PLASMACYTOID DENDRITIC CELL-MEDIATED HUMORAL AUTOIMMUNITY

Humoral autoimmunity is characterized by the breakdown of B cell immune tolerance to self-antigens and consequent production of pathogenic autoantibodies. Plasmacytoid dendritic cells (pDCs), a potent type I interferon (IFN-I) producer, have been linked to the pathogenesis of systemic lupus erythematosus (SLE), a prototypic systemic humoral autoimmune disease. However, the cellular events that stimulate the development of humoral autoimmunity as a result of pDC activation have not been characterized. Moreover, the B cell subset(s) responsible for the generation of autoantibodies remains to be clearly identified. The immunization of DNA-containing amyloids into non-autoimmune mice triggers the activation of pDCs and induction of lupus-like disease, characterized by the production of autoantibodies. Using this lupus model that is dependent on pDC activation and IFN-I production, we delineated the B cell responses elicited during the break of tolerance and characterized the key cellular players that may influence those responses. We found that, when IgM autoantibodies were induced, germinal centers were inhibited whereas immature B cells were activated and expanded. Such interesting observation suggested that humoral autoimmunity may arise from B cells outside germinal centers. While pDCs were involved in the overt activation of immature B cells, type II interferon (IFN-II) promoted their expansion. In addition, both IFN-I and IFN-II were required for isotype-class switch of autoantibodies thereby the generation of pathogenic subtypes. We further determined that IFN-II was produced by natural killer (NK) cells, which contributed to the development of humoral autoimmunity. In contrast, NKT cells suppressed the autoimmune B cell response. Last, we demonstrated that serum amyloid P-component, a humoral factor that binds to amyloids, prevented the activation of pDCs and IFN-I production thus may exert a protective role against humoral autoimmunity. Our results established a functional link between IFN-I and IFN-II, where IFN-I from pDCs and IFN-II from NK cells are essential in stimulating multiple types of adaptive immune cells to coordinate the differentiation and expansion of self-reactive B cells. Selective targeting of the key cellular and molecular players may lead to innovative therapies for SLE and other autoimmune diseases

Methylated BSA Mimics Amyloid-Related Proteins and Triggers Inflammation

The mechanistic study of inflammatory or autoimmune diseases requires the generation of mouse models that reproduce the alterations in immune responses observed in patients. Methylated bovine serum albumin (mBSA) has been widely used to induce antigen-specific inflammation in targeted organs or in combination with single stranded DNA (ssDNA) to generate anti-nucleic acids antibodies in vivo. However, the mechanism by which this modified protein triggers inflammation is poorly understood. By analyzing the biochemical properties of mBSA, we found that mBSA exhibits features of an intermediate of protein misfolding pathway. mBSA readily interact with a list of dyes that have binding specificity towards amyloid fibrils. Intriguingly, mBSA displayed cytotoxic activity and its binding to ssDNA further enhanced formation of beta-sheet rich amyloid fibrils. Moreover, mBSA is recognized by the serum amyloid P, a protein unanimously associated with amyloid plaques in vivo. In macrophages, we observed that mBSA disrupted the lysosomal compartment, signaled along the NLRP3 inflammasome pathway, and activated caspase 1, which led to the production of IL-1β. In vivo, mBSA triggered rapid and prominent immune cell infiltration that is dependent on IL-1β induction. Taken together, these data demonstrate that by mimicking amyloidogenic proteins mBSA exhibits strong innate immune functions and serves as a potent adjuvant. These findings advance our understanding on the underlying mechanism of how aberrant immune responses lead to autoimmune reactions

Is the combination of immunotherapy and radiotherapy in non-small cell lung cancer a feasible and effective approach?

For many years, conventional oncologic treatments such as surgery, chemotherapy, and radiotherapy (RT) have dominated the field of non-small-cell lung cancer (NSCLC). The recent introduction of immunotherapy (IT) in clinical practice, especially strategies targeting negative regulators of the immune system, so-called immune checkpoint inhibitors, has led to a paradigm shift in lung cancer as in many other solid tumors. Although antibodies against programmed death protein-1 (PD-1) and programmed death ligand-1 (PD-L1) are currently on the forefront of the immuno-oncology field, the first efforts to eradicate cancer by exploiting the host's immune system date back to several decades ago. Even then, researchers aimed to explore the addition of RT to IT strategies in NSCLC patients, attributing its potential benefit to local control of target lesions through direct and indirect DNA damage in cancer cells. However, recent pre-clinical and clinical data have shown RT may also modify antitumor immune responses through induction of immunogenic cell death and reprogramming of the tumor microenvironment. This has led many to reexamine RT as a partner therapy to immuno-oncology treatments and investigate their potential synergy in an exponentially growing number of clinical trials. Herein, the authors review the rationale of combining IT and RT across all NSCLC disease stages and summarize both historical and current clinical evidence surrounding these combination strategies. Furthermore, an overview is provided of active clinical trials exploring the IT-RT concept in different settings of NSCLC

mBSA shares properties with soluble amyloid precursor.

<p>(A) Gel shift analysis of salmon sperm ssDNA that was pre-mixed with mBSA in the presence of different amounts of heparin. (B) Assessment of cell death of RPMI 8226 cells cultured 24 hrs with different amounts of BSA or mBSA. Shown are representative results of PI staining from 2 independent experiments. (C) Assessment of dead RPMI 8226 cell population cultured with different amounts of BSA or mBSA that were pre-incubated with medium, DNA or heparin. Results shown are representative of 2 independent experiments. (D) Fluorescence emission profiles of bis-ANS obtained after incubation in PBS, with BSA or with mBSA.</p

Sequences of primers used for quantitative PCR analysis.

<p>Sequences of primers used for quantitative PCR analysis.</p

mBSA displays features of amyloid.

<p>(A) Fluorescence intensity of Thioflavin T at 480 nm in buffer, with BSA, mBSA, Aβ or with the reverse control peptide. Error bars are means ± SD of 3 independent experiments. **p<0.005. (B) Gel shift analysis of salmon sperm ssDNA pre-mixed with different amounts of BSA or mBSA. (C) Thioflavin S fluorescence in the presence of different BSA proteins or aggregates observed under fluorescence microscopy. Original magnification 100X. scale bar: 20 µm. (D) Fluorescence emission profiles of Congo Red in the presence of buffer, different proteins or aggregates. (E) Birefringence of Congo Red on mBSA or mBSA plus ssDNA aggregate observed by polarized light microscopy. Original magnification 40X. bar: 50 µm. (F) Transmission electron microscopy analysis of mBSA or mBSA plus ssDNA. Bar: 100 nm.</p

mBSA elicits IL-1β production by activating inflammasome.

<p>(A) Secretion of IL-1β by BMDM stimulated with different forms of BSA or Aβ peptides for 2 hours. Error bars are means ± SD of 5 independent experiments. ****p<0.0001. (B) Caspase 1 activation in BMDMs stimulated with different forms of BSA analyzed by FLICA assay. Representative result of 3 independent experiments is shown. Original magnification 100X. bar: 20 µm. (C) Acridine orange staining of BMDM stimulated by different forms of BSA to reveal lysosome integrity. Original magnification of fluorescence microscopy 100X. bar: 20 µm. (D) Secretion of IL-1β by BMDM induced by different forms of BSA in the presence of different inhibitors. Error bars are means ± SD of 2 independent experiments. *p<0.05, **p<0.005. (E) Secretion of IL-1β by BMDM induced by different forms of BSA or Aβ peptides in the presence or not of the NRLP3 inhibitor glybenclamide. Error bars are means ± SD of data obtained with cells from 4 different mice. *p<0.05, **p<0.005. (F) Secretion of IL-1β by BMDM induced by different forms of BSA in the presence of the caspase 1 inhibitor Ac-YVAD-CMK. Error bars are means ± SD of data obtained with cells from 3 different mice. *p<0.05, **p<0.005.</p

Serum amyloid P binds to mBSA.

<p>(A) Binding of SAP to different amounts of Aβ or the reverse control peptide was assessed by ELISA. Similar results were obtained from 2 independent experiments. (B) Binding of SAP to different amounts of BSA or mBSA with or without ssDNA was assessed by ELISA. Error bars are means ± SD of 2 independent experiments.</p

mBSA triggers inflammation in vivo.

<p> (A) Numbers of infiltrating macrophages (left), monocytes (middle) and neutrophils (right) in the peritoneum of mice 4 h after <i>i.p.</i> injection of different stimuli. Error bars are means ± SD of 4 mice per group. *p<0.05, **p<0.005. (B) Levels of IL-1α (left) and IL-1β (right) secreted in the peritoneal lavages. *p<0.05. (C) Gene expression of peritoneal exudate cells presented as a heat map. One BSA-injected animal was used as a reference. Each block represents one mouse. (D) Plot of induced transcript expression of the chemokines from the bottom cluster of *p<0.05, **p<0.005. (C). (E) Numbers of infiltrating macrophages (left), monocytes (middle) and neutrophils (right) in the peritoneum of wild-type or IL-1β^−/− mice 4 h after <i>i.p.</i> injection of different stimuli. Error bars are means ± SD of 3 mice per group. **p<0.005.</p