Positron emission tomography and single photon emission computed tomography imaging of tertiary lymphoid structures during the development of lupus nephritis.

Lymphoid neogenesis occurs in tissues targeted by chronic inflammatory processes, such as infection and autoimmunity. In systemic lupus erythematosus (SLE), such structures develop within the kidneys of lupus-prone mice ((NZBXNZW)F1) and are observed in kidney biopsies taken from SLE patients with lupus nephritis (LN). The purpose of this prospective longitudinal animal study was to detect early kidney changes and tertiary lymphoid structures (TLS) using in vivo imaging. Positron emission tomography (PET) by tail vein injection of 18-F-fluoro-2-deoxy-D-glucose (18F-FDG)(PET/FDG) combined with computed tomography (CT) for anatomical localization and single photon emission computed tomography (SPECT) by intraperitoneal injection of 99mTC labeled Albumin Nanocoll (99mTC-Nanocoll) were performed on different disease stages of NZB/W mice (n = 40) and on aged matched control mice (BALB/c) (n = 20). By using one-way ANOVA analyses, we compared two different compartmental models for the quantitative measure of 18F-FDG uptake within the kidneys. Using a new five-compartment model, we observed that glomerular filtration of 18FFDG in lupus-prone mice decreased significantly by disease progression measured by anti-dsDNA Ab production and before onset of proteinuria. We could not visualize TLS within the kidneys, but we were able to visualize pancreatic TLS using 99mTC Nanocoll SPECT. Based on our findings, we conclude that the five-compartment model can be used to measure changes of FDG uptake within the kidney. However, new optimal PET/SPECT tracer administration sites together with more specific tracers in combination with magnetic resonance imaging (MRI) may make it possible to detect formation of TLS and LN before clinical manifestations.

ICAM1 expression is induced by proinflammatory cytokines and associated with TLS formation in aggressive breast cancer subtypes.

Intratumoral formation of tertiary lymphoid structures (TLS) within the tumor microenvironment is considered to be a consequence of antigen challenge during anti-tumor responses. Intracellular adhesion molecule 1 (ICAM1) has been implicated in a variety of immune and inflammatory responses, in addition to associate with triple negative breast cancer (TNBC). In this study, we detected TLS in the aggressive tumor phenotypes TNBC, HER2+ and luminal B, whereas the TLS negative group contained solely tumors of the luminal A subtype. We show that ICAM1 is exclusively expressed in TNBC and HER2 enriched subtypes known to be associated with inflammation and the formation of TLS. Furthermore, cell from normal mammary epithelium and breast cancer cell lines expressed ICAM1 upon stimulation with the proinflammatory cytokines TNFα, IL1ß and IFNγ. ICAM1 overexpression was induced in MCF7, MDA-MB-468 and SK-BR-3 cells regardless of hormone receptor status. Taken together, our findings show that ICAM1 is expressed in aggressive subtypes of breast cancer and its expression is inducible by well-known proinflammatory cytokines. ICAM1 may be an attractive molecular target for TNBC, but further investigations elucidating the role of ICAM1 in targeted therapies have to take into consideration selective subtypes of breast cancer.

Mesenchymal stem cells and T cells in the formation of Tertiary Lymphoid Structures in Lupus Nephritis.

Tertiary lymphoid structures (TLS) develop in the kidneys of lupus-prone mice and systemic lupus erythematosus (SLE) patients with lupus nephritis (LN). Here we investigated the presence of mesenchymal stem cells (MSCs) in the development of TLS in murine LN, as well as the role of human MSCs as lymphoid tissue organizer (LTo) cells on the activation of CD4+ T cells from three groups of donors including Healthy, SLE and LN patients. Mesenchymal stem like cells were detected within the pelvic wall and TLS in kidneys of lupus-prone mice. An increase in LTß, CXCL13, CCL19, VCAM1 and ICAM1 gene expressions were detected during the development of murine LN. Human MSCs stimulated with the pro-inflammatory cytokines TNF-α and IL-1ß significantly increased the expression of CCL19, VCAM1, ICAM1, TNF-α, and IL-1ß. Stimulated MSCs induced proliferation of CD4+ T cells, but an inhibitory effect was observed when in co-culture with non-stimulated MSCs. A contact dependent increase in Th2 and Th17 subsets were observed for T cells from the Healthy group after co-culture with stimulated MSCs. Our data suggest that tissue-specific or/and migratory MSCs could have pivotal roles as LTo cells in accelerating early inflammatory processes and initiating the formation of kidney specific TLS in chronic inflammatory conditions.

Tertiary lymphoid structures are associated with higher tumor grade in primary operable breast cancer patients.

BACKGROUND: Tertiary lymphoid structures (TLS) are highly organized immune cell aggregates that develop at sites of inflammation or infection in non-lymphoid organs. Despite the described role of inflammation in tumor progression, it is still unclear whether the process of lymphoid neogenesis and biological function of ectopic lymphoid tissue in tumors are beneficial or detrimental to tumor growth. In this study we analysed if TLS are found in human breast carcinomas and its association with clinicopathological parameters. METHODS: In a patient group (n = 290) who underwent primary surgery between 2011 and 2012 we assessed the interrelationship between the presence of TLS in breast tumors and clinicopathological factors. Prognostic factors were entered into a binary logistic regression model for identifying independent predictors for intratumoral TLS formation. RESULTS: There was a positive association between the grade of immune cell infiltration within the tumor and important prognostic parameters such as hormone receptor status, tumor grade and lymph node involvement. The majority of patients with high grade infiltration of immune cells had TLS positive tumors. In addition to the degree of immune cell infiltration, the presence of TLS was associated with organized immune cell aggregates, hormone receptor status and tumor grade. Tumors with histological grade 3 were the strongest predictor for the presence of TLS in a multivariate regression model. The model also predicted that the odds for having intratumoral TLS formation were ten times higher for patients with high grade of inflammation than low grade. CONCLUSIONS: Human breast carcinomas frequently contain TLS and the presence of these structures is associated with aggressive forms of tumors. Locally generated immune response with potentially antitumor immunity may control tumorigenesis and metastasis. Thus, defining the role of TLS formation in breast carcinomas may lead to alternative therapeutic approaches targeting the immune system.

Nucleosomes contribute to increase mesangial cell chemokine expression during the development of lupus nephritis.

Nucleosomes represent a set of major autoantigens in the induction of systemic lupus erythematosus (SLE), and appear to be essential for the development of lupus nephritis. Deposition of nucleosome-containing immune complexes within the mesangial matrix and activation of mesangial cells may be important in the progression of lupus nephritis from a "sleeping" minimal change disease state to a full blown disease state. This study investigated the renal cytokine profile both in vivo during the development of the disease in (NZBxNZW)F1 (B/W) mice, and in vitro in primary B/W mesangial cells stimulated with nucleosomes, nucleosome-IgG complexes, and anti-dsDNA mAb respectively. Nucleosomes alone stimulated primary mesangial cells in a dose dependent manner. Of the chemokines produced by activated mesangial cells, CCL2, CCL7, CCL20, CXCL1, CXCL2 and CXCL5 were highly up-regulated compared to unstimulated cells. These chemokines were also increased in vivo in anti-dsDNA antibody positive and nephritic B/W kidneys, and was accompanied by infiltration of neutrophils, macrophages, T and B cells. This study provides a link between nucleosome-containing immune complexes, activation of mesangial cells, infiltration of effector cells and the development of lupus nephritis. Nucleosomes are pro-inflammatory and trigger innate immune responses, and thus are not only passive targets for autoantibodies but may play an active role in lupus pathogenesis. The removal or increased enzymatic destruction of nucleosomes, and/or the inhibition of mesangial cell activation may be possible treatment strategies in lupus nephritis.

Circulating levels of chromatin fragments are inversely correlated with anti-dsDNA antibody levels in human and murine systemic lupus erythematosus.

Anti-dsDNA antibodies represent a central pathogenic factor in Lupus nephritis. Together with nucleosomes they deposit as immune complexes in the mesangial matrix and along basement membranes within the glomeruli. The origin of the nucleosomes and when they appear e.g. in circulation is not known. Serum samples from autoimmune (NZBxNZW)F1 mice, healthy BALB/c mice, patients with SLE, RA and normal healthy individuals were analyzed for presence and amount of circulating anti-dsDNA antibodies and nucleosomal DNA. Here we use a quantitative PCR to measure circulating DNA in sera. We demonstrate an inverse correlation between anti-dsDNA antibodies and the DNA concentration in the circulation in both murine and human serum samples. High titer of anti-DNA antibodies in human sera correlated with reduced levels of circulating chromatin, and in lupus prone mice with deposition within glomeruli. The inverse correlation between DNA concentration and anti-dsDNA antibodies may reflect antibody-dependent deposition of immune complexes during the development of lupus nephritis in autoimmune lupus prone mice. The measurement of circulating DNA in SLE sera by using qPCR may indicate and detect the development of lupus nephritis at an early stage.

Nephritogenic antibodies bind in glomeruli through interaction with exposed chromatin fragments and not with renal cross-reactive antigens.

Cross-reactivity of anti-double stranded DNA (anti-dsDNA) antibodies with glomerular antigens has been postulated as a key factor in the development of lupus nephritis. Because no direct proof has been presented on anti-dsDNA antibodies binding in vivo to glomerular structures, we have analysed the binding of potentially nephritogenic anti-dsDNA antibodies to α-actinin and laminin. By enzyme-linked immunosorbent assay and surface plasmon resonance (SPR) analyses, we demonstrate that monoclonal antibodies (mAbs) bind both double-stranded DNA and α-actinin at high affinity. However, when added to nephritic kidney sections they did not bind to such structures, but rather to nucleosome-containing structures within the mesangial matrix or the glomerular basement membranes (GBMs). Nucleosomes, anti-nuclear antibodies and complexes of them were tested for their binding to glomerular components such as agrin, perlecan and laminin using SPR analysis. Nucleosomes bound to laminin, marginally to agrin, but not to perlecan or heparan sulphate-depleted agrin. Anti-histone H2B and anti-nucleosome antibodies in complex with nucleosomes slightly increased the binding of nucleosomes to agrin, while binding to laminin was slightly decreased compared to nucleosomes alone. In conclusion, the availability of nucleosomal antigens and the binding of these antigens to components of the mesangial matrix and GBM seem crucial for the glomerular deposition of immune complexes.

Heparin exerts a dual effect on murine lupus nephritis by enhancing enzymatic chromatin degradation and preventing chromatin binding in glomerular membranes.

OBJECTIVE: Association of nucleosome-IgG immune complexes with glomerular basement membranes (GBMs) is an important event in the development of lupus nephritis. Preventing this binding and/or increasing nuclease sensitivity of nucleosomes may be viable strategies for the prevention of the disease. Theoretically, heparin may alter nucleosomal structure and increase sensitivity to proteinases and nucleases, and may also inhibit binding of nucleosomes and nucleosome-IgG complexes to basement membrane structures. The aim of this study was to investigate whether and eventually how heparin prevents murine lupus nephritis. METHODS: Surface plasmon resonance was used to analyze if heparin inhibits binding of nucleosomes to laminin and collagen. The effect of heparin on nuclease- and proteinase-mediated degradation of nucleosomes was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and agarose gel electrophoresis. In vitro results were compared with analyses in vivo in heparin-treated (NZB × NZW)F(1) mice. Anti-double-stranded DNA antibody production, deposition of nucleosome-IgG complexes in GBMs, and development of proteinuria were monitored, and circulating chromatin fragments were quantified using quantitative polymerase chain reaction. RESULTS: In vitro studies demonstrated that heparin increased enzymatic degradation of nucleosomes and almost completely inhibited binding of nucleosomes to laminin and collagen. (NZB × NZW)F(1) mice treated with heparin demonstrated delayed or no antibody production and higher variation of circulating chromatin levels compared with untreated control mice. This effect was accompanied by highly reduced nucleosome-IgG complexes in GBMs and delayed development of nephritis. CONCLUSION: Increasing the degradation of nucleosomes, reducing their immunogenicity, and preventing binding of nucleosome-IgG complexes in glomeruli together provide an alternative basis for the treatment of lupus nephritis.

Deposition of chromatin-IgG complexes in skin of nephritic MRL-lpr/lpr mice is associated with increased local matrix metalloprotease activities.

Chromatin-IgG complexes appear as electron dense structures (EDS) in glomerular basement membranes in lupus nephritis. Here, we present results of comparative analyses of the composition of EDS in murine lupus dermatitis and nephritis. One focus was to perform an analytical approach to understand why such complex structures bind skin basement membrane components. Transcription of skin membrane-encoding genes was analysed to see if expression of such genes was increased, eventually indicating that binding capacity of immune complexes increased when dermatitis developed. Variations in matrix metalloprotease 2 (MMP2), MMP9 and Dnase1 mRNA levels and enzymatic activities were correlated with circulatory chromatin-IgG complexes and deposition in skin. We also examined if glomerular deposits of EDS predicted similar deposits in skin of (NZB x NZW)F1 or MRL-lpr/lpr mice, as we observed chromatin-IgG complexes in capillary lumina in skin and glomeruli in both strains. EDS consisting of chromatin fragments and IgG were found sub-epidermally in skin with LE-like lesions of end-stage nephritic MRL-lpr/lpr mice. Dermal MMP-encoding genes were up-regulated during disease progression, and gelatinolytic activity was increased in affected skin. Dnase1 mRNA level and total nuclease activity remained stable in skin during the disease, in contrast to progressive loss of renal Dnase1 mRNA and total renal nuclease activity during development of nephritis. Loss of renal Dnase1 may explain release of chromatin fragments, while increased MMP activity may disrupt membranes making them accessible for chromatin fragment-IgG complexes. Circulatory chromatin-IgG complexes, and up-regulated intradermal MMP activity may be crucial for deposition of immune complexes in skin of lupus-prone mice.

Lupus nephritis: the central role of nucleosomes revealed.

Systemic lupus erythematosus (SLE) is an autoimmune syndrome characterized by autoantibodies to nuclear constituents. Some of these antibodies are diagnostically important, whereas others act as disease-modifying factors. One clinically important factor is autoantibodies against dsDNA and nucleosomes, which have overlapping diagnostic and nephritogenic impact in SLE. Although a scientific focus for 5 decades, the molecular and cellular origin of these antibodies, and why they are associated with lupus nephritis, is still not fully understood. A consensus has, however, evolved that antibodies to dsDNA and nucleosomes are central pathogenic factors in the development of lupus nephritis. In contrast, no agreement has been reached as to which glomerular structures are bound by nephritogenic anti-nucleosome antibodies in vivo. Mutually contradictory paradigms and models have evolved simply because we still lack precise and conclusive data to provide definitive insight into how autoantibodies induce lupus nephritis and which specificity is critical in the nephritic process(es). In this review, data demonstrating the central role of nucleosomes in inducing and binding potentially nephritogenic antibodies to DNA and nucleosomes are presented and discussed. These autoimmune-inducing processes are discussed in the context of Matzinger's danger model (Matzinger P: Friendly and dangerous signals: is the tissue in control? Nat Immunol 2007, 8:11-13; Matzinger P: The danger model: a renewed sense of self. Science 2002, 296:301-305; Matzinger P: Tolerance, danger, and the extended family. Annu Rev Immunol 1994, 12:991-1045) and Medzhitov's and Janeway's (Medzhitov R, Janeway CA Jr: Decoding the patterns of self and nonself by the innate immune system. Science 2002, 296:298-300; Medzhitov R, Janeway CA Jr: How does the immune system distinguish self from nonself? Semin Immunol 2000, 12:185-188; Janeway CA Jr, Medzhitov R: Innate immune recognition. Annu Rev Immunol 2002, 20:197-216) distinction of noninfectious self (NIS) and infectious nonself (INS). The mechanisms leading to production of potentially nephritogenic anti-nucleosome antibodies and to overt lupus nephritis are interpreted in the context of these paradigms.

Glomerular expression of large polyomavirus T antigen in binary tet-off regulated transgenic mice induces apoptosis, release of chromatin and initiates a lupus-like nephritis.

Binary tetracycline-regulated polyomavirus large T antigen transgenic mice were generated to study immunological tolerance for nucleosomes. Expression of T antigen resulted in binding of the protein to chromatin, and released T antigen-nucleosome complexes from dying cells maintained anti-dsDNA and anti-nucleosome antibody-production by activating autoimmune nucleosome-specific B cells and CD4+ and CD8+ T antigen specific T cells. Glomerular T antigen expression was observed in these mice. Here, we demonstrate that this expression was linked to glomerular cell apoptosis, release of nucleosomes and association of nucleosomes with glomerulus basement membranes, detected as electron dense structures. Immune electron microscopy (IEM) revealed that these structures were glomerular targets for induced anti-dsDNA and anti-T antigen antibodies. Co-localization IEM demonstrated that in vivo-bound auto-antibodies co-localized with experimental monoclonal antibodies to dsDNA and to T antigen. A comparative analysis of glomeruli from nephritic (NZWxNZB)F1 and T antigen expressing transgenic mice revealed deposition of nucleosomes in glomerular capillary and mesangial matrix membranes and binding of anti-nucleosome antibodies in both mice strains. A controlled experimental model that may elucidate the initial events accounting for nucleosome-mediated nephritis has not been available. The transgenic mouse may be important to describe early immunological and cellular events accounting for the enigmatic lupus nephritis.

A central role of nucleosomes in lupus nephritis.

Lupus nephritis is characterized by the presence of subendothelial and subepithelial immune complexes and thickening of the glomerular basement membranes (GBM). Electron-dense structures (EDS) in mesangium and GBM have been demonstrated to constitute target structures for nephritogenic autoantibodies in vivo. Whether these antibodies bind nucleosomal antigens within the EDS or cross-react with components of the GBM has not been resolved. Data recently published point at intra-GBM-associated nucleosomes as target for the nephritogenic autoantibodies. Colocalization IEM has demonstrated that autoantibodies and experimental antibodies against DNA, histones, or transcription factors like TATA box-binding protein colocalize in the EDS. By using terminal transferase in situ nick-end labeling in combination with immune electron microscopy to detect DNA specifically in human and murine SLE kidneys, we were able to detect DNA within the EDS of nephritic glomeruli that corresponded with the detected autoantibodies.