Ranjit Sahu

Structure and function of renal macrophages and dendritic cells from lupus-prone mice.

To characterize renal macrophages and dendritic cells (DCs) in 2 murine models of lupus nephritis.

The Yaa Locus and IFN-α Fine-Tune Germinal Center B Cell Selection in Murine Systemic Lupus Erythematosus

Male NZW/BXSB.Yaa (W/B) mice express two copies of TLR7 and develop pathogenic autoantibodies, whereas females with only one copy of TLR7 have attenuated disease. Our goal was to analyze the regulation of the autoantibody response in male and female W/B mice bearing the autoreactive site-directed H chain transgene 3H9. Serum anti-dsDNA Abs appeared in males at 12 wk, and most had high-titer IgG anti-dsDNA and anti-cardiolipin Abs and developed >300 mg/dl proteinuria by 8 mo. Females had only low-titer IgG anti-cardiolipin Abs, and none developed proteinuria by 1 y. Males had a smaller marginal zone than females with a repertoire that was distinct from the follicular repertoire, indicating that the loss of marginal zone B cells was not due to diversion to the follicular compartment. Vk5-43 and Vk5-48, which were rare in the naive repertoire, were markedly overrepresented in the germinal center repertoire of both males and females, but the VJ junctions differed between males and females with higher-affinity autoreactive B cells being selected into the germinal centers of males. Administration of IFN-α to females induced anti-cardiolipin and anti-DNA autoantibodies and proteinuria and was associated with a male pattern of junctional diversity in Vk5-43 and Vk5-48. Our studies are consistent with the hypothesis that presence of the Yaa locus, which includes an extra copy of Tlr7, or administration of exogenous IFN-α relaxes the stringency for selection in the germinal centers resulting in increased autoreactivity of the Ag-driven B cell repertoire.

Anti–tumor necrosis factor α treatment of interferon‐α–induced murine lupus nephritis reduces the renal macrophage response but does not alter glomerular immune complex formation

OBJECTIVE To analyze the mechanism for the therapeutic effects of tumor necrosis factor α (TNFα) inhibition in a murine model of systemic lupus erythematosus. METHODS We used the (NZB × NZW)F(1) (NZB/NZW) mouse model of interferon-α-induced lupus nephritis and treated mice with TNF receptor type II (TNFRII) Ig after TNFα expression was detected in the kidneys. Autoantibodies were measured by enzyme-linked immunosorbent assay (ELISA), and autoantibody- forming cells were determined using an enzyme-linked immunospot assay. Activation of splenocytes was analyzed by flow cytometry. Kidneys were harvested and analyzed using flow cytometry, immunohistochemistry, ELISA, Western blotting, and real-time polymerase chain reaction. RESULTS TNFRII Ig treatment stabilized nephritis and markedly prolonged survival. Autoantibody production and systemic immune activation were not inhibited, but the renal response to glomerular immune complex deposition was attenuated. This was associated with decreases in renal production of chemokines, renal endothelial cell activation, interstitial F4/80(high) macrophage accumulation, tubular damage, and oxidative stress. In contrast, perivascular lymphoid aggregates containing B cells, T cells, and dendritic cells accumulated unabated. CONCLUSION Our data suggest that TNFα is a critical cytokine that amplifies the response of the nephron to immune complex deposition, but that it has less influence on the response of the systemic vasculature to inflammation.

BAFF/APRIL Inhibition Decreases Selection of Naive but Not Antigen-Induced Autoreactive B Cells in Murine Systemic Lupus Erythematosus

BAFF inhibition is a new B cell-directed therapeutic strategy for autoimmune disease. Our purpose was to analyze the effect of BAFF/APRIL availability on the naive and Ag-activated B cell repertoires in systemic lupus erythematosus, using the autoreactive germline D42 H chain (glD42H) site-directed transgenic NZB/W mouse. In this article, we show that the naive Vκ repertoire in both young and diseased glD42H NZB/W mice is dominated by five L chains that confer no or low-affinity polyreactivity. In contrast, glD42H B cells expressing L chains that confer high-affinity autoreactivity are mostly deleted before the mature B cell stage, but are positively selected and expanded in the germinal centers (GCs) as the mice age. Of these, the most abundant is VκRF (Vκ16-104*01), which is expressed by almost all IgG anti-DNA hybridomas derived from the glD42H mouse. Competition with nonautoreactive B cells or BAFF/APRIL inhibition significantly inhibited selection of glD42H B cells at the late transitional stage, with only subtle effects on the glD42H-associated L chain repertoire. However, glD42H/VκRF-encoded B cells were still vastly overrepresented in the GC, and serum IgG anti-DNA Abs arose with only a slight delay. Thus, although BAFF/APRIL inhibition increases the stringency of negative selection of the naive autoreactive B cell repertoire in NZB/W mice, it does not correct the major breach in B cell tolerance that occurs at the GC checkpoint.

TLR7 Influences Germinal Center Selection in Murine SLE

TLR7 enhances germinal center maturation and migration of B cells to the dark zone where proliferation and somatic hypermutation occur. Our goal was to determine how Tlr7 dose influences selection of the autoreactive B cell repertoire in NZW/BXSB. Yaa mice bearing the site-directed heavy chain transgene 3H9 that encodes for the TLR7 regulated anti-CL response. To create a physiologic setting in which autoreactive B cells compete for survival with non-autoreactive B cells, we generated bone marrow chimeras in which disease onset occurred with similar kinetics and the transferred 3H9+ female non-Yaa, male Yaa or male TLR7-/Yaa cells could be easily identified by positivity for GFP. Deletion of 3H9 B cells occurred in the bone marrow and the remaining 3H9 follicular B cells manifested a decrease in surface IgM. Although there were differences in the naïve repertoire between the chimeras it was not possible to distinguish a clear pattern of selection against lupus related autoreactivity in TLR7-/Yaa or female chimeras. By contrast, preferential expansion of 3H9+ B cells occurred in the germinal centers of male Yaa chimeras. In addition, although all chimeras preferentially selected 3H9/Vκ5 encoded B cells into the germinal center and plasma cell compartments, 3H9 male Yaa chimeras had a more diverse repertoire and positively selected the 3H9/Vκ5-48/Jκ4 pair that confers high affinity anti-cardiolipin activity. We were unable to demonstrate a consistent effect of Tlr7 dose or Yaa on somatic mutations. Our data show that TLR7 excess influences the selection, expansion and diversification of B cells in the germinal center, independent of other genes in the Yaa locus.

Early detection of colorectal cancer relapse by infrared spectroscopy in “normal” anastomosis tissue

Abstract. Colorectal cancer is one of the most aggressive cancers usually occurring in people above the age of 50 years. In the United States, colorectal cancer is the third most diagnosed cancer. The American Cancer Society has estimated 96,830 new cases of colon cancer and 40,000 new cases of rectal cancer in 2014 in the United States. According to the literature, up to 55% of colorectal cancer patients experience a recurrence within five years from the time of surgery. Relapse of colorectal cancer has a deep influence on the quality of patient life. Infrared (IR) spectroscopy has been widely used in medicine. It is a noninvasive, nondestructive technique that can detect changes in cells and tissues that are caused by different disorders, such as cancer. Abnormalities in the colonic crypts, which are not detectable using standard histopathological methods, could be determined using IR spectroscopic methods. The IR measurements were performed on formalin-fixed, paraffin-embedded colorectal tissues from eight patients (one control, four local recurrences, three distant recurrences). A total of 128 crypts were measured. Our results showed the possibility of differentiating among control, local, and distant recurrence crypts with more than a 92% success rate using spectra measured from the crypts’ middle sites.

Analysis of Renal Mononuclear Phagocytes in Murine Models of SLE

In this chapter we present methods for the isolation and characterization of mononuclear phagocytes from the kidneys of mice with SLE. Activation of these cells is associated with the onset of clinical disease in mice and infiltration with these cells is associated with poor prognosis in humans. Using magnetic beads followed by flow cytometric sorting, pure populations of cells are obtained that are functional in a variety of assays. Sufficient numbers of cells are obtained for genomic characterization. An analysis of the function of these cells should lead to a better understanding of the inflammatory processes that cause renal impairment in SLE and other renal inflammatory diseases.

Molecular studies of lupus nephritis kidneys

Lupus nephritis is a devastating complication of systemic lupus erythematosus (SLE) for which current therapies are insufficiently effective. Histologic evaluation of renal biopsies is a poor predictor of therapeutic response or outcome. Integrated immunologic, genomic and proteomic approaches may yield new insights into disease pathogenesis and thereby improve therapeutic strategies for lupus nephritis. Given the lack of sequential biopsies from humans, it also remains essential to study informative animal models of disease. Cross-species analyses can identify cells or pathways that are relevant to human disease and can be further studied in mouse models. Using a systems biology approach in which we compare molecular data from kidneys of three different mouse models of lupus nephritis with data from human lupus biopsies, we have found that inflammatory events escalate rapidly around the time of proteinuria onset. This is followed by hypoxia and metabolic stress, and by tubular and endothelial dysfunction. The failure of complete reversal of these abnormalities may increase the sensitivity of the kidney to further insult. We further found that renal macrophages and dendritic cells are key players in lupus nephritis both in mouse models and humans and that macrophages display a hybrid molecular profile that reflects incomplete resolution of inflammation and excessive tissue remodeling. Finally, our studies have suggested several new biomarkers for disease stage that can now be tested longitudinally in human SLE patients.