Katherine Miles

Apoptotic cells protect mice from autoimmune inflammation by the induction of regulatory B cells

The maintenance of immune tolerance to apoptotic cells (AC) within an inflammatory milieu is vital to prevent autoimmunity. To investigate this, we administered syngeneic AC i.v. into mice carrying a cohort of ovalbumin (OVA)-specific transgenic T cells (DO11.10) along with OVA peptide and complete Freunds adjuvant, observing a dramatic increase in OVA-specific IL-10 secretion. Activated splenic B cells responded directly to AC, increasing secretion of IL-10, and this programming by AC was key to inducing T cell-derived IL-10. We went on to ask whether AC are able to modulate the course of autoimmune-mediated, chronic inflammation. AC given up to 1 month before the clinical onset of collagen-induced arthritis protected mice from severe joint inflammation and bone destruction. Antigen-specific CD4+ T cells again secreted significantly more IL-10, associated with a reduced titer of pathogenic anti-collagen II antibodies. Inhibition of IL-10 in vivo reversed the beneficial effects of AC. Passive transfer of B cells from AC-treated mice provided significant protection from arthritis. These data demonstrate that AC exert a profound influence on an adaptive immune response through the generation of CD19+ regulatory B cells, which in turn are able to influence the cytokine profile of antigen-specific effector T cells.

Dying and necrotic neutrophils are anti-inflammatory secondary to the release of α-defensins

Neutrophils are recruited to sites of injury but their timely removal is thought to be vital to prevent exacerbating inflammation. In addition, the recognition of apoptotic cells by cells of the innate immune system provides potent anti-inflammatory and anti-immunogenic signals. In this article, we describe how human neutrophils dying by apoptosis or necrosis release anti-inflammatory peptides, the α-defensins. This family of small cationic peptides effectively inhibits the secretion of multiple proinflammatory cytokines and NO from macrophages, the main innate immune cell found at sites of chronic inflammation. In addition, the systemic administration of necrotic neutrophil supernatants and α-defensins protects mice from a murine model of peritonitis. Hence. their effects may be far-reaching and serve to kill microbes while regulating a potentially tissue-destructive inflammatory response.

A tolerogenic role for Toll-like receptor 9 is revealed by B-cell interaction with DNA complexes expressed on apoptotic cells.

Intracellular protein complexes containing nucleic acids are common targets of autoantibodies in many autoimmune diseases. Central tolerance to these antigens is incomplete, yet nucleosomal DNA is expressed on the surface of cells dying by apoptosis. It is commonly believed that autoimmunity is prevented by the rapid uptake of apoptotic cells (ACs) by neighbors or professional phagocytes to which they deliver anti-inflammatory signals. Self-reactive, innate-like B cells contact and are selected by intracellular antigens expressed on ACs; however, how self-tolerance is maintained is not well understood. Here we report that IL-10 production by B cells, stimulated by contact with ACs, results from the engagement of Toll-like receptor 9 (TLR9) within the B cell after recognition of DNA-containing complexes on the surface of ACs. Until now, TLR9 ligation has been considered an inflammatory signal, but we have confirmed a hitherto unexpected immunoregulatory role by demonstrating the absence of the protective effect of ACs during experimental autoimmune encephalitis (EAE) in TLR9-deficient mice. Human circulating CD27+ B cells also respond to DNA-bearing ACs, but not to DNase-treated cells, by secreting IL-10. Chronic autoimmune disease may arise if this tolerance mechanism is not reimposed after episodes of inflammation, or if the regulatory B-cell response is subverted.

Inflammation-induced formation of fat-associated lymphoid clusters

Fat-associated lymphoid clusters (FALCs) are a type of lymphoid tissue associated with visceral fat. Here we found that the distribution of FALCs was heterogeneous, with the pericardium containing large numbers of these clusters. FALCs contributed to the retention of B-1 cells in the peritoneal cavity through high expression of the chemokine CXCL13, and they supported B cell proliferation and germinal center differentiation during peritoneal immunological challenges. FALC formation was induced by inflammation, which triggered the recruitment of myeloid cells that expressed tumor-necrosis factor (TNF) necessary for signaling via the TNF receptors in stromal cells. Natural killer T cells (NKT cells) restricted by the antigen-presenting molecule CD1d were likewise required for the inducible formation of FALCs. Thus, FALCs supported and coordinated the activation of innate B cells and T cells during serosal immune responses.

Plasma Cell Homeostasis: The Effects of Chronic Antigen Stimulation and Inflammation

Long-lived plasma cells (LLPCs) that maintain humoral immunity to previously encountered Ags occupy a compartment in the bone marrow (BM). The rules and mechanisms by which cells enter (and leave) this compartment are poorly understood. We looked at what happens to the LLPC compartment and to plasma cell lifespan in general, in situations in which Ag stimulation and/or inflammation persist. We find that chronic Ag supply causes the generation of short-lived plasma cells in the local lymphoid organ, at the expense of any LLPC production. Furthermore, we find that inflammation caused by infection (mediated via TNF-α) causes a dramatic mobilization of LLPCs from the BM, with a concomitant reduction in circulating Ab levels against previously immunized Ags. These data are discussed in the context of the capacity of the BM LLPC compartment and competition for entry to it.

Neutrophil-derived alpha defensins control inflammation by inhibiting macrophage mRNA translation

Significance Neutrophils are the major effectors of acute inflammation responding to tissue injury or infection. The clearance of apoptotic neutrophils by inflammatory macrophages also provides a powerful proresolution signal. Apoptotic or necrotic neutrophils also release abundant amounts of the antimicrobial peptides alpha defensins. In this report, we show that the most abundant of these peptides, HNP1 (Human Neutrophil Peptide 1), profoundly inhibits protein translation. It achieves this without affecting mRNA stability or preventing mRNA polysomal association. This is, to our knowledge, the first demonstration of a peptide released from one cell, a leukocyte, entering and directly modulating the translatome of another cell. It alludes to a previously unidentified mechanism, driven by dying neutrophils, that ensures the timely resolution of macrophage-driven inflammation, without compromising antimicrobial function. Neutrophils are the first and most numerous cells to arrive at the site of an inflammatory insult and are among the first to die. We previously reported that alpha defensins, released from apoptotic human neutrophils, augmented the antimicrobial capacity of macrophages while also inhibiting the biosynthesis of proinflammatory cytokines. In vivo, alpha defensin administration protected mice from inflammation, induced by thioglychollate-induced peritonitis or following infection with Salmonella enterica serovar Typhimurium. We have now dissected the antiinflammatory mechanism of action of the most abundant neutrophil alpha defensin, Human Neutrophil Peptide 1 (HNP1). Herein we show that HNP1 enters macrophages and inhibits protein translation without inducing the unfolded-protein response or affecting mRNA stability. In a cell-free in vitro translation system, HNP1 powerfully inhibited both cap-dependent and cap-independent mRNA translation while maintaining mRNA polysomal association. This is, to our knowledge, the first demonstration of a peptide released from one cell type (neutrophils) directly regulating mRNA translation in another (macrophages). By preventing protein translation, HNP1 functions as a “molecular brake” on macrophage-driven inflammation, ensuring both pathogen clearance and the resolution of inflammation with minimal bystander tissue damage.

Plasmacytoid Dendritic Cells Respond Directly to Apoptotic Cells by Secreting Immune Regulatory IL-10 or IFN-α

Plasmacytoid dendritic cells (pDCs) play a pivotal role in driving the autoimmune disease systemic lupus erythematosus, via the secretion of IFN-α in response to nuclear self-antigens complexed with autoantibodies. Apoptotic cells, generated at sites of inflammation or secondary lymphoid organs, are exposed to activated pDCs and also express the same nuclear antigens on their cell surface. Here, we show that in the absence of autoantibodies, activated pDCs directly respond to apoptotic cell-expressed chromatin complexes by secreting IL-10 and IL-6, which also induces T cells to secrete IL-10. Conversely, when activated by the viral mimetic CpG-A, apoptotic cells enhance their secretion of IFN-α. This study demonstrates that activated pDCs respond directly to apoptotic cells and may maintain tolerance via IL-10, or promote inflammation through secretion of IFN-α, depending on the inflammatory context.

Immune Tolerance to Apoptotic Self is mediated primarily by Regulatory B1a cells

The chronic autoimmune inflammatory diseases, systemic lupus erythematosus and Sjogren’s syndrome, develop when tolerance to apoptotic cells (ACs) is lost. We have previously reported that this tolerance is maintained by innate-like, IL-10 secreting regulatory B cells. Two questions remained. First, do these regulatory B cells belong predominantly to a single subset of steady-state B cells and second, what is their specificity? We report here that innate-like B cells with markers characteristic for B1a cells (CD43+veCD19hiCD5+veIgMhiIgDlo) constitute 80% of splenic and 96% of peritoneal B cells that respond to ACs by secreting IL-10. AC responsive B1a cells secrete self-reactive natural antibodies (NAbs) and IL-10, which is augmented by toll-like receptor (TLR) 7 or TLR9 stimulation. In so doing, they both accelerate the clearance of dying cells by macrophages and inhibit their potential to mount proinflammatory immune responses. While B1a cells make prolonged contact with ACs, they do not require TIM1 or complement to mediate their regulatory function. In an animal model of neural inflammation (experimental autoimmune encephalomyelitis), just 105 activated B1a B cells was sufficient to restrain inflammation. Activated B1a B cells also induced antigen-specific T cells to secrete IL-10. Hence, regulatory B1a cells specifically recognize and augment tolerance to apoptotic self via IL-10 and NAbs; but once activated, can also prevent autoimmune mediated inflammation.