentation.

Proteasomes are the major source of proteases responsible for the generation of peptides bound to major histocompatibility complex class I molecules. Antigens, adjuvants, and cytokines can modulate the composition and enzymatic activity of proteasomes and thus alter the epitopes generated. In the present study, we examined the effect of human immunodeficiency virus type 1 (HIV-1) p24 on proteasomes from a dendritic cell line (JAWS II), from a macrophage cell line (C2.3), and from murine primary bone marrow-derived macrophages and dendritic cells. HIV-1 p24 downregulated PA28beta and the beta2i subunit of the immunoproteasome complex in JAWS II cells but did not decrease the immunoproteasome subunits in macrophages, whereas in primary dendritic cells, PA28alpha, beta2i, and beta5i were downregulated. Exposure of JAWS II cells and primary dendritic cells to HIV-1 p24 for 90 min significantly decreased the presentation of ovalbumin to a SIINFEKL-specific CD8(+) T-cell hybridoma. The decrease in antigen presentation and the downmodulation of the immunoproteasome subunits in JAWS II cells and primary dendritic cells could be overcome by pretreating the cells with gamma interferon for 6 h or by exposing the cells to HIV-1 p24 encapsulated in liposomes containing lipid A. These results suggest that early antigen processing kinetics could influence the immunogenicity of CD8(+) T-cell epitopes generated.

Modulation of immunoproteasome subunits by liposomal lipid A.

Liposomes are phospholipid vesicles that have been used as carriers of antigens and adjuvants. Lipid A, the endotoxic moiety of Gram-negative bacterial lipopolysaccharide is a potent adjuvant and incorporation into liposomes essentially reduces the endotoxic activity of lipid A. In this study, we analyzed the effect of liposomal lipid A [L(LA)] on the MHC class I antigen processing machinery in murine antigen presenting cells (APCs). L(LA) enhanced the surface expression of MHC class I, class II, CD80, and CD86 molecules, induced the secretion of IFN-gamma, IL-12p40, TNF-alpha and IL-10, and caused a shift in the proteasome profile from constitutive to immunoproteasomes as observed by the induction of beta2i, beta5i, PA28alpha, and PA28beta subunits. L(LA) acts through the production of IFN-gamma as demonstrated with APCs generated from IFN-gamma knockout mice. L(LA) therefore appears to act as an intracellular adjuvant by upregulating the antigen processing machinery, which could result in efficient antigen presentation.